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Protection against leishmaniasis by DNA injection of a plasmid encoding the Leishmania major Cpn60 gene Webber, Jane 2001

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PROTECTION AGAINST LEISHMANIASIS BY DNA INJECTION OF A PLASMID ENCODING THE Leishmania major CPN60 GENE by JANE WEBBER B. Sc. The University of Victoria, 1993 A THESIS SUBMITTED IN PARTIAL FULFILMEENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Pathology and Laboratory Medicine The University of British Columbia We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA i . AUGUST 2001 4)JANE WEBBER, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 A B S T R A C T The leishmaniases are parasitic protozoan infections caused by members of the genus Leishmania. Injection of Leishmania major into various strains of inbred mice cause disease symptoms which mimic the symptoms of a human leishmanial infection. Protection against an L. major infection in BALB/c mice depends on the generation of a macrophage-activating CD4 + Thl immune response with production of IL-2 and IFN-y, but not IL-4, a Th2 cytokine that results in disease exacerbation. Immunization using DNA vaccines is a promising new approach for the prevention of diseases caused by intracellular pathogens. The antigen of interest encoded in the plasmid is transcribed and translated by the host cellular machinery and presented to the immune system inducing specific cell-mediated and humoral immune responses against the encoded protein. In this study a eukaryotic expression vector, pcDNA3.1 encoding the L. major chaperonin 60 (Cpn60) was constructed and tested in vitro to confirm that the mammalian translational machinery could synthesize the protein. Cell-free coupled transcription/translation using rabbit reticulocyte lysate and transfection studies using COS-7 cells were carried out by SDS-PAGE and western blot analysis of protein products. A single protein of the correct size was produced in vaccine samples but not in the Controls. To test the vaccine efficacy, BALB/c mice were immunized with either the vaccine construct, empty plasmid control or PBS, and boosted twice two weeks apart. Spleen cells from mice immunized with the vaccine construct did not produce significant amounts of the Thl cytokines interleukin-2 (IL-2) or gamma interferon (IFN-y), or the I l l Th2 cytokine IL-4, when cultured with soluble Leishmania antigen (SLA) in vitro. Sera from each mouse group was pooled and used to probe immunoblots where protein from control organisms, purified human Cpn60 and L. major total protein was used as antigen. Sera from the vaccine mouse group did not detect the L. major Cpn60 protein. The results of these experiments indicated that the protein was expressed in vitro but immunization experiments were unsuccessful using this vaccination protocol. Review of vaccination techniques and a change in the immunization site may improve chances for a successful outcome. TABLE OF CONTENTS iv Page ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS xiii RATIONALE AND HYPOTHESIS xiv THESIS OBJECTIVES xvi I. INTRODUCTION 1 1.1. Leishmania 1 1.1.1 The parasite 1 1.1.2 Infectivity 2 1.1.3 Leishmania spp. and patterns of disease 3 1.1.3.1 Cutaneous leishmaniasis 3 1.1.3.2 Mucocutaneous leishmaniasis 4 1.1.3.3 Visceral leishmaniasis 4 1.1.4 Treatment of leishmaniasis 5 1.1.5 Murine models of leishmaniasis 6 1.1.5.1 Host-parasite factors 6 1.1.5.2 Susceptibility and resistance patterns in a mouse model 8 1.1.5.3 Role of IL-4 and IL-12 in CD4 + T cell development in the murine model of infection with L. major 9 1.1.6 Vaccine requirements for sustained cellular immunity to intracellular parasitic infection 12 1.2 DNA Vaccines 13 1.2.1 Characteristics and properties of a DNA vaccine 13 1.2.2 Vaccine strategies 15 1.2.2.1 Live attenuated vaccines 15 1.2.2.2 Killed or subunit vaccines 16 1.2.2.3 DNA vaccines vs. traditional vaccines 16 1.2.3 Limitations of DNA vaccines 17 1.2.4 Immunology of DNA vaccines 18 1.2.4.1 DNA vaccine intracellular route 18 1.2.4.2 Mechanism of immunity of DNA vaccines 19 1.2.4.3 Dendritic cells are the most potent antigen-presenting cells 21 1.2.5 The plasmid vector as an adjuvant 22 1.2.6 Modulating the immune response to DNA 25 1.2.6.1 Method and location of immunization 25 1.2.6.2 Immunization regimen 27 1.2.6.3 Co-administration of cytokines and co-stimulatory molecules 27 1.2.7 Leishmanial antigens employed in vaccine studies to date 29 1.3 Chaperonin60 30 1.3.1 Immunology of chaperonins 3 2 1.1.3.1 Chaperonin 60 and the immune response 32 1.1.3.2 T cell recognition of chaperonins 3 3 1.3.2 Vaccine studies with plasmids encoding Cpn60 (Hsp 65) as antigen 34 II. MATERIALS AND METHODS 35 2.1 X EMBL-3 Phage Preparation From Plate Lysate 35 2.1.1 Growth ofE. coli strain LE392 35 2.1.2 Plate preparation 35 2.1.3 Plate lysis method of phage recovery 36 2.2 PCR of L. major Cpn60 Gene 36 2.3 Agarose Gel Electrophoresis and Restriction Analysis of the L. major Cpn60 Gene 37 2.4 Subcloning of L. major Cpn60 into pcDNA3.1 38 2.4.1 pcDNA3.1 38 2.4.2 The 1.7 kb L. major Cpn60 gene 38 2.5 Transformation of E. coli Electrocompetent Cells 39 2.6 Sequencing of the E major 1.7 kb Cpn60 Gene 40 2.7 Site-Directed Mutagenesis 41 2.7.1 Primer design 41 2.7.2 Reaction mix and cycling parameters 42 2.7.3 Transformation of E. coli XL1 -Blue supercompetent cells 42 2.7.4 Sequencing of the corrected E major Cpn60 gene 43 2.8 Large Scale Plasmid Purification 43 2.9 In Vitro Coupled Transcription / Translation Using Rabbit Reticulocyte Lysate 44 2.9.1 Coupled transcription / translation 44 2.9.2 Denaturing gel analysis of translation products 44 2.9.3 Chemiluminescent detection of protein products 45 2.10 In Vitro Transfection of COS-7 Cells 46 2.10.1 Culture of COS-7 cells 46 2.10.2 Transfection using FuGene 6 transfection reagent 46 2.10.3 Harvesting of COS-7 cells following transfection 47 2.11 Antibodies and Reagents Used in Western Blots 47 2.11.1 Primary antibodies 47 2.11.1.1 Rabbit anti-Cpn60 47 2.11.1.2 Chicken mti-Leishmania Hsp60 48 2.11.1.3 Mouse sera " 48 2.11.2 Secondary antibodies 48 2.11.3 Reagents for immunoblots 49 2.12 Western Blot and Detection of Protein Products 49 2.13 Leishmania major Total Protein 50 2.14 Mouse Immunization Protocol 5 0 2.14.1 Vaccine mouse group 51 2.14.2 Empty plasmid control group 51 2.14.3 PBS control group 51 2.15 Cardiac Puncture Protocol 52 2.16 Spleen Removal and Culture of Mouse Lymphocytes 52 2.17 In Vitro Cytokine Assay 53 2.17.1 Cytokine assay from six mouse trial immunization experiment (exp. 1) 53 2.17.2 Cytokine assay from the second mouse immunization experiment (exp. 2) 54 2.18 RT-PCR 55 2.18.1 RNA preparation 55 2.18.2 Reverse transcription 56 2.18.3 PCR of RT samples 56 2.18.3.1 Cycling parameters (4F/4R and 5F/5R) 57 2.18.3.2 Cycling parameters (BAC-1005/BAC-1006) 57 RESULTS AND DISCUSSION 58 3.1 PCR Amplification, Cloning and Sequencing of the L. major Cpn60 Gene 58 3.1.1 Amplification of the L. major Cpn60 gene contained in the X EMBL-3 clone 58 3.1.2 Cloning of the L. major Cpn60 gene 59 3.1.3 Plasmid purification and sequencing of L. major Cpn60 61 3.1.4 Site-directed mutagenesis to correct the deletion in the L. major Cpn60 gene 62 3.2 In Vitro Experiments and Western Blot Analysis 63 3.2.1 Cell-free coupled transcription / translation using a TNT R Quick Coupled Transcription/Translation System 63 3.2.2 In vitro transfection of COS-7 cells with pcDNA3.1/Cpn60 and pcDNA3.1 control plasmid 66 3.3 RT-PCR: Generation of cDNA, Amplification and Analysis 70 3.4 In vivo Mouse Immunization Experiments 72 3.4.1 Trial immunization study 72 3.4.1.1 Immunoassay for IL-4 73 3.4.1.2 Immunoassay for IL-2 73 3.4.2 Serum antibody study in the trial immunization 74 3.4.3 Second immunization study 76 3.4.3.1 Immunoassay for IL-4 76 3.4.3.2 Immunoassay for IL-2 77 3.4.3.3 Immunoassay for IFN-y 78 3.4.3.4 Serum antibody results from the second immunization study 80 IV CONCLUSION 81 LITERATURE CITED 86 LIST OF TABLES Vlll TABLE I. TABLE II TABLE III TABLE IV TABLE V TABLE VI The effect of immunization site and delivery methods on the orientation of the immune response to DNA Vaccines. Effect of co-administration of plasmids encoding cytokines with DNA vaccines on the immune response. Cycling parameters for pcDNA3.1/Cpn60 In vitro production of IL-2 from spleen cells of vaccinated mice stimulated with L. major lysate or unstimulated in the trial immunization study In vitro production of IL-2 from spleen cells of vaccinated mice stimulated with Con A, L. major lysate, or unstimulated in the second immunization study Page 26 28 42 73 77 In vitro production of IFN-y from spleen cells of vaccinated mice stimulated with Con A, L. major lysate, or unstimulated following the second boost in the second immunization study 79 IX LIST OF FIGURES Page Figure 1. Life cycle of Leishmania. 2 Figure 2. PCR amplification of the putative 1.7 kb Cpn60 gene from L. major 58 Figure 3. Restriction analysis of the putative 1.7 kb fragment of the L. major Cpn60 gene using BamHl. 59 Figure 4 5.4 kb eukaryotic expression vector pcDNA3.1+ 60 Figure 5 Pst\ and BamHl digestion of the control and recombinant plasmid. 63 Figure 6 In vitro coupled transcription / translation of L. major Cpn60 using rabbit reticulocyte lysate. 65 Figure 7 Immunoblot No. 1 of protein products from COS-7 cells transfected with pcDNA3.1/Cpn60 or empty pcDNA3.1 and probed with antibody #85. 67 Figure 8 Immunoblot No. 2 of protein products from COS-7 cells transfected with pcDNA3.1/Cpn60 or empty pcDNA3.1 and probed with antibody #85. 67 Figure 9 Immunoblot No. 3 of protein products from COS-7 cells transfected with pcDNA3.1/Cpn60 or empty pcDNA3.1 probed with primary antibody mti-Leishmania Cpn60 (SPA 823). 68 Figure 10 Results of RT-PCR of mRNA from cells of pcDNA3.1/Cpn60 vaccinated mouse. 70 Figure 11 Serum antibody study of vaccine, empty plasmid and PBS mouse groups in the trial experiment 75 LIST OF ABBREVIATIONS x Ag Antigen A L M Whole cell heat-killed L. major antigen AP Alkaline phosphatase APC Antigen-presenting cell BGH Bovine growth hormone C. therm Reverse transcriptase Con A Concanavalin A COS-7 African green monkey kidney cells CpG Cytosine-phosphate-guanosine Cpn60 Chaperonin 60 CR3 Complement receptor 3 CTL Cytotoxic T lymphocyte DC Dendritic cell DCL Diffuse cutaneous leishmaniasis DMSO Dimethyl sulfoxide dNTP Deoxyribonucleotide 5'-triphosphate DTT Dithiothreitol EDTA Ethylenediaminetetra-acetic acid ELISA Enzyme-linked immunosorbent assay Endofree Bacterial endotoxin free ErBr Ethidium bromide Exo+ Exonuclease+ FCS Fetal calf serum GM-CSF Granulocyte macrophage- colony stimulating factor GpB Glycoprotein B Gp63 Glycoprotein 63 GroEL E. coli Cpn60 homologue HA Hemagglutinin HBsAg Hepatitis B small antigen HBSS Hanks balanced salt solution H 2 0 2 Hydrogen peroxide HCMV Human cytomegalovirus promoter HCV Hepatitis C virus HIV Human immunodeficiency virus HPV Human papilloma virus Hsp Heat shock protein Hsp65 Heat shock protein 65 IFN-y Interferon gamma IFN-yRa+p+ Interferon gamma receptor alpha positive, beta positive IgG Immunoglobulin IL-1 Interleukin-1 IL-ip Interleukin-1 beta IL-2 Interleukin 2 IL-4 Interleukin 4 IL-4R+ Ibterleukin-4 receptor positive IL-5 Interleukin-5 IL-6 Interleukin-6 IL-9 Interleukin-9 IL-10 Interleukin-10 IL-12Rpip2 Interleukin-12 receptor beta 1, beta 2 IL-12 Interleukin-12 IL-13 Interleukin-13 iNOS Inducible nitric oxide synthase Iscoms Immune system complexes ISS Immunostimulatory sequences JEV Japanese encephalitis virus Kb Kilobase Kda Kilodalton LACK Leishmania-activated C kinase LB Luria-Bertani LC Langerhans cell LMP Low melting point LPG Lipophosphoglycan LNC Lymph node cell LR Leishmaniasis recidiva MAF Macrophage activating factor MgS0 4 Magnesium sulfate MHC Major histocompatibility complex Min Minute mRNA messenger RNA NK Natural killer NO Nitric oxide NP Nucleoprotein ODN Oligodeoxyribonucleotide PBS Phosphate-buffered saline pcDNA3.1/Cpn60 Plasmid encoding the L. major Cpn60 pcDNA3.1 Empty control plasmid Pen-Strep Penicillin- Streptomycin Pfu Plaque-forming units PCR Polymerase chain reaction PIV Primary in vitro system PSA-2 Parasite surface antigen-2 PVDF Polyvinylidene difluoride ROI Reactive oxygen intermediates RNA Ribonucleic acid S Second SDS Sodium dodecyl sulfate SDS-PAGE SDS polyacrylamide gel electrophoresis SIV Simian Immunodeficiency virus SLA Soluble Leishmania antigen SRP-HRP Streptavidin-horseradish peroxidase TAE Tris - acetic acid - EDTA TAP-1 Transporter associated with antigen processing-1 TAP-2 Transporter associated with antigen processing-2 TBS Tris buffered saline TBST Tris buffered saline, Tween 20 TCA3 P-chemokine chemotactic for monocytes/nacrophages Thl T helper 1 Th2 T helper 2 TNF Tumor necrosis factor tRNA , Transfer RNA TSA Thiol-specific antioxidant Xlll ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Swee Han Goh, for all his advice and support during my time as a graduate student. Also, a thank you to Dr. Neil Reiner for his gift of the X EMBL-3 phage, Dr. S. Hemmingsen for his help and gift of the anti-Cpn60 antibody, and to Dr. R. McMaster for supplying me with L. major promastigotes from his laboratory. I would like to thank Dr. Ben Kelly for his help with L. major cultures and electroporation, and Spencer Kong and Dr. Marcel Bally for their help and advice in the cell culture lab at the Cancer Research Centre. Thanks to Jose Rey-Ladino for his helpful advice, time and for his gift of the COS-7 cells. Finally, a special thanks to the co-op students in Dr. Goh's laboratory, Cheng He and Sarah Allen, for their help with the mouse immunization work carried out at the Jack Bell Animal Care Facility. xiv RATIONALE AND HYPOTHESIS The development of vaccines over the last 100 years has effectively controlled some of the world's most devastating diseases by using attenuated or killed organisms or through subunit vaccines. Still for some diseases, in particular those caused by intracellular pathogens, effective vaccines have been elusive. In 1990 Wolff, demonstrated that injection of purified 'naked' DNA reporter constructs into mouse muscle resulted in an immune response being generated against the foreign DNA Since then genetic immunization has been applied to more than 30 systems involving a wide variety of viruses, bacteria and parasitic pathogens as well its use as a cancer vaccine. Genetic immunization involves introduction of non-replicative plasmids encoding a foreign protein under the control of a strong promoter directly into the host animal. This technology has many advantages over conventional approaches to immunization. DNA vaccines elicit antibodies and CD4 + responses in animals but their major advantage has been the capacity to induce specific CD8 + T cell responses, a major mechanism of protection against most intracellular pathogens. Unlike attenuated virus vaccines there is no risk of the vector reverting to pathogenicity. Any protein or protein variant can be tested using simple recombinant DNA technology. The plasmid construct is stable over a wide range of temperatures. In this study a DNA vaccine against the protozoan parasite Leishmania major was constructed using a plasmid expression vector encoding the L. major chaperonin 60 gene X V as antigen. L. major is an intracellular pathogen for which no highly effective and safe vaccine exists. Disease severity is dependent on the host genetics and the species of infecting parasite. Inbred strains of mice have proven to be a valuable model for studying host immune responses to the parasite, particularly the mechanism for the initiation of Thl vs. Th2 immune responses. Infection of BALB/c mice with L. major produces disease symptoms that mimic a human leishmanial infection. A CD4+ Thl response with IFN-y secretion is correlated with resolution of disease while a Th2 response with secretion of IL-4 promotes disease progression. The choice of Cpn60 as antigen in a vaccine preparation was made because of its abundance in stressed cells and its immunogenicity. The conservation of Cpn60 over time suggests that it is not as subject to antigenic variation as cell surface antigen such as gp63 might be. Research carried out on this project was intended to test the hypothesis that a DNA vaccine utilizing a eukaryotic expression vector pcDNA3.1 encoding the L. major antigen Cpn60 under control of the human CMV promoter would induce a protective CD4 + Thl immune response when injected into BA:LB/c mice. THESIS OBJECTIVES xvi 1. To produce a DNA vaccine A DNA vaccine is constructed by subcloning the Cpn60 gene from Leishmania major into a plasmid expression vector under the control of the human CMV promoter. 2. To test for antigen production by in vitro methods Use of a cell-free coupled transcription/translation system (rabbit reticulocyte lysate) and transfection studies with COS-7 cells to confirm that the mammalian translational machinery could produce the protein and protein levels produced are not toxic to the cells. 3. To examine the immune response to the vaccine in BALB/c mice The vaccine is injected into the anterior tibialis muscle of BALB/c mice to determine if a protective CD4 + Thl immune response is generated. The immune response is assessed by assaying for levels of cytokines IL-2, IL-4 and IFN-y in splenic lymphocytes from vaccinated mice by sandwich ELISA. I. INTRODUCTION 1.1 Leishmania 1.1.1 The Parasite Leishmania spp are parasitic protozoa of the family Trypanosomatidae and are found throughout the tropics and subtropics. Infection with these parasites cause disease with a wide range of clinical manifestations, from a self-healing cutaneous lesion to a potentially fatal visceral disease, depending on the infecting parasite species and host genetics. Leishmaniasis is present in 88 countries and it is estimated that 12 million people are affected, with 2 million new cases each year. Animal reservoirs of Leishmania pathogenic for humans include dogs and rodents, and in South America, arboreal mammals (17, 18, 39, 41). Leishmaniae are dimorphic, living in the gut of the sandfly vector as motile, flagellated promastigotes and inside macrophages of the vertebrate host as aflagellate, nonmotile amastigotes. The life cycle is illustrated in Figure 1. The promastigote form of the parasite is injected into the skin of a naive recipient by an infected Phlebotamus sandfly. Shortly after infection the parasite is phagocytosed by macrophages, and within 72 hours transformation from the promastigote to the amastigote takes place. The parasites are released from the macrophage and infect other cells disseminating the infection within the host (17, 39, 40, 41, 42). 1 Sandfly • 7. Blood meal 6. in new vertebrate host promastigote amastigote • 5. regurgitation into new host during blood meal A 4. sandfly mouthparts T promastigote migration 1. infectea macrophages ingested from vertebrate host 2. gutor sandfly amastigote -^ promastigote 3. multiplication by binary fission Figure 1.. Life cycle of Leishmania. 1. Amastigotes inside infected macrophages from a vertebrate host are ingested by the sandfly. 2, 3. The amastigotes transform to promastigotes and replicate in the sandfly gut. 4. After one week, the parasites migrate to the sandfly mouthparts. 5. The promastigotes are then injected under the skin of a new host by the bite of the sandfly. 