Biology subjects

Hensley, S. E.

Publications and source records attributed to Hensley, S. E..

At least 19 recordsLinked to original sources

bioRxiv2026

The transition to postpartum lactation establishes a state of maternal antiviral resilience

Influenza A virus (IAV) causes severe disease during late pregnancy, yet how maternal adaptations in host defense shape susceptibility following childbirth remains unknown. Here, we show that the postpartum period represents a state of enhanced maternal antiviral immunity. Across postpartum stages, dams infected before weaning exhibited increased survival and reduced disease following lethal IAV challenge compared with nonpregnant females. Postpartum protection was associated with enhanced viral clearance and increased pulmonary antigen-specific CD8+ T cells and IL-17A-producing gamma-delta T cells. We identify the transition into lactation as a critical determinant of this protection. Protection extended to highly pathogenic avian influenza. Together, these findings reveal that maternal antiviral immunity remains actively shaped after childbirth, establishing the postpartum lactational period as a distinct state of maternal antiviral resilience.

immunology↗

bioRxiv2026

Near real-time data on the human neutralizing antibody landscape to influenza virus in summer of 2026 shows antigenic advance of H3N2 subclade K region D mutants and H1N1 D.3.1.1 Sa mutants

Human seasonal influenza evolves rapidly, necessitating twice yearly decisions about whether to update the strains in the vaccine. To help inform this decision, we have been using high-throughput sequencing-based neutralization assays to make twice yearly measurements of how recent human sera neutralize current human H3N2 and H1N1 strains. Here we provide the third installment in this series of measurements by reporting 52,268 titers representing neutralization of 148 viral strains by 355 human sera collected between April and August of 2026. Our measurements show that new H3N2 subclade K strains with mutations in antigenic region D and new H1N1 subclade D.3.1.1 strains with mutations in antigenic region Sa (such as G155E) have reduced neutralization by human sera, with notable heterogeneity in the impact of some of these mutations across sera from different individuals. This paper is accompanied by an interactive summary (https://jbloomlab.github.io/flu-seqneut-2026/summary.html) that enables detailed exploration of the results, and all titer data are publicly available for further analysis to aid vaccine antigen selection and studies of viral evolution.

microbiology↗

bioRxiv2026

H5N1 influenza binding and cell entry via human class II MHC, and blocking by cross-reactive antibodies

Summary paragraphHighly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses are currently responsible for a multi-species outbreak affecting wild birds, poultry, numerous mammalian species, and humans. Influenza A viruses typically initiate infection through binding to sialic acid, although select bat and human influenza viruses can also exploit class II major histocompatibility complex (MHC-II) molecules for cell entry. Here we show that emerging H5N1 clade 2.3.4.4b viruses, but not historical H5 lineages, bind human MHC-II HLA-DR and mediate sialic acid-independent cell entry. Hemagglutinin binding to primary human immune cells varies with MHC-II expression and is further shaped by HLA-DR allelic variation, identifying host genetic determinants that may influence susceptibility to infection. Mammalian-adaptive substitutions within the hemagglutinin sialic acid receptor-binding domain reduce MHC-II binding, suggesting this interaction is remodeled during clade 2.3.4.4b H5 adaptation to a human host. Lastly, cross-reactive monoclonal antibodies isolated from clade 2.3.4.4b H5-naive humans can block the hemagglutinin-MHC-II interaction. These findings identify a previously unrecognized receptor pathway in contemporary H5N1 viruses and reveal that both human genetic variation and pre-existing humoral immunity can modulate this interaction, with implications for host range, cellular tropism, spillover risk, and therapeutic intervention.

immunology↗

bioRxiv2026

Clade 2.3.4.4b H5N1 influenza virus and SARS-CoV-2 seroprevalence among owned and feral cats in Philadelphia and surrounding communities

