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An analysis of the apparent neutralization of rous sarcoma virus with antiserum to normal chick tissues
VIROLOGY
2,
545-558
An Analysis
Biology
(1956)
of the Apparent Sarcoma Virus with Normal Chick
Division,
California
Neutralization Antiserum Tissues
HARRY
RUBIN’
Institute
of Technology,
Accepted
May
of Rous to
Pasadena,
California
21, 1956
The effect of antiserum to normal chick embryo tissues on the infectivity assay of the Rous sarcoma virus was investigated. It was found that such serum, when mixed with the virus and inoculated on the chorioallantoic membrane (CAM) of the developing chick embryo, caused a sharp reduction in the number of virus-induced tumors when compared with a control containing normal serum. In contrast to neutralization with specific antiviral serum, however, the reaction required complement and was completely reversible on dilution of the serum-virus mixture. Furthermore, the anticell serum suppressed tumor formation even when added after virus had adsorbed to susceptible cells, whereas the antiviral serum was effective only before adsorption. Most of the tumor-inhibiting activity of the antichick serum could be removed by absorption with normal chick cells and to a lesser degree by absorption with sheep red blood cells. Moreover, antiserum to boiled sheep red blood cell stroma was also capable of reducing the number of tumors. The antichick serum had no significant effect on the assay of vaccinia virus on the chorioallantoic membrane. It was concluded that the apparent neutralization of ROW sarcoma virus by antichick serum was due to impairment of the ability of the infected cells to multiply rather than to a direct neutralization of virus itself. This contradicts some earlier reports which indicated that unaltered normal cell protein forms an integral part of the functional surface of the Rous sarcoma virus. INTRODUCTION
There have been several contradictory reports dealing with the effect of antiserum to normal chick tissues on the infectivity of the Rous sarcoma virus. Gye and Purdy (1931) and Amies and Carr (1939) reported that such antiserum neutralized the virus, whereas Keogh ’ Research Fellow of the American Cancer Society on recommendation of the Committee on Growth, National Research Council. Aided in part by a grant from The American Cancer Society. 545
546
HARRY
RUBIN
(1938), Kabat and Furth (1941), and Barrett (1941) failed to confirm this finding. This question has important implications, since a positive result would suggest a very close structural relationship between the protein of the virus and of the host. The problem was therefore reinvestigated with the aim of resolving the contradictory claims. The findings of the disagreeing groups were not strictly comparable, since their methods differed. Thus, Gye and Purdy added guinea pig complement to the serum-virus mixtures, as did Amies and Carr when unpurified virus suspensions were used. Furthermore, both these groups incubated their virus with strong concentrations of serum, which were then inoculated into the assay animal without dilution. By contrast, neither Kabat and Furth nor Barrett used complement in their mixtures, and the former workers diluted their serum-virus mixtures before inoculation. In all cases but one, the assay methods (tumor size or endpoint dilution) have large innate variability. Keogh employed the CAM but gave no details as to procedure. The disparate results obtained with such different techniques suggested a possible explanation for the apparent contradiction; i.e., the antiserum might act by suppressing multiplication of infected cells in the indicator host rather than by inactivating the virus. This hypothesis would be consistent with the suggestion that complement might be required and that dilution of the virus-serum mixture before inoculation may eliminate the effect. Complement is known to be necessary for anticell serum to exert some of its cell-damaging effects (Kalfayan and Kidd, 1953; Morgan, 1955, Schrek and Preston, 1956), whereas dilution of the serum before inoculation might dilute it beyond a concentration at which it could act effectively in the host. The phenomenon may therefore be an apparent rather than a true neutralization. This interpretation would also explain why this neutralization is restricted to tumor viruses, although other viruses containing normal tissue precipitins are known (Knight, 1946). The experiments reported in this paper were designed to clarify this point. They show that the neutralization when tested on the CAM is indeed apparent and that it derives from a marked effect of the antichick serum on the infected cells. MATERIALS
AND
METHODS
