CD8 Cytotoxic T Cell Epitopes in Bovine Leukemia

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JVAC 17119 1–9 ARTICLE IN PRESS

Vaccine xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
1 Novel CD8+ cytotoxic T cell epitopes in bovine leukemia

2 virus with cattle
3 Q1 Lanlan Bai a,b , Shin-nosuke Takeshima a , Emiko Isogai b , Junko Kohara c , Yoko Aida a,∗
a
4 Viral Infectious Diseases Unit, RIKEN, Wako, Saitama 351-0198, Japan
b
5 Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi
6 981-8555, Japan
c
7 Animal Research Center, Hokkaido Research Organization, Shintoku, Hokkaido 081-0038, Japan
8
9
24 a r t i c l e i n f o a b s t r a c t
10
11 Article history: Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human
12 Received 28 August 2015 T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the pro-
13 Received in revised form 27 October 2015 gression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8+ CTL epitopes
14 Accepted 29 October 2015
remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env
15 Available online xxx
proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify
16
11 novel CD8+ T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16,
17 Keywords:
tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8+ T cell
18 Bovine leukemia virus
19 CD8+ CTL epitope mapping
epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among
20 Experimentally infected calves the 11 epitopes identified, only gp51N11 was capable of inducing CD8+ T cell-mediated cytotoxicity in
21 Bovine MHC class I all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism
22 CD8+ T cell proliferation according to the Wu–Kabat variability index. By contrast, no CTL epitopes were identified from the Gag
23 Cytotoxicity structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular
immunity against BLV is strongly targeted to these proteins. CD8+ CTL epitopes from gp30 and Tax were
less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-
rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide
contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell
activation. Moreover, at least one CD8+ CTL epitope derived from gp30 was identified in each of the four
calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV
vaccine.
© 2015 Published by Elsevier Ltd.
25 1. Introduction gp51 and gp30 are directly involved in infectivity events such as 37
receptor recognition and membrane fusion [5]. Unlike other retro- 38
26Q2 Bovine leukemia virus (BLV) is a member of the family Retro- viruses, BLV encodes at least two regulatory proteins, Tax and Rex. 39
27 viridae that the causative agent of enzootic bovine leukosis (EBL), Tax is a key contributor to the oncogenic potential, as well as a key 40
28 which is the most common neoplastic disease of cattle and is closely protein involved in the replication of the virus [1,2,6,7]. 41
29 related to human T cell leukemia virus (HTLV) [1,2]. BLV expresses The CD8+ cytotoxic T lymphocytes (CTLs) mediate cellular 42
30 the gag, pol, and env genes, which are both essential and common immunity by killing infected host cells and by producing cytokines 43
31 to retroviruses and are necessary for the production of infectious that can inhibit viral replication [8–10]. The major histocompat- 44
32 virions [3]. The gag gene of BLV is translated as the precursor Pr70 ibility complex (MHC) system in cattle is known as the bovine 45
33 Gag and is processed into three mature proteins: p15 is the matrix leukocyte antigen (BoLA) system, and is strongly involved in the 46
34 protein, p24 is the most abundant capsid protein, and p12 is the subclinical progression of BLV infection [10,11]. MHC molecules are 47
35 nucleocapsid protein [3,4]. The BLV env gene encodes a mature sur- grouped into two classes (I and II) that are highly polymorphic. The 48
36 face glycoprotein (gp51) and a transmembrane protein (gp30) [3]. ␣1 and ␣2 domains of MHC class I form a peptide binding groove 49
