The Impact of Antibody Biophysical Properties on Antigen Recognition and Fc Effector Functions

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2021

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Abstract

Vaccines save millions of lives every year primarily through the induction of antigen-specific antibodies; however, there is a critical lack of understanding of the mechanistic underpinings of vaccine-elicited, antibody-mediated protection. Vaccinologists have long since recognized neutralization as an important antibody function while comparatively neglecting the role of Fc functions of antibodies. Therefore, the goal of my dissertation was to dissect out the functional roles of different subpopulations of antibodies in preventing infection using a controlled human infection model (CHIM) of a pathogen with a mucosal and systemic phase of infection, Salmonella Typhi. The first objective was to determine which isotypes/subclasses within the polyclonal humoral immune response are protective against S. Typhi infection and through what functional mechanisms. The second objective focused on vaccine-derived monoclonal antibodies to identify biophysical properties that affect the ability to recognize S. Typhi Vi antigen and influence downstream antibody Fc-mediated function. Most immune correlates of vaccine-mediated protection focus on either total IgG responses or bulk polyclonal serum with little-to-no defined understanding of how the quality (avidity) and characteristics (epitope-specificity, functional potency) of specific subpopulations impact protection. In this dissertation, I interrogated the humoral response to vaccination with two Vi polysaccharide vaccine constructs: one plain polysaccharide vaccine (Vi-PS) and one Vi-protein conjugate vaccine (Vi-TT). In chapter 2 of this dissertation, I evaluated the binding and avidity of subclass-specific IgA and IgG antibodies to the Vi polysaccharide. Firstly, I demonstrated that Vi-specific IgA magnitude correlated with protection in the typhoid fever CHIM. In addition, Vi IgG1 and IgA2 avidity were higher amongst protected individuals of both vaccine groups. These findings suggest that a combined IgA, perhaps via IgA2, and IgG1 response may be critical in preventing infection. Although IgA magnitude was associated with protection from S. Typhi infection, I did not identify a threshold concentration of Vi-specific IgA that prevented infection. Therefore, I hypothesized that Fc functions, rather than neutralization, may be critical in the mechanism of IgA-mediated protection. Following the findings of the univariate analysis, a multivariate analysis was conducted as part of a wider scientific collaboration in which binding and avidity data, as well as functional data (including FcR binding and Fc-mediated functions), were included to identify the smallest set of immune measurements that could robustly predict protection. In a composite analysis with both vaccine regimens combined, we identified Vi specific IgA magnitude, IgG2 magnitude, and IgA2 avidity as the top features that were enriched among protected individuals. These responses were also linked to antibody-dependent neutrophil phagocytosis and oxidative burst. However, we found enriched antibody-dependent NK cell activation and complement deposition amongst diagnosed vaccinees. These data suggest that a highly specialized mechanism driven by IgA and IgG mediated neutrophil activation via FcαR and FcγRs impart protection whereas broad innate immune activation can be detrimental. In chapter 3 of this dissertation, I interrogated the humoral response to Vi immunization using recombinantly produced monoclonal antibodies derived from vaccinees. I identified a convergent B cell response amongst vaccinees with most antibodies targeting the immunodominant C3 O-acetyl group of the Vi polysaccharide. However, I also identified four unique subdominant epitopes that varied in their accessibility for antibody binding by performing a de-O-acetylation of the polysaccharide backbone and conducting epitope binning competitive assays. Furthermore, I demonstrated that specific subdominant epitopes on the Vi polysaccharide antigen can be effectively targeted by antibody for phagocytosis and complement deposition. I utilized a kinetics rates-based assay, BioLayer Interferometry, to assess the association rate, dissociation rate, and overall avidity of each monoclonal antibody to the Vi antigen. By conducting correlative analyses of kinetic parameters and functional outcomes, I demonstrated that association rate has no substantial association with Fc function, while dissociation rate is highly associated with Fc function. Taken together, these findings highlight a likely mechanism of protection from infection with S. Typhi through cellular phagocytosis mediated cooperatively by IgA and IgG antibodies. This study also highlights the key epitopes that antibodies elicited by intramuscular Vi vaccination target for functional Fc outcomes which is dependent on epitope exposure and on antibody-antigen binding stability (off-rate). Identifying these specific mechanisms of protective immunity against typhoid fever will facilitate evaluation and licensure of the many Vi conjugate vaccines in development that would otherwise require large scale trials, and also help guide any future development of Vi vaccines. More broadly, the analysis here highlights the importance of evaluating specific subpopulations of antibodies, and their quality, as potential mediators of protection for other vaccine constructs rather than focus only on magnitude of bulk polyclonal responses. This type of analysis deeply enhances our understanding of protective humoral immunity while also informing both the development and the evaluation of new vaccine candidates.

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Dahora, Lindsay Carvalho (2021). The Impact of Antibody Biophysical Properties on Antigen Recognition and Fc Effector Functions. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/22952.

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