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Multiplication of vaccinia virus in the Ehrlich ascites carcinoma
VIROLOGY
3, 514-526 (1957)
Multiplication
of Vaccinia
Virus
in the
Ehrlich
Ascites
Carcinomal” WILLIAM Langbord
Virus
A. CASSEL
Laboratory,
Department of Microbiology, College, Philadelphia, Pennsylvania Accepted February
Hahnemann
Medical
7, 1967
Although the IHD-E strain of vaccinia virus multiplies on the initial intmperitoneal inoculation of mice bearing the Ehrlich ascites tumor, the virus exerts no lethal effect on the host. In fact, the survival time of tumor-bearing mice is significantly increased when a large concentration of IHD-E virus is injected into the tumor. After 6 or more serial passages of IHD-E in tumorbearing mice, the derived virus IHD-T: (a) showed an increased ability to destroy tumor cells; (b) shortened the survival time of the tumor-bearing mice; (c) exhibited a toxicity for normal mice when injected intravenously; and (d) had lost the capacity to produce hemagglutinin. When IHD-T virus was passed serially in eggs, additional changes in the properties of the virus were noted, including: (a) a markedly reduced ability to grow in the tumor and, therefore, a loss of toxic manifestations in the tumor-bearing mice; (b) the recovery of the capacity to produce hemagglutinin; (c) a decreased capacity for multiplication in mouse brain; and (d) a diminished lethal effect in mice following intracerebral inoculation. INTRODUCTION
The effect of viruses on malignant tumors has been examined by a number of investigators (Moore, 1952). With the conversion of solid tumors into the ascites form (Klein, 1951), however, it has become possible to carry out a more definitive study of the growth of viruses in cancer cells. The present communication will deal with the changes which occur in the properties of vaccinia virus following serial passage of the virus in the Ehrlich ascites carcinoma of t,he mouse, and with the additional alterations observed when the derived virus is passed serially in eggs. MATERIALS
AND METHODS
‘ciirus. The virus employed was the mouse-neurotropic, IHD strain of vaccinia. It was procured from Dr. Randall L. Thompson, and had a 1 This study was aided by a grant from the American Cancer Society. 2 Studies carried out during the tenure of a Lederle Medical Faculty Award. 514
VSCCINIA
VIRUS
IN
THE
EHRLICH
TUMOR
315
history of passage in mouse brain. In our laboratory it was carried through 6 chorioallantoic egg passages, and is referred to as IHD-E. In making preparations from membranes they were ground in a mortar with 90 mesh Alundum. A 20 % virus suspension was prepared by adding 1 ml of 0.85 % NaCl per membrane. Passage of the virus in tumor-bearing mice involved the intraperitoneal inoculation of 0.2 ml of a given dilut,ion of virus. The volume of the virus dilution used for the intracerebral inoculation of mice was 0.02 ml. Tumor. The Ehrlich tumor, which was provided by Dr. T. S. Hauschka, was t’he Lettre, hyperdiploid line. After being aspirated from mice, the tumor fluid was transferred to a tube resting in an ice water bath. New tumors were initiated by the intraperitoneal inoculation of mice with 0.2 ml of fluid from ‘i-day tumors. This volume of the tumor contained approximately 40 million tumor cells. Mice so inoculated invariably died on the 14th day after inoculation. Mice. The National Institutes of Health strain of albino mouse was found to be the most expedient for carrying the tumor. Female mice weighing 23 to 26 g were employed. In studies involving intracerebral inoculations, 6-week-old mice were used. These had an average weight of 18 g. The mice were sacrificed by cervical fracture of the spinal cord. Virus assay. The viral content of suspensions of vaccinia virus was determined by chorioallantoic titration, 6 membranes being used for each tenfold dilution of the specimen in 0.85 % NaCl. The volume used to inoculate the lo- to 12-day chick embryos was 0.05 ml. The infected eggs were incubated for 2 days at 37”, examined for lesions, and the number of pocks per membrane recorded. Assuming that each pock was derived from I virus particle, then the number of infective units in the suspension was equal to the average number of pocks per membrane times the dilution factor. The membranes employed to determine the concentrat’ion of infective particles were those which averaged between 1 and 50 pocks. Virus distribution. In this study 200 pock-producing particles (p.p.p.) of 21st passage tumor virus were inoculated intraperitoneally into each of the mice bearing 5-day tumors. Three mice were sacrificed daily, and t’he corresponding organs or fluids pooled. The liver, spleen, lungs, and brain were assayed after they had been rinsed several times with 0.85 % XaCl, ground with Alundum and prepared as 20% suspensions by weight in 0.85% NaCl. The blood and the tumor fluid were assayed directly. Hemagglutination titers. Twenty per cent suspensions of freshly pre-
516
CASSEL
pared membranes with confluent lesions were diluted in twofold steps in a volume of 0.3 ml of 0.85 % NaCl. To each dilution was added 0.3 ml of. a 1% suspension of chicken erythrocytes. After mixing, the agglutination pattern was observed in about 25 minutes. The dilutions given in the data refer to the initial membrane dilutions. The erythrocytes employed were obtained from a single chicken. Cell counts. Standard blood cell counting procedures were employed. Total cell determinations were made by calculation, after the tumor volumes had been measured by aspiration. Immune serum. Rabbits were inoculated intradermally with IHD tumor-adapted virus (22nd passage) which had been passed once in eggs.
DAYS 12345
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1234567 IIIIIII
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12345676 llIIIIII
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I2345676 llllllII
i
106 IO5 IO4 IO3 IO2 IO’ lNOCULUM FIG.
through
1. Multiplication of IHD-T virus 14 serial passages in the tumor.
IO0
(WY?) X5.6 in 5-day
tumors.
