Identification and retrospective validation of T-cell epitopes in the hepatitis C virus genotype 4 proteome

Figures & data

Table 1. Five HLA class I candidate epitopes for HCV-4 vaccine design

Epitope sequence	Percent conservation	EpiMatrix Z-scores						Hits	Location
		A*01:01	A*02:01	A*03:01	A*24:02	B*07:02	B*44:03
DVVCCSMSY	98%	2.52	−0.14	2.42	0.19	−0.21	0.6	2	NS5A/NS5B
RLLAPITAY	98%	1.86	1.07	2.64	0.09	0.93	2.02	3	NS2/NS3
FWAKHMWNF	96%	−0.21	−0.01	−0.54	3.11	1.16	0.97	1	NS4B
DPRRRSRNL	98%	−1.56	−1.12	−1.46	0.45	2.81	−0.49	1	Core
YLVAYQATV	93%	0.71	3.2	0.6	0.27	0.56	0.76	1	NS3

EpiMatrix Z-scores ≥2.32 indicate that the epitope is from among the top 1% binders of that allele in a random set of peptides, those ≥1.64 indicate that the epitope is from among the top 5% binders. Binding scores ≥ 1.64 are emphasized in bold. The ‘Hits’ column indicates the number of HLA alleles each epitope is predicted to bind to with a Z-score ≥ 1.64, representing a measure of the epitope’s binding promiscuity.

Table 2. Retrospective validation of 6 predicted HLA class I epitopes

Epitope sequence	EpiMatrix Z-scores						Empirically tested HLA restrictions (Supertype)	Ref.	Type of assay	Assay result
	A*01:01	A*02:01	A*03:01	A*24:02	B*07:02	B*44:03
DVVCCSMSY	2.52	−0.14	2.42	0.19	-0.21	0.6	A26 (A01)	22	HLA Binding Assay	Positive
								22	CD8^+ T cell Assay	Positive
								23	CD8^+ T cell Assay	Positive
RLLAPITAY	1.86	1.07	2.64	0.09	0.93	2.02	A*03:01 (A03)	24	HLA Binding Assay	Positive
FWAKHMWNF	−0.21	−0.01	−0.54	3.11	1.16	0.97	A*24:02 (A24)	25, 26, 27	HLA Binding Assay	Positive
DPRRRSRNL	−1.56	−1.12	−1.46	0.45	2.81	-0.49	B7 (B07)	28–32	CD8^+ T cell Assay	Positive
								33	CD8^+ T cell Assay	Negative
YLVAYQATV	0.71	3.2	0.6	0.27	0.56	0.76	A*02:01 (A02)	24, 25, 34-43	HLA Binding Assay	Positive
								24	CD8^+ T cell Assay	Positive
								41, 44, 45	CD8^+ T cell Assay	Negative
							A*02:02 (A02)	38, 39, 43	HLA Binding Assay	Positive
							A*02:03 (A02)	38, 39, 43	HLA Binding Assay	Positive
							A*02:05 (A02)	39	HLA Binding Assay	Positive
							A*02:06 (A02)	38, 39, 43	HLA Binding Assay	Positive
							A*02:07 (A02)	39	HLA Binding Assay	Negative
							A*68:02 (A02)	38, 39, 43	HLA Binding Assay	Positive
							A2 (A02)	38, 46–49	CD8^+ T cell Assay	Positive
QYLAGLSTL	−0.5	0.95	−0.69	2.73	0.55	1.13	A*02:01 (A02)	24	HLA Binding Assay	Positive

The table lists the retrospective validation results for the same epitopes listed in Table 1, in addition to an epitope that was assigned a false negative score by EpiMatrix. The Z-scores of the supertype alleles whose EpiMatrix predictions have been empirically validated are underlined and emphasized in bold. The supertypes of the empirically determined HLA restrictions (between parentheses) follow the Sette and Sidney supertype classification.^19,50 The references listed in the “Ref.” column are the research articles listed on IEDB where the HLA restrictions have been empirically tested by either HLA binding or T cell assays, giving positive or negative results.

