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In principle, humans can produce an antibody response to any non-self-antigen molecule in the appropriate context. This flexibility is achieved by the presence of a large repertoire of naive antibodies, the diversity of which is expanded by somatic hypermutation following antigen exposure 1 . The diversity of the naive antibody repertoire in humans is estimated to be at least 10 12 unique antibodies 2 . Because the number of peripheral blood B cells in a healthy adult human is on the order of 5 × 10 9 , the circulating B cell population samples only a small fraction of this diversity. Full-scale analyses of human antibody repertoires have been prohibitively difficult, primarily owing to their massive size. The amount of information encoded by all of the rearranged antibody and T cell receptor genes in one person—the ‘genome’ of the adaptive immune system—exceeds the size of the human genome by more than four orders of magnitude. Furthermore, because much of the B lymphocyte population is localized in organs or tissues that cannot be comprehensively sampled from living subjects, human repertoire studies have focused on circulating B cells 3 . Here we examine the circulating B cell populations of ten human subjects and present what is, to our knowledge, the largest single collection of adaptive immune receptor sequences described to date, comprising almost 3 billion antibody heavy-chain sequences. This dataset enables genetic study of the baseline human antibody repertoire at an unprecedented depth and granularity, which reveals largely unique repertoires for each individual studied, a subpopulation of universally shared antibody clonotypes, and an exceptional overall diversity of the antibody repertoire. A genetic study of the baseline human antibody repertoire, based on the circulating B cell populations of ten subjects, reveals universally shared antibody clonotypes within repertoires that are largely unique to the individual.

A limited subset of HIV-1 infected adult individuals typically after at least 2-3 years of chronic infection, develop broadly neutralizing antibodies (bnAbs), suggesting that highly conserved neutralizing epitopes on the HIV-1 envelope glycoprotein are difficult for B cell receptors to effectively target, during natural infection. Recent studies have shown the evolution of bnAbs in HIV-1 infected infants. We used bulk BCR sequencing (BCR-seq) to profile the B cell receptors from longitudinal samples (3 time points) collected from a rare pair of antiretroviralnaïve, HIV-1 infected pediatric monozygotic twins (AIIMS_329 and AIIMS_330) who displayed elite plasma neutralizing activity against HIV-1. BCR-seq of both twins revealed convergent antibody characteristics including V-gene use, CDRH3 lengths and somatic hypermutation (SHM). Further, antibody clonotypes with genetic features similar to highly potent bnAbs isolated from adults showed ongoing development in donor AIIMS_330 but not in AIIMS_329, corroborating our earlier findings based on plasma bnAbs responses. An increase in SHM was observed in sequences of the IgA isotype from AIIMS_330. This study suggests that children living with chronic HIV-1 can develop clonotypes of HIV-1 bnAbs against multiple envelope epitopes similar to those isolated from adults, highlighting that such B cells could be steered to elicit bnAbs responses through vaccines aimed to induce bnAbs against HIV-1 in a broad range of people including children.

The generation of broadly neutralizing antibodies (bnAbs) to conserved epitopes on HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The animal models commonly used for HIV do not reliably produce a potent broadly neutralizing serum antibody response, with the exception of cows. Cows have previously produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models, other engineered immunogens were shown to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n = 4) with two regimens of V2-apex focusing Env immunogens to investigate whether antibody responses could be generated to the V2-apex on Env. Group 1 was immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 was immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows, respectively, and showed moderate breadth and potency. Potent and broad responses in this study developed much later than previous cow immunizations that elicited CD4bs bnAbs responses and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target more than one broadly neutralizing epitope on the HIV surface reveals the generality of elongated structures for the recognition of highly glycosylated proteins. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response.

The humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoires of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa light chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies.