6. Inside the mammalian host parasite transformation to the aflagellate amastigote form takes place inside macrophages. The parasite replicates inside the macrophage, then lyses the cell. Replication and cell lysis is repeated in other macrophages. 7. The infection cycle is repeated when a sandfly ingests infected macrophages during a blood meal (93). 1.1.2 Infectivity The infective form of Leishmania major is the mature parasite called a metacyclic (41). The discovery of a high molecular mass surface antigen, a component of lipophosphoglycan (LPG), specific for metacyclics was reported by Handman et al. (41). In L. major, LPG has been shown to function as a receptor for host macrophages. A membrane glycoprotein, gp63, which is up-regulated in metacyclics has also been shown to function as a macrophage receptor, interacting with both the complement receptor, CR3, and the mannose-fucose receptor on the macrophage surface. It has been hypothesized that the mannose-fucose 2 receptor and CR3 synergize to mediate parasite binding and uptake by macrophages (41). Once bound to the macrophage membrane the parasite is internalized into a phagolysosome. Survival inside the phagolysosome depends on the rapid transformation from promastigote to amastigote as promastigotes are rapidly killed inside macrophages, probably due to the oxidative burst (41, 42). The amastigote is better prepared to evade the host immune response because it stimulates less H2O2 from macrophages than the promastigote and produces its own superoxide dismutase (93). Survival inside the phagolysosome also depends on the presence of LPG on the parasite surface. LPG" variants transform normally but are quickly killed (41). 1.1.3 Leishmania spp. and Patterns of Disease 1.1.3.1 Cutaneous leishmaniasis Cutaneous leishmaniasis is caused by Leishmania major in the Old World (middle East, Africa and Mediterranean areas) and in the New World (Central and South America) by Leishmania mexicana. Old World leishmaniasis, or Oriental sore, is a self-limiting disease. A lesion develops at the bite site where multiplication of the parasite takes place. Over time lymphocytes and plasma cells accumulate at the infection site and this is usually accompanied by disappearance of the parasite. Healing begins spontaneously leaving behind a hypopigmented area and, in most cases, scarring at the lesion site (5, 16, 41). Recovery from disease results from cell-mediated immunity and can be measured in vitro by T cell assays. Two uncommon forms of cutaneous disease, diffuse cutaneous leishmaniasis (DCL) and leishmaniasis recidiva (LR) have been identified. DCL represents a defective host response to an otherwise normal cutaneous parasite infection. The host does not mount a T cell response to parasite antigen but does produce antibodies to the infection. In LR a cell 3 mediated response is induced but the immune response does not eliminate the parasite (39, 40) . 1.1.3.2 Mucocutaneous leishmaniasis Mucocutaneous leishmaniasis, or espundia, is caused by L. braziliensis (in the New World only). The course of this disease is much like cutaneous leishmaniasis with a lesion forming at the bite site and eventual healing. Months to years later, a new lesion forms at the original site or elsewhere on the body. The parasite then migrates to the oronasal and pharyngeal mucosa causing leprosy-like tissue destruction. In this disease a good cell-mediated and antibody response is generated so pathogenesis of the disease is not well understood (39, 40, 41) . 1.1.3.3 Visceral leishmaniasis Systemic visceral leishmaniasis, or kala-azar, is caused by L. donovani. The disease is characterized by prolonged fever and wasting, and is progressive, ultimately leading to death of the patient if left untreated. The liver, spleen, and bone marrow are the organs most often involved. There is a proliferation of infected macrophages accompanied by infiltration of lymphocytes, granuloma formation and necrosis. A strong antibody response is induced but cell-mediated immunity is greatly impaired. This is thought to be the reason for failure of disease control by the host (39, 40, 41). 4 1.1.4 Treatment of Leishmaniasis No effective vaccine exists against leishmaniasis. Disease control relies on chemotherapy, in the form of pentavalent antimony compounds (39, 41), and vector control to reduce transmission (90). Vaccination strategies to date have involved the use of infective material. One strategy, 'leishmanization', involves exposing the buttocks of uninfected children to the bite of a sandfly. The rationale behind this approach is that a lesion on the buttocks forms and with resolution protective immunity develops. Another vaccination strategy has been the transfer of material from an infected lesion to an uninfected individual. When cultured organisms became available, they were used in the Soviet Union, Israel and Iran to infect individuals, effectively immunizing them. Successful immunity was achieved by injecting approximately 106 organisms intradermally. These 'immunization' protocols have been curtailed due to associated high risks (41). Results of early studies using phenol-killed organisms as a possible vaccine were inconclusive. In vaccine experiments in the Soviet Union the use of non-infective L. major promastigotes proved ineffective as vaccines even though a cell-mediated response was generated (41). With the increasing prevalence of drug-resistance in organisms and the possibility of relapse, even after an initially successful course of chemotherapy, the need for a vaccine is vital (42). The immunity observed after a natural leishmanial infection indicates a prophylactic vaccine against leishmaniasis should be feasible (5, 16, 41). 5 1.1.5 Murine Model of Leishmaniasis 1.1.5.1 Host-parasite factors The genetics of the host has a great influence on both the susceptibility to and clinical manifestations of leishmaniasis. Genetics seems to influence the intensity of macrophage activation, the potency of the T cell response, and suppression of T cell activation (41). Infection of inbred strains of mice with L. major has proven to be a valuable model for studying the host immune response to the parasite; particularly the mechanisms for the initiation of Thl versus Th2 cell development from common CD4 + T cell precursors (38, 42). In the mouse model, as in human disease, CD4 + T cells play the dominant role in determining the outcome of infection. Healing of cutaneous leishmaniasis is dependent on the induction of a cell-mediated response (5, 16, 36, 41, 46). Injection of L. major into BALB/c susceptible mice results in uncontrolled growth of the parasite at the injection site with spread to local draining lymph nodes (21). In these mice the disease eventually involves the viscera causing the death of the animal (42). In susceptible mice, where parasite replication in the infected macrophage continues unhampered, there may be a defect in T cell recognition of parasite antigen as a result of downregulation of MHC Class II molecules on the macrophage surface (17, 41). C57BL/6 resistant mice, on the other hand, mount an effective immune response against the parasite very early in the infection process and are able to resolve their lesions (5, 16, 42). The involvement of T cells in resistance to disease has been demonstrated by immunosuppression treatment that blocks the cell-mediated response and by adoptive transfer of T cells (41). Menon et. al. (50), however, claimed that susceptibility or resistance to an L. major infection is not an absolute trait but one that is conditional on parasite strain and dose. 6 Infection of susceptible BALB/c mice with 102 or 103 Friedelin strain L. major parasites resulted in 0/10 mice producing lesions eight weeks post infection. Infection with 104 parasites resulted in 5/10 mice with lesions eight weeks post infection and infection with 106 parasites resulted in 10/10 mice producing lesions in the same time period. Infection of CBA resistant mice with 107 Freidelin strain parasites did not produce lesions but infection with 5 o x 10 parasites resulted in 6/10 mice producing lesions at eight weeks post infection. Infection of BALB/c mice with 10 parasites of the NIH 173 strain of L. major resulted in 1/6 mice with lesion development at eight weeks post infection, but all of these mice produced lesions eight weeks post infection when infected with 104 parasites. Infection of CBA mice with 104 parasites of the NIH 173 strain resulted in none of the mice producing lesions but on infection with 5 x 107 NIH 173 strain all of the CBA mice developed lesions in this eight week time period (50). This study showed that infection of BALB/c mice with about 1000-fold fewer L. major parasites than the dose used to define a susceptible phenotype did not cause progressive infection in these mice. Such an infection induced a cell-mediated Thl response with no detectable antibody production (50). Further, this Thl response to a low dose infection caused the immune system of these exposed mice to respond to challenge with parasite numbers that normally cause disease progression in unexposed mice with a Thl profile that defines a resistant phenotype. That susceptibility may be modifiable is important because it means vaccination protocols that are efficacious for everyone is possible despite host genetic diversity (50). 7 1.1.5.2 Susceptibility and resistance in a mouse model Evidence indicates that the patterns of susceptibility and resistance to leishmaniasis are due to activation and proliferation of distinct CD4 + T cell subsets (16, 38, 42,44, 46). These subsets are defined by the cytokines they produce following antigen recognition (5, 16, 36, 42, 46). In the genetically resistant individual there is a preferential induction of the Thl subset of CD4 + T cells producing a cell mediated response. It has been suggested that resistance to leishmaniasis involves the recruitment and activation of L3T5+ Thl cells (41). This response is characterized by the production of IL-2, IL-12, macrophage-activating factor (MAF), and IFN-y. IL-2 is involved in the differentiation and expansion of Thl cells. IL-12, a Thl growth factor produced by macrophages and dendritic cells (88), activates NK cells and ThO cells to produce IFN-y which in turn drives Thl differentiation and inhibits the expansion of Th2 cells. NK cell lytic activity is also enhanced by IL-12. IFN-y upregulates MHC expression and, alone or in synergy with TNF-a, activates infected macrophages to produce nitric oxide (NO) resulting in cell lysis (41, 86). The importance of the NO-dependent pathway in parasite killing in macrophages was demonstrated when C57BL/6 resistant mice were treated with NO inhibitors or were deficient for the NO gene in that these mice failed to heal their lesions when infected with L. major (85). It is known that L. major persists in low numbers in mice, even in resistant mice that have resolved an infection (85, 87). Treatment of these mice with iNOS (inducible nitrogen oxide synthase) inhibitors resulted in the expansion of parasites that had been quiescent or replicating very slowly (87). In genetically susceptible individuals there is a strong humoral response with preferential induction of Th2 cells and production of IL-4, -5, -6, -9, -10 and -13. IL-4 is involved in B 8 cell activation, inhibits the production of NO by infected macrophages causing an increase in parasite load (6, 15) and downregulates the IL-12 receptor (86). In mice IL-10 is involved in the inhibition of Thl expansion (16, 51) while IL-1, produced by infected macrophages, induces the expansion of Th2 cells (5, 16, 21). IL-13 has been shown to inhibit the production of IL-12 by macrophages and inhibits killing of L. major parasites in vitro. It has also been shown that IL-13 acts independently of IL-4 and is therefore a major factor in the immune response to leishmaniasis (88). The genetics of host resistance and susceptibility to infection with L. major has been well established in the mouse model but no comparable studies have been possible in humans (41). However, evidence for a polarized CD4 + T cell response does exist. A high level of patient-to-patient variability exists in the levels of Thl and Th2 cytokines produced in a human leishmanial infection. Studies have demonstrated that a weak or absent Thl response combined with high levels of anti-parasite antibody suggests a Th2 response in patients with non-healing cutaneous leishmaniasis. These patients often go on to develop DCL or LR (44). 1.1.5.3 Role of IL-4 and IL-12 in CD4+ T cell development in the murine model of infection with L. major Most immune responses involve contributions of both types of cytokines and Thl and Th2 responses tend to interact with each other in feedback loops (51). In the naive state all T cells are IL-4R+, IL-12Rprp2" and IFN-yRa+P+. Following activation with antigen and costimulatory molecules, the IL-4R is upregulated, T cells express IL-12Rpip2 and the IFN-yR is maintained. Cytokines now determine the T cell differentiation. IL-12 secreted by APCs binds to NK and undifferentiated (ThO) cells inducing the secretion of IFN-y. This 9 leads to stabilization of the IL-12R and a downregulation of the IFN-yRp subunit resulting in an IFN-y secreting Thl cell that is unresponsive to its own IFN-y, still responsive to IL-12, and potentially responsive to IL-4 through the IL-4 receptor (92). IL-4 inhibits IL-12 secretion, downregulates expression of the IL-12P2 subunit and has been reported to switch a Thl immune response to a Th2 response through activation of its own receptor on Thl cells (92). Evidence has been obtained for the role of IL-4 in Th2 cell development and IL-12 in Thl cell development in a murine model of infection with L. major (85, 86). IL-4 mRNA expression was examined in BALB/c and C57BL/6 mice during the first seven days of an L. major infection. Within 24 hours of infection BALB/c mice exhibited a peak of IL-4 mRNA expression in draining lymph nodes, however, IL-4 returned to baseline levels after 48 hours. From day five onward IL-4 mRNA levels increased again and continued throughout the course of infection. This early burst of IL-4 mRNA in BALB/c mice occurred in a subpopulation of Vp4Va8 CD4 + T cells. These T cells are specific for a single L. major antigen, LACK (Leishmania-activated C kinase), which has previously been identified as an important antigen in mice infected with this parasite (85). No early IL-4 mRNA was observed in C57BL/6 resistant mice. A slight elevation of IL-4 mRNA levels occurred around day five of infection but the levels returned to baseline by day seven (85). During the initiation of a leishmanial infection, treatment of BALB/c mice with anti-IL-4 monoclonal antibody, or with exogenous IL-12, has been shown to render normally susceptible BALB/c mice resistant to infection with L. major (85). Recent reports have also shown that BALB/c mice with complete disruption of the IL-4 gene are able to resist infection due to impaired Th2 cell development. Conversely, treating normally resistant C57BL/6 mice 10 with anti-IL-12 monoclonal antibody inhibited the development of a Thl response causing an expansion of Th2 cells and disease progression (38). The IL-4 produced 16-<48 hours after infection with L. major renders CD4 + T cells unresponsive to IL-12 and irreversibly drives the differentiation of precursor ThO cells toward a Th2 phenotype. Therefore, the effectiveness of treatment of BALB/c mice with exogenous IL-12 in either preventing IL-4 mRNA expression or inducing IFN-y mRNA expression is restricted to a very narrow time frame (38, 85). An important consideration in the development of a vaccine against leishmaniasis is whether an exclusive Thl response is necessary for protection, or whether a mixed Thl/Th2 response would be protective. An experiment was done in which an L. major surface antigen, PSA-2, was encoded in a plasmid and used to vaccinate C3H/He mice of intermediate susceptibility. PSA-2 in immune-stimulating complexes (iscoms), cage-like adjuvant structures that promote the development of a dual Thl/Th2 response, was also used as a vaccine. (Previously PSA-2 protein with Corynebacterium parvum as an adjuvant was shown to protect mice against infection with Leishmania). Iscoms induced a strong, but mixed Thl/Th2 response and although large numbers of CD4+ T cells secreting IFN-y were activated, the response did not protect against infection. In contrast, plasmid encoded PSA-2 activated low numbers of CD4 + T cells secreting IFN-y without detectable IL-4. This weak Thl response was sufficient to protect mice against infection. The results of this experiment suggest that a Th2 response nullifies the effect of that Thl response, even when a strong Thl response is present. This is further substantiated by the administration of anti-IL-4 antibody during the iscom immunization which led to a dampening of the Th2 response and concomitant protection against infection (46). Susceptibility to cutaneous leishmaniasis in mice is therefore correlated with the presence of IL-4 rather than the lack of IFN-y (46, 52). 11 1.1.6 Vaccine Requirements for Sustained Cellular Immunity to an Intracellular Parasitic Infection One of the problems with vaccination studies in humans is how to assess vaccine efficacy without challenge. It is important, therefore, to have a good animal model in which to measure vaccine effectiveness. Resistant mice, like humans, remain immune to reinfection. In BALB/c mice susceptibility is correlated with production of IL-4 and IL-10 which inhibit IL-12 responsiveness and the development of a protective Thl response (5, 16, 33, 36, 40, 41, 43, 45, 46, 47). Experiments by Gurunathan et al. (47) have provided strong evidence that long term cellular immunity to an intracellular parasite such as L. major requires continuous IL-12 production. In this study mice susceptible to infection with L. major were vaccinated with a plasmid encoding LACK DNA, or A L M (a whole cell killed L. major vaccine) plus recombinant IL-12, or A L M plus IL-12 DNA. All were effective in generating short term immunity against an L. major challenge. LACK DNA (without additional adjuvant) and A L M plus IL-12 DNA (but not A L M plus rIL-12 protein) were able to generate a long term immune response that controlled an L. major infection twelve weeks post vaccination. This data is consistent with results that show Thl cells are regulated continuously by IL-12 (48). It is likely that immunostimulatory sequences in the plasmid backbone also contribute to IL-12 persistence (47). It has been demonstrated in vitro that infection with the parasite reduces the capacity of macrophages to present exogenous antigen to CD4 + T cells. The suggested mechanism is the downregulation of MHC Class II molecules or other accessory molecules found on the surface of macrophages infected with the parasite (41). As well, Leishmania promastigotes and amastigotes have been shown to modify several signaling pathways including responsiveness to Thl cytokines (49). Data from Kima et. al. (17) have shown that after 12 transformation to amastigotes within a vacuole of an infected macrophage their antigens become sequestered from the MHC Class II pathway. Results suggested that amastigotes were able to internalize and degrade MHC class II molecules after fusion of the parasitophorous vacuole with other endocytic vesicles within the infected macrophage. Sequestration of antigen enhances the ability of these parasites to evade CD4 + T cell recognition, a major anti-leishmanial capacity of the host (17). However, it has been shown that immune manipulation of susceptible mice can overcome genetic susceptibility and experiments on vaccination against infection with L. major have demonstrated that resistance can be achieved in these mice (16, 36, 41, 42, 43, 45, 47, 53). 