Background
Cancer type | HPyV6 Positive | HPyV7 Positive | Reference |
---|---|---|---|
Verrucous keratosis | 75% | 75% | [14] |
Squamous cell carcinoma in chronic lymphocytic leukemia cases who had bone marrow transplantation | 33.3% | Not determined | [15] |
Squamous cell carcinoma | 36.4% | 54.5% | [14] |
Squamous cell carcinoma | 100% | 100% | [9] |
Keratoacanthoma | 42.3% | Not applicable | [10] |
Trichoblastoma | 22.2% | Not applicable | [10] |
Cutaneous T-cell lymphomas | 20% | 8.6% | [16] |
Methods
Retrieval and analysis of protein sequences
Identification and evaluation of Cytotoxic T-Lymphocyte (CTL) epitopes
Identification and analysis of HTL (Helper T-Lymphocyte) epitopes
Identification and analysis of LBL (Linear B-Lymphocyte) epitopes
Evaluation of the human homology and epitope conservancy
Molecular docking and peptide modeling analysis
Population coverage analysis
MHC cluster analysis
Designing and formulation of the multi-epitope vaccine
Antigenicity, allergenicity, solubility, and physicochemical property assessment
BLAST homology assessment
Prediction of the secondary structure
Homology modeling and 3D structure refinement and validation
Identification of discontinuous B-Cell epitopes
Molecular docking of the immune receptor (TLR4) and designed vaccine
In silico immune simulation
Molecular dynamics simulation
Results
Vaccine design
Retrieval and evaluation of protein sequences
Species | UniProt entry | protein name | abbreviation | Antigenicity (Probable antigen) | allergenecity | no. of TM helices |
---|---|---|---|---|---|---|
HPyV6 | D6QWG6 | Large T antigen | LTAg | 0.4928 | Probable non-allergen | 0 |
D6QWG0 | Viral Protein 1 | VP1 | 0.4555 | Probable non-allergen | 0 | |
D6QWF3 | Viral Protein 2 | VP2 | 0.4288 | Probable non-allergen | 0 | |
HPyV7 | D6QWJ6 | Large T antigen | LTAg | 0.4996 | Probable non-allergen | 0 |
D6QWJ5 | Viral Protein 1 | VP1 | 0.4281 | Probable non-allergen | 0 | |
D6QWJ3 | Viral Protein 2 | VP2 | 0.4323 | Probable non-allergen | 0 |
Identification and validation of CTL epitopes
specie | epitopes | protein | C-score | Immunogenicity score | antigenicity | allergenicity | toxicity | epitope conservancy hit (maximum identity %) | human homology | interacting MHC-I alleles |
---|---|---|---|---|---|---|---|---|---|---|
HPyV6 | LSHATLGNK | LTAg | 1.2320 | 0.10205 | 1.5211 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-A*11:01, HLA-A*03:01, HLA-A*30:01 |
FERWVSFGM | LTAg | 1.3474 | 0.24276 | 1.2787 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-B*18:01, HLA-B*40:01, HLA-B*40:02 | |
WLLFVLEEL | VP2 | 1.1038 | 0.28696 | 0.7654 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-A*02:01, HLA-A*02:06 | |
IYKVEAILL | VP1 | 1.5128 | 0.21171 | 0.4174 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-A*24:02, HLA-B*35:03 | |
