Induction of cross-protection against influenza A virus by DNA prime-intranasal protein boost strategy based on nucleoprotein

Abstract

Background: The highly conserved nucleoprotein (NP) is an internal protein of influenza virus and is capable of inducing cross-protective immunity against different influenza A viruses, making it a main target of universal influenza vaccine. In current study, we characterized the immune response induced by DNA prime-intranasal protein boost strategy based on NP (A/PR/8/34, H1N1) in mouse model, and evaluated its protection ability against a lethal dose challenge of influenza virus.

Results: The intranasal boost with recombinant NP (rNP) protein could effectively enhance the pre-immune response induced by the NP DNA vaccine in mice. Compared to the vaccination with NP DNA or rNP protein alone, the prime-boost strategy increased the level of NP specific serum antibody, enhanced the T cell immune response, and relatively induced more mucosal IgA antibody. The overall immune response induced by this heterologous prime-boost regimen was Th-1-biased. Furthermore, the immune response in mice induced by this strategy provided not only protection against the homologous virus but also cross-protection against a heterosubtypic H9N2 strain.

Conclusions: The NP DNA prime-intranasal protein boost strategy may provide an effective strategy for universal influenza vaccine development.

Figure 1

Survival rates (A) and body weight changes (B) after challenge with lethal homologous influenza virus. One hundred and fourteen mice were randomly divided into six groups. Group D1 received one dose of 100 μg NP DNA vaccine; group P1 received one dose of 50 μg rNP vaccine; group D2 received two doses of 100 μg NP DNA vaccine; group D1P1 received one dose of 100 μg NP DNA vaccine followed by one dose of 50 μg rNP; group D2P1 received two doses of NP DNA vaccine followed by one dose of rNP vaccine. 3 weeks after the last immunization, mice were challenged with a lethal dose (5×LD₅₀) of influenza PR8 virus. Survival rates (A) and body weight loss (B) were monitored for 21 days.

Figure 2

The numbers of INF-γ secreting splenic CD8⁺T cells in vaccinated mice. Mice were immunized as described in method section. 3 weeks after the last immunization, the number of IFN-γ secreting CD8⁺ T cells in the spleen from the different groups of mice was evaluated by ELISpot. The results represent the averages of triplicate wells of three mice, and are expressed as means ± SD, * Significant difference (P<0.05).

Figure 3

The numbers of IFN-γ / IL-4 secreting splenic CD4⁺T cells in vaccinated mice. Mice were immunized as described in method section. 3 weeks after the last immunization, the number of IFN-γ /IL-4 secreting CD4⁺ T cells in the spleen from the different groups of mice was evaluated by ELISpot. The results represent the averages of triplicate wells of three mice, and are expressed as means ± SD.

Figure 4

Survival rates (A) and body weight changes (B) after challenge with lethal heterologous influenza virus. Mice were immunized as described in method section. 3 weeks after the last immunization, mice were challenged with a lethal dose (5×LD₅₀) of influenza A/Chicken/JiangSu/07/2002(H9N2). Survival rate (A) and body weight loss (B) were monitored for 21 days.