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Enhancement in hamsters of virus oncogenesis attending vaccination procedures
DISCUSSION
AND
PRELIMINARY
estimated on the basis of a molecular weight of 80,000 per subunit (9). We would like to emphasize, therefore, that similar bodies can be constructed not only with P = 3, but also with several other values of P and f. Our choice of P = 3 was dictated by t.he relative ease in construction of adequately fitting models. The real number remains open for discussion. The precise knowledge of the crystallographic properties of the polyheads may help to impose further restriction on the choice of these parameters. Theoretical considerations and experiments on this problem are presently being pursued. The consequences of these variants for the understanding of the morphopoiesis of phage are discussed in more detail elsewhere (8). ACKNOWLEDGMENTS We are indebted to Dr. M. F. Moody and Mr. D. E. Bradley for communicating their results prior to publication. Our thanks go also to Dr. F. A. Eiserling, who made his enormous documentation available to us and participated with valuable discussions. REFERENCES 1. MOODY, M. F., ViroZog?/ 26, 567-576 (1965). 2. BRADLEY, D. E. J. Gen. Microbial. 38, 395-408 (1965). 3. GRABE, M.,Bou DE ~~4TOUR, E., EISERLING, F. A., and KELLENBERGER, E., unpublished observations (1964). 4. FAVRE, R., BOY DE LA TOUR, E., SEGR~, N. and KELLENBERGER, E., J. Ultrastruct. Res. 13, in press (1965). 5. FINCH, J. T., KLUG, A. and STRETTON, A. O., J. Mol. Biol. IO, 570-575 (1964). ~.KELLENBERGER, E.,and BOY DE LA TOUR, E., J. lXtrastrz&. Res. 13, in press (1965). 7. CASPAR, D. L. D., and KLUG, A., Cold Spring Harbor Symp. Quant. Biol. 27, l-24 (1962). 8. KELLENBERGER, E., Ciba Found. Symp. Principles Biomolecular Organization, in press. Churchill, London, 1965. 9. VAN VUNAKIR, H., BAKER, W. H. and BROWN, R. K., Virology 5, 327-336 (1958). E. BOY DE LA TOUR E. KELLENBERGER Znstitut de Bioiogie Mo/t?culaire, Laboratoire de Biophysique, Universitd de GenBve Accepted June 28, 1965
225
REPORTS
Enhancement Attending
in Hamsters Vaccination
of Virus Oncogenesis Procedures’
Two reports (1, 9) relating to tests of experimental vaccines against SV40 or polyoma virus-induced cancer have been published by our group. In the first series of tests (I), the vaccine was an antigen prepared from an extract of SV40 or polyoma virus-induced hamster tumor according to a method described by Bjorklund et al. (8) and in keeping with details furnished by Dr. D. Blaney, who prepared such antigen in Dr. Bjorklund’s laboratory in Sweden. In the studies, homologous or heterologous tumor antigens in Freund’s incomplete mineral oil adjuvant as well as the aqueous material were administered parenterally to the animals 7-56 days after neonatal inoculation with SV40 or polyoma virus and well in advance of first tumor appearance. The vaccines, in the regimen used, failed to protect against homologous virus-induced tumor. These experiments should not be confused with the later findings (2) from our laboratories in which a highly protective effect was obtained using X-irradiated whole tumor cells as vaccine. In the first report (1) of the tests using the tumor extract antigen, a small but statistically significant enhancement of tumor induction was noted in most instances to follow vaccination. The experiment,s did not establish the component or components of the vaccine responsible for enhancement, and it was noted that “the experiment did not exclude the possible role of mineral oil adjuvant in enhancing tumorigenesis.” Since that time, an additional experiment was performed which is pertinent to this point. Aqueous vaccine was prepared by blending 1 g of dried extract antigen (1) from SV40 primary hamster tumor in 20 ml of physiological saline solution at high speed in an Omnimix cup immersed in an ice bath. Adjuvant vaccine consisted of the aqueous vaccine suspensions emulsified with an equal 1 This work was supported in part by Contract PH43-64-55 from the National Cancer Institute, U.S. Public Health Service.