that accommodates antigen-derived peptides and presents them 50
to CD8+ T cells [10–12]. The binding of peptides to MHC class I pro- 51
∗ Corresponding author. Tel.: +81 48 462 4408; fax: +81 48 462 4399. teins is the most selective event in antigen presentation to CD8+ T 52
E-mail address: aida@riken.jp (Y. Aida). cells [13]. 53
http://dx.doi.org/10.1016/j.vaccine.2015.10.128
0264-410X/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Bai L, et al. Novel CD8+ cytotoxic T cell epitopes in bovine leukemia virus with cattle. Vaccine (2015),
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2 L. Bai et al. / Vaccine xxx (2015) xxx–xxx
54 Cellular immunity is induced during natural BLV infection and initial screening were tested for their ability to induce CD8+ T cell 72
55 plays a role in protecting hosts from BLV infection [9,14–16]. Previ- cytotoxicity in a non-radioactive cytotoxicity assay; (v) The con- 73
56 ous studies postulated that cellular immunity against BLV antigens servation of amino acids in CD8+ CTL epitopes was analyzed by the 74
57 contributes to the suppression of BLV replication, thereby leading Wu–Kabat variability index; and (vi) the expression of these epi- 75
58 to the delay in disease progression [9,14–22]. However, in these topes by cattle with different BLV proviral loads is described. These 76
59 previous studies, only a small number of peptides corresponding results may help to select the best candidates for synthetic peptide 77
60 to selected regions of the BLV Env and Tax proteins were used to vaccine against BLV infection. 78
61 characterize CD4+ and CD8+ T cell epitopes for BLV. Here, we iden-
62 tified CD8+ T cell-restricted epitopes for BLV antigens as follows: 2. Materials and methods 79
63 (i) First, a library of 115 overlapping peptides covering the entirety
64 of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, 2.1. Synthetic peptides 80
65 and p12), and the Tax protein was generated; (ii) Four calves with
66 defined BoLA class I genotypes were experimentally infected with A series of 20-mer peptides overlapping by 10 amino acids were 81
67 the amount of BLV taken from the same individual at the same designed based on the reported sequences of each mature pep- 82
68 time; (iii) Mapping of the CD8+ T cell epitopes was performed by tide encoded and expressed by genes in env, gag, and tax. All of 83
69 the water-soluble tetrazolium salt (WST-8) assay using the syn- peptides were synthesized and purified to >70% purity (Sigma). 84
70 thetic peptide library and CD8+ T cells from the four BLV-infected These peptides are divided into 23 pools were suspended in 80% 85
71 calves; (iv) CD8+ T cell epitopes that induced proliferation in the dimethylsulfoxide (DMSO) (Fig. 1). 86
Fig. 1. Synthetic peptides of 20-mer overlapping peptides corresponding to the Gag (p15, p24, and p12), Env (gp51 and gp30) and Tax proteins. These overlapping peptides
were made against each mature peptide encoded and expressed by genes in env, gag, and tax, which sequences of BLV Gag (GenBank accession no. LC057268), Env (GenBank
accession no. EF600696), and Tax (GenBank accession no. EF600696) were reported. (A) Schematic representation of genome organization of BLV. (B) The pool, name, sequence
and amino acid positions of peptide are shown in column (left to right). The pool contains five individual peptides. a 15 amino acids in length; b 19 amino acids in length; and
c
18 amino acids in length.
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L. Bai et al. / Vaccine xxx (2015) xxx–xxx 3
87 2.2. Experimental animals prepared by treating CD8− cells with 50 ␮g/ml mitomycin C (Sigma) 127
for 30 min. APCs (2 × 106 cells) and CD8+ T cells (2 × 106 cells) were 128
88 Four BLV-negative Japanese Black calves (6–8-months-old) mixed in the presence of either peptide pool containing individual 129
