Biology subjects

Baharani, V. A.

Publications and source records attributed to Baharani, V. A..

7 recordsLinked to original sources

bioRxiv2026

Multivalent Anti-ACE2 Nanobodies Confer Broad Pan-Sarbecovirus Protection

The continual emergence of SARS-CoV-2 variants that rapidly evade conventional spike-directed neutralizing antibodies, together with the ongoing risk of cross-species spillover and new sarbecovirus outbreaks, underscores the need to develop broadly acting, escape-resistant therapeutic agents. Here, we optimized a nanobody discovery pipeline incorporating competition-based yeast surface display assays to isolate single-chain variable heavy chain-only antibody domains (VHHs or nanobodies) that bind human ACE2 and inhibit SARS-CoV-2 entry. Dimeric VHHs, as well as bivalent and tetravalent Fc-fusion proteins exhibited markedly increased antiviral activity, blocking a broad panel of SARS-CoV-2 variants and diverse sarbecoviruses at low-nanomolar to picomolar concentrations. These agents did not affect ACE2 enzymatic function or cell surface expression. The VHH-Fc fusion proteins had favorable pharmacokinetics and conferred prophylactic protection in mouse models of both SARS-CoV-2 and SARS-CoV infection, showcasing their potential as broadly acting receptor-targeted biologics against pandemic-threat viruses.

immunology↗

bioRxiv2026

Structural and mutational analyses define distinct molecular routes to broad SARS-CoV-2 receptor-binding domain recognition

Broadly reactive antibodies elicited by SARS-CoV-2 infection or vaccination can reveal conserved viral vulnerabilities and inform vaccines with broad coronavirus coverage. Here, we characterize two human-derived monoclonal antibodies, B2014 and C5078, that recognize conserved epitopes on the SARS-CoV-2 RBD and retain activity across antigenically distinct variants. Notably, C5078 also recognizes diverse sarbecoviruses and remains active against currently circulating variants, including XFG and NB.1.8.1. Cryo-EM structures reveal that B2014 recognizes an epitope adjacent to the class 3 antibody site, whereas C5078 targets the highly conserved, cryptic site V epitope. Structural analysis defines how C5078 uses affinity-matured interactions to engage conserved RBD residues, providing a molecular basis for its exceptional breadth. Deep mutational scanning across multiple SARS-CoV-2 variant backgrounds further defines potential pathways of antibody escape, explaining the loss of B2014 activity against antigenically evolved variants while revealing a high barrier to escape from C5078. Together, these findings define distinct structural solutions for broad RBD recognition and highlight conserved, mutationally constrained epitopes that may serve as targets for vaccines designed to elicit antibody responses resilient to ongoing SARS-CoV-2 evolution and future sarbecovirus emergence.

biophysics↗

bioRxiv2026

A designed overlapping variant immunogen pool elicits broad sarbecovirus neutralization

A central problem in achieving vaccine-based protection against viral infections is eliciting antibodies that are resilient to viral variation. Successive waves of SARS-CoV-2 infection during the COVID19 pandemic were driven by variants that acquired resistance to neutralizing antibodies elicited by prior SARS-CoV-2 variants. To the extent that serum neutralization breadth occurs in individuals with multiple exposures to SARS-CoV-2 antigens, we and others find that it is largely comprised of antibodies that target the variable receptor binding domain (RBD), rather than more conserved spike protein domains. By designing synthetic dimeric RBD immunogens we show that limiting divergence in heterodimeric components favors the generation of cross-reactive B cells and antibodies. We thus devised a vaccine approach based on a two-dose immunization with a pool of five overlapping heterodimeric synthetic RBD variants. Collectively, the RBD heterodimer pool was designed to cover 10% sequence variation and elicited greater antibody cross-reactivity and neutralization breadth than homodimers or heterodimers with highly divergent components. Using an unconventional prospective challenge model in mice, we demonstrate the effectiveness of the RBD heterodimer pool in inducing antibody responses that attenuate infection by future SARS-CoV-2 variants, as well as protection in a challenge model based on a chimeric vesicular stomatitis virus bearing a spike protein from SARS-CoV-1. Significance statementViral antigenic escape undermines both vaccination efforts and the development of herd immunity, resulting in an enormous viral disease burden. A central problem in eliciting vaccine-based protection against some viral infections is achieving antibody neutralization breadth. To elicit collections of antibodies that overcome the problem of limited antibody tolerance of viral variation, we designed a novel strategy based on an overlapping series of immunogens. This immunogen pool conferred at least partial protection against subsequently prevalent SARS-CoV-2 variants as well as a chimeric SARS-CoV-1 based model virus.

