Design, Synthesis and In Vivo Evaluation of a Candidate Fusion Epitopic Construct Vaccine Based on M2e, HA1, HA2, NA and NP Fragments of the Highly Pathogenic Avian H5N1 Influenza Virus.

Abstract

The H5N1 subtype of the influenza virus is highly pathogenic and lethal to humans and animal. The necessity for the development of new vaccines with a broad spectrum of efficacy against this pathogen seems to be very crucial. One highly regarded solution to this problem is to design and production of recombinant vaccines using the conserved peptide of influenza viruses. A search of international databases yielded the peptide sequence of the M2e fragment of H5N1 viruses isolated from Iran, as well as a variety of conserved peptide sequences of fragments of HA1, HA2, NA and NP of other H5N1 viruses. These sequences were obtained for both MHC receptors in mice. Subsequently, these fragments, in conjunction with a PADRE sequence, were connected by bioinformatics to design a fusion epitopic construct. Subsequently, the construct was optimized for expression in E.coli BL21. Following the expression and purifications of the fusion epitopic construct, it was injected subcutaneously (SC) into the hindlimb muscles of 6-8 old week female BALB/c mice. Three weeks following the conclusion of the second immunization, the mice in both immunized and control groups were weighed and checked for any adverse effects at the injection sites. Subsequently, the mice were euthanized and blood was collected from their hearts to determine the total IgG antibody titer before and after immunization by ELISA. No evidence of local inflammation or complications was observed at the SC injection sites until the end of the experiment. Additionally, the autopsy of mice showed no bleeding or lesions in organs, particularly the liver and spleen. The mice exhibited no significant change in weight throughout the immunization period. The total IgG level, as determined by average OD value in the serum of immunized mice, was found to be five times higher (5.881 ng/ml) than that of the control group (1.143 ng/ml). The results demonstrated a highly significant IgG antibody response following SC administration of an immunogenic recombinant peptide in mice.