1.2 DNA Vaccines 1.2.1 Characteristics and Properties of a DNA Vaccine Vaccination with naked DNA involves a radical change in the method of antigen delivery. Typically, DNA vaccines are bacterial plasmids carrying genes that encode an antigen from a pathogen or tumor antigen under the control of a strong promoter. The promoter drives the expression of the encoded gene in a recipient host cell (84). The recombinant plasmid is administered, usually in saline solution, either by intramuscular or intradermal needle injection or by propelling DNA-coated gold particles through the dermis with a gene gun (2, 11). This DNA is taken up by host cells, either myocytes or antigen-presenting cells (APCs), or both, at the site of injection. Successful immunization depends on the plasmid DNA encoding the gene of interest being transcribed and translated by the host cellular machinery (3, 4, 5). Although circular DNA does not easily pass through cell membranes and very few 13 cells are transfected, the small amount of protein produced can lead to very strong immune responses in the absence of a secondary adjuvant. However, it is known that the bacterial DNA itself acts as a powerful immune stimulant (1). The plasmid DNA can persist in the cell as an episome for an extended period of time providing continuous low doses of mRNA leading to de novo protein synthesis (5,6). DNA vaccines have the potential to elicit protective immunity by inducing specific cell-mediated and humoral immune responses against the encoded antigen (23). In the last seven years protective cellular and humoral immunity has been demonstrated in over 30 studies involving a wide variety of pathogens in several different animal models. In rodent models these include protective responses against gpl60, env and rev from HIV; rabies surface glycoprotein; Mycobacterium tuberculosis Hsp65 and Ag85; core nucleoprotein (NP) and hemagglutinin from Influenza A; HBsAg of HBV; HCV core antigen; E6 of HPV; gpB from HSV; the malaria circumsporozoite; Nsl protein from Japanese encephalitis virus (JEV); ELI from Mycoplasma pulmonis, and SIV gag and env (1, 4, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16). Protection has also been demonstrated against influenza in chickens and cottontail virus in rabbits. (11, 13). Influenza HA or NP genes and HIV gp 120 encoded in plasmids have generated protection against challenge for at least six months in ferrets and rhesus monkeys (5, 8, 13, 17). DNA vaccines have also been used to generate immune responses against tumor antigens in a wide variety of animals such as mice and chimpanzees (8, 13). The efficacy of DNA immunization against tumor antigens for B cell lymphoma is currently being evaluated (18). Phase 1 clinical trials are now underway for Influenza A and HIV. Since DNA vaccines have been shown to be protective against a number of intracellular infections using a mouse model, this technology was used to test for the generation of a CD4 + 14 Thl response against Cpn60 from L. major in BALB/c mice susceptible to a leishmanial infection. 1.2.2 Vaccine Strategies To date, traditional vaccines have been very successful against many diseases. These successes include protection against diseases such as mumps, measles, rubella, polio, influenza A, smallpox, diptheria, tetanus, pertussis, typhoid, and hepatitis B to name just a few (2, 3, 19, 20, 91). Still, many pathogens lack an effective vaccine against them due to safety issues or lack of a potent immune response (2, 7, 22). Traditional vaccination strategies fall into two broad categories: (1) live, attenuated, vaccines and (2) killed, inactivated or subunit preparations. 1.2.2.1 Live attenuated vaccines (viruses and bacteria) Attenuated vaccines induce a diverse and long-lasting immunity while replicating in the host. These vaccines stimulate a protective response with a weakened version of the pathogen and because their pathogenicity is limited they do not routinely cause disease. However, there are possible disadvantages involving safety using live viruses as vaccines, (i) During viral replication in the host reversion to wild type or mutation to a more pathogenic organism is a major concern, (ii) A virus insertion into the host genome can result in induction of tumor formation, (iii) Interference with pre-existing immunity to the vector may be a problem, (iv) Even in the attenuated state viruses may still cause disease in the immunocompromised (2, 7). 15 1.2.2.2 Killed virus or subunit vaccines These vaccines do not replicate in the host and are therefore safe for immunocompromised individuals. They localize in the extracellular space only and generate a CD4 + T cell and humoral immune response but cannot induce a good CD8 + cytotoxic T lymphocyte (CTL) response against intracellular pathogens. The immunity induced by killed and acellular vaccines frequently wanes and repeated boosting may be required to achieve life-long immunity (2,1). 1.2.2.3 DNA vaccines vs. traditional vaccines DNA vaccines combine the positive features of live, attenuated vaccines with the safety of non-live vaccines. An important feature of DNA vaccine technology is that it offers strategies for protection against some diseases where protection has not been achieved by traditional vaccines (19). The advantages of a DNA vaccine preparation include the following: (i) DNA vaccines yield proteins that faithfully reproduce native conformation and contain mammalian post-translational modifications (2, 7). (ii) Like live attenuated vaccines, DNA vaccines produce long-lived host immunity and generate MHC Class I restricted host immune responses because the protein is synthesized endogenously. (iii) Unlike live attenuated vaccines, the possibility of pathogenicity with the vector is unlikely and therefore safety issues are no longer major concerns (1, 11, 12). (iv) DNA vaccines are simple and inexpensive to produce in large quantities using recombinant DNA technology which is now commonplace. These vaccines are easier to manufacture than are inactivated pathogens, subcellular fractions or recombinant protein vaccines because new technologies do not have to be invented for each new disease. Production of a DNA vaccine involves targeting different antigens for each 16 disease but the plasmid and basic molecular biology for production remain the same (20). (v) The bacterial plasmid serves as both the vector carrying the genes and as an adjuvant (1, 12). Genes inserted into a plasmid can be readily modified by site-directed mutagenesis. Amino acid modification can be designed to enhance antigenicity or to delete epitopes that trigger an unwanted immune response (3, 7, 17). (vi) DNA vaccines remain stable through a relatively wide range of ambient temperatures. The stability of the DNA at room temperature simplifies storage and transportation to remote areas. Currently, maintaining the chain of cold storage refrigeration accounts for 80% of the cost of vaccinating individuals in developing countries (2). (vii) DNA vaccines can be used to induce either a CD4+ Thl or Th2 immune response. The type of T helper cell induced with its associated cytokines is important in determining how effectively the body clears the infection (19, 22). (viii) It is relatively simple to combine different immunogens into a single vaccine preparation thereby decreasing the number of injections given (2, 17). (ix) Genes that encode important protective epitopes can be included whereas those that confer pathogenicity or virulence can be excluded (7). (x) Testing for efficacy of antigens is relatively easy in a DNA vaccine preparation whereas screening large numbers of purified proteins for a single disease can be difficult and time consuming (2). 1.2.3 Limitations of DNA Vaccines DNA vaccines share some of the limitations of traditional vaccine preparations. Like recombinant protein-based vaccines, the antigen conferring protection and the nucleotide sequence of the antigen must be known (1, 19). DNA vaccines target protein components of pathogens exclusively, therefore membrane-bound polysaccharides important in the control of certain pathogens lie outside the scope of these vaccines (19). Currently, DNA vaccines are 17 delivered most commonly into the muscle or skin by needle injection. Delivery of the vaccine by nasal spray or orally is desirable for some diseases. Most infectious agents enter the body through the respiratory, genital or urinary tracts. Vaccination for agents that cross mucosal surfaces is most effective when administered at the point of entry of the pathogen as memory cells patrol surfaces where the antigen is most often encountered (19). At this point there is a lack of experience using DNA vaccine technology in humans. Concerns about foreign DNA interacting with host DNA in instances where considerable sequence homology exists must also be addressed. Preliminary data suggests that the amount of DNA needed to vaccinate humans is much higher than the amount needed to successfully vaccinate rodents. 1.2.4 Immunology of DNA Vaccines 1.2.4.1 DNA vaccine intracellular route The plasmid encoding the gene for an antigenic peptide is taken up by host cells and the DNA is transported to the nucleus where transcription takes place. The mRNA is then translated into protein on ribosomes in the cytoplasm. As proteins are assembled a finite number undergo proteolysis into peptides in the proteasome. Al l peptides that enter the lumen of the ER are actively transported through association with TAP-1 and TAP-2 (transporters associated with antigen processing) proteins. Al l proteins produced within a cell are presented to the immune system in the context of MHC Class I molecules (7, 21). The antigen-MHC complex is directed to the cell surface where presentation of antigen to CD8 + cytotoxic T lymphocytes (CTL) takes place (7, 8, 21). The MHC class I recognition signal, along with secondary stimulation, in the form of co-stimulatory cell surface molecules from professional 18 APCs, and cytokines from T helper cells, drives the maturation of CTLs which are then able to recognize and destroy infected cells. Although host proteins are also presented in this same manner, normally the mechanism of self/non-self recognition suppresses CTL development against host proteins. CD4 + T lymphocytes are activated when proteins shed from infected cells into the extracellular space are internalized in professional APCs by endocytosis. The pH in endosomes containing protein antigen gradually decreases while progressing down the endocytic pathway and the antigen is degraded by acid proteases. MHC class II molecules pass through acidified endosomes and bind peptide fragments on their way to the cell surface where they induce T helper cells to secrete cytokines that activate other cells of the immune system (7, 21). 1.2.4.2 M e c h a n i s m o f i m m u n i t y o f D N A vacc ines The mechanism by which DNA vaccines stimulate the immune system is not completely clear (21). It was originally thought that muscle cells transfected with the recombinant plasmid presented antigen to T cells (94). Myocytes express low levels of MHC Class I molecules on their cell surface, but do not express MHC Class II and the costimulatory molecules needed for priming naive T cells and are therefore unlikely to function as antigen presenters (21, 95). It is more likely that bone-marrow derived APCs function to present plasmid-derived antigen to T cells after migration from the immunization site to local draining lymph nodes. Antibodies are generated when CD4 + T cells interact with B cells to produce an appropriate antibody response (7, 21). Two hypotheses have been proposed to explain antigen recognition by the immune system in response to DNA vaccines. The first hypothesis states that a small number of APCs 19 (dendritic cells or macrophages) are transfected directly with plasmid DNA. Upon migration to regional lymph nodes they activate CD8 + CTL. CD4 + T helper cells and B cells are activated by antigen shed from either transfected myocytes or other APCs (95). This mechanism may function in epidermal injection of DNA because the epidermis contains high levels of Langerhans cells, but resident dendritic cells and macrophages are scarce in muscle tissue (6,23,94). The second hypothesis proposes that antigen produced by transfected myocytes is transferred to APCs that have infiltrated the muscle as part of the inflammatory response associated with needle injection. The transferred protein could then 'cross over' into the MHC Class I pathway allowing the antigen presenting cell to prime CD8 + CTL responses (95). This theory contradicts the current understanding that only endogenously produced antigen can enter the MHC Class I pathway. However, researchers have recently described how antigen in particulate form can be taken up by APCs and presented in the context of MHC Class I molecules (23, 96). There is evidence to support both of the above hypotheses. Chattergoon et al. (7) have demonstrated that direct transfection of macrophages and dendritic cells can occur after intramuscular DNA injection. Plasmid DNA has been isolated from dendritic cells after intramuscular injection and from Langerhans cells after intradermal injection. Torres et al. (24) reported that complete surgical removal of injected muscle as early as ten minutes after injection did not prevent the induction of antigen-specific CTL or antibody responses. This suggests a limited role for myocytes in the induction of the immune response and gives support to the direct transfection of APCs by plasmid DNA. Ulmer et. al. (15) reported that transplantation of myoblasts stably transfected with influenza A nucleoprotein (NP) induced 20 both anti-NP antibodies and antigen-specific CTL responses, and protected against a lethal challenge with influenza A virus in recipient mice. This would suggest that the antigen transfer from muscle cells to APCs was the mechanism for the priming of CTL as there could be no direct transfection of APCs (15, 95) 1.2.4.3 Dendritic cells are the most potent antigen-presenting cells Dendritic cell progenitors originate in the bone marrow of mammalian adults, enter the bloodstream and seed non-lymphoid tissue where they develop optimal capability for antigen uptake and processing. These cells are localized in epithelial tissue, genitourinary tract, gut, lung, and in the interstitial spaces of solid organs. After maturity the cells enter secondary lymphoid tissue where they initiate primary T cell responses (21). Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced in response to inflammation or injury appears to recruit dendritic cells to local sites. Non-lymphoid dendritic cells synthesize and secrete IL-12, which is involved in the differentiation of T helper cells, and seem to perform a "sentinel" function for the immune system. As a rule, dendritic cells migrate from epithelia and solid organs to regional lymph nodes via afferent lymph vessels and from solid organs to the spleen via the blood. Migration from non-lymphoid to lymphoid tissue is promoted by IL-1 p and tumor necrosis factor (TNF) generated during an inflammatory response (26). The success of DNA vaccination appears to be linked to the activation of T cells initiated by DC. IL-12, secreted by DCs and other APCs, is a key link between the innate and cell-mediated immune systems. This cytokine induces IFN-y secretion from N K and Thl cells and enhances the proliferation of antigen-specific T cells (80). 21 Shankar et al. (25) examined the influence of dendritic cells, B cells, macrophages and IFN-y on priming of L. major specific T cells. In this study a primary in vitro (PIV) system was developed in which Leishmania wq/'or-specific T cells were derived by co-culturing naive murine lymphocytes with L. major. Spleen cells from naive BALB/c mice depleted of macrophages, B cells and dendritic cells prevented the priming of T cells. Purified APCs were then added back to the depleted spleen cells. Data showed that mixing purified dendritic cells, naive T cells and L. major resulted in priming of L. mq/or-specific T cells. It was also found that dendritic cells contributed most strongly to priming however maximum T cell priming occurred only when dendritic cells, B cells and macrophages were all present (25, 26). Given that macrophages are the host cell for Leishmania, it is possible that parasite antigens can be shed by infected macrophages and taken up and presented by dendritic cells and B cells. Evidence presented by Casares et al. (6) also suggested that uptake of DNA by dendritic cells triggers the immune response to DNA vaccines. Antigen presenting cells were isolated in vitro from draining lymph nodes or skin after intramuscular or intradermal injection of DNA. It was found that dendritic cells, but not B cells, carried plasmid DNA and presented antigen to T cells. 1.2.5. The Plasmid Vector as an Adjuvant The immune adjuvant properties of bacterial DNA were first discovered using mycobacterial DNA. Freund's adjuvant uses extracts from mycobacteria and the immunostimulatory effects are associated with severe inflammatory and toxic side effects which precludes their use in humans (34). Recent studies done using mycobacterial DNA 22 demonstrated that IFN-y production resulted from DNA motifs that have an unmethylated CpG core (29). Mammalian immune systems respond to bacterial infection by rapidly initiating an inflammatory reaction that limits the early spread of the pathogen and primes antigen-specific immunity (27). Immune recognition of unmethylated CpG dinucleotides in bacterial DNA, but not vertebrate DNA, contributes to the host inflammatory response. There is a difference in the frequency of unmethylated CpG dinucleotides in the two genomes. The expected frequency of CpG in bacterial DNA is 1/16, but in vertebrate DNA is 10-20 fold less, (a phenomenon known as CpG suppression), and the majority of CpG dinucleotides in vertebrate DNA contain a methylated cytosine (22, 28). The possibility exists that a role in innate immunity has led to maintenance of CpG suppression in mammalian DNA (29, 34). Immune activation by bacterial DNA is sequence dependent and is optimized when the CpG dinucleotides are flanked by two 5' purines and two 3' pyrimidines forming a 6 base immunostimulatory sequence (ISS) (22, 29. 30, 31, 32). Upon entering the cell, bacterial DNA may encounter intracellular regulatory proteins whose binding to CpG motifs leads to downstream activation events inducing an immune response (22, 29). The DNA appears to trigger an immune response by (i) activating B lymphocytes, inducing them to proliferate and become resistant to apoptosis; (ii) inducing the secretion of immunoglobulins (32, 33); (iii) inducing a rapid nuclear translocation of NF-KB with upregulation of TNF-a mRNA in antigen-presenting cells, specifically B cells and monocytes (33, 34); (iv) inducing the secretion of high concentrations of IL-12 by macrophages which regulates IFN-y production, IL-18, involved in T cell activation, and IL-6, involved in B cell activation (33, 34) and (v) inducing production of TNF-a and IFN-oc,p by monocytes and macrophages (32, 33, 34). Cytokines produced by macrophages in response to CpG also induce activation of IFN-y-23 secreting NK cells thereby enhancing the capacity of macrophages to eliminate intracellular and extracellular pathogens (32, 33, 35. 