HLA-A*0201, HLA-A*1101, and HLA-B*0702 Transgenic Mice Recognize Numerous Poxvirus Determinants from a Wide Variety of Viral Gene Products

Valerie Pasquetto; Huynh-Hoa Bui; Rielle Giannino; Fareed Mirza; John Sidney; Carla Oseroff; David C. Tscharke; Kari Irvine; Jack R. Bennink; Bjoern Peters; Scott Southwood; Vincenzo Cerundolo; Howard Grey; Jonathan W. Yewdell; Alessandro Sette

doi:10.4049/jimmunol.175.8.5504

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FIGURE 1.
Screening of pools of HLA-predicted binding peptides derived from VACV sequences. Positive pools of peptides selected for further analysis are indicated in black. The criteria of positivity were SI >1.4 and net SFCs/10⁶ cells >20. A, A total of 24 of 81 pools was identified in VACV-infected HLA-A*0201/K^b transgenic mice. B, A total of 6 of 103 pools was identified in VACV-infected HLA-A*1101/K^b transgenic mice. C, A total of 7 of 34 pools was identified in VACV-infected HLA-B*0702/K^b transgenic mice.
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FIGURE 2.
Screening of individual HLA-predicted binding peptides from immunogenic ORFs derived from VACV. Positive peptides selected are indicated in black. The criteria of positivity were SI >2.0 and net SFCs/10⁶ cells >20. An asterisk indicates epitopes for which a second overlapping peptide containing the same core sequence also induced a positive response. The amino acid sequence and the protein name from which each of the epitopes was derived are indicated. A, Fourteen positive peptides were identified in HLA-A*0201/K^b transgenic mice. B, four positive peptides were identified in HLA-A*1101/K^b transgenic mice. C, Three positive peptides were identified in HLA-B*0702/K^b transgenic mice.
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FIGURE 3.
CD8⁺ T cell response against HLA-A*0201-restricted VACV epitopes. Representative experiments using different stimulator cells and purified CD8⁺ T cells from VACV-infected HLA-A*0201/K^b transgenic mice are shown. The dotted line indicates the 20 net SFCs/10⁶cells threshold used to define positivity. A and B, Representative graphs of peptides for which reactivity was seen with HLA-A*0201/K^b-expressing stimulator cells, but not with target cells that lacked the HLA-A*0201 transgene. C and D, Representative graphs of peptides for which reactivity was seen with both HLA-A*0201 and H-2-expressing stimulator cells.
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FIGURE 4.
CD8⁺ T cell response against HLA-A*1101-restricted VACV epitopes. Titration experiments using stimulator cells from different sources with purified CD8⁺ T cells from VACV-infected HLA-A*1101/K^b transgenic mice are shown. The dotted line indicates the 20 net SFCs/10⁶cells threshold used to define positivity. A, Representative graph of a peptide for which reactivity was seen with HLA-A*1101/K^b-expressing stimulator cells, but not with target cells that lacked the HLA-A*1101 transgene. B, Representative graph of a peptide for which reactivity was seen with both HLA-A*1101 and H-2-expressing stimulator cells.
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FIGURE 5.
HLA-A*0201-restricted epitopes are naturally processed by VACV-infected human cells. Epitope-immunized CD8⁺ T cells were stimulated with the human Jurkat HLA-A*0201/K^b tumor cell line either infected with VACV at a multiplicity of infection = 9, or pulsed with different concentrations of the respective peptide and their response analyzed by ex vivo ELISPOT assay. The net SFCs/10⁶ CD8⁺ T cells is shown.

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Table I.

