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Different transplantation antigens in BHK21 cells transformed by four strains of polyoma virus
744 Different Cells
DISCUSSION
AND
Antigens
in BHK21
Transplantation Transformed Polyoma
by
Four
Strains
PRELIMINARY
of
Virus
In an analysis of new transplantation antigens in cells from tumors induced in mice by polyoma virus, Sjogren (1) used the transplant-rejection technique (2, 3) to test several different cell lines which were found to contain the same, polyoma-specific antigen. From this type of evidence it has been suggested that this antigen might be associated with some critical function in the conversion of normal to neoplastic cells by polyoma virus (4). Hare (5), however, described a strain of polyoma virus which produced tumors, cells from which did not appear to synthesize new transplantation antigen. This strain was a large-plaque type derived from stocks of Stewart-Eddy (SE) virus obtained from Dr. W. P. Rowe. The present results are of an investigation to determine whether hamster cells transformed in vitro by different strains of polyoma virus contain different transplantation antigens. Transformed cells were isolated from colonies which developed after infection of a single stock of a clone, Cl3 (8) of BHK21 cells, with each virus. Since the Cl3 clone is presumed to be genetically homogeneous, the transplantation antigens of different transformed cells derived from it could be compared directly. To ensure as far as possible that a transformed cell line consisted of a single population of cells containing the same antigenie determinants, each was grown from a single cell. The polyoma virus strains employed were: (a) a large-plaque SE strain (LP 39) believed to be the sameas that used by Hare (5), (b) a small-plaque mutant derived from stocks of LP 39 (PY ZOS), (c) a Iargeplaque Toronto strain (P 189) (7) and (d) a small-plaque mutant isolated from Toronto large-plaque stocks (P 231) (8). All the virus preparations had been cloned by plaque purification. The virions of these strains could not be distinguished from each other by the hemagglutination-inhibition (HAI) test. Sera prepared in rabbits by
REPORTS
inoculation of CsCl density gradient purified LP 39 and P231 viruses had titers of I : 64,000 and 1: 128,000, respectively, against 8 hemagglutinating doses (HAD) of each of the 4 viruses. Transformed Cl3 eelIs were prepared as follows: cell suspensions, 50 generations from cloning, were exposed for 1 hour at 37” to each of the viruses at a multiplicity of 100 plaque-forming units (PFU) per cell. Then 5 X lo4 infected cells were plated in Eagle’s medium supplemented with 10% tryptose phosphate broth and 10% calf serum (ETC) and containing 0.33 % agar (9). Seven days later colonies of transformed cells were picked from the agar, dispersed in ETC, and cloned in microdrops under oil. Ten clones of each type were grown in ETC to a population of lo8 and stored at -70” in aliquots until required. One clone corresponding to each virus strain was chosen at random for study; these were designated LP 39/8-a, PY 208/4-2, P 189/8-a, and P 231/6-l. The colonial morphology on glass of these four clones was identical. To determine whether LP 39/8-a cells contained polyoma-induced transplantation antigen, a transplant-rejection test was carried out in which two series of 6-week-old Syrian hamsters were inoculated subcutaneously (SC) with 2 X lo6 PFU of LP 39 and P 231; two series were retained as controls. Fourteen days later the animals in each series were arranged in 3 groups of 4 and challenged with dosesof 103, 104, lo5 of either LP 39/8-a or P 231/6-l cells, in a checkerboard type of experiment. At this time sera from all the members of a random sample of 25 % of the virus-inoculated animals contained HA1 antibodies to the homologous virus. These had titers of between 1:800 and 1: 1600 against 8 HAD. After 10 weeks the proportion of animals in each series bearing tumors was noted, and the median tumor dose (MTD), i.e., tumor dose 50 %, and its standard error were calculated by the method of SpaermanIGrber (10). The value of log,, MTD (immunized groups) - log10MTD (control groups) was expressed as the resistance index (RI) to provide a measure of the
DISCUSSION
TRANSPLANT
REJECTION
Expt.
Immunizing material
No.