36). Early IL-12 and IFN-y may be decisive in the induction of antigen-specific CD4 + Thl cells (1, 92). Vertebrate DNA lacks these immune stimulating properties (33). Evidence for activation of the immune system by ISS resulted from studies where immune stimulation was inhibited when DNA within the ISS region was mutated, yet mutations in the DNA outside the consensus region had little effect (28, 97). Methylation of bacterial DNA by CpG methylase also eliminated immune cell activation (97). Krieg et al. (32) recently reported that CpG acts as an immune enhancer when combined with antigen to promote an antigen-specific response. However, enhancement of the immune response may be dose dependent. In a mouse study, protection against Listeria monocytogenes was dependent upon the dose of CpG DNA administered. The optimal dose was 10 to 30 pg while higher doses reduced protection. Previous studies have demonstrated that high doses of CpG DNA, in the range of several hundred ug, cause mice to develop sepsis syndrome characterized by overwhelming inflammation (32). Sato et. al. (97) reported that plasmids containing ISS induced a more vigorous antibody and CTL immune response than an otherwise identical vaccine without ISS. Subsequent studies have confirmed that CpG motifs enhance immunity by preferentially inducing a Thl response (29, 37). Co-injection of an ISS-containing plasmid with a protein antigen that normally induces a Th2 response in mice shifted the immune response back toward a Thl response with IFN-y production. This Thl inducing capacity of bacterial DNA containing ISS may be a reason why most DNA vaccines studied to date induce a predominantly Thl profile when injected intramuscularly or intradermally (37). 24 Certain synthetic oligodeoxyribonucleotides (ODN) with core unmethylated CpG motifs within ISS mimic the immunostimulatory effects of bacterial DNA (30). These ODN activate APCs to upregulate the expression of costimulatory molecules B7.1 and B7.2, and secrete TNFa, IL-1, IL-6 and high levels of IL-12 in vivo (30). Recently, a study involving BALB/c mice infected with Leishmania major was carried out to determine if CpG-ODN promoted Thl development needed to resolve a L. major infection. In this study CpG-ODN treated mice developed resistance to infection while untreated animals developed severe disease and were euthanised eight weeks post-infection. Control ODN, without the core CpG motifs, failed to influence the infection outcome (34). Also, in a study testing CpG induced Thl responses in murine leishmaniasis, lymph node cells (LNCs) from ODN-treated mice produced high concentrations of IFN-y, but no IL-4, when stimulated with L. major antigen in vitro. Thl development may be induced through the ability of CpG-ODN to prevent L. major mediated suppression of IL-12 production (30, 38). Lipford et al. (34) identified a syn-thetic ODN sequence that would activate IL-12 while suppressing the toxic effects of TNF-oc cytokine production using L. major infected BALB/c mice as an animal model. 1.2.6 Modulating the Immune Response to DNA 1.2.6.1 Method and location of immunization In addition to using plasmids containing ISS that preferentially induce a Thl CD4 + T cell response, the route and method of delivery can affect the type of immunity that is induced (1). Table I (22) illustrates the type of immune response generated using different routes and methods of DNA delivery. Gene gun-mediated DNA delivery is much more efficient than needle injection. Intramuscular needle injection requires 10-100 u.g of DNA, while as little as 25 16 ng of plasmid DNA delivered intradermally with a gene gun induces similar levels of antibody and CD8 + CTL responses in mice (98). The large amount of plasmid DNA with its Thl-inducing properties delivered by injection could possibly be the reason why injected TABLE I. The effect of immunization site and delivery methods on the orientation of the immune response to DNA vaccines. Immunization Delivery vehicle T helper response generated site Muscle Needle injection Thl Muscle* Gene gun Th2 Skin Needle injection Thl/Th2 Skin Gene gun Th2 Mucosal Inhaled drops or Th2 liquid suspension Although both the gene gun and needle injection methods have been used for immunizations in skin and muscle, the gene gun has been more commonly used for epidermal rather than intramuscular injections DNA vaccines induce a Thl response. Gene gun immunization requires much less plasmid to be delivered because cells are transfected directly whereas needle injection delivers the vaccine to the extracellular space and only a small amount of the vaccine is actually taken up by cells. The uptake of plasmid by needle injection may trigger a set of intracellular signals that ultimately leads to Thl cytokine production while gene gun inoculation bypasses any uptake mechanism and may trigger a different set of intracellular signals (23). 26 1.2.6.2 Immunization regimen The dose, number, and frequency of injections of the vaccine may determine which T helper subset is induced. Immunizing mice with a plasmid containing the HIV-1 env delivered by a gene gun induced strong CTL responses when one to three immunizations were given. However, a fourth immunization caused a marked drop in CTL responses with a concomitant increase in antibody titers and a shift in the types of cytokines produced by antigen-stimulated splenocytes. A decline in IFN-y secretion has also been reported following the first and second boosts after injection of plasmid encoding the influenza nucleoprotein (NP) gene (99). Unlike the administration of HIV-1 env plasmid with a gene gun, the decrease in IFN-y levels in the NP experiment was not accompanied by an increase in the levels of IL-4 (99). Timing of immunizations may also influence the immune response in mice. A longer rest period between immunizations (6 weeks) has also been found to be immune enhancing (100). 1.2.6.3 Co-administration of cytokines and co-stimulatory molecules Co-injection of plasmids encoding cytokines with antigen-encoding plasmids has also been found to enhance an immune response (reviewed in 22). Table II lists various cytokines that have been encoded in plasmids and co-administered with a DNA vaccine to test for immune enhancing capabilities. The effectiveness of DNA vaccination can also be strengthened by co-delivery of plasmids encoding genes for costimulatory molecules B7.1 (CD80), B7.2 (CD86), or CD40, with the goal of enhancing the antigen-presenting capabilities of transfected cells (101). 27 T A B L E II. Effect of co-administration of plasmids encoding cytokines with D N A vaccines on the immune response Gene Effect on immune response Proinflammatory cytokines IL-1 Increased antibody production; Th unchanged; increased CTL response TNF-a Increased antibody, Th and significant increase in CTL response TNF-P Increased antibody, Th and CTL responses TCA3 1 Decreased antibody response; increased Th and CTL responses GM-CSF Increased antibody, Th and CTL responses Thl inducing cytokines IL-2 Increased antibody, Th and CTL responses IL-12 Decreased antibody response; increased Th and *CTL responses IL-15 Increased antibody response; Th unchanged; * increased CTL response IL-18 Increased antibody, Th and CTL responses IFN-y Decreased antibody and Th responses; CTL response unknown Th2 inducing cytokines IL-4 Increased * antibody and Th responses; decreased CTL response IL-5 Increased *antibody and Th responses, CTL response unchanged IL-10 Increased * antibody and Th responses; CTL response unchanged i TCA3 is a p-chemokine chemotactic for monocytes/macrophages and neutrophils. * Response markedly enhanced. To date these responses have only been studied in rodents. These co-stimulatory molecules provide a second signal necessary for efficient MHC-restricted T cell activation (84). Studies in mice have shown that co-delivery of B7.2-containing plasmids significantly enhances the CTL response (55, 84) when co-injected with plasmids encoding influenza A NP or HIV-1 proteins. Co-injection of plasmids encoding CD40 increased antibody and CTL responses (7, 35, 101). Manipulation of the immune response by these methods may allow researchers to tailor the immune responses to DNA-based vaccines based on the 'correlates of immunity' for a particular disease. This technology has been tested mainly on rodents and further work needs to be done in an animal model such as primates to determine if these immune modifying factors hold true (7, 35). 28 1.2.7 Leishmania Antigens Employed in Vaccine Studies to Date Primary immunization involves the introduction of antigens which will evoke an optimal immune response and generate memory cells for a secondary encounter with the same antigen. Leishmanial antigens important in the induction of a protective host immune response are not fully known (40). A number of studies have been done employing different leishmanial antigens in vaccine preparations to determine the protective efficacy of each of the test vaccines. A major surface glycoprotein, gp63, of L. major has been used in a number of vaccine preparations (16, 36, 43, 53). Xu and Liew (16) reported that mice immunized with gp63 encoded in a plasmid developed significant resistance to challenge compared to controls. Spleen cells from immunized mice produced IL-2 and IFN-y but no IL-4 when stimulated in vitro with leishmanial antigen. A similar experiment by Walker et al. (36) using a gp63 plasmid construct found that only 30% of susceptible mice were protected from challenge with L. major despite the fact that individual mice produced Leishmania-specifc T cells and IFN-y. It was hypothesized that the L. major parasites evaded gp63-specific T cells by down-regulating gp63 on their cell surface after intracellular transformation. The ability to evade the immune system by downregulating cell surface molecules is typical of other parasitic infections such as trypanosomiasis and malaria (36, 40). Gp63 protein used in a vaccine preparation was not protective (36). L. major gp63 transformed into the AroA" strain of Salmonella typhimurium (S13261) was used to immunize CBA mice orally. These orally immunized mice developed significant resistance to challenge with L. major (43, 53). A DNA vaccine preparation encoding LACK induced protection in BALB/c mice. Protection, however, was shown to be dependent on a CD8 + cytotoxic T cell response rather than a CD4 + 29 T cells response (47). Another DNA preparation encoding L. major PSA-2 (parasite surface antigen-2) primed a Thl response that protected C3H/He mice against challenge (46). BALB/c mice were protected from a fatal L. major infection by immunization with whole, irradiated promastigotes and these experiments concluded that the development of an antibody response to parasite surface antigens is not necessary for protection (45). Walker et al.(36), however, reported freeze-thawed parasites were not efficacious as a vaccine. L. major antigens used in protein based vaccines include a 22.1 kDa leishmanial protein referred to as TSA because of its significant homology to eukaryotic thiol-specific antioxidant proteins (42) and Hsp60, a member of the heat shock family of proteins (also known as chaperonin 60, Cpn60) (54). TSA was found to induce a strong cell-mediated response and conferred protective immunity to BALB/c mice against infection with L. major when the protein was co-delivered with IL-12. Immunization of BALB/c mice with the 60 kDa heat shock protein failed to protect these mice from challenge. As well, mice immunized with rLHsp60 plus IL-12 were not protected. At five weeks post-infection, lesions in these mice were the same size as lesions in mice immunized with IL-12 alone. In this study, immunization with IL-12 alone did not substantially control parasite load (54). 1.3 Chaperonin 60 The heat shock protein chaperonin 60 (Cpn60) is involved in the stress response, translocation of proteins and in the folding of nascent protein chains (56, 62). These proteins are known by a variety of names: 60 kDa chaperonin, Hsp60, Cpn60, GroEL, 60 - 65 kDa antigen, bacterial 'common antigen', and mitochondrial PI protein (57). The first member to 30 be identified was GroEL from E. coli. In eukaryotes Cpn60 is constituitively expressed and targeted to mitochondria by a positively charged amino terminal sequence. Heat shock causes a two to three-fold increase in expression of this protein. Cpn60 represents about 0.3% of total cell protein and is induced by heavy metals, amino acid analogs, fever, inflammation, injury, viral and bacterial infections, and aging (56, 58, 62, 81). Chaperonin proteins are highly conserved over a wide range of organisms. Al l prokaryotic and eukaryotic cells investigated so far contain a heat shock protein with a molecular weight of about 60 kDa and sequence analysis shows a high conservation between the different proteins (between 45 and 55% sequence identity). Al l available evidence suggests the mitochondrial Cpn60 appears to be a homo-oligmer like the GroEL complex (58, 62). Host cells and microbes are confronted with extreme alterations in their environment during infection. Under these conditions, upregulation of heat shock protein synthesis in pathogens is vital for their survival. Increased levels of Hsps in pathogens leads to rapid degradation of these Hsp proteins by the host cell processing machinery. Pathogen derived antigen is then efficiently presented to the host immune system (81). The 60 kDa protein is one of the most potent stimulators of the immune system known and constitutes the major antigenic protein of numerous pathogenic bacteria such as Mycobacterium tuberculosis, Mycobacterium leprae and Yersinia enterocolitica (56, 60, 61). A remarkable feature of the T lymphocyte response to chaperonins is its dominance over the response to other antigens. At the onset of infection chaperonins act directly on the innate immune system. The immune system then selects chaperonins from hundreds of other proteins and mounts a major adaptive immune response (56, 59). Bacterial chaperonins have a high sequence similarity to mammalian chaperonins. It is somewhat surprising that the immune system would invest 31 such a high proportion of its immune energy into T cells that may cross-react with self-chaperonins. Two possible reasons for this phenomenon are that these proteins are abundant in pathogens under stress conditions and frequent encounters with different microbes by the host generates immunological memory to cross-reactive epitopes in conserved Hsps. This prepares the immune system to respond more quickly to Hsps than to more pathogen-specific antigen (56, 62, 81). 1.3.1 Immunology of Chaperonins 1.3.1.1 Chaperonin 60 and the immune response The 65kDa antigen from M. tuberculosis induces the secretion of proinflammatory cytokines TNF-a and IL-1 from human monocytes and macrophages. TNF-a is involved in the localization of mycobacteria into granulomas and IL-1 is believed to be important in the differentiation and function of lymphocytes. However, MHC Class II molecules are not upregulated on the cell surface and there is no increase in reactive oxygen intermediates (ROI). Phagocytes, therefore, are not classically 'activated' (63, 64). It is hypothesized that chaperonin-monocyte interaction may provide a first line of defense for the innate immune system and may also function as an early warning system for the adaptive immune response (56). Immunization of animals with whole lysates of bacteria results in a powerful immune response mounted against chaperonins characterized by (i) anti-chaperonin antibodies and (ii) antichaperonin T lymphocytes. Antichaperonin antibodies may be incidental to a protective immune response since, primarily, chaperonins are intracellular proteins shielded from antibody binding. In Mycobacterium tuberculosis immunized mice, one in five of the reactive T lymphocytes responds to mycobacterial Hsp65. T helper lymphocytes (Th2) assist B 32 lymphocytes to make antibody and this may explain why anti-chaperonin antibodies are found with such high regularity in infections (56). 1.3.1.2 T c e l l r e c o g n i t i o n o f c h a p e r o n i n s The majority of T cells recognize antigen through the T cell receptor (TCR) which consists of two chains, a and p. These cells require peptide fragments to be presented to them bound to MHC Class I molecules found on the surface of most cells or bound to MHC Class II molecules found on the surface of professional antigen presenting cells (81,86). A smaller subset of T cells, about 1.5% of the total T cell population, expresses a TCR that has y8 chains. These cells usually do not express CD4 or CD8 molecules on their surface. Although they can contribute to immunity against invading pathogens, the mechanism of antigen recognition is not completely understood (81). Human T cells react with epitopes of chaperonins that are shared between self and non-self This suggests that self-chaperonin reactive T cells are not deleted in the thymus but are a normal component of the immune system (21, 56, 65). Ten to twenty percent of this T cell population in the adult mouse recognize Cpn60. Also, human peripheral y5 T cells recognize Cpn60 on the surface of human cells. There is evidence that these y8 T cells perform an immune surveillance role where they recognize and lyse infected, malignant, or senescent host cells. (56). Steinhoff et al. (66) showed that host Cpn60 is processed in stressed cells and that epitopes bound to MHC Class I molecules are presented to CD8 + T cells. These epitopes contain amino acid sequences that are located in highly conserved regions on heat shock proteins and are also present in microbial agents and in dietary protein. It can therefore be concluded that conserved peptides from chaperonins, whether from self, from infectious 33 agents, or from dietary protein, are processed in normal cells in MHC Class I and Class II pathways for presentation to T lymphocytes (56, 66). This appears to be a universal phenomenon that should be regarded as physiological (56, 65). Whether this increases the risk of autoimmune disease is unknown, but the likelihood is very small, as infectious disease is common whereas autoimmune disease is rather uncommon. 1.3.2 Vaccine Studies with Plasmids Encoding Cpn60 (Hsp65) as Antigen Vaccine studies have shown that immunization with plasmid encoded Cpn60 from different pathogens is protective upon challenge with the pathogen. Protection has been demonstrated using Cpn60 from the following organisms: Yersinia enterocolitica (61), Mycobacterium tuberculosis (60, 67, 68), Legionella pneumophila (82), and Histoplasma capsulatum (83). Because of the abundance of this protein in pathogens under stress conditions and because it serves as an important antigen in defense against infectious agents the Cpn60 protein appears to be a good candidate for use as a prophylactic vaccines against leishmaniasis caused by L. major. 34 II. METHODS AND MATERIALS 2.1 EMBL -3 Phage DNA Preparation From Plate Lysate The X EMBL-3 phage containing the coding sequence for the L. major Cpn60 gene was a gift from Dr. Neil Reiner (Health Sciences Center, Vancouver Hospital, Vancouver, B.C., Canada). 