Designing of CD8⁺ and CD8⁺-overlapped CD4⁺ epitope vaccine by targeting late and early proteins of human papillomavirus

Figures & data

Figure 1 Flowchart of the overall study.

Abbreviations: HPV, human papillomavirus; IFN-γ, interferon gamma.

Table 1 CD8⁺ CTL epitopes prediction from HPV45 early and late antigenic proteins

Predicted CTL epitopesa	NetCTL scorea	Proteasome cleavage scoreb	TAP scoreb	MHC binding prediction scoreb	Processing scoreb	Total scoreb	Interacting MHC class I allele with an affinity <200b
(A) Replication protein E1 of HPV45
ATDTGSDMV40–48	1.95	1.06	0.12	−2.16	1.18	−0.99	HLA-A*01:01 (145.9)
		1.06	0.12	−1.27	1.18	−0.09	HLA-C*05:01 (18.5)
MLAVFKDIY200–208	1.96	1.31	1.28	−2.23	2.58	0.35	HLA-A*30:02 (168.5)
		1.31	1.28	−2.28	2.58	0.3	HLA-A*29:02 (188.9)
FKDIYGLSF204–212	0.85	1.38	0.99	−1.98	2.36	0.39	HLA-C*05:01 (95)
FTDLVRNFK212–220	1.44	0.98	0.07	−1.36	1.05	−0.3	HLA-A*68:01 (22.8)
		0.98	0.07	−1.67	1.05	−0.62	HLA-A*11:01 (46.7)
CTDWVMAIF226–234	2.63	1.33	1.01	−1.81	2.35	0.54	HLA-A*01:01 (64.2)
		1.33	1.01	−1.63	2.35	0.72	HLA-C*05:01 (42.6)
TLIKPATLY246–254	1.02	1.22	1.29	−1.21	2.51	1.31	HLA-A*29:02 (16.1)
VLILALLRY266–274	0.85	1.45	1.31	−1.21	2.76	1.56	HLA-A*29:02 (15.7)
LRSSLAALY305–313	0.91	1.21	1.34	−2.17	2.56	0.39	HLA-A*30:02 (146.9)
		1.21	1.34	−2.27	2.56	0.29	HLA-A*29:02 (184.5)
		1.21	1.34	−1.92	2.56	0.64	HLA-B*27:05 (82.7)
SSLAALYWY307–315	1.22	1.28	1.31	−1.48	2.59	1.11	HLA-A*30:02 (30.4)
		1.28	1.31	−1.91	2.59	0.67	HLA-A*11:01 (81.8)
		1.28	1.31	−2.26	2.59	0.33	HLA-A*29:02 (182.9)
LSDMVQWAF350–358	3.01	1.31	1.05	−1.29	2.35	1.06	HLA-A*01:01 (19.5)
		1.31	1.05	−2.03	2.35	0.32	HLA-B*35:01 (107.2)
		1.31	1.05	−1.04	2.35	1.31	HLA-C*05:01 (11.1)
LTDESDMAF362–370	2.73	1.21	1.04	−1.15	2.25	1.11	HLA-A*01:01 (14.2)
		1.21	1.04	−2.19	2.25	0.06	HLA-A*02:06 (155.9)
		1.21	1.04	−1.78	2.25	0.47	HLA-B*35:01 (60.3)
		1.21	1.04	−0.91	2.25	1.35	HLA-C*05:01 (8)
MNMSQWIKY413–421	0.76	1.41	1.28	−1.71	2.68	0.97	HLA-A*29:02 (50.4)
		1.41	1.28	−2.15	2.68	0.52	HLA-B*35:01 (142.4)
LADTKVAML506–514	0.82	1.51	0.36	−0.93	1.86	0.94	HLA-C*05:01 (8.5)
		1.51	0.36	−1.89	1.86	−0.02	HLA-C*03:03 (76.9)
DATHTCWTY516–524	1.13	1.51	1.21	−1.37	2.7	1.33	HLA-B*35:01 (23.4)
ATHTCWTYF517–525	0.92	1.25	1.17	−1.97	2.42	0.45	HLA-A*32:01 (92.8)
YLESRVTVF571–579	0.85	1.42	1.00	−2.09	2.42	0.34	HLA-B*15:01 (122.9)
		1.42	1.00	−2.36	2.42	0.07	HLA-B*08:01 228.1
		1.42	1.00	−2.03	2.42	0.39	HLA-C*12:03 (107.5)
FTFPHAFPF579–587	0.79	0.97	1.10	−0.61	2.07	1.46	HLA-A*02:06 (4)
		0.97	1.10	−0.91	2.07	1.17	HLA-A*29:02 (7.9)
		0.97	1.10	−1.14	2.07	0.92	HLA-A*32:01 (13.9)
		0.97	1.10	−1.53	2.07	0.54	HLA-A*68:02 (33.5)
