Medicine, Faculty of
Medicine, Department of
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UBCV
Gelinas, Laura Clinckett
Master of Science - MSc
University of British Columbia
Vaccines are one of the greatest medical triumphs. Due to the empirical nature of vaccine development, however, it is still unclear how we maintain immunological memory and protection following vaccination. Using cells from a cohort of healthy infants and the hepatitis B virus (HBV) vaccine as a model, I developed a vaccine-specific stimulation assay that detects multiple immune readouts simultaneously from memory T cells in response to antigen stimulation in vitro. By stratifying my cohort by their anti-HBV surface antigen antibody (anti-HBs) titers, I determined how their HBV-specific memory T cell immune responses differed stratified by serological response. Based on my results, interferon (IFN)-γ appeared to be the most consistent predictor of a stronger serological response to HBV vaccination. The most striking trend was a positive correlation between bulk IFN-γ secretion and anti-HBs titers at 6 and 18 months following vaccination. A similar trend was noted for interleukin (IL)-10 secretion, but it did not extend past the 6-month time point. Tumor necrosis factor (TNF)-α, on the other hand, had a dynamic production profile. Overall, TNF-α production in proliferating CD8 T cells was increased in those subjects who produced lower levels of anti-HBs six months after vaccination. This observation was reversed by the 18-month time point—children who had anti-HBs titers less than 10 IU/l secreted lower levels of TNF-α. Many poor responders, as defined by their anti-HBs titers, demonstrated evidence of T cell memory either by HBsAg-specific proliferation, intracellular cytokine production, or cytokine secretion—indicating that an alternative or additional immunological mechanism might also correlate with vaccine-induced protection. The ultimate goal of this research project was to develop an in vitro antigen stimulation protocol that minimized sample usage and gathered data to help generate hypotheses regarding how the adaptive immune system, as a whole, responds to vaccination. The results from my study identified IFN-γ, TNF-α, as well as IL-10 as potential targets for continued investigation. However, in order to determine which immune parameters—whether humoral, cell-mediated, or even innate immunity—best predict strong and long-lasting immunological memory, we need to design more comprehensive longitudinal studies that extend beyond infancy.
THE RELATIONSHIP BETWEEN T AND B CELL HEPATITIS B VIRUS VACCINE- SPECIFIC RESPONSES IN A COHORT OF HEALTHY INFANTS by Laura Clinckett Gelinas B.Sc.(Hons), Dalhousie University, 2010 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2013 © Laura Clinckett Gelinas, 2013 ii ABSTRACT Vaccines are one of the greatest medical triumphs. Due to the empirical nature of vaccine development, however, it is still unclear how we maintain immunological memory and protection following vaccination. Using cells from a cohort of healthy infants and the hepatitis B virus (HBV) vaccine as a model, I developed a vaccine-specific stimulation assay that detects multiple immune readouts simultaneously from memory T cells in response to antigen stimulation in vitro. By stratifying my cohort by their anti-HBV surface antigen antibody (anti- HBs) titers, I determined how their HBV-specific memory T cell immune responses differed stratified by serological response. Based on my results, interferon (IFN)-γ appeared to be the most consistent predictor of a stronger serological response to HBV vaccination. The most striking trend was a positive correlation between bulk IFN-γ secretion and anti-HBs titers at 6 and 18 months following vaccination. A similar trend was noted for interleukin (IL)-10 secretion, but it did not extend past the 6-month time point. Tumor necrosis factor (TNF)-α, on the other hand, had a dynamic production profile. Overall, TNF-α production in proliferating CD8 T cells was increased in those subjects who produced lower levels of anti-HBs six months after vaccination. This observation was reversed by the 18-month time point—children who had anti-HBs titers less than 10 IU/l secreted lower levels of TNF-α. Many poor responders, as defined by their anti-HBs titers, demonstrated evidence of T cell memory either by HBsAg-specific proliferation, intracellular cytokine production, or cytokine secretion—indicating that an alternative or additional immunological mechanism might also correlate with vaccine-induced protection. iii The ultimate goal of this research project was to develop an in vitro antigen stimulation protocol that minimized sample usage and gathered data to help generate hypotheses regarding how the adaptive immune system, as a whole, responds to vaccination. The results from my study identified IFN-γ, TNF-α, as well as IL-10 as potential targets for continued investigation. However, in order to determine which immune parameters—whether humoral, cell-mediated, or even innate immunity—best predict strong and long-lasting immunological memory, we need to design more comprehensive longitudinal studies that extend beyond infancy. iv PREFACE This thesis is original, unpublished work by the author, L. C. Gelinas. All experiments and data analyses were designed and performed by the author with two exceptions. Cell isolation and cryopreservation (Section 2.2) was performed by Darren Blimkie, Bing Cai, and Nathan Corbett. The anti-Hepatitis B surface antigen antibody assays (Section 2.3) were completed by Yves Delmarcelle under the guidance of Dr. Arnaud Marchant at the Université Libre de Bruxelles in Belgium. This study was approved by the University of British Columbia Children and Women’s Health Centre Research Ethics Board (Certificate Number: H06-03339). v TABLE OF CONTENTS ABSTRACT................................................................................................................................... ii PREFACE..................................................................................................................................... iv TABLE OF CONTENTS..............................................................................................................v LIST OF TABLES ..................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... ix LIST OF ABBREVIATIONS ...................................................................................................... x ACKNOWLEDGEMENTS ...................................................................................................... xiii 1 INTRODUCTION .................................................................................................................. 1 1.1 Vaccines ............................................................................................................................ 1 1.1.1 Developing immunological memory........................................................................... 1 1.1.2 Mechanisms of protection ........................................................................................... 3 1.2 Methods used to assess vaccine-specific immune responses in vitro ........................... 5 1.2.1 Proliferation................................................................................................................. 8 1.2.2 Enzyme-linked immunosorbent assay (ELISA).......................................................... 9 1.2.3 Enzyme-linked immunosorbent spot (ELISPOT) ....................................................... 9 1.2.4 Flow cytometry ......................................................................................................... 10 1.3 Hepatitis B virus ............................................................................................................ 11 1.3.1 Epidemiology ............................................................................................................ 11 1.3.2 Prevention of HBV infection..................................................................................... 12 1.3.3 HBV vaccine as part of the EPI ................................................................................ 13 1.3.4 HBV vaccine efficacy ............................................................................................... 14 1.3.5 HBV vaccine non-responders.................................................................................... 15 1.4 Research project ............................................................................................................ 16 2 MATERIALS AND METHODS ......................................................................................... 17 2.1 Cohort description ......................................................................................................... 17 2.2 Sample processing.......................................................................................................... 18 2.3 HBsAg enzyme immunoassay....................................................................................... 19 2.4 Cell culture and proliferation assay............................................................................. 19 2.5 Measurement of bulk cytokine secretion in supernatant........................................... 21 2.6 Measurement of total immunoglobulin (Ig) isotypes and IgG subclasses ................ 21 2.7 Flow cytometry............................................................................................................... 22 2.8 Proliferation analyses .................................................................................................... 23 2.9 Statistical analyses ......................................................................................................... 23 3 RESULTS .............................................................................................................................. 25 3.1 Demographics................................................................................................................. 25 3.2 HBsAg-specific antibody titers and total immunoglobulin levels ............................. 26 3.3 HBsAg-stimulation assay: descriptive results ............................................................. 28 3.3.1 PBMC proliferation................................................................................................... 29 3.3.2 Bulk cytokine secretion............................................................................................. 31 vi 3.3.3 Intracellular cytokine production .............................................................................. 34 3.4 Comparing humoral and cell-mediated antigen-specific responses......................... 38 3.4.1 Low responder versus responder: year 1................................................................... 38 3.4.2 Top and bottom quartiles: year 1............................................................................... 40 3.4.3 Correlation between anti-HBs titer and HBsAg-specific T cell responses: year 1 ... 41 3.5 Comparing humoral and cell-mediated antigen-specific responses: extending to year 2 ........................................................................................................................................ 42 3.5.1 Seronegative versus seropositive: year 2 .................................................................. 43 3.5.2 Top and bottom quartiles: year 2............................................................................... 46 3.5.3 Correlation between anti-HBs titer and HBsAg-specific T cell responses: year 2 ... 48 3.6 Comparing humoral and cell-mediated antigen-specific responses: summary ....... 48 3.7 Demographics................................................................................................................. 50 4 DISCUSSION ........................................................................................................................ 51 4.1 HBsAg-specific proliferation ........................................................................................ 52 4.2 Cytokine Responses ....................................................................................................... 55 4.2.1 HBsAg-specific IL-10 production............................................................................. 55 4.2.2 HBsAg-specific IL-13 production............................................................................. 58 4.2.3 HBsAg-specific IL-21 production............................................................................. 60 4.2.4 HBsAg-specific TNF-α production........................................................................... 62 4.2.5 HBsAg-specific IFN-γ production ............................................................................ 64 4.3 Combination vaccines and concomitant vaccination.................................................. 67 4.4 Parallels to other vaccines............................................................................................. 69 4.4.1 Measles vaccine......................................................................................................... 69 4.4.2 Pertussis vaccine ....................................................................................................... 71 4.4.3 Smallpox vaccine (vaccinia) ..................................................................................... 72 4.4.4 Tetanus vaccine ......................................................................................................... 72 4.4.5 Hepatitis A virus vaccine .......................................................................................... 73 4.4.6 HBV infection ........................................................................................................... 73 5 CONCLUSION ..................................................................................................................... 75 5.1 Study limitations ............................................................................................................ 76 5.2 Future directions............................................................................................................ 78 5.3 Final remarks ................................................................................................................. 81 BIBLIOGRAPHY....................................................................................................................... 82 APPENDICES............................................................................................................................. 97 Appendix A: Gating strategy for flow cytometry data........................................................ 97 Appendix B: P-values from statistical tests for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers less than 100 IU/l and greater or equal to 100 IU/l at the Y1 time point.................................................................................................. 98 Appendix C: P-values from statistical tests for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers in the top and bottom quartile at the Y1 time point. ................................................................................................................................ 99 Appendix D: Correlation coefficients (r) and P-values from statistical tests for all correlation analyses between anti-HBs titers at Y1 time point and T cell HBsAg-specific responses. ............................................................................................................................... 100 vii Appendix E: P-values from statistical tests for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers less than 10 IU/l and greater or equal to 10 IU/l at the Y2 time point....................................................................................................... 101 Appendix F: P-values from statistical tests for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers in the top and bottom quartile at the Y2 time point. .............................................................................................................................. 102 Appendix G: Correlation coefficients (r) and P-values from statistical tests for all correlation analyses between anti-HBs titers at Y2 time point and T cell HBsAg-specific responses. ............................................................................................................................... 103 Appendix H: P-values from statistical tests for all comparison of HBsAg-specific CMI responses and anti-HBs titers between males and females. .............................................. 104 Appendix I: P-values from statistical tests for all comparison of HBsAg-specific CMI responses and anti-HBs titers between major racial groups in the cohort (Caucasian, Asian, South Asian, Asian-Caucasian)................................................................................ 105 viii LIST OF TABLES Table 1. Demographics of cohort with breakdown for humoral and cell-mediated immunity components. .......................................................................................................................... 25 Table 2. Phlebotomy and HBV vaccination schedule for all cohort subjects. ............................. 26 Table 3. HBsAg-specific antibody titers for cohort subjects and number of subjects with anti- HBs titers below 10 IU/l, below 100 IU/l, and equal or greater than 100 IU/l at one and two years of age. .......................................................................................................................... 27 Table 4. Number of cohort subjects with incomplete and complete paired data sets. ................. 27 Table 5. Levels of total immunoglobulin isotypes and IgG subclasses in cohort subjects. ......... 28 Table 6. Correlation analyses between levels of cytokine secreted into supernatant in response to HBsAg stimulation. .............................................................................................................. 33 Table 7. Correlation analyses between percentage of starting CD4 or CD8 that proliferated in response to HBsAg stimulation and the level of bulk cytokine secretion. ........................... 33 Table 8. Correlation analyses between percentage of cytokine positive proliferating CD4 T cells in response to HBsAg stimulation. ....................................................................................... 34 Table 9. Correlation analyses between percentage of cytokine positive proliferating CD8 T cells in response to HBsAg stimulation. ....................................................................................... 36 Table 10. Significant correlations between anti-HBs titers at one year of age and HBsAg-specific T cell responses..................................................................................................................... 42 Table 11. Significant differences in HBsAg-specific T cell responses between those with titers < 100 IU/l and ≥ 100 IU/l (at Y1 time point) and between those with titers < 10 IU/l and ≥10 IU/l (at Y2 time point). ......................................................................................................... 44 Table 12. Significant differences in HBsAg-specific T cell responses between those with anti- HBs titers that fall within the top and bottom quartile at the Y1 and Y2 time points. ......... 47 Table 13. Significant correlation analyses results between HBsAg-specific T cell responses and anti-HBs titers from Y1 and/or Y2 time points. ................................................................... 48 Table 14. Number of HBV vaccine non-responders, hypo-responders and responders with a positive cell-mediated immune response after HBsAg stimulation...................................... 50 ix LIST OF FIGURES Figure 1. A significant increase in the percentage of proliferating CD4 and CD8 T cells from the starting population was observed following stimulation with HBsAg and anti-CD3 compared to unstimulated controls. ...................................................................................... 30 Figure 2. A significant increase in levels of IFN-γ, IL-10, IL-13, and TNF-α, but not IL-17A was observed in response to HBsAg stimulation compared to unstimulated controls. ............... 32 Figure 3. A significant increase in percentage of IFN-γ, IL-13, and IL-21 positive proliferating CD4 T cells was observed following HBsAg stimulation. ................................................... 35 Figure 4. A significant increase in percentage of IFN-γ, IL-13, and TNF-α positive proliferating CD8 T cells was observed following HBsAg stimulation. ................................................... 37 Figure 5. Subjects with anti-HBs titers less than 100 IU/l at one year of age secreted less IL-10 and had a higher percentage of TNF-α positive proliferating CD8 T cells in response to HBsAg stimulation than individuals with anti-HBs titers equal or greater than 100 IU/l. ... 