Influenza viruses are deadly respiratory pathogens of special importance due to their long history of global pandemics. During influenza virus infections, the host responds by producing interferons, which activate interferon-stimulated genes (ISGs) inside target cells. One of these ISGs is inducible nitric oxide synthase (iNOS). iNOS produces nitric oxide (NO) from arginine and molecular oxygen inside the cell. NO can react with superoxide radicals to form reactive nitrogen species, principally peroxynitrite. While much work has been done studying the many roles of nitric oxide in influenza virus infections, the direct effect of peroxynitrite on influenza virus proteins has not been determined. Manipulations of NO, either by knocking out iNOS or chemically inhibiting NO, produced no change in virus titers in mouse models of influenza infection. However, peroxynitrite has a known antimicrobial effect on various bacteria and parasites, and the reason for its lack of antimicrobial effect on influenza virus titers in vivo remains unclear. Therefore, we wished to test the direct effect of nitration of influenza virus proteins. We examined the impact of nitration on virus infectivity, replication, and immunogenicity. We observed that the nitration of influenza A virus proteins decreased virus infectivity and replication ex vivo. We also determined that the nitration of influenza virus hemagglutinin protein can reduce antibody responses to native virus protein. However, our study also suggests that nitration of influenza virus proteins in vivo is likely not extensive enough to inhibit virus functions substantially. These findings will help clarify the role of peroxynitrite during influenza virus infections. IMPORTANCE Nitric oxide and peroxynitrite produced during microbial infections have diverse and seemingly paradoxical functions. While nitration of lung tissue during influenza virus infection has been observed in both mice and humans, the direct effect of protein nitration on influenza viruses has remained elusive. We addressed the impact of nitration of influenza virus proteins on virus infectivity, replication, and immunogenicity. We observed that ex vivo nitration of influenza virus proteins reduced virus infectivity and immunogenicity. However, we did not detect nitration of influenza virus hemagglutinin protein in vivo. These results contribute to our understanding of the roles of nitric oxide and peroxynitrite in influenza virus infections.

In pandemic scenarios involving novel human pathogenic viruses, it is highly desirable that vaccines induce strong neutralizing antibodies as quickly as possible. However, current vaccine strategies require multiple immunization doses to produce high titers of neutralizing antibodies and are poorly protective after a single vaccination. We therefore wished to design a vaccine candidate that would induce increased protective immune responses following the first vaccine dose. We hypothesized that antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein could be increased by drawing upon immunity to a previous infection. We generated a fusion protein containing the influenza H1N1 PR8 virus nucleoprotein (NP) and the SARS-CoV-2 spike RBD. Mice with or without preexisting immunity to PR8 were then vaccinated with NP/RBD. We observed significantly increased SARS-CoV-2 neutralizing antibodies in mice with PR8 immunity compared to mice without preexisting PR8 immunity. Vaccination with NP/RBD protected mice from SARS-CoV-2-induced morbidity and mortality after a single dose. Additionally, we compared SARS-CoV-2 virus titers in the lungs and nasal turbinates 4 days post-challenge of mice vaccinated with NP/RBD. SARS-CoV-2 virus was detectable in the lungs and nasal turbinate of mice without preexisting PR8 immunity, while SARS-CoV-2 virus was completely undetectable in mice with preexisting PR8 immunity. We also found that CD4-positive T cells in mice with preexisting immunity to PR8 play an essential role in producing the increased antibody response against RBD. This vaccine strategy potentially can be modified to target other pathogens of concern and offers extra value in future pandemic scenarios.IMPORTANCEIncreased globalization and changes in human interactions with wild animals has increased the likelihood of the emergence of novel viruses with pandemic potential. Vaccines can be effective in preventing severe disease caused by pandemic viruses. However, it takes time to develop protective immunity via prime-boost vaccination. More effective vaccine designs should quickly induce protective immunity. We propose leveraging preexisting immunity to a different pathogen to boost protection against emerging viruses. We targeted SARS-CoV-2 as a representative pandemic virus and generated a fusion protein vaccine that combines the nucleoprotein from influenza A virus and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Our vaccine design significantly increased the production of RBD-specific antibodies in mice that had previously been exposed to influenza virus, compared to those without previous exposure. This enhanced immunity reduced SARS-CoV-2 replication in mice. Our results offer a vaccine design that could be valuable in a future pandemic setting.