Vol. 66, No. 11
JOURNAL OF VIROLOGY, Nov. 1992, p. 6788-6793
0022-538X/92/116788-06$02.00/0 Copyright © 1992, American Society for Microbiology
Identification of T-Cell Epitopes on E2 Protein of Rubella Virus, as Recognized by Human T-Cell Lines and Clones DAWEI OU,1 PELE CHONG,2 YVONNE CHOI,2 PAUL McVEIGH 2 WILFRED A. JEFFERIES 3,4,5,6 GERASINOS KOLOITIS,3'4'5'6 AUBREY J. TINGLE,17 AND SHIRLEY GILLAM1* Department of Pathology' and Department of Paediatrics, 7 Research Centre, University of British Columbia, 950 West 28th Avenue, Vancouver, British Columbia VSZ 4H4, Connaught Centre for Biotechnology Research, Wllowdale, Ontario M2R 3T4,2 and Biotechnology Laboratory3 and Departments of Medical Genetics,4 Microbiology,5 and Zoology,6 University of British Columbia, Vancouver, British Columbia V6T 1 W5, Canada Received 1 April 1992/Accepted 10 August 1992
T-cell epitopes on the E2 protein of rubella virus were studied by using 15 overlapping synthetic peptides covering the E2 protein sequence. The most frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 tested T-cell lines responding to it. Two CD4+ cytotoxic T-cell clones isolated from one T-cell line responded strongly in proliferation assays with peptide E24 and were cytotoxic to target cells presenting the E2-4 determinant. Truncated peptides contained within the E2-4 peptide sequence were used to define the T-cell determinants. Results indicated that amino acid residues 54 to 65 were directly involved. Human cell lines with different HLA phenotypes were tested for the capacity to present the antigenic determinants. The results suggested that recognition of peptide E2-4 by T-cell clones was associated with HILA DR7.
Rubella virus (RV) is the causative agent of German measles. Immunization of infants and susceptible women of child-bearing age against RV is now a standard public health measure (1). RV vaccines in current use contain live, attenuated virus (29). The development of both acute and chronic forms of arthritis has been reported in association with both RV infection and immunization (7, 13). As well, potential concerns remain over the long-term consequences of RV vaccine infection in developing fetuses after accidental maternal immunization during pregnancy (31). A new approach using synthetic subunit vaccines is an alternative to the conventional, attenuated RV vaccines (24, 25). The development of a synthetic subunit vaccine requires the identification of T- and B-cell epitopes of RV structural proteins. RV is the sole member of the genus Rubivirus of the family Togaviridae (22). The virions contain three structural proteins, El (58 kDa), E2 (42 to 47 kDa), and C (33 kDa). El and E2 are membrane glycoproteins located on the virion exterior, and the capsid protein (C) is associated with the genomic RNA forming the nucleocapsid (26). The biological role of E2 is not well defined, although it has been reported to possess strain-specific epitopes (11) and possibly at least one neutralizing domain (16). B-cell epitopes of the E2 protein defined by monoclonal antibodies (MAbs) have been reported (34). We have recently demonstrated capsid antigen-specific T-cell responses in seropositive humans (27). The fine specificity and genetic restriction of the T-cell clones reactive to the three immunodominant T-cell epitopes on C protein of RV were characterized by using synthetic peptides of C protein (28). Little is known about the cell-mediated immune responses to the E2 protein of RV. In order to identify the T-cell epitopes on E2, 15 overlapping synthetic peptides covering the entire E2 protein sequence of RV strain M33 (9) were synthesized in an automated peptide synthesizer (23, 27) to
