Prediction of B-cell epitopes, MHC I Epitopes and MHC II Epitopes
The HA and NA proteins of A/Guangdong/801/2009 (pH1N1) contained 567 amino acids coded by 1701 nucleotides and 470 amino acids coded by 1410 nucleotides, respectively; each having the identical gene lengths of the sH1N1 virus. The HA and NA proteins of 68 pH1N1 viruses were aligned to illustrated the variations in the proteins.
The B-cell epitopes predicted by ABCpred with a threshold values of 0.51 were 330 in HA and 278 in NA. The MHC I molecules in HLA-A1, HLA-A0201, HLA-A3 and HLA-A1101 of HA and NA proteins predicted by BIMAS (number of top-scoring = 100) were 400 in HA and 400 in NA, meanwhile those in HLA-A*01, HLA-A*0201, HLA- A*03 and HLA-A*1101 predicted by SYFPEITHI were 400 in HA and 400 in NA. The MHC II molecules in HLA-DRB1*0101, HLA-DRB1*0301, HLA-DRB1* 0401 HLA- DRB1*0701, HLA-DRB1*1101, and HLA-DRB1*1501 of HA and NA proteins predicted by SYFPEITHI were 1423 in HA and 1146 in NA.
Gene alignment and IEDB epitope
The 1432 (95.8% of 1495) proteins of sH1N1 HA and the 1928 (96.2% of 2005) proteins of sH1N1 NA were aligned after the reduplications in downloaded sequences were discharged. According to the above predictive B-cell epitope, MHC I molecules and MHC II molecules of pH1N1, the conserved ratio of each sH1N1 epitope sequence was acquired. Forty-six epitope sequences of sH1N1 HA proteins were searched in IEDB (291 epitopes of sH1N1 HA in IEDB) for those which had 90%-100% of conserved ratios and twenty-one epitope sequences of NA proteins were done (80 epitopes of sH1N1 NA in IEDB). The epitopes downloaded in IEDB mixed both B-cell epitopes and T-cell epitopes.
There were twenty-nine conserved epitope sequences in HA proteins with 97.1%-100% of conserved ratios between sH1N1 and pH1N1. Epitope SVIEKMNTQFTAV (IEDB No.80042, aa398-410) was overlapped with epitope SVIEKMNTQFTAVGKE (IEDB No.127161, aa398-413), shown in Table . There were eight conserved epitope sequences in NA proteins with conserved ratios of 99.3-99.9% between sH1N1 and pH1N1, shown in Table .
Conservancy of hemagglutinin (HA) epitopes of sH1N1 and pH1N1 influenza viruses
Conservancy of neuraminidase (NA) epitopes of sH1N1 and pH1N1 influenza viruses
Only 62.1% (18/29) of predictive B-cell epitopes of pH1N1 HA proteins overlapped the conserved epitope sequences in sH1N1 HA proteins. The predictive sequences of MHC I molecules and MHC II molecules of pH1N1 covered almost all the conserved epitope sequences in sH1N1 HA proteins, but the positive ratios in HLA-A*0101, A*0201, A*03 and A*1101 were 37.9% (11/29), 62.1% (18/29), 48.3% (14/29) and 100% (29/29), respectively. Only 37.5% (3/8) of predictive B-cell epitopes of pH1N1 NA proteins overlapped the conserved epitope sequences in sH1N1 NA proteins. The predictive sequences of MHC I molecules and MHC II molecules of pH1N1 covered almost all the conserved epitope sequences in sH1N1 NA proteins, but the positive ratios in HLA-A*01, A*0201, A*03 and A*1101 were 62.5% (5/8), 75.0% (6/8), 50.0% (4/8) and 75.0% (6/8), respectively.
The predictive epitopes of MHC I and MHC II molecules of pH1N1 covered almost all the conserved epitope sequences in sH1N1 HA proteins, with the positive ratios in HLA-DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101 and DRB1*1501 being 86.2% (25/29), 79.3% (23/29), 93.1% (27/29), 82.8% (24/29), 55.2% (16/29) and 82.8% (24/29), respectively; and those in six HLA-DRB1 alleles were 75.0% (6/8), 50.0% (4/8), 87.5% (7/8), 62.5% (5/8), 75.0% (6/8) and 87.5% (7/8), respectively.
The H1 hemagglutinin trimer of strain A/801/2009 was obtained using SWISS-MODEL. The 64.2% identity of 2wr0B in the Protein Data Bank (PDB) spanned amino acid residues from 18 to 511 with 2.45 Å X-ray resolution. The epitope region aa22-47 of the pH1N1 HA protein referred to that of the sH1N1 HA protein in IEDB (No. 95458, 95880, 128470 and 128846) and the epitope region aa341-363 referred to that of the sH1N1 HA protein in IEDB (No.128623 and 128979), whose atoms/bonds structures are shown in Figure , and were positively predicted by all three methods (the B-cell epitope ABCpred, the MHC I molecule BIMAS and SYFPEITHI and the MHC II molecule SYFPEITHI. Both epitope regions were neighboring in close proximity in three-dimensional structure although the sequence positions spanned 294 amino acids, as shown in Figure .
HA tetramer decorated with two epitope regions. a. Side view; b. Vertical view. AA22-47 and AA341-363 are IGYHANNSTDTVDTVLEKNVTVTHSV in red and IQSRGLFGAIAGFIEGGWTGMVD in magenta.
The N1 neuraminidase tetramer of strain A/Guangdong/801/2009 was obtained using SWISS- MODEL. The model 3ti4B in the Protein Data Bank (PDB) had the highest sequence homology (99.5% identity) at amino acid residues from 82 to 468 with 1.60 Å X-ray resolution, as shown in Figure . There were the epitope region aa102-123 of pH1N1 HA proteins referred to that of sH1N1 HA proteins in IEDB (No.128924 and 129048) and the epitope region aa130-146 of pH1N1 HA proteins referred to that of sH1N1 HA proteins in IEDB (No.127810), whose atoms/bonds structures were shown in Figure , and were positively predicted by the three methods. Both epitope regions were close in three-dimensional structure and the sequence positions spanned seven amino acids.
NA tetramer decorated with two epitope regions. AA102-123 and AA 130–146 are KDNSIRIGSKGDVFVIREPFIS in red and RTFFLTQGALLNDKHSN in magenta.