39 Figure 6. Subjects ranked in the bottom quartile based on their anti-HBs titers at one year of age secreted less IL-10 and IFN-γ and had a higher percentage of TNF-α positive proliferating CD8 T cells in response to HBsAg stimulation than individuals ranked in the top quartile.41 Figure 7. Subjects with anti-HBs titers less than 10 IU/l at two years of age secreted less TNF-α, had a lower percentage of starting CD4 T cells that proliferated, a lower percentage of IFN- γ positive, IL-13 positive and IL-21 positive proliferating CD4 T cells and lower a percentage of IL-13 positive proliferating CD8 T cells in response to HBsAg stimulation than individuals with anti-HBs titers equal or greater than 10 IU/l. ..................................... 45 Figure 8. Subjects ranked in the bottom quartile based on their anti-HBs titers at two years of age had a lower percentage of IFN-γ positive proliferating CD4 T cells in response to HBsAg stimulation than individuals ranked in the top quartile............................................ 47 x LIST OF ABBREVIATIONS γδ T cell 3H anti-HBs ASC APC BCG BrdU CD CFSE CI CMI CO2 CTL DC DPBS EDTA EIA ELISA ELISPOT EPI FCS HBIG gamma delta T cell tritium antibodies specific against hepatitis B virus surface antigen antibody secreting cell antigen presenting cell bacillus Calmette-Guérin bromodeoxyuridine cluster of differentiation 5,6-carboxylfluorescein diacetate succinimidyl ester confidence interval cell mediated immunity carbon dioxide cytotoxic lymphocyte dendritic cell Dulbecco’s phosphate buffered saline ethylenediaminetetraacetic acid enzyme immunoassay enzyme-linked immunosorbent assay enzyme-linked immunosorbent spot Expanded Programme on Immunization fetal calf serum hepatitis B immune globulin xi HAV HBcAg HBeAg HBsAg HBV HCC HIV HLA ICS IFN Ig IL IQR IU LPS MBA MHC ml NCS rcf RPMI RT MMR hepatitis A virus hepatitis B core antigen hepatitis B e antigen hepatitis B surface antigen hepatitis B virus hepatocellular carcinoma human immunodeficiency virus human leukocyte antigen intracellular cytokine staining interferon immunoglobulin interleukin interquartile range international unit lipopolysaccharide multiplex bead array major histocompatibility complex millilitre newborn calf serum relative centrifugal force Roswell Park Memorial Institute room temperature mumps, measles, and rubella vaccine xii OG488 PAMP PBMC PHA PRN PRR SI TCR Tcm Tem Tfh Th TLR TNF WHO Y1 Y2 CellTrace™ Oregon Green® 488 Carboxylic Acid Diacetate, Succinimidyl Ester pathogen-associated molecular pattern peripheral blood mononuclear cell phytohaemagglutinin plaque reduction neutralization pattern recognition receptor stimulation index T cell receptor central memory T cell effector memory T cell follicular helper T cell helper T cell toll-like receptor tumor necrosis factor World Health Organization Year 1 Year 2 xiii ACKNOWLEDGEMENTS I would, first and foremost, like to thank and acknowledge my supervisor, Dr. Tobias Kollmann, for his inspiration and motivation over the last three years. In addition, I want thank Dr. Edgardo Fortuno III for his endless guidance and advice as well as my committee, Drs. David Scheifele, John Schrader and Simon Dobson, for their encouragement and helpful discussion. And, of course, I offer my enduring gratitude to all of the members of the Kollmann laboratory. I would especially like to thank Patricia Cho, Kinga Smolen and Bing Cai for their constant help, support, and friendship. Finally, I would like to acknowledge funding provided by the Canadian Institutes of Health Research through the Frederick Banting and Charles Best Canada Graduate Scholarship for the 2011/2012 scholastic year. 1 1 INTRODUCTION 1.1 Vaccines Vaccines remain, unequivocally, one of the greatest medical triumphs. Since the pioneering work of Edward Jenner in the late 1700s, vaccines have had a significant impact on the global incidence of many infectious diseases including their eradication (e.g., smallpox), regional elimination (e.g., poliomyelitis), and dramatic reduction of both morbidity and mortality (e.g., measles, yellow fever).1 Despite the advances made in the fields of immunology, microbiology, and vaccinology, many of the most successful vaccines were empirically derived with little to no understanding of a known mechanism of protection. Current efforts to develop effective vaccines against new targets, such as human immunodeficiency virus (HIV) and malaria, are not advancing as fast as hoped due to this lack of knowledge.2 1.1.1 Developing immunological memory Vaccines, similar to natural infections, initiate a highly organized and complex sequence of immunological events. While it is the innate immune system that represents the front line defence against invading microbes,3, 4 immunological memory is a unique feature of the adaptive immune system.2 Upon re-encounter with the same pathogen, it responds more rapidly and with greater intensity than the initial exposure. The duration of illness is often shorter and symptoms are less severe or can even go unnoticed. The primary immune response is initiated upon activation of dendritic cells (DC) at the site of vaccination and subsequent presentation of antigen (the inoculum) to naïve T cells 2 residing within secondary lymphoid organs. Naïve T cells require three separate signals in order to become fully activated. The first signal occurs when a membrane-bound T cell receptor (TCR) recognizes and binds to a specific peptide-major histocompatibility complex (MHC) on a DC. The second signal occurs through binding of co-stimulatory molecules CD28 and B7 (CD80 and CD86) on the T cell and DC, respectively. The third signal occurs through cytokine signalling and helps to fine-tune the T cell response. DCs are continually surveying and sampling the body for invading microbes.2 They express a wide variety of germ line-encoded pattern recognition receptors (PRR) that detect evolutionary conserved pathogen-associated molecular patterns (PAMP) broadly shared by pathogens.3, 5 In general, PRRs are capable of recognizing the category of invading pathogen— whether it is bacteria or virus, for instance. Binding of cognate PAMP to its PRR initiates a signalling cascade that activates the DC and helps it to instruct the antigen-specific naïve T cell to differentiate into the most appropriate effector T cell (e.g., Th1, Th2 or suppressor phenotype).2, 6 For B cells, the primary immune response begins when a DC-primed and activated T cell interacts with a specific B cell that has already processed and is presenting antigen. This induces rapid differentiation of the B cell into short-lived plasma cells which is followed by the formation of a germinal centre reaction leading to the eventual generation of both memory B cells and long-lived plasma cells. For polysaccharide antigens, it is also possible for B cells to differentiate into short-lived plasma cells without T cell presentation; however, this does not confer immunological memory.7 Once the inoculum (or infection) is cleared, the antigen-specific cells undergo clonal contraction leaving only a few remaining cells to differentiate into memory lymphocytes. For B 3 cells, long-lived plasma cells confer immediate protection by constant secretion of antibodies in an antigen-independent manner while memory B cells rapidly proliferate upon antigenic stimulation and subdivide into plasma cells to produce a rapid, but transient spike in antibodies.7 For T cells, effector memory T cells (Tem) are constantly circulating to survey blood and tissues and upon antigen recognition, quickly proliferate and provide immediate effector functions.7 Central memory T cells (Tcm) reside in secondary lymphoid organs and once presented with cognate peptide by antigen presenting cells (APC) rapidly divide to provide the recall response.7 1.1.2 Mechanisms of protection While our understanding of the innate and adaptive immune systems is continually increasing and evolving, we still remain unsure of how we maintain immunological memory and protection following most vaccinations.8 What are the relative contributions of plasma cells, T helper cells, or even innate immune cells towards long-lasting immunity? As we move towards the rational method of vaccine design, the identification of a mechanism or mechanisms of protection becomes an important concept to vaccinologists. There are two main terms used to define vaccine efficacy: correlate and surrogate of protection.9 A correlate of protection is an immune response that is responsible for protection; whereas, a surrogate is an immune response the is being used to substitute for a true immunological correlate (whether it be unknown or unmeasurable).9 These can also be referred to as mechanistic and non-mechanistic correlates of protection respectively.10 Determining a vaccine’s correlate(s) of protection is not a simple task. For instance, the mechanism of protection might not be the same as the mechanism for recovery from infection. Many of the current day vaccines rely on antibodies (functional or absolute titer) to determine its 4 efficacy. While antibodies perform important immune functions that expedite and improve pathogen clearance, in particular against extracellular pathogens or toxins, attempts to explain vaccine-mediated protection merely by the humoral response can be a bit simplistic for some vaccines.11 The immune system has been observed to be redundant and vaccines—like natural infection—might rely on numerous mechanisms. This is particularly true for vaccines such as hepatitis B virus (HBV) vaccine where a waning of antibodies over time is common yet individuals apparently remain protected against infection. In addition, those who remain unable to mount a humoral response (non-responders) are often still protected. For some vaccines, cellular mediated immunity (CMI) provides protection, such as the BCG vaccine.9 Another example is smallpox where antibody levels correlate best with protection, but CMI has been shown to influence the severity of infection regardless of the presence of antibodies.9 Another fitting example is the measles vaccine whose defined threshold for protection is a plaque reduction neutralization (PRN) titer equal or greater than 120 IU/l.12 Clinical observations, however, demonstrate that individuals with cellular immunodeficiencies are susceptible to severe and even fatal measles13-15 while those with congenital agammaglobulinemia recover with fewer complications and appear protected from re-infection when exposed by close contact to confirmed infective cases of measles.16 The examples above clearly illustrate that, for cases in which antibodies cannot completely opsonize or neutralize the pathogen allowing it to infect and replicate intracellularly, T cell mediated immune mechanisms become essential for clearance. As such, it is becoming increasingly apparent that co-correlates of protection might be a more fitting model for many vaccines used today. To accomplish this, however, we need to be able to accurately assess 5 vaccine-specific immune responses extending beyond antibody titers to include CMI and, considering its ability to modulate the ensuing adaptive response2, 6, 17, innate immunity as well. 1.2 Methods used to assess vaccine-specific immune responses in vitro There are limited options to assess vaccine responses in vivo in humans. Since many vaccines use antibody titer or function as their correlate or surrogate of protection,9, 12, 18 we often use this readout as a qualifier or quantifier of the in vivo vaccine response. When it is tested pre- and post-booster, it can be used to determine recall responses. The methods to describe in vitro vaccine-specific immune responses, however, are more numerous and continuously changing as technology advances. While it can be technically challenging,19-21 detection of antigen-specific memory B and T cells and description of their phenotypes can provide important information that further our understanding of how the adaptive immune system as a whole reacts to immunization as well as gain insight into long-term efficacy of vaccines. Before describing the various techniques used to identify and describe memory lymphocytes, there are certain variables that are independent of the type of assay used but can equally impact the results. Effect of cryopreservation on peripheral blood mononuclear cell function A common debate amongst scientists is whether it is advantageous to use freshly isolated cells compared to cryopreserved cells. While freshly isolated cells eliminate any artefact generated by freezing, the benefit of cryopreservation to reduce inter-assay variability as well as optimize use of available resources makes it an attractive option, especially for multi-centre trials 6 or longitudinal studies. As such, numerous groups have taken to determining the effects of freezing on function and phenotype of peripheral blood mononuclear cells (PBMCs). Function of cryopreserved cells, as measured by lymphoproliferation, has been observed to associate with viability after thaw.22 But in optimized conditions and post-thaw viability ≥ 75%, both lymphoproliferation and flow cytometric results between cryopreserved and freshly isolated cells are highly correlated.23, 24 It has been shown that cryopreserved cells have a marked decrease in CD62L23, 25 and CD45RO23 expression. These markers can be used to identify naïve T, Tcm, and Tem cells.23 However, using alternative markers such as CCR7 and CD45RA could be potentially used as substitutes to identify the same populations. Another group also found that when using anti-CD3/anti-CD28 as stimulation, there was no difference in level of proliferation or cytokine production between freshly isolated and cryopreserved PBMCs.26 Similar results were confirmed using the enzyme-linked immunosorbent spot (ELISPOT) method to detect cytokine secreting memory T cells.27 With respect to the effect of cryopreservation on B cells, one study found no difference in the number of antigen-specific antibody secreting cells in fresh and cryopreserved samples.28 It seems that cryopreservation may impact PBMC function or phenotype, but in situations where cryopreservation is the only feasible option (e.g., longitudinal or multi-centre trials) these effects can be greatly reduced or eliminated by optimized thawing procedures29 and selecting cell surface markers that are not labile in the freeze-thaw process. Length of stimulation To observe antigen-specific immune responses in vitro, length of stimulation is an extremely important variable. In literature, there are two general protocols that are used: short- term stimulation30-33 or long-term stimulation.34-41 Short-term stimulations are typically between 7 six and 18 hours while long-term stimulations can last anywhere from three to ten days. Both methods have benefits and disadvantages. A clear advantage to the short-term approach is the lesser time commitment, which is particularly useful during assay optimization when experiments are dependent on the previous results. Proponents of short-term stimulation argue that longer culture time increases background and can introduce artefact into the system.42, 43 Those in support of long-term culture, however, retort that antigen-specific cells are rare in peripheral blood, and it can be difficult to detect such small cell populations above background. In addition, allowing for in vitro expansion of antigen-specific cells during a long-term culture has been shown to increase statistical power to detect phenotypic differences.44 Both methods are popular in current literature, and as long as the limitations the selected assay are acknowledged—either can be acceptable. Type of stimulus It is becoming increasingly common to use overlapping peptide pools as source for antigenic stimulation over whole antigen or specific peptides. For those who argue that cryopreservation affects the ability to process and present antigen,33 use of peptide pools bypasses that step. It offers the advantage over single peptides or use of class I or class II tetramers by being able to stimulate CD4 and CD8 T cells and being independent of the human leukocyte antigen (HLA) haplotype.45 Its high cost and difficulty to generate peptide pools for complex virions can limit its widespread applicability.46 The use of whole antigen becomes favourable when you want to use a stimulant that is similar to the inoculum used for in vivo immunization. And as mentioned above, CD4 and CD8 cryopreserved T cells are still able to 8 respond to stimulation27 indicating that whole antigen can processed and presented through both MHC class I and II pathways. 1.2.1 Proliferation A critical measure of CMI is proliferation of antigen-specific T cells. The traditional method for measuring proliferation is pulsing cells for the last 6 to 18 hours of culture with tritiated- (3H) thymidine. The amount of labelled thymidine incorporated correlates with the number of cells synthesizing DNA during the S phase of cell division.47 This method becomes problematic, however, when trying to detect proliferation in rare cell populations.48 In addition, unless purified cell populations are pulsed, it is impossible to determine the phenotype of the responding cells.48 Finally, the 3H-thymidine assay can only detect cells that are proliferating during the pulse. If the cell culture is several days in length, it is possible to miss responding cells that proliferated earlier during the stimulation prior to the 3H-thymidine pulse. An alternative assay to 3H-thymidine was first described in 1994 and is based on dilution of the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE).49 Prior to antigen stimulation, cells are incubated with the dye, which is able to diffuse freely in and out of cells. Following a series of biochemical reactions, the dye becomes trapped, highly fluorescent and stably expressed within the cell.50 Upon stimulation, the fluorescence intensity of the dye is halved with each successive round of cell division. This allows for easy identification and enumeration of proliferating cells using flow cytometry. When combined with fluorochrome- conjugated antibodies to identify cell surface markers and effector molecules, such as cytokines, this assay can provide a much more comprehensive snapshot of antigen-specific responses in vitro.48, 50 9 1.2.2 Enzyme-linked immunosorbent assay (ELISA) The enzyme-linked immunosorbent assay (ELISA) was originally described in the early 1970s51, 52 and is still a very popular method to detect or quantify levels of specific proteins in cell culture or bodily fluids. For immunological studies, ELISAs are often used to detect levels cytokines, chemokines, growth factors or other inflammatory markers providing further insight into the quality of immune response generated in vitro or ex vivo. 41, 53, 54 Conversely, and important for vaccine-related studies, it can also quantify levels of antigen-specific antibodies.55, 56 A drawback to the ELISA is that a separate reaction is required for each analyte measured. If the sample size is small, it limits the number of ELISAs that can be performed. Multiplex bead arrays (MBA) offer an alternative to ELISAs which can circumvent sample limitations. Using MBA technology, there are over 40 cytokines, chemokines, effector molecules, or growth factors that can be measured simultaneously—making it is possible to include a wide range of analytes to capture as many effector subsets as needed without gratuitous sample usage.57 Since ELISAs and MBAs can only measure total protein levels, however, unless purified populations are used, the cellular source of the analyte is unknown. 1.2.3 Enzyme-linked immunosorbent spot (ELISPOT) The ELISPOT assay is conceptually similar to an ELISA, but enumerates antigen- specific memory lymphocytes instead of bulk protein levels. The original assay was designed to enumerate B cell secreting antigen-specific antibodies,58 and still remains a popular method due to its high sensitivity.21, 59-64 The assay has since evolved so that it can also detect cytokine secreting antigen-specific memory T cells. Currently, it is possible to measure up to three 10 different cytokines simultaneously allowing for both enumeration and description—at the polyfunctionality level—of antigen-specific memory T cells.65 1.2.4 Flow cytometry The concept of flow cytometry or cell sorting was originally described and developed in the 1950s.66 Since then, this technology has radically evolved and become one of the useful tools in an immunologist’s arsenal. With newer flow cytometers being able to detect up to 20 different fluorescent emissions, it is possible to describe the phenotype and function of multiple cell types at an unsurpassed level of detail.67 With its ability to measure intracellular cytokine production, polyfunctionality, activation status, proliferation, and cell subsets, flow cytometry is invaluable in vaccine research.67-71 While the techniques mentioned above are reflective of the popular experimental methodology described in current literature, the list is by no means exhaustive. In fact, a systems biology approach has even been used to generate a global picture of the immune responses following vaccination.72 It is now possible to describe, in detail, the antigen-specific immune response using multiple different methods. This is important considering the difficulties that have been encountered developing an effective vaccine against HIV or malaria, for instance.73 In order to begin to understand the immunological mechanisms by which vaccines confer protective immunity, we need to study the current vaccines. There are numerous vaccines that would be suitable models to pursue this avenue of research. In fact, the highly effective yellow fever vaccine, YF-17D, has been previously studied for that purpose.74 For numerous reasons described below, the HBV vaccine is another ideal candidate. 