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Neonatal immunization with Listeria monocytogenes induces T cells with an adult-like avidity, sensitivity,… Smolen, Kinga Krystyna 2009

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Neonatal immunization with Listeria monocytogenes induces T cells with an adult-like avidity, sensitivity, and TCR-Vβ repertoire, and does not adversely impact the response to boosting by Kinga Krystyna Smolen B.Sc., University of Waterloo, 2006  A THESIS SUBMITTED IN PARTIAL FUFUILLMENT OF THE REQUIERMENTS FOR THE DREGEE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February 2010  © Kinga Krystyna Smolen, 2009  Abstract Neonates are highly susceptible to infection and respond suboptimally to most vaccines. However, our previous studies have shown that a protective Th1-type immune response to neonatal Listeria monocytogenes (Lm) immunization can be induced. In this study, we investigated the impact of neonatal Lm immunization and boosting on the key parameters of the T cell response. We found that immunized murine neonates reached maximum antigen-specific CD8+ T cell expansion after only a single immunization, while adults required a booster dose to reach their maximal response. Antigen-specific CD4+ expansion in both age groups however required a booster dose to reach its peak. Neither functional avidity nor sensitivity of antigenspecific CD8+ and CD4+ T cells differed between mice immunized as neonates or adults. The range of different TCR Vβ chains employed in the response was also similar in both age groups. Lastly, neonatal immunization and subsequent boosting did not decrease protection, as both immunized neonates and adults remained well protected after one or after multiple doses. Overall, our data provide further evidence in support of immunization at birth as a feasible public health strategy to combat early life infections, and that Lm in particular represents an attractive vehicle to do so.  ii  Table of Contents ABSTRACT ................................................................................................................................................. ii TABLE OF CONTENTS........................................................................................................................... iii LIST OF TABLES ..................................................................................................................................... v LIST OF FIGURES .................................................................................................................................... ii ABBREVIATIONS ................................................................................................................................... vii ACKNOWLEDGMENTS ......................................................................................................................... ix DEDICATION............................................................................................................................................. x CO-AUTHORSHIP STATEMENT ......................................................................................................... xi CHAPTER I................................................................................................................................................. 1 1  INTRODUCTION ................................................................................................................................ 1 1.1 VACCINES ........................................................................................................................................ 1 1.1.1 Vaccination .............................................................................................................................. 1 1.1.2 Current pediatric vaccines....................................................................................................... 2 1.1.3 Neonatal vaccines .................................................................................................................... 3 1.1.4 Concerns with neonatal vaccines............................................................................................. 3 1.2 AN OVERVIEW OF THE T CELL IMMUNE SYSTEM ........................................................................... 4 1.2.1 T cell response ......................................................................................................................... 4 1.2.2 Effector functions of T cells ..................................................................................................... 5 1.2.3 Memory T cell response ........................................................................................................... 6 1.3 COMPARING THE NEONATAL TO THE ADULT T CELL IMMUNE RESPONSES ................................... 7 1.3.1 Environment ............................................................................................................................. 7 1.3.2 Neonatal T cell responses ........................................................................................................ 8 1.4 MODIFYING THE NEONATAL IMMUNE RESPONSE ......................................................................... 10 1.4.1 Dose ....................................................................................................................................... 10 1.4.2 Adjuvant ................................................................................................................................. 10 1.4.3 Prime-boost response............................................................................................................. 12 1.5 LISTERIA MONOCYTOGENES AS VACCINE MODEL .......................................................................... 12 1.5.1 Listeria monocytogenesis....................................................................................................... 12 1.5.2 Lm as a vaccine model ........................................................................................................... 14 1.5.3 Benefits of the Lm model........................................................................................................ 14 1.6 HYPOTHESIS AND OBJECTIVE ....................................................................................................... 15 1.7 REFERENCES .................................................................................................................................. 16  CHAPTER II ............................................................................................................................................. 21 2 MANUSCRIPT 1 NEONATAL IMMUNIZATION WITH LISTERIA MONOCYTOGENES INDUCES T CELLS WITH AN ADULT-LIKE AVIDITY, SENSITIVITY, AND TCR-VΒ REPERTOIRE .......................................................................................................................................... 21 2.1 INTRODUCTION .............................................................................................................................. 21 2.2 MATERIALS AND METHODS .......................................................................................................... 22 2.2.1 Animals................................................................................................................................... 22 2.2.2 Bacterial strains..................................................................................................................... 23 2.2.3 Immunization schedule........................................................................................................... 23  iii  2.2.4 Intracellular cytokine flow cytometry .................................................................................... 24 2.2.5 Surface tetramer staining....................................................................................................... 25 2.2.6 TCR Vβ chain usage analysis................................................................................................. 25 2.2.7 Protection............................................................................................................................... 26 2.2.8 Statistics ................................................................................................................................. 26 2.3 RESULTS ........................................................................................................................................ 27 2.3.1 Contrary to adults, boosting of mice immunized as neonates did not increase the ability of Ag-specific CD8+ T cells to expand further; however, boosting did increase the CD4+ T cell ability to expand. ........................................................................................................................................... 27 2.3.2 CD8+ and CD4+ T cell sensitivity and functional avidity did not differ between mice immunized as neonates or as adults.. ................................................................................................. 32 2.3.3 The diversity of the CD8+ TCR Vβ response of mice immunized as neonates or as adults did not differ, nor change in response to boosting................................................................................... 34 2.3.4 Boosting neonatally immunized mice did not adversely affect their protection against wildtype challenge..................................................................................................................................... 37 2.4 DISCUSSION ................................................................................................................................... 39 2.5 REFERENCES .................................................................................................................................. 45 CHAPTER III ........................................................................................................................................... 48 3  DISCUSSION...................................................................................................................................... 48 3.1 FUTURE DIRECTION ....................................................................................................................... 51 3.1.1 Immediate............................................................................................................................... 51 3.1.2 Long-term............................................................................................................................... 54 3.2 IMPACT OF DESCRIBED WORK ...................................................................................................... 56 3.3 REFERENCES .................................................................................................................................. 58  APPENDICES ........................................................................................................................................... 59 APPENDIX A ............................................................................................................................................ 59 APPENDIX B MANUSCRIPT 2: FINE-TUNING SAFETY AND IMMUNOGENICITY OF LISTERIA MONOCYTOGENES-BASED NEONATAL VACCINE PLATFORMS ................................................................. 60 Introduction ........................................................................................................................................ 60 Materials and methods ....................................................................................................................... 63 Results ................................................................................................................................................ 68 Discussion .......................................................................................................................................... 79 References .......................................................................................................................................... 85 APPENDIX C ............................................................................................................................................ 88 APPENDIX D ............................................................................................................................................ 88  iv  List of Tables Table 1. Mice immunized as neonates or as adults maintained a highly diverse CD8+ TCR Vβ repertoire directed against OVA257-264 after the primary, secondary, and tertiary response to ΔactA-OVA. ......................................................................................................................... 35 Table 2. Routine immunization schedual for infants and children up to the age of two. Suggestion made by the Public Health Agency of Canada................................................... 59  v  List of Figures Figure 1. Neonates generated a higher primary CD8+ T cell response but similar CD8+ and CD4+ secondary and tertiary responses as compared to adults....................................................... 29 Figure 2. The OVA-Tetramer+ CD8+ T cells response is similar in mice immunized as neonates or as adults ............................................................................................................................ 30 Figure 3. The amount of IFN-γ produced per cell does not differ between mice first immunized as neonates or as adults ........................................................................................................ 31 Figure 4. Functional avidity of Ag-specific T cells did not change in response to booster immunization in mice immunized as neonates or as adults.................................................. 33 Figure 5. Both, mice immunized as neonates or as adults maintain a highly diverse CD8 TCR Vβ repertoire directed against OVA257-264 after the primary, secondary, and tertiary immunizations with Lm ΔactA-OVA .................................................................................. 36 Figure 6. Neonatally immunized mice displayed the same protection pattern as mice immunized as adults, and did not lack a response to boosting. ............................................................... 38  vi  Abbreviations Ag  Antigen  APC  Antigen presenting cell  APC  Allophycocyanin  BCG  Bacillus Calmette-Guerin  BCR  B cell receptor  CFU  Colony forming units  CMV  Cytomegalovirus  CTL  Cytotoxic T lymphocytes  DC  Dendritic cells  DNA  Deoxyribonucleic acid  FACS  Fluorescence activated cell sorting  FBS  Fetal bovine serum  FITC  Fluorescein isothiocyanate  FDA  Food and Drug Adminstration  HepB  Hepatitis B  Hib  Haemophilus influenzae  IFN-γ  Interferon gamma  IgG  Immunoglobulin G  IL-2  Interleukin 2  IL-4  Interleukin 4  IL-5  Interleukin 5  IL-10  Interleukin 10  vii  IL-12  Interleukin 12  IL-13  Interleukin 13  IL-17  Interleukin 17  i.p.  Intraperitoneal  i.v.  Intravenous  Lm  Listeria monocytogenes  MHC  Major histocompatibility complex  OPV  Oral Polio Vaccine  OVA  Ovalbumin  PBS  Phosphate buffer solution  PE  Phycoerythrin  PerCP  Peridinin chlorophyll protein  TCR  T cell receptor  Th1  T helper cell 1  Th2  T helper cell 2  TNF-α  Tumor necrosis factor alpha  Tregs  Regulatory T cells  WT  Wild-type  viii  Acknowledgments First and foremost I would like to acknowledge the support and kindness of my supervisor Dr. Tobias Kollmann, and the members of my committee, Dr. Jan Dutz and Dr. Stuart Turvey. This would not have been possible without you. I am indebted to Dr. Tobias Kollmann for taking me into his lab and always providing insightful advice, expert guidance, encouragement to explore new directions, and most importantly inspiring my passion for research. I would like to thank all of the members of the Kollmann lab, and my CFRI and UBC colleagues for their encouragement and input throughout the duration of my degree. I would especially like to thank Dr. Daniela Loeffler and all the past and present members of the ‘mouse group’. Special thanks go to Mr. Nicholas Himmelman who provided encouragement and support throughout. I would also like to thank my family who has supported me throughout my academic endeavors; my gratitude to them extends beyond words.  ix  Dedication  To my parents  x  Co-Authorship Statement The research program from which these results were generated was collaboratively designed by Dr. T. R. Kollmann, D.I.M. Loeffle, and K.K. Smolen. The experiments were designed, preformed, and analyzed by K.K. Smolen. The manuscript was written by K.K. Smolen and Dr. T.R. Kollmann. A version of Chapter II has been accepted for publication. Smolen, KK., Loeffler, DIM., Reikie, BA, Aplin, L., Cai, B., Fortuno III, ES., and Kollmann, TR. Neonatal immunization with Listeria monocytogenes induces T cells with an adult-like avidity, sensitivity, and TCR-Vβ repertoire. Vaccine (Ms. Ref. No.: JVAC-D-09-00645R1). A version of Appendix B has been published. Loeffler, DIM. *, Smolen, K. *, Aplin, L., Cai, B., Kollmann, TR. 2009 Fine-tuning the safety and immunogenicity of Listeria monocytogenesbased neonatal vaccine platform. Vaccine. 178(6): 3695-701 * Equal contributing co-authors  xi  CHAPTER I 1 INTRODUCTION 1.1 Vaccines  Although the practice of vaccination originated in the 10th and 11th century in Central Asia, it was not until the 1980’s with the global eradication of smallpox (using vaccinia virus, hence the term ‘vaccination’) that the full potential of vaccination was recognized [1]. Vaccines have since become one of the most effective medical interventions, leading to a significant reduction in morbidity and mortality for many infectious diseases [2]. It is estimated that in 2002 alone, approximately 2 million childhood deaths were averted through vaccinations [2].  1.1.1  Vaccination  The immune system is a phenomenally efficient defense system, which functions to protect the host against invading disease-causing agents. Vaccines are meant to simulate natural infection, without the associated disease. In most cases, vaccines consist of at least two major components: 1) the immunogen - an antigen (Ag), the target for the immune system, and 2) the adjuvant substance used in combination with an immunogen to produce a stronger or longer lasting immune response [3]. Vaccinations involve the artificial induction of a host immune response against the immunogen, to induce host immunity, i.e. protection. The immune response following infection or immunization leads to activation of a cellular and a humoral arm of the immune system. The cellular response (or cell mediated immunity; CMI) is largely provided by T cells, which can be divided into at least two subsets, CD4+ and CD8+ T cells. The humoral response consists of antibody production by B cells. Both T and B cells contain unique receptors: the T cell receptor (TCR) and the B cell receptor (BCR). The TCR and BCR are able to 1  recognize a large array of Ags (up to 1015 different Ags). This antigenic stimulation is required to activate and expand the Ag-specific cell population. The ensuing clonal expansion of Ag or pathogen specific T and/or B cells leads to the eradication of pathogens. Some of these expanded cells mature into immune memory cells, protecting against re-infection with the same pathogen. Ag-specific memory cells differ from naïve cells by increased speed of expansion and more efficient effector function, such as killing of infected target cells [4]. The protective efficacy of vaccines depends on their success at inducing cellular and/or a humoral immune memory responses, that protect against subsequent infections with the same pathogen [3]. This ability to ‘learn’ from past exposure classifies the T and B cell immune response as the ‘adaptive arm’ of the immune system.  1.1.2  Current pediatric vaccines  Currently, there are 11 routine vaccinations that are given to infants in Canada, resulting in approximately 20 immunizations by the second year of life [5] (Appendix A). These vaccines include: 1) polio vaccine which uses killed intact virus; 2) mumps, measles, rubella, and chickenpox, all live attenuated virus; 3) hepatitis B (Hep B) which uses a recombinant protein produced in yeast; 4) influenza an inactivated virus; 5) diphtheria and tetanus are inactivated toxins (i.e. toxoids); and 6) Haemophilus influenza type b, pneumococcal, and meningococcal vaccines all consist of purified polysaccharide as their Ag, that is coupled to a protein carrier [2]. While all of these vaccines have proven to be safe and effective, and are administered routinely, the precise immune mediated mechanisms of protection are largely unknown. However, this mechanistic knowledge urgently needs to be generated in order to optimize existing and develop new vaccines [6].  