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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ItemSalmonella Typhimurium metabolism in the murine host and importance to virulence( 2014)The bacterial pathogen Salmonella enterica is responsible for considerable global morbidity and mortality, being the cause of several enteric and systemic diseases, including typhoid fever. Non- typhoidal Salmonella (NTS) serovars such as Typhimurium cause gastroenteritis in immunocompetent individuals, but in the context of HIV-co-infection can cause invasive disease with high fatality rates. Successive generations of antimicrobials have become ineffective against S. enterica pathogens due to the widespread development of resistance, and new drugs are critically needed. In addition, vaccines against typhoid fever have sub-optimal efficacy and no human NTS vaccines are currently available. Although the in vitro metabolism of S. enterica is relatively well defined, little is known about the specific nutrients that the pathogen consumes in its hosts, and the metabolic mechanisms by which S. enterica utilise these nutrients. Therefore, the central aim of this study was to investigate and characterise S. enterica serovar Typhimurium metabolism in the murine host. Attention was focused on two areas of S. Typhimurium metabolism which have been speculated to contribute to bacterial virulence: methylglyoxal detoxification and central carbon (sugar) catabolism. It was anticipated that this study may contribute to better defining the metabolic requirements of S. Typhimurium during infection and disease, and present opportunities for the identification of potential drug targets and metabolically-attenuated strains amenable to use as live vaccines. Several studies have ventured that methylglyoxal detoxification mechanisms are required for survival of bacterial pathogens in the mammalian host, but this hypothesis has not been thoroughly tested. In the current study, although S. Typhimurium mutants defective in the glutathione-dependent glyoxalase system (GDGS) or Kef-mediated potassium efflux were highly sensitive to methylglyoxal, they were not attenuated for intracellular replication and growth in mice, suggesting that these methylglyoxal detoxification mechanisms are not required for S.Typhimurium pathogenesis in the mammalian host and are not suitable targets for antimicrobial therapy against S. Typhimurium disease. While others have reported that the Embden-Meyerhof pathway (EMP) and the ability to utilise glucose are required for S. Typhimurium virulence in mice, the importance of other sugars and carbon catabolic pathways to S. enterica virulence is unclear. In this study, S. Typhimurium mutants blocked in several sugar catabolic pathways including the Entner-Doudoroff pathway (EDP) were found not to be attenuated for intracellular growth or fulminant infection of mice, demonstrating that gluconate, glucuronate, galacturonate are not essential carbon sources for S. Typhimurium in vivo. However, evidence was presented which suggested that the ability to utilise gluconate and glucose is required for optimal shedding of the pathogen in the murine faeces, revealing potential strategies for reducing the faecal-oral transmission of S. Typhimurium. EMP/EDP double mutants showed greater attenuation in mice than a EMP mutant, suggesting that the EMP mutant utilises the EDP in order to facilitate it’s modest growth in vivo. These findings demonstrated the functional redundancy of S. Typhimurium metabolism and suggested that combinational drug therapies targeting several bacterial pathways concurrently might be a viable option for treating S. Typhimurium disease. The S. Typhimurium EMP/EDP mutant TAS2010 was found to provide increased, long-term protection from virulent infection in a murine typhoid vaccination model than the prototypical aro-negative vaccine strain BRD509. Given that the lack of effective vaccines against S. enterica pathogens can be largely attributed to an insufficient understanding of the host immune response to the pathogen, the immunological response to the TAS2010 vaccine strain was characterised in mice to understand the mechanisms responsible for the increased protective capabilities of this strain. In comparison to BRD509, TAS2010 was found to replicate to higher numbers in murine organs and induce an increased immune response in the form of increased interferon-gamma secretion by splenic CD4+ T cells. Evidence was presented which suggested that the protection provided by TAS2010 is less reliant on T cells and more dependent on a CD4-CD8-Thy1+ lymphocyte subset, probably Thy1+ NK cells. In conclusion, this study has enhanced the understanding of S. Typhimurium metabolism in the murine host and introduced a live vaccine strain with improved protective and immunogenic properties.