Immunogenicity of vaccinia virus-derived HLA-A*0201-restricted peptides in different mouse strains

HLA-A2.1 Potential Epitopes			HLA-A2.1/K^b (CD 8⁺/LPS blasts) (n = 2)			Av. Net SFCs/10⁶ Cells^a
Peptide	Sequence	% of peptide response	Av. net SFCs/10⁶ cells^a	HHD A₂ mice (n = 1)^b	CB6F1 (CD8⁺/LPS blasts) (n = 2)	K^b−/−D^b−/− (CD8⁺/LPS blasts) (n = 2)	CBA/J (CD8⁺/LPS blasts) (n = 2)
A14L (51–59)	FILGIIITV	34	543	509	—	—	—
I1L (211–219)	RLYDYFTRV	8	130	290	—	—	—
A46R (142–150)	GLFDFVNFV	6	92	40	—	—	—
A17L (61–70)	RTLLGLILFV	2	34	153	—	—	—
H3L (184–192)^c	SLSAYIIRV	6.5	105	185	—	—	—
VACWR082 (18–26)	ILDDNLYKV	2	28	101	—	—	—
B14R (327–335)	HVDGKILFV	3	49	443	—	—	—
A6L (6–14)	VLYDEFVTI	5	81	524	—	—	—
D12L (251–259)	RVYEALYYV	2.5	40	181	—	—	—
G7L (250–258)	YLPEVISTI	4	66	—^d	—	—	—
VACWR050 (196–204)	FLIVSLCPT	3	50	—	—	—	—
M1L (374–383)	IIIPFIAYFV	15	245	478	1123	—	—
A17L (81–90)	ILMIFISSFL	5.5	88	395	243	—	—
B6R (108–116)	LMYDIINSV	3	54	—	136	—	—
HRP2 (74–82)	KVDDTFYYV	0	0	51	ND	ND	ND
Vaccinia-infected target cells	NA		1026		ND	1100	43	820

a Net SFCs/10⁶ cells are calculated according to the formula: [(number of spots against relevant peptide) − (number of spots against irrelevant peptides (HCV core 132)] × [(1 × 10⁶)/number of effector cells/well].
b Dumped peptide at 10 μg/ml.
c HLA-A2-restricted peptide described by Drexler et al. (13 ).
d (−) indicates <20 SFCs/10⁶ cells. Control-irrelevant peptides were on average 3.5 for CB6F1, 18.5 for HLA-A2.1/K^b, 3.35 for K^b−/−D^b−/−, and 15 for CBA/J.

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Table II.

Immunogenicity of vaccinia virus-derived HLA-A*1101-restricted peptides in different mouse strains

HLA-A11 Potential Vaccinia Virus Epitopes			HLA-A11/K^b (CD8⁺/LPS blasts) (n = 2)			Av. Net SFCs/10⁶ Cells^a
Peptide	Sequence	% of peptide response	Av. net SFCs/10⁶ cells	HLA-A11/K^b−/− (CD8⁺/LPS blasts) (n = 2)	CB6F1 (CD8⁺/LPS blasts) (n = 2)	K^b−/−D^b−/− (CD8⁺/LPS blasts) (n = 2)	CBA/J (CD8⁺/LPS blasts) (n = 2)
I3L (116–124)	AVYGNIKHK	16	82	565	—	—	—
E7R (130–138)	STLNFNNLR	15	78	—^b	—	—	—
A10L (171–180)	NTYLFNILYK	57	287	—	469	—	—
A47L (139–148)	AFEFINSLLK	11.5	58	—	45	—	—
Vaccinia-infected target cells	NA		1125		1455	1100	43	820

a NetSFCs/10⁶ cells are calculated according to the formula: [(number of spots against relevant peptide − (number of spots against irrelevant peptides (HCV core 132)] × [(1 × 10⁶)/number of effector cells/well]. Sample SFCs control irrelevant A11-peptide (Human Mage 3 69). NA, Not applicable. (−) indicates <20 SFCs/10⁶ cells.
b Control irrelevant peptide were on average 20 SFCs/10⁶ cells for HLA-A11/K^b, 15 for HLA-A11/K^b knockout, 15 for CB6F1; 14 for K^b−/−D^b−/−, and 57 for CBA/J.

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Table III.