*
screen for the peptide-specific T-cell lines and clones. Figure 1 shows the locations of these peptides and the profiles of the indices of a-helix and n-turn and the hydrophilicity plots calculated for E2. Using these overlapping synthetic peptides, we have studied the proliferative responses of 10 human RV-specific T-cell lines derived from seropositive healthy donors and RV vaccine recipients to E2 epitopes of RV. Donors 1 to 5 were healthy individuals (average age, 36 years) with seropositive responses to RV structural proteins in immunoblot analysis (data not shown). Donors 6 to 10 were RV vaccine (RA27/3) recipients (average age, 24 years) whose blood samples were collected 3 weeks after administration of RV vaccine. RV-specific T-cell lines were derived from the peripheral blood mononuclear cells (PBMC) of donors by stimulation with UV-inactivated RV and then expansion with interleukin-2 (27). The frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 T-cell lines responding, E2-13 (residues 218 to 239), with 4 of 10 lines responding, and E2-14 (residues 233 to 257), with 3 of 10 lines responding (Table 1). Peptides E2-1 (residues 1 to 20), E2-2 (residues 15 to 36), E2-8 (residues 124 to 146), E2-10 (residues 156 to 177), and E2-11 (residues 176 to 199) were also recognized by some of these T-cell lines (Table 1). Since, of 15 E2 peptides, E2-4 peptide was recognized at the
highest frequency by T-cell lines, T-cell clones (A3 and A9) specific for E2-4 peptide were established from T-cell line 3 by limiting dilution as previously described (5, 6, 28) for detailed analysis. Both clones showed specific proliferation with UV-inactivated RV strain M33 (data not shown). By using overlapping E2 synthetic peptides, T-cell clones A3 and A9 were found to respond strongly to the E2-4 peptide (residues 54 to 74) in proliferation assays (Fig. 2A). Surface phenotype analysis in a fluorescence-activated cell sorter showed that more than 95% of cells in T-cell lines and 99% of cells in T-cell clones were CD3+, CD4+, and CD8- (data not shown), demonstrating that these T-cell lines and clones belong to the T-helper cell class. The cytotoxic activities of T-cell clones A3 and A9 were studied by using autologous Epstein-Barr virus-transformed
Corresponding author. 6788
NOTES
VOL. 66, 1992
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Residue Number FIG. 1. Predicted structure of RV E2 protein by conventional structural analysis algorithms. (Top) Secondary-structure analysis of local a-helix and ,B-turn potentials according to the method of Chou and Fasman (8); (bottom) hydrophilicity plots predicted by the method of Hopp and Woods (17). The values are derived from the average of heptapeptide windows and are plotted at the midpoint of each segment. average
B-cell line (EBV-BL) AT cells as targets in a standard 51Cr release assay (5, 6, 28). EBV-BL AT cells were incubated with UV-inactivated RV strain M33 (106 focus-forming units per ml) or E24 peptide (5 p,g/ml) overnight before T cells were added. EBV-BL AT cells alone were used as control target cells. The results indicated that T-cell clones A3 and A9 had the capacity to kill autologous EBV-BL target cells binding with peptide E24 in a dose-dependent manner (Fig.
2B). These T-cell clones were also cytotoxic against the autologous target cells incubated with UV-inactivated RV, while control target cells were not killed (Fig. 2B). To localize the residues within peptide E24 (residues 54 to 74) necessary for recognition by T-cell clones A3 and A9, we synthesized a set of nested truncated peptides spanning this region and assessed their capacity to sensitize target cells or antigen-presenting cells (APCs) for recognition by T-cell
