Biology subjects

Pekosz, A.

Publications and source records attributed to Pekosz, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Influenza A virus H5N1 genotypes B3.13 and D1.1 show temperature-dependent restriction of replication in primary human respiratory epithelial cell cultures derived from the upper and lower respiratory tract.

H5N1 clade 2.3.4.4b avian influenza A viruses pose a significant threat to wild animal populations, domesticated animals, and potentially, the human population. For H5N1s to infect and transmit among mammalian species, mutations for improved utilization of mammalian receptors and enhanced replication at the lower temperatures of the upper respiratory tract need to be acquired. A human H1N1pdm09-like virus was compared to H5N1 genotypes B3.13 and D1.1 for replication at 33{o}C, 37{o}C, and 39{o}C - temperatures consistent with the upper and lower respiratory tract in humans, and dairy cow udder tissue. All H5N1 viruses had increased plaque sizes on MDCK cells at 37{o}C and 39{o}C compared to H1N1pdm09. In primary, differentiated human nasal and bronchial epithelial cultures, all H5N1 viruses show restricted infectious virus production compared to H1N1 at 33{o}C. While H5N1 D1.1 also showed restricted replication at 37{o}C and 39{o}C, the H5N1 B3.13 replicated to nearly equivalent titers as H1N1pdm09. All H5N1 viruses demonstrated similar cell tropism in cells from the upper and lower respiratory tract, infecting more ciliated than non-ciliated cells relative to H1N1pdm09. H1N1, H5N1 B3.13 D1.1 infection induced similar innate immune factors, with nasal epithelial cells producing higher levels compared to bronchial epithelial cells. These data suggest that genotype B3.13 and D1.1 H5N1 viruses show different temperature dependent replication patterns compared to H1N1pdm09.

microbiology↗

bioRxiv2026

Sex differences in vaccine-induced neuraminidase cross-recognition and protection against H5N1 in mice

H5N1 vaccines have been poorly immunogenic in humans, creating a challenge for vaccine development. Seasonal influenza vaccines offer some cross-protection against H5N1, but there has been no consideration of whether protection differs between the sexes. We investigated sex differences in antibody responses following receipt of either beta-propiolactone inactivated whole virus H1N1 or H5N1 (LAIV backbone) vaccines in C57BL/6 mice. Using systems serology assays, vaccination induced strong homologous and heterologous antibody responses, with females generating greater IgG titers than males against whole virus H1N1 and H5N1, which was primarily mediated by greater IgG responses to neuraminidase (NA) than hemagglutinin (HA) protein. Cross-reactive H5N1 IgG titers were greater among H1N1-vaccinated females, and primarily mediated by greater N1-specific IgG titers. IgG2b and IgG2c were the primary antibody isotypes generated in response to these vaccines, with females having greater IgG2b titers and enhanced binding to Fc{gamma}RIV for avian and human NA than males following either homologous or heterologous vaccination. Antibody-dependent complement deposition was measured as an FcR-mediated non-neutralizing response against HA and NA and was more robust among H1N1 and H5N1 vaccinated females than their male counterparts in response to homologous HA only. Vaccinated females tended to have greater neutralizing antibody titers than males against the homologous vaccine strain, with limited cross-neutralizing antibodies detected in either sexes. Neuraminidase inhibition titers were greater in vaccinated females than males against the heterologous virus following H1N1 vaccination and against both the vaccine and heterologous viruses following H5N1 vaccination. When H1N1 and H5N1 vaccinated mice were challenged with a lethal dose of A/Texas/37/2024 H5N1, all H5N1 vaccinated mice were protected, regardless of sex. Among H1N1 vaccinated mice, while both sexes were protected against disease, H1N1 vaccinated females restricted virus to the upper respiratory tract and had lower pulmonary virus titers than males at 3 days post challenge. These findings highlight that sex differences in vaccine-induced NA-specific antibody responses are associated with differential respiratory dissemination of H5N1 and that sex should be considered in studies of vaccine-induced cross-reactive influenza immunity.

