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Induction of specific antitumor immunity in hamsters with hydroxylamine-inactivated SV40 virus
DISCUSSION 2. SJ~GREN, H. O., HELLSTR~M,
AND PRELIMINARY
I., and KLEIN,
REPORTS
747
solution was adjusted to 6.1. The inactivation of the virus was carried out at 37” 3. HABEL, K., 106, by mixing the virus-containing culture fluid 722-725 (1961). free from cell debris with 5 M solution of Q. KLEIN, G., Cold Spring Harbor Symp. Quant. HA in the proportion 4: 1. Preliminary Biol. 27, 463-470 (1962). 5. HARE, J. D., Proc. Sot. Exptl. Biol. Med. 115, experiments revealed that the increase of pH to 7.0-9.1, the decrease of incubation 805-810 (1964). +,emperature to 4”, the decrease of the final 6. MACPHERSON, I., and STOKER, M., Virology PiA concentration to less than 1218sharply 16, 147-151(1962). 7. MCCULLOCH, E.A., HO~ATSON, A.F., SIMINOinhibited the inactivation of the virus. The VITCH, L., AXELRAD, A.A.,and HAM, A.W., samplesof virus taken after different periods Nature 183, 1535-1536 (1959). of incubation were freed from HA by 8. CRAWFORD, L. V., T/‘iroZogy 18, 177-181(1962). threefold dialysis against Earle’s solution, 9. MACPHERSON, I., and MONTAGNIER, L., pH 7.8 at 4”. Virology 23, 291-294 (1964). Each sample was tested for the infec10. FINIVEY, D. J., “Statistical Method in Biologitious activity of virus in tissue culture and cal Assay,” p. 524. Griffen, London, 1952. OSWALD JARRETT for capacity to induce antitumor immunity in hamsters. The infectivity was titrated Institute of Virology University of Glasgow using the method of limiting dilution, inGlasgow, Scotland fecting 4 to 6 tube tissue cultures of green Accepted September 9, 1966 monkey kidney with each IO-fold dilution of the virus. The final results were read on day 20-22 after infection. The test samples of virus were injected intraperiinduction of Specific Antitumor Immunity in toneally into adult hamsters in volumes of Hamsters with Hydroxylamine-Inactivated 1.0-2.0 ml. The resistance of virus inoculated animals to the cells of the SV40-induced SV40 Virus tumor was tested 7-11 days later using the method described previously (5, 6). The At present it is generally accepted that the live oncogenic virus induces specific index of resistance induced by the virus was antitumor immunity through the forma- estimated by determination of the TrDSo tion of a new transplantation antigen in (50 % t’ransplantable dose of tumor cells) in treated and control animals. Log of TrDso infected cells (1, 2). In this investigation we tried to answer the following question: was calculated by the Reed and Muench method. is it possible to induce antitumor immunity The indexes of resistance produced by using the SV40 virus with altered infecthe virus after treatment with HA were tious activity? To elucidate this question we stddied the compared with those produced by unrelationship between the inactivation of treated virus in IO-fold decreasing doses. The results of one experiment are prethe SV40 virus infectivity and the capacity of such treated virus preparations to induce sented in Table 1. The data including the antitumor immunity in hamsters. As an results of five experiments are summarized inactivating agent hydroxylamine (HA) was in Fig. 1. On the basis of these findings it may be used (3). A large-plaque strain of SV40 virus concluded that as a rule at least lo6 TCDbo (Rh2A 426) was used (4). The virus was are needed to induce antitumor immunity grown in green monkey kidney tissue cul- using the untreated virus (one resistanceture in medium 199 containing 2 % bovine inducing unit of the SV40 virus). Lower serum. A 5 M solution of HA (chemically doses are usually inactive. However, the pure) was prepared using Earle’s solution. HA-treated SV40 with residual infectivity 10 N YaOH was added so that after S-fold of 102.5-103.0TCDso/ml still induced a high level of resistance to tumor cells. A dilution with medium 199 pH of the &al G., Exptl.
Cell Res. 23, 204-208 (1961). Proc. Sot. Exptl. Biol. Med.
748
DISCUSSION
THE
Sample
XNACTIVATION
OF SV40 IMMUNITY
Time of treatment
No.0
\;tturf)*
1 2 3 4 5 6 7 8 9 10 (control)
AND
REPORTS
TABLE 1 INFECTIVITY AND ITS CAPACITY IN HAMSTER BY HYDROXYLAMINE
TO INDUCE
Transplantability of SV40 tumor cells used for challenge in the dose?
