A short peptide eluted from the H-2Kb molecule of a polyomavirus-positive tumor corresponds to polyomavirus large T antigen peptide at amino acids 578 to 585 and induces polyomavirus-specific immunity.

0022-538X/96/$04.0010

Copyrightq1996, American Society for Microbiology

A Short Peptide Eluted from the H-2K

b

Molecule of a

Polyomavirus-Positive Tumor Corresponds to Polyomavirus Large T Antigen

Peptide at Amino Acids 578 to 585 and Induces

Polyomavirus-Specific Immunity

ZSOFIA BERKE,

_{* SARAH PALMER,}

_{† TOMAS BERGMAN,}

_{DANIELE WESTER,}

JONATHAN SVEDMYR,

_{STIG LINDER,}

_{HANS JO}

_{¨ RNVALL,}

AND

TINA DALIANIS

Division of Clinical Virology, Karolinska Institutet, Huddinge University Hospital, F67, S-141 86 Huddinge,

_and

Department of Medical Biochemistry and Biophysics, Karolinska Institutet,

_{and Department of Experimental}

Oncology, Karolinska Hospital,

_{S-171 77 Stockholm, Sweden}

Received 4 December 1995/Accepted 20 February 1996

A short peptide in complex with the H-2K

molecule on PyRMA, a polyomavirus transfectant of the mouse

lymphoma cell line RMA, was identified as a polyomavirus tumor-specific transplantation antigen. The peptide

was obtained by affinity chromatography, acidic extraction, and reverse-phase high-pressure liquid

chroma-tography (HPLC). In one HPLC fraction, a peptide sequence in which 5 of 8 amino acids, GKxGLxxA,

corresponded to residues 578 to 585 of polyomavirus large T antigen was identified. In tumor rejection assays,

we therefore tested three related synthetic peptides, corresponding to the octapeptide LT 578–585,

GKTG-LAAA; the nonapeptide LT 578–586, GKTGLAAAL; and the decapeptide LT 578–587, GKTGLAAALI. The

octapeptide was found to give the most effective immunization against the outgrowth of the polyomavirus

DNA-positive PyRMA tumor. However, none of the three peptides immunized against the original

polyoma-virus-negative RMA line.

Murine polyomavirus, a potentially oncogenic, small DNA

virus, does not induce tumors in its natural host under normal

conditions because of T-cell-dependent immunity (2, 18). The

antigens recognized by the immune system are now suggested

to be peptide antigens derived from the three polyomavirus T

antigens, large T antigen (LT), middle T antigen, and small T

antigen (8, 24, 26).

Previously, it had been shown that immunization of mice

with irradiated polyomavirus tumor cells, wild-type

polyoma-virus, deletion mutants of polyomapolyoma-virus, or vaccinia virus

re-combinant mutants could prevent the outgrowth of

polyoma-virus tumors (9, 10, 17, 29). Later, we demonstrated that

purified middle or small T antigen and short synthetic peptides

derived from the amino acid sequences of the three T antigens

could evoke polyomavirus-specific immunity (23, 24, 26). Since

none of the three T antigens was detected on the outside of the

cell surface, it was suggested that the three T antigens were

processed intracellularly into peptides (24). These peptide

products could then be presented to the immune system

to-gether with determinants of the major histocompatibility

com-plex (MHC) (24, 26). This had been reported previously for the

influenza virus nucleoprotein by Townsend et al. (31) and later

by Ro

¨tzschke et al. (27) and for the vesicular stomatitis virus

nucleocapsid protein by Van Bleek et al. (32).

The aim of this study was to examine if polyomavirus

T-antigen-derived peptides could indeed be presented by MHC

class I molecules on the surfaces of polyomavirus tumor cells

and also if such peptides were immunogenic. The murine

lym-phoma cell line RMA and its polyomavirus transfectant

PyRMA were used for these studies. H-2K

_{MHC class}

I-bound peptides were eluted from both PyRMA and RMA cells

by affinity chromatography under acidic conditions. The

differ-ent peptides were then separated by reverse-phase

high-pres-sure liquid chromatography (HPLC). In one of the fractions, a

unique PyRMA peptide in which 5 of 8 amino acid residues

(GKxGLxxA) corresponded to amino acid residues 578 to 585

of polyomavirus LT was identified. In tumor rejection tests,

this LT 578–585 octapeptide, GKTGLAAA, was shown to

immunize efficiently against PyRMA cells, but not against

RMA cells.

