Immunization with DNA prime-subunit protein boost strategy based on influenza H9N2 virus conserved matrix protein M1 and its epitope screening

Abstract

Developing an effective universal influenza vaccine against influenza virus with highly conserved antigenic epitopes could induce a broad-spectrum immune response to prevent infection. The soluble protein M1 that can induce the M1 specific immune response was first confirmed in our previous study. In this study, we characterized the immune response induced by DNA prime-subunit protein boost strategy based on the relatively conserved matrix protein 1 (M1) in the BALB/c mouse model, and evaluated its protection ability against a lethal challenge of homologous H9N2 avian influenza virus (A/Chicken/Jiangsu/11/2002). The results showed that 100 μg DNA prime + 100 μg M1 subunit protein boost-strategy significantly increased antibody levels more than vaccination with M1 DNA or M1 subunit protein alone, and induced a more balanced Th1 / Th2 immune response, which not only can provide protection against the homologous virus but also can provide part of the cross-protection against the heterosubtypic PR8 H1N1 strain. In addition, we used an Elispot assay to preliminary screen the T cell epitope in M1 protein, and identified that p22 (M1_11-25 VLSIIPSGPLKAEIA) epitope was the only immunodominant M1-specific CD4⁺ T cell epitopes, which could be helpful in understanding the function of influenza virus T cell epitopes.

Figure 1

Survival rates (A) and body weight changes (B) after a challenge with lethal homologous influenza virus. Mice were immunized as described above. Two weeks post-vaccination, mice were challenged with a lethal dose (20 × LD₅₀) of the mouse-adapted A/Chicken/Jiangsu/7/2002 (H9N2) virus. The survival rates and body weights of the mice were measured daily from the date of the challenge to 21 days after the challenge. Values represent means ± SD of each group of mice.

Figure 2

The expression of cytokines in the lungs. Mice were immunized as described above. Two weeks after the last administration, mice were challenged with a lethal dose (20 × LD₅₀) of the mouse-adapted A/Chicken/Jiangsu/7/2002 (H9N2) virus. The BALFs were collected and the selected cytokines were quantitatively analyzed. Panels A, B and C represent data obtained from measurements of IFN-γ, IL-2, and IL-4, respectively. *Significant differences compared to the corresponding M1 protein immunization group alone (p < 0.05).

Figure 3

Mice primed with 100 μg DNA-boosted with 100 μg M1 protein generated more M1 specific IFN-γ producing T cells compared with mice immunized alone. Splenocytes harvested from mice 14 days after vaccination were stimulated with 50 μg/ml M1 pools for 22-24 h and scored in ELISPOT assays for IFN-γ producing cells. The values represent the averages of quadruplicate wells of 3 mice, and are expressed as means ± SD. The results were expressed as the number of SFC per 10⁶ input cells. *Significant differences between the treatment groups and the control group (p < 0.05).

Figure 4

Survival rates (A) and body weight changes (B) after challenge with lethal heterosubtypic influenza virus. Mice were immunized as described above. Two weeks after the last immunization, mice were challenged with a lethal dose (5 × LD₅₀) of mouse-adapted heterosubtypic PR8(H1N1) influenza A virus. The survival rates and body weights of the mice were measured daily from the date of the challenge to 21 days after the challenge. Values represent means ± SD of each group of mice.

Figure 5

T cell epitope screening of peptide library spanning the entire M1 protein of H9N2 avian influenza virus. (A) A library of overlapping peptides spanning the entire M1 protein divided into ten pools of 5 peptides each according to a 5 × 5 matrix. 25 peptides were randomly named with number 1 to 25 and listed according to the matrix above. Peptides in the same line or column mixed as one pool, respectively. Then each pool as a stimulus for ELISPOT assay. (B) Peptide pool detection. Two weeks after the DNA primary - 100 μg M1 intranasal boost immune strategy, the spleen lymphocytes were isolated, 2 × 10⁵ responding cells were incubated with each pool (50μg/ml) for 20-24 hrs in 96-well PVDF plates coated with anti-IFN-γ monoclonal antibody, then calculated the specific spots. The spots >10 were as the positive peptide pools. The values represent the averages of quadruplicate wells of 3 mice, and are expressed as means ± SD. The results were expressed as the number of SFC per 10⁶ input cells.

Figure 6

Peptide activity of M1 T cell epitope of H9N2 avian influenza virus. Two weeks after the DNA primary - 100 μg M1 intranasal boost immune strategy, the spleen lymphocytes were isolated, 2 × 10⁵ responding cells were incubated in 96-well PVDF plates coated with anti-IFN-γ monoclonal antibody. According to a 5 × 5 matrix, the single peptide of the intersection of two positive peptide pools may be the M1 specific T cell epitope peptide, the preliminarily determined positive single peptides were respectively used as ELISPOT reaction stimulus (50 μg/ml peptide), then to calculate the specific spots after 20-24 hrs. The peptide with spots >10 was considered positive peptides. The values represent the averages of quadruplicate wells of 3 mice, and are expressed as means ± SD. The results were expressed as the number of SFC per 10⁶ input cells.

Figure 7

CD4⁺T/CD8⁺T cell depletion ELISPOT assay. (1) CD4⁺T/CD8⁺T cell depletion. Two weeks after the DNA primary - 100 μg M1 intranasal boost immune strategy, the spleen lymphocytes were isolated and CD8 ⁺ T and CD4 ⁺ T cells were removed respectively by using MACs microbeads. (A) before the mixed lymphocyte sorting; (B) after removing the CD8⁺ T cells; (C) after removing the CD4⁺ T cells. (2) Collecting the MACs positive sorting cells. 2 × 10⁵ responding cells were incubated in 96-well PVDF plates coated with anti-IFN-γ monoclonal antibodies, the single peptide p22 was used to stimulate three kinds of different cells respectively, and the weakly positive single peptide p20 and p24 were used as the control (50 μg/ml peptide). (D) left-slash representing the whole spleen lymphocytes, cross hatch representing the spleen lymphocytes removing CD4⁺ T cells, right-slash representing the spleen lymphocytes removing CD8⁺ T cells. The values represent the averages of quadruplicate wells of 3 mice, and are expressed as means ± SD. The results were expressed as the number of SFC per 10⁶ input cells. *Significant differences compared to the mice in the whole splenocytes group (p < 0.05).