Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-ß. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

Impact of sex on humoral immunity with live influenza B virus vaccines in mice.

Influenza B virus (FLUBV) poses a significant infectious threat, with frequent vaccine mismatch limiting its effectiveness. Our previous work investigated the safety and efficacy of modified live attenuated FLUBV vaccines with rearranged genomes (FluB-RAM and FluB-RANS) or a temperature-sensitive PB1 segment with a C-terminal HA tag (FluB-att). In this study, we compared the immune responses of female and male DBA/2J mice vaccinated with these vaccines, including versions containing a chimeric HA segment with an N-terminal IgA-inducing peptide (IGIP). Importantly, both recombinant viruses with and without IGIP remained genetically stable during egg passage. We found that introducing IGIP strengthened vaccine attenuation, particularly for FluB-RAM/IGIP. Prime-boost vaccination completely protected mice against lethal challenge with a homologous FLUBV strain. Notably, recombinant viruses induced robust neutralizing antibody responses (hemagglutination inhibition titers ≥40) alongside antibodies against NA and NP. Interestingly, female mice displayed a consistent trend of enhanced humoral and cross-reactive IgG and IgA responses against HA, NA, and NP compared to male counterparts, regardless of the vaccine used. However, the presence of IGIP generally led to lower anti-HA responses but higher anti-NA and anti-NP responses, particularly of the IgA isotype. These trends were further reflected in mucosal and serological responses two weeks after challenge, with clear distinctions based on sex, vaccine backbone, and IGIP inclusion. These findings hold significant promise for advancing the development of universal influenza vaccines.

Use of Reverse Genetics for the Generation of Recombinant Influenza Viruses Carrying Nanoluciferase.

Influenza A (FLUAV) and influenza B (FLUBV) viruses are human and/or animal pathogens widely studied due to their importance to public health and animal production. Both FLUAV and FLUBV possess a genome composed of eight viral gene segments. For reverse genetics of influenza viruses, transcription of the mRNA for the viral proteins is typically done from a plasmid encoding an RNA polymerase II (pol II) promoter element upstream of cloned viral cDNA and expressed like host mRNA. On the other side, the synthesis of the negative-sense, single-stranded, uncapped vRNAs can be accomplished by the host's RNA polymerase I (pol I). The reverse genetics for influenza has allowed the manipulation of influenza genomes incorporating heterogeneous sequences into different segments of the influenza genome, such as reporter genes. In this chapter, we outline the protocol from the generation of reverse genetic plasmid that can be applied for the cloning of any of the segments of FLUAV or FLUBV. Furthermore, we describe a protocol for generating FLUAV or FLUBV recombinant viruses carrying Nanoluciferase (NLuc) in the PB1 gene using reverse genetics. Finally, we delineate a microneutralization protocol using FLUAV-NLuc or FLUBV-NLuc viruses optimized for the use of antibodies from different sources (mice, ferrets, avian, etc.), which provides a more sensitive, reliable, and avidity-independent method to assess the presence of neutralizing antibodies against FLUAV or FLUBV.

Antigenic mapping of the hemagglutinin of the H9 subtype influenza A viruses using sera from Japanese quail (Coturnix c. japonica).

IMPORTANCE: Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies.

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Characterizing a century of genetic diversity and contemporary antigenic diversity of N1 neuraminidase in influenza A virus from North American swine.

Influenza A viruses (IAVs) of the H1N1 classical swine lineage became endemic in North American swine following the 1918 pandemic. Additional human-to-swine transmission events after 1918, and a spillover of H1 viruses from wild birds in Europe, potentiated a rapid increase in genomic diversity via reassortment between introductions and the endemic classical swine lineage. To determine mechanisms affecting reassortment and evolution, we conducted a phylogenetic analysis of N1 and paired HA swine IAV genes in North America between 1930 and 2020. We described fourteen N1 clades within the N1 Eurasian avian lineage (including the N1 pandemic clade), the N1 classical swine lineage, and the N1 human seasonal lineage. Seven N1 genetic clades had evidence for contemporary circulation. To assess antigenic drift associated with N1 genetic diversity, we generated a panel of representative swine N1 antisera and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assays and antigenic cartography. Within the N1 genes, the antigenic similarity was variable and reflected shared evolutionary history. Sustained circulation and evolution of N1 genes in swine had resulted in a significant antigenic distance between the N1 pandemic clade and the classical swine lineage. Between 2010 and 2020, N1 clades and N1-HA pairings fluctuated in detection frequency across North America, with hotspots of diversity generally appearing and disappearing within 2 years. We also identified frequent N1-HA reassortment events (n = 36), which were rarely sustained (n = 6) and sometimes also concomitant with the emergence of new N1 genetic clades (n = 3). These data form a baseline from which we can identify N1 clades that expand in range or genetic diversity that may impact viral phenotypes or vaccine immunity and subsequently the health of North American swine.