MATERIALS AND METHODS

Tumor cells.The RMA cell line was derived from the Rauscher leukemia virus-induced mouse T-cell lymphoma RBL-5 of the H-2b_{haplotype (19). The} PyRMA cell line was obtained by transfecting RMA cells by electroporation (960

mF, 250 V) with plasmid construct Pywt-neo, a modified version of p1023 con-taining the polyomavirus genome cloned into the pBR322 plasmid at the BamHI sites (21). Transfected clones were selected on G418 and were identified by a polyomavirus-specific PCR (Fig. 1), as described previously (5). Expression of LT was confirmed by Northern (RNA) blotting (data not shown). No infectious virus is produced by using this protocol. All cells were grown in tissue culture (RPMI medium supplemented with 5% fetal calf serum,L-glutamine, penicillin, and streptomycin) or as ascites tumors in (C57BL/63A.SW)F1mice.

Peptide extraction.Cells (108_{) from ascites tumors were lysed in 10 ml of}

phosphate-buffered saline (PBS) containing 0.5% Nonidet P-40, 0.1% aprotinin (Boehringer Mannheim Scandinavia, Bromma, Sweden), and 0.1 mM AEBSF (Calbiochem, La Jolla, Calif.) and stirred for 30 min at 48C. Lysates were centrifuged for 30 min at 13,0003g. Supernatants were then subjected to affinity

prepurification on a Prosep A column coated with normal mouse serum and thereafter passed through a Prosep A column coated with anti-H-2Kb

antibodies (AF6-88.5.3; American Type Culture Collection, Rockville, Md.). Bound mate-rial was eluted from the column with citrate buffer at pH 3. Supernatants were centrifuged in Centriprep ultrafiltration tubes stepwise at 100, 30, and 10 kDa according to the manufacturer’s (Amicon; Grace AB, Helsingborg, Sweden) instructions. The filtrate volume was reduced to 3 ml by vacuum evaporation. The separation of peptides contained in the filtrate was performed by reverse-phase HPLC (16).

Reverse-phase HPLC.HPLC analysis were carried out with a Gilson Medical Electronics (Villiers-le-Bel, France) instrument equipped with a dual-wavelength monitor, set at A214and A280. All integrations were performed by using RAININ

* Corresponding author.

† Present address: Department of Virology, Swedish Institute for Infectious Disease Control, S-105 21 Stockholm, Sweden.

3093

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Dynamax HPLC method manager software. Sample volumes of 500 ml were injected onto the HPLC column. Peptides were separated on a Super Pac Pep-S (5mm; 250 by 4 mm) mixed-bed C18/C2column (Pharmacia LKB Biotechnology,

Uppsala, Sweden) by using a gradient of 0 to 60% acetonitrile in 0.1% triflu-oracetic acid for elution, at a constant flow of 1.0 ml/min. For amino acid analysis and peptide sequencing, 1-min fractions were collected.

Peptide analysis.The amino acid compositions of peptides were determined after hydrolysis with 6 M HCl–0.5% phenol in evacuated tubes for 24 h at 1108C. The released amino acids were analyzed by reverse-phase HPLC as phenylthio-carbamyl derivatives (3).

Sequence analysis was carried out with an Applied Biosystems 470A instru-ment. Phenylthiohydantoin derivatives from degradations were analyzed by re-verse-phase HPLC essentially as described by Bergman and Jo¨rnvall (4).

Synthetic peptides corresponding to polyomavirus LT 578–585 (GKTG LAAA), LT 578–586 (GKTGLAAAL), and LT 578–587 (GKTGLAAALI) were obtained from Scandinavian Peptide Synthesis AB, Ko¨ping, Sweden.

Mice.Inbred C57BL/6 (H-2b_{) and (C57BL/63A.SW)F}

1(H-2b3H-2s) mice

were used for tumor rejection tests.

