We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (ΔNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a “self-adjuvant effect”, in turn increasing vaccine immunogenicity. As a result, ΔNS1 viruses elicit strong B- and T- cell mediated immune responses. We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5∶3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log₁₀ TCID_50/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains. Our findings show that intranasal immunization with the replication deficient H5N1 ΔNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of ΔNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.

New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1–124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.

Tuberculosis is a major global threat to human health. Since the widely used BCG vaccine is poorly effective in adults, there is a demand for the development of a new type of boost tuberculosis vaccine. We designed a novel intranasal tuberculosis vaccine candidate, TB/FLU-04L, which is based on an attenuated influenza A virus vector encoding two mycobacterium antigens, Ag85A and ESAT-6. As tuberculosis is an airborne disease, the ability to induce mucosal immunity is one of the potential advantages of influenza vectors. Sequences of ESAT-6 and Ag85A antigens were inserted into the NS1 open reading frame of the influenza A virus to replace the deleted carboxyl part of the NS1 protein. The vector expressing chimeric NS1 protein appeared to be genetically stable and replication-deficient in mice and non-human primates. Intranasal immunization of C57BL/6 mice or cynomolgus macaques with the TB/FLU-04L vaccine candidate induced Mtb-specific Th1 immune response. Single TB/FLU-04L immunization in mice showed commensurate levels of protection in comparison to BCG and significantly increased the protective effect of BCG when applied in a “prime-boost” scheme. Our findings show that intranasal immunization with the TB/FLU-04L vaccine, which carries two mycobacterium antigens, is safe, and induces a protective immune response against virulent M. tuberculosis.

Influenza viruses with an impaired NS1 protein are unable to antagonize the innate immune system and, therefore, are highly immunogenic because of the self-adjuvating effect. Hence, NS1-mutated viruses are considered promising candidates for the development of live-attenuated influenza vaccines and viral vectors for intranasal administration. We investigated whether the immunogenic advantage of the virus expressing only the N-terminal half of the NS1 protein (124 a.a.) can be translated into the induction of protective immunity against a heterologous influenza virus in mice. We found that immunization with either the wild-type A/PR/8/34 (H1N1) influenza strain (A/PR8/NSfull) or its NS1-shortened counterpart (A/PR8/NS124) did not prevent the viral replication in the lungs after the challenge with the A/Aichi/2/68 (H3N2) virus. However, mice immunized with the NS1-shortened virus were better protected from lethality after the challenge with the heterologous virus. Besides showing the enhanced influenza-specific CD8⁺ T-cellular response in the lungs, immunization with the A/PR8/NS124 virus resulted in reduced concentrations of proinflammatory cytokines and the lower extent of leukocyte infiltration in the lungs after the challenge compared to A/PR8/NSfull or the control group. The data show that intranasal immunization with the NS1-truncated virus may better induce not only effector T-cells but also certain immunoregulatory mechanisms, reducing the severity of the innate immune response after the heterologous challenge.

Contemporary influenza B virus strains were generated encoding C-terminally truncated NS1 proteins. Viable viruses containing the N-terminal 14, 38, 57 or 80 aa of the NS1 protein were rescued in Vero cells. The influenza B virus NS1-truncated mutants were impaired in their ability to counteract interferon (IFN) production, induce antiviral pro-inflammatory cytokines early after infection and show attenuated or restricted growth in IFN-competent hosts. In Vero cells, all of the mutant viruses replicated to high titres comparable to the wild-type influenza B virus. Mice that received a single, intranasal immunization of the NS1-truncated mutants elicited an antibody response and protection against wild-type virus challenge. Therefore, these NS1-truncated mutants should prove useful as potential candidates for live-attenuated influenza virus vaccines.