2  1.1.3  Neonatal vaccines  Neonatal vaccination offers many advantages over infant or childhood vaccination, for instance, it provides earlier protection and ensures greater coverage, given that birth is the most reliable point of contact with the medical care world wide [2]. Unfortunately most of the above mentioned vaccines are not efficient in the most infection prone population, the neonate. But there are exceptions to this rule. The Bacillus Calmette-Guerin (BCG), oral poliovirus (OPV), and HepB vaccines have been shown to induce long-lasting protection when administered at birth [7-13], proving that neonatal vaccination is possible [2].  1.1.4  Concerns with neonatal vaccines  Although the neonate is able to mount a strong immune response within hours of birth [8], many potential concerns have been associated with neonatal immunization. For example, immunization of newborns has been shown to lead to inadequate immune responses [14, 15], weak responses to boosting [16, 17], impaired long-term immune memory [18-20], or dampening of responses to other co-administered vaccines [21]. The two best studied examples are OPV and HepB: Neonates immunized with OPV responded with high titers of neutralizing antibodies, but reduced proliferation and interferon (IFN)-γ responses upon re-stimulation when compared to subjects immunized later in life [22]. While neonatally HepB-immunized subjects appear to have a reduced anamnestic response 15 years after vaccination, compared to those immunized as children or adolescents [18, 20]. Together these data highlight potential pitfalls associated with neonatal vaccine strategies. We therefore set out to investigate the impact of neonatal immunization with Lm on several key parameters of the T cell response such as TCR repertoire  3  selection, functional avidity, and T cell sensitivity to recall stimulation, as well as the impact of boosting on these parameters.  1.2 An Overview of the T cell Immune System While I recognize the importance of B cells and antibodies in the protective immune response to many vaccines, my thesis focuses on CD4+ and CD8+ T cells, as little is known about CMI responses in early life, and some evidence points to a reduced CMI around birth. I will therefore restrict my background discussion to the T cell response to vaccination.  1.2.1  T cell response  The major subsets of T cells are defined by the presence of CD4+ or CD8+ co-receptors on their surface that are expressed in conjunction with the unique TCR, which consists of α- and βchains. A primary immune response of these T cells is initiated by the presentation of an Ag to naïve T cells. T cells are activated by an antigenic peptide present on the major histocompatibility complex (MHC) molecule on the surface of Ag presenting cells (APC). CD4+ T cells recognize antigenic peptides bound to a MHC class II molecule, while the CD8+ T cells recognize peptides bound to a MHC class I molecule.  MHC molecules are expressed on all nucleated cells, and are key to the function of APC such as dendritic cells (DC), macrophages, and B cells. During Ag presentation, Ags are taken up by APC and digested into peptides. Typically, MHC class I molecules present intracellular Ags while the MHC class II molecules present extracellular Ags. This is not always the case, as exogenous Ag can also be ‘cross-presented’ to CD8+ T cells via the MHC class I molecule [23]. TCR interactions with potentially small numbers of MHC-peptide complexes on infected cells  4  transmit signals that result in T cell expansion and activation of effector functions [24]. The synergistic strength of the total binding interaction, known as avidity, is defined as the total number of individual binding interactions (e.g. affinities) between the TCR and MHC-peptide complexes. If the TCR binds the peptide-MHC complex, the first (Signal 1) of three signals required to stimulate T cells is provided. This first signal initiates an activation program. The other two signals are provided by a) co-stimulatory molecules (Signal 2), such as CD80 and CD86 on APC binding molecules such as CD28 on the T cell, which lower the activation threshold of T cells by the MHC-TCR interaction (increase the quantity of the ensuing response), and b) production of cytokines (Signal 3), such as interleukin (IL)-6, IL-12, and type-IFN, which are produced by APCs, and direct the quality of the ensuing T cell response. Successful delivery of these signals also triggers the T cell to produce IL-2 and to express IL-2 receptors, providing an autocrine survival feedback loop, driving activated cells to proliferation. As mentioned above, some of the activated cells differentiate into effector T cells, while others differentiate into cells that will persist as memory T cells. The exact lineage relationship between effector and memory T cells however has not been adequately delineated.  1.2.2  Effector functions of T cells  CD4+ effector T cells are able to activate innate cells such as macrophages to kill (e.g. bacteria), enhance qualitative and quantitative aspects of B cell antibody production, and sustain or enhance CD8+ T cell function. CD4+ T cells can differentiate into various types of effector (and memory) cells depending on the type of Signal 3 [25], and can be divided into at least three main effector subtypes [26]; Type 1 helper T cells (Th1), Type 2 helper T cells (Th2), T-helper 17 cells (Th17). Th1 produce IL-2 and IFN-γ, and mainly protect against intracellular pathogens.  5  Th2 helper cells secrete IL-4, IL-5, and IL-13 to mediate a response against multicellular parasites and extracellular bacterial pathogens, with the help of Th17 cells which produce IL-17, leading to activation of neutrophils [27]. A further CD4+ T cell subset is the T regulatory cell (Tregs), which appears to protect the host from autoimmune disease by maintaining homeostasis and possibly self-tolerance [16].  CD8+ effector T cells eliminate intracellular pathogens. These cells kill directly (i.e. are cytotoxic) by releasing granule contents such as perforin (a complement-like protein which creates pores in the lipid membrane of infected target cells), and granzymes (proteases that induce apoptosis in target cells). CD8+ T cells may also use the FAS-ligand mediated apoptotic cascade to eliminate infected cells [28]. In addition to direct killing, with the help of Th1, CD8+ T cells make cytokines such as IFN-γ and Tumor necrosis factor (TNF)-α that reinforce immune defenses by rendering adjacent cells resistant to infection, as well as augmenting innate immune defenses [29].  1.2.3  Memory T cell response  Successfully activated Ag-specific T cells are believed to proceed through three phases in vivo: expansion, contraction, and memory. Even if initial Ag exposure is very short (less then 8 hours), once activated, the CD8+ T cells undergo a series of pre-programmed steps leading to multiple rounds of cell division (expansion) and activation induced cell death (contraction) to leave less than 5% of the total number of activated cells as long-lived memory cells [36-38]. During the expansion phase, a rapid increase in Ag-specific T cells can be measured, which is first detectable around day 5-7 during a primary response, and 2-3 days after secondary memory  6  responses [30, 35]. During the contraction phase, a process automatically initiated during priming leads to the disappearance of 95% of the initially expanded effector T cells. During the ensuing memory phase a small number of specialized T cells are maintained for a long period of time. Effector and memory T cells can be differentiated through either phenotypic marker expression or functional responses, ideally both [31].  1.3 Comparing the Neonatal to the Adult T cell Immune Responses Mortality due to intracellular viral, bacterial, fungal, or parasitic infections is highest in early life. This clinical observation clearly supports the notion that T cell-mediated immune responses are different in infants compared to adults [8]. Although it has long been believed that immune responses are inadequate in early life [16], in certain circumstances, neonates can overcome their limitations and mount adult-like T cell responses [32].  1.3.1  Environment  Neonates emerge from an immunosuppressive environment, otherwise, both mother and fetus would attack each other as they would a half-foreign (semi-allogeneic) transplanted organ [33]. For instance, T cell cytokines are known to be extremely ‘toxic’ to the placenta [34], thus cytokine release at the maternofetal interface is tightly controlled. This control allows the maternal immune system to successfully eradicate fetal cells from the peripheral circulation while remaining functionally tolerant of the fetus [35, 36]. The Th1 and Th2 cytokine balance is an important mechanism determining the survival of the fetus in the womb [37], as Th2 cytokines have been shown to favour the maintenance of pregnancy while Th1 cytokines are detrimental to the fetus and lead to rejection, pre-eclampsia [37], or miscarriage [38]. The immunosuppressive action of trophoblast cells [39] appears to inhibit T cell activation and  7  proliferation, largely through inducing or producing IL-10 [35, 40]. T cells that evaded the local suppression appear to be destined to die through maternal T cells using Fas Ligand [40, 41]. CD4+CD25+ Tregs are other important regulators maintaining an intact maternofetal interface: Tregs are up-regulated in the circulation during normal pregnancy but diminished in mice undergoing spontaneous abortion [42, 43]. Together these factors appear to maintain the pregnancy but in doing so strongly suppress the fetal immune system. As a result these factors could also be involved in dampening responses to vaccines given during the early neonatal period.  1.3.2  Neonatal T cell responses  Neonates have been described as being ‘immuno-deficient’ due to their functionally dampened immune responses, as measured by the heightened susceptibility to infections and their suboptimal response to existing vaccines. T cells of neonates exhibit a variety of qualitative and quantitative functional differences when compared to T cells of adults. For example, while the majority of circulating T cells in the adult have been previously exposed to their specific Ag, circulating neonatal T cells are mainly naïve [55, 56]. The activation of these naïve cells is dependent on the presentation of antigenic peptides by specialized APC, namely the DC [44, 45]. However, neonatal DC are present in lower numbers in the neonate, and also display different functional characteristics compared to adult DC [10]. Furthermore, the chemokine receptor expression profiles differs between adult and neonatal T cells, reflecting the neonate’s attenuated capacity to migrate in response to inflammatory signals [35] and expansion, with adult Agspecific T cells shown to expand to threefold higher levels as compared to neonates [46]. However, homeostatic T cell proliferation is higher at birth than at later stages in life [47, 48], but this is not sustained as activation of these T cells increases their susceptibility to apoptosis. 8  Despite all these inherent and environmental limitations, neonatal T lymphocytes can be induced to proliferate like adult T cells if proper in vitro conditions are provided [49, 50]. But even if successfully activated around birth, neonatal CD4+ T cells appear to lean toward a Th2 cell memory response when re-exposed to the Ag as adults, as early immunization is associated with lower IFN-γ and IL-2 cytokine production and high IL-5, IL-4, and IL-13 responses to most vaccines [8, 51-55]. Furthermore, several reports suggest that this Th2 bias may also contribute to the phenomenon of neonatal tolerance [56, 57].  The response of mature cytolytic CD8+ T memory cells can be detected in the cord blood after congenital infection [11, 12]. In human newborns, several studies have shown functional cytotoxic T lymphocytes (CTL) after congenital Cytomegalovirus (CMV) and Trypanosoma cruzi (T. cruzi) infection [11, 12]. CTLs have also been recovered from infants following an influenza infection but not after influenza immunization, indicating that the infection was able to overcome the neonatal Th2 bias, but the existing vaccine regimen was not [58].  The induction of adult like Th1 and CTL responses in the neonate has only recently been shown to reflect the relative capacity of vaccines to activate neonatal APC. If a sufficient threshold of APC activation is reached, activation of neonatal T cells may occur, suggesting that a higher level of activation in the neonate compared to the adult is required to stimulate the desired response. Importantly, under appropriate conditions, neonates have been shown to develop strong immune responses to vaccines [16, 49]. Based on these considerations we believe that effective neonatal vaccines can be developed. But to design successful neonatal vaccines, it is  9  important to dissect and understand the specific requirements that are involved, as well as the possible impact on the ensuing neonatal immune response.  1.4 Modifying the Neonatal Immune Response 1.4.1  Dose  As outlined above, neonatal T cells exhibit a Th2 type immune bias [35], but Th1 response may be elicited under the appropriate conditions [11]. Dose of either infectious agent or vaccine has been implicated in leading to the neonatal Th2 bias [59]. In certain cases a reduction of the dose appears to restore the induction of Th1 type neonatal responses, suggesting that neonatal ‘immune overload’ could be responsible for the development of preferential Th2 responses [8].  1.4.2  Adjuvant  An ideal adjuvant would provide the appropriate stimulus to obtain the desired immune response. Currently the FDA and Health Canada have approved only a limited number of adjuvants for human use [3, 60]. Furthermore, although of great interest, it is still unclear why some agents enhance the early life responses to vaccine Ags, whereas others fail to do so despite efficacy in adults. Forsthuber et al. described that a selective generation of neonatal Th1 vs. Th2 response can be obtained using different adjuvants [61]. For instance, incomplete Freund’s adjuvant elicited a Th2 response while complete Freund’s adjuvant initiated a Th1 response [62]. Other stimulators such as CpG ODN [63] or intracellular bacteria [64, 65] have also successfully induced strong early life Th1 responses.  10  In addition to conventional adjuvants, exogenously administered cytokines have been used experimentally as adjuvants when endogenous production in the neonatal host is presumed to be deficient. The co-administration of IL-12 and influenza subunit vaccine at birth elevates neonatal splenic expression of IFN-γ, IL-10, and IL-15 mRNA and the protective efficacy of the vaccination [66]. As neonatal T cells are hyper-responsive to IL-4 and hypo-responsive to IL-12, a complementary treatment with cytokines has also been shown to result in the desired Thresponse [35].  DNA vaccines have also been shown to enhance the neonatal murine immune response [8]. They have also been tested in humans [67]. In the mouse, single neonatal DNA vaccination is known to induce cellular and humoral immune responses, which are maintained for a significant part of the animal's life [68]. DNA vaccination at birth also has been shown to result in the rapid induction of Ag-specific CD8+ T cells [69] and may even be effective if given prior to birth [70]. Although DNA immunizations show promise, they are associated with more variable and often Th2 biased responses when given in the first 7 days of life compared to adult recipients [71]. Furthermore, mice immunized as neonates with DNA as an adjuvant mount a primary Th1 response equivalent to an adult response but upon boosting later in life appear to revert to a Th2 biased [71]. A variation of this approach involves the vaccine Ag expressed directly from a DNA inserted into bacterial or viral vectors. In this scenario the bacteria express the vaccine protein as the Ag while simultaneously serving as an adjuvant. Published bacterial carriers for protein vaccines include Salmonella enterica, Escherichia coli, Salmonella typhi, and Listeria monocytogenes (Lm). Such bacterial vectors appear to function as effective delivery vehicles even in neonates [72] and appear to elicit strong immediate and anamnestic immune responses in  11  neonatally immunized mice, as seen with Salmonella typhi expressing Yersinia pestis F1 Ag to elicit robust and long-lived mucosal and system immunity [72].  1.4.3  Prime-boost response  Follow-up immunizations (booster(s)), might be necessary to mount or to maintain an optimal immune response. For almost all existing vaccines, boosters are necessary to ensure optimal protection. In prime-boost regimens the quality of the initial priming event appears to imprint itself on the immune system. By boosting, the T cells can selectively be expanded, increasing their number over the threshold required to most effectively fight specific pathogens [73]. The general avidity of the boosted T cell populations also appear enhanced with boosting, which further increases the efficacy of the available T cells [74].  1.5 Listeria monocytogenes as Vaccine Model Certain attenuated strains of Lm have the capacity to induce a robust and protective immune response when administered to the neonate [64, 65]. Lm, intracellular bacteria, can be used as a strong adjuvant for initiating a Th1 immune response within the neonate, providing a great model to dissect the differences between neonate and adult, as many tools are available in this system [64, 75].  1.5.1  Listeria monocytogenesis  Lm is a gram positive, food-borne pathogen, causing listeriosis with clinical symptoms due to its capacity to cross human barriers, such as the intestinal barrier, the blood brain barrier, and the placental barrier [76]. Lm species are ubiquitous in nature and may be found in a variety of environments such as stream water, sewage, plants, food, soil, and the intestinal tract of humans  12  and animals [77]. Lm poses a threat mainly to individuals who are immunocompromised, the elderly, pregnant, or newly born.  Lm is an intracellular pathogen residing in the host cells, such as macrophages and hepatocytes. It invades the host cells by attaching to the surface-receptors of the cell membrane by a family of bacterial surface proteins, called internalins. Internalins are host and cell-specific. Once bound, the bacteria are phagocytosed into a phagolysosome within the host cell. In the phagolysosome Lm uses a pore-forming endolysin called listeriolysis O (LLO) to lyse the membrane of the phagolysosome and escape in to the host cytoplasm. Once in the cytosol Lm replicates. The bacterial surface protein ΔactA then induces polymerization of host cell actin filament at one pole of the bacteria. These filaments become organized creating a ‘rocket tail’, which provides propulsion for the organism to move through the host cell and into the adjacent cells. In the next host cell the process repeats [78-80].  In an immunocompetent individual, Lm are ingested, degraded, and presented on the cell surface. Lm peptides from the phagosomes are presented on the MHC class II to the CD4+ T lymphocytes [81]. Lm induction of Th1 development in vitro is believed to be mediated by IL-12 produced by macrophages. Cells with Th1 phenotype secrete IL-2 and IFN-γ during primary infection [82, 83]. The majority of the Lm peptides are presented on MHC class I molecule, and recognized by CD8+ T cells. With the Lm Ags and IL-2 present, CD8+ T cells proliferate and a robust response is induced during infection. Importantly, these CD8+ T cells are key in conferring protective immunity [84, 85].  13  1.5.2  Lm as a vaccine model  Lm has been used as a model to dissect various aspects of the adult and neonatal immune system. In our particular study, the Ovalbumin (OVA) protein was used as a vaccine model Ag. The expression of such recombinant protein results in the secretion of both Lm and OVA proteins into the host cells, inducing a strong Th1 biased immune response. Both can be measured simultaneously, providing opportunities to compare between the responses to different Ag.  