Virus. Frozen ampules of concentrated Rous sarcoma virus in 0.15 M Na citrate were kindly supplied by Dr. W. R. Bryan of the National
NEUTRALIZATION
OF
ROUS
SARCOMA
VIRUS
547
Institutes of Health. This virus titered about 8 X lo6 tumor-forming particles per milliliter and was used in varying dilutions in all the experiments. Antiviral serum. This was obtained from a chicken convalescing from an infection with the Lous sarcoma virus and had a high degree of neutralizing activity against the virus. It, too, was supplied by Dr. W. R. Bryan. Antichick serum. Six lo-day-old chick embryos were homogenized in 50 ml of phosphate-buffered saline in a Waring blendor. Two milliliters of (*rude homogenate was used to inoculate rabbits intramuscularly and subcutaneously twice a week for 2 months. The antichick serum was obtained 2 weeks after the final inoculation. Normal serum had been obtained from these rabbits before immunization. Owing to the requirement for concentrated serum in the experiments, it. was necessary to use three sera of varying activities in order to carry out all t,he required tests. CompEement. Complement was obtained from fresh guinea pig serum kept frozen at -20”. :7ledia. Phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954) was used as a diluent throughout all the experiments. Virus assay. The virus was assayed on the chorioallantoic membrane of the developing chick embryo, as described previously (Rubin, 1955). RESULTS
Apparent neutralization of Row sarcoma virus by antichick serum. The earlier reports of successful neutralization of the Rous sarcoma virus by antiserum to normal chick tissues arose from experiments assayed in adult chickens (Gye and Purdy, 1931; Amies and Carr, 1939). The first step in the present experiments was to determine whether analogous findings would be encountered when tumor counts on the CAM of the chick embryo were used for assay. Seven-tenths milliliter of virus containing about 2000 tumor-forming particles was mixed with 0.2 ml of undiluted antichick serum and 0.1 ml of guinea pig complement. A control WSLSset up in which normal prevaccination serum was substituted for the antichick serum. The mixtures were incubated for an hour at 5”, and 0.1 ml was inoculated directly on the CAM without further dilution. As shown in columns 1 and 2, Table 1, there were fewer than 10% as many tumors resulting from the mixture containing the antichick serum as from the control.
548
HARRY
RUBIN
TABLE APPARENT NORMAL
NEUTRALIZATION CHICK EMBRYOS,
1
OF Rous SARCOMA VIRUS WITH AND REVERSAL OF THE EFFECT
ANTISERUM ON DILUTION
TO
(Two-tenths milliliter of the appropriate serum (serum excess) was mixed with 0.7 ml of the indicated dilution of virus and 0.1 ml of guinea pig complement. The mixtures were incubated for 2 hours at 37”; 0.1 ml of each was then either inoculated directly into chick embryos or diluted 1:lOO before inoculation.)
co
(1) Normal rabbit serum undiluted + virus 1:670, incubated and inoculated directly
Apparent virus
1.5 x
106
(3)
Antichick serum undiluted + virus 1: 670, incubated and inoculated directly
1.3
x
106
Antichick serum undiluted + virus 1:6.7, dilutxl;; 100
(4)
I
Antivirus serum 1: 10 virus + 1:670
inoculation
(5) Antivirus serum 1: 10 + virus 1:6.7, dilut;;;; 100 inoculation
5 x
104
1.7
x
106
titer
E$ect of dilution on mixtures of the ROW sarcoma virus with antichick and antiviral sera. Careful kinetic study of neutralization of several viruses (poliomyelitis, Western equine encephalomyelitis, Newcastle disease virus) by specific antiviral sera has shown the inactivation to be largely irreversible (Dulbecco et al., 1956; Rubin and Franklin, unpublished observations). Consequently, dilution of a serum-virus mixture leads to no appreciable dissociation of serum-virus complex. The following experiment was carried out to determine the effect of dilution on mixtures of Rous sarcoma virus, and either antichick serum or antiviral sera. In each case, complement was added, the mixture incubated, and then diluted. The dilution was designed to bring the serum well beyond the concentration at which it would inhibit tumor formation on the CAM as previously determined. The original incubation was made in a region of great antibody excess, so the antibody concentration would not be limiting when mixed with the higher virus concentrations. It may be seen from Table 1 that all evidence of neutralization with the antichick sertim disappeared on dilution, the diluted mixture producing as many tumors as the mixture containing only normal serum. In contrast, about 90% of the virus mixed with specific antiviral serum was irreversibly neutralized, since this fraction remained inactive after dilution. There wax a suggestion of reactivation of about 8 % of the inactivated virus by dilution in the latter case. This may be due to the