11 1.3 Hepatitis B virus Hepatitis B virus is a partially double stranded DNA virus located within the hepadnaviridae family. It infects and replicates in hepatocytes interfering with liver function. The infectious particle (also referred to as Dane particle) is 42 nm in diameter and composed of two major proteins: HB surface antigen (HBsAg) and HB core antigen (HBcAg). The viral genome along with a DNA-dependent polymerase is packaged within an isometric nucleocapsid made of the core protein (HBcAg), which is then surrounded by an outer envelope composed of protein (HBsAg) and lipids. During infection, HBsAg is often produced in excess and forms non- infectious particles (either spherical or filamentous) that are smaller (22 nm) than the Dane particle. These non-infectious particles can be found in excess of 10 000 to 1 000 000 times to the infectious HB virion.75 1.3.1 Epidemiology According to the World Health Organization (WHO), approximately two billion people have been infected with HBV at some point in their lives.75, 76 Acute infection with HBV can have numerous clinical outcomes ranging from asymptomatic infection, acute self-limited hepatitis, or fulminant infection requiring liver transplantation. Those infected with HBV can also become chronically infected. Currently, 350 million people remain chronically infected and become carriers for life.75-77 Chronic HBV infection is a major global cause of hepatocellular carcinoma (HCC) and liver cirrhosis. HCC ranks fifth in overall global frequency of malignant diseases with an annual mortality rate that often matches its annual incidence.76 Between 50 and 80% of all cases of HCC can be attributed to chronic HBV infection75, 76 making HBV (along with tobacco) one of the most important environmental carcinogens to humans.76 12 Age at time of infection plays a major role in the outcome of HBV infection. The likelihood of developing symptoms during acute infection increases with age while there is an inverse relationship between age and risk for developing chronic infection.75 Up to 90% of HBV- infected infants will develop a chronic infection compared to 6-10% of older children (≥5 years) and adults.77 Of those infected with HBV during infancy or childhood, up to 25% develop and die prematurely from HBV-associated sequelae, such as cirrhosis or HCC.77 As such, to reduce overall morbidity and mortality associated with HBV infection, prevention of transmission and infection in infants and young children is essential. 1.3.2 Prevention of HBV infection Passive immunization Prior to the introduction of the HBV vaccine, the only available option for HBV immune prophylaxis was administration of high-titred human hepatitis B immunoglobulin (HBIG), which became available in 1974. HBIG is an effective form of passive immunization that is administered shortly following exposure to HBV and provides immediate and short-lived (3 to 6 months) protection against infection.75 Candidates for HBIG treatment include infants (immediately following birth) born to HBV-positive mothers and health-care workers that are exposed to HBV in workplace (e.g., needle prick). Due to several factors, including high cost and limited availability, HBIG treatment is generally not an affordable option in developing countries. 13 Hepatitis B virus vaccine Vaccination remains the most effective method for preventing transmission of HBV. The HBV vaccine represents the first vaccine against a major human cancer. While the HBV vaccine has evolved substantially since its licensing in the early 1980s, the immunogenic component has always been HBsAg. First generation vaccines consisted of inactivated, alum-adsorbed non- infectious HBV subparticles (composed only of HBsAg protein) derived from the plasma of HBsAg-positive donors. Second generation HBV vaccines were developed following the advancement of recombinant DNA technology in the mid-1980s.75 Using this new technology, the HBsAg gene was isolated and cloned into expression plasmids and introduced into either yeast or mammalian cells. Similar to the plasma-derived vaccine, the recombinant HBsAg protein self assembles into non-infectious particles. Benefits of the second generation HBV vaccine include shorter production time and increased consistency between lots. Studies have shown that both vaccine formulations are effective at preventing HBV infection55, 78 although there have been some studies that have observed differences in peak anti-HBs titers and rates of anamnestic response between the two types of vaccines.79, 80 1.3.3 HBV vaccine as part of the EPI The WHO established the Expanded Programme on Immunization (EPI) in 1974 with its with primary goal to provide universal access to life-saving vaccines in order reduce morbidity and mortality associated with vaccine-preventable diseases.81, 82 The HBV vaccine was integrated into national EPI schedules during the early 1990s. By the end of 2011, HBV vaccine was part of the routine childhood vaccination schedule in 180 countries and worldwide coverage with 3 doses of HBV vaccine was 75%.81 The vaccination schedule for HBV vaccine varies 14 depending on the country. The WHO recommends that all infants, particular those in areas of intermediate to high HBV endemicity, should receive 3 or 4 doses of the HBV vaccine with their first dose soon after birth (<24 hours).83 Since Canada is a country with low HBV endemicity, the province of British Columbia follows a 3-dose HBV vaccine schedule administered at 2, 4, and 6 months of age for infants born to HBV-uninfected mothers.84 1.3.4 HBV vaccine efficacy Anti-HBs titers are currently the only established correlate of vaccine-induced protection against HBV.9, 12, 18, 77 Early studies demonstrated that those who develop anti-HBs titers equal or greater than 10 IU/l one month after their final dose of HBV vaccine were protected against becoming chronic carriers of HBsAg.85 Those who do not manage to develop anti-HBs titers above 10 IU/l following vaccination are at increased risk for HBV infection.86 While the levels of anti-HBs antibodies can decline rapidly following completion of the immunization series, multiple long-term studies (5-23 years after vaccination) in various geographical locations have confirmed most individuals whose titers fall below 10 IU/l are still protected against infection86, 87 and mount a strong anamnestic response following a booster dose of HBV vaccine.62, 78, 80, 88-92 It is likely that anamnestic response is highly protective against infection due to the long incubation period of HBV. Although there have been studies that show in areas with low HBV endemicity (without natural boosting) up to half of vaccine recipients fail to have an anamnestic response following a booster dose of HBV vaccine.93-96 Breakthrough infections have been reported even in locations with high HBV endemicity.79, 88, 96 15 1.3.5 HBV vaccine non-responders Between 5 – 10% of healthy adult vaccine recipients fail to produce protective levels of antibodies to HBV vaccine after the standard immunization schedule and are referred to as non- responders.97 While some non-responders mount protective responses as measured by serology upon additional boosters98 or re-vaccination with vaccines of different adjuvant formulation38 or antigen composition,99 others remain susceptible to infection with HBV. A genetic predisposition—HLA type100-102 or cytokines103-105— has also been shown to influence the strength of the immune response to HBV vaccine. A twin study in China was designed to compare the effects of genetics and perinatal environment factors on development of anti-HBs titers. They found that genetic factors had a dominating role (91%) in the development of humoral responses against HBsAg.106 Other factors that have been associated with reduced immunogenicity of the HBV vaccine or vaccine failure include health status (hepatitis C virus or HIV infection), gender, tobacco smoking, obesity, age, birth weight (if vaccinated as infant), and site of injection.55, 87, 91, 98, 103, 107, 108 Studies that have tried to elucidate the biological mechanisms behind the non- responsiveness to HBV vaccine have made some significant findings. An elegant study used HLA-matched pairs of individuals with discordant reactions to HBV vaccine to investigate the cellular or molecular basis for non-responsiveness to this antigen. Their experiments demonstrated that the defect in non-responders is in their T cells, not APC nor from suppression of cytotoxicity directed against the immune system (i.e. cytokines).109 16 1.4 Research project The HBV vaccine presents as an ideal model to attempt to dissect the biological mechanisms that contribute towards immunological memory. This is a big undertaking, as genetics, innate as well as adaptive immune responses surely all play a role in conferring long- term immunity to infectious diseases. To begin, I propose studying the relationship between vaccine-specific humoral and CMI responses. Take for example the HBV vaccine, we continue to rely on anti-HBs titer as correlate of protection while non-responders appear protected and breakthrough cases of HBV have been reported. It is becoming increasingly clear that we might be missing a piece of the puzzle that determines our response to vaccination. Using PBMC samples from a cohort of healthy Canadian infants, my goal was to generate a detailed and descriptive method to assess memory T cell immune responses to the HBV vaccine and compare that with the gold standard assay for HBV vaccine responsiveness (anti-HBs titers). Combining these two sets of data together will hopefully provide further insight into how immune system interacts to develop immunological memory. 17 2 MATERIALS AND METHODS 2.1 Cohort description This study was approved by the Institutional Ethics Review Board at the University of British Columbia and the University of Washington. This cohort was designed to study the ontogeny of the immune system from birth until two years of age; as such, recruitment of study participants began at birth with follow-up at one and two years of age. During initial recruitment, parents were contacted prior to giving birth at the British Columbia Women’s Hospital and written informed consent was obtained from both parents. As part of the study design to minimize variables, only healthy full-term newborns after elective Caesarean sections without labor were enrolled in this study. Recruitment began in 2005 and was completed in 2008. Prior to postnatal blood draws at year one and year two, written informed consent was re-obtained from the parent or legal guardian. All the participants in the study were considered healthy based on history-driven health assessment. Permanent exclusion criteria from the study included: (1) significant chronic medical condition in the infant, including HIV infection, immune deficiency, immunosuppression by disease or medication, cancer, bone marrow or organ transplantation, blood transfusion or intravenous immunoglobulin administration within 3 months, bleeding disorder or major congenital malformation or genetic disorder; (2) if the parent or legal guardian was unable to read or comprehend English; (3) if the family moved outside the Greater Vancouver Area during the study period. Temporary exclusion criteria (resulting in a delayed blood draw) included: (1) febrile illness within 24 hours; (2) recent immunization (within 4 weeks after MMR or varicella vaccine, or 7 days after other vaccinations); (3) brief (<1 month) 18 immunosuppressive medication use within one month. All blood draws were performed in the hospital by a trained pediatric phlebotomist prior to any vaccinations received on the same day. Participants in this cohort received the pediatric formulation of the Recombivax HB® vaccine (Merck & Co., INC.) by intramuscular injection at 2, 4, and 6 months of age. Each 0.5 ml dose contained 5 µg of recombinant HBsAg (adw subtype) adjuvanted with amorphous aluminium hydroxyphosphate sulphate (alum). In compliance with the British Columbia childhood immunization schedule, Recombivax HB® was co-administered with Pentacel® (Sanofi Pasteur) at 2, 4, and 6 months of age, Prevnar® (Pfizer) at 2 and 4 months of age, and NeisVac-C (GlaxoSmithKline) at 2 months of age. 2.2 Sample processing Peripheral blood (7-10 ml) was obtained via sterile venipuncture into vacutainers containing sodium-heparin (Becton Dickinson). Tubes were initially spun at 300 rcf for 10 minutes to produce the blood-plasma interface. Plasma was collected and frozen at -80°C until further analysis. The remaining blood products (white and red blood cells, platelets) were diluted with Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies, Burlington, ON) at a 1:1 ratio and layered on Ficoll-Paque Plus (GE Healthcare, Baie d’Urfe, QC). Cells were spun at 900 rcf for 25 minutes with no brake. PBMCs were isolated as per manufacturer’s protocol. Cells were cryopreserved as previously described110 using Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies), 40% heat-inactivated newborn calf serum (HI-NCS) (Life Technologies), 10% dimethyl sulfoxide (Sigma-Aldrich, Oakville, ON) as freezing medium. PBMCs were stored in a liquid nitrogen storage unit (vapour phase) until further analysis. 19 2.3 HBsAg enzyme immunoassay HBsAg-specific antibodies were detected in plasma using an enzyme immunoassay (EIA) from Diasorin (Brussels, Belgium) (catalog number ETI-AB-AUK3) as per manufacturer’s protocol. The standard curve for the assay ranged from 1.25 IU/l to 1000 IU/l. All plasma samples were originally tested at 1:2 dilution, and samples that exceeded the upper limit of the standard curve were retested at a 1:10 dilution. Samples with optical densities below the lower limit of detection were assigned a value of 0 IU/l. 2.4 Cell culture and proliferation assay PBMCs were thawed and washed twice with pre-warmed R10 medium (RPMI 1640 medium, 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Logan, UT), penicillin (100 units/ml) (Life Technologies), and streptomycin (100 µg/ml) (Life Technologies), 50 µM β- mercaptoethanol (Sigma-Aldrich)). Cells were treated with DNase I (60 µg/ml) (Sigma-Aldrich) for 5 minutes at 37°C with 5% CO2. Cells were then washed with RPMI 1640 medium and resuspended in 5 ml of R10 medium and transferred to a 6-well polystyrene culture plate (Becton Dickinson, Franklin Lakes, NJ) and incubated overnight at 37°C with 5% CO2. The following day, ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, Oakville, ON) was added to the cells at a final concentration of 2 mM and incubated for 10 minutes at 37°C with 5% CO2. Cells were then harvested, washed and counted. Cells were resuspended in 500 µL of staining buffer (DPBS with 5% FCS) and passed through a 70-µm cell strainer (Becton Dickinson). CellTrace™ Oregon Green® 488 Carboxylic Acid Diacetate, Succinimidyl Ester (OG488) (Life Technologies), a derivative of CFSE, was added to the cell suspension at a final concentration of 5 µM. For unstained controls, an equal volume of staining buffer was 20 added to cells and treated identically as stained samples. Cells were mixed thoroughly, shielded from light, and incubated with OG488 for 5 minutes at room temperature (RT). After 5 minutes, an equal volume of FCS was added to the cell suspension and mixed thoroughly to quench any residual OG488. Cells were washed 3 times with staining buffer to remove excess OG488. Cells were resuspended in R10 at a final concentration of 4 x 106 cells/ml and plated at 0.5 x 106 cells/well in a 96-well U-bottom polystyrene tissue culture plate (Becton Dickinson). Unstimulated controls were incubated with anti-CD28 (1 µg/ml) and anti-CD49d (1 µg/ml) monoclonal antibodies (eBioscience, San Diego, CA) in R10 medium. Antigen-specific samples were incubated with recombinant Hepatitis B surface antigen (HBsAg) (5 µg/ml) (Aldevron, Fargo, ND) and anti-CD28 (1 µg/ml) and anti-CD49d (1 µg/ml) monoclonal antibodies (eBioscience) in R10 medium. Positive controls were stimulated with anti-CD3 (30 ng/ml) and anti-CD28 (1 µg/ml) monoclonal antibodies (eBioscience) in R10 medium. Unstimulated and HBsAg conditions were plated with technical duplicates when possible. Plates were incubated for 7 days at 37°C with 5% CO2. After 7 days, plates were spun at 300 rcf for 10 minutes to pellet cells and 50 µl/well of cell-free supernatant (for detection of bulk cytokine secretion) was transferred to a clean plate and frozen at -80°C until further analysis. Cells were then resusupended and re-stimulated with a cocktail containing phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) (Sigma-Aldrich), ionomycin (1 µM) (Sigma-Aldrich), and brefeldin A (BFA, 2.5 µg/ml) (Sigma-Aldrich). Plates were returned to the incubator (37°C and 5% CO2) for an additional 6 hours. After 6 hours, EDTA (2 mM) was added to each well and incubated for an additional 10 minutes at 37°C with 5% CO2. The plates were then spun and supernatants were removed. Cells were fixed in a 1x FACSlyse solution (Becton Dickinson) and frozen at -80°C until further analysis. 21 2.5 Measurement of bulk cytokine secretion in supernatant The cell-free supernatant was thawed at RT, and the levels of interleukin (IL)-10, IL-13, IL-17A, IL-21, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were measured by Luminex assay (Millipore, Billerica, MA) as per manufacturer’s instructions. The assays were read using the Luminex 200 Total System (Luminex, Austin, TX), and data were acquired and analyzed using MasterPlex softwares (MiraiBio Group, San Francisco, CA). The standard curve for the Luminex assay ranged from 10 to 40 000 pg/ml for IFN-γ, 1 to 5 000 pg/ml for IL-10, 7 to 30 000 pg/ml for IL-13, 12 to 50 000 pg/ml for IL-17A, 5 to 20 000 pg/ml for IL-21, and 2.5 to 10 000 pg/ml for TNF-α. Cytokines expressed below the lower limit of detection were assigned a value of 0 pg/ml. 2.6 Measurement of total immunoglobulin (Ig) isotypes and IgG subclasses Previously frozen plasma was thawed at RT, and the concentration of total immunoglobulin (Ig)G1, IgG2, IgG3, IgG4, IgA, and IgM were measured by Luminex assay (Millipore) as per manufacturer’s instructions. The assays were read using the Luminex 200 Total System (Luminex), and data were acquired and analyzed using MasterPlex softwares (MiraiBio Group). The standard curve for the Luminex assay ranged from 13.72 to 10 000 ng/ml for IgG1, 41.45 to 30 000 ng/ml for IgG2, 0.21 to 150 ng/ml for IgG3, 0.41 to 300 ng/ml for IgG4, 2.06 to 1 500 ng/ml for IgA, and 3.43 to 2 500 ng/ml for IgM. Titers measured below the lower limit of detection were assigned a value of 0 ng/ml. 22 2.7 Flow cytometry Previously frozen plates containing cells fixed in FACSLyse solution were thawed in 37°C incubator for 10 minutes. Cells were washed once in PBSAN solution (PBS (Lonza, Basel, Switzerland), 0.5% bovine serum albumin (Sigma-Aldrich), 0.1% sodium azide (Teknova, Hollister, CA) and resuspended in 1X FACS Permeabilization Solution (Becton Dickinson) and incubated for 10 minutes at RT. Following one wash with PBSAN, cells were stained in a final volume of 100 µl/well of PBSAN for 30 minutes at RT. The following antibodies were used: anti-CD3-APCeFluor780 (clone UCHT1; eBioscience), anti-CD4-eFluor450NC (clone RPA-T4; eBioscience), anti-CD8-PE-Cy7 (clone SK1; eBioscience), anti-IFN-γ-PE-CF594 (clone B27; Becton Dickinson), anti-IL-2-PerCPeFluor710 (clone MA1-17H12; eBioscience), anti-IL-13- APC (clone JES10-5A2; Becton Dickinson), anti-IL-21-PE (clone eBio3A3-N2; eBioscience), and anti-TNF-α-AlexaFluor700 (clone MAb11; eBioscience). All antibodies were used at a 1:100 dilution. OG488 was detected using the same filter as FITC. Following two washes with PBSAN, cells were resuspended in 200 µl/well of PBSAN and immediately analyzed using a LSRII Flow Cytometer (Becton Dickinson). FACSDiva (Version 9) (Becton Dickinson) was used as acquisition software. Compensation beads (CompBeads) (Becton Dickinson) were used as single stain controls for all purchased antibodies. For OG488, an equal number of unstained and stained PBMCs were combined and used as single stain control for compensation purposes. Optimal cytometer settings were determined in preliminary experiments and saved in FACSDiva. Prior to each acquisition, Cytometer Set-up and Tracking (CST) beads (Becton Dickinson) were run on the LSRII and previously standardized and saved cytometer settings were applied. Cells and beads were acquired uncompensated using a high-throughput sampler (sample volume and flow rate were set at 190 µl and 1.5 µl/second, respectively; and mixing 23 volume and speed were set at 100 µl and 200 µl/second, respectively). Maximum number of cells were acquired from each well and ranged from 144 000 (116 000 – 179 000) (median (interquartile range (IQR))) for unstimulated and HBsAg-stimulated samples and 389 000 (365 000 – 410 500) for anti-CD3-stimulated cells. Five thousand CompBeads and 50 000 OG488 single stain control cells were acquired per well. Compensation matrices were calculated and applied using FlowJo (Version 9.4) (TreeStar, Ashland, OR). All samples were analyzed compensated. Biological controls were used to set appropriate gates for analyses. 