89 were experimentally challenged intravenously with blood con- peptides at 20 ␮M, 20 ␮M positive peptide, or 80% DMSO (negative 130
90 taining a proviral load of 4 × 107 proviral copies per 105 control) at once, and were co-incubated at 37 ◦ C in a total volume 131
91 cells, as determined by the BLV-coordination of common of 110 ␮l of cell medium. After 109 h of incubation, 10 ␮l of WST-8 132
92 motifs-quantitative real-time polymerase chain reaction-2 (BLV- solution (Dojindo Molecular Technologies) was added to each well, 133
93 CoCoMo-qPCR-2) assay from BLV-infected Holstein-Friesian cattle and then the cells were incubated for 4 h. The microplate was read 134
94 (Table 1). This study was approved by the Animal Ethical Commit- at an optical density (OD) of 450 nm. 135
95 tee and the Animal Care and Use Committee of the Animal Research
96 Center, Hokkaido Research Organization (approval number 1302). 2.7. CytoTox96 non-radioactive cytotoxicity assay 136
97 2.3. Amplification, cloning, and DNA sequencing of BoLA class I The CD8+ cytotoxicity were performed by non-radioactive cyto- 137
toxicity assay. It is a colorimetric alternative to 51 Cr release 138
98 RNA was extracted from peripheral blood mononuclear cells cytotoxicity assays. It quantitatively measures lactate dehydroge- 139
99 (PBMCs) obtained from each of experimantal calves and were nase (LDH), a stable cytosolic enzyme that is released upon cell lysis. 140
100 reverse-transcribed into cDNA to obtain BoLA class I heavy Released LDH in culture supernatant is measured with a 30 min 141
101 chain cDNA clones. cDNAs were amplified by PCR using primers coupled enzymatic assay, which results in the conversion of a tetra- 142
102 BoLA 1F (5 -ATGCGAGTYATGGGGCCGCGAGCCC-3 ) and BoLA 1R zolium salt (INT) into a red formazan product. The amount of color 143
103 (5 -TCAMMCTTTAGGAACYGTGAGA-3 ). The PCR products were formed is proportional to the number of lysed cells. Effector cells 144
104 subcloned into the pGEM-T easy vector (Promega). The sequences (CD8+ T cells; 1 × 107 cells/50 ␮l or 2 × 107 cells/50 ␮l) were plated 145
105 were analyzed using softwares, Sequencher ver. 4.0.5 (Gene Codes), in the experimental wells to measure spontaneous LDH release by 146
106 and Genetyx ver. 10, software (Genetyx). effector cells. Meanwhile, different concentrations of peptide were 147
added to the experimental wells, and target cells (PBMCs; 1 × 106 148
107 2.4. Measurement of the BLV proviral load and anti-BLV cells/50 ␮l) were added to the experimental wells and the wells 149
108 antibodies measuring minimum (spontaneous) and maximum LDH release 150
and mixed at once. The microplate was incubated for 48 h that 151
109 The BLV proviral load was measured using the BLV-CoCoMo- the effector cells were sufficiently activated by peptide at 37 ◦ C. 152
110 qPCR-2 method [23–26]. An anti-BLV antibody ELISA kit (JNC) was Ten ␮l of Lysis solution was added to the maximum LDH release 153
111 used according to the manufacturer’s instructions. wells 45 min prior to the end of incubation period. After incuba- 154
tion, 50 ␮l of supernatant was transferred from each well into a 155
112 2.5. Preparation of PBMCs and CD8+ T lymphocytes fresh microplate for the enzymatic assay. Next, 50 ␮l of the recon- 156
stituted substrate mix was added to each well and incubated at 157
113 PBMCs were separated according to the method of Miyasaka and room temperature for 30 min. Finally, 50 ␮l of stop solution was 158
114 Trunka [27]. CD8+ cells were purified using the 7C2B monoclonal added and was recorded the absorbance at 492 nm after 45 min 159
160
115 antibody (MAb) (mouse anti-bovine CD8; VMRD) and anti-mouse incubation. The percent cytotoxicity was calculated as follows:
161
116 IgG Mab by the MACS System (Miltenyi Biotech). The CD8+ T
117 lymphocyte-depleted PBMCs were used as the CD8− cells in this Percent cytotoxicity 162
118 study. experimental − effector spontaneous − target spontaneous