Clade 2.3.4.4b H5NX influenza viruses have spread widely in birds since 2020. In addition to causing disease in birds, these viruses have infected a variety of mammals, including humans. Clade 2.3.4.4b H5N1 viruses are currently causing an outbreak among dairy cattle in the United States, and it is important to determine if other mammals have been exposed to H5NX viruses. Cats, specifically outdoor and feral cats, frequently predate wild birds. Recent studies have shown that cats living on dairy cattle farms can be infected with H5N1. Here, we completed serological studies to determine if owned and feral cats living in an urban environment in the United States have evidence of past H5N1 exposures. We used multianalyte bead-based assays to measure clade 2.3.4.4b hemagglutinin (HA) antibody levels in serum samples collected in July 2023 to June 2025 from 417 feral and 228 owned cats from the greater Philadelphia area. We also measured antibody levels against a panel of HAs from other human and non-human influenza viruses, and the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We completed additional H5N1 and SARS-CoV-2 neutralization assays using samples that had detectable antibodies in the multianalyte bead-based assays. One cat (0.16%) was positive for H5 antibodies and twenty cats (3.1%) were positive for SARS-CoV-2 antibodies in both binding and neutralization assays. These data suggest that cats in the Philadelphia area have not been routinely exposed to clade 2.3.4.4b H5N1 viruses but have been more commonly exposed to SARS-CoV-2. ImportanceHighly pathogenic H5NX avian influenza viruses have caused widespread disease and mortality in both wild and domestic birds, and have infected marine mammals, cattle, and humans. Recent studies have demonstrated that cats can also be infected with H5NX viruses, but it is unknown if these infections are common. In this report, we completed a seroprevalence study and found that H5NX exposure in feral and owned cats in the Philadelphia area appears to be rare. We also completed SARS-CoV-2 serological experiments and found that 3.1% of cats possessed antibodies reactive to this virus.

microbiology↗

bioRxiv2026

A multivalent mRNA-lipid nanoparticle vaccine containing eight hemagglutinin antigens elicited broad neutralizing antibody responses and protected against influenza A virus challenge in swine.

The diversity within H1 and H3 subtype influenza A viruses (IAV) in swine prevents effective vaccine control approaches with inactivated whole-virus vaccines. We addressed the challenge of controlling co-circulating hemagglutinin (HA) clades of swine IAV with the development of a multivalent mRNA-lipid nanoparticle (LNP) vaccine expressing 8 HA proteins to maximize genetic coverage. We applied a computational approach to select eight HA genes that represented 95% of the observed IAV detected in the United States between 2022 and 2025. Piglets were vaccinated and boosted intramuscularly with either individual HA mRNA-LNP or an 8-HA multivalent mRNA-LNP. Serum was collected to evaluate systemic antibody levels. Twenty-one days post-boost, pigs were challenged with a field relevant H1 1A.3.3.3-c3 IAV strain. The 8-HA multivalent mRNA-LNP vaccine induced neutralizing antibodies against all eight antigens and vaccinees were protected against lung lesions, with lesion scores similar to non-challenged animals. Homologous monovalent vaccination significantly reduced IAV detection in nasal secretions and in the lungs. Heterologous monovalent vaccination was not cross-protective but did not induce vaccine-associated enhanced respiratory disease. We provide evidence that monovalent and multivalent mRNA-LNP influenza vaccines elicited neutralizing antibody responses in pigs and protected against viral challenge. The versatility and capacity for rapidly updating the mRNA-LNP vaccine platform make it an appealing tool to improve animal health and minimize the circulation and diversity of IAV in swine. ImportanceInfluenza A virus is an important respiratory pathogen in swine, and zoonotic transmission of swine strains to humans remains a public health risk. Control strategies against IAV in swine herds rely heavily on biosecurity measures and vaccination. However, the antigenic diversity of IAV circulating in swine challenges current vaccination programs, and there is a need for broadly protective vaccines or platforms that can rapidly update components to reflect circulating diversity. mRNA-LNP vaccines have emerged as promising vaccine platforms, offering simultaneous delivery of multiple antigens, rapid development, scalable manufacturing, and potent immunogenicity. In this study, we assessed the immunogenicity and protective capacity of monovalent and multivalent mRNA-LNP vaccines encoding eight representative IAV HA antigens. To our knowledge, this is the first study to objectively select multiple representative endemic swine IAV strains by quantifying genetic diversity within the phylogeny and to apply this selection to rationally design and evaluate a multivalent HA mRNA-based influenza vaccine in the swine model.