Several virulence-attenuated strains of Lm have been used as a delivery system for recombinant proteins of interest. Our Lm attenuated mutant strain contains a deletion in the ΔactA gene. The ΔactA deletion results in the inactivation of actin-based mobility preventing the bacterial cell to cell spread. This attenuated Lm strain has proven to be an effective yet safe delivery system even in humans, and tested as a vaccine delivery vehicle for cancer [86] and other diseases [87]. Under normal circumstances Lm causes listeriosis and death in neonates, but this ΔactA deletion attenuated strain is safe and effective in neonatal vaccination models, as we have previously shown [64].  1.5.3  Benefits of the Lm model  Attenuated Lm has been shown to be an effective vaccine model [87]. As described above, Lm is an intracellular bacterium with strong adjuvant activity initiating a Th1 immune response. This allows neonatal T cells to mount an effective adult-like CD8+ and CD4+ T cell response, and mediated protection against Lm infection [64, 75]. An additional aspect we took full advantage of was that Lm allows the dissection of the precise immunologically relevant mechanisms responsible for the variation between neonatal and adult immune responses [88]. Knowledge of  14  these is essential for the rational design of effective neonatal vaccines. As we have published, using a plasmid-based Lm system, different combinations of attenuations can easily be compared and evaluated in order to determine the optimal response [65]. Furthermore, recombinant Lm bacteria carrying vaccine Ag can easily be mass-produced and transported without a cold-chain (lyophilized). Ultimately this model may also provide a needle-free delivery system, as it could be given orally to humans. Together, Lm thus offers to become an effective and affordable worldwide vaccine vehicle.  1.6 Hypothesis and Objective The hypothesis is that the neonatal immune response toward Lm based vaccination and subsequent boosting would not differ from the adult immune response. The underlying objective of this study was to obtain a greater understanding of the neonatal T cell immune response, specifically, using the Lm model to dissect potential differences between neonates and adults at primary, secondary, and tertiary immune responses using a variety of tools. Clearly, my studies only represent the first descriptive part of this investigative path, but they focused the mechanistic questions that now can be asked.  We want to explore the use of Lm vaccine delivery as a means of overcoming Th2 bias and impaired Th1 immune response. Our approach: Using Lm as a strong Th1 adjuvant, I compared the newborn and the adult in prime-boost combination vaccination schemes to clearly delineate difference in key T cell response parameters. 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Kollmann, T.R., et al., Induction of protective immunity to Listeria monocytogenes in neonates. J Immunol, 2007. 178(6): p. 3695-701. Loeffler, D.I., et al., Fine-tuning the safety and immunogenicity of Listeria monocytogenes-based neonatal vaccine platforms. Vaccine, 2009. 27(6): p. 919-27. Arulanandam, B.P., et al., Neonatal administration of IL-12 enhances the protective efficacy of antiviral vaccines. J Immunol, 2000. 164(7): p. 3698-704. Drape, R.J., et al., Epidermal DNA vaccine for influenza is immunogenic in humans. Vaccine, 2006. 24(21): p. 4475-81. Hassett, D.E., et al., Immune responses following neonatal DNA vaccination are longlived, abundant, and qualitatively similar to those induced by conventional immunization. J Virol, 2000. 74(6): p. 2620-7. Zhang, J., et al., Neonates mount robust and protective adult-like CD8(+)-T-cell responses to DNA vaccines. J Virol, 2002. 76(23): p. 11911-9. Gerdts, V., et al., Fetal immunization by a DNA vaccine delivered into the oral cavity. Nat Med, 2000. 6(8): p. 929-32. Bot, A., S. Antohi, and C. Bona, Immune response of neonates elicited by somatic transgene vaccination with naked DNA. Front Biosci, 1997. 2: p. d173-88. Ramirez, K., et al., Mucosally delivered Salmonella typhi expressing the Yersinia pestis F1 antigen elicits mucosal and systemic immunity early in life and primes the neonatal immune system for a vigorous anamnestic response to parenteral F1 boost. J Immunol, 2009. 182(2): p. 1211-22. Seder, R.A. and A.V. Hill, Vaccines against intracellular infections requiring cellular immunity. Nature, 2000. 406(6797): p. 793-8. Estcourt, M.J., et al., Prime-boost immunization generates a high frequency, high-avidity CD8(+) cytotoxic T lymphocyte population. Int Immunol, 2002. 14(1): p. 31-7. Mombaerts, P., et al., Different roles of alpha beta and gamma delta T cells in immunity against an intracellular bacterial pathogen. 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Patel, Enhanced production of murine interferon gamma by T cells generated in response to bacterial infection. J Exp Med, 1982. 156(1): p. 112-27.  19  83. 84. 85.  86. 87. 88.  Hsieh, C.S., et al., Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science, 1993. 260(5107): p. 547-9. Harty, J.T. and M.J. Bevan, CD8 T-cell recognition of macrophages and hepatocytes results in immunity to Listeria monocytogenes. Infect Immun, 1996. 64(9): p. 3632-40. Conlan, J.W. and R.J. North, Early pathogenesis of infection in the liver with the facultative intracellular bacteria Listeria monocytogenes, Francisella tularensis, and Salmonella typhimurium involves lysis of infected hepatocytes by leukocytes. Infect Immun, 1992. 60(12): p. 5164-71. Starks, H., et al., Listeria monocytogenes as a vaccine vector: virulence attenuation or existing antivector immunity does not diminish therapeutic efficacy. J Immunol, 2004. 173(1): p. 420-7. Ikonomidis, G., et al., Delivery of a viral antigen to the class I processing and presentation pathway by Listeria monocytogenes. J Exp Med, 1994. 180(6): p. 2209-18. Zenewicz, L.A. and H. Shen, Innate and adaptive immune responses to Listeria monocytogenes: a short overview. Microbes Infect, 2007. 9(10): p. 1208-15.  20  CHAPTER II 2  Manuscript 1: Neonatal immunization with Listeria monocytogenes induces T cells with an adult-like avidity, sensitivity, and TCR-Vβ repertoire1  2.1 Introduction Compared to adults, neonates display suboptimal responses to many vaccines and have a higher risk of suffering from infection [1]. A functional impairment of Ag presentation [2] and an overall decrease in T cell-mediated immune responses [3] appear to be among several possible underlying factors associated with decreased immune-mediated defenses in early life. However, in certain circumstances neonates can mount an adult-like Th1-type T cell response [4-8]. This suggests that the newborn, given the appropriate stimulus, is fully capable of priming Agspecific Th1-type T cell immunity [8-10]. The factors that allow an adult-like immune response to be generated in early life are of great interest to public health immunization strategies but are only now beginning to be understood [11].  We have previously shown that certain attenuated strains of Lm have the capacity to induce a robust and protective immune response when administered to murine neonates [12, 13]. While these observations provide hope for improved neonatal vaccination strategies, neonatal immunization also has the potential to adversely affect the developing immune system, as immunization of newborns can potentially lead to inadequate primary immune responses [14,  1  A version of this chapter has been accepted for publication. Smolen, KK., Loeffler, DIM., Reikie, BA., Aplin, L., Cai, B., Fortuno, ES., and Kollmann, TR. Neonatal immunization with Listeria monocytogenes induces T cells with an adult-like avidity, sensitivity, and TCR-Vβ repertoire, and does not adversely impact the response to boosting. Vaccine (Ms. Ref. No.: JVAC-D-09-00645R1) 21  15],[16], and most concerning, a weakened responses to boosting [1, 17] or even long-term suppressed vaccine-specific immune memory [18-20]. We therefore set out to investigate the impact of neonatal immunization with Lm on several key parameters of the T cell response such as TCR repertoire selection, and T cell avidity and sensitivity to recall stimulation. We also examined the impact of neonatal immunization on subsequent booster responses. We confirmed our previous findings that neonatal immunization leads to a higher expansion of OVA-specific CD8+ T cells during the primary response compared to adult immunization. We also found that the functional avidity and sensitivity of Ag-specific T cells did not differ between neonatally immunized mice and mice immunized as adults. Interestingly, the number of TCR Vβ chains involved in the OVA-specific CD8+ T cell response did not differ between the two age groups, nor did it change in either group in response to booster immunization. Most importantly, we show that neonatal immunization with our attenuated Lm ΔactA-OVA strain did not adversely affect the ability to protect against WT Lm. Overall, our data failed to show any adverse effect of neonatal Lm immunization on long-term T cell responses. Instead, our data provide further evidence in support of immunization at birth as a feasible public health strategy to combat early life infections.  2.2 Materials and Methods 2.2.1  Animals  Lm class I immunodominant peptides have been described only in the murine H-2d haplotype and class II immunodominant peptides only in the H-2b haplotype. Therefore, we used neonatal and adult (6-week-old) F1 mice (H-2b x H-2d) derived from mating C57BL/6 (H-2b) and C57B10.D2 (H-2d) mice (both from The Jackson Laboratories) [21]. Previous experiments indicated that there was no difference in the Lm-specific T cell responses in C57BL/6 (H-2b) or C57B10.D2 22  (H-2d) compared with F1 (H-2b x H-2d) mice for the respective immunodominant epitopes [12]. Because our experiments were meant to provide insight into mechanisms possibly underlying the human neonate’s reduced response to vaccines, we chose 5- to 7-day-old murine pups since they are the closest to human newborns with respect to the maturation status of their immune system [22]. All animals were housed under specific pathogen-free conditions at the Child and Family Research Institute of the University of British Columbia. All animal experiment protocols were approved by the Institutional Animal Care and Use Committee.  2.2.2  Bacterial strains  An attenuated strain of Lm ΔactA-OVA, as described in [12], was used for immunization and boosting. Wild-type Lm-OVA, as described in [12], was used to challenge the mice. All strains were grown as described previously [13]. In brief, Lm were grown to mid-log phase (OD600 = 1) at 37°C, washed and resuspended in endotoxin-free isotonic saline solution at a concentration of 107 CFU/ml for the attenuated strain and 105 CFU/ml for the wild-type. One hundred microliters of the Lm suspensions were injected either intraperitoneally (i.p.) or intravenously (i.v.) as outlined below.  2.2.3  Immunization schedule  Mice were immunized once, twice, or three times as part of this study. In order to evaluate their primary response, adult and newborn mice were immunized once i.p. with Lm ΔactA-OVA. The i.p. route was used for the primary immunization as i.v. injection in neonatal mice is too inconsistent given the technical challenges. The splenocytes were harvested and analyzed 10 days later as outlined below. To assess their secondary response, mice were initially immunized  23  i.p. and boosted i.v. 6 weeks later. The splenocytes were harvested and analyzed 5 days postboost as outlined below. For the tertiary response, mice were initially immunized i.p., boosted i.v. 6 weeks later, and again after an additional 4 weeks. The splenocytes were harvested and analyzed 5 days after the second boost as outlined below. The immunization and challenge methods used in this study were i.p. and i.v., both of which are comparable to the i.m. route [23]. Our current model did not allow us to study oral delivery of Lm based vaccines. However, Lm strains capable of infecting mice via the oral route have recently been developed [24], and we are currently in the process of exploring the oral Lm immunization route in neonates in more detail.  2.2.4  Intracellular cytokine flow cytometry  To obtain single cell suspensions, spleens were homogenized between two sterile glass slides, subjected to RBC lysis, and filtered through a 70µm cell strainer. Two million splenocytes per well of a 96-well plate were incubated for 5 h in 200 µl of RPMI 1640 medium supplemented with 10% FCS (HyClone), L-glutamine, penicillin, streptomycin, and 10µg/ml Brefeldin A with or without the peptides OVA257-264 or LLO189-201 (0.5 µM unless otherwise noted). To detect intracellular cytokine production, cells were permeabilized with Cytofix/Cytoperm solution (BD Pharmingen) and stained with allophycocyanin-labeled anti-IFN-γ (BD Pharmingen) for 45 min at room temperature (RT) together with surface staining for CD3, CD4 and CD8 with FITClabeled anti-CD3 (eBioscience) and either PerCP-labeled anti-CD8 (BD Pharmingen) or antiCD4 (BD Pharmingen). Stained cells were acquired on a FACSAria flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). The flow cytometry experiments are detailed in the Supplementary File in compliance with the MiFlowCyt standards [25] (see Supplementary File).  24  2.2.5  Surface tetramer staining  Splenocyte suspensions were prepared by homogenizing spleens between two sterile glass slides, subjected to RBC lysis, and filtered through a 70µm cell strainer. For surface staining using tetramers, two million splenocytes per well of a 96-well plate were resuspended in 100 µl of PBSAN (PBS containing 10% NaN3, 5% BSA). To detect the Ag-specificity of the T cells, tetramers specific for the OVA257-264 epitope were used. The cells were stained for 45 minutes at RT in the dark with PE-labeled PE-H-2Kb/OVA257–264 tetramer (Beckman Coulter), Alexa750labeled anti-CD3 (eBioscience), PerCP-labeled anti-CD8 (BD Pharmingen), and one of the 15 different FITC-labeled anti-TCR Vβ in our panel (BD Pharmingen; see below). Stained cells were acquired on a FACSAria flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). The flow cytometry experiments are detailed in the Supplementary File in compliance with the MiFlowCyt standards [25](see Supplementary File).  2.2.6  TCR Vβ chain usage analysis  TCR Vβ chain usage by Ag-specific CD8+ T cells was determined by staining cells with a panel of FITC-conjugated anti-TCR Vβ antibodies that included Vβ2, Vβ3, Vβ4, Vβ5.1/5.2, Vβ6, Vβ7, Vβ8.1/8.2, Vβ8.3, Vβ9, Vβ10b, Vβ11, Vβ12, Vβ13, Vβ14, and Vβ17 according to the manufacturer’s instructions (BD Pharmingen). In order to determine if the Ag-specific CD8+ T cells were positive or negative for a respective TCR Vβ chain, a specific cut-off threshold was used. The threshold for each specific TCR Vβ response from the immunized groups (tetramer+TCR-Vβ+) was the corresponding TCR Vβ response plus three times the standard deviation of the average naïve response at each immunization level. A TCR Vβ response in the  25  immunized group that was above this threshold was counted as positive.  2.2.7  Protection  Groups of 3-5 mice were immunized i.p. with 1x106 CFU Lm ΔactA-OVA, resuspended in endotoxin-free isotonic saline solution. The mice were either boosted twice as described above or not boosted at all (i.e., they only received the primary immunization). Four to six weeks after the last injection of the attenuated Lm ΔactA-OVA, both groups were challenged i.v. with 105 CFU of wild-type Lm-OVA. Spleens and livers of infected mice were harvested on day 4 postchallenge to determine the CFU number of Lm in each organ by plating the cell suspensions on Brain Heart Infusion (BHI) agar.  2.2.8  Statistics  The Mann-Whitney test was used for statistical analysis to determine if the T cell response was significant. The one-way ANOVA (Kruskal-Wallis test) was used when comparing the T cell response between primary, secondary and tertiary immunizations. The one-way ANOVA with Dunnett’s multiple comparisons was used to assess if the protection based on CFU was significant.  26  2.3 Results 2.3.1  Contrary to adults, boosting of mice immunized as neonates did not increase the ability  of Ag-specific CD8+ T cells to expand further; however, boosting did increase the CD4+ T cell ability to expand. To determine if boosting differentially affects the magnitude of the T cell recall response between mice initially immunized as neonates or adults, we analyzed the CD8+ and CD4+ functional T cell response in each group after boosting. Mice were immunized as neonates or adults with 106 CFU of Lm ΔactA-OVA, and then boosted with the same dose once or twice, 6 weeks and 10 weeks after the primary immunization. Ten days after the initial immunization or 5 days after either the first or second boost, splenocytes were isolated and stimulated with OVA257-264 in vitro. Intracellular IFN-γ production by CD8+ T cells was then measured by flow cytometry. As the total number of T cells, including CD8+ T cells, in the spleen is low in early life [12], the absolute number of OVA-specific T cells per spleen was also lower in earlier life as compared to the adult. But surprisingly, mice immunized as neonates reached their highest expansion of Ag specific IFN-γ+ CD8+ T cells already after the primary immunization, (Figure 1A). Boosting of neonatally immunized mice once or twice did not lead to a further increase in the magnitude of the overall OVA specific IFN-γ+ CD8+ T cell expansion. In contrast, mice immunized as adults achieved only a modest primary Ag-specific IFN-γ+ CD8+ T cell expansion. Only after an additional booster immunization did the mice immunized as adults reach the same magnitude of functional IFN-γ+ CD8+ T cell expansion as the mice immunized as neonates. Naïve controls were not shown due to marginal detection above zero. The expansion and contraction phase kinetics of Ag-specific IFN-γ+ producing CD4+ and CD8+ T cells during the primary response is known to be the same in mice immunized with Lm ΔactA as neonates or as adults; different kinetics can thus not explain our observations [12]. Employing  27  OVA257-264-specific tetramers yielded similar trends as the IFN-γ+ CD8+ T cell read-out, confirming that the difference in the primary response between neonate and adult immunization was not due to variation in specific effector function; however the tetramer analysis also suggested that for both neonatally and adult immunized mice, not all OVA-specific CD8+ T cells produced IFN-γ+ (Figure 2). We are currently in the process of investigating the functional status of the non- IFN-γ+ producers.  We also analyzed the CD4+ T cell response by IFN-γ production, employing the Lm-specific LLO189-201 peptide for in vitro restimulation. Mice immunized either as neonates or as adults achieved only a modest primary response after the initial immunization (Figure 1B). Only with boosting did both groups reach their peak CD4+ T cell recall expansion to LLO189-201. Similar to the CD8+ T cell response, boosting mice primed either as neonates or as adults twice did not lead to any further increase in the magnitude of the overall Ag-specific CD4+ T cell expansion. Analysis of the mean-fluorescent-intensity (MFI), as a measure of the amount of IFN-γ produced per cell, detected no significant difference between neonatally and adult induced OVA257-264-specific CD8+ or LLO189-201-specific CD4+ T cells response (Figure 3).  28  A!  B!  Figure 1. Neonates generated a higher primary CD8+ T cell response but similar CD8+ and CD4+ secondary and tertiary responses as compared to adults. Adult (dark grey) and neonatal (light grey) splenocytes are depicted at day 10 post initial immunization or day 5 post-boost (secondary or tertiary immunization). The functional CD8+ (A.) or CD4+ (B.) T cell response was determined by measuring intracellular IFN-γ production in CD8+ or CD4+ splenic T cells following OVA257-264 (for CD8 T cells) or LLO189-201 (for CD4 T cells) peptide re-stimulation in vitro. The results show the average and SEM of between 2-6 mice per group. Statistical analysis: Mann-Whitney.  29  Figure 2. The OVA-Tetramer+ CD8+ T cells response is similar in mice immunized as neonates or as adults (Sup. Fig. 1). Tetramer-staining captures all cells specific for the given epitope, irrespective of their functional status. We found that the percent of OVA-Tetramer+ CD8+ T cells was not statistically different between neonate and adult immunized mice at any stage of the immune response. The results show the average and SEM of six mice per group. Statistical analysis: Mann-Whitney.  