1
LP
39 virus
P 231 virus None
TESTS
x-LP
39/8-ab
x-P
231/6-lb
x-c
13b
PRELIMINARY
a/l Challenge
cells
745
REPORTS
TABLE 1 TO DEMONSTRATE POLYOMA TRANSPLANTATION VIRUS-TRANSFORMED Cl3 CELLS
ANTIGENS
IN POLYOMA
at these log10 doses5 Log,, median tumor dose 3.0 4.0 5.0
Resistance index
Significance (P 5 0.05)
LP 3918-a
213
314
3/3
3.08
0.09
-
P 231/6-l LP 39/8-a P 231/6-l LP 39/8-a P 231/6-l
2/4 O/3 O/4 l/3 4/4 ______-
414 414 O/3 414 414
212 414 4/4 414 414"
3.00 3.50 4.50 3.17 2.50 __-----___---__
0.50 0.33 2.00
+
LP 39/8-a P 231/6-l LP 39/8-a
114 414 l/3
414 313 414
414 414 414
3.25 2.50 3.17
0.25 0.00 0.17
-
P 231/6-l LP 39/8-a P 231/6-l
O/3 2/4 4/4
114 4/4 4/4
3/4 414 4/4"
4.50 3.00 2.50
2.00
+
of animals
inoculated).
--__-2
AND
a a/b = (number of animals with tumors)/(number b Cells irradiated with 3 kr. c O/4 at lo2 assumed, based on previous results.
degree of resistance to challenge of the immunized groups and the significance of the RI was evaluat’ed at P I 0.05 by a t test. The results of this experiment, shown in Table 1, indicated that hamsters inoculated with P 231 virus were resistant to challenge by P 231-transformed cells; LP 39 virus had no significant immunizing effect against either of the cell lines nor were LP 39.transformed cells rejected in P 231 inoculated animals. Thus, in this test, transplantation antigen was demonstrable in Cl3 cells transformed by P 231 virus, but not in those transformed by LP 39 virus. To determine whether this result might be due to the inability of LP 39 virus to stimulate transplantation immunity in vivo, a direct t,ransplant rejection test was carried out using 10’ X-irradiated (3 Kr) cells as the immunizing material (Jarrett, O., in preparation). The results, in the second part of Table 1 are similar to those obtained using virus. The study was then extended to include Cl3 cells transformed by the related Py 208 and P 189 strains and a similar, larger checkerboard transplant-rejection test was carried out, the results of which are shown in Table 2. Summarizing these, it appeared
Ghat again no antigen could be detected in the LP 39-transformed clone but that cells of the other 3 clones contained antigen. Moreover, there was cross-reactivity between P 231/6-l and P 189/8-l and between the latter and Py 208/4-a. This suggested that there were at least two separate antigens or antigen complexes and that P 189/ 8-l cells contained both; whereas both the Toronto strain-transformed clones appeared to share a common antigen, the clones transformed by the SE strains did not. These results may be interpreted in two ways. First, the polyoma transplantation antigen is not only virus specific but strain of polyoma virus specific; since variants induce different antigens, further evidence is provided that the genetic information for the synthesis of transplantation antigens is supplied by the virus, not by the cell. Second, two (or more) transplantation antigens may be present in each of the clones tested, but in different proportions in each clone. In this case, P 189/8-l may contain both, Py 208/4-a more of one and P 231/6-l more of the other, while LP 39/8-a has little oFnone of either antigen. It is not possible to exclude the presence of an antigen where none is detected owing to the
746
DISCUSSION
AND
PRELIMINARY TABLE
REPORTS
2
TKANSPLANT REJECTION TESTS TO DEMONSTRATE POLYOMA TRANSPLANTATION ANTIGENS IS POLYOMA VIRUS-TRANSFORMED Cl3 CELLS Immunizing strain
virus
LP 39
PY
a/b at these log10 dosesa Challenge
LP 39/8-a PY 208/4-2 P 189/8-l P 231/6-l LP 39/8-a PY 208/4-a P 189/8-l P 231/6-l LP 39/8-a PY 208/4-2 P 189/8-l P 231/6-l LP 39/8-a PY 208/4-2 P 189/8-l P 231/6-l LP 39/8-a PY 208/4-2 P 189/8-l P 231/6-l
208
P 189
P 231
None
a a/b = (number of animals b O/4 at lo2 assumed, based
Resistance index
Significance (P I O.CS)
3/3 4/4 4/4 414 414 313 414 414 4/4 3/3 114 4/4 414 414 O/4 4/4 414 313 4/4b 4/4b
3.25 4.00 3.00 2.50 3.50 4.50 3.50 3.00 4.00 4.50 5.25 3.50 3.50 3.75 5.50 3.50 3.50 3.50 2.50 2.50
0.25 0.50 0.50 0.00 0.00
-
1.00 1.00
+ +
1.00
+
of animals
inoculated).