Immunization with polyomavirus, synthetic peptides, and tumor cells.Mice were immunized four times at weekly intervals intraperitoneally with either 53

106_{PFU of large-plaque strain PA2 of polyomavirus (14) or 6mg of peptide}

dissolved in 100ml of PBS or subcutaneously (s.c.) with 23106_irradiated

(10,000 R) RMA cells.

Transplantation rejection tests.Living RMA or PyRMA tumor cells (53103

) were inoculated s.c. 1 week after the last immunization in different groups of control or immunized mice. Each group contained five mice. At least three separate experiments were performed for each tumor. In order to exclude non-specific boosting of the immune system by polyomavirus, animals were subjected to 400 R of whole-body irradiation prior to tumor cell inoculation (9). Animals were checked for tumor growth, and each tumor diameter was measured every second or third day for 3 weeks. When tumor diameters were approximately 15 mm, animals were sacrificed in order to avoid dissemination of the tumor and further suffering of animals. The mean tumor load (MTL) was calculated for each group of animals by adding the individual tumor diameters (d11d21dn) and dividing the sum by the total number of animals within the group (n), as described previously (26). The significance of a decrease in MTL was calculated according to the Wilcoxon-Mann-Whitney test (28).

RESULTS

Identification of a polyomavirus-specific H-2K

-restricted

peptide.

Short peptides bound to the H-2K

_{MHC class I}

molecule were extracted from PyRMA and RMA cells under

acidic conditions. The extracted material was separated by

reverse-phase HPLC. Comparison of the chromatograms from

RMA and PyRMA cell extracts revealed one PyRMA-specific

peak (compare Fig. 2A and B). Corresponding fractions,

frac-tions 22, from both RMA and PyRMA extracts were subjected

to amino acid sequence and compositional analysis and

pep-tide sequencing. The results of analysis indicated the following

specific amino acid sequence in PyRMA fraction 22: GKxGLxxA,

with 5 of 8 amino acids common to residues 578 to 585 of

polyomavirus LT (30). The amino acid sequence identified in

RMA fraction 22 was ANEQNEQG. Since the exact length of

the peptide was not determined and since H-2K

_-restricted

peptides have been suggested to be 8 or 9 residues long (for

review, see references 11 and 12), three peptides

correspond-ing to this part of the polyomavirus LT were synthesized. These

peptides were 8, 9, and 10 amino acids long with a common N

terminus at residue 578 of LT and different C termini: LT

578–585, GKTGLAAA; LT 578–586, GKTGLAAAL; and LT

578–587, GKTGLAAALI. These peptides were then analyzed

by HPLC, using the same protocol as that for cellular eluates.

Of the three peptides tested, the synthetic nonamer, LT 578–

586, was the one observed to coelute with the specific PyRMA

peak in fraction 22 (Fig. 2C).

A synthetic octamer peptide corresponding to polyomavirus

LT 578–585 immunizes against PyRMA cells, not against

RMA cells.

Groups of C57BL/6 or (C57BL/6

3

A.SW)F

mice

were immunized with either peptide LT 578–585, LT 578–586,

or LT 578–587, polyomavirus, or irradiated RMA cells and

challenged with living PyRMA or RMA tumor cells. Three

separate experiments were performed for each tumor. In each

experiment, the outgrowth of tumors was monitored and

tu-mor diameters were measured. The MTL was calculated. The

cell number inoculated was determined as the lowest dose

required for 100% tumor outgrowth in control animals and was

5

3

10

_{cells per inoculum.}

Figure 3 illustrates the outgrowth of PyRMA in immunized

and control mice in one experiment. Immunization with

wild-type polyomavirus or the synthetic octapeptide inhibited

growth of the PyRMA tumor efficiently (Fig. 3). Tumor

out-growth was observed in nonimmunized controls and in mice

inoculated with the nonapeptide (Fig. 3). In this particular

experiment, immunization with the decapeptide provided some

protection, but tumor outgrowth was still extensive (Fig. 3).