30  OVA257-264 40000 30000 20000 10000 0  Primary  Secondary  Tertiary  LLO189-201 40000 30000 20000 10000 0  Primary  Secondary  Tertiary  Figure 3. The amount of IFN-γ produced per cell does not differ between mice first immunized as neonates or as adults (Sup. Fig. 2). The Mean Fluorescent Intensity (MFI) measures the amount of IFNγ produced per cell. We found no statistically significant difference between neonatally and adult immunized mice after re-stimulation in vitro with OVA257-264-specific and LLO189-201-specific peptide for the CD8+ T cell (A) and CD4+ T cell (B) response, respectively. The data represents an average of 3 mice from one of two independent experiments, with the SEM. Statistical analysis: One-way ANOVA (Kruskal-Wallis).  31  2.3.2  CD8+ and CD4+ T cell sensitivity and functional avidity did not differ between mice  immunized as neonates or as adults. Sensitivity and functional avidity are two critical parameters of a T cell response. Sensitivity is defined as the lowest concentration of peptide necessary to induce a measurable functional response, while functional avidity is the concentration of peptide at which the half-maximal response (ED50) has been reached [26, 27]. We investigated if differences exist in the sensitivity and functional avidity of Ag-specific T cells of mice immunized either as neonates or as adults, and how these parameters were affected by boosting. To achieve this, we isolated splenocytes from mice immunized either as neonates or as adults, and either boosted once, twice, or not at all. Next we re-stimulated the cells in vitro with the respective CD4+- or CD8+-restricted peptide, and stained for IFN-γ producing CD4+ or CD8+ T cells as the functional read-out. We were surprised to find no change in either sensitivity (5 × 10-4 µM) or functional avidity (5 × 10-3 µM) of the OVA-specific CD8+ T cells of each group (Figure 4A). Interestingly, neither sensitivity nor functional avidity of Ag-specific CD8+ T cells increased with boosting. Similarly, the functional avidity of CD4+ T cells was also the same for mice immunized as neonates and as adults (5 × 10-2 µM), and did not change with boosting. But contrary to CD8+ T cells, the sensitivity of Ag-specific CD4+ T cells from mice either immunized as adults or as neonates increased from 5 × 10-3 to 5 × 10-4 µM with one boost (Figure 4B), but remained the same with additional boosting. Importantly, there was no difference in CD4+ or CD8+ sensitivity or functional avidity between mice first immunized as neonates or as adults.  32  OVA 257-264!  B!  LLO 189-201!  3-"0%"&'  ,-12.*%"&'  !"#$%"&'  A!  ()#*#+&'  ,-./#0)#+&'  Figure 4. Functional avidity of Ag-specific T cells did not change in response to booster immunization in mice immunized as neonates or as adults. At day 10 post-initial immunization or day 5 post-boost (secondary or tertiary immunization), the functional responsiveness of CD8+ or CD4+ T cells was determined by measuring intracellular IFN-γ production following stimulation with increasing doses of peptide (5 × 10-6 to 5 × 10-3 µM). The connected line shows the CD8+ or CD4+ T cell sensitivity for each experimental group. The open arrow marks the beginning of the sensitivity response for each group and the closed arrow marks the functional avidity for each experimental group. A. Percentage of OVA257+ + 264 -specific IFN-γ CD8 T cells in the spleen following re-stimulation with peptide. B. Percentage of LLO189-201-specific IFN-γ+CD4+ T cells in the spleen following re-stimulation with peptide. The results shown in A and B depict the average and SEM of between 2-6 mice per group.  33  2.3.3  The diversity of the CD8+ TCR Vβ response of mice immunized as neonates or as  adults did not differ, nor change in response to boosting. To determine if Ag-specific CD8+ T cells generated in early or adult stages of life exhibit similar TCR repertoire usage, we compared the TCR Vβ usage of OVA-specific CD8+ T cells in splenocytes by flow cytometry (Figure 5). We also set out to follow the impact of boosting on TCR Vβ usage in mice first immunized either as neonates or as adults (Table 1). The large variation between mice in each group (i.e. the relatively large standard deviation) is a reflection of the known fact that at most only 20-25% of Ag-specific TCR-Vβ restricted responses are shared within individual mice even within the same inbred strain [28]. We did detect selected differences in the Vβ repertoire of OVA-specific CD8+ T cells in mice first immunized as neonates versus those first immunized as adults; first, the presence of TCR Vβ 5.1/5.2 in the primary neonatal response. Second, the TCR Vβ 8.1/8.2 is observed in all neonatal response while in adults it is observed post-boosting, as is TCR Vβ 11. Third, the presence of TCR Vβ 4 and 9 is observed in the tertiary neonatal response while the TCR Vβ 2 is observed in the tertiary adult response (Table 1). However, the overall number of different Vβ chains involved in the OVA257-264-specific CD8+ T cell response was the same between mice first immunized either as neonates or as adults. This number of different Vβ chains employed in the response also did not appear to change appreciably between primary, secondary, or tertiary responses in mice first immunized either as neonates or as adults. The most striking difference again was the overall higher magnitude of the T cell response of mice immunized as neonates vs. those first immunized as adults.  34  Table 1. Mice immunized as neonates or as adults maintained a highly diverse CD8+ TCR Vβ repertoire directed against OVA257-264 after the primary, secondary, and tertiary response to ΔactAOVA.  This table shows the average neonatal and adult TCR Vβ response on OVA257-264 tetramer-CD8+ T cells during primary (1o), secondary (2o), and tertiary (3o) immune responses. The threshold to be considered positive for a specific TCR Vβ response was calculated from the corresponding response in the naïve mice plus three times their standard deviation. A value of zero represents groups that did not reach above this threshold. Shown are the average and the SD of six mice per group.  35  OVA257-264 Tetramer+  A! #"  !"  !"  $" %"  B! #" $" %" V!2" V!3" V!4" V!5.1"V!6" V!7" V!8.1"V!8.3" V!9" V!10" V!11" V!12" V!13" V!14" V!17" /5.2" /8.2"  TCR V!  Figure 5. Both, mice immunized as neonates or as adults maintain a highly diverse CD8 TCR Vβ repertoire directed against OVA257-264 after the primary, secondary, and tertiary immunizations with Lm ΔactA-OVA (Sup. Fig. 3). Representative FACS plots indicating the percent of OVA257-264 tetramer+ TCR-Vβ+ of CD8+ T cell in adult (A.) and neonate (B.) at primary, secondary, and tertiary immune responses to 106 Lm ΔactA-OVA. This graph represents a single mouse from each of the immunization levels.  36  2.3.4  Boosting neonatally immunized mice did not adversely affect their protection against  wild-type challenge. To determine if boosting of mice immunized as neonates adversely affects their protective ability, we immunized neonatal and adult groups with 106 CFU of Lm ΔactAOVA once or three times, and challenged them 3 months after the initial immunization with 105 CFU of wild-type Lm-OVA. Four days after challenge the spleens and livers were harvested, and the CFUs per organ were counted. No death occurred in any of the immunized mice, and none of the mice in the immunized groups, boosted or not displayed signs of morbidity at any time. This high level of protection from challenge was confirmed quantitatively by CFU counts in the Lm target organs-spleen (Figure 6A) and liver (Figure 6B). As we had reported previously, mice initially immunized either as neonates or as adults were already significantly protected after only a single immunization. Strikingly, the level of protection in mice first immunized as neonates or as adults significantly increased with boosting, to provide sterilizing protection already 4 days post-challenge. Neonatal immunization did not prohibit this increase in sterilizing immunity with subsequent booster doses.  37  Figure 6. Neonatally immunized mice displayed the same protection pattern as mice immunized as adults, and did not lack a response to boosting. Naïve (checkered), neonate (light grey), and adult (dark grey) CFU per organ counts in the spleen (A.) and liver (B.) for all three immunization groups; nonimmunized mice (Naïve), singly immunized mice (1 dose), and immunized and subsequently twiceboosted mice (i.e. total of 3 doses). After immunization (1 or 3 doses) or non-immunization (naïve) the mice were challenged with 1x105 CFU of wild-type Lm-OVA. 4 days post challenge the CFU counts were conducted for the spleen and liver of each mouse. Using a one-way ANOVA statistical analysis with Dunnett’s multiple comparison, both adults and neonates for the spleen (A.) and liver (B.) immunized once (1 dose) or three times (3 doses) were statistically significant when compared to the naïve group. There was no significant difference between neonatal and adult groups immunized once for the spleen or liver. The results show the average and SEM of 3-12 mice per group. Statistical analysis: One-way ANOVA (Dunnett’s multiple comparison)  38  2.4 Discussion Neonatal immunization offers many important advantages over immunization later in life, but it also has the potential to negatively impact the developing immune system [1, 14, 15, 18-20, 29]. We have previously shown that immunization with an attenuated strain of Lm induces protective immune responses in early life [12, 13]. We now set out to investigate potential negative aspects of Lm-based neonatal immunization. We found that 1) neonatal immunization induced maximal Ag-specific CD8+ T cell expansion after a single dose already, while adult immunization required a booster dose to reach the same level of response; 2) once the expansion potential had been reached, expansion did not increase further with additional boosting; 3) sensitivity of CD4+ T cells, but not CD8+ T cells, increased with one booster dose in mice immunized either as adults or as neonates; 4) functional avidity of CD4+ and CD8+ T cells did not increase with boosting in mice immunized either as adults or as neonates; 5) the number of different TCR Vβ chains employed in the response to one specific epitope (OVA) was similar between mice immunized as neonates or as adults, and did not change with booster vaccination; 6) neonatal immunization and subsequent boosting did not lead to a loss but an increase in protection. In summary, the data presented fail to show any negative impact of neonatal immunization on the immune response; instead, our data add further evidence to our hypothesis that the neonatal period is an optimal time to immunize.  Although neonatal vaccination presents an optimal strategy to prevent infections in early life, caution must be employed. There are several examples of the negative impact of neonatal immunization on vaccine-mediated immune responses. For example, vaccination of humans with conjugated Haemophilus influenza type b (Hib) induces a T cell-dependent B cell response and  39  protective immunity after a single dose given at 2 months of age [15, 17]. However, this conjugate vaccine fails to induce a measurable immune response when given to neonates, and even prevents subsequent booster responses for the first six months of life [1, 17]. Similar to this Hib study, the whole-cell pertussis vaccine if given in the first 24 hours of life results in an inadequate serological response and hampered booster responses [14, 30], and while acellular pertussis vaccine administered at birth leads to adequate serological responses [30], it dampens the responses to co-administered Hib and Hepatitis B (HepB) vaccines. Studies investigating the T cell response raise similar concerns. For instance, the neonatal response to oral polio vaccination results in high titers of neutralizing antibodies, but reduced proliferation and IFN-γ responses to polio Ag restimulation compared to immunization later in life [31]. Additionally, HepB vaccine long-term follow-up studies showed that a higher number of subjects initially immunized at birth have reduced anamnestic responses as adolescents compared to those immunized later in life [18, 20]. In mice, it was also shown that while neonatal immunization with DNA [32] or protein [33] can lead to an initial Th1 response, neonatally immunized mice revert to a Th-2 type T cell response upon boosting[34].  In light of these findings, we investigated whether neonatal immunization with Lm ΔactA-OVA would negatively impact Ag-specific recall responses later in life. We found that the primary CD8+ T cell expansion in response to Lm immunization reached significantly higher levels in the neonate compared to the adult, confirming our previous findings where CD8+ T cells in mice immunized as neonates responded strongly and equally to Lm specific-Ag (LLO91-99, P60217-225) as well as OVA257-264 specific peptides [12, 13]. The OVA257-264, LLO91-99, and P60217-225 are presented via MHC class I, while the LLO189-201 peptide is presented via MHC class II. Contrary  40  to the CD8+ T cell response, Ag-specific primary CD4+ T cell expansion did not differ between the two age groups, suggesting that a relative vaccine dose difference compared to body weight of the Lm vaccine inoculums could not explain the more pronounced neonatal primary CD8+ T cell expansion. This also had been shown in our dose-response experiments as previously described [13]. The higher expansion potential of neonatal CD8+ T cells may result from the higher spontaneous proliferation of T cells in early life [35, 36]. Interestingly, mice first immunized as adults reached the same magnitude of Ag-specific CD8+ T cell expansion as mice immunized as neonates, but only after an additional booster dose administration. And although neonates reached their maximal expansion level earlier than adults, expansion during secondary and tertiary responses for CD8+ and CD4+ T cells were similar in mice initially immunized as adults or neonates. This has important implications for vaccine strategies targeting early life, in that while multiple doses of inactivated vaccines appear to be required to achieve protection especially if given in early life [11, 37], immunization with live vectors may generate substantial immune memory and protection without need for booster doses [38-40]. From our data, it appeared that an epitope-specific maximum level of CD8+ and CD4+ T cell expansion exists, and that neonates reach this maximum CD8+ T cell response after a single dose. However, contrary to our findings with Lm, virally infected neonates develop fewer Ag-specific CD8+ T cells after primary infection than adults, and reach adult-like responses only after re-infection [41]. This difference could be due to limited spread of our hyper-attenuated Lm compared to viral infections. It will be of interest to determine how generally applicable our observation of this ‘glass ceiling’ for T cell expansion is. Importantly, neonatal immunization with Lm did not negatively impact the ability of a recall response, nor the ability to reach this apparent ‘glass ceiling’ of epitope-specific T cell expansion.  41  The priming event elicited by the initial exposure to the Ag appears to be imprinted on the immune system, particularly in the T cell [42]. This concept is exploited in prime-boost strategies to increase subsequent memory T cell responses as measured in avidity and sensitivity [43]. Functional avidity maturation is believed to be mediated through selective expansion of T cells bearing receptors with higher affinity for Ags [44], e.g. avidity maturation has been postulated to select high avidity and affinity CD8+ T cells with enhanced protective capabilities in influenza prime-boost strategies [45, 46]. Contrary to these observations, we found no difference in functional avidity between primary, secondary, or tertiary responses in mice immunized first as neonates or as adults for either CD4+ or CD8+ Ag-specific T cells. We were also unable to detect any difference in T cell sensitivity of Ag-specific CD4+ or CD8+ T cells in mice immunized as neonates or as adults. But while sensitivity of the CD8+ T cell response did not change in either group between the primary response or secondary and tertiary responses, we did detect an increase in CD4+ T cell sensitivity from primary to secondary, but not from secondary to tertiary responses. However, we measured these parameters not at the single cell level, but in bulk for the entire Ag-specific T cell population at the height of expansion, i.e. at a point in time when most cells represent recently expanded effector T cells. We thus can not comment on changes in either avidity or sensitivity at the single-cell level, nor on the development of these parameters in memory T cells over time in individual mice. This work is currently in progress. Nevertheless, our current findings indicate that the neonate was capable of developing Ag-specific CD4+ and CD8+ T cells in response to Lm immunization that displayed functionally similar patterns as those of an adult.  42  TCR Vβ usage in Ag-specific CD8+ T cells is a measure of repertoire diversity, with a higher diversity generally believed to indicate a higher ability to respond to variation in Ags and, with that, presumably provide better protection [28]. We were unable to detect any striking difference in the number of different Vβ chains detected on OVA-specific CD8+ T cells between mice immunized as neonates or as adults. Interestingly, there also appeared to be no change in the number of TCR Vβs used in either age group between primary, secondary, or tertiary responses. The TCR repertoire is believed to become more restricted upon successive rounds of Ag exposure in a given individual [47, 48], but even in genetically identical mice, TCR repertoires of single epitope-specific CD8+ T cells display a high diversity between individual mice with as little as 20% overlap [28]. Due to the small neonatal blood volume obtainable during the primary response, we did not attempt to analyze the development of a TCR Vβ repertoire in a single mouse over time, and can thus not comment on the development of the TCR Vβ repertoire in single mice over time.  The fact that there was no detectable negative influence of neonatal immunization with Lm  ΔactA-OVA on recall responses, and an apparent stable maximum level of CD8+ T cell effector cell expansion after boosting, prompted us to investigate the impact of boosting on protection. Our results showed that both single and multiple immunizations protect both the neonatally immunized mice and mice immunized as adults equally well. The data indicated, however, that immunizing mice multiple times resulted in a faster protection from challenge than single immunizations. Mice immunized with multiple exposures showed complete clearance of the bacteria in both the spleen and liver at day 4 post-challenge [18, 20]. Interestingly, the increase in speed of protection with boosting that we observed in both groups of mice occurred in the  43  absence of an apparent increase in the number of OVA-specific T cells. This argues that with boosting, there is either an increase in non-OVA-specific T cells or more efficient effector functions at the single-cell level. For example, polyfunctionality of Ag-specific T cells may differ between CD8+ and CD4+ T cell responses after a single dose or following multiple booster doses. As polyfunctionality has been proposed to be a correlate of protection [49-53], this may explain the increase in protection we observed. We are currently in the process of comparing the degree of polyfunctionality in mice immunized as neonates or as adults, and how this is impacted by booster doses.  Additionally, recent modifications of the Lm vaccine vehicle allow for the exploration of various vaccine delivery routes. The development of transgenic mice, which express the human Ecadherin receptor [54], or a ‘murinized’ Lm, which binds to the mouse E-cadherin [55, 56], allow for oral application of Lm in mice. We are currently exploring this model system to test if oral delivery of Lm-vaccines could offer a safer (needle-free) and cost-effective approach to neonatal vaccine delivery worldwide.  In summary, employing the widely accepted model system of murine listeriosis to dissect important immunological principles [57], we were unable to detect any negative impact of neonatal immunization with Lm ΔactA-OVA on Ag-specific T cell avidity and sensitivity, TCR Vβ selection, and on the ability to respond to boosting. Most importantly, neonatal immunization did not adversely affect the ability to increase protection with subsequent boosting. This supports our hypothesis that neonatal immunization is feasible, and that Lm in particular offers a potential vaccine vehicle to do so.  44  2.5 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.  