NEUTRALIZATION
OF
ROWS
SARCOMA
549
VIRUS
continued action of the concentrated serum in the host until the virus is adsorbed, rather than to the reversible inactivation of this small fraction (Dulbeceo et al., 1956). Eflect of serum concentration in vitro and in viva on the degree of’ apparent neutralization. Results of the previous section suggested that the effect of the antichick serum was independent of the serum concentration during in vitro incubation, depending only on the concentration inoculated into the host. The following experiment was carried out to learn more about the quantitative aspects of this dependence. Two series of four tubes each were set up. In the first series the undiluted serum was mixed with serial fourfold dilutions of the virus and incubated for 1 hour at, 5”. Each of the tubes was then diluted to give an equal concentration of virus in all tubes before inoculation. Thus, the virus of all the tubes was incubated with the same concentration of serum but was inoculated on the CAM with successive fourfold dilutions of this serum. The second series was incubated with concentrations of serum and virus which were identical to the first series after its final dilution. The second series was then inoculated without further dilution. Thus both series were identical in serum concentration on inoculation but differed during incubation. The results are present,ed in Table 2, and t’hey show t,hat the degree of apparent neutralization was strictly dependent, on the concentration TABLE EFFECT
6~
VARYING
SERUM
2
CONCENTRATIONS
APPARENT
I;itro
in.
AND
in
Viva
ON
NEUTRALIZATIOS
(Series A consisted of four tubes with successive fourfold dilutions of virus and undiluted serum, which were incubated at 37” for 2 hours, in t,he presence of excess complement. The contents of each was then diluted to give equal concentrations of virus, with successive fourfold dilutions of serum, and inoculated onto the CAM. Series B consisted of four tubes which were &o&ted with the same concentrations of virus and serum as w-ere present in series A after final dilution. These were then inoculated without further dilution. The results are expressed as number of tumors on the CAM.) Series
A B
Apparent
virus
Undiluted
5.7 5.7
x x
titer
at preliminary
1:4
104 104
5.6 4.2
X lo5 x 105
serum concentration
of:
1:16
7..5 x 8.3 x
1:64
105 105
1.3 9.6
x 10” X IO5
550
HARRY
RUBIN
of serum inoculated into the host, being completely independent of the concentration during incubation. Role of complement. Although complement may enhance the neutralization of certain viruses by their specific antisera (Leymaster and Ward, 1949), it is not an essential component (Dulbecco et al., 1956). It is, however, required to produce certain types of cell damage (Kalfayan and Kidd, 1953; Morgan, 1955; Schrek and Preston, 1956). The earliest report of neutralization by antichick serum explicitly stated a requirement for complement (Gye and Purdy, 1931), whereas a later report indicated that it was required with one type of virus preparation but not with another (Amies and Carr, 1939). Requirement for complement would lend weight to the theory that host cell damage is responsible for apparent neutralization. To learn whether complement is necessary to obtain this effect on the CAM, the antichick serum was heated to destroy complement (56” for 30 minutes) and the serum used in the experiments described in Table 3 to determine the role of complement. A parallel study was made with antiviral serum. The results in the table show that complement is necessary in order to obtain any evidence of apparent neutralization with antichick serum. On the other hand, it did not prove to be essential for neutralization with the antiviral serum, although perhaps it exerted some slight enhancing effect. E$ect of adding serum after the virus has adsorbed to the host cell. The experiments already reported suggested that the antichick serum reduced the number of tumors either by damaging the cells or by neutralizing the virus in a completely reversible way. To distinguish between these alternatives, the action of the antiserum on previously infected cells was determined. Once a virus particle has irreversibly adsorbed TABLE ROLE
OF COMPLEMENT
IN
(Both rabbit antichick serum and 30 minutes to destroy complement. 0.2 ml was mixed with 0.7 ml of virus. of 0.1 ml of complement or 0.1 ml of
7.8 titer
x
106
4.4
x
NEUTRALIZATION
chick antiviral serum were heated at 56” for These were then appropriately diluted, and The mixtures were incubated in the presence buffer andtheninoculated onto the CAM.)