immunology↗

bioRxiv2026

Antibody Transcytosis and Neutralizing Activity in Respiratory Epithelial Cells

The neutralizing activity present in human serum is considered a correlate of protection against SARS-CoV-2 infection and disease but the mechanisms by which serum antibodies are transported into the lumen of the respiratory tract, where they are required to interact with virus particles and infected cells remain incompletely understood. The transcytosis and neutralizing activity of serum-derived IgG and IgA antibodies was investigated using an in vitro SARS-CoV-2 infection model with primary differentiated human nasal and basal epithelial cells (hNECs and hBECs) cultures. Expression of the antibody transport receptors neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) in hNECs cultures was confirmed by qPCR, immunofluorescence microscopy, and flow cytometry. Both receptors were expressed throughout the epithelial cultures, with enriched expression observed in ciliated cells compared with goblet and basal cells. Purified IgG and IgA isolated from convalescent plasma demonstrated specificity for SARS-CoV-2 spike protein and inhibited ACE2-Spike interactions, although activity was reduced against later variants. Purified IgG contained higher anti-spike antibody titers than purified IgA. Functional neutralization assays showed that transcytosed IgG and IgA significantly reduced SARS-CoV-2 infection compared with untreated controls. However, serial dilution studies demonstrated that IgG-mediated neutralization was more potent than IgA-mediated neutralization. Similar results were determined for influenza A virus H3N2 subtype. The transcytosis of IgG was more efficient in hBEC cultures while IgA transcytosis was higher in hNEC cultures, reflecting the levels of the corresponding transport proteins. Together, these findings demonstrate that serum-derived IgG and IgA can undergo transepithelial transport across human nasal epithelium while retaining SARS-CoV-2 or influenza A virus neutralizing activity in vitro. These results suggest that FcRn- and pIgR-mediated antibody transport may contribute to mucosal protection following vaccination or infection and may help identify antibody responses associated with protection against SARS-CoV-2. ImportanceSerum antibody levels are considered correlates of protection for SARS-CoV-2 and Influenza A virus but its unclear how those antibodies are transported to the apical surface of respiratory epithelial cells, where they must be present for optimal activity. We show that IgG and IgA specific for SARS-CoV-2 or influenza A virus is transcytosed across respiratory epithelial cell cultures, the efficiency of which reflects the level of FcRn or pIgR expression levels, suggesting that cells of the upper and lower respiratory tract transport different antibodies from the blood.

microbiology↗

bioRxiv2026

A continuous viral vaccine biomanufacturing platform utilizing multiple bioreactor configurations

Scalable, continuous biomanufacturing processes have grown in importance to meet demand for smaller bioreactor sizes, lowered production costs, and improved quality attributes. The Sf9/recombinant baculovirus (rBV) expression system demonstrates promise for virus-like particle (VLP) vaccine and gene therapy production. Here, we present a continuous rBV platform integrating an infection plug flow reactor (PFR) between stirred tank growth (gCSTR) and production (pCSTR) bioreactors. Cell expansion in the gCSTR included a ramp-up stage followed by continuous growth, reaching a steady state of 5x106 cells/mL and >90% viability. Peclet number-fit tracer studies confirmed near-ideal plug flow in the PFR, yielding a 10 h residence time and progressive infection as measured by gp64 signaling. Finally, a pCSTR with a residence time of 48 h exhibited sustained recombinant protein production. An integrated pilot cascade incorporating all reactors ran continuously for 5 days, maintaining stable CSTR cell densities and a measurable increase in infected cell diameter from 14.5 m to 16.1 m. Western blotting and EM of [~]100 nm VLPs in pCSTR effluent demonstrated platform success. Digital twin mechanistic models across four distinct stages of bioreactor operation and Hill-type relationships for rBV infection kinetics predicted cell growth and death for a 7-day run, demonstrating promise for designing continuous systems in silico and building a quantitative framework for scale-up and optimization. Our multi-stage reactor configuration represents a cell host- and product-agnostic production scheme, particularly for processes prone to product heterogeneity, and paves the way towards a true end-to-end continuous platform for myriad modalities in the future.

