J Vet Sci.  2016 Mar;17(1):71-78. 10.4142/jvs.2016.17.1.71.

A novel M2e-multiple antigenic peptide providing heterologous protection in mice

• Wen F
• Ma JH
• Yu H
• Yang FR
• Huang M
• Zhou YJ
• Li ZJ
• Wang XH
• Li GX
• Jiang YF
• Tong W
• Tong GZ

• KMID: 2363337
• DOI: http://doi.org/10.4142/jvs.2016.17.1.71

Abstract

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.

Keyword

H3N2; M2e-multiple antigenic peptide; high-yield; inactivated vaccine; swine influenza virus

MeSH Terms

Animals
Antibodies, Viral/blood
Antigens, Viral/genetics/*immunology
Body Weight
Cross Protection/*immunology
Disease Models, Animal
Epitopes, T-Lymphocyte/genetics/immunology
Female
Influenza A Virus, H3N2 Subtype/genetics/*immunology
Influenza Vaccines/*immunology
Mice
Mice, Inbred BALB C
Orthomyxoviridae Infections/*immunology/mortality/pathology/prevention & control
Peptides/genetics/*immunology
Random Allocation
Survival Analysis
Vaccines, Synthetic/immunology
Virus Replication
Antibodies, Viral
Antigens, Viral
Epitopes, T-Lymphocyte
Influenza Vaccines
Peptides
Vaccines, Synthetic

Figure

• 

  Fig. 1 Structure and sequence of synthetic M2e-multiple antigenic peptide (M2e-MAP).

• 

  Fig. 2 Antibody response induced by M2e-MAP combined vaccine. M2e-specific antibody and rgH3N2-specific antibody titers were determined by end-point ELISA. Mice were vaccinated with M2e-MAP+rgH3N2+Freund, rgH3N2+Freund and rgH3N2 subcutaneously. Mice receiving Freund were used as negative controls. Sera were collected at 1, 2, 3 and 4 weeks after the first immunization. The titers were expressed as the highest serum dilution greater than twice the average absorbance value at OD450 nm of pre-vaccination sera. The data are expressed as the geometric mean titer ± standard deviation (SD) of 5 mice per group. The lower limit of detection (1 : 20 in A and 1 : 50 in B) is indicated by a dotted line. Experiments were repeated three times.

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  Fig. 3 Viral amounts in lungs on 3 dpi. Five mice in every challenge group were euthanized on 3 dpi and the viral amounts of lungs were determined by real time PCR. The values were expressed as the mean log10 viral copies/µL ± SD of 5 mice per challenge group. *Means in M2e-MAP+rgH3N2+Freund immunized and PR8 challenged group p < 0.001 compared to the other 4 immunized groups. **Means in M2e-MAP+rgH3N2 +Freund immunized and SwGD96 challenged group p < 0.001 compared to the remaining 4 groups. ***Indicates p < 0.001 for M2e-MAP+rgH3N2+Freund immunized and SwHLJ1 challenged group compared to the other 4 groups.

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  Fig. 4 Histopathological changes in the lungs of virus challenged mice. Immunized mice were challenged by PR8 (A, D, G, and J), SwGD96 (B, E, H, and K) and SwHLJ1 (C, F, I, and L) and lungs were collected for histopathological analysis on 3 dpi. The figure indicates the representative imagines of histopathological observations of M2e-MAP+rgH3N2+Freund, rgH3N2+Freund, rgH3N2 and Freund, respectively. H&E stain. Scale bars = 50 µm.

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  Fig. 5 Survival and body weight curve in PR8 challenged mice. Mice were challenged with 10LD50 of PR8 virus intranasally and monitored daily for 2 weeks post challenge. (A) Survival rate. The difference among the four groups is significant (p = 0.0002). (B) Percentage of mouse body weight. Each point represents the mean of 5 mice per group.

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