Background/Objectives: Intranasal vaccination enhances protection against respiratory viruses by providing stimuli to the immune system at the primary site of infection, promoting a balanced and effective response. Influenza vectors with truncated NS1 are a promising vaccine approach that ensures a pronounced local CD8+ T-cellular immune response. Here, we describe the protective and immunomodulating properties of an influenza vector FluVec-N carrying the C-terminal fragment of the SARS-CoV-2 nucleoprotein within a truncated NS1 open reading frame. Methods: We generated several FluVec-N recombinant vectors by reverse genetics and confirmed the vector’s genetic stability, antigen expression in vitro, attenuation, and immunogenicity in a mouse model. We tested the protective potential of FluVec-N intranasal immunization in naïve mice and seropositive Th2-prone mice, primed with aluminium-adjuvanted inactivated SARS-CoV-2. Immune response in immunized and challenged mice was analyzed through serological methods and flow cytometry. Results: Double intranasal immunization of naïve mice with FluVec-N reduced weight loss and viral load in the lungs following infection with the SARS-CoV-2 beta variant. Mice primed with alum-adjuvanted inactivated coronavirus experienced substantial early weight loss and eosinophilia in the lungs during infection, demonstrating signs of enhanced disease. A single intranasal boost immunization with FluVec-N prevented the disease enhancement in primed mice by modulating the local immune response. Protection was associated with the formation of specific IgA and the early activation of virus-specific effector and resident CD8+ lymphocytes in mouse lungs. Conclusions: Our study supports the potential of immunization with influenza vector vaccines to prevent respiratory diseases and associated immunopathology.

Background/Objectives: The current strategy for seasonal influenza prophylaxis relies on updating the vaccine components annually to account for the rapid antigenic drift of viruses and the low cross-protective efficacy of available vaccines. Mutant influenza viruses with truncated or deleted NS1 protein are known to stimulate cross-specific T-cell immune response and provide protection against heterosubtypic influenza A and B viruses. Methods: We generated NS1ΔC influenza A and B viruses with C-terminal NS1 deletions by reverse genetics. In a mouse model, we assessed the safety and immunogenicity of the B/Lee/NS1ΔC strain upon intranasal administration, as well as the mechanism of its cross-protective efficacy against sublethal B/Victoria and B/Yamagata challenges. We then investigated the potential of the intranasal Flu/NS1ΔC vaccine–a trivalent formulation of NS1ΔC A/H1N1, A/H3N2, and B influenza viruses–to protect mice from lethal influenza infection with homologous, heterologous, and antigenically drifted influenza A and B viruses. Results: Intranasal immunization with the B/Lee/NS1ΔC strain was safe in mice. It activated cross-specific T-cell responses in the lungs and protected animals against heterologous challenge by reducing viral load, inflammation, and lung pathology. Immunization with the trivalent Flu/NS1ΔC vaccine formulation improved survival and reduced weight loss and viral load upon challenge with A/H1N1pdm, A/H2N2, A/H5N1, and B/Victoria viruses. Conclusions: The trivalent intranasal Flu/NS1ΔC influenza vaccine is a promising tool to improve seasonal influenza protection and preparedness for an influenza pandemic.

Background/Objectives: The current strategy for seasonal influenza prophylaxis relies on updating the vaccine components annually to account for the rapid antigenic drift of viruses and the low cross-protective efficacy of available vaccines. Mutant influenza viruses with truncated or deleted NS1 protein are known to stimulate cross-specific T-cell immune response and provide protection against heterosubtypic influenza A and B viruses. Methods: We generated NS1ΔC influenza A and B viruses with C-terminal NS1 deletions by reverse genetics. In a mouse model, we assessed the safety and immunogenicity of the B/Lee/NS1ΔC strain upon intranasal administration, as well as the mechanism of its cross-protective efficacy against sublethal B/Victoria and B/Yamagata challenge. We then investigated the potential of the intranasal Flu/UniVec vaccine–a trivalent formulation of NS1ΔC A/H1N1, A/H3N2, and B influenza viruses–to protect mice from lethal influenza infection with homologous, heterologous, and antigenically drifted influenza A and B viruses. Results: Intranasal immunization with the B/Lee/NS1ΔC strain was safe in mice. It activated cross-specific T-cell responses in the lungs and protected animals against heterologous challenge by reducing viral load, inflammation, and lung pathology. Immunization with the trivalent Flu/UniVec vaccine formulation improved survival and reduced weight loss and viral load upon challenge with A/H1N1pdm, A/H2N2, A/H5N1, and B/Victoria viruses. Conclusions: The trivalent intranasal replication-deficient Flu/UniVec influenza vaccine is a promising tool to improve seasonal influenza protection and preparedness for an influenza pandemic.