		0.97	1.10	−1.89	2.07	0.18	HLA-A*68:01 (77.5)
		0.97	1.10	−2.08	2.07	0.97	HLA-A*26:01 (119.6)
		0.97	1.10	−1.65	2.07	0.41	HLA-B*15:01 (45)
		0.97	1.10	−0.91	2.07	1.17	HLA-B*35:01 (7.9)
		0.97	1.10	−0.71	2.07	1.35	HLA-C*03:03 (5.2)
		0.97	1.10	−0.79	2.07	1.28	HLA-C*12:03 (6.1)
		0.97	1.10	−1.46	2.07	0.61	HLA-C*07:02 (29)
		0.97	1.10	−1.53	2.07	0.54	HLA-C*14:02 (33.6)
		0.97	1.10	−1.95	2.07	0.11	HLA-C*15:02 (89.6)
(B) Regulatory protein E2 of HPV45
NTEPSQCFK109–117	0.82	0.99	0.19	−1.16	1.19	0.02	HLA-A*68:01 (14.5)
YVVWDSIYY137–145	1.15	1.34	1.29	−0.55	2.64	2.09	HLA-A*29:02 (3.6)
		1.34	1.29	−1.72	2.64	0.91	HLA-A*68:01 (53)
		1.34	1.29	−1.94	2.64	0.71	HLA-A*26:01 (87.6)
		1.34	1.29	−2.03	2.64	0.61	HLA-A*30:02 (107.7)
		1.34	1.29	−0.74	2.64	1.91	HLA-B*35:01 (5.5)
MTETGIWEK146–154	1.75	0.92	0.15	−1.24	1.07	−0.18	HLA-A*68:01 (17.5)
		0.92	0.15	−1.73	1.07	−0.66	HLA-A*11:01 (53.5)
CVSYWGVYY158–166	2.19	1.38	1.28	−0.8	2.66	1.86	HLA-A*29:02 (6.3)
		1.38	1.28	−1.96	2.66	0.71	HLA-A*30:02 (90.5)
		1.38	1.28	−2.03	2.66	0.63	HLA-A*11:01 (107.4)
		1.38	1.28	−2.21	2.66	0.45	HLA-A*01:01 (162.3)
		1.38	1.28	−2.22	2.66	0.44	HLA-A*68:01 (166.6)
		1.38	1.28	−2.07	2.66	0.59	HLA-B*35:01 (117.8)
YIKDGDTTY166–174	1.15	1.51	1.26	−1.66	2.76	1.11	HLA-A*29:02 (45.8)
		1.51	1.26	−1.72	2.76	1.04	HLA-B*15:01 (53.1)
		1.51	1.26	−1.36	2.76	1.41	HLA-B*35:01 (22.9)
		1.51	1.26	−1.62	2.76	1.14	HLA-B*15:02 (41.8)
NSNTWEVQY187–195	2.17	1.52	1.31	−2.09	2.82	0.74	HLA-A*01:01 (121.9)
		1.52	1.31	−2.26	2.82	0.57	HLA-B*58:01 (180.4)
TSDDTVSAT209–217	0.92						HLA-C*05:01 (153)
NTHVHNSLL265–273	0.81						HLA-A*30:01 (168.1)
RLRKYADHY309–317	0.85	1.31	1.34	−1.75	2.65	0.89	HLA-B*15:01 (56.5)
NTGILTVTY332–340	2.47	1.59	1.25	−2.21	2.84	0.63	HLA-A*01:01 (162.1)
LTVTYNSEV336–344	0.98	0.96	0.18	−1.75	1.15	−0.61	HLA-A*68:02 (55.9)
		0.18	−1.98	1.15	−0.83	0.18	HLA-A*02:06 (94.7)
		0.96	0.18	−2.27	1.15	−1.12	HLA-C*15:02 (186.7)
NSVQISVGY187–195	1.31	1.37	1.37	−2.19	2.74	0.55	HLA-B*35:01 (155.9)
(C) Protein E4
CAVPVTTRY4–12	1.16	1.38	1.3	−2.00	2.68	0.68	HLA-A*29:02 (101)
		1.38	1.3	−1.03	2.68	1.65	HLA-B*58:01 (10.8)
		1.38	1.3	−1.17	2.68	1.51	HLA-B*35:01 (14.9)
		1.38	1.3	−2.13	2.68	0.55	HLA-A*30:02 (135.3)
(D) Protein E6
RTEVYQFAF41–49	1.55	1.25	1.09	−1.32	2.34	1.02	HLA-A*32:01 (20.8)
		1.25	1.09	−2.21	2.34	0.13	HLA-B*58:01 (163.7)
YSRIRELRY72–80	1.69	1.41	1.27	−2.05	2.68	0.63	HLA-A*29:02 (113)
(E) Protein E7
STLSFVCPW93–101	1.77	1.06	0.43	−0.92	1.49	0.57	HLA-A*32:01 (8.4)
		1.06	0.43	−0.97	1.49	0.52	HLA-B*58:01 (9.3)
		1.06	0.43	−1.16	1.49	0.33	HLA-B*57:01 (14.3)
LQQLFLSTL87–95	1.08	1.59	0.44	−1.96	2.03	0.07	HLA-A*02:06
QLFLSTLSF89–97	1.47	1.3	1.14	−2.18	2.44	0.26	HLA-B*15:02
