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A Caulobacter crescentus microbicide prevents vaginal infection with HIV-1 and HSV-2 in preclinical models Farr, Christina 2016

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A Caulobacter crescentus microbicide prevents vaginal infection with HIV-1 and HSV-2 in preclinical models by  Christina Farr B.H.Sc. (Hons), McMaster University, 2008 M.Sc., University of Toronto, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Doctor of Philosophy in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2016  © Christina Farr, 2016 ii  Abstract  Over 2 million people are infected with HIV each year. The majority of these infections occur in women residing in low-income countries, where their access to and control over preventative measures is often limited. This suggests that female-controlled prevention options for HIV-1 are urgently needed. Microbicides, which can be topically applied to the vaginal tract in advance of sexual activity to protect from HIV-1 infection, represent a promising female-controlled prevention option. We have investigated the development of an HIV-1 specific microbicide using the non-pathogenic, freshwater bacterium Caulobacter crescentus. C. crescentus contains a Surface or S-layer that is easily modified for high-density display of recombinant proteins. We have developed 18 recombinant C. crescentus that display anti-HIV proteins, including decoy receptors and ligands, anti-viral lectins and fusion inhibitors, that can prevent various steps of the HIV-1 attachment and entry process. In vitro testing with these recombinant C. crescentus indicated that 15 were able to provide substantial protection from HIV-1 infection. Studies with immune-competent mice demonstrated that application of C. crescentus to the vaginal tract does not induce the production of inflammatory cytokines or recruitment of immune cells. To test for protection against HIV-1 in vivo we have combined the implantation of human fetal liver and thymus tissue with the intravenous injection of autologous CD34+ cells into NOD-scid IL2Rγnull mice to create humanized Bone Marrow-Liver-Thymus (BLT) mice and we have demonstrated that the peripheral blood of these mice contains human CD45+ cells, including CD4+ and CD8+ T cells, B cells, myeloid cells, and NK cells. Our data indicates that vaginal application of recombinant C. crescentus at the time of HIV-1JR-CSF infection provides protection from HIV-1 infection.  iii   Seven of the recombinant C. crescentus were predicted to also prevent infection with herpes simplex virus 2 (HSV-2). HSV-2 is a major co-morbidity for HIV-1 infection, contributing to a 2-4 fold increase in acquisition. Four recombinant C. crescentus provided significant protection from HSV-2 infection in vivo.  Taken together this data suggests that a C. crescentus based microbicide could be a safe and effective method to prevent infection with HIV-1 and HSV-2, having considerable impact on public health. iv  Preface  All of the work presented henceforth was conducted in the Life Sciences Institute and the Modified Barrier Facility at the University of British Columbia, Point Grey Campus, and the Child and Family Research Institute at BC Children’s Hospital. Dr. Horwitz and Dr. Smit were the supervisory authors on this thesis and were involved throughout for all the projects in concept formation and manuscript composition.  A version of the research presented in Chapter 2 has been published (Farr C, Nomellini JF, Ailon E, Shanina I, Sangsari S, Cavacini, LA, Smit J, Horwitz MS. 2013. Development of an HIV-1 Microbicide Based on Caulobacter crescentus: Blocking Infection by High-Density Display of Virus Entry Inhibitors. PLoS ONE 8(6): e65965. doi:10.1371/journal.pone.0065965). I was responsible for designing all experiments described in this manuscript. Dr. John Nomellini developed the recombinant C. crescentus and provided a figure demonstrating S-layer protein expression. Mr. Evan Ailon, Ms. Iryna Shanina and Mr. Sassan Sangsari provided preliminary data. Dr. Lisa Cavacini provided reagents and technical advice. I wrote the first draft of the manuscript, which was revised by Dr. Nomellini, Dr. Cavacini, Dr. John Smit and Dr. Marc Horwitz.  Portions of the research presented in Chapters 2, 3 and 5 has been submitted for publication (Farr Zuend C, Nomellini JF, Smit, J, Horwitz MS. Generation of a dual-target, safe, inexpensive microbicide that protects against HIV-1 and HSV-2, Submitted). For this work I was the lead investigator, responsible for experimental design, data collection and analysis. I wrote the first draft of this manuscript, which was revised together with Dr. Nomellini, Dr. Smit and Dr. Horwitz. Dr. Nomellini developed the recombinant C. crescentus used in this manuscript and provided a figure demonstrating S-layer protein expression. v    I was the lead investigator, responsible for experimental design, data collection and analysis for the work presented in Chapter 4. Ms. Iryna Shanina and Ms. Ana Citlali Marquez performed CD34+ cell isolation from fetal liver tissue.  Dr. Nomellini developed all recombinant C. crescentus used in this project. He performed the low pH extraction to verify expression of the recombinant S-layer protein and provided Figure 2.2.  Dr. Michael Jones provided the illustration demonstrating the HIV-1 attachment and entry process (Figure 1.1). This figure was adapted for Figure 2.1. Dr. Jones has made this illustration freely available on Wikipedia: https://en.wikipedia.org/wiki/HIV#/media/File:HIV_Membrane_fusion_panel.svg  All reagents provided by external research groups are indicated in the materials and methods.   All animal work was approved by the University of British Columbia Animal Care Committee, Protocol Numbers A13-0055, A12-0245 and A12-0234. Human ethics approval was obtained from the University of British Columbia Clinical Ethics Review board, certificate number H12-02480.  This work was supported by a grant from CIHR to Dr. Horwitz and Dr. Smit. Graduate student funding was provided by the University of British Columbia Four Year Fellowship and two Mitacs-Accelerate internships.  vi  Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ......................................................................................................................... vi List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiv List of Abbreviations ............................................................................................................... xviii Acknowledgements .................................................................................................................. xxiii Dedication .................................................................................................................................. xxv Chapter 1: Introduction ............................................................................................................... 1 1.1 Human Immunodeficiency Virus ................................................................................... 1 1.1.1 HIV-1 ....................................................................................................................... 1 1.1.2 HIV-1 transmission and pathogenesis ..................................................................... 4 1.1.2.1 HIV-1 transmission ........................................................................................... 4 1.1.2.2 HIV-1 pathogenesis ........................................................................................... 6 1.2 HIV prevention .............................................................................................................. 8 1.2.1 Microbicides ............................................................................................................ 9 1.2.1.1 Microbicide candidates under development ...................................................... 9 1.2.1.2 Bacteria based microbicides ............................................................................ 11 1.2.1.3 Tenofovir ......................................................................................................... 13 1.2.2 Pre-Exposure Prophylaxis ...................................................................................... 15 vii  1.2.3 Vaccination ............................................................................................................ 21 1.2.3.1 rgp120 HIV Vaccine Study (VAX004, AIDSVAX B/B) ............................... 22 1.2.3.2 Bivalent recombinant glycoprotein 120 (AIDSVAX B/E) ............................. 22 1.2.3.3 DNA/rAd5 Vaccine Study (HVTN 505) ......................................................... 23 1.2.3.4 MRKAd5 HIV-1 gag/pol/nef vaccine (Step Study) ........................................ 23 1.2.3.5 ALVAC-HIV and AIDSVAX B/E (RV144) ................................................... 23 1.3 Caulobacter crescentus ................................................................................................ 24 1.4 Humanized mice .......................................................................................................... 27 1.4.1 Mouse strains and early experiments ..................................................................... 27 1.4.2 Graft versus Host Disease in BLT mice ................................................................. 35 1.4.3 HIV infection in BLT mice .................................................................................... 36 1.5 Herpes simplex virus 2 ................................................................................................. 38 1.5.1 HSV-2 life cycle .................................................................................................... 39 1.5.2 HSV-2 pathogenesis ............................................................................................... 40 1.5.3 HSV-2 prevention .................................................................................................. 41 1.5.3.1 HSV-2 microbicides ........................................................................................ 41 1.5.3.2 HSV-2 vaccines ............................................................................................... 43 1.6 Research hypothesis and rationale ............................................................................... 43 1.6.1 Aim 1 ..................................................................................................................... 44 1.6.2 Aim 2 ..................................................................................................................... 45 1.6.3 Aim 3 ..................................................................................................................... 45 1.6.4 Aim 4 ..................................................................................................................... 46 viii  Chapter 2: In vitro protection from HIV-1 infection using recombinant Caulobacter crescentus ..................................................................................................................................... 47 2.1 Introduction .................................................................................................................. 47 2.2 Materials and methods ................................................................................................. 49 2.3 Results .......................................................................................................................... 55 2.3.1 Expression of recombinant proteins in the S-layer of C. crescentus ..................... 55 2.3.2 Inhibition of HIV-1 pseudovirus infection with Cc-MIP1α .................................. 63 2.3.3 Inhibition of HIV-1 pseudovirus infection with recombinant C. crescentus expressing domain 1 of CD4 or CD4-mimetic with the S-layer ....................................... 64 2.3.4 Anti-HIV lectins block HIV-1 pseudovirus infection ............................................ 66 2.3.5 Fusion inhibitors block HIV-1 pseudovirus infection ........................................... 69 2.3.6 GB virus C E2 protein blocks infection with HIV-1 pseudovirus ......................... 71 2.3.7 Cc-BmKn2 block HIV-1 pseudovirus infection while Cc-Mucroporin provides minimal inhibition ............................................................................................................. 72 2.3.8 Cc-Elafin has inconsistent activity against HIV-1 infection in vitro ..................... 75 2.3.9 Cc-A1AT has limited activity against HIV-1 infection ......................................... 76 2.3.10 Cc-gp120M15D and Cc-gp120NB did not provide protection from HIV-1 infection in vitro ................................................................................................................ 78 2.3.11 Comparison of C. crescentus JS4026 and JS4038 strains .................................... 79 2.3.12 Cc-Indo and Cc-Indo2 prevent HIV-1 infection in some HIV strains ................. 82 2.3.13 Many recombinant C. crescentus block infection with live HIV-1 ..................... 84 2.4 Discussion .................................................................................................................... 97 ix  Chapter 3: Caulobacter crescentus does not induce an immune response in the mouse vagina ......................................................................................................................................... 102 3.1 Introduction ................................................................................................................ 102 3.2 Materials and methods ............................................................................................... 103 3.3 Results ........................................................................................................................ 106 3.3.1 Intraperitoneal injection of C. crescentus induces low production of inflammatory cytokines ......................................................................................................................... 106 3.3.2 Low production of cytokines after vaginal application of C. crescentus ............. 108 3.3.3 No immune cell recruitment to the vaginal tract after C. crescentus application 109 3.3.4 No change in cells in the vaginal lavage fluid after topical application of C. crescentus ........................................................................................................................ 113 3.3.5 No antibodies produced in the vaginal lavage fluid or blood after vaginal application of C. crescentus ............................................................................................ 114 3.4 Discussion .................................................................................................................. 117 Chapter 4: Investigating whether Humanized Bone Marrow-Liver-Thymus (BLT) mice are protected from vaginal infection with HIV-1JR-CSF by a recombinant C. crescentus based microbicide ................................................................................................................................ 119 4.1 Introduction ................................................................................................................ 119 4.2 Materials and methods ............................................................................................... 121 4.3 Results ........................................................................................................................ 124 4.3.1 Development of humanized BLT mice ................................................................ 124 4.3.2 Analysis of HIV-1JR-CSF infection in humanized BLT mice ................................. 126 x  4.3.3 Cc-MIP1α may provide protection of NSG-BLT mice from vaginal infection with HIV-1JR-CSF ...................................................................................................................... 133 4.3.4 Cc-CD4 and Cc-CD4M33F23 may provide some measure of protection from vaginal infection with HIV-1JR-CSF in BLT mice ............................................................ 138 4.3.5 Recombinant C. crescentus expressing anti-viral lectins may provide variable levels of protection from vaginal infection with HIV-1JR-CSF in BLT mice ................... 144 4.3.6 Recombinant C. crescentus expressing fusion inhibitors may provide variable protection from vaginal infection with HIV-1JR-CSF in BLT mice .................................. 153 4.3.7 Cc-GBVCE2 may provide protection from vaginal infection with HIV-1JR-CSF . 161 4.3.8 Cc-BmKn2 does not protect from vaginal infection with HIV-1JR-CSF ................ 165 4.3.9 Cc-Elafin may protect from vaginal infection with HIV-1JR-CSF ......................... 168 4.3.10 Cc-A1AT may protect from vaginal infection with HIV-1JR-CSF ....................... 171 4.3.11 Cc-Indo may protect from vaginal infection with HIV-1JR-CSF .......................... 175 4.4 Discussion .................................................................................................................. 180 Chapter 5: Recombinant C. crescentus can provide protection from vaginal infection with Herpes simplex virus 2 .............................................................................................................. 184 5.1 Introduction ................................................................................................................ 184 5.2 Materials and methods ............................................................................................... 187 5.3 Results ........................................................................................................................ 190 5.3.1 Cc-Cyanovirin and Cc-Griffithsin protect from vaginal HSV-2 infection in a mouse model ................................................................................................................... 190 5.3.2 Cc-A1AT and Cc-Indo provide protection from HSV-2 infection ...................... 193 5.3.3 Protective C. crescentus induce early cytokine production in vaginal tract ........ 196 xi  5.4 Discussion .................................................................................................................. 202 Chapter 6: Conclusions and future directions ....................................................................... 206 6.1 Discussion .................................................................................................................. 206 6.2 C. crescentus expressing anti-HIV proteins provides significant protection from HIV-1 infection in vitro .................................................................................................................. 210 6.3 C. crescentus appears safe for topical application to the vaginal tract ...................... 212 6.4 Some recombinant C. crescentus provide protection from vaginal infection with HIV-1JR-CSF in the humanized BLT mouse .................................................................................. 213 6.5 Recombinant C. crescentus can prevent infection with HSV-2 ................................. 215 6.6 Conclusion and future direction ................................................................................. 217 6.6.1 How long does protection last? ............................................................................ 217 6.6.2 Will the microbicide provide protection during co-infections? ........................... 219 6.6.3 Will seminal plasma impact efficacy of the microbicide? ................................... 221 6.6.4 Conclusion ........................................................................................................... 223 Bibliography .............................................................................................................................. 225 Appendices ................................................................................................................................. 289  xii  List of Tables Table 1.1 PrEP clinical trials ..................................................................................................... 16 Table 1.2 HSV-2 microbicides ................................................................................................... 41 Table 2.1 Recombinant C. crescentus display constructs ........................................................ 60 Table 2.2 Comparing C. crescentus strains with and without the EPS layer ........................ 81 Table 2.3 HIV-1 viral blocking summary data ........................................................................ 95 Table 4.1 HIV-1JR-CSF infection in the BLT mouse model ..................................................... 128 Table 4.2 HIV-1JR-CSF + Cc-Control ........................................................................................ 131 Table 4.3 HIV-1JR-CSF + Cc-MIP1α ......................................................................................... 136 Table 4.4 HIV-1JR-CSF + Cc-CD4 .............................................................................................. 139 Table 4.5 HIV-1JR-CSF + Cc-CD4M33F23 ............................................................................... 142 Table 4.6 HIV-1JR-CSF + Cc-Cyanovirin .................................................................................. 148 Table 4.7 HIV-1JR-CSF + Cc-Microvirin ................................................................................... 150 Table 4.8 HIV-1JR-CSF + Cc-Griffithsin ................................................................................... 151 Table 4.9 HIV-1JR-CSF + Cc-Fuzeon ......................................................................................... 157 Table 4.10 HIV-1JR-CSF + Cc-T1249 ......................................................................................... 159 Table 4.11 HIV-1JR-CSF + Cc-C52 ............................................................................................ 160 Table 4.12 HIV-1JR-CSF + Cc-GBVCE2 ................................................................................... 163 Table 4.13 HIV-1JR-CSF + Cc-BmKn2 ...................................................................................... 167 xiii  Table 4.14 HIV-1JR-CSF + Cc-Elafin ......................................................................................... 170 Table 4.15 HIV-1JR-CSF + Cc-A1AT ......................................................................................... 173 Table 4.16 HIV-1JR-CSF + Cc-Indo ........................................................................................... 177 Table 4.17 BLT mice summary data ....................................................................................... 179 Table 5.1 HSV-2 summary data .............................................................................................. 200  Table B.2 Summary of BLT mouse cohorts .......................................................................... 294   xiv  List of Figures Figure 1.1 HIV-1 attachment and entry ..................................................................................... 3 Figure 1.2 HIV-1 transmission across the vaginal mucosa ....................................................... 6 Figure 2.1 Recombinant C. crescentus targets all aspects of HIV-1 attachment and entry . 56 Figure 2.2 Low pH demonstrating expression of recombinant S-layer proteins .................. 57 Figure 2.3 Inhibition of HIV-1 pseudovirus infection with Cc-MIP1α .................................. 64 Figure 2.4 Inhibition of HIV-1 pseudovirus infection with Cc-CD4 and Cc-CD4M33F23 . 65 Figure 2.5 Inhibition of HIV-1 pseudovirus infection with Cc-Cyanovirin, Cc-Microvirin and Cc-Griffithsin ....................................................................................................................... 68 Figure 2.6 Inhibitory activity against HIV-1 pseudovirus infection of Cc-Fuzeon, Cc-T1249 and Cc-C52 .................................................................................................................................. 70 Figure 2.7 Inhibitory activity against HIV-1 pseudovirus infection of Cc-GBVCE2 ........... 72 Figure 2.8 Cc-BmKn2 may block HIV-1 pseudovirus infection while Cc-Mucroporin provides minimal protection ...................................................................................................... 74 Figure 2.9 Cc-Elafin has inconsistent activity against HIV-1 infection ................................. 76 Figure 2.10 Cc-A1AT has limited activity against HIV-1 pseudovirus infection .................. 77 Figure 2.11 Cc-gp120M15D and Cc-gp120NB do not protect from HIV-1 pseudovirus infection ........................................................................................................................................ 78 Figure 2.12 Cc-Indo and Cc-Indo2 may block HIV-1 infection ............................................. 83 xv  Figure 2.13 Live virus blocking assays ...................................................................................... 85 Figure 3.1 Intraperitoneal injection of C. crescentus induces low production of inflammatory cytokines ............................................................................................................ 107 Figure 3.2 C. crescentus does not induce significant production of inflammatory cytokines after vaginal application ........................................................................................................... 109 Figure 3.3 No recruitment of immune cells to vaginal tissue following application of Cc-Control ....................................................................................................................................... 111  Figure 3.4 Vaginal cytology following application of Cc-Control ....................................... 113 Figure 3.5 No production of anti-Caulobacter antibodies after vaginal application of Cc-Control ....................................................................................................................................... 115 Figure 4.1 Human immune cell reconstitution in BLT mice ................................................ 125 Figure 4.2 HIV-1JR-CSF infection after vaginal exposure ....................................................... 128 Figure 4.3 HIV-1JR-CSF + Cc-Control ...................................................................................... 131 Figure 4.4 Cc-MIP1α may provide 71% protection from vaginal infection with HIV-1JR-CSF..................................................................................................................................................... 135 Figure 4.5 Cc-CD4M33F23 may provide some protection from vaginal infection with HIV-1JR-CSF but Cc-CD4 may not ..................................................................................................... 141 Figure 4.6 Recombinant C. crescentus expressing anti-viral lectins may provide variable levels of protection from vaginal infection with HIV-1JR-CSF in BLT mice .......................... 145 xvi  Figure 4.7 Recombinant C. crescentus expressing fusion inhibitors may provide variable protection from vaginal infection with HIV-1JR-CSF in BLT mice ........................................ 154 Figure 4.8 Cc-GBVCE2 may provides partial protection from vaginal infection with HIV-1JR-CSF ......................................................................................................................................... 162 Figure 4.9 Cc-BmKn2 does not appear to protect from vaginal infection with HIV-1JR-CSF..................................................................................................................................................... 166 Figure 4.10 Cc-Elafin may provide partial protection from vaginal infection with HIV-1JR-CSF ............................................................................................................................................... 169 Figure 4.11 Cc-A1AT may provide partial protection from vaginal infection with HIV-1JR-CSF ............................................................................................................................................... 172 Figure 4.12 Cc-Indo may provide partial protection from vaginal infection with HIV-1JR-CSF ............................................................................................................................................... 176 Figure 5.1 Cc-Griffithsin and Cc-Cyanovirin protect from HSV-2 infection ..................... 191 Figure 5.2 Cc-A1AT and Cc-Indo protection from HSV-2 infection ................................... 194 Figure 5.3 Protective C. crescentus induce early cytokine production in the vaginal tract ..................................................................................................................................................... 198 Figure A.1 C. crescentus titration ............................................................................................ 289 Figure A.2 HIV-1 clades A/D pseudovirus data ..................................................................... 290 Figure B.3 Representative flow cytometry staining of peripheral blood from a BLT mouse..................................................................................................................................................... 296 xvii  Figure B.4 Preliminary rectal microbicide testing ................................................................. 297 Figure C.1 Comparing dual protein expression in RsaA or combination of individual C. crescentus ................................................................................................................................... 298 Figure C.2 Preliminary combination data .............................................................................. 299 Figure C.3 Preliminary timed viral blocking assays .............................................................. 302  xviii  List of Abbreviations ACC – Animal Care Committee AIDS – Acquired Immunodeficiency Syndrome ANOVA – analysis of variance ART – anti-retroviral therapy BAT24 – before and after sex dosing, no more than 2 doses in 24 hours BLT mouse – bone marrow-liver-thymus humanized mouse Bp – base pair CD – cluster of differention cDC – conventional dendritic cell cDNA – complementary DNA CFRI – Child and Family Research Institute CCL – C-C chemokine ligands CCR – C-C chemokine receptor CPE – cytopathic effect CRM1 – chromosomal maintenance 1 CXCR – C-X-C chemokine receptor DC – dendritic cell DMEM – Dulbecco’s modified Eagle Medium DMSO – dimethyl sulfoxide DNA – deoxyribonucleic acid E - early EBV – Epstein-Barr Virus xix  EDTA - Ethylenediaminetetraacetic acid ELISA – enzyme linked immunosorbent assay EPS – extracellular polysaccharide ESCRT – endosomal sorting complexes required for transport FBS – fetal bovine serum FTC – emtricitabine G-CSF – granulocyte colony stimulating factor GM-CSF – granulocyte macrophage colony stimulating factor GALT – gut associated lymphoid tissue GI – gastro-intestinal tract gp – glycoprotein GvHD – graft versus host disease HEC - hydroxyethylcellulose HIV – Human Immunodeficiency Virus HPV – human papillomavirus HSC – hematopoietic stem cell HSV – herpes simplex virus, HSV-1, HSV-2 Hu-mice/hu-mouse – Humanized mice/mouse IE – immediate early IFN – interferon Ig – Immunoglobulin, IgD, IgM, IgG, IgA, IgE IL – interleukin IL2Rγc – interleukin 2 receptor gamma chain xx  kDa – kilo Dalton L – late LAT – latency associated transcript LEDGF – lens epithelium-derived growth factor LOD – limit of detection LPS – lipopolysaccharide MCP-1 – monocyte chemoattractant protein-1 MEM – Minimum essential media MHC – major histocompatibility complex, MHC I and MHC II MOI – multiplicity of infection mRNA – messenger RNA NIAID – National Institute of Allergy and Infectious Diseases NIH – National Institutes of Health NK cell – natural killer cell NKT cell – natural killer T cell NOD – non-obese diabetic mouse NOD-SCID – non-obese diabetic-severe combined immunodeficiency mouse NSG – NOD-SCID IL-2 receptor γ chain null mouse (NOD-scid IL2Rγnull) (complete deletion IL2Rγc) PAP – prostatic acid phosphotase PBMC – peripheral blood mononuclear cell PBS – phosphate buffered saline pDC – plasmacytoid dendritic cell xxi  pfu – plaque forming unit PHA - phytohemagglutinin PrEP – pre-exposure prophylaxis PrKdc – Protein kinase, DNA activated, catalytic polyprotein PYE – peptone yeast extract media R5 – HIV-1 that uses CCR5 coreceptor rAd5 – recombinant Adenovirus type 5 RANTES - Regulated on Activation, Normal T Expressed and Secreted (CCL5) RNA – ribonucleic acid RPMI 1640 – Roswell Park Memorial Institute media 1640 RT-qPCR – reverse transcriptase quantitative real time polymerase chain reaction SDS – sodium dodecyl sulphate SDS-PAGE – sodium dodecyl sulphate-polyacrylamide gel electrophoresis SCF – stem cell factor SCID – severe combined immunodeficiency SCID-hu – SCID-humanized mouse SCID-hu-PBL – SCID-humanized-peripheral blood leukocyte mouse SCID-hu-thy/liv – SCID-humanized-liver/thymus mouse SEM – standard error of the mean SEVI – semen enhancer of virus infection SHIV – simian-human immunodeficiency virus SIRPα – signal regulatory protein alpha TCID50 – 50% tissue culture infectious dose xxii  TDF – tenofovir disproxil fumurate TFV – tenofovir TKO-BLT – triple knockout bone marrow-liver-thymus mouse TNF – tumour necrosis factor TSST-1 – Toxic Shock Syndrome Toxin 1 UBC - University of British Columbia Vif – viral infectivity factor Vpr – viral protein R Vpu – viral protein U X4 – HIV-1 that uses CXCR4 coreceptor  xxiii  Acknowledgements  I would like to thank my supervisors for giving me the opportunity to work on this project. Thank you for the opportunity to learn and grow as a scientist, and for allowing me to work independently and guide this project in directions that were of interest to me.  I would like to thank the members of the Horwitz lab for their help and support over the years. I’d particularly like to thank Iryna Shanina and Costanza Casiraghi for helping me get started with the mouse work. I’d also like to thank Iryna and Citlali Marquez for help with the CD34+ cell isolation for the humanized mice, and for always lending a hand when I needed it. Finally, I’d like to thank Citlali for helping to make my time in the Horwitz lab enjoyable.  I would like to thank the members of the Smit lab for all their help and support over the years. A special thank you to John Nomellini for making the Caulobacters for this project, and for always helping me out when I needed it. I’d also like to thank Mike Jones for making the figure that I’ve used for 4 years to explain my research, and for always keeping me on my toes with the newest HIV research being published.  A thank you to Marc for sending me to San Francisco for 2 weeks, and thank you to Dr. Cheryl Stoddart at USCF for providing my training on the surgical procedures for making BLT mice. I would not have been able to do any of the HIV-1 mouse work without this opportunity.   To my committee, Dr. Kenneth Harder, Dr. Georgia Perona-Wright and Dr. Hélène Côte – thank you for your advice and guidance over the years.  Thank you to Dr. Rusung Tan, Dr. John Priatel, Keven Tsai and Dr. Megen Levings at CFRI for letting me start making BLT mice in your labs. Thank you to Ashley Hilchie (Hancock Lab) and Jessica Tunengel (Gantt Lab) for providing surplus PBMCs.  xxiv   Thank you to the staff at MBF, CDM, CFRI Animal Unit and FINDER for all your help and support with the mouse and HIV work.   Thank you to the members of the Jean, Abraham, Perona-Wright, Harder, Gold and Johnson labs for all your help and discussions over the years.   To my wonderful husband Darryl, thank you for all your support. You’ve sacrificed so much for me to get here and words can’t describe how grateful I am. Thank you for being by my side through all these years of grad school, for being so positive when I am down, and for bringing the kitties into my life. xxv  Dedication To Darryl1  Chapter 1: Introduction 1.1 Human Immunodeficiency Virus  Over 36 million people worldwide are estimated to be living with Human Immunodeficiency Virus 1 (HIV-1), the causative agent of Acquired Immunodeficiency Syndrome (AIDS) (1). There are two million new infections occurring annually, and 1.2 million people die AIDS-related deaths every year (1). As such, HIV-1 has enormous social, political and economic impact. Although sexual transmission of HIV-1 can be efficiently blocked by the use of condoms, in Sub-Saharan Africa the distribution and use of condoms has not been a successful option to prevent HIV transmission, particularly for young women (2). With the difficulty in developing an efficacious vaccine, it is apparent that we currently lack a solution for the HIV-1 pandemic. There is a need for development of prevention strategies that are safe, inexpensive to produce and distribute, and are acceptable in the social context present in developing countries. An HIV-1 specific microbicide is a potential solution to this problem. 1.1.1 HIV-1  HIV-1 is an enveloped, single-stranded RNA retrovirus of the lentivirus family (3-12). The HIV-1 virion is composed of two identical copies of the RNA genome as well as the integrase, reverse transcriptase and protease proteins carried inside a capsid composed of the protein p24 (3). The capsid is surrounded by a p17 protein matrix, which in turn is surrounded by the viral envelope (3). The envelope is embedded with viral glycoprotein spikes (3). The glycoprotein spikes are composed of three gp120 “caps” on a gp41 “stem” (3, 13, 14). There are 8-14 functional membrane spikes on a virion (15-17). It is not known how many membrane spikes are necessary to elicit viral entry (15).  2   The main receptor that HIV-1 uses for attachment to target cells is CD4 (18-22). Binding of gp120 to CD4 induces a conformational change in gp120 that exposes the co-receptor binding site present on gp120 (18, 19). The main co-receptor used during sexual transmission is the chemokine receptor CCR5, but CXCR4 can also be used (18, 23-26). Co-receptor binding causes an additional conformational change that exposes the fusion peptide gp41 (19, 27). gp41 contains two heptad repeats, HR1 and HR2, that bring the viral membrane and the target cell into close proximity to allow fusion to occur (15, 28). The HR2 region “zips” around preformed trimers of HR1 to form a 6-helix bundle that draws the cellular and viral membranes together (28). The zipping process takes several minutes to occur, and formation of the 6-helix bundle completes the fusion of the membranes, allowing the viral core to enter the target cell (28, 29). (Figure 1.1)  Once inside a target cell, the virus uncoats and the pre-integration complex composed of the RNA genome, matrix protein, Vpr, reverse transcriptase and integrase is formed (19, 30, 31). The HIV-1 Vif protein is crucial for HIV-1 proviral DNA synthesis, by allowing the processing and transport of the HIV-1 core (31, 32). The RNA genome begins to be reverse transcribed to double-stranded DNA by reverse transcriptase in the cytoplasm and this continues during transport to the nucleus (3, 19, 31). The viral cDNA is integrated into the host cell chromosome by the action of the viral integrase (19, 31, 33). Integrase cuts the cellular DNA and joins the viral DNA to it (31, 33). The host cell protein LEDGF/p75 tethers HIV-1 integrase to the chromatin and protects it from degradation (33, 34). Host cell enzymes complete the integration by filling in the DNA gaps (35).   Viral transcription is activated by binding of host cell transcription factors to the HIV-1 long terminal repeats (19, 36). Transcription requires the viral trans-activator protein Tat for efficient elongation (37). Viral mRNAs are produced as a variety of multiply spliced species 3  (38). The mRNA transcripts are transported to the cytoplasm by the HIV Rev protein binding to the Rev response element in the mRNA with the help of the host nuclear export factor CRM1 (38-41). The structural proteins, capsid, matrix and nucleocapsid are synthesized as parts of the precursor polyprotein Gag (42). Gag is responsible for particle assembly and release (43). Budding of the assembled virion from the cells is mediated by Gag and endosomal sorting complex required for transport (ESCRT) pathway components (43-45). HIV-1 Gag recruits the ESCRT-I machinery, which then recruits ESCRTs II and III to the membrane, mediating viral budding (36, 45). Cellular tetherin inhibits the release of budding particles by retaining them on the membrane (46, 47). HIV-1 Vpu neutralizes tetherin, allowing virion release to occur (46, 48-51). The final step occurs at or just after budding when the virus matures to an infectious virion via proteolysis of the Gag and Gag-Pol proteins to matrix, capsid, nucleocapsid, protease, reverse transcriptase and integrase (52).  Figure 1.1 HIV-1 attachment and entry  Figure 1.1 1. HIV-1 gp120 binds to CD4 on a target cell. 2. This causes a conformational change in gp120 that exposes the co-receptor binding site. The main co-receptor used during sexual transmission of HIV-1 is CCR5. 3. gp120-coreceptor binding causes a second conformational HIVCD4+ T-Cell CD4+ T-Cell CD4+ T-Cell CD4+ T-Cell1.gp120gp41Ccr5 Ccr5gp120Ccr5gp120gp41Cellular membraneHIVHIV HIV2. 3. 4.CD4 CD4gp41CD4gp414  change to expose the fusion peptide, gp41. 4. gp41 mediates fusion of HIV-1 with the target cell, allowing virus entry to occur.  Image courtesy of Michael Jones  1.1.2 HIV-1 transmission and pathogenesis 1.1.2.1 HIV-1 transmission  Although heterosexual transmission, primarily male-to-female, is the most common mode of transmission for HIV-1, little is known about the mechanisms that allow HIV-1 to breach the vaginal epithelial cell barrier and cause infection (53, 54). Several mechanisms have been proposed for HIV-1 transmission across the vaginal epithelium, including direct infection of epithelial cells, transcytosis of viral particles across the epithelium, entrance through breaches in the epithelium, or direct infection of cells sampling the vaginal tract (54-63). Langerhans cells, dendritic cells (DC), macrophages and CD4+ T cells are known to be very early targets for the virus (62). (Figure 1.2)   HIV-1 can be taken up by genital epithelial cells via endocytosis and can be transcytosed to the basolateral side (55-58). In ex vivo genital epithelial cell culture there is no evidence of productive infection of epithelial cells or integration of the virus (55). Virus particles that are taken up by epithelial cells are released into both the apical and basolateral compartments and the virus is capable of infecting CD4+ T cells (55). Transcytosis is inefficient, with less than 0.5% of the initial cell-free viral inoculum crossing polarized epithelial monolayers (59). The virus that does pass through the cells is significantly less infectious than the initial viral inoculum, with a 132-143 fold reduction in integration of HIV-1 in cells with virus collected from the basolateral side (59, 64). 