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Anthrax Vaccine Adsorbed (AVA) immunization protects against anthrax disease by eliciting a neutralizing antibody response. However, antigen-specific antibody concentrations are not observed in high quantities until three immunizations have been administered over six months. Even then, humoral responses to AVA do not provide long-term immunity without an annual booster. We followed six healthy volunteers over the five-dose, 18-month AVA schedule to characterize the genetics of the immunoglobulin repertoire during the vaccination series. Two tiers of data were collected: 1) Immunoglobulin variable region genes (IgVRG) from bulk sorted naïve, memory and plasmablast (PB) B cells and 2) single cell sorted and sequenced IgVRG from plasmablasts. Samples were collected prior to and one and two weeks following each immunization. Our initial analyses indicated that technical error, the variation introduced by biological sampling and standard sample preparation, resulted in skewed output, and we developed a model to better estimate quantitative values from Ig-seq. We also utilized unique molecular identifiers to correct for nucleotide errors and PCR over-amplification. Our analysis of IgVRG following AVA administration reveals that the population of peripheral PBs following primary immunization is not distinguishable from the pre-immune peripheral PB repertoire. These PBs have more somatic mutations than expected for newly activated and differentiated naïve B cells, and are unlikely to be vaccine-elicited. In contrast, PBs observed following the 2nd dose have low mutation frequencies that increase upon subsequent vaccination. These clones are more persistent than clones first observed following any other immunization, but still make up a very small proportion of the overall repertoire. At no time is the clonal repertoire consistently dominated by a few clones, and the total and plasmablast repertoires are highly transient, even after the elicitation of vaccine-specific antibodies. AVA immunization thus results in a polyclonal B cell response which is not dominated by one or a few highly specific, strongly-elicited clones. We conclude that primary immunization by AVA is not sufficiently immunogenic to elicit vaccine-responsive, class-switched PBs to the periphery, nor is complete AVA immunization able to sustain proliferation of individual clones, providing insight into why AVA may require regular boosts.

Affinity maturation increases the affinity of B-cell derived antibodies to their cognate antigens. In this study, we characterized the kinetic, structural, dynamic and thermodynamic evolution of antibodies during affinity maturation. Through single B-cell cell sorting, paired heavy and light chain sequencing, phylogenetic analysis, antibody expression, and physicochemical characterization, we were able to longitudinally analyze the stages of affinity maturation of anti-PA (B.anthracis protective antigen) antibodies. Following repeated immunizations, we observed up to an 10,000-fold increase in antibody affinity, mainly through a decrease in the off-rates. For detailed maturation analysis, we chose three antibodies lying along a single clonal branch--the clone’s unmutated common ancestor (UCA), a medium affinity antibody (MAAb) appearing after second immunization, and a high-affinity antibody (HAAb) appearing after third immunization. Most of the mutations that occur between the UCA and HAAb resulted in key changes to structural conformation. In particular, mutations change residues in the CDR-H3 region inducing the folding of the CDR-loops into a conformation that is more complementary to PA. This advantageous new antibody conformation is preserved in the unbound state, indicating that though the UCA and MAAb appear to use an induced fit and/or conformational selection mechanism, the HAAb is more rigidly lock-and-key. Thermodynamic results support this interpretation. In the first maturation step from UCA to MAAb, enthalpic improvement indicates optimization of noncovalent interactions. The second step from MAAb to HAAb predominantly involves entropic improvement by which the advantageous conformation made accessible in the first step is made more dominant via the narrowing of effectively accessible conformations, which allows better contact with PA. This is also reflected by a less significant improvement in the enthalpic component of PA-binding. Studies examining the evolving protein-dynamic characteristics further support this interpretation. In summary, we observed that a single energetic component is not responsible for increased affinity in the maturation pathways we studied. From UCA to MAAb, affinity increases through optimization of noncovalent interactions. From MAAb to HAAb, affinity increase is achieved through changes that stabilize the favorable conformation in the unbound state. A better understanding of affinity maturation can have implications for antibody engineering and vaccine development.