HPyV7 | MELTDVLLI | LTAg | 1.2565 | 0.08008 | 1.3253 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-B*44:02, HLA-B*44:03, HLA-B*40:01, HLA-B*18:01, HLA-B*53:01, HLA-B*40:02, HLA-B*51:01 |
TPKRRNLLF | LTAg | 1.3998 | 0.01055 | 1.7138 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-B*08:01, HLA-B*07:02 | |
GPRIGSTTM | VP2 | 0.08343 | 0.7918 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-B*07:02 | ||
LWLPQAWPW | VP2 | 1.0974 | 0.08973 | 0.5232 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-A*24:02, HLA-B*58:01, HLA-B*57:01, HLA-A*23:01, HLA-B*53:01 | |
DTMIVWEAY | VP1 | 2.0643 | 0.43299 | 0.5828 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-A*26:01, HLA-A*25:01, HLA-A*01:01, HLA-B*35:01, HLA-B*18:01, HLA-A*29:02, HLA-A*30:02, HLA-B*46:01 | |
TELLFAPQM | VP1 | 1.0319 | 0.05903 | 0.8241 | probable non-allergen | non-toxic | 100 | non-homologue | HLA-B*18:01, HLA-B*44:02, HLA-B*40:02, HLA-B*44:03, HLA-B*40:01 |
Identification and validation of HTL epitopes
specie | epitopes | protein | antigenicity | allergenicity | IFN-γ | IL4 | toxicity | epitope conservancy hit (maximum identity %) | human homology | interacting MHC-II alleles |
---|---|---|---|---|---|---|---|---|---|---|
HPyV6 | VAAQRRVLMLESTRQ | LTAg | 0.8394 | probable non-allergen | positive | inducer | non-toxic | 100 | non-homologue | HLA-DRB1*04:10, HLA-DRB1*04:04, HLA-DRB1*04:05, HLA-DRB1*04:08 |
GLVNLVNYAVNYNRQ | VP2 | 0.7212 | probable non-allergen | positive | inducer | non-toxic | 100 | non-homologue | HLA-DRB1*08:06, HLA-DRB1*01:02, HLA-DRB1*08:13, HLA-DRB1*13:04, HLA-DRB1*08:01, HLA-DRB1*04:23 | |
GRFFRVHFRQRRVKH | VP1 | 0.4931 | probable non-allergen | positive | inducer | non-toxic | 100 | non-homologue | HLA-DRB1*11:20, HLA-DRB1*11:14, HLA-DRB1*13:23, HLA-DRB1*08:04, HLA-DRB5*01:01, HLA-DRB5*01:05 | |
SAGYIRAQGTPAGVE | VP1 | 0.8598 | probable non-allergen | positive | inducer | non-toxic | 100 | non-homologue | HLA-DRB1*09:01 | |
HPyV7 | DWNFVADFAADMELT | LTAg | 1.2109 | probable non-allergen | positive | inducer | non-toxic | 100 | non-homologue | HLA-DRB3*01:01, HLA-DRB1*03:09, HLA-DRB1*03:05, HLA-DRB1*04:21 |
FEPGGVVMYDTQNLP | VP2 | 0.9125 | probable non-allergen | negative | inducer | non-toxic | 100 | non-homologue | HLA-DRB1*15:06, HLA-DRB1*04:02, HLA-DRB1*08:06, HLA-DRB1*15:02, HLA-DRB1*08:04, HLA-DRB1*08:13, HLA-DRB1*04:10, HLA-DRB1*08:01 | |
FFRVHCRQRRIKHPY | VP1 | 0.4266 | probable non-allergen | negative | inducer | non-toxic | 100 | non-homologue | HLA-DRB5*01:05, HLA-DRB1*11:20, HLA-DRB1*11:14, HLA-DRB1*13:23 |
Identification and validation of linear B‑cell epitope (LBL)
specie | epitopes | protein | protein probability score | antigenicity | allergenicity | toxicity | human homology |
---|---|---|---|---|---|---|---|