bioengineering↗

bioRxiv2025

Mechanisms of sex differences in acute and long COVID sequelae in mice

Males are more likely to suffer severe outcomes during acute COVID-19 and a greater proportion of females develop post-acute sequalae of COVID-19 (PASC). To identify mechanisms of PASC, mice were infected with SARS-CoV-2 and viral, inflammatory, and neurocognitive outcomes were evaluated through 84 days post-infection. Sex differences were not observed in virus replication or persistence of viral RNA in pulmonary or extrapulmonary tissues. After infectious virus clearance, females exhibited persistent neurocognitive impairments, and greater frequencies of inflammatory myeloid cells, neuroinflammation, and dysregulated T cells, including effector memory and Tregs. Sex differences in persistent inflammation and cognitive impairments during PASC were mediated by the presence of two X chromosomes and infection-induced upregulation of the X-linked genes Xist and Tlr7, which was reversed by pharmacological inhibition of TLR7 activity during the PASC phase. Sustained X-linked TLR7-mediated inflammation underlies how the burden of PASC is greater for females and provides a therapeutic target.

immunology↗

bioRxiv2025

Emergence of an Antigenically Drifted and Reassorted Influenza B Virus at the end of the 2024-25 Influenza Season

Influenza B virus (IBV) is a significant contributor to annual and severe cases of influenza, particularly in the young and elderly. Late in the 2024-25 Northern Hemisphere influenza season, a surge of IBV cases were identified in the Johns Hopkins Hospital Systems. The IBV responsible for the surge, C.3.1/re, was a clade C.3 virus that had reassorted with clade C.5.1 viruses and acquired the D197N mutation in hemagglutinin restoring a putative N-linked glycan predicted to mask a key neutralizing antibody epitope. The C.3.1/re viruses preferentially infected children but showed no significant change in disease severity. C.3.1/re viruses were poorly neutralized by pre- and post- influenza vaccination serum in a human cohort. Removal of the glycan at residue 197 restored neutralizing antibody recognition. The C.3.1/re IBV genotype that emerged late in the 2024-25 influenza season was antigenically mismatched with IBV vaccine strains for the 2025 and 2026 Southern hemisphere, as well as the 2025-26 Northern Hemisphere influenza seasons. While the 2026-27 Northern Hemisphere vaccine strain is a C.3.1/re, the egg adapted isolate selected (B/Tokyo/EIS13-175/2025) lacks the 197 glycosylation which is predicted to have poor recognition with circulating IBV clades. Phylogenetic analysis of currently circulating IBVs shows a diversification of circulating C.3 clades with multiple reassortment events between C.3 and C.5 clades in addition to independent acquisitions of D197N mutations, suggesting IBV is going through a period of significant antigenic and genetic expansion. IMPORTANCEInfluenza B viruses are undergoing a period of antigenic and genetic expansion, with several reassorted viruses emerging that also contain point mutations in key hemagglutinin antigenic sites proximal to the receptor binding domain. This has important impacts on vaccine strain choice, as only one IBV component is included in current influenza vaccines. We demonstrate a significant shift in the demographics of IBV infected individuals with the emergence of the antigenically drifted and reassorted IBV C.3.1/re. Furthermore, we show that 197 glycosylation of hemagglutinin is critical for C.3.1/re antigenic drift and we document several emergent C.3 reassortments encoding the D197N mutation. With the IBV vaccine component for the Northern Hemisphere 2026-27 season having lost a key N-linked glycan on the hemagglutinin protein, and multiple independent emergences of antigenically drifted and reassorted viruses, attention to IBV infections should be increased in the upcoming Southern and Northern hemisphere influenza seasons.

microbiology↗

bioRxiv2025

Low levels of H5N1 HA and NA antibodies in the human population are boosted by seasonal A/H1N1 infection but not by A/H3N2 infection or influenza vaccination