18. 19.  Wilson, C.B. and T.R. Kollmann, Induction of antigen-specific immunity in human neonates and infants. 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Badovinac, V.P., et al., Accelerated CD8+ T-cell memory and prime-boost response after dendritic-cell vaccination. Nat Med, 2005. 11(7): p. 748-56. Pamer, E.G., Immune responses to Listeria monocytogenes. Nat Rev Immunol, 2004. 4(10): p. 812-23. Kaech, S.M., E.J. Wherry, and R. Ahmed, Effector and memory T-cell differentiation: implications for vaccine development. Nat Rev Immunol, 2002. 2(4): p. 251-62. Siegrist, C.A., et al., Induction of neonatal TH1 and CTL responses by live viral vaccines: a role for replication patterns within antigen presenting cells? Vaccine, 1998. 16(14-15): p. 1473-8.  46  41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57.  Fadel, S.A., D.A. Ozaki, and M. Sarzotti, Enhanced type 1 immunity after secondary viral challenge in mice primed as neonates. J Immunol, 2002. 169(6): p. 3293-300. Woodland, D.L., Jump-starting the immune system: prime-boosting comes of age. Trends Immunol, 2004. 25(2): p. 98-104. Appay, V., D.C. Douek, and D.A. Price, CD8+ T cell efficacy in vaccination and disease. Nat Med, 2008. 14(6): p. 623-8. Slifka, M.K. and J.L. Whitton, Functional avidity maturation of CD8(+) T cells without selection of higher affinity TCR. Nat Immunol, 2001. 2(8): p. 711-7. Woodberry, T., et al., Prime boost vaccination strategies: CD8 T cell numbers, protection, and Th1 bias. J Immunol, 2003. 170(5): p. 2599-604. Estcourt, M.J., et al., Prime-boost immunization generates a high frequency, high-avidity CD8(+) cytotoxic T lymphocyte population. Int Immunol, 2002. 14(1): p. 31-7. Naylor, K., et al., The influence of age on T cell generation and TCR diversity. J Immunol, 2005. 174(11): p. 7446-52. Haynes, L. and S.M. Eaton, The effect of age on the cognate function of CD4+ T cells. Immunol Rev, 2005. 205: p. 220-8. Ciuffreda, D., et al., Polyfunctional HCV-specific T-cell responses are associated with effective control of HCV replication. Eur J Immunol, 2008. 38(10): p. 2665-77. Duvall, M.G., et al., Polyfunctional T cell responses are a hallmark of HIV-2 infection. Eur J Immunol, 2008. 38(2): p. 350-63. Sun, Y., et al., Magnitude and quality of vaccine-elicited T-cell responses in the control of immunodeficiency virus replication in rhesus monkeys. J Virol, 2008. 82(17): p. 88129. Nebbia, G., et al., Polyfunctional cytomegalovirus-specific CD4+ and pp65 CD8+ T cells protect against high-level replication after liver transplantation. Am J Transplant, 2008. 8(12): p. 2590-9. Makedonas, G. and M.R. Betts, Polyfunctional analysis of human t cell responses: importance in vaccine immunogenicity and natural infection. Springer Semin Immunopathol, 2006. 28(3): p. 209-19. Lecuit, M., et al., A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science, 2001. 292(5522): p. 1722-5. Niemann, H.H., et al., Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB. Cell, 2007. 130(2): p. 235-46. Wollert, T., et al., Extending the host range of Listeria monocytogenes by rational protein design. Cell, 2007. 129(5): p. 891-902. Zenewicz, L.A. and H. Shen, Innate and adaptive immune responses to Listeria monocytogenes: a short overview. Microbes Infect, 2007. 9(10): p. 1208-15.  47  CHAPTER III 3 DISCUSSION In this thesis I compared the impact of neonatal and adult immunization with attenuated Lm on the ensuing T cell response. I found that attenuated Lm immunized neonatal mice reached maximal Ag-specific CD8+ T cell expansion after only a single immunization, while adults required two doses. Ag-specific CD4+ T cell expansion was observed in both age groups, though a boost was required to reach peak expansions for both. Neither functional avidity, sensitivity, nor the TCR Vβ repertoire of the Ag-specific T cells differed between mice immunized as neonates or adults. Lastly, neonatal immunization and subsequent boosting with attenuated Lm did not adversely affect the ability to increase protection in response to boosting. Overall, my findings provide further evidence in support of immunization at birth as a feasible public health strategy to combat early life infections.  Complementary to this study, my co-author paper (see Appendix B) showed confirmatory results with Lm recombinant for four different constructs. The Appendix B study described similar results in the variation of effector T cells seen between neonates and adults, as measured with IFNγ readout. Mice immunized as neonates reached a higher number of Ag-specific IFNγ+ CD8+ T cell after primary immunization, while adults responded with a modest response to all four recombinants. Similarly Appendix B also showed a modest CD4+ T cell response after primary immunization. Both studies describe similar results indicating that recombinant Lm, offers great potential as a safe and effective vaccine vehicle for neonatal vaccination.  48  The hypothesis of this study was that the neonatal immune response to vaccination and subsequent boosting would not differ from the adult immune response. In the various T cell based read-outs most of the parameters I investigated did not show any difference. However, a difference was seen between the neonatal and adult CD8+ T cell expansion after the first immunization: The neonatally immunized mice were able to reach maximal expansion after a single immunization while adults required two doses, indicating that perhaps neonates respond better to initial immunization than adults.  Although neonatal immunization offers many important advantages over immunization later in life, it has the potential to negatively impact the developing immune system. Proof of concept for neonatal vaccination exists [1], but many vaccines still fail to induce an appropriate immune response. Why some vaccines work when given early in life, and others do not, is unclear. My studies represent our ongoing effort to delineate the underlying mechanisms responsible for this, with their final aim to establish a rational basis on which to design optimal neonatal vaccines.  Attenuated Lm is not only an effective neonatal vaccine vehicle [3], but also allows for the pinpointing of immunological differences between neonates and adults on the cellular and molecular level [2]. This study is the first in-depth investigation of the neonatal T cell immune response to Lm focused on the impact of neonatal boosting. The focus was chosen, as boosting would likely be desired for several specific vaccine targets. In other model systems, boosting neonatally immunized mice revealed several shortcomings of the vaccination strategy.  49  Directly comparing the adult to the neonatal response provides this study with a number of strengths. The most important component of this study is the ability to analyze the similarities and differences between neonates and adults in response to identical vaccination schemes. Most vaccines have been designed with a certain degree of trial-and-error for the adult population, and then adapted to the neonate – mostly unsuccessful. By understanding the initial differences between the adult and neonatal immune systems we hope that a rational method of neonatal vaccine design can be developed. In this study, the initial immune response as well as the subsequent response to boosting were examined, which allowed for the observation of the immune system as the mice matured. An additional advantage of this study is the analysis of both CD4+ and CD8+ T cell subsets in the same mice. Both CD4+ and CD8+ T cell subsets are known to have crucial roles in the immune response to Lm. Thus, my concurrent analysis of both CD8+ and CD4+ T cells in the immune response provides further insight through the detailed contrasting of the complexities underlying each neonatal and adult immunization.  In addition to the strengths, this study also had a number of weaknesses. First is the limited scope of this thesis. Only a fraction of the entire immunological response was examined, namely the T cells. The T cell focus allowed for great in-depth analysis of T cell involvement as described above, but limited the examination of other elements of the immune response. Additionally this study is further limited in scope by the use of a limited number of antigenic targets (peptides) to analyze the immune response. Through the use of one CD8+ and one CD4+ T cell peptide I was able to focus on a representative of each major subtype of T cell. Finally, this study focused on only one immunizing dose. The dose used was an effective dose for assessing the immune  50  response, but immunizing with different doses could uncover potential dose related discrepancies, as was mentioned above.  3.1 Future direction This study further supported that neonatal Lm immunization is an effective and safe vaccination strategy, and eventually could be developed to combat early life infections in humans. Although effective, it is important to understand the entire immunological response within the neonate, to predict side-effects and further optimize the response. In order to do so, follow up studies, asking narrowly focused mechanistic questions can now be carried out.  3.1.1  Immediate  First, this study focused on the T cell response, providing an in-depth analysis of T cell involvement but a limited examination of other immunological elements. Although the T cell response is a crucial component of the immune response, other aspects, such as the APC, must be considered to understand the complete response of Lm neonatal vaccination. What roles do the differences between neonatal and adult APC function play in the ensuing responses to vaccination? An experimental method using adoptive transfers would allow for the investigation of APC involvement as well as the APC-T cell interaction during neonatal immunization. This experiment could involve specific knock-out (KO) mice (e.g. type-1 IFN or IL-12 deficient mice) to narrow down the molecular mechanisms. Such studies of the APC within the Lm model system should provide insight into the underlying innate-adaptive interactions directing the response to Lm vaccination.  51  Secondly, the study showed that the CD8+ T cell population in neonatally immunized mice was able to reach maximal expansion after a single immunization while adults required two doses. The CD8+ and CD4+ T cell populations were investigated through the use of specific epitopes OVA257-264 and LLO189-201 respectively. Although these epitopes are good representatives for each subset, other immunodominant peptides should be incorporated. In our previous studies neonates have been shown to respond to other Lm peptides [3] such as; LLO91-99, p60217-225, LLO296-304, p60449-457, and Mpl184-192, while adults were focused on only one dominant epitope. What are the underlying Ag processing and presentation mechanisms that lead to this difference between neonatal and adult immunization? The investigation of Ag-specific CD8+ and CD4+ T cell expansion after immunization based on additional epitopes could depict further variation between the neonate and adult populations. Again, using specific KO mice to examine individual mechanisms, such as the immunoproteasome, we now will be able to dissect the precise molecular “cause and effect” interactions.  Additionally, IFN-γ, an immunomodulator, was used to detect the differences between the two T cell populations. With the use of additional cytokine read-outs such as IL-2 or TNF-α the experiments could be used to provide a poly-functional analysis of the immune response at each of the specified conditions. Specifically, we can now ask: Does the impact of neonatal Lm based vaccination differ for different read outs or immune targets? For example, how does neonatal Lm immunization impact the development of Th-17 cells? Analyzing the IL-17 production in the system described in my thesis should shed significant light on this interesting population.  52  TCR Vβ which provides the specific T cell response showed no difference between groups of neonatal and the adult mice. However we did not investigate the development of this aspect within the same mouse over time. How does neonatal immunization impact the development Agspecific T cell clones in individual mice over time? The use of an ‘immunoscope’ or a spectratyping analysis technique to assess the TCR Vβ response for either mono- or poly-clonal populations, as described in [4], would help assess the differences between the neonatal and adult response at the individual TCR clone level and individual level. Understanding the individual TCR clonal involvement would provide information about the specifics of the memory CD8+ T cell response after vaccination that is essential in evaluating the overall strategy of neonatal versus adult immunization.  This study showed that the neonates were protected at initial immunization and after boosting. Both neonatally and adult immunized mice increased the speed with which subsequent challenging inocula were cleared and both responded well to boosting. This implies that early vaccination and boosting do not negatively affect long-term protection. In this study, two epitopes were considered but many more were involved, and as mentioned above, investigating all immunodominant epitopes involved in parallel would provide further insight into the T cell response. In particular, the immune response to intracellular pathogens is characterized by massive activation and expansion of CD8+ T cells [5]. These CTL are responsible for the Agspecific recognition of infected cells. Failure to develop CTL activity has been linked to the neonatal immune response. Does the neonatally immunized mouse develop and maintain as broad a CTL activity as the IFN- γ response suggests? Identifying the CTL response to specific peptides through in vivo CTL assays, and contrasting this to the adult, would answer this  53  question. Additionally through the use of specific KO mice (perforin, granzyme or FasL), the molecular contribution of each could be precisely ascribed.  Several other questions can now also be addressed based on the knowledge my work has provided (i.e. specific read-outs and longitudinal development). These are: How does the humoral response to neonatal Lm immunization develop over time? How do variations in the maternal environment from which the infant emerges influence the final outcome (e.g. congential infection; prematurity; etc.)? And most importantly, does neonatal Lm-mediated delivery of reallife, and relevant pathogens (e.g. pertussis, malaria) or allergens lead to the expected level of protection? Studies addressing the above listed questions are currently underway.  3.1.2 Long-term Vaccines are one of the most effective medical interventions, leading to a significant reduction in morbidity and mortality for many infectious diseases. Despite the overwhelming success of vaccination, conflicting opinions still exist regarding potential negative side-effects of vaccination. Although opposition to vaccination has existed since its inception, most accept that vaccination is beneficial so long as the benefits outweigh the risks. However, serious disputes have arisen, shaking public trust in vaccination when the risk-benefit analysis shows a small margin. Given that most childhood infections have been drastically reduced due to immunizations, the benefit of immunizing your child is no longer as apparent to parents as it was 50 years ago. Many negative and misinformed campaigns, largely Internet-based, have broadcast negative images and amplified public concerns. These concerns are now real, however scientifically unfounded they may be, and need to be addressed if there is hope to ever introduce novel vaccination for the most vulnerable population, the neonate [6]. In the current climate, it  54  would be extremely difficult to convince parents to allow their newborn infants to be vaccinated with any form of live attenuated Lm. Especially since Lm has been the recent infectious culprit in outbreaks in meat factories, such as in Ontario. To alleviate these concerns, the signaling pathways responsible for the success of our Lm based approach need to be identified in order to target the development of non-live approaches, possibly synthetic adjuvants focused on the same pathways. Studies specifically investigating this have been initiated in our lab.  Clearly, knowledge of possible vaccine associated adverse events is limited, even for vaccines that have been in clinical use for decades. This is largely due to the logistical difficulties of tracking the millions of recipients over long periods of time, and the nearly impossible task of determining the cause-and-effect between a specific vaccine and clinical impact. Only recently have databases been setup (e.g. IMPACT in Canada) that allow a statistically and scientifically valid approach to this difficult, but extremely important topic. But basic science also has to play a major role in this endeavor. For example, possible bystander-effects of the vaccine, where unwanted immune reactions are triggered, need to be studied to increase our mechanistic understanding of how adverse events might occur [7]. Specifically, adjuvants used in vaccines to augment the immune response, elicit an inflammatory reaction which directly influence the effector and memory T cells of Ag-specific and non-specific cells [8]. Further research is required to assess this potential risk in all vaccines, especially in the neonatal setting.  In addition to the fear of vaccine associated side-effects, another point of concern often raised by the public is the idea that the immune system has ‘limited space’. Although many studies have shown that the immune system is malleable, responsive, and today is exposed to fewer Ag in a  55  vaccine than 100 years ago [9], concerns about overwhelming the limited resources of the immune system still exist [6]. Further public education needs to be undertaken to explain the specifics of Lm vaccination, and the falsehood of the ‘immune system overload’ hypothesis. Again, our Lm model should allow this question to be addressed in the necessary detail.  3.2 Impact of Described Work Identifying the potential differences between the neonatal and adult immune responses to vaccination is essential in creating and designing optimal neonatal vaccines. Neonates can mount an adult-like Th1-type T cell response [10-14], indicating that newborns are fully capable of priming Ag-specific T cell immunity under the appropriate stimulus [14-16]. Pinpointing the factors that allow a complete immune response to be generated in early life will provide the information needed to design the optimal neonatal vaccine.  My study failed to identify any negative impacts of neonatal immunization on the developing immune system. Contrary to the idea that neonates have a weaker response, this Lm vaccine model showed that immunized neonates are protected and are perhaps better able to mount an immune response upon initial immunization compared to adults. This suggests that neonatal vaccination is potentially a valuable public health intervention. Although there are only a limited number of vaccines that are effective in neonates, certain similarities between them help in identifying the crucial mechanisms for success. The two most effective neonatal vaccines are BCG and Hep B; like Lm, both are based on an intracellular pathogen. Additionally all three vaccine models stimulate a strong Th1 response [17, 18], resulting in stimulation of an effective immune response against the pathogen. These data suggest that activation of the neonatal APC response is essential in eliciting a strong immune response in early life. Adjuvants (or delivery  56  systems) for neonatal immunization need to accomplish this. This strongly argues, that the first step towards optimal neonatal vaccine design aimed to induce robust, long-lived, protective adaptive immune memory, is to gain a better understanding of the neonatal innate immune system.  In conclusion, the implementation of this Lm vaccine delivery system would have a far-reaching impact on global health. But beyond this translational application, our Lm model offers the opportunity to dissect the precise immunological mechanisms responsible for the variation in neonatal and adult immune responses, and will aid in the rational design of effective neonatal vaccines.  