HPyV6 | SSEVRPPPQYGSPGWE | LTAg | 0.84 | 1.0311 | probable non-allergen | non-toxic | non-homologue |
PSKENQPSVAGIKATR | VP1 | 0.88 | 1.3222 | probable non-allergen | non-toxic | non-homologue | |
SNKKRRSGGYGNSATF | VP2 | 0.80 | 0.7625 | probable non-allergen | non-toxic | non-homologue | |
HPyV7 | DSKYSATPPKQKKPNP | LTAg | 0.90 | 1.1870 | probable non-allergen | non-toxic | non-homologue |
PATIPPTVEGGLGFAP | VP1 | 0.88 | 1.1122 | probable non-allergen | non-toxic | non-homologue | |
DQRGGFHDEGTWVSFQ | VP2 | 0.81 | 1.1021 | probable non-allergen | non-toxic | non-homologue |
Vaccine evaluation
Evaluation of human homology and epitope conservancy
Molecular docking analyses of the CTL epitopes and HLA alleles
alleles | epitopes | HADDOCK score | Van der Waals energy | Electrostatic energy | Desolvation energy | Restraints violation energy | Buried Surface Area | RMSD from the overall lowest-energy structure | Z-Score |
---|---|---|---|---|---|---|---|---|---|
HLA-A*03:01 | LSHATLGNK | -66.5 ± 3.3 | -28.3 ± 3.5 | -206.2 ± 23.6 | 0.2 ± 1.9 | 28.5 ± 24.3 | 1006.2 ± 77.1 | 1.2 ± 0.1 | -1.7 |
HLA-B*18:01 | FERWVSFGM | -101.3 ± 6.3 | -45.5 ± 6.9 | -112.0 ± 25.7 | -39.3 ± 3.5 | 58.6 ± 38.3 | 1442.7 ± 27.8 | 0.5 ± 0.3 | -1.6 |
HLA-A*02:01 | WLLFVLEEL | -85.0 ± 0.4 | -39.3 ± 3.4 | -97.8 ± 4.0 | -29.8 ± 1.6 | 36.2 ± 21.4 | 1408.9 ± 38.7 | 2.2 ± 0.1 | -1.4 |
HLA-A*24:02 | IYKVEAILL | -72.3 ± 2.8 | -39.3 ± 5.0 | -69.2 ± 14.9 | -22.7 ± 1.9 | 35.1 ± 19.9 | 1143.8 ± 17.1 | 1.5 ± 0.1 | -1.1 |
HLA-B*08:01 | TPKRRNLLF | -99.3 ± 2.1 | -43.4 ± 2.8 | -220.7 ± 11.5 | -12.0 ± 4.6 | 2.8 ± 1.2 | 1447.9 ± 117.0 | 0.6 ± 0.4 | -1.6 |
HLA-B*07:02 | GPRIGSTTM | -62.2 ± 0.8 | -35.0 ± 1.6 | -169.5 ± 9.4 | 5.9 ± 1.6 | 8.0 ± 7.7 | 1242.8 ± 24.3 | 0.2 ± 0.1 | -1.7 |
HLA-B*58:01 | LWLPQAWPW | -93.9 ± 1.8 | -48.4 ± 2.6 | -29.3 ± 8.5 | -43.7 ± 2.3 | 40.4 ± 34.9 | 1296.0 ± 22.3 | 0.6 ± 0.1 | -1.5 |
HLA-A*01:01 | DTMIVWEAY | -108.1 ± 3.0 | -52.8 ± 5.8 | -181.1 ± 25.2 | -19.3 ± 2.8 | 3.4 ± 3.5 | 1485.3 ± 18.1 | 0.4 ± 0.3 | -2.3 |
HLA-B*44:03 | MELTDVLLI | -70.2 ± 3.1 | -47.4 ± 3.9 | -63.0 ± 25.0 | -10.5 ± 1.0 | 2.9 ± 1.7 | 1331.2 ± 24.4 | 0.3 ± 0.2 | -1.2 |
HLA-B*40:02 | TELLFAPQM | -82.9 ± 2.5 | -46.1 ± 3.2 | -87.9 ± 12.5 | -20.9 ± 2.4 | 17.2 ± 13.6 | 1362.8 ± 30.6 | 0.4 ± 0.3 | -1.5 |
Population coverage analysis
Population/Area | MHC I | MHC II | MHC I and MHC II Combined | ||||||
---|---|---|---|---|---|---|---|---|---|
Coverage(%)a | Average hitb | PC90c | Coverage(%)a | Average hitb | PC90c | Coverage(%)a | Average hitb | PC90c | |
World | 97.74% | 3.2 | 1.55 | 26.99% | 0.34 | 0.14 | 98.35% | 3.54 | 1.77 |
East Asia | 96.82% | 3.29 | 1.62 | 53.39% | 0.66 | 0.21 | 96.82% | 3.29 | 1.62 |
Northeast Asia | 91.63% | 2.34 | 1.06 | 33.41% | 0.39 | 0.15 | 91.63% | 2.34 | 1.06 |