Immunogenicity of vaccinia virus-derived HLA-B*0702-restricted peptides in different mouse strains

HLA-B7 Potential Epitopes			HLA-B7/K^b (CD8⁺/LPS blasts) (n = 2)			Av. Net SFCs/10⁶ Cells^a
Peptide	Sequence	% of peptide response	Av. net SFCs/10⁶ cells^a	CB6F1 (CD8⁺/LPS blasts) (n = 2)	K^b−/−D^b−/− (CD8⁺/LPS blasts) (n = 2)	CBA/J (CD8⁺/LPS blasts) (n = 2)
A34R (82–90)	LPRPDTRHL	75	285	198	—	—
J2R (116–124)	KPFNNILNL	17	66	—^b	—	—
D1R (808–817)	RPSTRNFFEL	7	28	—	—	—
Vaccinia-infected target cells	NA		367		1100	43	820

a Net SFCs/10⁶ cells are calculated according to the formula: [(number of spots against relevant peptides − (number of spots against irrelevant peptides (HCV core 132)] × [(1 × 10⁶)/number of effector cells/well] (HBV envelope 378).
b (-) indicates <20 SFCs/10⁶ cells. Control irrelevant peptide were on average 6 SFCs/10⁶ cells for CB6F1, 5 for HLA-B7/K^b, 2 for K^b−/−D^b−/−, and 90 for CBA/J.

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Table IV.

HLA class I supertype binding capacity of vaccinia-derived T cell peptides

A. A2-Supertype Epitopes
Epitope	HLA-A2 Supertype Binding Capacity (IC₅₀ nM)^a
Epitope	A*0201	A*0202	A*0203	A*0206	A*6802
A14L (51–59)	0.11	+	+	+	+
VACWR050 (196–204)	1.5	+	+	+	−
A46R (142–150)	0.97	+	+	+	+
B14R (327–335)	124	-	-	+	-
M1L (374–383)	9.5	+	+	+	+
VACWR082 (18–26)	2.2	+	+	+	-
B6R (108–116)	1.8	+	+	+	+
I1L (211–219)	11	+	+	+	-
A17L (61–70)	20	+	+	+	-
D12L (251–259)	1.5	+	+	+	+
H3L (184–192)	2	+	+	+	-
A6L (6–14)	4.6	+	+	+	-
G7L (250–258)	2	+	+	+	+
A17L (61–70)	22	+	+	+	+

B. A3-Supertype Peptides
Epitope	HLA-A3 Supertype Binding Capacity (IC₅₀ nM)
Epitope	A*1101	A*0301	A*3101	A*3301	A*6801
I3L (116–124)	18	+	+	−	+
A10L (171–180)	11	+	+	−	+
E7R (130–138)	0.3	−	+	+	+
A47L (139–148)	470	−	−	−	−

C. B7-Supertype Peptides
Epitope	HLA-B7 Supertype Binding Capacity (IC₅₀ nM)
Epitope		B*0702	B*3501	B*5101	B*5301	B*5401
J2R (116–124)	202	−	−	−	−
D1R (808–817)	11	−	−	−	−
A34R (82–90)	14	−	−	−	−

a Bold font highlights intermediate affinity binders (IC₅₀<500 nM). A dash (-) indicates IC₅₀ >500 nM, and (+) indicates IC₅₀ <500 nM.

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Table V.