5   Sexual intercourse can create breaches in the vaginal epithelium (59). Although vaginal mucus and other antimicrobial factors present in the vaginal tract can prevent HIV-1 infection, these breaches make it easier for HIV-1 to enter the body (59). In the stratified epithelium of the lower female reproductive tract the superficial epithelial cells are not bound together by tight junctions (59). They have gaps or pores between adjacent cells filled with interstitial fluid and vaginal secretions (59). Although the diffusion of HIV-1 across the vaginal mucosa is slowed by vaginal mucus, HIV-1 infection can still occur (60).   Langerhans cells below the vaginal epithelium may be the first cell type infected with HIV-1 (61). However, it is unknown if Langerhans cells are productively infected with HIV-1, allowing virus replication and spread, or if they transcytose the virus and infect CD4+ T cells via formation of a virological synapse (54, 62). Ex vivo human organ culture indicates that HIV-1 can penetrate intraepithelial vaginal Langerhans cells and CD4+ T cells (63). HIV entered CD1a+ Langerhans cells by endocytosis using multiple receptors, and the virions persisted intact within the cytoplasm for several days (61, 63). Viral entry into Langerhans cells was partially dependent on CD4 and CCR5, with C-type lectins having a more marginal role (63). HIV-1 entered CD4+ T cells without requiring passage from Langerhans cells, meaning direct viral fusion occurred (63). Productive HIV-1 infection of CD4+ T cells was not dependent on stable conjugation to Langerhans cells, but Langerhans cells appeared to have an enhancing effect (63). Langerhans cells can pass infectious virus to CD4+ T cells without being productively infected (61).   Following HIV-1 transmission, plasmacytoid dendritic cells (pDC) are recruited to the vaginal tract by CCL20 secreted by vaginal epithelial cells (62). The pDCs secrete chemokines which cause the influx of CD4+ T cells to the vaginal tract and aids in the establishment of 6  productive infection (62). Syncytia can form between infected cells, which helps establish a tethering interaction that may facilitate cell-to-cell transmission of HIV-1 from the first cells infected to CD4+ T cells (65). Once productive infection is established in the vaginal tract, the virus can be spread to the draining lymph nodes where it undergoes amplification and begins to disseminate (54).   Figure 1.2 HIV-1 transmission across the vaginal mucosa  Figure 1.2 HIV-1 can cross the vaginal epithelial cell barrier by direct infection of epithelial cells, transcytosis of viral particles across the epithelium, entrance through breaches in the epithelium, or direct infection of cells sampling the vaginal tract.  1.1.2.2 HIV-1 pathogenesis 1.1.2.2.1 Viral load  In the first weeks following infection, patients develop acute HIV-1 syndrome (66). This is characterized by a rapid increase in viremia, peaking 3-4 weeks post-infection, before viral load drops to reach a steady-state (66-68). The viral load decreases over 12-20 weeks to reach a stable level viral set point (60). Viral diversification occurs during this decrease in viral load, as                                             Mucus Epithelial	  cells	    HIV Transcytosis	  through	  epithelial	  cell Entry	  through	  microabrasion Langerhans cell Uptake	  by	  Langerhans	  cell Direct	  infection	  of	  epithelial	  cell Vaginal	  lumen  HIV  HIV  HIV 7  multiple escape mutants are selected under pressure of the adaptive immune response (60, 68). Eventually the viral load will begin to increase and CD4+ T cell numbers will drop significantly, leading to AIDS, presence of opportunistic infections and death (69). With daily, life-long antiretroviral therapy (ART), a person with HIV-1 may have an undetectable viral load and can expect a relatively normal life span (70).  1.1.2.2.2 Immune response to HIV-1 infection  Within a few days of infection HIV-1 travels to the gut-associated lymphoid tissues (GALT), where there is a large number of CD4+ T cells (68, 71). There is a rapid expansion of HIV-1 in the GALT, and massive CD4+ T cell death occurs (66, 72-75). Direct infection with HIV-1 is not the main mediator of cell death, with most CD4+ T cells undergoing bystander apoptosis or pyroptosis (66, 73, 76-78).   CD8+ T cell responses correlate with the initial decline in HIV-1 replication and help maintain a lowered viral set point (60, 66, 79-81). The earliest CD8+ T cell responses are usually specific for the viral envelope and Nef (60, 68, 82). After the peak in the CD8+ T cell response, the virus sequence begins to change dramatically (60, 68, 83, 84). The first CD8+ T cell responses decline rapidly when the viral escape mutations are selected (60, 68, 83, 84). The CD8+ T cell response evolves over the course of HIV-1 infection (68).  The first antibody response that develops is non-neutralizing, and is primarily directed towards the gp41 region of HIV-1 (85, 86). Twelve weeks post-infection neutralizing antibodies will be developed (66). However, the majority of these antibodies are targeted to epitopes that are shielded by envelope glycans or can be rapidly mutated, leading to the neutralization sensitive virus rapidly being replaced with resistant variants (66, 87).    8  1.1.2.2.3 HIV-1 latency  HIV-1 can remain latent in an infected host for many years, allowing chronic infection to occur (88, 89). Resting memory CD4+ T cells and monocytes or macrophages can be a reservoir for HIV infection, with the virus stably integrated into the genome but no gene expression occurring (88, 90-92). As resting memory CD4+ T cells are not readily infected by HIV-1, it is thought that activated cells are infected as they are transitioning back to the resting state (91, 93). The factors that determine whether a virus establishes active or latent infection are not well characterized (94). The latent reservoir is established very early during acute infection, which limits the efficacy of ART (89, 95). HIV-1 latency occurs at a low frequency (95).  1.1.2.2.4 AIDS  AIDS occurs when the CD4+ T cell count drops below 200 cells per µL of blood, or the occurrence of specific diseases in association with an HIV-1 infection. In the absence of treatment, about half of the people infected with HIV develop AIDS within ten years. The most common conditions that indicate AIDS has developed are pneumocystis pneumonia, wasting syndrome and esophageal candidiasis. (96, 97)  1.2 HIV prevention  Each year 2 million new HIV-1 infections occur (1). The majority of these new HIV-1 infections occur through sexual transmission, with young women disproportionally affected (1, 2). Although condoms can provide protection from sexual transmission of HIV-1, they have had minimal impact on stopping the HIV-1 pandemic. As such, alternative prevention options are urgently needed. New prevention options should focus on development of female controlled prevention options – options that do not require the consent or knowledge of the male partner for 9  use so a woman has the opportunity to protect herself from HIV-1 exposure. Microbicides, pre-exposure prophylaxis, and vaccination are potential female-controlled prevention options. 1.2.1 Microbicides  Microbicides, which are drug products that can be topically applied to mucosal surfaces to prevent infection (98), have long been considered a promising option for female-controlled prevention of HIV-1 (2). Although no microbicides are currently on the market, several are under development (98-100). These microbicides use a wide variety of strategies to prevent HIV-1 infection.  Microbicides have been developed that target most steps of the HIV-1 life cycle, including entry blockers (101-106), reverse transcriptase inhibitors (2, 107-113), integrase inhibitors (114), and protease inhibitors (115, 116). Some non-specific chemical compounds have also been tested (117-122). To minimize the risk of side effects some groups have tried engineering species of the natural vaginal bacteria Lactobacillus for microbicide development (123-126). The topic of HIV-1 microbicides has been reviewed in depth by others (127, 128) so I will focus on an overview of some potential candidates, the use of bacterial based microbicides, and the nucleoside reverse transcriptase inhibitor tenofovir, which has undergone extensive clinical testing. 1.2.1.1 Microbicide candidates under development  The HIV-1 coreceptor CCR5 has been a key target for entry inhibition using decoy ligands to block CCR5 (105). Topical application of high doses of PSC-RANTES has provided potent protection against vaginal simian-human immunodeficiency virus (SHIV) challenge in rhesus macaques (103). All 5 animals that received a high dose of PSC-RANTES were protected from SHIV infection, whereas a lower dose protected 4 of 5 macaques from infection (103). 10  Tests in humanized SCID-hu-PBL mice indicated that the mice were protected from parenteral HIV-1 challenge, but in some cases escape was observed with both R5 and X4 viruses (103). An orally delivered CCR5 small molecule inhibitor can provide substantial protection from SHIV in rhesus macaques for 10-14 days after the dose is given (106). Macaques that did get infected had reduced plasma viremia during the early stages of infection (106).  UC781 is a reverse transcriptase inhibitor that has undergone phase I testing for use as a rectal microbicide (109). Although UC781 was able to suppress HIV infection by 100% in vitro and had a good safety profile with no changes in mucosal immunoinflammatory markers, no epithelial sloughing, no histology changes and no changes in rectal microflora, further development was not pursued because there were problems with solubility and stability (108). Dapivirine and rilpivirine are non-nucleotide reverse transcriptase inhibitors with high potency and long-half life that are also being evaluated as prevention options (110, 129).  Integrase inhibitors and protease inhibitors have been proposed as options for microbicide development (114, 115). These act later in the virus life cycle, after the virus enters the cell and reverse transcribes the RNA, and relies on inducing an abortive infection instead of preventing the initial infection from occurring, which may be problematic if there is not a complete block in infection (114, 115). However, given the fact that these inhibitors act later in the life cycle of HIV-1, they may have a wider window for protection compared to inhibitors of HIV-1 entry since they have a longer length of time to act (114, 115). Integrase inhibitors and protease inhibitors may be an excellent prevention option when used in combination with other drugs that act earlier in the infection cycle (114, 115).  Nonoxynol-9 is a non-ionic surfactant used as a spermicide (117). Cellulose sulphate is a high molecular weight sulphated carboxymethylcellulose polymer (117). Nonoxynol-9 and 11  cellulose sulphate were promising microbicide candidates in initial studies, but clinical trials found increased HIV-1 infection when they were used frequently (117). In particular, a phase 3 trial examined the use of 6% cellulose sulphate or 4% nonoxynol-9 and was stopped before completion because HIV transmission was increased with microbicide use (117). These candidates damaged the vaginal epithelium making it easier for HIV-1 to enter the body (122).   Vaginal applications of broadly neutralizing monoclonal antibodies has been used as a potential microbicide option. The b12 antibody, which is a broadly neutralizing monoclonal antibody to gp120 has been studied for ability to prevent SHIV-162P4 infection in rhesus macaques (104). Six of eight macaques that received b12 prior to SHIV infection were protected whereas nine of ten control animals became infected (104). The macaques that became infected had lower viral load and delayed peak viral loads (104).  1.2.1.2 Bacteria based microbicides  As women in Sub-Saharan Africa are at high risk for HIV-1 infection, a microbicide must not only be effective at preventing HIV-1 infection, it must also be affordable. One of the barriers to microbicide use is the need to purchase a product on a regular basis. Another concern with microbicides is negative impacts on the vaginal microenvironment, either by shifting the vaginal microbiome or causing damage to the epithelial barrier. To counteract these difficulties, some groups have attempted to engineer bacteria to display HIV-1 blocking agents (123-125). As the vaginal tract is naturally colonized with commensal bacteria, primarily of the Lactobacillus genus (124, 130-138), many groups have focused on isolates of this bacterium for microbicide development. Lactobacillus strains have been engineered to express proteins that could interfere with HIV-1 attachment or entry into cells to prevent infection (123-125).  12   Lactobacillus jensenii, a natural human vaginal isolate, has been engineered to secrete a 2-domain CD4 (124). The secreted protein recognized a conformation-dependent anti-CD4 antibody and bound to gp120 (124). By binding to CD4 this bacterium was able to inhibit in vitro entry of HIV-1HXB2 and HIV-1JR-FL into target cells by 95% and 55% respectively (124). Additional studies investigated expressing the 2-domain CD4 on the surface of Lactobacillus jensenii (139). There were 1250 correctly folded 2-domain CD4 molecules per bacterium (139). Additional studies with Lactobacillus jensenii expressing 2-domain CD4 have not been published.  Lactobacillus jensenii has also been modified to express the anti-viral lectin cyanovirin-N (125, 126). The cyanovirin-N expressed by Lactobacillus was able to inhibit HIV-1BaL infection in vitro (125). The recombinant Lactobacillus was able to colonize the vaginal tract of mice, and full-length cyanovirin-N could be detected at six days post-application (125). There was 63% reduction in SHIV transmission after repeated vaginal challenges of macaques treated with Lactobacillus jensenii expressing cyanovirin-N (126). Peak viral loads were reduced by 6-fold in those macaques that received the microbicide but were still infected (126). The vaginal tract of the macaques was colonized by the recombinant Lactobacillus jensenii and prolonged antiviral protein secretion did not cause an increase in proinflammatory markers (126). Secretion of full-length cyanovirin-N was detected in vaginal lavage in as little as 24 hours and was maintained for 6 weeks (126).   Lactobacillus reuteri RC-14 is a probiotic that has been shown to colonize the human vagina and restore a healthy microbiome (123). Recombinant bacteria expressing CD4 2-domain, MIP-1β, and T-1249 were created (123). The recombinant bacteria were able to express the proteins as cell wall associated or secreted protein (123). Expression of T-1249 affected the 13  growth rate of the recombinant bacteria and peptide expression was undetectable (123). The CD4 2-domain was able to bind to single or dual-tropic co-receptor viruses using an HIV-1 primary isolate (123). The proteins were expressed as cell-wall associated or secreted, with the cell-wall associated CD4 2-domain able to bind a wide variety of viruses (123). The secreted proteins reduced HIV and SHIV infection of human peripheral blood mononuclear cells by 20-100% depending on the assay used (123).   Although these bacteria have shown some efficacy, several difficulties could arise from the use of Lactobacillus, including lack of sustained expression of the protein and challenges in eradication of the bacteria without damaging natural vaginal microflora should unwanted side effects occur. 1.2.1.3 Tenofovir  Tenofovir (TFV) is an adenosine nucleotide analog reverse transcriptase inhibitor that has potent activity against retroviruses, and has been approved for the treatment of HIV ((140) and Gilead.com). TFV competitively inhibits HIV-1 reverse transcriptase by being incorporated into the DNA chain and causing termination (140). TFV is available as a treatment for HIV-1 and has undergone extensive testing for microbicide development and pre-exposure prophylaxis (discussed in Section 1.2.2).   The Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 microbicide trial was a two-arm, double-blind, randomized, placebo-controlled trial conducted from 2007-2010 in KwaZulu-Natal, South Africa (2). Women received either 1% TFV gel or the “universal” hydroxyethylcellulose (HEC) placebo gel, which has minimal impact on HIV acquisition (2). The CAPRISA 004 trial used a “before and after” sex dosing strategy, a strategy termed BAT24, with one dose of gel applied within 12 hours before sex and a second dose of gel 14  applied as soon as possible within 12 hours after sex, with no more than 2 doses in a 24 hour period (2). In the women with the highest adherence, protection was no higher than 54% (2, 141).  The 1% TFV gel reduced HIV acquisition by 38% in intermediate adherers and 28% in low adherers (2, 141). Over the course of the study 94.3% of participants reported at least one adverse event, with diarrhea being a common adverse event (2). In the CAPRISA 004 trial, women who acquired HIV had higher systemic innate immune activation prior to infection as well as activation of soluble and cellular immune mediators (141). These women had higher concentration of tumour necrosis factor α (TNFα), interleukin-2 (IL-2), IL-7, and IL-12p70, as well as higher proportions of activated natural killer (NK) cells and spontaneous cytotoxic T cell degranulation (141).   The FACTS 001 trial, a follow-up to the CAPRISA 004 trial, examined the efficacy of the BAT24 strategy on prevention of vaginal infection with HIV-1 (142). The 1% TFV gel provided no protection from HIV-1 infection in this trial, and low adherence seemed to be responsible for the lack of efficacy (142).  The Vaginal and Oral Interventions to Control the Epidemic (VOICE) trial (MTN-003) took place from 2009 to 2011 in South Africa, Uganda and Zimbabwe (143). Women were randomly assigned to one of five regimens: oral tenofovir disoproxil fumarate, a prodrug of TFV (TDF), oral TDF plus emtricitabine (FTC), oral placebo, vaginal 1% TFV gel, or vaginal placebo gel (143). In September 2011 the data and safety monitoring board recommended that the oral TDF treatment be discontinued due to futility (143). In November 2011 the TFV gel arm was stopped due to futility (143). Based on returned-product counts the mean rate of adherence was 86%, however TFV was detected in vaginal swabs of women in the gel group only 25% of the time, suggesting adherence may be an issue (143). No regimen in this trial significantly reduced 15  the risk of HIV acquisition (143). These conflicting results indicate that more research needs to be undertaken to determine if TFV gel is able to prevent HIV infection, with an emphasis on product adherence.  Ex vivo studies using colonic biopsy tissue have indicated that single and 7 day rectal exposure to TFV gel showed significant suppression of infection (109). Rectal dosing of TFV gel in a phase I trial (RMP-02/MTN-006) had greater tissue detection than oral TDF, but caused adverse events in the gastrointestinal tract, indicating that it is not entirely safe for rectal use (109). Rectal secretion of some cytokines including IL-1β, IL-6 and MIP1α were reduced in TFV treated groups (109).  Few gel recipients liked the product, which may impact adherence (109). Given that TFV gel has been shown to suppress anti-inflammatory mediators, increase T cell densities, cause mitochondrial dysfunction, alter regulatory pathways of cell differentiation and survival and stimulate epithelial cell proliferation in the rectum, the long-term safety of rectal use of TFV gel needs to be investigated (144).  A phase 1 randomized rectal safety and acceptability study of 1% TFV gel (MTN-007) demonstrated that there was no difference in fecal calprotectin, microflora or epithelial cell sloughing between treatment arms (145). Evidence suggested there was a difference in histology, mucosal gene expression, T cell phenotype, and protein expression (145). 1.2.2 Pre-Exposure Prophylaxis  The prophylactic use of antiretroviral therapy to prevent HIV infection is a promising new strategy in the HIV/AIDS epidemic (146, 147). Several lines of evidence indicate that prophylactic treatment with TFV can reduce HIV-1 transmission, a strategy termed Pre-Exposure Prophylaxis (PrEP) (148-157). PrEP is used by uninfected people that are at high risk of HIV-1 exposure. PrEP uses oral TDF alone or in combination with FTC (149-151, 154, 155). Studies 16  have demonstrated that efficacy is increased when both TDF and FTC are used in combination (149-151, 154, 155). Five clinical trials have demonstrated that PrEP is effective, with efficacy ranging from 39-75% (148-152). However, 2 trials conducted among African women did not demonstrate protection (143, 158). The seven PrEP clinical trials that have been reported to date are summarized below. Table 1.1 PrEP clinical trials Trial Target Population Treatment Effectiveness Problems Reference FEM-PrEP HIV negative women in Kenya, South Africa and Tanzania Daily oral TDF-FTC or placebo Did not significantly reduce the rate of HIV, study stopped early due to lack of efficacy Low drug adherence (40% had evidence of recent pill use), increased rates of side effects (159) Bangkok Tenofovir Study Injection drug users in Bangkok, Thailand Daily oral TDF or placebo Reduced risk of infection by 48.9%, included prevention services Similar rates of adverse events, slightly more nausea and vomiting in TDF group (148) 17  Trial Target Population Treatment Effectiveness Problems Reference iPrEX Men or transgender women who have sex with men in Peru, Ecuador, Brazil, USA, Thailand, South Africa Daily oral FTC-TDF or placebo  44% reduction in the incidence of HIV when provided as part of a prevention package (condoms, counselling, STI testing and treatment) Similar rates of adverse events, more nausea with FTC-TDF in first 4 weeks (149) Partners PrEP HIV-1 serodiscordant heterosexual couples from Kenya and Uganda, HIV-1 seropositive  Oral daily TDF, TDF-FTC or placebo  67% reduction in HIV infection with TDF and 75% reduction with TDF- Adverse events were similar among all groups (150) 18  Trial Target Population Treatment Effectiveness Problems Reference Partner’s PrEP (continued) Partner not eligible for ART  FTC; among women TDF 71%, TDF-FTC 66%, among men TDF 63%, TDF-FTC 84%, as part of a prevention package that included condoms, counselling, STI testing and treatment, access to PEP, hepatits B vaccination circumcision   19  Trial Target Population Treatment Effectiveness Problems Reference TDF2 HIV seronegative men and women in Botswana Daily oral TDF-FTC or placebo 62.2% reduction in HIV infection as part of a prevention package including counselling, STI testing and treatment,  Higher rates of nausea, dizziness and vomiting in TDF-FTC group (151) VOICE African women in South Africa, Uganda, Zimbabwe,  Oral daily TDF, TDF-FTC or placebo. Trial also contained a 1% tenofovir vaginal gel or placebo    TDF arm stopped early due to futility. -49% efficacy for TDF,  -4.4% efficacy for TDF-FTC  Elevated serum creatinine levels among TDF-FTC group, low adherence (143)  20  Trial Target Population Treatment Effectiveness Problems Reference ANRS IPERGAY Men who have sex with men, at high risk for HIV-1 exposure Oral TDF-FTC or placebo before and after sexual activity (approximately 15 pills taken per month) 82% reduction in HIV-1 infection as part of a prevention package including condoms, testing and counselling  Increased gastrointestinal adverse events and renal adverse events in TDF-FTC group  (152)   PrEP is only recommended for people that are confirmed to be HIV-1 negative, and people taking PrEP should receive HIV-1 testing every 3 months to verify they continue to remain HIV-1 negative (160). A major concern with PrEP use is drug resistance, which can emerge if PrEP drugs continue after treatment failure (105). While on PrEP people will be using weeks of mono or dual agent ART before seroconversion is detected and PrEP can be discontinued, which could select for resistant viral variants (111). This has been rare in the PrEP trials (111). There has been no evidence of increased risk behaviours while on PrEP, and self-reported condom use increased during the studies (111). Mathematical modelling that takes into account age, gender, sexual activity, HIV infection status, stage of disease, PrEP 21  coverage/discontinuation, and HIV drug susceptibility was designed to simulate the impact of PrEP on drug resistance in sub-Saharan Africa (161). Analyses suggested that the prevalence of HIV drug resistance would be influenced most by the extent and duration of inadvertent PrEP use in individuals already infected with HIV-1, with drug resistance predicted to be 2.5% to 40% over 10 years based on different scenarios that take into account PrEP efficacy, coverage and frequency of inadvertent use (161). 1.2.3 Vaccination  There have been 187 separate vaccine trials for HIV, with only 5 candidates advancing to efficacy testing (162). The difficulty in developing an HIV vaccine partially lies in the viral diversity (162). With 9 different subtypes, 35 circulating recombinant forms, a 20% difference within a subtype and 35% difference between subtypes as well as the high mutation rate of the virus, it is difficult to develop a vaccine that will protect from all HIV-1 subtypes (162). In addition, there is a lack of understanding of the immune response correlates for protection from HIV-1 infection in humans (162). The natural immune responses to HIV-1 do not prevent or eradicate infection so there is no disease free state to emulate (162). Neutralizing antibodies rarely occur in individuals that are HIV-1 positive, which makes it difficult to determine which viral epitopes should be included in a vaccine (162). It takes many years and multiple rounds of selection must occur in the germinal centres before broadly neutralizing antibodies are produced by HIV-1 infected individuals (163).  In addition, the integration event that establishes the latent pool of virus infected cells occurs within hours to days after transmission, requiring a quick immune response to be protective (164). A successful vaccine needs to provide sterilizing immunity at the time of 22  exposure, which is unique in the field of vaccine research as all other vaccines rely upon secondary T and B cell responses to prevent disease (164). 1.2.3.1 rgp120 HIV Vaccine Study (VAX004, AIDSVAX B/B)  A double-blind, randomized trial of a recombinant HIV-1 envelope glycoprotein subunit vaccine was conducted among men who have sex with men, and women at high risk for heterosexual transmission of HIV-1. The study vaccine contained 2 recombinant gp120 HIV-1 envelope antigens that had been derived from 2 different subtype B strains adsorbed onto alum. The volunteers received 7 injections (months 0, 1, 6, 12, 18, 24 and 30) of vaccine or placebo. HIV-1 acquisition rates were 6.7% in those that received the vaccine and 7.0% in the placebo recipients, with vaccine efficacy estimated at 6% (95% confidence interval, -17%-24%) . There were no significant differences in viral load or genetic characteristics of the HIV-1 infecting strains between treatment groups. The vaccinees had HIV-1-specific antibody responses that were neutralizing and CD4-blocking antibody responses, but this was not sufficient for protection. (165) 1.2.3.2 Bivalent recombinant glycoprotein 120 (AIDSVAX B/E)  A randomized, double-blind placebo controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine was tested among injection drug users in Bangkok. The vaccine was injected at months 0, 1, 6, 12, 18, 24 and 36. The vaccine contained 2 recombinant gp120 HIV-1 envelope antigens, 1 from a clade B strain and one from a CRFO1_AE isolate. There was no difference between the vaccine and placebo arms, and vaccine efficacy was estimated at 0.1%. All sampled vaccinees developed antibodies to gp120. (166) 23  1.2.3.3 DNA/rAd5 Vaccine Study (HVTN 505)  A randomized, double-blind placebo-controlled trial of a DNA prime-recombinant adenovirus type 5 boost (DNA/rAd5) vaccine regimen was tested in people with increased risk for HIV-1 infection in the United States. The vaccine contained 6 plasmids with DNA expressing clade B Gag, Pol, and Nef as well as envelope proteins from clades A, B, and C, and was administered at weeks 0, 4 and 8. The rAd5 vector boost, expressing clade B Gag-Pol fusion proteins and envelope glycoproteins from clades A, B, and C was administered at week 24. The vaccine trial was halted for lack of efficacy. The vaccine efficacy was -25% (95% confidence interval, −121.2%- 29.3%), with 27 HIV infections in vaccinees and 21 infections in the placebo group. Viral load was the same in each group. The vaccine induced CD4+ T cell responses, CD8+ T cell responses and IgG and IgA antibody responses, but theses results were not associated with protection. (167) 1.2.3.4 MRKAd5 HIV-1 gag/pol/nef vaccine (Step Study)  A double-blind, randomized, placebo-controlled vaccine study assessed the MRKAd5 HIV-1 gag/pol/nef vaccine compared to placebo. Participants received 3 injections of the vaccine, on day 1, week 4 and week 26. More HIV-1 infections occurred in the vaccinees compared to those that received the placebo, and the viral load was not lowered. The goal of this vaccine was to induce cell-mediated immunity to control viral replication instead of preventing infection. The trial was stopped early due to futility. (168) 1.2.3.5 ALVAC-HIV and AIDSVAX B/E (RV144)  The RV144 trial combined the unsuccessful ALVAC, vCP1521 and AIDSVAXB/E r-gp120. This was a randomized, double-blind, placebo-controlled efficacy trial evaluating four priming injections of a recombinant canarypox vector vaccine (ALVAC-HIV (vCP1521)) plus 24  two booster injections of a recombinant glycoprotein 120 subunit vaccine (AIDSVAX B/E). ALVAC-HIV was given at day 0, week 4, 12 and 24 and AIDSVAX B/E was given at weeks 12 and 24. There was a trend towards HIV-1 prevention among the vaccine recipients, with a vaccine efficacy of 26.4%. In the modified intention-to-treat analysis the vaccine efficacy was 31.2%. Vaccination did not affect the degree of viremia or the CD4+ T cell counts in vaccinees. (169)   1.3  Caulobacter crescentus C. crescentus is a non-pathogenic, gram-negative freshwater bacterium that is readily isolated from most soil and aquatic sources, including drinking water (170, 171). The bacterium cannot grow at temperatures above 33°C and does not grow at the salt concentrations found in human blood and tissues (172, 173).  C. crescentus has a surface (S)-layer, a crystalline protein layer composed of a monomer protein that is secreted at very high levels and self-assembles into a six-member unit lattice attached to the cell surface (outer membrane) (174-176). The S-layer is composed of a single protein, RsaA, with a mass of approximately 100kDa (177). C. crescentus is covered by the S-layer at all times (175, 178). It is estimated that 40,000 RsaA monomers are interlinked to form the S-layer, and the S-layer proteins constitutes more than 30% of the total cell protein (171, 174). Secretion of the S-layer relies on a stable C-terminal secretion signal and a three-component ABC-transporter system (176, 179). The S-layer attaches to the surface of C. crescentus by the O-polysaccharide of a smooth lipopolysaccharide (LPS), with a core sugar and fatty acid complement identical to those of rough LPS (180).  25  A system to insert foreign protein sequences into the S-layer protein RsaA has previously been developed (171, 176, 179, 181, 182). BamHI linker mutagenesis was performed to introduce foreign peptides into rsaA, and incorporated an antibiotic resistance gene for selection of linker insertions (171). There were 146 different target sites in rsaA that were capable of receiving linker insertions (171). 195 BamHI linker mutated genes were found in 195 rsaA plasmids, and 49 of these were able to produce RsaA protein that could be released from the cell surface with 100mM Hepes at pH 2, indicating surface expression (171). There was a lack of permissive sites in the C-terminus of the protein, which is where RsaA carries a C-terminal secretion signal (179, 183). There were permissive sites in the N-terminal portion of the protein, which is responsible for membrane anchoring, suggesting that some sites can accommodate additional mass without serious disruption of S-layer biogenesis (171, 179). There were 12 unique insertion sites that were able to synthesize S-layer protein carrying an inserted peptide, corresponding to amino acids: 69, 277, 353, 450, 467, 485, 551, 574, 622, 690, 723 and 944 (171). Shed S-layer protein was evident in the culture fluid of cells with insertions at amino acids 69, 277, 353, 450, 485, 551, and 622 (171). Insertions at amino acids 723 and 944 created cell-anchored S-layer protein secreted at a similar level to control cells with no proteolytic degradation of RsaA (171). The foreign proteins had no impact on the secretion and expression of the S-layer, and the proteins retained their function once expressed on the surface of the bacteria (171, 176, 179, 181, 182). As such, S-layer mediated display provided high expression of functional proteins, which can be utilized for many biotechnological applications. RsaA must pass through both the inner and outer membranes to form the S-layer on the outer surface of the bacteria, and does this using type 1 secretion (176). A type 1 secretion apparatus consists of three components – the ABC-transporter embedded in the inner membrane, 26  with an ATP-binding region, which recognizes the C-terminal secretion sequence of the substrate protein; a membrane fusion protein, that is anchored to the inner membrane and spans the periplasm; and an outer membrane protein that is thought to interact with the membrane fusion protein to form a channel that extends from the cytoplasm through the two membranes to the outside of the cell (176). In C. crescentus RsaD is the ABC-transporter and RsaE is the membrane fusion protein (176). Two proteins (RsaFa and RsaFb) have been identified to play a role in the outer membrane component in the RsaA secretion machinery (184). When the genes, rsaFa and rasFb are knocked out there is no RsaA secretion (184). A metalloprotease gene designated sap was found to cause proteolytic activity that affected S-layer monomers with foreign inserts (185). By inactivating sap the likelihood that heterologous insertions will be efficiently presented in the S-layer of C. crescentus in an intact form has been improved (185). The RsaA anchoring region lies within the first 225 amino acids and this RsaA anchoring region requires a smooth LPS species found in the outer membrane, as loss of this region resulting in loss of anchoring (186).  C. crescentus appears to be safe for use as a biotechnology platform. Collaborators have demonstrated that C. crescentus has no adverse effects when injected intraperitoneally into mice (172).  In particular, injection of 2 x 107 C. crescentus intraperitoneally into mice were able to produce only 40,000 colony forming units after five days and by day 10 there were no viable bacteria detected in the peritoneum (172). Further studies have shown that the lipid A portion of the LPS of C. crescentus is unique, significantly different than the typical lipid A of enteric bacteria, and does not cause sepsis, even when injected at high levels (187). The lipid A has a unique structure with respect to the combination of backbone and fatty acyl groups, and a lack of phosphates (187). In addition, the TNF response that is stimulated by the C. crescentus LPS is 27  more than 100-fold less than that observed for E. coli, with the maximum response that could be elicited about 3-fold less than the maximum possible with E. coli LPS, suggesting that C. crescentus does not induce an immune response and may be safe for use in biotechnology applications (187).   1.4 Humanized mice  Since HIV-1 is a human specific pathogen, it is difficult to work with the virus in a physiologically relevant system. The development of small animal models of HIV-1 infection has been important for the study of HIV-1 prophylaxis and virus pathogenesis. Chimeric mouse models with human immune cells have been developed that recapitulate the mucosal transmission of HIV-1, permitting studies of early events in HIV pathogenesis and evaluation of prevention and treatment options. 1.4.1 Mouse strains and early experiments  In 1983 the CB17-SCID mouse strain was established (188). This strain has a spontaneous autosomal recessive mutation in the PrKdc (protein kinase, DNA activated, catalytic polypeptide) gene, resulting in a T-to-A point mutation creating a stop codon, causing severe combined immunodeficiency (SCID) (188-190). PrKdc functions in DNA non-homologous end-joining and is important for V(D)J recombination (191). This defect makes the mice hypersensitive to radiation due reduced repair of double-strand DNA breaks (192). There is an absence of B cells and T cells in these mice, although some immunoglobulins and Thy-1+ cells are present, which indicates that B and T cell development may occur at low levels (188). The bone marrow environment of these mice appears to be functional as transfer of bone marrow from congenic BALB/c mice leads to the production of immunoglobulins of BALB/c allotype 28  within 4 weeks (188). CB17-SCID mice had a rudimentary thymic medulla without cortex, relatively empty splenic follicles and lymph nodes, and undeveloped bronchial and gastrointestinal lymphocytic foci (193). While the mice fail to develop T or B cells because of a defect in VDJ somatic recombination, the myeloid lineage appears normal (193, 194). The CB17-SCID mice were able to accept skin grafts from C57Bl/6 mice, an unrelated strain (188). Taken together, this suggested that the CB17-SCID mice might represent an excellent model for human cell and tissue engraftment. Many studies have attempted to engraft a functional human immune system into these mice.  The SCID-hu mouse was described by McCune et al in 1988 (195). SCID mice were implanted with human fetal thymus then given an intravenous or intrathymic injection of human fetal liver cells as the fetal liver is the main site for hematopoiesis (195). The fetal thymus tissue was able to grow in the mice (195). The microscopic anatomy of the implanted thymus resembled human thymus, with cortical and medullary compartments (195). The human fetal liver cells that were injected intravenously into the mice could be detected in the peripheral blood at day 42 and 49 post-injection (195). However, human immune cells were transient as no human cells were present in the blood by 10-12 weeks post-injection (195).  In 1988 Mosier et al created the SCID- hu-PBL mouse by transplanting human PBMCs into SCID mice (189, 196, 197). The PMBCs needed to be injected intraperitoneally as intravenous injection did not lead to development of human cells (196). The human PBMCs were able to survive for at least 6 months in the mice, and there was generation of human immunoglobulins (196). Human cells were found in the spleen, lymph nodes and blood of the mice (196). Human T cells, B cells and myeloid cells were present, but the relative proportions of these cells differed from normal peripheral blood (196). There was mild and transient graft 29  versus host disease, which may have contributed to spontaneous activation of B cells, but the researchers did not consider this to be a significant problem (196).   The SCID-hu thy/liv mouse was described in 1990 (198, 199). The SCID mouse was surgically co-implanted with small pieces of human fetal thymus and liver under the kidney capsule, resulting in chimeric mice that could support long-term generation of human T cells, for at least 6 months, and up to 15 months in some mice (189, 198, 199). The fetal liver and thymus acted as a microenvironment for the development of human cells of the myelomonocytic, erythroid and megakaryocytic lineages (198). The T cells that developed were functional and able to respond to mitogens and CD3 antibody in vitro (198, 200). There were no pathological changes characteristic of graft versus host disease in the mice (198).  While these initial studies with human cell engraftment in SCID mice were promising, these mice can spontaneously generate murine T and B cells as the mice age, and they have high levels of NK cell activity, which can have an impact on longevity of the mice and functionality of the human immune cells (197, 201). In an effort to create an improved mouse model, SCID mice were backcrossed onto the NOD/Lt strain background to create the NOD/SCID mouse (202). These mice are deficient in T cells and B cells, with little to no serum immunoglobulins present (202). The NOD/SCID mice have a reduction of NK cell numbers and function, defects in macrophage function, impaired DC maturation, and lack the haemolytic complement compartment due to a 2 base pair deletion in the coding region of the gene that encodes the C5 complement component, which prevents the formation of the C5b-9 membrane attack complex (202-204). The decrease in NK cell numbers should lead to increased immune cell engraftment in the NOD/SCID mice compared to SCID mice (205). In addition to these deficiencies, the SIRPα membrane glycoprotein expressed on myeloid cells in the NOD strain is similar to human 30  SIRPα (206, 207). The ligand for SIRPα, CD47, is expressed on all cells (207). The SIRPα/CD47 ligation inhibits macrophage phagocytosis and mediates xenograft rejection (207). By closely resembling human SIRPα the macrophages in the NOD mice will interact with the human CD47 on transplanted cells and phagocytosis will be inhibited.  Human cell engraftment is significantly increased in NOD/SCID mice compared to SCID mice (207-209). Engraftment of human PBMCs in the spleen of NOD/SCID mice is increased by 5- to 10-fold compared to SCID mice, and there are more human cells in the blood of the mice (208). In NOD/SCID mice the proportion of human PBMCs increases over time compared to SCID mice (208). Similar results were observed when human cord blood hematopoietic stem cells were injected into the mice (209). There was a higher level of engraftment in the NOD/SCID mice compared to SCID mice, although there was no change in the distribution of the cells (209).  