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Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI., Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells., Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors., Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Koch, Shehata, Müller-Ruttloff, Gouda, Wetzstein, Patyna, Scholz, Schmid, Dietrich, Münch, Ziebuhr, Geiger, Martinez-Sobrido, Baer, Mostafa and Pleschka.)

Background: The continuous evolution of drug-resistant influenza viruses highlights the necessity for repurposing naturally-derived and safe phytochemicals with anti-influenza activity as novel broad-spectrum anti-influenza medications., Methods: In this study, nitrogenous alkaloids were tested for their viral inhibitory activity against influenza A/H1N1 and A/H5N1 viruses. The cytotoxicity of tested alkaloids on MDCK showed a high safety range (CC 50  > 200 µg/ml), permitting the screening for their anti-influenza potential., Results: Herein, atropine sulphate, pilocarpine hydrochloride and colchicine displayed anti-H5N1 activities with IC 50 values of 2.300, 0.210 and 0.111 µg/ml, respectively. Validation of the IC 50 values was further depicted by testing the three highly effective alkaloids, based on their potent IC 50 values against seasonal influenza A/H1N1 virus, showing comparable IC 50 values of 0.204, 0.637 and 0.326 µg/ml, respectively. Further investigation suggests that colchicine could suppress viral infection by primarily interfering with IAV replication and inhibiting viral adsorption, while atropine sulphate and pilocarpine hydrochloride could directly affect the virus in a cell-free virucidal effect. Interestingly, the in silico molecular docking studies suggest the abilities of atropine, pilocarpine, and colchicine to bind correctly inside the active sites of the neuraminidases of both influenza A/H1N1 and A/H5N1 viruses. The three alkaloids exhibited good binding energies as well as excellent binding modes that were similar to the co-crystallized ligands. On the other hand, consistent with in vitro results, only colchicine could bind correctly against the M2-proton channel of influenza A viruses (IAVs). This might explicate the in vitro antiviral activity of colchicine at the replication stage of the virus replication cycle., Conclusion: This study highlighted the anti-influenza efficacy of biologically active alkaloids including colchicine. Therefore, these alkaloids should be further characterized in vivo (preclinical and clinical studies) to be developed as anti-IAV agents., (© 2024. The Author(s).)

Lung infections in Influenza-Like Illness (ILI) are triggered by a variety of respiratory viruses. All human pandemics have been caused by the members of two major virus families, namely Orthomyxoviridae (influenza A viruses (IAVs); subtypes H1N1, H2N2, and H3N2) and Coronaviridae (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). These viruses acquired some adaptive changes in a known intermediate host including domestic birds (IAVs) or unknown intermediate host (SARS-CoV-2) following transmission from their natural reservoirs (e.g. migratory birds or bats, respectively). Verily, these acquired adaptive substitutions facilitated crossing species barriers by these viruses to infect humans in a phenomenon that is known as zoonosis. Besides, these adaptive substitutions aided the variant strain to transmit horizontally to other contact non-human animal species including pets and wild animals (zooanthroponosis). Herein we discuss the main zoonotic and reverse-zoonosis events that occurred during the last two pandemics of influenza A/H1N1 and SARS-CoV-2. We also highlight the impact of interspecies transmission of these pandemic viruses on virus evolution and possible prophylactic and therapeutic interventions. Based on information available and presented in this review article, it is important to close monitoring viral zoonosis and viral reverse zoonosis of pandemic strains within a One-Health and One-World approach to mitigate their unforeseen risks, such as virus evolution and resistance to limited prophylactic and therapeutic interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Khalil, Martinez-Sobrido and Mostafa.)

