Biology subjects

Raju, N.

Publications and source records attributed to Raju, N..

9 recordsLinked to original sources

bioRxiv2025

Non-human primate LIBRA-Seq accelerates neutralizing antibody discovery in RM vaccinated against HIV-1

Broadly neutralizing antibodies (bNAbs) exhibit protective efficacy against HIV-1 infection making them an ideal archetype for HIV-1 vaccine design. Presently, no vaccine candidate has induced antibody responses capable of meaningful protection against the swathe of circulating, difficult to neutralize tier 2 HIV-1 viruses. However, the development of stabilized, native-like envelope (Env) trimers such as BG505.SOSIP.664.T332N (BG505 SOSIP) has marked a significant advancement in vaccine design, due to their ability to elicit NAbs that neutralize tier 2 viruses in rhesus macaques (RM). NAb development following envelope trimer immunization in RM remains poorly understood, with hypothesized contributions from genetic variation at the IG loci, naive B cell repertoire, and differential gene expression in B cell lineages. To address these knowledge gaps, we have developed a set of BG505 SOSIP probes capable of recovering paired clonotype identity, antigen specificity, and gene expression of B cells in a high throughput fashion. These probes were constructed by conjugating biotinylated BG505 SOSIP to streptavidin covalently linked to both sc-RNA-Seq compatible DNA oligonucleotides and flow cytometry compatible fluorophores. Using these reagents, we isolated and sequenced BG505 SOSIP specific memory B cells from the PBMCs of an RM that developed high titers of neutralizing antibodies. To benchmark the accuracy of our technology, we compared our recovered heavy and light chain sequences to those identified from the same animal using conventional methodology and recovered 100% of previously identified NAbs. We then applied this technology to recover BG505 SOSIP specific memory B cells from five additional vaccinated RMs, cloned 34 antibodies for functional characterization, and identified ten antibodies with autologous neutralizing activity. Author SummaryUnderstanding how effective antibodies arise after HIV vaccination is essential for developing a protective vaccine, yet studying these responses in non-human primates has been limited by low- throughput methods. In this study, we adapted a high-throughput single-cell sequencing approach to identify HIV envelope-specific antibodies from vaccinated rhesus macaques. This method allowed us to recover paired antibody sequences together with their antigen specificity from thousands of individual B cells. We successfully identified known neutralizing antibodies and discovered additional antibodies capable of neutralizing HIV across multiple animals. Our analysis revealed that vaccine-elicited antibody responses were dominated by a small number of expanded lineages, with shared genetic features among animals with stronger neutralization. These findings demonstrate that this approach can efficiently define the antibody repertoires generated by HIV vaccines and provide a powerful tool for evaluating and improving immunogens in preclinical vaccine studies.

immunology↗

bioRxiv2025

A Broad Survey and Functional Analysis of Immunoglobulin Loci Variation in Rhesus Macaques

Rhesus macaques (RMs) are a vital model for studying human disease and invaluable to pre-clinical vaccine research, particularly for the study of broadly neutralizing antibody responses. Such studies require robust genetic resources for antibody-encoding genes within the immunoglobulin (IG) loci. The complexity of the IG loci has historically made them challenging to characterize accurately. To address this, we developed novel experimental and computational methodologies to generate the largest collection to date of integrated antibody repertoire and long-read genomic sequencing data in 106 Indian origin RMs. We created a comprehensive resource of IG heavy and light chain variable (V), diversity (D), and joining (J) alleles, as well as leader, intronic, and recombination signal sequences (RSSs), including the curation of 1474 novel alleles, unveiling tremendous diversity, and expanding existing IG allele sets by 60%. This publicly available, continually updated resource (https://vdjbase.org/reference_book/Rhesus_Macaque) provides the foundation for advancing RM immunogenomics, vaccine discovery, and translational research.

immunology↗

bioRxiv2023

Development of LIBRA-seq for the Guinea Pig Model System as a Tool for the Evaluation of Antibody Responses to Multivalent HIV-1 Vaccines