= 163
target maximum − target spontaneous
119 2.6. WST-8 assay
× 100 164
165
120 The cell proliferation response against peptides was performed
121 by WST-8 assay [44]. It is a determination of cell viability in cell
122 proliferation that was reduced by dehydrogenase activities in cells 2.8. Statistical analysis 166
123 to give a yellow-color formazan dye, which was soluble in the tissue
124 culture media. The amount of the formazan dye, generated by the The conservation of epitopes was analyzed by Wu–Kabat vari- 167
125 activities of dehydrogenases in cells, was directly proportional to ability index [28]. The function program in Microsoft Excel was used 168
126 the number of living cells. Antigen-presenting cells (APCs) were to conduct F-tests and t-tests for all results of cytotoxicity. 169
Table 1
Japanese black calves examined in the studya .
Animal no. Proviral loadb Breed Age (month) Sex WBCsc (cells/␮l) PBLsd (cells/␮l) BoLA class 1e Allele Antibodies
(CoCoMo-qPCR-2) to BLVf
A-JB 72 Japanese black 6 ♀ 7480 4520 BoLA-N*02604 +

B-JB 58,501 Japanese black 7 ♂ 13400 8650 BoLA-N*02702 +
C-JB 67,729 Japanese black 6 ♀ 13500 9250 BoLA-N*02604 +
D-JB 76,839 Japanese black 8 ♂ 14100 8820 BoLA-N*02701 +
a
Blood samples (used for DNA and serum isolation) were collected for 10 weeks after the first inoculation.
b
Proviral load (expressed as the number of copies of provirus per 105 peripheral blood mononuclear cells) was evaluated using CoCoMo-qPCR-2.
c
White blood cells (WBCs) were measured at a single time point in all calves during the experiment.
d
Peripheral blood lymphocytes (PBLs) were measured at a single time point in all calves during the experiment.
e
BoLA class1 alleles are according to the BoLA nomenclature committee of the IPD–MHC database, calves A-JB and C-JB carried allele (GenBank accession no. LC055984)
which is identical to the previously reported allele, BoLA-N*02604, D-JB carried a novel allele, BoLA-N*02701 (GenBank accession no. LC055985), and B-JB carried allele
(GenBank accession no. LC055986) which is identical to the previously reported allele, BoLA-N*02702. The amino acid sequences of BoLA class were determined from cDNA
clone sequences using the pGEM® -T Easy Vector System. The allele was considered one of the predominantly expressed types in each experiment animal.
f
ELISA (enzyme-linked immunosorbent assay) to detect anti-BLV antibodies was performed using the BLV ELISA kit + BLV positive serum.
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170 3. Results gp30N16 and gp51N11, induced particularly high CD8+ T cell pro- 205
liferation in calf A-JB, which had 72 copies BLV proviral load per 206
171 3.1. Preparation of synthetic peptides and BLV-infected 105 cells (Fig. 3A). By contrast, the three other calves with higher 207
172 experimental calves proviral loads than A-JB responded to a greater number of pep- 208
tides. In calf C-JB, which had 67,729 copies BLV proviral load 209
173 To characterize CD8+ T cell epitopes for BLV, previous studies per 105 cells significant CD8+ T cell proliferation was observed 210
174 [15,20,22,29] used a small number of peptides derived from por- in response to stimulated with five peptides (gp30N6, gp30N8, 211
175 tions of the Env and Tax proteins. To conduct a more extensive gp51N11, gp51N12, and tax19) (Fig. 3C). Interestingly, calf D-JB 212
176 screen for CTL epitopes, we prepared a library of 115 synthetic pep- which proviral load was 76,839 copies per 105 cells, showed CD8+ 213
177 tides derived from the entire Env, Gag, and Tax proteins (Fig. 1). T cell proliferative responses to the largest number (seven) of 214
178 Mapping of CD8+ T cell epitopes was previously performed using peptides (gp30N6, gp51N5, gp51N11, gp51N12, tax16, tax18, and 215
179 lymphocytes from mice, sheep and cattle [15,20,22,29]. Here, four tax20) (Fig. 3D). Peptides gp30N5, gp30N6, gp30N16, and gp51N11 216
180 calves of the same age were experimentally infected with the same increased the CD8+ T cell proliferation for calf B-JB, which proviral 217
181 amount of BLV taken from a single individual infected cow (Table 1). load was 58,501 copies per 105 cells (Fig. 3B). 218
182 In addition, the BoLA class I alleles of experimental calves were The number of CD8+ T cell epitopes was correlated with the 219
183 determined. According to the BoLA nomenclature committee of proviral load in BLV-infected calves. In addition, all four calves 220
184 the IPD–MHC database, calves A-JB and C-JB carried allele, BoLA- responded to peptide gp51N11, indicating that this peptide was the 221
185 N*02604, B-JB carried allele, BoLA-N*02702, and D-JB carried a novel strongest at inducing CD8+ T cell proliferation. Notably, no epitopes 222
186 allele, BoLA-N*02701. The proviral loads of infected calves were were found from Gag structural proteins. 223
187 estimated by BLV-CoCoMo-qPCR-2 [23–26]. These infected calves