Pathobiology and dysbiosis of the respiratory and intestinal microbiota in 14 months old Golden Syrian hamsters infected with SARS-CoV-2.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS2) affected the geriatric population. Among research models, Golden Syrian hamsters (GSH) are one of the most representative to study SARS2 pathogenesis and host responses. However, animal studies that recapitulate the effects of SARS2 in the human geriatric population are lacking. To address this gap, we inoculated 14 months old GSH with a prototypic ancestral strain of SARS2 and studied the effects on virus pathogenesis, virus shedding, and respiratory and gastrointestinal microbiome changes. SARS2 infection led to high vRNA loads in the nasal turbinates (NT), lungs, and trachea as well as higher pulmonary lesions scores later in infection. Dysbiosis throughout SARS2 disease progression was observed in the pulmonary microbial dynamics with the enrichment of opportunistic pathogens (Haemophilus, Fusobacterium, Streptococcus, Campylobacter, and Johnsonella) and microbes associated with inflammation (Prevotella). Changes in the gut microbial community also reflected an increase in multiple genera previously associated with intestinal inflammation and disease (Helicobacter, Mucispirillum, Streptococcus, unclassified Erysipelotrichaceae, and Spirochaetaceae). Influenza A virus (FLUAV) pre-exposure resulted in slightly more pronounced pathology in the NT and lungs early on (3 dpc), and more notable changes in lungs compared to the gut microbiome dynamics. Similarities among aged GSH and the microbiome in critically ill COVID-19 patients, particularly in the lower respiratory tract, suggest that GSHs are a representative model to investigate microbial changes during SARS2 infection. The relationship between the residential microbiome and other confounding factors, such as SARS2 infection, in a widely used animal model, contributes to a better understanding of the complexities associated with the host responses during viral infections.

Rational design of a deuterium-containing M2-S31N channel blocker UAWJ280 with in vivo antiviral efficacy against both oseltamivir sensitive and -resistant influenza A viruses.

Seasonal influenza A virus (IAV) infections are among the most important global health problems. FDA-approved antiviral therapies against IAV include neuraminidase inhibitors, M2 inhibitors, and polymerase inhibitor baloxavir. Resistance against adamantanes (amantadine and rimantadine) is widespread as virtually all IAV strains currently circulating in the human population are resistant to adamantanes through the acquisition of the S31N mutation. The neuraminidase inhibitor-resistant strains also contain the M2-S31N mutant, suggesting M2-S31N is a high-profile antiviral drug target. Here we report the development of a novel deuterium-containing M2-S31N inhibitor UAWJ280. UAWJ280 had broad-spectrum antiviral activity against both oseltamivir sensitive and -resistant influenza A strains and had a synergistic antiviral effect in combination with oseltamivir in cell culture. In vivo pharmacokinetic (PK) studies demonstrated that UAWJ280 had favourable PK properties. The in vivo mouse model study showed that UAWJ280 was effective alone or in combination with oseltamivir in improving clinical signs and survival after lethal challenge with an oseltamivir sensitive IAV H1N1 strain. Furthermore, UAWJ280 was also able to ameliorate clinical signs and increase survival when mice were challenged with an oseltamivir-resistant IAV H1N1 strain. In conclusion, we show for the first time that the M2-S31N channel blocker UAWJ280 has in vivo antiviral efficacy in mice that are infected with either oseltamivir sensitive or -resistant IAVs, and it has a synergistic antiviral effect with oseltamivir.

Development of a Novel Live Attenuated Influenza A Virus Vaccine Encoding the IgA-Inducing Protein.

Live attenuated influenza virus (LAIV) vaccines elicit a combination of systemic and mucosal immunity by mimicking a natural infection. To further enhance protective mucosal responses, we incorporated the gene encoding the IgA-inducing protein (IGIP) into the LAIV genomes of the cold-adapted A/Leningrad/134/17/57 (H2N2) strain (caLen) and the experimental attenuated backbone A/turkey/Ohio/313053/04 (H3N2) (OH/04att). Incorporation of IGIP into the caLen background led to a virus that grew poorly in prototypical substrates. In contrast, IGIP in the OH/04att background (IGIP-H1att) virus grew to titers comparable to the isogenic backbone H1att (H1N1) without IGIP. IGIP-H1att- and H1caLen-vaccinated mice were protected against lethal challenge with a homologous virus. The IGIP-H1att vaccine generated robust serum HAI responses in naïve mice against the homologous virus, equal or better than those obtained with the H1caLen vaccine. Analyses of IgG and IgA responses using a protein microarray revealed qualitative differences in humoral and mucosal responses between vaccine groups. Overall, serum and bronchoalveolar lavage samples from the IGIP-H1att group showed trends towards increased stimulation of IgG and IgA responses compared to H1caLen samples. In summary, the introduction of genes encoding immunomodulatory functions into a candidate LAIV can serve as natural adjuvants to improve overall vaccine safety and efficacy.