Table 3. Four EpiAssembler-constructed Immunogenic Consensus Sequence (ICS) peptides

ICS sequence	% ORF coverage	EpiMatrix cluster score	JanusMatrix cluster score	Location	Region
SQGYKVLVLNPSVAATLGFG	100	35.24	14.23	1246–1265	NS3
VSGIQYLAGLSTLPGNPA	100	18.78	4.58	1769–1786	NS4B
STQQTLLFNILGGWVAAQI	100	11.32	2.23	1802–1820	NS4B
EGAVQWMNRLIAFASRGNHVA	100	23.46	19.42	1910–1930	NS4B

The 9-mer frames of the core sequences of the ICS peptides (excluding the 3 AA of the N- and C-termini) are emphasized in bold and underlined in the ICS sequence column; the 3 terminal AA residues at the N- and C-termini of the peptides are involved in assisting peptide binding to the HLA class II binding cleft. The Open Reading Frame (ORF) coverage column displays the percentage of the analyzed HCV-4 sequences covered by each ICS sequence in terms of the conservation of its core 9-mer frames collectively across the 46 sequences.

Table 4. Percent Conservation and EpiMatrix Z-scores of the component 9-mer frames of ICS peptide NS3 (1246–1265)

Frame Sequence	% Conservation	EpiMatrix Z-Scores								Hits
		DRB1*01:01	DRB1*03:01	DRB1*04:01	DRB1*07:01	DRB1*08:01	DRB1*11:01	DRB1*13:01	DRB1*15:01
SQGYKVLVL	02%	−0.08	−0.37	0.14	0.89	0.26	−0.08	1.1	0.45	0
QGYKVLVLN	98%	−1.14	−0.83	−1.37	−0.81	0.73	−0.66	−0.37	−1.09	0
GYKVLVLNP	98%	−0.45	−0.47	0.36	0.15	−0.6	0.24	−0.11	−0.69	0
YKVLVLNPS	98%	1.81	1.93	2.02	1.43	2.6	3.08	1.35	2.1	6
KVLVLNPSV	100%	1.59	0.97	1.31	1.33	0.87	1.61	1.26	1.9	1
VLVLNPSVA	100%	1.9	1.39	1.73	1.48	1.31	1.74	1.49	1.58	3
LVLNPSVAA	100%	2.15	2.04	2.28	1.76	1.6	1.59	2	1.77	6
VLNPSVAAT	100%	0.62	0.02	1.15	0.41	0.35	0.64	0.9	0.19	0
LNPSVAATL	100%	1.97	1.83	1.39	1.9	0.82	1.45	1.68	1.38	4
NPSVAATLG	100%	0.22	−0.39	0.08	−0.69	−0.18	−0.65	−0.18	−0.06	0
PSVAATLGF	100%	−0.51	0.19	0.67	0.24	−0.34	−0.29	0.32	−0.25	0
SVAATLGFG	100%	−0.44	0.95	−0.35	−0.68	0.85	0.22	−0.17	1.05	0

Z-scores ≥2.32 indicate that the 9-mer frame is from among the top 1% binders of that allele in a random set of peptides, those ≥1.64 indicate that the frame is from among the top 5% binders Binding scores ≥ 1.64 are emphasized in bold.The 3 N- and C-terminal AA residues are underlined within the 9-mer frames that harbor them.

Table 5. Retrospective validation of the ICS peptide NS3 (1246–1265)

Type of Assay	Test peptide sequence	Validation results and references
		DRB1*01:01	DRB1*03:01	DRB1*04:01	DRB1*07:01	DRB1*08:01	DRB1*11:01	DRB1*13:01	DRB1*15:01
HLA Binding Assay	GYKVLVLNPSVAAT	TP ^52 ^, ^53	—	TP ^53	TP ^53	TP ^53	TP ^53	TP ^53	TP ^53
	GYKVLVLNPSV	—	—	—	—	—	TP ^54	—	—
	GYKVLVLNPSVAATL	—	—	TP ^55	—	—	—	—	—
	KVLVLNPSVAATLGF	TP ^56	FP ^56	TP ^56	TP ^56	—	TP ^56	—	TP ^56
	GYKVLVLNPSVAATLGFGAY	TP ^57	—	TP ^57	TP ^57	TP ^57	TP ^57	TP ^57	TP ^57
CD4+ T cell Assay	QGYKVLVLNPSVAATLGFGA	—	—	—	—	—	TP ^58	—	—
	GYKVLVLNPSVAATL	—	—	TP ^55 ^, ^59	—	—	—	—	—
	GYKVLVLNPSVAAT	—	—	TP ^52	—	—	TP ^52	TP ^52	—
	VLVLNPSVAATLGFGAYM	—	—	TP ^60	—	—	—	—	—
Overall validation results		TP	FP	TP	TP	TP	TP	TP	TP

The parts of the test peptide sequences that are identical to the core sequence of the ICS peptide are underlined. True positive (TP) and false positive (FP) assignments listed in the table validate/refute the EpiMatrix binding predictions outlined in Table 4. Cells without assignments have no corresponding empirical data for comparison.