microbiology↗

bioRxiv2025

Clonal Expansion and Diversification of Germinal Center and Memory B Cell Responses to Booster Immunization in Primates

Effective vaccines elicit B cell clonal expansion in germinal centers (GCs) that produce memory B cells and antibody secreting plasma cells. Studies in mice indicate that, whereas the plasma cell compartment is enriched for cells producing high affinity antibodies, the memory pool is more diverse and contains only a relatively small proportion of higher affinity cells. Upon boosting, murine memory B cells producing high affinity antibodies tend to develop into plasma cells but few if any re-enter GCs. However, mice live for only a few weeks in nature, and in keeping with the rather limited requirement for immune memory, this compartment comprises only 1-2% of all B cells. In contrast, memory accounts for nearly 50% of all B cells in primates. Here we examine memory and GC B cell responses in rhesus macaques immunized and boosted ipsilaterally or contralaterally with an mRNA vaccine encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein. The neutralizing activity of antibodies cloned from the memory compartment, and the size of the compartment, was independent of the site of boosting. Moreover, in primates, memory B cells enter and undergo iterative expansion in newly developing GCs when boosting is at a site distal to the site of priming. Thus, in primates, high affinity memory B cells constitute a reservoir that actively participates in further development of immunity irrespective of the anatomical site of vaccine boosting. Highlights- Clonal overlap between primate memory and germinal center B cell compartments following booster immunization. - Neutralization activity of the memory and germinal center compartments are independent of the boost site. - Relationship between site of booster immunization (ipsilateral versus contralateral) and development of memory and germinal center (GC) responses in primates

immunology↗

bioRxiv2025

Cross-reactive sarbecovirus antibodies induced by mosaic RBD-nanoparticles

Broad immune responses are needed to mitigate viral evolution and escape. To induce antibodies against conserved receptor-binding domain (RBD) regions of SARS-like betacoronavirus (sarbecovirus) spike proteins that recognize SARS-CoV-2 variants of concern and zoonotic sarbecoviruses, we developed mosaic-8b RBD-nanoparticles presenting eight sarbecovirus RBDs arranged randomly on a 60-mer nanoparticle. Mosaic-8b immunizations protected animals from challenges from viruses whose RBDs were matched or mismatched to those on nanoparticles. Here, we describe neutralizing mAbs isolated from mosaic-8b-immunized rabbits, some on par with Pemgarda, the only currently FDA-approved therapeutic mAb. Deep mutational scanning, in vitro selection of spike resistance mutations, and single-particle cryo-electron microscopy structures of spike-antibody complexes demonstrated targeting of conserved RBD epitopes. Rabbit mAbs included critical D-gene segment RBD-recognizing features in common with human anti-RBD mAbs, despite rabbit genomes lacking an equivalent human D-gene segment, thus demonstrating that the immune systems of humans and other mammals can utilize different antibody gene segments to arrive at similar modes of antigen recognition. These results suggest that animal models can be used to elicit anti-RBD mAbs with similar properties to those raised in humans, which can then be humanized for therapeutic use, and that mosaic RBD-nanoparticle immunization coupled with multiplexed screening represents an efficient way to generate and select broadly cross-reactive therapeutic pan-sarbecovirus and pan-SARS-CoV-2 variant mAbs. Significance StatementSARS-CoV-2 variants and potential zoonotic sarbecovirus infections continue to threaten human health. Anti-SARS-CoV-2 mAbs that recognize conserved epitopes could be used prophylactically or therapeutically. We present approaches to elicit and identify cross-reactive mAbs using immunizations in animals with mosaic RBD-nanoparticles. We show that human and other mammalian immune systems can utilize different antibody gene segments to arrive at similar modes of antigen recognition, underscoring the flexibility of mammalian antibody repertoires and suggesting that experimental animals can be used to generate therapeutically-useful cross-reactive anti-RBD mAbs. The combination of mosaic-8b RBD-nanoparticles to focus the immune response and a multiplexed assay to select cross-reactive mAbs can be applied at larger scale, or against other pathogens, to identify mAbs of therapeutic and scientific potential.