2.1.1 Growth ofE. coli Strain LE392 E. coli strain LE392 was provided by Dr R. McMaster (Dept. of Medical Genetics, University of British Columbia, Vancouver, B.C). The bacteria were grown to saturation overnight in a shaking water bath (225 rpm) at 37°C in Luria-Bertani (LB) broth supplemented with 0.2% maltose and lOmM MgS04. Growth in maltose induces production of the X receptor in E. coli and M g + + aids in phage adsorption. 2.1.2 Plate Preparation LB agar (100 x 15 mm. culture dishes) was prepared by standard protocol (69). Electrophoresis grade agarose (Sigma, Oakville, ONT) was substituted for Bacto-agar in the preparation. If agar is replaced with agarose in both the plate recipe and in the top agar then DNA extracted from phage in the lysate is an acceptable substrate for restriction enzymes and ligases (69). 35 2.1.3 Plate Lysis Method 1 x IO5 pfu (from a stock of 1.3 x 1010 pfu/ml) were mixed with 0.1 ml of an overnight culture of E. coli and incubated at 37°C for 20 min. Freshly prepared top agarose (3.0 ml) was added to the phage^acteria mixture and poured onto freshly prepared LB agarose plates. After the top agarose was firm, the plates were inverted and incubated for 10 - 12 hours until lysis was confluent. Five (5) ml of diluent buffer (20 mM Tris, pH 7.4, 10 mM MgS04) was added to the plate and incubated at 4°C for several hours with intermittent shaking. This was followed by harvesting of the phage. Fresh buffer (1.0 ml) was added to the plate and stored at 4°C for 15 minutes in a tilted position. This buffer was then added to the first harvest. The lysate was treated with chloroform (1 ul/ml) to lyse any residual bacteria and centrifuged at 4000 g for 10 min to remove bacterial debris and residual agarose. The plate lysis procedure yields approximately 4 - 5 ml of a phage stock containing 1010- 1011 phage/ml. Following centrifugation, the X DNA was extracted from the lysate using a Qiagen Lambda starter kit (Qiagen, Chatsworth, CA). 2.2 PCR of the L. major Cpn60 Gene Primers for amplification of the L. major Cpn60 gene were designed from the initiation and termination sequences of the gene (sequences shown below). A Kozak consensus sequence (underlined) was introduced and restriction sites (bold letters) for Hindlll and EcoRI were introduced into the 5' ends of the forward and reverse primers respectively. Additional nucleotides were also added to the 5' ends of the primers for more efficient endonuclease cleavage. L. major Cpn60 sequences are shown over the broken line. 36 Forward Primer : 42 bases 5 ' CCC A A G C T T GCC A 3 C C ATG C + 4 TT TCT CGT ACT GTG CCT CGT TGT 3 ' Reverse Primer: 39 bases 3 ' GTT CTA GGC CGG CGG CTA CTT CAA ACT GGC C T T A A G GCC 5 ' < - -Amplification of the gene was carried out using a Robocycler, (Stratagene, La Jolla, CA). Components of the reaction mix in a total volume of 50 ul were as follows: Vent DNA polymerase exo+ (1.25U) and Vent reaction buffer (lx) (New England Biolabs, Mississauga, ONT), forward and reverse primers: 1 uM (BioCan, Mississauga, ONT) and dNTPs: 200 uM. Thermocycling conditions: initial denaturation 94°C for 5 min, cycle denaturation 94°C for 1 min, cycle annealing 68°C for 2 min, cycle elongation 72°C for 10 min (30 cycles) and a final extension of 72°C for 10 min. 2.3 Agarose Gel Electrophoresis and Restriction Analysis of the L. major Cpn60 1.7 kb Gene The PCR products were loaded in sample buffer (10% glycerol, 0.04% bromophenol blue in lx TAE buffer, 40 mM Tris, 20 mM acetic acid, 2mM EDTA (pH 8.0) and run on a 0.8% agarose gel containing 0.1 |ig/ml of ethidium bromide (EtBr) in lx TAE. A PCR product in the expected size range of 1.7 kb was detected. The remaining PCR products were cut with BamRl and digestion products were run on a 0.8% agarose gel against a 100 bp DNA ladder to confirm the correct PCR fragment. 37 2.4 Subcloning of the L. major Cpn60 into pcDNA 3.1 pcDNA 3.1+ (Invitrogen, San Diego,CA) is a 5.4 kb eukaryotic expression vector carrying the human cytomegalovirus (CMV) promoter, a T7 promoter/priming site, BGH reverse priming site and BGH polyadenylation signal, ampicillin resistance gene and a multiple cloning site. Both plasmid and insert were cut with restriction enzymes Hindlll and EcoRl (Gibco/BRL, Burlington, ONT) as follows: a one-step double digestion was done in which both restriction enzymes were added in one reaction. 2.4.1 pcDNA3.1 Plasmid DNA (10 ug) was cut with£coRI (10 U) and Hindlll (10 U) in lx React 3 reaction buffer (Gibco/BRL) in a total reaction volume of 20 al and incubated according to manufacturer's instructions. A 10 ul volume of the cut plasmid was run on a 0.8% low melting point (LMP) agarose gel without ethidium bromide. Following electrophoresis, the gel was incubated briefly (25 min) with ethidium bromide (0.1 pg/ml) in 30 ml of lx TAE buffer with gentle shaking. The 5.4 kb band was cut out of the gel under UV light and purified with a Qiaquick Gel Extraction kit (Qiagen, Chatsworth, CA). 2.4.2 The 1.7 kb Cpn60 Gene The 1.7 kb DNA fragment from the PCR reaction (10 pi) was cut with the same restriction enzymes and purified in the same manner as the plasmid DNA. The concentration of both the plasmid DNA and the amplified Cpn60 DNA was determined by running serial dilutions of a known concentration of X DNA ranging from 0.5 pg to 15 ng on a 1.0 % agarose gel alongside the purified DNA. The concentration of plasmid DNA was determined to be 38 approximately 50 ng/p.1 and the concentration of purified Cpn60 DNA was approximately 10 ng/ul. Molar ratios of plasmid to insert were calculated as follows: Mass ratio of plasmid: insert was 5.4 kb:1.7 kb respectively, which gives a 3:1 mass ratio A 1:1 molar ratio would contain 50 ng of plasmid:16 ng of insert.. Molar ratios of 1:1, 1:3 and 3:1 of vector: insert (50:16 ng, 50:48 ng, and 50:5.3 ng plasmid:insert were used in ligation reactions. Ligation reactions with T4 DNA ligase (1U) in lx T4 DNA ligase buffer (Gibco/BRL, Burlington, ONT) were prepared in a total volume of 10 ul. Following an overnight incubation at 4°C the reactions were incubated at 65°C for 10 min. to inactivate the ligase. Samples were stored at -20°C. 2.5 Transformation of E. coli Electrocompetent Cells Transformation of E. coli TOP 10 F' electrocompetent cells (Invitrogen, Carlsbad, CA) with the pcDNA/Cpn60 recombinant plasmid and the pcDNA3.1 empty plasmid was carried out according to manufacturer's directions. Briefly, DNA (50 ng) was added to three separate microcentrifuge tubes on ice. Electrocompetent cells were thawed on ice, tapped gently to mix, and an aliquot added to each of the microcentrifuge tubes containing the DNA. The cell/DNA mixture was incubated on ice for 10 minutes then transferred to a chilled microelectroporation chamber. Electroporation was carried out at 300 V. Cells were immediately removed from the chamber and added to 1 ml of room temperature SOC. A control transformation was carried out using empty pCDNA 3.1 according to the same protocol. Following electroporation, cells were grown for one hour in SOC medium (Gibco/BRL, Burlington, ONT), plated on LB-ampicillin plates, (0.06 mg/ml ampicillin) and 39 incubated overnight at 37°C. Ten colonies were selected, grown overnight in 6 ml of LB inoculated with ampicillin (0.06 mg/ml) in a rotary shaking water bath (225 rpm) at 37° C. One (1) ml from each of the ten overnight cultures was stored in glycerol at -80°C. The remaining 5 ml of each culture was then purified using a plasmid purification kit (Qiagen, Chatsworth, CA). Purity and concentration of the recombinant plasmid was measured by spectrophotometric methods. The samples were cut with EcoRl and Hindlll to mobilize the 1.7 kb fragment from the recombinant plasmid. 2.6 Sequencing of the L. major 1.7 kb Cpn60 Gene Six sets of overlapping primers (BioCan, Mississauga, ONT) were designed to fully sequence the insert in the forward and reverse directions. There were approximately 350 bases between primer pairs with an overlap of approximately 50 bases with the neighbouring primer pair. Primer sequences are as follows: Set # 1 (IF) 5 C C C A A G CTT GCC ACC ATG CTT TCT CGT ACT GTG CCT CGT TGT 3 (IR) 5 T C G CCC CGG AGA GGA C A G T A G 3 ' Set # 2 (2F) 5 A C G ACA ACG CCG GTG ACG G C A 3 ' (2R)SCCC ACG CTC A A A GGA CAT CCC 3 ' Set #3 (3F)5'CGC TTA ATA CGG AGC TCG AGC 3 ' (3R) 5CGA CCC CGT GAA GAC GGC G A T 3 ' Set #4 (4F)5'GCG ACA TGC GCA TCA ACC AGC 3 ' (4R) 5GAT GCG GTC CTT CTT CTC A T T 3 Set #5 (5F)5'GAT CAA GGT CGG TGG CGC C T C 3 ' (5R)5 ATT CAC ATA CTC GCC GGT C T G 3 ' 40 Set #6 (6F)5'CGC GCG CAA GGA TCC GAG CTT 3 ' (BGHR) 5 TAG A A G GCA CAG TCG AGG CTG v Note: Primer IF is the original PCR forward primer. BGHR is the sequence of the BGH reverse priming site on the plasmid. Primers were diluted to a concentration of 3.2 uM for the sequencing reactions. Al l sequencing was done by the DNA sequencing laboratory at UBC. 2.7 Site-Directed Mutagenesis To correct the base sequence of L. major Cpn60 for the deletion of the 'G ' 45 bases from the 3' end of the gene, site-directed mutagenesis was carried out using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA) according to manufacturer's directions. 2.7.1 Primer Design Primer sequences were designed to incorporate the desired mutation (bold underline) with 15 correct bases on either side of the base to be inserted. Primers anneal to the same sequence on opposite strands of the plasmid. Both primers (BioCan, Mississauga, ONT) were 33 bases in length, with a T m = 81.7°C. (It is recommended that primer T m be equal to or greater than 78°C.) SDM Forward primer: 5 C A A GCA GGT A A A TGA GGC GGG ACG CAC CG TGC 3 SDM Reverse primer 5 G C A TCG GTG CGT CCC GCC TCA TTT ACC TGC TTG 3 41 2.7.2 Reaction Mix and Cycling Parameters Reaction mix: (sample) 1 x Reaction buffer (Stratagene) 5 - 50 ng of dsDNA template 125 ng forward primer 125 ng reverse primer dNTP mix ((Stratagene) ddFfiO to a final volume of 50 pi Pfu DNA polymerase (2.5 U) Control reaction (pWhitescript) lx Reaction buffer 10 ng pWhitescript 125 ng #1 control primer 125 ng #2 control primer dNTP mix (Stratagene) ddH^O to final volume of 50 ul Pfu DNA polymerase (2.5 U). TABLE III. Cycling parameters for pcDNA3.1/Cpn60 gene Cycles Segment Temperature Time 1 Initial denaturation 95°C 4 min 16 Cycle denaturation 95°C. 1 min Annealing 68°C 2 min Elongation 72°C. 2 min/kb of plasmid length Note: Cycling parameters for the pWhitescript control plasmid as per Stratagene protocol. Following temperature cycling the reactions were cooled to <37°C by placing on ice for 2 minutes. Digestion of the methylated parental DNA strand was carried out by adding Dpnl, 10 U, (recognition site 5 G m e t A T C 3 ) directly to each reaction mixture below the mineral oil overlay and each reaction was mixed by gently pipetting up and down several times. The reaction mixtures were centrifuged, then incubated at 37°C for one hour before using in the E. coli X L 1-Blue transformation reactions. 2.7.3 Transformation of E. coli XLl-Blue Supercompetent Cells Briefly, XLl-Blue supercompetent cells were thawed on ice and 50 ul of the cells were aliquotted into prechilled Falcon 2059 tubes (Becton Dickinson, Lincoln Park, NJ). One ul 42 from each Dpnl treated reaction was added to the aliquot of XLl-Blue supercompetent cells. The tubes were gently swirled to mix and incubated on ice for 30 minutes. The reactions were heat-pulsed for 45 s at exactly 42°C, then placed on ice for two min. SOC broth (0.5 ml) was preheated to 42°C and added to the transformation reactions, which were then incubated at 37°C for one hour with shaking at 225 rpm. The entire volume of each reaction was plated on LB-ampicillin plates and incubated at 37° C for 16 hours. Transformed E. coli were selected on LB-ampicillin plates and purified as described previously. 2.7.4 Sequencing of the Corrected L. major Cpn60 Gene The 3'end of the Cpn60 gene from each of three samples selected was sequenced using the 6F (forward) and BGH (reverse) primers to ensure the correct base had been added. The entire gene was again sequenced using the same six sequencing primer pairs previously described to confirm that the entire gene sequence was correct following site directed mutagenesis. 2.8 Large Scale Plasmid Purification Large scale plasmid purification for both the vaccine plasmid, pcDNA3.1/Cpn60, and the empty plasmid, pcDNA3.1, were carried out as follows: 250 ml of LB containing 0.06 mg/ml of ampicillin was inoculated with 2.5 ml of an overnight culture of clone pcDNA3.1/Cpn60 or pcDNA3.1. The cultures were incubated in a rotary shaker at 37°C overnight. Bacteria were pelletted by centrifugation and the plasmids purified using an Endofree Plasmid Purification 43 Mega/Giga kit (Qiagen, Chatsworth, CA). The purified plasmids were then resuspended in lx endofree PBS. DNA purity and concentration were measured by spectrophotometric methods. Following purification procedures the pcDNA3.1/Cpn60 and the pcDNA3.1 control plasmid were digested with restriction enzymes Pst\ and BamHl TO confirm the correct insert. 2.9 In Vitro Coupled Transcription/Translation Using Rabbit Reticulocyte Lysate 2.9.1 Coupled Transcription/Translation In vitro transcription and translation was carried out using a TNT R (T7) Quick Coupled Transcription/Translation System (Promega, Madison, Wl) in a translation extract of rabbit reticulocyte lysate, and the Transcsend™ Non-Radioactive Translation Detection Systems (Promega) according to the manufacturer's protocol. Briefly, 1-6 pg of recombinant plasmid DNA and 1-2 p.g of control plasmid (1.0 ug/ul) were added to each reaction mix of Biotin-Lysyl tRNA, TNT R Quick Master Mix (Promega), and nuclease-free H2O to a final volume of 50 ul. Control reactions without DNA were also carried out as above. The tubes were incubated at 30° C for 90 minutes. 2.9.2 Denaturing Gel Analysis of Translation Products Following incubation, a 1 pl aliquot was removed from each reaction and added to SDS sample buffer (2.0 ml glycerol, 2.0 ml 10% SDS, 0.25 mg bromophenol blue, 2.5 ml 2M Tris-HCl (pH 6.8), 0.5 ml P-mercaptoethanol and 3.0 ml deionized H2O) in a ratio of 1:4 sample:buffer. The remainder of the sample was stored at -20°C. Samples were denatured by 44 heating to 95°C for 2 min and loaded on a discontinuous 10% SDS polyacrylamide gel (acrylamide/bis 30%) using a Mini-PROTEAN II Electrophoresis Cell (Bio-Rad, Hercules, CA). Electrophoresis was carried out according to manufacturers directions. Briefly, 20 pl of each sample and control was loaded onto the gel and electrophoresis carried out in 5x electrode running buffer, pH 8.3 (Tris base 15g/L, glycine 72g/L, SDS 5g/L) until the dye front ran off the end of the gel (approximately 1.5 hours at 190V). The gel was removed and equilibrated in transfer buffer pH 9.2 (Tris 5.82g/L, glycine 2.93 g/L, SDS 0.0375 g/L) for 15 minutes and then blotted to a PVDF (polyvinylidene difluoride) membrane using a Trans-Blot semi-dry transfer cell (Bio-Rad, Hercules, CA.) according to manufacturer's directions. The PVDF membrane was equilibrated with 100% methanol with gentle shaking, then H20 was added until the methanol was 20% of the total volume. The membrane was rinsed twice with H20 and then blocked by incubation in 15 ml of TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCI, and 0.5% TweenR 20) for one hour with gentle shaking. 2.9.3 Chemiluminescent Detection of Protein Products. Biotinylated lysines incorporated into the nascent proteins were detected by binding of Streptavidin-Horseradish peroxidase (SRP-HRP) and the addition of Transcend™ chemiluminescent substrate (Promega, Madison Wl) according to manufacturer's directions. Briefly, the membrane was incubated in a 1:10,000 dilution of the Streptavidin-HRP conjugate in TBST for 1 hour, washed 3 times with TBST, then 3 times with TBS (20 mM Tris-HCl, 150 mM NaCI). The membrane was then incubated with the chemiluminescent substrate mixture for 1 min in dim light and the blot exposed to x-ray film for 3 minutes. 45 2.10 In Vitro Transfection of COS-7 Cells 2.10.1 Culture of COS-7 Cells COS-7 cells were a gift from Dr. Jose Rey-Ladino (Terry Fox Laboratory, Vancouver, B.C). The cells were cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine and 1% PenStrep. Cells were seeded in T25 flasks containing 5 ml of media and incubated at 37°C with 5% CO2. Passage of cells was carried out 5 times before use in transfection experiments. The COS-7 cells were cultured as follows: spent media was removed and cells were rinsed with sterile Hanks balanced salt solution (HBSS). One ml of 0.25% Trypsin-EDTA was added to the flask which was gently rocked 1 to 2 times to ensure all the cells were covered. The trypsin was then taken off and the flask placed back in the 37°C incubator until the cells were loose on gentle tapping (approximately 3 - 5 minutes). The cells were then suspended in fresh media (5 ml). One (1) ml of the resuspended cells was removed and added to 4 ml of fresh media in new T25 flasks and returned to the cell culture incubator. The COS-7 cells became confluent every two to three days. 2.10.2 Transfection using FuGene 6 Transfection Reagent The transfection of COS-7 cells with pcDNA3.1/Cpn60 or empty pcDNA3.1 using FuGene 6 (Roche Molecular Biochemicals, Laval, QUE) transfection reagent was carried out according to the manufacturer's protocol. Briefly, COS-7 cells were passaged, counted using a hemocytometer, and plated in a 6-well plate at a density of 2x 106 cells in 2 ml. of fresh media/well. In a small sterile tube 3 pi of FuGene 6 reagent was added to 97 ul of serum-free media and incubated at room temperature for 5 min. DNA (at concentrations of 1.0 and 1.5 pg) was added to separate sterile tubes (the total volume of DNA in each tube should not 46 exceed 10 ul). The diluted FuGene 6 was added dropwise to the tube containing the DNA, tapped to mix, and incubated for 15 minutes at room temperature. The DNA/FuGene 6 was then added dropwise to the COS-7 cells that were 60 - 80% confluent. The plates were gently swirled to ensure even dispersal around each well and then returned to the cell culture incubator and incubated for 48 hours. The plates were checked microscopically on a daily basis for signs of cytotoxicity. 2.10.3 Harvesting of COS-7 cells Following Transfection Media was removed from each well and the cells were rinsed twice with sterile HBSS. Following the final rinse, 200 pl of sample buffer was added to each well and the cells were scraped from the surface of the plate with a cell scraper. The contents of each well were transferred to a 1.5 ml Eppendorf tube and heated to 94°C for 10 min. The samples were then stored at -30°C until immunoblotting was performed. 2.11 Antibodies and Reagents Used in Western Blots 2.11.1 Primary Antibodies 2.11.1.1 Rabbit anti-Cpn60 The primary antibody used for probing western blots was provided by Dr. S. Hemmingsen (PBI/NRC, Saskatoon, SASK.). Plant plastid Cpn60 is composed of two subunits, a and p. cDNA genes were constructed from the a and P subunits for expression in E. coli. The protein products from the cDNA genes produced in E. coli accumulated to levels similar to that of GroEL, the E. coli homologue of Cpn60. Purified GroEL + beta 14mers or GroEL 14 47 alpha protein was used as antigen to produce rabbit antisera #85, #86, and #87. In each case the antiserum recognized chaperonin proteins from a broad range of sources. Antiserum #85 was used to probe immunoblots of protein products from the transfection protocol and was found to detect the Cpn60 protein from the following organisms: African green monkey, L. major, E. coli, S. aureus, mouse, and human. 