South Asia | 88.69% | 1.96 | 0.88 | 24.5% | 0.29 | 0.13 | 88.69% | 1.96 | 0.88 |
Southeast Asia | 94.27% | 3.05 | 1.26 | 41.54% | 0.46 | 0.17 | 94.27% | 3.05 | 1.26 |
Southwest Asia | 89.37% | 2.16 | 0.94 | 18.19% | 0.21 | 0.12 | 89.37% | 2.16 | 0.94 |
Europe | 99.55% | 3.86 | 2.18 | 20.76% | 0.28 | 0.13 | 99.55% | 3.86 | 2.18 |
East Africa | 88.85% | 2.18 | 0.9 | 28.6% | 0.39 | 0.14 | 88.85% | 2.18 | 0.9 |
West Africa | 92.69% | 2.38 | 1.11 | 52.54% | 0.66 | 0.21 | 92.69% | 2.38 | 1.11 |
Central Africa | 84.65% | 1.94 | 0.65 | 24.28% | 0.35 | 0.13 | 84.65% | 1.94 | 0.65 |
North Africa | 92.84% | 2.38 | 1.11 | 37.7% | 0.51 | 0.16 | 92.84% | 2.38 | 1.11 |
South Africa | 91.08% | 2.16 | 1.04 | 1.79% | 0.02 | 0.1 | 91.08% | 2.16 | 1.04 |
West Indies | 98.25% | 3.4 | 1.77 | 27.52% | 0.37 | 0.14 | 98.25% | 3.4 | 1.77 |
North America | 98.19% | 3.37 | 1.7 | 24.95% | 0.32 | 0.13 | 98.19% | 3.37 | 1.7 |
Central America | 9.07% | 0.14 | 0.11 | 16.53% | 0.2 | 0.12 | 9.07% | 0.14 | 0.11 |
South America | 80.56% | 2.05 | 0.51 | 27.31% | 0.35 | 0.14 | 80.56% | 2.05 | 0.51 |
Oceania | 95.27% | 3.08 | 1.35 | 35.77% | 0.4 | 0.16 | 95.27% | 3.08 | 1.35 |
MHC cluster analysis
Formulation of the vaccine construct
Vaccine assessment
Assessment of the antigenicity, allergenicity, and physicochemical properties of the final vaccine protein
characteristics | findings | remarks |
---|---|---|
Number of amino acids | 501 | suitable |
molecular weight | 54,059.97 Da | average |
Theoretical pI | 9.41 | basic |
chemical formula | C2455H3814N662O694S11 | –- |
extinction coefficient (at 280 nm in H2O) | 77,810 M−1 cm−1 | |
estimated half-life (mammalian reticulocytes, in vitro) | 30 h | satisfactory |
estimated half-life (yeast cells, in vivo) | > 20 h | satisfactory |
estimated half-life (E. coli, in vivo) | > 10 h | satisfactory |
instability index of vaccine | 34.63 | Stable |
aliphatic index of vaccine | 72.95 | thermostable |
grand average of hydropathicity (GRAVY) | -0.317 | hydrophilic |
antigenicity | 0.6455 (VaxiJen v2.0) 0.897722 (ANTIGENPro) | antigenic |
allergenicity | No (AllergenFP v1.0) No (AlgPred) No (AllerTop v2.0) | non-allergen |
solubility | 0.766196 (SolPro) 0.504 (Protein-Sol) | soluble |
TM helices | No | suitable |
BLAST homology assessment
Secondary structure extrapolation
Tertiary structure modeling, refinement, and validation of the multi‑epitope vaccine
Model | GDT-HA | RMSD | MolProbity | Clash score | Poor rotamers | Rama favored |
---|---|---|---|---|---|---|
Initial | 1.0000 | 0.000 | 3.074 | 5.4 | 18.2 | 71.5 |
MODEL 1 | 0.9097 | 0.506 | 2.216 | 14.1 | 0.8 | 89.6 |
MODEL 2 | 0.9087 | 0.504 | 2.240 | 15.4 | 0.3 | 90.0 |
MODEL 3 | 0.9037 | 0.517 | 2.195 | 14.4 | 0.8 | 90.6 |
MODEL 4 | 0.9142 | 0.498 | 2.338 | 15.3 | 1.3 | 89.6 |
MODEL 5 | 0.9077 | 0.515 | 2.230 | 14.2 | 0.5 | 89.2 |
