Biology subjects

Bhavsar, D.

Publications and source records attributed to Bhavsar, D..

11 recordsLinked to original sources

bioRxiv2026

N1 neuraminidase monoclonal antibodies 2H08 and 3H03 have decreased binding and inhibition properties against H1N1 influenza viruses circulating after 2018

Influenza viruses are global public health problem. Vaccines are available but have to be updated annually due to antigenic drift of the main vaccine component, the surface glycoprotein hemagglutinin (HA). The second surface glycoprotein of the virus, neuraminidase (NA), has recently moved into the spotlight of vaccine development. However, its antigenic evolution is understudied. We found that two broadly protective N1 NA antibodies, which had previously been shown to bind and inhibit most N1 NA, lost activity to recent N1s from seasonal H1N1 viruses. Here, we show that this drop in activity can mostly be attributed to a change in electrostatic surface potential from neutral to positively charged. This effect was not observed when historic human plasma samples were tested, suggesting that the drop of activity is specific to the two tested mAbs but does not broadly impact the polyclonal human response. Finally, we confirmed that the tested mAbs were fully active against recently emerged pan-zootic clade 2.3.4.4b H5N1 NA.

microbiology↗

bioRxiv2025

A clade 2.3.4.4b H5N1 virus vaccine that elicits cross-protective antibodies against conserved domains of H5 and N1 glycoproteins

The continuous evolution and widespread dissemination of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose critical challenges to global pandemic preparedness. In this study, we assessed a low-dose inactivated split virus vaccine derived from clade 2.3.4.4b H5N1, formulated with an Alum/CpG adjuvant, using a preclinical mouse model. This vaccine induced potent humoral and cellular immune responses, generating high titers of cross-reactive antibodies targeting both hemagglutinin (HA) and neuraminidase (NA) glycoproteins across homologous and heterologous H5 clades. The Alum/CpG adjuvant enabled significant antigen dose-sparing while promoting a balanced Th1/Th2 immune profile. Functional analyses demonstrated strong virus neutralization, neuraminidase inhibition, and potent antibody-dependent cellular cytotoxicity activity. Additionally, the vaccine elicited robust antigen-specific CD4+ and CD8+ T cell responses and effectively controlled viral replication in the lungs, accompanied by reduced lung inflammation. Importantly, vaccinated mice were fully protected against lethal challenges with both the homologous clade 2.3.4.4b and heterologous clade 1 H5N1 viruses, despite low hemagglutination inhibition titers. Electron microscopy polyclonal epitope mapping revealed serum antibodies targeting multiple epitopes on homologous HA and NA, with some cross-reacting to conserved epitopes on heterologous proteins, underscoring broad immune recognition. Collectively, these results highlight the potential of this vaccine candidate to provide broad, multifunctional, and durable immunity against both current and emerging H5N1 threats, supporting its further development for pandemic preparedness.

microbiology↗

bioRxiv2025

Local B-cell immunity and durable memory following live-attenuated influenza intranasal vaccination of humans

Seasonal influenza vaccines are most frequently delivered as intramuscular inactivated vaccines which elicit systemic responses against the immunodominant hemagglutinin (HA) head domain. An intranasally administered, live-attenuated influenza vaccine designed to stimulate mucosal immunity, FluMist, is the sole intranasal vaccine approved in the United States. However, FluMist has lower systemic immunogenicity and efficacy in adults compared to intramuscular formulations. In this study, human mucosal and systemic immunity were examined following seasonal intramuscular or intranasal vaccination. Nasopharyngeal swabs of adenoid tissue were used to longitudinally sample the upper airway. Notably, FluMist induced substantial increases in upper respiratory tract IgG+ and IgA+ HA-specific memory B cells, which displayed an activated CD27+CD21- phenotype. H1, H3, and influenza B virus HA-specific memory B cells were all detected in the upper airway after intranasal immunization and remained elevated at 6-months post-vaccination. Recently activated upper airway memory B cells were not readily detected in intramuscular vaccinees, despite marked elevation of systemic antibody and memory B cells. Thus, despite minimal immune response detected in circulation, live-attenuated influenza vaccine can generate substantial local antigen-specific memory B cell responses in adults. These findings have implications for improving influenza vaccines and for mucosal vaccination against other respiratory pathogens. One Sentence SummaryLongitudinal nasopharyngeal sampling reveals local influenza-specific B cell responses following intranasal but not intramuscular vaccination.

immunology↗

bioRxiv2025

Structural and functional characterization of the antigenicity of influenza A virus hemagglutinin subtype H15

Avian H15 influenza viruses are closely related to H7 viruses, but feature a unique 9-amino acid insertion in their hemagglutinin head domain, creating an additional site for antigenic variation. Here, we characterized a panel of mouse monoclonal antibodies (mAbs) raised against the A/wedge-tailed shearwater/Western Australia/2576/1979 ancestral strain, and a human mAb isolated from an H7N9 vaccinee. We found differences in binding and neutralization profiles against the ancestral strain and drifted strains of H15 isolated after 2008. MAbs that have hemagglutination inhibition activity against the ancestral strain do not show binding to drifted strains, hinting at antigenic differences in the receptor binding site. We show that the mAbs protect in vivo and elucidate mAb-antigen interactions using negative stain and cryo-electron microscopy. The characterization of H15 antigenicity and mechanisms of antibody-mediated neutralization expands our knowledge of this rare avian influenza virus and informs our understanding of immune pressures on viral surface glycoproteins.

