Biology subjects

Gnanapragasam, P. N. P.

Publications and source records attributed to Gnanapragasam, P. N. P..

14 recordsLinked to original sources

bioRxiv2026

Stabilized gp120-specific CD4 for next-generation HIV-1 inhibitors

HIV-1 Envs gp120 subunit uses the T-cell coreceptor CD4 to enter host cells in a manner that prevents the evolution of host resistance by sharing the binding epitope with the footprint of CD4s natural ligands, class II MHC proteins1,2. Consequently, CD4-containing biologics, such as CD4-Ig3,4 and derivatives5-9, benefit from this conserved relationship and are promising broad-acting anti-HIV-1 agents that are resistant to viral mutational escape10. However, these biologics suffer from short serum half-lives in humans11,12 and animals3,13, likely due to CD4s poor thermostability14 and/or off-target class II MHC binding15. This latter property also warrants caution for CD4-containing biologics that could indiscriminately recruit Fc-dependent effector functions against uninfected cells and/or compete with host CD4 for class II MHC during T cell interactions with antigen-presenting cells. Here, we describe gp120-specific CD4 (gCD4), which exhibits enhanced thermostability and retains Env, but not class II MHC, binding. CD4-Ig variants incorporating gCD4 did not bind class II MHC on human B cells, displayed greater longevity in human tonsil organoid cultures, showed half-lives equivalent to therapeutic IgG antibodies in mice, and neutralized HIV-1 more broadly and potently compared to the original CD4-Ig molecules. Encouragingly, one variant neutralized 100% of a panel of clinically-relevant HIV-1 strains at titers correlating to infection prevention in humans, outperforming known broadly neutralizing antibodies16,17. Thus, gCD4 holds promise for the development of new CD4-containing biologics with best-in-class specificity, pharmacokinetic properties, and neutralization breadth and potency.

bioengineering↗

bioRxiv2025

Single administration of mosaic-8b RBD-nanoparticle vaccine prepared with atomic layer deposition technology elicits broadly neutralizing anti-sarbecovirus responses

Atomic layer deposition (ALD), a new vaccine technology, permits multiple dosing with a single administration by pulsatile release of one or more immunogens. We evaluated ALD delivery of mosaic-8b [60-mer nanoparticles presenting 8 different SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] that elicits broadly cross-reactive antibodies and protects against mismatched sarbecoviruses not represented by RBDs on mosaic-8b. Compared with conventional prime-boost immunizations, ALD-delivered mosaic-8b RBD-nanoparticles elicited antibodies in both naive and pre-vaccinated mice with improved mismatched binding and neutralization. Results of RBD epitope mapping of serum antibodies from ALD-delivered mosaic-8b were consistent with broader coverage of RBD epitopes compared to conventional immunizations, and systems serology revealed distinct IgG subclass and Fc{gamma}R-binding IgG distributions. These results suggest that ALD is a promising technology for use with mosaic-8b RBD-nanoparticle vaccines to protect against future sarbecovirus spillovers and support applications for ALD vaccine delivery to elicit cross-reactive antibodies against rapidly mutating or diverse pathogens.

immunology↗

bioRxiv2025

Design and characterization of HIV-1 vaccine candidates to elicit antibodies targeting multiple epitopes

A primary goal in the development of an AIDS vaccine is the elicitation of broadly neutralizing antibodies (bNAbs) that protect against diverse HIV-1 strains. To this aim, germline-targeting immunogens have been developed to activate bNAb precursors and initiate the induction of bNAbs. While most pre-clinical germline-targeting HIV-1 vaccine candidates only include a single bNAb precursor epitope, an effective HIV-1 vaccine will likely require bNAbs that target multiple epitopes on Env. Here, we report a newly designed germline-targeting Env SOSIP trimer, named 3nv.2, that presents three bNAb epitopes on Env: the CD4bs, V3, and V2 epitopes. 3nv.2 forms a stable trimeric Env and binds to bNAb precursors from each of the desired epitopes. Immunization experiments in rhesus macaques and mice demonstrate 3nv.2 elicits the combined effects of its parent immunogens. Our results provide proof-of-concept for using a germline-targeting immunogen presenting three or more bNAb epitopes and a framework to develop improved next-generation HIV-1 vaccine candidates.

bioengineering↗

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

Mapping essential somatic hypermutations in a CD4-binding site bNAb informs HIV-1 vaccine design

