Microbiology & Immunology - Theses
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ItemFunctional antibody immunity in SARS-CoV-2 infection and vaccination( 2023-01)Since the emergence of SARS-CoV-2 in late-2019, the virus has caused substantial mortality in humans, with the confirmed death-toll exceeding 6.5 million lives and causing over-600 million confirmed cases. The introduction of COVID-19 vaccines has been instrumental in reducing the risk of hospitalisation and death globally. However, with the rise of novel variants, SARS-CoV-2 continues to cause high levels of infections, and thus the impact of the COVID-19 pandemic remains far from over. Antibodies play crucial roles in the protective immunological response to SARS-CoV-2. As such, this MPhil thesis explores the antibody response to SARS-CoV-2 in the context of both infection and vaccination using high-throughput multiplexing technologies. We began by investigating the systemic (plasma) and mucosal (saliva) antibody responses during acute (first-fourteen days) infection in adults and children, shown in Chapter 3 of this thesis. We used a cohort of individuals recruited from household clusters of COVID-19 infections during the first waves of ancestral-strain SARS-CoV-2 in Melbourne, Australia. Using systems serology analysis, we found that households with higher rates of secondary transmission to household contacts display greater levels of virus in the nasopharynx; and correspondingly display enhanced systemic antibody responses, suggesting a relationship between viral load and seroconversion. Additionally, we observed differential antibody response induced during acute infection between adults and children. Levels of IgA antibodies to SARS-CoV-2 spike (S)- and nucleocapsid-proteins were substantially lower, notably in the saliva, of children compared to adults. We speculate this in part may reflect that adults have greater prior exposure to endemic human coronaviruses. Given little information is known regarding the mucosal response in children, future studies are needed to profile the mucosal response more extensively in this population. The emergence of variants of SARS-CoV-2 are a challenge to public health. Many variants harbor mutations in the RBD of the SARS-CoV-2 S, a dominant target for neutralising and non-neutralising antibodies. In Chapter 4, we comprehensively characterised the effect of RBD mutations, including mutations present in the Omicron (BA.2) variant, on ACE2-binding affinity, as well as the functional antibody response induced in BNT162b2-vaccinated recipients and mild-convalescent SARS-CoV-2 infected individuals. The RBD of variants of concern (VOCs) displayed greater affinity to ACE2 than ancestral SARS-CoV-2 RBD. Furthermore, using a surrogate neutralisation assay that examined 39-naturally occurring RBD mutations, we showed reduced capacity of plasma antibodies to block ACE2-binding to VOCs. Additionally, we show a reduced capacity by RBD-binding plasma antibodies to engage Fc-receptors (FcRs), suggesting the potential for RBD-binding antibodies to recruit Fc effector functions are compromised by mutations in the RBD. These findings highlight the capacity of viral variants with RBD mutations to subvert host antibody responses. Lastly, with the observation that S-binding antibodies are attenuated by mutations found in variants, it is thought enhancing the magnitude of the antibody response may be useful in offering protection against SARS-CoV-2. As such, in Chapter 5 we investigated the effect of administration of a novel priming vaccine strategy designed to augment antibody responses – Bacillus Calmette-Guerin (BCG) immunisation – prior to immunisation with two homologous doses of either COVID-19 vaccine BNT162b2 or ChadOx1 on the antibody responses to the SARS-CoV-2 S. No differences in the SARS-CoV-2 antibody levels between BCG- and non-BCG-vaccinated individuals was observed, suggesting this strategy may not substantially improve the efficacy of current COVID-19 vaccines. Further studies are needed to determine whether this reflects the time window (>1 year) between BCG-vaccination and COVID-19 vaccines, or whether BCG- priming is not a suitable option for augmenting COVID-19 antibody responses. In summary, our work provides greater insights into the antibody response to SARS-CoV-2, of which can be further explored in prospective work.
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ItemThe dynamics of the B cell response during influenza A virus infection( 2020)Although vaccination remains the most effective method of managing influenza epidemics, there is still much that remains to be characterized about humoral immunity against the varying contexts in which influenza infection can occur. With the continuous subversion of humoral immunity by seasonal influenza through antigenic drift and the potential of zoonotic influenza viruses adapting and spreading through human populations through antigenic shift, improving our understanding of B cell immunity against different types of influenza infection could provide important insights into improving management of epidemics and vaccine formulations. In order to understand B cell responses during influenza infection, the well-characterized C57BL/6 mouse model was used to investigate and compare humoral responses in the context of different influenza infection histories. Markers that identified specific B cell subsets such as germinal centre (GC) B cells and plasmablasts were analysed by flow cytometry paired with influenza virus-specific B cell ELISPOT assays to investigate strain-specific antibody secreting responses within the same experiment. As the surface glycoprotein HA is thought to be the immunodominant response for B cell responses against influenza virus, the prediction is that the greater the antigenic differences between the HA of the first and second infecting strains, the more primary-like the response to the second strain would be. Primary and homologous secondary B cell responses in the mediastinal lymph node (MLN) and spleen were first characterized using this model to establish baseline responses against influenza virus before heterosubtypic infection was studied through infection of mice with H1N1A/Puerto Rico/8/34 (PR8) virus followed by H3N2 A/Udorn/305/72 (Udorn) virus 7 weeks later. Unexpectedly, a secondary-like plasmablast, GC B cell and Udorn-specific antibody secreting cells was observed during heterosubtypic infection, with earlier and higher magnitude B cell responses. These findings suggested a possible role for cross-subtype T cell memory in modulating B cell responses. The effect of antigenic drift on the B cell responses during influenza infection was then analysed with the same model. Mice were infected with H3N2 strains isolated between 1972 and 1979, representing different antigenic distance from a virus isolated in 1982 (Ph82). Seven weeks post infection mice were reinfected with Ph82 and the B cell response over the course of infection examined. It was found that infection of strains up to 10 years apart appeared to induce a secondary-like B cell response in the secondary lymphoid organs when compared to baseline primary and secondary responses against Ph82 virus. Prior infection with any H3N2 strain also resulted in minimal viral replication during the secondary challenge when compared to primary infection groups. However, data from both primary antibody inhibition and HA-specific B cell responses appears to suggest a narrower threshold of recognition, around a maximum of 3 years drift before serum and HA-specific responses cease to bind with other strains. Taken together, secondary-like B cell responses in both heterosubtypic and drift models of infection and in the case of drift responses, irrespective of reactivity of HA-specific B cells, appear to refute the hypothesis that virus-specific B cell responses would reflect antigenic relatedness between the HA of the infecting strains. Overall, data from this study identifies the diversity of the overall B cell response against influenza infection in the context of prior exposure to strains of different antigenic properties. Further study into the reactivity of these B cells against different influenza virus components and the role of memory T cells in the observed responses may provide important insights into the nature of host immunity against the ever-shifting target of influenza virus.