2.8 Proliferation analyses Proliferation data were analyzed using the Proliferation platform on FlowJo. Positive controls were used to set the gates identifying successive rounds of cell division (generation gates) and subsequently copied to unstimulated and HBsAg-stimulated samples on a per subject basis. Number of cells in each generation gate were determined and used to calculate the percentage of the starting population that divided. 2.9 Statistical analyses Where applicable, technical duplicate values were averaged and unstimulated values were subtracted from stimulated values. For the anti-HBs EIA and the total Ig isotypes and IgG subclasses Luminex assay, all longitudinal samples from the same donor were run together to minimize inter-assay variability. Non-parametrical tests were used for all analyses. Wilcoxon matched pairs statistical test was used to test for differences between unstimulated and stimulated values from the same subject. Mann-Whitney test was used to test for differences in CMI responses between subjects with low (< 10 IU/l, <100 IU/l, or bottom quartile) or high (≥ 24 10 IU/l, ≥ 100 IU/l, or top quartile) anti-HBs titers. Correlations between anti-HBs titers and CMI responses were studied by use of Spearman’s rank analysis. All demographic data and antibody data are presented as median and IQR. All CMI data are presented as box and whisker plots with the whiskers representing 5 and 95% confidence intervals (CI). Since positive HBsAg- specific CMI responses for MBA or flow cytometry assays have not been defined, approximate cut-offs were calculated based on literature.111 A positive response was defined as greater than the mean plus two times the standard deviation of the unstimulated control. Data were analysed using Excel 2008 (Version 12.3.5) and Prism (Version 4.0). An alpha level of 0.05 was considered statistically significant. 25 3 RESULTS 3.1 Demographics Phlebotomy visits were conducted for a total of 104 subjects at one-year of age (Y1) and 100 subjects at two-years of age (Y2). Due to limited blood collection, PBMCs were not collected from every individual. For this study, anti-HBs titers were determined from plasma collected at the Y1 and Y2 time points, but only PBMCs harvested from the Y1 time point were used for HBsAg stimulation experiments. In addition, intracellular cytokine data from 14 subjects were excluded because they failed to meet quality control criteria. The demographic details of this cohort are described in detail in Table 1. Please note that racial groups in which the numbers were very low were grouped together (referred to as “Other”) and were subsequently excluded from any statistical analyses in which race was a variable. Table 1. Demographics of cohort with breakdown for humoral and cell-mediated immunity components. Humoral Immunity (anti-HBs titers) Cell-Mediated Immunity (Year 1 only) (T cell responses to HBsAg) Year 1 (Y1) Year 2 (Y2) Proliferation and secreted cytokines Intracellular cytokine production Total (N) 104 100 77 63 Male, n (%) 50 (48.1) 49 (49) 36 (46.8) 29 (46) Female, n (%) 54 (51.9) 51 (51) 41 (53.2) 34 (54) Caucasian, n (%) 55 (52.8) 53 (53) 38 (49.3) 31 (49.2) Asian, n (%) 12 (11.5) 11 (11) 9 (11.7) 7 (11.1) South Asian, n (%) 14 (13.5) 13 (13) 11 (14.3) 10 (15.9) Asian-Caucasian, n (%) 9 (8.7) 9 (9) 6 (7.8) 5 (7.9) Other, n (%) 14 (13.5) 14 (14) 13 (16.9) 10 (15.9) 26 The vaccination schedule for HBV vaccine in British Columbia is 2, 4, and 6 months of age.84 Since this study was investigating immune memory responses to HBsAg, the immunization history of each subject was examined to ensure they received their vaccines at the appropriate ages and intervals. Table 2 describes the number of days deviated from the vaccine schedule (based on a 30 day-month). In addition, the date of their phlebotomy appointments was recorded to determine if their Y1 and Y2 appointments deviated from their actual birthdays. These details are also recorded in Table 2 and presented as median and IQR. The phlebotomy visits and immunization records showed that all participants received three doses of HBV vaccine and no major deviation from schedules were noted (Table 2). Table 2. Phlebotomy and HBV vaccination schedule for all cohort subjects. Phlebotomy Hepatitis B Vaccination Schedule Time point Year 1 Year 2 2-Month 4-Month 6-Month # of days deviated (median (IQR)) 6 (4-11) 11 (5-25) 4 (1-9) 7 (3-12) 8 (2-17) 3.2 HBsAg-specific antibody titers and total immunoglobulin levels Titers of HBsAg-specific antibodies were quantified from blood plasma for study participants at year one and year two. The data are presented as median and IQR in Table 3. Anti-HBs titers above 10 IU/l were considered protective against infection.75 In this cohort five subjects had titers below 10 IU/l at the Y1 time point and 20 at the Y2 time point. Out of the five subjects at Y1 with titers below 10 IU/l, three subjects continued to have titers below 10 IU/l at the Y2 time point. Due to relatively low number of subjects with anti-HBs titers below 10 IU/l, the number of subjects with titers below 100 IU/l were also determined (Table 3). 27 Table 3. HBsAg-specific antibody titers for cohort subjects and number of subjects with anti-HBs titers below 10 IU/l, below 100 IU/l, and equal or greater than 100 IU/l at one and two years of age. Time Point N anti-HBs titer (IU/l) <10 IU/l (n) <100 IU/l (n) ≥100 IU/l (n) Y1 104 234.2 (99.9–427.4) 5 26 78 Y2 100 64.43 (15.8–199.6) 20 59 41 Anti-HBs titers are presented as median (IQR). PBMCs were not collected from 27 subjects at the Y1 time point—limiting the number of possible individuals with paired data sets (HBsAg-specific T cell and humoral immune responses) to 77 (Table 4). In addition, HBsAg-specific intracellular cytokine production was excluded for 14 subjects resulting in 63 subjects with a complete paired data set (i.e. HBsAg- specific proliferation, bulk cytokine secretion, intracellular cytokine production and anti-HBs titers) (Table 4) at the Y1 time point. At the Y2 time point, the number of subjects with incomplete and complete paired data sets decreased to 70 and 58, respectively due to study attrition (Table 4). Table 4. Number of cohort subjects with incomplete and complete paired data sets. Y1 Y2 Type of data set N <10 IU/l ≥10 IU/l <100 IU/l ≥100 IU/l N <10 IU/l ≥10 IU/l <100 IU/l ≥100 IU/l Incomplete paired 77 3 74 19 58 70 13 57 41 29 Complete paired 63 2 61 15 48 58 10 48 35 23 Paired data sets refer to subjects with anti-HBs titers and HBsAg-specific T cell immune responses. Incomplete data sets include HBsAg-specific proliferation and bulk cytokine secretion. Complete data sets include HBsAg-specific proliferation, bulk cytokine secretion, and intracellular cytokine production. 28 Levels of total IgG1, IgG2, IgG3, IgG4, IgA, and IgM were also determined for the cohort at both one and two years of age. All isotypes and subclasses were found to be within the normal range for their age groups and presented as median (IQR) in Table 5.112, 113 To determine whether there was a relationship between total immunoglobulin and HBsAg-specific immunoglobulins, subjects were ranked based on their anti-HBs titers at Y1 and Y2 time points and divided into quartiles. The concentration of IgG1, IgG2, IgG3, IgG4, IgA, and IgM were then compared between top and bottom quartile, and no significant differences were found (data not shown). Table 5. Levels of total immunoglobulin isotypes and IgG subclasses in cohort subjects. Time Point IgG1 (mg/dl) IgG2 (mg/dl) IgG3 (mg/dl) IgG4 (mg/dl) IgA (mg/dl) IgM (mg/dl) Year 1 301.6 (223.8 – 420.2) 143.4 (62.6 – 178.0) 33.0 (22.3 – 48.1) 0.7 (0.3 – 3.3.) 14.9 (9.7 – 25.2) 71.9 (50.4 – 100.9) Year 2 375.8 (293.4 – 478.2) 143.4 (127.5 – 178.0) 33.4 (21.7 – 44.9) 6.4 (0.8 – 26.6) 28.5 (18.6 – 41.2) 92.4 (61.5 – 135.3) Data are presented as median (IQR). 3.3 HBsAg-stimulation assay: descriptive results Due to limited PBMCs available from each study participant, it was necessary to develop an assay that could maximize the quantity and breadth of data produced with minimal cell usage. As such, this HBsAg-stimulation protocol generated data with regards to HBsAg-specific cell proliferation, bulk cytokine secretion and intracellular cytokine production from a single well of cell culture using flow cytometry and Luminex systems. The gating strategy for the flow cytometric data can be found in Appendix A. 29 3.3.1 PBMC proliferation Cell proliferation was measured using the intracellular fluorescent dye, OG488. Based on decreasing fluorescence intensity, it is possible to determine how many rounds of division a particular cell underwent – making it possible to calculate how many cells from the original population proliferated in response to stimulation. When comparing unstimulated cells to HBsAg-stimulated cells, there were significant increases in the percentages of starting CD4 (Figure 1A) and CD8 (Figure 1B) T cells that proliferated in response to HBsAg. In addition, the percentages of starting CD4 (Figure 1C) and CD8 (Figure 1D) T cells that proliferated in response to the positive control (anti-CD3/anti-CD28) were significantly higher than the unstimulated cells. There also was a significantly greater percentage of proliferating CD4 T cells compared to CD8 T cells following HBsAg stimulation (P-value < 0.0001, data not shown). In addition, there was a significant positive correlation between the percentage of CD4 and percentage of CD8 T cells that proliferated in response to HBsAg stimulation (r = 0.3431, P- value = 0.0023, data not shown). 30 Figure 1. A significant increase in the percentage of proliferating CD4 and CD8 T cells from the starting population was observed following stimulation with HBsAg and anti-CD3 compared to unstimulated controls. Cryopreserved PBMCs were thawed and stimulated with HBsAg (A and B) or anti-CD3 (C and D) for seven days. Proliferation was determined for CD3+CD4+ (A and C) and CD3+CD8+ (B and D) T cells based on fluorescence intensity of OG488. (N = 77) Data presented as median (lines), IQR (boxes), and 5 and 95% confidence interval (CI) (whiskers). *** P < 0.001 31 3.3.2 Bulk cytokine secretion Stimulation of PBMCs with HBsAg induced significantly increased secretion of IFN-γ (Figure 2A), IL-10 (Figure 2B), IL-13 (Figure 2C), and TNF-α (Figure 2D) compared to unstimulated cells. There was no difference observed in IL-17A secretion between unstimulated and HBsAg-stimulated cells (Figure 2E). IL-21 secretion was not detected in any conditions (data not shown). To determine whether there was a relationship between the levels of cytokine secreted following HBsAg stimulation, correlation analyses were performed. There was a positive and significant correlation observed between all cytokines except TNF-α and IL-10 secretion (Table 6). Similarly, additional correlation analyses were performed between the percentage of starting CD4 or CD8 T cells that divided in response to HBsAg stimulation and the amount of cytokine secreted into the supernatant. There was a positive and significant correlation between percentage of responding CD4 T cells and levels of IL-10 and TNF-α (Table 7). There was a positive correlation between the concentration of IL-13 detected in the supernatant and percentage of CD4 T cells that divided, but it did not reach the alpha level of significance. There were no significant correlations noted between any cytokine and CD8 proliferation (Table 7). 32 Figure 2. A significant increase in levels of IFN-γ, IL-10, IL-13, and TNF-α, but not IL-17A was observed in response to HBsAg stimulation compared to unstimulated controls. Cryopreserved PBMCs were thawed and stimulated with HBsAg for seven days. Levels of IFN-γ (A), IL-10 (B), IL-13 (C), TNF-α (D) and IL-17A (E) in the supernatant were measured using Luminex. (N = 77) Data presented as median (lines), IQR (boxes), and 5 and 95% CI (whiskers). n.s. not significant, *** P < 0.001 33 Table 6. Correlation analyses between levels of cytokine secreted into supernatant in response to HBsAg stimulation. IFN-γ IL-10 IL-13 TNF-α IFN-γ IL-10 0.2575 (0.0248) IL-13 0.3459 (0.0022) 0.2399 (0.0369) TNF-α 0.2895 (0.0112) 0.1958 (0.09) 0.2719 (0.01075) Data presented as correlation coefficient (P-value). Statistically significant (p < 0.05) correlations are indicated by red font. (N = 77) Table 7. Correlation analyses between percentage of starting CD4 or CD8 that proliferated in response to HBsAg stimulation and the level of bulk cytokine secretion. IFN-γ IL-10 IL-13 TNF-α CD4 T cells 0.1670 (0.1523) 0.3247 (0.0045) 0.2225 (0.055) 0.4032 (0.0003) CD8 T cells 0.1592 (0.1696) 0.08867 (0.446) 0.05047 (0.6650) 0.05422 (0.642) Data presented as correlation coefficient (P-value). Statistically significant (p < 0.05) correlations are indicated by red font. (N = 77) 34 3.3.3 Intracellular cytokine production For analysis of intracellular cytokine production via flow cytometry, CD4 and CD8 T cells were separated two groups: proliferating (i.e. HBsAg responders) and non-proliferating (i.e. HBsAg non-responders) based on their OG488 fluorescence intensity (please refer to Appendix A for more details on the gating strategy). Proliferating CD4 T cells CD4 T cells that proliferated in response to HBsAg also had an increase in percentage of IFN-γ positive (Figure 3A), IL-13 positive (Figure 3C) and IL-21 positive (Figure 3D) cells compared to unstimulated cells. There was no observed increase in the percentage of IL-2 positive (Figure 3B) or TNF-α positive (Figure 3E) cells following HBsAg stimulation. Correlation analyses were performed for the cytokines in which there was a significant increase of percentage positive cells following HBsAg stimulation (IFN-γ, IL-13 and IL-21). There were significant positive correlations observed between all three cytokines (Table 8), particularly between IL-13 and IL-21 (r = 0.7532). There were no correlations found between percentage of starting CD4 population that proliferated in response to HBsAg stimulation and the percentages of cytokine positive proliferating CD4 T cells (data not shown). Table 8. Correlation analyses between percentage of cytokine positive proliferating CD4 T cells in response to HBsAg stimulation. IFN-γ IL-13 IL-21 IFN-γ IL-13 0.319 (0.0108) IL-21 0.4181 (0.0007) 0.7532 (<0.0001) Data presented as correlation coefficient (P-value). Statistically significant (p < 0.05) correlations are indicated by red font. (N = 63) 35 Figure 3. A significant increase in percentage of IFN-γ, IL-13, and IL-21 positive proliferating CD4 T cells was observed following HBsAg stimulation. Cryopreserved PBMCs were thawed and stimulated with HBsAg for seven days. Percentage of IFN-γ (A), IL-2 (B), IL-13 (C), IL-21 (D), and TNF-α (E) positive proliferating CD4 T cells were detected by flow cytometry. (N = 63) Data presented as median (lines), IQR (boxes), and 5 and 95% CI (whiskers). n.s. not significant, *** P < 0.001 36 Proliferating CD8 T cells CD8 T cells that proliferated in response to HBsAg also showed an increase in percentage of IFN-γ positive (Figure 4A), IL-13 positive (Figure 4C) and TNF-α positive (Figure 4E) cells compared to unstimulated controls. There was no observed increase in the percentage of IL-2 positive (Figure 4B) or IL-21 positive (Figure 4D) cells following HBsAg stimulation. Correlation analyses were performed for the cytokines in which there was a significant increase of percentage positive cells following HBsAg stimulation (IFN-γ, IL-13 and TNF-α). There was positive correlation observed between the percentages of IFN-γ positive and TNF-α positive proliferating CD8 T cells only (Table 9). There was no relationship detected between percentage of IL-13 positive proliferating CD8 T cells with either cytokine (IFN-γ or TNF-α) (Table 9). There were no correlations found between percentage of starting CD8 population that proliferated in response to HBsAg stimulation and the percentages of cytokine positive proliferating CD8 T cells (data not shown). Table 9. Correlation analyses between percentage of cytokine positive proliferating CD8 T cells in response to HBsAg stimulation. IFN-γ IL-13 TNF-α IFN-γ IL-13 n.s. TNF-α 0.4505 (0.0002) n.s. Data presented as correlation coefficient (P-value). Statistically significant (p < 0.05) correlations are indicated by red font. (N = 63) n.s. not significant 37 Figure 4. A significant increase in percentage of IFN-γ, IL-13, and TNF-α positive proliferating CD8 T cells was observed following HBsAg stimulation. Cryopreserved PBMCs were thawed and stimulated with HBsAg for seven days. Percentage of IFN-γ (A), IL-2 (B), IL-13 (C), IL-21 (D), and TNF-α (E) positive proliferating CD8 T cells were detected by flow cytometry. (N = 63) Data presented as median (lines), IQR (boxes), and 5 and 95% CI (whiskers). n.s. not significant, ** P < 0.01, *** P < 0.001 38 Non-proliferating T cells Both CD4 and CD8 T cells that did not proliferate in response to HBsAg stimulation were also analyzed for intracellular cytokine production. However, there was either no significant difference in percentage of cells positive for cytokines between unstimulated and HBsAg-stimulated samples or there was an increase in percentage of cells positive for cytokines in unstimulated cells compared to HBsAg-stimulated (data not shown). These results indicated that cells that did not proliferate in response to antigen were not producing cytokines in an antigen-specific manner. As such, non-proliferating cells were excluded from further analyses. 