Immunogenic Peptide Identification in H1N1 Virus as Target for Vaccine Design

Abstract

Last 100 years have witnessed two pandemic outbreaks of H1N1 influenza viz. Spanish flu in 1918 and swine flu in 2009. It has been the most prevalent subtype of influenza virus in humans responsible for significant morbidity and mortality worldwide. In case of unforeseen outbreaks of influenza, antiviral prophylaxis is chosen to put an immediate check on its spread, However, the efficacy of antiviral drugs in treating influenza seems uncertain due to the rise of drug resistant strains. Further, influenza vaccines presently available are strain specific and require periodic reformulation to include combination of circulating influenza A and B virus strains. The problem associated with such vaccines is the induction of humoral immune response directed against the highly mutable hemagglutinin protein. Further, their timely and adequate availability is difficult in case of a sudden outbreak. Thus, there is a pressing need to develop a refined vaccine approach which can offer universal or at least broad protection against the ever mutating influenza virus strains in the population distributed worldwide. Highly conserved T cell epitopes of influenza A protein are known to be cross protective. Such T cell epitopes which have the capacity to bind to an array of HLA molecules are anticipated to serve as candidates for universal or broadly reactive influenza vaccine. In light of the above facts, the present study focused on identifying highly conserved promiscuous peptides containing multiple overlapping CD8+ and CD4+ T cell epitopes from hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) protein of H1N1 influenza virus using different epitope prediction tools and molecular docking. Further, peripheral blood mononuclear cells from healthy volunteers were subjected to repetitive stimulation by these chemically synthesized peptides and their proliferation and IFN-γ level was measured by MTT and ELISA assay respectively to assess immunogenic response. Epitope prediction tools identified 24 CD8+ and 11 CD4+ T cell epitopes of HA, 13 CD8+ and nine CD4+ T cell epitopes of NA and ten CD8+ and nine CD4+ T cell epitopes of the M1 protein. Overlapping epitopes were merged to generate 14 peptides containing multiple CD8+ and CD4+ T cell epitopes from the HA, NA and M1 protein (five HA, six NA and three M1 peptides) belonging to H1N1 subtype of influenza virus. Four peptides (two each from NA and HA) were excluded from current study as these peptides were reported to induce immunogenic response in previous studies. Ten peptides (PH1, PH3 and PH4 of HA, PN2, PN4, PN5 and PN6 of NA and PM1, PM2 and PM3 of M1 protein) were selected for further evaluation. Population coverage analysis of the selected peptides revealed that five peptides viz. PH1, PN2, PN6, PM1 and PM2 showed excellent coverage in Asia, Europe, Africa, North America, South America and Oceania countries. In order to confirm the unrestrained binding capacity of selected peptides with multiple HLA molecules, docking was carried out by AutoDock vina. The binding energy obtained after docking of most of the CD8+ T cell epitopes and peptides containing multiple CD4+ T cell epitopes with various HLA class I and II molecules was comparable to that of the native peptides except for three CD8+ T cell epitopes (two of HA and one of M1 protein). Interestingly, binding energy obtained for some of epitopes/peptides was higher than the native peptides of corresponding HLA. In vitro peptide stimulation assays revealed that HA and M1 peptides elicited better immunogenic response in terms of proliferation and IFN-γ secretion among various PBMC samples as compared to NA peptides. Higher number of positive responders were observed in case of HA and M1 peptides. Peptides PH1, PH4, PN5, PN6, PM1 and PM2 induced proliferative response in most of the healthy samples (≥5). Similarly, significantly higher secretion of IFN-γ level was observed in PH1, PH4, PM2 and PM3 peptide stimulated cells in most of the healthy samples (≥5). The results suggested that the in silico approach applied in combination with in vitro experimentation successfully identified peptides of hemagglutinin, neuraminidase and matrix 1 protein which were capable of eliciting immune response, thus making them potential candidates for universal influenza vaccine development.