Figure 4 summarizes the results of three PyRMA tumor

rejection tests performed in C57BL/6 or (C57BL/6

3

A.SW)F

mice. The MTLs of PyRMA in different groups of mice given

are those at the time when the MTLs in the controls of all three

experiments were about 5 (Fig. 4A) or 11 (Fig. 4B) mm.

Im-munization with polyomavirus induced a significant reduction

in tumor progression of PyRMA (Fig. 4). In the groups of mice

immunized with polyomavirus, decreases in the MTLs of 90 (P

#

0.0015) and 80% (P

#

0.0015) were observed when the

MTLs of the controls were 5 (Fig. 4A) and 12 (Fig. 4B) mm,

respectively. Immunization with the octapeptide LT 578–585

also inhibited tumor progression of PyRMA. In the groups of

mice immunized with the octapeptide LT 578–585, significant

reductions in the MTLs, 67 (P

#

0.002) and 68% (P

#

0.0035),

were observed when the MTLs of the controls were 5 (Fig. 4A)

and 12 (Fig. 4B) mm, respectively. Both the nonapeptide LT

578–586 and the decapeptide LT 578–587 were less efficient in

their inhibition of tumor outgrowth. The nonapeptide LT 578–

586 induced nonsignificant reductions in the MTLs, 30% when

the MTL of controls was 5 mm (Fig. 4A) and 45% (P

#

0.03)

when the MTL of controls was 12 mm (Fig. 4B). The

decapep-tide LT 578-587 induced reductions in the MTL of 55% (P

#

0.0075) when that of the controls was 5 mm (Fig. 4A) and of 45%

(P

#

0.03) when that of the controls was 12 mm (Fig. 4B).

Outgrowth of the RMA tumor was inhibited efficiently only

in the group of mice immunized with RMA cells (Fig. 5).

Polyomavirus and the three LT peptides had no significant

effect (Fig. 5).

Figure 6 illustrates the results of three RMA tumor rejection

tests at the time point when the MTLs of the three control

FIG. 1. Detection of polyomavirus DNA in the polyomavirus-transfected PyRMA cell line, not in the RMA cell line. The size of the polyomavirus-specific PCR product was 650 bp, and that of the myogenin-specific product was 245 bp (5). MW, molecular weight; neg., negative.

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groups were around 14 mm. Only immunization with

irradi-ated RMA cells reduced the MTL significantly, 90% (P

#

0.005) (Fig. 6). Immunization with polyomavirus or any of the

polyomavirus LT peptides had no significant (

,

23%)

inhibi-tory effect on RMA tumor outgrowth (Fig. 6).

DISCUSSION

Naturally occurring peptides associated with the H-2K

MHC class I molecule were eluted off a

polyomavirus-trans-fected RMA (H-2

_{) tumor, PyRMA. In one HPLC fraction, a}

peptide sequence in which 5 of 8 amino acids (GKxGLxxA)

corresponded to residues 578 to 585 of polyomavirus LT was

identified. Although it is not absolute evidence, this indicates

that a peptide presented on the cell surface of a

polyomavirus-positive tumor in association with the H-2K

_{MHC class I}

molecule may originate from polyomavirus LT. It is likely that

FIG. 3. In vivo growth of PyRMA in different groups of immunized and control mice after inoculation with an s.c. inoculum of 53103

cells per mouse. MTLs in mice immunized with polyomavirus, LT 578–585, LT 578–586, and LT578–587 and in controls are indicated. The MTL was calculated for five mice at different time points after tumor cell inoculation.

FIG. 2. Separation of peptides by reverse-phase HPLC. Peptides were puri-fied by affinity chromatography on an anti-H-2Kb

-coated Prosep A column from 108

RMA (A) and PyRMA (B) cells. (C) Three synthetic polyomavirus LT peptides, octapeptide LT 578–585, nonapeptide LT 578–586, and decapeptide LT 578–587.

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the peptide presented on the surface of PyRMA is a

nonapep-tide, since this peptide coeluted with the PyRMA-specific

HPLC fraction in which the sequence was identified (Fig. 2B

and C). Furthermore, it has a leucine at position 9, which is in

line with previous reports suggesting that peptides associated

with the H-2K

_{molecule are 8 or 9 amino acids long with an}

anchoring leucine at the last position (11, 12).