Titer of virus (log TCDac/ml)
0 0 0 0 18 24 44 70 90 0
PRELIMINARY
7.5 6.5 5.5 4.5 5.2 4.4 3.0 1.2 0.2 -
104.5
103.E
102J
101.t
l/6 2/g 516
0 l/6
O/6 ‘W 016 O/6
‘W O/6 O/6 O/6
O/5 O/5 O/5
O/5 O/5 O/5
O/6
O/6
5/6 O/5 l/5 O/5 5/6 5/5
4/6 5/6 O/5 l/5 O/5 5/6 5/5
6/6
f-v3
l/5
O/5
216
O/f3
ANTITUMOR
Log TrD60
4.8 4.6 3.4 3.2 5.0 4.8 5.0 3.2 2.9 2.8
Log re;fr
of c
2.0 1.8 0.6 0.4 2.2 2.0 2.2 0.4 0.1 0
a The first four samples represent the untreated original virus preparation and its tenfold dilutions; the nex$ five samples represent the undiluted virus preparation treated with HA during different intervals of time; the tenth (control) sample consists of medium 199. b These doses of tumor cells were injected subcutaneously to hamsters 11 days after the inoculation of the appropriate samples of the virus. Numerator gives number of hamsters bearing tumors 40 days after challenge. Denominator gives number of animals inoculated. c Log TrDSo (for each virus sample) minus log TrDSO (for medium 199).
8
?
6
5
4
3
2
4
FIG. 1. Induction of antitumor immunity in hamsters using the SV40 virus inactivated by hydroxylamine. Ordinate = log of resistance index. Abscissa = log TCDso per hamster. (A) Control (the gradient of virus doses is obtained using tenfold dilutions of the original virus preparation). (B) Test: the undiluted virus treated with HA is inoculated to hamsters; the gradient of residual infectivity is obtained using different time of inactivation. i, 2, 3, 4, 5 denote the numbers of individual experiments.
more complete inactivation of the virus resulted in the loss of the ability to induce immunity. Thus, it was found that the SV40 virus preparation having the infectivity significantly reduced by HA was capable of inducing specific antitumor immunity in hamsters. When treated for a longer period the virus loses not only its infectivity, but also the capacity to induce immunity. The published data point out that virusinactivating activity of HA is connected with its action on the viral nucleic acids (7-9). It is known that HA inactivates the infectivity of DNA and RNA viruses without noticeable alteration of their antigenic characteristics (10). Some evidence was presented that at pH 6.1 HA reacts predominantly with cytosine, while at pH 9.1 it reacts with uracil (8). Nevertheless the alteration of viral capsid in HA treated SV40 preparation in our experiments cannot be excluded. However, this possibility seems to be unlikely as: (a) the HA-treated SV40 after two injections into guinea pigs induced the production of virus-neutralizing antibodies which reached the same titer
DISCUSSION
AND
PRELIMINARY
as in experiment with the native virus preparation; (b) the HA-treated SV40 injected into newborn hamsters was oncogenic (to be published). It appears that this latter phenomenon could not take place if the inactivation were connected predominantly with the damage of viral protein coat. Apparently the inactivation rates of SV40 infectious and transplantation antigeninducing activities are different. If one assumes the linear character of inactivation of resistance inducing activity of the virus treated with HA it is possible to calculate tentatively that the infectivity target is 3.5-5.5 times more sensitive to HA than the target of resistance-inducing activity of the virus. The multiplicity reactivation of SV40 virus treated with HA is also one of the possible explanations of the data. To test this possibihty we studied with immunofluorescent method the ability of HAtreated SV40 to induce T-antigen in the cells of green monkey kidney cultures 48 hours after infection and in the hamster kidney cultures 72 hours after infection. A parallelism was noted between the inactivation of infectivity and T-antigen inducing activity (to be published). Thus the multiplicity reactivation of SV40 virus apparently is not involved in tissue cultures. Nevertheless the partial reactivation of the HA-treated virus in hamsters cannot be excluded completely. The most likely explanation of the data is the suggestion that only a part of SV40 genome is necessary for induction of transplantation antigen synthesis while the infectious activity is conditioned by an intact viral genome. This suggestion is in agreement with the data concerning the ability of the adenoSV40 hybrid virus to induce resistance of hamsters to SV40 tumor cells (11). The same explanation was given for the experimental data obtained in com-