microbiology↗

bioRxiv2026

Serial vaccination expands and refines human CD4+ T cell memory

CD4+ T cells coordinate protective immunity against pathogens. However, a major unresolved question is how human CD4+ T cell memory is established and evolves following primary and repeated vaccination. Using COVID-19 mRNA vaccination as a model, we tracked 50 distinct antigen-specific populations directly ex vivo with peptide-MHC class II tetramers in eight SARS-CoV-2-naive individuals from pre-vaccine baseline through memory time points after three mRNA doses. Our findings identify the primary vaccine series as the main driver of memory pool size. It leverages pre-existing memory while preferentially recruiting high-avidity T cells, establishing an immunodominance hierarchy dominated by a small subset of precursors. Booster vaccination refines both the magnitude and quality of T cell memory. It increases select populations and enhances differentiation of subdominant CD4+ T cells. Populations that did not become more abundant after boosting retained their polyfunctional potential. Beyond establishing memory to the ancestral spike, vaccinations broadened responses by recruiting cross-reactive T cells recognizing viral variants. Collectively, these findings reveal how human CD4+ T cell memory evolves through sequential immunizations to generate a functionally diverse and broadly responsive memory repertoire against future viral challenges.

immunology↗

bioRxiv2026

Evaluation of an H5 influenza virus mRNA-lipid nanoparticle (LNP) vaccine in lactating dairy cows

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4b H5N1 virus has recently emerged in dairy cattle in the United States. The virus replicates primarily in the mammary gland of infected cattle, leading to dramatic reductions in milk production. It is thought that the virus transmits from animal to animal through viral shedding in milk, and therefore, vaccines that decrease the amount of virus in milk can potentially limit the current outbreak and reduce the risk of H5N1 spillover into humans. Here, we assess the immunogenicity and efficacy of a clade 2.3.4.4b H5 mRNA-LNP vaccine in lactating dairy cows. We found that the H5 mRNA-LNP vaccine elicited robust antibody responses in sera and milk and significantly reduced viral replication and disease caused by clade 2.3.4.4b H5N1 intramammary infection.

microbiology↗

bioRxiv2026

Strain-specific differences in the response to egg-derived versus recombinant protein influenza vaccines

The 2023/2024 influenza vaccine included an updated H1N1 component designed to better match a new clade of H1N1 that had multiple mutations in antigenic epitopes of hemagglutinin. Despite this update, the vaccine trended towards being less effective against the vaccine-matched H1N1 clade than the parental H1N1 clade lacking the new antigenic mutations. Here we measure neutralization titers of serum antibodies from individuals who had received either a recombinant protein or an egg-derived vaccine against a set of viruses with hemagglutinins from 58 H1N1 strains representative of the diversity during the 2023/2024 season. We find that egg-derived vaccine recipients, but not recombinant protein vaccine recipients, had a relatively lower boost in neutralizing titers to the new clade that the updated vaccine was designed to target. We suggest that the difference in the extent that the egg-derived versus recombinant protein vaccines boosted neutralizing titers to the new H1N1 clade is because the seed strain for the egg-derived vaccine strain had acquired a reversion of a key antigenic mutation (K142R) present in that clade. Our results show how egg-derived versus recombinant protein vaccines can elicit different relative titer boosts against different subsets of viral strains, a phenomenon that could impact vaccine effectiveness. ImportanceInfluenza vaccines can be produced from virus grown in eggs, or grown in cells or made with recombinant protein. Egg-derived influenza vaccines often contain egg-adaptive mutations in the viral antigen hemagglutinin (HA) which can impact the antigenicity or immunogenicity of the HA. In this study, we compare neutralization titers from egg-derived and recombinant protein vaccine recipients against recently circulating influenza A(H1N1) strains. We find that the egg-derived vaccine induces less of a boost in titers than the recombinant protein vaccine to the new clade of viral strains that the vaccine was designed to target.

immunology↗

bioRxiv2026

Near real-time data on the human neutralizing antibody landscape to influenza virus as of early 2026 to inform vaccine-strain selection