Since the exact length of the peptide was not determined

after amino acid analysis, three synthetic peptides, an

octapep-tide, a nonapepoctapep-tide, and a decapeptide with the N terminus at

amino acid position 578 of LT, were synthesized and analyzed

further. The octapeptide, GKTGLAAA, was shown to

immu-nize efficiently against a polyomavirus-positive tumor cell

(PyRMA) inoculum in vivo (Fig. 3 and 4). The other two

peptides were not as immunogenic as was the octapeptide. The

immune response to PyRMA cells after peptide immunization

seemed to be polyomavirus specific, since none of the synthetic

LT peptides immunized significantly against the control RMA

tumor (Fig. 5 and 6).

The fact that immunization with the octapeptide resulted in

better protection against the outgrowth of a PyRMA inoculum

than did immunization with the nonapeptide or the

decapep-tide provides an additional aspect to consider when using

syn-thetic peptides as immunogens in vaccine trials. It indicates

that an abundant naturally presented peptide may not be the

most immunogenic. Existing T cells may be triggered more

efficiently by modified versions of the naturally presented

pep-tide. Potentially, by identifying the sequence of naturally

oc-curring peptides and modifying them, we may be able to

im-FIG. 4. In vivo growth of PyRMA in different groups of immunized and control mice after inoculation with an s.c. inoculum of 53103

cells per mouse. The total combined MTLs in different groups of immunized mice and controls from three experiments are given at the time when the MTL was 5 (A) and 12 (B) mm in the controls.

FIG. 5. In vivo growth of RMA in different groups of immunized and control mice after inoculation with an s.c. inoculum of 53103

cells per mouse. MTLs in mice immunized with irradiated RMA cells, polyomavirus, LT 578–585, LT 578–586, and LT 578–587 and in controls are indicated. The MTL was calculated for four mice at different time points after tumor cell inoculation.

FIG. 6. In vivo growth of RMA in different groups of immunized and control mice after inoculation with an s.c. inoculum of 53103

cells per mouse. The total combined MTLs in different groups of immunized mice and controls from three experiments are given at the time when the MTL was 14.2 mm in the controls.

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prove the immune response of the host to certain tumors. This

strategy is an improvement over those of earlier trials, in which

peptide immunization in vivo was less efficient than was

im-munization with wild-type virus at inducing the rejection of

polyomavirus tumors (26). This may have been due to the fact

that the peptides used previously were not the naturally

occur-ring ones or were the wrong size. Similar findings have been

reported earlier by others (25, 27, 32).

Even though the in vivo protective effect of certain

polyoma-virus-specific peptides against polyomavirus tumor challenge is

clear, the exact mechanisms of immune recognition and tumor

rejection are still not understood. The immune response

against polyomavirus tumors is believed to be mediated by T

cells (2, 18, 22, 33). In fact, we have previously shown that both

CD4

and CD8

T cells are important for the rejection of

polyomavirus tumor cells (18). So far, only two reports have

indicated the presence of polyomavirus-specific cytotoxic T

lymphocytes in mice (15, 20). However, in vitro cytotoxicity has

been demonstrated in several other systems, not only against

virus-infected cells (1, 27, 32, 34) but also against

virus-trans-formed cells (7, 13).

Antiviral and antitumor immunities may demand a wide

spectrum of immune mechanisms, but the observation that

immunity to polyomavirus protects against polyomavirus

tu-mor growth suggests a link between the two. It is also likely

that certain MHC class I-presented antigenic peptides are

shared by polyomavirus-infected cells and

polyomavirus-in-duced tumor cells. Still, there must be some differences. Both

CD4

and CD8

cells are necessary for polyomavirus tumor

rejection (19). However, a permanent polyomavirus infection

is eliminated in both CD4

_{and CD8}

_{single knockout}

mice, not in CD4

_CD8

_{double negative mice (6).}

In conclusion, in vivo protection against a polyomavirus

tu-mor cell inoculum can be obtained after immunization with a

synthetic polyomavirus LT 578–585 peptide. This sequence was

identified by amino acid analysis of naturally occurring

pep-tides on the H-2K

_{MHC class I molecule on a polyomavirus}

tumor cell. However, it seems that the most efficient

immuno-gen was a shorter version of the naturally occurring peptide.

