- Email: [email protected]
A single peptide derived from the sequence common to polyoma small and middle t-antigen induces immunity against polyoma tumors
VIROLOGY 172,359-X2
(1989)
A Single Peptide Derived from the Sequence Common to Polyoma Small and Middle T-Antigen Induces Immunity against Polyoma Tumors TORBJ~~RN RAMQVIST,*
GIT
REINtioLDssON,*~t MATS CARLQUIST,+
TOMAS
BERGMAN,§
AND TINA DALIANIS*+’
Departments of *Tumor Biology, *Biochemistry II, and SChemistry I, Karolinska Institute, Box 60 400, S- 104 0 1 Stockholm; and tStockholm City Council, Microbiology Central Laboratory, Box 70 470, 10726 Stockholm, Sweden Received February
10, 1989; accepted May 8, 1989
Polyoma virus, an oncogenic virus, fails to induce tumors in immunocompetent rodents due to T cell-dependent mechanisms. The target recognized by the immune system has been functionally defined as polyoma tumor-specific transplantation antigen (TSTA) and has been postulated to be related to the virus three early proteins small T (ST), middle T (MT), and large T (LT) antigens. We show here that immunization with a synthetic peptide corresponding to amino acids 162-176 of polyoma MT and ST was able to decrease tumor progression of polyoma tumors, but not of nonpolyoma tumors. This indicated that these amino acids constitute an epitope of the polyoma tumor-specific (0 1989 Academic Press, Inc. transplantation antigen.
Polyoma-specific immunity has been obtained after immunization of mice and rates with wild-type (wt) or deletion mutants of polyoma virus (I-4), with cells transformed with wt or mutants of polyoma virus (2, 5) as well as with vaccinia recombinant vectors expressing middle T (MT) or large T (LT) (6). These results have therefore suggested that the expression of products of the virus early region are important, either directly or indirectly, for the induction of tumor-specific transplantation antigen (TSTA). Recently we have shown that an anti-TSTA response could be induced when mice were immunized with purified MT or small T (ST) (7), and that tumor cells that lack evident T antigen expression do not express TSTA (8). Since T antigens, however, are not detected on the outside of the cell surface it was suggested that they were processed and copresented to the immune system together with major histocompatibility complex (MHC) determinants as has been described earlier for the influenza nucleoprotein by Townsend et a/. (9- 1 1). Assuming that it is less likely that the purified T antigens entered the antigen-presenting cells in an intact and/or biologically active form, and thus inducing new cellular antigens, these findings argued that antigenic epitopes of polyoma MT and ST antigens themselves have direct TSTA activity. Together with the previous results (6, 8), they suggested that epitopes on all three T antigens may act directly as TSTAs.
To define epitopes of polyoma TSTA in detail, peptides corresponding to different regions of MT were synthesized. Our aim was to see if it was possible to obtain a rejection response against polyoma tumors after immunization of mice with a single synthetic T antigen-derived peptide, and if so to use peptides to map polyoma TSTA epitopes. To reduce the number of different peptides produced, an algorithm devised by Margalit et al. (12) that selects for amphipathic a-helices was employed for prediction of likely T helper cell
TABLE 1 THE AMINO ACID COMPOSITION
OF THE SIX MT-DERIVED
AND THEIR PRESENCE IN THE DIFFERENTT
PEPTIDES
ANTIGENS
Present in Label
MT,-,9 MT,, 38 MTs, 66 MT,,,-,,, MT,wo/ MTm-m
aasequence MDRVLSRADKERLLELLKL+ C WGDFGRMQQAYKQQS + C LMQELNSLWGTFKTEV + C TRDVLNLYADFIASM RRSEELRRAATVHYT + C RAHSMQRHLRRLGRTL
ST
MT
LT
t t t t -
+ + + + + +
+ t +-
Note. The figures In the pepttde labels denote the corresponding posltion of the first and last aa on MT. An extra cysteine was added to four of the peptides. Peptlde synthesis: The protected peptldes were assembled by solid-phase synthesis on a cross-linked polystyrene support, using t-Boc amino acids. The syntheses were performed on a peptlde synthesizer (Applied Blosystems 430A) utilizing a standard program. Peptides were cleaved from the resin and deprotected by hydrogen fluoride. Final products were analyzed by reverse-phase HPLC and verified through amino aclcl analysis.