226
DISCUSSION
AND
PRELIMINARY TABLE
REPORTS
1
TUMOIS APPEARANCE IS HOLSTERS INOCULATED WTTH SV40 S:IRUS DURING THE NEON.ITAL PERIOD AND VACCINATED, PRETUMOR APPEARANCE, WITH HOMOLOGOUS HI\MSTER TUXOR ANTIGEN OR PLACEBO CONTROL MATERL~L Tumor appearance, cumulative Time post virus (month)
Nonvaccinated controls (group A)
Hamster tumor antigen (group B)
Difference a~~mW~O confidence Placebo control (group Cl
A-B
A-C
3 4 5
o/39” (OS)* 7/37 (13%) 16/36 (43% )
6/33 23/32 24/32
(18%) (71%) (74% )
g/30 z/29 23/29
(39%) (71%) (78%)
18 l 13 53 It 20 31 + 22
30 f 53 f 35 f
16 21 22
6 7 8
24/36 31/36 33/35
25/32
(77Yo)
24/29 W29 26/27
(82%) (89%) (96% )
11 * 21 7 & 14 5 i 11
16 f 3 f 2 f
21 16 15
(66%) (8647,) (92% :
30/32 (93% ) Q’32 (97%)
n Number with tumor/total in group less nonspecific deaths. * Statistical calculations made in accordance with Cutler et al. (8). Computations A. Itkin.
were made by Mr.
emulsified in Freund’s adjuvant as described above. Newborn hamsters less than 24 hours of age were each inoculated subcutaneously into the interscapular area with 106.5TCDSO of SV40 virus in 0.2 ml volume. Fourteen days later, each litter was divided into three groups: one group received 1 ml of extract antigen in Freund’s adjuvant subcutaneously; a second group was injected with the same volume of Hanks’ BSS in Freund’s adjuvant; and a third group was kept untreated. Fourteen days later, the animals in the extract antigen and placebo control groups were given 1 ml of corresponding aqueous
MONTHSFOLLOWING SV,VlRUS INOCULATION FIG. 1. Time of tumor appearance in vaccinated and nonvaccinated animals in the SV40hamster model. O- -0, Vaccinated with Hanks’ balanced salt solution, adjuvant and aqueous (placebo control; group C). a--@, Vaccinated with SV40 hamster tumor extract, adjuvant and aqueous (group B). A- - -A, Nonvaccinated controls (group A). (See Table 1.)
volume of Freund’s incomplete mineral oil adjuvant (90 % Drakeol, 10% Arlacel A). Placebo vaccine consisted of Hanks’ balanced salt solution in aqueous form or
material
via
the
intraperitoneal
route. Animal care and observations were as recorded previously (1). All animals were palpated at least one time each week until tumors were detected and more frequently thereafter. All animals which died without palpable tumors were autopsied and examined for neoplastic lesions. Suspect tissues were studied microscopically. Table 1 and Figure 1 record the cumulative occurrence of tumors in the three groups of hamsters according to the time following inoculation of SV40 virus. There was a statist’ically significant increase in the rate of tumor appearance during the early period in animals which had received either
DISCUSSION
AND PRELIMINARY
the extract antigen or the placebo. The difference was no longer apparent 6 months after the time of inoculation of virus. This single experiment indicated that enhancement of oncogenesis was obtained in animals which had received the placebo as well as in those which were given extract antigen. The extent to which the extract antigen itself may or may not have contributed to the overall enhancement effect in the present and previous (1) experiments remains to be determined. Enhancement of tumor formation is a well extablished phenomenon and has been the subject of many reports (4-7). The present report presents additional information as to the kinds of materials which may cause enhancement and emphasizes the possible dangers and complications which might attend prophylaxis in human cancer. REFERENCES 1. GOLDNER, H., GIRARDI, A. J., and HILLEMAN, M. R., Proc. Sot. Exptl. Biol. Med. 114, 456467 (1963). 2. GOLDNER, H., GIRARDI, A. J., LARSON, V.M., and HILLEMAN, M. R., Proc. Sot. Exptl. Biol. Med. 117, 851-857 (1964). 3. BJORELUND, B., LUNDBLAD, G., and BJORKLUND, V., Intern. Arch. Allergy Appl. Immunol. 12, 241-261 (1958). 4. KALISS, N., Cancer Res. 18, 992-1003 (1958). 5. MOLLER, G., J. Natl. Cancer Inst. 30, 11531175 (1963). 6. MOLLER, G., J. Natl. Cancer Inst. 30, 11771203 (1963). 7. MOLLER, G., J. Natl. Cancer Inst. 30, 1205 1226 (1963). 8. CUTLER, S. J., and EDERER, F., J. Chronic Diseases 8, 699-712 (1958). H. GOLDNER~ A, J. GIRARDI~
M. R. HILLEMAN Division of Virus and Cell Biology Research Merck Institute for Therapeutic Research West Point, Pennsylvania Accepted July 23, 1965 2 Present address: South Jersey Medical Research Foundation, Camden, New Jersey. 3 Present address: The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania.