Antichick St!llUD + buffer
Apparent virus
3 APPARENT
106
Antichick SWUIII + complement
2.5
X lo4
Antiviral S~KUlll + buffer
1.5
x
Antiviral StTlUll + complement
10’
1.2
x
104
NEUTRlLIZATION
OF
ROW
TABLE ~';FFE('T
OF ADDISC:
SERUM
S’:ARCOMA
1
AFTER
I.VFECTION
(Virus was diluted to give about 200 tumors on the CAM of 36 embryos. At, appropriate serum mixture (5 parts serum, 2 parts buffer, in groups of 6 embryos.) Hours after infection
1 ‘l 48
Nomal rabbit serum
~~~-~~
8.1
x
10”
531
VIRI:S
HAS
(kCIIRRED
per 0.1 ml of inoculum and placed intervals, 0.1 ml of the indicated 1 part complement.) was inoculated
Rabbit
antichick Serum
-2.3 7.8 5.0
X IO” x 106 x 105
Chirken
antiviral
1:lO serum -
4.5 x X.8 x
IO” IO”
to a cell, it is no longer susceptible to neutralization by a specific antiviral serum (Rubin and Franklin, unpublished observations). The cells themselves, however, may be just as sensitive to damage with anticell antibody after infection as they had been before infection. If this serum decreased the number of tumors by damaging t’he cells, then it might be expected to act just as effectively soon after infect,ion as before. Accordingly, either antiviral or antichick serum was added to the CAM at appropriate intervals after virus inoculation. The results in Table 4 show that the antiviral serum, capable of neutralizing over 90% of t’he virus in z&o, had no effect on the number of tumors when added 4 hours after the virus. The ant’ichick serum retained its effectiveness, even if added as late as 2 days after infection. This result favored the interpretation that the effect of the serum was on t’he cells. It should be noted that there was about 50 % reduction in the number of tumors when the antiviral serum was added 1 hour after infection. This suggests that virus adsorption was only half-complete at this t,ime. Absorptio?z of sera with chick embryo and sheep red blood cells. Roth antichick and antiviral sera were incubated with chick embryo cells to remove antibodies to normal chick cells. The neutralizing capacities of the sera were assayed before and after this Absorption, and the results are presented in Table 5. The antiviral serum was unaffected by this treatment, but the antichick serum lost almost all it,s capacity for apparent neutralization, clearly indicating t)hat two unrelated antibodies were involved in direct neutralization and apparent neutraliaation. Thus, it appears that no anticell antibody was active in the direct neutralization with antiviral serum, whereas the effective component in apparent neutralization with antichick serum was antibody against
552
HARRY
RUBIN
.TABLE ABSORPTION
OF SERA
WITH
CHICK
5
EMBRYO
BLOOD
TISSIJES
AND
WITH
SHEEP
RED
CELLS
(A suspension of lo-day-old chick embryo cells at a concentration of 108 cells/ml was made (Dulbecco, 1954) in phosphate buffer. A 10% suspension of sheep red blood cells was also prepared (6 X lo8 cells/ml). One-milliliter cell aliquots were centrifuged and resuspended in the appropriate serum. The mixtures were incubated at 37” for 2 hours, and the cells were again centrifuged. The superantant serum was absorbed once more and then used to test for neutralizing behavior.)