Due to antigenic drift and shift of influenza A viruses (IAV) and the tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to new strains of seasonal IAV and is at risk from viruses with pandemic potential for which limited or no immunity may exist. The genetic drift of H3N2 IAV is specifically pronounced, resulting in two distinct clades since 2014. Here, we demonstrate that immunization with a seasonal inactivated influenza vaccine (IIV) results in increased levels of H3N2 IAV-specific serum antibodies against hemagglutinin (HA) and neuraminidase (NA). Detailed analysis of the H3N2 B cell response indicated expansion of H3N2-specific peripheral blood plasmablasts 7 days after IIV immunization which expressed monoclonal antibodies (MAbs) with broad and potent antiviral activity against many H3N2 IAV strains as well as prophylactic and therapeutic activity in mice. These H3N2-specific B cell clonal lineages persisted in CD138 + long-lived bone marrow plasma cells. These results demonstrate that IIV-induced H3N2 human MAbs can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IAV H3N2-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza A virus (IAV) infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets within the influenza virus hemagglutinin and neuraminidase proteins. We have demonstrated that seasonal immunization with inactivated influenza vaccine (IIV) stimulates H3N2-specific monoclonal antibodies in humans that are broad and potent in their neutralization of virus in vitro . These antibodies also provide protection from H3N2 IAV in a mouse model of infection. Furthermore, they persist in the bone marrow, where they are expressed by long-lived antibody-producing plasma cells. This significantly demonstrates that seasonal IIV can induce a subset of H3N2-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine., Competing Interests: The authors declare a conflict of interest. M.S.P., A.N., L.M.-S., and J.J.K. are inventors with patent applications (US 11,168,129 B2,: 62/666,180) held/submitted by the University of Rochester that covers the antibodies described in this manuscript.

Influenza B virus (IBV) contributes to substantial influenza-mediated morbidity and mortality, particularly among children. Similar to influenza A viruses (IAV), the hemagglutinin (HA) and neuraminidase (NA) of IBV undergo antigenic drift, necessitating regular reformulation of seasonal influenza vaccines. NA inhibitors, such as oseltamivir, have reduced activity and clinical efficacy against IBV, while M2 channel inhibitors are only effective against IAV, highlighting the need for improved vaccine and therapeutics for the treatment of seasonal IBV infections. We have previously described a potent human monoclonal antibody (hMAb), 1092D4, that is specific for IBV NA and neutralizes a broad range of IBVs. The anti-viral activity of MAbs can include direct mechanisms such as through neutralization and/or Fc-mediated effector functions that are dependent on accessory cells expressing Fc receptors and that could be impacted by potential host-dependent variability. To discern if the in vivo efficacy of 1092D4 was dependent on Fc-effector function, 1092D4 hMAb with reduced ability to bind to Fc receptors (1092D4-LALAPG) was generated and tested. 1092D4-LALAPG had comparable in vitro binding, neutralization, and inhibition of NA activity to 1092D4. 1092D4-LALAPG was effective at protecting against a lethal challenge of IBV in mice. These results suggest that hMAb 1092D4 in vivo activity is minimally dependent on Fc-effector functions, a characteristic that may extend to other hMAbs that have potent NA inhibition activity.

Epidemiological studies associate biomass smoke with an increased risk for respiratory infections in children and adults in the developing world, with 500,000 premature deaths each year attributed to biomass smoke-related acute respiratory infections including infections caused by respiratory viruses. Animal dung is a biomass fuel of particular concern because it generates more toxic compounds per amount burned than wood, and is a fuel of last resort for the poorest households. Currently, there is little biological evidence on the effects of dung biomass smoke exposure on immune responses to respiratory viral infections. Here, we investigated the impact of dung biomass exposure on respiratory infection using a mouse model of dung biomass smoke and cultured primary human small airway epithelial cells (SAECs). Mice infected with influenza A virus (IAV) after dung biomass smoke exposure had increased mortality, lung inflammation and virus mRNA levels, and suppressed expression of innate anti-viral mediators compared to air exposed mice. Importantly, there was still significant tissue inflammation 14 days after infection in dung biomass smoke-exposed mice even after inflammation had resolved in air-exposed mice. Dung biomass smoke exposure also suppressed the production of anti-viral cytokines and interferons in cultured SAECs treated with poly(I:C) or IAV. This study shows that dung biomass smoke exposure impairs the immune response to respiratory viruses and contributes to biomass smoke-related susceptibility to respiratory viral infections, likely due to a failure to resolve the inflammatory effects of biomass smoke exposure., (Copyright © 2022. Published by Elsevier Inc.)