This virus
had been
VACCIKIA
VIRUS
IN
THE
EHRLICH
517
TUMOR
This was done to minimize or prevent the carry over to the rabbit of any material other than the virus. Toxicity testing. When mice bearing 5-day tumors are inoculated with 5 X lo6 p.p.p. of the tumor-adapted virus, 50% of the mice will die in 24 to 48 hours. At this point the tumor fluid from the surviving mice was pooled, the cellular debris centrifuged out at’ low speed, and the supernate containing the virus employed as the “toxic” tumor fluid. Toxicity tests were carried out by the conventional method of inoculating 12 to 15 g mice intravenously with 0.5 ml of the preparation to be examined. RESULTS
Multiplication
of the Virus in the Tumor
When large doses of virus were used for serial passage in mice bearing j-day tumors, the animals died prematurely from what appeared to be a toxic effect. In order to circumvent this difficulty, the infected tumor was assayed for its viral content after each passage, and the next serial passage was initiated by injecting only 200 pock-producing particles (p.p.p.). At each passage the tumor fluid was harvested 6 days following inoculation of the virus. TABLE TPMOR
CELL DESTRUCTIUN 200 POCK-PRODWING
1
THE INW~IIATI~~ OF TUMORS OF IHD-E AND IHD-T VIRUSES
FOLLOWING
PARTICLES
WITH
Total tumor cells X 108” Day after virus inoculationb
--~ ControlsC
.4fter infection with IHD-E virus
After infection with IHD-T virusd
8.4 9.4
10.0 10.3 12.7 16.1 15.8 16.6 14.4 11.2
10.0 10.4 12.0 8.1 4.1 4.2 3.4
10.2 14.9 15.8 15.6 13.6 10.6 a Equal volumes of tumor fluid from b The tumors were g-days old at the oculum was contained in 0.2 ml. c These tumors were not, inoculated d IHD-T virus had been through 21
9.3
3 mice were pooled, and then counted. time of virus inoculation. The virus inw-ith virus. serial passages
in the tumor.
518
CASSEL
The data in Fig. 1 show the multiplication of virus in the tumor. In this experiment each different concentration of virus was inoculated intraperitoneally into a group of 30 tumor mice. Three mice from each group were sacrificed daily, and equivalent volumes of the infected tumor fluid were pooled and assayed for virus. Although not depicted in Fig. 1, it should be noted that when high concentrations of virus (greater than lo5 p.p.p.) are inoculated into tumor mice, the animals generally die in 2 to 3 days. On the other hand, when inject,ed wit,h lower concentrations of virus t,he mice do not, usually die in less than 7 days. It was on the
FIG. 2. Multiplication of the egg line (IHD-E) and the tumor line (IHD-T-PI passages) of vaccinia virus in tumor-bearing mice. The lower dose of virus was injected into 6-day tumor mice, and the higher dose of virus into 5-day tumor mice. Also plotted is the multiplication in g-day t.umors of the egg line of virus which had been passed 15 t,imes in the tumor, and then 16 times in eggs (IHDE-T-E). Equal volumes of tumor fluid from 3 mice were pooled daily, and assayed for virus.
VACCINI.4
VIRCS
IX
THE
EHRLICH
519
TUMOR
basis of these observations that the passage procedure described above was employed. On initial intraperitoneal inoculation of the egg line of virus (IHD-E) into tumor mice, the virus multiplies in the cancer cells without producing a lethal effect. When 200 p.p.p. of IHD-E virus are inoculated, there is no detectable destruct’ion of the tumor cells (Table 1) even though the concentration of virus in the tumor fluid is greater than lo6 p.p.p. per milliliter on the 7th day (Fig. 2). An equivalent inoculation (200 p.p.p.) with virus which has had 21 serial passages in the tumor (IHD-T) resulted in a marked reduction in the number of tumor cells, which began on the 5th day following the inoculation of the virus and continued progressively (Table 1). The full measure of cell damage is not necessarily reflected by the total number of tumor cells destroyed, since many of the remaining intact cells show signs of advanced deterioration. With this inoculum the concentration of IHD-T virus reached a level of greater than lo7 p.p.p. per milliliter on the 5th day, and the t’iter continued to rise to a level of more than lo8 p.p.p. per milliliter on the 7th day (Fig. 2). If tumor mice are injected with 5 X lo6 p.p.p. of IHD-E, destruction of the tumor cells is evident on the 3rd day, and by the 5th day approximately 90% of the cells have been destroyed (Table 2). Despite the marked destructSion of the cancer cells induced by high concentrations TABLE TUMOR 5 X
CELL
~)ESTRWTIOS 10” POCK-PROI)C:CING
2 THE ~NOC~I~ATIOS OF TUMORS OF IHD-E ASD IHI)-T VIRUSES
FOLLOWISG
PARTICLES
Total tumor cells X Day after virus inoculationb
1 2 3 4 5
WTH
lO@
-~-~ControlsC
6.4 8.4 9.4 10.2 14.9
I’ Equal volumes of tumor fluid from b The tumors were B-days old at the orulum was contained in 0.2 ml. c These tumors were not inoculated d IHWT virus had been through 21 e No mice survived beyond the 2nd
After infection IHD-E virus
with
4.4 8.6 5.8 3.6 1.4
After
infection
IHD-T
with
vird
3.5 4.3”
3 mice were pooled, and then counted. time of virus inoculation. The virlls inwith virus. serial passages day.
in the tumor.