3.0
4.0
5.0
l/4 O/4 214 313 O/4 O/3 O/4 214 O/4 O/4 O/4 O/4 O/4 114 O/4 O/4 O/4 2/4 414 4/4
4/4 2/4 4/4 414 414 O/4 4/4 414 214 O/4 O/4 414 414 2/4 O/4 414 4/4 214 414 4/4
wit,h tumors)/(number on previous results.
0.50 0.50
-
2.75
+
1.00 0.00
+ -
0.25 3.00 1.00
+ +
transplant rejection The transplantation antigens of more be excluded that there clones of Cl3 cells transformed by each of were two viruses in the P 189 stock, each the viruses require to be examined to test of which induced a different antigen. This the generality of the pattern of antigens is considered unlikely since this stock was found in this study. Work is in progress to produced from cloned virus. determine whether this pattern can be The work of Hare (5) has been confirmed, demonstrated in vitro by cytotoxic, imthat certain large-plaque stocks derived mune adherence, or complement fixation from SE polyoma virus do not induce tests. ACKNOWLEDGMENT detectable transplantation antigens in hamstier cells. Assuming that polyoma virus The author is grateful to Professor Michael codes for a change in the structure of the Stoker and Dr. Ian Macpherson for their genercell which is manifest as a new transplantaous advice and encouragement and to Dr. H. for help in the interpretation of tion antigen, there are three explanations Subak-Sharpe Dulbecco kindly supplied the for the absence of antigen in LP 39-trans- data. Dr. Renato virus. This work was supformed cells: (a) the LP 39 genome does stocks of SE polyoma not include the information which directs ported in part by a grant from the British Empire Cancer Campaign, and was carried out while the the synthesis of antigen; (b) this informa- author was in receipt of a Training Scholarship of tion is present in the viral genome but is the Horserace Betting Levy Board. not expressed in the transformed cell; or REFERENCES (c) the expression of the information does not result, in this case, in an antigenic 1. SJ~GREN, H. O., J. Arc&. Cancer Inst. 32, 361-374 (1964). change in the transformed cell. insensitivity test. Also, it
of the
Log10 median tumor dose
cells
cannot
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.
DISCUSSION
AND
Antigens
in BHK21
Transplantation Transformed Polyoma
by
Four
Strains
PRELIMINARY
of
Virus
In an analysis of new transplantation antigens in cells from tumors induced in mice by polyoma virus, Sjogren (1) used the transplant-rejection technique (2, 3) to test several different cell lines which were found to contain the same, polyoma-specific antigen. From this type of evidence it has been suggested that this antigen might be associated with some critical function in the conversion of normal to neoplastic cells by polyoma virus (4). Hare (5), however, described a strain of polyoma virus which produced tumors, cells from which did not appear to synthesize new transplantation antigen. This strain was a large-plaque type derived from stocks of Stewart-Eddy (SE) virus obtained from Dr. W. P. Rowe. The present results are of an investigation to determine whether hamster cells transformed in vitro by different strains of polyoma virus contain different transplantation antigens. Transformed cells were isolated from colonies which developed after infection of a single stock of a clone, Cl3 (8) of BHK21 cells, with each virus. Since the Cl3 clone is presumed to be genetically homogeneous, the transplantation antigens of different transformed cells derived from it could be compared directly. To ensure as far as possible that a transformed cell line consisted of a single population of cells containing the same