Convergent antibody responses are associated with broad neutralization of hepatitis C virus

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development

CHARACTERIZATION OF BROADLY NEUTRALIZING ANTIBODIES INDUCTION AND RESISTANCE IN HUMAN SUBJECTS WHO REPEATEDLY CLEAR HEPATITIS C VIRUS INFECTION

Development of a prophylactic hepatitis C virus (HCV) vaccine is necessary for disease eradication. One of the challenges to the development of a successful HCV vaccine is the worldwide genetic diversity of the virus. Approximately 25% of HCV infected individuals can clear HCV infection spontaneously. 80% of these can also clear subsequent infections (reinfections), which have shorter duration and lower peak viremia, indicating protection by adaptive immunity. Over the years, multiple broadly neutralizing antibodies (bNAbs) have been identified that block infection by diverse HCV strains in vitro and infusion of bNAbs is protective against HCV infection in animal models. Therefore, a prophylactic HCV vaccine that elicits bNAbs could be key to HCV eradication. However, the induction of anti-HCV bNAbs in humans and the effect of repeated infections on anti-HCV bNAb responses remain not well characterized. Here, we characterize the antibody response of eight reinfected individuals in a cohort of people who inject drugs. These reinfected individuals provide a unique opportunity to study the immune response to repeated challenges with heterologous viral strains. The overall goal of this project is to study these rare HCV reinfections to better understand the mechanisms of antibody-mediated control of HCV reinfection, and the effect of repeated infections with diverse viruses on antibody neutralizing breadth. By measuring neutralizing breadth of plasma antibodies in these participants, we identified key features of the antigenic stimuli associated with the induction of potent anti-HCV bNAbs in humans. By measuring plasma antibody neutralization of autologous longitudinal HCV variants from the same subjects, we found that in people who spontaneously clear HCV reinfections, bNAbs can drive evolution of the virus, selecting viral variants with decreasing E2 fitness over time. This work illustrates that an antibody-based HCV vaccine should be a goal of future vaccine development efforts. This thesis provides a novel approach for vaccine antigen selection that could also be useful for antigen design for other genetically diverse pathogens. Finally, it suggests that optimal presentation of residues targeted by protective antibodies should improve vaccine efficacy

Expression cloning and analysis of a putative Brugia malayi POU-homeodomain transcription factor

Neglected tropical diseases (NTDs) are a series of diseases that afflict individuals living predominantly in developing countries. One of the most impactful NTDs is lymphatic filariasis (LF), a disease caused by filarial parasitic nematodes. Current efforts to combat these diseases have been met with considerable success, but the concern of possible drug resistance underscores a need for the development of novel anthelmintic therapeutics that cause minimal harm to the human host. Expression of the parasite’s genome undergoes dramatic changes when the parasite is transmitted from the mosquito vector into the human host. Despite the critical need to understand these gene expression changes, little is known about promoters and transcription factors in filarial parasites. The aim of this study is to identify transcription factors in our model organism, B. malayi, that could serve as potential drug targets to help combat filariasis. Through a comparative analysis between whole genome datasets of B. malayi and C. elegans, we were able to identify the putative transcription factor Bma-UNC-86 as the best candidate for this initial project. Bioinformatics analyses revealed high homology between the binding sequences of UNC-86 in the promoter region of its target gene, mec-3, in both C. elegans and B. malayi. The putative transcription factor Bma-UNC-86 was cloned and expressed in E. coli. The purified UNC-86 protein will be used in future studies to investigate gene regulation at the protein level by analyzing the interaction of the transcription factor with the promoter region of its target gene in B. malayi. Application of this approach will further expand the data available to investigate the biology of parasitic helminths and lead to the development of novel therapeutics

Neutralizing antibodies evolve to exploit vulnerable sites in the HCV envelope glycoprotein E2 and mediate spontaneous clearance of infection.