REPORTS
227
Evidence for the Exponential Reproduction of the Genome
of Encephalomyocarditis Virus
Recent genetic work has shown the mutation rate for the r+ + r character in encephalomyocarditis (EMC) virus to be 1 to 2 X 10e4 per particle per duplication (1, and to be published). The data for this determination were obtained by the analysis of virus clones for large (r) and small (r+) plaque-forming virus. It has become evident that these data may also be used for determining the mode of replication of EMC virus RNA. Three models of RNA replication, elucidated by Luria (5’) for the T2 DNA phage, will be considered. These have been termed geometric, follow-the-leader, and stamping machine replication. Model I, Geometric reproduction: All genomes may serve as templates for the formation of progeny genomes. Model II, Follow-the-leader reproduction: Replica genomes arise by successive replications of the last genome to be formed. Model III, Stamping-machine reproduction: Only the initial genome can form replica genomes. According to model I, any mutation would at once perpetuate itself and generate a clone of mutant genomes, if the mutations occurred randomly throughout the clones with a small, constant probability per duplication. The size of any mutant clone would depend on how early or late in the cell infection that the mutation took place. In model II, clones of mutants of all sizes would occur with equal probability, whilst in model III the number of mutant genomes would be distributed at random over all clones. When the number of mutants (X) in a clone is plotted against t’he number of clones with X or more mutants, each model predicts a particular frequency distribution of the number of mutants per clone (a, 3). Figure 1 shows the distribution of the total data, 61 clones (circles) and the 20 clones containing between 3000 and 10,000 plaque-forming units (triangles). The two
AND
PRELIMINARY
estimated on the basis of a molecular weight of 80,000 per subunit (9). We would like to emphasize, therefore, that similar bodies can be constructed not only with P = 3, but also with several other values of P and f. Our choice of P = 3 was dictated by t.he relative ease in construction of adequately fitting models. The real number remains open for discussion. The precise knowledge of the crystallographic properties of the polyheads may help to impose further restriction on the choice of these parameters. Theoretical considerations and experiments on this problem are presently being pursued. The consequences of these variants for the understanding of the morphopoiesis of phage are discussed in more detail elsewhere (8). ACKNOWLEDGMENTS We are indebted to Dr. M. F. Moody and Mr. D. E. Bradley for communicating their results prior to publication. Our thanks go also to Dr. F. A. Eiserling, who made his enormous documentation available to us and participated with valuable discussions. REFERENCES 1. MOODY, M. F., ViroZog?/ 26, 567-576 (1965). 2. BRADLEY, D. E. J. Gen. Microbial. 38, 395-408 (1965). 3. GRABE, M.,Bou DE ~~4TOUR, E., EISERLING, F. A., and KELLENBERGER, E., unpublished observations (1964). 4. FAVRE, R., BOY DE LA TOUR, E., SEGR~, N. and KELLENBERGER, E., J. Ultrastruct. Res. 13, in press (1965). 5. FINCH, J. T., KLUG, A. and STRETTON, A. O., J. Mol. Biol. IO, 570-575 (1964). ~.KELLENBERGER, E.,and BOY DE LA TOUR, E., J. lXtrastrz&. Res. 13, in press (1965). 7. CASPAR, D. L. D., and KLUG, A., Cold Spring Harbor Symp. Quant. Biol. 27, l-24 (1962). 8. KELLENBERGER, E., Ciba Found. Symp. Principles Biomolecular Organization, in press. Churchill, London, 1965. 9. VAN VUNAKIR, H., BAKER, W. H. and BROWN, R. K., Virology 5, 327-336 (1958). E. BOY DE LA TOUR E. KELLENBERGER Znstitut de Bioiogie Mo/t?culaire, Laboratoire de Biophysique, Universitd de GenBve Accepted June 28, 1965
225
REPORTS
Enhancement Attending
