Microbiology & Immunology - Theses
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ItemGenerating an effective T cell-based influenza vaccine( 2023)Protective immunity against influenza virus is heavily dependent on humoral and cellular immune responses. Current influenza vaccines predominantly utilise antibody immunity, but as this is strain-specific, it leaves the population vulnerable to antigenic drift by influenza virus and importantly fails to protect against novel pandemic strains. CD8+ T cell immunity, on the other hand, due to its ability to recognise highly conserved antigenic determinants of influenza virus, enables the possibility of broadly protective universal immunity. In particular, CD8+ T cells that reside within the lung called tissue-resident memory T cells (TRM) are the responsible subset mediating cross-protection against influenza virus. However, current vaccines either do not or poorly generate CD8+ T cell responses. Therefore, this PhD thesis investigated the capacity of a novel T cell-based vaccine candidate to elicit lung CD8+ TRM and critical parameters required for the optimal induction of cross-protective influenza-specific lung CD8+ TRM. We investigated the cellular immune response evoked following a single-cycle replication-incompetent influenza vaccine candidate called S-FLU. Intranasal S-FLU immunisation generated lung CD8+ T cells and CD8+ TRM of reduced magnitude and functional avidity relative to natural influenza virus infection controls. Interestingly, the limited inflammatory profile of S-FLU immunisation conferred a clonally diverse CD8+ T cell and TRM profile in the lung. As a result, a greater propensity of these cells cross-reacted against a naturally occurring variant and prevented the development of T cell escape mutants. Our findings suggest the inflammatory milieu of a vaccine is an important consideration as this may influence the T cell receptor repertoire, resulting in downstream alterations in the cross-reactivity and capacity to subvert viral variants. Vaccine studies investigating protective efficacy must take into consideration pre- existing influenza-specific immunity generated by prior infections and the annual vaccination regime that occur over the course of an individual’s lifetime. Using a panel of live attenuated influenza virus vaccine candidates (cold-adapted and single-cycle), we next investigated the capacity of live attenuated influenza vaccines to elicit lung CD8+ TRM responses in the face of pre-existing immunity against the vaccine backbone. We determined that pre-existing antibodies specific for the vaccine backbone inhibited CD8+ T cell priming and therefore memory CD8+ T cell development and lung CD8+ TRM populations. Importantly, high dose vaccination could mitigate the impairment in CD8+ T cell priming, for which the resultant lung CD8+ TRM were protective against heterologous influenza virus challenge. Influenza infection can result in a transient depot of antigen long after viral clearance that influences influenza-specific CD8+ T cell responses, but it is unclear how this antigenic stimulation impacts the local cognate antigen-requisite lung CD8+ TRM compartment. Our studies suggest that residual antigen persistence is likely applicable to only certain epitopes. Furthermore, persistence of residual antigen activated naive CD8+ T cells that then formed CD8+ TRM populations in the lung, however these cells exhibited reduced polyfunctionality and longevity. Our results thus imply that lung CD8+ TRM generated from residual antigen following influenza viral clearance are unlikely to meaningfully participate in protection against re-infection with influenza virus. Overall, we show vaccines that evoke lung CD8+ T cells and CD8+ TRM of broad repertoire diversity are valuable against influenza virus variants and that this local immunity may be compromised in hosts with pre-existing humoral immunity against the vaccine backbone. As such, our work uncovered several insights in the optimal implementation of T cell-based vaccines aiming to induce universal protective immunity against influenza virus.