Table 6. HLA class II binding assay results compared with the highest calculated Z-score per allele of the 9-mer frames of the core sequences of each ICS peptide

ICS sequence	DRB1*01:01		DRB1*04:01		DRB1*07:01		DRB1*15:01
	Highest Z-Score	IC50 (nM)	Highest Z-Score	IC50 (nM)	Highest Z-Score	IC50 (nM)	Highest Z-Score	IC50 (nM)
SQGYKVLVLNPSVAATLGFG	2.15	44	2.28	123	1.9	3,010	2.1	711
VSGIQYLAGLSTLPGNPA	2.54	148	2.25	694	1.93	Non-binder	2.25	1239
STQQTLLFNILGGWVAAQI	2.33	215	1.94	721	1.77	122 087	1.6	782
EGAVQWMNRLIAFASRGNHVA	2.54	2726	1.52	20 416	1.55	44 713	2.56	103

The binding affinity scale is divided as follows: (1) Strong Binder: IC₅₀ < 10 000 nM, (2) Moderate Binder: 10 000 < IC₅₀ < 100 000 nM, (3) Weak binder: IC₅₀ > 100 000 nM.

Figure 1. Flowchart summarizing the identification and analysis of HLA class I and II T cell epitopes from HCV-4 and the retrospective validation of the results using the IEDB.

Table 7. Summary of the results of the in silico epitope identification and retrospective validation process

In silico analysis results	HLA class I	HLA class II
Total 9-mer frames scored by EpiMatrix and Conservatrix	32 286	32 286
Epitopes selected for further analysis	151	14*
Epitopes with no homologous hits in the human genome	90	0
Epitopes with matches on IEDB	70	14
Novel Epitopes (0 IEDB Matches)	20	0
Epitopes with retrospectively validated binding predictions	19	8

* HLA class II epitopes are the ICS peptides.

Neumann-Haefelin C, Killinger T, Timm J, Southwood S, McKinney D, Blum HE, Thimme R. Absence of viral escape within a frequently recognized HLA-A26-restricted CD8+ T-cell epitope targeting the functionally constrained hepatitis C virus NS5A/5B cleavage site. J Gen Virol 2007; 88:1986 - 91; http://dx.doi.org/10.1099/vir.0.82826-0; PMID: 17554032

 PubMed Web of Science ®Google Scholar

Neumann-Haefelin C, Timm J, Spangenberg HC, Wischniowski N, Nazarova N, Kersting N, Roggendorf M, Allen TM, Blum HE, Thimme R. Virological and immunological determinants of intrahepatic virus-specific CD8+ T-cell failure in chronic hepatitis C virus infection. Hepatology 2008; 47:1824 - 36; http://dx.doi.org/10.1002/hep.22242; PMID: 18454507

 PubMed Web of Science ®Google Scholar

Wentworth PA, Sette A, Celis E, Sidney J, Southwood S, Crimi C, Stitely S, Keogh E, Wong NC, Livingston B, et al. Identification of A2-restricted hepatitis C virus-specific cytotoxic T lymphocyte epitopes from conserved regions of the viral genome. Int Immunol 1996; 8:651 - 9; http://dx.doi.org/10.1093/intimm/8.5.651; PMID: 8671652

 PubMed Web of Science ®Google Scholar

Mashiba T, Udaka K, Hirachi Y, Hiasa Y, Miyakawa T, Satta Y, Osoda T, Kataoka S, Kohara M, Onji M. Identification of CTL epitopes in hepatitis C virus by a genome-wide computational scanning and a rational design of peptide vaccine. Immunogenetics 2007; 59:197 - 209; http://dx.doi.org/10.1007/s00251-006-0185-3; PMID: 17225159

 PubMed Web of Science ®Google Scholar

Nakamoto Y, Kaneko S, Takizawa H, Kikumoto Y, Takano M, Himeda Y, Kobayashi K. Analysis of the CD8-positive T cell response in Japanese patients with chronic hepatitis C using HLA-A*2402 peptide tetramers. J Med Virol 2003; 70:51 - 61; http://dx.doi.org/10.1002/jmv.10349; PMID: 12629644

 PubMed Web of Science ®Google Scholar

Kaji K, Nakamoto Y, Kaneko S. Analysis of hepatitis C virus-specific CD8+ T-cells with HLA-A*24 tetramers during phlebotomy and interferon therapy for chronic hepatitis C. Oncol Rep 2007; 18:993 - 8; PMID: 17786365