These results further support the feasibility of using antiviral or

antitumor vaccines based on synthetic peptides.

ACKNOWLEDGMENTS

This work was supported by the Karolinska Institute, Swedish Med-ical Research Council project no. 13x-10832 (T.B. and H.J.), and Swedish Cancer Society project no. 1806 (T.B. and H.J.) and 1753 (T.D.).

REFERENCES

1. Aichele, P., H. Hengartner, R. M. Zinkernagel, and M. Schulz. 1990. Anti-viral cytotoxic T cell response induced by in vivo priming with a free synthetic peptide. J. Exp. Med. 171:1815–1820.

2. Allison, A. C. 1980. Immune responses to polyoma virus and polyoma virus induced tumors, p. 481–487. In G. Klein (ed.), Viral oncology. Raven Press, New York. 3. Bergman, T., M. Carlquist, and H. Jo¨rnvall.1986. Amino acid analysis by

high performance liquid chromatography of phenylthiocarbamyl derivatives, p. 45–55. In B. Wittmann-Liebold, J. Salnikow, and V. A. Erdmann (ed.), Advanced methods in protein microsequence analysis. Springer, Berlin. 4. Bergman, T., and H. Jo¨rnvall.1987. Electroblotting of individual

polypep-tides from SDS-polyacrylamide gels for direct sequence analysis. Eur. J. Biochem. 169:9–12.

5. Berke, Z., and T. Dalianis. 1993. Persistence of polyomavirus in mice in-fected as adults differs from that observed in mice inin-fected as newborns. J. Virol. 67:4369–4371.

6. Berke, Z., T. Wen, S. Jin, G. Klein, and T. Dalianis. 1995. Polyomavirus persists in CD42/282/2double knockout, but not in CD42/2or CD82/2 single knockout mice. Virology 212:268–271.

7. Bright, R. K., M. H. Shearer, and R. C. Kennedy. 1994. Immunization of Balb/c mice with recombinant simian virus 40 large tumor antigen induces antibody-dependent cell-mediated cytotoxicity against simian virus 40-trans-formed cells. J. Immunol. 153:2064–2071.

8. Dalianis, T. 1990. Studies on the polyoma virus tumour-specific transplan-tation antigen (TSTA). Adv. Cancer Res. 55:57–85.

9. Dalianis, T., G. Magnusson, Y. Ito, and G. Klein. 1982. Immunization against the polyoma virus-induced tumor-specific transplantation antigen by early region mutants of the virus. J. Virol. 43:772–777.

10. Dalianis, T., T. Ramqvist, and G. Klein. 1984. Studies on the polyoma-virus-induced tumor specific transplantation antigen (TSTA)—does middle or large T-antigen play a role? Int. J. Cancer 34:403–406.

11. Engelhard, V. H. 1994. Structure of peptides associated with MHC class I molecules. Curr. Opin. Immunol. 6:13–17.

12. Falk, K., O. Ro¨tzschke, S. Stevanovic, G. Jung, and H.-G. Rammensee.1991. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature (London) 351:290–296.

13. Feltkamp, M. C. W., H. L. Smiths, M. P. M. Vierboom, R. P. Minnaar, B. M.

de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. M. Melief, and W. M. Kast.

1993. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16 trans-formed cells. Eur. J. Immunol. 23:2242–2249.

14. Fried, M., B. E. Griffin, E. Lund, and D. L. Robberson. 1974. A study of wild type mutant and defective DNAs. Cold Spring Harbor Symp. Quant. Biol.

39:45–52.

15. Greene, M., L. Perry, E. Kinney-Thomas, and T. Benjamin. 1982. Specific thymus-derived (T) cell recognition of papova virus transformed cells. J. Immunol. 128:732–736.

16. Joyce, S., and S. G. Nathenson. 1994. Methods to study peptides associated with MHC class I molecules. Curr. Opin. Immunol. 6:24–29.