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ItemMucosal-associated invariant T (MAIT) cells and their function in bacterial infection( 2021)Mucosal-associated invariant T (MAIT) cells are a subset of innate-like alpha/beta T cells that recognize riboflavin metabolites presented by the monomorphic major histocompatibility complex (MHC) class I related protein-1 (MR1). The most potent antigen known to date is 5-(2-oxopropylidineamino)-6-D- ribitylaminouracil (5-OP-RU). MAIT cells are abundant in mucosal tissues and blood in humans. Upon bacterial infection, MAIT cells expand rapidly, with production of cytokines, including interferon-gamma (IFN gamma), tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-17 (IL-17), and cytotoxic granzymes. Their phenotype indicates that they play an important role in immunity. Previous studies showed a protective role in local infections, involving a single organ or tissue, but the mechanisms of MAIT cell-mediated protection in systemic infections are not fully understood. This study aimed to elucidate MAIT cell activation, protective role in primary infection and potential for a MAIT cell-based systemic vaccination to protect against Francisella tularensis live vaccine strain (LVS) and Legionella longbeachae. F. tularensis is a gram-negative intracellular bacterium which can cause systemic infection, in mice and humans. In this study, F. tularensis LVS was used to induce systemic infection in C57BL/6 (wild type) mice and in Mr1 -/- mice, which lack MAIT cells. A combination of CpGcombo (fused oligos for CpG-B and CpG-P) and synthetic 5-OP-RU antigen was used to vaccinate mice. Bacterial load, survival rate, and MAIT cell response kinetics and post-infection cytokine profiles were examined. MAIT cells expanded systemically and showed Th1-like cellular profile after infection with F. tularensis LVS. In several organs, C57BL/6 mice showed better control of bacterial burden compared with Mr1 -/- mice. Vaccination of MAIT cells with CpGcombo plus 5-OP-RU, but not CpGcombo alone, protected mice from infections by an otherwise lethal dose of F. tularensis LVS and L. longbeachae. Thus, MAIT cells displayed a protective role against systemic infections and potential to be boosted to protect against local and systemic infections. This study also showed that post infection, MAIT and non-MAIT alpha/beta-T cells manifest different contraction kinetics, indicating that these two groups could be regulated by different viability mechanisms. Specifically, the in vivo data illustrated that loss of receptor-interacting serine/threonine-protein kinase 3 (RIPK3, a key regulator of apoptosis and necroptosis), but not mixed-lineage kinase domain like pseudokinase (MLKL, a signaling molecule involved in necroptosis), preferentially increased MAIT cell abundance in a cell-intrinsic manner. In summary, the results in this thesis demonstrated that MAIT cells are critical in immune protection against systemic F. tularensis LVS infection, and are long lived with a differently regulated cell death and survival. These findings may inform the future development of vaccination strategies targeting MAIT cells.
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ItemUnderstanding human B cell and antibody responses against seasonal influenza viruses( 2020)Vaccination is the best available means to reduce the burden of seasonal influenza. However, current influenza vaccines need to be updated frequently to keep up with evolution among circulating viruses. Antigenic evolution, otherwise termed drift, is most rapid among A/H3N2 viruses, and the A/H3N2 component of vaccines is frequently updated. Despite this, influenza vaccine effectiveness against the A/H3N2 subtype has been poor in recent years, especially among previously vaccinated individuals. Protection induced by inactivated influenza vaccines is largely mediated by B cells and antibodies reactive against the head of the hemagglutinin (HA) protein, with help from T follicular helper cells. The cellular and molecular mechanisms that underlie the attenuating effects of prior vaccination and existing immunity are largely undefined. It has been suggested that existing antibodies clear or mask antigen, or that memory B cells induced by prior exposures competitively dominate responses so that B cells and antibodies become focused on epitopes that are shared between prior and prevailing vaccine strains. The aim of the work presented in this PhD thesis was to examine the impact of pre-existing immune responses induced by prior infection with different A/H3N2 strains on influenza vaccine immunogenicity. In depth antibody as well as B cell assessments were performed to understand the impact of existing antibodies and memory B cells following vaccination and provide insights into the design of new vaccine strategies. As a lead up to the ex vivo analysis of B cells from vaccinees, we first sought to understand how human naive versus memory B cells differentiate in vitro. Experiments were conducted in Chapter 3 to compare the stimuli required for their differentiation into plasmablasts, and subsequently understand how they change phenotypically once stimulated. Specifically, sorted human naive and memory B cells from healthy individuals were stimulated in vitro to induce differentiation into plasmablasts. Data obtained in this PhD thesis showed that stimulation with the Toll-like receptor (TLR) 7/8 agonist R848 in the presence of monocytes induced the highest activation of both naive and memory B cells. Conversely, stimulation with the TLR9 agonist CpG or with R848 in the absence of monocytes induced little to no differentiation of naive B cells but were able to stimulate memory B. cell differentiation. Despite robust differentiation into antibody secreting plasmablasts, naive-derived B cells remained phenotypically distinct from memory-derived B cells up to day 6 after in vitro activation, with differential expression of CD27, CD38 and CD20. This work resulted in a first-author publication in Clin Transl Immunol, 2019. The focus of Chapters 4 and 5 was to understand how prior influenza virus infection affects antibody and B cell responses to influenza vaccination. To address this