M2e Subunit Vaccines Of Avian Influenza Virus

Highly pathogenic avian influenza (HPAI) has been recognized as a devastatingviral disease of poultry since 1878. Currently, vaccination s remain the most effectivemeasure to prevent avian influenza, but it is becoming more and more difficulty tosurmount the epidemic situations in the context of antigenic variations of influenzavirus and the mismatch of between vaccines and field viruses, therefore, it isnecessary and urgent to develop"universal vaccines"which bring about broadcross-protection against avian influenza.The in fluenza Matrix protein 2 (M2), especially, the extracellular domain of M2(M2e) is highly conserved, M2e-based vaccines are considered as promisingcandidates to develop in fluenza"universal vaccine". Moreover, several constructedvaccines on the basis of M2 or M2e of human influenza virus have shown that theycan elicit protective efficacy in vary degrees in mice model, however, it has littleresearch about the protective efficacy of avian-type M2-based or M2e-based vaccinesin birds model until now.In order to determine the protective efficacy of avian-typed M2e-based subunitvaccines in SPF chickens, primers were designed according to the sequences of M2gene of avian influenza virus (AIV) H5N1 subtype available in GenBank, theoptimized avian-type d M2e gene was amplified by polymerase chain reaction (PCR) ,then the recombinant plasmids involving different M2e tandem copies wereconstructed with the pMAL-c2X vector and expressed in Escherichia coli. . Thefusion proteins were induced with 0.3mmol/L isopropylβ-D-1-thiogalactopyranoside(IPTG ). The expressions of fusion proteins were identified by SDS-PAGE andWestern blot. All fusion proteins were emulsified with with Freud's adjuvant andMONTANIDE ISA70M adjuvant to generate subunit vaccines , respectively, thenBALB/c mice and SPF chickens were immunized to valuate the discrepancy inantibody levels with indirect ELISA, the bioactivity of immunized animals sera wereidentified by immunofluorescence assay (IFA) , challenges with A/Chicken/Guang -dong/04(H5N1) virus were pursued to detect the protective efficacy of differentsubunit vaccines and the discrepancy of protective efficacy between SPF chickensand BALB/c mice . The results showed that plasmids were constructed successfully;fusion proteins were soluble and had good immunogenicity and bioactivity. Thequantities of protein expressions were 58%,49.8%,55%,52.4%,62.9% by thin-layerscanning analysis, respectively. The indirect ELISA s showed that higher epitopedensity engendered higher antibody levels but not in serial increase, namely,MBP-3M2e group produced the highest antibody level compared to negative control (p < 0.05) , in which antibody response was intensively enhanced compared withothers in different immunized groups were not significant in chickens and micemodel (p >0.1) , moreover, the specific IgG antibody levels in chickens were higherbut not significant than in mice (p >0.1) . Challenge showed the subunit vaccinesproduced better protective efficacy in SPF chickens than in BALB/c mice . Theprotective rate was 0%(MBP-M2e),37.5%(MBP-3M2e),12.5% (MBP-6M2e),12.5%(MBP-9M2e),12.5%(MBP-12M2e) in SPF chickens, respectively, however,only MBP-3M2e group engendered 16.7% in mice model.The results showed that avian-typed M2e subunit vaccines could induce specificimmune response and confer partial protection against the challenge of H5N1in fluenza A virus in SPF chickens.