The first description of humanized bone marrow-liver-thymus (BLT) mice was the NOD/SCID-hu BLT model (210, 211). These mice combined the SCID-hu thy/liv transplantation and the NOD/SCID hematopoietic stem cell (HSC) injection models, resulting in NOD/SCID mice that were first transplanted with human fetal thymus and liver tissue under the kidney capsule before subsequently receiving intravenous injection of human CD34+ cells isolated from autologous fetal liver (210, 211). The mice received sublethal irradiation prior to injection of CD34+ cells (210, 211). Human cells were detected in the peripheral blood, liver, lung, bone marrow, spleen, lymph node, and within the implanted organoid of the mice (210, 211). Human T cells, B cells, monocytes and macrophages, and DCs were present in all tissues (210, 211). Analysis of the implanted organoid showed long-term sustained thymopoieses, contributing to a diverse Vβ-T-cell antigen receptor repertoire (210). The spleens of the mice had 31  B cell zones and T cell zones, whereas the lymph nodes had rudimentary secondary lymphoid structures (210). Human IgM and IgG were present in the mice (211). In this model the human T cells develop in an autologous human thymic environment, leading to T cell restriction for human major histocompatibility (MHC) proteins (210).   To establish the functionality of the human immune cells that developed in the NOD/SCID-BLT mouse fetal liver B cells were infected with Epstein-Barr Virus (EBV) to establish autologous lymphoblastoid cell lines, then the mice were infected with EBV (210). The peripheral blood T cells showed an increase in the percentage of CD45RA-CD27+ memory T cells, consistent with the pattern of expansion of human T cells in peripheral blood during acute EBV infection in humans (210). The lymphoblastoid cell lines acted as antigen presenting cells, and the increase in memory T cells indicated that the human T cells that developed in the mice were able to respond to antigen in the context of human MHC (210).   To determine if human T cells and human DCs were able to interact with each other the NOD/SCID BLT mice were infected with Toxic Shock Syndrome Toxin 1 (TSST-1) (210). After infection with TSST-1, there was an expansion of Vβ2+ T cells in the bone marrow and spleen of the mice, as well as an increase in the systemic concentrations of human IFNγ, IL-10, IL-2, IL-6 and TNFα (210). Before administration of TSST-1 the DCs had an immature resting phenotype, but after administration they had a marked upregulation of CD40, CD80, CD86 and CD83 (210).   The NOD/SCID BLT mice were able to reject skin grafts, indicating donor antigen presentation to T cells, T cell activation and expansion, and trafficking of effector cells to the graft (211). 32   BLT mice have robust, long-term, systemic immune reconstitution with a full complement of functional human lymphoid and myeloid cells, robust immunoglobulin production, T cells educated in human thymus organoid, production of human inflammatory cytokines and upregulation of key activation and maturation markers on DCs after antigen exposure (205, 212, 213). The mice have human progenitor cells in the bone marrow and the thymic graft contains CD4+CD8+ (double positive) thymocytes (214). The human immune reconstitution can be maintained in these mice for up to 9 months (205).  Human B cells are detected in the blood of the mice 1 month after HSC transplantation, and numbers gradually increase over the next 3 months (206). In the bone marrow there are early B cells, pro-B cells, pre-B cells and immature B cells, with numbers comparable between BLT mice and humans (206, 214). Immunization induces antigen-specific IgM but antigen-specific IgG responses are very weak due to defects in class-switching, suggesting that there are poor interactions between T cell and B cells (206, 212). The differentiation of B cells into plasmablasts occurred at lower levels in BLT mice (214). Some groups have reported that BLT mice have strong antigen-specific T cell responses, T cell dependent production of IgG including IgG1, IgG2, IgG3, meaning there are functional human T-B cell interactions capable of causing B cell activation, antibody production and class switching (215).   Small populations of NK, NKT and γδ T cells have been reported in these mice (205, 216). However, others have not observed γδ T cells (217). The NKT cells are functional and able to produce IFNγ and IL-4 (215).   Conventional DC (cDC) and pDC are present in BLT mice (214). cDC and pDC are found in the bone marrow, spleen and liver (206). The DCs are functional and can induce activation of allogenic human T cells or produce IFNα, but the cell frequency is low, at less than 33  1% (206). Human monocytes and macrophages can be detected in the blood, the lymphoid organs and in some tissues (206). There is little evidence for mast cells, but some groups report their presence (206).  Red blood cells and platelets are almost undetectable (215). While there are large numbers of human CD45-CD71+ nucleated immature erythroid cells in the bone marrow, red blood cells are undetectable in the blood (218). Red blood cells are detected upon macrophage depletion (218). Since the NOD SIRPα closely mimics human SIRPα, it is thought that red blood cell rejection is independent of CD47-SIRPα interaction (218).   While some groups have reported that neutrophils are not developed in humanized mice, others can detect neutrophils in BLT mice (206, 219).    The human immune cells reconstitution of BLT mice is not limited to the peripheral blood. Human CD45+ cells have been found in the bone marrow, thymic organoid, spleen, lymph nodes, liver, lung, and the gastrointestinal tract (205, 210, 214, 217). Human immune cells are also found in the female reproductive tract of BLT mice (205, 216). There are human T cells, macrophages and DCs in the vagina, ectocervix, endocervix and uterus, with distribution very similar to humans (205, 216). The oral cavity and upper GI tract of BLT mice is reconstituted with human leukocytes including DCs, macrophages and CD4+ T cells (220). The gut contains B cells, T cells, macrophages, NK cells and DCs, with the gut CD4+ and CD8+ T cells expressing a memory phenotype (213). CD4+CD8+ intraepithelial and lamina propria T cells are present in the small intestine (217). CD4+ T cells are found throughout the rectum and colon (217).  There are a number of factors important in cell development and function that differ between mouse and human, which can have an impact on innate immune cell development in 34  BLT mice (203, 206, 221-223). Several studies have been undertaken to provide human cytokines and growth factors to BLT mice in an attempt to increase innate cell development. Providing recombinant human granulocyte colony stimulating factor (G-CSF) to humanized mice increased human myeloid cells in the circulation, particularly granulocytes and macrophages (224). Transgenic mice expressing human IL-3/GM-CSF (granulocyte macrophage colony stimulating factor) have enhanced levels of human myeloid cells in the circulation (225). Provision of IL-4 and GM-CSF increased the number of DCs present in humanized mice (226). Providing human stem cell factor (SCF) or thrombopoietin enhanced HSC engraftment and myeloid differentiation (227-229).  NOD/SCID mice have high incidence of spontaneous thymic lymphomas and shortened life spans (197, 202). The thymic lymphomas are associated with expression of a NOD mouse-unique endogenous ecotropic murine leukemia provirus locus (Emv-30) that is proposed to interact with the scid mutation to initiate thymomagenesis (230). In NOD/SCID mice T cell differentiation is blocked at an early stage of maturation, leading to a large population of pre-T cells with increased replication capacity (230). Activation of Emv-30 in these cells is proposed to lead to thymomagenesis (230). NK cells are still present in these mice, and there is a “leakiness” that leads to development of mouse T cells and B cells as the mice age (197, 202). Mouse models have been developed that seek to further reduce the NK cell numbers and prevent B cell and T cell development.  The IL-2 receptor gamma chain is a crucial component of the high-affinity receptor for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 (219). The targeted mutation of the IL-2Rγ chain leads to severe impairments in B and T cell development and function, due to the lack of IL-2 signalling, and completely prevents NK cell development due to the lack of IL-15 signalling (197, 222). 35  Mice have been generated that lack the IL-2Rγ chain on the NOD/SCID background (231). The resulting NOD-scid-IL2Rγnull (NSG) mouse lacks mature lymphocytes and NK cells, expresses severe impairments in innate immunity, does not develop thymic lymphomas, and are long-lived, with a median survival 89 weeks, compared to 37 weeks for NOD/SCID mice (231). There was poor lymph node development and organization in the NSG mice (222). There was no evidence of serum immunoglobulin in the NSG mice (231). The spleens from poly(I:C)-stimulated NSG mice exhibited extremely low levels of NK cell cytotoxic activity (231). DC cultures from NSG mice generate abnormally low percentages of TNFα+ and CD86+ DCs (231). The NSG mice support heightened levels of human CD34+ cell engraftment compared to NOD/SCID mice, with percentages of human cells in the bone marrow of the NSG mice 5 to 6 fold higher than in the bone marrow of NOD/SCID mice (222, 231).   Both the NOD/SCID and the NSG model have been used for the development of BLT mice (NOD/SCID-BLT and NSG-BLT, respectively). While some groups have reported no differences between the strains in the extent of human immune system reconstitution (214, 232), others have observed poor reconstitution and susceptibility to mucosal HIV infection with NOD/SCID-BLT mice (219). Stoddart et al found that NSG-BLT mice had substantially higher levels of human leukocyte reconstitution in peripheral blood, with a mean of 13 times more human CD4+ T cells, 8 times more CD8+ T cells, 4 times more monocytes, 3 times more B cells and similar numbers of human NK cells and neutrophils (219). Female NSG mice are the best recipients for small numbers of HSCs (233).   1.4.2 Graft versus Host Disease in BLT mice  A form of Graft versus Host Disease (GvHD) has been described in BLT mice. In NOD/SCID-BLT mice this is characterized by progressive inflammation and sclerosis of the 36  skin, lungs, gastrointestinal tract, and parotid glands (234). All of the NOD/SCID-BLT mice showed involvement of at least one organ site, with the incidence of overt clinical disease reaching 35% by 22 weeks post-surgery (234). NSG-BLT mice have a delayed onset of GvHD by about 6 weeks, but by 35 weeks post-surgery 35% of NSG-BLT mice develop GvHD (234). GvHD was characterized by infiltration of CD4+ T cells into the skin and a shift towards Th1 cytokine production (234). There is mixed M1/M2 polarization phenotype in dermal murine CD11b+MHCII+ macrophages (234). Others have reported GvHD involving multi-organ cellular infiltrates containing human T and B cells and macrophages (235). Fibrosis was present in the lungs and liver, and the skin was thickened with alopecia (235, 236). Skin and liver cell death was associated with lymphocytes that were reactive with anti-human CD45, CD3 and CD8 (236).  The BLT triple knockout humanized mice (C57Bl/6Rag2-/-γc-/-CD47-/-) mice (TKO-BLT) have high levels of multilineage hematopoiesis, an intact complement system, and show no signs of GvHD for 29 weeks post-transplantation, and are susceptible to HIV-1 infection (237). Further work needs to be undertaken to determine if the TKO-BLT mouse model is superior to the NSG-BLT model. 1.4.3 HIV infection in BLT mice  While earlier models of humanized mice have demonstrated some utility for in vivo studies of HIV-1 (199, 238-241), BLT mice currently represent the best small animal model for HIV-1 transmission, pathogenesis, treatment, and prevention research.  BLT mice have a functional human immune system containing T cells, B cells, DCs and monocytes/macrophages (214). Importantly, the female reproductive tract is reconstituted with human immune cells and the distribution of cells closely resembles the human female 37  reproductive tract (205, 216). This makes the mice susceptible to intravaginal infection with HIV-1 (214, 216, 219, 242). Abrasions are not necessary for vaginal transmission of HIV-1 in BLT mice (197, 216). HIV-1 infection in these mice closely recapitulates the acute stage of HIV-1 that occurs in humans (205, 219, 222). There is a rapid increase in viremia peaking 4-6 weeks post-infection, followed by a decline in virus levels (205, 219, 222). This is accompanied by a decline in CD4+ T cells, including in the GALT (214, 216).   BLT mice are susceptible to HIV-1 infection via all major physiological routes of viral transmission – vaginal (205, 216, 219, 243-247), rectal (217, 232), intravenous or intraperitoneal injection (214, 232, 248, 249) and oral (220). BLT mice are able to sustain a high level, disseminated HIV-1 infection (214, 219, 220, 243, 244). HIV is rapidly disseminated throughout all tissues in the BLT mouse (205, 216, 217). The GI tract of the mice has human lymphoid cells, which are rapidly depleted after HIV infection (205, 216, 217). CD4+ T cell depletion also occurs in the blood (207, 214, 216, 232). There is production of HIV-specific human antibodies (217). Humoral specific immune responses are present in these mice by three months post-infection (214, 222). There are HIV specific CD4+ and CD8+ T cell responses by 9 weeks post-infection (214). The CD8+ T cell responses in these mice closely resemble those observed in humans in terms of specificity, kinetics and immunodominance (249). There is rapid viral escape (249). Productively infected cells are found in the lymph nodes, spleen, human thymic tissue, lung, GI tract and reproductive tracts (217).   Mice that receive ART harbour latently infected resting human CD4+ T cells, which can be induced ex vivo to produce HIV-1 (250). The latent reservoirs consist of resting CD4+ T cells harbouring integrated provirus expressing little or no viral gene products (248). If the latently infected cells are stimulated, productive infection occurs (248). The viral reservoir is formed 38  soon after primary infection occurs, and the latently infected cells have a long half-life, which can maintain life-long infection (248).  A wide variety of PrEP and microbicide studies have been undertaken with BLT mice. PrEP studies have demonstrated protection from vaginal transmission using oral raltegravir and maraviroc (243), protection from rectal and intravenous HIV-1 transmission using systemic administration of FTC-TDF (232), protection from vaginal HIV-1 transmission with systemic administration of FTC-TDF (216), and that systemic administration of antiretrovirals can prevent oral transmission of HIV-1 (220). Microbicide studies have demonstrated protection from vaginal transmission using a rilpivirine encapsulated nanoparticles as a gel (251), 1% TFV gel applied following the CAPRISA 004 guidelines protects BLT mice from vaginal HIV-1 infection (246), and topical application of maraviroc protects from vaginal HIV-1 infection (247). BLT mice have represented an excellent model to study HIV-1 prevention.  1.5 Herpes simplex virus 2  Herpes simplex virus 2 (HSV-2) is a common genital infection, with a prevalence of 10-20% of people living in the USA and 30%-80% of people in some parts of Asia, Eastern Europe and Sub-Saharan Africa (252, 253). Up to 75% of people that have HSV-2 are unaware that they have the virus (254). The seroprevalence of HSV-2 in women is up to twice as high as men (255-257). HSV-2 is a major risk factor for HIV-1 acquisition, contributing to a 2-4 fold increase in risk of HIV-1 infection (258-261). There are several mechanisms that contribute to this increase in HIV-1 acquisition (255, 258). Genital ulceration caused by HSV-2 creates breaches in the epithelium that can aid HIV-1 entry (255, 262). In addition, HSV-2 lesions are associated with persistent infiltration of CD4+ T cells and CD8+ T cells in the vaginal tract, leading to a chronic 39  inflammatory state in the genital region that can last for months after lesion resolution (258, 259, 262-265).  1.5.1 HSV-2 life cycle  HSV-2 is a large, enveloped double-stranded DNA virus from the Herpesviridae family (266). The genome contains two regions, the unique long (UL) and unique short (Us) regions and encodes for 84 different proteins (266, 267).   HSV-2 enters epithelial cells through at least two steps, attachment and fusion (267). HSV-2 attaches to cells by binding to glycosaminoglycans on the cell surface proteoglycans, most frequently heparin sulphate or chondroitin sulphate (268). Attachment is mediated by HSV-2 gC and gB (268, 269). Viral fusion requires gH/gL and gB, which are the core machinery of fusion, plus gD (268, 270-273). gD interacts with alternative receptors, nectin 1, HVEM and modified heparin sulphate (268, 270, 274, 275). gD is a determinant of viral tropism and a key player in triggering fusion (270). Entry can occur at the plasma membrane or following endocytosis, or the virus may enter by macropinocytosis (270). The specific pathway differs from cell-to-cell (270). Following fusion, the capsid is released into the cytoplasm and transported to the nucleus by cell microtubule proteins (266, 276). The DNA is released into the nucleus and circularizes before gene expression can occur (266).   All viral transcripts are produced by cellular RNA polymerase (266). Gene expression occurs in waves, beginning with immediate early (IE) genes, followed by early (E) genes, “leaky-late” (L1) genes, DNA replication and lastly, late (L2) gene expression (277). Almost all of the IE gene products are involved in gene regulation, whereas E genes are involved in DNA replication and L genes are structural proteins required for assembly of infectious virions (252, 277). 40  1.5.2 HSV-2 pathogenesis   In most cases, initial infection with HSV-2 leads to the formation of lesions in the genital mucosa (252, 259). Virus pathology is mainly caused by direct cytopathic effects of the virus, resulting in epithelial cell lysis and focal necrosis of the infected area (255). Although these lesions will resolve, the virus is not cleared, and undergoes latency in the nerves (252). The virus can periodically reactivate, leading to shedding of infectious virions, and lesions may be formed (252).   HSV-2 infects and replicates in epithelial cells and undergoes latency in the nervous system (252). There is no detectable viral replication during latency (266). The only genes readily detectable are the latency associated transcripts (LATs) (252). The function of the LATs is not well understood, but it is unlikely these genes are translated into protein (252). Lytic cell viral genes are not generally detected during latency, so the reactivation of the virus seems to occur in the absence of pre-existing viral proteins (252). The mechanism of reactivation from latency is unclear, with many groups reporting differing roles for viral proteins (266, 278-281). Cellular factors may also play a role in reactivation (266).   HSV establishes latency within sensory neurons by entering via the nerve terminus during primary infection (252). The virus infects the neuronal dendrites of sensory ganglia that innervate the mucosa, and are transported to the nerve cell body by retrograde microtubule-associated transport (252). During primary infection the virus replicates within mucosal epithelial cells (252). During latent infection no infectious virus is produced from infected cells and the disease is absent, with no transmission occurring (252). Reactivation occurs occasionally, allowing the virus to re-enter the lytic phase of its life cycle (252).  41  1.5.3 HSV-2 prevention  While condoms can provide some measure of protection against HSV-2 infection, there is no highly effective method to prevent HSV-2 transmission (282). Microbicides and vaccines have been investigated for HSV-2 prevention with little success. 1.5.3.1 HSV-2 microbicides  Microbicides have been investigated as a potential option for HSV-2 prevention. Table 1.2 HSV-2 microbicides Microbicide Target Efficacy Reference Zinc acetate and carrageenan Binding of zinc to viral membrane glycoproteins impairing virus entry Decreases infection by 20%-80% in macaques; efficacy for HIV-1 prevention  (283-286) Acyclovir and the prodrug valacyclovir Nucleotide analogs that prevent viral replication by inhibiting DNA polymerase When given to HSV-2 seropositive person valacylovir can prevent HSV-2 transmission, acyclovir is ineffective (287-289) Nonoxonyl-9 Spermicide, inactivates HSV-2 Anti-HSV-2 activity in vitro. Increased HSV-2, HPV and HIV-1 infection in animals and people by disrupting the epithelium  (122, 290-293) 42  Microbicide Target Efficacy Reference VivalGel (SPL7013) Inhibits viral entry Efficacy in vitro for HSV-2 and HIV-1 (294) Surfactants SDS and C31G (Savvy) Disrupt the membrane proteins of viruses Toxicity in vivo, increased susceptibility to HSV-2 infection, caused adverse events such as vulvovaginitis (295-297) Cellulose sulphate and Pro 2000 Entry blockers that mimic heparin sulphate and bind gB Not protective for HSV-2 and increase HIV-1 infection (121, 298-301) 1% TFV gel Prevents viral DNA replication CAPRISA 004 sub-study found TFV gel caused a 71% reduction in HSV-2 acquisition (2, 302) Griffithsin Prevents cell-to-cell spread of HSV-2 Protected mice from HSV-2 infection (303) Elafin Unclear, seems to act indirectly on cells, targeting viral attachment and post-entry cellular responses Attenuates HSV-2 pathology and trend towards better survival in mice (304)   43  1.5.3.2 HSV-2 vaccines  There are two approaches to HSV-2 vaccination, prophylactic vaccination and therapeutic vaccination (282). Many vaccines that have been developed for HSV-2 were successful in preventing infection in animal models, but had no efficacy in people (258). Since the rate of seroconversion is estimated to be 5% per year, vaccine trials are expensive to conduct as large numbers of participants are necessary to show efficacy, so few HSV-2 vaccine clinical trials have been conducted (282).   Recombinant glycoprotein subunit vaccines have received the most attention for HSV-2. A recombinant gB and gD subunit vaccine was safe for use and induced high levels of neutralizing antibodies and CD4+ T cell responses (305, 306). However, this was not successful at preventing HSV-2 infection (305, 306).  A glycoprotein D subunit vaccine was tested in people whose regular sexual partners had a history of genital herpes (307). The vaccine induced both cellular and humoral immune responses (307). The efficacy of the vaccine was 73% for women that were seronegative for both HSV-1 and HSV-2 (307). The vaccine did not work in women who were seropositive for HSV-1, or in men regardless of HSV-2 serostatus (307). A follow-up study found that the vaccine was not efficacious, with protection for HSV-2 at 20% (308). However, efficacy was 58% against HSV-1 genital disease (308).  1.6 Research hypothesis and rationale  Vaginal transmission accounts for approximately half of all new HIV-1 infections. Although condoms can provide a measure of protection against HIV-1 infection, campaigns focused on abstinence and condom use have not been completely successful at lowering HIV-1 44  infection rates for women (2). This is at least partially due to the fact that condom use relies on the consent of a partner, which may not always be possible. Nevertheless, a new female controlled prevention option for HIV-1 is urgently needed. With the difficulty in developing an efficacious vaccine, and the lack of efficacy of a daily PrEP routine in women in Sub-Saharan Africa, it is clear that an alternative option is needed for HIV-1 prevention. Microbicides represent a promising option for female-controlled prevention of HIV-1.  As the S-layer of C. crescentus is easily modifiable to display a wide variety of functional proteins (171, 176, 179, 181, 182), we hypothesized that displaying proteins that could interfere with HIV-1 attachment or entry in the S-layer of C. crescentus would prevent HIV-1 infection. We have previously demonstrated a proof-of-concept of this hypothesis, showing that CD4 and MIP1α can be displayed at high density in the S-layer of C. crescentus, and that these recombinant C. crescentus are able to provide significant protection from HIV-1 pseudovirus infection in vitro (309). In this thesis, these studies were expanded to include additional inhibitors of HIV-1 infection that target all aspects of the attachment and entry process, additional in vitro testing using live HIV-1, and in vivo testing with humanized BLT mice. In addition, since HSV-2 is a major risk factor for HIV-1 acquisition, testing was undertaken in a mouse model of HSV-2 to determine if a dual-target microbicide could be developed. 1.6.1 Aim 1   As microbicides represent a promising option for female controlled prevention of HIV-1, we hypothesized that expressing anti-HIV proteins that target HIV-1 attachment or entry in the S-layer of C. crescentus would provide protection from HIV-1 infection for microbicide development. As mentioned above, it has previously been demonstrated that expressing CD4 or MIP1α in the S-layer could provide protection from HIV-1 infection in vitro. To expand these 45  studies we identified 16 additional proteins from the literature that could interfere with various aspects of the HIV-1 attachment and entry process. In Chapter 2 we describe the creation of the recombinant C. crescentus that will form the basis of this microbicide. The recombinant C. crescentus were first tested in vitro using the enveloped pseudo-typed HIV-1 (pseudovirus) representing viral clades B and C and the TZM-bl reporter cell line. Those candidates that were successful in the pseudovirus assays moved on to testing with live, replication competent HIV-1 using both the TZM-bl cell line and human PBMCs as appropriate.  1.6.2 Aim 2  In Aim 1 we demonstrated that anti-HIV proteins could be expressed in the S-layer of C. crescentus and were able to prevent HIV-1 infection in vitro. In Aim 2 we sought to demonstrate that C. crescentus is safe for topical application to the vaginal tract. As C. crescentus is a gram-negative bacterium, there is the risk of development of an immune response, which could enhance HIV-1 infection. Although collaborators have demonstrated that C. crescentus is safe for intraperitoneal injection into mice (172), no research has been undertaken on mucosal application. Based on this intraperitoneal data we hypothesized that C. crescentus would be safe for topical application to the vaginal tract. Chapter 3 describes the cytokine response following intraperitoneal injection and vaginal application of C. crescentus. Additional studies investigated immune cell recruitment to the vaginal tract and production of antibodies after topical application of C. crescentus.  1.6.3 Aim 3  In Aims 1 and 2 we demonstrated that recombinant C. crescentus can prevent HIV-1 infection in vitro, and that C. crescentus appears safe for vaginal application. Although in vitro screening represents an excellent tool for rapid determination of HIV-1 protection against a wide 46  variety of viruses, it does not fully recapitulate HIV-1 transmission in a physiologically relevant system. As the in vitro studies demonstrated that recombinant C. crescentus could prevent HIV-1 infection, I hypothesized that these results would be maintained in an in vivo model of HIV-1 infection. In this aim we describe the generation of the BLT mouse model at the University of British Columbia and testing of the recombinant C. crescentus in this model for ability to prevent vaginal infection with HIV-1. 1.6.4 Aim 4  HSV-2 is a major risk factor for HIV-1 acquisition. A dual target microbicide could have enormous impact on preventing both HSV-2 and HIV-1 infection. As seven of the microbicide candidates have been demonstrated or suggested to provide protection against HSV-2 infection, I hypothesized that these seven recombinant C. crescentus would provide protection from infection with both HIV-1 and HSV-2. Chapter 5 describes in vivo testing of these candidates for the ability to prevent HSV-2 infection in C57Bl/6 mice.    47  Chapter 2: In vitro protection from HIV-1 infection using recombinant Caulobacter crescentus 2.1 Introduction The HIV/AIDS pandemic is one of the largest global health concerns, with over 36.9 million people worldwide living with an HIV infection (1). The majority of new infections occur through sexual transmission, with 2 million new infections annually (1, 2). Sexual transmission of HIV can be prevented by the use of condoms, but women in developing countries do not always have the access to or option of insisting on condom use, often due to cultural or religious practices (2). As such, women over the age of 15 in developing countries account for the majority of new HIV-1 infections (1, 2). The development of female-controlled prevention strategies for HIV-1 is urgently needed. With long-standing difficulties in developing an efficacious vaccine, alternative prevention options are required. Microbicides are drug products that are topically applied to mucosal surfaces to prevent infection (98). They may be able to fill the need for female controlled prevention, as they can often be administered without partner knowledge, and are able to maintain efficacy for extended periods of time. Currently, no microbicides for HIV-1 are on the market, but approximately 50 candidates are under development (98-100). Many of these are non-specific and/or expensive to produce, which may limit the clinical effectiveness or practicality for delivery to low income populations (98). To counteract these difficulties, some efforts have focused on engineering bacteria to display HIV blocking agents (123-125). While most of these approaches have used commensal vaginal bacteria, such as species of Lactobacillus, use of these recombinant bacteria would rely on their ability to colonize the vaginal tract and maintain expression of the protein for 48  extended periods of time, which has shown limited success (123-125). Issues with expression and secretion of larger proteins, as well as concerns about eradication of the bacteria should unwanted side effects occur, suggest the use of Lactobacillus species in microbicide formulations may be problematic (123-125).    In contrast, we have engineered an alternative bacterium-based microbicide strategy that does not require colonization or maintenance of the bacteria in the patient. Using non-pathogenic Caulobacter crescentus (309, 310) we have generated a system to insert foreign protein sequences into the surface (S)-layer protein RsaA of C. crescentus (171, 176, 179, 181, 182). Native expression levels of RsaA exceeds 30% of total cell protein and the insertion of foreign proteins often has minimal impact on secretion and assembly of RsaA into the ordered S-layer structure (174). Although difficult to assess in every instance, the proteins generally retain their function when displayed within the S-layer (171, 176, 179, 181, 182). Proteins of a wide range of sizes can all be expressed and displayed within the S-layer (171, 181). In addition to the protein expression capabilities, C. crescentus cannot grow at the temperature or salt concentrations found in human tissue (172, 173). Further, the lipopolysaccharide of C. crescentus has an unusually low endotoxin response, and no obvious adverse effects when injected intraperitoneally into mice (172, 187). In our view these features make it an ideal candidate for development into a microbicide (172, 173, 187).   The C. crescentus protein display system was utilized previously to express MIP1α or domain 1 of CD4 in the S-layer (309). Expression of either MIP1α or CD4 individually provided significant protection from infection with several variants of HIV-1 pseudovirus representing clade B in a standard single cycle infection assay (309). In this chapter we test the hypothesis that alternative inhibitors of infection may be more effective at preventing infection by 49  introducing C. crescentus constructs that express a diverse array of proteins that could prevent HIV-1 attachment or entry. These agents represent a range in potential with both high and low expectations for successful display as well as blocking HIV-1 infection. Herein, these findings are further refined to show protection from infection with an HIV-1 pseudovirus representing clade C. Together, they have allowed us to better define the limitations of agents that can be utilized within C. crescentus based microbicides.   All the proteins were expressed individually within the S-layer, and each C. crescentus construct provided variable, yet significant protection from HIV-1 pseudoviruses representing clades B and C, uncovering the large potential for efficacy and function using this C. crescentus based microbicide strategy. The anti-HIV lectins were the most effective at preventing HIV infection, providing up to a 70% decrease in infection levels. Those recombinant C. crescentus that were able to provide protection from pseudovirus infection underwent subsequent testing with live, replication competent HIV-1 and protection levels were generally maintained with live HIV-1.  2.2 Materials and methods Bacterial strains and growth conditions - Escherichia coli strain DH5α (Invitrogen, Carlsbad, CA) was grown at 37°C in Luria Broth (1% tryptone, 0.5% NaCl, 0.5% yeast extract). C. crescentus strains were grown in PYE medium (0.2% peptone, 0.1% yeast extract, 0.01% CaCl2, 0.02% MgSO4) at 30°C.  For growth on solid medium, agar was added to 1.3%.  JS4026 is a derivative of CB2 that is Sap negative and contains repBAC inserted into xylX (174, 175). C. crescentus strain JS4038 was made defective in capsular polysaccharide synthesis (311).  It is a modification of JS4026 in which a GDP-L-fucose synthase gene (comparable to the gene 50  annotated as CCNA00471 in C. crescentus NA1000) (312) has been inactivated by introduction of an internal deletion. The oligos JN471-1 (CCGCATCGAGCATATTTATCAAGATCC) and JN471-52 (GTATCGGTGATGATCTCGCCCTTG) were used to amplify by PCR a 1721 base pair (bp) fragment from C. crescentus strain CB2A.  This PCR product contained 900 bp upstream of the gene and the first 821 bp of the 967 bp GDP-L-fucose synthase gene.  After blunt ligation into EcoRV cut pBSKIIEEH (184) the 215 bp Bstx1 – Sma1 segment was removed, blunted and religated to make an internal deletion in the fucose synthase gene.  This ~1500 bp segment was moved into pKmobSacB (313) as an EcoR1/HindIII fragment.  Clones were screened for first and second crosses with the oligos JN 471-31 (CTGCTGATCGGTGATCCGACGAAG) and JN 471-52. A clone demonstrating a ~750 bp PCR product (indicating replacement of the chromosomal gene with the deletion version) and loss of mucoid colony appearance on PYE medium supplemented with 3% sucrose (312) was named JS4038. Unless otherwise indicated, JS4038 was used for all studies. When needed, media contained chloramphenicol (CM) at 20 µg/ml (E. coli) or 2 µg/ml (C. crescentus).  Electroporation of C. crescentus was performed as previously described (314).  Preparation of C. crescentus displaying chimeric S-layer proteins  - The MIP1α and CD4 gene segments have been described (309). All other gene segments were synthesized by commercial sources with codon usage adapted for C. crescentus. The amino acid sequence specified for each are indicated in Table 2.1. In addition, the synthesized DNA segments specified BglII and SpeI restriction sites on the 5’ side and an NheI site on the 3’ end. This permitted directional cloning into p4ARsaA(723)/GSCC digested with BglII and NheI (181). Following ligation, plasmids were introduced into E. coli by electroporation. Inserted sequences were confirmed by DNA sequencing before transfer to C. crescentus by electroporation.   51  Protein analysis - S-layer proteins were prepared by a low-pH extraction method (315). Proteins were visualized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% separating gels and staining with Coomassie Brilliant Blue R. Preparation of C. crescentus cells for binding assay. - C. crescentus S-layer display constructs were grown in PYE medium to an optical density at 600nm of approximately 1 (3.1 x 109 cells/mL).  Cells were centrifuged and suspended in water. This was repeated and cell density was adjusted to 1 x 1010 cells/mL for binding experiments. Plasmids and DNA manipulations - The Standard Reference Panel of Subtype B HIV-1 Env Clones was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Drs. David Montefiori, Feng Gao and Ming Li: PVO, clone 4 (SVPB11); TRO, clone 11 (SVPB12), and the env-deleted backbone plasmid pSG3Δenv from Drs. J.C. Kappes and X. Wu (86, 316, 317). The Subtype C HIV-1 Reference Panel of Env Clones was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Drs. D. Montefiori, F. Gao, S. Abdool Karim and G. Ramjee: Du156.12 (SVPC3) and Du172.17 (SVPC4) (318, 319). E. coli with plasmids containing the viral DNA cassettes were grown as described (309). Plasmids were purified using a large scale plasmid isolation procedure (320). The Nucleic Acid Protein Service Unit of the University of British Columbia provided DNA sequencing.  Small-scale plasmid isolations were done using EZ-10 spin mini plasmid kits (Bio Basic Inc). DNA fragments were recovered from agarose gels using a QIAEX II gel extraction kit (QIAGEN).   Cell lines – 293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 7.5% fetal bovine serum (FBS), 100U/mL Penicillin and 0.1mg/mL Streptomycin. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: 52  TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc (317, 321-324). TZM-bl cells were maintained in DMEM with 7.5% FBS (Gibco), 100U/mL Penicillin and 0.1mg/mL Streptomycin (Gibco). The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: 174xCEM Cells from Dr. Peter Cresswell (325). 174xCEM cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640) supplemented with 10% FBS.  Primary cells – Human PBMCs were obtained from healthy donors after informed, written consent. Whole blood was collected and processed using Lymphoprep (Stem Cell Technologies). PBMCs were grown in RPMI 1640 supplemented with 20% FBS. All procedures were approved by the University of British Columbia Clinical Research Ethics Board certificate H12-02480. Pseudovirus transfection - One day prior to transfection 3.6 x 106 293T cells were seeded in a 10 cm Corning plate, using complete DMEM (1% penicillin/streptomycin and 7.5% FBS). Transfection was performed using the jetPrime kit (Polyplus Transfection). 12µg env clone and 24µg pSG3Δenv were diluted in 1.5mL buffer. After vortex mixing, 30µL of jetPrime reagent was added and the mixture was incubated for 10 minutes at room temperature, added to the 293T cells in FBS- and antibiotic-free medium and incubated for 4-6 hours. The medium was then replaced with antibiotic-free DMEM containing 7.5% FBS and the cells were incubated for 48 hours at 37°C. The virus produced was harvested and collected using a 0.45µm syringe filter and stored at -80°C. HIV-1 – The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-189.6 from Dr. Ronald Collman (326); pYK-JRCSF (Cat# 2708) from Dr. Irvin SY Chen and Dr. Yoshio Koyanagi (327-329) and was a gift from Dr. Zabrina Brumme (Simon Fraser University); HIV-1SF162 from Dr. Jay Levy (330); HIV-1BaL 53  from Dr. Suzanne Gartner, Dr. Mikulas Popovic and Dr. Robert Gallo (331, 332); HIV-1JR-FL from Dr. Irvin Chen (328, 333, 334). HIV-1 propagation - HIV-189.6 was propagated in 174xCEM cells with 7.5µg/mL DEAE-dextran (Sigma) and was harvested at peak CPE between 7-10 days. HIV-1pykJR-CSF was propagated in 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. HIV-1JR-FL was propagated in PBMCs. HIV-1JR-FL was added to 5 x 106 PHA-stimulated PBMCs with 10µg/mL polybrene (Hexadimethrine bromide, Sigma) for 2 hours at 37°C with occasional shaking. PBMCs were washed and grown in RPMI 1640 + 20% FBS for 4 days before the media was changed and replaced with fresh RPMI 1640 + 20% FBS. Virus was harvested on days 5, 6 and 7 post-infection. HIV-1BaL was propagated in PHA-stimulated PBMCs with 7.5µg/mL DEAE-dextran for 10 days. HIV-1SF162 was propagated in PHA-stimulated PBMCs with 10µg/mL polybrene for 10 days. Virus titration - Serial dilutions of virus were prepared in 96-well plates using medium containing 7.5µg/mL DEAE-dextran. TZM-bl cells were adjusted to a concentration of 2 x 105 cells/mL and added to each well. The plates were maintained at 37°C and 5% CO2 for 48 hours. Infection of cells was measured indirectly using a Mammalian β-galactosidase assay kit (Pierce) followed by absorbance reading at 415 nm. An absorbance of greater than 0.2 was considered a positive infection. The 50% Tissue Culture Infectious Dose (TCID50) per mL was determined for each viral stock by identifying the dilution of virus in which 50% of the TZM-bl cells were infected, as measured by the presence of β-galactosidase.  Virus blocking experiments - The C. crescentus constructs were grown at 30°C in PYE medium with 2µg/ml chloramphenicol 1-2 days before the experiment, prepared in sterile water as described above and diluted to 5 x 109 cells/mL immediately prior to experimental setup. 54  Experiments were carried out in quadruplicate wells of 96-well plates. The volume of virus added was determined by calculating the 200TCID50 value. 1 x 108 C. crescentus cells were added to each well. The virus and C. crescentus constructs were incubated for 1 hour at 37°C before adding 10,000 TZM-bl cells or PBMCs and 7.5µg/ml DEAE-dextran to each well. Cc-MIP1α was incubated with the TZM-bl cells or PBMCs for 1 hour before virus addition. After an overnight incubation at 37°C and 5% CO2 the plates were centrifuged at 800 rpm for 5 minutes and medium was changed in all wells (pseudovirus or PBMCs only). After 24-48 hours the level of infection was determined by β-galactosidase assay kit (TZM-bl cells) or p24 ELISA (ZeptoMetrix Corporation and ProSci Incorporated) according to the manufacturer’s instructions (PMBCs). For β-galactosidase assays data are presented and determined as a percentage of infection of the untreated control infection wells with the background from uninfected TZM-bl cells subtracted. For p24 ELISAs data are normalized to infection of the untreated control wells as 100%.  