520
CASSEL
of IHD-E virus, no lethal effect was observed. On the contrary, it was noted that the survival time of tumor mice infected with IHD-E virus was significantly increased. The average survival time of 21 tumor mice infected with IHD-E virus was increased 6 days in comparison with a similar group of noninfected tumor mice. When tumor mice were injected with 5 X lo6 p.p.p. of IHD-T virus, extensive cell destruction was evident (Table 2), and in addition the virus had a marked lethal effect. Of the 22 tumor mice inoculated with IHD-T, 3 mice were sacrificed on the 1st day, and 15 of the 19 remaining mice died on the 2nd day. At the time the mice died the virus infectivity levels in the tumor fluid had reached approximately lo9 p.p.p. per milliliter (Fig. 2). In comparison, it will be recalled that no lethal
15
0
I
2 DAYS
AFTER
3 VIRUS
4
I 5
INOCULATION
FIG. 3. The effect of IHD virus on the weight of tumor-bearing mice. Five-day tumor mice were inoculated with 6 X lo4 p.p.p. of virus from tumor passages 1 to 10. In each case, the weights are an average of 5 mice.
VACCINIA
VIRUS
IN
THE
EHRLICH
521
TUMOR
effect was noted with IHD-E virus, and the maximal infectivity titer of about 10’ p.p.p. per milliliter was reached on the 5th day (Fig. 2). Mice inoculated intraperitoneally with 40 million tumor cells will about double their preinoculation weight in 14 days. During the course of serial passage of vaccinia virus in the tumor it was observed that the mice not only failed to gain weight, but actually lost weight. The data included in Fig. 3 show the changes in weight of tumor mice infected with 6 X lo4 p.p.p. of virus from each passage. Distribution
of the Virus in Infected Tumor Mice
In evaluating the over-all picture of infection it was of interest to determine the distribution of IHD-T virus in several sites other than the
TUMOR
/ LIVER SPLEEN
LUNGS
A BLOOD -\
I
2
3
4
5
\
‘\
6
,‘-\
7
BRAIN \
8
9
DAYS
4. Distribution ing the intraperitoneal FIG.
of IHD-T virus (21st passage) in 5-day tumor mice followinoculation of 200 p.p.p.
522
CASSEL
tumor. As the data in Fig. 4 indicate, the growth of virus in the liver and spleen closely parallels the growth of the virus in the tumor. Somewhat lower levels of virus were found in the lungs, and still lower concentrations of virus were present in the blood. The low infectivity of IHD-T virus in the brain prompted a comparison of the growth of the IHD-E and IHD-T viruses in mice following intracerebral inoculation. As is apparent from Fig. 5, the growth of IHD-E and IHD-T viruses in the brain is not significant,ly different. Thus, it is probable that the lower level of IHD-T virus in the brain following inoculation of the tumor was associated with the route of inoculation. Loss and Recovery of Viral Hemagglutinin Although virus which has been passed serially 14 times in the tumor multiplies to a high titer (Fig. l), no hemagglutinin can be detected in
FIG. 5. Multiplication of IHD-E, IHD-T, and IHD-E-T-E viruses in the brains of mice following intracerebral inoculation with 100 p.p.p. IHD-T had 21 tumor passages. IHD-E-T-E had 15 tumor passages followed by 16 egg passages.
VACCINIA
VIRUS
IS
THE
EHRLICH
TCMOK
52.3
the tumor fluid. When this IHD-T virus is passed once in eggs, preparations of infected membranes wit,h confluent lesions contain about lo8 or more p.p.p. per milliliter. These were also negative when tested for hemagglut,ination wit’h highly susceptible erythrocytes from 11 chickens. Membranes which are infected with vaccinia virus and contain 10’ or more p.p.p. per milliliter have been reported to produce agglutination of chicken erythrocyt,es (Briody and Stannard, 1951). Therefore, the results obtained with the IHD-T virus suggested an important change had occurred during the course of serial passage of the virus in the tumor. In order to ascertain if the ability to produce hemagglutinin was lost on initial passage of the virus in t,he t’umor or disappeared more slowly, 200 p.p.p. of IHD-E virus were passed serially in tumor mice. The reTABLE; I,oss
OF HEMAM;LUTINIS DURISG OF HEMAGGLUTISIS
WITH
Hemagglutinin tit&
0 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15
3
TUMOR PASSAGE
160 80 80 20 10
SUBSEQUENT Subsequent egg passa&
1 2 3 4 5 6 7 8 0 10 11 12 13 14 15
OF IHI) VIRUS ANI) RECOVERY EGG PASSAGE Hemagglutinin titer
u Two hundred p.p.p. of virus were passed serially in mice bearing 5-day tumors, and the tumor fluids w-ere harvested 6 days later. * The tumor-passed virus was inoculated on the chorioallantois of the chick embryo, and the confluent membranes harvested and examined for hemagglutinin. When tested directly, infected tumor fluid failed t,o agglutinate chicken erythrocytes. c The egg passage series was initiated with virus which had 15 tumor passages. One thousand p.p.p. were inoculated at each passage.