antigenie determinants, each was grown from a single cell. The polyoma virus strains employed were: (a) a large-plaque SE strain (LP 39) believed to be the sameas that used by Hare (5), (b) a small-plaque mutant derived from stocks of LP 39 (PY ZOS), (c) a Iargeplaque Toronto strain (P 189) (7) and (d) a small-plaque mutant isolated from Toronto large-plaque stocks (P 231) (8). All the virus preparations had been cloned by plaque purification. The virions of these strains could not be distinguished from each other by the hemagglutination-inhibition (HAI) test. Sera prepared in rabbits by
REPORTS
inoculation of CsCl density gradient purified LP 39 and P231 viruses had titers of I : 64,000 and 1: 128,000, respectively, against 8 hemagglutinating doses (HAD) of each of the 4 viruses. Transformed Cl3 eelIs were prepared as follows: cell suspensions, 50 generations from cloning, were exposed for 1 hour at 37” to each of the viruses at a multiplicity of 100 plaque-forming units (PFU) per cell. Then 5 X lo4 infected cells were plated in Eagle’s medium supplemented with 10% tryptose phosphate broth and 10% calf serum (ETC) and containing 0.33 % agar (9). Seven days later colonies of transformed cells were picked from the agar, dispersed in ETC, and cloned in microdrops under oil. Ten clones of each type were grown in ETC to a population of lo8 and stored at -70” in aliquots until required. One clone corresponding to each virus strain was chosen at random for study; these were designated LP 39/8-a, PY 208/4-2, P 189/8-a, and P 231/6-l. The colonial morphology on glass of these four clones was identical. To determine whether LP 39/8-a cells contained polyoma-induced transplantation antigen, a transplant-rejection test was carried out in which two series of 6-week-old Syrian hamsters were inoculated subcutaneously (SC) with 2 X lo6 PFU of LP 39 and P 231; two series were retained as controls. Fourteen days later the animals in each series were arranged in 3 groups of 4 and challenged with dosesof 103, 104, lo5 of either LP 39/8-a or P 231/6-l cells, in a checkerboard type of experiment. At this time sera from all the members of a random sample of 25 % of the virus-inoculated animals contained HA1 antibodies to the homologous virus. These had titers of between 1:800 and 1: 1600 against 8 HAD. After 10 weeks the proportion of animals in each series bearing tumors was noted, and the median tumor dose (MTD), i.e., tumor dose 50 %, and its standard error were calculated by the method of SpaermanIGrber (10). The value of log,, MTD (immunized groups) - log10MTD (control groups) was expressed as the resistance index (RI) to provide a measure of the
DISCUSSION
TRANSPLANT
REJECTION
Expt.
Immunizing material
No.
1
LP
39 virus
P 231 virus None
TESTS
x-LP
39/8-ab
x-P
231/6-lb
x-c
13b
PRELIMINARY
a/l Challenge
cells
745
REPORTS
TABLE 1 TO DEMONSTRATE POLYOMA TRANSPLANTATION VIRUS-TRANSFORMED Cl3 CELLS
ANTIGENS
IN POLYOMA
at these log10 doses5 Log,, median tumor dose 3.0 4.0 5.0
Resistance index
Significance (P 5 0.05)
LP 3918-a
213
314
3/3
3.08
0.09
-
P 231/6-l LP 39/8-a P 231/6-l LP 39/8-a P 231/6-l
2/4 O/3 O/4 l/3 4/4 ______-
414 414 O/3 414 414
212 414 4/4 414 414"
3.00 3.50 4.50 3.17 2.50 __-----___---__
0.50 0.33 2.00
+
LP 39/8-a P 231/6-l LP 39/8-a
114 414 l/3
414 313 414
414 414 414
3.25 2.50 3.17
0.25 0.00 0.17
-
P 231/6-l LP 39/8-a P 231/6-l
O/3 2/4 4/4
114 4/4 4/4
3/4 414 4/4"
4.50 3.00 2.50
2.00
+
of animals
inoculated).
--__-2
AND
a a/b = (number of animals with tumors)/(number b Cells irradiated with 3 kr. c O/4 at lo2 assumed, based on previous results.