’ To whom requests for reprints should be addressed at present address: Laboratory for Eukaryotic Molecular Genetics, National Institute for Medical Research, The Ridgeway. Mill Hill, London NW7 IAA, UK.
359
0042.6822/89
$3.00
CopyrIght 0 1989 by Academic Press, inc All rIghIs of reproduction I” any form reserved
SHORT COMMUNICATIONS
360
12s
30~ 7 r
25..
: :;; E 2
0-O
py virus Freund’s
*-*
c, /
lo..
i v
MT162-176
!
i
6--
lo--
z 5
4-
I” 5--
9
o-o 0-O
none pyvirus
A-.
MT162-176
0
a--
15.-
0
A
none
A-A
20..
s 5
o-o
/
/ 0 /
2--
./ ./e’
11
13
15 Days After
O@-==U--, 0 10
l 17 Challenge
19
21
23
H-t/-
;/ 12
B
16.
o-o O-0 A-A
-2 5
12--A-A
14 Days After
16 Challenge
18
20
:
none s/Y cells Freund’s MT152-176
z
= D
’
lo
b4ys
= Afte~8Chollen;:
=
=
*
26
FIG. 1. In viva growth (measured as MTL) of (A) polyoma tumor SEBB/YACUT (8) with an inoculum of 100,000 cells/mouse in (A X CBA)F, mice; (B) polyoma tumor SEBA (4) with an inoculum of 1000 cells/mouse in CBA mice; (C) nonpolyoma tumor SBfnHDneo-ras (7) with an inoculum of 50,000 cells in (CBA X BALB/c)F, mice; and (D) polyoma TSTA negative variant tumor SEBBNACUT B (8) with an inoculum of 100,000 cells/ mouse in (A X CBA)F, mice, after immunization with peptide MT ,62- ,76, polyoma wt virus, irradiated SBfnHDneo-ras cells, irradiated SEBB/ YACUT (S/Y) cells, Freund’s adjuvant, and after no immunization. Methods: Mice were immunized in (A), (C), and (D) with 10 pglmouse of MT,62-,76 three times subcutaneously (s.c.), once with Freund’s complete adjuvant and twice with Freund’s incomplete adjuvant. In (B) 15 rg/ mouse of MT,62-,76 in PBS was administered S.C. three times. The 20 HAU of polyomavirus was inoculated three times, the first two times intraperitoneally and the third time S.C. All immunizations were performed at 3-week intervals. In order to exclude nonspecific boosting of the immune defense by the polyoma virus all mice received 400 rad whole-body irradiation prior to live-cell challenge (4). MTL is defined as the sum of the mean diameters of the tumors present in a group of mice divided by the total number of mice in the group.
epitopes. Although it is not known if T helper cells specifically participate in the rejection response against polyoma-induced tumors, it has been demonstrated that T cells are required for this response (1, 13, 14). Six peptides with a length of 15-l 9 amino acids were produced. Their sequences, their occurrence in T antigens, and their nomenclature are presented in Table 1. The peptides were tested for their ability to induce an anti-TSTA response in several independent experiments. Mice were immunized with the peptides as described in Fig. 1, followed by live-cell challenge with polyoma or nonpolyoma tumors. Protective immunity was assayed for by calculation of the mean tumor load (MTL) (7). Immunization of mice with wt virus or peptide MT162-176resulted in a reduced tumor progression after challenge with the polyoma tumors SEBB/YACUT (of A X CBA origin) (8) or SEBA (of CBA origin) (4) as compared to tumor progression in nonimmunized mice, or mice immunized with Freund’s adjuvant alone (Figs. 1A and 1 B). As shown in Fig. 1C the non polyoma tumor
SBfnHDneo-ras (7) grew equally well in unimmunized mice and mice immunized with peptide MT,62-176 or with Freund’s adjuvant, but a decrease in tumor progression was observed in mice immunized with SBfnHDneo-ras. Furthermore, a polyoma TSTA loss variant, SEBBNACUT B (8) grew equally well in unimmunized mice, mice immunized with Freund’s adjuvant alone, or in mice immunized with peptide MT162-176, while immunization with the original SEBB/YACUT tumor line resulted in reduced tumor progression (Fig. 1D). Although peptide MT,62-,76 induced a consistent immunization against polyoma tumors, the immunization achieved was weaker when compared to that obtained by wt virus. In order to compare the results between the individual experiments and to detect possible weaker immunization effects induced by the other peptides used, we have compared the effects on MTL at specific stages of tumor progression, i.e., when the MTL of the control groups were 10, 15, and 20 mm. As demonstrated in Fig. 2, when the results of six different
361
SHORT COMMUNICATIONS
12345670
9
10
