The COVID-19 pandemic, caused by SARS-CoV-2, has had a devastating global impact. While existing vaccines have significantly reduced severe disease and mortality, the emergence of highly transmissible and immune-evasive variants necessitates the development of next-generation vaccines. These new vaccines should ideally induce robust mucosal immunity in the upper respiratory tract to prevent or minimize infection. Intranasal vaccines offer a promising approach because they can stimulate local immunity at the site of initial viral entry. The high transmission rates and immune evasion capabilities of variants such as Omicron continue to pose challenges, leading to hospitalizations and deaths, especially among vulnerable populations. Current intramuscular vaccines induce high serum antibody and circulating T cell responses, but the extent of upper respiratory tract immunity remains less clear. Breakthrough infections and repeated infections suggest a need for alternative approaches that enhance upper respiratory tract immunity. A key focus for next-generation vaccines is enhancing immunity in the upper respiratory tract to reduce SARS-CoV-2 circulation. Intranasal administration has shown advantages in preclinical studies for inducing robust local immunity against viral pathogens. Given the likelihood of continued co-circulation of SARS-CoV-2 and seasonal influenza, a dual-function vaccine could improve vaccine acceptance and cost-effectiveness. This study builds upon previous research on live-attenuated influenza viruses (LAIVs) with a deleted NS1 gene, demonstrating their potential for cross-protection against SARS-CoV-2. Previous studies have shown promising results with DeINS1-RBD and DeINS1-RBD-OPT1, prompting this investigation into an improved version, DelNS1-RBD4N-DAF, tested against current variants in animal models.

The literature review extensively cites previous research on the development and efficacy of various COVID-19 vaccines, highlighting the limitations of existing intramuscular vaccines in preventing infection and the need for mucosal immunity. Several studies are referenced demonstrating the potential benefits of intranasal vaccines in inducing robust local immunity against viral pathogens. Previous work by the authors on live-attenuated influenza viruses (LAIVs) with deleted NS1 genes is also detailed, emphasizing their potential for cross-protection and the safety profile of a related vaccine candidate in human clinical trials. The review further discusses the challenges posed by the emergence of highly transmissible SARS-CoV-2 variants and the need for vaccines that can provide broader protection against these variants. The role of T cell responses and mucosal IgA in providing protective immunity is also highlighted.

The study involved the development and testing of a novel intranasal vaccine candidate, DelNS1-RBD4N-DAF, based on a live-attenuated influenza virus (LAIV) with a deleted NS1 gene and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The vaccine was engineered to enhance RBD surface expression and target specific N-glycosylation sites to optimize immunogenicity. The immunogenicity of DelNS1-RBD4N-DAF was evaluated in mice and hamsters using a prime-boost immunization regimen. Various assays were used to assess immune responses, including ELISA for measuring antibody titers, neutralization assays (using both pseudoviruses and live viruses) to evaluate neutralizing antibody activity, and flow cytometry to assess T cell responses. Mice were immunized intranasally with DelNS1-RBD4N-DAF encoding different RBD variants (Beta, Delta, Omicron BA.1) and compared to intramuscular administration of the BNT162b2 mRNA vaccine in hamsters. Following immunization, animals were challenged with mouse-adapted or live SARS-CoV-2 variants, and viral replication in respiratory tissues was assessed. Histopathological examinations were also performed to assess tissue damage. Detailed procedures are described for the construction and generation of the LAIV vaccines, including gene modifications and virus rescue. Methods for ELISA, virus neutralization assays (using both pseudoviruses and live viruses), and flow cytometry for T cell analysis are meticulously explained. The generation of mouse-adapted SARS-CoV-2 viruses for the mouse challenge experiments is also detailed, including the methods used for passaging the viruses and determining their titers. The study also includes rigorous statistical analysis of the data obtained using methods such as one-way ANOVA followed by Dunnett's multiple comparisons test.

The study's key findings demonstrate that the intranasal DelNS1-RBD4N-DAF LAIV vaccine effectively induced high levels of neutralizing antibodies against various SARS-CoV-2 variants in both mice and hamsters. Importantly, this vaccine stimulated robust T cell responses, including both CD4+ and CD8+ T cells, in mice. The intranasal administration of DelNS1-RBD4N-DAF LAIV significantly prevented the replication of SARS-CoV-2 variants, including Delta and Omicron BA.2, in the respiratory tissues of both mice and hamsters. In contrast, the intramuscular BNT162b2 mRNA vaccine did not provide this level of protection in the respiratory tract. The inclusion of DAF in the vaccine significantly enhanced the levels of anti-RBD antibodies and neutralizing activity compared to the vaccine without DAF. Targeting N-glycosylation sites in the RBD further enhanced the immunogenicity of the vaccine. The study also demonstrated that the DelNS1-RBD4N-DAF LAIV induced mucosal IgA responses in mice. These results suggest that this intranasal vaccine candidate could offer significant advantages over currently available intramuscular vaccines in preventing SARS-CoV-2 infection and transmission. Further, it shows strong potential as a dual-function vaccine to protect against both influenza and SARS-CoV-2. The study also demonstrated protection against a mouse-adapted Omicron BA.1 variant in mice and the Omicron BA.2 variant in hamsters. In hamsters, even a single intranasal booster dose of Omni-DAF with a two-dose BNT162b2 mRNA regimen significantly reduced viral titers in the lungs and nasal turbinates. Finally, the study showed that the vaccine retains the ability to induce immunity to influenza components, making it a potential dual-function vaccine.

The findings of this study strongly support the potential of intranasal DelNS1-RBD4N-DAF LAIV as a highly effective vaccine against SARS-CoV-2. The ability of this vaccine to prevent viral replication in the respiratory tissues of animals, unlike the intramuscular mRNA vaccine, is a significant advance. The induction of both humoral and cellular immune responses, coupled with the observed mucosal IgA production, suggests a mechanism for potent protection against infection and transmission. The vaccine’s effectiveness against multiple SARS-CoV-2 variants, including Omicron, is crucial given the ongoing evolution of the virus. The potential of DelNS1-RBD4N-DAF as a dual-function vaccine against both influenza and SARS-CoV-2 is a key contribution, potentially offering a simplified and more cost-effective vaccination strategy. These results have significant implications for public health strategies to control SARS-CoV-2 and influenza, potentially reducing transmission and preventing severe disease.

This study successfully demonstrates the potential of an intranasal, live-attenuated influenza virus-vectored vaccine (DelNS1-RBD4N-DAF) to prevent SARS-CoV-2 replication in respiratory tissues of animal models, offering superior protection compared to an intramuscular mRNA vaccine. The vaccine's effectiveness against multiple variants, along with its potential for dual-functionality against influenza and SARS-CoV-2, makes it a promising candidate for further clinical development. Future research should focus on human clinical trials to assess the safety and efficacy of this vaccine in preventing SARS-CoV-2 infection and transmission, potentially as a stand-alone or booster vaccine.

The study was conducted in animal models (mice and hamsters), and the findings may not fully translate to human responses. The mouse-adapted SARS-CoV-2 strains used might exhibit different characteristics compared to the naturally occurring human strains. The sample sizes in some experiments are relatively small, which could affect the statistical power of certain findings. The long-term efficacy and durability of the immune response elicited by the vaccine are not yet known and require further investigation.