57  3.3 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.  Demirjian, A. and O. Levy, Safety and efficacy of neonatal vaccination. Eur J Immunol, 2009. 39(1): p. 36-46. Loeffler, D.I., et al., Fine-tuning the safety and immunogenicity of Listeria monocytogenes-based neonatal vaccine platforms. Vaccine, 2009. 27(6): p. 919-27. Kollmann, T.R., et al., Induction of protective immunity to Listeria monocytogenes in neonates. J Immunol, 2007. 178(6): p. 3695-701. Blattman, J.N., et al., Evolution of the T cell repertoire during primary, memory, and recall responses to viral infection. J Immunol, 2000. 165(11): p. 6081-90. Ahmed, R. and D. Gray, Immunological memory and protective immunity: understanding their relation. Science, 1996. 272(5258): p. 54-60. Hilton, S., M. Petticrew, and K. Hunt, 'Combined vaccines are like a sudden onslaught to the body's immune system': parental concerns about vaccine 'overload' and 'immunevulnerability'. Vaccine, 2006. 24(20): p. 4321-7. Wraith, D.C., M. Goldman, and P.H. Lambert, Vaccination and autoimmune disease: what is the evidence? Lancet, 2003. 362(9396): p. 1659-66. Cui, W., et al., Effects of Signal 3 during CD8 T cell priming: Bystander production of IL-12 enhances effector T cell expansion but promotes terminal differentiation. Vaccine, 2009. 27(15): p. 2177-87. Offit, P.A., et al., Addressing parents' concerns: do multiple vaccines overwhelm or weaken the infant's immune system? Pediatrics, 2002. 109(1): p. 124-9. Fadel, S.A., et al., Neonate-primed CD8+ memory cells rival adult-primed memory cells in antigen-driven expansion and anti-viral protection. Int Immunol, 2006. 18(2): p. 24957. Adkins, B., C. Leclerc, and S. Marshall-Clarke, Neonatal adaptive immunity comes of age. Nat Rev Immunol, 2004. 4(7): p. 553-64. Siegrist, C.A., The challenges of vaccine responses in early life: selected examples. J Comp Pathol, 2007. 137 Suppl 1: p. S4-9. Marchant, A. and M. Goldman, T cell-mediated immune responses in human newborns: ready to learn? Clin Exp Immunol, 2005. 141(1): p. 10-8. Marchant, A., et al., Mature CD8(+) T lymphocyte response to viral infection during fetal life. J Clin Invest, 2003. 111(11): p. 1747-55. Dadaglio, G., et al., Efficient in vivo priming of specific cytotoxic T cell responses by neonatal dendritic cells. J Immunol, 2002. 168(5): p. 2219-24. Hermann, E., et al., Human fetuses are able to mount an adultlike CD8 T-cell response. Blood, 2002. 100(6): p. 2153-8. Marchant, A., et al., Newborns develop a Th1-type immune response to Mycobacterium bovis bacillus Calmette-Guerin vaccination. J Immunol, 1999. 163(4): p. 2249-55. Velu, V., et al., Relationship between T-lymphocyte cytokine levels and sero-response to hepatitis B vaccines. World J Gastroenterol, 2008. 14(22): p. 3534-40.  58  APPENDICES Appendix A Table 2. Routine immunization schedual for infants and children up to the age of two. Suggestion made by the Public  Health Agency of Canada. Diphtheria, Tetanus, acellular Pertussis and Inactive Polio virus  Haemophilus influenzae type b conjugate  2 months  √  √  4 months  √  √  6 months  √  √  12 months 18 months  √  Age at Vaccine  Measles Mumps and Rubella  Varicella  Hepatitis B  Pneumococcal conjugate  Meningococcal C conjugate  √  √  √  √  Influenza  Birth  √  √ (3doses)  √ √  √  √  √  √  √  √  59  Appendix B: Manuscript 2: Fine-Tuning Safety and Immunogenicity of Listeria monocytogenes-based Neonatal Vaccine Platforms2 Introduction Neonates and infants have an increased susceptibility to infection and respond sub-optimally to most vaccines, resulting in over 2.2 million deaths due to vaccine preventable infections per year (reviewed in [1, 2]). The urgent need for vaccines that induce protection early in life has been recognized for many years [2-4]. The challenge to develop effective neonatal vaccines arises from what some consider inherent limitations of the neonatal immune system [5]. It is believed that neonates exhibit functionally impaired Ag presentation, shorter-lived and lower level antibody responses, and a Th2-type immune response bias and decreased cell-mediated immune responses overall when compared to adults [6]. However a number of studies have shown that neonates are able to generate an adult-like T cell response by using, for instance, a strong activator of the cell-mediated immune response or delivering protein Ag directly to the professional APC [1, 7-10]. These observations suggest that under appropriate conditions, neonates can develop immune responses to vaccination that are similar in quality and quantity to their adult counterparts [5].  Vaccines based on recombinant live virulence-attenuated microorganisms have proven to induce long-lasting protective immunity, wherein both humoral and cell-mediated immune responses are often efficiently generated [11, 12]. Particularly, Lm-mediated delivery of Ag has been established as a functional and versatile approach for vaccination against allergies, or 2  A version of this chapter has been published. Loeffler, DIM, Smolen K, Aplin L, Cia, B. Kollmann, TR. Fine-Tuning Safety and Immunogenicity of Listeria monocytogenes-based Neonatal Vaccine Platform. Vaccine 2009. Feb 5;27(6):919-27 60  malignancies in adult mice (reviewed in [13, 14]). But particularly for infectious diseases, Lm has been successfully used as a vaccine carrier to deliver bacterial, viral, or parasitic Ag [15-18]. This model has been so successful that human clinical trials are already under way (Advaxis, Inc.; Cerus Corporation). The great appeals of Lm as a vaccine carrier are its intracellular life cycle and its strong associated immunomodulatory abilities. This Gram-positive bacterium escapes the phagolysosome, through a process facilitated by the secreted pore-forming protein, listeriolysin O (LLO). After its escape, Lm replicates efficiently within the cytosol of many host cells including macrophages and DC [19]. In addition, Lm spreads from cell to cell via an ActAmediated process (ActA, actin nucleator protein of Lm), thereby evading the extracellular milieu where antibodies are found. Protective immunity against Lm is thus almost entirely cellmediated, depending on both cytotoxic CD8 and CD4 Th1 T cells [20]. Our group published recently that a virulence-attenuated strain of Lm (Lm ΔactA) is safe and well tolerated in newborn mice and that immunization with Lm ΔactA strain carrying protein Ag directly into the cytosol of neonatal host cells was successful in eliciting a life-long protective immune response in murine neonates after only a single immunization [9].  The use of a robust listerial promoter, which is activated within the host cell, is required to optimally express a heterologous vaccine Ag in an Lm vaccine carrier strain. Overall, there are two strategies used to introduce heterologous Ag into Lm. The first is by insertional integration of the expression cassette within the Lm chromosome; the second is by cloning an expression cassette into a multi-copy replicating vector that remains extra- chromosomal (reviewed in [13]). In the first strategy, the integration of the heterologous Ag expression cassette into the bacterial chromosome increases its stability but lowers the Ag expression levels [21]. Moreover, the  61  integration of an expression cassette into the chromosome of the bacterial carrier is timeconsuming and labour-intensive. On the other hand, extra-chromosomal multi-copy plasmids are often afflicted by instability, with resulting plasmid loss and marginal Ag expression diminishing the efficacy of these recombinant vaccines [22]. We have previously developed a balanced-lethal plasmid system Lm Δ(trpS) that represents a multi-copy, stable, high-expression extrachromosomal vaccine platform that has proven to be a superb vaccine carrier in adult mice [23].  Both subsets of T cells, CD8 and CD4, are often required for efficient protection against pathogens [24]. Therefore, Ag must have access to both MHC class I (for CD8) and class II (for CD4) presentation pathways. The presence of Lm as a vaccine carrier in both phagosomal (i.e. MHC-II) and cytosolic (i.e. MHC-I) host cell compartments, as well as its inherent immunostimulatory capacities, gives the Ag direct access to both MHC molecules for Ag presentation and stimulation of CD4 and CD8 T cells [20]. However, where the Ag should first be expressed (phagosome or cytosol) to have an optimal impact on primary and secondary T cell-mediated responses and on protective efficacy by bacterial vaccine carriers in neonatal mice has, to our knowledge never been investigated.  In this report, we describe a crucial improvement of our previously published system [9] by using the balanced-lethal plasmid system Lm Δ(trpS actA) which adds additional attenuation and safety check-points, and is easily manipulated to carry heterologous vaccine Ags into Agpresenting cells. Using this system, we compared the relative vaccine efficacy in neonatal and adult mice which were immunized with several virulence-attenuated Lm Δ(trpS actA) strains that express and secrete multiple copies of the model vaccine, chicken egg albumin (ovalbumin,  62  OVA). These vaccine strains express and secrete OVA protein under the control of a predominantly phagosomal (Phly) or cytosolic (PactA) listerial promoter [19]. We found that immunization with Lm Δ(trpS actA) secreting OVA into the phagosomal compartment elicited levels of Ag-specific primary and secondary CD8 and CD4 T cell responses comparable to Lm Δ(trpS actA) strains secreting OVA into the cytosol. But neonatal and adult mice immunized with Lm Δ(trpS actA) secreting OVA into the phagosome were better protected against wild-type Lm challenge after only a single immunization. Interestingly, only neonatal mice immunized with the phagosomal expression strain developed anti-OVA antibodies, while no antibodies were detected in adults immunized in the same manner. Our results with this Lm-based neonatal vaccine platform represent a major step forward in the overall goal of a single-dose neonatal vaccination able to induce protection from infectious diseases early in life.  Materials and Methods Animals: For all our animal experiments we used 5- to 7-day old mouse pups (Neonates) and 6to 12-week old (Adult) F1 mice (H-2b × H-2d) derived from matings between C57BL/6 (H-2b) and C57B10.D2 (H-2d), which were bred in our animal facility. H-2b × H-2d F1 mice were used because Lm class I immunodominant peptides have been described only in the mouse H-2d haplotype and class II immunopeptides only in the H-2b haplotype. All animals were housed under specific pathogen-free conditions at the Child and Family Research Institute of the University of British Columbia. All animal experiments were approved by the Institutional Animal Care and Use Committee.  63  Bacterial strains, plasmids, media, and growth conditions: The construction of the plasmids and Lm strains (kindly provided by W. Goebel (University of Wuerzburg, Germany) has been described in detail previously [23]. The recombinant bacterial strains used in this work are listed in Table 1. Competent Lm cells were transformed by electroporation as described by Park and Stewart [25]. After transformation of Lm Δ(trpS actA)/pTRPS with the expression plasmids, the resulting recombinant Lm strains were cultured in an erythromycin-containing medium without tetracycline to remove the plasmid pTRPS. For immunization and infection experiments, Lm strains were grown to the late logarithmic phase (optical density at 600 nm (OD600), 1.0) at 37°C in brain-heart infusion (BHI) medium, washed twice with endotoxin-free isotonic saline (0.9% NaCl), resuspended in 20% (vol/vol) glycerol in 0.9% NaCl, and stored at -80°C prior to injection as described below.  Preparation of supernatant and cellular proteins of L. monocytogenes strains: For preparation of protein extracts of L. monocytogenes, all strains were grown to the logarithmic phase (OD600, 1.0) in BHI medium supplemented with 1% (w/v) AmberliteTM XAD-4. Addition of AmberliteTM XAD-4 into the BHI broth leads to the activation of the PrfA-dependent virulence gene expression [19, 26]. Supernatants were precipitated on ice with 10% trichloroacetic acid, pelleted by centrifugation (5,000 ×g at 4°C), washed in acetone, and resuspended in phosphatebuffered saline (PBS) to obtain a volume that was 0.2% of the original culture volume. For preparation of cellular proteins, the cell pellet was washed twice in PBS, resuspended in cold lysis buffer (PBS supplemented with protease inhibitors (Sigma)), and transferred into a 2-ml BLUE TUBE (Q-Biogene) filled with silica sand. The tube was shaken 3 times for 45 s each in a mini-beadbeater (Biospec Products). This was followed by ultrasonification for 45 s. The cell  64  debris was removed by centrifugation at 14,000 rpm for 30 min at 4°C. Total protein concentrations were determined by defining the amount of proteins at the wavelength-ratio of 260/280 in a spectrophotometer (Bio-Rad). A total amount of 150 µg/0.025 ml protein suspension in SDS-PAGE loading buffer was heated to 110°C for 7 min before they were loaded on sodium dodecyl sulphate (SDS) gels.  SDS-PAGE and immunoblotting: SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to standard protocols [27]. After SDS-PAGE, cellular proteins and proteins from the culture supernatant were subjected to Western blotting onto nitrocellulose membranes. OVA proteins were detected using rabbit polyclonal anti-OVA antibody (Sigma), peroxidaseconjugated secondary goat anti-rabbit antibody (Sigma) and ECL chemiluminescent kit (GE Healthcare).  Immunization and Infection of animals: Groups of 3 to 5 mice were immunized intraperitoneally (i.p.) with 1 × 107 bacteria (unless otherwise noted), resuspended in 0.1 ml endotoxin-free 0.9% NaCl. Ten days post immunization, spleens were collected for flow cytometric analysis. In protection experiments, groups of 3 to 5 mice were infected intravenously with 5 × 106 wild-type Lm-OVA in 0.1 ml endotoxin-free 0.9% NaCl 6 weeks after immunization. Spleens and livers of infected mice were harvested on day 3 and 5 after challenge to determine the number of Lm in each organ (day 3 post-challenge (p.c.)) and for the enumeration of Ag-specific T cells in spleens of immunized mice (day 5 p.c.). Viable bacterial counts of intracellular bacteria (colony-forming units, (CFU)) were determined by plating on BHI agar serial dilutions of mechanically lysed cell suspensions as described above.  65  Enumeration of Ag-specific T cells: Ag-specific T cells were detected as described [9]. Briefly, splenocyte suspensions were prepared by homogenizing spleens between two sterile glass slides, subjected to red blood cell lysis, and filtered through a 70 µm cell strainer. A total of 2 × 106 splenocytes was cultured for 5 h in 200 µl of complete medium (RPMI 1640 supplemented with 10% FCS, streptomycin, penicillin), along with Brefeldin A (10µg/ml) in either the presence or absence of 0.5 µM LLO91-99, P60217-225, OVA257-264 (SIINFEKL) or LL0190-201 peptide. Cells were then incubated with Cytofix/Cytoperm (BD PharMingen) to permeabilize the plasma membrane. Staining for intracellular cytokines was performed using allophycocyanin-labeled anti-IFN-γ for 30 min at room temperature together with surface staining for CD3, CD8 and CD4 using FITClabeled anti-CD3 and PerCP-labeled anti-CD8 or anti-CD4 (BD PharMingen). Stained cells were acquired on a FACSAria flow cytometer and analyzed using FlowJo software (Tree Star).  Enzyme-linked immunosorbent assay (ELISA) for mouse IgG antibodies: ELISA assays were performed two weeks after challenge with WT Lm-OVA. Briefly, maxisorb 96-well ELISA plate (Nunc Inc.) were coated overnight at 4°C with 2 mg/ml OVA (Worthington, Biochemical Corporation) in 100 µl of freshly prepared carbonate buffer (pH 9.6 at 4°C). The wells were washed 5 times with 0.05% (vol/vol) Tween-20 in PBS (PBST) and blocked for 2 h at room temperature with 200 µl of 10% FCS in PBS. After an additional 5 washes, 100 µl of two-fold serial dilutions (in PBS with 10% FCS) of the serum aliquots were transferred to the coated and blocked wells and incubated for 2 h at room temperature. Measurements were obtained in duplicate. Naïve mouse and anti-OVA serum were included as negative and positive controls, respectively. After this, the plates were washed 5 times with PBST, incubated with 1:5000 of  66  peroxidase-conjugated AffiniPure Goat anti-mouse IgG antibody (JacksonImmunoResearch) for 1 h at RT. After five washes in PBST, bound antibody was exposed to 100 µl/well of TMB substrate solution (eBioscience), the reaction was stopped with 50 µl 2M H2SO4, and then measured by subtracting the optical readings at 570 nm from those at 450 nm.  Statistics: Data are expressed as mean ± standard deviation. One-way ANOVA followed by Dunnett’s post-test was used for statistical analysis. P-values < 0.05 were considered statistically significant.  67  Results Multi-copy plasmid-containing Lm Δ (trpS actA) strains produce more OVA proteins in comparison to integrated, single-copy Lm Δ actA-OVA strains. Based on our previous data [9], we hypothesized that Lm Δ(trpS actA), a derivative of Lm ΔactA that is more easily manipulated to carry heterologous vaccine Ags on an extra-chromosomal multi-copy expression plasmid, might serve as a vaccine vehicle for neonatal immunization that is at least as effective as the Lm ΔactA with a chromosomally-integrated, single-copy, Ag-expressing casette. As a first step towards testing this hypothesis, we compared the new strain Lm Δ(trpS actA)/pSP0-PSactAOVA with Lm ΔactA-OVA through in vitro expression profiling. Expression of OVA protein was detected by Western Blot analysis in supernatants (i.e. secreted form) and cell pellets (i.e. nonsecreted form) only in strains expressing OVA, not in the control strains Lm Δ(trpS actA)/pSP0 and Lm ΔactA (Fig. 1). The new strain, Lm Δ(trpS actA)/pSP0-PSactAOVA, displayed a higher level of OVA production as compared to Lm ΔactA-OVA, both in the cell-bound as well as in the secreted fraction.  Appendix Figure 1. Multi-copy plasmid-containing Lm Δ(trpS actA)/pSP0-PSactAOVA strains produce more OVA protein (truncated OVA ∼25kD) in comparison to chromosomally-integrated single-copy Lm ΔactAOVA strains (full-length ∼50 kD). OVA protein expression and secretion of indicated Lm strains was determined by Western blot analysis using a purified rabbit anti-ovalbumin antiserum. Shown here are total cellular proteins and secreted proteins in supernatants of Lm cultures grown in BHI broth supplemented with 1% (w/v) AmberliteTM XAD-4. Lm, Listeria monocytogenes.  68  A high dose of Lm Δ (trpS actA)/pSP0-PSactAOVA induces primary total CD8 T cell responses in mouse neonates similar to a low dose of the Lm Δ actA-OVA strain. We next compared the capacity of the new strain Lm Δ(trpS actA)/pSP0-PSactAOVA to induce a vaccine-specific primary TC1 response in mouse neonates to that of the older Lm ΔactA-OVA strain in a direct, dose-dependent analysis. For this we injected 104, 105, 106 or 107 CFU of either strain intraperitoneally (i.p.) into neonatal mice and analyzed their splenocytes for OVA- and Lm peptide-specific CD8 T cells 10 days post infection. At immunization doses of 104-105 CFU, neonates immunized with Lm ΔactA-OVA contained a higher number of Ag-specific IFN-γ secreting CD8 T cells as mice immunized with the same dose of Lm Δ(trpS actA)/pSP0PSactAOVA (Fig. 2). Since we discovered that 107 CFU of Lm ΔactA-OVA per mouse was the 50% lethal dose (LD50) for neonates, we were not able to determine the amount of CD8 T cells in neonatal mice immunized with a dose higher than 106 CFU of Lm ΔactA-OVA. While always displaying lower OVA reactive fraction, neonatal mice immunized with Lm Δ(trpS actA)/pSP0PSactAOVA obtained similar overall percentages of Lm- and OVA-reactive CD8 T cells as mice immunized with Lm ΔactA-OVA when they were immunized with 106 or 107 CFU of Lm ΔactA-OVA. Thus, our new strain Lm ΔtrpS/pSP0-PSactAOVA is comparable to the Lm ΔactAOVA strain as a neonatal vaccine carrier when used at a dose of 107 CFU.  69  Appendix Figure 2. A dose of 107 CFU Lm Δ(trpS actA)/pSP0-PSactAOVA induces similar primary CD8 T cell responses in mouse neonates as the Lm ΔactA-OVA strain. Six-day old mice were immunized i.p. with respective strains at the indicated dose or with saline (Naïve). On day 10 after immunization, spleens were harvested to obtain splenocytes. Splenocytes were stimulated for 5 hours with Lm- and OVA- peptides, fixed and stained for IFN-γsecreting CD8 T cells. These data represent means ± standard deviation for groups of 3-5 mice from one representative out of two experiments.  