There is a critical need for physiologically relevant, robust, and ready-to-use in vitro cellular assay platforms to rapidly model the infectivity of emerging viruses and develop new antiviral treatments. Here we describe the cellular complexity of human alveolar and tracheobronchial air liquid interface (ALI) tissue models during SARS-CoV-2 and influenza A virus (IAV) infections. Our results showed that both SARS-CoV-2 and IAV effectively infect these ALI tissues, with SARS-CoV-2 exhibiting a slower replication peaking at later time-points compared to IAV. We detected tissue-specific chemokine and cytokine storms in response to viral infection, including well-defined biomarkers in severe SARS-CoV-2 and IAV infections such as CXCL10, IL-6, and IL-10. Our single-cell RNA sequencing analysis showed similar findings to that found in vivo for SARS-CoV-2 infection, including dampened IFN response, increased chemokine induction, and inhibition of MHC Class I presentation not observed for IAV infected tissues. Finally, we demonstrate the pharmacological validity of these ALI tissue models as antiviral drug screening assay platforms, with the potential to be easily adapted to include other cell types and increase the throughput to test relevant pathogens., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Children and young adults with mutant forms of ataxia telangiectasia mutated (ATM), a kinase involved in DNA damage signaling and mitochondrial homeostasis, suffer from recurrent respiratory infections, immune deficiencies, and obstructive airways disease associated with disorganized airway epithelium. We previously showed in mice how Atm was required to mount a protective immune memory response to influenza A virus [IAV; Hong Kong/X31 (HKx31), H3N2]. Here, Atm wildtype (WT) and knockout (Atm-null) mice were used to investigate how Atm is required to regenerate the injured airway epithelium following IAV infection. When compared with WT mice, naive Atm-null mice had increased airway resistance and reduced lung compliance that worsened during infection before returning to naïve levels by 56 days postinfection (dpi). Although Atm-null lungs appeared pathologically normal before infection by histology, they developed an abnormal proximal airway epithelium after infection that contained E-cadherin + , Sox2 + , and Cyp2f2 + cells lacking secretoglobin family 1 A member 1 (Scgb1a1) protein expression. Patchy and low expression of Scgb1a1 were eventually observed by 56 dpi. Genetic lineage tracing in HKx31-infected mice revealed club cells require Atm to rapidly and efficiently restore Scgb1a1 expression in proximal airways. Since Scgb1a1 is an immunomodulatory protein that protects the lung against a multitude of respiratory challenges, failure to efficiently restore its expression may contribute to the respiratory diseases seen in individuals with ataxia telangiectasia.

Secondary experimental procedures such as immunostaining have been utilized to study wild-type influenza A viruses (IAV) but are inadequate to rapidly determine the virus in infected cells or for the high-throughput screening (HTS) of antivirals or neutralizing antibodies. Reverse genetics approaches have allowed the generation of recombinant IAV expressing bioluminescent (BL) reporters or fluorescent proteins (FPs). These approaches can easily track viral infections in cultured cells and in validated animal models of infection using in vivo imaging systems (IVIS). Here, we describe the experimental procedures to generate recombinant monomeric (m)Cherry-expressing influenza A/Puerto Rico/8/34 (PR8-mCherry) H1N1 by altering the non-structural (NS) vRNA segment and its use in mCherry-based microneutralization assays to assess antivirals and neutralizing antibodies. The experimental procedures could be used for the generation of other recombinant influenza virus types (e.g., influenza B) or IAV subtypes (e.g., H3N2) expressing mCherry or other BL reporters or FPs from the NS or other vRNA segment. These recombinant reporter-expressing viruses represent an excellent toolbox for the identification of prophylactics or therapeutics for the treatment of influenza viral infections in HTS settings as well as to study different aspects related with the biology of influenza viruses and/or its interaction with the host., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections.

