Abstract
The study was aimed to evaluate the elicitation of highly pathogenic avian influenza (HPAI) virus (AIV) M2e and HA2-specific immunity in chicken to develop broad protective influenza vaccine against HPAI H5N1. Based on the analysis of Indian AIV H5N1 sequences, the conserved regions of extracellular domain of M2 protein (M2e) and HA2 were identified. Synthetic gene construct coding for M2e and two immunodominant HA2 conserved regions was designed and synthesized after codon optimization. The fusion recombinant protein (~38 kDa) was expressed in a prokaryotic system and characterized by Western blotting with anti-His antibody and anti-AIV polyclonal chicken serum. The M2e–HA2 fusion protein was found to be highly reactive with known AIV-positive and -negative chicken sera by ELISA. Two groups of specific pathogen-free (SPF) chickens were immunized (i/m) with M2e synthetic peptide and M2e–HA2 recombinant protein along with one control group with booster on the 14th day and 28th day with the same dose and route. Pre-immunization sera and whole blood were collected on day 0 followed by 3, 7, 14, 21, and 28 days and 2 weeks after the second booster (42 day). Lymphocyte proliferation assay by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method revealed that the stimulation index (SI) was increased gradually from days 0 to 14 in the immunized group (p < 0.05) than that in control chicken. Toll-like receptor (TLR) mRNA analysis by RT-qPCR showed maximum upregulation in the M2e–HA2-vaccinated group compared to M2e- and sham-vaccinated groups. M2e–HA2 recombinant protein-based indirect ELISA revealed that M2e–HA2 recombinant fusion protein has induced strong M2e and HA2-specific antibody responses from 7 days post-primary immunization, and then the titer gradually increased after booster dose. Similarly, M2e peptide ELISA revealed that M2e–HA2 recombinant fusion protein elicited M2e-specific antibody from day 14 onward. In contrast, no antibody response was detected in the chicken immunized with synthetic peptide M2e alone or control group. Findings of this study will be very useful in future development of broad protective H5N1 influenza vaccine targeting M2e and HA2.
Highlights
Influenza A viruses have been isolated from a wide range of animals including poultry, wild and cage birds, pigs, horses, dogs, sea mammals, and humans, ducks are considered the natural reservoirs of avian influenza viruses (AIVs)
Synthetic gene construct in pET32b(+) coding for M2 protein (M2e)-HA2 was transformed into the host, E. coli Rosetta Blue (DE3)pLysS
The addition of IPTG induced the overexpression of ∼38-kDa molecular weight recombinant protein, which was confirmed by Western blotting with anti-His and anti-AIV antibody (Figure 1)
Summary
Influenza A viruses have been isolated from a wide range of animals including poultry, wild and cage birds, pigs, horses, dogs, sea mammals, and humans, ducks are considered the natural reservoirs of avian influenza viruses (AIVs). In South Asia, the H5N1 virus was first reported in domestic poultry in India and Pakistan during February 2006 and followed by Bangladesh, Nepal, and Bhutan in March 2007, January 2009, and February 2010, respectively [4]. The first introduction of clade 2.3.2 H5N1 virus to South Asia was reported from Nepal in February 2010 [9, 10], followed by in India in February 2011 [11]. Between November 2014 to March 2015, clade 2.3.2.1c has been reported as the new introduction to India [12], followed by worldwide circulation of clade 2.3.4.4 including India [13,14,15,16,17]. Due to the continuous change of clades, cross protection between the clades become uncertain
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