FIG. 2. In viva growth of polyoma tumor SEBB/YACUT with an inoculum of 2-10 x 1O4cells/mouse in six different experiments (assayed for by the combined total MTL) In different groups of immunized mice. The combined total MTL of the different groups of immunized mice are indicated at the time pornt when the MTL of nonimmunized controls wrthin each experiment were 15 mm. It was calculated as the sum of the mean tumor diameters of the tumors present within each specific type of group, divided by the total number of mice. The different bars correspond to groups of nonimmunized mice (1) Freund’s adjuvant (2), polyoma virus (3) peptide MT,-,9 (5) MT24-38 0311MT,, 66 (7) MT,,2-,,6 (8). MTlg3 207 (9) PeptIde MT380-395 (lo), and a mrxture of these peptrdes (4). Error bars denote the standard error. The combined total MTL of the virus or MT,62.,,6 groups were significantly lower than In the control group as evaluated with the Student’s t test (P < 0.0001). lmmunrzation was performed with the different peptides In the same way as described in Fig. 1 with doses from 3-l 0 ~glrnoculatronimouse together with Freund’s adjuvant.
experiments were summarized at the time point when the MTL of nonimmunized mice were 15 mm, no peptide other than MT,eZm,,7 induced a significant decrease in polyoma tumor progression. In mice which had received this peptide a 47% decrease of the MTL was obtained. No reduction in tumor takes was observed in any of the groups with the exception of the polyoma virus-immunized group. Similar results were obtained for the time points when the MTL of the control groups were 10 and 20 mm (data not shown). In conclusion we have observed that it is possible to evoke an antipolyoma TSTA response by immunizing with 1O-30 /*g/mouse of a single uncoupled synthetic peptide. This peptide corresponds to amino acids 162-l 76 of MT and ST and has a sequence which is most likely to form an amphipathic a-helix. Since it is acidic and did not dissolve well at a neutral pH, the injected solution contained peptide aggregates which may have facilitated its detection by the immune system. These results strongly suggest that amino acids 162-l 76 of MT and ST constitute a TSTA epitope. The anti-TSTA response induced by this peptide was much weaker than the response obtained after polyoma virus immunization, but similar to that induced by purified MT or ST (7). The weaker anti-TSTA response induced by a single peptide as compared to immunization with
wt virus is possibly due to the fact that several target epitopes can be generated by wt virus, and that continuous virus replication in viva is likely to result in a more efficient response. Thus, although it is possible for the immune system to detect and respond to this epitope in CBA or A x CBA mice, it is not evident that this epitope is the dominant target during an anti-TSTA response after immunization with polyoma virus. The reason for why the mixture of peptides did not give any protection is unknown. It could, however, be due to a competition between the different peptides for binding to antigens of the MHC. These MHC-binding peptides may not necessarily be capable of inducing a response in CBA and A X CBA mice, and thus the end result would be an abrogation of the immune response as was observed. The existence of at least one other TSTA epitope was demonstrated earlier by Reinholdsson el al. (15), where the presence of an epitope within the first 1 13 amino acids of MT and ST was established. This epitope would clearly not overlap with MT,6Z_,76. The studies of Reinholdsson et al. (15) were performed in rats and it is possible that different epitopes are recognized in different strains of rats and mice. Therefore, although a significant immune response after immunization with the other five peptides was not obtained we can not definitely conclude that they do not constitute TSTA epitopes in other strains of mice. There could be other reasons for the lack of response. The MHC of the mouse strains tested may not bind these peptides or the dose used for immunization or the immunization procedure may not have been optimal. We are presently investigating whether these latter peptides can confer an antVTSTA response with other immunization protocols and in other mouse strains.