524
CASSEL
sults recorded in Table 3 show that the capacity to produce hemagglutinin gradually diminished on serial passage of the virus in the tumor, and was not detectable after the fifth passage. The data included in Table 3 also reveal that when virus which has had 15 serial passages in the tumor is passed serially in eggs, the infected membranes were positive for hemagglutinin at the eighth passage. With continued egg passage the hemagglutinin titer of the infected membranes gradually increased and attained high levels. Characteristics of Virus Passed Serially in the Tumor and Subsequently Passed Serially in Eggs (IHD-E-T-E) The loss of hemagglutinin on serial passage of IHD virus in the tumor and recovery of hemagglutinin on subsequent egg passage suggested that other properties of this virus (IHD-E-T-E) should be examined. Of primary interest was the ability of IHD-E-T-E virus to proliferate in the tumor. As may be observed from the results depicted in Fig. 2, the IHD-E-T-E virus shows a markedly reduced capacity for multiplication in the tumor. The maximal infectivity titers in the tumor of IHD-E TABLE MORTALITY AMONG POCK-PRODUCING
4
MICE AFTER INTRACEREBRAL PARTICLES OF THREE LINES
INOCULATION OF VACCINIA
WITH VIRUF
100
Number of mice dead after infection with: Day HID-Eb
1 2 3 4 5 6
0 0 0 16 6 3
IHD-‘I?
0 0 0 9 7 5
IHD-E-T-E)
0 0 0 0 0 0
KEY: IHD-E-The egg line of virus. IHD-T-Virus which had 21 passages in the tumor. IHD-E-T-E-Virus which had 15 passages in the tumor, then 16 passages in eggs. a Identical preparations of the three lines of virus were used to obtain the data included in Table 4 and Fig. 5. b A total of 45 mice was inoculated with each virus. Four mice were sacrificed daily for 6 days. In the case of IHD-E infected mice none was sacrificed on the 6th day since they had all died by this time. No deaths were seen in mice infected with IHD-E-T-E during the g-week observation period.
VACCINIA
VIRUS
IN
THE
EHRLICH
525
TUMOR
and IHD-T viruses are 1,000 and 100,000 times higher than that of IHD-E-T-E virus (Fig. 2). Another characteristic of IHD-E-T-E virus worthy of investigation was the ability of the virus to grow in mouse brain following intracerebra1 inoculation. As is apparent from Fig. 5, the multiplication of IHD-E-T-E virus occurred at a lower level than that observed with IHD-E or IHD-T virus. Correlated with the lower infectivity level of IHD-E-T-E virus was the absence of any lethal effect on mice following the intracerebral inoculation of 100 p.p.p. of the virus. In order to produce a 50% mortality, 800 p.p.p. of IHD-E-T-E virus were required. On the other hand, the intracerebral inoculation of 100 p.p.p. of IHD-E or IHD-T virus in mice produced a marked lethal effect from which all the mice died in 6 days (Table 4). TABLE
5
TOXIC EFFECT OF THE TUMOR LINE
OF IHD
VIRUS”
Number deaths occurring in inoculated mice Pock-producing particles per 0.05 ml of test mat&a 1
-1 ‘otal Inice
Material testedb 411
2 3 5 9 202112 -
Test 1 2.3 x 2.3 X 2.3 X 2.0 x None
106 lo6 lo6 106
Test 2 5.0 x 106 5.0 x 106 5.0 x 106
1 1 2
Virus + normal tumor fluid Virus + normal rabbit serum Virus + immune rabbit serum Virus infected membranesc Disintegrated tumor cell&
Virus Virus Virus
+ normal tumor fluid + normal rabbit serum + immune rabbit serum
- -
2 1
6 1 5 1 1 2
4
3 5 231 3 2
13 I2 - -1a The virus had been through 21 passages in the tumor. b Ten per cent by volume of normal tumor fluid, normal rabbit serum, or anti-vaccinial serum was added to the virus-infected, cell-free fluid. The preparations were held at room temperature for 30 minutes, and 0.5 ml injected intravenously into 12 to 15 g mice. c This virus had 21 serial passages in the tumor followed by 1 egg passage. d More than 99% of the cells from uninfected g-day t,umors were disintegrated by shaking with glass beads (Nossal, 1953), and the supernat’e remaining after low speed centrifugation was employed.
526 Toxicity of IHD-T
CASSEL
Virus
In view of the early deaths in tumor mice infected with high concentrations of IHD-T virus, infected tumor fluids were examined for their ability to kill mice following intravenous inoculation. From the data included in Table 5 it is evident that virus-infected tumor fluid is toxic for mice. It may also be seen that normal rabbit serum has little effect on the toxicity whereas anti-vaccinial immune serum significantly reduces the toxicity. Although not included in Table 5, it should be noted that when IHD-T virus is removed from the tumor fluid by centrifugation the fluid is no longer toxic. ,If, however, the sedimented virus was resuspended in an equivalent volume of tumor fluid from uninfected mice, the preparation was toxic. A control preparation of mechanically disintegrated tumor cells had no lethal effect on the mice. The over-all picture strongly suggests that the toxicity is associated with the virus particle. The diminution of toxicity of the IHD-T virus following one passage in eggs may be a reflection of the total virus, in contrast with the infective virus. It is probable that the total virus per unit of volume present in the tumor is higher than in the egg. ACKNOWLEDGMENTS The author is indebted to Miss Barbara Fater and Mrs. Mary Fearing Downing for technical assistance, and to Dr. B. A. Briody for discussion of the work during its progress. REFERENCES BRIODY, B. A., and STANNARD, C. (1951). Studies on vaccinia virus I. J. Immunol., 67, 403-411. KLEIN, G. (1951). Comparative studies of mouse tumors with respect to their
capacity for growth as “ascites tumors” and their average nucleic acid content per cell. Exptl. Cell Research 2, 518-573. MOORE, A. E. (1952). Viruses with oncolytic properties and their adaptation to tumors. Ann. N. Y. Acad. Sci. 64, 945-952. NOSSAL, P. M. (1953). A mechanical cell disintegrator. Australian J. Exptl. Biol. Med. Sci. 31, 583-589.