Statistical analysis - Statistical analysis was performed with Prism GraphPad software. Results are reported as mean + standard error of the mean (SEM). Student’s t test (Cc-Control and 1 recombinant C. crescentus) or ANOVA with Bonferroni’s correction for multiple comparisons (Cc-Control and 2+ recombinant C. crescentus, each compared to the Cc-Control and each other) were used to evaluate the significance of differences between groups as appropriate. A p value of <0.05 was considered statistically significant. Experiments were conducted in quadruplicate wells and repeated in a minimum of three independent experiments, unless indicated otherwise.  55  2.3 Results 2.3.1 Expression of recombinant proteins in the S-layer of C. crescentus  To expand the number of HIV inhibitors tested in a recombinant C. crescentus microbicide, I first identified several classes of anti-HIV proteins from the literature, including natural proteins, antiviral lectins and synthetic fusion inhibitors. These proteins target various parts of the HIV attachment and entry process, and are diverse in size and composition (Figure 2.1 and Table 2.1). Using an expression vector for C. crescentus carrying restriction sites for insertion of genetic material at a site corresponding to amino acid 723 of the S-layer protein (181), recombinant C. crescentus that display each of these proteins within the S-layer were generated. The sequence of each expression vector was confirmed prior to transformation into C. crescentus. A low pH extraction method (which has been a reliable method to monitor export, assembly and surface attachment of S-layer proteins) and SDS-PAGE was used to measure the expression levels for the chimeric S-layer proteins. Although not quantified, all constructs resulted in expression of substantial quantities of protein and demonstrated a size shift by protein gel analysis, indicating that the recombinant protein was expressed. In most cases, the level of recombinant chimeric S-layer protein was comparable to native protein expression (Figure 2.2, Dr. John Nomellini). We previously demonstrated surface expression of recombinant S-layer protein and display of inserted proteins with antibodies directed to CD4 and MIP1α (309).    56  Figure 2.1: Recombinant C. crescentus target all aspects of HIV-1 attachment and entry  Figure 2.1 HIV-1 gp120 attaches to CD4 on a target cell. This leads to a conformational change that exposes the co-receptor binding site on gp120. Co-receptor binding (CCR5 or CXCR4) triggers an additional conformational change that exposes the fusion peptide gp41. gp41 mediates fusion of HIV with the target cell. Cyanovirin-N, microvirin and Griffithsin interact with glycosylations on the gp120 protein. gp120 nanobody binds to gp120. gp120M15D acts as a decoy ligand for CCR5. CD4 and CD4M33F23 act as decoy receptors for HIV-1. MIP1α acts as a decoy ligand for CCR5. α-1 antitrypsin prevents virus fusion. Fuzeon, T1249 and C52 prevent fusion of HIV-1 with the target cell. BmKn2, Elafin, Indolicidin and mucroporin M1 act on the HIV-1 attachment and entry process but the site of interaction is unknown. Image courtesy of Dr. Michael Jones and adapted by Christina Farr.      HIVCD4+ T-Cell CD4+ T-Cell CD4+ T-Cell CD4+ T-Cell1.gp120gp41Ccr5 Ccr5gp120Ccr5gp120gp41Cellular membraneHIVHIV HIV2. 3. 4.CD4 CD4gp41CD4gp41CD4,	  CD4M33F23 MIP1α	  gp120M15D  Fuzeon,	   T1249,	   C52 Cyanovirin, Microvirin,	   Griffithsin BmKn2,	  Elafin,	  GBVCE2,	  Indolicidin,	  mucroporin	  M1 gp120	  nanobody α-­‐1	  antitrypsin 57  Figure 2.2: Low pH demonstrating expression of recombinant S-layer protein A)  B)        1	  	  	  	  	  2	  	  	  	  3	  	  	  	  	  4	  	  	  	  	  5	  	  	  	  6	  	  	  	  	  	  7	  	  	  	  8	  	  	  	  	  9	  	  	  10	  	  	  	  11 98	  kDa	  kDa66	  kDa 44	  kDa   	  	  	  	  1	  	  	  	  	  	  	  	  2	  	  	  	  	  	  	  3	  	  	  	  	  	  4	  	  	  	  	  	  5	  	  	  	  	  	  	  6	  	  	  	  	  	  	  	  7 98	  kDa 66	  kDa   RsaA 58  C)  D)         RsaA 98	  kDa 66	  kDa 44	  kDa 1 2 3 4 5 98	  kDa 66	  kDa RsaA 	  	  1	   2	   	  	  	  3	  	  	  	  	  	  	  4	   	  	  	  	  	  5	   	  	  	  	  6	   	  	  	  	  	  	  	  7	   8 59  E)  F)  Image courtesy of Dr. John Nomellini Figure 2.2 Representative Coomassie blue stained 7.5% SDS-PAGE of normalized low pH extracted RsaA protein from C. crescentus strain JS 4038 containing RsaA plasmids. Arrows indicate the location of the S-layer protein. A. Low pH of C. crescentus S-layer. 1) Cc-Control (no insert); 2) Cc-MIP1α; 3) Cc-CD4; 4) Cc-CD4M33F23; 5) Cc-Cyanovirin; 6) Cc-Microvirin; 98	  kDa 66	  kDa 44	  kDa RsaA 1	   	  	  	  2	   	  	  	  	  3	  	  	  	  	  	  	  4	   	  	  	  	  	  	  	  5	  	  	  	  	  	  	  	  	  6 98	  kDa 66	  kDa 44	  kDa RsaA 	  	  1	   	  	  	  	  	  	  	  	  2	   	  	  	  	  	  3	   	  	  	  	  	  	  	  	  	  	  	  4 60  7) Cc-Griffithsin; 8) Cc-Fuzeon; 9) Cc-T1249; 10) Cc-C52; 11) Cc-Control (no insert). B. 1) Cc-Control (no insert); 2) Cc-A1AT; 3) Cc-BmKn2; 4) Cc-Elafin; 5) Cc-Indo; 6) Cc-Indo2; 7) Cc-Control. C. 1) Cc-Control; 2) Cc-GBVCE2; 3-5) S-layer expression controls. D. 1) Cc-Control; 2) S-layer expression controls; 3) Cc-gp120M15D; 4-6) S-layer expression controls; 7-8) Empty wells. E. 1) Cc-Control; 2) S-layer expression control; 3) Cc-Mucroporin; 4-6) S-layer expression controls. F. 1) Cc-Control; 2) Cc-gp120NB; 3-4) S-layer expression control.  Table 2.1: Recombinant C. crescentus display constructs Displayed Segment Sequence Size (# of Amino Acids) Name of Recombinant C. crescentus Reference MIP1α APLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSA 70 Cc-MIP1α (309, 335) CD4 GDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQ 81 Cc-CD4 (309, 335) CD4M33F23 NLHFCQLRCKSLGLLGKCAGSFCACV  26 Cc-CD4M33F23 (336) 61  Displayed Segment Sequence Size (# of Amino Acids) Name of Recombinant C. crescentus Reference Cyanovirin-N LGKFSQTCYNSAIQGSVLTSTCERTNGGYNTSSIDLNSVIENVDGSLKWQGSNFIETCRNTQLAGSSELAAECKTRAQQFVSTKINLDDHIANIDGTLKYE 101 Cc-Cyanovirin (337) Microvirin MPNFSHTCSSINYDPDSTILSAECQARDGEWLPTELRLSDHIGNIDGELQFGDQNFQETCQDCHLEFGDGEQSVWLVCTCQTMDGEWKSTQILLDSQIDNNDSQLEIG 108 Cc-Microvirin (338) Griffithsin SLTHRKFGGSGGSPFSGLSSIAVRSGSYLDAIIIDGVHHGGSGGNLSPTFTFGSGEYISNMTIRSGDYIDNISFETNMGRRFGPYGGSGGSANTLSNVKVIQINGSAGDYLDSLDIYYEQY 121 Cc-Griffithsin (339) Fuzeon (T-20, enfuvirtide) MYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFM 38 Cc-Fuzeon (340) 62  Displayed Segment Sequence Size (# of Amino Acids) Name of Recombinant C. crescentus Reference T-1249 WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF 39 Cc-T1249 (341) C52 NHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNI 52 Cc-C52 (342) GB virus C E2 protein WDRGNVTLLCDCPNGPWVWVPAFCQAVG 28 Cc-GBVCE2 (343) BmKn2 FIGAIARLLSKIF 13 Cc-BmKn2 (344) Elafin AQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ 57 Cc-Elafin (304, 345) Indolicidin ILPWKWPWWPWRR 13 Cc-Indo (346, 347) Indolicidin dimer ILPWKWPWWPWRRASILPWKWPWWPWRR 28 Cc-Indo2  α1-antitrypsin LEAIPCSIPPEFLFGKPFVFLMIEQNTKSPLFMG 34 Cc-A1AT (348) MucroporinM1 LFRLIKSLIKRLVSAFK  17 Cc-Mucroporin (344) gp120 M15D IRKAHCNISRADWND  15 Cc-gp120M15D (349) 63  Displayed Segment Sequence Size (# of Amino Acids) Name of Recombinant C. crescentus Reference gp120 nanobody EVQLVESGGGLVQAGGFLRLSCELRGSIFNQYAMAWFRQAPGKEREFVAGMGAVPHYGEFVKGRFTISRDNAKSTVYLQMSSLKPEDTAIYFCARSKSTYISYNSNGYDYWGRGTQVTVSS 121 Cc-gp120NB (350, 351)  2.3.2 Inhibition of HIV-1 pseudovirus infection with Cc-MIP1α  MIP1α is the natural ligand for the HIV-1 coreceptor CCR5, which is the most common coreceptor used during initial HIV-1 infection (18). We have previously generated recombinant Cc-MIP1α and found that it was sufficient to block infection with an HIV-1 pseudovirus representing clade B, which is the most common clade of HIV-1 found in North America and Europe, by 35-78% in single cycle infection assays (309, 352). To further investigate the relevance of C. crescentus display for HIV-1 prevention we measured inhibition of infection with HIV-1 pseudovirus representing clade C, which is most common in Sub-Saharan Africa and the major cause of the HIV-1 pandemic (352). 108 Cc-MIP1α cells were preincubated with TZM-bl cells, then clade C pseudovirus SVPC3 or SVPC4 was added. Previously, dose response experiments demonstrated that 108 C. crescentus was sufficient to block HIV-1 infection with clade C viruses (Appendix A, Figure A.1). We found that Cc-MIP1α provided statistically significant protection from infection when compared to Cc-Control with each clade C 64  pseudovirus (Figure 2.3, Table 2.3). In particular, Cc-MIP1α reduced infection by 52-84% across the two strains. These findings are similar to what we have previously observed using clade B viruses, and indicate that our previous work with Cc-MIP1α can be extended to include protection from clade C viruses.  Figure 2.3 Inhibition of HIV-1 pseudovirus infection with Cc-MIP1α  Figure 2.3 Cc-Control and Cc-MIP1α were incubated with TZM-bl cells and 200TCID50 SVPC3 or SVPC4 pseudovirus for 48 hours. HIV-1 infection was measured indirectly using a mammalian β-galactosidase assay. Infection rate is shown as a percentage and was normalized to virus + TZM-bl cells minus the background of uninfected TZM-bl cells. The assay was performed in quadruplicate and repeated in 4-5 independent experiments. Statistics between each C. crescentus construct for each pseudoviurs were calculated by Student’s t test. ***p<0.001  2.3.3 Inhibition of HIV-1 pseudovirus infection with recombinant C. crescentus expressing domain 1 of CD4 or CD4-mimetic with the S-layer  Previously, we generated recombinant Cc-CD4 and demonstrated Cc-CD4 was able to provide 22-56% inhibition from infection with HIV-1 pseudovirus representing clade B (309). SVPC3 SVPC40255075100125150175Cc-ControlCc-MIP1αHIV-1 Pseudovirus% Infection    *** *** 65  Cc-CD4M33F23 was generated and compared to Cc-CD4 using clade B and C pseudoviruses. CD4M33F23 is a CD4 mimetic that has been modified to optimize interaction with gp120 and inhibits infection of a wide range of HIV-1 isolates (336). Co-incubating Cc-CD4 with HIV-1 pseudovirus representing clade C resulted in a significant inhibition of infection of TZM-bl cells (Figure 2.4). Infection was inhibited at levels that were similar to those previously observed with the clade B pseudoviruses. Cc-CD4M33F23 also provided significant protection from infection with either clade B or clade C viruses (Figure 2.4, Table 2.3). With most of the pseudoviruses, Cc-CD4M33F23 was more effective than Cc-CD4 in preventing HIV-1 infection, but this was not statistically significant.  Figure 2.4: Inhibition of HIV-1 pseudovirus infection with Cc-CD4 and Cc-CD4M33F23    A SVPB11 SVPB120255075100125 Cc-ControlCc-CD4Cc-CD4M33F23HIV-1 Pseudovirus% Infection * * ** ** 66   Figure 2.4 Cc-Control, Cc-CD4 and Cc-CD4M33F23 were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and was normalized to wells containing virus + TZM-bl cells minus the background for uninfected TZM-bl cells. The assay was performed in quadruplicate wells and repeated in 3-4 independent experiments. Statistics were performed to compare all C. crescentus with each pseudovirus using one-way ANOVA with Bonferroni’s correction for multiple comparisons. p values represent the comparison between each recombinant C. crescentus and Cc-Control. *p<0.05, **p<0.01, ***p<0.001; All comparisons between Cc-CD4 and Cc-CD4M33F23 p>0.05  2.3.4 Anti-HIV lectins block HIV-1 pseudovirus infection  Cyanovirin-N, microvirin and Griffithsin are carbohydrate binding agents that have been demonstrated to have anti-HIV-1 activity. Cyanovirin-N was isolated from the prokaryote Nostoc ellipsosporum, and binds to the mannose repeats on the HIV gp120 protein, potently blocking entry of most HIV clades and subtypes (337, 353, 354). Microvirin is also a mannose specific SVPC3 SVPC40255075100125HIV-1 Pseudovirus% InfectionCc-ControlCc-CD4Cc-CD4M33F23*** ***  ** *** B 67  lectin, isolated from Microcystis aeruginosa, that has been shown to have anti-HIV activity similar to cyanovirin-N, and a better safety profile, with lower production of cytokines and no induction of activation markers on PBMCs (338, 355).  Griffithsin was identified and isolated from the red alga Griffithsia sp. and has been shown to bind to monosaccharides on the HIV viral envelope, preventing HIV-1 infection (339, 356-358).  Each of the anti-HIV lectins were expressed individually on the surface of C. crescentus and their ability to prevent HIV-1 infection was measured in vitro using HIV-1 pseudovirus. All three anti-HIV lectins provided significant inhibition of infection with both clade B and clade C viruses (Figure 2.5, Table 2.3). Cc-Griffithsin was the most effective at preventing infection, with protection ranging from 39.8-84.2%. Although Cc-Griffithsin provided the most protection from infection, it was not significantly better than Cc-Cyanovirin or Cc-Microvirin at preventing HIV-1 infection. It is interesting to note that Cc-Cyanovirin is more effective at inhibiting infection with clade B viruses compared to clade C viruses, which may be due to binding affinity to the mannose repeats present on viruses from this clade.         68  Figure 2.5: Inhibition of HIV-1 pseudovirus infection with Cc-Cyanovirin, Cc-Microvirin and Cc-Griffithsin   Figure 2.5 Cc-Control, Cc-Cyanovirin, Cc-Microvirin and Cc-Griffithsin were combined with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus and incubated for 48 hours. Infection rate was measured indirectly using a mammalian β-galactosidase assay and is given as a percentage, normalized to wells containing virus + TZM-bl cells, minus background for uninfected TZM-bl cells. The assay was performed SVPB11 SVPB120255075100Cc-ControlCc-CyanovirinCc-MicrovirinCc-GriffithsinHIV-1 Pseudovirus% Infection** ** *** *** *** *** A SVPC3 SVPC40255075100 Cc-ControlCc-CyanovirinCc-MicrovirinCc-GriffithsinHIV Pseudovirus% Infection** ** *** *** *** *** B 69  in quadruplicate wells and repeated in 3-6 independent experiments. Statistics for each pseudovirus were calculated using one-way ANOVA with Bonferroni’s correction for multiple comparisons. p values represent comparison between Cc-Control and each lectin individually. **p<0.01, ***p<0.0001. All comparisons between Cc-Cyanovirin, Cc-Microvirin and Cc-Griffithisn p>0.05  2.3.5 Fusion inhibitors block HIV-1 pseudovirus infection  Fuzeon (enfuvirtide, T-20) is a 36 amino acid peptide that corresponds to residues 643-678 in the HR2 domain of HIV-1 env protein (340). Fuzeon competitively binds to HR1 and prevents HR2 from binding, which blocks the formation of the six-helix bundle, thereby preventing fusion of the gp41 protein with the cell surface (359). T-1249 is a second-generation variant of Fuzeon, which is more potent and shows a wider activity range against clinical and laboratory isolates (341, 360). The C52 peptide contains 52 residues of the HIV-1 gp41 glycoprotein, including the region of Fuzeon that has been shown to prevent infection, and C34, a pocket binding region that is active in vitro against Fuzeon-resistant viruses (342). By combining these two regions it is anticipated that greater inhibition of infection will be observed.  All three fusion inhibitors were expressed individually on the surface of C. crescentus to determine if they could prevent infection with HIV-1, and whether any one provided more protection than the others. All three fusion inhibitors were very effective at preventing HIV-1 pseudovirus infection, with protection ranging from 24-84.8% (Figure 2.6, Table 2.3). There was no significant difference between each fusion inhibitor in this assay, and protection levels were similar with both viral clades.  70  Figure 2.6: Inhibitory activity against HIV-1 pseudovirus infection of Cc-Fuzeon, Cc-T1249 and Cc-C52   Figure 2.6 Cc-Control, Cc-Fuzeon, Cc-T1249 and Cc-C52 were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and normalized to wells containing virus + TZM-bl cells minus the background for uninfected TZM-bl cells. The assay was performed in quadruplicate wells in 3-6 independent experiments. Statistics were calculated for each pseudovirus using one-way ANOVA with Bonferroni’s correction for multiple comparisons. p SVPB11 SVPB120255075100125 Cc-ControlCc-FuzeonCc-T1249Cc-C52HIV-1 Pseudovirus% Infection** ** ** *** ***  *** A SVPC3 SVPC40255075100125HIV-1 Pseudovirus% InfectionCc-ControlCc-FuzeonCc-T1249Cc-C52*** *** *** ** *** ** B 71  values represent the comparison between Cc-Control and each construct individually. **p<0.01, ***p<0.001. For comparisons between Cc-Fuzeon, Cc-T1249 and Cc-C52 p>0.05  2.3.6 GB virus C E2 protein blocks infection with HIV-1 pseudovirus  GB virus C (formerly hepatitis G virus) is a persistent viral infection that does not cause any known disease pathology (361). It has been demonstrated that GB virus C infection has a beneficial effect for people that are HIV positive (362). Those people that are co-infected with HIV and GB virus C have improved survival and delayed progression to AIDS (362-365). Many factors are linked to this favourable survival including inhibition of HIV-1 replication (362, 366, 367), production of HIV-suppression factors including chemokines (362, 366, 368, 369), and decreasing expression of HIV-1 co-receptors (366, 368, 370). The E2 protein of GB virus C is a putative fusion peptide that interferes with HIV binding or fusion (369, 371-373). It has been suggested that the E2 protein interferes with late viral entry after engagement of gp120 with CD4 and the coreceptor (343). The sequence similarities between the N-terminus of E2 and the HIV-1 gp120 enables E2 to interact with the gp41 disulfide loop (343). The E2 protein of GB virus C was expressed in the S-layer of C. crescentus and used in in vitro viral blocking assays with HIV-1 pseudoviruses representing viral clades B and C. Cc-GBVCE2 provided approximately 50% reduction in HIV-1 infection with both viral clades (Figure 2.7, Table 2.3).      72  Figure 2.7: Inhibitory activity against HIV-1 pseudovirus infection of Cc-GBVCE2  Figure 2.7 Cc-Control or Cc-GBVCE2 were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and normalized to wells containing virus + TZM-bl cells minus the background for uninfected TZM-bl cells. The assay was performed in quadruplicate wells in 2-5 independent experiments. Statistics for each pseudovirus were calculated using Student’s t test. p values represent the comparison between Cc-Control and each construct individually. *p<0.05, **p<0.01  2.3.7 Cc-BmKn2 block HIV-1 pseudovirus infection while Cc-Mucroporin provides minimal inhibition  Anti-microbial peptides are widely expressed and rapidly induced to repel invasion from diverse infectious agents, including bacteria, viruses, fungi and parasites (344). Anti-microbial peptides from scorpion venom have demonstrated biological activity against viral pathogens SVPB11 SVPB12 SVPC3 SVPC40255075100125150Cc-ControlCc-GBVCE2HIV-1 Pseudovirus% Infection* * ** ** 73  such as herpes simplex virus, hepatitis C virus and measles virus (344). Mucroporin, cloned from the venon of the scorpion Lychas mucronatus, and its optimized derivative mucroporin-M1 have demonstrated antimicrobial activity against bacteria and measles virus (344). BmKn2 was cloned from the venom of the scorpion Mesobuthus martensii and has demonstrated strong antimicrobial activity against bacteria (344).  Both mucroporin-M1 and BmKn2 demonstrated anti-HIV activity in vitro, likely by interacting with the viral particle (344). Mucroporin-M1 and BmKn2 were each expressed in the S-layer of C. crescentus and used in in vitro viral blocking assays. Cc-BmKn2 has performed inconsistently with both clade B and clade C pseudoviruses. In some assays it provides up to a 50% reduction in HIV-1 infection, but in other assays infection is reduced by as little as 10%, or is actually enhanced (Figure 2.8). With some replicates the same C. crescentus constructs have inhibited infection with one pseudovirus, but have provided no protection when tested with a different pseudovirus on the same 96-well plate. This variability may be due to differences between the env protein of each pseudovirus. As the viral target of each of these proteins is not known, it is possible that the target is not present consistently on the pseudovirus particles. Although the pseudovirus attaches and enters cells using the same mechanism as live HIV-1, it is possible that production of the pseudovirus in 293T cells does not fully mimic all aspects of live HIV-1 propagation in PBMCs, particularly in regards to viral egress from the host cell. If the target of these proteins is obtained from the host cell during egress, it may not be present on the pseudovirus. Cc-Mucroporin caused an approximately 50% decrease in HIV-1 infection across all clades (Figure 2.8, Table 2.3). However, Cc-Mucroporin has extremely low expression of RsaA, suggesting it may be RsaA negative (Figure 2.2E). Preliminary studies with an RsaA negative C. crescentus have indicated that there is non-specific 74  inhibition of HIV-1 infection (data not shown). No further studies were undertaken with Cc-Mucroporin.  Figure 2.8: Cc-BmKn2 may block HIV-1 pseudovirus infection while Cc-Mucroporin provides minimal protection   SVPB11 SVPB12 SVPC3 SVPC40255075100125150HIV-1 Pseudovirus% InfectionCc-ControlCc-BmKn2* SVPB11 SVPB12 SVPC3 SVPC40255075100125150HIV-1 Pseudovirus% InfectionCc-ControlCc-Mucroporin* 75  Figure 2.8 Cc-Control, Cc-BmKn2 and Cc-Mucroporin were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and normalized to wells containing virus + TZM-bl cells minus the background for uninfected TZM-bl cells. The assay was performed in quadruplicate in 2-4 independent experiments. Statistics were calculated using Student’s t test. p values represent the comparison between Cc-Control and each construct individually. *p<0.05  2.3.8 Cc-Elafin has inconsistent activity against HIV-1 infection in vitro  Elafin is a member of the whey acidic protein family that has antiproteolytic, immunomodulatory and antimicrobial properties (345). It has been shown that elafin is elevated in cervicovaginal lavage fluid collected from commercial sex workers that are HIV-1 resistant compared to those that are HIV-1 susceptible (374). Pretreatment of R5 viruses with elafin caused a reduction in infection of TZM-bl cells (304). Elafin was expressed in the S-layer protein of C. crescentus and tested in vitro in the pseudovirus blocking assay. As with Cc-BmKn2, results with Cc-Elafin were inconsistent from one experiment to the next, although a trend towards protection from infection was observed (Figure 2.9, Table 2.3). As with Cc-BmKn2, it is possible that the target for elafin is not present on the pseudovirus, so only preliminary studies were conducted with the pseudoviruses, and Cc-Elafin was subsequently tested with live HIV-1 (Figure 2.13).    76  Figure 2.9: Cc-Elafin has inconsistent activity against HIV-1 infection   Figure 2.9 Cc-Control and Cc-Elafin were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and normalized to wells containing virus + TZM-bl cells minus the background for uninfected TZM-bl cells. The assay was performed in quadruplicate in 1 (SVPC3), 2 (SVPB11) or 3 (SVPB12, SVPC4) independent experiments. When applicable, statistical analysis indicated that the difference between Cc-Control and Cc-Elafin was not statistically significant.  2.3.9 Cc-A1AT has limited activity against HIV-1 infection  α1-antitrypsin is a serpin that plays a role in preventing the fusion of HIV-1 with the target cell (348). α1-antitrypsin interacts with its target protease, resulting in the cleavage of a C-terminal fragment, designated C-36 (375). This fragment was identified as a CD4 competitive binding inhibitor for gp120 (375). Cervicovaginal lavage fluid was collected from 293 HIV-1 SVPB11 SVPB12 SVPC3 SVPC40255075100125150HIV-1 Pseudovirus% InfectionCc-ElafinCc-Control77  resistant, uninfected and infected sex workers that are part of the Pumwani Sex Worker cohort and analyzed by proteomics (376). α1-antitrypsin was found to be significantly over-abundant in HIV-1 resistant women, suggesting it may have some role in protection from infection (376). α1-antitrypsin was expressed in the S-layer of C. crescentus and used in in vitro viral blocking assays. Cc-A1AT provided low level of protection from HIV-1 infection in the pseudovirus assays with clade C virus but provided no protection with clade B virus (Figure 2.10, Table 2.3).  Figure 2.10: Cc-A1AT has limited activity against HIV-1 pseudovirus infection  Figure 2.10 Cc-Control and Cc-A1AT were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and normalized to wells containing virus + TZM-bl cells minus the background for uninfected TZM-bl cells. The assay was performed in quadruplicate wells in 2-3 independent experiments. p>0.05  SVPB11 SVPB12 SVPC3 SVPC40255075100125150175200HIV-1 Pseudovirus% InfectionCc-A1ATCc-Control78  2.3.10 Cc-gp120M15D and Cc-gp120NB did not provide protection from HIV-1 infection in vitro  Cc-gp120M15D is a gp120 fragment with a lysine to aspartic acid switch that blocks the interaction between HIV-1 gp120 and the coreceptor (349). Cc-gp120NB is derived from an HIV-1 anti-gp120 antibody raised in llamas (350, 351). Cc-gp120M15D and Cc-gp120NB were used in preliminary studies and both constructs were unable to provide any protection from HIV-1 infection in the viral blocking assay (Figure 2.11, Table 2.3). This may be due to improper folding of the proteins in the S-layer or reversible binding between the constructs and their targets. No additional studies were undertaken with these C. crescentus.   Figure 2.11: Cc-gp120M15D and Cc-gp120NB do not protect from HIV-1 pseudovirus infection  SVPB11 SVPB12 SVPC3 SVPC40255075100125150175200HIV-1 Pseudovirus% InfectionCc-ControlCc-gp120M15D79   Figure 2.11 Cc-Control, Cc-gp120M15D and Cc-gp120NB were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and normalized to wells containing virus + TZM-bl cells with the background for uninfected TZM-bl cells subtracted out. The assay was performed in quadruplicate in 1-3 independent experiments.   2.3.11 Comparison of C. crescentus JS4026 and JS4038 strains  The C. crescentus protein display system was developed in the Smit lab as a means of expressing useful peptides in high quantities in the S-layer. We have had promising but variable results using this system, so we aimed to improve the S-layer display capabilities. Some difficulties were previously encountered in binding of displayed protein to the intended target, especially in cases where affinities were expected to be much less than a typical antibody-antigen interaction. It was believed that this may be caused by the existence of an extracellular SVPB11 SVPB12 SVPC3 SVPC40255075100125150175200HIV-1 Pseudovirus% InfectionCc-ControlCc-gp120NB80  polysaccharide layer (EPS) located exterior to the S-layer (311), which interferes with recombinant protein interactions. To this end, we created knockout mutants of the fucose synthase gene, because fucose was known to be present in the EPS but not the LPS, which is required for S-layer attachment, expecting to see a major modification of the EPS layer. PCR was used to verify that the bacteria were deficient for the fucose synthase gene. Strain JS4038 contains the EPS knockout and produces less or no EPS, as judged by loss of colony mucoidy on sucrose containing solid media. A general trend of an increase in binding efficacy of proteins displayed on the surface of several of our C. crescentus constructs has been observed. Specifically, the use of JS4038 strains, including Cc-MIP1α, Cc-CD4, Cc-Cyanovirin and Cc-Fuzeon, have shown consistently improved viral blocking compared to JS4026 expressing the same peptides. In particular, Cc-MIP1α displayed on JS4038 provided a statistically significant increase in viral blocking (Table 2.2). Differences between many of the other constructs approached statistical significance. In almost every case, the JS4038 strain construct was shown to function at least as well as, if not better than, the JS4026 equivalent (Table 2.2) indicating that the JS4038 strain is a more effective protein display system for these studies.   81  Table 2.2: Comparing C. crescentus strains with and without the EPS layer Construct	   Control	   MIP1α	   CD4	   Cyanovirin-­‐N	   Fuzeon	  Strain	   B5	   Δ471	   B5	   Δ471	   B5	   Δ471	   B5	   Δ471	   B5	   Δ471	  SVPB11	  121.9	  	  	  	  	  	  	  	  (98.1-­‐148.2)	  	  	  	  	  114.9	  	  	  	  	  	  	  	  	  	  	  	  	  	  (93.5-­‐138.7)	  50.3	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (35.6-­‐60)	  43.1	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (27.5-­‐69)	  70.4	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (59.7-­‐79.3)	  67.7	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (62.4-­‐71.5)	  36.3	  	  	  	  	  	  	  	  	  	  (28.1-­‐51.7)	  28.5	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (18.9-­‐37.2)	  45.8	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (22.4-­‐60.4)	  52.7	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (34-­‐78.6)	  SVPB12	  118.2	  	  	  	  	  	  	  	  	  (92.4-­‐131)	  111	  	  	  	  	  	  	  	  	  	  	  	  	  	  (92.4-­‐131)	  44.7	  	  	  	  	  	  	  	  	  	  	  	  	  (30.3-­‐67.5)	  48.2	  	  	  	  	  	  	  	  	  	  	  	  (42.2-­‐54.5)	  67.2	  	  	  	  	  	  	  	  	  	  	  (52.4-­‐86.5)	  71.5	  	  	  	  	  	  	  	  	  	  (63.5-­‐78)	  34.7	  	  	  	  	  	  	  	  	  	  	  	  	  	  (18-­‐61.8)	  23.5	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (14-­‐35.1)	  50	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (34.3-­‐81.8)	  47.8	  	  	  	  	  	  	  	  	  	  	  	  	  (34.5-­‐58.7)	  SVPC3	  116.2	  	  	  	  	  	  	  	  	  	  	  	  (90.9-­‐162)	  72.3	  	  	  	  	  	  	  	  	  	  	  	  	  (46.2-­‐87.2)	  53.2	  	  	  	  	  	  	  	  	  	  	  	  	  (41.6-­‐71.8)	  32.4	  	  	  	  	  	  	  	  	  	  	  	  	  (15.9-­‐44.8)	  72.3	  	  	  	  	  	  	  	  	  	  	  	  	  (46.2-­‐87.2)	  74.2	  	  	  	  	  	  	  	  	  	  	  	  	  	  (54.5-­‐88)	  40.9	  	  	  	  	  	  	  	  	  	  	  	  (27.3-­‐45.7)	  29.6	  	  	  	  	  	  	  	  	  	  	  	  	  (19.1-­‐40)	  59.9	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (30.5-­‐87.1)	  43.2	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (19.6-­‐61.1)	  SVPC4	  122.7	  	  	  	  	  	  	  	  (91.9-­‐164.6)	  118.5	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (97.4-­‐136.6)	  56.7	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (44.5-­‐66.8)	  33.9	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (31.9-­‐37.1)	  70.7	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (61.2-­‐79.1)	  64.3	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (61-­‐67.5)	  44.5	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (27.4-­‐56.1)	  25.8	  	  	  	  	  	  	  	  	  	  	  	  	  	  (21-­‐30.8)	  59.5	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  (49.1-­‐76.8)	  56.6	  	  	  	  	  	  	  	  	  	  (36.9-­‐70.3)	   Table 2.2 JS4026 and JS4038 strains were compared side-by-side in the described viral blocking assay. Results represent mean percent infection with the range provided. Experiments were undertaken in quadruplicate and performed a minimum of 3 times. 82  2.3.12 Cc-Indo and Cc-Indo2 prevent HIV-1 infection in some HIV strains  Indolicidin is an antimicrobial cationic peptide that has been described to have direct anti-viral activity against HIV-1, thought to be related to its membrane disruptive properties (346, 347). Indolicidin was expressed within the S-layer of C. crescentus. As indolicidin is a small protein (13 amino acids) an indolicidin multimer, containing 2 copies of indolicidin for every 1 RsaA protein was also created. Cc-Indo and Cc-Indo2 were the last candidates developed for this project. At the time of development of these candidates the viral blocking assays had transitioned from pseudovirus to live, replication competent virus, so Cc-Indo and Cc-Indo2 have been tested with live HIV-1 only. The live HIV-1 assays were undertaken to determine if the results that were observed with the pseudovirus were maintained in the presence of replication competent HIV-1 and physiologically relevant cell lines, as applicable. As there was some variability in protection levels within and between clades, several different HIV-1 strains were used for the live virus assays. 108 recombinant Cc-Indo or Cc-Indo2 were incubated with HIV-189.6 for 1 hour before adding TZM-bl cells, and HIV-1 infection was measured 48 hours later. Significant protection from HIV-189.6 infection was observed with Cc-Indo (44.8% protection) and Cc-Indo2 (44.9% protection) (Figure 2.12, Table 2.3). There was no difference in inhibition of HIV-189.6 infection between Cc-Indo and Cc-Indo2. Although Cc-Indo2 contained two tandem copies of indolicidin per each RsaA monomer, the overall expression level of recombinant RsaA on Cc-Indo2 was less than the level of Cc-Indo (Figure 2.2B), which may account for the same degree of inhibition. Interestingly, Cc-Indo and Cc-Indo2 were unable to block infection of TZM-bl cells with HIV-1JR-CSF. Although Cc-Indo2 did lower HIV-1 infection by approximately 30%, this was not statistically significant (Figure 2.12). Preliminary studies with HIV-189.6 and 83  PBMCs yielded similar results to HIV-189.6 in TZM-bl cells. Preliminary studies with HIV-1BaL and TZM-bl cells showed no protection from infection (data not shown).  Figure 2.12: Cc-Indo and Cc-Indo2 may block HIV-1 infection  Figure 2.12 Cc-Control Cc-Indo and Cc-Indo2 were incubated for 48 hours with TZM-bl cells and 200TCID50 HIV-189.6 or HIV-1pykJRCSF. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and normalized to wells Cc-ControlCc-IndoCc-Indo2050100150% Infection* * HIV-­‐189.6	  +	  TZM-­‐bl	  cells % InfectionCc-ControlCc-IndoCc-Indo2050100150HIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells 84  containing virus + TZM-bl cells with the background for uninfected TZM-bl cells subtracted out. The assay was performed in quadruplicate in 3-5 independent experiments. *p<0.05  2.3.13 Many recombinant C. crescentus block infection with live HIV-1   Those recombinant C. crescentus that blocked HIV-1 infection in vitro using the HIV-1 pseudovirus assay underwent subsequent testing with live, replication competent HIV-1 in both TZM-bl cells and PHA-stimulated PBMCs, as applicable. These assays were used as an additional screening tool to verify that similar levels of inhibition were observed with different cell types and various HIV-1 strains. The live virus assays are more physiologically relevant as the HIV-1 used is able to replicate, and some of the HIV-1 strains used can infect primary peripheral blood mononuclear cells (PBMCs). Cc-MIP1α (Figure 2.13A) provided 32%-90% inhibition from infection based on the cell type and virus used. Cc-CD4 provided 33%-97% inhibition from HIV-1 infection based on the cell type and virus used whereas Cc-CD4M33F23 provided 36%-82% inhibition from infection (Figure 2.13B). Cc-Cyanovirin provided 35%-84% inhibition from HIV-1 infection, Cc-Microvirin provided 48%-64% inhibition from HIV-1 infection, and Cc-Griffithsin provided 37%-69% inhibition from HIV-1 infection depending on the cell type and virus used (Figure 2.13C). Cc-Fuzeon provided 47%-81% inhibition from HIV-1 infection, and Cc-T1249 provided 32%-63% inhibition from infection. Interestingly, no inhibition was observed with Cc-T1249 with HIV-1JR-FL in PBMCs, even though inhibition was observed with HIV-1JR-FL in TZM-bl cells. Cc-C52 provided 44%-98% inhibition from infection depending on the cell type and virus used (Figure 2.13D). Cc-GBVCE2 provided 54%-87% inhibition from HIV-1 infection depending on the virus and cell type used (Figure 2.13E). Cc-BmKn2 provided 37%-89% inhibition from HIV-1 infection depending on the virus and cell type 85  used (Figure 2.13F). Cc-Elafin provided 29%-77% inhibition from HIV-1 infection depending on the virus and cell type used (Figure 2.13G). Cc-A1AT provided 34%-77% inhibition from HIV-1 infection depending on the virus and cell type used (Figure 2.13H)(Table2.2).  Figure 2.13: Live virus blocking assays A) Cc-MIP1α   % InfectionCc-ControlCc-MIP1α050100150% InfectionCc-ControlCc-MIP1α050100150 HIV-­‐189.6	  +	  TZM-­‐bl	  cells ** HIV-­‐189.6	  +	  PBMCs * % InfectionCc-ControlCc-MIP1α020406080HIV-­‐1BaL	  +	  TZM-­‐bl	  cells % InfectionCc-ControlCc-MIP1α050100150HIV-­‐1JR-­‐FL	  +	  TZM-­‐bl	  cells n=4 n=4 n=2 n=1 86           % InfectionCc-ControlCc-MIP1α050100150 HIV-­‐1JR-­‐FL	  +	  PBMCs *** % InfectionCc-ControlCc-MIP1α050100150 HIV-­‐1SF162	  +	  PBMCs % InfectionCc-ControlCc-MIP1α050100150* HIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells n=3 n=2 n=4 87  B) Cc-CD4 and Cc-CD4M33F23    % InfectionCc-ControlCc-CD4Cc-CD4M33F23050100150% InfectionCc-ControlCc-CD4020406080HIV-­‐1BaL	  +	  TZM-­‐bl	  cells HIV-­‐1JR-­‐FL	  +	  TZM-­‐bl	  cells n=2 n=2 n=2 Cc-ControlCc-CD4Cc-CD4M33F23050100150% Infectionn=2 n=2 % InfectionCc-ControlCc-CD4Cc-CD4M33F23050100150HIV-­‐1JR-­‐FL	  +	  PBMCs n=3 * n=3 * HIV-­‐1SF162	  +	  PBMCs % InfectionCc-ControlCc-CD4Cc-CD4M33F23050100150n=4 * n=5 * HIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells 88  C) Cc-Cyanovirin, Cc-Microvirin and Cc-Griffithsin     Cc-ControlCc-CyanovirinCc-MicrovirinCc-Griffithsin050100150% InfectionHIV-­‐189.6	  +	  TZM-­‐bl	  cells n=4 * n=7 **** n=3** % InfectionCc-ControlCc-CyanovirinCc-MicrovirinCc-Griffithsin050100150 HIV-­‐189.6	  +	  PBMCs 	  	  n=6 n=6 * * Cc-ControlCc-CyanovirinCc-MicrovirinCc-Griffithsin050100150% Infection% InfectionCc-ControlCc-CyanovirinCc-MicrovirinCc-Griffithsin020406080100HIV-­‐1BaL	  +	  TZM-­‐bl	  cells HIV-­‐1JR-­‐FL	  +	  PBMCs n=3 n=1 n=2 n=2 n=2 n=2 % InfectionCc-ControlCc-CyanovirinCc-MicrovirinCc-Griffithsin050100150HIV-­‐1SF162	  +	  PBMCs Cc-ControlCc-CyanovirinCc-MicrovirinCc-Griffithsin050100150% InfectionHIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells n=5 ** n=5 ** n=3 *   n=2 n=1 n=1 89  D) Cc-Fuzeon, Cc-T1249 and Cc-C52     Cc-ControlCc-FuzeonCc-T1249Cc-C52050100150% InfectionHIV-­‐189.6	  +	  TZM-­‐bl	  cells n=5 *** n=5 ** n=4 *** % InfectionCc-ControlCc-FuzeonCc-T1249Cc-C52050100150HIV-­‐189.6	  +	  PBMCs n=4 n=3 * n=3 % InfectionCc-ControlCc-FuzeonCc-T1249Cc-C52020406080 HIV-­‐1BaL	  +	  TZM-­‐bl	  cells % InfectionCc-ControlCc-FuzeonCc-T1249Cc-C52050100150 HIV-­‐1JR-­‐FL	  +	  TZM-­‐bl	  cells n=2 n=1 n=1 n=1 n=2 n=2 % InfectionCc-ControlCc-FuzeonCc-T1249Cc-C52050100150200250 HIV-­‐1JR-­‐FL	  +	  PBMCs % InfectionCc-ControlCc-FuzeonCc-T1249Cc-C52050100150 HIV-­‐1SF162	  +	  PBMCs n=2 n=2 n=2 n=1 n=1 n=1 90   E) Cc-GBVCE2   % InfectionCc-ControlCc-FuzeonCc-T1249Cc-C52050100150HIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells n=5 * n=3 n=4 * % InfectionCc-ControlCc-GBVCE2050100150 HIV-­‐189.6	  +	  TZM-­‐bl	  cells n=5 ** % InfectionCc-ControlCc-GBVCE2050100150HIV-­‐189.6	  +	  PBMCs n=7 ** % InfectionCc-ControlCc-GBVCE2020406080HIV-­‐1BaL	  +	  TZM-­‐bl	  cells % InfectionCc-ControlCc-GBVCE2050100150HIV-­‐1JR-­‐FL	  +	  PBMCs n=1 n=2 91   F) Cc-BmKn2     Cc-ControlCc-GBVCE2050100150% InfectionHIV-­‐1SF162	  +	  PBMCs Cc-ControlCc-GBVCE2050100150% InfectionHIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells n=1 n=5 * Cc-ControlCc-BmKn2050100150% InfectionHIV-­‐189.6	  +	  TZM-­‐bl	  cells    n=5    ** % InfectionCc-ControlCc-BmKn2050100150 HIV-­‐189.6	  +	  PBMCs n=4 ** % InfectionCc-ControlCc-BmKn2050100150 HIV-­‐1JR-­‐FL	  +	  PBMCs % InfectionCc-ControlCc-BmKn2050100150 HIV-­‐1SF162	  +	  PBMCs n=2 n=1 92   G) Cc-Elafin   % InfectionCc-ControlCc-BmKn2050100150HIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells n=5 Cc-ControlCc-Elafin050100150% InfectionHIV-­‐189.6	  +	  TZM-­‐bl	  cells n=4 ** Cc-ControlCc-Elafin050100150% InfectionHIV-­‐189.6	  +	  PBMCs n=4 *** % InfectionCc-ControlCc-Elafin020406080100 HIV-­‐1BaL	  +	  TZM-­‐bl	  cells % InfectionCc-ControlCc-Elafin050100150 HIV-­‐1JR-­‐FL	  +	  TZM-­‐bl	  cells n=2 n=2 93    H) Cc-A1AT  % InfectionCc-ControlCc-Elafin050100150 HIV-­‐1JR-­‐FL	  +	  PBMCs % InfectionCc-ControlCc-Elafin050100150 HIV-­‐1SF162	  +	  PBMCs n=1 n=1 % InfectionCc-ControlCc-Elafin050100150HIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells n=5 * Cc-ControlCc-A1AT050100150% InfectionHIV-­‐189.6	  +	  TZM-­‐bl	  cells n=7 ** % InfectionCc-ControlCc-A1AT050100150 HIV-­‐189.6	  +	  PBMCs n=5** 94    Figure 2.13 Recombinant C. crescentus were incubated for 48 or 72 hours with TZM-bl cells or PHA-stimulated PBMCs and 200TCID50 live HIV-1. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay (TZM-bl cells) or p24 ELISA (PBMCs). For TZM-bl cells infection rate is given as a percentage and normalized to wells containing virus + TZM-bl cells minus the background for uninfected TZM-bl cells. For PBMCs values were normalized to virus + cells. The assay was performed in quadruplicate wells in 1-7 independent replicates, as indicated on each figure. When assays were performed 3 or more times statistics were calculated by Student’s t test (2 groups) or one-way ANOVA with Bonferroni’s correction for multiple comparisons (3 or more groups). p values represent the comparison between Cc-% InfectionCc-ControlCc-A1AT050100150 HIV-­‐1BaL	  +	  TZM-­‐bl	  cells % InfectionCc-ControlCc-A1AT050100150HIV-­‐1JR-­‐FL	  +	  PBMCs n=2 n=2 % InfectionCc-ControlCc-A1AT050100150HIV-­‐1SF162	  +	  PBMCs % InfectionCc-ControlCc-A1AT050100150HIV-­‐1pykJRCSF	  +	  TZM-­‐bl	  cells n=2 * n=1 95  Control and each recombinant C. crescentus. *p<0.05, **p<0.01, ***p<0.001. Within inhibitor groups (graphed together) for comparison between each recombinant C. crescentus p>0.05.   