Structural and phylogenetic characteristics of VACWR immunogenic Ags

VACV-WR Gene Name	Ag Name^a	Protein Size	Time of Expression	Description	Function Category	Epitope	Restriction Element	% of Similarity with VAR-Major^b	% of Similarity with MVA^b
VACWR063	E7R	166	Late	Soluble, myristylprotein has N-myristyltransferase target	—	E7R (130–138)	HLA-A*1101	88.8 (E7R-VAR-IND)	100 (MVA054R)
VACWR094	J2R	177	Early	Thymidine kinase	Viral regulation	J2R (116–124)	HLA-B*0702	88.8 (L2R)	100 (MVA086R)
VACWR106	D1R	844	Early	Large subunit of mRNA capping enzyme	Viral regulation	D1R (808–817)	HLA-B*0702	100 (F1R)	90 (MVA098R)
VACWR157	A34R	168	Late	EEV glycoprotein	Virion structure	A34R (82–90)	HLA-B*0702	100 (A37R)	100 (MVA145R)
VACWR172	A46R	240	—	Toll/IL1-receptor [TIR]-like	Virulence factor	A46R (142–150)	HLA-A*0201	100 (A52R-VAR-IND; A49R-VAR-BSH)	100 (MVA159R)
VACWR188	B6R	173	—	Ankyrin-like	—	B6R (108–116)	HLA-A*0201	55.5 (A2L)	100 (MVA174R)
VACWR195	B14R	345	Early	Serpin, SPI-2/CrmA	Virulence factor	B14R (327–335)	HLA-A*0201	100 (B13R-VAR-IND; B12R-VAR-BSH)	55.5 (MVA025L)
VACWR030	M1L	472	Early	Ankyrin-like	—	M1L (374–383)	HLA-A*0201	100 (O1L)	≤50
VACWR070	I1L	312	Late	Virion core DNAbinding protein	Viral regulation	I1L (211–219)	HLA-A*0201	100 (K1L)	100 (MVA062L)
VACWR072	I3L	269	Early	ssDNA-binding phosphoprotein	Viral regulation	I3L (116–124)	HLA-A*1101	100 (K3L)	100 (MVA064L)
VACWR085	G7L	371	Late	IMV protein, proteolytic process	Virion structure	G7L (250–258)	HLA-A*0201	100 (H7L)	100 (MVA077L)
VACWR117	D12L	287	Early	Small subunit of mRNA capping enzyme	Viral regulation	D12L (251–259)	HLA-A*0201	100 (N2L)	100 (MVA109L)
VACWR125	A6L	372	—	—	—	A6L (6–14)	HLA-A*0201	100 (A6L-VAR-IND; A7L-VAR-BSH)	100 (MVA117L)
VACWR129	A10L	891	Late	Precursor p4a of core protein 4a	Virion structure	A10L (171–180)	HLA-A*1101	100 (A10L-VAR-IND; A11L-VAR-BSH)	100 (MVA121L)
VACWR133	A14L	90	Late	IMV membrane protein	Virion structure	A14L (51–59)	HLA-A*0201	100 (A14L-VAR-IND; A15L-VAR-BSH)	100 (MVA125L)
VACWR137	A17L	203	Late	IMV surface membrane protein	Virion structure	A17L (61–70)	HLA-A*0201	100 (A17L-VAR-IND; A18L-VAR-BSH)	100 (MVA128L)
VACWR137	A17L	203	Late	IMV surface membrane protein	Virion structure	A17L (81–90)	HLA-A*0201	100 (A17L-VAR-IND; A18L-VAR-BSH)	100 (MVA128L)
VACWR173	A47L	252	—	—	—	A47L (139–148)	HLA-A*1101	100 (J1L)	100 (MVA160L)
VACWR082	—	434	—	—	—	VACWR082 (18–26)	HLA-A*0201	100 (H5R)	100 (MVA074R)
VACWR050	—	348	—	—	—	VACWR050 (196–204)	HLA-A*0201	100 (C15L)	55.5 (MVA056L)
VACWR101	H3L	324	Late	IMV heparin binding surface protein	Virion structure	H3L (184–192)	HLA-A*0201	100 (I3L)	100 (MVA093L)

a Name from VACV-Cop. GenBank accession no. M35027.
b Percentage similarity of the determinant.
^cPercentage of similarity with ACAM3000 MVA176 (AAT10572 in GenBank protein database).

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Table VI.
Characteristics of the Ags recognized: Fisher exact statistical analysis

a. Size
ORFs Average Size
>100 aa ≤100 aa Total
Recognized 20 1 21
Other 163 74 237
Total 183 75 258
p = 0.0049.
b. Time of Expression
ORFs Time of expression
Early/Intermediate Late Total
Recognized 6 9 15
Other 50 49 99
Total 56 58 114
p = 0.1658.
c. Function Category
ORFs Virulence Factors Structural Viral Regulation Total
Recognized 2 7 5 14
Other 19 23 45 87
Total 21 30 50 101
p = 0.0176
d. Homology of VACWR-Epitope with Variola Major India and MVA Sequences
% Homology Variola MVA
100 18 17
80–99 2 1
51–79 1 2
≤50 0 1
Total 21 21