3, 514-526 (1957)
Multiplication
of Vaccinia
Virus
in the
Ehrlich
Ascites
Carcinomal” WILLIAM Langbord
Virus
A. CASSEL
Laboratory,
Department of Microbiology, College, Philadelphia, Pennsylvania Accepted February
Hahnemann
Medical
7, 1967
Although the IHD-E strain of vaccinia virus multiplies on the initial intmperitoneal inoculation of mice bearing the Ehrlich ascites tumor, the virus exerts no lethal effect on the host. In fact, the survival time of tumor-bearing mice is significantly increased when a large concentration of IHD-E virus is injected into the tumor. After 6 or more serial passages of IHD-E in tumorbearing mice, the derived virus IHD-T: (a) showed an increased ability to destroy tumor cells; (b) shortened the survival time of the tumor-bearing mice; (c) exhibited a toxicity for normal mice when injected intravenously; and (d) had lost the capacity to produce hemagglutinin. When IHD-T virus was passed serially in eggs, additional changes in the properties of the virus were noted, including: (a) a markedly reduced ability to grow in the tumor and, therefore, a loss of toxic manifestations in the tumor-bearing mice; (b) the recovery of the capacity to produce hemagglutinin; (c) a decreased capacity for multiplication in mouse brain; and (d) a diminished lethal effect in mice following intracerebral inoculation. INTRODUCTION
The effect of viruses on malignant tumors has been examined by a number of investigators (Moore, 1952). With the conversion of solid tumors into the ascites form (Klein, 1951), however, it has become possible to carry out a more definitive study of the growth of viruses in cancer cells. The present communication will deal with the changes which occur in the properties of vaccinia virus following serial passage of the virus in the Ehrlich ascites carcinoma of t,he mouse, and with the additional alterations observed when the derived virus is passed serially in eggs. MATERIALS
AND METHODS
‘ciirus. The virus employed was the mouse-neurotropic, IHD strain of vaccinia. It was procured from Dr. Randall L. Thompson, and had a 1 This study was aided by a grant from the American Cancer Society. 2 Studies carried out during the tenure of a Lederle Medical Faculty Award. 514
VSCCINIA
VIRUS
IN
THE
EHRLICH
TUMOR
315
history of passage in mouse brain. In our laboratory it was carried through 6 chorioallantoic egg passages, and is referred to as IHD-E. In making preparations from membranes they were ground in a mortar with 90 mesh Alundum. A 20 % virus suspension was prepared by adding 1 ml of 0.85 % NaCl per membrane. Passage of the virus in tumor-bearing mice involved the intraperitoneal inoculation of 0.2 ml of a given dilut,ion of virus. The volume of the virus dilution used for the intracerebral inoculation of mice was 0.02 ml. Tumor. The Ehrlich tumor, which was provided by Dr. T. S. Hauschka, was t’he Lettre, hyperdiploid line. After being aspirated from mice, the tumor fluid was transferred to a tube resting in an ice water bath. New tumors were initiated by the intraperitoneal inoculation of mice with 0.2 ml of fluid from ‘i-day tumors. This volume of the tumor contained approximately 40 million tumor cells. Mice so inoculated invariably died on the 14th day after inoculation. Mice. The National Institutes of Health strain of albino mouse was found to be the most expedient for carrying the tumor. Female mice weighing 23 to 26 g were employed. In studies involving intracerebral inoculations, 6-week-old mice were used. These had an average weight of 18 g. The mice were sacrificed by cervical fracture of the spinal cord. Virus assay. The viral content of suspensions of vaccinia virus was determined by chorioallantoic titration, 6 membranes being used for each tenfold dilution of the specimen in 0.85 % NaCl. The volume used to inoculate the lo- to 12-day chick embryos was 0.05 ml. The infected eggs were incubated for 2 days at 37”, examined for lesions, and the number of pocks per membrane recorded. Assuming that each pock was derived from I virus particle, then the number of infective units in the suspension was equal to the average number of pocks per membrane times the dilution factor. The membranes employed to determine the concentrat’ion of infective particles were those which averaged between 1 and 50 pocks. Virus distribution. In this study 200 pock-producing particles (p.p.p.) of 21st passage tumor virus were inoculated intraperitoneally into each of the mice bearing 5-day tumors. Three mice were sacrificed daily, and t’he corresponding organs or fluids pooled. The liver, spleen, lungs, and brain were assayed after they had been rinsed several times with 0.85 % XaCl, ground with Alundum and prepared as 20% suspensions by weight in 0.85% NaCl. The blood and the tumor fluid were assayed directly. Hemagglutination titers. Twenty per cent suspensions of freshly pre-
516
CASSEL
pared membranes with confluent lesions were diluted in twofold steps in a volume of 0.3 ml of 0.85 % NaCl. To each dilution was added 0.3 ml of. a 1% suspension of chicken erythrocytes. After mixing, the agglutination pattern was observed in about 25 minutes. The dilutions given in the data refer to the initial membrane dilutions. The erythrocytes employed were obtained from a single chicken. Cell counts. Standard blood cell counting procedures were employed. Total cell determinations were made by calculation, after the tumor volumes had been measured by aspiration. Immune serum. Rabbits were inoculated intradermally with IHD tumor-adapted virus (22nd passage) which had been passed once in eggs.
DAYS 12345
1234567 llIIIlI
1234567 IIIIIII
12345678 IIIIIIII
12345676 llIIIIII
12345676 llllllll
I2345676 llllllII
i
106 IO5 IO4 IO3 IO2 IO’ lNOCULUM FIG.
through
1. Multiplication of IHD-T virus 14 serial passages in the tumor.
IO0
(WY?) X5.6 in 5-day
tumors.
