Protective Immunorespone Agaist Influenza Virus Induced By Immunization With M₂e Gene

ObjectivesInfluenza virus is one of the most important respiratory pathogens in the worldwide. It has been confirmed that the pathogen was the influenza A virus for the worldwide pandemics in three times. It was proved that immunization was the most effective measure in preventing pathogens infection. Current influenza vaccines mainly aimed at inducing neutralizing antibodies against one of surface glycoproteins, hemagglutinin (HA). However, HA was easily happen antigenic drift or shift (10^-3 substitutions per nucleotide per year), which made the influenza virus easy to escape immune system surveillance. As a result, it was necessary to inject the influenza vaccines with updated information each year to prevent influenza epidemic in humans as suggested by the World Health Organization. As we all know, no commercial human influenza vaccine with broad-spectrum protective activities has been developed so far.Recently, some researchs demonstrated that the M₂e (the extracellular domain of M₂ protein,M₂e) of influenza virus was highly conserved.M₂e sequences from 716 influenza A viruse strains were aligned and compared according to host restriction specificities in GenBank. The results indicated that there was no amino acid change among the first 9 N-terminal amino acid (aa) of M₂e. In the other hand, one region (aa 10-20) of M₂e protein had higher varation rates than other regions on M₂e protein particularly. But we should also notice that there was one evolutionarily constant residue, W (aa 15), in the middle of this high varation sequence. EVETPIRN peptide (aa 6-13), one of the epitope sequence of the influenza A virus M₂ protein, could induce specific antibodies, which can reduce the the replication level of influenza A virus in the lungs of infected mice. M₂ protein, containing the highly conservative epitope (EVETPIRN. aa 6-13) in influenza A virus, was immunogenic and conferred immune protection in mice. All the evidences suggested that the M₂e protein could be one attractive immune target for a broad spectrum influenza vaccine design.Above all, in order to study the broad spectrum influenza vaccine providing cross-protection against different influenza variants, we constructed an eukaryotic expressing vector pcDNA3.1(+)-M₂e containing M₂e gene from influenza virus and designed it as a DNA vaccine, and observed the M₂e-specific immune respone induced by the DNA vaccine and its protection against influenza virus challenge.MethodsTwo oligonucleotides were designed and synthesized according to the M₂e DNA sequence in GenBank. The two oligonucleotides were annealed and formed the doubble strands DNA, the M₂e gene. After that, the M₂e gene was inserted into the eukaryotic expressing vector pcDNA3.1(+) for constructing the recombinant plasmid pcDNA3.1(+)-M₂e. The recombinant plasmid pcDNA3.1(+)-M₂e were identified by restriction enzymes digestion and sequencing analysis. Afterwards, the recombinant plasmid was transfcted into HEK293 cells with Lipofectamine^TM 2000 Transfection Reagent. The transfected HEK293 cells were harvested after the selecting by 400μg/ml G418 for about 10 days. The M₂e protein expressed in tranfected cell was investigated by Immunoflourescence. Whereafter, the pcDNA3.1(+)-M₂e in E.coli JM109 was amplified, and extracted by through hydroxybenzenechloroform. The plasmid was identified by restriction enzymes digestion again. After that, BALB/c mice were immunized intramuscularly with 100μg plasmids DNA three times at two weeks intervals. Seven days after the first immunization and boosting immunization, the sera of the mice were collected. Then, anti-M₂e antibody and cell mediated immune respose were checked. Anti-M₂e antibody elicited in the mice immunized with pcDNA3.1(+)-M₂e were tested by ELISA. The cell mediated immunity was tested by lymphocytes multiplication. The immuned BALB/c mice were challenged by 10×LD⁵⁰ influenza virus (A/PR/8/34, H1N1) one week after last immunization.ResultsThe doubble strands DNA, the M₂e gene, were identified by 2.5% agrose electrorhoresis, and the result was just as expected. Restrictive enzyme identification demonstrated that the M₂e gene was inserted into pcDNA3.1(+). The right sequences and ORF were confirmed by DNA sequencing analysis. After the selecting by 400μg/ml G418 for about 10 days, we gained the stable tranfected HEK293 cell. We could see the fluorescence on the transfected cell membrane under the fluorescence microscope. Using the culture supernatants as the stimulants, the lymphocytes multiplication stimulanted with M₂e was higher than that of control. The difference among them were distinguished by LSD analysis of variance (P＜0.05). The amplified plasmid pcDNA3.1(+)-M₂e extrated by hydroxybenzene-chloroform was correct through restriction enzymes digestion identification. The results of ELISA detecting the anti-M₂e antibody in the mice immunized with pcDNA3.1(+)-M₂e were positive, and the level of anti-M₂e antibody was remarkably rosen after the boosting immunization. The difference among the 3 groups were distinguished by LSD analysis of variance(P＜0.05). The results of the lymphocytes multiplication indicated that the lymphocytes of the BALB/c mice immuned with pcDNA3.1(+)-M2e was very sensitive to influenza A virus M₂e protein, and multiplication was easily induced. The multiplication index was 2.35, whereas the multiplication index of the pcDNA3.1(+) group was only 1.03. The immuned BALB/c mice were challenged by 10×LD⁵⁰ influenza virus. Three mice survived in the pcDNA3.1(+)-M₂e group, but all the mice were died in control groups within 7 days.ConclusionsThe eukaryotic expressing plasmid containing M₂e gene was constructed successfully. The tranfected HEK293 cell expressing M₂e protein steadily were gained through G418 selection. The M₂e protein expressed by the transfected cells not only existed on the tranfected cell membrane, but also secreted into the supernatants. After immunization by plasmid pcDNA3.1(+)-M₂e, both humoral immunity and cell mediated immunity against M2e could be induced in the BALB/c mice. The protection against the challenge with lethal dose of influenza virus in mice could be achieved by the immunization with plasmid pcDNA3.1(+)-M₂e. All these results suggested the M₂e could induce protective immunoresponse against influenza virus infection. These data would benefit for the study of the broad spectrum influenza vaccine providing cross-protection against different influenza variants.