REPORTS
749
parative studies on inactivation of polyoma virus infectivity and transformation activity (19, 15) and the inactivation of SV40 virus infectivity and capacity to induce T-antigen (14). The transforming activit,y of HA-treated SV40 preparations in tissue cultures and the oncogenic activity in hamsters are under study. REFERENCES 1. HABEL, K., Proc. Sot. Exptl. Biol. Med. 106, 722-725 (1961). 2. SJ~~GREN, H. O., HELLSTROM, J., and KLEIN, G., Cancer Res. 21, 329-337 (1961). 3. FRANKLIN, R. M., and WECKER, E., Nature 184, 343-345 (1959). ..& ALTSTEIN, A. D., DODONOVA, N. N., and VASSILJEVA, N. N., Nature 209, 1048 (1966). 5. DEICHMAN, G. I., and KLUCHAREVA, T. E., Virology 24, 131-137, (1964). 6. VASSILJEVA, N. N., and ALTSTEIN, A. D., Proc. Tarassevitch Memorial Session, State Control Institute, Moscow 178-179 (1964). 7. FREESE, E., BAUTZ-FREESE, E., and BAUTZ, E., J. Mol. Biol. 3, 133-143 (1961). 8. SCHUSTER, H., J. Mol. Biol. 3, 447-457 (1961). 9. BROWN, D. M., and SCHELL, P., J. Mol. Biol. 3, 709-710 (1961). 10. GENDON, Yu. Z., Probl. Viral. (USSR) (English Transl.), 11, 483487 (1966). II. RAPP, F., TEVETHIA, S. S., MELNICK, J. L., J. Null. Cancer Inst. 36, 703-708 (1966). 12. BENJAMIN, T., Proc. NatZ. Acad. Sci. U.S. 54, 121-124 (1965). 13. BASILICO, C., and Dr MAYORCA, G., Proc. NatZ. Acad. Sci. U.S. 54, 125-127 (1965). 14. CARP, R. I., and GILDEN, R. V., Virology 27, 639-641 (1965). A. D. ALTSTEIN~ G. I. DEICHMAN$ N. N. DODONOVA~ L. A. Tarassevich State Control Institute Moscow, U.S.S.R.t Institute of Experimental and Clinical OncoZogyJ Academy of Medical Sciences Accepted September 13, 1966
AND PRELIMINARY
I., and KLEIN,
REPORTS
747
solution was adjusted to 6.1. The inactivation of the virus was carried out at 37” 3. HABEL, K., 106, by mixing the virus-containing culture fluid 722-725 (1961). free from cell debris with 5 M solution of Q. KLEIN, G., Cold Spring Harbor Symp. Quant. HA in the proportion 4: 1. Preliminary Biol. 27, 463-470 (1962). 5. HARE, J. D., Proc. Sot. Exptl. Biol. Med. 115, experiments revealed that the increase of pH to 7.0-9.1, the decrease of incubation 805-810 (1964). +,emperature to 4”, the decrease of the final 6. MACPHERSON, I., and STOKER, M., Virology PiA concentration to less than 1218sharply 16, 147-151(1962). 7. MCCULLOCH, E.A., HO~ATSON, A.F., SIMINOinhibited the inactivation of the virus. The VITCH, L., AXELRAD, A.A.,and HAM, A.W., samplesof virus taken after different periods Nature 183, 1535-1536 (1959). of incubation were freed from HA by 8. CRAWFORD, L. V., T/‘iroZogy 18, 177-181(1962). threefold dialysis against Earle’s solution, 9. MACPHERSON, I., and MONTAGNIER, L., pH 7.8 at 4”. Virology 23, 291-294 (1964). Each sample was tested for the infec10. FINIVEY, D. J., “Statistical Method in Biologitious activity of virus in tissue culture and cal Assay,” p. 524. Griffen, London, 1952. OSWALD JARRETT for capacity to induce antitumor immunity in hamsters. The infectivity was titrated Institute of Virology University of Glasgow using the method of limiting dilution, inGlasgow, Scotland fecting 4 to 6 tube tissue cultures of green Accepted September 9, 1966 monkey kidney with each IO-fold dilution of the virus. The final results were read on day 20-22 after infection. The test samples of virus were injected intraperiinduction of Specific Antitumor Immunity in toneally into adult