Table 2.3 Chapter 2 summary Name Pseudovirus Clade B (mean and range HIV-1 inhibition) Pseudovirus Clade C (mean and range HIV-1 inhibition) Live virus TZM-bl cells (mean and range HIV-1 inhibition) Live virus PBMCs (mean and range HIV-1 inhibition) Cc-Control % infection: 106.3% Range 85.2%-137.6% % infection: 112.1% Range 95.4%-136.7% % infection: 110.1%  Range 80.8%-137.7% % infection: 100% Range 99.9%-100% Cc-MIP1α Not tested by CF 68.5%  (51.9%-84.1%) 60.3% (23%-83%) 69.2% (11.2%-97.2%) Cc-CD4 Not tested by CF 56.8% (35%-77.6%) 55.5% (10.6%-72.5%) 74.6% (18%-97.5%) Cc-CD4M33F23 50.3% (36.7%-66.4%) 67.5% (48%-80.7%) 36.2% (16%-74.3%) 26.9% (36.5%-93.2%) Cc-Cyanovirin 72.4% (64.4-83.7%) 64.8% (42.2%-80.9%) 51.6%  (21.6%-87.6%) 71.6% (15.9%-100%) Cc-Microvirin 60.9% (49.2%-79.8%) 52.9% (30%-81.3%) 61.8% (11.9%-82.4%) 56% (-7.9%-100%) 96  Name Pseudovirus Clade B (mean and range HIV-1 inhibition) Pseudovirus Clade C (mean and range HIV-1 inhibition) Live virus TZM-bl cells (mean and range HIV-1 inhibition) Live virus PBMCs (mean and range HIV-1 inhibition) Cc-Griffithsin 72.2% (61.7%-82.7%) 67.4% (39.8%-84.2%) 61% (19.6%-85.2%) 56.7% (25.4%-100%) Cc-Fuzeon 48.7% (34.2%-72.7%) 51.9% (14%-80.4%) 58.8% (29.3%-78.9%) 62% (8.3%-100%) Cc-T1249 47.7% (32%-75.3%) 57.7%  (23%-84.8%) 40% (4.9%-54.2%) 27.6% (-30.9%-100%) Cc-C52 48.2% (28.2%-73.6%) 58.2% (26%-7.1%) 50.7% (12.2%-81.3%) 86.8% (46.5%-100%) Cc-GBVCE2 50.1% (25.8%-81.7%) 61.1% (38.2%-89.3%) 64.9% (19.5%-86.4%) 75.7% (27.2%-87.9%) Cc-BmKn2 24.9% (13.3%-31.8%) 32.3% (21.7%-33.8%) 38.3% (14.1%-84.3%) 78% (61.2%-83.5%) Cc-Mucroporin 33.1% (12.6%-56.5%) 50.6% (46.6%-57.7%) Not tested Not tested Cc-Elafin 28.6% (10%-63.6%) 51.4% (33.6%-69.8%) 44.8% (15.5%-80.4%) 76.5% (57.7%-80.2%)  97  Name Pseudovirus Clade B (mean and range HIV-1 inhibition) Pseudovirus Clade C (mean and range HIV-1 inhibition) Live virus TZM-bl cells (mean and range HIV-1 inhibition) Live virus PBMCs (mean and range HIV-1 inhibition) Cc-A1AT -18.8% (-78.8%-33.7%) 15.6% (-26.5%-49.5%) 39.6% (9.9%-87.8%) 56.2% (28.1%-73.6%) Cc-gp120M15D -48.5% (-68.7%- -27%) -13.3% (-27.3%- 0%) Not tested Not tested Cc-gp120NB -8.2% (-38.6%-14.3%) -36.6% (-53.6%-3%) Not tested Not tested Cc-Indo Not tested Not tested 20.6% (-2.2%-52.7%) Not tested Cc-Indo2 Not tested Not tested 63.3% (23.9%-62.9%) Not tested Table 2.3: Summary of in vitro viral blocking data. Values represent the average for all experiments, with range representing the lowest and highest percent inhibition observed across all individual experiments.  2.4 Discussion Although use of condoms remains an effective means to prevent HIV-1 infection, cultural or societal norms or a lack of access, often prevent women from determining condom use. Development of prevention methods that women can control remains an urgent issue. This issue 98  has created interest in the development of microbicides, specifically microbicides that can be topically applied by women in advance of sexual activity and maintain efficacy. Some microbicide formulations have shown promise in a laboratory setting but failed during clinical trials (98). Recently some success has been observed with the use of tenofovir, a nucleotide reverse transcriptase inhibitor, formulated as a microbicide gel. An initial clinical trial with tenofovir showed promise, providing 39% protection from HIV infection (2), but a second trial was stopped due to inefficacy relative to placebo (377). The tenofovir trials highlight the potential power of microbicides but also the difficulties in translating laboratory success to clinical use. It was previously demonstrated that C. crescentus expressing MIP1α or CD4 could provide protection from infection with HIV-1 pseudovirus representing clade B (309). In this chapter we extended these experiments by testing HIV-1 pseudovirus representing clade C and observed similar results (335). In addition, we have generated a variety of novel display constructs, and have shown that C. crescentus expressing MIP1α, CD4, CD4 mimetic CD4M33F23, anti-viral lectins cyanovirin-N, microvirin and Griffithsin, fusion inhibitors Fuzeon, T-1249 and C52, GB virus C E2 protein, Elafin, and α-1-antitrypsin provide significant protection from HIV-1 pseudovirus infection (335). C. crescentus expressing Mucroporin M1, gp120 mimetic M15D, and gp120 nanobody were unable to prevent HIV-1 pseudovirus infection in vitro, and subsequent studies were not undertaken. The protection level observed with each construct was variable, with protection levels similar among each different pseudovirus tested. Finally, we compared C. crescentus strains JS4026 and JS4038, and demonstrated that the JS4038 strain represents the best viral blocking potential. 99  The pseudovirus assay was used to allow rapid screening for protection from infection with a wide variety of HIV-1 viral clades. Preliminary data using viral clades A and D indicated that the protection levels observed with clades B and C were maintained (Appendix A, Figure A.2). Those recombinant C. crescentus that were able to prevent HIV-1 pseudovirus infection underwent additional testing with live, replication competent HIV-1 in both TZM-bl cells and human PBMCs depending on the virus strain. Protection levels were generally maintained from the pseudovirus assays. Results were generally similar across viral strains and cell types.   In vitro we have been able to show up to 97% reduction in HIV infection with a single recombinant C. crescentus, and it is possible that combining our constructs could provide still higher levels of protection. In addition, it is conceivable that our system combined with antiretroviral based microbicides could further increase efficacy. While we cannot predict the effectiveness of our approach in people, even partial protection will likely make a significant impact. For example, estimates have shown that a microbicide with 60% effectiveness as a single strategy could prevent over 1 million new infections each year (378).  Many microbicides under development for HIV-1 prevention have a high production cost, which could limit their use, particularly in the low-income countries where they are most urgently needed. Bacteria-based microbicides have been under investigation for many years. Since Lactobacilli are part of the natural flora of the vagina, it was elegantly proposed that engineered Lactobacillus would be an excellent choice for this purpose. Various groups have engineered different strains of Lactobacillus to express CCR5/RANTES (379), CD4 (124), cyanovirin-N (125), and fusion inhibitors (123), and tested their ability to prevent HIV-1 infection. Efforts to achieve high levels of surface expression or secretion of recombinant proteins in Lactobacillus have met with limited success, and the ability of engineered 100  Lactobacillus to persist long term as a commensal bacterium in the competitive microbial milieu of the human urogenital system has not been established. In short, additional microbicide strategies seem warranted.  A bacterium based system that can secrete or display blocking proteins at high levels but does not need to compete with other genital tract bacteria, because it is expected to be used at high concentrations just before sexual activity, may be more successful. Ideally, such a bacterium must be easily and inexpensively produced, safe to use, and able to block infection in a biologically inactive form. The C. crescentus display system has these characteristics. A wide variety of peptides or proteins, with sizes ranging from 13-121 amino acids, have been successfully expressed in the S-layer of C. crescentus and surface displayed. Although some of the chimeric S-layer proteins did result in lowered expression of the S-layer, this generally did not impact the ability of those proteins to prevent HIV-1 infection. In particular, the anti-viral lectins lowered S-layer protein levels but were one of the groups that provided the best protection from HIV infection, indicating that maximum protein expression is not necessarily needed for anti-viral activity in this system. Part of the reason for this lies in the extreme levels of native RsaA secretion, which accounts for as much as 31% of total protein expression in C. crescentus (174). Thus even a significant (e.g., 90%) reduction in secretion levels as the result of a foreign insertion still results in thousands of copies of blocking protein displayed per cell.  This high level expression alone suggests that C. crescentus deserves serious consideration as an expression platform for microbicide development. Clade B and C pseudoviruses were initially used for these studies because these clades account for approximately 62% of worldwide HIV-1 infections, including sub-Saharan Africa (clade C), as well as Western Europe and North and South America (clade B). The efficacy of 101  the C. crescentus display system was maintained between clades B and C, so it can be expected that this system will provide protection from additional HIV-1 clades with relatively similar efficacy. Although the HIV-1 pseudoviruses are only capable of a single-cycle infection they use CD4 and CCR5 to enter cells and are an appropriate and accepted method for viral entry studies. These studies were expanded to undertake trials of C. crescentus display constructs using live HIV-1 and PBMCs and results were generally maintained.  In this chapter we have described the creation of many new C. crescentus display constructs. Creating constructs displaying a diverse array of anti-HIV proteins was an important step for a number of reasons. First, it provides further evidence that C. crescentus appears to be easily modified to express a variety of structurally different anti-HIV peptides. Further, a large reduction in HIV-1 infection occurred using individually displayed peptides. When C. crescentus expressing CD4 or MIP1α were combined, a significant increase in protection from infection occurs (309). This finding leads us to anticipate that combining multiple constructs, either on a single C. crescentus or several strains mixed together, might further reduce infection rates (Appendix C, Figure C.2).   In summary, the C. crescentus display system represents an exciting new approach in the HIV-1 microbicide field. Many constructs that target all aspects of the HIV-1 attachment and entry process have been created, and most of these recombinant C. crescentus are able to provide significant protection from HIV-1 infection in vitro. In the following chapters I will test the clinical relevance and safety of this approach in animal studies. 102  Chapter 3: Caulobacter crescentus does not induce an immune response in the mouse vagina 3.1 Introduction  In Chapter 2 we described the generation of a recombinant C. crescentus based microbicide using S-layer display technology, and demonstrated that 15 of the recombinant C. crescentus candidates were able to provide significant protection from HIV-1 infection in vitro. This in vitro data is promising that a recombinant C. crescentus based microbicide could prevent HIV-1 infection, the safety of using C. crescentus in the vaginal tract should be investigated before undertaking microbicide testing in a mouse model.  Although C. crescentus is a non-pathogenic, freshwater bacterium primarily found in lakes and rivers, and thus not expected to induce a strong immune response, it is a gram-negative bacterium (170). The C. crescentus S-layer is attached to the surface of the bacterium by LPS (187). While there is little concern for C. crescentus to grow due to the temperature and salt concentrations found in the human body, it is a possibility that the LPS of C. crescentus could stimulate an immune response which could lead to CD4+ T cell recruitment and potentially enhance HIV-1 infection.  Work by collaborators using a mouse model of bladder cancer has demonstrated that intraperitoneal injection of C. crescentus does not cause any observable disease pathology in mice (172). Within 7-10 days post-injection C. crescentus could not be cultured from the peritoneal cavity of the mice (172). Additional research has demonstrated that the lack of an extensive innate immune response to C. crescentus seems to be mediated by the unique composition of the LPS lipid A (187). The lipid A has an unusual backbone structure with a lack 103  of phosphates and unusual fatty acids present (187). Studies in murine macrophages have demonstrated that C. crescentus LPS stimulates 100-1000 times less TNF compared to LPS from pathogenic bacteria such as E. coli (187).   This intraperitoneal work is a promising indication that recombinant C. crescentus might be safe for in vivo use. However, the response to mucosal application may be different from the systemic immune system. In this chapter we describe experiments investigating the immune response to both intraperitoneal injection and vaginal application of C. crescentus. C. crescentus did not induce inflammatory cytokine production in the vaginal tract, immune cell recruitment, or production of antibodies, suggesting that C. crescentus will be safe for topical application.   3.2 Materials and methods Bacterial strain and growth conditions - C. crescentus (Cc-Control) was grown in PYE medium (0.2% peptone, 0.1% yeast extract, 0.01% CaCl2, 0.02% MgSO4) with chloramphenicol at 2 µg/mL at 30°C.  Preparation of C. crescentus displaying chimeric S-layer proteins – The DNA segments specifying proteins of interest were constructed to have BglII and SpeI restriction sites on the 5’ side and an NheI site on the 3’ end. This permitted directional cloning into p4ARsaA(723)/GSCC digested with BglII and NheI (181). Following ligation, plasmids were introduced into E. coli by electroporation. Inserted sequences were confirmed by DNA sequencing before transfer to C. crescentus by electroporation.   Preparation of C. crescentus cells - C. crescentus S-layer display constructs were grown in PYE medium to an optical density at 600nm of approximately 1 (3.1 x 109 cells/ml).  Cells were 104  centrifuged and suspended in water. This was repeated three times and cell density was adjusted to 1 x 1010 cells/mL for binding experiments. Cytokine analysis - For intraperitoneal studies, female 6 week old C57Bl/6 mice were obtained from The Jackson Laboratory and maintained at MBF. The mice were injected with 100µL PBS, 20µg LPS from Salmonella enterica Minnesota serotype, or 108 C. crescentus. Blood was collected from the saphenous vein 5 days before injection then 4 and 24 hours after injection. Blood was centrifuged at 8000RPM for 13 minutes then serum was aliquoted and stored at -80°C for analysis.  For vaginal studies 6 week old female C57Bl/6 mice were injected subcutaneously with 2mg Medroxyprogesterone 17-acetate (Sigma) 4 days prior to experimental start. Vaginal lavage was performed by twice pipetting 30µL PBS in and out of the vaginal tract, 4 days prior to intravaginal inoculation with 20µg LPS from Salmonella enterica serotype Minnesota (Sigma), 108 recombinant C. crescentus, or PBS. Vaginal lavage fluid was collected at 4, 24 and 48 hours post-inoculation. Vaginal lavage fluid was centrifuged at 400 x g and the supernatant was stored at -80°C until analysis. 5µL serum or 25µL vaginal lavage fluid was analyzed using a Cytometric Bead Array kit for mouse inflammatory cytokines, IFNγ, TNF, IL-6, IL-10, MCP-1, IL-12p70 (BD Biosciences) according to the manufacturer’s instructions. Data was acquired on an LSRII and analyzed using FlowJo (TreeStar) and GraphPad Prism. Caulobacter ELISA - Female C57Bl/6 mice were inoculated either once or biweekly intravaginally with 108 Cc-Control for 2 months. Vaginal lavage fluid was collected as described above before inoculation and 28 days (one inoculation) or 67 days (biweekly inoculation) post-inoculation. Vaginal lavage fluid was centrifuged at 400 x g for 5 minutes and diluted in blocking buffer containing 1% BSA, 10% FBS and 0.05% Tween-20. An antibody against RsaA 105  raised in rabbits was used as an assay control. 96 well Nunc plates were coated with 2 x 108 Cc-Control diluted in carbonate buffer and incubated overnight at 4°C. Plates were washed and blocked with 1% bovine serum albumin. Diluted vaginal lavage fluid was added to each well. After incubation at room temperature for 2 hours the plates were washed and 1:1000 anti-mouse IgG-HRP (Sigma) or 1:1000 anti-mouse IgM-HRP (Sigma) were added to each well. The plates were incubated at room temperature for 2 hours then 1:10 O-Phenylenediamine dihydrochloride (Sigma, 2mg/mL) and 1:200 Urea hydrogen peroxide (Sigma, 40mg/mL) were diluted in citrate phosphate buffer and 100µL was added to each well. Twelve minutes later 50µL 25% H2SO4 was added to each well and the OD490 was determined using a plate reader. Data is presented as OD490 with the background subtracted out. Histology - Mice were inoculated intravaginally with 108 Cc-Control, PBS or 20µg LPS from Salmonella enterica Minnesota subtype as previously described. Four hours and 48 hours later vaginal tissue was harvested and placed in buffered formalin. Fixed vaginal tissue was embedded in paraffin, cut into 7µM slices, and mounted on slides. Tissue was deparafinized in xylene and re-hydrated in ethanol before being stained with Harris hematoxylin and eosin Y solution.  Vaginal cytology - Mice were inoculated intravaginally with 108 Cc-Control, PBS or 20µg LPS from Salmonella enterica serotype Minnesota as previously described. Vaginal lavage fluid was collected 4, 24 and 48 hours later as described above. Vaginal lavage fluid was centrifuged at 400 x g and the cell pellet was stored at -80°C in 90% FBS/10% DMSO until analysis. The cell pellet was thawed on ice, washed with PBS and placed on a microscope slide. Slides were dipped in 1% Crystal Violet for 1 minute then washed twice with distilled water. Ethics statement – All animal work was approved by the University of British Columbia Animal Care Committee (Protocols A13-0055, A12-0245).  106  Statistical analysis - Statistical analysis was performed with Prism GraphPad software. Results are reported as mean + SEM. Student’s t test or two-way ANOVA with Bonferroni’s correction for multiple comparisons were used to evaluate the significance of differences between groups as appropriate. p<0.05 was considered statistically significant.  3.3 Results 3.3.1 Intraperitoneal injection of C. crescentus induces low production of inflammatory cytokines  It has previously been demonstrated that intraperitoneal inoculation of C. crescentus causes no adverse events on immune-competent mice (172). To confirm and expand these results before moving into a vaginal inoculation model, a preliminary intraperitoneal experiment was undertaken. Female C57Bl/6 mice were injected intraperitoneally with PBS, LPS from Salmonella enterica serotype Minnesota or 108 Cc-Control. Blood was collected prior to injection, and then 4 and 24 hours after injection and analyzed by a cytometric bead array for mouse inflammatory cytokines. LPS from Salmonella induced significant production of TNF, IL-6, MCP-1 and IL-10. There was no significant difference between PBS and Cc-Control for any of the cytokines tested, although low levels of TNF, IL-6, MCP-1 and IL-10 were produced, these were only present at 4 hours post-infection and are not expected to be problematic as the levels of these cytokines were less than those produced after LPS application. Levels of IFNγ were below the limit of detection of the assay. (Figure 3.1)    107  Figure 3.1: Intraperitoneal injection of C. crescentus induces low production of inflammatory cytokines      IL-12p70Baseline4 hours 24 hours0200400600pg/mLTimePBSLPSCc-ControlIL-6Baseline4 hours24 hours0501001502005000100001500020000Timepg/mL PBSLPSCc-ControlTNFBaseline4 hours24 hours0100200300400500pg/mLTimePBSLPSCc-Control* **** IL-10Baseline4 hours24 hours0200400600800pg/mLTimePBSLPSCc-ControlMCP-1 Baseline4 hours24 hours0200400600800100010000200003000040000Timepg/mL PBSLPSCc-Control**** * 108  Figure 3.1 Cytokine bead array analysis of serum collected from mice injected intraperitoneally with PBS, LPS or Cc-Control. Statistics were performed using a two-way ANOVA with Bonferroni’s correction for multiple comparisons. *p<0.05, ****p<0.0001. n=4 (LPS, Cc-Control) or 6 (PBS)  3.3.2 Low production of cytokines after vaginal application of C. crescentus  Although previous work by our laboratory (187) and collaborators (172) suggested that C. crescentus would be safe for inoculation, the mucosal response to C. crescentus has not been studied. 108 Cc-Control, PBS or LPS isolated from Salmonella enterica serotype Minnesota were applied to the vaginal tract of progesterone treated C57Bl/6 mice and vaginal lavage fluid was collected from the mice 5 days prior to application, then 4, 24 and 48 hours post-inoculation. It has been suggested that immune response in the vaginal tract peaks within 4-6 hours after stimulation (380). Subsequent time points were chosen to determine if any cytokine production was transient. Vaginal lavage fluid was analyzed using a cytometric bead array kit for mouse inflammatory cytokines. There was no significant difference between PBS, Salmonella LPS and 108 Cc-Control for IL-10, IFNγ and IL-12p70 (Figure 3.2). Salmonella LPS induced an increase in MCP-1 and IL-6 production at 4 hours post-application compared to both PBS and Cc-Control, but there was no significant difference observed between PBS and Cc-Control (Figure 3.2). Although Cc-Control did cause a small increase in TNF at 4 hours post-application, this difference was not statistically significant compared to PBS and about a third of the amount of TNF that was produced after application of Salmonella LPS. Taken together these results indicate that C. crescentus does not induce inflammatory cytokines in a manner similar to pathogenic gram-negative bacteria (Figure 3.2).  109  Figure 3.2: C. crescentus does not induce significant production of inflammatory cytokines after vaginal application  Figure 3.2 PBS, 20µg LPS from Salmonella enterica serotype Minnesota or 108 Cc-Control were applied to the vaginal tract of C57Bl/6 mice. Vaginal lavage fluid was collected prior to application then 4, 24 and 48 hours post-application and analyzed by cytometric bead array for mouse inflammatory cytokines. Statistics were calculated using a two-way ANOVA with Bonferroni’s correction for multiple comparisons. *p<0.05, **p<0.01. n=4-5 (TNF), n=7-11 (remaining cytokines).  3.3.3 No immune cell recruitment to the vaginal tract after C. crescentus application Although C. crescentus application did not lead to cytokine production in the vaginal tract, it is possible that immune cells could be recruited to the vaginal tract following C. crescentus application. Female C57Bl/6 mice were treated with progesterone to sync the estrous IFNγBaseline4 hours24 hours48 hours02468pg/mLTimePBSLPSC. crescentusMCP-1Baseline4 hours24 hours48 hours0200400600800pg/mLTimePBSLPSC. crescentusIL10Timepg/mLBaseline4 hours24 hours48 hours0200400600800PBSLPSC. crescentus* IL6Timepg/mLBaseline4 hours24 hours48 hours02004006008001000PBSLPSC. crescentus** IL12p70Baseline4 hours24 hours48 hours0102030pg/mLTimePBSLPSC. crescentusTNFTimepg/mLBaseline4 hours24 hours48 hours0100200300400500 C. crescentusPBSLPS** 110  cycle before intravaginal inoculation with PBS, LPS from Salmonella enterica serotype Minnesota or 108 Cc-Control. Four and forty-eight hours later vaginal tissue was excised and fixed. After paraffin embedding, tissue was cut to a thickness of 7µM and stained with Harris hematoxylin and eosin Y. Evaluation by microscopy revealed no evidence of immune cell infiltration to the vaginal tract at 4 or 48 hours post-application of Cc-Control (Figure 3.3).                   111  Figure 3.3: No recruitment of immune cells to vaginal tissue following application of Cc-Control A      PBS	  4	  hours LPS	  4	  hours Cc-­‐Control	  4	  hours Progesterone	  only	  control Epithelium Mucosa Lamina	  Propria 112  B  Figure 3.3 Harris hematoxylin and eosin Y stained vaginal tissue 4 hours (A) and 48 hours (B) after application of PBS, LPS from Salmonella enterica serotype Minnesota or 108 C. crescentus, or progesterone treated control. Representative images of 4 mice.    PBS	  48	  hours LPS	  48 Cc-­‐Control	  48	  hours 113  3.3.4 No change in cells in the vaginal lavage fluid after topical application of C. crescentus The cellular portion of the vaginal lavage fluid was analyzed to confirm cell typology did not change after application of C. crescentus. Progesterone treated, female C57Bl/6 mice were inoculated intravaginally with PBS, LPS from Salmonella enterica Minnesota serotype or 108 Cc-Control. Vaginal lavage fluid was collected prior to application and then 4, 24 and 48 hours post-inoculation. Cells were isolated from vaginal lavage fluid and stained with Crystal Violet and analyzed for changes in presence of epithelial cells and leukocytes. There was no evidence of immune cell infiltration in the vaginal lavage fluid at any time post-application (Figure 3.4).  Figure 3.4: Vaginal cytology following application of Cc-Control  0	  hours PBS 4	  hours 24	  hours 48	  hours LPS Cc-­‐Control    Leukocyte Nucleated	  Epithelial	  Cell Cornified	  Epithelial	  Cell 114  Figure 3.4 Progesterone treated mice were inoculated intravaginally with PBS, LPS from Salmonella enterica serotype Minnesota or 108 C. crescentus. Vaginal lavage fluid was collected 4, 24 and 48 hours after application. Cells were stained with Crystal Violet. Representative images of 3 mice.   3.3.5 No antibodies produced in the vaginal lavage fluid or blood after vaginal application of C. crescentus Finally, we investigated whether antibodies against C. crescentus would be produced in the vaginal tract after vaginal application of Cc-Control. Mice were inoculated intravaginally with PBS, LPS from Salmonella enterica Minnesota serotype or 108 C. crescentus. Vaginal lavage fluid and serum was collected 28 days later and analyzed by ELISA for antibodies against whole C. crescentus (Figure 3.5A). IgG is the predominant antibody found in the vaginal tract (381). Although there are low levels of IgA in the vaginal tract it is difficult to detect. After application of Cc-Control there was no detectable production of IgM or IgG antibodies in the vaginal lavage fluid. While a signal was detected on the ELISA for both IgM and IgG when serum was analyzed, this was present for each experimental group and the comparison between groups was not statistically significant.  As a C. crescentus based microbicide would need to be applied on multiple occasions to prevent HIV-1 infection, additional experiments were undertaken to mimic this use pattern. C57Bl/6 mice were inoculated intravaginally with 108 Cc-Control, PBS or LPS from Salmonella enterica serotype Minnesota bi-weekly for 9 weeks. Vaginal lavage fluid was collected and analyzed for the presence of antibodies against whole C. crescentus by ELISA. No IgG or IgM antibodies against C. crescentus could be detected in the vaginal fluids (Figure 3.5B). The results 115  in the serum were similar after multiple applications of C. crescentus compared to single application. In both experiments, an antibody against C. crescentus S-layer that was raised in rabbits was used as an ELISA control. Taken together, these results suggest that C. crescentus does not elicit a humoral immune response in the vaginal tract and hence may be safe for multiple vaginal applications.  Figure 3.5: No production of anti-Caulobacter antibodies after vaginal application of Cc-Control A)   PBSCc 10^8 LPSControl0.00.51.01.5OD490PBSCc 10^8 LPSControl0.00.51.01.52.02.5OD490IgG IgM Vaginal	  Fluid ns ns 1:4 1:8 1:161:321:640.00.51.01.5DilutionOD490PBSCc-ControlLPSSerum 1:4 1:8 1:161:321:640.00.51.01.52.0DilutionOD490PBSCc-ControlLPSns ns 116  B)  Figure 3.5 Mice were inoculated intravaginally once or biweekly for 9 weeks with 108 C. crescentus, PBS or LPS from Salmonella enterica serotype Minnesota before vaginal lavage fluid and serum was collected and analyzed by ELISA for antibodies against C. crescentus. An antibody against C. crescentus S-layer protein raised in rabbits was used as a positive control. Data is presented as OD490 with the background subtracted out, and represents mean + SEM. n= 3-5 (1 dose); for biweekly experiments n=1 (LPS), 2 (PBS) or 3 (Cc-Control). ns is p>0.05 IgM DayOD490Day 0Day 670.00.51.01.5PBSLPSC. crescentusControlIgGTimeOD490Day 0Day 670123PBSLPSCc-ControlControlIgMOD4901/161/321/641/1281/2561/5121/10241/20480.00.51.01.5PBSLPSC. crescentusControlVaginal	  Fluid Serum IgG1/161/321/641/1281/2561/5121/10241/20480.00.51.01.52.02.5DilutionOD490PBSLPSCc-ControlControlDilution 117  3.4 Discussion  In Chapter 2 we described the generation of 15 recombinant C. crescentus candidates that were able to provide significant protection from HIV-1 infection in vitro. While this work was promising for the development of a C. crescentus based HIV-1 specific microbicide, in vivo studies are necessary to continue microbicide testing in a physiologically relevant system. However, C. crescentus is a gram-negative bacteria, which may limit its use as a microbicide because the LPS of C. crescentus may induce an inflammatory immune response in the vaginal tract, which could enhance HIV-1 acquisition.   While the possibility for induction of an immune response by C. crescentus exists, this seemed unlikely based on work by collaborators in a mouse model of bladder cancer (172). In this model there were no adverse events following intraperitoneal injection of C. crescentus, and C. crescentus could not be cultured from the peritoneal cavity 10 days after injection (172). Additional studies indicated that the LPS of C. crescentus has a unique lipid A structure and stimulates 100-1000 fold less TNF compared to LPS from E. coli (187). While these results suggest that C. crescentus would be safe for in vivo use, the mucosal immune response has not been investigated, so additional experiments were warranted.  In the first part of this chapter we investigated the cytokine response to intraperitoneal injection of C. crescentus, to confirm and expand the results from Bhatnagar et al (172). There were no adverse events observed in the mice over the course of these experiments. While there was low production of inflammatory cytokines after injection of C. crescentus this was not statistically significant compared to PBS and is not expected to be problematic, as LPS isolated from Salmonella enterica Minnesota serotype induced significantly greater production of many inflammatory cytokines. The remainder of this chapter characterized the immune response to 118  vaginal application of C. crescentus. While low levels of TNF were observed in the vaginal tract after application of C. crescentus, this did not appear to be biologically significant as there was no evidence of immune cell recruitment to the vaginal tissue or lavage fluid after application of C. crescentus. Finally, no antibodies were present in the vaginal fluid or blood of the mice after both single dose and multiple doses of C. crescentus. Taken together, these studies suggest that C. crescentus may be safe for topical application to the vaginal tract.   These results were not surprising since C. crescentus is normally found in freshwater sources and has been isolated from drinking water, suggesting that people are regularly exposed to C. crescentus with no adverse effects. These results provide some confirmation that C. crescentus should be safe for mucosal application. The goal of the studies presented in this chapter was to identify any major immunological concerns that would hinder the in vivo use of C. crescentus. These results were sufficient for us to move on to in vivo testing with recombinant C. crescentus. However, before a recombinant C. crescentus based microbicide could move to testing in humans some additional safety testing is warranted. This would include ensuring that C. crescentus does not disrupt the epithelial barrier, verifying the vaginal microflora is not altered, and more extensive testing for changes in soluble and cellular immune mediators in the vaginal fluid. We do not anticipate changes in the vaginal microenvironment after C. crescentus application, and our HIV-1 mouse data supports that C. crescentus does not increase HIV-1 infection. 119  Chapter 4: Investigating whether Humanized Bone Marrow-Liver-Thymus (BLT) mice are protected from vaginal infection with HIV-1JR-CSF by a recombinant C. crescentus based microbicide 4.1 Introduction  Each year 2 million new HIV-1 infections occur (1). Approximately half of all new infections occur in women, the majority of whom reside in low-income countries, where lack of access to prevention services, social factors and gender inequalities make young women more likely to acquire HIV-1 through sexual transmission compared to young men (2). This suggests that our current prevention options have not been successful for prevention of HIV-1 infection in young women. With the difficulties in developing an HIV-1 vaccine, and the lack of success of PrEP in African women (143, 159, 382), new female controlled prevention options for HIV-1 are urgently needed.  Microbicides, which are drug products that can be topically applied to mucosal surfaces in advance of sexual activity, are an excellent option for female controlled prevention of HIV-1 (98). While tenofovir gel has shown some efficacy as an HIV-1 microbicide in the CAPRISA 004 trial, the tenofovir gel arm of the VOICE trial was stopped due to futility (2, 143). While adherence was a concern, follow-up studies have indicated that tenofovir gel caused side effects that may prevent long-term use (141, 144), indicating that alternative microbicide options need to be developed.   In Chapter 2 we described the development of an HIV-1 specific microbicide using the non-pathogenic, freshwater bacterium C. crescentus. Eighteen different recombinant C. crescentus that displayed proteins with the potential to prevent the attachment or entry of HIV-1 120  into a target cell were described in Chapter 2. These recombinant C. crescentus were tested in vitro for ability to prevent HIV-1 infection of TZM-bl cells and human PBMCs using both HIV-1 pseudovirus representing viral clades B and C, and replication competent HIV-1. It was demonstrated that 15 recombinant C. crescentus were able to provide significant protection from HIV-1 infection in vitro while three recombinant C. crescentus were unable to prevent HIV-1 pseudovirus infection and did not undergo additional testing. In Chapter 3 it was demonstrated that C. crescentus does not induce significant inflammatory cytokine production, immune cell recruitment, or antibody production after topical application to the mouse vaginal tract. This data, combined with the published intraperitoneal injection data (172), suggests that C. crescentus may be safe for use as a topical mucosal agent. In this chapter the generation of humanized BLT mice and in vivo testing for ability to prevent vaginal infection of humanized BLT mice with HIV-1 is described.  BLT mice represent a useful model for pre-clinical microbicide testing. These mice are individually created for each experiment by implanting human fetal liver and thymus tissue under the kidney capsule of immunodeficient mice, followed by intravenous injection of autologous CD34+ cells (210, 211). BLT mice have a functional human immune system, including reconstitution of the mucosa with human immune cells (205, 210, 211, 216). This renders the mice susceptible to vaginal infection with HIV-1 (205, 214, 219, 242). Disease characteristics in the mice mimic what is observed in humans, making the BLT mice a useful model to study HIV-1 (205, 216, 219, 222). While there are several strains of immunodeficient mice that can be used for development of BLT mice, the NSG strain was used in these experiments. The NSG mouse is severely immunodeficient, lacking B cells T, cells, NK cells and haemolytic complement (231). It has been reported that the NSG strain has better human 121  immune cell engraftment and greater susceptibility to HIV-1 infection compared to the NOD/SCID strain (214, 219, 232).  4.2 Materials and methods Bacterial strains and growth conditions - Escherichia coli strain DH5α (Invitrogen, Carlsbad, CA) was grown at 37°C in Luria Broth (1% tryptone, 0.5% NaCl, 0.5% yeast extract). C. crescentus strains were grown in PYE medium (0.2% peptone, 0.1% yeast extract, 0.01% CaCl2, 0.02% MgSO4) with 2 µg/ml chloramphenicol at 30°C. C. crescentus strain JS4038 was described in Chapter 2.  Preparation of C. crescentus displaying chimeric S-layer proteins - All gene segments were synthesized by commercial sources with codon usage adapted for C. crescentus. The amino acid sequence specified for each are indicated in Chapter 2, Table 2.1. In addition, the synthesized DNA segments specified BglII and SpeI restriction sites on the 5’ side and an NheI site on the 3’ end. This permitted directional cloning into p4ARsaA(723)/GSCC digested with BglII and NheI (181). Following ligation, plasmids were introduced into E. coli by electroporation. Inserted sequences were confirmed by DNA sequencing before transfer to C. crescentus by electroporation.   Preparation of C. crescentus cells - C. crescentus S-layer display constructs were grown in PYE medium to an optical density at 600nm of approximately 1 (3.1 x 109 cells/ml).  Cells were centrifuged and suspended in water. This was repeated three times and cell density was adjusted to 1 x 1010 cells/mL for experiments. Preparation of HIV-1JR-CSF – HIV-1pykJR-CSF was a gift from Dr. Zabrina Brumme (Simon Fraser University) and originally obtained from the NIH AIDS Reagent and Reference Program 122  (described in Chapter 2). 24µg pyk-JRCSF was transfected into 293T cells using Lipofectamine 2000 and harvested after 48 hours. HIV-1JR-CSF was concentrated using Amicon-15 filters and titrated on TZM-bl cells as described in Chapter 2.  Preparation of humanized Bone Marrow-Liver-Thymus (BLT) mice – NSG mice were obtained from The Jackson Laboratory and maintained in the Modified Barrier Facility at the University of British Columbia. Six to twelve week old mice were used in experiments. Second trimester human fetal liver and thymus tissue was obtained after informed written consent from women undergoing elective abortion and procured by Advanced Biosciences Resources Inc (Alameda, California). The fetal liver was split and 1mm3 pieces of liver and thymus were implanted under the kidney capsule of mice in a liver-thymus-liver tissue sandwich. The remaining fetal liver tissue was used for CD34+ cell isolation by using a CD34+ cell positive selection kit from Stemcell Technology Inc according to the manufacturer’s instructions. CD34+ cells were frozen in 90% human serum/10% DMSO. Three weeks following the surgical implantation, mice received 225cGy irradiation from an X-ray source followed by intravenous injection of 100,000-200,000 CD34+ cells within 30 hours. Mice were incubated for 9 weeks before blood was collected from the saphenous vein and characterized for human immune cell reconstitution. Flow cytometry - 100µL peripheral blood was collected from the saphenous vein and treated with EDTA. Red blood cell lysis buffer was added and samples were incubated for 15 minutes before washing with FACS buffer (PBS + 2% FBS). Cells were stained with anti-mouse CD45 Pacific Blue (eBioscience), mouse anti-human CD45 Pe-Cy7 (BD Pharminogen), mouse anti-human CD3 Alexa 700 (eBioscience), mouse anti-human CD4 PE (BD Pharminogen), mouse anti-human CD8 PE-Cy5 (BD Pharminogen), mouse anti-human CD14 APC-Cy7 (BD 123  Pharminogen), mouse anti-human CD19 APC (BD Pharminogen), and mouse anti-human CD56 FITC (BD Pharminogen). Data was acquired on an LSRII and analyzed using FlowJo (TreeStar). HIV-1JR-CSF infection – Mice were anaesthesized with isoflurane before atraumatic vaginal infection with 10,000TCID50 HIV-1JR-CSF in the presence or absence of 108 C. crescentus. C. crescentus was pre-mixed with HIV-1JR-CSF less than 5 minutes before inoculation into the vaginal tract in a volume of 20µL using a sterile p200 pipette tip. Mice remained anaesthesized for 4 minutes and were checked to ensure no leakage occurred before being recovered. HIV-1JR-CSF infection analysis – Blood was collected from the retro-orbital sinus on days 0, 14, 28, 42 and 56 post-infection. Blood was centrifuged at 8000RPM for 13 minutes and serum was aliquoted and frozen at -80°C before being analyzed by p24 ELISA and RT-qPCR. p24 ELISA was performed using a kit from ZeptoMetrix (LOD 3.9pg/mL), according to the manufacturer’s instructions. Viral RNA was extracted from equal volumes of serum using the QIAamp Viral RNA Mini Kit (Qiagen). Equal volumes of RNA were converted to cDNA using the Applied Biosystems cDNA kit (Thermo Fisher Scientific). Equal volumes of cDNA were run in duplicate on the Mx3005p PCR multiplex quantitative PCR instrument (Stratagene) with Sybr Green (BioRad), forward primer ATCAAGCAGCTATGCAAATGCT, reverse primer CTGAAGGGTACTAGTAGTCCCTGCTATGTC, settings are 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, 60°C for 1 minute. The limit of detection was 500 copies/mL. Additional RT-qPCR studies would need to be performed to confirm results. For both p24 ELISA and RT-qPCR the reported limit of detection was verified during each p24 ELISA and each RT-qPCR by serial dilution. Statistical analysis –HIV-1 infection data was graphed using a modified survival curve with each event representing the detection of HIV-1 infection in a mouse by p24 ELISA or RT-qPCR 124  and was analyzed using the log-rank (Mantel-Cox) test. p<0.05 was considered statistically significant. p>0.05 unless otherwise indicated. Experimental groups were pooled across mouse cohorts. Ethics statement – All animal work was approved by the University of British Columbia Animal Care Committee (Protocols A13-0055, A13-0234, A12-0245). Human ethics approval was obtained from the University of British Columbia Clinical Ethics Board (H12-02480).  4.3 Results 4.3.1 Development of humanized BLT mice  Humanized BLT mice were individually created for each experiment by implanting human fetal liver and thymus tissue, gestational age 16-24 weeks, under the kidney capsule of NSG mice. CD34+ cells were isolated from autologous fetal liver and were injected intravenously after the mice received sublethal irradiation.   Nine weeks after CD34+ cell injection (12 weeks post-surgery) blood was collected from the saphenous vein of the mice and analyzed for the presence of human immune cells by flow cytometry. On average there were 25.8% human CD45+ cells in the blood of the mice. These human immune cells were characterized by the expression of CD3.  On average 15.5% of the CD3+ cells were CD4+ T cells while 8.7% were CD8+ T cells. The CD3- cells were further analyzed for the presence of myeloid cells, B cells and NK cells. 