3.4 Comparing humoral and cell-mediated antigen-specific responses 3.4.1 Low responder versus responder: year 1 The initial approach to compare T cell responses (CMI) to HBsAg between those who have low and high anti-HBs titers was to divide the cohort into two groups: those with anti-HBs titers less than 10 IU/l (< 10 IU/l) and those with titers equal or greater than 10 IU/l (≥ 10 IU/l) at the Y1 time point. However, because of sample limitations, and there were only two subjects with anti-HBs titers less than 10 IU/l that also had a complete set of paired CMI data at Y1 (Table 4). It was not possible to perform proper statistics on such unbalanced groups. As such, I modified my grouping criteria as follows: those who had anti-HBs titers less than 100 IU/l (< 100 IU/l) and those who had anti-HBs titers equal or greater than 100 IU/l (≥ 100 IU/l) at the Y1 time point. Individuals with anti-HBs titers less than 100 IU/l have been classified as hypo- responders or low responders by others.97 Please refer to Table 4 for the number of hypo- responders and responders with incomplete and complete paired T cell data sets. 39 In this cohort, for those individuals who had anti-HBs titers less than 100 IU/l at one year of age also secreted significantly less IL-10 in response to HBsAg stimulation than those with anti-HBs titers equal or greater than 100 IU/l (Figure 5A). In contrast, those who had anti-HBs titers less than 100 IU/l had significantly higher percentage of TNF-α positive proliferating CD8 T cells than those with anti-HBs titers equal or greater than 100 IU/l (Figure 5B). Please refer to Appendix B for statistical results for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers less than 100 IU/l and equal or greater than 100 IU/l. Figure 5. Subjects with anti-HBs titers less than 100 IU/l at one year of age secretes less IL- 10 and had a higher percentage of TNF-α positive proliferating CD8 T cells in response to HBsAg stimulation than individuals with anti-HBs titers equal or greater than 100 IU/l. Cryopreserved PBMCs were thawed and stimulated with HBsAg for seven days. (A) Levels of IL-10 in the supernantant were measured by Luminex. (N: < 100 IU/l = 19, ≥ 100 IU/l = 58) (B) Percentages of TNF-α positive proliferating CD8 T cells were detected by flow cytometry. (N: < 100 IU/l = 15, ≥ 100 IU/l = 48) Data presented as median (lines), IQR (boxes), and 5 and 95% CI (whiskers). * P <0.05 40 3.4.2 Top and bottom quartiles: year 1 In order to find potential differences in T cell responses to HBsAg between those who respond poorly or very well to the HBV vaccine, the cohort was also ranked and divided into quartiles based on their anti-HBs titers at the Y1 time point. The antigen-specific T cell responses were then compared between those in the bottom and top quartiles. Similar strategies have been used by others.114 Similarly to those with less than 100 IU/l of anti-HBs, those in the bottom quartile also secreted less IL-10 (Figure 6A) as well as less IFN-γ (Figure 6B) following HBsAg stimulation compared to those in the top quartile. Once again, those in the bottom quartile also had a higher percentage of TNF-α positive proliferating CD8 T cells following HBsAg stimulation compared to those in the top quartile (Figure 6C). Please refer to Appendix C for statistical results for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers in the top and bottom quartile at one year of age. 41 Figure 6. Subjects ranked in the bottom quartile based on their anti-HBs titers at one year of age secreted less IL-10 and IFN-γ and had a higher percentage of TNF-α positive proliferating CD8 T cells in response to HBsAg stimulation than individuals ranked in the top quartile. Cryopreserved PBMCs were thawed and stimulated with HBsAg for seven days. Levels of IL-10 (A) and IFN-γ (B) in the supernantant were measured by Luminex. (N: bottom quartile = 19, top quartile = 19) (B) Percentages of TNF-α positive proliferating CD8 T cells were detected by flow cytometry. (N: bottom quartile = 15, top quartile = 14) Data presented as median (lines), IQR (boxes), and 5 and 95% CI (whiskers). * P <0.05, ** P < 0.01 3.4.3 Correlation between anti-HBs titer and HBsAg-specific T cell responses: year 1 To identify any relationship between anti-HBs titers (at Y1 time point) and T cell responses, a correlation analysis was performed. Similar to the results from the quartile comparison, there were significant positive relationships between IFN-γ and IL-10 secretion and anti-HBs titers at the first year of age (Table 10). Similarly, there was a significant negative 42 relationship (r = -0.2665) between the percentage of TNF-α positive proliferating CD8 T cells following HBsAg stimulation and anti-HBs titers (Table 10). Please refer to Appendix D for statistical results (correlation coefficient and P-value) for all correlation analyses between anti- HBs titers at one year of age and all T cell HBsAg-specific responses. Table 10. Significant correlations between anti-HBs titers at one year of age and HBsAg- specific T cell responses. T cell response N Correlation coefficient (r) P-value IFN-γ secretion 77 0.2578 0.0246 IL-10 secretion 77 0.299 0.0087 %age of TNF-α+ proliferating CD8 T cells 63 -0.2665 0.0348 Statistically significant correlations were defined as p < 0.05. 3.5 Comparing humoral and cell-mediated antigen-specific responses: extending to year 2 As an extension to the comparative analysis completed between the HBsAg-specific T cell responses and anti-HBs titers at one year of age, a similar comparison was done at the Y2 time point. The main purpose of this extended analysis was to identify any potential T cell responses (using PBMCs from the Y1 time point) that might associate with lower titers in the future. To begin, a correlation analysis between anti-HBs titers measured at the Y1 and Y2 time points was completed. There was a positive relationship (r = 0.5392, P-value < 0.0001) between data sets indicating that those with higher titers at one year of age are likely to have higher titers at two years of age. In general, the antibody titers at the Y2 time point were lower than the Y1 time point (Table 3) and as such there was a much larger group of individuals who had antibody titers less 43 than 10 IU/l. To this end, the analyses that were performed using the anti-HBs titers from one year of age were repeated using the anti-HBs titers from the Y2 time point. For clarification purposes, individuals with anti-HBs titers < 10 IU/l and ≥ 10 IU/l at the Y2 time point are referred to as “seronegative” and “seropositive”, respectively. 3.5.1 Seronegative versus seropositive: year 2 Similar to Section 3.4.1, the cohort was divided into two groups and the HBsAg-specific responses were compared. However, in this case the groups were split into those with anti-HBs titers less than 10 IU/l (< 10 IU/l) and those with titers equal or greater than 10 IU/l (≥ 10 IU/l). One of the goals of this analysis was to identify T cell responses measured shortly after immunization that could potentially predict anti-HBs titers at later time points. As such, individuals with paired T cell data sets (both incomplete and complete) and anti-HBs titers less than 10 IU/l at both Y1 and Y2 time points (n=3) were excluded from this analysis. Please refer to Table 4 for the number of seronegative and seropositive subjects at the Y2 time point with incomplete and complete paired T cell data sets that were included in this analysis. Similar to the results from the Y1 time point, there was an increase in IL-10 secretion in seropositive individuals compared to seronegative subjects following HBsAg stimulation, but at the Y2 time point this difference was not statistically significant (P-value = 0.0748) (Figure 7A). In addition, there was a significant increase in TNF-α secretion (Figure 7B) and percentage of proliferating CD4 T cells (Figure 7C) following HBsAg stimulation for those with anti-HBs titers equal or greater than 10 IU/l at the Y2 time point. Finally, looking at intracellular cytokine production, those with anti-HBs titers less than 10 IU/l also had significantly lower percentages of IFN-γ positive (Figure 7D), IL-13 positive (Figure 7E), IL-21 positive (Figure 7F) 44 proliferating CD4 T cells and lower percentage of IL-13 positive proliferating CD8 T cells (Figure 7G) than those with anti-HBs titers equal or greater than 10 IU/l. Table 11 displays the P-values from the statistical tests comparing T cell responses that were significantly different at the Y1 and/or Y2 time point. While there are no parameters that are significantly different at both time points, IL-10 secretion appears to have the strongest signal. Please refer to Appendix E for statistical results comparing all of HBsAg-specific CMI responses between those with anti-HBs titers less than 10 IU/l and equal or greater than 10 IU/l at the Y2 time point. Table 11. Significant differences in HBsAg-specific T cell responses between those with titers < 100 IU/l and ≥ 100 IU/l (at Y1 time point) and between those with titers < 10 IU/l and ≥10 IU/l (at Y2 time point). T cell Response P-value (Year 1) P-value (Year 2) IL-10 secretion 0.0249 0.0748 TNF-α secretion 0.1029 0.0415 %age of starting CD4 T cells that proliferated 0.2626 0.0297 %age of IFN-γ+ proliferating CD4 T cells 0.1628 0.0307 %age of IL-13+ proliferating CD4 T cells 0.6226 0.0139 %age of IL-21+ proliferating CD4 T cells 0.815 0.0315 %age of IL-13+ proliferating CD8 T cells 0.7653 0.0242 %age of TNF-α+ proliferating CD8 T cells 0.0255 0.5575 Data presented as correlation coefficient (P-value). Statistically significant (p ≤ 0.05) correlations are indicated by red font. Refer to Figure 5 for Ns for Year 1 and Figure 7 for Ns for Year 2. 45 Figure 7. Subjects with anti-HBs titers less than 10 IU/l at two years of age secreted less TNF-α, had a lower percentage of starting CD4 T cells that proliferated, a lower percentage of IFN-γ positive, IL-13 positive and IL-21 positive proliferating CD4 T cells and lower a percentage of IL-13 positive proliferating CD8 T cells in response to HBsAg stimulation than individuals with anti-HBs titers equal or greater than 10 IU/l. Cryopreserved PBMCs were thawed and stimulated with HBsAg for seven days. Levels of IL-10 (A) and TNF-α (B) in the supernantant were measured by Luminex. (C) Percentage of starting CD4 T cells that proliferated was measured by flow cytometry (N: < 10 IU/l = 13, ≥ 100 IU/l = 57). Percentages of IFN-γ positive (D), IL-13 positive (E), IL-21 (F) proliferating CD4 T cells and IL-13 positive proliferating CD8 T cells (G) were detected by flow cytometry (N: < 10 IU/l = 10, ≥ IU/l = 48). Data presented as median (lines), IQR (boxes), and 5 and 95% CI (whiskers). * P <0.05 46 3.5.2 Top and bottom quartiles: year 2 Similar to Section 3.4.2, the cohort was ranked and divided into quartiles based on their anti-HBs titers from the Y2 time point. The antigen-specific T cell responses were then compared between those in the bottom and top quartiles. Subjects within the bottom quartile had lower percentages of IFN-γ positive proliferating CD4 T cells compared to those within the top quartile (Figure 8). Table 12 displays the P-values from the statistical tests comparing T cell responses that were significantly different between the bottom and top quartile from either Y1 and Y2 time points or both. Based on these data, there did not appear to be a consistent trend in HBsAg- specific T responses between those that rank in the bottom and to quartile from one to two years of age. Please refer to Appendix F for statistical results for all comparison between those with anti-HBs titers in the top and bottom quartile based on their anti-HBs titers at two years of age. 47 Figure 8. Subjects ranked in the bottom quartile based on their anti-HBs titers at two years of age had a lower percentage of IFN-γ positive proliferating CD4 T cells in response to HBsAg stimulation than individuals ranked in the top quartile. Cryopreserved PBMCs were thawed and stimulated with HBsAg for seven days. Percentages of IFN-γ positive proliferating CD4 T cells were detected by flow cytometry. (N: bottom quartile = 14, top quartile = 16) Data presented as median (lines), IQR (boxes), and 5 and 95% CI (whiskers). * P <0.05 Table 12. Significant differences in HBsAg-specific T cell responses between those with anti-HBs titers that fall within the top and bottom quartile at the Y1 and Y2 time points. T cell Response P-value (Year 1) P-value (Year 2) IL-10 secretion 0.0131 0.7791 IFN-γ secretion 0.0121 0.117 %age of IFN-γ+ proliferating CD4 T cells 0.2136 0.034 %age of TNF-α+ proliferating CD8 T cells 0.0056 0.1767 Data presented as correlation coefficient (P-value). Statistically significant (p < 0.05) correlations are indicated by red font. Refer to Figure 6 for Ns for Year 1 and Figure 8 for Ns for Year 2. 48 3.5.3 Correlation between anti-HBs titer and HBsAg-specific T cell responses: year 2 To identify any relationship between anti-HBs titers (at Y2 time point) and T cell responses, a correlation analysis was performed. Table 13 outlines the correlation coefficients (r) and P-values from the T cell responses that were found to have a significant relationship with Y1 and/or Y2 anti-HBs titers. Based on these sets of data, IFN-γ secretion following HBsAg stimulation had a positive correlation with anti-HBs titer at both Y1 and Y2 time points. In addition there appeared to be a strong trend of a negative relationship between anti-HBs titer and percentage of TNF-α positive proliferating CD8 T cells at both time points. Finally, there was a positive relationship between the percentage of IFN-γ positive proliferating CD4 T cells and anti-HBs titers at the Y2 time point. Please refer to Appendix G for statistical results (r coefficient and P-value) for all correlation analyses between anti-HBs titers at two years of age and all T cell antigen responses. Table 13. Significant correlation analyses results between HBsAg-specific T cell responses and anti-HBs titers from Y1 and/or Y2 time points. T cell response Year 1 Year 2 IFN-γ secretion 0.2578 (0.0246) 0.2984 (0.0121) IL-10 secretion 0.299 (0.0087) 0.05871 (0.6292) %age of IFN-γ+ proliferating CD4 T cells 0.1681 (0.1878) 0.3658 (0.0048) %age of TNF-α+ proliferating CD8 T cells -0.2665 (0.0348) -0.2441 (0.0649) Data presented as correlation coefficient (P-value). Statistically significant (p < 0.05) correlations are indicated by red font. Please refer to Table 9 for Ns for correlation analyses with Y1 anti-HBs titers. For correlation analyses with anti-HBs titers from Y2, cytokine secretion (N = 70), intracellular cytokine production (N = 58). 3.6 Comparing humoral and cell-mediated antigen-specific responses: summary The primary purpose of this project was to compare T and B cell memory responses to HBsAg in a cohort of healthy infants. T cell memory responses were measured by HBsAg- 49 specific proliferation and intracellular cytokine production detected by flow cytometry as well as bulk cytokine secretion detected by Luminex. B cell memory was assessed by quantifying titers of anti-HBs antibodies from plasma collected at one year of age (Y1; 6 months following final HBV dose) and two years of age (Y2; 18 months following final HBV dose). T cell memory was determined using PBMCs collected at one year of age only. There were several T cell parameters that were different when the cohort was divided into groups based on their anti-HBs titers from either Y1 or Y2 time points. Increased secretion of IL- 10 and IFN-γ in response to HBsAg as well as higher percentages of IFN-γ positive proliferating CD4 T cells associated with higher anti-HBs titers. Conversely, higher percentages of TNF-α positive proliferating CD8 T cells were observed in subjects with lower anti-HBs titers. The initial approach of this project was to look at differences in HBsAg-specific T cell responses based their anti-HBs titers. However, I also wanted to see whether individuals who had lower titers (either < 10 or <100 IU/l at Y1 or Y2 time points) had a positive T cell response following HBsAg stimulation. Since there was no established cut-offs to define a positive CMI response after HBsAg stimulation, I did an approximate calculation and defined a positive response as greater than the mean plus two times the standard deviation of the unstimulated control. The number of individuals (grouped by their anti-HBs titer at Y1 and Y2 time points) with a positive CMI response can be found in Table 14. There were multiple individuals who did not have seroprotective titers (≥ 10 IU/l), but did have one or multiple positive CMI responses to HBsAg stimulation (Table 14)—indicating the presence of T cell memory. 50 Table 14. Number of HBV vaccine non-responders, hypo-responders and responders with a positive cell-mediated immune response after HBsAg stimulation. Number of subjects with positive CMI response, n (%) anti-HBs titers (IU/l) at Y1 anti-HBs titers (IU/l) at Y2 T cell immune response <10 <100 ≥100 <10 <100 ≥100 IFN-γ 2 (67) 5 (26) 23 (40) 4 (31) 10 (24) 15 (52) IL-10 0 (0.0) 5 (26) 34 (59) 4 (31) 21 (51) 16 (55) IL-13 2 (67) 12 (63) 49 (85) 9 (69) 32 (78) 24 (83) Bulk cytokine secretion TNF-α 1 (33) 16 (84) 9 (16) 1 (7.7) 8 (20) 4 (14) CD4 2 (67) 6 (32) 17 (29) 1 (7.7) 12 (29) 9 (31) Proliferation CD8 1 (33) 2 (11) 11 (19) 1 (7.7) 5 (12) 7 (24) IFN-γ 0 (0.0) 4 (27) 21 (44) 4 (40) 12 (34) 12 (52) IL-13 2 (100) 10 (67) 41 (85) 6 (60) 29 (83) 20 (87) Intracellular cytokine production (CD4) IL-21 2 (100) 8 (53) 27 (56) 2 (20) 19 (54) 15 (65) IFN-γ 1 (50) 5 (33) 10 (21) 1 (10) 8 (23) 6 (26) IL-13 0 (0.0) 2 (13) 5 (10) 1 (10) 5 (14) 2 (8.6) Intracellular cytokine production (CD8) TNF-α 0 (0.0) 2 (13) 2 (4.2) 1 (10) 2 (5.7) 1 (4.3) A positive cell-mediated immune response was defined as an HBsAg-specific response greater than the mean plus two times the standard deviation of the unstimulated control. For this table, HBV vaccine non-responder: anti-HBs titers < 10 IU/l, hypo-responder: anti-HBs titers <100 IU/l, and responder: anti-HBs titers ≥ 100 IU/l. Please refer to Table 4 for total number of cohort subjects with incomplete and complete paired data sets—the values used as denominators to calculate the percentages in this table. 3.7 Demographics To verify whether there were any other factors that might have influenced the results above, the cohort was stratified by either sex or race (limited to the 4 racial groups that composed the majority of the cohort: Caucasian, Asian, South Asian and Asian-Caucasian) and was analysed for differences in the antibody or T cell responses. There were no significant differences between either sex or race noted in any of the results from the previous sections. Please refer to Appendix H and I for statistical results from all comparisons between sexes and races, respectively. 51 4 DISCUSSION The purpose of this research project was two-fold. Firstly, to develop a sensitive assay to detect HBsAg-specific T cells, and secondly to describe and compare the T and B cell immune responses to the HBV vaccine in a cohort of healthy infants. As low cell numbers is a common limitation when studying a cohort of such a young age, the assay was designed to maximize data output with minimal cell usage. Using the supernatant to detect bulk cytokine secretion from the same cell culture that was used to detect proliferation and intracellular cytokine production by flow cytometry both minimized the amount of cells required and enabled me to compare directly between T cell parameters–a task that becomes difficult when separate cultures are used for separate readouts (e.g., when 3H-thymidine is used to measure cell proliferation and multiple ELISAs are used to detect cytokine secretion). Overall, the cohort showed an excellent response to the HBV vaccine at 12 months of age (approximately six months following their last dose). Only 4.8% of subjects had titers below 10 IU/l at the Y1 time point. Considering that the standard time to check for peak antibody titers following HBV vaccination is 4-5 weeks following the final dose, it is possible that those who had titers below 10 IU/l at one year of age actually developed a seroprotective response following vaccination. The Luminex and flow cytometry panels were designed to include the effector cytokines of the major T cell helper subtypes: Th1 (IFN-γ), Th2 (IL-13), Th17 (IL-17A, IL-21), Tfh (IL- 21), pro-inflammatory and CTL (TNF-α), immunoregulatory (IL-10) as well as general T cell proliferative response (IL-2). Luminex experiments detected increased levels of IFN-γ, IL-13, TNF-α and IL-10 secreted following HBsAg stimulation. In addition, there was a positive and 52 significant correlation observed between all cytokines except TNF-α and IL-10 secretion. Intracellular cytokine production detected by flow cytometry showed significant increase in percentages of IFN-γ positive, IL-13 positive, IL-21 positive proliferating CD4 T cells following HBsAg stimulation. For proliferating CD8 T cells, the cytokine profile consisted of IFN-γ, IL-13 and TNF-α. Luminex and intracellular cytokine staining (ICS) were not independent assays, but they appeared to be complementary and confirmatory with both showing a balanced cytokine response to HBsAg stimulation (i.e. no obvious polarization towards a Th1 or Th2 response). This has also been shown by others,115 but conflicting results with vaccine recipients showing a Th1-skewed response in response to HBsAg stimulation have also been published.59 It should be noted that there are several factors that make it difficult to compare my results with previously published studies. I was unable to assess peak anti-HBs titers (one month following final HBV vaccine booster), and thus I could not classify my cohort into the standard groupings of non-responders and responders. Another factor to take into consideration is the age difference between study participants. The vast majority of studies on HBV vaccine responses have concentrated on older children, adolescents or adults. Of the studies that look at children less than two years of age, very few use a 2-, 4-, and 6-month HBV vaccination schedule. Given that there are differences in both the innate116-118 and adaptive118, 119 immune responses between adults and infants until around two years of age—observations noted from adult cohorts may not be applicable to younger subjects. 