TABLE 1. Proliferative responses to synthetic peptides of E2 by RV-specific T-cell lines from seropositive healthy donors and vaccine recipients Cell proliferation index3 with: Peptide
Vaccine recipient no.:
Seropositive healthy donor no.:
Position 1
2
3
4
5
6
7
8
9
10
0.98 1.94 0.78 0.80 2Q .Q2 1.70 0.83 1.17 1.56 1-20 E2-1 1.03 1.46 2Q 1.90 0.87 1.46 1.13 0.84 0.54 E2-2 15-36 9.66 0.84 1.25 1.48 0.73 1.36 0.62 0.56 0.82 0.73 0.91 33-57 E2-3 1.04 0.62 1.80 0.93 0.86 126 Z&i 54-74 E2-4 L:_ 22 Lo 0.56 0.89 0.61 1.03 0.80 0.60 1.69 1.23 0.53 0.83 69-91 E2-5 1.05 0.88 1.08 1.67 1.82 1.01 0.92 0.87 1.56 0.76 87-107 E2-6 0.87 1.18 1.33 0.66 0.69 0.48 1.70 0.93 1.10 0.65 E2-7 104-124 0.83 1.11 1.45 1.20 1.67 0.59 1.29 0.91 1.25 E2-8 124-146 2.3Q 1.03 0.95 1.80 1.41 0.84 0.76 1.21 0.91 0.81 0.84 E2-9 139-159 0.66 1.12 0.85 1.53 1.16 0.86 0.92 0.65 2.Q E2-10 156-177 &Q1 0.91 0.76 1.42 1.15 1.22 1.97 1.54 0.73 E2-11 176-199 3.am m22 0.83 1.95 0.66 1.56 1.21 1.50 0.93 1.02 1.45 E2-12 195-220 0.32 0.69 0.65 0.91 1.48 23Q 1.94 0.74 E2-13 218-239 2mQ 2532 0.84 1.61 1.61 0.51 2.a2 2.Q 0.72 1.27 1.21 E2-14 233-257 LO 1.10 1.38 0.61 0.63 1.44 1.65 1.97 0.88 1.07 0.75 254-281 E2-15 a A total of 2 x 104 T celis from each cell line was tested for proliferative response to each E2 synthetic peptide at a final concentration of 15 pg/ml in the presence of gamma-irradiated (3,000 rads) autologous PBMC, with 5 x 104 used as APCs. Results are presented as the mean counts per minute obtained from triplicate determinations. Underlined numbers represent significant cell proliferation indices (>2).
6790
NOTES
J. VIROL.
A
B
7f
40r
A3
A3
6
30 -
5 4
20-
E 0.
3 2
co
._
0
10-
0 I1
0 a
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 N
0 ._
a
-
0
0.3
0
1
----------W--------md. 10 3
1*
0
4-
Co
6
80
'40
A9
._
A9
ca)
0. at)
0
60 -
CPO
4
0
40 -
2 201-
Km1
I
EE..-EE-E--
L-
2 3 4
6
0
0.3
3
10
6 7 8 9 10 11 12 13 14 16 N
Effector/Target Ratio
E2 peptide of RV
FIG. 2. Proliferative and cytotoxic responses of RV-specific T-cell clones. (A) RV-specific T-cell clones A3 and A9 were tested for their proliferative responses to 15 overlapping E2 peptides at a final concentration of 5 pg/ml in the presence of gamma-irradiated (3,000 rads) autologous PBMC from donor 3 as APCs. Significant cell proliferation was considered to have occurred when the ratio of the counts per minute obtained in the presence of antigen to the counts per minute in the absence of antigen was >2. (B) Cytotoxic activities of E2-4 peptide-specific T-cell clones A3 and A9 were tested in a 51Cr release assay. Target cells used were autologous EBV-BL AT cells incubated overnight with corresponding E2-4 peptide at 5 pg/ml (0), EBV-BL AT cells incubated with UV-inactivated RV at 106 focus-forming units per ml (A), and EBV-BL cells alone (-).
clones A3 and A9. As shown in Table 2, besides the parent antigenic 21-mer peptide E2-4 (E2-4A, residues 54 to 74), only peptide E2-4C (residues 54 to 65) was efficient in stimulating the proliferative response of T-cell clone A3 and induced a cytotoxic response from T-cell clones A3 and A9. Other peptides from this region, E2-4B (residues 54 to 61), E2-4D (residues 59 to 70), and E2-4E (residues 63 to 74), gave negative results in both T-cell functional assays. It is possible that clones A3 and A9 are separate isolates of clones of identical specificities. Studies of T-cell epitopes indicated that the minimum length of a T-cell epitope is 8 to 12 amino acid residues (30). Results of this study indicate that amino acid residues 54 to 65 of E2 are directly involved in the E2-4 T-cell determinant.
