Microbiology & Immunology - Theses
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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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ItemElucidating the role of T-box protein family members in influenza-specific T cell immunity( 2017)CD8+ T lymphocytes are specialised cells responsible for immunity to intracellular pathogens and cancer. Upon exposure to its cognate antigen, a naïve CD8+ T cell undergoes a program of differentiation and proliferation into a pool of effector CD8+ T cells. Following clearance of infection, this pool contracts and a pool of long lived memory CD8+ T cells remain. Transcription factors (TFs) from the T-box group of proteins are known to play a key role in differentiation but their molecular mechanism remains unclear. This thesis aims to understand the molecular mechanism, that T-box members T-bet and Eomes play in CD8+ T cell differentiation and also the role that the epigenetic modifier KDM6b plays. Utilising a model of influenza A virus (IAV) infection and adoptive transfer of Tbx21-deficient (-/-) cells, we show that while T-bet is dispensable for early CD8+ T cell participation and cellular division, it is essential for full expansion of a virus-specific effector CD8+ T cell response. While Tbx21-/- IAV-specific CD8+ T cells are able to divide early after infection, Tbx21-/- CD8+ T cells are unable to sustain CD8+ T cell lineage specific cytokine expression. Whole transcriptome analysis showed a global dysregulation in early programming events where IAV-specific Tbx21-/- CD8+ T cells expressed genes associated with various CD4+ T cell lineages. Finally, analysis of histone signatures within the Ifng locus demonstrated that T-bet deficient CD8+ T cells were unable to activate “poised” enhancer elements compared to wild-type (WT) CD8+ T cells, correlating with diminished Ifng transcription. In all, these data support a model whereby CD8+ T cell activation induces rapid induction of T-bet. This ultimately leads to correct CD8+ T cell programming and differentiation via T- bet interactions with regulatory genetic elements that promote appropriate chromatin remodelling events at key gene loci. KDM6b is a lysine demethylase that has specific activity against histone 3, lysine 27 di-, and tri-methylation (H3K27me2/3). This histone modification is associated with a repressive transcriptional state, or, when found co-located with the active H3K4me3 mark, marks a bivalent epigenetic state. Resolution of bivalency at fundamental TF loci has been shown to be a key mechanism for the initiation of CD8+ T cell differentiation. This thesis aimed to understand if KDM6b was required for resolution of bivalency at the loci of key TFs involved in CD8+ T cell differentiation. To address this question, we developed a conditional short hairpin RNA (shRNA) mouse model targeting KDM6b. In this model, the Kdm6b shRNA will target the Kdm6b locus and result in degradation of Kdm6b mRNA and subsequently decrease KDM6b activity. Preliminary data using this mouse model showed that the endogenous CD8+ T cell response to IAV was impeded with KDM6b knockdown. Moreover, the Tbx21 promoter remains bivalent in these mice, compared to luciferase hairpin controls. Surprisingly, this is not a general mechanism as other TF promoters were resolved, suggesting there is specific targeting of KDM6b to the T-bet locus in IAV-specific CD8+ T cells. Eomesodermin (Eomes) has previously been reported to play a role in development of the memory CD8+ T cell pool, specifically central memory (TCM) CD8+ T cells. Analysis of endogenous IAV-specific Eomes-/- CD8+ T cell responses demonstrated that this TF is essential for the development of the memory CD8+ T cell pool, particularly TCM. Interestingly, secondary challenge showed that Eomes is dispensable for a secondary IAV-specific effector CD8+ T cell response but essential for maintenance of the secondary memory CD8+ T cell pool. Furthermore, transcriptome analysis of primary memory CD8+ T cells showed surprisingly few differentially expressed genes in the absence of this TF. Taken together, this data suggests a role for Eomes in the development and long term maintenance of memory CD8+ T cells, but not the programming and function of these cells. These data show that the T-box proteins play specific roles in different stages of T cell differentiation. T-bet plays a role rapidly following initiation of differentiation, likely by interacting with regulatory genetic elements at key CD8+ T cell signature genes to maintain CD8+ T cell identity and fate. Eomes is involved later during the immune response to shape the memory CD8+ T cell pool and maintain these populations. Finally, KDM6b appears to be involved in the resolution of bivalency at fundamental TF loci. This information can be used to inform future vaccine or drug target strategies that enhance or modulate the CD8+ T cell response.