HIV-1 broadly-neutralizing antibodies (bNAbs) targeting the CD4-binding site (CD4bs) contain rare features that pose challenges to elicit these bNAbs through vaccination. The IOMA-class of CD4bs bNAbs includes fewer rare features and somatic hypermutations (SHMs) to achieve broad neutralization, thus presenting a potentially accessible pathway for vaccine-induced bNAb development. Here, we created a library of IOMA variants in which each SHM was individually reverted to the inferred germline (iGL) counterpart to investigate the roles of SHMs in conferring IOMAs neutralization potency and breadth. Impacts on neutralization for each variant were evaluated, and this information was used to design minimally-mutated IOMA-class variants (IOMAmin) that incorporated the fewest SHMs required for achieving IOMAs neutralization breadth. A cryo-EM structure of an IOMAmin variant bound to Env was used to further interpret characteristics of IOMA variants to elucidate how IOMAs structural features correlate with its neutralization mechanism, informing the design of IOMA-targeting immunogens. HighlightsIOMAmin variants with reduced SHMs retain neutralization potency and breadth. Cryo-EM structure reveals IOMAmin preserves key Env interactions despite fewer SHMs. IOMA variants with mutations in CDRH3 and CDRL1 fail to improve neutralization.

immunology↗

bioRxiv2024

Designed mosaic nanoparticles enhance cross-reactive immune responses in mice

Using computational methods, we designed 60-mer nanoparticles displaying SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs) by (i) creating RBD sequences with 6 mutations in the SARS-COV-2 WA1 RBD that were predicted to retain proper folding and abrogate antibody responses to variable epitopes (mosaic-2COMs; mosaic-5COM), and (ii) selecting 7 natural sarbecovirus RBDs (mosaic-7COM). These antigens were compared with mosaic-8b, which elicits cross-reactive antibodies and protects from sarbecovirus challenges in animals. Immunizations in naive and COVID-19 pre-vaccinated mice revealed that mosaic-7COM elicited higher binding and neutralization titers than mosaic-8b and related antigens. Deep mutational scanning showed that mosaic-7COM targeted conserved RBD epitopes. Mosaic-2COMs and mosaic-5COM elicited higher titers than homotypic SARS-CoV-2 Beta RBD-nanoparticles and increased potencies against some SARS-CoV-2 variants than mosaic-7COM. However, mosaic-7COM elicited more potent responses against zoonotic sarbecoviruses and highly mutated Omicrons. These results support using mosaic-7COM to protect against highly mutated SARS-CoV-2 variants and zoonotic sarbecoviruses with spillover potential.

immunology↗

bioRxiv2024

Mosaic sarbecovirus vaccination elicits cross-reactive responses in pre-immunized animals

Immunization with mosaic-8b [60-mer nanoparticles presenting 8 SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate-mapping in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19 vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

immunology↗

bioRxiv2022

Mosaic RBD nanoparticles protect against multiple sarbecovirus challenges in animal models

To combat future SARS-CoV-2 variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles presenting randomly-arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against conserved/relatively-occluded, rather than variable/immunodominant/exposed, epitopes. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD-nanoparticles in mice and macaques, observing stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants including Omicron and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest mosaic-8 RBD-nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers.

microbiology↗

bioRxiv2022

A naturally arising broad and potent CD4-binding site antibody with low somatic mutation

The induction of broadly neutralizing antibodies (bNAbs) is a potential strategy for a vaccine against HIV-1. However, most bNAbs exhibit features such as unusually high somatic hypermutation, including insertions and deletions, which make their induction challenging. VRC01-class bNAbs exhibit extraordinary breadth and potency, but also rank among the most highly somatically-mutated bNAbs. Here we describe a VRC01-class antibody isolated from a viremic controller, BG24, that has less than half the mutations of most other relatives of its class, while achieving comparable breadth and potency. A 3.8 [A] X-ray crystal structure of a BG24-BG505 Env trimer complex revealed conserved contacts at the gp120 interface characteristic of the VRC01-class Abs, despite lacking common CDR3 sequence motifs. The existence of moderately-mutated CD4-binding site (CD4bs) bNAbs such as BG24 provides a simpler blueprint for CD4bs antibody induction by a vaccine, raising the prospect that such an induction might be feasible with a germline-targeting approach. TeaserAn anti-HIV-1 antibody with comparable neutralization breadth and potency to similarly-classed antibodies, with half as many mutations.

immunology↗

bioRxiv2021

Rapid Identification of Neutralizing Antibodies against SARS-CoV-2 Variants by mRNA Display

The increasing prevalence of SARS-CoV-2 variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly-reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identified a set of antibodies against SARS-CoV-2 spike (S) proteins and characterized the structures of nAbs that recognized epitopes in the S1 subunit of the S glycoprotein. These structural studies revealed distinct binding modes for several antibodies, including targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interacts with angiotensin- converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. A potent ACE2-blocking nAb was further engineered to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is a promising approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.