The role of HLA class II molecules in the proliferative responses to epitope E2-4 by T-cell clone A9 was studied by using a series of anti-HLA MAbs in inhibition assays. MAbs
specific for monomorphic regions were tested for the ability to block presentation of the relevant E2-4 peptide epitope by
autologous PBMC. Stimulation of clone A9 by peptide E2-4 clearly inhibited in the presence of the anti-HLA-DR and -DQ MAb 9.3 F10 (33) and the anti-HLA-DR MAb L243 (19) but not in the presence of the anti-HLA-A.B.C. MAb W6/32 (3) and anti-HLA-DQ MAb IVD12 (15) (data not shown). Therefore, the restrictive element of A9 reactive to E2-4 peptide is of the HLA-DR phenotype. In order to further identify HLA-restricting determinants presenting the E2-4 peptide epitope, we used eight EBV-BLs
was
NOTES
VOL. 66, 1992
6791
TABLE 2. Antigenicity of truncated E2-4 peptides for T-cell clones % Specific 5"Cr release0
Position
Sequence"
A3
A9
Cell proliferation of A3
54-74 54-61 54-65 59-70 63-74
ASDVLPGHWLQGGWGCYNLSD ABDVLPGH
23.1 2.1 19.8 0 1.0 0
45.3 0.5 50.4 0 1.2 0
7,734C 173 3,663 715 337 249
Amino acid Peptide
E2-4A E2-4B E2-4C E2-4D E2-4E None
ASDVLPGHWLQG PGHWLQGGWGCY LQGGWGCYNLSD
a Autologous EBV-BL AT cells were incubated with individual truncated E24 peptides at 5 p.g/ml overnight before T cells were added as targets. Cytotoxicity was determined by using T-cell clones A3 and A9 at an effector/target ratio of 3. bThe underlined sequence indicates the truncated E24 peptide with significant cell proliferation and cytotoxicity. c Proliferative responses of T-cell clone A3 to truncated E24 peptides were tested at a peptide concentration of 5 FM in the presence of gamma-irradiated autologous PBMC as APCs. Results are presented as the mean counts per minute obtained from triplicate determinations.
DR7-associated haplotype. Similar results were obtained in the dose-response analysis of cytotoxicity assays. E2-4 peptide induced strong cytotoxic reactions of T-cell clone A9 to the MANN and EBV-BL AT cell targets but no responses to human cell lines MAJA, MST, WT-20, PRIESS, or Lg-38
with different HLA phenotypes as APCs in proliferation assays and used six of them as targets in cytotoxicity assays. EBV-BLs were established from donors with different HLA phenotypes by the method described previously (6). Six human cell lines with homozygous HLA phenotypes (provided kindly by Roberto Acolla) were also used as target cells in 51Cr release assays. In proliferation assays, the E2-4 epitope was presented only by EBV-BL AT cells but not by seven other EBV-BLs without the HLA-DR7 phenotype for stimulation of the responses of T-cell clones A3 and A9 (data not shown). In cytotoxicity assays, T-cell clone A9 was cytotoxic to the MANN and EBV-BL AT cell targets that presented E2-4 epitopes (Fig. 3A). MANN is a human cell line expressing HLA-DR7. The HLA-DR phenotypes of autologous EBV-BL AT cells are DR4 and DR7. Both positive targets share DR7, suggesting that the restriction element used to present the E2-4 determinant is of the
(Fig. 3B).