In a challenge trial, “healthy volunteers are intentionally exposed to pathogens in a controlled environment, in order to promote understanding of the pathogenesis, transmission, prevention and treatment of infectious diseases in humans.” (WHO 2021, Preface). Intentional infection is an uncomfortable concept, and as a result there is a widely held belief amongst research ethics scholars and commentators that a significant ethical concern with challenge trials is their potential to negatively impact the public’s trust in the institution of medical research (Eyal 2022, 4). However, the relationship between public trust and the ethics of conducting and assessing challenge trials is complex and existing literature on the subject does not sufficiently clarify it. This paper will begin by examining the ethical permissibility of challenge trials. Once these trials are shown to be ethically permissible under particular circumstances, I will explore how concerns about the way these trials allegedly exacerbate public mistrust largely result from ambiguities in the terms ‘public’ and ‘trust’. After both terms are defined, I will formulate my own account of how public trust should apply to a risk/benefit analysis for the purpose of trial design selection called the community engagement account, which argues that trial design selection policy should focus on demonstrating trustworthiness rather than garnering trust. Because demonstrating trustworthiness requires meeting a set of known expectations, this account identifies local, specific publics as those whose expectations should be of concern when discussing public trust and trial design selection. To examine the expectations of these publics, this account defends community engagement as the measure which should be used to acquire evidence of harmful public mistrust towards the institution of science that could potentially result from conducting a challenge trial. / Thesis / Candidate in Philosophy / In a challenge trial, “healthy volunteers are intentionally exposed to [diseases] in a controlled environment,” to give researchers a better understanding of a disease in order to develop cures or preventative measures for it (WHO 2021, Preface). Many research ethics scholars believe that conducting challenge trials could negatively impact the public’s faith in the institution of medical research, but the relationship between public trust and conducting challenge trials is complex and existing literature on the subject does not sufficiently clarify it. This paper begins by exploring whether or not challenge trials can be ethically conducted. Once I show that they can be under particular circumstances, I examine how public trust concerns largely result from the fact that ‘public’ and ‘trust’ are not well defined. After defining them, I formulate my own account of how public trust should apply to a risk/benefit analysis for the purpose of trial design selection.

The Inhibitor of Apoptosis (IAP) proteins are a highly conserved group of anti-apoptotic proteins that regulate various pathways, particularly those that affect proliferation and cell death. Smac mimetics compounds (SMCs) are IAP antagonists that induce the degradation of two IAPs, cellular IAP 1 (cIAP1) and cellular IAP 2 (cIAP2). cIAP1 and cIAP2 are negative regulators of the alternative NF-κB pathway, which is critical to the regulation, activation, proliferation, and survival of immune cells. Consequently, SMCs can affect immunological responses by providing co-stimulatory signals for antigen-presenting cells or promoting proliferation and activation of T cells. Due to their potent immunomodulatory properties, SMCs are an ideal candidate for new vaccine adjuvants. I sought to demonstrate the potential of SMCs as a vaccine adjuvant by evaluating SMCs effects on dendritic cells (DCs). I demonstrated that SMC treatment of bone marrow derived dendritic cells (BMDCs) induces degradation of both cIAP1 and cIAP2 and leads to activation of the alternative NF-κB signalling pathway. Furthermore, SMC treatment led to upregulation of proteins associated with DC maturation, as well as secretion of pro-inflammatory cytokines. Despite the activating effects elicited by SMCs in vitro, the use of SMCs as an adjuvant for peptide vaccination failed to prevent tumour growth. Further work to determine the best use of SMCs as adjuvants in vivo needs to be done to explore the potential of this class of drugs. Thus, these findings will guide the use of SMCs in adjuvant vaccine therapies for robust protective immunity.

Mycoplasma gallisepticum (MG) is a major and economically significant pathogen of avian species. Different strains of MG have been used as vaccines in multiple-age commercial layer farms in an effort to protect the birds against more virulent field strains. The lower level of protection afforded by the low virulent MG strain vaccines provides an opportunity to the use of an overlay (revaccination) with an F-strain M. gallisepticum (FMG) later in their production cycles. In the present study, three trials were conducted to investigate the effects of prelay vaccinations of ts-11 strain MG, MG-Bacterin, or their combination, in conjunction with a FMG overlay after peak production in commercial layers. The following treatments were utilized at 10 wk of age (woa): 1) Control (no vaccinations); 2) ts-11 MG vaccine; 3) MG-Bacterin vaccine; and 4) ts-11 MG and MG-Bacterin combination. At 45 woa, all the birds in trial 1 and half of the birds in each treatment group in trials 2 and 3 were overlaid with an FMG vaccine. Various parameters including performance, internal egg and eggshell quality, blood, and visceral characteristics of the birds were evaluated. In this study, the ts-11 MG vaccination at 10 woa was shown to increase shell weight and the yolk lipid content of the eggs laid by the birds without affecting their performance. On the other hand, a prelay vaccination with MG-Bacterin did not prevent a drop in egg production in response to an overlay with FMG at 45 woa. A decrease in proportional infundibulum length in the control birds and MG-Bacterin vaccinated birds after being overlaid with FMG might be the underlying factor responsible for the observed decrease in egg production. Nevertheless, the prelay use of MG-Bacterin together with ts-11 MG didn’t appear to provide any additional benefit over the ts-11 MG vaccine alone on any of the parameters investigated. In conclusion, these results establish the potentially effective prelay use of the ts-11 MG vaccine in combination with an FMG overlay for the provision of continual protection against field strain MG infections, without eliciting any subsequent suppressive effects on the performance of commercial layers.

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