Live Attenuated Influenza Virus Expressing Human Interleukin-2 Reveals Increased Immunogenic Potential in Young and Aged Hosts

Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinant ca parent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8(+) T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8(+) T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8(+) T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups

Immunogenicity and Protection Efficacy of Replication-Deficient Influenza A Viruses with Altered NS1 Genes

We explored the immunogenic properties of influenza A viruses with altered NS1 genes (NS1 mutant viruses). NS1 mutant viruses expressing NS1 proteins with an impaired RNA-binding function or insertion of a longer foreign sequence did not replicate in murine lungs but still were capable of inducing a Th1-type immune response resulting in significant titers of virus-specific serum and mucosal immunoglobulin G2 (IgG2) and IgA, but with lower titers of IgG1. In contrast, replicating viruses elicited high titers of serum and mucosal IgG1 but less serum IgA. Replication-deficient NS1 mutant viruses induced a rapid local release of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6. Moreover, these viruses also elicited markedly higher levels of IFN-α/β in serum than the wild-type virus. Comparable numbers of virus-specific primary CD8(+) T cells were determined in all of the groups of immunized mice. The most rapid onset of the recall CD8(+)-T-cell response upon the wild-type virus challenge was detected in mice primed with NS1 mutant viruses eliciting high levels of cytokines. It is noteworthy that there was one NS1 mutant virus encoding NS1 protein with a deletion of 40 amino acids predominantly in the RNA-binding domain that induced the highest levels of IFN-α/β, IL-6 and IL-1β after infection. Mice that were immunized with this virus were completely protected from the challenge infection. These findings indicate that a targeted modification of the RNA-binding domain of the NS1 protein is a valuable technique to generate replication-deficient, but immunogenic influenza virus vaccines