2.11.1.2 Chicken anti-Leishmania Hsp60 (SPA-823) Chicken anti-Leishmania Hsp60 polyclonal antibody (Stressgen, Victoria, B.C.) was used to probe transfection samples previously probed with Ab #85. This antibody reacts weakly with human Cpn60 at elevated temperatures but does not react with African green monkey Cpn60. 2.11.1.3 Mouse sera Sera prepared from blood obtained by cardiac puncture of BALB/c mice that had been immunized with pcDNA3.1/ L. major Cpn60, empty pcDNA 3.1 or PBS only was used to probe western blots of L. major total protein, total protein from control organisms 5". aureus and E. coli, purified human Cpn60 or total protein from HeLa cells. 2.11.2 Secondary Antibodies Alkaline phosphatase conjugated goat anti-rabbit IgG Fab fragment (Sigma, Saint Louis, MI) was used on all western blots where antibody #85 was used as a primary antibody. Alkaline phosphatase conjugated goat anti-mouse IgG Fab fragment (Sigma) was used on all western blots where mouse serum prepared from the blood of immunized mice was used as a 48 primary antibody. Alkaline phosphatase conjugated rabbit anti-chicken (Sigma) was used to probe western blots where chicken anti-Leishmania Cpn60 was used as a primary antibody. 2.11.3 Reagents for Immunoblots Reagents used in western blots included recombinant human Hsp60 (Cpn60) (Stressgen, Victoria, B.C.), kaleidoscope prestained standards and PVDF membrane (Bio-Rad, Hercules, CA). Biotinylated molecular weight markers, Avidix-AP streptavidin-alkaline phosphatase conjugate, I-Block, lOx Assay Buffer and CDP-STAR substrate solution were all obtained from Tropix (Bedford MA). 2.12 Western Blot and Detection of Protein Products Protein samples were run on a 10% SDS-polyacrylamide gel and blotted to a PVDF membrane as described previously. Chemiluminescent detection using a Tropix Western Star detection system (Bedford, MA) was carried out as follows: the blot was washed briefly in lx PBS and incubated in blocking buffer (I-Block 1.8 gm, lx PBS and 0.3% Tween 20) in a total volume of 600 ml at room temperature for 30 min with gentle rocking. The appropriate primary antibody was diluted in 12 - 15 ml of blocking buffer, at a concentration of 1 pi of antibody per 1 ml of blocking buffer and incubated with the blot for 45 min at room temperature. The membrane was then washed twice in blocking buffer (100 ml/wash) for 30 min each. The secondary antibody, diluted in 30 - 40 ml of blocking buffer at a concentration of 1 ul/4 ml of buffer, was incubated with the blot for 1 hour at room temperature. Following incubation, the blot was washed 3 times in blocking buffer for 30 min followed by washing 49 twice with lx assay buffer (50 ml) for 10 min/wash. Al l antibody incubations and washings were done with gentle rocking on a shaker (Life Science Inc., Greensboro, NC). The blot was then drained, placed on a sheet of plastic wrap and CDP-Star substrate solution (1.5-2 ml) was pipetted onto the membrane. The blot was drained after 5 min, placed in a development folder and exposed to x-ray film. 2.13 Leishmania major Total Protein Leishmania major (Friedlin strain) stationery phase promastigotes were obtained from Dr. R. McMaster. Leishmania total protein used in SDS-PAGE was prepared as follows: promastigotes (1x10 equivalents/ml) were spun down at 1500 rpm in a Beckman centrifuge, rinsed twice with lx sterile PBS, resuspended in sample buffer and stored at -20°C. Leishmania soluble antigen used to stimulate mouse splenocytes in culture was obtained in the following manner: L. major promastigotes (2 x 107 equivalents/ml) were spun down, rinsed twice in sterile lx PBS and resuspended in RPMI 1640 supplemented with 10% FCS, 2mM L-glutamine, 1% Pen-Strep and 50 uM 2-mercaptoethanol. The pellet was subjected to nine freeze-thaw cycles on dry ice/ethanol, centrifuged at 8000 x g for 10 min in a microcentrifuge, filtered through a sterile 0.45 um filter and stored at -80°C. 2.14 Mouse Immunization Protocol Five week old female BALB/c mice, obtained from either the animal care facility at UBC or from Charles River, were housed at the Jack Bell animal care facility. Al l mice were 50 shaved over the injection site prior to immunization with pcDNA 3.1/Z. major Cpn60 vaccine, empty pcDNA 3.1, or sterile endofree lx PBS. Before injection the mice were anaesthetized by inhalation with halothane and the area to be injected was swabbed with 70% ethanol. 2.14.1 Vaccine Mouse Group 100 pg of the vaccine plasmid resuspended in sterile, endofree lx PBS (1 u.g/u.1) was delivered intramuscularly into either the thigh muscle or the anterior tibialis muscle of the hindlimb of each mouse bilaterally (50 u.g/muscle) using a 3/10 cc. insulin syringe with a 29G x V2" needle. The needle was covered with polyethylene tubing leaving approximately 3mm exposed. 2.14.2 Empty Plasmid Control Group 100 pg or 76 pg of empty pcDNA3.1 was resuspended in 100 pi of sterile endofree lx PBS (0.76 pg/ul or 1 p.g/ui) and delivered bilaterally (38 p,g or 50 pg/muscle) in the same manner as the vaccine plasmid. 2.14.3 PBS Control Group 100 ul of sterile, endofree lx PBS (50 ul/muscle bilaterally) was administered in the manner described for the other two groups. Each of the three groups of mice was housed in separate cages and only one group was handled at any one time. 51 2.15 Cardiac Puncture Protocol Mice were anaesthetized by inhalation with halothane. When reflexes were absent to toe pinch the mouse was placed on its back and a 21G x 1" needle was inserted through the chest wall at a slight angle just below the xyphoid process. Blood was collected in a 3 ml syringe, dispensed into a sterile 15 ml conical tube and incubated at 4° overnight. Following incubation the clotted blood was removed and the serum was transferred to a sterile 1.5 ml Eppendorf tube and centrifuged to pellet any red blood cells remaining in the serum. The serum was then passed through a sterile 0.45 pM filter and stored at 4°C. 2.16 Spleen Removal and Culture of Mouse Lymphocytes Spleens were removed from all euthanized mice in the following manner: the mouse was turned with the left side facing up and the area flooded with 70% ethanol. An incision was made through the skin and underlying tissues and the area again flooded with 70% ethanol to remove any loose hair. The spleen was exposed, removed with sterile scissors and immediately placed in a sterile 15 ml conical polyethylene tube containing 5 ml of sterile lx PBS. The spleens were then moved to a tissue culture hood. Single cell suspensions of splenocytes were made by cutting the spleen at one end and passing the cells through a sterile mesh filter, gently 'scrubbing' the cells back and forth over the filter and washing intermittently with lx sterile PBS. The splenocytes were pelleted by spinning at 1200 rpm in a Beckman centrifuge, rinsed twice with sterile PBS, then resuspended in RPMI 1640 (Gibco/BRL, Burlington, ONT)) supplemented with 10% FCS, 2mM L-glutamine, 1% Pen-Strep and 50 uM 2-mercaptoethanol. Lymphocytes were isolated with Ficoll-Paque 52 (Pharmacia Biotech AB, Uppsala, Sweden) by adding Ficoll solution (3 ml) to a sterile 15 ml conical tube, then adding 4 ml of the cell suspension on top of the Ficoll to get distinct layers. The cells were centrifuged for 25 min at 1500 rpm with the brake off. The milky interface containing the lymphocytes was removed with a sterile transfer pipette, placed in a sterile 15 ml centrifuge tube, and the cells were pelletted by spinning at 1000 rpm for 5 min. The old media was taken off and the cells were washed twice and then resuspended in RPMI 1640. Cell density was determined by counting with a hemacytometer using trypan blue to stain the cells. 2.17 In Vitro Cytokine Assay 2.17.1 Cytokine Assays from a Six Mouse Trial Immunization Experiment (Experiment I) Six 5-week-old female BALB/c mice were obtained from the animal care facility at the University of British Columbia. The mice were separated into three groups (2 mice/ group) and each group was immunized with either pcDNA3.1/Cpn60, empty pcDNA3.1, or PBS alone according to the immunization protocol previously described. The mice were boosted twice using the same protocol with injections given 15 days apart. Seven (7) days after the last boost cardiac puncture was performed as described to obtain serum for antibody testing. Spleens were removed as described and splenocytes from each group were pooled and prepared as described above. Cell suspensions were diluted to 2.5 x 106/ml, plated in 24 well plates at a density of 5 x 106 cells/well. Cells from all three groups were either stimulated with soluble leishmania antigen (SLA), 1 x 107 equivalents, or unstimulated, plated in triplicate and cultured at 37°C with 5% CO2. The supernatants were harvested at 48 and 96 53 hours as follows: cell suspensions were centrifuged at 1000 rpm for 5 min, the supernatant transferred to 1.5 ml Eppendorf tubes and stored at -20°C. Supernatants were assayed for IL-2 and IL-4 using the Biotrak mouse ELISA system (Amersham Pharmacia Biotech, Oakville, ONT) and carried out according to manufacturer's directions. Briefly, samples, controls and standards were added to the wells of a microtitre plate coated with capture antibody specific for the cytokine of interest. Following a wash to remove unbound antibody, a second antibody conjugated to alkaline phosphatase was added to the wells and incubated. Unbound conjugate was washed away and substrate added to the wells. The degree of substrate hydrolysis was measured colourimetrically. and the reactions were read using an ELISA plate reader. The colour intensity that develops is directly proportional to the amount of cytokine bound to the microtiter plate. Sample values were determined by comparison with values from a standard curve prepared by plotting the mean absorbance (y axis) against concentration (x axis) of mouse cytokine standards included with each kit. 2.17.2 Cytokine Assays from a Twenty-One Mouse Immunization Experiment (Experiment 2) Twenty-two 4-week-old female BALB/c mice were obtained from Charles River. One of the mice was sacrificed at the beginning of the experiment and blood for serum was obtained by cardiac puncture. The spleen was removed and splenocytes were prepared as previously described. The remainder of the mice were divided into three groups of seven and designated as a vaccine, an empty plasmid or a PBS group. Each of the three groups were housed in separate cages. The mice were immunized as described previously and were boosted twice, two weeks apart. Three mice from each group were sacrificed 11 days after the first boost. Blood was obtained by cardiac puncture, spleens were removed and lymphocytes were 54 cultured as described. The remainder of the mice were sacrificed ten days following the second boost. Lymphocytes within each group were pooled, plated at a density of 6.3 x ft 1 10 /well in a 6 well plate, and either stimulated with SLA (1x10 equivalents/well), Con A (2.5 pg/ml), or unstimulated. Cells were harvested after 72 hours and assayed for cytokines IL-2, IL-4 and IFN-y using the Quantikine M Immunoassay system ( R & D Systems, Minneapolis, MN) according to the manufacturer's protocol. 2.18 RT-PCR 2.18.1 RNA Preparation Six female BALB/c mice, 5 weeks of age, were obtained from the animal care facility at UBC. The mice were divided into two groups of three mice. Each mouse was shaved over the anterior tibialis muscle on both hindlimbs and injected with either 100 pg of pcDNA3.1/Cpn60 or 100 pg of empty pcDNA3.1 as per the immunization protocol. Forty-eight hours later the mice were sacrificed by CO2 inhalation. Approximately 30 mg of muscle tissue around the injection site was removed aseptically and immediately placed in a 1.5 ml Eppendorf tube containing a small amount of liquid nitrogen which was then placed on dry ice and ethanol. RNA was prepared using an RNeasy Total RNA Kit (Qiagen, Chatsworth CA). Briefly, the frozen tissue was placed in RNAse free 2 ml microcentrifuge tubes containing lysis buffer RLT from the Qiagen RNeasy kit plus p-mercaptoethanol. The tissue was ground with disposable tissue grinders (Kontes, Vineland, NJ) until no particulate was visible. The remainder of the RNA preparation was carried out according to Qiagen RNeasy protocol. The eluate was treated with RNase-free DNase. One-half of the DNase treated sample was stored 55 at -30°C and the other half was repurified using RNeasy cleanup protocol following DNase digestion. 2.18.2 Reverse Transcription A two-step RT-PCR was carried out using the RT-PCR kit and protocols from Roche Molecular Biochemicals. Briefly, RT was done using C. therm polymerase and antisense primers 5R or 6R, (sequences previously shown) or control antisense primer, mouse p-actin BAC-1006 (Baxim Biotech Inc., San Francisco, CA): 5 TAA AAC GCA GCT CAG TAA CAGTCCG 3 , T m =69.3°C. RT was performed on samples in a Perkin-Elmer thermocycler at 65°C for 30 min. Samples to be used as controls were not incubated but went directly from the RT reaction to the PCR reaction. Final concentration of reaction components in the RT reaction were as follows: lx RT buffer, 5mM DTT, 0.8 mM each dNTP, 5% DMSO, 6U C. therm polymerase, 1 uM primer, and up to 1 pg of RNA in a final volume of 20 ul. 2.18.3 PCR of RT Samples PCR of the samples subjected to RT was carried out using Taq DNA polymerase. Primer pair 5F/5R were used on samples where 6R was used as antisense RT primer and primer pair 4F/4R were used where 5R was used as antisense RT primer. Primer sequences have been previously shown, p actin primer pair BAC-1005 / BAC-1006 were used in PCR to amplify control RT samples where BAC-1006 was used as the antisense RT primer (BAC-1005 sequence: 5 ' TGG AAT CCT GTG GCA TCC ATG A A A C 3 ) . The final concentration of components of the reaction mix were as follows: cDNA from step #5 (5 pl), dNTP 0.2 mM, 56 forward and reverse primers 0.3 uM each, Taq DNA polymerase 2.5 units, lx PCR buffer, 1.5M Betain to a final volume of 50 ul. 2.18.3.1 Cycling parameters (4F/4R and 5F/5R) Initial denaturation: 94°C for 4 min, cycle denaturation 94°C for 90 s, cycle annealing: 57°C for 90 s, cycle extension: 72°C for 90 s, (40 cycles), final extension 72°C for 7 min. 2.18.3.2 Cycling parameters (BAC-1005/BAC-1006) Initial denaturation: 94°C for 3 min, cycle denaturation: 94°C for 30 s, cycle annealing 60°C for 90 s, cycle extension: 72°C for 90 s, (35 cycles), final extension: 72°C for 7 min. 57 III. RESULTS AND DISCUSSION 3.1 PCR Amplification, Cloning and Sequencing of the L. major Cpn60 Gene 3.1.1 Amplification of the L. major Cpn60 Gene Contained in the A EMBL-3 Clone The first step in the production of a DNA vaccine in this study was the amplification of the Leishmania major Cpn60 gene contained in the X EMBL-3 clone by polymerase chain reaction (PCR). Figure 2. PCR amplification of the putative 1.7 kb Cpn60 gene from L. major. An 8 pl sample from the PCR reaction was run on a 0.8% agarose gel. Lane 8 shows a band of approximately 1.76 kb (arrow), the expected size of the L. major Cpn60 gene. Molecular size markers (500 bp ladder) are shown on the extreme left and right (lanes 1 and 12). The 1.76 kb Leishmania major Cpn60 gene contained in the X EMBL-3 clone was amplified enzymatically using primers designed from the 5' and 3' termini of the gene previously described. PCR products run on a 0.8% agarose gel with ethidium bromide showed amplification of a DNA fragment in Figure 2, lane 8 (arrow) corresponding to the expected size of the L. major 1.76 kb Cpn60 gene. 58 The 1.7 kb fragment was confirmed by restriction analysis using BamHl. BamRl is expected to digest the gene into two fragments, a 5' fragment of 1473 base pairs and a 3' fragment of 297 base pairs (Figure 3). Based on the BamHl restriction digest and the resulting Figure 3. Restriction analysis of the putative 1.7 kb fragment of the L .major Cpn60 gene using BamXW The BamHl restriction pattern for the putative 1.7 kb Cpn60 gene of L. major shows two bands: the larger is approximately 1500 bp and the smaller approximately 300 bp. A 100 bp molecular size marker is shown on the left. fragment size it was concluded that the L. major Cpn60 gene had been successfully amplified (Figure 3). 3.1.2 Cloning of the L. major Cpn 60 gene pcDNA3.1, a eukaryotic expression vector, was used for transient expression of the L. major Cpn60 gene. This plasmid contains the human cytomegalovirus (hCMV) promoter which allows for high level expression of the protein in mammalian cells. The use of two restriction sites, HindlU and EcoRI, on the plasmid allowed for unidirectional cloning with the result that all of the E. coli clones with the recombinant plasmid contained the gene in the 59 proper orientation. Since pcDNA3.1 is a 'non-fusion' vector, a Kozak consensus sequence was introduced into the 5' primer at the transcriptional start site to improve translation efficiency. Figure 4. 5.4 kb eukaryotic expression vector pcDNA3.1+ The vector contains (i) the hCMV promoter for efficient expression of the protein in mammalian cells, (ii) T7 promoter/priming site for in vitro transcription, (iii) a multiple cloning site (iv) BGH reverse priming site and polyadenylation signal, (v) ColEl origin of replication and (vi) an ampicillin resistance gene for selection of the vector in E. coli. 60 To direct the position of translation initiation in vertebrate mRNAs both the ATG initiation codon and specific sequences flanking the initiation codon are required. The Kozak consensus sequence GCC A/G"3CC AUG G + 4) introduced into the 5' primer does not have a purine at the +4 position (GCC A" 3CC AUG C + 4). However, the Kozak rule states that for practicality, an initiation codon can be designated either strong or weak by considering only positions -3 and +4. As long as a purine occupies the -3 position, other deviations only slightly impair translation efficiency. In the absence of a purine in position -3, a "G" must occupy +4. (It should be noted that in mammals the purine at -3 is an " A " 97% of the time (69, 89). 3.1.3 Plasmid Purification and Sequencing of the L. major Cpn60 Gene The plasmid DNA and insert were then confirmed by digesting with Hindlll and EcoKl to mobilize the 1.7 kb fragment. Three clones were selected and sent to the DNA sequencing laboratory at UBC for full sequencing using six overlapping sets of primers. The nucleotide sequences of the three clones were edited by comparing the gene sequences of each of the three samples with the L. major Cpn60 gene sequence submitted to GenBank (Accession No. U59320). It was discovered that each of the Cpn60 clones selected had a deletion of one nucleotide, a guanosine (G) 45 bases from the 3' end of the gene. Sequencing in the reverse direction confirmed a cytosine (C) missing at the same location. This deletion introduced a premature stop codon in the amino acid sequence with the loss of 15 amino acids at the COOH terminus. 