Twice each year, a decision is made on whether to update the strains included in the seasonal influenza vaccine to better match the most recent circulating viral strains. To characterize the antigenic properties of current seasonal influenza A strains to inform the upcoming decision about which strains to include in the 2026-2027 Northern Hemisphere vaccine, here we perform high-throughput sequencing-based neutralization assays using a library of 57 H3N2 and 34 H1N1 influenza hemagglutinins reflecting the circulating diversity of strains in late 2025 to early 2026. We assay this library against 302 human sera collected in late 2025. The resulting data set encompasses 27,409 titers, and provides a near real-time portrait of the human neutralizing antibody landscape against influenza virus. We find that many human sera have lower titers against the K subclade of H3N2 and the D.3.1.1 subclade of H1N1; these subclades have recently become dominant among their respective subtypes. Our measurements also reveal variability in titers to different subvariants within the K subclade of H3N2, with titers especially low to subclade K strains with additional mutations in antigenic regions D and E. We make all our data and accompanying visualizations publicly available to enable their use in vaccine-strain selection and analyses of influenza evolution and immunity.

microbiology↗

bioRxiv2025

Fatal Human H3N8 Influenza Virus has a Moderate Pandemic Risk

In China, low pathogenic avian influenza (LPAI) H3N8 virus is widespread among chickens and has recently caused three zoonotic infections, with the last one in 2023 being fatal. Here we evaluated the relative pandemic risk of this 2023 zoonotic H3N8 influenza virus, utilizing our previously published decision tree. Serological analysis indicated that a large proportion of the human population does not have any cross-neutralizing antibodies against this H3N8 strain. LPAI H3N8 displayed a dual affinity for 2-3 and 2-6 sialic acids and replicated efficiently in human bronchial epithelial cells. Furthermore, we observed H3N8 transmission via direct contact but not aerosols to ferrets with pre-existing H3N2 immunity. Although pre-existing H3N2 immunity resulted in a shortened disease course in ferrets, it did not reduce disease severity or replication in the respiratory tract. This study suggests that this zoonotic H3N8 strain has moderate pandemic potential and emphasizes the continued need for avian influenza surveillance.

microbiology↗

bioRxiv2025

H5 influenza virus mRNA-lipid nanoparticle (LNP) vaccination elicits adaptive immune responses in Holstein calves

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4b H5N1 is circulating widely in lactating cows in the United States. Due to the critical need for intervention strategies for this outbreak, we evaluated antibody and cellular immune responses of a clade 2.3.4.4b H5 mRNA-LNP vaccine in calves. We found that the H5 mRNA-LNP vaccine induced a robust antibody and CD8+ T cellular-mediated immune response and conferred protection against clade 2.3.4.4b H5N1 infection.

microbiology↗

bioRxiv2025

High-throughput neutralization measurements correlate strongly with evolutionary success of human influenza strains

Human influenza viruses rapidly acquire mutations in their hemagglutinin (HA) protein that erode neutralization by antibodies from prior exposures. Here, we use a sequencing-based assay to measure neutralization titers for 78 recent H3N2 HA strains against a large set of children and adult sera, measuring [~]10,000 total titers. There is substantial person-to-person heterogeneity in the titers against different viral strains, both within and across age cohorts. The growth rates of H3N2 strains in the human population in 2023 are highly correlated with the fraction of sera with low titers against each strain. Notably, strain growth rates are less correlated with neutralization titers against pools of human sera, demonstrating the importance of population heterogeneity in shaping viral evolution. Overall, these results suggest that high-throughput neutralization measurements of human sera against many different viral strains can help explain the evolution of human influenza.

evolutionary biology↗

bioRxiv2024

Bovine H5N1 influenza virus binds poorly to human-type sialic acid receptors

Clade 2.3.4.4b highly pathogenic H5N1 avian influenza (HPAI) viruses started circulating widely in lactating dairy cattle in the United States at the end of 2023. Avian influenza viruses enter cells after binding to glycan receptors with terminally linked 2-3 sialic acid, whereas human influenza viruses typically bind to glycan receptors terminally linked 2-6 sialic acid in the upper respiratory tract. Here, we evaluated the receptor binding properties of hemagglutinin (HA) trimers from a clade 2.3.4.4b avian isolate (A/American Wigeon/South Carolina/22-000345-001/2021) and a cattle isolate (A/dairy cattle/Texas/24-008749-002-v/2024). Using two different methods, we found that both of the 2.3.4.4b H5s bound efficiently to glycan receptors with terminally linked 2-3 sialic acid with no detectable binding to glycan receptors with terminally linked 2-6 sialic acid. Our data suggest that clade 2.3.4.4b H5N1 viruses bind poorly to human receptors. It will be important to continue evaluating receptor binding properties of these viruses as they evolve in cattle.