In terms of vaccine design, it is imperative that a synthetic T-cell determinant prime a memory T-cell response that can be recalled by a determinant on the pathogen (24). Multiple antigens on both structural and nonstructural viral proteins may serve as inducers of T-helper cells, targets for cytotoxic T-lymphocytes (CD4+ and CD8+), and sites for neutralizing antibodies. Our present study was undertaken to characterize the specificity and genetic restriction of an immunodominant T-cell epitope, E2-4, on E2 protein of RV. E2-4 peptide stimulated significant proliferative responses of T-cell lines 1, 3, 4, 6, and 9 as well as T-cell clones A3 and A9 (Table 1
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JOURNAL OF VIROLOGY, Nov. 1992, p. 6788-6793
0022-538X/92/116788-06$02.00/0 Copyright © 1992, American Society for Microbiology
Identification of T-Cell Epitopes on E2 Protein of Rubella Virus, as Recognized by Human T-Cell Lines and Clones DAWEI OU,1 PELE CHONG,2 YVONNE CHOI,2 PAUL McVEIGH 2 WILFRED A. JEFFERIES 3,4,5,6 GERASINOS KOLOITIS,3'4'5'6 AUBREY J. TINGLE,17 AND SHIRLEY GILLAM1* Department of Pathology' and Department of Paediatrics, 7 Research Centre, University of British Columbia, 950 West 28th Avenue, Vancouver, British Columbia VSZ 4H4, Connaught Centre for Biotechnology Research, Wllowdale, Ontario M2R 3T4,2 and Biotechnology Laboratory3 and Departments of Medical Genetics,4 Microbiology,5 and Zoology,6 University of British Columbia, Vancouver, British Columbia V6T 1 W5, Canada Received 1 April 1992/Accepted 10 August 1992
T-cell epitopes on the E2 protein of rubella virus were studied by using 15 overlapping synthetic peptides covering the E2 protein sequence. The most frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 tested T-cell lines responding to it. Two CD4+ cytotoxic T-cell clones isolated from one T-cell line responded strongly in proliferation assays with peptide E24 and were cytotoxic to target cells presenting the E2-4 determinant. Truncated peptides contained within the E2-4 peptide sequence were used to define the T-cell determinants. Results indicated that amino acid residues 54 to 65 were directly involved. Human cell lines with different HLA phenotypes were tested for the capacity to present the antigenic determinants. The results suggested that recognition of peptide E2-4 by T-cell clones was associated with HILA DR7.
Rubella virus (RV) is the causative agent of German measles. Immunization of infants and susceptible women of child-bearing age against RV is now a standard public health measure (1). RV vaccines in current use contain live, attenuated virus (29). The development of both acute and chronic forms of arthritis has been reported in association with both RV infection and immunization (7, 13). As well, potential concerns remain over the long-term consequences of RV vaccine infection in developing fetuses after accidental maternal immunization during pregnancy (31). A new approach using synthetic subunit vaccines is an alternative to the conventional, attenuated RV vaccines (24, 25). The development of a synthetic subunit vaccine requires the identification of T- and B-cell epitopes of RV structural proteins. RV is the sole member of the genus Rubivirus of the family Togaviridae (22). The virions contain three structural proteins, El (58 kDa), E2 (42 to 47 kDa), and C (33 kDa). El and E2 are membrane glycoproteins located on the virion exterior, and the capsid protein (C) is associated with the genomic RNA forming the nucleocapsid (26). The biological role of E2 is not well defined, although it has been reported to possess strain-specific epitopes (11) and possibly at least one neutralizing domain (16). B-cell epitopes of the E2 protein defined by monoclonal antibodies (MAbs) have been reported (34). We have recently demonstrated capsid antigen-specific T-cell responses in seropositive humans (27). The fine specificity and genetic restriction of the T-cell clones reactive to the three immunodominant T-cell epitopes on C protein of RV were characterized by using synthetic peptides of C protein (28). Little is known about the cell-mediated immune responses to the E2 protein of RV. In order to identify the T-cell epitopes on E2, 15 overlapping synthetic peptides covering the entire E2 protein sequence of RV strain M33 (9) were synthesized in an automated peptide synthesizer (23, 27) to