immunology↗

bioRxiv2024

Bispecific antibodies with broad neutralization potency against SARS-CoV-2 variants of concern

The ongoing emergence of SARS-CoV-2 variants of concern (VOCs) that reduce the effectiveness of antibody therapeutics necessitates development of next-generation antibody modalities that are resilient to viral evolution. Here, we characterized N-terminal domain (NTD) and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from COVID-19 convalescent donors for their activity against emergent SARS-CoV-2 VOCs. Among these, the NTD-specific antibody C1596 displayed the greatest breadth of binding to VOCs, with cryo-EM structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite. Given C1596s favorable binding profile, we designed a series of bispecific antibodies (bsAbs) termed CoV2-biRNs, that featured both NTD and RBD specificities. Notably, two of the C1596-inclusive bsAbs, CoV2-biRN5 and CoV2-biRN7, retained potent in vitro neutralization activity against all Omicron variants tested, including XBB.1.5, EG.5.1, and BA.2.86, contrasting the diminished potency of parental antibodies delivered as monotherapies or as a cocktail. Furthermore, prophylactic delivery of CoV2-biRN5 significantly reduced the viral load within the lungs of K18-hACE2 mice following challenge with SARS-CoV-2 XBB.1.5. In conclusion, our NTD-RBD bsAbs offer promising potential for the design of resilient, next-generation antibody therapeutics against SARS-CoV-2 VOCs. One Sentence SummaryBispecific antibodies with a highly cross-reactive NTD antibody demonstrate resilience to SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2024

Delineating the functional activity of antibodies with cross-reactivity to SARS-CoV-2, SARS-CoV-1 and related sarbecoviruses

The recurring spillover of pathogenic coronaviruses and demonstrated capacity of sarbecoviruses, such SARS-CoV-2, to rapidly evolve in humans underscores the need to better understand immune responses to this virus family. For this purpose, we characterized the functional breadth and potency of antibodies targeting the receptor binding domain (RBD) of the spike glycoprotein that exhibited cross-reactivity against SARS-CoV-2 variants, SARS-CoV-1 and sarbecoviruses from diverse clades and animal origins with spillover potential. One neutralizing antibody, C68.61, showed remarkable neutralization breadth against both SARS-CoV-2 variants and viruses from different sarbecovirus clades. C68.61, which targets a conserved RBD class 5 epitope, did not select for escape variants of SARS-CoV-2 or SARS-CoV-1 in culture nor have predicted escape variants among circulating SARS-CoV-2 strains, suggesting this epitope is functionally constrained. We identified 11 additional SARS-CoV-2/SARS-CoV-1 cross-reactive antibodies that target the more sequence conserved class 4 and class 5 epitopes within RBD that show activity against a subset of diverse sarbecoviruses with one antibody binding every single sarbecovirus RBD tested. A subset of these antibodies exhibited Fc-mediated effector functions as potent as antibodies that impact infection outcome in animal models. Thus, our study identified antibodies targeting conserved regions across SARS-CoV-2 variants and sarbecoviruses that may serve as therapeutics for pandemic preparedness as well as blueprints for the design of immunogens capable of eliciting cross-neutralizing responses. AUTHOR SUMMARYThere is a large collection of sarbecoviruses related to SARS-CoV-2 circulating in animal reservoirs with the potential to spillover into humans. Neutralizing antibodies have the potential to protect against infection, although viral escape is common. In this study, we isolated several monoclonal antibodies that show broad activity against different sarbecoviruses. The antibodies target epitopes in the core of the receptor binding domain that are highly conserved in sequence across sarbecoviruses and emerging SARS-CoV-2 variants. One antibody showed remarkable breadth against both SARS-CoV-1 variants as well as diverse sarbecoviruses. The results of deep mutational scanning suggest that mutations at these predicted sites of escape may functionally constrain viral fitness. Our functional profiling of cross-reactive antibodies highlights vulnerable sites of sarbecoviruses, with some antibodies poised as broadly neutralizing candidates for therapeutic use against future sarbecovirus emergence.

immunology↗