This virus
had been
VACCIKIA
VIRUS
IN
THE
EHRLICH
517
TUMOR
This was done to minimize or prevent the carry over to the rabbit of any material other than the virus. Toxicity testing. When mice bearing 5-day tumors are inoculated with 5 X lo6 p.p.p. of the tumor-adapted virus, 50% of the mice will die in 24 to 48 hours. At this point the tumor fluid from the surviving mice was pooled, the cellular debris centrifuged out at’ low speed, and the supernate containing the virus employed as the “toxic” tumor fluid. Toxicity tests were carried out by the conventional method of inoculating 12 to 15 g mice intravenously with 0.5 ml of the preparation to be examined. RESULTS
Multiplication
of the Virus in the Tumor
When large doses of virus were used for serial passage in mice bearing j-day tumors, the animals died prematurely from what appeared to be a toxic effect. In order to circumvent this difficulty, the infected tumor was assayed for its viral content after each passage, and the next serial passage was initiated by injecting only 200 pock-producing particles (p.p.p.). At each passage the tumor fluid was harvested 6 days following inoculation of the virus. TABLE TPMOR
CELL DESTRUCTIUN 200 POCK-PRODWING
1
THE INW~IIATI~~ OF TUMORS OF IHD-E AND IHD-T VIRUSES
FOLLOWING
PARTICLES
WITH
Total tumor cells X 108” Day after virus inoculationb
--~ ControlsC
.4fter infection with IHD-E virus
After infection with IHD-T virusd
8.4 9.4
10.0 10.3 12.7 16.1 15.8 16.6 14.4 11.2
10.0 10.4 12.0 8.1 4.1 4.2 3.4
10.2 14.9 15.8 15.6 13.6 10.6 a Equal volumes of tumor fluid from b The tumors were g-days old at the oculum was contained in 0.2 ml. c These tumors were not, inoculated d IHD-T virus had been through 21
9.3
3 mice were pooled, and then counted. time of virus inoculation. The virus inw-ith virus. serial passages
in the tumor.
518
CASSEL
The data in Fig. 1 show the multiplication of virus in the tumor. In this experiment each different concentration of virus was inoculated intraperitoneally into a group of 30 tumor mice. Three mice from each group were sacrificed daily, and equivalent volumes of the infected tumor fluid were pooled and assayed for virus. Although not depicted in Fig. 1, it should be noted that when high concentrations of virus (greater than lo5 p.p.p.) are inoculated into tumor mice, the animals generally die in 2 to 3 days. On the other hand, when inject,ed wit,h lower concentrations of virus t,he mice do not, usually die in less than 7 days. It was on the
FIG. 2. Multiplication of the egg line (IHD-E) and the tumor line (IHD-T-PI passages) of vaccinia virus in tumor-bearing mice. The lower dose of virus was injected into 6-day tumor mice, and the higher dose of virus into 5-day tumor mice. Also plotted is the multiplication in g-day t.umors of the egg line of virus which had been passed 15 t,imes in the tumor, and then 16 times in eggs (IHDE-T-E). Equal volumes of tumor fluid from 3 mice were pooled daily, and assayed for virus.
VACCINI.4
VIRCS
IX
THE
EHRLICH
519
TUMOR
basis of these observations that the passage procedure described above was employed. On initial intraperitoneal inoculation of the egg line of virus (IHD-E) into tumor mice, the virus multiplies in the cancer cells without producing a lethal effect. When 200 p.p.p. of IHD-E virus are inoculated, there is no detectable destruct’ion of the tumor cells (Table 1) even though the concentration of virus in the tumor fluid is greater than lo6 p.p.p. per milliliter on the 7th day (Fig. 2). An equivalent inoculation (200 p.p.p.) with virus which has had 21 serial passages in the tumor (IHD-T) resulted in a marked reduction in the number of tumor cells, which began on the 5th day following the inoculation of the virus and continued progressively (Table 1). The full measure of cell damage is not necessarily reflected by the total number of tumor cells destroyed, since many of the remaining intact cells show signs of advanced deterioration. With this inoculum the concentration of IHD-T virus reached a level of greater than lo7 p.p.p. per milliliter on the 5th day, and the t’iter continued to rise to a level of more than lo8 p.p.p. per milliliter on the 7th day (Fig. 2). If tumor mice are injected with 5 X lo6 p.p.p. of IHD-E, destruction of the tumor cells is evident on the 3rd day, and by the 5th day approximately 90% of the cells have been destroyed (Table 2). Despite the marked destructSion of the cancer cells induced by high concentrations TABLE TUMOR 5 X
CELL
~)ESTRWTIOS 10” POCK-PROI)C:CING
2 THE ~NOC~I~ATIOS OF TUMORS OF IHD-E ASD IHI)-T VIRUSES
FOLLOWISG
PARTICLES
Total tumor cells X Day after virus inoculationb
1 2 3 4 5
WTH
lO@
-~-~ControlsC
6.4 8.4 9.4 10.2 14.9
I’ Equal volumes of tumor fluid from b The tumors were B-days old at the orulum was contained in 0.2 ml. c These tumors were not inoculated d IHWT virus had been through 21 e No mice survived beyond the 2nd
After infection IHD-E virus
with
4.4 8.6 5.8 3.6 1.4
After
infection
IHD-T
with
vird
3.5 4.3”
3 mice were pooled, and then counted. time of virus inoculation. The virlls inwith virus. serial passages day.
in the tumor.