 PubMed Web of Science ®Google Scholar

Gruener NH, Jung MC, Ulsenheimer A, Gerlach JT, Zachoval R, Diepolder HM, Baretton G, Schauer R, Pape GR, Schirren CA. Analysis of a successful HCV-specific CD8+ T cell response in patients with recurrent HCV-infection after orthotopic liver transplantation. Liver Transpl 2004; 10:1487 - 96; http://dx.doi.org/10.1002/lt.20300; PMID: 15558593

 PubMed Web of Science ®Google Scholar

Grüner NH, Gerlach TJ, Jung MC, Diepolder HM, Schirren CA, Schraut WW, Hoffmann R, Zachoval R, Santantonio T, Cucchiarini M, et al. Association of hepatitis C virus-specific CD8+ T cells with viral clearance in acute hepatitis C. J Infect Dis 2000; 181:1528 - 36; http://dx.doi.org/10.1086/315450; PMID: 10823750

 PubMed Web of Science ®Google Scholar

Lauer GM, Ouchi K, Chung RT, Nguyen TN, Day CL, Purkis DR, Reiser M, Kim AY, Lucas M, Klenerman P, et al. Comprehensive analysis of CD8(+)-T-cell responses against hepatitis C virus reveals multiple unpredicted specificities. J Virol 2002; 76:6104 - 13; http://dx.doi.org/10.1128/JVI.76.12.6104-6113.2002; PMID: 12021343

 PubMed Web of Science ®Google Scholar

Grey HM, Ruppert J, Vitiello A, Sidney J, Kast WM, Kubo RT, Sette A. Class I MHC-peptide interactions: structural requirements and functional implications. Cancer Surv 1995; 22:37 - 49; PMID: 7536628

 PubMedGoogle Scholar

Ishizuka J, Grebe K, Shenderov E, Peters B, Chen Q, Peng Y, Wang L, Dong T, Pasquetto V, Oseroff C, et al. Quantitating T cell cross-reactivity for unrelated peptide antigens. J Immunol 2009; 183:4337 - 45; http://dx.doi.org/10.4049/jimmunol.0901607; PMID: 19734234

 PubMed Web of Science ®Google Scholar

Battegay M, Fikes J, Di Bisceglie AM, Wentworth PA, Sette A, Celis E, Ching WM, Grakoui A, Rice CM, Kurokohchi K, et al. Patients with chronic hepatitis C have circulating cytotoxic T cells which recognize hepatitis C virus-encoded peptides binding to HLA-A2.1 molecules. J Virol 1995; 69:2462 - 70; PMID: 7884894

 PubMed Web of Science ®Google Scholar

Anthony DD, Valdez H, Post AB, Carlson NL, Heeger PS, Lehmann PV. Comprehensive determinant mapping of the hepatitis C-specific CD8 cell repertoire reveals unpredicted immune hierarchy. Clin Immunol 2002; 103:264 - 76; http://dx.doi.org/10.1006/clim.2001.5193; PMID: 12173301

 PubMed Web of Science ®Google Scholar

Fitzmaurice K, Petrovic D, Ramamurthy N, Simmons R, Merani S, Gaudieri S, Sims S, Dempsey E, Freitas E, Lea S, et al. Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection. Gut 2011; 60:1563 - 71; http://dx.doi.org/10.1136/gut.2010.228403; PMID: 21551190

 PubMed Web of Science ®Google Scholar

Scognamiglio P, Accapezzato D, Casciaro MA, Cacciani A, Artini M, Bruno G, Chircu ML, Sidney J, Southwood S, Abrignani S, et al. Presence of effector CD8+ T cells in hepatitis C virus-exposed healthy seronegative donors. J Immunol 1999; 162:6681 - 9; PMID: 10352286

 PubMed Web of Science ®Google Scholar

Sidney J, Southwood S, Mann DL, Fernandez-Vina MA, Newman MJ, Sette A. Majority of peptides binding HLA-A*0201 with high affinity crossreact with other A2-supertype molecules. Hum Immunol 2001; 62:1200 - 16; http://dx.doi.org/10.1016/S0198-8859(01)00319-6; PMID: 11704282

 PubMed Web of Science ®Google Scholar

Tsai SL, Sheen IS, Chien RN, Chu CM, Huang HC, Chuang YL, Lee TH, Liao SK, Lin CL, Kuo GC, et al. Activation of Th1 immunity is a common immune mechanism for the successful treatment of hepatitis B and C: tetramer assay and therapeutic implications. J Biomed Sci 2003; 10:120 - 35; http://dx.doi.org/10.1007/BF02256004; PMID: 12566993