*
screen for the peptide-specific T-cell lines and clones. Figure 1 shows the locations of these peptides and the profiles of the indices of a-helix and n-turn and the hydrophilicity plots calculated for E2. Using these overlapping synthetic peptides, we have studied the proliferative responses of 10 human RV-specific T-cell lines derived from seropositive healthy donors and RV vaccine recipients to E2 epitopes of RV. Donors 1 to 5 were healthy individuals (average age, 36 years) with seropositive responses to RV structural proteins in immunoblot analysis (data not shown). Donors 6 to 10 were RV vaccine (RA27/3) recipients (average age, 24 years) whose blood samples were collected 3 weeks after administration of RV vaccine. RV-specific T-cell lines were derived from the peripheral blood mononuclear cells (PBMC) of donors by stimulation with UV-inactivated RV and then expansion with interleukin-2 (27). The frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 T-cell lines responding, E2-13 (residues 218 to 239), with 4 of 10 lines responding, and E2-14 (residues 233 to 257), with 3 of 10 lines responding (Table 1). Peptides E2-1 (residues 1 to 20), E2-2 (residues 15 to 36), E2-8 (residues 124 to 146), E2-10 (residues 156 to 177), and E2-11 (residues 176 to 199) were also recognized by some of these T-cell lines (Table 1). Since, of 15 E2 peptides, E2-4 peptide was recognized at the
highest frequency by T-cell lines, T-cell clones (A3 and A9) specific for E2-4 peptide were established from T-cell line 3 by limiting dilution as previously described (5, 6, 28) for detailed analysis. Both clones showed specific proliferation with UV-inactivated RV strain M33 (data not shown). By using overlapping E2 synthetic peptides, T-cell clones A3 and A9 were found to respond strongly to the E2-4 peptide (residues 54 to 74) in proliferation assays (Fig. 2A). Surface phenotype analysis in a fluorescence-activated cell sorter showed that more than 95% of cells in T-cell lines and 99% of cells in T-cell clones were CD3+, CD4+, and CD8- (data not shown), demonstrating that these T-cell lines and clones belong to the T-helper cell class. The cytotoxic activities of T-cell clones A3 and A9 were studied by using autologous Epstein-Barr virus-transformed
Corresponding author. 6788
NOTES
VOL. 66, 1992
6789
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Residue Number FIG. 1. Predicted structure of RV E2 protein by conventional structural analysis algorithms. (Top) Secondary-structure analysis of local a-helix and ,B-turn potentials according to the method of Chou and Fasman (8); (bottom) hydrophilicity plots predicted by the method of Hopp and Woods (17). The values are derived from the average of heptapeptide windows and are plotted at the midpoint of each segment. average
B-cell line (EBV-BL) AT cells as targets in a standard 51Cr release assay (5, 6, 28). EBV-BL AT cells were incubated with UV-inactivated RV strain M33 (106 focus-forming units per ml) or E24 peptide (5 p,g/ml) overnight before T cells were added. EBV-BL AT cells alone were used as control target cells. The results indicated that T-cell clones A3 and A9 had the capacity to kill autologous EBV-BL target cells binding with peptide E24 in a dose-dependent manner (Fig.