Individuals who clear primary hepatitis C virus (HCV) infections clear subsequent reinfections more than 80% of the time, but the mechanisms are poorly defined. Here, we used HCV variants and plasma from individuals with repeated clearance to characterize longitudinal changes in envelope glycoprotein E2 sequences, function, and neutralizing antibody (NAb) resistance. Clearance of infection was associated with early selection of viruses with NAb resistance substitutions that also reduced E2 binding to CD81, the primary HCV receptor. Later, peri-clearance plasma samples regained neutralizing capacity against these variants. We identified a subset of broadly NAbs (bNAbs) for which these loss-of-fitness substitutions conferred resistance to unmutated bNAb ancestors but increased sensitivity to mature bNAbs. These data demonstrate a mechanism by which neutralizing antibodies contribute to repeated immune-mediated HCV clearance, identifying specific bNAbs that exploit fundamental vulnerabilities in E2. The induction of bNAbs with these specificities should be a goal of HCV vaccine development

Image_3_Convergent antibody responses are associated with broad neutralization of hepatitis C virus.pdf

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.</p

Repeated exposure to heterologous hepatitis C viruses associates with enhanced neutralizing antibody breadth and potency

A prophylactic hepatitis C virus (HCV) vaccine that elicits neutralizing antibodies could be key to HCV eradication. However, the genetic and antigenic properties of HCV envelope (E1E2) proteins capable of inducing anti-HCV broadly neutralizing antibodies (bNAbs) in humans have not been defined. Here, we investigated the development of bNAbs in longitudinal plasma of HCV-infected persons with persistent infection or spontaneous clearance of multiple reinfections. By measuring plasma antibody neutralization of a heterologous virus panel, we found that the breadth and potency of the antibody response increased upon exposure to multiple genetically distinct infections and with longer duration of viremia. Greater genetic divergence between infecting strains was not associated with enhanced neutralizing breadth. Rather, repeated exposure to antigenically related, antibody-sensitive E1E2s was associated with potent bNAb induction. These data reveal that a prime-boost vaccine strategy with genetically distinct, antibody-sensitive viruses is a promising approach to inducing potent bNAbs in humans

Table_1_Convergent antibody responses are associated with broad neutralization of hepatitis C virus.docx

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.</p

Image_1_Convergent antibody responses are associated with broad neutralization of hepatitis C virus.pdf

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.</p

SARS-CoV-2-specific immune responses in boosted vaccine recipients with breakthrough infections during the Omicron variant surge

BACKGROUND. Breakthrough SARS-CoV-2 infections in vaccinated individuals have been previously associated with suboptimal humoral immunity. However, less is known about breakthrough infections with the Omicron variant. METHODS. We analyzed SARS-CoV-2???specific antibody and cellular responses in healthy vaccine recipients who experienced breakthrough infections a median of 50 days after receiving a booster mRNA vaccine with an ACE2 binding inhibition assay and an ELISpot assay, respectively. RESULTS. We found that high levels of antibodies inhibited vaccine strain spike protein binding to ACE2 but that lower levels inhibited Omicron variant spike protein binding to ACE2 in 4 boosted vaccine recipients prior to infection. The levels of antibodies that inhibited vaccine strain and Omicron spike protein binding after breakthrough in 18 boosted vaccine recipients were similar to levels seen in COVID-19???negative boosted vaccine recipients. In contrast, boosted vaccine recipients had significantly stronger T cell responses to both vaccine strain and Omicron variant spike proteins at the time of breakthrough. CONCLUSION. Our data suggest that breakthrough infections with the Omicron variant can occur despite robust immune responses to the vaccine strain spike protein. FUNDING. This work was supported by the Johns Hopkins COVID-19 Vaccine-related Research Fund and by funds from the National Institute of Allergy and Infectious Disease intramural program as well as awards from the National Cancer Institute (U54CA260491) and the National Institutes of Allergy and Infectious Disease (K08AI156021 and U01AI138897)