4.1 HBsAg-specific proliferation When looking at proliferation as an immune response following stimulation with HBsAg, there was a significantly greater percentage of proliferating CD4 T cells compared to CD8 T 53 cells (P-value < 0.0001). There was also a significant positive correlation between the percentage of proliferating CD4 and CD8 T cells in response to HBsAg stimulation—indicating that those with a greater percentage of proliferating CD4 T cells were likely to have a greater percentage of proliferating CD8 T cells as well. When comparing the percentages of proliferation according to anti-HBs titers, there were no differences between hypo-responders (< 100 IU/l) and responders (≥ 100 IU/l) Y1, nor in the top and bottom quartiles at either time point. However, when the cohort was divided based on their anti-HBs titers at the Y2 time point (< 10 IU/l or ≥ 10 IU/l), there was a significantly greater percentage of CD4 T cells that divided in response to HBsAg stimulation for seropositive subjects (≥ 10 IU/l) than seronegative subjects (< 10 IU/l). This suggested that the antigen- specific proliferative response measured in our studies might correspond to longer-lasting immune memory. Despite having a poor humoral response, 2 out 3 individuals with anti-HBs titers below 10 IU/l at the Y1 time point had a positive response for proliferation in their CD4 T cells following HBsAg stimulation. In addition, there was a greater percentage of hypo-responders (< 100 IU/l) at Y1 with a positive response than responders (32% versus 29%). At the Y2 time point, the percentage of seropositive individuals (≥ 10 IU/l) with a positive response for proliferation in their CD4 T cells was greater than that of seronegative individuals with only one seronegative subject having a positive response. When looking at their CD8 T cell proliferation, regardless of anti-HBs titers, there were fewer individuals with a positive response following HBsAg stimulation. I did still observe, however, hypo-responders at Y1 and seronegative individuals at Y2 that had a positive response for CD8 proliferation. Taken together, these data demonstrated that despite having lower anti- 54 HBs titers, subjects appeared to have T cell memory towards HBsAg as measured by antigen- specific proliferation. Correlations between anti-HBs titer and T cell proliferation have been shown by others.19, 53, 107, 120 Previous studies that evaluated T cell parameters between adult responders and non- responders shortly after an HBV vaccine booster found that non-responders lack a proliferative response.121, 122 An earlier study also had similar findings and followed up with CD8 depletion experiments to confirm that CD8 T cells do not have a suppressive phenotype and are not the reason for lack of response in the non-responder subjects.120, 123 Larsen et al., did an extensive study to describe the differences between adult responders and non-responders to HBV vaccine. They found that subjects who had protective anti-HBs titers demonstrated a variable range of proliferation with stimulation indices (SI) ranging from 0.7 to 55. They did find a positive correlation between anti-HBs titer and SI, but they limited their correlation analysis to only include the SI from subjects who proliferated in response to HBsAg stimulation (defined by a SI >2.0).53 Interestingly, the group noted that those with an SI > 2.0 following stimulation with HBsAg (regardless of antibody titer) had a cytokine profile similar to that of strong responders (i.e. balanced Th1/Th2 cytokine profile). While cells with an SI ≤ 2.0 in response to HBsAg produced no Th1 and a weak Th2 cytokine response.53 These data from previous papers strongly suggest that T cell function, in this case, assessed by level of proliferation at approximately one month following booster or completion of HBV vaccination series, does relate to the humoral response to HBV vaccine. It should be noted that the majority of studies assess proliferation using 3H-thymidine while my project used the fluorescent dye dilution assay. These methods have some inherent differences that may make it difficult for direct comparisons. Firstly, the dye dilution assay used 55 in my study in conjunction with cell surface markers allowed me to quantify proliferation in specific T cell subsets; whereas 3H-thymidine labelling can only measure total proliferation of the cell culture. Because mixed cell cultures (either PBMC or whole blood) are most often used for antigen-specific stimulations, proliferation levels using the 3H-thymidine method may be higher since it is detecting proliferation from all cells as opposed to only CD4 or CD8 T cells. Secondly, the dye dilution method allows me to determine how many cells from the original population divided in response to stimulation. Techniques that use radioactive tracers (3H- thymidine) or other markers detectable by fluorochrome-conjugated antibodies (Ki67 or bromodeoxyuridine (BrdU)) can only identify cells that are proliferating for a particular window of time. For 3H-thymidine and BrdU, the window is often the final 6-18 hours of culture, and for Ki67 it is at the time of harvest. Antigen-specific cells that divided earlier in the culture would not be detected using these methods; whereas, the dye dilution assay can capture the entire proliferation response. 4.2 Cytokine Responses 4.2.1 HBsAg-specific IL-10 production Because almost all of the cohort developed sufficient humoral immunity towards the HBV vaccine, there were too few subjects at the Y1 time point to compare between those with anti-HBs titers above and below 10 IU/l. As an alternative, the cohort was divided into those with titers above and below 100 IU/l, and T cell responses to HBsAg stimulation were compared. At the Y1 time point, individuals who had anti-HBs titers less than 100 IU/l also secreted significantly less IL-10 in response to HBsAg stimulation. The trend was also observed when the top and bottom quartiles of the cohort based on their anti-HBs titers were compared. In addition, 56 when a correlative analysis was done between anti-HBs titers at the Y1 time point and HBsAg- specific T cell responses, there was a positive relationship between antibody titers and IL-10 secretion. When humoral responses 18 months after the final dose (Y2 time point) were examined and compared to T cell responses at Y1, we continued to see a difference in IL-10 secretion with lower amounts being secreted by those with titers less than 10 IU/l, but it did not reach statistical significance (P-value = 0.0748). There were no differences in IL-10 secretion between individuals in the top and bottom quartiles based on their Y2 anti-HBs titers, and when the correlative analysis was repeated, there were no significant relationships found. Even though responders at Y1 (≥ 100 IU/l) secreted significantly more IL-10, approximately 26% of hypo-responders at Y1 and 31% of seronegative individuals at Y2 also had a positive response for IL-10 secretion following HBsAg stimulation, At both time points, there was still a greater percentage of responders and seropositive individuals with a positive response for IL-10 secretion (59% and 57%, respectively). Nevertheless, the fact that some hypo- responders and seronegative subjects had a positive response lends support to the idea that they still have T cell memory towards HBV despite weaker humoral responses. The cellular sources for IL-10 are predominantly monocytes, macrophages and Th cells, but many other cell types have been shown capable of producing it.124 While it was originally presumed that Th2 cells were the main source of IL-10 within the T helper cell subset, it has since been shown that many subsets including Th1, Th17, and Th22 cells are capable of producing detectable amounts of IL-10.124 IL-10 has pleiotropic roles in the immune response. Notably, it has immunoregulatory functions through inhibition of pro-inflammatory cytokine synthesis and downregulation of MHC II molecules on activated monocytes.125 Direct IL-10 57 signalling in T cells can reduce proliferation and production of effector cytokines;126 although I observed a positive correlation between IL-10 secretion and CD4 T cell proliferation in response to HBsAg stimulation. Pre-treatment of APCs with IL-10 also shows inhibition of IFN-γ production by Th1 cells.127 IL-10 can also act, however, as a potent growth and differentiation factor for activated B cells inducing secretion of immunoglobulins.128 Given the important role that IL-10 plays in the stimulation of B cells and subsequent immunoglobulin secretion, it follows logically that those individuals who secrete lower levels of anti-HBs might also secrete lower levels of IL-10. Other groups have studied the role of IL-10 on the immune response to HBV vaccination. Studies investigating the role of genetic variability of IL-10 in the generation of the humoral response against HBV vaccine have published differing results with some finding an association and others not.103, 129 Similar to my study, others have shown that individuals who do not mount a protective humoral response following HBV vaccination secrete significantly lower levels of IL-10, and in some cases, similar levels to HBV vaccine naïve individuals, in response to HBsAg stimulation.53, 130-132 This was also noted in an infant cohort as well.133 For all of these studies, non-responders mounted cytokine responses comparable to responders when stimulated with mitogens or other antigens (tetanus toxoid), excluding generic immune defects or differences. Curiously, my study observed that there was a positive correlation between IL-10 secretion and IFN-γ and IL-13 secretion. In other words, the more IL-10 secreted, the more IFN- γ or IL-13 was secreted. While IL-13 is considered a Th2 effector cytokine, the positive correlation with IFN-γ secretion indicates that there was a balanced Th1/Th2 cytokine profile in response to HBsAg stimulation. Others have also noted a balanced production of Th1/Th2 cytokines in HBV vaccine responders.41, 53 58 4.2.2 HBsAg-specific IL-13 production I detected significantly higher levels of IL-13 in the cell culture supernatant as well as in both CD4 and CD8 proliferating T cells following antigen stimulation. There was also a strong positive correlation between IL-13 secretion and IL-13 positivity in proliferating CD4 T cells (r = 0.7771, P-value <0.0001). IL-13 is a type 2 cytokine that is also important in the activation of B cells and production of immunoglobulin. IL-13 is classically associated with CD4 Th2 cells, but CD8 T cells are also capable of producing IL-13134 (as seen in our study). In fact, cytotoxic CD8 T cells, like CD4 T cells, mature into functionally different subsets, Tc1 and Tc2, for example. Tc2 cells have been shown to produce Th2-type cytokines, including IL-13 135,134, 136 In this study, while IL-13 was strongly induced following HBsAg stimulation, there was no difference in levels (in the supernatant or intracellularly) of IL-13 when stratifying the Y1 samples by their anti-HBs titers. However, when Y2 samples were divided according to their anti-HBs titers (greater or less than 10 IU/l) there were significantly higher percentages of IL-13 positive CD4 and CD8 proliferating T cells in individuals who had anti-HBs titers equal or greater than 10 IU/l. At the Y1 time point, 66% and 100% of non-responders had a positive response for IL-13 secretion and intracellular production (CD4 T cells), respectively. In addition, 63% and 67% of hypo-responders (< 100 IU/l) at Y1 and 69% and 83% of seronegative individuals (< 10 IU/l) at Y2 had positive responses when looking at bulk cytokine secretion or intracellular production of IL-13 in CD4 T cells, respectively. Even though there were fewer individuals with positive responses for IL-13 production detected in CD8 T cells by intracellular staining, there still were non-responders, hypo-responders, and seronegative subjects with positive responses in their CD8 T cells. 59 There is limited knowledge regarding the role of IL-13 in the response to the HBV vaccine. A paper published by Chen et al., found certain genotypes in the IL13 gene to associate with non-response to HBV vaccine in an adult population.105 However, another group did not find associations between anti-HBs titers and genetic variation in IL13 or IL4R genes in their cohort of patients undergoing hemodialysis.137 Those studies looking at immunological differences between HBV vaccine responders and non-responders have found that non- responders have lower percentage of IL-13 and/or IL-4 positive CD4 T cells or secrete lesser or undetectable amounts.53, 121, 133 Using a slightly different approach to assess HBV immunity, one group measured T cell memory responses in adult non-, low- and high-responders using the ELISPOT assay. They found that non- and low-responders had significantly less IL-4 secreting HBsAg-specific memory T cells than high-responders.138 Another group looked at cellular and humoral immune responses to HBV vaccination in adults and neonates responders.41 They found that adults had increased levels of IL-13 following HBsAg stimulation and a trend towards increased IL-5 (another classic Th2 effector cytokine). Neonates, on the other hand, showed significant increase of IL-5 following the full vaccination series but no difference in IL-13,41 a finding which differed from my study. Based on the data available from published studies, it appears that the Th2 response is important for an effective protective response following HBV vaccination—whether in adult or infant. This appears to be logical since Th2 immune responses are important for B cell activation and immunoglobulin production. While I did not observe a difference in IL-13 intracellular production or secretion at the Y1 time point, when the cohort was divided into groups of greater or less than 10 IU/l based on their Y2 time point there was a significantly higher percentage of IL-13 positive proliferating CD4 and CD8 T cells. This may indicate that a stronger Th2 60 response soon after vaccination might predict which vaccine recipients will have waning antibody titers in the future. This needs to be tested in follow up studies. 4.2.3 HBsAg-specific IL-21 production While IL-21 was not detected in the supernatant by Luminex assay, it was detected by ICS. There are a number of possible explanations for this observation. It could be that IL-21 was being produced but not secreted, or it was produced earlier during the culture period and has low stability after secretion, or is bound to cells in the culture. At this time, I am unable to determine what factors might have caused this discrepancy. There were no observed differences in percentage of IL-21 positive proliferating cells between individuals at the Y1 time point. Both non-responders at the Y1 time point had a positive response for intracellular production of IL-21 in their CD4 T cells, and the percentages of hypo-responders and responders with positive responses were very similar (53% and 56%, respectively) There was a significant difference, however, between individuals with anti-HBs titers greater or less than 10 IU/ml at the Y2 time point. Percentage of IL-21 positive CD4 proliferating T cells was increased in individuals who had titers equal or greater than 10 IU/ml. This trend was not observed when stratified by quartiles or in correlation analyses. When looking at the number of individuals with a positive response for intracellular IL-21 production at the Y2 time point, 65% of seropositive and 20% of seronegative subjects had a positive response in their CD4 T cells following HBsAg stimulation. IL-21 has multiple roles in the immune system. A recent paper describing a loss of function mutation in the IL-21 receptor—causing primary immunodeficiency syndrome as well as liver disease—highlights the critical role of IL-21.139 Exposure of antigen-specific CD8 T cells to IL-21 improved their cytolytic and anti-viral responses126 as well as promotes antigen 61 specific proliferation and sustains long-term CD8 responses.140 IL-21 production, along with IL- 17A, has also been shown in the Th17 subset of T helper cells.141 Most notably, however, it is a distinguishing feature of follicular helper T cells (Tfh), a subset of helper T cells that are responsible for the formation and maintenance of germinal centres—the location of B cell differentiation into plasma and memory cells.142 Others have shown that IL-21 has been shown to enhance IL-10 production.124 Given the somewhat similar roles Th2 and Tfh cells play in the humoral response, it would be logical to observe a coordinated production of both types of cytokines and that there would be higher levels detected in subjects with higher antibody titers (as seen above). This was observed in my cohort with a strong, positive relationship between the percentage of IL-13 and IL-21 positive proliferating CD4 T cells (r = 0.7532, P-value < 0.0001) as detected by intracellular cytokine staining. The role of IL-21 in the response to HBV vaccine or infection in humans remains relatively unknown. A very interesting study by Publicover et al., describes a murine model that mimics the age-related differences in the outcome of primary HBV infection observed in humans—infection in young mice results in chronic disease whereas infection during adulthood is cleared. They found that IL-21 plays a key role in this age-dependent process. Adult mice with HBV infection had higher levels of IL-21 in the liver compared to young mice. In addition, IL-21 receptor knockout adult mice developed chronic infection, similar to that of young mice.143 There is still limited data concerning the role of IL-21 in the response to HBV vaccination. But given its pleiotropic roles in the immune system, its apparent role in the outcome of HBV infection in mice, and the antigen-specific production observed in this study including a higher percentage of IL-21 positive CD4 T cells in responders at the Y2 time point— this area of research merits further attention. 62 4.2.4 HBsAg-specific TNF-α production In my cohort, an increase in TNF-α secretion and percentage TNF-α positive proliferating CD8 T cells was observed following stimulation with HBsAg compared to unstimulated controls. The level of bulk TNF-α secretion positively correlated with secretion of bulk IFN-γ and IL-13. As expected, IFN-γ and IL-13 were the other two cytokines detected intracellularly at higher levels in proliferating CD8 T cells following HBsAg stimulation. In addition, there was a positive correlation between intracellular TNF-α detected by flow cytometry and intracellular IFN-γ. While TNF-α was not detected at higher levels than background in proliferating CD4 T cells, there was a positive correlation between bulk TNF-α secretion and percentage of starting CD4 T cells that proliferated in response to HBsAg stimulation. According to the Y1 anti-HBs titers, those with titers below 100 IU/l and within the bottom quartile have a significantly higher percentage of TNF-α positive proliferating CD8 T cells than those with titers equal or greater to 100 IU/l and found within the top quartile. When a correlative analysis between Y1 anti-HBs titers and T cell immune response was performed, a similar trend was observed with a significant negative relationship (r = -0.2665) between these two parameters. On a similar note, 13% of hypo-responders at Y1 (< 100 IU/l) had a positive response for intracellular TNF-α production in their CD8 T cells while only 4.2% of responders did. A more striking observation, however, was that 84% of hypo-responders at Y1 had a positive response for TNF-α secretion (detected by Luminex) compared to 16% of responders. This pattern did not continue when T cell immune responses at the Y1 time point were compared to humoral responses at the Y2 time point. In fact, there was a significant increase in TNF-α secretion, detected by Luminex, in individuals with anti-HBs titers equal or greater than 63 10 IU/l at the Y2 time point. This trend was not observed when the bottom and top quartiles were compared or when correlative analyses were performed. TNF-α is a pro-inflammatory cytokine that is primarily secreted by activated monocytes and macrophages as well as lymphocytes amongst a wide variety of cell types.144 It has strong activation and differentiation effects on monocytes and macrophages. TNF-α acts only on activated T cells and induces expression of CD25 thereby increasing proliferative responses to IL-2 as well as production of IL-2.144 Others have shown that TNF-α increases generation of cytotoxic lymphocytes (CTL) as well.145 CD8 T cells have been shown to play an essential role in controlling HBV infection, not only through cytolysis of infected cells, but also with non-cytolitic mechanisms, such as cytokine production. In fact, TNF-α as well as IFN-γ produced by HBV-specific CD8 T cells was observed to be necessary for controlling HBV infection.146-148 An animal study showed TNF-α knockout mice were unable to develop HBV-specific CTL cells following primary immunization with an HBsAg-encoding plasmid compared to their wild-type controls.149 Despite the important role that TNF-α appears to play in controlling HBV infection, there is limited information regarding its role in the response to HBV vaccination—particularly in infants. Both arms of the adaptive immune system are important for clearance of HBV. The humoral response is responsible for recognizing circulating viral particles by their envelope antigens. The CMI response clears infected cells through both cytolitic and non-cytolitic mechanisms. One study found that adult non-responders produced significantly less TNF-α than strong responders following re-stimulation with HBsAg.132 While these data agree with the results using the Y2 humoral data, there was a clear opposite trend when looking at the Y1 anti- HBs titers. Hypotheses to explain the Y1 humoral and CMI data could state that the increase in 64 TNF-α response is to compensate for the lower levels of antibodies, or alternatively that TNF-α positive proliferating CD8 T cells have an immunosuppressive phenotype. Another explanation could be that the differences in TNF-α production might reflect differences in innate immunity since innate immune cells can also produce TNF-α. Regardless, TNF-α remains an interesting target for future studies in order to dissect the relationship between TNF-α and the pro- inflammatory response to HBV vaccination. 