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus. IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination., (Copyright © 2019 American Society for Microbiology.)

Although most seasonal inactivated influenza vaccines (IIV) contain neuraminidase (NA), the extent and mechanisms of action of protective human NA-specific humoral responses induced by vaccination are poorly resolved. Due to the propensity of influenza virus for antigenic drift and shift and its tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to waves of new strains of seasonal viruses and is at risk from viruses with pandemic potential for which limited or no immunity may exist. Here we demonstrate that the use of IIV results in increased levels of influenza B virus (IBV) NA-specific serum antibodies. Detailed analysis of the IBV NA B cell response indicates concurrent expansion of IBV NA-specific peripheral blood plasmablasts 7 days after IIV immunization which express monoclonal antibodies with broad and potent antiviral activity against both IBV Victoria and Yamagata lineages and prophylactic and therapeutic activity in mice. These IBV NA-specific B cell clonal lineages persisted in CD138 + long-lived bone marrow plasma cells. These results represent the first demonstration that IIV-induced NA human antibodies can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IBV NA-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza virus infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets such as regions within the influenza neuraminidase protein. We have demonstrated that seasonal immunization stimulates neuraminidase-specific antibodies in humans that are broad and potent in their protection from influenza B virus when tested in mice. These antibodies further persist in the bone marrow, where they are expressed by long-lived antibody-producing cells, referred to here as plasma cells. The significance in our research is the demonstration that seasonal influenza immunization can induce a subset of neuraminidase-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine., (Copyright © 2019 Piepenbrink et al.)

In 2009, a pandemic H1N1 influenza A virus (IAV) (pH1N1) emerged in the human population from swine causing a pandemic. Importantly, this virus is still circulating in humans seasonally. To analyze the evolution of pH1N1 in humans, we sequenced viral genes encoding proteins inhibiting general gene expression (nonstructural protein 1 [NS1] and PA-X) from circulating seasonal viruses and compared them to the viruses isolated at the origin of the pandemic. Recent pH1N1 viruses contain amino acid changes in the NS1 protein (E55K, L90I, I123V, E125D, K131E, and N205S), as previously described (A. M. Clark, A. Nogales, L. Martinez-Sobrido, D. J. Topham, and M. L. DeDiego, J Virol 91:e00721-17, 2017, https://doi.org/10.1128/JVI.00721-17), and amino acid changes in the PA-X protein (V100I, N204S, R221Q, and L229S). These amino acid differences between early and more recent pH1N1 isolates are responsible for increased NS1-mediated inhibition of host gene expression and decreased PA-X-mediated shutoff, including innate immune response genes. In addition, currently circulating pH1N1 viruses have acquired amino acid changes in the PA protein (V100I, P224S, N321K, I330V, and R362K). A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from currently circulating viruses is fitter in replication in cultured cells and in mice and is slightly more pathogenic than the original ancestor pH1N1 virus. These results demonstrate the need to monitor the evolution of pH1N1 in humans for mutations in the viral genome that could result in enhanced virulence. Importantly, these results further support our previous findings suggesting that inhibition of global gene expression mediated by NS1 and PA-X proteins is subject to a balance which can determine virus pathogenesis and fitness. IMPORTANCE IAVs emerge in humans from animal reservoirs, causing unpredictable pandemics. One of these pandemics was caused by an H1N1 virus in 2009, and this virus is still circulating seasonally. To analyze host-virus adaptations likely affecting influenza virus pathogenesis, protein amino acid sequences from viruses circulating at the beginning of the pandemic and those circulating currently were compared. Currently circulating viruses have incorporated amino acid changes in two viral proteins (NS1 and PA-X), affecting innate immune responses, and in the PA gene. These amino acid differences led to increased NS1-mediated and decreased PA-X-mediated inhibition of host gene expression. A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from recently circulating viruses is fitter in replication in tissue culture cells and in mice, and the virus is more pathogenic in vivo Importantly, these results suggest that a balance in the abilities of NS1 and PA-X to induce host shutoff is beneficial for IAVs., (Copyright © 2018 American Society for Microbiology.)