Millions of people continue to die from HIV despite wide-spread use of antiretroviral drugs, signaling the need to develop an effective vaccine. It is well established that neutralizing antibodies play a large role in anti-viral protection and will need to be induced by successfully designed immunogens. Nevertheless, extreme diversity of circulating HIV strains has made this goal difficult to achieve. Therefore, the reliable vaccine elicitation of broadly neutralizing antibodies (bnAbs) that work to confer protection against vastly diverse viral strains represents a promising contributor to ending or controlling the HIV pandemic. Additionally, although a small subset of naturally infected individuals develops potent bnAbs, the antibodies themselves are often associated with rare features of the antibody response to infections which may act as a barrier for successful vaccine design. Thus, the focus of this thesis is to identify and characterize components of the antibody repertoire that are important for the elicitation of bnAbs.In Chapter 1, I use high-throughput antibody next-generation sequencing to characterize the baseline antibody repertoire of an immunoglobulin-loci knock-in rodent, the OmniRat. The goal of this study is to understand its relevance for modeling the human antibody repertoire for vaccination studies. By leveraging our previously published largest single collection of human antibody gene sequences I was able to make comparisons to humans and demonstrate that although the OmniRat may be useful for monoclonal antibody discovery, it has a distinct repertoire from that of humans. Therefore, it is not well suited for use in approximating the human antibody response to candidate vaccine immunogens. Chapter 2 is a longitudinal follow-up to our original description of the baseline human antibody repertoire. Here I aim to describe how features of the baseline human antibody repertoire change over time. To do this, I re-sequenced the antibody repertoires of two healthy human donors from our original study. I show that after ~4 years within a single individual, features of the antibody repertoire had shifted enough to look like that of a different individual. Additionally, we found that most antibody sequences and clonotypes were replaced within that time and characterized a small number of persistent public clonotypes. Our results reveal a stable size and overall diversity of the antibody repertoire, but a significant turnover in the fine composition of the repertoire over time.In Chapter 3 I studied the case of an individual living with HIV-1 who was infected with two similar related founder viruses and went on to mount a broadly neutralizing immune response after 9–11 months of infection. From this individual, a potent lineage of bnAbs was isolated that features different lengths of genetic insertions within a single position. The phenotype of memory B cells from this lineage was also described. By characterizing the antibody-virus co-evolution that occurs within this individual, I offer insight into how a bnAb response might be facilitated through vaccination.Chapter 4 addresses the need for rapid development of neutralizing antibodies against diseases like SARS-CoV-2 and HIV through in vitro affinity maturation. To achieve this end, I wrote an open-source Python package, 'NNK', which is used for processing next-generation sequencing data derived from in vitro affinity maturation experiments and for the identification of enriched mutations. Its application is demonstrated by improving the potency of the anti-HIV bnAb, N49P7. This system will be useful for future pandemic preparedness and the elucidation of features important for bnAb breadth.Chapter 5 explores vaccine induced genetic insertions and deletions (indels) from somatic hypermutation. These indels are rare in memory B cells in part due to the risk of non-productive frameshift mutations. Here through the use two antibody heavy and light chain double knock-in mouse models I show that genetic insertions can be readily induced by immunization and begin to explore the genetic origins of these insertions. These findings, although preliminary, indicate that the creation of indels through vaccination may be less challenging for generating bnAbs than previously thought, significantly impacting future HIV bnAb vaccine design.In conclusion, this thesis provides a comprehensive exploration of the complexities inherent in the antibody repertoire and its potential applications in the design and prediction of effective responses to HIV immunogens. Through a series of detailed investigations, it underscores the significant challenges, as well as the promising opportunities, in the quest to develop a reliable HIV vaccine.

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population “arms” that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes.

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response.

Conventional vaccine design has been based on trial-and-error approaches, which have been generally successful. However, there have been some major failures in vaccine development and we still do not have highly effective licensed vaccines for tuberculosis, HIV, respiratory syncytial virus, and other major infections of global significance. Approaches at rational vaccine design have been limited by our understanding of the immune response to vaccination at the molecular level. Tools now exist to undertake in-depth analysis using systems biology approaches, but to be fully realized, studies are required in humans with intensive blood and tissue sampling. Methods that support this intensive sampling need to be developed and validated as feasible. To this end, we describe here a detailed approach that was applied in a study of 15 healthy adults, who were immunized with hepatitis B vaccine. Sampling included ~350 mL of blood, 12 microbiome samples, and lymph node fine needle aspirates obtained over a ~7-month period, enabling comprehensive analysis of the immune response at the molecular level, including single cell and tissue sample analysis. Samples were collected for analysis of immune phenotyping, whole blood and single cell gene expression, proteomics, lipidomics, epigenetics, whole blood response to key immune stimuli, cytokine responses, T cell responses, antibody repertoire analysis and the microbiome. Data integration was undertaken using different approaches-NetworkAnalyst and DIABLO. Our results demonstrate that such intensive sampling studies are feasible in healthy adults, and data integration tools exist to analyze the vast amount of data generated from a multi-omics systems biology approach. This will provide the basis for a better understanding of vaccine-induced immunity and accelerate future rational vaccine design.

The humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoire of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies. Footnotes * Author affiliations and label for figure S6e

ABSTRACT The emergence of SARS-CoV-2 underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing neutralizing antibody to a related but resistant virus. Using a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1 neutralizing antibody, to bind SARS-CoV-2 receptor binding domain with >1000-fold improved affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of an enhanced antibody elucidated the molecular mechanisms by which engineered CR3022 can accommodate sequence differences in the epitope between SARS-CoV-1 and SARS-CoV-2. The workflow described provides a blueprint for rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses. Competing Interest Statement J.G.J., D.R.B., and F.Z. are listed as inventors on pending patent applications describing the eCR3022 antibodies.