Apparent virus titer
I
I
5.72
&xlO’
Antichick
Antichick serum absorbed with shee red bloo B calls
4.9 x 106 2.1 x
Antiviral se*um 1:20
Antiviral serum 1: 20 absorbed with chick tissues
105
normal cell antigens. A similar finding was made by Amies and Carr (1939). The antichick serum was similarly treated with sheep red blood cells to remove heterogenetic Forssmann-type antibody. Serum treated in this way permitted the formation of eight times as many tumors as the unabsorbed serum. The sheep cell-absorbed serum retained the capacity to reduce the number of tumors by more than 60% when compared with normal serum, however, indicating that not all the apparent neutralizing antibody was of the Forssmann type. Effect of antiserum to boiled sheepred cell stroma. Since absorption of the antichick serum with sheep red blood cells results in some loss of its capacity for apparent neutralization, then antiserum to sheep cells may simulate to some degree the action of the antichick serum. Antisera to sheep red blood cells made by subcutaneous injection of rabbits in this laboratory exhibited only a low order of activity. A potent antiserum to boiled sheep red blood cell stroma was provided by Dr. Jose Vinas of Johns Hopkins University, however. A 1: 19,500 dilution of this serum gave 50% lysis of a suspensioncontaining 5 X lo* sheep red blood cells per milliliter. At a final dilution of 1:5 it reduced the number of tumors on the CAM to about 4%; a powerful antichick serum at the same concentration reduced it to 1%. (Table 6). Action of anticell serum on pock production by vaccinia virus. The results presented up to this point indicate that the phenomenon of
NEUTRALIZATION
OF
ROUS
TABLE EFFECT
OF ANTISERUM
TO SHEEP
RED
SARCOMA
riS3
VIRUS
6
BLOOD SARCOMA
CELL
STROMA
ON THE
ASSAY
OF THE
Rous VIRUS (Two-tenths milliliter of the appropriate serum was mixed with 0.7 ml of diluted ROW sarcoma virus plus 0.1 ml of complement and incubated for 1 hour at 5”. Then 0.1 ml of each mixture was inoculated onto the CAM of 6 embryos.) Antichick SCNm
Apparent virus
infectivity
4 x 106
of Rous sarcoma
TABLE EFFECT
OF ANTICHICK
SERUM
ON THE
4 x 104
Antisheep stroma
RBC Serum
1.7 x 105
7 ASSAY
OF VACCINIA
VIRUS
(Two-tenths milliliter of the appropriate serum was mixed with 0.7 ml of diluted vaccinia virus and 0.1 ml of complement and incubated for 1 hour at 5”. Then 0.1 ml of each mixture was inoculated onto the CAM of 6 embryos.) Normal
serum
-__--
Pock titer of vaccinia
virus
2.5 X lo6
Antichick - ~-.-l__l_-~.
S~TUIII
1.9 x 106
apparent neutralization of the ROW sarcoma virus is mediated through action of the antichick serum on the infected cell rather than on the virus. Does the mechanism of this action consist in int’erference with the ability of the infected cell to multiply or, alternatively, in impairment, of its ability to produce virus? If the latter explanation were correct, it would seem likely that impairment of the cell’s ability to produce nontumor viruses as well would result. For this reason, the effect of the antichick serum on the production of pocks on the CAM by vaccinia virus was tested. The results are presented in Table 7. The data show that there was no significant decrease in the number of vaccinia pocks, nor was there any apparent difference in t,heir size. This indicates that antiserum-treated cells are capable of producing vaccinia virus and favors the hypothesis that the serum *acts by interfering with cell multiplication. The over-all thickened appearance of antiserum-treated membranes, when compared to the controls, indicates some damage to normal as well as to infected cells. It is evident that the cells are not totally destroyed, however, since they can produce virus. In addition, the pocks made up of cells destroyed by vaccinia infection can be clearly distiu-
554
HARRY
guished against the background tions suggest that the anticell but does not destroy them.