188 were proviral loads ranged from 72 to 76,839 copies per 105 cells 3.3. Non-radioactive cytotoxicity assay 224
189 (Table 1).
To identify the immunodominant epitope, all 11 peptides that 225
190 3.2. Proliferation of CD8+ T cells from BLV-infected calves induced a CD8+ T cell proliferative response were tested for their 226
ability to induce cytotoxicity. First, the optimal conditions for cyto- 227
191 CD8+ T cells were stimulated with the peptide pools that individ- toxicity assay were determined for using autologous PBMCs as 228
192 ual peptide at 20 ␮M and proliferations were measured by WST-8 supplemental APCs, and the optimal ratio of effector cells and tar- 229
193 assay. APCs were prepared by the CD8+ T cell-depleted PBMCs that get cells was determined as 10:1 for calf A-JB and 20:1 for the 230
194 treating with mitomycin C for 60 min before to inhibit cells prolif- other three calves when using 5 × 104 PBMCs per well (data not 231
195 eration. Fig. 2 shows that peptide pools 9, 10, 11, 13, 14, 15, and shown). 232
196 21 induced higher CD8+ T cell proliferation for calf A-JB; pools 8, 9, Next, non-radioactive cytotoxicity assays were carried out by 233
197 11, 14, and 21 induced higher CD8+ T cell proliferation for calf B-JB; incubating APCs with CD8+ T cells in the presence of different con- 234
198 pools 9, 11, 14, 20, and 21 induced higher CD8+ T cell proliferation centrations (0, 0.5, 5, or 20 ␮M) of peptides. Fig. 4A shows the 235
199 for calf C-JB; and pools 9, 13, 14, 15, and 21 induced higher CD8+ T results obtained from calf A-JB, gp30N16 and gp51N11 induced 236
200 cell proliferation for calf D-JB. cytotoxic activity in a dose-dependent manner. By contrast, in calf 237
201 To further map the CD8+ T cell epitopes recognized by exper- C-JB, all five peptides significantly increased the CTL activity com- 238
202 imental calves, CD8+ T cell proliferative responses to the each pared to the negative control, but none displayed dose-dependent 239
203 peptide within positive peptide pools were examined in the WST- activity (Fig. 4C); a concentration of 0.5 ␮M, which was the low- 240
204 8 assay (Fig. 3). On this second screening, only two peptides, est concentration tested for each peptide, induced near-maximal 241
Fig. 2. CD8+ T cell proliferative responses to 23 peptide pools. CD8+ T cells were separated from PBMCs from four BLV-infected calves [A-JB (A), B-JB (B), C-JB (C), and D-JB (D)]
and used as effector cells. CD8− cells were treated with 50 ␮g/ml mitomycin C for 60 min at 37 ◦ C and used as APCs. CD8+ T cells (1 × 105 cells/50 ␮l) and CD8− cells (1 × 105
cells/50 ␮l) were treated with 80% DMSO (negative control) or with 23 peptide pools, each containing five individual peptides at 20 ␮M of each peptide. After incubation for
109 h at 37 ◦ C, the CD8+ T cell proliferation responses were examined in a WST-8 assay. The bars represent the mean ± standard deviation (S.D.) of triplicate wells.
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Fig. 3. CD8+ T cell proliferative responses to individual peptides within positive peptide pools. CD8+ T cells (effector cells; 1 × 105 cells/50 ␮l) from four BLV-infected calves
[A-JB (A), B-JB (B), C-JB (C), and D-JB (D)] and CD8− cells (APCs; 1 × 105 cells/50 ␮l) were treated with mitomycin C and incubated with 80% DMSO (negative control) or 20 ␮M
of each peptide from pools 9, 10, 11, 13, 14, 15, and 21 for calf A-JB, pools 8, 9, 11, 14, and 21 for calf B-JB, pools 9, 11, 14, 20, and 21 for calf C-JB, and pools 9, 13, 14, 15, and
21 for calf D-JB. The cells were treated with peptide for 109 h at 37 ◦ C and the CD8+ T cell proliferation responses were examined in a WST-8 assay. The bars represent the
mean ± standard deviation (S.D.) of triplicate wells.
242 cytotoxicity. In contrast to A-JB and C-JB, D-JB showed differing pat- and tax20 peptides induced cytotoxicity in a dose-dependent man- 246
243 terns of CTL activity depending on the peptides (Fig. 4). However, ner in D-JB, while gp51N12 had little effect. As shown in Fig. 4B, 247
244 CTL lysed target cells in response to gp51N11 only at the highest the gp30N16 peptide showed the cytotoxic activity in a dose- 248
245 concentration tested (20 ␮M). On the other hand, the tax16, tax18, dependent manner, reaching 80.9% and 77.9% cytotoxicity at 20 ␮M 249
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Fig. 4. Cytotoxic activity of CD8+ cytotoxic T cells against autologous PBMCs in response to peptides. These peptides had CD8+ T cell proliferative response in the WST-8 assay.
CD8+ T cells (effector cells, EC) were separated from PBMCs from four BLV-infected calves [A-JB (A), B-JB (B), C-JB (C), and D-JB (D)] and incubated with autologous PBMCs