20.2%% of the cells were myeloid cells (CD45+CD3-CD14+CD19-), 4.5%% were B cells (CD45+CD3-CD19+CD14-) and 4.8% were NK cells (CD45+CD3-CD56+CD14-CD19-). (Figure 4.1 and Appendix B, Table B.1)  125  Figure 4.1: Human immune cell reconstitution in BLT mice   Figure 4.1 NSG-BLT mice were created by implanting human fetal liver and thymus tissue under the kidney capsule of NSG mice followed by sublethal irradiation and intravenous injection of autologous CD34+ cells. Peripheral blood was collected 12 weeks post-transplant and analyzed for presence of human immune cells by flow cytometry. n=183 mice  Human CD45+0102030%% human CD45+ CellsCD3+ CellsCD4+ T cellsCD8+ T cells05101520B cellsMonocytesNK Cells0510152025% of CD45+CD3- Cells  %CD45+CD3+	  cells %CD3-­‐CD14-­‐CD19-­‐ 126  4.3.2 Analysis of HIV-1JR-CSF infection in humanized BLT mice    In this chapter, the primary research question addressed was whether recombinant C. crescentus could block vaginal infection with HIV-1JR-CSF in the humanized BLT mice. HIV-1JR-CSF infection was measured by analyzing serum levels of p24 and HIV-1JR-CSF RNA. Mice were considered HIV-1 positive provided HIV-1 infection was detected above the limit of detection of either the p24 ELISA assay (>3.9pg/mL) or the RT-qPCR assay (>500 copies/mL). If the levels of p24 or HIV-1JR-CSF RNA was at the limit of detection (ex. 500 copies/mL) this was considered negative, as it cannot be determined if this sample contains HIV-1JR-CSF. Successful blocking of infection was defined by a lack of p24 or HIV-1JR-CSF RNA (when available) in the blood. Overall results presented for each recombinant C. crescentus candidate represent a summary of the complete set of data that is currently available, and it is possible that this may change as further RNA analysis is completed. Due to the small cohort sizes (10-24 mice per organ set) it was necessary to pool cohorts for these experiments. Each cohort contained at least two “positive control” mice: one mouse that received HIV-1JR-CSF and one mouse that received HIV-1JR-CSF + Cc-Control. Each recombinant C. crescentus was tested in groups of 3-4 mice in a preliminary experiment. If the recombinant C. crescentus provided greater than 40% protection from HIV-1 infection the experiment was repeated with 3-4 additional mice from a different cohort. Only cohorts with at least one of the positive control mice measured as HIV-1 positive were included in the analysis to ensure that HIV-1 infection was able to occur in every cohort.  When this project was initiated it was common to measure HIV-1 infection in BLT mice by p24 ELISA alone. Recently, the field has shifted to measure HIV-1 infection in the mice by RT-qPCR.  Initially, HIV-1 infection in the BLT mice were measured using p24 ELISA and the p24 data presented in this chapter represents a “complete” data set, in that serum has been 127  analyzed for p24 at every experimental time point for each mouse presented. To stay current with the publication standard, and support the p24 ELISA data, for HIV-1 experiments in BLT mice, analysis by RT-qPCR has been initiated. When available, RNA data is reported in addition to p24 ELISA data. In some cases there is a difference between the p24 ELISA and RNA results. RNA analysis is more sensitive than p24 ELISA and may be detecting HIV-1 infection that was below the limit of detection of the p24 assay.   It is important to note that although the BLT mouse model represents a useful small animal model of HIV-1 infection, HIV-1 infection in these mice does not fully recapitulate human HIV-1 infection. Although some mice have sustained viremia following vaginal infection with HIV-1, it is common for mice to have a “transient” infection course (Figure 4.2, Table 4.1, Figure 4.3, Table 4.2). For the purposes of these experiments, the courses of HIV-1 infection is less relevant than whether mice that are exposed to HIV-1 vaginally ever have detectable HIV-1 in the blood.           128  Figure 4.2: HIV-1JR-CSF infection after vaginal exposure  Figure 4.2 HIV-1JR-CSF infection in BLT mice. Mice were infected vaginally with 10,000TCID50 HIV-1JR-CSF. Blood was collected from the retro-orbital sinus every other week and analyzed by p24 ELISA. Each line represents one mouse. n=13  Table 4.1 HIV-1JR-CSF infection in the BLT mouse model Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected F11 p24 - + - + n/a ✔ RNA ND ND ND ND n/a F3 p24 - + + + - ✔ RNA ND ND ND ND ND 101 p24 - + - - - ✔ RNA ND ND ND ND ND 0 14 28 42 56020406080pg/mlDay129  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected F4 p24 - + - - - ✔ RNA - ND ND ND ND 34 p24 - - - - + ✔ RNA - ND ND ND ND 136 p24 - - - - n/a ✔ RNA - ND + ND n/a 160 p24 - - - - - ✔ RNA - - - - + 280 p24 - - - - + ✔ RNA ND ND ND ND ND 287 p24 - - - + + ✔ RNA ND ND ND ND ND 314 p24 - + - - + ✔ RNA ND ND ND + ND 324 p24 - + + + + ✔ RNA ND ND ND ND ND 35 p24 - - - - - ? RNA ND ND ND ND ND   130  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected F33 p24 - - - - - ? RNA ND ND ND ND ND  Table 4.1 Summary of p24 ELISA data and RNA data for BLT mice infected intravaginally with 10,000 TCID50 HIV-1JR-CSF. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below the LOD of the assay); ND – Analysis currently not done or single replicate; n/a – Mouse euthanized early. Mice euthanized upon reaching the humane endpoint due to complications such as eye infection from retro-orbital bleeding, gastrointestinal problems, graft versus host disease; ? can not determine at this time if mouse is HIV-1 positive. Mouse has been reported as HIV-1 negative in analysis           131  Figure 4.3 HIV-1JR-CSF + Cc-Control  Figure 4.3 HIV-1JR-CSF + Cc-Control infection in BLT mice. Mice were infected vaginally with 10,000TCID50 HIV-1JR-CSF + 108 Cc-Control. Blood was collected from the retro-orbital sinus every other week and analyzed by p24 ELISA. Each line represents one mouse. n=13  Table 4.2 HIV-1JR-CSF + Cc-Control   Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected F7 p24 - + - - n/a ✔ RNA - + ND + n/a F6 p24 - + - - n/a ✔ RNA ND ND ND ND n/a 54 p24 - - - - + ✔ RNA ND ND ND ND ND 0 14 28 42 560204060pg/mlDay132  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected 103 p24 - + - - - ✔ RNA ND ND ND ND ND 110 p24 - - + ND + ✔ RNA - - ND ND ND 141 p24 - - - - FD ✖ RNA - - - - FD 176 p24 - - - - - ✔ RNA ND ND ND ND + F21 p24 - - + - - ✔ RNA ND ND ND ND ND F22 p24 - + - - - ✔ RNA ND ND ND ND ND 281 p24 - + - + - ✔ RNA ND ND ND ND ND 288 p24 - - - + - ✔ RNA ND ND ND ND ND 315 24 - + - - - ✔ RNA ND ND ND ND ND  133  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected 327 p24 - + + + - ✔ RNA - ND + ND -  Table 4.2 Summary of p24 ELISA and RNA data for mice infected intravaginally with HIV-1JR-CSF + Cc-Control. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate; n/a – Mouse euthanized early. Mice euthanized upon reaching the humane endpoint due to complications such as eye infection from retro-orbital bleeding, gastrointestinal problems, graft versus host disease; FD – Mouse found dead  4.3.3 Cc-MIP1α may provide protection of NSG-BLT mice from vaginal infection with HIV-1JR-CSF  Having established the NSG-BLT model, I first verified that these mice were susceptible to vaginal infection with HIV-1. Once I confirmed that the mice could successfully be infected intravaginally with HIV-1 I then used this model to test in vivo efficacy of the microbicide. NSG-BLT (subsequently referred to as BLT) mice were used to determine if Cc-MIP1α could provide protection from vaginal infection with HIV-1JR-CSF. In vitro, Cc-MIP1α resulted in up to 86% inhibition of HIV-1 infection (Chapter 2). BLT mice were infected intravaginally with 10,000 TCID50 HIV-1JR-CSF, HIV-1JR-CSF + Cc-Control, or HIV-1JR-CSF + Cc-MIP1α. C. crescentus was pre-mixed with HIV-1JR-CSF and immediately inoculated atraumatically into the vaginal tract of anaesthesized mice. 108 recombinant C. crescentus were used for all HIVJR-CSF 134  experiments in BLT mice. In Appendix A this dose was shown to be sufficient to prevent HIV-1 infection in cell culture without causing non-specific blocking of infection. In Chapter 5, 108 recombinant C. crescentus was demonstrated to be sufficient to prevent HSV-2 infection in vivo.   Blood was collected from the retro-orbital sinus prior to HIV-1JR-CSF infection and then every other week for 6-8 weeks and analyzed by p24 ELISA and RT-qPCR (See Appendix B for Additional Details). As detailed above, all BLT mouse samples discussed in this section have been analyzed by p24 ELISA while analysis by RT-qPCR is incomplete.   The primary research question addressed was if recombinant C. crescentus could block vaginal infection with HIV-1JR-CSF in the humanized mice. As described above, mice were considered HIV-1 positive provided HIV-1 infection was detected above the limit of detection of either the p24 ELISA assay (>3.9pg/mL) or RT-qPCR (>500 copies/mL). Successful blocking of infection was defined by a lack of p24 and HIV-1JR-CSF RNA in the blood (Figure 4.4). In 6 of 7 mice receiving HIV-1JR-CSF + Cc-MIP1α, p24 was not detected in the blood of the mice over the course of the experiment (Table 4.3). One mouse had detectable p24 in the blood at day 14 post-infection, but had no detectable p24 at any other time (Table 4.3). During preliminary analysis of HIV-1JR-CSF RNA levels, one mouse (BLT-173) that had no detectable p24 in the blood had detectable HIV-1 RNA (Table 4.3). At this time, this mouse has been included in the analysis and considered HIV-1 positive. This RNA analysis would need to be repeated to confirm the conflicting p24 and RNA results. RT-qPCR has indicated that one mouse that received HIV-1JR-CSF + Cc-MIP1α (BLT-174) is negative for both p24 and HIV-1JR-CSF RNA in the blood at all time points (Table 4.3). RNA analysis on the remaining 5 mice that received HIV-1JR-CSF + Cc-MIP1α is incomplete. Based on the available data, 5 of 7 (71%) mice that received HIV-1JR-CSF + Cc-MIP1α appeared protected from vaginal infection with HIV-1JR-CSF. 135  Figure 4.4: Cc-MIP1α may provide 71% protection from vaginal infection with HIV-1JR-CSF   0 14 28 42 56050100% HIV-1 NegativeDayHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-MIP1a* HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-MIP1aHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-MIP1a05101520# MiceHIV positiveHIV negativeDay 14 Day 56 136   Figure 4.4 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Blood was collected biweekly for 8 weeks and analyzed by p24 ELISA and RT-qPCR. Mice are considered HIV-1 infected the first day they gave a positive test result by either p24 ELISA or RT-qPCR, statistical analysis performed by Log-rank (Mantel-Cox) test. n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), 7 (HIV-1JR-CSF + Cc-MIP1α). *p<0.05  Table 4.3 HIV-1JR-CSF + Cc-MIP1α Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13 HIV-1 + CcControl p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 0 14 28 42 560246810pg/mlDayHIV-1 + Cc-MIP1a HIV negativeHIV-1 + Cc-MIP1a HIV positive137  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected 152 p24 - - - - - ✖ RNA - - - ND ND 173 p24 - - - FD FD ✔ RNA ND ND + FD FD 174 p24 - - - - - ✖ RNA ND ND ND ND ND 175 p24 - - - - - ✖ RNA ND ND ND ND ND F23 p24 - + - - - ✔ RNA ND ND ND ND ND F24 p24 - - - - - ✖ RNA ND ND ND ND ND F25 p24 - - - - - ✖ RNA ND ND ND ND ND  Summary 5/7 HIV-  Table 4.3 Summary of p24 ELISA and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-MIP1α. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 138  p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate; FD – Mouse found dead  4.3.4 Cc-CD4 and Cc-CD4M33F23 may provide some measure of protection from vaginal infection with HIV-1JR-CSF in BLT mice  As described above, we investigated if the mice that were infected vaginally with HIV-1JR-CSF + Cc-CD4 or HIV-1JR-CSF + Cc-CD4M33F23 were protected from HIV-1JR-CSF infection. Mice were considered HIV-1 positive if HIV-1 infection was detected at any time point post-infection. Mice are reported as HIV-1 negative if no detectable p24 or HIV-1JR-CSF RNA was measured in the blood.   Pilot experiments using 3-4 mice were undertaken to determine if the recombinant C. crescentus acting as decoy receptors for HIV-1 were able to protect BLT mice from vaginal infection with HIV-1JR-CSF. Cc-CD4 was tested in 4 BLT mice. While the Cc-CD4 experiments were ongoing, there were health complications within the BLT mouse colony. Necropsy and PCR analysis of fecal pellets suggested that the mice were infected with Helicobacter, which impacted the health and survival of the mice. One mouse that received HIV-1JR-CSF + Cc-CD4 was euthanized at day 14 post-infection. Blood was collected from this mouse and analyzed by p24 ELISA at this time. There was no detectable p24 in the blood of this mouse. Since seroconversion was not observed in some mice until after day 14, it was not known if this mouse was HIV-1 infected, but with p24 levels below the limit of detection at the time of death, so this mouse was excluded from the analysis. Of the three remaining mice, two mice were euthanized prematurely due to health concerns, one at day 28 post-infection and the other at day 42 post-infection. Prior to death, both of these mice had detectable p24 in the blood (Table 4.2). The 139  remaining mouse survived to the end of the experiment and p24 was not detected in the blood of this mouse at any time post-infection (Table 4.2).   In preliminary BLT mouse experiments with Cc-CD4M33F23, more than half of the mice were protected from vaginal infection with HIV-1JR-CSF, so additional experiments were undertaken. Of the 10 mice that received HIV-1JR-CSF + Cc-CD4M33F23, one died before day 14 post-infection and one died between day 28 and 42 post-infection. These mice did not have detectable p24 in the blood and were excluded from the analysis. Three mice that were infected intravaginally with HIV-1JR-CSF + Cc-CD4M33F23 did not have detectable p24 in the blood over the course of the experiment (Figure 4.5). In particular, p24 could not be detected in the blood of four of eight mice (Table 4.5). However, using RT-qPCR, HIV-1JR-CSF was detected in the blood of one of the mice that was measured as HIV-1 negative by p24 ELISA (Table 4.5).   Table 4.4 HIV-1JR-CSF + Cc-CD4 Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total: 11/13 RNA 0/13 0/13 1/13 1/13 1/13 HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total: 12/13 RNA 0/13 1/13 1/13 1/13 1/13 97 p24 - + - - FD ✔ RNA ND ND ND ND FD 140  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV-1 Infected 93 p24 - - + FD FD ✔ RNA - + ND FD FD 94 p24 - - - - - ✖ RNA ND ND ND ND ND 92 p24 - - FD FD FD Excluded RNA - - FD FD FD  Summary 1/3 HIV-  Table 4.4 Summary of p24 ELISA and RNA data for mice infected intravaginally with HIV-1JR-CSF + Cc-CD4.; + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate; FD – Mouse found dead        141  Figure 4.5: Cc-CD4M33F23 may provide some protection from vaginal infection with HIV-1JR-CSF    Figure 4.5 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Mice are considered HIV-1 infected the first day either p24 ELISA or RT-qPCR gave a positive result, statistical analysis performed by Log-rank (Mantel-Cox) test. 0 14 28 42 56050100% HIV-1 NegativeDayHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-CD4M33F23ns # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-CD4M33F23HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-CD4M33F2305101520HIV positiveHIV negativeDay 14 Day 56 142  n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), 8 (D-F HIV-1JR-CSF + Cc-CD4M33F23). p>0.05  Table 4.5 HIV-1JR-CSF + Cc-CD4M33F23 Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13  HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total  12/13 RNA 0/13 1/13 1/13 1/13 1/13 166 p24 - FD FD FD FD Excluded RNA - FD FD FD FD 167 p24 - - FD FD FD Excluded RNA - - FD FD FD 308 p24 - + - + - ✔ RNA ND ND ND ND ND 346 p24 - + + + + ✔ RNA - ND ND ND ND   143  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected 347 p24 - + - + - ✔ RNA - ND ND ND ND 307 p24 - - - + - ✔ RNA ND ND ND ND ND 165 p24 - - - - - ✔ RNA - - - - + F30 p24 - - - - - ✖  RNA ND ND ND ND ND  F31 p24 - - - - - ✖  RNA ND ND ND ND ND  F32 p24 - - - - - ✖  RNA ND ND ND ND ND   Summary 3/8 HIV-  Table 4.5 Summary of p24 ELISA data and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-CD4M33F23. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate; FD – Mouse found dead 144  4.3.5 Recombinant C. crescentus expressing anti-viral lectins may provide variable levels of protection from vaginal infection with HIV-1JR-CSF in BLT mice  In this section we determined if the recombinant C. crescentus were able to provide protection from vaginal infection with HIV-1JR-CSF. Mice were considered HIV-1 positive if HIV-1 infection was detected by either p24 ELISA (>3.9pg/mL) or RT-qPCR (>500 copies/mL) at any time point. Mice are reported as HIV-1 negative if no detectable p24 and/or HIV-1JR-CSF RNA was measured in the blood.    In vitro assays indicated that all three anti-viral lectins, Cc-Cyanovirin, Cc-Microvirin, and Cc-Griffithsin were able to provide significant protection from HIV-1 infection. While the most effective anti-viral lectin varied depending on the cell type and virus used, none were significantly better than the others at preventing HIV-1 infection in vitro.   In a pilot experiment, Cc-Cyanovirin did not appear to provide protection of BLT mice from vaginal infection with HIV-1JR-CSF (Figure 4.6A). Although all three mice were positive for HIV-1 infection, as measured by p24 levels in the serum, there appeared to be a delay in seroconversion, with no detectable p24 until day 42 post-infection in one mouse and day 56 post-infection in the remaining two mice (Table 4.6). This is in comparison to mice that received HIV-1JR-CSF alone or HIV-1JR-CSF + Cc-Control, which have detectable viremia at day 14 post-infection, the earliest time point analyzed. RT-qPCR analysis would need to be performed for these mice to support and confirm the results.   Cc-Microvirin appeared to provide protection from infection of one of four mice (25%) that received HIV-1JR-CSF + Cc-Microvirin (Figure 4.6B). Two mice were measured as HIV-1 negative by lack of detection of p24 in the blood and two mice were measured as HIV-1 positive by presence of p24 in the blood (Table 4.7). BLT-114 was measured as HIV-1 positive by both 145  p24 ELISA and RT-qPCR (Table 4.7). One mouse that was not measured as HIV-1 positive by p24 ELISA currently has an incomplete RNA data set, but HIV-1JR-CSF RNA has been detected in the blood of this mouse at day 14 post-infection. This mouse has been considered to be HIV-1 positive, but assays would need to be repeated to confirm the differing p24 ELISA and RT-qPCR results. RT-qPCR analysis would need to be repeated for the remaining two mice.   Cc-Griffithsin appeared to protect 9 of 10 mice (90%) from vaginal infection with HIV-1JR-CSF (Figure 4.6C). Over the course of the experiment none of the mice (10 of 10) that received HIV-1JR-CSF + Cc-Griffithsin had detectable p24 in the blood (Table 4.8). However, one mouse that was measured as HIV-1 negative by p24 ELISA had HIV-1JR-CSF RNA detected in the blood by RT-qPCR (Table 4.8). RT-qPCR analysis has been completed for 4 mice and all 4 mice were measured as HIV-1 negative by both p24 ELISA and RT-qPCR.  Figure 4.6: Recombinant C. crescentus expressing anti-viral lectins may provide variable levels of protection from vaginal infection with HIV-1JR-CSF in BLT mice A)  0 14 28 42 56050100% HIV-1 NegativeDayHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Cyanovirinns 146    B)  # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-CyanovirinHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Cyanovirin05101520HIV positiveHIV negativeDay 14 Day 56 0 14 28 42 56050100% HIV-1 NegativeDayHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Microvirinns 147    C)  # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-MicrovirinHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Microvirin05101520HIV positiveHIV negativeDay 14 Day 56 0 14 28 42 56050100% HIV-1 NegativeDayHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Griffithsin*** 148   Figure 4.6 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Mice were considered HIV-1 positive the first time either test result was positive, statistical analysis performed by Log-rank (Mantel-Cox) test. n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), A) 3 (Cc-Cyanovirin), B) 4 (Cc-Microvirin), C) 10 (Cc-Griffithsin). ***p<0.001  Table 4.6 HIV-1JR-CSF + Cc-Cyanovirin Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total  11/13 RNA 0/13 0/13 1/13 1/13 1/13   # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-GriffithsinHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Griffithsin0510152025HIV positiveHIV negativeDay 14 Day 56 149  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total  12/13 RNA 0/13 1/13 1/13 1/13 1/13 12 p24 - - - + - ✔ RNA - - - + - 10 p24 - - - - + ✔ RNA ND ND ND ND ND 13 p24 - - - - + ✔ RNA ND ND ND ND ND  Summary 0/3 HIV-  Table 4.6 Summary of p24 ELISA and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-Cyanovirin. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate      150  Table 4.7 HIV-1JR-CSF + Cc-Microvirin Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13 HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 114 p24 - - + + n/a ✔  RNA - + ND + n/a 112 p24 - - - - - ✖ RNA ND ND ND ND ND 113 p24 - + - - + ✔ RNA ND ND ND ND ND 115 p24 - - - - - ✔ RNA - + ND ND ND  Summary 1/4 HIV- Table 4.7 Summary of p24 ELISA data and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-Microvirin. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate; n/a – Mouse euthanized early. Mice euthanized upon reaching the humane 151  endpoint due to complications such as eye infection from retro-orbital bleeding, gastrointestinal problems, graft versus host disease  Table 4.8 HIV-1JR-CSF + Cc-Griffithsin Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total  11/13 RNA 0/13 0/13 1/13 1/13 1/13 HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13  F9 p24 - - - - - ✔ RNA - - - + + 145 p24 - - - - - ✖ RNA - - - - - 147 p24 - - - - - ✖ RNA - - - - - 149 p24 - - - - FD ✖ RNA - - - - FD 153 p24 - - - - FD ✖ RNA - - - - FD 152  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected F26 p24 - - - - - ✖ RNA ND ND ND ND ND F28 p24 - - - - - ✖ RNA ND ND ND ND ND 312 p24 - - - - - ✖ RNA - - - - - 342 p24 - - - - - ✖ RNA - - ND - - F27 p24  - - - FD FD ✖ RNA ND ND ND FD FD  Summary 9/10 HIV-  Table 4.8 Summary of p24 ELISA and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-Griffithsin. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate; FD – Mouse found dead  153  4.3.6 Recombinant C. crescentus expressing fusion inhibitors may provide variable protection from vaginal infection with HIV-1JR-CSF in BLT mice  As described above, these experiments sought to determine if vaginal application of recombinant C. crescentus could prevent vaginal infection with HIV-1JR-CSF. p24 and RNA levels were measured in the serum and mice were considered HIV-1 positive if HIV-1 infection was detected by either p24 ELISA (>3.9pg/mL) or RT-qPCR (>500 copies/mL) at any time point. Mice are reported as HIV-1 negative if no detectable p24 or RNA was measured in the blood.   Cc-Fuzeon consistently provided approximately 50% inhibition from in vitro infection with HIV-1, while Cc-T129 provided inhibition ranging from 0%-63% depending on the virus and cell type used, and Cc-C52 consistently provided the best inhibition of HIV-1 infection, with levels exceeding 90% in preliminary experiments with HIV-1JR-FL and HIV-1SF162 in PMBCs. Preliminary experiments have been undertaken to determine if these recombinant C. crescentus can protect BLT mice from vaginal infection with HIV-1JR-CSF.   Overall, Cc-Fuzeon provided 50% protection from vaginal infection with HIV-1JR-CSF (Figure 4.7A). The p24 ELISA results suggested that Cc-Fuzeon provided 100% protection (4/4 mice) from vaginal infection with HIV-1JR-CSF based on a lack of detection of p24 in the blood of the mice (Table 4.9). One mouse had no detectable p24 or HIV-1JR-CSF RNA in the blood at any time point post-infection (Table 4.9). However, 2 of these mice had HIV-1JR-CSF RNA detected in the blood by RT-qPCR (Table 4.9). These analyses would need to be repeated to confirm the results.   Cc-T1249 provided 29% protection from vaginal infection with HIV-1JR-CSF (Figure 4.7B). Four of the mice did not have p24 detectable in the blood (Table 4.10). The three mice that did have p24 detected in the blood had lower levels of p24 in the blood at early time points 154  after infection, but had higher levels of p24 detected in the blood by day 56 post-infection compared to HIV-1JR-CSF alone and HIV-1JR-CSF + Cc-Control (Table 4.10). One mouse was measured as HIV-1 positive by both p24 ELISA and RT-qPCR (Table 4.10). One mouse that had undetectable p24 in the blood had HIV-1JR-CSF RNA detected by RT-qPCR (Table 4.10).   Cc-C52 appeared to protect 1 of 4 mice from vaginal infection with HIV-1JR-CSF in a preliminary experiment (Figure 4.7C). There was no difference in peak viremia based on p24 levels in those mice that were infected with HIV-1JR-CSF in the presence of Cc-C52, although those mice that did seroconvert only had detectable p24 at day 14 post-infection, compared to control mice, which had sustained detectable p24 until the end of the experiment (Table 4.11). One mouse had no detectable p24 in the blood, but HIV-1JR-CSF RNA was detected by RT-qPCR (Table 4.11).  Figure 4.7: Recombinant C. crescentus expressing fusion inhibitors may provide variable protection from vaginal infection with HIV-1JR-CSF in BLT mice A)  Day% HIV-1 Negative0 14 28 42 56050100 HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Fuzeonns 155    B)  # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-FuzeonHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Fuzeon05101520HIV positiveHIV negativeDay 14 Day 56 Day% HIV-1 Negative0 14 28 42 56050100 HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-T1249ns 156    C)  # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-T1249HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-T124905101520HIV positiveHIV negativeDay 14 Day 56 Time% HIV-1 Negative0 14 28 42 56050100 HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-C52ns 157   Figure 4.7 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Blood was collected biweekly and analyzed by p24 ELISA and RT-qPCR. A), B), C) Mice were considered HIV-1 positive the first time either test result was positive, statistical analysis Log-rank (Mantel-Cox) test. n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), A) 4 (Cc-Fuzeon) B) 7 (Cc-T1249), C) 4 (Cc-C52).   Table 4.9 HIV-1JR-CSF + Cc-Fuzeon Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13   # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-C52HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-C5205101520HIV positiveHIV negativeDay 14 Day 56 158  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 1 p24 - - - - - ✖ RNA ND ND ND ND ND 2 p24 - - - - - ✔ RNA - - + + + 3 p24 - - - - - ✖ RNA ND ND ND ND ND 4 p24 - - - - - ✔ RNA - - - + -  Summary 2/4 HIV-  Table 4.9 Summary of p24 ELISA and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-Fuzeon. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate    159  Table 4.10 HIV-1JR-CSF + Cc-T1249 Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV infection HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13 HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 116 p24 - - - - - ✖ RNA ND ND ND ND ND 123 p24 - - - - - ✖ RNA ND ND ND ND ND 124 p24 - - - - - ✖ RNA ND ND ND ND ND 125 p24 - - - - - ✔ RNA - + ND ND ND 275 p24 - + - - - ✔ RNA - - - ND ND 276 p24 - + - + - ✔ RNA - ND + ND -   160  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected 277 p24 - + - + + ✔ RNA - - - ND ND  Summary 3/7 HIV-  Table 4.10 Summary of p24 ELISA data and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-T1249. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate  Table 4.11 HIV-1JR-CSF + Cc-C52 Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total  11/13 RNA 0/13 0/13 1/13 1/13 1/13 HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 127 p24 - + - - - ✔ RNA ND ND ND ND ND   161  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected 128 p24 - + - - - ✔ RNA - + ND ND ND 126 p24 - - - - - ✖ RNA ND ND ND ND ND 129 p24 - - - - - ✔ RNA - + ND ND ND  Summary 1/4 HIV-  Table 4.11 Summary of p24 ELISA data and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-C52. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate  4.3.7 Cc-GBVCE2 may provide protection from vaginal infection with HIV-1JR-CSF  The primary research question addressed in this section was if the mice that were infected vaginally with HIV-1JR-CSF + Cc-GBVCE2 had detectable levels of HIV-1 in the serum by p24 ELISA and RT-qPCR over the course of the experiment. Mice were considered HIV-1 positive if HIV-1 infection was detected by p24 ELISA (>3.9pg/mL) and/or RT-qPCR (>500 copies/mL) at any time point. Mice are reported as HIV-1 negative if no detectable p24 and/or RNA was measured in the blood.  162   Cc-GBVCE2 provided up to 87% protection from HIV-1 infection in vitro. Experiments were undertaken to determine if Cc-GBVCE2 could provide protection from vaginal infection with HIV-1JR-CSF in the BLT mouse model. Preliminarily, Cc-GBVCE2 appeared to 43% protection from vaginal infection with HIV-1JR-CSF (Figure 4.8). Using the p24 ELISA, p24 was not detected in the blood of 3 of 7 mice (43%) over the course of the experiment (Table 4.12). Those mice that did become HIV-1 positive in the presence of Cc-GBVCE2 had similar infection characteristics to HIV-1JR-CSF alone or HIV-1JR-CSF + Cc-Control mice.   Figure 4.8: Cc-GBVCE2 may provide partial protection from vaginal infection with HIV-1JR-CSF  Day% HIV-1 Negative0 14 28 42 56050100 HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-GBVCE2ns 163   Figure 4.8 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Blood was collected biweekly and analyzed by p24 ELISA and RT-qPCR. Mice were considered HIV-1 positive the first time either test result was positive, statistical analysis performed by Log-rank (Mantel-Cox) test. n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), 7 (HIV-1JR-CSF + Cc-GBVCE2)  Table 4.12 HIV-1JR-CSF + Cc-GBVCE2 Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13   # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-GBVCE2HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-GBVCE205101520HIV positiveHIV negativeDay 14 Day 56 164  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 20 p24 - - - No data* - ✖ RNA ND ND ND No data* ND 21 p24 - - - - - ✖ RNA ND ND ND ND ND 304 p24 - - - - - ✖ RNA - - - - ND 305 p24 - - - + - ✔ RNA - - ND ND - 306 p24 - - - + - ✔ RNA - - - ND ND 348 p24 - + + + - ✔ RNA ND ND ND ND ND 349 p24 - + + - - ✔ RNA ND ND ND ND ND  Summary 3/7 HIV- Table 4.12 Summary of p24 ELISA data and RNA data from BLT mice infected intravaginally with HIV-1JR-CSF + Cc-GBVCE2. + HIV-1 p24 or RNA detected (above the LOD of the assay); - 165  HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done; No data* - mouse had severe ocular swelling and no blood could be collected  4.3.8 Cc-BmKn2 does not protect from vaginal infection with HIV-1JR-CSF  To determine if Cc-BmKn2 was able to provide protection from vaginal infection with HIV-1JR-CSF, BLT mice were infected with HIV-1JR-CSF in the presence of Cc-BmKn2 and HIV-1 infection was measured in the serum by p24 ELISA and RT-qPCR. Mice were considered HIV-1 positive if HIV-1 infection was detected by either p24 ELISA (>3.9pg/mL) or RT-qPCR (>500 copies/mL) at any time point. Mice are reported as HIV-1 negative if no detectable p24 and/or RNA was measured in the blood.   Cc-BmKn2 did not consistently prevent HIV-1 pseudovirus infection in vitro, but was able to provide 37%-84% protection from replication competent HIV-1 in vitro, depending on the cell type and virus used. A preliminary experiment was undertaken to determine if Cc-BmKn2 was able to prevent vaginal infection with HIV-1JR-CSF in BLT mice. Three mice were infected with HIV-1JR-CSF + Cc-BmKn2, and all mice were measured as HIV-1 positive by day 42 post-infection (Figure 4.9). One of three mice (33%) had no detectable p24 in the blood over the course of the experiment (Table 4.13). By p24 ELISA, the two mice that were infected with HIV-1JR-CSF in the presence of Cc-BmKn2 had similar infection progression as HIV-1JR-CSF and HIV-1JR-CSF + Cc-Control mice (Table 4.13). However, while the third mouse did not have detectable p24 in the blood, HIV-1JR-CSF RNA was detected by RT-qPCR (Table 4.13). As Cc-BmKn2 does not provide protection from HIV-1 infection, and seemed to enhance HSV-2 infection (Chapter 5, Figure 5.2), no additional experiments were undertaken with this recombinant C. crescentus. 166  Figure 4.9: Cc-BmKn2 does not appear to protect against vaginal infection with HIV-1JR-CSF   Figure 4.9 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Blood was collected biweekly and analyzed by p24 ELISA and RT-qPCR. Mice were considered HIV-1 positive the first time either test result was positive, Day% HIV-1 Negative0 14 28 42 56050100 HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-BmKn2ns # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-BmKn2HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-BmKn205101520HIV positiveHIV negativeDay 14 Day 56 167  statistical analysis by Log-rank (Mantel-Cox) test. n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), 3 (HIV-1JR-CSF + Cc-BmKn2)  Table 4.13 HIV-1JR-CSF + Cc-BmKn2 Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13 HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13  26 p24 - + - + - ✔ RNA - ND ND + ND 28 p24 - - - - - ✔ RNA - - - + - 29 p24 - - + - - ✔ RNA ND ND ND ND ND  Summary 0/3 HIV-  Table 4.13 Summary of p24 ELISA and RNA data from BLT mice infected intravaginally with HIV-1JR-CSF + Cc-BmKn2. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done 168  4.3.9 Cc-Elafin may protect from vaginal infection with HIV-1JR-CSF  To determine if the mice that were infected vaginally with HIV-1JR-CSF + Cc-Elafin were HIV-1 positive, p24 ELISA and RT-qPCR were used to measure viral load in serum. Mice were considered HIV-1 positive if HIV-1 infection was detected by either p24 ELISA (>3.9pg/mL) or RT-qPCR (>500 copies/mL) at any timepoint. Mice are reported as HIV-1 negative if no detectable p24 or RNA was measured in the blood.   Cc-Elafin provided 29%-77% protection from HIV-1 infection in vitro, suggesting it may be a suitable candidate for microbicide development. BLT mice were infected with HIV-1JR-CSF in the presence of Cc-Elafin. Three of seven mice (43%) were protected from vaginal infection with HIV-1JR-CSF when Cc-Elafin was provided at the time of HIV-1 infection (Figure 4.10). Those mice that were infected with HIV-1JR-CSF had a delay in detectable levels of p24 in the blood until day 42 (Table 4.14). However, at day 42 and day 56 there were higher levels of p24 detected in the blood of the mice that received HIV-1JR-CSF + Cc-Elafin compared to the peak levels reached with HIV-1JR-CSF alone.          169  Figure 4.10: Cc-Elafin may provide partial protection from vaginal infection with HIV-1JR-CSF   Figure 4.10 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Blood was collected biweekly and analyzed by p24 ELISA and RT-qPCR. Mice were considered HIV-1 positive the first time either test result was positive, Day% HIV-1 Negative0 14 28 42 56050100 HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Elafinns % HIV-1 NegativeHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-ElafinHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Elafin05101520HIV positiveHIV negativeDay 14 Day 56 170  statistical analysis performed by Log-rank (Mantel-Cox) test. n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), 7 (HIV-1JR-CSF + Cc-Elafin)  Table 4.14 HIV-1JR-CSF + Cc-Elafin Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13 HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 17 p24 - - - FD FD ✖ RNA ND ND ND FD FD 18 p24 - - - - - ✖ RNA ND ND ND ND ND 19 p24 - - - - - ✖ RNA ND ND ND ND ND 293 p24 - + - - - ✔ RNA - ND ND - - 294 p24 - - - + + ✔ RNA - - - + ND 171  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected 295 p24 - - - + - ✔ RNA - - - ND ND 296 p24 - - - + - ✔ RNA ND ND ND ND ND  Summary 3/7 HIV-  Table 4.14 Summary of p24 ELISA and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-Elafin. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate; FD – Mouse found dead  4.3.10 Cc-A1AT may protect from vaginal infection with HIV-1JR-CSF  As described previously, the primary research question addressed was if the mice that were infected vaginally with HIV-1JR-CSF + Cc-A1AT had detectable viremia measured by serum levels of p24 and HIV-1 RNA. Mice were considered HIV-1 positive if HIV-1 infection was detected by either p24 ELISA (>3.9pg/mL) or RT-qPCR (>500 copies/mL) at any time point. Mice are reported as HIV-1 negative if no detectable p24 or RNA was measured in the blood.   Although Cc-A1AT appeared to provide inconsistent protection from infection with HIV-1 pseudovirus, it provided 34%-77% protection from infection with replication competent HIV-1 in vitro. Overall, Cc-A1AT protected 40% of the mice from vaginal infection with HIV-1JR-CSF 172  (Figure 4.11). Cc-A1AT was able to protect 5 of 10 mice (50%) from vaginal infection with HIV-1JR-CSF based on p24 detection in the blood (Table 4.15). RNA analysis has confirmed that one mouse is HIV-1 negative by both RNA and p24 (Table 4.15). The five mice that were HIV-1 positive by p24 ELISA were also HIV-1 positive determined by presence of HIV-1JR-CSF in the blood.   Figure 4.11: Cc-A1AT may provide partial protection from vaginal infection with HIV-1JR-CSF  Day% HIV-1 Negative0 14 28 42 56050100 HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-A1ATns 173   Figure 4.11 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Blood was collected biweekly and analyzed by p24 ELISA and RT-qPCR. Mice were considered HIV-1 positive the first time either test result was positive, statistical analysis performed by Log-rank (Mantel-Cox). n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), 10 (HIV-1JR-CSF + Cc-A1AT)  Table 4.15 HIV-1JR-CSF + Cc-A1AT Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13   # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-A1ATHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-A1AT05101520HIV positiveHIV negativeDay 14 Day 56 174  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 131 p24 - - - - - ✖ RNA ND ND ND ND ND 132 p24 - - - - - ✖ RNA ND ND ND ND ND 133 p24 - - - - - ✖ RNA ND ND ND ND ND 134 p24 - - - - - ✔ RNA - + ND ND ND 289 p24 - - - + - ✔ RNA - + - ND - 290 p24 - + - - + ✔ RNA - ND - - + 291 p24 - - - - - ✖ RNA - - - - -   175  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected 333 p24 - + - - - ✔ RNA - + - - - 335 p24 - + + + n/a ✔ RNA - + ND ND n/a 336 p24 - + - + - ✔ RNA ND ND ND ND ND  Summary 4/10 HIV-  Table 4.15 Summary of p24 ELISA and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-A1AT. + HIV-1 p24 or RNA detected (above the LOD of the assay); - HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done or single replicate; n/a – no sample available because the mouse had severe swelling of both eyes and blood could not be collected  4.3.11 Cc-Indo may protect from vaginal infection with HIV-1JR-CSF  In this section the primary research question addressed was if the mice that were infected vaginally with HIV-1JR-CSF + Cc-Indo had detectable viremia over the course of the experiment.  Levels of p24 and HIV-1 RNA in the serum were measured. Mice were considered HIV-1 positive if HIV-1 infection was detected by either p24 ELISA (>3.9pg/mL) or RT-qPCR (>500 176  copies/mL) at any time point. Mice are reported as HIV-1 negative if no detectable p24 or RNA was measured in the blood.   Cc-Indo was able to provide up to 77% protection from infection with replication competent HIV-1 in vitro, suggesting it may be a good candidate for further testing as an HIV-1 microbicide. BLT mice were infected intravaginally with HIV-1JR-CSF in the presence of Cc-Indo. Three of seven mice (43%) were protected from vaginal infection with HIV-1JR-CSF (Figure 4.12). Two of the infected mice had similar infection characteristics as HIV-1JR-CSF alone mice, with peak detectable p24 observed at Day 14 post-infection, while 2 mice had a delay in detectable viremia by p24 levels until day 42 post-infection (Table 4.16).   Figure 4.12: Cc-Indo may provide partial protection from vaginal infection with HIV-1JR-CSF  Day% HIV-1 Negative0 14 28 42 56050100 HIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Indons 177   Figure 4.12 BLT mice were infected intravaginally with HIV-1JR-CSF in the presence or absence of recombinant C. crescentus. Blood was collected biweekly and analyzed by p24 ELISA and RT-qPCR. Mice were considered HIV-1 positive the first time either test result was positive, statistical analysis performed using log-rank (Mantel-Cox) test. n= 13 (HIV-1JR-CSF), 13 (HIV-1JR-CSF + Cc-Control), 7 (HIV-1JR-CSF + Cc-Indo)  Table 4.16 HIV-1JR-CSF + Cc-Indo Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV infected HIV-1 alone p24 0/13 6/13 2/13 4/13 5/13 Total 11/13 RNA 0/13 0/13 1/13 1/13 1/13   # MiceHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-IndoHIV-1HIV-1 + Cc-ControlHIV-1 + Cc-Indo05101520HIV positiveHIV negativeDay 14 Day 56 178  Mouse ID Assay Day 0 Day 14 Day 28 Day 42 Day 56 HIV Infected HIV-1 + Cc-Control p24 0/13 7/13 3/13 3/13 2/13 Total 12/13 RNA 0/13 1/13 1/13 1/13 1/13 156 p24 - - - FD FD ✖ RNA - - - FD FD 158 p24 - - - FD FD ✖ RNA - - - FD FD 159 p24 - - - - - ✖ RNA - - - - - 297 p24 - - - + - ✔ RNA - - - ND ND 298 p24 - - - + - ✔ RNA - - - ND ND 299 p24 - + - + - ✔ RNA - ND ND ND ND 300 p24 - + - + - ✔ RNA - ND ND ND ND  Summary 3/7 HIV- Table 4.16 Summary of p24 ELISA data and RNA data for BLT mice infected intravaginally with HIV-1JR-CSF + Cc-Indo. + HIV-1 p24 or RNA detected (above the LOD of the assay); - 179  HIV-1 p24 or RNA not detected (below LOD of the assay); ND – Analysis currently not done; FD – Mouse found dead  Table 4.17 BLT mice summary data Microbicide construct applied with HIV-1 % Protection from HIV-1JR-CSF infection Cc-Control 8% Cc-MIP1α 71% Cc-CD4 33% Cc-CD4M33F23 38% Cc-Cyanovirin 0% Cc-Microvirin 25% Cc-Griffithsin 90% Cc-Fuzeon 50% Cc-T1249 29% Cc-C52 25% Cc-GBVCE2 43% Cc-Elafin 43% Cc-BmKn2 0% Cc-Indo 43% Cc-A1AT 40%  180  4.4 Discussion  In Chapter 2 it was demonstrated that recombinant C. crescentus expressing a wide variety of anti-HIV proteins targeting the attachment and entry steps of the viral life cycle were able to prevent infection of both TZM-bl cells and PBMCs from HIV-1 pseudovirus and replication-competent virus infection in vitro. In Chapter 3 it was demonstrated that mucosal application of C. crescentus did not induce the production of inflammatory cytokines or immune cells recruitment to the vaginal tract after topical application of C. crescentus. Taken together these studies suggested that a C. crescentus based microbicide could be safe and effective at preventing HIV-1 infection, and that further studies were warranted. In this chapter, these studies were expanded to examine the ability of the recombinant C. crescentus to prevent vaginal infection with HIV-1 in the humanized BLT mouse model of infection.   