An increase in the number of human cases of influenza A/H5N1 infection in the US has raised concerns about the pandemic potential of the virus. Preexisting population immunity is a key determinant for risk assessment and pandemic potential for any virus. Antibody responses against the bovine A/H5N1 hemagglutinin (HA) and neuraminidase (NA) proteins were measured among a population of influenza-vaccinated or influenza-infected individuals. Modest titers of bovine A/H5N1 HA-binding antibodies and low to undetectable neutralizing antibody responses were detected in a cohort of 73 individuals. Conversely, bovine A/H5N1 NA binding and neuraminidase-inhibiting antibody responses were comparable to those against a human A/H1N1 NA at baseline. Seasonal influenza vaccination failed to significantly increase antibody titers against both HA and NA glycoproteins of bovine A/H5N1. Recent infection with human A/H1N1 but not A/H3N2 viruses induced significant increases in bovine A/H5N1 neutralizing antibody, as well as increases in NA-binding and NA-inhibiting antibodies to bovine A/H5N1 NA. While the degree of protection afforded by these A/H5N1 cross-reactive antibodies is not known, incorporating NA or enhancing current seasonal vaccine formulations to increase NA-specific antibody responses may increase antibody breadth and protection against both seasonal and pandemic influenza viruses.

immunology↗

bioRxiv2025

Vertical Transmission of Bovine H5N1 during Pregnancy and Lactation in Mice

Avian influenza H5N1 has pandemic potential and historically has caused more severe disease in pregnant women than the general population. With increasing transmission of H5N1 detected among placental mammals, animal models are necessary for testing countermeasures, including during pregnancy. Pregnant outbred mice were infected with a contemporary strain of bovine H5N1 during the second or third trimester equivalent. Second trimester infection caused in utero transmission, with infectious virus detected in the uterus, placenta, and fetus. Birth following third trimester infection resulted in offspring with decreased size, neurodevelopmental delays, and adolescent behavioral impairments, with infectious virus detected in the neonatal milk ring and lungs, as well as mammary tissues. H5N1 viral protein colocalized with trophoblast cells in the placenta and epithelial cells in mammary tissue that spatially overlapped with lectins for 2,3-linked SA. With the pandemic potential of H5N1, our vertical transmission model in placental mammals is essential for understanding viral spread and evaluating treatments during pregnancy.

microbiology↗

bioRxiv2025

Acute induction of IFNα is responsible for the attenuation of the live measles vaccine

The live-attenuated measles vaccine (LAMV) protects against measles in a safe, efficacious and durable manner. However, the mechanism by which LAMV is attenuated is not well understood, contributing to the increased vaccine hesitancy that has caused a worldwide resurgence of measles. Here, we provide a molecular model of attenuation that implicates increased host innate immune responses and increased LAMV susceptibility to those responses. Our study leverages both in vitro and in vivo models of infection to find that acute induction of innate immunity in immune cells is the critical determinant of LAMV attenuation. We show that LAMV, in contrast to pathogenic wild type measles virus (MeV), causes a strong IFN response in primary human peripheral blood mononuclear cells, which restricts viral replication and spread in a post-entry manner. LAMV can enter, transcribe viral mRNA and translate viral proteins in immune cells, but is not able to form infectious particles and spread from cell to cell. Specifically, we find that mutations conserved across LAMV strains in the P/V/C and H genes are responsible for the induction of IFN and the restriction of infectious virus production. In PBMC cultures and in rhesus macaques, LAMV infection also results in the acute induction of proinflammatory cytokines that likely play a role in the immunogenicity of the vaccine. Overall, our study provides a virological and immunological framework for LAMV attenuation which can be leveraged in repurposing the LAMV backbone and in future vaccine design.