61 3.1.4 Site-directed Mutagenesis to Correct the Deletion in the L. major Cpn60 Gene It was decided to employ site directed mutagenesis to correct the one nucleotide deletion in the gene rather than reamplifying the gene by PCR from the original X clone. It was not known whether the deletion was present in the original clone or if the error occurred during the first PCR amplification using Vent exo+ DNA polymerase (with a 3 ' ^ 5'proofreading capability). The site-directed mutagenesis kit (Stratagene) allows the introduction of a specific mutation in double-stranded plasmids without the need for single-stranded DNA rescue. Pfu DNA polymerase (theoretical error rate approximately 1/580,000) has a 12-fold lower error rate than conventional thermostable enzymes and replicates both DNA strands with high fidelity and without primer displacement. Lack of extendase activity ensures blunt ends with no 3' overhangs. During temperature cycling the primers will anneal to each corrected strand as well as the parental strand but only the primer annealing to the parental strand will be extended. Since DNA isolated from E. coli is dam methylated, treatment with Dpn\, which recognizes methylated and hemimethylated DNA, will digest the defective parental DNA strand in the plasmid, leaving the unmethylated newly synthesized strand containing the desired nucleotide insertion intact. Mutagenesis efficiencies are reported to be 83 - 100% effective. The nicks in the plasmid are repaired intracellularly. Following large-scale plasmid purification, both the recombinant and control plasmids were cut with Pstl and BamHl restriction enzymes and run on a 0.8% agarose gel (Figure 5). Pstl digests the plasmid plus insert (pcDNA3.1/Cpn60) into three fragments of 4965, 1357 and 887 bp and digests the control plasmid (pcDNA3.1) into two fragments of 4043 and 1357bp. BamHl linearizes the 7.1 kb pcDNA3.1/Cpn60 and linearizes the 5.4 kb empty 62 pcDNA3 .1 . Following plasmid purification the concentration and purity of the DNA was determined by spectrophotometric methods. 5b 6 7 8 9 10 1500 bp 1000 bp-*-Figure 5. Pstl and BamHl digestion of the control and recombinant plasmids. Figure 5a. pcDNA3.1/Cpn60 recombinant plasmid digested with restriction enzymes Pstl and BamHl. Lanes 1 and 5: 500 bp marker; lane 2 uncut recombinant plasmid; lane 3: recombinant plasmid cut with Pstl; lane 4: recombinant plasmid cut with BamHl. Figure 5b: empty pcDNA3.1 digested with Pstl and BamHl. Lanes 6 and 10: 500 bp ladder; lane 7: uncut pcDNA3.1; lane 8: pcDNA3.1 cut with Pstl; lane 9: pcDNA3.1 cut with BamHl. Resequencing of the gene following transformation of E. coli XLl-Blue showed the defect had been corrected in all samples selected for sequencing. One of the samples selected was used in the vaccine preparation for all of the in vitro and in vivo experiments that followed. 3.2 In Vitro Experiments and Western Blot Analysis 3.2.1 Cell-free Coupled Transcription/Translation using a TNT* Quick Coupled Transcription/ Translation System To ensure that the mammalian transcriptional machinery could synthesize the L major Cpn60 gene, in vitro coupled transcription/translation was carried out. In this non-radioactive translation detection system biotinylated lysines are added to the translation reaction as a pre-63 charged ^-labeled biotinylated lysine-tRNA complex and incorporated into nascent proteins. The intensity of the bands is due to the number of biotinylated lysines incorporated into the protein. Although each sample may contain slightly differing amounts of biotinylated lysines, researchers have shown that between 25-33% of lysine residues are biotinylated in translation products that contain 4-5 lysine residues. Lysine residues are common in most proteins, accounting on average for 6-7% of a protein's total amino acid content (72). In the L. major Cpn60, 6% (36/589) of the amino acids are lysines. The specificity of the T7 RNA polymerase for transcriptional initiation from their promoters is very high, so the RNA synthesized in the transcription/ translation reactions should yield a single species that encodes the desired protein (72). One of the most critical parameters for synthesizing mRNA is the quality of the DNA template. With high quality DNA, transcription of full length mRNA is rarely a problem. A potential problem, however, is that contaminating ribonucleases will easily destroy transcription products (71). Figure 6 shows the results of an in vitro transcription/translation experiment carried out using concentrations of pcDNA3.1/Cpn60 ranging from 1-3 ug (Fig.6a: lanes 1, 2, 3) and 4-6 ug (Fig. 6b: lanes 6, 7, 8) on a discontinuous SDS-polyacrylamide gel. Empty pcDNA3.1 1 and 2 pg, (Fig. 6a: lane 4 and Fig. 6b: lane 9) and lysate alone (Fig. 6a: lane 5 and Fig. 6b: lane 10) were run as controls. Al l samples containing the pcDNA 3.1/Cpn60 (lanes 1-3 and 4-6) showed protein products whose molecular weight corresponds to the molecular weight of the L. major Cpn60 protein (approximately 65kDa). No bands were detected in any of the controls (lanes 4, 5, 9 and 10). Band intensity appears to be fairly uniform in all of the samples. Studies have shown that the use of more than 2 p,g of plasmid does not necessarily increase the amount of protein produced (72). 64 45 Figure 6. In vitro coupled transcription and translation of L. major Cpn60 using rabbit reticulocyte lysate. Molecular size markers are shown to the left. 6a: Lanes 1, 2, and 3, contain protein products from pcDNA3.1/Cpn60 (1, 2, 3 ug respectively); Lane 4: control empty pcDNA 3.1 (1 pg);. Lane 5: lysate without DNA. 6b: Lanes 6, 7, and 8 contain protein products from pcDNA3.1/Cpn60 (4, 5. 6 fig respectively); Lane 9: control empty pcDNA3.1 (2 ug); Lane 10: 1 rabbit reticulocyte lysate only. It was concluded that DNA template quality in this construct was good and that the L. major Cpn60 gene encoded in the plasmid was successfully transcribed and translated by the mammalian translation machinery, albeit in a cell-free system. 65 3 . 2 . 2 In vitro Transfection of COS-7 Cells with pcDNA3.1/Cpn60 and pcDNA3.1 Control Plasmid Following successful in vitro transcription and translation in a cell free system, the next step was to carry out in vitro transfection of mammalian cells and western blot. Immunoblotting is a rapid and sensitive assay for the detection of proteins by exploiting the specificity of antigen-antibody recognition. This technique is useful in identifying specific antigens which are recognized by monoclonal or polyclonal antibodies. As little as 1 ng of antigen can be detected (70). In vitro transfection experiments were done to ensure that the plasmid vector could direct protein synthesis inside cells and that the levels of protein produced were not toxic to the transfected cells. Both of the immunoblots (Figures 7 and 8) were probed with antibody #85. A mammalian Cpn60 and L. major total protein were run along with protein products from both transfected cells and controls. Immunoblots in Figures 7 and 8 revealed that transfection of COS-7 cells with pcDNA3.1/Cpn60 resulted in the expression of a protein of approximately 65 kDa (Figure 7: lanes 4 and 5; Figure 8: lanes 8 and 9). This corresponds to the size of a protein expressed from L. major (Figure 7: lane 3; Figure 8: lane 2). Moreover this protein was not present in cells transfected with empty plasmid (Figure 7: lane 6; Figure 8: lanes 6 and 7) or in the COS-7 cells (Figure 7: lane 7; Figure 8: lane 4). Additionally, antibody #85 also recognized a slightly smaller protein in all lanes where COS-7 cells protein were part of the lysate (Figure 7: lanes 4, 5, 6, 7 and Figure 8: lanes 4, 5, 6, 7, 8, 9)., but not in the L. major lysate (Figure 7: lane 3; Figure 8: lane 2). The molecular weight of the purified human Cpn60 appears to be larger than the size predicted for human Cpn60 (Figure 7: lane 2). The molecular weight of the band from HeLa cell total protein (Figure 8: lane 1) corresponds to 66 the size of the predicted band for human Cpn60 which is approximately 2.5 kDa smaller than the predicted size of the Leishmania Cpn60, 64.5 kDa Figure 7. Immunoblot No. 1 of protein products from COS-7 cells transfected with pcDNA3.1/Cpn60 or empty pcDNA3.1 using transfection reagent FuGene 6. This western blot was probed with primary antibody #85 and secondary antibody goat anti-rabbit IgG. Lane 1: kaleidoscope size marker; lane 2: human Cpn60; lane 3: L. major total protein; lanes 4 and 5: pcDNA3.1/Cpn60 (1.0 and 1.5 ug respectively); lane 6: control empty pcDNA3.1 (1.5 ug); lane 7: COS-7 cells only. Figure 8 Immunoblot No. 2 of protein products from COS-7 cells transfected with pcDNA3.1/Cpn60 or empty pcDNA3.1 using transfection reagent FuGene 6. This western blot was probed with primary antibody #85 and secondary antibody goat anti-rabbit IgG. Lane 1: HeLa cells total protein; lane 2: L. major total protein; lane 3: kaleidoscope size marker; lane 4: COS-7 cells only; lane 5: COS-7 cells + FuGene 6 only; lanes 6 and 7: COS-7 cells transfected with control plasmid pcDNA3.1 (1.0 and 1.5 pg respectively); lanes 8 and 9: COS-7 cells transfected with pcDNA3.1/Cpn60 (1.0 and 1.5 ug respectively). 67 65 kDa 5 l i/r7 J ° f P r ° t e i n p r ° d U C t S f r 0 m C O S " 7 c e , , s fansfected with SfSff,I-e,n|1? PCDNA3.1 probed with primary antibody ^-Leishmania Cpn60 , 8 2 3 ) a n d r a b b , t a n t l - d " c k e n IgG secondary antibody. Lane 1: COS-7 cells + FuGene 6 anes 2 and 3: COS-7 cells transfected with control plasmid pcDNA3.1 (1.0 and 1.5 ug respectively)' lanes 4 and 5: COS-7 cells transfected with pcDNA3.1/Cpn60 (1.0 and 1.5 pg respectively) The immunoblot in Figure 9, probed with leishmania anti-Cpn60 polyclonal antibody #823, picked up bands of approximately 65kDa where COS-7 cells were transfected with pcDNA3.1/Cpn60 (lanes 4 and 5). No bands were seen in any of the lanes where COS-7 cells were transfected with control empty pcDNA3.1 (lanes 2 and 3) or in lanes where proteins from COS-7 cells plus FuGene 6 (lane 1) were run. Moreover, the bands that were in the size range of 62 kDa evident in the two previous blots (Figures 7 and 8) probed with antibody #85 were not in evidence on this blot. These in vitro translation experiments utilized COS-7 cells (African green monkey kidney cells) and the transfection reagent FuGene 6, a non-liposomal lipid formulation in 80% ethanol. In the immunoblots where primary antibody #85 was used, the two bands in lanes where COS-7 cells were transfected with the recombinant plasmid pcDNA3.1/Cpn60 are analogous to the size of the Cpn60 protein from L. major (64.5 kDa) and COS 7 cells. The exact size of he Cpn60 protein from green monkey cells is unknown. In lanes where COS-7 68 cells were transfected by empty plasmid only, or were not transfected, a single band corresponding to the Cpn60 protein from the COS-7 cells was seen. The size of the Cpn60 from African green monkey cells is slightly smaller than the L. major Cpn60 when comparing the protein sizes on western blots, and is probably closer to the molecular weight of the human Cpn60 precursor which is reported to be 61 kDa. The size of the purified human Cpn60 (Figure 7, lane 2) appears to be slightly larger than the L. major Cpn60 but this may be due to post-translational modifications. The calculated molecular weight of a protein (from standard curve comparisons with other known protein molecular weights) may not always agree with molecular weights estimated by SDS-PAGE. Typically, the apparent molecular weight is within 10% of the calculated molecular weight but may differ as much as 20 - 30% for proteins that are heavily glycosylated or phosphorylated. Additionally, SDS may not be able to remove all variables such as shape and charge completely because many factors such as hydrophobicity and protein preparation may affect binding (73). These results indicate that Ab #85 detects the Cpn60 protein from the COS-7 cells as well as the L. major and human Cpn60. The immunoblot probed with leishmania anti-Cpn60 (Figure 9) showed only the L. major Cpn60 protein was detected in the lanes where COS-7 cells transfected with pcDNA3.1/Cpn60 protein were run. The green monkey kidney cell Cpn60 protein was not recognized by this antibody. This agrees with the published literature from Stressgen, (Victoria, B.C.) indicating that this antibody is specific for leishmania Cpn60. Results from transfection experiments and immunoblots, which utilized either antibody #85 or SPA 823 as primary antibody, indicated that synthesis of the L. major Cpn60 encoded 69 in the pcDNA3.1 was successful and that the levels of protein produced did not appear to be toxic to the cells. 3.3 RT-PCR: Generation of cDNA, Amplification and Analysis This RT-PCR procedure was done to examine whether mouse cells at the immunization site were taking up the vaccine construct and whether the gene was being transcribed in vivo. RT-PCR was carried out as described on approximately 30 mg of fresh tissue excised from the vaccination site of six BALB/c mice. The tissue was transported from the animal care facility on dry ice to the laboratory for extraction of total RNA. RT was done using gene specific primers, 5R, 6R, or P-actin antisense primer. Control samples were prepared (1) where incubation for RT was not done but the samples went directly from the RT reaction to the PCR reaction and (2) samples were not subjected to RT. Figure 10. Results of RT-PCR from cells of pcDNA 3.1/Cpn60 vaccinated mouse. Lanes 1 and 12: 100 bp ladder, lane 2: empty, lane 3: PCR without RT incubation -primers and 4F/4R, lane 4: RT-PCR using 5R (RT) and 4F/4R (PCR), lane 5: RT-PCR using 6R (RT) and 5F/5R (PCR), lane 6 PCR without RT incubation - primers 5F/5R, lane 7: PCR without template using primers 4F/4R), lane 8: PCR without template using primers 5F/5R, lane 9 empty, lane 10: positive PCR control using pcDNA3.1/Cpn60 (1 ng) and primers 4F/4R), lane 11: positive PCR control using pcDNA 3.1/Cpn60 (1 ng) and primers 5F/5R). 70 . Samples from all mice vaccinated with either pcDNA3.1 (control group A, 1-6) and pcDNA3.1/Cpn60 (vaccine group B, 7-12) were subjected to RT-PCR. DNA was detected on a 2% agarose gel with ethidium bromide. None of the samples from mice immunized with pcDNA3.1 control plasmid showed amplification of DNA (data not shown). Only one sample from the pcDNA3.1/Cpn60 immunized mice (B9) showed amplification of a 350 bp fragment (Figure 10, lanes 4 and 5). RT-PCR using P-actin-specific primers showed that one sample only (B9) amplified a 350 bp P-actin fragment (data not shown). In a typical mammalian cell mRNA constitutes only 1 - 5% of total RNA. The expected yield of total RNA from mouse muscle is approximately 1 pg/mg of tissue, therefore total yield of mRNA will be 0.01 - 0.05 pg/mg of tissue (77). The difficulty in RNA isolation is that most ribonucleases are very stable, active enzymes and do not require cofactors to work (77, 78, 79). The amplification of DNA in the appropriate size range in one sample only (out of a possible 6 samples) indicated that in the other five samples (a) the quality of the RNA was poor or there was not enough RNA present, (b) the myocytes were not transfected by the recombinant vaccine plasmid, or (c) the cells carrying the plasmid were missed on excision of the tissue. As the control B-actin gene was amplified in only one sample, it would seem that the quality and the RNA concentration were too low in the other samples. Since even single molecules of mRNA can be amplified by PCR efficiently (79), the conclusion must be that the quality of the RNA in the non-amplified samples was poor or the RNA was absent. Although it is possible that the muscle cells were not transfected in the other samples, all cells contain P-actin and this gene would have been amplified by RT-PCR if high quality P-actin mRNA had been present. 71 3.4 In Vivo Mouse Immunization Experiments T cell function can be studied by measuring the amount and type of cytokines that are produced (21). Detection and analysis of cytokine production are necessary to determine the success or failure of immune responses to pathogenic antigens contained in vaccine preparation (46). Sandwich ELISA is a rapid and sensitive method to assay for biological molecules using antibody/antigen interactions. Antigen that may cross-react with one antibody is highly unlikely to cross-react with the other increasing assay specificity. Cytokines of interest in this study include IL-4, IL-2 and IFN-y. Elevated levels of IL-2 and IFN-y and little, or no, IL-4 are expected from T cells isolated from mice successfully vaccinated against L. major following antigen stimulation. These results are consistent with work done by other researchers testing vaccine preparations against leishmaniasis (5, 16, 36, 38,41,44, 45,46, 47). 3.4.1 Trial Immunization Study Six BALB/c mice, five weeks of age, were injected bilaterally in the thigh muscle (50 ug DNA or 50 ul PBS in each muscle). Mice were sacrificed ten days following the second boost. Cardiac puncture was performed and splenic lymphocytes, stimulated with L. major lysate or unstimulated, were grown in culture. Supernatants were harvested at 48 and 96 hours and assayed for cytokines IL-2 and IL-4. This system can detect concentrations of antigen as low as 14 pg/ml (74). 72 3.4.1.1 Immunoassay for IL-4 The results of the IL-4 irnmunoassay (not shown) indicated that.IL-4 values for all groups were lower than the minimum detection range of the standard for IL-4, 15 pg/ml, and values for stimulated and unstimulated cells did not differ appreciably. In a DNA vaccine study involving protection against leishmaniasis, Xu and Liew (16) and Walker et al. (36) reported IL-4 levels in lymphocytes cultured with soluble leishmania antigen at less than 100 pg/ml in gp63/pcDNA 3, empty plasmid and PBS vaccinated BALB/c mice. 3.4.1.2 Immunoassay for IL-2 Table IV shows the results of the IL-2 immunoassay in the trial immunization study. The levels of IL-2 measured in the vaccine and PBS mouse lymphocytes did not change significantly over the 48 and 96 hour harvest, nor was there any appreciable difference between the stimulated and unstimulated cells in these two groups. The empty plasmid mouse Table IV. In vitro production of IL-2 from spleen cells of vaccinated mice stimulated with L. major lysate or unstimulated in the trial immunization study. Mouse group 48 hour harvest Stimulated(pg/ml) 96 hour harvest Stimulated(pg/ml) Unstimulated(pg/ml) Vaccine Empty plasmid PBS 52± 0.7 43±0.8 22+0.5 40+0.9 170+0.7 30+0.3 55+0.6 146+0.7 30+0.5 Standards low range: 14 pg/ml 73 group produced comparable levels of IL-2 to the vaccine and PBS mouse groups at 48 hours. At 96 hours, however, the levels of this cytokine increased slightly in both the soluble leishmania antigen (SLA) stimulated and unstimulated cells in this group, but no appreciable difference in cytokine levels between the stimulated and unstimulated cells was noted. IL-2 levels in the vaccine group more closely parallel the low levels in the PBS group at 96 hours. It was expected that IL-2 values for the pcDNA3.1/Cpn60 vaccinated mice would have been significantly elevated over background and that IL-2 levels for both of the control groups would not have differed significantly from background levels. The levels of IL-2 produced by the empty plasmid mouse group, in both stimulated and unstimulated cells, was 4 - 5 times higher than either the vaccine or PBS group. Reasons for the higher levels in the empty plasmid group are not known. It cannot be attributed to the vaccination regime as all mouse groups were treated in the same manner. These results suggest that a Thl response, with increased levels of IL-2, was not generated. Xu and Liew (16) reported elevated IL-2 levels (100 ng/ml) after 48 hours in culture from spleen and lymph node cells stimulated with SLA in plasmid encoded go63 vaccinated BALB/c mice. IL-2 levels in control mice were less than 0.1 ng/ml. (Similar results were recorded after 24 and 72 hours in culture in this same experiment). Elevated levels of IL-2 have also been measured in other studies where a Thl response was necessary for protection against an intracellular pathogen (4, 13). 