microbiology↗

bioRxiv2025

Characterization of the glycoproteins of novel fish influenza B-like viruses

Novel influenza-like virus sequences previously identified in fish and amphibians were found to cluster as a sister clade of influenza B viruses, but have thus far remained uncharacterized. We demonstrate that salamander influenza-like virus (SILV) HA is functionally divergent from influenza B virus HA and does not bind to 2,3- and 2,6-linked sialic acids. However, the HAs of Siamese algae-eater influenza-like virus (SAEILV) and chum salmon influenza-like virus (CSILV) bind to 2,3 linked sialic acid. Furthermore, SAEILV HA binds to sialyated Lewis X, is activated by human airway enzymes and is fusogenic at a wide range of pH conditions. SAEILV NA has a highly conserved active site and a similar structure to other known NAs. We also determined the cryo-electron microscopy structure of the HA of a previously described virus from the same sister clade, the Wuhan spiny eel influenza virus (WSEIV). Importantly, no cross-reactive antibodies against these HAs or NAs were found in the human serum, suggesting that humans are immunologically naive to these viruses. One sentence summaryNovel influenza like-viruses, displayed different target receptor specificity and limited antigenic conservation of the HA and NA relative to influenza B Viruses.

microbiology↗

bioRxiv2025

Immunogenicity and protective efficacy of an intranasal neuraminidase-based influenza virus vaccine adjuvanted with bacterial cell membrane-derived adjuvants

Influenza virus neuraminidase (NA) has emerged as a promising vaccine candidate due to its relatively stable antigenic structure and the ability of NA-specific antibodies to provide cross-protection within influenza virus subtypes. Since the influenza virus causes respiratory infections in humans, developing mucosal vaccines to protect the entry site of the virus is of high importance. Recombinant NA requires adjuvants to induce a protective immune response after mucosal administration. In the current study, we analyze the immunogenicity and protective efficacy of a recombinant NA-based influenza virus vaccine administered intranasally in combination with adjuvants consisting of outer membrane proteins from Neisseria meningitidis complexed with exogenous lipopolysaccharides (LPS) from Shigella flexneri or endogenous LPS from N. meningitidis. We evaluated the local and systemic humoral and cellular immune responses to adjuvanted recombinant N1 NA, analyzing the dynamics of local follicular T-helper (Tfh) cells and germinal center B cells (GCB) in nasal-associated lymphoid tissue (NALT) and tissue-resident memory T cells in lungs, as well as the levels of IgA and IgG in the upper and lower respiratory tracts. Finally, we performed a heterologous challenge study to test the ability of the investigated vaccine formulations to induce cross-protection. The study demonstrates that bacterial cell membrane-derived adjuvants significantly improve the immunogenicity and protective efficacy of the recombinant N1 NA-based influenza vaccine leading to protection against clade 2.3.4.4b H5N1 challenge. This finding supports the potential of these adjuvanted vaccines in providing effective mucosal immunity against influenza virus.

microbiology↗

bioRxiv2025

Human monoclonal antibodies that target clade 2.3.4.4b H5N1 hemagglutinin

The highly pathogenic avian influenza H5N1 virus clade 2.3.4.4b has been spreading globally since 2022, causing mortality and morbidity in domestic and wild birds and mammals, including infection in humans, raising concerns about its pandemic potential. We aimed to generate a panel of anti-hemagglutinin (HA) human monoclonal antibodies (mAbs) against the H5 protein of clade 2.3.4.4b. H2L2 Harbour Mice(R), which express human immunoglobulin germline genes, were immunized with H5 and N1 recombinant proteins from A/mallard/New York/22-008760-007-original/2022 H5N1 virus, enabling the generation of human chimeric antibodies. Through hybridoma technology, sixteen full human mAbs were generated, most of which showed cross-reactivity against H5 proteins from different virus variants. The functionality of the sixteen mAbs was assessed in vitro using hemagglutination inhibition and microneutralization assays with viruses containing a clade 2.3.4.4b HA. Fourteen out of the sixteen mAbs neutralized the virus in vitro. The mAbs with the strongest hemagglutination inhibition activity also demonstrated greater neutralizing capacity and showed increased protective effects in vivo when administered prophylactically or therapeutically in a murine H5N1 challenge model. Using cryo-electron microscopy, we identified a cross-clonotype conserved motif that bound a hydrophobic groove on the head domain of H5 HA. Akin to mAbs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the coronavirus 2019 (COVID-19) pandemic, these mAbs could serve as important treatments in case of a widespread H5N1 epidemic or pandemic.

microbiology↗

bioRxiv2024

Functional and epitope specific monoclonal antibody discovery directly from immune sera using cryoEM

