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Abstract As the new SARS-CoV-2 Omicron variants and subvariants emerge, there is an urgency to develop intranasal, broadly protective vaccines. Here, we developed highly efficacious, intranasal trivalent SARS-CoV-2 vaccine candidates (TVC) based on three components of the MMR vaccine: measles virus (MeV), mumps virus (MuV) Jeryl Lynn (JL1) strain, and MuV JL2 strain. Specifically, MeV, MuV-JL1, and MuV-JL2 vaccine strains, each expressing prefusion spike (preS-6P) from a different variant of concern (VoC), were combined to generate TVCs. Intranasal immunization of IFNAR1−/− mice and female hamsters with TVCs generated high levels of S-specific serum IgG antibodies, broad neutralizing antibodies, and mucosal IgA antibodies as well as tissue-resident memory T cells in the lungs. The immunized female hamsters were protected from challenge with SARS-CoV-2 original WA1, B.1.617.2, and B.1.1.529 strains. The preexisting MeV and MuV immunity does not significantly interfere with the efficacy of TVC. Thus, the trivalent platform is a promising next-generation SARS-CoV-2 vaccine candidate.

Summary: Influenza A virus (IAV) and SARS-CoV-2 virus are both acute respiratory viruses currently circulating in the human population. This study aims to determine the impact of IAV infection on SARS-CoV-2 pathogenesis and cardiomyocyte function. Infection of human bronchial epithelial cells (HBEC), A549 cells, lung fibroblasts (HLF), monocyte derived macrophages (MDMs), cardiac fibroblasts (HCF) and hiPSC-derived cardiomyocytes with IAV enhanced the expression of ACE2, the SARS-CoV-2 receptor. Similarly, IAV infection increased levels of ACE2 in the lungs of mice and humans. Of interest, we detected heavily glycosylated form of ACE2 in hiPSC-CMs and poorly glycosylated ACE2 in other cell types. Also, prior IAV infection enhances SARS-CoV-2 spike protein binding and viral entry in all cell types. However, efficient SARS-CoV-2 replication was uniquely inhibited in cardiomyocytes. Glycosylation of ACE2 correlated with enzymatic conversion of its substrate Ang II, induction of eNOS and nitric oxide production, may provide a potential mechanism for the restricted SARS-CoV-2 replication in cardiomyocytes.

Toll-like receptor 3 (TLR3) induces host innate immune response on recognition of viral double-stranded RNA (dsRNA). Although several studies of avian TLR3 have been reported, none of these studies used a gene knockout (KO) model to directly assess its role in inducing the immune response and effect on other dsRNA receptors. In this study, we determined the coding sequence of quail TLR3, identified isoforms, and generated TLR3 KO quail fibroblast (QT-35) cells using a CRISPR/Cas9 system optimized for avian species. The TLR3-mediated immune response was studied by stimulating the wild-type (WT) and KO QT-35 cells with synthetic dsRNA or polyinosinic:polycytidylic acid [poly(I:C)] or infecting the cells with different RNA viruses such as influenza A virus, avian reovirus, and vesicular stomatitis virus. The direct poly(I:C) treatment significantly increased IFN-β and IL-8 gene expression along with the cytoplasmic dsRNA receptor, melanoma differentiation–associated gene 5 (MDA5), in WT cells, whereas no changes in all corresponding genes were observed in KO cells. We further confirmed the antiviral effects of poly(I:C)-induced TLR3-mediated immunity by demonstrating significant reduction of virus titer in poly(I:C)–treated WT cells, but not in TLR3 KO cells. On virus infection, varying levels of IFN-β, IL-8, TLR3, and MDA5 gene upregulation were observed depending on the viruses. No major differences in gene expression level were observed between WT and TLR3 KO cells, which suggests a relatively minor role of TLR3 in sensing and exerting immune response against the viruses tested in vitro. Our data show that quail TLR3 is an important endosomal dsRNA receptor responsible for regulation of type I interferon and proinflammatory cytokine, and affect the expression of MDA5, another dsRNA receptor, most likely through cytokine-mediated communication.