Rapid changes in influenza A virus (IAV) antigenicity create challenges in surveillance, disease diagnosis, and vaccine development. Further, serological methods for studying antigenic properties of influenza viruses often rely on animal models and therefore may not fully reflect the dynamics of human immunity. We hypothesized that arrays of human monoclonal antibodies (hmAbs) to influenza could be employed in a pattern-recognition approach to expedite IAV serology and to study the antigenic evolution of newly emerging viruses. Using the multiplex, label-free Arrayed Imaging Reflectometry (AIR) platform, we have demonstrated that such arrays readily discriminated among various subtypes of IAVs, including H1, H3 seasonal strains, and avian-sourced human H7 viruses. Array responses also allowed the first determination of antigenic relationships among IAV strains directly from hmAb responses. Finally, correlation analysis of antibody binding to all tested IAV subtypes allowed efficient identification of broadly reactive clones. In addition to specific applications in the context of understanding influenza biology with potential utility in "universal" flu vaccine development, these studies validate AIR as a platform technology for studying antigenic properties of viruses and also antibody properties in a high-throughput manner. We further anticipate that this approach will facilitate advances in the study of other viral pathogens.

Cigarette smokers and people exposed to second-hand smoke are at an increased risk for pulmonary viral infections, and yet the mechanism responsible for this heightened susceptibility is not understood. To understand the effect of cigarette smoke on susceptibility to viral infection, we used an air-liquid interface culture system and exposed primary human small airway epithelial cells (SAEC) to whole cigarette smoke, followed by treatment with the viral mimetic polyinosinic polycytidylic acid (poly I:C) or influenza A virus (IAV). We found that prior smoke exposure strongly inhibited production of proinflammatory (interleukin-6 and interleukin-8) and antiviral [interferon-γ-induced protein 10 (IP-10) and interferons] mediators in SAECs in response to poly I:C and IAV infection. Impaired antiviral responses corresponded to increased infection with IAV. This was associated with a decrease in phosphorylation of the key antiviral transcription factor interferon response factor 3 (IRF3). Here, we found that cigarette smoke exposure inhibited activation of Toll-like receptor 3 (TLR3) by impairing TLR3 cleavage, which was required for downstream phosphorylation of IRF3 and production of IP-10. These results identify a novel mechanism by which cigarette smoke exposure impairs antiviral responses in lung epithelial cells, which may contribute to increased susceptibility to respiratory infections.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo ., (Copyright © 2017 American Society for Microbiology.)

Background: Numerous pre-clinical studies and clinical trials demonstrated that induction of antibodies to the β-amyloid peptide of 42 residues (Aβ42) elicits therapeutic effects in Alzheimer's disease (AD). However, an active vaccination strategy based on full length Aβ42 is currently hampered by elicitation of T cell pathological autoreactivity. We attempt to improve vaccine efficacy by creating a novel chimeric flu vaccine expressing the small immunodominant B cell epitope of Aβ42. We hypothesized that in elderly people with pre-existing memory Th cells specific to influenza this dual vaccine will simultaneously boost anti-influenza immunity and induce production of therapeutically active anti-Aβ antibodies., Methods: Plasmid-based reverse genetics system was used for the rescue of recombinant influenza virus containing immunodominant B cell epitopes of Aβ42 (Aβ1-7/10)., Results: Two chimeric flu viruses expressing either 7 or 10 aa of Aβ42 (flu-Aβ1-7 or flu-Aβ1-10) were generated and tested in mice as conventional inactivated vaccines. We demonstrated that this dual vaccine induced therapeutically potent anti-Aβ antibodies and anti-influenza antibodies in mice., Conclusion: We suggest that this strategy might be beneficial for treatment of AD patients as well as for prevention of development of AD pathology in pre-symptomatic individuals while concurrently boosting immunity against influenza.