microbiology↗

bioRxiv2024

Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance

H5 influenza is a potential pandemic threat. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic risk, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. Here we use pseudovirus deep mutational scanning to measure how all mutations to a clade 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind 2-6-linked sialic acids, and show that some viruses already carry mutations that stabilize HA. We also identify recent viral strains with reduced neutralization to sera elicited by candidate vaccine virus. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive characterization of mutations to inform surveillance of H5 influenza.

microbiology↗

bioRxiv2024

Influenza virus antibodies inhibit antigen-specific de novo B cell responses in mice

Antibody responses to influenza vaccines tend to be focused on epitopes encountered during prior influenza exposures, with little production of de novo responses to novel epitopes. To examine the contribution of circulating antibody to this phenomenon, we passively transferred a hemagglutinin (HA)-specific monoclonal antibody (mAb) into mice before immunizing with whole inactivated virions. The HA mAb inhibited de novo HA-specific antibodies, plasmablasts, germinal center B cells, and memory B cells, while responses to a second antigen in the vaccine, neuraminidase (NA), were uninhibited. The HA mAb potently inhibited de novo antibody responses against epitopes near the HA mAb binding site. The HA mAb also promoted IgG1 class switching, an effect that, unlike the inhibition of HA responses, relied on signaling through Fc-gamma receptors. These studies suggest that circulating antibodies inhibit de novo B cell responses in an antigen-specific manner, which likely contributes to differences in antibody specificities elicited during primary and secondary influenza virus exposures.

immunology↗

bioRxiv2023

Development of a nucleoside-modified mRNA vaccine against clade 2.3.4.4b H5 highly pathogenic avian influenza virus

Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. We generated an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. We show that the vaccine is immunogenic in mice and ferrets and prevents morbidity and mortality of ferrets following 2.3.4.4b H5N1 challenge.

microbiology↗

bioRxiv2023

Evaluation of mRNA-LNP and adjuvanted protein SARS-CoV-2 vaccines in a maternal antibody mouse model

Maternal antibodies (matAbs) protect against a myriad of pathogens early in life; however, these antibodies can also inhibit de novo immune responses against some vaccine platforms. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) matAbs are efficiently transferred during pregnancy and protect infants against subsequent SARS-CoV-2 infections. It is unknown if matAbs inhibit immune responses elicited by different types of SARS-CoV-2 vaccines. Here, we established a mouse model to determine if SARS-CoV-2 spike-specific matAbs inhibit immune responses elicited by recombinant protein and nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccines. We found that SARS-CoV-2 mRNA-LNP vaccines elicited robust de novo antibody responses in mouse pups in the presence of matAbs. Recombinant protein vaccines were also able to circumvent the inhibitory effects of matAbs when adjuvants were co-administered. While additional studies need to be completed in humans, our studies raise the possibility that mRNA-LNP-based and adjuvanted protein-based SARS-CoV-2 vaccines have the potential to be effective when delivered very early in life.

immunology↗

bioRxiv2023

Prior vaccination enhances immune responses during SARS-CoV-2 breakthrough infection with early activation of memory T cells followed by production of potent neutralizing antibodies

SARS-CoV-2 infection of vaccinated individuals is increasingly common but rarely results in severe disease, likely due to the enhanced potency and accelerated kinetics of memory immune responses. However, there have been few opportunities to rigorously study early recall responses during human viral infection. To better understand human immune memory and identify potential mediators of lasting vaccine efficacy, we used high-dimensional flow cytometry and SARS-CoV-2 antigen probes to examine immune responses in longitudinal samples from vaccinated individuals infected during the Omicron wave. These studies revealed heightened Spike-specific responses during infection of vaccinated compared to unvaccinated individuals. Spike-specific CD4 T cells and plasmablasts expanded and CD8 T cells were robustly activated during the first week. In contrast, memory B cell activation, neutralizing antibody production, and primary responses to non-Spike antigens occurred during the second week. Collectively, these data demonstrate the functionality of vaccine-primed immune memory and highlight memory T cells as rapid responders during SARS-CoV-2 infection.

immunology↗