		1.3	1.14	−1.32	2.44	1.12	HLA-B*15:01
		1.3	1.14	−1.38	2.44	1.05	HLA-A*32:01
(F) Major capsid protein L1
HVEEYDLQF51–59	1.28	1.32	1.04	−2.15	2.36	0.21	HLA-B*35:01 (142.7)
TLTAEVMSY67–75	1.33	1.3	1.25	−2.21	2.55	0.34	HLA-B*15:01 (161.8)
LTAEVMSYI68–76	0.84	0.92	0.25	−0.48	1.17	0.69	HLA-A*68:02 (3)
		0.92	0.25	−0.97	1.17	0.21	HLA-A*02:06 (9.2)
		0.92	0.25	−1.81	1.17	−0.63	HLA-A*02:01 (63.9)
		0.92	0.25	−1.77	1.17	−0.59	HLA-B*58:01 (58.3)
		0.92	0.25	−2.13	1.17	−0.96	HLA-C*12:03 (134.1)
		0.92	0.25	−2.28	1.17	−1.11	HLA-C*15:02 (190.3)
(G) Minor capsid protein L2
KRASATDLY10–18	0.78	1.2	1.38	−2	2.58	0.58	HLA-A*30:02 (99.8)
		1.2	1.38	−2.3	2.58	0.28	HLA-B*27:05 (199.2)
STSFTNPAF154–162	0.96	1.3	1.11	−2.04	2.41	0.38	HLA-A*32:01 (108.8)
		1.3	1.11	−2.08	2.41	0.33	HLA-B*15:01 (121)
		1.3	1.11	−2.23	2.41	0.18	HLA-C*14:02 (171.6)
FSDPSIIEV162–170	2.22	1.14	0.02	−0.75	1.16	0.41	HLA-A*02:06 (5.6)
		1.14	0.02	−1.67	1.16	−0.51	HLA-A*02:01 (46.3)
		1.14	0.02	−2.17	1.16	−1.01	HLA-A*01:01 (148.5)
		1.14	0.02	−2.17	1.16	−1.01	HLA-A*68:02 (148.8)
		1.14	0.02	−1.13	1.16	0.03	HLA-C*12:03 (13.6)
		1.14	0.02	−1.05	1.16	0.1	HLA-C*05:01 (11.4)
		1.14	0.02	−2	1.16	−0.84	HLA-C*15:02 (99.2)
		1.14	0.02	−2.03	1.16	−0.87	HLA-C*08:02 (106)
		1.14	0.02	−1.71	1.16	−0.56	HLA-C*03:03 (51.8)
LVTFDNPAY248–256	0.88	1.32	1.29	−1.62	2.61	1	HLA-A*30:02 (41.3)
		1.32	1.29	−1.72	2.61	0.89	HLA-A*29:02 (52.4)
		1.32	1.29	−0.9	2.61	1.71	HLA-B*35:01 (8)
HSDFMDIIR273–281	0.83	1.09	0.56	−1.78	1.66	−0.13	HLA-A*68:01 (60.9)
QIGGRVHFY312–320	1.08	1.4	1.19	−2.03	2.59	0.56	HLA-A*29:02 (107.9)
		1.4	1.19	−2.07	2.59	0.52	HLA-A*30:02 (117.1)
ATDLYKTCK14–22	1.06	0.98	0.19	−1.91	1.16	−0.75	HLA-A*11:01 (80.8)
STINKSFTY362–370	2.39	1.4	1.32	−1.24	2.72	1.48	HLA-A*11:01 (17.2)
		1.4	1.32	−1.28	2.72	1.44	HLA-A*29:02 (19.3)
		1.4	1.32	−1.59	2.72	1.13	HLA-A*32:01 (39)
		1.4	1.32	−1.67	2.72	1.05	HLA-A*30:02 (46.9)
		1.4	1.32	−1.72	2.72	1	HLA-A*26:01 (52.6)
		1.4	1.32	−2.26	2.72	0.46	HLA-A*68:01 (183.1)
MPSTAASSY377–385	0.76	1.3	1.14	−2.16	2.45	0.29	HLA-A*30:02 (145.1)
		1.3	1.14	−0.47	2.45	1.97	HLA-B*35:01 (3)
		1.3	1.14	−1.71	2.45	0.74	HLA-B*53:01 (50.7)
TSAWDVPIY393–401	2.04	1.43	1.29	−1.85	2.72	0.87	HLA-A*30:02 (23)
		1.43	1.29	−1.85	2.72	0.87	HLA-B*35:01 (70.3)
PTNAATSTY420–428	2.67	1.35	1.16	−2.24	2.51	0.27	HLA-A*01:01 (172.4)
		1.35	1.16	−2.28	2.51	0.23	HLA-A*30:02 (188.7)
QYYLWPWYY435–443	0.82	1.31	1.35	−1.54	2.66	1.12	HLA-A*23:01 (34.8)
		1.31	1.35	−0.55	2.66	2.11	HLA-A*29:02 (3.5)
		1.31	1.35	−1.64	2.66	1.02	HLA-A*30:02 (43.6)
		1.31	1.35	−1.66	2.66	1.01	HLA-C*07:02 (45.3)
		1.31	1.35	−1.85	2.66	0.82	HLA-C*14:02 (70.1)

