Development of the polyvalent human influenza vaccine to include avian influenza subunits"

Abstract

Although administration of the currently licensed seasonal trivalent influenza vaccine is effective in eliciting protective immunity against seasonal influenza, this approach is associated with a number of insurmountable problems for preventing an avian influenza pandemic specially H5N1 infection (Tang et al., 2009). Quadrivalent formulations including avian influenza subunit represent a next logical step for seasonal influenza vaccines. This polyvalent vaccine would more accurately reflect the current epidemiology of influenza and would allow vaccination campaigns to more effectively protect their target populations (Ambrose and Levin, 2012). From a regulatory standpoint, this new formulation would not present many challenges, since the components would simply be augmented by a new component, probably with the same antigenic content as the other components (Monto, 2010). Adding a hemagglutinin 5 (HA5) to the seasonal vaccine would, however, constitute a “new” vaccine and would need to be clinically tested for efficacy and safety. In the present study, an attempt has been made to assess a quadrivalent influenza vaccine containing the 3 seasonal strains (H1N1, H3N2 and B) in addition to the 4th H5N1 strain. Two forms of this polyvalent vaccine were designed; one with cloned HA5 subunit protein and the other with split H5N1 virus. Both of polyvalent vaccines were evaluated in mice in parallel with seasonal vaccine and each prepared monovalent H5 vaccine alone. The results obtained from this study can be summarized in the following points:  For production of the HA5 protein subunit, the target HA5 gene was isolated from A/Duck/Egypt/M2583A/2010 (H5N1) RNA by RT-PCR technique. The amplification of 1745 bp fragment corresponded with the expected molecular weights of HA5 gene was demonstrated.  The purified HA5 fragments were inserted into pCR2.1-TOPO- plasmid as a transfer vector producing pCR2.1-TOPO/HA5 construct which used directly for transformation of competent E. coli (DH5α) cells. After plasmid isolation, positive plasmid samples were recorded having higher molecular weights (of plasmid plus the inserted HA5 fragment) than the negative samples. The insertion of the target fragment into the pCR2.1-TOPO vector was further confirmed by PCR amplification of 1759 bp fragment.  The amplified HA5 full length gene tagged with 6-his was subcloned as NotI-BamHI fragment into the purified digested mammalian expression vector pcDNA3.1. Subcloning was done at the multiple cloning site (MCS) of the pcDNA3.1 vector between the CMV promoter and the BGH polyadenylation termination sequence.  The recombinant destination plasmids (pcDNA3.1/HA5) were transformed into E. coli (DH5α) for propagation. Transformation was confirmed by colony PCR giving 1759 bp as the predictable M.Wt and by plasmid isolation in which positive samples recorded more molecular weights than the negative control one. Furthermore, verification by PCR amplification of HA5 from isolated plasmids was followed.  Large scale propagation and purification of the recombinant expression vector pcDNA3.1/HA5 were carried out. The concentration of the purified DNA was found to be 632 ng/μl.  The identity of a HA5 fragment was further verified using sequence analysis. The sequenced fragment was shown to be identical to that sequence in gene bank which confirms that the insert is in sequence with the vector for subsequent translation.  Baby hamster kidney cells (BHK) were used for the transfection and expression of the constructed pcDNA3.1/HA5. Cytotoxicity assay was performed and a concentration of 1000 μg/ml of geneticin (G418) was used for efficient selection of BHK transfected cells which took 10-14 days. The selected stable integrants were 100% homogenous.