An Influenza A Vaccine Based on the Extracellular Domain of Matrix 2 Protein Protects BALB/C Mice Against H1N1 and H3N2

Abstract

Current seasonal influenza A virus (IAV) vaccines are strain-specific and require annual reconstitution to accommodate the viral mutations. Mismatches between the vaccines and circulating strains often lead to high morbidity. Hence, development of a universal influenza A vaccine targeting all IAV strains is urgently needed. In the present study, the protective efficacy and immune responses induced by the extracellular domain of Matrix 2 protein (M2e) displayed on the virus-like particles of Macrobrachium rosenbergii nodavirus (NvC-M2ex3) were investigated in BALB/c mice. NvC-M2ex3 was demonstrated to be highly immunogenic even in the absence of adjuvants. Higher anti-M2e antibody titers corresponded well with increased survival, reduced immunopathology, and morbidity of the infected BALB/c mice. The mice immunized with NvC-M2ex3 exhibited lower H1N1 and H3N2 virus replication in the respiratory tract and the vaccine activated the production of different antiviral cytokines when they were challenged with H1N1 and H3N2. Collectively, these results suggest that NvC-M2ex3 could be a potential universal influenza A vaccine.

Keywords: H1N1; H3N2; Macrobrachium rosenbergii nodavirus; extracellular domain of matrix 2 protein; universal influenza A vaccine; virus-like particle.

Figure 1

Immunogenicity of the chimeric protein in BALB/c mice. Serum samples of BALB/c mice immunized with NvC or NvC-M2ex3 were collected at three weeks after the primary injection, three weeks after the first booster, and one week after the second booster by submandibular bleeding. Serum samples (1:2000 dilution) from mice (n = 8) of respective groups were pooled and analyzed by enzyme-linked immunosorbent assay (ELISA). The microtiter plate wells were coated with the M2e synthetic peptide and the anti-M2e antibody reacted with the peptide was detected with the anti-mouse antibody conjugated to alkaline phosphatase. a, b, and c indicate statistical significance (p < 0.001) of the results within each time point and when compared to other time points.

Figure 2

Mortality and morbidity of BALB/c mice after lethal influenza A virus infections. Survival rate of mice infected with (A) H1N1 and (B) H3N2. Solid and dashed lines indicate the survival rates of the mice immunized with NvC and NvC-M2ex3, respectively. Percentage of body weight of mice infected with (C) H1N1 and (D) H3N2. Dashed lines indicate endpoint of the animals. Morbidity of mice infected with (E) H1N1 and (F) H3N2. A complete protection against H1N1 or H3N2 infection was observed in the mice immunized with NvC-M2ex3. After infection with mouse-adapted H3N2, the mice vaccinated with NvC-M2ex3 did not demonstrate significant weight loss and morbidity. On the contrary, all mice immunized with NvC succumbed to the infections. (●) and (■) indicate the percentage of body weight and morbidity of the mice immunized with NvC and NvC-M2ex3, respectively. The asterisks (* p < 0.01, ** p < 0.001, *** p < 0.0001) indicate statistical significance of the results between different treatment groups.

Figure 3

Viral RNA copy numbers in the lungs and oropharynges of infected mice. At day five post-infection, the lungs and oropharyngeal swabs of the infected mice (n = 3) were obtained. The viral RNA was then extracted, reverse transcribed, and quantified using quantitative polymerase chain reaction (qPCR). (A) Higher viral RNA copy numbers (10-fold) were detected in the lungs of the mice immunized with NvC relative to those immunized with NvC-M2ex3 after being challenged with H1N1 or H3N2. These results demonstrate the potential of NvC-M2ex3 in limiting virus replication. (B) Detection of the viral RNA of H1N1 or H3N2 in the oropharynges of the mice suggests viral shedding and possible transmission between animals. All the NvC-M2ex3-immunized mice exhibited a reduced viral shedding in both lethal H1N1 and H3N2 infections. The differences between treatment groups are significant with p < 0.05.

Figure 4

Concentration of cytokines in the lungs of mice (n = 3) infected by H1N1 and H3N2. The mice vaccinated with NvC-M2ex3 demonstrated different cytokine profiles after being challenged with H1N1 and H3N2. (A) A higher level of IFN-γ and IL-12 was detected in the lungs of the mice immunized with NvC-M2ex3 compared to the control group after being challenged with H1N1. (B) On the contrary, when the mice were challenged with H3N2, they exhibited lower concentrations of IFN-γ and IL-6, although a higher level of IL-12 was observed. The asterisks (* p < 0.05, ** p < 0.01,) indicate statistical significance of the results between different treatment groups.

Figure 5

Hematoxylin and Eosin (H and E) staining of the lungs of mice (n = 3) immunized with the chimeric proteins and challenged with influenza A virus H1N1 or H3N2. (A) The lungs of mice immunized with (i) NvC-M2ex3 or (ii) NvC at day five post-infection with H1N1. (B) The lungs of mice immunized with (i) NvC-M2ex3 or (ii) NvC at day five post-infection with H3N2. Insets with letters (a, b, c) are projected at 400x magnification as (a), (b), and (c). Dashed and solid arrows indicate bronchiolitis and interstitial inflammation respectively, and triangles indicate edema, n = 3.

Figure 6

Lung inflammatory scores of the mice (n = 3) challenged with influenza A virus. A lower lung inflammatory score was observed in the mice immunized with NvC-M2ex3 after being challenged with (A) H1N1 or (B) H3N2. All mice immunized with NvC experienced a higher lung inflammatory score which corresponded well with increased morbidity and reduced survival. The differences between the treatment groups are significant with p < 0.01, n = 3.