Notes:

a CTL prediction by NetCTL 1.2 (threshold >0.75).

b Prediction by NetMHC ANN 3.4; proteasome cleavage score is interpreted as logarithm of the total amount of cleavage site usage liberating the peptide C-terminus; TAP score estimates an effective log(IC₅₀) value for binding to TAP of a peptide or its N-terminal prolonged precursors; MHC binding prediction score is identical to the class I −log(IC₅₀) values; processing score combines the proteasomal cleavage and TAP transport predictions, and predicts a quantity proportional to the amount of peptide present in the endoplasmic reticulum, where a peptide can bind to multiple MHC molecules; total score combines the proteasomal cleavage, TAP transport, and MHC-binding predictions and predicts a quantity proportional to the amount of peptide presented by MHC molecules on the cell surface.

Abbreviations: CTL, cytotoxic T lymphocyte; HLA, human leukocyte antigen; HPV, human papillomavirus; MHC, major histocompatibility complex; TAP, transporter associated with antigen processing.

Figure 2 Conservation across hrHPV strains by the CD8⁺ HPV45 immunogenic epitopes.

Abbreviations: HPV, human papillomavirus; hrHPV, high-risk human papillomavirus; MHC, major histocompatibility complex.

Figure 3 PC of the HPV45 epitopes based on the MHC class I restriction data.

Notes: The top four geographical regions with the highest PC of the pooled HPV45 epitopes: (A) Europe, (B) North America (C), West Indies, and (D) Northeast Asia. The line (-o-) represents the cumulative percentage of PC of the epitopes; the bars represent the PC for the individual epitopes.
Abbreviations: HLA, human leukocyte antigen; HPV, human papillomavirus; MHC, major histocompatibility complex; PC, population coverage.