(target cells, TC) with different concentrations of peptide (0, 0.5, 5, or 20 ␮M), and the cytotoxic activity of the CD8+ T cells was examined in a CytoTox96 non-radioactive
cytotoxicity assay. A-JB was tested at an EC:TC ratio of 10:1, while B-JB, C-JB, and D-JB were used at an EC:TC ratio of 20:1. The bars represent the mean ± standard deviation
(S.D.) of triplicate wells. Significant differences between the groups are indicated by asterisks (* p < 0.05, ** p < 0.01, and *** p < 0.001).
250 in calves B-JB and A-JB, respectively, while 0.5 ␮M of each of the as T cell and B cell epitopes [15,20,22,29], but so far no T cell or 277
251 other three peptides was sufficient to kill target cells in B-JB. B cell epitopes from gp30 have been reported. On the contrary, 278
no epitopes were found in Gag proteins. Second, fewer CD8+ CTL 279
252 3.4. Estimation of the Wu–Kabat variability index for positive epitopes were identified from calf A-JB with the low proviral load, 280
253 peptides and more CD8+ CTL epitopes were identified from higher provi- 281
ral load than A-JB. This result strongly suggests that the number 282
254 The conservation of amino acids in the 11 CD8+ CTL epitopes of CD8+ T cell epitopes correlates with the proviral load in BLV- 283
255 was evaluated using the Wu–Kabat variability index. Fig. 5 shows infected cattle. Third, although several different CD8+ CTL epitopes 284
256 the Wu–Kabat variability indexs of the 11 CD8+ CTL epitopes based were identified from individual calf, a common CD8+ CTL epitope, 285
257 on information on Env and Tax amino acid sequence variants gp51N11, has capability of inducing cytotoxicity in all four calves. 286
258 obtained from the Genbank database. A value of 1 indicates that Thus, the predominant CD8+ CTL epitope may be located in the cen- 287
259 an amino acid site is monomorphic, while a value of >2 indicates tral portion of gp51. However, gp51N11 is not considered a suitable 288
260 a polymorphic site [28]. Interestingly, four residues of gp51N11 target for a vaccine because it shows high variability according to 289
261 and three residues of gp51N12 had values exceeding 4 that the the Wu–Kabat variability index. Fourth, Wu–Kabat variability index 290
262 highest variability values (Fig. 5A). By contrast, the other epitopes analysis clearly showed that CD8+ CTL epitopes corresponding to 291
263 of gp30 were slightly less polymorphic than the epitopes within gp30 and Tax were less polymorphic than those of gp51. In addition, 292
264 the gp51 (Fig. 5B). Particularly, the gp30N16 contains a proline- CD8+ CTL epitopes from all experimental calves were derived from 293
265 rich region upstream of immunoreceptor tyrosine-based inhibition the gp30 protein but not the Tax protein, indicating that the BLV 294
266 motifs (ITAMs) and immunoreceptor tyrosine-based inhibition gp30 region may be the best candidate for vaccine development. 295
267 motif (ITIMs) [30]. In addition, identified tax epitope region, which The number of CD8+ T cell epitopes appeared to correlate with 296
268 include a leucine-rich activation domain that encompasses a trans- the proviral load in BLV-infected cattle as evidence by: (i) The 297
269 criptional activation region [31], are less polymorphic (Fig. 5C). number of peptides that induced good CD8+ T cells proliferation 298
270 Indeed, only one residue in each of epitopes gp30N5 and tax16 had increased with the BLV proviral load in the experimental calves; 299
271 values exceeding 4 (Fig. 5A and B). (ii) Four epitopes on the Tax protein were detected in only two 300
cattle with viral loads of 67,729 and 76,839 copies per 105 cells. 301
272 4. Discussion This suggests that Tax-specific CTL may be induced in animals with 302
higher (>58,501 copies per 105 cells) proviral loads because the 303
273 Here, we determined 11 novel CD8+ CTL epitopes and produced expression of Tax protein is very weak compared with the expres- 304
274 four major conclusions. First, we found it interesting that this is sion of the Env proteins; (iii) gp30N16-specific CTL was only found 305
275 the first time T cell epitopes from the gp30 have been identified. in the calves A-JB and B-JB. gp30N16 encompasses a proline-rich 306
276 In previous reports, some regions of gp51 and Tax were identified region upstream of the ITAMs and ITIMs of the cytoplasmic tail 307
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Fig. 5. Wu–Kabat variability index of the CD8+ CTL epitopes on all sequences of gp51, gp30, and Tax. The 517 sequences of gp51, 118 sequences of gp30, and 90 sequences
of Tax were obtained from the GenBank database. The vertical axis indicates the Wu–Kabat index at each amino acid position and the horizontal axis shows the amino acid
