Microbiology & Immunology - Theses
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ItemFc-effector functions and plasma IgA in viral pandemics (HIV-1 and SARS-CoV-2)( 2023)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - the causative agent of the COVID-19 pandemic- has caused over 6 million deaths globally since late 2019. Mutations in the receptor binding domain (RBD) of SARS-CoV-2 have resulted in the emergence of variants of concern (VOC). Following infection and/or vaccination SARS-CoV-2-specific antibodies are generated with neutralising and/or Fc functional properties. Neutralising antibodies can protect from subsequent infections. While Fc-effector functions, including antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent cellular cytotoxicity (ADCC) are important for control and resolution of many infectious diseases including SARS-CoV-2. Notably, the neutralising antibody response wanes rapidly following infection with SARS-CoV-2, however, the durability of antibody mediated Fc-effector functions including ADCP remains largely unknown. To investigate the SARS-CoV-2 Fc functional antibody response, we developed two in vitro cell based assays to assess SARS-CoV-2 specific phagocytosis and cell association (trogocytosis) over time in mild-moderate convalescent COVID-19 individuals (Chapter 2). Interestingly, we observed evidence of SARS-CoV-2 specific trogocytosis occurring during the cell association assay using confocal microscopy. We demonstrate that the SARS-CoV-2 Fc functional antibody response, specifically ADCP and cell association, were more durable than the neutralizing antibody response. All COVID-19 individuals retained detectable antibody mediated phagocytosis and cell association at ~4 months post symptom onset, while 30% of the cohort lost detectable neutralization. Therefore, highlighting the potential importance of Fc-effector functions in long-term immunity from SARS-CoV-2 reinfection. The importance of IgG antibodies for protection and control of SARS-CoV-2 has been extensively reported. However, other antibody isotypes including IgA have been poorly characterized. We aimed to examine the functional contributions of plasma IgA to neutralisation and Fc-effector functions following SARS-CoV-2 infection (Chapter 3). Using a multiplex surrogate neutralisation assay, we assessed the neutralising capacity of IgA and IgG depleted plasma and purified antibody fractions against ancestral SARS-CoV-2 Spike receptor binding domain (RBD) and RBDs with common single amino acid mutations. Notably, more than 60% of the cohort showed significantly reduced neutralising capacity following IgA depletion (p = 0.0001). Furthermore, 30% of the cohort induced stronger IgA-mediated neutralization than IgG when purified antibody fractions were tested at equivalent concentrations. Moreover, convalescent purified IgA and IgG recognized similar RBD mutations and showed comparable neutralisation of RBD mutants. Depletion of IgG significantly reduced Fc-effector functions (ADCP and cell association) of convalescent plasma, in contrast no change was observed with depletion of IgA. We demonstrate that plasma IgA has the capacity to neutralize ancestral SARS-CoV-2 RBD, however, IgA contributes minimally to SARS-CoV-2 plasma Fc-effector function. Overall, neutralizing IgA and duel functional IgG contributes to the COVID-19 antibody response after infection. A constellation of RBD mutations have resulted in enhanced transmission and/or immune escape of SARS-CoV-2 circulating strains, giving rise to new variants of concern (VOCs). Mutations within the RBD can reduce antibody recognition, leading to reduced neutralising potency and potentially altering vaccine efficacy. However, the impact of RBD mutations on Fc-effector functions following vaccination remains unknown. We examined the capacity for SARS-CoV-2 Pfizer (BNT162b2) vaccine (2 weeks post second dose) and infection induced antibodies to mediate Fc-effector functions against SARS-CoV-2 VOCs (Chapter 4). We measured IgG binding to RBDs and engagement of RBD specific antibodies with Fc gamma receptors (FcyRs) via multiplex for 6 historical VOCs (Alpha, Beta, Gamma, Delta, Kappa and Omicron BA.2). Notably, FcyRIIa and FcyRIIIa engagement was significantly reduced for the VOCs Beta, Gamma, and Omicron BA.2. Furthermore, we confirmed that reduced FcyR engagement to RBD mutants resulted in reduced cellular Fc-effector functions, via a novel competitive SARS-CoV-2 duplex ADCP assay. This novel SARS-CoV-2 ADCP duplex assay enables assessment of the functional capacity of the same pool of antibodies to two different SARS-CoV-2 variants in a competitive high throughput assay. Taken together, we successfully optimised a novel SARS-CoV-2 ADCP assay and show that mutations within the SARS-CoV-2 RBD may have consequences on the Fc functional capacity of vaccine induced antibodies. The human immunodeficiency virus (HIV-1) is the causative agent of the acquired immune deficiency syndrome (AIDS) epidemic which has resulted in an estimated 40.1 million deaths globally since the early 1980s. Antibodies including IgG and IgA can recognise HIV-1 to elicit antiviral functions such as neutralisation and Fc effector functions. Plasma IgA can engage with the Fc alpha receptor (FcaR) to activate Fc-effector functions including phagocytosis. However, IgA can also mediate inhibition of Fc effector functions via FcaR and potentially interfere with protective antibody functions during viral infections. Notably, elevated IgA levels were associated with reduced vaccine efficacy and inhibited ADCC in the RV144 HIV-1 human vaccine trial. We investigated the Fc functional contributions of plasma IgA to HIV-1 phagocytosis during early and chronic HIV-1 infection (Chapter 5). We depleted IgA from plasma at two early and one chronic HIV-1 timepoint and assessed the IgA functional contribution. Notably, depletion of IgA at early timepoints resulted in significantly reduced ADNP (p < 0.05), suggesting IgA contributes to HIV-1 phagocytosis during early infection. However, depletion of IgA during chronic HIV-1 plasma enhanced ADNP, suggesting the IgA at this timepoint interferes with Fc-effector functions. Furthermore, we also observed IgA mediated inhibition of ADCP for various HIV-1 mAbs with purified IgA from people living with or without HIV-1, although the magnitude of inhibition is heterogenous amongst mAbs. We suggest this inhibitory effect is at least partially mediated via the FcaRI. However, this is a preliminary study and future studies are essential to investigate this phenomenon in greater depth with a larger cohort. Overall, our studies highlight the importance of Fc-effector functions and the complexity of the functional IgA response during SARS-CoV-2 and HIV-1 infections. Plasma IgA can induce potent neutralisation and contribute to ADNP during acute/ early infections. However, plasma IgA may interfere with Fc-effector functions during chronic HIV-1. Future studies should investigate the effect of IgA on other Fc-effector functions such as trogocytosis, complement activation and ADCC in different acute and chronic viral infections.