 PubMed Web of Science ®Google Scholar

Guo Z, Zhang H, Rao H, Jiang D, Cong X, Feng B, Wang J, Wei L, Chen H. DCs pulsed with novel HLA-A2-restricted CTL epitopes against hepatitis C virus induced a broadly reactive anti-HCV-specific T lymphocyte response. PLoS One 2012; 7:e38390; http://dx.doi.org/10.1371/journal.pone.0038390; PMID: 22701633

 PubMedGoogle Scholar

Sette A, Sidney J. Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 1999; 50:201 - 12; http://dx.doi.org/10.1007/s002510050594; PMID: 10602880

 PubMed Web of Science ®Google Scholar

Sidney J, Peters B, Frahm N, Brander C, Sette A. HLA class I supertypes: a revised and updated classification. BMC Immunol 2008; 9:1; http://dx.doi.org/10.1186/1471-2172-9-1; PMID: 18211710

 PubMed Web of Science ®Google Scholar

Diepolder HM, Gerlach JT, Zachoval R, Hoffmann RM, Jung MC, Wierenga EA, Scholz S, Santantonio T, Houghton M, Southwood S, et al. Immunodominant CD4+ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection. J Virol 1997; 71:6011 - 9; PMID: 9223492

 PubMed Web of Science ®Google Scholar

Pape GR, Gerlach TJ, Diepolder HM, Grüner N, Jung M, Santantonio T. Role of the specific T-cell response for clearance and control of hepatitis C virus. J Viral Hepat 1999; 6:Suppl 1 36 - 40; http://dx.doi.org/10.1046/j.1365-2893.1999.00006.x; PMID: 10760033

 PubMed Web of Science ®Google Scholar

Shoukry NH, Sidney J, Sette A, Walker CM. Conserved hierarchy of helper T cell responses in a chimpanzee during primary and secondary hepatitis C virus infections. J Immunol 2004; 172:483 - 92; http://dx.doi.org/10.4049/jimmunol.172.1.483; PMID: 14688358

 PubMed Web of Science ®Google Scholar

Day CL, Seth NP, Lucas M, Appel H, Gauthier L, Lauer GM, Robbins GK, Szczepiorkowski ZM, Casson DR, Chung RT, et al. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. J Clin Invest 2003; 112:831 - 42; http://dx.doi.org/10.1172/JCI200318509; PMID: 12975468

 PubMed Web of Science ®Google Scholar

Castelli FA, Leleu M, Pouvelle-Moratille S, Farci S, Zarour HM, Andrieu M, Auriault C, Ménez A, Georges B, Maillere B. Differential capacity of T cell priming in naive donors of promiscuous CD4+ T cell epitopes of HCV NS3 and Core proteins. Eur J Immunol 2007; 37:1513 - 23; http://dx.doi.org/10.1002/eji.200636783; PMID: 17492804

 PubMed Web of Science ®Google Scholar

Lamonaca V, Missale G, Urbani S, Pilli M, Boni C, Mori C, Sette A, Massari M, Southwood S, Bertoni R, et al. Conserved hepatitis C virus sequences are highly immunogenic for CD4(+) T cells: implications for vaccine development. Hepatology 1999; 30:1088 - 98; http://dx.doi.org/10.1002/hep.510300435; PMID: 10498664

 PubMed Web of Science ®Google Scholar

Harcourt GC, Lucas M, Sheridan I, Barnes E, Phillips R, Klenerman P. Longitudinal mapping of protective CD4+ T cell responses against HCV: analysis of fluctuating dominant and subdominant HLA-DR11 restricted epitopes. J Viral Hepat 2004; 11:324 - 31; http://dx.doi.org/10.1111/j.1365-2893.2004.00516.x; PMID: 15230855

 PubMed Web of Science ®Google Scholar

Ebinuma H, Nakamoto N, Li Y, Price DA, Gostick E, Levine BL, Tobias J, Kwok WW, Chang KM. Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T cells in hepatitis C virus infection. J Virol 2008; 82:5043 - 53; http://dx.doi.org/10.1128/JVI.01548-07; PMID: 18337568

 PubMed Web of Science ®Google Scholar

Eckels DD, Bian T, Gill JC, Sønderstrup G. Epitopes of the NS3 protein of hepatitis C virus: recognition in HLA-DR4 transgenic mice. Immunol Cell Biol 2002; 80:106 - 12; http://dx.doi.org/10.1046/j.1440-1711.2002.01053.x; PMID: 11869368

 PubMed Web of Science ®Google Scholar