microbiology↗

bioRxiv2025

A conserved region T-cell vaccine for Sarbecoviruses

The rapid development of vaccines was a critical part of the global response to the COVID-19 pandemic. SARS-CoV-2 (a Sarbecovirus and member of the Betacoronavirus genus responsible for the pandemic) virus was first detected in Wuhan, China in late 2019. Effective mRNA vaccines based on the viral Spike protein were designed from the earliest isolates and available by December of 2020. SARS-CoV-2 has continued to evolve in the human population, accruing neutralizing antibody resistance mutations that have necessitated updating the vaccine periodically to better match contemporary variants. Neutralizing antibody cross-reactivity is generally very limited among the diverse members of the betacoronavirus genus that are of clinical importance in people. Here, we present an alternative vaccine strategy based on eliciting T-cell responses targeting four highly conserved regions shared across the betacoronavirus proteomes. We hypothesized that cross-reactive responses to these regions could temper disease severity. Focusing immune responses on highly conserved epitopes could be beneficial as SARS-CoV-2 continues to evolve, or if a novel betacoronavirus should enter the human population. Vaccination with these highly conserved regions induced robust T-cell responses in mice and rhesus macaques. Vaccinated hamsters were significantly protected against weight loss and lung inflammation after challenge with the SARS-CoV-2 Omicron variant. After a SARS-CoV-2 Delta challenge in rhesus macaques, 3 out of 4 animals in the control group had infectious virus in their bronchoalveolar lavage samples, while the 4 animals in the vaccinated group did not.

immunology↗

bioRxiv2025

The proton-activated chloride channel inhibits SARS-CoV-2 spike protein-mediated viral entry through the endosomal pathway.

SARS-CoV-2 binds to its obligatory receptor, angiotensin-converting enzyme 2 (ACE2) and capitalizes on decreasing endosomal acidity and cathepsin-mediated spike protein cleavage to enter cells. Endosomal acidification is driven by V-ATPase which pumps protons (H+) into the lumen. The driving force for H+ is maintained by the import of chloride (Cl-) which is mediated by intracellular CLC transporters. We have recently identified the Proton-Activated Chloride (PAC) channel as a negative regulator of endosomal acidification. PAC responds to low pH and releases Cl- from the lumen to prevent endosomal hyperacidification. However, its role in SARS-CoV-2 viral entry remains unexplored. Here, we show that overexpressing the PAC channel in ACE2 expressing HEK 293T cells markedly inhibited the SARS-CoV-2 spike-mediated viral entry. Several lines of evidence suggest that this effect was due to the suppression of the endosomal entry pathway. First, the abilities of PAC to regulate endosomal acidification and inhibit pseudoviral entry were both dependent on its endosomal localization and channel activity. Second, the inhibitory effect on viral entry was similar to the suppression mediated by E64-d, a cathepsin inhibitor, while no major additive effect for both treatments was observed. Third, this inhibition was also attenuated in cells expressing TMPRSS2, which provides the alternative entry pathway through cell surface. Importantly, PAC overexpression also inhibited the number and size of plaques formed by two live SARS-CoV-2 isolates (B.1 and Omicron XBB.1.16) in Vero E6 cells. Altogether, our data indicates that PAC plays a vital role in inhibiting SARS-CoV-2 viral entry and identifies this endosomal channel as a potential novel target against the infection of SARS-CoV-2 and other viruses, which rely on the endosomal pathway.

physiology↗

bioRxiv2024

Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

SARS-CoV-2 infection poses a major threat to public health, and understanding the mechanism of viral replication and virion release would help identify therapeutic targets and effective drugs for combating the virus. Herein, we identified E3 ubiquitin-protein ligase Itchy homolog (ITCH) as a central regulator of SARS-CoV-2 at multiple steps and processes. ITCH enhances the ubiquitination of viral envelope and membrane proteins and mutual interactions of structural proteins, thereby aiding in virion assembly. ITCH-mediated ubiquitination also enhances the interaction of viral proteins to the autophagosome receptor p62, promoting their autophagosome-dependent secretion. Additionally, ITCH disrupts the trafficking of the protease furin and the maturation of cathepsin L, thereby suppressing their activities in cleaving and destabilizing the viral spike protein. Furthermore, ITCH exhibits robust activation during the SARS-CoV-2 replication stage, and SARS-CoV-2 replication is significantly decreased by genetic or pharmacological inhibition of ITCH. These findings provide new insights into the mechanisms of the SARS-CoV-2 life cycle and identify a potential target for developing treatments for the virus-related diseases.