Background/Objectives: Influenza B virus infection contributes substantially to annual morbidity and mortality, accounting for 20% to 30% of influenza-associated deaths worldwide. Although vaccination reduces the risk of severe disease, widely used inactivated influenza vaccines are often insufficient to prevent virus transmission. Moreover, influenza B viruses are less susceptible to commonly used antivirals than influenza A viruses. New approaches are therefore required to decrease disease burden and limit virus spread. Neomycin, an aminoglycoside antibiotic, was recently shown to mitigate SARS-CoV-2 transmission in a hamster model. Here, we conducted an exploratory study to assess the effect of neomycin on influenza B virus transmission. Methods: Contact transmission was evaluated using a guinea pig model (n = 4 per group), and aerosol transmission was assessed using a ferret model (n = 6 per group). Animals in the experimental groups received neomycin sulfate (5 mg/guinea pig, 20 mg/ferret) or placebo intranasally, starting one day before exposure to infected animals and continuing for four days thereafter. In the guinea pig study, an additional control group received intranasal interferon alpha. Viral transmission to contact animals was assessed by RT-PCR and virus culture of nasal washes collected over two weeks. Clinical signs and body weight were monitored daily. Results: In the guinea pig model, 75% of contact animals became infected with influenza B virus regardless of treatment. Neither neomycin nor interferon alpha prevented infection, although both delayed the onset of viral shedding in contact animals. In the ferret model, infection occurred in 33% of placebo-treated contact animals, whereas no viral shedding was detected in the neomycin-treated group. Conclusions: Prophylactic intranasal neomycin treatment has the potential to protect exposed individuals from aerosol transmission of influenza B virus during influenza outbreaks.

Background/Objectives: Influenza B virus contributes substantially to annual morbidity and mortality, accounting for 20% to 30% of all influenza-associated deaths globally. Vaccination prevents severe disease, yet widely used inactivated influenza vaccines fail to reduce virus transmission. Also, influenza B viruses are less susceptible to commonly used antivirals than influenza A viruses. Therefore, new approaches are needed to decrease disease burden and limit virus spread. Neomycin, an aminoglycoside antibiotic, has recently been shown to mitigate SARS-CoV-2 transmission in a hamster model. In this study, we investigated the impact of neomycin on the transmission of influenza B virus. Methods: We used an influenza contact transmission model in guinea pigs and an aerosol transmission model in ferrets. Animals in experimental groups received intranasal neomycin sulphate (5 mg/guinea pig, 20 mg/ferret) or placebo one day before contact with infected animals and for four days thereafter. In the guinea pig experiment, an additional control group of animals was treated intranasally with interferon alpha. The virus spread from infected to contact animals was assessed by RT-PCR and viral culture of nasal washes during two weeks. Clinical signs and body weight were monitored daily. Results: In the guinea pig model, 75% of contact animals became infected with influenza B virus regardless of treatment. Neither neomycin nor interferon alpha prevented infection, although both delayed the onset of viral shedding in contact animals. In the ferret model, 33% of contact animals in the placebo group became infected, whereas no viral shedding was detected in the neomycin-treated group. Conclusions: Prophylactic intranasal neomycin treatment has the potential to protect exposed subjects from aerosol transmission of influenza B virus during flu outbreaks.

Introduction: The development of bivalent mucosal vaccines capable of providing protection against both influenza and SARS-CoV-2 is a major public health focus. While most COVID-19 vaccines target the spike (S) protein, the highly conserved nucleocapsid (N) protein represents a strategic target for cross-reactive, cell-mediated immunity. This study evaluates Corfluvec, an intranasal vaccine candidate based on an attenuated NS1-truncated influenza vector expressing a fragment of the SARS-CoV-2 N protein. Methods: Protective efficacy, including viral load and pathomorphological changes in the lungs and vessels, was evaluated in Syrian hamsters challenged with high and low doses of SARS-CoV-2 (lineage B.1.1). Cross-protective efficacy against homologous and heterologous influenza A strains (H1N1pdm09, H3N2, and A/PR/8/1934) was tested in a lethal murine model. Additionally, immunogenicity of Corfluvec applied via human-compatible delivery device was tested in cynomolgus macaques (Macaca fascicularis). Results: In Syrian hamsters, vaccination significantly reduced viral loads in the lungs and nasal turbinates. Histopathological analysis revealed a preservation of lung vascular integrity: vaccinated animals showed stable CD31 expression and controlled Ki-67 proliferative activity, accompanied by a marked reduction in vasculitis and perivascular edema compared to placebo controls. In mice, the vaccine provided 100% protection against homologous and heterologous influenza virus challenges. In macaques, the two-dose intranasal immunization was well-tolerated and induced significant systemic IgG and mucosal sIgA responses, alongside robust N-specific IFNγ+ T-cell activation. Conclusions: Corfluvec is a promising bivalent vaccine candidate that provides dual protection against influenza and COVID-19. Its ability to limit viral shedding from the upper respiratory tract and to mitigate SARS-CoV-2-induced pulmonary pathology, contributing to the preservation of lung vascular integrity, underscores the utility of mucosal immunization with Corfluvec as a valuable intranasal complement to current systemic vaccination strategies.