Screening for conformational B-Cell epitopes
no | residues | no. of residues | score |
---|---|---|---|
1 | B:S492, B:G493, B:G494, B:Y495, B:G496, B:N497, B:S498, B:A499, B:T500, B:F501 | 10 | 0.975 |
2 | B:M1, B:A2, B:K3, B:L4, B:S5, B:T6, B:D7, B:E8, B:L9, B:L10, B:D11, B:A12, B:F13, B:K14, B:E15, B:M16, B:T17, B:L18, B:L19, B:E20, B:L21, B:S22, B:D23, B:F24, B:V25, B:K26, B:K27, B:F28, B:E29, B:E30, B:T31, B:F32, B:E33, B:V34, B:T35, B:A36, B:A37, B:A38, B:P39, B:V40, B:A41, B:V42, B:A43, B:A44, B:A45, B:G46, B:A47, B:P49, B:A50, B:G51, B:A52, B:A53, B:V54, B:E58, B:S61, B:E62, B:F63, B:D64, B:V65, B:I66, B:L67, B:E68, B:A69, B:A70, B:G71, B:D72, B:K73, B:K74, B:I75, B:G76, B:V77, B:I78, B:K79, B:V80, B:V81, B:R82, B:E83, B:I84, B:V85, B:S86, B:G87, B:L88 | 82 | 0.773 |
3 | B:Q353, B:G354, B:P355, B:P357, B:G378, B:S379, B:A380, B:G381, B:R384, B:A385, B:T388, B:P389, B:G391, B:V392, B:K394, B:K395, B:D396, B:K398, B:Y399, B:S400, B:A401, B:T402, B:P403, B:K407, B:I417, B:P419, B:T420, B:V421, B:E422, B:G423, B:G424, B:L425, B:G426, B:F427, B:A428, B:P429, B:K430, B:K431, B:D432, B:Q433, B:R434, B:G435, B:G436, B:F437, B:H438, B:D439, B:E440, B:G441, B:T442, B:W443, B:V444, B:S445, B:F446, B:Q447, B:K448, B:K449, B:S450, B:S451, B:E452, B:V453, B:R454, B:P455, B:P456, B:P457, B:Q458, B:Y459, B:G460, B:S461, B:P462, B:G463, B:W464, B:E465, B:K466, B:K467, B:P468, B:S469, B:K470, B:E471, B:N472, B:Q473, B:P474, B:S475, B:V476, B:A477, B:G478, B:I479, B:K480, B:A481, B:T482, B:R483, B:K484, B:K485, B:S486, B:N487, B:K488, B:K489, B:R490, B:R491 | 98 | 0.737 |
4 | B:G275, B:P276, B:G277, B:F278 | 4 | 0.696 |
5 | B:F261, B:D264, B:F265 | 3 | 0.639 |
6 | B:D268, B:L271, B:T272 | 3 | 0.632 |
7 | B:A122, B:A123, B:G124, B:A125 | 4 | 0.582 |
8 | B:W221, B:P223, B:Q224, B:A225, B:W228 | 5 | 0.568 |
9 | B:L186, B:R199, B:R200, B:N201, B:L202, B:L203, B:Y207, B:R210, B:P250, B:Q251, B:M252, B:G253, B:P254, B:G255, B:P256, B:G257, B:D258, B:W259 | 18 | 0.563 |
10 | B:K107, B:V108, B:E111 | 3 | 0.561 |
11 | B:A100, B:P101, B:K102, B:P103, B:L104, B:L105 | 6 | 0.555 |
12 | B:P337, B:G338, B:G339, B:N342 | 4 | 0.545 |
13 | B:P297, B:G298, B:F299 | 3 | 0.53 |
Molecular docking of the vaccine protein and TLR complex
TLR4 (chain A) | Vaccine | Bond length (Å) |
---|---|---|
Tyr207 | His431 | 3.02 |
Arg363 | Asn51 | 2.67 |
Asp268 | Arg606 | 2.87 |
Arg360 | Asn51 | 2.62 |
Arg360 | Glu27 | 2.76 |
Arg324 | Glu27 | 2.73 |
Arg367 | Glu27 | 2.87 |
Gln224 | Glu605 | 2.88 |
His290 | Glu31 | 3.14 |
Glu111 | Arg355 | 2.72 |
Arg150 | Glu425 | 2.74 |
Leu18 | Lys150 | 2.53 |
Phe13 | Lys150 | 2.67 |
Lsy480 | Gln91 | 2.50 |
Glu15 | Gly124 | 2.89 |
Glu15 | Ser126 | 2.85 |
Lys14 | Ser127 | 2.76 |
Trp237 | Val32 | 2.86 |
Arg332 | Pro28 | 2.86 |
Arg332 | Cys29 | 2.81 |