molecular biology↗

bioRxiv2024

Mitochondrial morphodynamics are modulated by physiological range of temperature and influence host cell outcomes during influenza infection

Influenza viruses replicate in both the cooler, upper portions of the airway and the warmer, lower portions of the respiratory tract. This study investigates how physiological ranges of temperature, specifically 33{degrees}C and 37{degrees}C, impact host cell biology and how temperature-dependent differences in host cells influence outcomes during influenza A virus infection. This study prioritizes describing mitochondrial networks due to their importance in maintaining cellular homeostasis and mediating immune responses to viral infection. The temperature at which cells are incubated significantly influences mitochondrial network morphology and mitochondrial function. Moreover, temperature-dependent changes to mitochondrial networks prior to infection result in temperature-specific changes to host cell outcomes during infection. These findings indicate that mitochondrial structure alone can modulate host cell outcomes during viral infection and that both the form and function of mitochondria directly impact influenza A virus production. While not all mitochondrial processes were shown to be affected by temperature or infection, these results highlight the importance of using physiologically relevant temperatures in respiratory pathogen research and elucidate how mitochondrial dynamics contribute to host cell outcomes during influenza A virus infection. ImportanceRespiratory viruses infect the upper and lower respiratory tract but rarely is the impact of physiological ranges of temperature (33{degrees}C to 37{degrees}C) considered. Mitochondria are central mediators of numerous physiological pathways, and their functions are often modified by virus infection. Physiological ranges of temperature can alter mitochondrial form and function, which is further impacted by virus infection. The study sheds light on how temperature can impact mitochondrial form and function in concert with virus infection.

microbiology↗

bioRxiv2024

A Third COVID-19 Vaccine Dose in Kidney Transplant Recipients Induces Antibody Response to Vaccine and Omicron Variants but Shows Limited Ig Subclass Switching

Solid organ transplant recipients (SOTRs) suffer more frequent and more severe infections due to their compromised immune responses resulting from immunosuppressive treatments designed to prevent organ rejection. Pharmacological immunosuppression can adversely affect immune responses to vaccination. A cohort of kidney transplant recipients (KTRs) received their third dose of ancestral, monovalent COVID-19 vaccine in the context of a clinical trial and antibody responses to the vaccine strain, as well as to Omicron variants BA.1 and BA.5 were investigated and compared with healthy controls. Total IgG and live virus neutralizing antibody titers were reduced in KTRs compared to controls for all variants. KTRs displayed altered IgG subclass switching, with significantly lower IgG3 antibodies. Responses in KTRs were also very heterogeneous, with some individuals showing strong responses but a significant number showing no Omicron-specific neutralizing antibodies. Taken together, immune responses after COVID-19 vaccination in KTRs were not only lower than healthy controls but highly variable, indicating that simply increasing the number of vaccine doses alone may not be sufficient to provide greater protection in this population. ImportanceThis study addresses the challenges faced by kidney transplant recipients (KTRs) in mounting effective immune responses against COVID-19. By evaluating the antibody responses to a third dose of monovalent mRNA COVID-19 vaccine and its effectiveness against Omicron subvariants (BA.1 and BA.5), this study reveals significant reductions in both binding and neutralizing antibodies in KTRs compared to healthy controls. The research highlights altered IgG subclass switching and heterogeneous responses within the KTR population. Reduced recognition of variants, coupled with differences in IgG subclasses, decreases both the quality and quantity of protective antibodies after vaccination in KTRs. These findings underscore the need for tailored vaccination strategies for immunosuppressed populations such as KTRs. Alternative formulations and doses of COVID-19 vaccines should be considered for people with severely compromised immune systems, as more frequent vaccinations may not significantly improve the response, especially regarding neutralizing antibodies.

immunology↗

bioRxiv2024

SARS-CoV-2 variants from long-term, persistently infected immunocompromised patients have altered syncytia formation, temperature-dependent replication, and serum neutralizing antibody escape

SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates from two COVID-19 patients undergoing immunosuppressive cancer therapy, with all isolates obtained greater than 100 days after the initial COVID-19 diagnoses and compared to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient - who did mount a SARS-CoV-2 IgM response - showed temperature dependent changes in plaque size as well as increased syncytia formation and escape from serum neutralizing antibody. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.

microbiology↗

bioRxiv2024

Clade-defining mutations in human H1N1 hemagglutinin protein from 2021-2023 have opposing effects on in vitro fitness and antigenic drift

Seasonal influenza viruses frequently acquire mutations that have the potential to alter both virus replication and antigenic profile. Recent seasonal H1N1 viruses have acquired mutations to their hemagglutinin (HA) protein receptor binding site (RBS) and antigenic sites, and have branched into the clades 5a.2a and 5a.2a.1. Both clades demonstrated improved in vitro fitness compared with the parental 5a.2 clade as measured through plaque formation, infectious virus production in human nasal epithelial cells, and receptor binding diversity. Both clades also showed reduced neutralization by serum from healthcare workers vaccinated in the 2022-23 Northern Hemisphere influenza season compared to the vaccine strain. To investigate the phenotypic impact of individual clade-defining mutations, recombinant viruses containing single HA mutations were generated on a 5a.2 genetic background. The 5a.2a mutation Q189E improved plaque formation and virus replication, but was more efficiently neutralized by serum from individuals vaccinated in 2022-23. In contrast, the 5a.2a mutation E224A and both 5a.2a.1 mutations P137S and K142R impaired aspects of in vitro fitness but contributed significantly to antigenic drift. Surprisingly, the E224A mutation and not Q189E caused broader receptor binding diversity seen in clinical isolates of 5a.2a and 5a.2a.1, suggesting that receptor binding diversity alone may not be responsible for the phenotypic effects of the Q189E mutation. These data document an evolutionary trade-off between mutations that improve viral fitness and those that allow for the evasion of existing host immunity. ImportanceSeasonal influenza is a significant contributor to global morbidity and mortality. To better understand how these viruses evolve, recent seasonal H1N1 viruses were characterized and the impact of individual mutational differences between the hemagglutinin proteins of these viruses was assessed. Mutations that contribute to escape from preexisting immunity were deleterious to virus replication in human nasal epithelial cell cultures, requiring the accumulation of additional mutations to compensate for this loss of fitness. This adds to our understanding of how influenza evolution balances replication fitness with antigenic drift.

microbiology↗

bioRxiv2024

ZFAND6 is a subunit of a TRAF2-cIAP E3 ubiquitin ligase complex essential for mitophagy

The A20 ubiquitin-editing enzyme is a critical negative regulator of NF-{kappa}B signaling and inflammation. While the mechanisms by which A20 restricts inflammation have been extensively studied, the physiological functions of other A20-like proteins are largely unknown. Here, we report a previously unknown function of the A20 family member ZFAND6 as a novel regulator of mitophagy. Deletion of ZFAND6 in bone marrow-derived macrophages (BMDMs) promotes the upregulation of reactive oxygen species (ROS) and the accumulation of damaged mitochondria due to impaired mitophagy. Consequently, mitochondrial DNA (mtDNA) is released into the cytoplasm, triggering the spontaneous expression of interferon-stimulated genes (ISGs) in a cGAS-STING dependent manner, which leads to enhanced viral resistance in vitro. However, mice lacking ZFAND6 exhibit increased morbidity and mortality upon challenge with a sublethal dose of influenza A virus (IAV) due to impaired myeloid cell activation and diminished type I IFN signaling. Mechanistically, ZFAND6 bridges a TRAF2-cIAP1 interaction, which is required for the initiation of ubiquitin-dependent mitophagy. Our results suggest that ZFAND6 is a subunit of a TRAF2-cIAP E3 ligase complex that promotes the clearance of damaged mitochondria by mitophagy to maintain mitochondrial homeostasis.

immunology↗

bioRxiv2024

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

microbiology↗