520
CASSEL
of IHD-E virus, no lethal effect was observed. On the contrary, it was noted that the survival time of tumor mice infected with IHD-E virus was significantly increased. The average survival time of 21 tumor mice infected with IHD-E virus was increased 6 days in comparison with a similar group of noninfected tumor mice. When tumor mice were injected with 5 X lo6 p.p.p. of IHD-T virus, extensive cell destruction was evident (Table 2), and in addition the virus had a marked lethal effect. Of the 22 tumor mice inoculated with IHD-T, 3 mice were sacrificed on the 1st day, and 15 of the 19 remaining mice died on the 2nd day. At the time the mice died the virus infectivity levels in the tumor fluid had reached approximately lo9 p.p.p. per milliliter (Fig. 2). In comparison, it will be recalled that no lethal
15
0
I
2 DAYS
AFTER
3 VIRUS
4
I 5
INOCULATION
FIG. 3. The effect of IHD virus on the weight of tumor-bearing mice. Five-day tumor mice were inoculated with 6 X lo4 p.p.p. of virus from tumor passages 1 to 10. In each case, the weights are an average of 5 mice.
VACCINIA
VIRUS
IN
THE
EHRLICH
521
TUMOR
effect was noted with IHD-E virus, and the maximal infectivity titer of about 10’ p.p.p. per milliliter was reached on the 5th day (Fig. 2). Mice inoculated intraperitoneally with 40 million tumor cells will about double their preinoculation weight in 14 days. During the course of serial passage of vaccinia virus in the tumor it was observed that the mice not only failed to gain weight, but actually lost weight. The data included in Fig. 3 show the changes in weight of tumor mice infected with 6 X lo4 p.p.p. of virus from each passage. Distribution
of the Virus in Infected Tumor Mice
In evaluating the over-all picture of infection it was of interest to determine the distribution of IHD-T virus in several sites other than the
TUMOR
/ LIVER SPLEEN
LUNGS
A BLOOD -\
I
2
3
4
5
\
‘\
6
,‘-\
7
BRAIN \
8
9
DAYS
4. Distribution ing the intraperitoneal FIG.
of IHD-T virus (21st passage) in 5-day tumor mice followinoculation of 200 p.p.p.
522
CASSEL
tumor. As the data in Fig. 4 indicate, the growth of virus in the liver and spleen closely parallels the growth of the virus in the tumor. Somewhat lower levels of virus were found in the lungs, and still lower concentrations of virus were present in the blood. The low infectivity of IHD-T virus in the brain prompted a comparison of the growth of the IHD-E and IHD-T viruses in mice following intracerebral inoculation. As is apparent from Fig. 5, the growth of IHD-E and IHD-T viruses in the brain is not significant,ly different. Thus, it is probable that the lower level of IHD-T virus in the brain following inoculation of the tumor was associated with the route of inoculation. Loss and Recovery of Viral Hemagglutinin Although virus which has been passed serially 14 times in the tumor multiplies to a high titer (Fig. l), no hemagglutinin can be detected in
FIG. 5. Multiplication of IHD-E, IHD-T, and IHD-E-T-E viruses in the brains of mice following intracerebral inoculation with 100 p.p.p. IHD-T had 21 tumor passages. IHD-E-T-E had 15 tumor passages followed by 16 egg passages.
VACCINIA
VIRUS
IS
THE
EHRLICH
TCMOK
52.3
the tumor fluid. When this IHD-T virus is passed once in eggs, preparations of infected membranes wit,h confluent lesions contain about lo8 or more p.p.p. per milliliter. These were also negative when tested for hemagglut,ination wit’h highly susceptible erythrocytes from 11 chickens. Membranes which are infected with vaccinia virus and contain 10’ or more p.p.p. per milliliter have been reported to produce agglutination of chicken erythrocyt,es (Briody and Stannard, 1951). Therefore, the results obtained with the IHD-T virus suggested an important change had occurred during the course of serial passage of the virus in the tumor. In order to ascertain if the ability to produce hemagglutinin was lost on initial passage of the virus in t,he t’umor or disappeared more slowly, 200 p.p.p. of IHD-E virus were passed serially in tumor mice. The reTABLE; I,oss
OF HEMAM;LUTINIS DURISG OF HEMAGGLUTISIS
WITH
Hemagglutinin tit&
0 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15
3
TUMOR PASSAGE
160 80 80 20 10
SUBSEQUENT Subsequent egg passa&
1 2 3 4 5 6 7 8 0 10 11 12 13 14 15
OF IHI) VIRUS ANI) RECOVERY EGG PASSAGE Hemagglutinin titer
u Two hundred p.p.p. of virus were passed serially in mice bearing 5-day tumors, and the tumor fluids w-ere harvested 6 days later. * The tumor-passed virus was inoculated on the chorioallantois of the chick embryo, and the confluent membranes harvested and examined for hemagglutinin. When tested directly, infected tumor fluid failed t,o agglutinate chicken erythrocytes. c The egg passage series was initiated with virus which had 15 tumor passages. One thousand p.p.p. were inoculated at each passage.