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ItemUnderstanding mechanisms of influenza A virus escape from CD8+ T cells in mouse and human models of infection( 2011)CD8+ T cells can provide cross-reactive immunity between different influenza strains and subtypes, when there is sufficient sequence or structural homology of the pMHC-I. Otherwise escape from pre-existing immunity can occur. The focus of this PhD thesis is understanding whether existing CD8+ T cell responses are able to recognise and protect against new antigenic variants, such as those derived from the A(H1N1)-2009 pandemic strain. Additionally, this thesis examines factors that lead to immune escape of CD8+ T cell recognition, informing vaccine development about diverse targets of the immune response. A panel of engineered influenza viruses were mutated at a prominent TCR contact residue in the B6 model of influenza infection. Results highlighted that standard modes of TCR recognition and the number of naïve precursors are critical in establishing an endogenous primary CD8+ T cell response. Furthermore, analysis of viral species within influenza infected lungs showed the emergence of de novo escape variants within CD8+ T cell targetted peptides. Mutation was common for influenza viruses despite the acute nature of the disease, and occured as early as 5 days after infection. Viral escape was driven by CD8+ T cell mediated immune pressure, as no mutants were found in MHC-I mis-matched mice and mutants reverted to wild type where there was no immune response. The higher frequency of mutation within different epitopes correlated with limited TCR repertoire diversity. The M158 peptide is prime vaccine candidate due to its high sequence conservation and eliciting an immunodominant CD8+ T cell response in HLA-A2+ humans. The public A2-M158-specific TCR recognised naturally occurring M158 peptide variants, which possibly plays a key role in limiting variation. Additionally, the A2-M158 CD8+ T cell response only selected a very low level of de novo M158 mutants during acute infection of HHD HLA-A2+ mice. Conversely, other human influenza peptides, such as the B7/B35-restricted NP418 epitope have frequent variation in prominent residues involved in TCR recognition. Importantly, the A(H1N1)2009 pandemic influenza virus was able to evade existing memory CD8+ T cell responses due to NP418 sequence variation from recent seasonal influenza viruses. The TCRαβ repertoire of the NP418-specific CD8+ T cell response was diverse with no public or shared TCR capable of recognising newly emerged peptide variants. The difference in the features of the TCR repertoire that recognise the conserved A2-M158 and variable B35/B7-NP418 CD8+ T cell responses reflected their ability to deal with newly emerged peptide variants. The capacity to respond to new infection is severely reduced in the elderly, due to both functional compromise and the emergence of ‘repertoire holes’ arising from the loss of low frequency clonotypes. Aged mice showed that early vaccination prior to the attrition of low frequency anti-influenza CD8+ T cells was important to maintain a diverse array of TCRs. The TCR repertoire in extremely aged mice was ‘locked’ early following vaccination, which is advantageous for high avidity repertoires and maintaining response magnitude. Whilst vaccination and then challenge at an extreme age resulted in equivalent CD8+ T cell response magnitude, the TCR repertoire was perturbed. Therefore, it is important to establish influenza-specific CD8+ T cell responses early in life to preserve optimal, influenza-specific CD8+ T-cell responsiveness and protect against the age-related attrition of naïve T-cell precursors. Overall, this thesis has expanded our understanding of heterologous CD8+ T cell immunity and the capacity for the influenza virus to escape CD8+ T cells during acute infection. Establishing broad CD8+ T cell responses that recognise peptide variants by pre-emptive vaccination will be critical for combating future unpredicted pandemic influenza viruses and other influenza strains that could be encountered during a lifetime.