Compared to adult mice, neonatally immunized mice develop a stronger Lm- and OVAspecific primary CD8 and CD4 T cell response. Given the above observation, we next assessed in detail the capacity of the Lm ΔtrpS/pSP0-PSactAOVA strain to generate an efficient Lm- and OVA-specific primary CD8 and CD4 T cell response in neonatal and adult mice by carrying out a dose-dependent analysis. To evaluate the optimal immunization dose of multi-copy plasmid strain Lm ΔtrpS/pSP0-PSactAOVA, mice were immunized with 104-108 CFU. Ten days after immunization, we enumerated Lm- and OVA-specific CD8 and CD4 T cells by intracellular IFN-γ staining of splenocytes after restimulation in vitro with Lm-specific MHC-class-I LLO9199,  p60217-225 and OVA257-264 or MHC-class-II LLO189-201 restricted peptides. We observed a dose-  70  dependent increase in Lm- and OVA-specific primary CD8 and CD4 T cells in adult as well as in neonatally immunized mice (Fig. 3). Remarkably, neonatally immunized mice developed a higher percentage of Lm- and OVA-specific primary CD8 (Fig.3B) and CD4 T cells (Fig. 3C) than mice immunized as adults. We also determined that 107 CFU as an immunizing dose led to the highest percentage of reactive T cells during the primary T cell response for both adult as well as neonatally immunized mice. We detected no pathologically appreciable side effects at any of the administered doses.  Appendix Figure 3. Neonatally immunized mice develop a stronger Lm- and OVA-specific CD8 (A, B) and CD4 (C) T cell primary response than mice immunized as adults. Neonatal and adult mice were immunized i.p. with increasing doses (104 to 108 CFU) of Lm Δ(trpS actA)/pSP0-PSactAOVA. Ten days after immunization, spleens were obtained from these mice, and splenocytes were stimulated with the indicated MHC class I- and MHCclass IIrestricted Lm- and OVA-specific peptides before analysis of intracellular IFN-γ cytokine secretion by flow cytometry. Shown in A for CD8 and CD4 are examples of the flow cytometric analysis. The mean percentage of IFN-γ-producing splenocytes are shown in (B) for CD8 and in (C) for CD4. Unstimulated controls are included in each experiment. These data present means ± standard deviation for 3-5 mice per age-group from one representative out of two experiments. n, neonates; a, adults.  71  Independent of in vivo subcellular location of Ag expression, neonatally immunized mice develop a broader primary T cell response than adult immunized mice. To further define the use of Lm Δ(trpS actA) as a neonatal vaccine platform, we determined the impact of in vivo subcellular location of vaccine Ag expression on primary T cell-mediated responses in neonatal and adult mice. To our knowledge, there are no published reports on the impact of subcellular location of vaccine Ag expression on primary T cell-mediated responses by Lm vaccine carriers in neonates. We constructed variants of the Lm Δ(trpS actA) (Table 1), each with a different Lm promoter that is predominantly active in the phagosomal (Phly) or cytosolic (PactA) compartment of the host cell [28, 29]. We also set out to test the impact of an added virulence-attenuation (Lm-specific phagelysin Ply118 under the control of PactA) on the primary response [23], with the goal of adding additional safeguards for future potential clinical applications. We thus compared a total of four different bacterial expression plasmids. Both neonatal and adult mice were immunized i.p. with 107 CFU of Lm Δ(trpS actA)/pSP0-PSactAOVA, Lm Δ(trpS actA)/pSP118PSactAOVA, Lm Δ(trpS actA)/pSP0-PShlyOVA or Lm Δ(trpS actA)/pSP118-PShlyOVA. Ten days after immunization splenocytes were stimulated with Lm- or OVA-specific peptides before analysis of intracellular IFN-γ secretion by flow cytometry. Strikingly, neonatally immunized mice generated significantly more Ag-specific CD8 and CD4 T cells in comparison to adult immunized mice in response to all of the four different Lm strains (Fig. 4A and B). In addition, neonatal immunized mice developed a broader spectrum of Ag specific CD8 T cells than adult mice immunized in the same manner: neonates recognized multiple epitopes of the heterologous Ag (LLO91-99, p60217-225 or OVA257-264) in a co-dominant fashion, while mice immunized as adults generated predominantly OVA257-264 reactive CD8 T cells (Fig. 4A). Detection of the subdominant epitopes LLO296-304, p60449-457 and mpl184-192 was similarly low in both adult and  72  neonatally immunized mice (Fig. 4A). The MHC-class-II restricted peptide OVA323-339 was not recognized by adult and neonatal CD4 T cells, whereas the dominant CD4 epitope LLO189-201 was strongly and equally recognized by both (Fig. 4B). Furthermore, mice immunized with selfdestructing strains Lm Δ(trpS actA)/pSP118-PSactAOVA and Lm Δ(trpS actA)/pSP118PShlyOVA harboured similar amounts of IFN-γ secreting CD8 and CD4 T cells compared to mice immunized with the non-self-destructing strains Lm Δ(trpS actA)/pSP0-PSactAOVA and Lm Δ(trpS actA)/pSP0-PShlyOVA (Fig. 4A and B). There was no significant difference detectable between the levels Ag-specific CD8 and CD4 T cells induced in mice immunized with Lm secreting OVA into the phagosomal compartment vs. those that express OVA predominantly in the cytosol of the host cell. Taken together, neonatal immunized mice developed a stronger and broader Lm- and OVA-specific primary T cell response than adult immunized mice. The selfdestructing Ply118-mediated lysis did not negatively impact the induction of a primary immune response. Furthermore, the secretion of the Ag by the respective carrier strains into phagosomal vs. cytosolic compartment had no appreciable impact on the generation of primary CD8 and CD4 T cell responses in either neonates or adults.  73  Appenxid Table 1: Strains and plasmids used in this work Strains and plasmids  Relevant genotype  References or source  Δ(trpS actA)/ pTRPS  [30]  ΔtrpS / pTRPS  [30]  Δ(trpS actA)/ pSP0-PSactAOVA  This work  Δ(trpS actA)/ pSP118-PSactAOVA  This work  Δ(trpS actA)/ pSP0-PShlyOVA  This work  Δ(trpS actA)/ pSP118-PShlyOVA  This work  ΔactA-OVA  [9]  -OVA  Dr. H. Shen  pSP0- PSactAOVA  EmR, trpS, (PS)actA-ova-TinlA  [23]  pSP118- PSactAOVA  EmR, trpS, PactA-ply118, (PS)actA-ova-TinlA  [23]  pSP0- PShlyOVA  EmR, trpS, (PS)hly-ova-TinlA  [46]  pSP118 -PShlyOVA  EmR, trpS, PactA-ply118, (PS)hly-ova-TinlA  [46]  Listeria monocytogenes EGDe strains  Listeria monocytogenes strain 10403s  Plasmids  74  Appendix Figure 4. Neonatally immunized mice develop a broader Lm- and OVA-specific primary T cell response than adult immunized mice, which is not dependent on subcellular location of Ag expression. Neonatal and adult mice were immunized i.p. with 107 CFU of Lm Δ(trpS actA)/pSP0-PSactAOVA, Lm Δ(trpS actA)/pSP118-PSactAOVA, Lm Δ(trpS actA)/pSP0-PShlyOVA or Lm Δ(trpS actA)/pSP118-PShlyOVA. Ten days after immunization, spleens were removed and the splenocytes stimulated with the indicated MHC class I- and MHC class II-restricted Lm- and OVA-specific peptides before analysis of intracellular IFN-γ cytokine secretion by flow cytometry. Unstimulated controls are included. CD8 T cell responses are shown in (A) and CD4 T cell responses in (B). These data are expressed as means ± standard deviation and represent 3-5 mice per age-group from one representative out of two experiments.  Neonatally immunized mice develop protective Lm- and OVA-specific secondary response that is dependent on the subcellular location of Ag expression. We next wished to characterize which of the four strains - Lm Δ(trpS actA)/pSP0-PSactAOVA, Lm Δ(trpS actA)/pSP118-  75  PSactAOVA, Lm Δ(trpS actA)/pSP0-PShlyOVA and Lm Δ(trpS actA)/pSP118-PShlyOVA - would provide the most efficient protection against challenge with wild-type Lm. To this end, we immunized neonates and adults with 107 CFU and challenged them, along with naïve control mice, six weeks later with 5 × 106 CFU of Lm-OVA. Three days after challenge, we determined CFUs in spleen and liver. Five days after challenge we also enumerated Lm- and OVA-specific T cells in splenocytes by intracellular IFN-γ staining. As shown in Fig. 5A and 5B, both immunized adults and neonates displayed a robust CD8 and CD4 T cell secondary response. There was no appreciable difference in immunodominance for the secondary response between mice immunized as neonates or as adults. Neonatal and adult Lm Δ(trpS actA)/pSP0-PShlyOVAand Lm Δ(trpS actA)/pSP118-PShlyOVA-immunized mice displayed a CD8 T cell secondary response completely dominated by the OVA257-264 response, whereas neonatal and adult immunized mice immunized with Lm Δ(trpS actA)/pSP0-PSactAOVA, Lm Δ(trpS actA)/pSP118PSactAOVA elicited a broader CD8 T cell memory response to LLO91-99, p60217-225 and OVA257264  (Fig. 5A1 and 5A2). In summary, subcellular location of expression strongly affects  immunodominance. Strikingly, mice immunized as neonates with Lm Δ(trpS actA)/pSP0PShlyOVA and Lm Δ(trpS actA)/pSP118-PShlyOVA displayed significantly better protection than mice immunized as neonates with Lm Δ(trpS actA)/pSP0-PSactAOVA, Lm Δ(trpS actA)/pSP118PSactAOVA (Fig. 5C1 and 5C2) in direct comparison to the naive control group. Furthermore, only the adult mice immunized with Lm Δ(trpS actA)/pSP0-PShlyOVA showed a significant reduction in bacterial counts in liver and spleen in comparison to the naïve control group (Fig. 5C2). In conclusion, expression of bacterial Ag into the phagosomal compartment promoted protective immune responses more effectively than expression in the cytosol.  76  Appendix Figure 5. Neonatally immunized mice develop an Lm- and OVA-specific secondary response that provides protection, which is dependent on subcellular location of Ag expression. Mice immunized i.p. with 107 of Lm Δ(trpS actA)/pSP0-PSactAOVA, Lm Δ(trpS actA)/pSP118-PSactAOVA, Lm Δ(trpS actA)/pSP0-PShlyOVA or Lm Δ(trpS actA)/pSP118-PShlyOVA on day 6 of life (Neonate) or at 6 weeks of age (Adult) were infected i.v. with 5 × 106 CFU of wild-type Lm-OVA 6 weeks after immunization. Splenocytes were obtained from the indicated mice 5 days after infection along with age-matched non-immune mice (Naïve) and stimulated with the indicated MHC class I- and MHC class II-restricted Lm- and OVA-specific peptides before analysis of intracellular IFN-γ cytokine secretion by flow cytometry. Unstimulated controls are included. CD8 T cell responses are shown in (A), CD4 T cell responses in (B) and bacterial counts in liver and spleen in (C). These data represent 3-5 mice per age-group from one representative out of two experiments. The results are expressed as means ± standard deviation. An asterisk indicates a statistically significant difference (*=p<0.05 and **=p<0.01 as determined by one-way ANOVA) between experimental and control group.  77  Neonates, not adults, generate a strong IgG antibody response against ovalbumin after immunization with Lm. Finally, we investigated if our two most promising strains, Lm Δ(trpS actA)/pSP0-PShlyOVA and Lm Δ(trpS actA)/pSP118-PShlyOVA, i.e. those that protected neonatal mice most efficiently in vivo, could also generate a humoral immune response against the model vaccine Ag ovalbumin. Serum from mice immunized as neonates or adult with 107 CFU of Lm Δ(trpS actA)/pSP0-PShlyOVA or Lm Δ(trpS actA)/pSP118-PShlyOVA was examined by ELISA for the presence of IgG antibodies against OVA 14 days after challenge with 5 × 106 CFU of wild-type Lm-OVA 6 weeks after primary immunization. Surprisingly, only neonates immunized with Lm Δ(trpS actA)/pSP0-PShlyOVA and Lm Δ(trpS actA)/pSP118-PShlyOVA developed significant titers of IgG antibodies against ovalbumin (Fig. 6).  Appendix Figure 6. Only neonatally immunized mice, not mice immunized as adults, generate a strong IgG antibody response against ovalbumin. Mice were immunized i.p. with 107 CFU of Lm Δ(trpS actA)/pSP0PShlyOVA and Lm Δ(trpS actA)/pSP118-PShlyOVA, six weeks later challenged with 5 × 106 CFU of wild-type LmOVA given i.v. and sera were collected and analyzed for anti-OVA IgG antibodies 14 days after challenge. Absorbance at 450 nm minus at 570 nm of different serial dilutions of serum is shown. Serum from mice boosted three times with WT Lm-OVA served as the positive control, and serum from a non-infected mouse served as negative control. Each symbol indicates the means for the experimental groups (n=4 each). An asterisk indicates a statistically significant difference (p<0.05 as determined by one-way ANOVA) between experimental and control groups. The results of one representative out of two experiments are shown.  78  Discussion Effective yet safe vaccines to be administered in the first days to weeks of life are urgently needed. This report confirms that virulence-attenuated strains of Lm can be used as safe and effective vaccine carriers for newborns. Our present study also showed the following: First, Lm Δ(trpS actA)-based strains, which express and secrete multiple copies of OVA either under the control of a phagosomal (Phly)- or cytosolic (PactA)-driven listerial promoter, elicited similarly high levels of Ag-specific primary CD8 and CD4 T cell responses with just a single immunization. Second, neonatally immunized mice developed a stronger and broader dosedependent Ag-specific primary T cell response than mice immunized as adults. Third, both neonatal and adult mice immunized with the phagosomal-driven strains were significantly better protected against a lethal wild-type Lm challenge as compared to mice immunized with the cytosolic-driven strains. Lastly, only neonatal (not adult) mice immunized with the phagosomaldriven strains generated high IgG antibody responses against the model vaccine Ag OVA.  We have recently shown that Lm is suitable as a neonatal vaccine vehicle, requiring only a single immunization at birth to induce life-long protection [9]. We now wished to develop highly attenuated Lm strains to increase safety for clinical applications of Lm as a vaccine vehicle in neonates. Furthermore, the integration of vaccine Ag expression cassettes into the chromosome as required for Lm ΔactA is time-consuming and labour-intensive. To increase ease of manipulation, we set out to develop a plasmid-encoded Lm-based vaccine Ag expressing system. For these purposes (safety and ease of vaccine Ag expression) we focused on the virulenceattenuated Lm Δ(trpS actA) carrier strains that harbour the Ag expression cassette as well as the essential listerial trpS gene (TrpS, tryptophanyl-tRNA-synthetase) on a balanced-lethal plasmid.  79  A plasmid-based, balanced-lethal system absolutely requires the stability of the plasmid since the essential protein required by Listeria to survive is expressed on the plasmid. The loss of the plasmid results in cell death [22]. The Lm Δ(trpS actA) balanced-lethal plasmid system has already proven its stability and ability as a vaccine carrier in adult mice [23, 30]. We found that carrier strains of Lm ((trpS actA)/pSP0-PSactAOVA were indeed able to elicit primary Agspecific CD8 T cell responses in neonatal and adult mice comparable to strain Lm (actA–OVA). A comparable primary immune response required a higher immunizing dose for Lm ((trpS actA)/pSP0-PSactAOVA (Fig. 2). This dose-dependent difference was expected, indeed desired, since the Lm ((trpS)/pSP0-PSactAOVA parental strain is anticipated to be more attenuated than the Lm (actA -OVA’s parental strain Lm-OVA. The higher level of Ag expression driven by the multi-copy plasmids creates a metabolic burden reducing the overall fitness of Lm ((trpS actA) as compared to Lm (actA (reviewed in [31]). We confirmed these assumptions by Western blot analysis of cell pellet and supernatant fractions of both Lm Δ(trpS actA)/pSP0-PSactAOVA and Lm ΔactA–OVA strains (Fig. 1), and by detecting lower numbers of bacteria three days after infection with the respective parental strains, Lm Δ(trpS)/pSP0-PSactAOVA in comparison to Lm– OVA (data not shown). We thus had found what we were looking for: an easy to manipulate, high-level vaccine Ag expressing strain of Lm that is greatly attenuated yet effective as a neonatal vaccine carrier.  During these initial investigations, we also determined that an immunizing dose of 107 CFU of Lm Δ(trpS actA)/pSP0-PSactAOVA leads to the most efficient primary T cell response for both adult and neonatally immunized mice (Fig. 3). These results are in accordance with literature in that the CD8+ T cell expansion after Lm infection is primarily dependent on the initial infection  80  dose or amount of Ag displayed [32, 33]. Similarly to what we had described for Lm ΔactA– OVA already, mice immunized with Lm Δ(trpS actA)/pSP0-PSactAOVA as neonates developed stronger Lm- and OVA-specific primary CD8 and CD4 T cell responses than mice immunized as adults (Fig. 3).  The use of Lm strains that secrete the foreign protein under the transcriptional control of a phagosomal (Phly)- or cytosolic (PactA)-driven listerial promoter results in bacterial proteins having access to both MHC class II and class I molecules for Ag presentation to CD4 and CD8 T cells, respectively [20, 28, 34]. For many pathogens, both subsets of T cells are needed to provide optimal protective immunity [35]. Therefore, we investigated the impact the subcellular location of Ag expression would have on the subsequent immune response. Surprisingly, the subcellular location of Ag expression had no impact at all on the generation of the primary immune response in all four strains tested (Fig. 4). In contrast, immunodominance of the secondary response and, more importantly, protection were significantly affected by phagosomal vs. cytosolic Ag expression (Fig. 5A and 5C). In comparison to the naive control group, neonates, which had been immunized with Lm Δ(trpS actA) strains that secrete OVA into the phagosome, were much better protected against wild-type Lm challenge than neonatal mice immunized with strains that secrete OVA into the cytosol (Fig. 5C1). We are currently in the process of elucidating which of the Ag-specific immune responses (Lm or OVA) are primarily responsible for this surprising observation. We hypothesize that the various Lm carrier strains may have triggered different signalling pathways in the infected Ag-presenting cell, which in turn promoted a differential CD8 T cell response. For example, bacterial ligands generated in the phagosomal compartment are also targets of the cytosolic innate immune system receptors, such  81  as NOD2 [36], and distinct differences in signalling patterns, intensities, enhancement of DC maturations and T cell differentiation and function after infection with phagosomal vs. cytosolic localized Lm have been demonstrated before [37-40].  The subcellular location of Ag expression did affect the epitope hierarchy of the CD8 T cell response to Lm. CD8 T cells often focus on a few epitopes out of thousands available. In this study, we confirmed our previous findings that neonates have broader epitope recognition. LLO91-99, p60217-225, and OVA257-264 were recognized similarly by neonatal CD8 T cells in the primary response. On the other hand, the adult response was severely restricted to OVA257-264, with LLO91-99, p60217-225, p60449-457, and mpl84-92 barely recognized at all. This type of adult response is in accordance with the published record [41]. Adult IFN-γ KO mice also develop a broader CD8 T cell response [42, 43] akin to the broad neonatal response we have observed. Neonates are known to produce reduced levels of IFN-γ as compared to their adult counterparts [44]. We are currently in the process of identifying the responsible mechanisms for these observations, but hypothesize that IFN-γ is centrally involved and may influence e.g. T cell repertoire selection, T cell sensitivity, or specific T cell effector functions involved in the neonate’s broader and wider epitope recognition.  Surprisingly, we found that immunized neonates are able to generate IgG antibodies against OVA compared to immunized adults (Fig. 6). Why adult mice immunized in the same manner did not mount a detectable humoral response is not clear. It is known that exposure to Ag during the neonatal period leads to ‘imprinting’ of Th2 dominance that is maintained into adulthood. Th2 cytokines promote the preferential production of Th2-associated IgG1 [45]. Thus, the net  82  effector function associated with different Ig isotypes is expected to be different in mice immunized as adults vs. those immunized as neonates [45]. The possibility that our Lm Δ(trpS actA) strains secreting vaccine Ags into the phagosome induces both protective CD4 and CD8 T cell memory response, as well as a robust antibody response is of great importance in our long term goal of developing a single-dose, broadly protective neonatal vaccine platform. Further studies on the subclass, affinity, affinity maturation, and neutralizing capacity of these neonatal antibody responses are currently under way.  