ACKNOWLEDGMENTS We thank Dr. M. Troye-Blomberg and Professors P. Penman, H. Wig&l, and G. Klern for valuable discussons. We thank Mrs. M.-L. Sohlberg and Mrs. M. Hagelrn for excellent technical assistance, This investigation was supported by the Swedish Cancer Society, the Swedish Society of Medicine, together with the Medical Research Councrl (M.C.), the Nordrc lnsulrn Fund (M.C.), and the Foundations of Alex and Eva Wallstrom and of L. Hrertas Minne (T.D.). T.R. is a recrpient of a fellowshrp from the Cancer Research Institute and the Concern Foundatron. T.D. IS a recrprent of a Travelling Research Fellowshrp from Wellcome Trust
REFERENCES 7. ALLISON, A. C., in “Vlral
Oncology”
Raven Press, New York, 1980. 2. SJBGREN, H. O., J. Nat/. Cancerlnst.
(G. Klein, Ed.), pp. 481-487
32, 645-649
(1964).
362
SHORT COMMUNICATIONS
3. SJGGREN,H. O., J. Nqtl. Cancerlnst, 32, 361-374 (1964). 4. DALIANIS, T., MAGNUSSON, G., ITO, Y., and KLEIN, G. J., Virology 43,772-777 (1982). 5. DALIANIS, T., RAMQVIST,T., and KLEIN, G., ht. J. Cancer34, 403406 (1984). 6. LATHE, R., KIENY, M. P., GERLINGER,P., CLERTANT, P., GUIZANI, I., CUZIN, F., and CHAMBON, P., Nature (London) 326, 878-880 (1987). 7. RAMQVIST, T., PALLAS, D. O., DEANDA, J., HRLUND-RICHTER, L., REINHOLDSSON,G., ROBERTS, T. M., SCHAFFHAUSEN, B., and DALIANIS, T. ht. J. Cancer42, 123-l 28 (1988). 8. RAMQVIST,T., REINHOLDSSON,G., SZIGETI, R., KLEIN, G., and DALIANIS, T. ht. /. Cancer 40, 74-80 (1987).
9. TOWNSEND,A. R. M., GOTCH, F. M., and DAVEY, J., Cell42,457467 (1985). 10. TOWNSEND, A. R. M., ROTHBARD,J., GOTCH, F. M.. BAHADUR, G., WRAITH, D., and MCMICHAEL, A. J., Cell44, 959-968 (1986). 11. TOWNSEND,A. R. M., lmmunol. Res. 6,80-l 00 (1987). 72. MARGALIT, H., SPOUGE,J. L., CORNE~E, J. L.. CEASE, K., DELISI, C., and BERZOFSKY,J. A., J. lmmunol. 138,2213-2229 (1987). 13. DE LAPEYRIERE,O., IMBERT, J., and MEYER, G., Bull. Cancer 68, lOl-108(1981). 14. GAUGAS,J. M., ALLISON, A. C., CHESTERMAN,F. C., REES, R. J. W., and HIRSCH, M. S., Bit. f. Cancer 27, 1O-l 7 (1973). 15. REINHOLDSSON,G., RAMQVIST.R., BRANDBERG,J., and DALIANIS,T., virology166,616-619(1989).
(1989)
A Single Peptide Derived from the Sequence Common to Polyoma Small and Middle T-Antigen Induces Immunity against Polyoma Tumors TORBJ~~RN RAMQVIST,*
GIT
REINtioLDssON,*~t MATS CARLQUIST,+
TOMAS
BERGMAN,§
AND TINA DALIANIS*+’
Departments of *Tumor Biology, *Biochemistry II, and SChemistry I, Karolinska Institute, Box 60 400, S- 104 0 1 Stockholm; and tStockholm City Council, Microbiology Central Laboratory, Box 70 470, 10726 Stockholm, Sweden Received February
10, 1989; accepted May 8, 1989
Polyoma virus, an oncogenic virus, fails to induce tumors in immunocompetent rodents due to T cell-dependent mechanisms. The target recognized by the immune system has been functionally defined as polyoma tumor-specific transplantation antigen (TSTA) and has been postulated to be related to the virus three early proteins small T (ST), middle T (MT), and large T (LT) antigens. We show here that immunization with a synthetic peptide corresponding to amino acids 162-176 of polyoma MT and ST was able to decrease tumor progression of polyoma tumors, but not of nonpolyoma tumors. This indicated that these amino acids constitute an epitope of the polyoma tumor-specific (0 1989 Academic Press, Inc. transplantation antigen.