3.4.2 Serum Antibody Results in the Trial Immunization Study Cardiac puncture was performed under anaesthesia on all mice and approximately 0.5 - 1.0 ml of blood was collected from each mouse. Serum samples from the mice within each group 74 Protein Sfase markers (kj»a) ! 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Figure 11a Figure l i b Figure 11c Figure 11. Serum antibody studies of vaccine, empty plasmid and PBS mouse groups. Figure 11a.: Western blot probed with vaccine group serum. Figure l ib: Western blot probed with empty plasmid group serum. Figure 11c: Western blot probed with PBS group serum. All groups: lane 1: molecular weight markers; lane 2: E. coli total protein; lane 3: S. aureus total protein; lane 4 purified human Cpn60; lane 5 L major total protein were pooled and used as a primary antibody to probe immunoblots of protein from controls and L. major total protein (Figures 11a, l ib , and 11c). Sera from the vaccine group (Fig. 1 la) reacted with a single E. coli protein (lane 2) that is smaller than 45 kDa in size, S. aureus proteins from approximately 50 kDa and smaller (lane 3), and with a leishmanial protein of approximately 70 - 80 kDa (lane 5). Sera from the empty plasmid and PBS mouse groups (Figures l i b and 11c respectively) reacted with the same S. aureus proteins (lane 3) and E major protein (lane 5) as the vaccine group. Sera from the empty plasmid group and the PBS group failed to react with the E. coli protein (Figs, l i b and 11c: lane 2). No cross-reactivity with human Cpn60 (Fig. 11a, l ib , 11c lane 4) was observed in any of the blots. These results 75 suggest that an antibody response to L. major Cpn60 was not generated in this vaccine experiment. 3.4.3 Second immunization study Each of the three groups of seven mice was immunized with either 100 pg of vaccine plasmid (vaccine group), 76 pg of empty plasmid (control group) or 100 pl of lx sterile PBS in the anterior tibialis muscle bilaterally. Three mice from each group were sacrificed after the first boost, and the remaining four mice in each group were sacrificed ten days after the second boost. Splenocytes were stimulated with either Concanavalin A, SLA, or unstimulated. All supernatants were harvested after 72 hours. Immunoassays for IFN-y, IL-4 and IL-2 were performed on samples pooled within each group. 3.4.3.1 Immunoassay for IL-4 IL-4 concentrations in all samples (after both the first and second boost) were below the minimum detection range of 7.8 pg/ml on the standard curve prepared for mouse IL-4. Results of the immunoassay for IL-4 in both the 6 mouse and 21 mouse experiments were as expected with no detectable levels of IL-4 being produced by antigen stimulated T-cells. Con A stimulated cells, however, produced very inconsistent values between samples. It should be noted that immunoassay kits from different companies were used in the six-mouse and 21-mouse experiment, therefore standard curve values are not exactly the same between the trial and second immunization study. 76 3.4.3.2 Immunoassay for IL-2 Sample values for IL-2 after the first boost showed no appreciable difference between unstimulated and stimulated cells. IL-2 levels in the empty plasmid group were approximately 3-5 times higher than either the vaccine or PBS groups in both the unstimulated (145, 52, and 29 pg/ml respectively) and SLA stimulated cells (165, 39, and 29 pg/ml respectively). Higher values for the empty plasmid group was also noted in the trial immunization study for IL-2. Con A values ranged from 16-150 pg/ml. Table V. In vitro production of IL-2 from spleen cells of vaccinated mice stimulated with Con A, L. major lysate, or unstimulated in the second immunization study. Immunization Group Con A Stimulated Unstimulated After first boost Vaccine SLA Stimulated Empty plasmid PBS 31±0.9 150+1.1 16+1.8 52+0.6 145±0.8 29+0.5 39+0.9 165±0.8 29+0.5 After second boost Vaccine 65±L9 Empty plasmid 44+1.0 PBS 39+4.6 59±0.7 100+0.01 45+1.5 35±_0.5 37.5±1.0 <15.6 Minimum detection range: 15.6 pg/ml (75). Sample values shown are an average between duplicates. After the second boost Con A stimulated samples produced values that were essentially equal to background levels in all three groups (Table V). As in the trial immunization study IL-2 levels in SLA stimulated cells were not significantly different from background levels in 77 any of the three mouse groups, and, in fact, were slightly lower in the SLA stimulated cells than in any of the non-stimulated cells. IL-2 levels in the unstimulated empty plasmid group (lOOpg/ml) were twice as high as in both the PBS group (45 pg/ml) and the vaccine group (59 pg/ml). IL-2 levels in the SLA stimulated vaccine and empty plasmid groups were essentially the same (35 pg/ml and 37 pg/ml respectively), and in the SLA stimulated PBS group were below detectable range (15.6 pg/ml). Even though the biological controls failed, these results again suggest that the absence of elevated levels of the Thl immune enhancing cytokine IL-2 in the vaccine mouse group indicates that a protective Thl immune response against L. major Cpn60 was not generated. 3.4.3.3 Immunoassay for IFN-y Table VI shows results of the assay for IFN-y in the mouse splenocytes after the second boost. IFN-y levels after the first boost were below the minimum range detected of 9.4 pg/ml in all samples. Con A values ranged from 18-810 pg/ml. After the second boost here was no significant difference between IFN-y levels in the Con A stimulated cells and the unstimulated cells in the empty plasmid (842 pg/ml and 795 pg/ml respectively) and PBS groups (860pg/ml and 870pg/ml respectively). Why background levels of IFN-y are high in these two groups and only 55 pg/ml in the vaccine mouse group cannot be explained. Xu and Liew (16) recorded IFN-y background levels of less than 0.1 ng/ml after 48 hours (and unchanged after 72 hours) 78 T a b l e V I . In vitro p r o d u c t i o n o f I F N - y f r o m spleen cells o f v a c c i n a t e d m i c e s t i m u l a t e d w i t h C o n A, L. major lysate , o r u n s t i m u l a t e d i n the second i m m u n i z a t i o n s tudy f o l l o w i n g the second boost . I m m u n i z a t i o n G r o u p s C o n A s t i m u l a t e d U n s t i m u l a t e d SLA s t i m u l a t e d Vaccine 813±0.5 55±0.6 <9.4 Empty plasmid 842+0.1 795±1.6 <9.4 PBS 860±0.3 870+1.3 22+2.1 • Minimum detection range of assay: 9.4 pg/ml (76). Values shown are an average between duplicates. for empty plasmid and PBS control groups, and IFN-y levels of 200 ng/ml in the vaccine group in an L. major study using gp63 as a vaccine. In a similar study, also using L. major gp63 as a vaccine, Walker et al. (36) recorded no detectable levels of IFN-y in the empty plasmid group after 72 hours in culture (sensitivity level=10 pg/ml and IFN-y levels in the vaccine mouse group were reported as 65 ng/ml. In a Mycobacterium tuberculosis vaccine study (4) IFN-y levels in splenocytes stimulated with recombinant mycobacterial hsp65 from BALB/c mice vaccinated with a plasmid encoding M. leprae hsp65 were measured at 7 ng/ml while IFN-y levels in control mice were approximately 500 pg/ml. In this study SLA stimulated cells in the vaccine mouse group produced no detectable IFN-y. SLA appears to depress cytokine secretion in the empty plasmid and PBS groups as IFN-y measured was much higher in the unstimulated cells than in the stimulated cells. The expectation was that IFN-y secretion would be elevated over the empty plasmid and PBS controls in the SLA treated cells from the vaccine mouse group. The lack of any significant amount of IL-2 and IFN-y production from SLA stimulated cells in the vaccine mouse group may possibly be because (a) the pcDNA3.1/Cpn60 was not taken up by myocytes, (b) the 79 plasmid was taken up but the antigen was not produced, or (c) the antigen was not presented to professional antigen presenting cells resulting in the lack of activation of antigen-specific T lymphocytes. Another possibility is that the immunizations were not optimal and the injected plasmid did not find the muscle. 3.4.3.4 Serum antibody results in the second immunization study Blood was collected as before by cardiac puncture and sera within each group was pooled. This sera was used to probe western blots of Leishmania total protein. Control antibody, SPA 823 (anti-leishmanial Cpn60 antibody), was used to probe the same total protein from L. major. None of the sera from any group reacted with any of the leishmanial protein. The anti-leishmania Cpn60 antibody recognized a protein in the region of 65 kDa (data not shown). The absence of increased levels of Thl cytokines in mice vaccinated with the pcDNA3.1/Cpn60, and the absence of an antibody response to L. major Cpn60 indicates that this vaccination schedule did not support the hypothesis put forward in this study. 80 IV. CONCLUSION The induction of distinct subsets of CD4 + T cells in strains of mice susceptible or resistant to a leishmanial infection makes it possible to determine the efficacy of a potential vaccine against cutaneous leishmaniasis caused by L. major. Immunization with DNA represents a new approach in which long-lasting cell-mediated and humoral immune responses are generated. Plasmids do not replicate in host cells due to the lack of a functional origin of replication nor do they integrate into the host chromosomal DNA (82). Injection of L. major into BALB/c mice results in an uncontrolled growth of the parasite with spread to the local draining lymph nodes. If the infection is left to run its course in these mice they eventually succumb to visceralizing disease (41). Resistant mouse strains, on the other hand, mount an effective immune response to the parasite early in the infection cycle and are able to resolve their lesions. Although these mice are considered clinically cured, viable parasites can be found in lymph nodes that drain the site of infection, therefore, leishmaniasis is considered to be a chronic disease (44). In this study, a DNA vaccine was developed using pcDNA3.1 encoding the L. major Cpn60 gene under the control of a CMV promoter and regulatory elements known to mediate high levels of gene expression under mammalian cell culture conditions. This vector has been used in other successful DNA vaccine experiments (4,11,16, 36, 47). The plasmid acts as an adjuvant to drive the differentiation of CD4 + T cells toward a Thl phenotype due to ISS in the plasmid backbone. The pcDNA3.1 has 331 CpG and ten percent of these dinucleotides are found within ISS as defined by Kreig et al. (32). The vaccine plasmid was tested for expression in a cell-free transcription translation system using rabbit reticulocyte lysate, and in 81 transfection studies using COS-7 cells. A prominent product of the expected molecular mass of 65 kDa was produced by pcDNA3.1/Cpn60, but not by empty pcDNA3.1. These data show that the mammalian translational machinery could express the Cpn60 encoded in the plasmid and that expression levels did not appear to be toxic to the cells. Following successful expression of the protein in vitro, the vaccine was injected into BALB/c mice and splenic lymphocytes were assayed for the appropriate cytokines to test for vaccine efficacy. Assays for IL-2 and IFN-y (indicative of a Thl protective response), and IL-4 (indicative of a Th2 response) were done using ELISA. Cytokine profiles were not as expected. Elevated levels of Thl cytokines were not expressed by T lymphocytes in vivo from mice immunized with the vaccine construct after stimulation with L. major lysate. As well, serum antibodies to the antigen were not detected in vaccine mice. It is not clear why this vaccine experiment failed. There are many factors that can affect the outcome of a DNA vaccine study. The most important are: (1) the site and method of vaccine delivery, (2) transfer of plasmid DNA into the mouse muscle, (3) age and genotype of the host animal, and (4) the intracellular biology, expression levels, and toxicity of the antigen (23). The site and method of injection is of utmost importance in DNA based immunizations because the first step in this process is transfection of cells at the injection site. Gene gun delivery is much more efficient than needle injection, requiring nanogram amounts of DNA to generate a comparable immune response. DNA delivered by gene gun penetrates the cell membrane without killing the cell (88). Intramuscular and intradermal needle injections place the DNA extracellularly and most of this extracellular DNA is rapidly degraded by nucleases (23). The site of injection, i.e. muscle or epidermis, can make a marked difference in the outcome off a DNA vaccine study. One researcher vaccinated mice with the same vaccine 82 construct in three different sites, skeletal muscle, dermis of abdominal skin and the ear pinna to compare vaccination efficiencies. Although muscle tissue continued to express antigen longer than the other two sites, length of expression time did not correlate with the intensity of the immune response. The ear pinna was the most effective in generating specific humoral and CTL responses and muscle the least effective (102). In the trial immunization study mice were vaccinated by intramuscular injection in the quadriceps muscle and in the second immunization study injections were given in the anterior tibialis muscle. The quadriceps is composed of four separate muscles with a common tendon of insertion. Each of the individual muscle bellies of the quadriceps is smaller than the muscle belly of the anterior tibiaalis and there is a risk of injecting between the muscles into the interstitial space. Also, the skin of the mouse over the thigh is loose and fatty so there is a high possibility of inserting the DNA subcutaneously. The skin over the anterior tibialis is taut and thin and it is possible to see the muscle belly expanding as you inject the DNA correctly (65). Studies done by Davis et al have determined that endotoxin-free normal saline is a reasonable carrier for DNA resulting in the transfection of between 1 - 5% of myofibrils in the injection site area (10, 82). TE (Tris-EDTA) and water are not recommended as EDTA chelates the Ca 2 + needed for normal functioning of muscle fibres and water can cause the DNA to relax from the preferred supercoiled form. It has also been reported that muscle cells made edematous by an intramuscular injection of hypertonic 25% sucrose 20 minutes prior to DNA injection increased reporter gene expression (82), however, this strategy may not be feasible in human vaccination. The preferred volume for i.m. injections given bilaterally is 50 ul/injection as expression is less variable if the DNA is injected in a larger rather than a smaller volume (10). However, Barry et al. (23) recommends dividing the total volume given 83 to each mouse into four equal parts to decrease the chance of a mouse 'missing' an injection round completely. It has also been determined that younger mice (5 to 8 weeks old) exhibit a stronger immune response than older mice (10 weeks or more). In one study where antibody response was measured in 4, 5, and 10 week old mice it was found that antibody production was inversely proportional to age (23). Female mice also give a better immune response and are easier to group-house than their more aggressive male counterparts (65). The effect of levels of gene expression on the immune response is dependent on the antigen. Where toxicity is not a problem because the gene products are being continuously secreted out of the cell, then more is better. In contrast, increasing the expression of non-secreted proteins does not necessarily increase the immune response but does increase the possibility of the protein accumulating to toxic levels. Over-expression of the antigen may kill the transfected cells too early to maintain an immune response (23). If epitopes from the antigenic protein have been mapped, then the protein may be broken down into subunits to avoid toxicity before inserting them into a plasmid (23). If antigen expression levels are too low, inserting an intron between the promoter and the encoded protein may increase expression levels. Barry et al. (23) reported that inserting an inron 5' to luciferase in a CMV plasmid backbone increased expression levels 5-fold compared to the same CMV plasmid without the intron. Also, antigens from intracellular pathogens that are used in genetic immunization experiments may modify or even shut down host cell function. The efficiency of post-translational modifications of encoded proteins and the ability of the host immune system to recognize the available epitopes also is important in the successful outcome of vaccine experiments. 84 Whether failure to stimulate an immune response in this study was due to overexpression or underexpression of antigen is not known. It is possible that toxicity due to overexpression of the Cpn60 gene may have contributed to the inability to generate an immune response in mice immunized with this vaccine construct. Given that few cells are transfected by plasmid, even under optimal conditions, and that some injections may have missed their target, it is more likely that too little protein was produced to stimulate a good immune response. Future work could address some of these problems. An intron inserted in the plasmid 5' to the encoded protein could increase protein expression levels. If toxicity is a problem then using a plasmid under control of a SV40 promoter would decrease protein expression levels. Immune enhancement by co-injection of plasmids encoding Thl cytokines such as IL-12 may also be beneficial. Also, intradermal injections may be more efficient routes for uptake and presentation of DNA by dendritic cells as dendritic cells are abundant in skin. Further practice of injection techniques to optimize delivery of the vaccine is important to ensure a good response after all other factors have been considered. It has recently been reported by Zucchelli et al. (90) that increased expression of gene products using a DNA vaccine encoding hepatitis C virus E2 was achieved by intramuscular injection of the recombinant plasmid followed by electrical stimulation of the injected muscle fibers. Improved antibody response (up to 30 times greater than naked DNA injection without electrical stimulation) and enhanced CD4 + and CD8 + T cell induction were recorded. This electrical gene transfer (EGT) procedure has been used to provide efficient in vivo expression of plasmid encoded genes in the skin, corneal endometrium, brain, liver and muscle (90). This may be a useful procedure for any further experiments using pcDNA3.1/Cpn60 assuming 85 that in vivo expression of the encoded Cpn60 was very low due to poor transfection of the muscle fibres. LITERATURE CITED 1. Whalen, R. G. 1996. DNA vaccines, cyberspace and self-help programs. Intervirology 39: 120-125. 2. Liu, M . A . 1998. Vaccine developments. Nature Med. Vaccine Supplement. 4':515 -519. 3. Ertl, H. C , and Z. Xiang. 1996. Novel vaccine approaches. J. Immunol 156:3579-3582. 4. Tascon, R. E., M . J. Colston, S. Ragno, E. Stavropoulos, D. Gregory and D. B. Lowrie. 1996. Vaccination against tuberculosis by DNA injection. 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