4.2.5 HBsAg-specific IFN-γ production There was a significant production of IFN-γ following antigen stimulation detected by both Luminex and intracellularly in CD4 and CD8 T cells. This has been noted by others as well.41, 86 Similar to IL-13, there was a positive correlation between IFN-γ secretion and intracellular IFN-γ production (r = 0.6197, P-value <0.0001). I also observed a significant increase in IFN-γ secretion in the top quartile of the cohort compared to the bottom quartile based on their Y1 anti-HBs titers. Similarly, there was a positive correlation between anti-HBs titer at both the Y1 and Y2 time points with IFN-γ secretion. This was the only correlation that remained significant at both time points. In addition, when split into seropositive and seronegative individuals at the Y2 time point, there was a significantly greater percentage of IFN-γ positive proliferating CD4 T cells in the responder group compared to non-responders. Despite the observations above showing an association of higher anti-HBs titers with increased IFN-γ levels—at the Y1 time point, two out of three non-responders had a positive response for IFN-γ secretion. Hypo-responders at Y1 also demonstrated evidence of T cell memory towards HBV with 26%, 27%, and 33% of subjects having a positive response for IFN- γ secretion or intracellular production response in CD4 and CD8 T cells, respectively. There was 65 a greater percentage of positive IFN-γ responders as detected intracellularly in CD8 T cells in hypo-responders than responders at Y1 (33% versus 21%). At the Y2 time point, 31%, 40%, 10% of seronegative subjects had a positive response for IFN-γ secretion or intracellular production response in CD4 and CD8 T cells, respectively. IFN-γ is a Th1 cytokine and is produced by CD4 and CD8 T cells as well as macrophages and monocytes. IFN-γ synergizes with IL-12 to act as a potent growth factor for Th1 CD4 cells while repressing the development of Th2 CD4 T cells.150 It is a major activating factor for macrophages151, 152 and neutrophils by increasing antimicrobial activity and killing of intracellular pathogens.150 It causes the upregulation of numerous genes associated with immune activation including MHC class I and II, IL-2 receptor, and complement proteins. Neutralization of IFN-γ results in abrogated T cell proliferation. 150 Out of all the cytokines included in the Luminex and ICS panels for this study, IFN-γ and its role in the response to HBV vaccination is the best characterized. Multiple studies have published low or undetectable expression or secretion of IFN-γ in infant and adult low- or non- responders.53, 121, 122, 132, 133 Several groups have also seen a correlation between anti-HBs titer and IFN-γ production132 while others have not.41, 86 Other groups have used the ELISPOT method to assess immune memory to the HBV vaccine. Similar to what others have found using either ELISA or flow cytometry, adult non- or low-responders had significantly less IFN-γ secreting HBsAg-specific memory T cells than high- responders.138 A long-term follow-up (20 years) study in an area with high HBV endemicity looked at IFN-γ-secreting HBsAg-specific memory T cells before and after an HBV booster dose in a group of adults who had been vaccinated as infants.86 Regardless of anti-HBs titers before the boost, subjects who had titers greater than 10 IU/l after the booster shot also had HBsAg- 66 specific IFN-γ producing T cells. Approximately 60% of individuals who had anti-HBs titers less than 10 IU/l before and after the booster also had HBsAg-specific IFN-γ producing T cells. This indicates that even though anti-HBs titers did not increase after boosting—suggesting either a non-responder or lost immunity—there was T cell memory present for more than half of the individuals. They did not, however, observe a correlation between anti-HBs titer and frequency of IFN-γ secreting HBsAg-specific memory T cells. As the study locale was an area of high HBV endemicity they also looked for natural infection as a way for natural boosting by looking for the presence of HBeAg-specific T cells. They found that on average 87% of participants had HBeAg-specific T cells (HBeAg is secretory variant of HBcAg).86 These results might indicate that natural boosting by HBV exposure might help to sustain immunological memory long after vaccination, and perhaps T cell memory is retained longer than B cell memory. To further this point, another study examined T cell memory to HBsAg in a cohort of adults 4-8 years following vaccination whose titers had fallen below 10 IU/l. They observed that all individuals had IFN-γ secreting HBsAg-specific memory T cells even though their anti-HBs titers had declined below the level of protection.62 A recent paper published by Carollo et al., compared humoral and CMI responses in a cohort of children who received two different HBV vaccines 5 years prior (at approximately 11 months of age). Recipients of both vaccines were capable of generating T cell recall responses as determined by increased proliferation, intracellular production of IFN-γ and IL-2, and secretion of IFN-γ. While there were differences in the mean anti-HBs titers between the two vaccine groups, they did observe that the vaccine which induced lower antibody titers had a stronger T cell recall response.36 This publication is similar to my study as we both compared HBV-specific serological immunity and CMI as measured by proliferation and intracellular cytokine 67 production. Carollo et al., however, is looking at a later time point (5 years post vaccination) and also focused on identification of memory T cell subsets – as such, they have relatively limited CMI parameters (proliferation measured crudely by blast cell formation, intracellular cytokine staining for IFN-γ and IL-2, and bulk secretion of IFN-γ). The main focus of their paper was to compare immune responses between two HBV vaccines used in Italy: Hexavac® and Infanrix- Hexa®. My study, on the other hand, is looking at the immune response shortly after vaccination and has a more comprehensive assay to assess CMI responses to HBsAg stimulation. Another study described the importance of IFN-γ to the humoral response to HBV vaccine. They showed that the addition of IFN-γ to cell culture medium caused a dose-dependent increase in anti-HBs secreting B cells. The addition of monoclonal antibodies against IFN-γ or CD3 negated the effect of IFN-γ indicating the importance of the activation of T cells through IFN-γ in the B cell response to HBsAg.59 It is clear that IFN-γ and a Th1 response is important for a strong protective response against HBV infection. The fact that there was a significant correlation between IFN-γ secretion and anti-HBs titers at both time points in my study suggested that IFN-γ might be a suitable predictor for both magnitude and duration of immunity to HBV, and that cellular immune response should be considered as a surrogate of protection alongside serological markers. Additional long-term experiments are required in order to confirm this prediction. 4.3 Combination vaccines and concomitant vaccination Combination vaccines are becoming more popular as new vaccines are being added to immunization schedules. Advantages of combination (or multi-valent) vaccines include reducing number of injections (particularly beneficial for younger vaccine recipients) and improving 68 timeliness as well as completion of immunization schedules.153 Even with combination vaccines available, this study cohort still required co-administration of Pentacel® (a pentavalent vaccine against diphtheria, tetanus, acellular pertussis, (collectively referred to as DTaP), inactivated polio virus (IPV), and Haemophilius influenzae B (Hib)) at 2, 4, and 6 months of age, Prevnar® (Streptococcus pneumoniae conjugate vaccine) at 2 and 4 months of age, and NeisVac-C (Meningococcal C conjugate vaccine) at 2 months of age. The difference in immunogenicity using combination versus monovalent vaccines and the effects of concomitant vaccination in separate limbs has been investigated previously. A meta- review looking at the clinical utility of Pentacel® in the pediatric population found that its immunogenicity is comparable to its component vaccines. It is also not affected by co- administration with the mumps, measles, rubella (MMR) vaccine, pneumococcal conjugate vaccine or the HBV vaccine.154 Groups have observed that anti-Hib titers and percentage of subjects achieving seroprotective levels are lower following administration of a combination vaccine containing Hib compared to the monovalent Hib vaccine. The combination vaccine, despite reduced anti-Hib titers, still generated a humoral response that is considered protective against invasive Hib disease.155, 156 Another group looked at the differences in antibody responses to a hexavalent vaccine (DTaP-IPV-Hib-HBV), Hexaxim®, and co-administration of the pentavalent vaccine (Pentaxim®) with a separate HBV vaccine (Engerix B Pediatrico®) at 2, 4, and 6 months of age with a booster of Pentaxim® (for both groups) at 18 months. When they initially looked at antibody responses one month after the first 3 doses, the antibody titers were comparable between the two groups157—a finding that has been observed elsewhere.158 During the follow-up, they still observed that antibody titers were comparable before and after the booster with the exception of HBV. The percentage of participants that were seropositive (anti- 69 HBs titers ≥ 10 IU/l) was greater in the group receiving the pentavalent DTaP-IPV-Hib and separate HBV vaccines.159 Based on the literature available it appears that there is more likely to be a reduced humoral immune response when vaccines are combined together, but even vaccines associated with decreased titers still manage to generate a response that is considered protective against infection. Co-administration of vaccines in separate limbs appears to have a very minimal effect on vaccine immunogenicity. 4.4 Parallels to other vaccines We have entered the age of rational vaccine design, where successful development of the next generation vaccines depends on an in-depth understanding of how we respond to immunization and develop protective immunity. There have been studies that attempt to understand how human biology influences the immune response to vaccination. Similar to the HBV vaccine, host genetic factors have been shown to play an important role in explaining heterogeneity of other vaccine-specific immune responses. And while most studies focus on the humoral response to vaccination, some also attempt a more comprehensive description of the immune response by looking at CMI or innate immunity as well. 4.4.1 Measles vaccine Genetic differences in host proteins used by the measles virus to infect cells have been shown to associate with differences in the humoral response to measles vaccine.160 In addition, genetic variability in the TLR signalling pathways has been linked to both the humoral and CMI 70 response against measles following vaccination161— clearly demonstrating the important role that PRRs and the innate immune system can have on the modulation of the adaptive immune response. An interesting study by Bautista-Lopez et al., investigated the development of humoral immunity and CMI against measles in prospective and cross-sectional cohorts to examine short and long term protection, respectively.162 Six months following vaccination, a large proportion (~93%) of children in the prospective cohort had PRN titers that were considered protective against disease (PRN ≥ 120). Participants in the cross-sectional cohort (5-8 or 9-13 years following measles vaccination) had statistically lower PRN titers than those in the prospective study. Approximately 29% and 34% of individuals, 5-8 years and 9-13 years following measles vaccination, respectively, had PRN titers less than 120. They also looked at lymphoproliferation, measured by 3H-thymidine incorporation, as an additional vaccine response and compared it to the humoral response. Interestingly, approximately 60% of individuals regardless of cohort (prospective or cross-sectional) had proliferation responses defined as significant (3 times higher than background). These results indicate that measles-specific cellular immunity may be better sustained than humoral. They did notice, however, that for those individuals who had a both a protective humoral and a detectable lymphoproliferative response, their PRN titers tended to lower than those individuals with a humoral response only. While this observation was not significant, the trend was observed in both cohorts as well as both time points in the cross- sectional cohort. A similar observation was noted in a recent study from Italy comparing immune responses between two HBV vaccines where subjects that received the vaccine associated with lower anti-HBs titers produced a stronger T cell memory response.36 Finally, Bautista-Lopez et al., noted that looking at the cross-sectional cohort, the percentage of individuals not protected 71 (either through primary or secondary vaccine failure) against measles infection is much closer to actual measles incidence rates when they combined both PRN titers and lymphoproliferation (11%) compared to PRN titers alone (36.5%).162 Another group looked at response to measles re-vaccination in bone marrow transplant patients. They found that pre-existing lymphoproliferative memory to measles antigens interferes with subsequent vaccination; whereas, pre-existing PRN titers had no effect.163 A study looking at both CMI (measured by lymphoproliferation and IFN-γ production) and humoral immunity following measles vaccination in 6-, 9-, or 12-month olds found that CMI responses were equivalent across all age groups regardless of the presence of passive antibodies during immunization. Six-month olds, on the other hand, had lower levels of antibodies to measles virus compared to older groups.164 Taken together, these data indicate that cell-mediated immunity— in particular lymphoproliferation—might be an important measure to take into account when assessing vaccine success and should be potentially considered a surrogate of protection for the measles vaccine as well. 4.4.2 Pertussis vaccine Genetic variability in the TLR signalling pathways has been linked to the humoral response against pertussis toxin following vaccination.114 Once again, this demonstrates the importance of the innate immune system in the modulation of the adaptive immune response. A study by Hendrikx et al., looked at the relationship between plasma antibody levels against components of the pertussis vaccine and B cell memory towards the pertussis vaccine in children vaccinated with whole cell pertussis but boosted with the acellular pertussis vaccine.60 While the majority of subjects had both antibodies against pertussis proteins as well as antigen-specific 72 memory B cells, there were those with neither as well as those with high levels of antibodies towards pertussis antigens but no memory cells—indicating long-lived plasma cells which maintain antibody levels and memory B cells are distinct populations. These data may indicate that both memory B cells as well as antibodies contribute to protection against pertussis infection.60 Another group managed to study pertussis-specific cell mediated immunity in infants vaccinated with the acellular pertussis vaccine. While antigen-specific antibody titers continually declined after primary vaccination until booster dose almost 11 months later, cell mediated immune responses (measured by proliferation and production of IFN-γ and IL-2) remained stable.165 4.4.3 Smallpox vaccine (vaccinia) Numerous studies have looked at the factors impacting immune response to vaccinia, the vaccine against smallpox. Genetic variability in the IL-18 cytokine signalling pathway has been shown to associate with different humoral responses to smallpox vaccination.166 A study by Umlauf et al., looked for correlations between humoral and CMI response following administration of the vaccinia vaccine, and found that neutralizing antibody titer correlated with IL-2 and IFN-γ secretion following re-stimulation with live vaccinia virus in vitro. In addition, they observed a strong positive correlation in secretion of numerous pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, ad IL-12p40) following stimulation with vaccinia virus.54 4.4.4 Tetanus vaccine A study in Cameroon assessed long-term immunity against tetanus in children and adolescents. Individuals were given a booster of tetanus toxoid and antibody titers were 73 measured seven days later to determine their immune memory. Approximately half of the participants did not have an anamnestic response following the booster shot, and the only predictor was sufficiently high residual tetanus-specific antibody titers just prior to boosting. These data indicate the presence and role of long-term effector cells in immune memory towards tetanus as opposed to memory cells.167 4.4.5 Hepatitis A virus vaccine A group studying the immunological differences between non-, intermediate and high responders to the Hepatitis A virus (HAV) vaccine saw no differences in T or B cell populations as well as co-stimulatory or inhibitory marker expression between the groups. Nor was there a difference in CD25 and FoxP3 expression (to identify regulatory T cells). There was an increase in IFN-γ and IL-10 production seven days after booster, and this increase was not noted in the low and non-responders. Another difference was significantly lower levels of HAV cell receptor- 1 in CD4 T cells in the non- and intermediate responders compared to the high responders.168 Another group looked at ratio of IL-10 to IFN-γ production upon re-stimulation with HAV and found a positive relationship between HAV antibodies and IL:10:IFN-γ ratio.39 4.4.6 HBV infection The immune response to HBV infection is not identical to that of HBV vaccination. The outcome of infection, whether it remains acute and resolves (similar to vaccination) or becomes chronic, has been looked at extensively in order to understand the differences in immune status between those two groups. A study that compared antigen specific responses to HBsAg between those who had cleared the virus 1-5 years prior and those who were chronically infected found 74 that proliferation was only detected in individuals who had previously cleared the virus but not in those chronically infected. However IFN-γ was not detected in response to HBsAg stimulation for either group. The addition of IL-12 to culture medium rescued proliferation for those chronically infected.169 Secretion of IFN-γ by CD4 T cells into supernatant has been shown to inhibit HBV transcription and replication without significant cytotoxicity to hepatocytes.170 Looking at using HBV as a therapy for chronic HBV, another group showed that it was anti-viral cytokines (IFN-γ and TNF-α) (not IL-10 or IL-4) produced by CD4 T cells were responsible for controlling HBV replication.171 While the current standards for assessing vaccine efficacy still have merit, the small sampling of research cited above demonstrates that it is becoming increasing clear that vaccine response is not just reaching an antibody titer threshold—immunological memory is a complex interaction between the innate and adaptive, as well as memory and effector subsets of B and T cells. Loss of protective antibodies does not always lead to loss of protective immunity. The next generation of vaccines will rely on rational design, requiring an in-depth and complete understanding of the biology that drives the response to vaccination, including not only humoral, but CMI, innate immunity and even genetics as well. 75 5 CONCLUSION By developing a sensitive antigen-specific stimulation assay, I was able to assess multiple immune parameters simultaneously, including proliferation, intracellular cytokine production, and bulk cytokine secretion of memory T cells in response to HBsAg stimulation in vitro. When I stratified the cohort according to their respective anti-HBs titers 6 and 18 months after their last HBV vaccine dose, I was able to compare how memory T cell responses differed based on their serological response. Based on my results, IFN-γ appeared to be the most consistent predictor of a strong, sustained serological response to HBV vaccination. The most striking trend was a positive correlation between bulk IFN-γ secretion and anti-HBs titers at both time points following vaccination. A similar trend was noted for IL-10 secretion, but it did not extend to the Y2 serological samples. Interestingly, TNF-α had an opposite production profile. Overall, TNF-α production in proliferating CD8 T cells was higher in those who produced lower levels of anti- HBs at Y1 time point. This observation was reversed by Y2—children who had anti-HBs titers less than 10 IU/l also secreted lower levels of TNF-α. After calculating approximate cut-offs for positive CMI responses, I observed that many hypo-responders at Y1 and seronegative individuals at Y2 demonstrated evidence of cellular immunity either by HBsAg-specific proliferation, intracellular cytokine production, or cytokine secretion. This included the parameters that also associated with stronger humoral responses— IFN-γ secretion, for instance. A striking observation was that the percentage of hypo-responders at Y1 with a positive response for TNF-α secretion was over 5 times greater than that of responders (84% versus 16%). These data support the literature regarding alternative immunological mechanisms contributing towards protection following vaccination. 