question, vaccine responses were investigated in a unique influenza vaccine-naive cohort in Viet Nam, that had been monitored for both clinical and asymptomatic influenza virus infection for more than 9 years. In 2016, twenty-eight participants without documented A/H3N2 virus infection (since 2007) and 72 participants who had been infected with A/H3N2 viruses, belonging to a range of genetic clades, received an inactivated trivalent influenza vaccine containing an A/Hong Kong/4801/2014-like (H3N2) antigen. This work investigated whether influenza vaccination induced naive B cell responses specific for new epitopes or largely recalled B cells specific for conserved epitopes, common to the vaccine A/H3N2 component and prior infecting strains. Hemagglutination inhibition antibody titres were measured in pre- and serial post-vaccination sera against 40 A/H3N2 viruses spanning 1968-2018 to understand how the titre and cross-reactivity of antibodies against the HA head evolve. B cells were assessed by flow cytometry using a panel of phenotypic markers in addition to recombinant HA probes representing the vaccine and recently infecting strains (A/Perth/16/2009, A/Victoria/361/2011 and A/Switzerland/9715293/2013). Participants who had at least one pre-vaccination A/H3N2 virus infection had on average 2 to 3-fold higher vaccine-specific antibody titres, steeper titre rises in the weeks following vaccination (mean peak on day 14), and less titre decay by days 21 and 280 compared to participants without prior infection. Moreover, participants with prior infection exhibited greater and better-maintained titre rises against viruses that circulated a year after vaccination, indicating that prior infection extends the strain coverage of antibodies induced by vaccination. Notably, A/H3N2 viruses that circulated 275-340 days after vaccination caused illness in only 1.4% of participants with infection prior to vaccination and in 14% of participants without prior infection. This suggests that vaccine effectiveness can be enhanced by pre-existing immunity. However, it was also clear that the range of strains against which antibodies were induced was dictated by the strain with which participants were previously infected, indicating that vaccination may simply recall rather than update antibody-mediated immunity. HA-probe reactive B cell frequencies and activation status increased substantially after vaccination. The greatest increases in HA probe-reactive B cells were detected among participants who had recent prior infection, with the majority of B cells exhibiting cross-reactivity with prior strains. A modest but significant increase in the frequency of B cells that reacted with the HA of the vaccine strain, but not of past strains, could be detected in participants who lacked prior infection. The phenotype of vaccine HA single-positive B cells, including increased IgM expression, indicated that they may have been naive-derived B cells. Vaccination induced B cells that preferentially reacted with the HA of A/Perth/16/2009 and/or A/Victoria/361/2011 viruses, but not A/Switzerland/9715293/2013 viruses, among participants who had prior A/Perth/16/2009-like virus infection. However, B cells induced by vaccination in participants who had prior A/Switzerland/9715293/2013-like virus infection were equally cross-reactive with HA of all tested viruses. These results support the inference that immune responses to standard inactivated influenza vaccines are dominated and shaped by recalled memory B cells with limited activation of naive B cells to update immunity. Overall, this PhD thesis investigated how pre-existing immunity induced by documented influenza virus infection affected the humoral response to seasonal influenza vaccines in healthy adults. This work provides new insights into the capacity of influenza vaccines to stimulate naive B cells, which may be limited due to memory B cell dominance and to a lack of sufficient stimulation to activate naive B cells. This knowledge could be used to design new vaccine strategies and improve influenza vaccine-induced protection.
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ItemNeuroimmune responses in viral infection( 2020)The regulation of the immune responses is important in maintaining good health. Interactions between the nervous and immune systems are increasingly studied and widely appreciated to be influential in orchestrating immune responses. T cells express adrenergic receptors (AR) that enable them to respond to neurotransmitters produced by the sympathetic nervous system (SNS), noradrenaline (NA) and adrenaline, inducing downstream signalling and modulating cell functions, although whether this is stimulatory or inhibitory in T cell antiviral responses is unclear. In this thesis, I examine the effects of SNS in various models of viral infection through chemical sympathectomy and treatment with AR agonists. Modulation of sympathetic signals in systemic infections with LCMV had minor influences on T cell responses but resulted in increased viral loads. Notably, the infection results in a loss of tyrosine hydroxylase (TH) positive sympathetic fibres in the spleen as early as day 3 post infection and is reflected by decreased NA splenic NA. The immune response may play a role with interferon g partially contributing to the depletion. Additionally, this thesis also investigates the capability of long-lasting resident memory T cell (TRM) responses in the highly innervated, immune-privileged cornea. Using a model of herpes infection of the cornea, I showed that T cells are effectively recruited to the cornea with a small heterogenous population able to persist following cessation of immune responses. These cells express CD69 and CD103, canonical markers of tissue residency, to varying degrees. Persistence, but not recruitment, of these cells is dependent on antigen availability at the cornea. These memory cells are capable of responding to secondary encounters with antigen. Moreover, circulating memory cells are also able to infiltrate the immune-privileged cornea more efficiently following infection. Together, these results highlight the important nuances in the regulation of immune responses by the nervous system.