Humanized BLT are individually created for each experiment by implanting human fetal liver and thymus tissue under the kidney capsule of immunodeficient mice followed by sublethal irradiation and intravenous injection of CD34+ cells isolated from autologous fetal liver (210, 211). Twelve weeks after tissue transplantation the mice are reconstituted with human immune cells, including CD4+ T cells, CD8+ T cells, B cell, myeloid cells and NK cells (210, 211). Importantly, the mucosal immune system is reconstituted in BLT mice, with cell distribution closely resembling the human vaginal tract (205, 216). BLT mice are the only humanized mouse strain that are susceptible to HIV-1 infection via all major physiological routes, including vaginally and rectally (214, 217, 219, 242). While several different immunodeficient mouse strains have been used to create BLT mice, the NSG strain was used in these experiments. It has been reported that the NSG strain has better immune cell reconstitution and greater susceptibility to vaginal infection with HIV-1 (219). 181   Although BLT mice are a useful small animal model of HIV-1 infection, there are drawbacks to this model. In addition to the cost, time involvement, need for several organ sets of human fetal tissue and a highly skilled mouse surgeon, the immune system that develops in these mice is not completely analogous to a human immune system. There are differences in the types and numbers of human immune cells that develop, which can impact the functionality of the transplanted human immune system. While the BLT mice can be infected mucosally with HIV-1, the virus pathogenesis is not identical to HIV-1 pathogenesis in humans. Furthermore, although the NSG strain is an inbred mouse strain, the transplanted human immune system differs between mice, not only between mice created from different sets of human organs, but also within groups of mice that all receive tissue from the same donor, making the mice diverse. While some mice have sustained HIV-1 viremia, other mice have what appears to be a transient infection with virus only detected at one time post-infection. This may be an artefact of the blood collection timing, as it is restricted to just once every other week due to animal welfare concerns. Regardless, mice that were given HIV-1 alone have detectable virus at at least one time post-infection in 85-92% of the mice, which suggests that even though the mice appear to have “transient” infection, we are still able to detect infection. For the purposes of these experiments, the pathogenesis of HIV-1 infection in the mice is less relevant than the fact that the mice can be infected vaginally with HIV-1.  Several cohorts of BLT mice were created for these experiments. Due to the time involved in creating a BLT mouse, the cost associated with the mice, and the increasing difficulty in obtaining human fetal tissue for research purposes, each BLT mouse needed to be used in the best way possible. In addition to these complications, several issues were encountered within the vivarium that lead to sudden, unexpected death in the mice, seemingly caused by 182  contamination of some cages with Helicobacter. As there were 15 recombinant C. crescentus that were able to prevent HIV-1 infection in vitro, with no indication of in vivo functionality, the testing of the large number of microbicide candidates needed to be optimized. Pilot experiments were conducted with three or four mice per group for each recombinant C. crescentus. If the candidate was able to protect half of the mice in the pilot experiment additional experiments were undertaken. Priority was given to those candidates that provided the highest level of protection in vitro.    HIV-1 infection in the mice was measured in the blood by p24 ELISA. RT-qPCR analysis has recently become the publication standard for HIV-1 infection in BLT mice. RT-qPCR analysis of this data is incomplete. The conclusions presented here take into consideration all data (both p24 ELISA and RT-qPCR) currently available, and the results presented here must therefore be viewed as only preliminary.   Cc-Griffithsin appeared to provide 90% protection from vaginal infection with HIV-1JR-CSF in the BLT mouse. Griffithsin is an anti-viral lectin isolated from the red algae Griffithsia sp, that has demonstrated anti-HIV activity in vitro by binding to sugar moieties present on the surface of the virion and exposing the CD4 binding site on gp120 (339, 356-358, 383-388). When expressed as a recombinant protein in the S-layer of C. crescentus, Griffithsin performed well in in vitro viral blocking assays, with consistent results across a wide variety of viral clades and cell types. Cc-Griffithsin represents an excellent candidate for additional investigation as a microbicide option. Cc-MIP1α is another promising candidate as it appeared to protect 71% of mice from vaginal infection with HIV-1JR-CSF. To fully confirm and support the p24 ELISA results, RT-qPCR analysis should be completed. 183   As young women are disproportionally affected by HIV-1, there is an urgent need to develop HIV-1 prevention options that can be used by women to protect themselves from HIV-1 infection. While PrEP has shown success among intravenous drug users (148), men who have sex with men (149, 152), and serodiscordant heterosexual couples (150), trials with PrEP in African women have shown little efficacy in preventing infection (143, 159). While tenofovir gel was able to provide up to 54% protection from infection among women with high adherence to the BAT24 strategy (2), the VOICE trial using daily application of tenofovir gel was stopped due to futility, suggesting that a daily treatment routine may not be an effective option for HIV-1 prevention (143). While more success was observed with the BAT24 strategy, this relies on a 2-dose strategy per exposure, which may not be feasible. With a C. crescentus based microbicide we propose a strategy that involves dosing prior to sexual activity. Preliminary data indicates that C. crescentus is able to prevent infection up to 8 hours after application, and additional studies are currently ongoing to see if this range can be extended (Appendix C, Figure C.3). In addition, preliminary work in an HSV-2 mouse model suggests that C. crescentus may have some ability to prevent infection when applied after exposure.  While vaginal transmission is responsible for the majority of HIV-1 infections, it is possible that a C. crescentus based microbicide may have the ability to prevent infection by other routes, such as rectal transmission. Preliminary studies with male BLT mice have indicated that rectal transmission of HIV-1 can be prevented by recombinant C. crescentus at similar levels to vaginal prevention (Appendix B, B.4). These studies will be expanded and additional studies will be undertaken to verify that C. crescentus is safe for topical application to the rectum. 184  Chapter 5: Recombinant C. crescentus can provide protection from vaginal infection with Herpes simplex virus 2 5.1 Introduction  Herpes simplex virus 2 (HSV-2) is the major cause of genital herpes (262, 282, 389). HSV-2 has a prevalence of 10-20% in North America and 30-80% in some developing countries and Sub-Saharan Africa, making it one of the most common sexually transmitted infections (252, 256). One in five North American women are infected with HSV-2 compared to one in ten men (390). HSV-2 infection is a major co-morbidity for HIV-1 acquisition. HSV-2 infection increases the risk of HIV acquisition by 2-4 fold (258-261). There are several possible mechanisms that potentially contribute to this increase in HIV-1 infection. HSV-2 creates breaches in the genital epithelium, allowing easier entry of HIV-1 into the body (262, 282, 389). In addition, HSV-2 infection recruits CD4+ and CD8+ T cells to the genital mucosa, creating a state of chronic inflammation, with many HIV-1 target cells present in the vaginal mucosa for several months after infection occurs (259, 262-265, 282, 389).   While condom use and disclosure of serostatus results in a 50% reduction in HSV-2 infection, there is no highly effective method to prevent HSV-2 infection (282). Vaccines developed for HSV-2 have been promising in animal models, but these results have not translated into clinical success (282, 305-308). Complicating matters, HSV-2 infection is life-long, with viral latency in the nervous system and periodic reactivation and virus transmission (252). HSV-2 reactivation can be asymptomatic, allowing transmission to occur in the absence of symptoms (391).   185   Given the lack of effectiveness of HSV-2 prevention options and enhancement of HIV-1 infection that occurs in those that are infected with HSV-2, the development of a dual target prevention option for HIV-1 and HSV-2 could be an efficient means to halt infection of both viruses.   Microbicides that are topically applied to the vaginal tract or rectum are an excellent option for female-controlled prevention of HIV-1 and HSV-2 infection (98). Although over 50 microbicide candidates have been evaluated for HIV-1 prevention and at least 10 for HSV-2 prevention, there are currently no commercially available microbicides for HIV-1 or HSV-2 (98-100). A formulation of 1% tenofovir gel provided 39% protection from HIV-1 infection and 51% reduction in HSV-2 infections using the BAT24 strategy in the CAPRISA 004 trial (2, 302). However, additional testing of daily application of 1% tenofovir gel in the VOICE trial resulted in no protective effect against HIV-1, and the trial was discontinued (382). A microbicide that can provide protection from HIV-1 and HSV-2 and is administered in a single dose using a per exposure strategy may be more successful.   Chapters 2, 3 and 4 describe the development of a microbicide utilizing engineered, C. crescentus for prevention of HIV-1 infection (309, 310, 335). A C. crescentus based microbicide gel would be inexpensive to produce and would rely on a per exposure strategy to prevent infection. We have created 15 different microbicide candidates that significantly reduce HIV-1 infection in vitro, with several of these candidates providing greater than 40% protection from HIV-1 infection in vivo. Seven of these candidates have demonstrated or are expected to be dual-target against both HIV-1 and HSV-2.   Cyanovirin-N, microvirin and Griffithsin are lectin-binding proteins that bind to lectins expressed on enveloped viruses, such as HIV-1. Cyanovirin-N is a virucidal protein isolated 186  from the cyanobacterium Nostoc ellipsosporum (337). Cyanovirin-N has been demonstrated to inhibit HIV-1 infection by interacting with glycans on the envelope of the HIV-1 virion (337, 353, 392). Studies have suggested that Cyanovirin-N is able to block HSV-1 cell entry by interaction with sugar residues on the glycoproteins (393, 394). As HSV-2 has 83% sequence homology with HSV-1 we hypothesized that Cyanovirin-N may also be able to prevent HSV-2 infection. Microvirin is a mannose-specific lectin isolated from Microcystis aeruginosa that binds to similar carbohydrate structures as Cyanovirin-N (338, 355). Microvirin has lower toxicity compared to Cyanovirin-N (338). As microvirin functions similarly to Cyanovirin-N we hypothesized it might also prevent HSV-2 infection. Griffithsin is an anti-viral lectin isolated from the red alga Griffithsia sp that has demonstrated anti-viral activity against HIV-1 and HSV-2 (303, 339, 356-358). Elafin is a member of the whey acidic protein family that has antiproteolytic, immunomodulatory and antimicrobial properties (345). It has been shown that elafin is elevated in cervicovaginal lavage fluid collected from commercial sex workers that are HIV-1 resistant compared to those that are HIV-1 susceptible (374). It has been demonstrated that elafin reduces HSV-2 replication in genital epithelial cells and attenuates clinical scores of mice infected with HSV-2, indicating it may be a suitable candidate for microbicide testing (304). BmKn2 was cloned from scorpion venom and has demonstrated anti-HIV activity, likely by interacting with the viral particle (344). α-1-antitrypsin is a serpin that plays a role in preventing the fusion of HIV-1 with the target cell (348). Indolicidin is an antimicrobial cationic peptide that has been described to have direct anti-viral activity against HIV-1 and HSV-2, thought to be related to its membrane disruptive properties (346, 347).   We therefore tested each of these 7 recombinant C. crescentus for the ability to prevent HSV-2 infection in vivo. Four of the seven recombinant C. crescentus tested were able to prevent 187  HSV-2 infection in vivo. HSV-2 protection seems to be mediated by initiation of an early immune response, with those recombinant C. crescentus that protect the mice having much earlier production of IFNγ, TNF and IL-6 in the presence of HSV-2 when compared to HSV-2 alone.   5.2 Materials and methods Cell lines and viruses - Vero cells were obtained from the American Tissue Culture Collection and maintained in Minimum Essential Media (MEM) supplemented with 7.5% FBS, 5mM sodium pyruvate, 1x MEM non-essential amino acids, 1U/mL penicillin and 1µg/mL streptomycin (Gibco). HSV-2 (strain 333) was a gift from Dr. Charu Kaushic, McMaster University. HSV-2 was propagated and titred on Vero cells. Briefly, Vero cells were grown to 70% confluence then infected with MOI 0.05 HSV-2. Viral supernatant was harvested when CPE reached 60%. Viral stocks were determined by plaque assay using Vero cells grown to confluence in 6 well plates. Virus was serially diluted in serum-free medium and incubated with Vero cells for 1 hour at 37°C with shaking, then 2% agar (Sigma) and 2x MEM (Gibco) supplemented with 10% FBS was added to each well at a 1:1 ratio. Plates were incubated at 37°C for 6 days before being fixed with 25% formaldehyde and stained with 1% Crystal Violet. Viral stocks were stored at -80°C.  Preparation of C. crescentus displaying chimeric S-layer proteins - Gene segments were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa) with codon usage adapted for C. crescentus. The amino acid sequence for each clone is indicated in Chapter 2, Table 2.1. The synthesized DNA segments also specified BglII and SpeI restriction sites on the 5’ side and an NheI site on the 3’ end to facilitate directional cloning into p4BRsaA(723)/GSCC digested 188  with BglII and NheI (181). p4BRsaA(723)/GSCC is identical to p4ARsaA(723)/GSCC except an extra, irrelevant BamH1 site has been removed by digesting with the enzyme, filling in ends with Klenow DNA polymerase and blunt self ligation (174). The 2X indolicidin was made in a fashion similar to other tandem repeat multimer clones (181). Caulobacter crescentus - C. crescentus S-layer display constructs were grown in PYE medium (0.2% peptone, 0.1% yeast extract, 0.01% CaCl2, 0.02% MgSO4) with 2µg/mL chloramphenicol to an optical density at 600 nm of approximately 1 (3 x 109 cells/mL). Cells were centrifuged and suspended in water three times and cell density was adjusted to 5 x 109 cells/mL for experiments. HSV-2 in vivo studies - C57Bl/6 mice were purchased from the Jackson Laboratory and bred and maintained in the Modified Barrier Facility at the University of British Columbia. Female, 6 week old mice were given subcutaneous injection of 2mg Medroxyprogesterone 17-acetate (Sigma) 4-5 days before infection with HSV-2. 108 recombinant C. crescentus cells, prepared as described above, were mixed with 105 plaque forming units (pfu) HSV-2 to a final volume of 20µL and immediately inoculated intravaginally into mice using a sterile p200 pipette tip. Vaginal lavage fluid was collected 24, 48 and 72 hours post-infection by pipetting twice consecutively with 30µL PBS. Vaginal lavage fluid was centrifuged at 400 x g for 5 minutes and the supernatant was stored at -80°C until analysis. Mice were scored daily for disease progression using a 5 point scale: 1 is slight redness of external vagina; 2 is swelling and redness of external vagina; 3 is severe swelling and redness of both vagina and surrounding tissue and hair loss in genital area; 4 is genital ulcerations with severe redness, swelling and hair loss of genital and surrounding tissue; 5 is severe genital ulceration and hind limb paralysis. Mice were euthanized when they reached a score ≥4. 189  Plaque assays - Vero cells were grown to confluence in 6-well plates. Vaginal lavage fluid was diluted in MEM and a limiting dilution was prepared. Diluted lavage fluid was incubated with Vero cells for 1 hour at 37°C with shaking then 2% agar and 2x MEM supplemented with 10% fetal bovine serum, 1U/mL penicillin and 1µg/mL streptomycin was added to each well at a 1:1 ratio. Plates were incubated for 4-6 days at 37°C then fixed with 25% formaldehyde (Sigma). Cells were stained with 1% Crystal Violet (Sigma) and plaques were counted. Cytokine analysis – Female 6 week old C57Bl/6 mice were injected subcutaneously with 2mg Medroxyprogesterone 17-acetate and vaginal lavage was performed as described above 4 days prior to intravaginal inoculation with HSV-2 alone, 108 recombinant C. crescentus alone, or HSV-2 plus C. crescentus. Vaginal lavage fluid was collected at 4, 24 and 48 hours post-inoculation. Vaginal lavage fluid was centrifuged at 400 x g and the supernatant was stored at     -80°C until analysis. 25µL vaginal lavage fluid was analyzed using a Cytometric Bead Array kit for mouse inflammatory cytokines, IFNγ, TNF, IL-6, IL-10, MCP-1, and IL-12p70 (BD Biosciences) according to the manufacturer’s instructions. Data was acquired on an LSRII and analyzed using FlowJo (TreeStar) and Prism. Statistical analysis - Statistical analysis was performed with Prism GraphPad software. As indicated in the text, Student’s t test or ANOVA with Bonferroni’s correction for multiple comparisons were used as appropriate to evaluate the significance of differences between groups. Kaplan-Meier survival curves were compared by log rank tests. A p value of <0.05 was considered significant. Ethics statement - All animal work was performed under strict accordance with the regulations of the Canadian Council for Animal Care. The protocols were approved by the Animal Care Committee of the University of British Columbia (certificate numbers A13-0055 and A12-0245). 190  5.3 Results 5.3.1 Cc-Cyanovirin and Cc-Griffithsin protect from vaginal HSV-2 infection in a mouse model  C. crescentus expressing Cyanovirin-N, microvirin and Griffithsin were shown in Chapter 2 to be able to provide significant protection from HIV-1 infection in vitro (335). Based on published work with HSV-1 and HSV-2 it was hypothesized that these recombinant C. crescentus might also provide protection from HSV-2 infection. To test this a murine model of HSV-2 infection was used. C57Bl/6 mice were infected intravaginally with HSV-2 in the presence of Cc-Control, Cc-Cyanovirin, Cc-Microvirin or Cc-Griffithsin. All mice that received HSV-2 alone reached the humane endpoint by day 11 post-infection (Figure 5.1A). Ten of twelve mice (83%) that were infected with HSV-2 in the presence of Cc-Control reached the humane endpoint by day 9 post-infection (Figure 5.1A). The remaining two mice developed symptoms of HSV-2 infection, between days 6-14 post-infection but never progressed to the humane endpoint, indicating that all mice that received HSV-2 + Cc-Control were infected. Cc-Griffithsin provided significant protection from HSV-2 infection with 4 of 7 mice (57%) protected from HSV-2 infection (Figure 5.1, Table 5.1). Cc-Cyanovirin also protected mice from HSV-2 infection with 3 of 7 mice (43%) protected from HSV-2 infection (Figure 5.1A). However, Cc-Microvirin provided no protection from HSV-2 infection, with 6 of 7 mice reaching the humane endpoint at the same time as mice that received HSV-2 only (Figure 5.1A). Cc-Griffithsin application at the time of HSV-2 infection led to a significant decrease in severity of HSV-2 symptoms beginning at day 9 post-infection and lasting until the experimental endpoint at day 18 (Figure 5.1B). Mice that received Cc-Cyanovirin had a significant delay in developing symptoms of HSV-2 infection up until day 12 post-infection (Figure 5.1B). There 191  was no difference in the development or severity of HSV-2 symptoms between mice that received virus only, Cc-Control and Cc-Microvirin mice (Figure 5.1B).  All recombinant C. crescentus tested, including those that did not prevent development of HSV-2 symptoms, provided a significant but not complete reduction in viral load in the vaginal lavage fluid at day 2 post-infection (Figure 5.1C). Although Cc-Griffithsin and Cc-Cyanovirin are protecting mice from HSV-2 infection and attenuating the severity of herpetic symptoms, these candidates are not completely suppressing viral replication in the vaginal tract. This suggests that they must be providing protection from HSV-2 infection by a different mechanism.  Figure 5.1: Cc-Griffithsin and Cc-Cyanovirin protect from HSV-2 infection A)      DaysPercent survival0 5 10 15 20050100 HSV-2 333HSV-2 + Cc-ControlHSV-2 + Cc-CyanovirinHSV-2 + Cc-MicrovirinHSV-2 + Cc-Griffithsin* * ns ns 192  B)  C)  Figure 5.1 Progesterone treated C57Bl/6 mice were infected intravaginally with 105 pfu HSV-2 strain 333 in the presence or absence of 108 recombinant C. crescentus. Mice were scored daily HSV-2 333HSV-2 + Cc-ControlHSV-2 + Cc-CyanovirinHSV-2 + Cc-MicrovirinHSV-2 + Cc-Griffithsin10-1100101102103104105106107pfu/mL* * * * 193  and euthanized if they reached a score ≥4. A) Survival curve B) Herpetic infection in the mice was scored daily using a 5-point scale. Data is shown as the average for each treatment group C) Vaginal lavage fluid collected on day 2 post-infection was cultured on Vero cells to determine the amount of infectious HSV-2 present. Plaques were counted after 6 days. Each data point represents an individual animal. N=7-12 mice per group. Statistics were performed as two-way ANOVA with Bonferroni’s correction for multiple comparisons or log-rank test as appropriate. *p<0.05, **p<0.01, ***p<0.001  5.3.2 Cc-A1AT and Cc-Indo provide protection from HSV-2 infection  Elafin has been demonstrated to have in vitro and in vivo anti-viral activity against HSV-2 (304) and indolicidin has been demonstrated to reduce HSV-2 infectivity in vitro (346). α-1-antitrypsin is a serpin antiprotease that is a CD4 competitive binding inhibitor for HIV-1 gp120 and an HIV-1 gp41 fusion peptide inhibitor (375). Serpin antiproteases are key players in the regulation of inflammatory responses (375), so α-1-antitrypsin may be able to protect from HSV-2 infection by regulating inflammation in the vaginal tract. The exact anti-viral mechanism of BmKn2 has yet to be elucidated, so it was possible that it may have broad anti-viral activity. These candidates were tested in vivo for ability to protect from vaginal infection with HSV-2.   Cc-A1AT provided significant protection from HSV-2 infection with 6 of 7 mice (86%) protected from infection (Figure 5.2A, Table 5.1). Cc-Indo protected 4 of 7 mice (57%) from HSV-2 infection but this was not statistically significant (Figure 5.2A). Cc-Elafin protected 3 of 7 mice (43%) from HSV-2 infection, however this was not statistically significant (Figure 5.2A). Preliminary experiments indicated that Cc-BmKn2 enhanced HSV-2 infection so additional experiments were not undertaken (Figure 5.2A). There was a slight delay in the development of 194  symptoms and a significant reduction in symptom severity with the mice that received Cc-A1AT compared to HSV-2 alone (Figure 5.2B). From day 10 until the end of the experiment at day 18 there was a significant decrease in HSV-2 scores for mice that received Cc-Indo compared to HSV-2 alone (Figure 5.2B). Although the disease score for mice receiving Cc-Elafin was lower, there was no significant difference between these mice and HSV-2 only mice. There was a significant difference between HSV-2 alone and HSV-2 plus Cc-Control; Cc-Elafin; Cc-A1AT; and Cc-Indo in regards to virus pfu in vaginal lavage fluid (Figure 5.2C). There was no difference between Cc-BmKn2 and HSV-2 alone (Figure 5.2C). As with the anti-viral lectins, this suggests that the recombinant C. crescentus were not protecting from HSV-2 infection by suppressing viral replication in the vaginal tract.  Figure 5.2: Cc-A1AT and Cc-Indo protect from HSV-2 infection A)    DaysPercent survival0 5 10 15 20050100 HSV-2 333HSV-2 + Cc-ControlHSV-2 + Cc-ElafinHSV-2 + Cc-BmKn2HSV-2 + Cc-A1ATHSV-2 + Cc-Indo*** ns ns ** 195  B)  C)  Figure 5.2 Progesterone treated C57Bl/6 mice were infected intravaginally with 105 pfu HSV-2 strain 333 in the presence or absence of 108 recombinant C. crescentus. Mice were scored daily HSV-2 333HSV-2 + Cc-ControlHSV-2 + Cc-ElafinHSV-2 + Cc-BmKn2HSV-2 + Cc-A1ATHSV-2 + Cc-Indo100101102103104105106107pfu/mL* * * * 196  and euthanized if they reached a score ≥4. A) Survival curve B) Herpetic infection in the mice was scored daily using a 5-point scale. Data is shown as the average for each treatment group. C) Vaginal lavage fluid collected on day 2 post-infection was cultured on Vero cells to determine the amount of infectious HSV-2 present. Plaques were counted after 6 days. Each data point represents an individual animal. N=7-12 mice per group. Statistics were performed as two-way ANOVA with Bonferroni’s correction for multiple comparisons or log-rank test as appropriate. *p<0.05, **p<0.01, ***p<0.001, #p<0.0001  5.3.3 Protective C. crescentus induce early cytokine production in vaginal tract  Given that viral loads were similar between the Cc-Control and each recombinant C. crescentus after HSV-2 infection, it was hypothesized that the recombinant C. crescentus were inducing different inflammatory cytokine profiles in the vaginal tract to protect or lower HSV-2 infection rates or severity. Mice were inoculated intravaginally with HSV-2 alone, recombinant C. crescentus alone, or HSV-2 plus recombinant C. crescentus. Vaginal lavage fluid was collected 4 days prior to inoculation then at 4, 24 and 48 hours post-inoculation and analyzed by cytometric bead array for mouse inflammatory cytokines. Cc-A1AT was analyzed because it was the most successful at preventing HSV-2 infection, Cc-Griffithsin because it protected approximately half of the mice from HSV-2 infection, Cc-Control because it did not protect from HSV-2 infection and Cc-BmKn2 because it seemed to enhance HSV-2 infection.  At 24 hours post-infection, mice that received HSV-2 plus Cc-A1AT had significantly more IFNγ in the vaginal lavage compared to mice that were infected with HSV-2 only (Figure 5.3A, Table 5.1). In comparison, although HSV-2 plus Cc-Griffithsin had elevated IFNγ in the vaginal tract at 24 hours post-infection, this was not significantly greater than HSV-2 only, and 197  about a third the amount of IFNγ that the HSV-2 plus Cc-A1AT mice produced (Figure 5.3A). Furthermore, mice that received HSV-2 plus Cc-BmKn2 had no increase in IFNγ in the vaginal tract at 24 hours post-infection (Figure 5.3A). At 4 hours post-infection, all recombinant C. crescentus tested caused a significant increase in the amount of TNF present in the vaginal lavage when given at the same time as HSV-2 (Figure 5.3B). Cc-A1AT and Cc-Griffithsin produced similar levels of TNF. This was about a third more than Cc-Control and Cc-BmKn2 and ten times more than HSV-2 alone. HSV-2 plus Cc-A1AT produced significantly more IL-6 at 4 hours post-infection than HSV-2 alone (Figure 5.3C). HSV-2 plus Cc-Griffithsin also produced significantly more IL-6 at 4 hours post-infection compared to virus alone (Figure 5.3C). This was about 20% less IL-6 than that produced by HSV-2 plus Cc-A1AT. Although HSV-2 plus Cc-BmKn2 did cause a slight elevation in IL-6 at 4 hours post-infection, this was not statistically significant (Figure 5.3C). Cc-A1AT alone did not produce significantly more IFNγ, TNF or IL-6 in the vaginal tract compared to Cc-Control (Figure 5.3). There was no significant difference between levels of IL-12p70, IL-10 and MCP-1 for any of the combinations at any of the time points (data not shown). Taken together, these results suggests that an earlier cytokine response was protective for HSV-2 infection and that Cc-A1AT protects mice from HSV-2 infection by inducing this earlier cytokine response in the vaginal tract during HSV-2 infection, with IFNγ production 24 hours earlier, TNF about 20 hours earlier, and IL-6 produced 2 days earlier than HSV-2 alone.     198  Figure 5.3: Protective C. crescentus induce early cytokine production in the vaginal tract  A1AT IFNgammapg/mlBaseline4 hours24 hours48 hours0100200300400500HSV-2Cc-ControlCc-A1ATHSV-2 + Cc-ControlHSV-2 + Cc-A1ATGriff IFNgammapg/mlBaseline4 hours24 hours48 hours0100200300400500HSV-2Cc-ControlCc-GriffithsinHSV-2 + Cc-ControlHSV-2 + Cc-GriffithsinBmKn2 IFNgammapg/mlBaseline4 hours24 hours48 hours0100200300400500HSV-2Cc-ControlCc-BmKn2HSV-2 + Cc-ControlHSV-2 + Cc-BmKn2A1AT TNFBaseline4 hours24 hours48 hours0100200300400500pg/mlHSV-2Cc-ControlCc-A1ATHSV-2 + Cc-ControlHSV-2 + Cc-A1ATGriff TNFpg/mlBaseline4 hours24 hours48 hours0100200300400500HSV-2Cc-ControlCc-GriffithsinHSV-2 + Cc-ControlHSV-2 + Cc-GriffithsinBmKn2 TNFpg/mlBaseline4 hours24 hours48 hours0100200300400500HSV-2Cc-ControlCc-BmKn2HSV-2 + Cc-ControlHSV-2 + Cc-BmKn2A B * * **** *** ** **** ** *** ** ** ** **** **** ** ** 199   Figure 5.3 Progesterone treated C57Bl/6 mice were infected intravaginally with 105 pfu HSV-2 A1AT IL6pg/mlBaseline4 hours24 hours48 hours050100150Cc-ControlHSV-2Cc-A1ATHSV-2 + Cc-ControlHSV-2 + Cc-A1ATGriff IL6pg/mlBaseline4 hours24 hours48 hours050100150HSV-2Cc-ControlCc-GriffithsinHSV-2 + Cc-ControlHSV-2 + Cc-GriffithsinBmKn2 IL6Baseline4 hours 24 hours 48 hours0100200300pg/mlHSV-2Cc-ControlCc-BmKn2HSV-2 + Cc-ControlHSV-2 + Cc-BmKn2C **** ** *** ** *** 200  strain 333 in the presence or absence of individual recombinant C. crescentus Cc-Control, Cc-BmKn2, Cc-Griffithsin or Cc-A1AT. Vaginal lavage fluid was collected prior to infection then 4, 24 and 48 hours post-infection and analyzed in a cytometric bead array for mouse inflammatory cytokines. A) IFNγ B) TNF C) IL-6. N=4-11 mice per group. Statistics were performed using two-way ANOVA with Bonferroni’s correction for multiple comparisons. *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001.  Table 5.1: HSV-2 summary data Name % Protected from HSV-2 infection PFU/mL in vaginal fluids (mean) Score at experiment end (mean) Notes HSV-2 0% 5.9x105 4 IFNγ produced at 48 hours; TNF produced at 24 hours; IL-6 produced at 48 hours HSV-2 + Cc-Control 0% 5.4x104 3 IFNγ produced at 48 hours; significant TNF production at 4 hours (33% less than HSV-2 + Cc-A1AT); significant IL-6 production at 4 hours  201  Name % Protected from HSV-2 infection PFU/mL in vaginal fluids (mean) Score at experiment end (mean) Notes HSV-2 + Cc-Cyanovirin 43% 4x104 2.6  HSV-2 + Cc-Microvirin 14% 1.2x105 3.4  HSV-2 + Cc-Griffithsin 57% 3.0x103 1.7 IFNγ produced at 24 hours (66% less than HSV-2 + Cc-A1AT); significant TNF production at 4 hours (10x more than HSV-2); significant IL-6 production at 4 hours (20% less than HSV-2 + Cc-A1AT) HSV-2 + Cc-Elafin 43% 1.5x104 2.3  HSV-2 + Cc-A1AT 86% 9.4x104 1 Significant IFNγ production at 24 hours; significant TNF production at 4 hours ( 202  Name % Protected from HSV-2 infection PFU/mL in vaginal fluids (mean) Score at experiment end (mean) Notes HSV-2 + Cc-A1AT (cont’d)    33% less than HSV-2 + Cc-A1AT); significant IL-6 production at 4 hours HSV-2 + Cc-Indo 57% 5.8x103 1.7  HSV-2 + Cc-BmKn2 0% 3.3x103 4 IFNγ produced at 48 hours; significant TNF production at 4 hours; low IL-6 production at 48 hours  5.4 Discussion  Women are disproportionally affected by sexually transmitted infections. While approximately half of all new HIV-1 infections worldwide occur in women, 2 out of 3 new HIV-1 infections that occur in Sub-Saharan Africa are in women (390). Furthermore, the prevalence for HSV-2 infection is 1 in 5 for women compared to 1 in 10 for men (390). Although condom use can provide a measure of protection against HSV-2 and HIV-1 infection, it is often difficult for women to negotiate condom use, because it relies on the consent of a male partner. This suggests that female-controlled prevention options for sexually transmitted infections are urgently needed. Since HSV-2 and HIV-1 are major co-morbidities, a dual-target prevention 203  option is ideal.  HSV-2 infection is a major risk factor for HIV-1 acquisition, leading to a 2-4 fold increase in HIV-1 infection (258). HSV-2 is a lifelong infection, with periodic outbreaks in the absence of symptoms, leading to herpes transmission and increased susceptibility to HIV-1 infection (391). No effective prevention option for HSV-2 currently exists. The use of condoms, disclosure of serostatus and anti-viral therapy are 50% effective at preventing HSV-2 infection (282). The antiviral drugs acyclovir and valacyclovir can prevent the virus from multiplying and lead to a shorter outbreak, but do not prevent future outbreaks (258). Several attempts have been made to develop a vaccine against HSV-2, with no reports of success (282, 305-308).  HIV-1 infection impairs immunity to HSV-2 with HIV-1 positive women having impaired mucosal immunity to HSV-2 infection and diminished anti-HSV activity of cervico-vaginal secretions (262). The depletion of CD4+ T cells by HIV-1 leads to an impaired HSV-2 response (262). There is currently no vaccine for HIV-1, and prevention efforts focused on condom use have not been successful in protecting women from HIV-1 infection. A dual-target microbicide could have a major impact on women’s sexual health.  Microbicides represent a promising option for female-controlled prevention. These are topical products that can be applied in advance of sexual activity without the knowledge or consent of a partner. Many microbicides have been under development for prevention of HIV-1 or HSV-2, but few have had success in clinical settings, and none have been specifically designed for dual prevention. 1% Tenofovir gel did provide 39% protection from HIV-1 infection and had the unexpected benefit of providing 51% protection against HSV-2 in the CAPRISA 004 trial (2, 302), but had no success in HIV-1 prevention in the VOICE trial (143, 302). By focusing all microbicide efforts on one option we are limiting our potential prevention 204  opportunities.    In Chapters 2, 3 and 4 we have described the development of an HIV-1 specific microbicide using C. crescentus S-layer mediated display of anti-HIV-1 proteins. Seven of the C. crescentus candidates have been demonstrated or are expected to prevent HSV-2 infection. In this chapter it is demonstrated that C. crescentus expressing α-1-antitrypsin, Griffithsin, cyanovirin-N and indolicidin are able to provide protection from vaginal infection with HSV-2. This protection seemed to be independent of viral load as C. crescentus alone was able to induce a significant reduction in HSV-2 virions in the vaginal tract. When cytokine production in the vaginal tract after inoculation with HSV-2 in the presence or absence of recombinant C. crescentus was measured, it was found that the protective C. crescentus induced an earlier production of IFNγ, TNF and IL-6 compared to virus alone. In particular, the amount of IFNγ produced at 24 hours post-infection seemed to correlate with protection, with Cc-A1AT producing 15 times more IFNγ than virus alone and protecting 86% of mice, Cc-Griffithsin producing 10 times more IFNγ and protecting 57% of mice, and Cc-BmKn2 providing no protection and producing twice the amount of IFNγ.   During an HSV-2 infection the virus will trigger pattern recognition receptors in the vaginal tract, leading to production of inflammatory cytokines and eventual recruitment of immune cells to control viral infection. The cytokine data presented in Figure 5.3 indicates that HSV-2 infection produces a slow rise in levels of IL-6 and TNF post-infection. The levels of these cytokines are first detected at 4 hours post-infection and slowly increase until 48 hours, which is the last time measured. IFNγ production is not detected until 48 hours post-infection. In contrast, HSV-2 infection in the presence of Cc-A1AT leads to a rapid and substantial increase in these cytokines within a day following infection. This suggests that the immune response to 205  HSV-2 infection begins 1-2 days earlier when Cc-A1AT is present. This cytokine response should cause an earlier recruitment of immune cells to the vaginal tract of the mice, and these immune cells are able to respond to and contain the viral infection before the virus has the chance to cause pathology. It would be interesting to harvest the vaginal tissue at various time points post-infection to determine if immune cells appear earlier in the presence of Cc-A1AT.   Both HIV-1 and HSV-2 cause significant morbidity each year. By preventing HSV-2 infection we may be able to prevent numerous HIV-1 infections. A microbicide that is targeted to two sexually transmitted viruses could have a major impact on the HIV-1 epidemic, and could prevent HSV-2 infections, having an enormous impact on public health.   206  Chapter 6: Conclusions and future directions 6.1 Discussion  Despite effective prevention options, 2 million new HIV-1 infections occur annually (1). The majority of these new infections occur in women, with young women residing in developing countries disproportionally affected by HIV-1 infection (2). Although condoms can protect from HIV-1 infection, this has not had an impact on preventing HIV-1 infection among women, particularly in Sub-Saharan Africa where 2 out of every 3 new HIV-1 infections occur in women (390). Due to societal norms, gender inequalities, and a lack of access to prevention services, many women are unable to use condoms, suggesting that female-controlled prevention options for HIV-1 are urgently needed.   A major risk factor for HIV-1 acquisition is existing sexually transmitted infections, in particular HSV-2 (258-261, 390). HSV-2, the common cause of genital herpes can increase the risk of HIV-1 acquisition in several ways. HSV-2 is a life-long infection, with phases of latency and active replication (252). Active infection is not always accompanied by the development of symptoms, but the virus can still be transmitted during this time (391). It has been estimated that HSV-2 infected people can be shedding the virus up to 28% of the time with no clinical symptoms present (282). Both clinical and subclinical HSV-2 infection can create breaches in the vaginal epithelium that make it easier for HIV-1 to enter the body (258, 262, 282). In addition, HSV-2 leads to immune cell recruitment to the vaginal tract that can be maintained for months following the resolution of an HSV-2 outbreak, facilitating HIV-1 infection (258, 259, 262-265, 282). While condoms can provide a measure of protection against HSV-2 infection, there is no effective method to prevent or treat HSV-2 infection (282).  207   While a single-dose vaccine strategy for prevention of HIV-1 infection in combination with a vaccine to prevent HSV-2 infection would be an ideal option for prevention, vaccine strategies have not yet been effective for either virus. HIV-1 is a diverse, rapidly mutating RNA virus that infects cells of the immune system, primarily via mucosal routes (53, 54, 162). While close to 200 different HIV-1 vaccines have been tested, only 5 candidates have progressed to clinical efficacy testing (162). Only one vaccine candidate, the RV144 vaccine, has been able to provide 39% protection from HIV-1 infection (169). However, protection from infection seems to be transient, with many vaccinees having rapidly decreasing levels of protection after one year (169). Some HSV-2 vaccines have been effective in animal models but these results have not translated to clinical success (282, 305-308, 389).  With the lack of success in developing a vaccine for HIV-1, scientists have turned to alternative options for HIV-1 prevention. Building from the successful prophylaxis campaigns for malaria (395), and efficacy of post-exposure prophylaxis for HIV-1 (396-401), it was proposed that prophylactic use of antiretrovirals may be an effective method to prevent HIV-1 infection (146, 147). In this strategy HIV-1 negative people that are at high risk of HIV-1 infection take daily ART to prevent HIV-1 infections before the virus can be established (148-157). The ART tenofovir was tested alone or in combination with emtricitabine for PrEP. It was found that the combination of TFV + FTC was more effective at preventing HIV-1 infection than TFV alone (149-151, 154, 155). In intravenous drug users (148), men that have sex with men (149, 152) and serodiscordant heterosexual couples (150, 151) PrEP was able to significantly reduce the risk of HIV-1 infection. However, two trials conducted among African women had no protection from HIV-1 infection (143, 159), indicating that daily PrEP may not be a suitable option in some populations. While the ANRS IPERGAY study has suggested that PrEP does not 208  need to be taken every day to be effective, this trial was conducted among men who have sex with men in Canada and France, and it is not yet known if the results can be translated to African women (152).   While PrEP is successful at preventing HIV-1 infection in a clinical trial setting, there are several concerns with widespread use of PrEP, primarily related to the need for high adherence and frequent HIV-1 testing (105). As those taking PrEP will be on prolonged mono- or dual-therapy, there is a concern that if HIV-1 infection does occur, the virus will be able to develop resistance to these drugs rapidly, which can be problematic for treatment options (129). Finally, adverse events ranging from diarrhea and nausea to kidney dysfunction have been reported among PrEP users in trials (143, 148, 149, 151, 152, 159). The mild side effects such as diarrhea may lower adherence, whereas the more severe side effects could cause long-term harm to health (402).    