Consistent elicitation of serum antibody responses that neutralize diverse clades of HIV-1 remains a primary goal of HIV-1 vaccine research. Prior work has defined key features of soluble HIV-1 Envelope (Env) immunogen cocktails that influence the neutralization breadth and potency of multivalent vaccine-elicited antibody responses including the number of Env strains in the regimen. We designed immunization groups that consisted of different numbers of Env strains to be used in a cocktail immunization strategy: the smallest cocktail (group 2) consisted of a set of two Env strains, which were a subset of the three Env strains that made up group 3, which in turn were a subset of the six Env strains that made up group 4. Serum neutralizing titers were broadest in guinea pigs that were immunized with a cocktail of three Envs compared to cocktails of two and six, suggesting that multivalent Env immunization provides a benefit but may be detrimental when the cocktail size is too large. We then adapted the LIBRA-seq platform for antibody discovery to be compatible with guinea pigs, and isolated several tier 2 neutralizing monoclonal antibodies. Three antibodies isolated from two separate guinea pigs were similar in their gene usage and CDR3s, establishing evidence for a guinea pig public clonotype elicited through vaccination. Taken together, this work investigated multivalent HIV-1 Env immunization strategies and provides a novel methodology for screening guinea pig B cell receptor antigen specificity at a high throughput level using LIBRA-seq. IMPORTANCEMultivalent vaccination with soluble Env immunogens is at the forefront of HIV-1 vaccination strategies, but little is known about the influence of the number of Env strains included in vaccine cocktails. Our results suggest that adding more strains is sometimes beneficial but may be detrimental when the number of strains is too high. Additionally, we adapted the LIBRA-seq platform to be compatible with guinea pig samples and isolated several tier 2 neutralizing monoclonal antibodies, some of which share V and J gene usage and >80% CDR3 identity, thus establishing the existence of public clonotypes in guinea pigs elicited through vaccination.

immunology↗

bioRxiv2021

Neutralization Fingerprinting Technology for Characterizing Polyclonal Antibody Responses to Dengue Vaccines

Dengue is a major public health threat. There are four serotypes of dengue virus (DENV), therefore efforts are focused on development of safe and effective tetravalent DENV vaccines. While neutralizing antibodies contribute to protective immunity, there are still important gaps in understanding of immune responses elicited by dengue infection and vaccination, including defining immune correlates of protection. To that end, here we present a computational modeling framework for evaluating the specificities of neutralizing antibodies elicited to tetravalent DENV vaccines, based on the concept of antibody-virus neutralization fingerprints. We developed and applied this framework to samples from clinical studies of TAK-003, a tetravalent vaccine candidate currently in phase 3 trials, to characterize the effect of prior dengue infection (baseline) on the specificities of vaccine-elicited antibody responses. Our results suggested a similarity of neutralizing antibody specificities in baseline-seronegative individuals. In contrast, amplification of pre-existing neutralizing antibody specificities was predicted for baseline-seropositive individuals, thus quantifying the role of immunologic imprinting in driving antibody responses to DENV vaccines. The analysis framework proposed here can apply to studies of sequential dengue infections and other tetravalent DENV vaccines and can contribute to understanding dengue immune correlates of protection to help guide further vaccine development and optimization.

immunology↗

bioRxiv2021

Single-Cell Profiling of the Antigen-Specific Response to BNT162b2 SARS-CoV-2 RNA Vaccine

RNA-based vaccines against SARS-CoV-2 are critical to limiting COVID-19 severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. We used single-cell technologies to identify and characterized antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 in longitudinal samples from a cohort of healthy donors. Mass cytometry and machine learning pinpointed a novel expanding, population of antigen-specific non-canonical memory CD4+ and CD8+ T cells. B cell sequencing suggested progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlated with eventual SARS-CoV-2 IgG and a donor lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms reveal an antigen-specific cellular basis of RNA vaccine-based immunity. ONE SENTENCE SUMMARYSingle-cell profiling reveals the cellular basis of the antigen-specific response to the BNT162b2 SARS-CoV-2 RNA vaccine.

immunology↗

bioRxiv2021

Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking

SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody-ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen-ligand interactions.

immunology↗

bioRxiv2021

Sequence and functional characterization of a public HIV-specific antibody clonotype

Public antibody clonotypes shared among multiple individuals have been identified for several pathogens. However, little is known about the limits of what constitutes a public clonotype. Here, we characterize the sequence and functional properties of antibodies from a public HIV-specific clonotype comprising sequences from 3 individuals. Our results showed that antigen specificity for the public antibodies was modulated by the VH, but not VL, germline gene. Non-native pairing of public heavy and light chains from different donors resulted in antibodies with consistent antigen specificity, suggesting functional complementation of sequences within the public antibody clonotype. The strength of antigen recognition appeared to be dependent on the specific antibody light chain used, but not on other sequence features such as germline or native-antibody sequence identity. Understanding the determinants of antibody clonotype "publicness" can provide insights into the fundamental rules of host-pathogen interactions at the population level, with implications for clonotype-specific vaccine development.

immunology↗

bioRxiv2021

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with a unique genetic signature and structural mode of spike recognition

The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.

immunology↗

bioRxiv2020

Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions

The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.

immunology↗