degree of resistance to challenge of the immunized groups and the significance of the RI was evaluat’ed at P I 0.05 by a t test. The results of this experiment, shown in Table 1, indicated that hamsters inoculated with P 231 virus were resistant to challenge by P 231-transformed cells; LP 39 virus had no significant immunizing effect against either of the cell lines nor were LP 39.transformed cells rejected in P 231 inoculated animals. Thus, in this test, transplantation antigen was demonstrable in Cl3 cells transformed by P 231 virus, but not in those transformed by LP 39 virus. To determine whether this result might be due to the inability of LP 39 virus to stimulate transplantation immunity in vivo, a direct t,ransplant rejection test was carried out using 10’ X-irradiated (3 Kr) cells as the immunizing material (Jarrett, O., in preparation). The results, in the second part of Table 1 are similar to those obtained using virus. The study was then extended to include Cl3 cells transformed by the related Py 208 and P 189 strains and a similar, larger checkerboard transplant-rejection test was carried out, the results of which are shown in Table 2. Summarizing these, it appeared
Ghat again no antigen could be detected in the LP 39-transformed clone but that cells of the other 3 clones contained antigen. Moreover, there was cross-reactivity between P 231/6-l and P 189/8-l and between the latter and Py 208/4-a. This suggested that there were at least two separate antigens or antigen complexes and that P 189/ 8-l cells contained both; whereas both the Toronto strain-transformed clones appeared to share a common antigen, the clones transformed by the SE strains did not. These results may be interpreted in two ways. First, the polyoma transplantation antigen is not only virus specific but strain of polyoma virus specific; since variants induce different antigens, further evidence is provided that the genetic information for the synthesis of transplantation antigens is supplied by the virus, not by the cell. Second, two (or more) transplantation antigens may be present in each of the clones tested, but in different proportions in each clone. In this case, P 189/8-l may contain both, Py 208/4-a more of one and P 231/6-l more of the other, while LP 39/8-a has little oFnone of either antigen. It is not possible to exclude the presence of an antigen where none is detected owing to the
746
DISCUSSION
AND
PRELIMINARY TABLE
REPORTS
2
TKANSPLANT REJECTION TESTS TO DEMONSTRATE POLYOMA TRANSPLANTATION ANTIGENS IS POLYOMA VIRUS-TRANSFORMED Cl3 CELLS Immunizing strain
virus
LP 39
PY
a/b at these log10 dosesa Challenge
LP 39/8-a PY 208/4-2 P 189/8-l P 231/6-l LP 39/8-a PY 208/4-a P 189/8-l P 231/6-l LP 39/8-a PY 208/4-2 P 189/8-l P 231/6-l LP 39/8-a PY 208/4-2 P 189/8-l P 231/6-l LP 39/8-a PY 208/4-2 P 189/8-l P 231/6-l
208
P 189
P 231
None
a a/b = (number of animals b O/4 at lo2 assumed, based
Resistance index
Significance (P I O.CS)
3/3 4/4 4/4 414 414 313 414 414 4/4 3/3 114 4/4 414 414 O/4 4/4 414 313 4/4b 4/4b
3.25 4.00 3.00 2.50 3.50 4.50 3.50 3.00 4.00 4.50 5.25 3.50 3.50 3.75 5.50 3.50 3.50 3.50 2.50 2.50
0.25 0.50 0.50 0.00 0.00
-
1.00 1.00
+ +
1.00
+
of animals
inoculated).
3.0
4.0
5.0
l/4 O/4 214 313 O/4 O/3 O/4 214 O/4 O/4 O/4 O/4 O/4 114 O/4 O/4 O/4 2/4 414 4/4
4/4 2/4 4/4 414 414 O/4 4/4 414 214 O/4 O/4 414 414 2/4 O/4 414 4/4 214 414 4/4
wit,h tumors)/(number on previous results.
0.50 0.50
-
2.75
+
1.00 0.00
+ -
0.25 3.00 1.00
+ +
transplant rejection The transplantation antigens of more be excluded that there clones of Cl3 cells transformed by each of were two viruses in the P 189 stock, each the viruses require to be examined to test of which induced a different antigen. This the generality of the pattern of antigens is considered unlikely since this stock was found in this study. Work is in progress to produced from cloned virus. determine whether this pattern can be The work of Hare (5) has been confirmed, demonstrated in vitro by cytotoxic, imthat certain large-plaque stocks derived mune adherence, or complement fixation from SE polyoma virus do not induce tests. ACKNOWLEDGMENT detectable transplantation antigens in hamstier cells. Assuming that polyoma virus The author is grateful to Professor Michael codes for a change in the structure of the Stoker and Dr. Ian Macpherson for their genercell which is manifest as a new transplantaous advice and encouragement and to Dr. H. for help in the interpretation of tion antigen, there are three explanations Subak-Sharpe Dulbecco kindly supplied the for the absence of antigen in LP 39-trans- data. Dr. Renato virus. This work was supformed cells: (a) the LP 39 genome does stocks of SE polyoma not include the information which directs ported in part by a grant from the British Empire Cancer Campaign, and was carried out while the the synthesis of antigen; (b) this informa- author was in receipt of a Training Scholarship of tion is present in the viral genome but is the Horserace Betting Levy Board. not expressed in the transformed cell; or REFERENCES (c) the expression of the information does not result, in this case, in an antigenic 1. SJ~GREN, H. O., J. Arc&. Cancer Inst. 32, 361-374 (1964). change in the transformed cell. insensitivity test. Also, it
of the
Log10 median tumor dose
cells
cannot
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.