immunology↗

bioRxiv2021

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

Many anti-SARS-CoV-2 neutralizing antibodies target the ACE2-binding site on viral spike receptor-binding domains (RBDs). The most potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly-emergent zoonotic sarbecoviruses and variants, but usually show only weak neutralization potencies. We characterized two class 4 anti-RBD antibodies derived from COVID-19 donors that exhibited broad recognition and potent neutralization of zoonotic coronavirus and SARS-CoV-2 variants. C118-RBD and C022-RBD structures revealed CDRH3 mainchain H-bond interactions that extended an RBD {beta}-sheet, thus reducing sensitivity to RBD sidechain changes, and epitopes that extended from the cryptic epitope to occlude ACE2 binding. A C118-spike trimer structure revealed rotated RBDs to allow cryptic epitope access and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.

immunology↗

bioRxiv2020

Mosaic RBD nanoparticles elicit neutralizing antibodies against SARS-CoV-2 and zoonotic coronaviruses

Protection against SARS-CoV-2 and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor-binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles; 4-8 distinct RBDs). Mice immunized with RBD-nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic-RBD-nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs compared to sera from immunizations with homotypic SARS-CoV-2-RBD-nanoparticles or COVID-19 convalescent human plasmas. Moreover, sera from mosaic-RBD-immunized mice neutralized heterologous pseudotyped coronaviruses equivalently or better after priming than sera from homotypic SARS-CoV-2-RBD-nanoparticle immunizations, demonstrating no immunogenicity loss against particular RBDs resulting from co-display. A single immunization with mosaic-RBD-nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses. One sentence summaryNanoparticle strategy for pan-sarbecovirus vaccine 125-character summary for online ToCImmunizing with nanoparticles displaying diverse coronavirus RBDs elicits cross-reactive and neutralizing antibody responses.

immunology↗

bioRxiv2020

Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate

Fusion of HIV-1 with the membrane of its target cell, an obligate first step in virus infectivity, is mediated by binding of the viral envelope (Env) spike protein to its receptors, CD4 and CCR5/CXCR4, on the cell surface. The process of viral fusion appears to be fast compared with viral egress and has not been visualized by electron microscopy (EM). To capture fusion events for EM, the process must be slowed or stopped by trapping Env-receptor binding at an intermediate stage. Here we describe using fusion inhibitors to trap HIV-1 virions attached to target cells by Envs in an extended pre-hairpin intermediate state. Electron tomography revealed HIV-1 virions bound to TZM-bl cells by 2-4 narrow spokes, with slightly more spokes present when evaluated with mutant virions that lacked the Env cytoplasmic tail. These results represent the first direct visualization of the hypothesized pre-hairpin intermediate and improve our understanding of Env-mediated HIV-1 fusion and infection of host cells.

cell biology↗

bioRxiv2018

Quantifying the Entropic and Energetic Effects of Linker Length and Rigidity within Synthetic HIV-1 Antibodies designed to Bind Bivalently to Env Spikes

Due to the low density of envelope (Env) spikes on the surface of HIV-1, neutralizing IgG antibodies rarely bind bivalently using both antigen-binding arms (Fabs) to crosslink between spikes (inter-spike crosslinking), instead resorting to weaker monovalent binding that is more sensitive to Env mutations. Synthetic antibodies designed to bivalently bind a single Env trimer (intra-spike crosslinking) were previously shown to exhibit increased neutralization potencies. In initial work, diFabs joined by varying lengths of rigid double-stranded DNA (dsDNA) were considered. Anticipating future experiments to improve synthetic antibodies, we investigate whether linkers with different rigidities could enhance diFab potency by modeling DNA-Fabs containing different combinations of rigid dsDNA and flexible single-stranded DNA (ssDNA) and characterizing their neutralization potential. Model predictions suggest that while a long flexible polymer may be capable of bivalent binding, it exhibits weak neutralization due to the large loss in entropic degrees of freedom when both Fabs are bound. In contrast, the strongest neutralization potencies are predicted to require a rigid linker that optimally spans the distance between two Fab binding sites on an Env trimer, and avidity can be further boosted by incorporating more Fabs into these constructs. These results inform the design of multivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.\n\nSignificanceIgG antibodies utilize avidity to increase their apparent affinities through simultaneous binding of two antigen-binding Fabs - if one Fab dissociates from an antigen, the other Fab can remain attached, allowing rebinding. HIV-1 foils this strategy by having few, and highly-separated, Envelope spike targets for antibodies, forcing most IgGs to bind monovalently. Here we develop a statistical mechanics model of synthetic diFabs joined by DNA linkers of different lengths and flexibilities. This framework enables us to translate the energetic and entropic effects of the linker into the neutralization potency of a diFab. We demonstrate that the avidity of multivalent binding is enhanced by using rigid linkers or including additional Fabs capable of simultaneous binding, providing the means to quantitatively predict the potencies of other antibody designs.

immunology↗