hamsters in volumes of Hamsters with Hydroxylamine-Inactivated 1.0-2.0 ml. The resistance of virus inoculated animals to the cells of the SV40-induced SV40 Virus tumor was tested 7-11 days later using the method described previously (5, 6). The At present it is generally accepted that the live oncogenic virus induces specific index of resistance induced by the virus was antitumor immunity through the forma- estimated by determination of the TrDSo tion of a new transplantation antigen in (50 % t’ransplantable dose of tumor cells) in treated and control animals. Log of TrDso infected cells (1, 2). In this investigation we tried to answer the following question: was calculated by the Reed and Muench method. is it possible to induce antitumor immunity The indexes of resistance produced by using the SV40 virus with altered infecthe virus after treatment with HA were tious activity? To elucidate this question we stddied the compared with those produced by unrelationship between the inactivation of treated virus in IO-fold decreasing doses. The results of one experiment are prethe SV40 virus infectivity and the capacity of such treated virus preparations to induce sented in Table 1. The data including the antitumor immunity in hamsters. As an results of five experiments are summarized inactivating agent hydroxylamine (HA) was in Fig. 1. On the basis of these findings it may be used (3). A large-plaque strain of SV40 virus concluded that as a rule at least lo6 TCDbo (Rh2A 426) was used (4). The virus was are needed to induce antitumor immunity grown in green monkey kidney tissue cul- using the untreated virus (one resistanceture in medium 199 containing 2 % bovine inducing unit of the SV40 virus). Lower serum. A 5 M solution of HA (chemically doses are usually inactive. However, the pure) was prepared using Earle’s solution. HA-treated SV40 with residual infectivity 10 N YaOH was added so that after S-fold of 102.5-103.0TCDso/ml still induced a high level of resistance to tumor cells. A dilution with medium 199 pH of the &al G., Exptl.
Cell Res. 23, 204-208 (1961). Proc. Sot. Exptl. Biol. Med.
748
DISCUSSION
THE
Sample
XNACTIVATION
OF SV40 IMMUNITY
Time of treatment
No.0
\;tturf)*
1 2 3 4 5 6 7 8 9 10 (control)
AND
REPORTS
TABLE 1 INFECTIVITY AND ITS CAPACITY IN HAMSTER BY HYDROXYLAMINE
TO INDUCE
Transplantability of SV40 tumor cells used for challenge in the dose?
Titer of virus (log TCDac/ml)
0 0 0 0 18 24 44 70 90 0
PRELIMINARY
7.5 6.5 5.5 4.5 5.2 4.4 3.0 1.2 0.2 -
104.5
103.E
102J
101.t
l/6 2/g 516
0 l/6
O/6 ‘W 016 O/6
‘W O/6 O/6 O/6
O/5 O/5 O/5
O/5 O/5 O/5
O/6
O/6
5/6 O/5 l/5 O/5 5/6 5/5
4/6 5/6 O/5 l/5 O/5 5/6 5/5
6/6
f-v3
l/5
O/5
216
O/f3
ANTITUMOR
Log TrD60
4.8 4.6 3.4 3.2 5.0 4.8 5.0 3.2 2.9 2.8
Log re;fr
of c
2.0 1.8 0.6 0.4 2.2 2.0 2.2 0.4 0.1 0
a The first four samples represent the untreated original virus preparation and its tenfold dilutions; the nex$ five samples represent the undiluted virus preparation treated with HA during different intervals of time; the tenth (control) sample consists of medium 199. b These doses of tumor cells were injected subcutaneously to hamsters 11 days after the inoculation of the appropriate samples of the virus. Numerator gives number of hamsters bearing tumors 40 days after challenge. Denominator gives number of animals inoculated. c Log TrDSo (for each virus sample) minus log TrDSO (for medium 199).
8
?
6
5
4
3
2
4
FIG. 1. Induction of antitumor immunity in hamsters using the SV40 virus inactivated by hydroxylamine. Ordinate = log of resistance index. Abscissa = log TCDso per hamster. (A) Control (the gradient of virus doses is obtained using tenfold dilutions of the original virus preparation). (B) Test: the undiluted virus treated with HA is inoculated to hamsters; the gradient of residual infectivity is obtained using different time of inactivation. i, 2, 3, 4, 5 denote the numbers of individual experiments.