76 The purpose of this research project was to generate hypotheses regarding how the adaptive immune system, as a whole, responds to vaccination. I have identified concrete avenues of research, focused on IFN-γ, IL-10, and TNF-α, that merit continued investigation. However, in order to determine which immune parameters best predict strong, long-lasting immunological memory, we need to design longitudinal studies that extend beyond infancy. Other groups have managed to investigate long-term vaccine efficacy, but they predominantly focus on the serological response. Now that it is possible to test multiple immune parameters with relatively limited sample, as done with my protocol, we should be assaying for both CMI and humoral, as well as innate immune status over longer periods of time to determine the best correlates of protection. 5.1 Study limitations This study has inherent limitations that should be addressed. First and foremost, as I mentioned earlier, the phlebotomy visits for this cohort did not capture the peak anti-HBs response following the last booster dose of HBV vaccine. Typically, anti-HBs titers are checked four weeks after vaccination in order to identify those who did not develop protective anti-HBs titers (≥ 10 IU/l). Since the Y1 samples were collected approximately 6 months after the last dose, I was unable to divide the cohort accordingly. This makes it more difficult to compare with other studies that correctly identified non-responders. In order to minimize inter-assay variability, PBMCs were harvested and cryopreserved so that all samples could be run at the same time with the same reagents. With that said, the effects of cryopreservation on PBMC adaptive immune function remain relatively unknown, and most groups prefer the use of freshly isolated PBMCs to eliminate any artefact that results from 77 cryopreservation. This was unavoidable for my study, and should be taken into account in the interpretation of the data. A very common limitation when studying a cohort of such a young age is the amount of blood drawn. At one year of age, 10 ml of venous blood is the upper limit of sample collected. For this cohort, numerous assays (innate stimulation, genotyping) were performed for other studies prior to cryopreservation of PBMCs leaving very few cells for the adaptive immune assays. When these types of experiments are performed on adult cells, they are often plated in triplicate or more with a dose curve. This was not possible for my experiments. As such, it is possible that the dose of HBsAg used may not have been the optimal dose for all subjects in my cohort. In flow cytometry, it is preferable to include a viability stain in order to gate out any dead cells to reduce non-specific binding.46 However, while I was developing my stimulation protocol it was logistically unfeasible to include a viability dye without significant loss of cells in the process (due to additional spins and transfers). I included a size gate using side scatter versus forward scatter to exclude as much debris and dead cells as possible, but it should be recognized that this technique is not as effective as a viability dye. Finally, as with any in vitro experiments, one should use caution to extrapolate data and apply it to the in vivo model. Obviously, cells respond differently in a culture dish than in the body. And while in vitro models provide important insight into the in vivo system, we should remain constantly aware that it might not translate directly from one to the other. 78 5.2 Future directions This project is not the first to look at antigen-specific proliferation as well as effector functions using flow cytometry34, but it is one of the more comprehensive studies done using a cohort close to the age to which they receive their standard vaccine schedule. Nonetheless, there are additional aspects that merit attention in the future. For instance, while this project focused entirely on the adaptive immune response, there is additional data on this cohort’s innate immune responses as well. PBMCs from the same cohort were stimulated with a panel of TLR ligands and assayed for both bulk and cell-specific cytokines. In addition, this cohort was also extensively genotyped for approximately 500 single nucleotide polymorphisms in immune genes. Looking for associations between all three levels (genetics, innate, and adaptive) would provide insight that, to my understanding, has not been studied. The following study highlights the importance of innate immune response in guiding the adaptive immune response. Jacques et al. compared two groups of adult HBV vaccine non- responders. One group received an additional dose of the traditional HBV vaccine formulation (with alum as the adjuvant). The other group received the HBV vaccine with a new adjuvant formulation containing alum as well as a derivative of lipid A, which has been shown to interact with TLR4 signalling group. The group of non-responders which received the adjuvant containing the lipid A had a statistically higher increase in geometric mean titer following vaccination. They also looked at CMI responses and found after the full series of vaccinations, that more individuals with the newer adjuvant formulation had stimulation indices of 3 or greater than those with the traditional formulation.38 Similarly, adding an innate immune stimulant (CpG) increased the concentration of anti-HBs in animal studies.172 79 In the consideration of the role of innate immune stimulation on the development of immunological memory, it is interesting to note that a study comparing whole cell and acellular pertussis vaccines found that the acellular vaccine generates higher antibody titers against all three proteins. This is contrary to what would be assumed since the whole cell vaccine should contain more PAMPs than that of the acellular formulation.40 A study that looked at immune responses to the influenza A vaccine in a cohort of the young and elderly found that there was an inverse correlation with IL-10 production induced by non-specific stimulation with phytohaemagglutinin (PHA) as well as a positive correlation with TNF-α secretion by lipopolysaccharide (LPS) stimulation with antibodies against influenza A.173 These results may indicate that there may be a predictive relationship between non-specific as well as innate immune responses with the ensuing adaptive immune response following vaccination. Other papers have found reduced expression levels of cell activation markers (CD69), or other functional markers (CD40L) in HBV vaccine adult non-responders.121 This, to the best of my knowledge, has not been studied in infants. Looking at these markers, as well as memory subsets might provide further insight into mechanisms leading to different immune responses to vaccination. γδT cells have been shown to respond to vaccination stimulation through production of IFN-γ34 and significant population expansion35 in response to live vaccine stimulation. Considering that γδT cells numbers are highest at birth and then decline with age, determining the role of γδT cells in the infant response to vaccination should be a focus of future studies. Another study looked at the response after each dose of the HBV vaccine and found that infants had a defective IFN-γ production during the primary phase of the response compared to adults.41 It would be interesting to look at the immune response—both antigen specific and innate—following each dose of the vaccine to see if there are biological predictors that could 80 indicate after a single dose or early on those who may not respond well to HBV and require a different adjuvant or increased or additional doses. While we were not able to look at B cell function in vitro, others have been able to study the HBs-specific B cell response. An early paper described the Spot-ELISA to identify IgM- and IgG-specific HBsAg B cells between responders and non-responders.61 They found that non- responders with the exception of one did not produce any IgG-anti-HBs spots. The one non- responder that did produce spots seroconverted after an additional dose while the remaining non- responders failed to seroconvert after the additional booster. They concluded that the ability to produce anti-HBs spots might indicate those who would seroconvert following additional boosters and those who are true non-responders.61 Another group found all individuals had memory HBsAg-specific B cells even though their anti-HBs titers had fallen below detection. Interestingly, they observed that depleting T cells from cell culture abrogated any anti-HBs secreting B cells.62 However, they did not observe a correlation between anti-HBs titers and frequency of memory B or T cells.62 If possible, it would be interesting to include an ELISPOT assay in future studies to compare T cell and B cell function in vitro to see how it correlates with vaccine response. Also, considering the important role that CD8 cytotoxic T cells play in the clearance of virally infected cells, it would also be very interesting to include CD8 effector proteins such as granzyme A and B as well as perforin in future studies assessing vaccine responses. Finally, there have been multiple studies investigating the effects of multivalent vaccines or concomitant vaccinations on the ensuing humoral immune response compared to either monovalent vaccines or single administrations. There are no studies, to my knowledge, however, 81 that have looked for differences in cell-mediated immunity. This would be an interesting topic to pursue in future studies as well. 5.3 Final remarks While this study focused on HBV vaccine, there is a need to understand how humans, and in particular infants, respond to all vaccinations. 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Non-proliferatingProliferating Oregon GreenOregon Green Proliferating Non-proliferating Positive for cytokine production FSC-A F S C -H CD8 C D 4 FSC-A S S C -A CD3 S S C -A Singlet Gate (to eliminate doublets) Size Gate (to eliminate dead cells and debris) CD3 Gate (to identify CD3+ cells) CD4 and CD8 Gate (to identify CD3+CD4+ andCD3+CD8+ cells) Divide between Peak 0 and Peak 1 (to identify proliferating and non-proliferating cells) 1 0 Intracellular Cytokine Negative for cytokine production Determine % positive for intracellular cytokine production for CD4+ and CD8+ proliferating and non-proliferating cells (based on biological negative control, greyed population) 1 0 98 Appendix B: P-values from statistical tests for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers less than 100 IU/l and greater or equal to 100 IU/l at the Y1 time point. Parameter P-value Percentage of starting CD4 T cells that proliferated 0.2804 Percentage of starting CD8 T cells that proliferated 0.2751 Percentage of starting CD4 T cells that proliferated (anti-CD3 stimulated control) 0.7542 Percentage of starting CD8 T cells that proliferated (anti-CD3 stimulated control) 0.8362 IFN-γ secretion (pg/ml) 0.1323 IL-10 secretion (pg/ml) 0.0249 IL-13 secretion (pg/ml) 0.2212 TNF-α secretion (pg/ml) 0.1029 Percentage of IFN-γ+ proliferating CD4 T cells 0.1628 Percentage of IL-13+ proliferating CD4 T cells 0.6226 Percentage of IL-21+ proliferating CD4 T cells 0.815 Percentage of IFN-γ+ proliferating CD8 T cells 0.7408 Percentage of IL-13+ proliferating CD8 T cells 0.7653 Percentage of TNF-α+ proliferating CD8 T cells 0.0255 Unless otherwise indicated, parameters described are in response to HBsAg stimulation. 99 Appendix C: P-values from statistical tests for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers in the top and bottom quartile at the Y1 time point. Parameter P-value Percentage of starting CD4 T cells that proliferated 0.3812 Percentage of starting CD8 T cells that proliferated 0.2314 Percentage of starting CD4 T cells that proliferated (anti-CD3 stimulated control) 0.4655 Percentage of starting CD8 T cells that proliferated (anti-CD3 stimulated control) 0.5019 IFN-γ secretion (pg/ml) 0.0121 IL-10 secretion (pg/ml) 0.0131 IL-13 secretion (pg/ml) 0.2801 TNF-α secretion (pg/ml) 0.5019 Percentage of IFN-γ+ proliferating CD4 T cells 0.2136 Percentage of IL-13+ proliferating CD4 T cells 0.8103 Percentage of IL-21+ proliferating CD4 T cells 0.8103 Percentage of IFN-γ+ proliferating CD8 T cells 0.7767 Percentage of IL-13+ proliferating CD8 T cells 0.7107 Percentage of TNF-α+ proliferating CD8 T cells 0.0056 Unless otherwise indicated, parameters described are in response to HBsAg stimulation. 100 Appendix D: Correlation coefficients (r) and P-values from statistical tests for all correlation analyses between anti-HBs titers at Y1 time point and T cell HBsAg- specific responses. Parameter r P-value Percentage of starting CD4 T cells that proliferated 0.1175 0.3121 Percentage of starting CD8 T cells that proliferated 0.189 0.102 Percentage of starting CD4 T cells that proliferated (anti-CD3 stimulated control) 0.02845 0.806 Percentage of starting CD8 T cells that proliferated (anti-CD3 stimulated control) -0.02999 0.7998 IFN-γ secretion (pg/ml) 0.2578 0.0246 IL-10 secretion (pg/ml) 0.299 0.0087 IL-13 secretion (pg/ml) 0.1145 0.3249 TNF-α secretion (pg/ml) 0.09363 0.4211 Percentage of IFN-γ+ proliferating CD4 T cells 0.1681 0.1878 Percentage of IL-13+ proliferating CD4 T cells 0.09745 0.4474 Percentage of IL-21+ proliferating CD4 T cells 0.06159 0.6316 Percentage of IFN-γ+ proliferating CD8 T cells 0.01181 0.9268 Percentage of IL-13+ proliferating CD8 T cells 0.1531 0.231 Percentage of TNF-α+ proliferating CD8 T cells -0.2665 0.0348 Unless otherwise indicated, parameters described are in response to HBsAg stimulation. 101 Appendix E: P-values from statistical tests for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers less than 10 IU/l and greater or equal to 10 IU/l at the Y2 time point. Parameter P-value Percentage of starting CD4 T cells that proliferated 0.0297 Percentage of starting CD8 T cells that proliferated 0.1349 Percentage of starting CD4 T cells that proliferated (anti-CD3 stimulated control) 0.649 Percentage of starting CD8 T cells that proliferated (anti-CD3 stimulated control) 0.7781 IFN-γ secretion (pg/ml) 0.6289 IL-10 secretion (pg/ml) 0.0748 IL-13 secretion (pg/ml) 0.1514 TNF-α secretion (pg/ml) 0.0415 Percentage of IFN-γ+ proliferating CD4 T cells 0.0307 Percentage of IL-13+ proliferating CD4 T cells 0.0139 Percentage of IL-21+ proliferating CD4 T cells 0.0315 Percentage of IFN-γ+ proliferating CD8 T cells 0.1947 Percentage of IL-13+ proliferating CD8 T cells 0.0242 Percentage of TNF-α+ proliferating CD8 T cells 0.5575 Unless otherwise indicated, parameters described are in response to HBsAg stimulation. 102 Appendix F: P-values from statistical tests for all comparison of HBsAg-specific CMI responses between those with anti-HBs titers in the top and bottom quartile at the Y2 time point. Parameter P-value Percentage of starting CD4 T cells that proliferated 0.478 Percentage of starting CD8 T cells that proliferated 0.1249 Percentage of starting CD4 T cells that proliferated (anti-CD3 stimulated control) 0.8301 Percentage of starting CD8 T cells that proliferated (anti-CD3 stimulated control) 0.8819 IFN-γ secretion (pg/ml) 0.117 IL-10 secretion (pg/ml) 0.7791 IL-13 secretion (pg/ml) 0.3303 TNF-α secretion (pg/ml) 0.2549 Percentage of IFN-γ+ proliferating CD4 T cells 0.034 Percentage of IL-13+ proliferating CD4 T cells 0.6929 Percentage of IL-21+ proliferating CD4 T cells 0.2362 Percentage of IFN-γ+ proliferating CD8 T cells 0.253 Percentage of IL-13+ proliferating CD8 T cells 1.00 Percentage of TNF-α+ proliferating CD8 T cells 0.1767 Unless otherwise indicated, parameters described are in response to HBsAg stimulation. 103 Appendix G: Correlation coefficients (r) and P-values from statistical tests for all correlation analyses between anti-HBs titers at Y2 time point and T cell HBsAg- specific responses. Parameter r P-value Percentage of starting CD4 T cells that proliferated 0.0638 0.5998 Percentage of starting CD8 T cells that proliferated 0.2122 0.0778 Percentage of starting CD4 T cells that proliferated (anti-CD3 stimulated control) -0.06216 0.6066 Percentage of starting CD8 T cells that proliferated (anti-CD3 stimulated control) -0.05845 0.6283 IFN-γ secretion (pg/ml) 0.2984 0.0121 IL-10 secretion (pg/ml) 0.05871 0.6292 IL-13 secretion (pg/ml) -0.04945 0.6844 TNF-α secretion (pg/ml) 0.185 0.3712 Percentage of IFN-γ+ proliferating CD4 T cells 0.3658 0.0048 Percentage of IL-13+ proliferating CD4 T cells 0.1178 0.3784 Percentage of IL-21+ proliferating CD4 T cells 0.1937 0.1452 Percentage of IFN-γ+ proliferating CD8 T cells 0.119 0.3735 Percentage of IL-13+ proliferating CD8 T cells 0.01232 0.9269 Percentage of TNF-α+ proliferating CD8 T cells -0.2441 0.0649 Unless otherwise indicated, parameters described are in response to HBsAg stimulation. 104 Appendix H: P-values from statistical tests for all comparison of HBsAg-specific CMI responses and anti-HBs titers between males and females. Parameter P-value Percentage of starting CD4 T cells that proliferated 0.9521 Percentage of starting CD8 T cells that proliferated 0.1604 Percentage of starting CD4 T cells that proliferated (anti-CD3 stimulated control) 0.4409 Percentage of starting CD8 T cells that proliferated (anti-CD3 stimulated control) 0.9146 IFN-γ secretion (pg/ml) 0.3398 IL-10 secretion (pg/ml) 0.5504 IL-13 secretion (pg/ml) 0.345 TNF-α secretion (pg/ml) 0.3883 Percentage of IFN-γ+ proliferating CD4 T cells 0.804 Percentage of IL-13+ proliferating CD4 T cells 0.4862 Percentage of IL-21+ proliferating CD4 T cells 0.2852 Percentage of IFN-γ+ proliferating CD8 T cells 0.3276 Percentage of IL-13+ proliferating CD8 T cells 0.1294 Percentage of TNF-α+ proliferating CD8 T cells 0.5955 anti-HBs titers at Y1 time point 0.4616 anti-HBs titers at Y2 time point 0.8496 Unless otherwise indicated, parameters described are in response to HBsAg stimulation. 105 Appendix I: P-values from statistical tests for all comparison of HBsAg-specific CMI responses and anti-HBs titers between major racial groups in the cohort (Caucasian, Asian, South Asian, Asian-Caucasian). Parameter P-value Percentage of starting CD4 T cells that proliferated 0.1014 Percentage of starting CD8 T cells that proliferated 0.1604 Percentage of starting CD4 T cells that proliferated (anti-CD3 stimulated control) 0.6198 Percentage of starting CD8 T cells that proliferated (anti-CD3 stimulated control) 0.9146 IFN-γ secretion (pg/ml) 0.7855 IL-10 secretion (pg/ml) 0.5293 IL-13 secretion (pg/ml) 0.4856 TNF-α secretion (pg/ml) 0.603 Percentage of IFN-γ+ proliferating CD4 T cells 0.804 Percentage of IL-13+ proliferating CD4 T cells 0.4862 Percentage of IL-21+ proliferating CD4 T cells 0.2852 Percentage of IFN-γ+ proliferating CD8 T cells 0.3276 Percentage of IL-13+ proliferating CD8 T cells 0.1294 Percentage of TNF-α+ proliferating CD8 T cells 0.5955 anti-HBs titers at Y1 time point 0.805 anti-HBs titers at Y2 time point 0.4215 Unless otherwise indicated, parameters described are in response to HBsAg stimulation.
Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivs 3.0 Unported
Graduate
The relationship between T and B cell hepatitis B virus vaccine-specific responses in a cohort of healthy infants
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