in Hamsters Vaccination
of Virus Oncogenesis Procedures’
Two reports (1, 9) relating to tests of experimental vaccines against SV40 or polyoma virus-induced cancer have been published by our group. In the first series of tests (I), the vaccine was an antigen prepared from an extract of SV40 or polyoma virus-induced hamster tumor according to a method described by Bjorklund et al. (8) and in keeping with details furnished by Dr. D. Blaney, who prepared such antigen in Dr. Bjorklund’s laboratory in Sweden. In the studies, homologous or heterologous tumor antigens in Freund’s incomplete mineral oil adjuvant as well as the aqueous material were administered parenterally to the animals 7-56 days after neonatal inoculation with SV40 or polyoma virus and well in advance of first tumor appearance. The vaccines, in the regimen used, failed to protect against homologous virus-induced tumor. These experiments should not be confused with the later findings (2) from our laboratories in which a highly protective effect was obtained using X-irradiated whole tumor cells as vaccine. In the first report (1) of the tests using the tumor extract antigen, a small but statistically significant enhancement of tumor induction was noted in most instances to follow vaccination. The experiment,s did not establish the component or components of the vaccine responsible for enhancement, and it was noted that “the experiment did not exclude the possible role of mineral oil adjuvant in enhancing tumorigenesis.” Since that time, an additional experiment was performed which is pertinent to this point. Aqueous vaccine was prepared by blending 1 g of dried extract antigen (1) from SV40 primary hamster tumor in 20 ml of physiological saline solution at high speed in an Omnimix cup immersed in an ice bath. Adjuvant vaccine consisted of the aqueous vaccine suspensions emulsified with an equal 1 This work was supported in part by Contract PH43-64-55 from the National Cancer Institute, U.S. Public Health Service.
226
DISCUSSION
AND
PRELIMINARY TABLE
REPORTS
1
TUMOIS APPEARANCE IS HOLSTERS INOCULATED WTTH SV40 S:IRUS DURING THE NEON.ITAL PERIOD AND VACCINATED, PRETUMOR APPEARANCE, WITH HOMOLOGOUS HI\MSTER TUXOR ANTIGEN OR PLACEBO CONTROL MATERL~L Tumor appearance, cumulative Time post virus (month)
Nonvaccinated controls (group A)
Hamster tumor antigen (group B)
Difference a~~mW~O confidence Placebo control (group Cl
A-B
A-C
3 4 5
o/39” (OS)* 7/37 (13%) 16/36 (43% )
6/33 23/32 24/32
(18%) (71%) (74% )
g/30 z/29 23/29
(39%) (71%) (78%)
18 l 13 53 It 20 31 + 22
30 f 53 f 35 f
16 21 22
6 7 8
24/36 31/36 33/35
25/32
(77Yo)
24/29 W29 26/27
(82%) (89%) (96% )
11 * 21 7 & 14 5 i 11
16 f 3 f 2 f
21 16 15
(66%) (8647,) (92% :
30/32 (93% ) Q’32 (97%)
n Number with tumor/total in group less nonspecific deaths. * Statistical calculations made in accordance with Cutler et al. (8). Computations A. Itkin.
were made by Mr.
emulsified in Freund’s adjuvant as described above. Newborn hamsters less than 24 hours of age were each inoculated subcutaneously into the interscapular area with 106.5TCDSO of SV40 virus in 0.2 ml volume. Fourteen days later, each litter was divided into three groups: one group received 1 ml of extract antigen in Freund’s adjuvant subcutaneously; a second group was injected with the same volume of Hanks’ BSS in Freund’s adjuvant; and a third group was kept untreated. Fourteen days later, the animals in the extract antigen and placebo control groups were given 1 ml of corresponding aqueous
MONTHSFOLLOWING SV,VlRUS INOCULATION FIG. 1. Time of tumor appearance in vaccinated and nonvaccinated animals in the SV40hamster model. O- -0, Vaccinated with Hanks’ balanced salt solution, adjuvant and aqueous (placebo control; group C). a--@, Vaccinated with SV40 hamster tumor extract, adjuvant and aqueous (group B). A- - -A, Nonvaccinated controls (group A). (See Table 1.)
