Microbiology & Immunology - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemUnderstanding immune responses to severe influenza disease( 2021)Respiratory viral infections caused by Influenza A viruses (IAVs) are responsible for annual seasonal epidemics that cause mild, severe or fatal pulmonary disease and estimated to result in 243,000-640,000 deaths globally every year. Severe infections can result in lung tissue damage, acute respiratory distress syndrome (ARDS) as well as secondary bacterial pneumonia, all of which are detrimental to the host and can potentially lead to a fatal disease outcome. Many of those at high-risk of developing severe disease include children, the elderly and immunocompromised individuals. Although current influenza vaccines are the best way to combat the seasonal influenza epidemics, the vaccines need to be updated annually to match strains predicted to circulate in the upcoming influenza season. Frequent mutations in the virus can lead to strain mismatches, which can affect the protective efficacy of these vaccines and result in significant increases in infection rates, putting those who are most vulnerable at risk for developing severe disease. The aim of this PhD thesis was to investigate host factors and mechanisms associated with severe influenza disease to provide insights into identification and prediction of severe disease outcomes. Co-expression of surface CD38 and MHC-II on CD8+ T cells is recognized as a classical hallmark of activation in many acute and chronic viral infection settings. However, high and prolonged CD38+MHC-II+ expression can be also associated with severe and fatal disease outcomes. Our previous studies have found that the persistence of CD38+MHC-II+ CD8+ T cells is associated with impaired IFN-gamma production in H7N9-infected patients with fatal outcomes. In Chapter III, we sought to investigate the mechanisms underpinning the expression of CD38+MHC-II+ markers using wild type and transgenic mouse models of influenza infection. We provided evidence that CD38+MHC-II+ CD8+ T cells are recruited early to site of infection during severe influenza A virus infection and can be activated in an antigen-driven as well as bystander manner. Our adoptive transfer experiments also demonstrated that MHC-II molecules were not expressed intrinsically by CD38+CD8+ T cells, but acquired from antigen presenting cells by trogocytosis, and the CD38+MHC-II+ phenotype was crucial for optimizing T cell recall ability. In Chapter IV, we investigated disease signatures during the early phases of infection to identify features associated with clinical outcomes. While comparable viral titres were observed between H7N9 patients who survived or succumbed to infection, there was a trend of higher level of inflammation responses in those who died. Our transcriptome analyses of blood samples from fatal and recovered H7N9 patients found that the gene Olah, encoding for a fatty acid hydrolase, was expressed more than 80-fold higher in fatal H7N9 patients compared to those that recovered at early days after disease onset. We established an Olah-overexpressing A549 cell line, which allowed us to develop a qPCR platform to screen Olah expression in human tissues and cells. Our analyses of Olah distribution across different anatomical sites found Olah expression in human lung, spleen and PBMCs, with the most prominent expression detected in CD14+ cells within the human lungs. Subsequently in Chapter V, we generated and established OLAH knockout (OLAH KO) mice to further investigate the role of Olah during severe influenza A virus infection. Compared to wild-type mice, OLAH KO mice displayed milder disease symptoms, less body weight loss and were protected from severe and fatal disease outcomes following IAV infection. In addition, OLAH KO lungs exhibited lower viral titres, less tissue damage and had significantly reduced levels of inflammatory cytokines and infiltrating innate immune cells, indicating the importance of Olah in controlling early excessive host responses and disease severity. These effects were associated with reduced lipid droplets in virus-infected epithelial cells lining the bronchioles, thus defining a role for OLAH in driving lipid metabolism during IAV disease. Overall, the findings from this PhD thesis highlight the importance of CD38+MHC-II+ CD8+ T cells during severe influenza A virus infection and identified the mechanisms underlying severe IAV disease. We also identified Olah as a potential biomarker of severe disease and demonstrated its role in modulating early host immune responses via the formation of lipid droplets and defined its impact on disease outcomes. Overall, data generated in this PhD thesis advance our understanding of factors underlying fatal clinical outcomes and provide insights into the development of novel treatment strategies and biomarkers against severe influenza disease.
-
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.