2B). These T-cell clones were also cytotoxic against the autologous target cells incubated with UV-inactivated RV, while control target cells were not killed (Fig. 2B). To localize the residues within peptide E24 (residues 54 to 74) necessary for recognition by T-cell clones A3 and A9, we synthesized a set of nested truncated peptides spanning this region and assessed their capacity to sensitize target cells or antigen-presenting cells (APCs) for recognition by T-cell
TABLE 1. Proliferative responses to synthetic peptides of E2 by RV-specific T-cell lines from seropositive healthy donors and vaccine recipients Cell proliferation index3 with: Peptide
Vaccine recipient no.:
Seropositive healthy donor no.:
Position 1
2
3
4
5
6
7
8
9
10
0.98 1.94 0.78 0.80 2Q .Q2 1.70 0.83 1.17 1.56 1-20 E2-1 1.03 1.46 2Q 1.90 0.87 1.46 1.13 0.84 0.54 E2-2 15-36 9.66 0.84 1.25 1.48 0.73 1.36 0.62 0.56 0.82 0.73 0.91 33-57 E2-3 1.04 0.62 1.80 0.93 0.86 126 Z&i 54-74 E2-4 L:_ 22 Lo 0.56 0.89 0.61 1.03 0.80 0.60 1.69 1.23 0.53 0.83 69-91 E2-5 1.05 0.88 1.08 1.67 1.82 1.01 0.92 0.87 1.56 0.76 87-107 E2-6 0.87 1.18 1.33 0.66 0.69 0.48 1.70 0.93 1.10 0.65 E2-7 104-124 0.83 1.11 1.45 1.20 1.67 0.59 1.29 0.91 1.25 E2-8 124-146 2.3Q 1.03 0.95 1.80 1.41 0.84 0.76 1.21 0.91 0.81 0.84 E2-9 139-159 0.66 1.12 0.85 1.53 1.16 0.86 0.92 0.65 2.Q E2-10 156-177 &Q1 0.91 0.76 1.42 1.15 1.22 1.97 1.54 0.73 E2-11 176-199 3.am m22 0.83 1.95 0.66 1.56 1.21 1.50 0.93 1.02 1.45 E2-12 195-220 0.32 0.69 0.65 0.91 1.48 23Q 1.94 0.74 E2-13 218-239 2mQ 2532 0.84 1.61 1.61 0.51 2.a2 2.Q 0.72 1.27 1.21 E2-14 233-257 LO 1.10 1.38 0.61 0.63 1.44 1.65 1.97 0.88 1.07 0.75 254-281 E2-15 a A total of 2 x 104 T celis from each cell line was tested for proliferative response to each E2 synthetic peptide at a final concentration of 15 pg/ml in the presence of gamma-irradiated (3,000 rads) autologous PBMC, with 5 x 104 used as APCs. Results are presented as the mean counts per minute obtained from triplicate determinations. Underlined numbers represent significant cell proliferation indices (>2).
6790
NOTES
J. VIROL.
A
B
7f
40r
A3
A3
6
30 -
5 4
20-
E 0.
3 2
co
._
0
10-
0 I1
0 a
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 N
0 ._
a
-
0
0.3
0
1
----------W--------md. 10 3
1*
0
4-
Co
6
80
'40
A9
._
A9
ca)
0. at)
0
60 -
CPO
4
0
40 -
2 201-
Km1
I
EE..-EE-E--
L-
2 3 4
6
0
0.3
3
10
6 7 8 9 10 11 12 13 14 16 N
Effector/Target Ratio
E2 peptide of RV
FIG. 2. Proliferative and cytotoxic responses of RV-specific T-cell clones. (A) RV-specific T-cell clones A3 and A9 were tested for their proliferative responses to 15 overlapping E2 peptides at a final concentration of 5 pg/ml in the presence of gamma-irradiated (3,000 rads) autologous PBMC from donor 3 as APCs. Significant cell proliferation was considered to have occurred when the ratio of the counts per minute obtained in the presence of antigen to the counts per minute in the absence of antigen was >2. (B) Cytotoxic activities of E2-4 peptide-specific T-cell clones A3 and A9 were tested in a 51Cr release assay. Target cells used were autologous EBV-BL AT cells incubated overnight with corresponding E2-4 peptide at 5 pg/ml (0), EBV-BL AT cells incubated with UV-inactivated RV at 106 focus-forming units per ml (A), and EBV-BL cells alone (-).
clones A3 and A9. As shown in Table 2, besides the parent antigenic 21-mer peptide E2-4 (E2-4A, residues 54 to 74), only peptide E2-4C (residues 54 to 65) was efficient in stimulating the proliferative response of T-cell clone A3 and induced a cytotoxic response from T-cell clones A3 and A9. Other peptides from this region, E2-4B (residues 54 to 61), E2-4D (residues 59 to 70), and E2-4E (residues 63 to 74), gave negative results in both T-cell functional assays. It is possible that clones A3 and A9 are separate isolates of clones of identical specificities. Studies of T-cell epitopes indicated that the minimum length of a T-cell epitope is 8 to 12 amino acid residues (30). Results of this study indicate that amino acid residues 54 to 65 of E2 are directly involved in the E2-4 T-cell determinant.