volume of Freund’s incomplete mineral oil adjuvant (90 % Drakeol, 10% Arlacel A). Placebo vaccine consisted of Hanks’ balanced salt solution in aqueous form or
material
via
the
intraperitoneal
route. Animal care and observations were as recorded previously (1). All animals were palpated at least one time each week until tumors were detected and more frequently thereafter. All animals which died without palpable tumors were autopsied and examined for neoplastic lesions. Suspect tissues were studied microscopically. Table 1 and Figure 1 record the cumulative occurrence of tumors in the three groups of hamsters according to the time following inoculation of SV40 virus. There was a statist’ically significant increase in the rate of tumor appearance during the early period in animals which had received either
DISCUSSION
AND PRELIMINARY
the extract antigen or the placebo. The difference was no longer apparent 6 months after the time of inoculation of virus. This single experiment indicated that enhancement of oncogenesis was obtained in animals which had received the placebo as well as in those which were given extract antigen. The extent to which the extract antigen itself may or may not have contributed to the overall enhancement effect in the present and previous (1) experiments remains to be determined. Enhancement of tumor formation is a well extablished phenomenon and has been the subject of many reports (4-7). The present report presents additional information as to the kinds of materials which may cause enhancement and emphasizes the possible dangers and complications which might attend prophylaxis in human cancer. REFERENCES 1. GOLDNER, H., GIRARDI, A. J., and HILLEMAN, M. R., Proc. Sot. Exptl. Biol. Med. 114, 456467 (1963). 2. GOLDNER, H., GIRARDI, A. J., LARSON, V.M., and HILLEMAN, M. R., Proc. Sot. Exptl. Biol. Med. 117, 851-857 (1964). 3. BJORELUND, B., LUNDBLAD, G., and BJORKLUND, V., Intern. Arch. Allergy Appl. Immunol. 12, 241-261 (1958). 4. KALISS, N., Cancer Res. 18, 992-1003 (1958). 5. MOLLER, G., J. Natl. Cancer Inst. 30, 11531175 (1963). 6. MOLLER, G., J. Natl. Cancer Inst. 30, 11771203 (1963). 7. MOLLER, G., J. Natl. Cancer Inst. 30, 1205 1226 (1963). 8. CUTLER, S. J., and EDERER, F., J. Chronic Diseases 8, 699-712 (1958). H. GOLDNER~ A, J. GIRARDI~
M. R. HILLEMAN Division of Virus and Cell Biology Research Merck Institute for Therapeutic Research West Point, Pennsylvania Accepted July 23, 1965 2 Present address: South Jersey Medical Research Foundation, Camden, New Jersey. 3 Present address: The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania.
REPORTS
227
Evidence for the Exponential Reproduction of the Genome
of Encephalomyocarditis Virus
Recent genetic work has shown the mutation rate for the r+ + r character in encephalomyocarditis (EMC) virus to be 1 to 2 X 10e4 per particle per duplication (1, and to be published). The data for this determination were obtained by the analysis of virus clones for large (r) and small (r+) plaque-forming virus. It has become evident that these data may also be used for determining the mode of replication of EMC virus RNA. Three models of RNA replication, elucidated by Luria (5’) for the T2 DNA phage, will be considered. These have been termed geometric, follow-the-leader, and stamping machine replication. Model I, Geometric reproduction: All genomes may serve as templates for the formation of progeny genomes. Model II, Follow-the-leader reproduction: Replica genomes arise by successive replications of the last genome to be formed. Model III, Stamping-machine reproduction: Only the initial genome can form replica genomes. According to model I, any mutation would at once perpetuate itself and generate a clone of mutant genomes, if the mutations occurred randomly throughout the clones with a small, constant probability per duplication. The size of any mutant clone would depend on how early or late in the cell infection that the mutation took place. In model II, clones of mutants of all sizes would occur with equal probability, whilst in model III the number of mutant genomes would be distributed at random over all clones. When the number of mutants (X) in a clone is plotted against t’he number of clones with X or more mutants, each model predicts a particular frequency distribution of the number of mutants per clone (a, 3). Figure 1 shows the distribution of the total data, 61 clones (circles) and the 20 clones containing between 3000 and 10,000 plaque-forming units (triangles). The two