17. Lathe, R., M. P. Kieny, P. Gerlinger, P. Clertant, I. Guizani, F. Cuzin, and

P. Chambon.1987. Tumor prevention and rejection with recombinant vac-cinia. Nature (London) 326:378–380.

18. Ljunggren, G., H.-G. Ljunggren, and T. Dalianis. 1994. T cell subsets involved in immunity against polyoma virus-induced tumors. Virology 198:714–716. 19. Ljunggren, H.-G., and K. Ka¨rre.1985. Host resistance directed selectively

against H-2 deficient lymphoma variants. Analysis of the mechanism. J. Exp. Med. 162:1745–1759.

20. Lukacher, A. E., Y. Ma, J. P. Carroll, S. R. Abromson-Leman, J. C. Laning,

M. E. Dorf, and T. Benjamin.1995. Susceptibility to tumors induced by polyoma virus is conferred by an endogenous mouse mammary tumor virus superantigen. J. Exp. Med. 181:1683–1692.

21. Luthman, H., M.-G. Nilsson, and G. Magnusson. 1982. Non-contiguous segments of the polyoma genome required in cis for DNA replication. J. Mol. Biol. 161:533–550.

22. Miller, J. F. A. P., R. C. Ting, and L. W. Law. 1964. Influence of thymectomy on tumor induction by polyoma virus in C57BL mice. Proc. Soc. Exp. Biol. Med. 116:323–328.

23. Ramqvist, T., D. O. Pallas, J. DeAnda, L. A¨hrlund-Richter, G. Reinholdsson,

T. Roberts, B. S. Schaffhausen, and T. Dalianis.1988. Immunisation against the polyoma tumor specific transplantation antigen (TSTA) with polyoma T-antigens. Int. J. Cancer 42:123–128.

24. Ramqvist, T., G. Reinholdsson, M. Carlquist, T. Bergman, and T. Dalianis. 1989. A single peptide derived from the sequence common to polyoma small-and middle-T antigen induces immunity against polyoma tumors. Virology

172:359–362.

25. Reddehase, M. J., J. B. Rothbard, and U. H. Koszinowski. 1989. A pen-tapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes. Nature (London) 337:651–653.

26. Reinholdsson-Ljunggren, G., T. Ramqvist, L. A¨hrlund-Richter, and T. Dalianis.1992. Immunization against polyoma tumors with synthetic pep-tides derived from the sequences of middle- and large-T antigens. Int. J. Cancer 50:142–146.

27. Ro¨tzschke, O., K. Falk, K. Deres, H. Schild, M. Norda, J. Metzger, G. Jung, and H.-G. Rammensee.1990. Isolation and analysis of naturally processed viral peptides as recognised by cytotoxic T cells. Nature (London) 348:252–254. 28. Siegel, S., and N. J. Castellan. 1988. Nonparametric statistics for the

behav-ioral sciences, 2nd ed., p. 128. McGraw-Hill, Singapore.

29. Sjo¨gren, H. O.1964. Studies on specific transplantation resistance to poly-omavirus-induced tumors. I. Transplantation resistance induced by polyoma-virus infection. J. Natl. Cancer Inst. 32:361–374.

30. Soeda, E., J. R. Arrand, N. Smolar, J. E. Walsh, and B. E. Griffin. 1980. Coding potential and regulatory signals of the polyoma virus genome. Nature (London) 283:445–453.

31. Townsend, A. R. M., J. Rothbard, F. M. Gotch, G. Bahadur, D. Wraith, and

A. J. McMichael.1986. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell

44:959–968.

32. Van Bleek, G. M., and S. G. Nathenson. 1990. Isolation of an endogenously processed immunodominant viral peptide from the class I H-2Kb_molecule.

Nature (London) 348:213–216.

33. Vandeputte, M. 1968. The effects of heterologous antilymphocytic serum on the oncogenic activity of polyoma virus. Life Sci. 7:855–865.

34. Zhou, X., L. Berg, U. M. Abdel Motal, and M. Jondal. 1992. In vivo primary induction of virus-specific CTL by immunisation with 9-mer synthetic pep-tides. J. Immunol. Methods 153:193–200.

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http://jvi.asm.org/