positions. (A) The Wu–Kabat index of the gp51 region (amino acids 1-301). Red, blue, and yellow blocks indicate the CD8+ CTL epitopes gp51N5, gp51N11, and gp51N12,
respectively. (B) The Wu–Kabat index of the gp30 region (amino acids 302–515). The blue, brown, green, and pink blocks indicate the CD8+ CTL epitopes gp30N5, gp30N6,
gp30N8, and gp30N16, respectively. (C) The Wu–Kabat index of Tax region (amino acids 1–309). The blue, red, green, and pink blocks indicate the CD8+ CTL epitopes tax16,
Q4 tax18, tax19, and tax20, respectively. (D) The sequences and Wu–Kabat index at each position of the CD8+ CTL epitopes (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.).
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308 of the envelope protein [30], which is known to be an SH3 bind- Stability is an important factor for developing epitope- 374
309 ing site [32]. In addition, the down-regulatory phosphatase SHP-1 driven vaccines. In previous study [46], the peptide 121-145 of 375
310 acts as a critical negative regulator of B-cell receptor signaling, and PPE68 (Rv3873) which was a major antigenic protein encoded 376
311 gp30 was thought be may act as a decoy to sequester SHP-1, Thus, by Mycobacteriun tuberculosis-specific genomic region of dif- 377
312 gp30N16 was thought maybe relevant in the context of T and B ference 1 was identified by antigen-specific T-cell lines from 378
313 mediated immunity [45]. Therefore, CTL targeting this domain may HLA-heteregeneous subjects, and conserved in all sequenced 379
314 be efficient at eliminating BLV-infected cells by cytotoxic activity. strains/species of M. tuberculosis and M. tuberculosis complex, and 380
315 In another previous study, synthetic peptides corresponding to several other pathogenic mycobacterial species; the peptide aa 381
316 the gp51 were used to test helper T cell epitopes, and peptide 121–145 was thought to be exploited as a peptide-based vaccine 382
317 98–117 was able to induce B cell neutralizing responses in mice candidate against tuberculosis and other mycobacterial diseases. 383
318 and a particularly strong helper T cell response in BLV-infected Therefore, we analyzed the conservation of amino acids in the 384
319 sheep [15]. The 17 residues within gp51N11, which was identi- 11 CD8+ CTL epitopes using the Wu–Kabat variability index. All 385
320 fied in this study, correspond to peptide 98–117. Fig. 4 clearly three epitopes from gp51 showed significantly higher variability 386
321 illustrates that gp51N11 has cytotoxic activity on autologous BLV- than epitopes from gp30 and Tax. In particular, gp51N11 which 387
322 infected cells from all four calves. The BoLA class I allele is highly induced responses in four all experimental calves, had the highest 388
323 polymorphic [10,11]. Indeed, the major BoLA class I allele was dif- Wu–Kabat variability value. The epitope gp51N12 showed a sim- 389
324 ferent in each of the four calves. The gp51N11 epitope may contain ilar pattern as gp51N11, in that it induced cytotoxic activity on 390
325 recognition site that binds to one or more BoLA class I alleles. autologous BLV-infected cells and had a high Wu–Kabat variability 391
326 Peptides 169–188 and 177–192 of gp51 induce good proliferation value. These data indicate that gp51N11 and gp51N12 are more 392
327 of bovine PBMCs depleted of B cells [14]. These peptides corre- variable at amino acid positions 141 and 155, respectively. The 393
328 spond to 12 residues within gp51N17, full length gp51N18, and gp51 mediates binding of virions to cell surface receptors [42], and 394
329 12 residues within gp51N19. Another peptide derived from Env, induces the expression of specific antibodies in infected animals 395
330 pep61, contains some sequences that induce T cell proliferation [1]. Variability within the gp51N11 and gp51N12 epitopes allow for 396
331 or mitogenicity in mice [21]. This peptide corresponds closely to efficient modification of host immune responses. Although many 397
332 gp51N7, which did not show CD8+ T cell proliferative activity in this vaccine trials have been undertaken [43], an effective vaccine has 398
333 study. Thus, the epitopes gp51N17–19 may be significant recogni- yet not been developed against BLV maybe because vaccine strate- 399
334 tion sites in some cattle, but were not identified as such in the four gies have focused on the Env protein gp51. In contrast to gp51, 400
335 experimental cattle in our study. epitopes derived from gp30 and Tax are less polymorphic. These 401
336 We identified four tax CD8+ CTL epitopes that correspond to the results provide useful information for BLV vaccines development. 402
337 region approximately halfway between amino acids 151 and 210
338 of the BLV Tax protein. Interestingly, this region of Tax contains
5. Conclusions 403