Antibodies are crucial therapeutics, comprising a significant portion of approved drugs due to their safety and clinical efficacy. Traditional antibody discovery methods are labor-intensive, limiting scalability and high-throughput analysis. Here, we improved upon our streamlined approach combining structural analysis and bioinformatics to infer heavy and light chain sequences from electron potential maps of serum-derived polyclonal antibodies (pAbs) bound to antigens. Using ModelAngelo, an automated structure-building tool, we accelerated pAb sequence determination and identified sequence matches in B cell repertoires via ModelAngelo derived Hidden Markov Models (HMMs) associated with pAb structures. Benchmarking against results from a non-human primate HIV vaccine trial, our pipeline reduced analysis time from weeks to under a day with higher precision. Validation with murine immune sera from influenza vaccination revealed multiple protective antibodies. This workflow enhances antibody discovery, enabling faster, more accurate mapping of polyclonal responses with broad applications in vaccine development and therapeutic antibody discovery.

immunology↗

bioRxiv2023

Mucosal antibody responses to SARS-CoV-2 booster vaccination and breakthrough infection

Coronavirus disease 2019 (COVID-19) vaccines have saved millions of lives. However, variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged causing large numbers of breakthrough infections. These developments necessitated the rollout of COVID-19 vaccine booster doses. It has been reported that mucosal antibody levels in the upper respiratory tract, especially for secretory IgA (sIgA), correlate with protection from infection with SARS-CoV-2. However, it is still unclear how high levels of mucosal antibodies can be induced. In this study, we measured serum IgG, saliva IgG and saliva sIgA responses in individuals who received COVID-19 mRNA booster vaccinations or who experienced breakthrough infections. We found that mRNA booster doses could induce robust serum and saliva IgG responses, especially in individuals who had not experienced infections before, but saliva sIgA responses were weak. In contrast, breakthrough infections in individuals who had received the primary mRNA vaccination series induced robust serum and saliva IgG as well as saliva sIgA responses. Individuals who had received a booster dose and then had a breakthrough infection showed low IgG induction in serum and saliva but still responded with robust saliva sIgA induction. These data suggest that upper respiratory tract exposure to antigen is an efficient way of inducing mucosal sIgA while exposure via intramuscular injection is not. ImportanceAntibodies on mucosal surfaces of the upper respiratory tract have been shown to be important for protection from infection with SARS-CoV-2. Here we investigate the induction of serum IgG, saliva IgG and saliva sIgA after COVID-19 mRNA booster vaccination or breakthrough infections.

immunology↗

bioRxiv2022

Immunity to seasonal coronavirus spike proteins does not protect from SARS-CoV-2 challenge in a mouse model but has no detrimental effect on protection mediated by COVID-19 mRNA vaccination

Seasonal coronaviruses have been circulating widely in the human population for many years. With increasing age, humans are more likely to have been exposed to these viruses and to have developed immunity against them. It has been hypothesized that this immunity to seasonal coronaviruses may provide partial protection against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been shown that coronavirus disease 2019 (COVID-19) vaccination induces a back-boosting effects against the spike proteins of seasonal betacoronaviruses. In this study, we tested if immunity to the seasonal coronavirus spikes from OC43, HKU1, 229E or NL63 would confer protection against SARS-CoV-2 challenge in a mouse model, and whether pre-existing immunity against these spikes would weaken the protection afforded by mRNA COVID-19 vaccination. We found that mice vaccinated with the seasonal coronavirus spike proteins had no increased protection as compared to the negative controls. While a negligible back-boosting effect against betacoronavirus spike proteins was observed after SARS-CoV-2 infection, there was no negative original antigenic sin-like effect on the immune response and protection induced by SARS-CoV-2 mRNA vaccination in animals with pre-existing immunity to seasonal coronavirus spike proteins. ImportanceThe impact that immunity against seasonal coronaviruses has on both susceptibility to SARS-CoV-2 infection as well as on COVID-19 vaccination is unclear. This study provides insights into both questions in a mouse model of SARS-CoV-2.

microbiology↗

bioRxiv2021

Functionality of the putative surface glycoproteins of the Wuhan spiny eel influenza virus

A panel of novel influenza-like virus sequences were recently documented in jawless fish, ray-finned fish, and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been historically documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterized the functionality and antigenicity of the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV to better understand this virus and its pandemic potential. Upon functional characterization of NA, we identified that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that could be inhibited by NA inhibitors. Testing of the functionality of HA was carried out including receptor specificity, stability, and preferential airway protease cleavage and showed very specific binding to monosialic ganglioside 2 (GM2). To probe the degree of conservation of target epitopes, binding of known broadly cross-reactive monoclonal antibodies targeting the influenza B HA and NA, respectively, were assessed through enzyme linked immunosorbent assays against recombinant WSEIV glycoproteins. Human serum samples of patients with antibodies to influenza B viruses were used to determine the cross-reactivity against these novel glycoproteins. Very few monoclonal antibodies - notably including pan NA antibody 1G01 - showed cross-reactivity and reactivity from human sera was basically absent. In summary, we have conducted a functional and antigenic characterization of the glycoproteins of the novel WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish.

microbiology↗