Polyoma-specific immunity has been obtained after immunization of mice and rates with wild-type (wt) or deletion mutants of polyoma virus (I-4), with cells transformed with wt or mutants of polyoma virus (2, 5) as well as with vaccinia recombinant vectors expressing middle T (MT) or large T (LT) (6). These results have therefore suggested that the expression of products of the virus early region are important, either directly or indirectly, for the induction of tumor-specific transplantation antigen (TSTA). Recently we have shown that an anti-TSTA response could be induced when mice were immunized with purified MT or small T (ST) (7), and that tumor cells that lack evident T antigen expression do not express TSTA (8). Since T antigens, however, are not detected on the outside of the cell surface it was suggested that they were processed and copresented to the immune system together with major histocompatibility complex (MHC) determinants as has been described earlier for the influenza nucleoprotein by Townsend et a/. (9- 1 1). Assuming that it is less likely that the purified T antigens entered the antigen-presenting cells in an intact and/or biologically active form, and thus inducing new cellular antigens, these findings argued that antigenic epitopes of polyoma MT and ST antigens themselves have direct TSTA activity. Together with the previous results (6, 8), they suggested that epitopes on all three T antigens may act directly as TSTAs.
To define epitopes of polyoma TSTA in detail, peptides corresponding to different regions of MT were synthesized. Our aim was to see if it was possible to obtain a rejection response against polyoma tumors after immunization of mice with a single synthetic T antigen-derived peptide, and if so to use peptides to map polyoma TSTA epitopes. To reduce the number of different peptides produced, an algorithm devised by Margalit et al. (12) that selects for amphipathic a-helices was employed for prediction of likely T helper cell
TABLE 1 THE AMINO ACID COMPOSITION
OF THE SIX MT-DERIVED
AND THEIR PRESENCE IN THE DIFFERENTT
PEPTIDES
ANTIGENS
Present in Label
MT,-,9 MT,, 38 MTs, 66 MT,,,-,,, MT,wo/ MTm-m
aasequence MDRVLSRADKERLLELLKL+ C WGDFGRMQQAYKQQS + C LMQELNSLWGTFKTEV + C TRDVLNLYADFIASM RRSEELRRAATVHYT + C RAHSMQRHLRRLGRTL
ST
MT
LT
t t t t -
+ + + + + +
+ t +-
Note. The figures In the pepttde labels denote the corresponding posltion of the first and last aa on MT. An extra cysteine was added to four of the peptides. Peptlde synthesis: The protected peptldes were assembled by solid-phase synthesis on a cross-linked polystyrene support, using t-Boc amino acids. The syntheses were performed on a peptlde synthesizer (Applied Blosystems 430A) utilizing a standard program. Peptides were cleaved from the resin and deprotected by hydrogen fluoride. Final products were analyzed by reverse-phase HPLC and verified through amino aclcl analysis.
’ To whom requests for reprints should be addressed at present address: Laboratory for Eukaryotic Molecular Genetics, National Institute for Medical Research, The Ridgeway. Mill Hill, London NW7 IAA, UK.
359
0042.6822/89
$3.00
CopyrIght 0 1989 by Academic Press, inc All rIghIs of reproduction I” any form reserved
SHORT COMMUNICATIONS
360
12s
30~ 7 r
25..
: :;; E 2
0-O
py virus Freund’s
*-*
c, /
lo..
i v
MT162-176
!
i
6--
lo--
z 5
4-
I” 5--
9
o-o 0-O
none pyvirus
A-.
MT162-176
0
a--
15.-
0
A
none
A-A
20..
s 5
o-o
/
/ 0 /
2--
./ ./e’
11
13
15 Days After
O@-==U--, 0 10
l 17 Challenge
19
21
23
H-t/-
;/ 12
B
16.