Turkey respiratory and gut microbiota play important roles in promoting health and production performance. Loss of microbiota homeostasis due to pathogen infection can worsen the disease or predispose the bird to infection by other pathogens. While turkeys are highly susceptible to influenza viruses of different origins, the impact of influenza virus infection on turkey gut and respiratory microbiota has not been demonstrated. In this study, we investigated the relationships between low pathogenicity avian influenza (LPAI) virus replication, cytokine gene expression, and respiratory and gut microbiota disruption in specific-pathogen-free turkeys. Differential replication of two LPAI H5N2 viruses paralleled the levels of clinical signs and cytokine gene expression. During active virus shedding, there was significant increase of ileal and nasal bacterial contents, which inversely corresponded with bacterial species diversity. Spearman’s correlation tests between bacterial abundance and local viral titers revealed that LPAI virus-induced dysbiosis was strongest in the nasal cavity followed by trachea, and weakest in the gut. Significant correlations were also observed between cytokine gene expression levels and relative abundances of several bacteria in tracheas of infected turkeys. For example, interferon γ/λ and interleukin-6 gene expression levels were correlated positively with Staphylococcus and Pseudomonas abundances, and negatively with Lactobacillus abundance. Overall, our data suggest a potential relationship where bacterial community diversity and enrichment or depletion of several bacterial genera in the gut and respiratory tract are dependent on the level of LPAI virus replication. Further work is needed to establish whether respiratory and enteric dysbiosis in LPAI virus-infected turkeys is a result of host immunological responses or other causes such as changes in nutritional uptake.

Outbreaks of novel highly pathogenic avian influenza viruses have been reported in poultry species in the United States since 2014. These outbreaks have proven the limitations of biosecurity control programs, and new tools are needed to reinforce the current avian influenza control arsenal. Some enzootic countries have implemented inactivated influenza vaccine (IIV) in their control programs, but there are serious concerns that a long-term use of IIV without eradication may result in the selection of novel antigenically divergent strains. A broadly protective vaccine is needed, such as live-attenuated influenza vaccine (LAIV). We showed in our previous studies that pc4-LAIV (a variant that encodes a C-terminally truncated NS1 protein) can provide significant protection against heterologous challenge virus in chickens vaccinated at 2-4 weeks of age through upregulation of innate and adaptive immune responses. The current study was conducted to compare the performances of pc4-LAIV and IIV in young chickens vaccinated at 1 day of age. A single dose of pc4-LAIV was able to induce stronger innate and mucosal IgA responses and protect young immunologically immature chickens better than a single dose of IIV. Most importantly, when 1-day-old chickens were intranasally primed with pc4-LAIV and subcutaneously boosted with IIV three weeks later, they showed a rapid, robust, and highly cross-reactive serum antibody response and a high level of mucosal IgA antibody response. This vaccination regimen warrants further optimization to increase its range of protection.

Interferon‐induced transmembrane protein 3 (IFITM3) is an antiviral protein that alters cell membranes to block fusion of viruses. Conflicting reports identified opposing effects of IFITM3 on SARS‐CoV‐2 infection of cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with SARS‐CoV‐2 experience extreme weight loss and lethality compared to mild infection in wild‐type (WT) mice. KO mice have higher lung viral titers and increases in inflammatory cytokine levels, immune cell infiltration, and histopathology. Mechanistically, we observe disseminated viral antigen staining throughout the lung and pulmonary vasculature in KO mice, as well as increased heart infection, indicating that IFITM3 constrains dissemination of SARS‐CoV‐2. Global transcriptomic analysis of infected lungs shows upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS‐CoV‐2 infection and overall demonstrate that IFITM3 is protective in SARS‐CoV‐2 infections in vivo. Synopsis: IFITM3 limits severity of SARS‐CoV‐2 infections by restricting viral replication and spread, thereby reducing lung inflammation, immune cell infiltration, angiogenesis, and pathology. IFITM3 KO mice are a new model for studying severe SARS‐CoV‐2 infections. IFITM3 limits morbidity and mortality following SARS‐CoV‐2 infection in two mouse models.Enhanced viral replication in IFITM3 KO mice is accompanied by increased lung pathology and extrapulmonary virus dissemination.IFITM3 KO mice upregulate inflammatory, angiogenic, and pro‐thrombotic gene programs following SARS‐CoV‐2 infection.IFITM3 KO mice provide a tool for studying severe SARS‐CoV‐2 infections with likely relevance to human populations with IFITM3 deficiencies. [ABSTRACT FROM AUTHOR]

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