524
CASSEL
sults recorded in Table 3 show that the capacity to produce hemagglutinin gradually diminished on serial passage of the virus in the tumor, and was not detectable after the fifth passage. The data included in Table 3 also reveal that when virus which has had 15 serial passages in the tumor is passed serially in eggs, the infected membranes were positive for hemagglutinin at the eighth passage. With continued egg passage the hemagglutinin titer of the infected membranes gradually increased and attained high levels. Characteristics of Virus Passed Serially in the Tumor and Subsequently Passed Serially in Eggs (IHD-E-T-E) The loss of hemagglutinin on serial passage of IHD virus in the tumor and recovery of hemagglutinin on subsequent egg passage suggested that other properties of this virus (IHD-E-T-E) should be examined. Of primary interest was the ability of IHD-E-T-E virus to proliferate in the tumor. As may be observed from the results depicted in Fig. 2, the IHD-E-T-E virus shows a markedly reduced capacity for multiplication in the tumor. The maximal infectivity titers in the tumor of IHD-E TABLE MORTALITY AMONG POCK-PRODUCING
4
MICE AFTER INTRACEREBRAL PARTICLES OF THREE LINES
INOCULATION OF VACCINIA
WITH VIRUF
100
Number of mice dead after infection with: Day HID-Eb
1 2 3 4 5 6
0 0 0 16 6 3
IHD-‘I?
0 0 0 9 7 5
IHD-E-T-E)
0 0 0 0 0 0
KEY: IHD-E-The egg line of virus. IHD-T-Virus which had 21 passages in the tumor. IHD-E-T-E-Virus which had 15 passages in the tumor, then 16 passages in eggs. a Identical preparations of the three lines of virus were used to obtain the data included in Table 4 and Fig. 5. b A total of 45 mice was inoculated with each virus. Four mice were sacrificed daily for 6 days. In the case of IHD-E infected mice none was sacrificed on the 6th day since they had all died by this time. No deaths were seen in mice infected with IHD-E-T-E during the g-week observation period.
VACCINIA
VIRUS
IN
THE
EHRLICH
525
TUMOR
and IHD-T viruses are 1,000 and 100,000 times higher than that of IHD-E-T-E virus (Fig. 2). Another characteristic of IHD-E-T-E virus worthy of investigation was the ability of the virus to grow in mouse brain following intracerebra1 inoculation. As is apparent from Fig. 5, the multiplication of IHD-E-T-E virus occurred at a lower level than that observed with IHD-E or IHD-T virus. Correlated with the lower infectivity level of IHD-E-T-E virus was the absence of any lethal effect on mice following the intracerebral inoculation of 100 p.p.p. of the virus. In order to produce a 50% mortality, 800 p.p.p. of IHD-E-T-E virus were required. On the other hand, the intracerebral inoculation of 100 p.p.p. of IHD-E or IHD-T virus in mice produced a marked lethal effect from which all the mice died in 6 days (Table 4). TABLE
5
TOXIC EFFECT OF THE TUMOR LINE
OF IHD
VIRUS”
Number deaths occurring in inoculated mice Pock-producing particles per 0.05 ml of test mat&a 1
-1 ‘otal Inice
Material testedb 411
2 3 5 9 202112 -
Test 1 2.3 x 2.3 X 2.3 X 2.0 x None
106 lo6 lo6 106
Test 2 5.0 x 106 5.0 x 106 5.0 x 106
1 1 2
Virus + normal tumor fluid Virus + normal rabbit serum Virus + immune rabbit serum Virus infected membranesc Disintegrated tumor cell&
Virus Virus Virus
+ normal tumor fluid + normal rabbit serum + immune rabbit serum
- -
2 1
6 1 5 1 1 2
4
3 5 231 3 2
13 I2 - -1a The virus had been through 21 passages in the tumor. b Ten per cent by volume of normal tumor fluid, normal rabbit serum, or anti-vaccinial serum was added to the virus-infected, cell-free fluid. The preparations were held at room temperature for 30 minutes, and 0.5 ml injected intravenously into 12 to 15 g mice. c This virus had 21 serial passages in the tumor followed by 1 egg passage. d More than 99% of the cells from uninfected g-day t,umors were disintegrated by shaking with glass beads (Nossal, 1953), and the supernat’e remaining after low speed centrifugation was employed.
526 Toxicity of IHD-T
CASSEL
Virus
In view of the early deaths in tumor mice infected with high concentrations of IHD-T virus, infected tumor fluids were examined for their ability to kill mice following intravenous inoculation. From the data included in Table 5 it is evident that virus-infected tumor fluid is toxic for mice. It may also be seen that normal rabbit serum has little effect on the toxicity whereas anti-vaccinial immune serum significantly reduces the toxicity. Although not included in Table 5, it should be noted that when IHD-T virus is removed from the tumor fluid by centrifugation the fluid is no longer toxic. ,If, however, the sedimented virus was resuspended in an equivalent volume of tumor fluid from uninfected mice, the preparation was toxic. A control preparation of mechanically disintegrated tumor cells had no lethal effect on the mice. The over-all picture strongly suggests that the toxicity is associated with the virus particle. The diminution of toxicity of the IHD-T virus following one passage in eggs may be a reflection of the total virus, in contrast with the infective virus. It is probable that the total virus per unit of volume present in the tumor is higher than in the egg. ACKNOWLEDGMENTS The author is indebted to Miss Barbara Fater and Mrs. Mary Fearing Downing for technical assistance, and to Dr. B. A. Briody for discussion of the work during its progress. REFERENCES BRIODY, B. A., and STANNARD, C. (1951). Studies on vaccinia virus I. J. Immunol., 67, 403-411. KLEIN, G. (1951). Comparative studies of mouse tumors with respect to their
capacity for growth as “ascites tumors” and their average nucleic acid content per cell. Exptl. Cell Research 2, 518-573. MOORE, A. E. (1952). Viruses with oncolytic properties and their adaptation to tumors. Ann. N. Y. Acad. Sci. 64, 945-952. NOSSAL, P. M. (1953). A mechanical cell disintegrator. Australian J. Exptl. Biol. Med. Sci. 31, 583-589.