Microbicides are an alternate option for female controlled prevention of HIV-1. These are drug products that can be topically applied to the vaginal tract or rectum to prevent infection (98). There have been many strategies undertaken to develop microbicides to prevent HIV-1 infection. Initial microbicide studies used spermicides or other non-specific gels to attempt to prevent HIV-1 infection, but in most cases HIV-1 infection increased (117-122). Entry inhibitors (101-106), reverse transcriptase inhibitors (2, 107-113, 143), integrase inhibitors (114), and protease inhibitors (115, 116) have all been investigated as microbicides with varying levels of success. Some groups have focused on developing bacteria-based strategies using commensal vaginal bacteria (123-126, 139, 379), and others on developing current ART options into gel formulations (2, 143). Some microbicides with acyclovir to prevent HSV-2 infection in combination with HIV-1 prevention have been investigated (403).  209   There have been varying levels of success in HIV-1 prevention using microbicides. Few candidates have made it to clinical efficacy testing, with mixed results. While 39% protection was observed in the CAPRISA 004 trial using a 1% tenofovir gel following a before and after sex dosing strategy (2), the tenofovir gel arm of the VOICE trial was stopped early due to futility (143, 377, 382). The VOICE trial focused on daily application of the gel and adherence to this strategy seems to have been problematic (143).  As an alternative, we proposed the development of a bacteria based microbicide that would be applied using a per exposure strategy to prevent HIV-1 infection (309, 335). The aim of this work was to develop a microbicide to prevent infection with both HIV-1 and HSV-2. We used the non-pathogenic, freshwater bacterium C. crescentus for microbicide development (170, 171). There are several features of C. crescentus that make it an excellent candidate for microbicide development. C. crescentus is not able to grow in humans, due to both the temperature and salt concentration present in the human body (172, 173), which means that C. crescentus would be easy to eradicate in the event of unwanted side effects. In addition, work by collaborators has indicated that C. crescentus does not induce any adverse events when injected intraperitoneally into mice, suggesting that C. crescentus will be safe for microbicide development (172). The most important feature of C. crescentus is that it expresses a Surface or S-layer at very high levels (174-178). The S-layer is composed of up to 40,000 copies of a single protein, RsaA, that coats the surface of the bacterium (171, 174-178). The S-layer is easily modified to express a wide variety of recombinant proteins (171, 176, 179, 181, 182). The insertion of these recombinant proteins into the S-layer generally has minimal impact on expression of the S-layer, and the inserted proteins typically retain their function (171, 176, 179, 210  181, 182). This leads to high-density display of a recombinant protein on the surface of the bacterium, which has enormous potential for biotechnological applications.   6.2 C. crescentus expressing anti-HIV proteins provides significant protection from HIV-1 infection in vitro  In Chapter 2 we described the creation of 18 different recombinant C. crescentus that expressed proteins that are able to interfere with all aspects of the HIV-1 attachment and entry process (Figures 2.1 and 2.2), including decoy receptors and ligands, anti-viral lectins, and fusion inhibitors. These recombinant C. crescentus were tested in vitro to determine if they could prevent infection with HIV-1 env pseudotyped virus. The pseudovirus allows rapid efficacy testing with a wide variety of HIV-1 clades. Levels of protection tended to be consistent across clades B and C (Figures 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10). Preliminary testing with pseudovirus from virus clades A and D (Appendix A, Figure A.2) demonstrated similar results. Pseudovirus testing eliminated three recombinant C. crescentus candidates that were unable to prevent HIV-1 infection (Figures 2.8, 2.11).   While the pseudovirus is an excellent screening tool, it does not fully recapitulate all aspects of an HIV-1 infection, so additional in vitro experiments were undertaken using replication competent HIV-1. The fifteen remaining candidates underwent efficacy testing with live HIV-1 and either the TZM-bl cell line or human PBMCs, as appropriate. Results from the pseudovirus assays were generally maintained with the live viruses and PBMCs (Figures 2.12, 2.13). Although some recombinant C. crescentus had no efficacy with certain viruses (ex. Cc-T1249 with HIV-1JR-FL in PBMCs, Figure 2.13D), all fifteen candidates provided some measure of protection from live HIV-1 infection in vitro.  211   As HIV-1 is a rapidly mutating virus, there is always the risk that resistance to a candidate could occur. Since a recombinant C. crescentus based microbicide aims to prevent HIV-1 infection before entry into a target cell occurs, it seems unlikely that resistance will develop because the virus will not have the opportunity to replicate in the presence of the microbicide. However, given that there was some variations between HIV-1 subtypes with each candidate, and that no candidate provided 100% protection from infection in vitro, a microbicide arising from this research should contain several different recombinant C. crescentus that target different steps of the attachment and entry process. Thus, the microbicide will have several opportunities to prevent infection. If a transmitted virus is resistant to, for example, Fuzeon, there are other candidates, such as an anti-viral lectin, present in the microbicide to prevent infection. We have previously demonstrated that this strategy is able to increase protection from HIV-1 infection (309). By combining the individual C. crescentus Cc-MIP1α and Cc-CD4 protection from infection was increased from 35-50% to 97% (309). Preliminary experiments have investigated two different strategies for combination prevention. In the first, two different recombinant proteins are expressed for every 1 RsaA protein of C. crescentus. In the second strategy two individual C. crescentus each expressing a different recombinant protein are combined. Preliminary studies have indicated that two individual C. crescentus combined are better at preventing infection compared to two recombinant proteins expressed on the same bacterium (Appendix C, Figure C.1). In addition, preliminary experiments have supported the original work with Cc-MIP1α and Cc-CD4, demonstrating that combination of two different recombinant C. crescentus increases protection from HIV-1 infection beyond what is observed for each alone (Appendix C, Figure C.2).  212  6.3 C. crescentus appears safe for topical application to the vaginal tract  The work presented in Chapter 2 indicated that we had developed a promising candidate microbicide for HIV-1 prevention. However, as C. crescentus is a gram-negative bacterium, it was possible that it would induce an immune response in the vaginal tract, which could enhance HIV-1 infection by recruiting immune cells to the vaginal tract in advance of HIV-1 exposure. While work by collaborators indicated that C. crescentus had no adverse effects following intraperitoneal injection into mice (172), the vaginal immune response to C. crescentus has not been investigated.   In Chapter 3 we applied C. crescentus (Cc-Control), which contains the restriction sites that are used for cloning but no inserted protein in the S-layer, to the vaginal tract of progesterone treated C57Bl/6 mice and examined the response. Vaginal lavage at 4, 24 and 48 hours post-application was performed and the lavage fluid was analyzed for mouse inflammatory cytokines (Figure 3.2). While C. crescentus did induce low production of TNF at 4 hours post-application, this was not significantly more TNF than that produced when PBS was applied, and significantly less TNF produced after application of LPS from a pathogenic bacteria, Salmonella enterica serotype Minnesota. C. crescentus did not induce the production of any other inflammatory cytokine tested. Histology of the vaginal tissue at 4 and 48 hours post-application of C. crescentus as well as analysis of the cellular portion of vaginal lavage fluid, did not indicate any recruitment of immune cells (Figures 3.3 and 3.4). Vaginal fluid and serum was analyzed after both single and bi-weekly application of Cc-Control. In both situations there was no production of antibodies against whole C. crescentus in the vaginal fluid, measured by ELISA (Figure 3.5). While a positive signal was observed on the ELISA plates for the serum after vaginal application, this signal was present at similar levels in all mice, including mice that were 213  taken directly from the breeding colony with no experimental exposure to C. crescentus, suggesting that this effect was non-specific.   Taken together these results suggested that C. crescentus may be safe for topical application to the vaginal tract. While we have been utilizing live C. crescentus in these studies, this is not necessary for protection from HIV-1 infection. We have previously demonstrated that heat-inactivated C. crescentus were able to provide a similar level of protection from HIV-1 infection in vitro compared to live C. crescentus (309). This indicated that inactivating the bacteria does not impact efficacy, which should further increase the safety of C. crescentus for topical use.  6.4 Some recombinant C. crescentus provide protection from vaginal infection with HIV-1JR-CSF in the humanized BLT mouse  Chapters 2 and 3 have demonstrated that a recombinant C. crescentus based microbicide could be a safe and effective strategy to prevent HIV-1 infection. While in vitro testing is an excellent option to determine efficacy of a candidate, it does not fully capture the complex environment present in the vaginal tract, which could impact the ability of a candidate to prevent HIV-1 infection. As such, we established a colony of humanized BLT mice to undertake in vivo testing of the recombinant C. crescentus from Chapter 2. BLT mice contain a functional human immune system and are susceptible to vaginal infection with HIV-1, making them an excellent preclinical model for testing HIV-1 microbicides.  BLT mice were exposed vaginally to HIV-1JR-CSF in the presence of each different recombinant C. crescentus. Cc-Griffithsin was able to provide 90% protection from vaginal infection with HIV-1, and Cc-MIP1α provided 71% protection from infection (Figures 4.2 and 214  4.4C). While many of the other recombinant C. crescentus were able to protect approximately half of the mice from HIV-1 infection (Figures 4.3D, 4.4B, 4.5B, 4.5C, 4.6, 4.8, 4.9), some provided minimal protection (Figures 4.3A-C, 4.4A, 4.7, 4.10). Interestingly, some of the candidates did not prevent infection but did lead to a delay in developing detectable viremia in the mice. It would be interesting to more closely monitor HIV-1 infection in these mice to determine how these recombinants are delaying infection.  While HIV-1JR-CSF and the recombinant C. crescentus were premixed immediately before inoculation for technical reasons, this is not analogous to a “real world” situation. When in use, it is more likely that the recombinant C. crescentus would be applied in advance of HIV-1 exposure. As C. crescentus is unlikely to survive long term in the competitive vaginal microenvironment, this means that the microbicide will need to be reapplied for protection to be maintained. We have been attempting to determine how long C. crescentus is present in the vaginal tract, but have encountered difficulties retrieving C. crescentus from the vagina after application. We do not know if this is due to the fact that there is no live bacteria to recover, or if the recombinant C. crescentus are not removed by lavaging. When a vaginal lavage is performed on mice within an hour of application of C. crescentus and the lavage fluid is plated on PYE-agar plates, less than 5 C. crescentus colonies have been detected (C Farr, unpublished). As it is unlikely that one hour is sufficient to kill the applied C. crescentus, since the bacteria can survive for at least 24 hours at 37°C in a cell culture incubator (C Farr, unpublished), and can be recovered and cultured after 5 days in the peritoneal cavity (172), we suspect that we are unable to collect the bacteria by lavage. We have recently created recombinant C. crescentus that express the mCherry fluorophore in the S-layer. We plan to inoculate these bacteria into the 215  vaginal tract of mice and harvest vaginal tissue at various time points post-application for examination by fluorescence microscopy to determine if the C. crescentus are still present.  In addition to these in vivo assays, we have been undertaking in vitro viral blocking assays to determine how long the recombinant C. crescentus are able to provide protection from HIV-1 infection. For these experiments the C. crescentus is incubated with the TZM-bl cells for various times before addition of HIV-189.6. Preliminary data indicated that the recombinant C. crescentus were able to provide protection from infection when added 8 hours before viral exposure, with minimal change in efficacy (Appendix C, Figure C.3).    6.5 Recombinant C. crescentus can prevent infection with HSV-2  HSV-2 is a major risk factor for HIV-1 infection. By preventing both HSV-2 and HIV-1 infection, a microbicide could have enormous impact on global public health. Several of the candidates for the HIV-1 microbicide have been suggested to be able to prevent infection with HSV-2. In Chapter 5 we investigated the ability of any potential broad-spectrum inhibitors to prevent infection with HSV-2. Cc-A1AT, Cc-Griffithsin, Cc-Elafin, Cc-Indo and Cc-Cyanovirin were able to prevent vaginal HSV-2 infection in a mouse model, with Cc-A1AT protecting 86% of the mice from HSV-2 infection (Figures 5.1 and 5.2). Interestingly, when the vaginal lavage fluid was analyzed by plaque assay for the number of infectious particles in the vaginal tract, it was found that all C. crescentus, including Cc-Control, induced at least a 1 log decrease in the plaque forming units in the vaginal tract (Figures 5.1, 5.2). This suggested that C. crescentus alone has some ability to lower viral replication of HSV-2 in the vaginal tract, but that this was not sufficient to protect the mice from developing symptoms of infection, since all Cc-Control 216  mice developed symptoms of HSV-2 infection, whereas not all recombinant C. crescentus mice did.   We decided to investigate the vaginal lavage fluid for inflammatory cytokines in mice that received Cc-Control, Cc-A1AT, Cc-Griffithsin or Cc-BmKn2 in the presence or absence of HSV-2 (Figure 5.3). We found the Cc-A1AT, the best candidate at protecting the mice from HSV-2 infection, induced earlier and greater amounts of TNF, IL-6 and IFNγ in the vaginal tract during HSV-2 infection compared to HSV-2 alone. Cc-Griffithsin was able to protect approximately half of the mice from HSV-2 infection. In the presence of HSV-2, Cc-Griffithsin induced increased levels of these same cytokines in the vaginal lavage, but not to the same level as Cc-A1AT. Cc-Control and Cc-BmKn2 did not significantly change the cytokine profile in the vaginal tract following HSV-2 infection. This suggested that Cc-A1AT was protecting the mice from infection by inducing an earlier immune response in the vaginal tract. This earlier cytokine response likely recruits immune cells to the vaginal tract and these cells may be able to clear the HSV-2 infection before it can be established.   It would be interesting to see if HSV-2 still undergoes latency in the mice that were protected from HSV-2 infection. While one group has described the detection of HSV-2 from the spinal cord of infected mice (304), we have not been able to detect HSV-2 in the spinal cord. It has been demonstrated that viral latency and spontaneous reactivation occurs in the guinea pig model of HSV-2 infection (404, 405), so it would be interesting to repeat these experiments in guinea pigs to determine if protection from primary infection prevents HSV-2 from undergoing latency.   217  6.6 Conclusion and future direction  The aim of this thesis was to develop a microbicide to prevent infection with HIV-1 and HSV-2. We have described the development and testing of a recombinant C. crescentus based microbicide in vitro and in vivo. The anti-viral lectin Griffithsin (Cc-Griffithsin) appears to be the best candidate as it was able to prevent 90% of infections with HIV-1 and 57% of infections with HSV-2. Cc-MIP1α provided 71% protection from vaginal infection with HIV-1JR-CSF in the humanized mouse model, and was not tested against HSV-2 because it is not anticipated to have any ability to prevent HSV-2 infection. While the serpin α-1-antitrypsin (Cc-A1AT) was able to provide 50% protection from HIV-1 infection, it could provide 86% protection from HSV-2 infection, making it the best candidate tested against HSV-2. As the final microbicide product would be expected to contain more than one candidate inhibitor, a microbicide containing Cc-Griffithin, Cc-A1AT and Cc-MIP1α would likely be the most effective at preventing infection with both HIV-1 and HSV-2.  Ideally, a C. crescentus based microbicide would be manufactured as a vaginal ring that could provide long-term (1+ week) protection from HIV-1 and HSV-2 infection. It is possible that a gel-based C. crescentus microbicide could be combined with personal lubricants, spermicides or condoms to provide greater protection from HIV-1 and HSV-2 infections.  While the research presented in Chapters 2, 3, 4 and 5 suggest that a C. crescentus based microbicide may be a safe and effective method to prevent HIV-1 and HSV-2 infection, several unanswered questions remain.  6.6.1 How long does protection last?  In the in vitro viral blocking assays described in Chapter 2 the recombinant C. crescentus was incubated with HIV-1 (cells for Cc-MIP1α) for 1 hour before addition of the cells (HIV-1 218  for Cc-MIP1α). This incubation allowed time for the recombinant C. crescentus to interact with its’ target before infection could occur. Due to the need for prolonged anaesthesia to perform intravaginal infection of mice, and the small size of the mouse vaginal tract, this timing of recombinant C. crescentus application and viral exposure was modified in Chapters 4 and 5. For in vivo experiments the recombinant C. crescentus was mixed with the virus then immediately applied to the vaginal tract. While this experimental design eliminated the difficulties with performing intravaginal infection of mice, it is unlikely to recapitulate the “real world” use of a microbicide. In addition, these experiments do not investigate the duration of protection. While the VOICE trial indicated that adherence to a daily microbicide regimen is low, both the CAPRISA 004 microbicide trial and the ANRS IPERGAY PrEP trial suggested that an “on demand” strategy may have greater adherence and thus be more successful at preventing infection (2, 143, 152). While we anticipate a C. crescentus based microbicide would be used in an “on demand” strategy, it would be useful to know how frequently application should occur. For example, would application of the microbicide provide protection on the day of exposure and the following day? Would 1-2 uses of the microbicide per week be sufficient for week-long protection from infection?  We are currently performing in vitro experiments to investigate the duration of protection from infection. In these experiments C. crescentus is incubated with the TZM-bl cells for increasing lengths of time before addition of HIV-1. Preliminary experiments (Appendix C, Figure C.3) indicate that protection from infection is maintained at a similar level for 8 hours post-application. Additional experiments are planned that will increase this time to 12 and 24 hours.  219   Once the in vitro experiments provide a time frame of protection these results will be translated into a preclinical model. While the in vitro experiments are being undertaken with HIV-1, the in vivo experiments will use HSV-2, primarily because C57Bl/6 mice, the mouse model of HSV-2, are readily available while humanized BLT mice are not. Despite this switch in virus, we believe this will not impact the results. For these experiments progesterone treated C57Bl/6 mice will be given the recombinant C. crescentus prior to HSV-2 infection, then the mice will be monitored daily for development of herpetic lesions.  6.6.2 Will the microbicide provide protection during co-infections?  The aim of this project was to develop a microbicide to prevent infection with HIV-1 and HSV-2. In Chapters 4 and 5 we demonstrated in vivo protection from HIV-1 alone or HSV-2 alone. We have not investigated protection from simultaneous viral exposure, as well as protection from HIV-1 infection in the presence of an active HSV-2 infection, or protection from HSV-2 infection in an HIV-1 infected mouse. There are several difficulties inherent with these experiments, including rapid progression of HSV-2 infection in mice and the need for humanized mice for the HIV-1 experiments. To overcome these difficulties other groups have developed two transgenic mouse models.  A transgenic mouse model has been described that is transgenic for HIV-1 provirus and human cyclin T1 under the control of the mouse CD4 promoter/enhancer (406). The CD4+ cells in these mice produce infectious HIV-1JR-CSF (406). These mice have been infected intravaginally with HSV-2 (407). There was a trend towards greater HSV-2 disease in these mice (407). In addition, HSV-2 infection increased vaginal viral load of HIV-1 and expression of HIV-1 RNA in vaginal tissue (407).  220   A second transgenic mouse has been described by this group. These transgenic mice contain a transgene for human CD4, CCR5 and cyclin T1 that is expressed in mouse CD4+ cells (408, 409). The combination of this gene expression allows HIV-1 infection and replication in mouse CD4+ cells (408). The mice developed disseminated infection of the spleen, small intestine, lymph nodes and lungs after intravenous injection with HIV-1, and vaginal transmission of HIV-1 was possible (408). Recently, these mice have been utilized for HIV-1/HSV-2 co-infection experiments (409). The mice were infected vaginally with HSV-2, then vaginal infection with a luciferase expressing HIV-1 occurring two days later (409). Six days after HIV-1 infection the vaginal mononuclear cells were examined for HIV-1 infection (409). HSV-2 infection caused a 4-fold increase in HIV-1 infection in these mice (409). Furthermore, TDF gel was able to prevent HIV-1 infection in 50% of the HSV-2 infected mice (409).   Both of these transgenic mouse strains could be used to test the utility of the C. crescentus microbicide during a co-infection situation. We could determine if HSV-2 infection can be prevented in the presence of an HIV-1 infection using the HIV-1 provirus transgenic mouse. The CD4/CCR5/cT1 mouse represents an excellent option to determine if the C. crescentus based microbicide can prevent HIV-1 infection in the presence of HSV-2. Neuronal HSV-2 was also detected in the HIV-1 provirus transgenic mouse, so it may be possible to determine if the C. crescentus microbicide can prevent HSV-2 latency in addition to primary infection using this model (407).  Our data suggested that Cc-A1AT was providing protection from HSV-2 infection by inducing an earlier immune response in the vaginal tract. I hypothesized that this early cytokine production leads to earlier immune cell recruitment to the vaginal tissue to control HSV-2 infection. This cytokine production could be problematic for HIV-1 prevention, as an increase in 221  immune cells in the vaginal tract is likely to increase HIV-1 infection. However, I do not expect this to be the case. When Cc-A1AT alone was applied to the vaginal tract of C57Bl/6 mice there was no production of inflammatory cytokines. In addition, when Cc-A1AT was given to the BLT mice at the same time as HIV-1JR-CSF infection, 43% of the mice were protected from HIV-1 infection and there were minimal differences in viremia in those mice that received Cc-A1AT and seroconverted. This suggests that it is the combination of Cc-A1AT plus HSV-2 that is responsible for the earlier cytokine response, and that in the absence of HSV-2 there is no concern for an increased immune response in the vaginal tract. However, it would be interesting to investigate what occurs immunologically in the vaginal tract when Cc-A1AT, HSV-2 and HIV-1 are all present simultaneously. While the HSV-2 data suggests that HIV-1 infection would be enhanced in this situation due to the presence of both Cc-A1AT and HSV-2 inducing TNF, IL-6 and IFNγ, it would be interesting to see if this is the case. This also raises the question if HSV-2 infection will be prevented. The described transgenic HIV-1 provirus and CD4/CCR5/cyclin T1 transgenic mice would be an ideal model to study this because HIV-1 infection can occur in these mice and HSV-2, HIV-1 and Cc-A1AT could all be applied simultaneously. 6.6.3 Will seminal plasma impact efficacy of the microbicide?  The in vitro and in vivo studies described in this thesis investigated the ability of the recombinant C. crescentus to prevent vaginal transmission of HIV-1 and HSV-2. Although the in vivo experiments aimed to closely recapitulate the physiologically relevant transmission of HIV-1 and HSV-2, there was one key component missing. All experiments were conducted in the absence of seminal plasma. There have been conflicting reports on the role of seminal plasma on 222  HIV-1 transmission and microbicide efficacy so it is unclear if the inclusion of seminal plasma into these experiments will change the results.  Some groups have reported that seminal plasma increases HIV-1 transmission (410-415). This appears to be caused by semen-derived amyloid fibrils (412). These amyloid fibrils are termed semen-derived enhancer of virus infection (SEVI) and are composed of fragments of the prostatic acid phosphatase (PAP) found in semen (412). SEVI has a cationic property to capture virus and promote viral attachment to cells, enhancing infectious virus titer by several orders of magnitude (412, 413). The semen enhancing effect seems to be donor dependent, with some donors enhancing HIV-1 infection by 50-fold, while others only causing a 2-fold enhancement in infection (415).  Other groups have reported that the presence of semen had no discernable effect on HIV-1 infection in vitro and in the humanized BLT mouse (247, 415-417). While SEVI seems to enhance HIV-1 infection, semen also contains a specific inhibitor that overcomes the enhancing effect of SEVI in the case of DC-SIGN mediated virus transmission, because semen has been reported to inhibit the DC-SIGN mediated transmission of HIV-1 (415).   In addition to these HIV-1 enhancing effects, the presence of semen also impairs the antiviral efficacy of microbicides (418). Both SPL7013 and cellulose sulphate were 20-fold less effective in the presence of semen (418). Neutralizing antibodies were 10-fold less effective in the presence of semen (418). TFV, the NNRTI nevirapine, the integrase inhibitor elvitegravir and the protease inhibitor indinavir had 8-13-fold decreased efficacy in the presence of semen (418). Interestingly, efficacy of maraviroc was not affected by the presence of semen, with it providing similar levels of protection in the presence or absence of semen (247, 418). The mechanism by which semen decreases microbicide efficacy is unclear (418). For polyanions it is possible that 223  SEVI neutralizes the negative charge, which decreases their efficacy (418). SEVI may also competitively bind to the viral envelope to prevent the ability of the virus to interact with the microbicide (418). SEVI could also alter the HIV-1 infection characteristics by shortening the time of microbicide exposure to HIV-1 or increasing the number of virions infecting each cell (418). It is not known why maraviroc, which targets the host cell CCR5 was not impacted by the presence of semen.  As the majority of the recombinant C. crescentus target the virus it is possible that semen could decrease their efficacy. Since the mechanism of action of semen enhancement of infection is unclear, it is possible that semen will have no impact on efficacy. In particular, while semen decreased effectiveness of TFV by 8-13-fold in vitro, TFV gel was able to prevent 54% of infections in high adherers in a clinical trial setting (2). These conflicting in vitro and clinical results indicate that additional studies examining the impact of semen on microbicide effectiveness are warranted. 6.6.4 Conclusion  A C. crescentus based microbicide represents a promising option for female controlled prevention of HIV-1 and HSV-2. It has been demonstrated that C. crescentus was easily modified for high-density peptide or protein display on the surface of the bacterium. These recombinant proteins were able to provide significant protection from HIV-1 infection in vitro and in vivo. Some recombinant C. crescentus were also able to provide significant protection from vaginal infection with HSV-2. C. crescentus appears safe for topical application to the vaginal tract. Further studies are warranted to investigate the utility of a C. crescentus based microbicide in a clinical setting. 224   A multi-component strategy is likely to have the greatest impact on decreasing HIV-1 infection. While condoms are appropriate in some settings, a full-prevention package for HIV-1 prevention should include topical agents and PrEP, allowing people the opportunity to choose the prevention option that works best for them. A C. crescentus based microbicide could be combined with other prevention strategies for STIs and pregnancy, as part of a comprehensive reproductive health program.  One of the biggest lessons that has been learned from the microbicide and PrEP trials is that adherence is key to success. 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Journal of virology 83:7783-7788.  289  Appendices Appendix A  Chapter 2 supplementary figures Figure A.1 C. crescentus titration   Figure A.1 C. crescentus titration using pseudovirus from clade C (SVPC3). 200TCID50 pseudovirus was incubated with 10,000 TZM-bl cells and the indicated numbers of recombinant C. crescentus. HIV-1 infection was measured after 48 hours by β-galactosidase assay. Infection rate is shown as a percentage and was normalized with virus + TZM-bl cells minus the 10^610^72.5x10^75x10^77x10^77.5x10^78x10^79x10^710^81.5x10^80100200300Cc-ControlCc-Griffithsin# Caulobacter% Infection# Caulobacter% Infection10^610^72.5x10^75x10^78x10^79x10^710^82x10^8050100150200Cc-ControlCc-Cyanovirin290  background for uninfected TZM-bl cells. Results are presented as mean + SEM. Experiments were performed in quadruplicate and repeated in 1-2 independent experiments.  Figure A.2 HIV-1 clade A/D pseudovirus data  Figure A.2 Preliminary viral blocking data using pseudovirus from clade D (D, D-912, D-911) and a clade A/D recombinant virus. 200TCID50 pseudovirus was incubated with 10,000 TZM-bl cells and 108 recombinant C. crescentus. HIV-1 infection was measured after 48 hours by β-galactosidase assay. Infection rate is shown as a percentage and was normalized with virus + TZM-bl cells minus the background for uninfected TZM-bl cells. Results are presented as mean + SEM. Experiments were performed in quadruplicate and repeated in 1-2 independent experiments. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: QA790.2041.ENV.A4 (A/D); QAO13.701.EBV.H1 (D-911); HIV-1 Pseudovirus% InfectionA/D D050100150200250Cc-ControlCc-MIP1αHIV-1 Pseudovirus% InfectionA/D DD-912D-911050100150200250Cc-ControlCc-CyanovirinCc-MicrovirinCc-GriffithsinHIV-1 Pseudovirus% InfectionA/D DD-912D-911050100150200250Cc-ControlCc-FuzeonCc-T1249Cc-C52HIV-1 Pseudovirus% InfectionD-912D-911050100150200Cc-ControlCc-CE2291  QAO13.701.ENV.M12 (D-912); QA465.59M.ENV.A1 (D) from Dr. Julie Overbaugh (419, 420).                      292  Appendix B  Chapter 4 supplementary information B.1 Development of humanized BLT mice at the University of British Columbia  In May 2012 I travelled to Dr. Cheryl Stoddart’s laboratory at the University of California San Francisco to receive training on the development of BLT mouse. I spent two weeks in her laboratory and learned how to successfully perform the thy/liv implant surgery, how to infect the mice intravaginally with HIV-1, and how to perform retro-orbital bleeding. When I returned to UBC I spent several months practicing the different procedures and preparing the animal protocols for approval by the UBC animal care committee (ACC). There were several difficulties encountered during the approval process. The veterinary staff at the animal facility I was working in at this time did not agree with the post-surgical care I planned to do and insisted that the analgesia be increased. I consulted with Dr. Stoddart’s lab and was told that they have the best post-surgery survival when handling of the mice post-surgery is minimized. It was their opinion that the suggestions our veterinary staff were proposing would significantly decrease post-surgery survival of the mice. Furthermore, the ACC would not approve a surgical protocol using wound clips and required me to use sutures to close the skin incision. After close to a year of consultations with the facility vet and numerous training sessions to demonstrate my mastery of the surgical skills, the protocol was still not approved. Dr. Rusung Tan at the Child and Family Research Institute (CFRI) had a humanized mouse surgery protocol approved at their facility but no personnel able to perform the surgery. We collaborated with them and in June 2013 I was finally able to begin making BLT mice at CFRI. Eventually we were able to get an animal protocol approved for work in the Modified Barrier Facility at UBC, so I was able to begin the protocol on campus. 293   Obtaining human fetal tissue for research has been difficult. We have been working with Advanced Bioscience Resources Inc to obtain the tissue. All fetal tissue for this project was obtained in the US. Many problems have been encountered importing the tissue into Canada, including timely delivery of the tissue by FedEx and release of the tissue by Canadian Customs. Many early shipments were received late in the day, lowering the usefulness. We developed a strategy to eliminate these problems.  Recently, we have had difficultly obtaining any fetal tissue and suspect the “Planned Parenthood sting videos” have played a role (http://www.nature.com/news/fetal-tissue-research-under-threat-1.18967).  In addition to these difficulties in establishing the BLT mouse model, another difficulty was encountered with receiving approval to collect blood samples from HIV-1 infected mice. The Public Health Agency of Canada classifies HIV-1 as a risk group 3 pathogen and states that the use of sharps should be strictly limited (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/hiv-vih-eng.php). To avoid the use of needles for blood collection, we proposed to use the retro-orbital route for blood collection. While the Canadian Council on Animal Care states that retro-orbital bleeding can be performed in exceptional circumstances (http://www.ccac.ca/Documents/Standards/Guidelines/Vol2/mice.pdf) the UBC ACC does not approve protocols with retro-orbital bleeding (http://www.ors.ubc.ca/sites/ors.drupalprod.webi.it.ubc.ca/files/uploads/documents/ORS/animalcare/006_Rodent_blood_withdrawal.pdf). After numerous consultations with the UBC Research Safety Advisor and veterinary staff we were able to receive approval to perform retro-orbital bleeding with the following conditions: Christina Farr may perform retro-orbital bleeding on HIV exposed mice only; Christina must demonstrate mastery of this technique to the veterinary 294  staff; retro-orbital bleeding may be performed at maximum every 14 days, using alternating eyes, for 8 weeks. Although these conditions limited the amount of work that could be done with HIV infected mice, this was sufficient for determining HIV-1 infection after microbicide treatment.  B.2 Summary of BLT mouse cohorts  Cohort ID Date Number of Mice %CD45 (human) Use 1 2013-08-01 5 46.4% Females – HIV Pilot Males – Tan Lab 2 2013-09-05 10 21.8% Females – HIV Pilot Males – EBV Pilot 3 2013-10-16 5 N/A Tissue held at customs, not viable 4 2013-12-06 15 29.2% Microbicide testing 5 2014-06-06 15 27.5% Microbicide testing 6 2014-07-03 4 (of 11) 21.1% Poor recovery of CD34+ cells; Microbicide testing 7 2014-09-05 4 (of 16) 71.5% Poor recovery of CD34+ cells; Microbicide testing 8 2014-09-12 24 47.3% Microbicide testing 9 2014-09-19 19 39.2% Microbicide testing 295  Cohort ID Date Number of Mice %CD45 (human) Use 10 2014-10-24 20  16.6% 4 mice not used, reconstitution too low; Microbicide testing 11 2015-02-27 10 (of 24) 32.4% Poor recovery of CD34+ cells; Microbicide testing 12 2015-03-13 18 (of 22) 17.4% 5 mice not used, reconstitution too low; Microbicide testing 13 2015-03-19 14 (of 18) 12.9% 4 mice not used, reconstitution too low; Microbicide testing 14 2015-07-31 18 In progress Poor recovery of CD34+ cells; Microbicide testing 15 2015-09-10 13 In progress Few CD34+ cells isolated 16 2015-10-15 22 In progress  17 2015-11-06 24 In progress  18 2016-01-22 N/A N/A Shipping delay due to winter storm. Tissue arrived 2016-01-25  296  B.3 Representative flow cytometry staining of peripheral blood from a BLT mouse   Figure B.3 Representative flow cytometry staining of peripheral blood from BLT mice. Red is unstained control, blue is cells from mouse blood. Human	  CD45 CD3 CD4 CD8 CD19	  (CD3-­‐,	  CD14-­‐) CD14	  (CD3-­‐,	  CD19-­‐) CD56	  (CD3-­‐,	  CD14-­‐,	  CD19-­‐) 297  B.4 Preliminary rectal microbicide testing  Figure B.4 BLT mice were infected rectally with 10,000TCID50 HIV-1JR-CSF in the presence or absence of 108 C. crescentus. Blood was collected from the retro-orbital sinus on days 0, 14, 28 and 42 post-infection and analyzed by p24 ELISA. HIV-1JR-CSF 2/2 infected; HIV-1JR-CSF + Cc-Control 3/3 infected; HIV-1JR-CSF + Cc-CD4M33F23 2/3 infected; HIV-1JR-CSF + Cc-T1249 2/3 infected.          Day 0Day 14Day 28Day 420510152025100200300400[p24] pg/mLHIV-1HIV-1 + Cc-ControlHIV-1+ Cc-CD4M33F23HIV-1 + Cc-T1249298  Appendix C  Chapter 6 supplementary figures C.1 Comparing dual protein expression in RsaA or combination of individual C. crescentus   Figure C.1 Preliminary combination viral blocking data using pseudotyped virus from clade B and C. 200TCID50 pseudovirus was incubated with 10,000 TZM-bl cells and 108 recombinant C. crescentus (5x107 of each for a total of 108 when individual C. crescentus were used). HIV-1 SVPB11SVPB12SVPC3SVPC4050100150200Cc-ControlCc-GriffithsinCc-FuzeonCc-GriffithsinFuzeonCc-Griffithsin + Cc-FuzeonHIV-1 Pseudovirus% InfectionSVPC3SVPC4050100150200Cc-FuzeonCc-ControlCc-CD4Cc-CD4FuzeonCc-CD4 + Cc-FuzeonHIV-1 Pseudovirus% Infection299  infection was measured after 48 hours by β-galactosidase assay. Infection rate is shown as a percentage and was normalized with virus + TZM-bl cells with the background for uninfected TZM-bl cells subtracted out. Results are presented as mean + SEM. Experiments were performed in quadruplicate and repeated in 1-3 independent experiments. Preliminary experiments suggested that combining two different recombinant C. crescentus provided better protection from HIV-1 infection compared to expressing two proteins simultaneously in the S-layer, so this strategy was used for subsequent combination experiments.  C.2 Preliminary combination data    MIP1a + CD4sHIV-1 Pseudovirus% InfectionSVPB11SVPB12SVPC3SVPC4050100150Cc-ControlCc-MIP1αCc-CD4Cc-CD4M33F23Cc-MIP1a + Cc-CD4Cc-MIP1a + Cc-CD4M33F23Griffithsin + FuzeonHIV-1 Pseudovirus% InfectionSVPB11SVPB12SVPC3SVPC4050100150200Cc-ControlCc-GriffithsinCc-FuzeonCc-Griffithsin + Cc-FuzeonCc-GriffithsinFuzeonFuzeon + CyanovirinHIV-1 Pseudovirus% InfectionSVPB11SVPB12SVPC3SVPC4050100150200Cc-ControlCc-FuzeonCc-CyanovirinCc-Fuzeon + Cc-CyanovirinCc-FuzeonCyanovirinMIP1a + Cyanovirin% InfectionCc-ControlCc-MIP1aCc-CyanovirinCc-MIP1a + Cc-Cyanovirin050100150300     MIP1a + Microvirin% InfectionCc-ControlCc-MIP1aCc-MicrovirinCc-MIP1a + Cc-Microvirin050100150MIP1a + Griffithsin% InfectionCc-ControlCc-MIP1aCc-GriffithsinCc-MIP1a + Cc-Griffithsin050100150T1249 + CyanovirinHIV-1 Pseudovirus% InfectionSVPB11SVPB12SVPC3SVPC4D - 912D - 911050100150200Cc-ControlCc-T1249Cc-CyanovirinCc-T1249CyanovirinCc-T1249 + Cc-CyanovirinT1249 + MicrovirinHIV-1 Pseudovirus% InfectionSVPB11SVPB12050100150Cc-ControlCc-T1249Cc-MicrovirinCc-T1249 + Cc-MicrovirinGBVCE2 + CyanovirinHIV-1 Pseudovirus% InfectionSVPB11SVPB12SVPC3SVPC4D - 912D - 911050100150200Cc-ControlCc-GBVCE2Cc-CyanovirinCc-GBVCE2 + Cc-CyanovirinMIP1a + GBVCE2HIV-1 Pseudovirus% InfectionSVPB11SVPB12050100150Cc-ControlCc-MIP1aCc-GBVCE2Cc-MIP1a + Cc-GBVCE2301    Figure C.2 Preliminary combination viral blocking data using pseudotyped virus from clade B, C and D. 200TCID50 pseudovirus was incubated with 10,000 TZM-bl cells and 108 recombinant C. crescentus (5x107 of each for a total of 108 when individual C. crescentus were used). HIV-1 infection was measured after 48 hours by β-galactosidase assay. Infection rate is shown as a percentage and was normalized with virus + TZM-bl cells with the background for uninfected TZM-bl cells subtracted out. Results are presented as mean + SEM. Experiments were performed in quadruplicate and repeated in 1-3 independent experiments.  MIP1a + T1249HIV-1 Pseudovirus% InfectionSVPB11SVPB12050100150Cc-ControlCc-MIP1aCc-T1249Cc-MIP1a + Cc-T1249GBVCE2 + T1249HIV-1 Pseudovirus% InfectionSVPB11SVPB12SVPC3SVPC4D - 912D - 911050100150Cc-ControlCc-GBVCE2Cc-T1249Cc-GBVCE2 + Cc-T1249GBVCE2 and GriffithsinHIV-1 Pseudovirus% InfectionSVPB11SVPB12SVPC3SVPC4D - 912D - 911050100150Cc-ControlCc-GBVCE2Cc-GriffithsinCc-GBVCE2 + Cc-GriffithsinGBVCE2 and CD4M33F23HIV-1 Pseudovirus% InfectionSVPB11SVPB12050100150Cc-CCc-GBVCE2Cc-CD4M33F23Cc-GBVCE2 + Cc-CD4M33F23302  C.3 Preliminary timed viral blocking assays  Figure C.3 Preliminary timed viral blocking data. 10,000 TZM-bl cells and 108 recombinant C. crescentus were incubated for the indicated times before addition of 200TCID50 HIV-189.6. HIV-1 infection was measured after 48 hours by β-galactosidase assay. Infection rate is shown as a percentage and was normalized with virus + TZM-bl cells with the background for uninfected TZM-bl cells subtracted out. Results are presented as mean + SEM. Experiments were performed in quadruplicate and repeated in 1-6 independent experiments. No data is currently available for Cc-Griffithsin at 8 hours.  % Infection0 hours0.5 hours1 hour2 hours8 hours050100150Cc-ControlCc-CD4M33F23Cc-GriffithsinCc-T1249Cc-A1AT

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