more complete inactivation of the virus resulted in the loss of the ability to induce immunity. Thus, it was found that the SV40 virus preparation having the infectivity significantly reduced by HA was capable of inducing specific antitumor immunity in hamsters. When treated for a longer period the virus loses not only its infectivity, but also the capacity to induce immunity. The published data point out that virusinactivating activity of HA is connected with its action on the viral nucleic acids (7-9). It is known that HA inactivates the infectivity of DNA and RNA viruses without noticeable alteration of their antigenic characteristics (10). Some evidence was presented that at pH 6.1 HA reacts predominantly with cytosine, while at pH 9.1 it reacts with uracil (8). Nevertheless the alteration of viral capsid in HA treated SV40 preparation in our experiments cannot be excluded. However, this possibility seems to be unlikely as: (a) the HA-treated SV40 after two injections into guinea pigs induced the production of virus-neutralizing antibodies which reached the same titer
DISCUSSION
AND
PRELIMINARY
as in experiment with the native virus preparation; (b) the HA-treated SV40 injected into newborn hamsters was oncogenic (to be published). It appears that this latter phenomenon could not take place if the inactivation were connected predominantly with the damage of viral protein coat. Apparently the inactivation rates of SV40 infectious and transplantation antigeninducing activities are different. If one assumes the linear character of inactivation of resistance inducing activity of the virus treated with HA it is possible to calculate tentatively that the infectivity target is 3.5-5.5 times more sensitive to HA than the target of resistance-inducing activity of the virus. The multiplicity reactivation of SV40 virus treated with HA is also one of the possible explanations of the data. To test this possibihty we studied with immunofluorescent method the ability of HAtreated SV40 to induce T-antigen in the cells of green monkey kidney cultures 48 hours after infection and in the hamster kidney cultures 72 hours after infection. A parallelism was noted between the inactivation of infectivity and T-antigen inducing activity (to be published). Thus the multiplicity reactivation of SV40 virus apparently is not involved in tissue cultures. Nevertheless the partial reactivation of the HA-treated virus in hamsters cannot be excluded completely. The most likely explanation of the data is the suggestion that only a part of SV40 genome is necessary for induction of transplantation antigen synthesis while the infectious activity is conditioned by an intact viral genome. This suggestion is in agreement with the data concerning the ability of the adenoSV40 hybrid virus to induce resistance of hamsters to SV40 tumor cells (11). The same explanation was given for the experimental data obtained in com-
REPORTS
749
parative studies on inactivation of polyoma virus infectivity and transformation activity (19, 15) and the inactivation of SV40 virus infectivity and capacity to induce T-antigen (14). The transforming activit,y of HA-treated SV40 preparations in tissue cultures and the oncogenic activity in hamsters are under study. REFERENCES 1. HABEL, K., Proc. Sot. Exptl. Biol. Med. 106, 722-725 (1961). 2. SJ~~GREN, H. O., HELLSTROM, J., and KLEIN, G., Cancer Res. 21, 329-337 (1961). 3. FRANKLIN, R. M., and WECKER, E., Nature 184, 343-345 (1959). ..& ALTSTEIN, A. D., DODONOVA, N. N., and VASSILJEVA, N. N., Nature 209, 1048 (1966). 5. DEICHMAN, G. I., and KLUCHAREVA, T. E., Virology 24, 131-137, (1964). 6. VASSILJEVA, N. N., and ALTSTEIN, A. D., Proc. Tarassevitch Memorial Session, State Control Institute, Moscow 178-179 (1964). 7. FREESE, E., BAUTZ-FREESE, E., and BAUTZ, E., J. Mol. Biol. 3, 133-143 (1961). 8. SCHUSTER, H., J. Mol. Biol. 3, 447-457 (1961). 9. BROWN, D. M., and SCHELL, P., J. Mol. Biol. 3, 709-710 (1961). 10. GENDON, Yu. Z., Probl. Viral. (USSR) (English Transl.), 11, 483487 (1966). II. RAPP, F., TEVETHIA, S. S., MELNICK, J. L., J. Null. Cancer Inst. 36, 703-708 (1966). 12. BENJAMIN, T., Proc. NatZ. Acad. Sci. U.S. 54, 121-124 (1965). 13. BASILICO, C., and Dr MAYORCA, G., Proc. NatZ. Acad. Sci. U.S. 54, 125-127 (1965). 14. CARP, R. I., and GILDEN, R. V., Virology 27, 639-641 (1965). A. D. ALTSTEIN~ G. I. DEICHMAN$ N. N. DODONOVA~ L. A. Tarassevich State Control Institute Moscow, U.S.S.R.t Institute of Experimental and Clinical OncoZogyJ Academy of Medical Sciences Accepted September 13, 1966