In this study we also characterized the use of self-destructing carrier strains in order to provide additional safeguards for employing Lm-based vaccine vehicles for neonatal immunization. The use of bacterial carriers that destroy themselves after several rounds of replication via Ply118 listerial phage-mediated lyses [23] did not affect the primary or secondary CD8 and CD4 T cell response, or the resulting protection (Fig. 4, 5). We believe the Lm-specific phagelysin Ply118 under the control of PactA encoded on the balanced-lethal plasmid designed to lyse bacteria upon entry of the cytosolic compartment of the infected cell adds an important safety component to this platform.  In summary, we identified Lm Δ(trpS actA) as an ideal vaccine vehicle for neonatal immunization. It is safe at high doses, and induces a strong primary and secondary immune response. Most importantly, it induces protection from challenge with wild-type Lm after only one immunization given around birth. In this report, we confirm and extend our previous observation of broader epitope recognition by CD8 T cells in neonates as compared to adults, and add to the advantages of neonatal Lm-based vaccination a stronger IgG response to the  83  vaccine Ag in neonates as compared to adults. Lm Δ(trpS actA) is highly attenuated, and given the multi-copy balanced-lethal plasmid platform available in this strain, allows easy manipulation to rapidly fine-tune the desired vaccine response. In this study, we have shown this advantage and demonstrated that a) a predominantly phagosomal vaccine Ag plasmid expression cassette provides the most optimal protection, and; b) additional attenuation through a plasmidencoded phage-mediated suicide vector does not negatively affect the immune response or protection. We believe that Lm Δ(trpS actA) will rapidly allow dissection of many parameters important for successful neonatal immunization, and will promote the development of rational vaccine design for early life immunization against infectious diseases such as Malaria or Whooping cough.  84  References: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.  Siegrist, C.A., The challenges of vaccine responses in early life: selected examples. J Comp Pathol, 2007. 137 Suppl 1: p. S4-9. Adkins, B., C. Leclerc, and S. Marshall-Clarke, Neonatal adaptive immunity comes of age. Nat Rev Immunol, 2004. 4(7): p. 553-64. Siegrist, C.-A., Vaccination in the Neonatal Period and Early Infancy. International Reviews of Immunology, 2000. 19(2): p. 195 - 219. Siegrist, C.-A., Neonatal and early life vaccinology. Vaccine, 2001. 19(25-26): p. 33313346. Wilson CB and K. TR., Induction of antigen-specific immunity in human neonates and infants. Nestle Nutr Workshop Ser Pediatr Program, 2008. 61: p. 183-95. Wilson CB, Lewis DB, and E. BK., T cell development in the fetus and neonate. Adv Exp Med Biol. , 1991. 310: p. 17-27. Marchant, A., et al., Mature CD8(+) T lymphocyte response to viral infection during fetal life. J Clin Invest, 2003. 111(11): p. 1747-55. Marchant, A. and M. Goldman, T cell-mediated immune responses in human newborns: ready to learn? Clin Exp Immunol, 2005. 141(1): p. 10-8. Kollmann, T.R., et al., Induction of Protective Immunity to Listeria monocytogenes in Neonates. J Immunol, 2007. 178(6): p. 3695-3701. Fadel, S.A., D.A. Ozaki, and M. Sarzotti, Enhanced type 1 immunity after secondary viral challenge in mice primed as neonates. J Immunol, 2002. 169(6): p. 3293-300. Kotton, C.N. and E.L. Hohmann, Enteric pathogens as vaccine vectors for foreign antigen delivery. Infect Immun, 2004. 72(10): p. 5535-47. Fouts, T.R., et al., Progress toward the development of a bacterial vaccine vector that induces high-titer long-lived broadly neutralizing antibodies against HIV-1. FEMS Immunol Med Microbiol, 2003. 37(2-3): p. 129-34. Bruhn, K.W., N. Craft, and J.F. Miller, Listeria as a vaccine vector. Microbes Infect, 2007. 9(10): p. 1226-35. Brockstedt, D.G. and T.W. Dubensky, Promises and challenges for the development of Listeria monocytogenes-based immunotherapies. Expert Rev Vaccines, 2008. 7(7): p. 1069-84. Soussi, N., et al., Listeria monocytogenes as a short-lived delivery system for the induction of type 1 cell-mediated immunity against the p36/LACK antigen of Leishmania major. Infect Immun., 2000. 68(3): p. 1498-506. Angelakopoulos, H., et al., Safety and shedding of an attenuated strain of Listeria monocytogenes with a deletion of actA/plcB in adult volunteers: a dose escalation study of oral inoculation. Infect. Immun., 2002. 70(7): p. 3592-3601. Lieberman, J. and F.R. Frankel, Engineered Listeria monocytogenes as an AIDS vaccine. Vaccine, 2002. 20(15): p. 2007-2010. Stevens, R., et al., Pre-existing immunity to pathogenic Listeria monocytogenes does not prevent induction of immune responses to feline immunodeficiency virus by a novel recombinant Listeria monocytogenes vaccine. Vaccine, 2005. 23(12): p. 1479-90. Vazquez-Boland, J.A., et al., Listeria Pathogenesis and Molecular Virulence Determinants. Clin. Microbiol. Rev., 2001. 14(3): p. 584-640.  85  20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.  Pamer, E.G., Immune responses to Listeria monocytogenes. Nat. Rev. Immunol., 2004. 4(10): p. 812-23. Bauer, H., et al., Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids. Gene Ther, 2005. 12(4): p. 364-72. Spreng, S. and J.-F. Viret, Plasmid maintenance systems suitable for GMO-based bacterial vaccines. Vaccine, 2005. 23(17-18): p. 2060-2065. Loeffler, D.I., et al., Comparison of different live vaccine strategies in vivo for delivery of protein antigen or antigen-encoding DNA and mRNA by virulence-attenuated Listeria monocytogenes. Infect Immun, 2006. 74(7): p. 3946-57. Khanolkar, A., V. Badovinac, and J. Harty, CD8 T cell memory development: CD4 T cell help is appreciated. Immunologic Research, 2007. 39(1): p. 94-104. Park, S.F. and G.S. Stewart, High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells. Gene, 1990. 94(1): p. 129-32. Ermolaeva, S., et al., Negative control of Listeria monocytogenes virulence genes by a diffusible autorepressor. Molecular Microbiology, 2004. 52(2): p. 601-611. Laemmli, U.K., Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 1970. 227(5259): p. 680-685. Moors, M.A., et al., Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes. Infect. Immun., 1999. 67(1): p. 131-9. Bubert, A., et al., Differential expression of Listeria monocytogenes virulence genes in mammalian host cells. Mol Gen Genet, 1999. 261(2): p. 323-36. Pilgrim, S., et al., Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery. Gene. Ther., 2003. 10(24): p. 2036-45. Galen, J.E. and M.M. Levine, Can a `flawless' live vector vaccine strain be engineered? Trends in Microbiology, 2001. 9(8): p. 372-376. Badovinac, V.P., B.B. Porter, and J.T. Harty, Programmed contraction of CD8+ T cells after infection. Nat. Immunol., 2002. 3(7): p. 619-26. Montoya, M. and M. Del Val, Intracellular rate-limiting steps in MHC class I antigen processing. J. Immunol., 1999. 163(4): p. 1914-1922. Bubert, A., et al., Differential expression of Listeria monocytogenes virulence genes in mammalian host cells. Mol. Gen. Genet., 1999. 261(2): p. 323-36. Janssen, E.M., et al., CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature, 2003. 421(6925): p. 852-6. Herskovits, A.A., V. Auerbuch, and D.A. Portnoy, Bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system. PLoS Pathog, 2007. 3(3): p. e51. Bahjat, K.S., et al., Cytosolic entry controls CD8+-T-cell potency during bacterial infection. Infect Immun, 2006. 74(11): p. 6387-97. Brzoza, K.L., A.B. Rockel, and E.M. Hiltbold, Cytoplasmic entry of Listeria monocytogenes enhances dendritic cell maturation and T cell differentiation and function. J Immunol, 2004. 173(4): p. 2641-51. McCaffrey, R.L., et al., A specific gene expression program triggered by Gram-positive bacteria in the cytosol. Proc Natl Acad Sci U S A, 2004. 101(31): p. 11386-91. Leber, J.H., et al., Distinct TLR- and NLR-mediated transcriptional responses to an intracellular pathogen. PLoS Pathog, 2008. 4(1): p. e6.  86  41. 42. 43. 44. 45. 46.  Busch, D.H. and E.G. Pamer, MHC Class I/Peptide Stability: Implications for Immunodominance, In Vitro Proliferation, and Diversity of Responding CTL. J Immunol, 1998. 160(9): p. 4441-4448. Badovinac, V.P. and J.T. Harty, Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells. J Immunol Methods, 2000. 238(1-2): p. 107-17. Skoberne, M. and G. Geginat, Efficient in vivo presentation of Listeria monocytogenesderived CD4 and CD8 T cell epitopes in the absence of IFN-gamma. J Immunol, 2002. 168(4): p. 1854-60. Byun, H.J., et al., An evaluation of the neonatal immune system using a listeria infection model. Neonatology, 2007. 92(2): p. 83-90. Adkins, B., Neonatal T cell function. J Pediatr Gastroenterol Nutr, 2005. 40 Suppl 1: p. S5-7. Schoen, C., et al., Listeria monocytogenes as novel carrier system for the development of live vaccines. International Journal of Medical Microbiology, 2008. 298(1-2): p. 45-58.  87  Appendix C Introduction This report is a supplementary file to the “Neonatal immunization with Listeria monocytogenes induces T cells with an adult-like avidity, sensitivity, and TCR-Vβ repertoire, and does not adversely impact the response to boosting” paper by Smolen, K et al. 2009, describing the flow cytometry experimental details in compliance with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard. 1.  Experimental Overview 1.1. Purpose The purpose of these experiments was to determine the difference in the adult and neonatal T cell response after Lm-ΔactA OVA immunizations. - Experiment 1: Flow cytometric analysis was used to compare the T cell functional response measured using IFN-γ production between adults and neonates after primary immunization and/or subsequent boosts. - Experiment 2: Flow cytometric analysis was used to identify the T cell receptor Vβ repertoire usage in the adult and neonate after primary immunization and/or subsequent boosts. 1.2. Keywords T cell, neonatal immunology, vaccination, boost response, TCR, rodent 1.3. Experimental Variable 1.3.1. Splenocytes from mice immunized once, twice, or three times were used to measure the T cell response. There were 3 mice per group plus a naïve control mouse, and a total of two groups per immunization level. 1.4. Organization 1.4.1. Name: Child and Family Research Institute 1.4.2. Address: 938 West 28th Ave., Vancouver, BC, V5Z 4H4, Canada (Rm A4-104) 1.5. Primary Contact 1.5.1. Name: Kinga Smolen 1.5.2. Email: ksmolen@interchange.ubc.ca 1.6. Data Experiments and data collection ran from Dec 2007 to Jan 2009, while flow cytometry analysis preformed on various dates between Jan 2008 and Jan 2009. 1.7. Conclusion 1.7.1. Experiment 1: Mice immunized as neonates reached their maximum magnitude of functional CD8+ T cell expansion after the primary immunization. Subsequent boosts did not increase the magnitude of the overall CD8+ T cell expansion. In contrast, mice immunized as adults achieved modest functional CD8+ T cell primary  88  expansion. After one additional boost, mice immunized as adults reached the same maximal magnitude of functional CD8+ T cell expansion as mice immunized as neonates. 1.7.2. Experiment 2: Number of different Vβ chains involved in the OVA257-264 specific CD8+ T cell response was roughly the same between mice first immunized as neonates and as adults. The number of Vβ chains employed in the specific CD8+ T cell response did not change appreciably between primary, secondary, or tertiary responses in mice first immunized either as neonates or as adults. 1.8. Quality Control Measures Experiment 1: Splenocytes were collected from immunized and naïve mice, the naïve controls were used to establish baseline-level response and staining control. Experiment 2: Splenocytes were collected from immunized and naïve mice, the naïve controls were used to establish base-level response and staining control. In addition, to the naïve mice a non-tetramer stain was incorporated to help distinguish the positive and negative tetramer responses within the immunized mice group. 2.  Flow Sample Details 2.1. Sample Material Description 2.1.1. Biological Samples 2.1.1.1. Biological Sample Description: Whole spleens were collected, homogenized, and aliquoted into 96-well plates (for ICC Staining (Experiment 1) or Surface Staining (Experiment 2)). 2.1.1.2. Biological Sample Source Description: Mus musculus. F1 mice (H-2b x H2d) derived from mating between C57BL/6 (H-2b) and C57B10.D2 (H-2d) mice 2.1.1.3. Biological Sample Source Organism Description: - Taxonomy: Mus musculus: F1 from the cross between C57BL/6 (H-2b) and C57B10.D2 (H-2d) - Age: Primary Immunization is 10d (neonate) or 6 wks (adult) Secondary Immunization is primary plus 6wks Tertiary Immunization is secondary plus 4 wks - Gender: female and male - Phynotype: cross: C57BL/6 (H-2b) and C57B10.D2 (H-2d) - Treatment: o Mice were immunized once, twice, or three times o Primary response: adult and newborn mice were immunized i.p. once with 106 CFU Lm ΔactA-OVA. 10 days post infection the splenocytes were harvested and analyzed as outlined below (i.p. infection was used for the primary immunization as i.v. is too difficult in neonatal mice). o Secondary response: mice were initially immunized i.p. and 6 weeks post initial infection the mice were boosted i.v. Five days post boost the splenocytes were harvested and analyzed as outlined below. o Tertiary response: mice were initially immunized i.p.; at 6 weeks the mice were boosted i.v. and after another 4 weeks boosted i.v.  89  again. Five days after the second boost the splenocytes of the mice were harvested and analyzed as outlined below. - Other Relevant Biological Sample Source Organism Information: Cage numbers and mouse ID have been used as sample identifier. 2.1.1.4. Other Relevant biological Sample Source Organism Information: All animals were housed under specific pathogen-free conditions at the Child and Family Research Institute of the University of British Columbia. All animal experiment protocols were approved by the Institutional Animal Care and Use Committee. 2.1.2. Environmental Samples N/A 2.1.3. Other Samples N/A 2.2. Sample Characteristics The whole mouse spleen tissue was used for these experiments. 2.3. Sample Treatment Description 2.3.1. Experiment 1: Intracellular Cytokine Stain - Single cell suspensions: spleens were homogenized between two sterile glass slides, subjected to RBC lysis, and filtered through a 70-µm cell strainer. - Two million splenocytes were incubated for 5 h in 200 µl of RPMI 1640 medium supplemented with 10% FCS (HyClone), L-glutamine, penicillin, and streptomycin in the presence or absence of 0.5 µM (unless otherwise noted) OVA257-264 or LLO190-201 and 10 µg/ml Brefeldin A. - Cells were permeabilized with Cytofix/Cytoperm solution (BD Pharmingen) and stained with APC-labeled anti-IFN-γ for 45 min at RT together with surface staining for CD3, CD4, and CD8 with FITC-labeled anti-CD3 and either PerCP-labeled anti-CD8 or anti-CD4 - Stained cells were acquired on a FACSAria flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). 2.3.2. Experiment 2: Surface Stain - Splenocyte suspensions: spleens were homogenized between two sterile glass slides, subjected to RBC lysis, and filtered through a 70-µm cell strainer. - Two million splenocytes were resuspended in100 µl of PBSAN (PBS, 10% NaN3, 5% BSA). To detect the Ag-specificity of the T cells, tetramers specific for the OVA257-264 epitope were used. - Cells were stained with PE-labeled PE-H-2Kb/OVA257–264 tetramer, Alexa75-labeled anti-CD3, PerCP-labeled anti-CD8, and FITC-labeled anti-TCR Vβ panel out of 15 different Vβ chains for 45 minutes at RT in the dark. - Stained cells were acquired on a FACSAria flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).  90  2.4. Fluorescence Reagent Description 2.4.1. Experiment 1 Each sample for the ICC Stain has been stained with the following: Reporter FITC PerCP APC Sample +Ab +Ab +Ab  Reporter Tube #1 Tube #2 Tube #3  Compensation tubes: FITC +Ab -  Reagents used: Characteristic Analyte T cell CD3 T cell suppressor CD8 T helper CD4 IFN- γ cytokine IFN-γ  PerCP +Ab Detector Anti-CD3 Anti-CD8 Anti-CD4 Anti-IFN-γ  APC +Ab Reporter FITC PerCP PerCP APC  Manufacture eBioscience BD BD BD  Cat #. 11003185 553036 553052 554431  2.4.2. Experiment 2 Each sample for the ICC Stain has been stained with the following: Reporter FITC PerCP Alexa-750 PE Sample +Ab +Ab +Ab +Ab Reporter Tube #1 Tube #2 Tube #3 Tube #4  Compensation tubes: FITC +Ab -  Reagents used: Characteristic Analyte T cell CD3 T cell suppressor CD8 TCR Vβ TCR OVA257-264 Tetramer Tetramer  PerCP +Ab Detector Anti-CD3 Anti-CD8 Anti-TCR Vβ MHC class I OVA complex  Alexa-750 +Ab Reporter Alexa-750 PerCP FITC PE  PE +Ab  Manufacture eBioscience BD BD BD  Cat #. 27003282 553036 557004 T03000  91  3.  Instrumental Details 3.1. Instrument Manufacturer BD Biosciences http://www.bdbiosciences.com/home/ 3.2. Instrumental Model BD FACSAria Flow Cytometer http://www.bdbiosciences.com/immunocytometry_systems/products/display_product.ph p?keyID=53 Serial number: P22300055 Technical specification at: http://www.bdbiosciences.com 3.3. Instrument Configuration and Setting 3.3.1. Flow Cell and Fluidics The instrument has not been altered. 3.3.2. Light Sources The instrument has not been altered; three-laser base configuration with ACDU. - 488nm Coherent Sapphire solid state - 633nm JDS Uniphase HeNe air-cooled - 407nm Point Source Violet solid state 3.3.3. Excitation Optics Configuration The instrument has been altered 3.3.4. Optical Filters 720/40 BP filter has been added to the red laser 3.3.5. Optical Detectors Optical Paths Instrument has not been altered 3.3.6. Other Relevant Instrument Details  Detector Array (Laser) Octogen (488nm blue laser)  Trigon (633nm red laser) Trigon (407nm violet laser)  PMT A B C D E F A B C A B  LP Mirror 735 655 595 556 502 735 685 502 -  BP Filter 780/60 695/40 610/20 575/26 530/30 488/10 780/60 720/40 660/20 530/30 450/40  Intended Dye PE-Cy7 PerCP-Cy5.5 PE-Texas Red PE FITC SSC APC-Cy7 APC Alexa-430, Hoeschst, DAPI Cascade blue, Pacific blue, Alexa 405  92  4.  Data Analysis Details 4.1. List-mode Data Files FCS data files can be obtained by contacting Kinga Smolen after this work has been published. 4.2. Compensation Description Shown below are the compensations for representative experiments. Compensations from all the experiments are available upon request after this work has been published.  Avidity Experiment compensation: FITC-A PerCP-A FITC-A 100 12.46 PerCP-A 0 100 APC-A 0 0.934  APC-A 2.795 3.317 100  TCR Experiment compensation: FITC-A PerCP-A FITC-A 100 0.0329 PerCP-A 0.00526 100 Alexa-750-A 0.00403 .00175 PE-A 0.01630 0.1850  Alexa-750-A 0.0 0.0157 100 .00156  PE-A 0.18600 0.00242 0.00138 100  4.3. Data Transformation Details 4.3.1. Purpose of Data Transformation Visualization and gating 4.3.2. Data Transformation Description FlowJo default visualization settings have been used for gating. 4.4. Gating (Data Filtering) Details Same gating strategy has been used for all the files in the respective experiments. 4.4.1. Gating boundaries and statistics Experiment 1: Avidity experiment gating strategy:  93  Experiment 2: TCR experiment gating strategy:  94  Appendix D https://rise.ubc.ca/rise/Doc/0/NNAKVTP7U23K3AFB0CI4CMEB1B/fromString.html  22/02/10 10:30 AM  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE BREEDING PROGRAMS  Application Number: A06-0023 Investigator or Course Director: Tobias Kollmann Department: Paediatrics Animals:  Mice F1 500 Mice C57Bl.6 30 Mice B10D.2 150  Approval Date: February 8, 2007 Funding Sources: Funding Agency: Funding Title:  Unfunded title:  Burroughs Wellcome Fund Induction of protective immunity to listeria in neonates  N/A  The Animal Care Committee has examined and approved the use of animals for the above breeding program. This certificate is valid for one year from the above approval date provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  Page 1 of 2  95  https://rise.ubc.ca/rise/Doc/0/NNAKVTP7U23K3AFB0CI4CMEB1B/fromString.html  22/02/10 10:30 AM  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  Page 2 of 2  96  

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