o-o O-0 A-A
-2 5
12--A-A
14 Days After
16 Challenge
18
20
:
none s/Y cells Freund’s MT152-176
z
= D
’
lo
b4ys
= Afte~8Chollen;:
=
=
*
26
FIG. 1. In viva growth (measured as MTL) of (A) polyoma tumor SEBB/YACUT (8) with an inoculum of 100,000 cells/mouse in (A X CBA)F, mice; (B) polyoma tumor SEBA (4) with an inoculum of 1000 cells/mouse in CBA mice; (C) nonpolyoma tumor SBfnHDneo-ras (7) with an inoculum of 50,000 cells in (CBA X BALB/c)F, mice; and (D) polyoma TSTA negative variant tumor SEBBNACUT B (8) with an inoculum of 100,000 cells/ mouse in (A X CBA)F, mice, after immunization with peptide MT ,62- ,76, polyoma wt virus, irradiated SBfnHDneo-ras cells, irradiated SEBB/ YACUT (S/Y) cells, Freund’s adjuvant, and after no immunization. Methods: Mice were immunized in (A), (C), and (D) with 10 pglmouse of MT,62-,76 three times subcutaneously (s.c.), once with Freund’s complete adjuvant and twice with Freund’s incomplete adjuvant. In (B) 15 rg/ mouse of MT,62-,76 in PBS was administered S.C. three times. The 20 HAU of polyomavirus was inoculated three times, the first two times intraperitoneally and the third time S.C. All immunizations were performed at 3-week intervals. In order to exclude nonspecific boosting of the immune defense by the polyoma virus all mice received 400 rad whole-body irradiation prior to live-cell challenge (4). MTL is defined as the sum of the mean diameters of the tumors present in a group of mice divided by the total number of mice in the group.
epitopes. Although it is not known if T helper cells specifically participate in the rejection response against polyoma-induced tumors, it has been demonstrated that T cells are required for this response (1, 13, 14). Six peptides with a length of 15-l 9 amino acids were produced. Their sequences, their occurrence in T antigens, and their nomenclature are presented in Table 1. The peptides were tested for their ability to induce an anti-TSTA response in several independent experiments. Mice were immunized with the peptides as described in Fig. 1, followed by live-cell challenge with polyoma or nonpolyoma tumors. Protective immunity was assayed for by calculation of the mean tumor load (MTL) (7). Immunization of mice with wt virus or peptide MT162-176resulted in a reduced tumor progression after challenge with the polyoma tumors SEBB/YACUT (of A X CBA origin) (8) or SEBA (of CBA origin) (4) as compared to tumor progression in nonimmunized mice, or mice immunized with Freund’s adjuvant alone (Figs. 1A and 1 B). As shown in Fig. 1C the non polyoma tumor
SBfnHDneo-ras (7) grew equally well in unimmunized mice and mice immunized with peptide MT,62-176 or with Freund’s adjuvant, but a decrease in tumor progression was observed in mice immunized with SBfnHDneo-ras. Furthermore, a polyoma TSTA loss variant, SEBBNACUT B (8) grew equally well in unimmunized mice, mice immunized with Freund’s adjuvant alone, or in mice immunized with peptide MT162-176, while immunization with the original SEBB/YACUT tumor line resulted in reduced tumor progression (Fig. 1D). Although peptide MT,62-,76 induced a consistent immunization against polyoma tumors, the immunization achieved was weaker when compared to that obtained by wt virus. In order to compare the results between the individual experiments and to detect possible weaker immunization effects induced by the other peptides used, we have compared the effects on MTL at specific stages of tumor progression, i.e., when the MTL of the control groups were 10, 15, and 20 mm. As demonstrated in Fig. 2, when the results of six different
361
SHORT COMMUNICATIONS
12345670
9
10
FIG. 2. In viva growth of polyoma tumor SEBB/YACUT with an inoculum of 2-10 x 1O4cells/mouse in six different experiments (assayed for by the combined total MTL) In different groups of immunized mice. The combined total MTL of the different groups of immunized mice are indicated at the time pornt when the MTL of nonimmunized controls wrthin each experiment were 15 mm. It was calculated as the sum of the mean tumor diameters of the tumors present within each specific type of group, divided by the total number of mice. The different bars correspond to groups of nonimmunized mice (1) Freund’s adjuvant (2), polyoma virus (3) peptide MT,-,9 (5) MT24-38 0311MT,, 66 (7) MT,,2-,,6 (8). MTlg3 207 (9) PeptIde MT380-395 (lo), and a mrxture of these peptrdes (4). Error bars denote the standard error. The combined total MTL of the virus or MT,62.,,6 groups were significantly lower than In the control group as evaluated with the Student’s t test (P < 0.0001). lmmunrzation was performed with the different peptides In the same way as described in Fig. 1 with doses from 3-l 0 ~glrnoculatronimouse together with Freund’s adjuvant.