339 a leucine-rich activation domain between residues 157 and 197
340 that contains 24% of the protein’s leucine residues and is possibly
Eleven novel CD8+ CTL epitopes were identified. However, no 404
341 involved in heterologous protein interactions [31]. The leucine-rich
CD8+ CTL epitopes were found from the Gag proteins. Interestingly, 405
342 activation domain also encompasses a transcriptional activating
several epitopes were obtained from Tax and gp30, indicating that 406
343 region that plays an important role in viral replication [31]. In
cellular immunity against BLV is strongly targeted to these proteins. 407
344 addition, these CD8+ CTL epitopes on the Tax protein have few poly-
Tax protein has a few polymorphic sites, but tax epitopes just were 408
345 morphic sites (Fig. 5C). Various studies showed that the HTLV Tax
identified in two calves. At least one epitope from gp30 region was 409
346 protein contains several CTL epitopes [33–38] and induces strong
identified in all four calves, and these epitopes have high conserva- 410
347 CTL responses against HTLV-1-infected patients [39–41]. These
tion than epitopes of gp51. Although CTL epitopes were identified 411
348 results suggest that the tax CD8+ CTL epitope may be a potential
from the gp51, they have high variability and therefore not a suit- 412
349 candidate for an anti-BLV vaccine.
able target for designing a vaccine. These results show that the gp30 413
350 By contrast, Sakakibara et al. reported that peptides of the BLV
region may be the best candidate for BLV vaccine development. 414
351 Tax protein at amino acids 111–130 and 131–150, and at 111–130
352 and 261–280, are T cell epitopes and B cell epitopes for mice,
353 respectively [20]. These sequences of Tax corresponded to pep- Acknowledgments 415
354 tides tax11–13, tax13–15, and tax 26–28 in our study, which did
355 not induce CD8+ T cell proliferation. We identified CD8+ CTL epi- We thank Mr. Sunao Hirai, Mr Susumu Ogawa, Mr. Kenji 416
356 topes of Tax that induced high cytotoxic activity against autologous Mizushiri, Mr. Naoya Takahashi, and Mr. Yoshihiro Okada of Ani- 417
357 BLV-infected cells. The T cell epitopes that have been identified in mal Research Center, Hokkaido Research Organization, for help 418
358 mice by Sakakibara et al. may therefore not be CD8+ CTL epitopes with drawing blood. We also thank Mis. Yuki Matsumoto, Mrs 419
359 in cattle, indicating that there are differences in CD8+ CTL epitopes Mari Kikuya, Mis. Yuka Takahashi, Dr. Ayumu Ohno, and other 420
360 between mice and cattle. members of the Viral Infectious Diseases Unit, RIKEN, for excellent 421
361 The transmembrane protein gp30 is a glycosylated polypeptide technical assistance, kind help, and suggestions. We are grateful 422
362 [1,2] that can induce the fusion of viral and cellular membranes to the Support Unit for Biomaterial Analysis, RIKEN BSI Research 423
363 in infected cells [42]. However, it was unclear whether CD8+ CTL Resources Center, for help with sequence analysis. This work was 424
364 epitopes are present in the gp30. In this paper, four CD8+ CTL supported by a grant from the Program for the Promotion of Basic Q3 425
365 epitopes were identified in gp30 for the first time. In particular, and Applied Research for Innovations in Bio-oriented Industry and 426
366 gp30N16 showed strong cytotoxic activity in calves A-JB and B-JB, by a grant from Integration Research for Agriculture and Interdis- 427
367 respectively. In addition, the cytotoxic activity at 0.5 ␮M of gp30N6 ciplinary Fields in Japan. 428
368 was 35.2–68.6% in calves B-JB, C-JB, and D-JB. Thus, CD8+ CTL epi-
369 topes from the gp30 were identified in all four BLV-infected calves,
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CD8 Cytotoxic T Cell Epitopes in Bovine Leukemia

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CD8 Cytotoxic T Cell Epitopes in Bovine Leukemia

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JVAC 17119 1–9 ARTICLE IN PRESS

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1 Novel CD8+ cytotoxic T cell epitopes in bovine leukemia

receptor recognition and membrane fusion [5]. Unlike other retro- 38

that accommodates antigen-derived peptides and presents them 50

to CD8+ T cells [10–12]. The binding of peptides to MHC class I pro- 51

E-mail address: aida@riken.jp (Y. Aida). cells [13]. 53

toxicity assay. It is a colorimetric alternative to 51 Cr release 138

tion, 50 ␮l of supernatant was transferred from each well into a 155

118 study. experimental − effector spontaneous − target spontaneous

A-JB 72 Japanese black 6 ♀ 7480 4520 BoLA-N*02604 +

187 estimated by BLV-CoCoMo-qPCR-2 [23–26]. These infected calves

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