Table 2 Molecular docking interactions of CD8⁺ MHC class I-restricted epitopes

S.no.	Peptides	sOPEP energy (PEP- FOLD3)	Rank (PatchDock)	Solution numbera	Global energy (kcal/ mol)a	aVDW energy (kcal/ mol)a	rVDW energy (kcal/ mol)a	AC energya	CD8^+ epitopes- restricted MHC class I allotypes	HB length (Å)b
1	MLAVFKDIY	−14.7991	1	6	−28.42	−15.17	2.17	−3.17	HLA-A*30:02	−0.75
			1	9	−53.92	−24.43	2.28	−10.72	HLA-A*29:02	0.00
2	FKDIYGLSF	−8.9529	1	2	−40.80	−30.45	12.25	−2.53	HLA-C*05:01	−1.32
3	FTDLVRNFK	−12.9293	1	5	−34.46	−23.88	8.49	0.65	HLA-A*68:01	−0.64
			1	9	−59.20	−32.69	27.35	−6.35	HLA-A*11:01	−2.30
4	CTDWVMAIF	−15.3595	1	7	−37.07	−20.97	3.94	−4.04	HLA-A*01:01	−1.48
			1	5	−38.23	−34.27	72.23	−9.94	HLA-C*05:01	−3.04
5	VLILALLRY	−19.3886	1	8	−54.59	−25.17	15.10	−5.46	HLA-A*29:02	−2.96
6	SSLAALYWY	−15.3223	1	7	−53.50	−19.29	7.82	−9.60	HLA-A*30:02	−2.83
			1	3	−73.99	−26.76	7.50	−15.73	HLA-A*11:01	−2.16
			1	5	−62.48	−27.77	12.07	−13.80	HLA-A*29:02	−2.69
7	DATHTCWTY	−5.18894	1	7	−23.19	−22.44	30.05	2.14	HLA-B*35:01	−3.39
8	ATHTCWTYF	−6.8348	1	7	−66.46	−25.61	6.88	−15.59	HLA-A*32:01	−1.86
9	YLESRVTVF	−8.16618	1	5	−46.77	−19.67	9.72	−7.24	HLA-B*15:01	−2.27
			1	2	−82.17	−36.57	39.79	−13.20	HLA-B*08:01	−5.55
			1	2	−61.57	−36.74	40.88	−11.30	HLA-C*12:03	−2.95
10	FTFPHAFPF	−8.80536	1	1	−14.11	−10.87	14.37	−1.92	HLA-A*02:06	0.00
			1	10	−43.09	−19.23	10.19	−10.34	HLA-A*29:02	−0.26
			1	8	−75.39	−32.86	40.24	−23.69	HLA-A*32:01	−2.38
			1	8	−49.87	−24.76	10.15	−9.86	HLA-A*68:02	0
			1	8	−49.87	−24.76	10.15	−9.86	HLA-A*68:01 (double)	0
			1	8	−60.03	−23.34	8.08	−13.61	HLA-A*26:01	−0.26
			1	6	−64.50	−26.23	18.01	−19.18	HLA-B*15:01	−0.22
			1	1	−34.92	−20.16	9.60	−9.95	HLA-B*35:01	−0.71
			1	9	−39.04	−16.21	7.20	−11.25	HLA-C*03:03	−0.87
			1	8	−46.09	−19.88	9.32	−7.35	HLA-C*12:03	−1.29
			1	8	−60.03	−23.34	8.08	−13.61	HLA-C*07:02	−0.26
			1	10	−63.01	−27.71	18.93	−15.47	HLA-C*14:02	−1.47
			1	2	−56.97	−28.87	11.24	−5.23	HLA-C*15:02	−1.67
11	YVVWDSIYY	−9.63342	1	8	−66.91	−26.31	24.91	−15.82	HLA-A*29:02	−2.35
			1	4	−65.84	−27.39	18.99	−13.88	HLA-A*68:01	0
			1	4	−66.62	−28.44	13.61	−19.13	HLA-A*26:01	−2.23
			1	10	−59.08	−29.94	12.45	−7.94	HLA-A*30:02	−2.03
			1	9	−58.47	−27.03	15.18	−4.47	HLA-B*35:01	−2.59
12	MTETGIWEK	−−5.25678	1	9	−49.67	−27.06	2.62	−7.34	HLA-A*68:01	−3.54
			1	1	−53.39	−31.89	14.13	−6.49	HLA-A*11:01	−0.73
13	CVSYWGVYY	−9.91873	1	10	−74.14	−27.18	7.95	−14.20	HLA-A*29:02	−5.35