The role of HLA class II molecules in the proliferative responses to epitope E2-4 by T-cell clone A9 was studied by using a series of anti-HLA MAbs in inhibition assays. MAbs
specific for monomorphic regions were tested for the ability to block presentation of the relevant E2-4 peptide epitope by
autologous PBMC. Stimulation of clone A9 by peptide E2-4 clearly inhibited in the presence of the anti-HLA-DR and -DQ MAb 9.3 F10 (33) and the anti-HLA-DR MAb L243 (19) but not in the presence of the anti-HLA-A.B.C. MAb W6/32 (3) and anti-HLA-DQ MAb IVD12 (15) (data not shown). Therefore, the restrictive element of A9 reactive to E2-4 peptide is of the HLA-DR phenotype. In order to further identify HLA-restricting determinants presenting the E2-4 peptide epitope, we used eight EBV-BLs
was
NOTES
VOL. 66, 1992
6791
TABLE 2. Antigenicity of truncated E2-4 peptides for T-cell clones % Specific 5"Cr release0
Position
Sequence"
A3
A9
Cell proliferation of A3
54-74 54-61 54-65 59-70 63-74
ASDVLPGHWLQGGWGCYNLSD ABDVLPGH
23.1 2.1 19.8 0 1.0 0
45.3 0.5 50.4 0 1.2 0
7,734C 173 3,663 715 337 249
Amino acid Peptide
E2-4A E2-4B E2-4C E2-4D E2-4E None
ASDVLPGHWLQG PGHWLQGGWGCY LQGGWGCYNLSD
a Autologous EBV-BL AT cells were incubated with individual truncated E24 peptides at 5 p.g/ml overnight before T cells were added as targets. Cytotoxicity was determined by using T-cell clones A3 and A9 at an effector/target ratio of 3. bThe underlined sequence indicates the truncated E24 peptide with significant cell proliferation and cytotoxicity. c Proliferative responses of T-cell clone A3 to truncated E24 peptides were tested at a peptide concentration of 5 FM in the presence of gamma-irradiated autologous PBMC as APCs. Results are presented as the mean counts per minute obtained from triplicate determinations.
DR7-associated haplotype. Similar results were obtained in the dose-response analysis of cytotoxicity assays. E2-4 peptide induced strong cytotoxic reactions of T-cell clone A9 to the MANN and EBV-BL AT cell targets but no responses to human cell lines MAJA, MST, WT-20, PRIESS, or Lg-38
with different HLA phenotypes as APCs in proliferation assays and used six of them as targets in cytotoxicity assays. EBV-BLs were established from donors with different HLA phenotypes by the method described previously (6). Six human cell lines with homozygous HLA phenotypes (provided kindly by Roberto Acolla) were also used as target cells in 51Cr release assays. In proliferation assays, the E2-4 epitope was presented only by EBV-BL AT cells but not by seven other EBV-BLs without the HLA-DR7 phenotype for stimulation of the responses of T-cell clones A3 and A9 (data not shown). In cytotoxicity assays, T-cell clone A9 was cytotoxic to the MANN and EBV-BL AT cell targets that presented E2-4 epitopes (Fig. 3A). MANN is a human cell line expressing HLA-DR7. The HLA-DR phenotypes of autologous EBV-BL AT cells are DR4 and DR7. Both positive targets share DR7, suggesting that the restriction element used to present the E2-4 determinant is of the
(Fig. 3B).
In terms of vaccine design, it is imperative that a synthetic T-cell determinant prime a memory T-cell response that can be recalled by a determinant on the pathogen (24). Multiple antigens on both structural and nonstructural viral proteins may serve as inducers of T-helper cells, targets for cytotoxic T-lymphocytes (CD4+ and CD8+), and sites for neutralizing antibodies. Our present study was undertaken to characterize the specificity and genetic restriction of an immunodominant T-cell epitope, E2-4, on E2 protein of RV. E2-4 peptide stimulated significant proliferative responses of T-cell lines 1, 3, 4, 6, and 9 as well as T-cell clones A3 and A9 (Table 1
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