experiments were summarized at the time point when the MTL of nonimmunized mice were 15 mm, no peptide other than MT,eZm,,7 induced a significant decrease in polyoma tumor progression. In mice which had received this peptide a 47% decrease of the MTL was obtained. No reduction in tumor takes was observed in any of the groups with the exception of the polyoma virus-immunized group. Similar results were obtained for the time points when the MTL of the control groups were 10 and 20 mm (data not shown). In conclusion we have observed that it is possible to evoke an antipolyoma TSTA response by immunizing with 1O-30 /*g/mouse of a single uncoupled synthetic peptide. This peptide corresponds to amino acids 162-l 76 of MT and ST and has a sequence which is most likely to form an amphipathic a-helix. Since it is acidic and did not dissolve well at a neutral pH, the injected solution contained peptide aggregates which may have facilitated its detection by the immune system. These results strongly suggest that amino acids 162-l 76 of MT and ST constitute a TSTA epitope. The anti-TSTA response induced by this peptide was much weaker than the response obtained after polyoma virus immunization, but similar to that induced by purified MT or ST (7). The weaker anti-TSTA response induced by a single peptide as compared to immunization with
wt virus is possibly due to the fact that several target epitopes can be generated by wt virus, and that continuous virus replication in viva is likely to result in a more efficient response. Thus, although it is possible for the immune system to detect and respond to this epitope in CBA or A x CBA mice, it is not evident that this epitope is the dominant target during an anti-TSTA response after immunization with polyoma virus. The reason for why the mixture of peptides did not give any protection is unknown. It could, however, be due to a competition between the different peptides for binding to antigens of the MHC. These MHC-binding peptides may not necessarily be capable of inducing a response in CBA and A X CBA mice, and thus the end result would be an abrogation of the immune response as was observed. The existence of at least one other TSTA epitope was demonstrated earlier by Reinholdsson el al. (15), where the presence of an epitope within the first 1 13 amino acids of MT and ST was established. This epitope would clearly not overlap with MT,6Z_,76. The studies of Reinholdsson et al. (15) were performed in rats and it is possible that different epitopes are recognized in different strains of rats and mice. Therefore, although a significant immune response after immunization with the other five peptides was not obtained we can not definitely conclude that they do not constitute TSTA epitopes in other strains of mice. There could be other reasons for the lack of response. The MHC of the mouse strains tested may not bind these peptides or the dose used for immunization or the immunization procedure may not have been optimal. We are presently investigating whether these latter peptides can confer an antVTSTA response with other immunization protocols and in other mouse strains.
ACKNOWLEDGMENTS We thank Dr. M. Troye-Blomberg and Professors P. Penman, H. Wig&l, and G. Klern for valuable discussons. We thank Mrs. M.-L. Sohlberg and Mrs. M. Hagelrn for excellent technical assistance, This investigation was supported by the Swedish Cancer Society, the Swedish Society of Medicine, together with the Medical Research Councrl (M.C.), the Nordrc lnsulrn Fund (M.C.), and the Foundations of Alex and Eva Wallstrom and of L. Hrertas Minne (T.D.). T.R. is a recrpient of a fellowshrp from the Cancer Research Institute and the Concern Foundatron. T.D. IS a recrprent of a Travelling Research Fellowshrp from Wellcome Trust
REFERENCES 7. ALLISON, A. C., in “Vlral
Oncology”
Raven Press, New York, 1980. 2. SJBGREN, H. O., J. Nat/. Cancerlnst.
(G. Klein, Ed.), pp. 481-487
32, 645-649
(1964).
362
SHORT COMMUNICATIONS
3. SJGGREN,H. O., J. Nqtl. Cancerlnst, 32, 361-374 (1964). 4. DALIANIS, T., MAGNUSSON, G., ITO, Y., and KLEIN, G. J., Virology 43,772-777 (1982). 5. DALIANIS, T., RAMQVIST,T., and KLEIN, G., ht. J. Cancer34, 403406 (1984). 6. LATHE, R., KIENY, M. P., GERLINGER,P., CLERTANT, P., GUIZANI, I., CUZIN, F., and CHAMBON, P., Nature (London) 326, 878-880 (1987). 7. RAMQVIST, T., PALLAS, D. O., DEANDA, J., HRLUND-RICHTER, L., REINHOLDSSON,G., ROBERTS, T. M., SCHAFFHAUSEN, B., and DALIANIS, T. ht. J. Cancer42, 123-l 28 (1988). 8. RAMQVIST,T., REINHOLDSSON,G., SZIGETI, R., KLEIN, G., and DALIANIS, T. ht. /. Cancer 40, 74-80 (1987).
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