			1	2	−59.36	−21.40	8.84	−12.75	HLA-A*30:02	−0.53
			1	3	−65.00	−26.68	41.07	−16.85	HLA-A*11:01	−0.49
			1	8	−15.92	−3.94	1.41	−3.39	HLA-A*01:01	−0.36
			1	2	−56.89	−25.93	6.28	−8.42	HLA-A*68:01	−2.53
			1	10	−34.57	−31.92	62.47	−11.31	HLA-B*35:01	−2.77
14	YIKDGDTTY	−4.48098	1	4	−24.36	−21.96	32.98	−4.51	HLA-A*29:02	−0.78
			1	5	−36.94	−30.71	10.27	−4.00	HLA-B*15:01	−1.80
			1	9	−43.84	−30.30	37.11	3.99	HLA-B*35:01	−7.73
			1	5	−36.94	−30.71	10.27	−4.00	HLA-B*15:02	−1.80
15	NSNTWEVQY	−3.9109	1	9	−29.71	−21.51	7.81	2.21	HLA-A*01:01	−0.83
			1	2	−34.59	−34.99	59.58	−5.59	HLA-B*58:01	−2.11
16	NTGILTVTY	−1.78903	1	6	−22.26	−19.65	21.64	−2.30	HLA-A*01:01	−0.52
17	CAVPVTTRY	−2.66502	1	8	−57.77	−23.51	8.23	−11.81	HLA-A*29:02	−0.87
			1	6	−67.29	−35.78	16.36	−7.84	HLA-A*30:02	−0.58
			1	2	−76.86	−26.91	13.47	−13.80	HLA-B*35:01	−6.25
			1	1	−86.71	−35.97	14.82	−11.52	HLA-B*58:01	−2.43
18	RTEVYQFAF	−12.2878	1	2	−61.05	−25.12	13.43	−15.85	HLA-A*32:01	−1.68
			1	2	−58.52	−24.39	16.56	−11.95	HLA-B*58:01	0.00
19	YSRIRELRY	−15.6298	1	8	−45.61	−25.84	12.89	−0.66	HLA-A*29:02	−1.28
20	HVEEYDLQF	−10.4675	1	8	−40.31	−24.51	22.28	−8.43	HLA-A*32:01	−2.43
			1	10	−58.45	−24.21	7.58	−9.82	HLA-B*15:01	−0.8
			1	9	−37.57	−20.01	3.85	−1.18	HLA-C*14:02	−0.88
21	STSFTNPAF	−2.87049	1	8	−50.44	−27.80	21.59	−8.84	HLA-A*02:06	−0.59
22	FSDPSIIEV	−6.00242	1	2	−59.10	−36.24	51.37	−11.39	HLA-A*02:01	−6.68
			1	6	−56.82	−23.20	20.57	−14.78	HLA-A*01:01	−1.13
			1	2	−52.17	−23.48	6.37	−8.66	HLA-A*68:02	−2.18
			1	8	−32.08	−24.27	19.26	−7.93	HLA-C*12:03	−1.97
			1	9	−40.61	−18.74	10.10	−6.26	HLA-C*05:01	−1.39
			1	5	−59.37	−26.36	12.50	−14.73	HLA-C*15:02	−0.64
			1	9	−40.75	−20.95	4.09	−2.37	HLA-C*08:02	0.00
			1	5	−39.06	−23.67	9.54	1.35	HLA-C*03:03	−1.02
23	LVTFDNPAY	−4.47434	1	6	−14.89	−7.75	4.51	−1.10	HLA-A*30:02	−0.47
			1	2	−23.99	−35.19	48.13	0.63	HLA-A*29:02	−6.19
			1	10	−50.65	−19.95	5.64	−11.11	HLA-B*35:01	0.00
24	HSDFMDIIR	−11.7285	1	3	−31.62	−20.20	7.02	−3.59	HLA-A*68:01	0.00
25	QIGGRVHFY	−4.88567	1	5	−48.36	−23.16	11.36	−3.14	HLA-A*29:02	−4.11
			1	4	−56.64	−32.72	44.20	−8.67	HLA-A*30:02	−4.21
26	TSAWDVPIY	−5.85866	1	7	−53.89	−24.78	7.43	−8.41	HLA-A*30:02	−2.69
27	QYYLWPWYY	−12.2259	1	10	−24.74	−13.38	3.85	−2.27	HLA-A*30:02	0.00
			1	7	−31.88	−21.33	6.39	3.31	HLA-A*23:01	−0.52
			1	2	−35.97	−21.52	11.11	−1.89	HLA-C*07:02	−1.89
			1	9	−39.41	−28.13	6.83	2.01	HLA-C*14:02	−1.08

Notes:

a FireDock is a rigid body scoring energy for best-ranked solution number, global energy, attractive and repulsive van der Waals energy, atomic contact energy, and hydrogen bond energy of the peptide and MHC receptor protein complex.

b Amino acid residues of peptide and MHC receptor protein docked complex and the initial distance between hydrogen bond donor and acceptor analyzed by Chimera.

Abbreviations: AC, atomic contact; aVDW, attractive van der Waals; HB, hydrogen bond; HLA, human leukocyte antigen; MHC, major histocompatibility complex; rVDW, repulsive van der Waals.

Table 3 CD8⁺ epitope sequences overlapped in CD4⁺ and B-cell epitope segments (indicated in bold)

S.no.	Protein	Start	End	Sequence	AllergenFP v.1.0	VaxiJen	IFN-γ production (hybrid method)	Immunogenicity
1	E1	306	320	RSSLAALYWYRTGIS	Non-allergen	Antigen	0.326	0.4023
2	E1	575	589	RVTVFTFPHAFPFDK	Non-allergen	**	0.307	0.5941
3	E1	306	321	RSSLAALYWYRTGISN	Non-allergen	Antigen	0.295	0.3448
4	E1	578	593	VFTFPHAFPFDKNGNP	Non-allergen	**	-0.029	0.2367
5	E1	199	214	AMLAVFKDIYGLSFTD	Non-allergen	**	2	0.1155
6	E2	137	151	YVVWDSIYYMTETGI	*	Antigen	6	0.2328
7	E2	157	171	ACVSYWGVYYIKDGD	Non-allergen	Antigen	18	0.0902
8	E2	160	174	SYWGVYYIKDGDTTY	Non-allergen	Antigen	17	0.1895
9	E2	184	198	KYGNSNTWEVQYGGN	Non-allergen	**	0.511	0.1604
10	E2	141	156	DSIYYMTETGIWEKTA	Non-allergen	Antigen	0.591	0.3512
11	E2	158	173	CVSYWGVYYIKDGDTT	*	Antigen	16	0.2388
12	E2	331	346	KNTGILTVTYNSEVQR	Non-allergen	Antigen	0.131	0.1717
13	E5	22	36	LVQSVYVCAFAWLLV	Non-allergen	**	0.538	0.2031
14	E6	72	86	YSRIRELRYYSNSVY	Non-allergen	**	1	0.0483
15	E6	66	81	HKCIDFYSRIRELRYY	*	Antigen	-0.153	0.4463
16	E6	38	53	TLERTEVYQFAFKDLC	Non-allergen	Antigen	0.777	0.2190
17	L1	273	287	HSDFMDIIRLHRPAL	Non-allergen	**	1.000	0.3681
18	L1	50	65	RHVEEYDLQFIFQLCT	Non-allergen	Antigen	0.046	0.3138
19	L2	435	449	QYYLWPWYYYFPKKR	*	Antigen	0.647	0.1930
20	L2	265	280	GKQIGGRVHFYHDISP	Non-allergen	Antigen	18	0.4629
21	L2	387	402	NVTVPLTSAWDVPIYT	Non-allergen	**	-0.228	0.3246
22	L2	248	263	LVTFDNPAYEPLDTTL	Non-allergen	Antigen	-0.288	0.3216
23	L2	156	167	SFTNPAFSDPSIIEVP	Non-allergen	Antigen	0.521	0.1995

Notes:

* Epitope sequence below the antigenic threshold 0.4.

** Epitope sequence below the allergenic threshold 0.4.

Abbreviation: IFN-γ, interferon gamma.

Figure 4 Biochemical pathway and pharmacokinetic studies on proposed peptides. (A) The panel represents the biochemical pathway of proposed peptides (μg) interaction with HPV45. (B) The graph represents the HPV45 time course simulation with proposed peptides (μg), where X axis represents the time and Y axis in left side represents the concentration and Y axis in right side indicates the entities which represent the molecules that interact with HPV45; peaks represent their biochemical activity during time course simulation.

Abbreviation: HPV, human papillomavirus.