RUBIN
of serum-damaged cells. These observaserum probably damages normal cells
DISCUSSION
The original finding of Gye and Purdy that antiserum to normal chick cells, when mixed with the Rous sarcoma virus in the presence of complement, reduces the apparent infectivity of the virus has been confirmed. The effect of the antichick serum depended only on the concentration of that serum at the moment of inoculation into the host; the effect was independent of the serum concentration during the period of incubation in vi&o with the virus. This contrasts with neutralization by a convalescent antiviral serum, which is strongly dependent on the in v$ro serum concentration. It corresponds to Amies and Carr’s finding (1939) that the virus could be centrifuged from an antichick serumvirus mixture and its full infectivity recovered. Antiviral serum could no longer neutralize virus which had already infected cells of the CAM. However, the antichick serum was just as effective in reducing the number of tumors if applied shortly after infection as it had been when incubated with the virus in vitro before simultaneous inoculation of both components. This showed that the antichick serum exerted its effect through damage inflicted on the cells. The possible occurrence of a completely reversible neutralization as an explanation for the dilution-reactivation phenomenon was thus ruled out. The absence of neutralization with antiviral serum after the virus had adsorbed to susceptible cells, when considered in the light of the observations presented below, suggested that cell-to-cell transmission of the virus was a minor factor in formation of the tumor. It implies that the tumor is formed chiefly through the increased multiplication rate of the originally infected cell and its progeny. This is consistent with the finding that vir.us is produced by sarcoma cells at a very slow rate (Rubin, 1955). It is also consistent with the fact that the height of the CAM tumors is ap.proximately equal to their width. If cell-tocell transmission of the virus occurred at a significant rate, the lateral spread of the virus to adjacent ectodermal cells would be much more rapid than the multiplication of the cells, and the lesion, as in the early proliferative stage of vaccinia infection, would be much wider than it is high. That some limited reinfection does occur, however, is clear from
PU’EUTRALIZATIOK
OF
ROUS
SARCOMA
VIRUS
503
the fact that mesodermal involvement occurs in the late stages of tumor development. Experiments with a nontumor virus, vaccinia, showed that serumtreated cells could produce virus. Therefore the most likely mechanism of action of anticell serum in apparent neutralization is to impair multiplication of the infected cells themselves, rather than to inhibit their ability to produce virus. This would explain why this phenomenon has been uniquely described with tumor viruses and not with necrotizing viruses (Knight, 1946). Antiserum to boiled sheep red blood cells simulates the tumor-suppressing activity of the antichick cell serum to a limited degree. Moreover, a portion of the activity of the antichick serum can be absorbed out with sheep red blood cells. This indicates that some of the cell antigens involved may be of the Forssmann heterogenetic type. Before these findings can be generalized to cover tumor viruses as a class, some observations made by other workers with other tumor viruses have to be considered. Gye and Purdy (1933) reported that antichick serum, although it apparently neutralized Fujinami myxosarcoma virus of chicken origin, failed to do so with that of duck origin, whereas antiduck serum gave the reverse effect. This relationship held true whether the assay was done in chicks or ducklings. This would seem to establish a strong case for true neutralization by the homologous tissue antiserum. The following observations cast doubt on this interpret,ation, however. The neutralization with antiserum to normal tissues always required complement and was species-specific. Antiserum against> the myxosarcoma itself did not require complement for neutralization and was not, species-specific. The latter antiserum always resulted in complete absence of t,umors; the former very rarely did. Normal tissues failed t,o absorb the antibodies from the antimyxosarcoma serum, but they were capable of absorbing homologous antibodies. Taken at face value, these findings suggest that the virus can be inactivated by t’wo unrelated antibodies, one virus-specific and the other species-specific, the latter having an absolute requirement for complement, a unique condition iu virus neutralization. The work also implies that only the species-specific antigens are uniquely thus involved, and not the many antigens which are common to both ducks and chickens. In the present work, by way of contrast, it is shown that even the ubiquitous Forssmann antigens are concerned in apparent neutralization of Rous sarcoma virus. In addition, the possibility of aggregation or coprecipitation, particu-
556
HARRY
RUBIN
larly when crude filtrates are used, cannot be excluded. Knight (1946) has found that antiserum to normal tissues can precipitate purified influenza virus derived from the homologous tissues without neutralizing the virus. Aggregation of a tumor virus, however, might easily result in an apparent reduction in infectivity, if infectivity is measured by tumor size, as it was in the aforementioned experiments, since the number of tumor-initiating foci would then be decreased. The assay method of tumor size which was used is subject to large variation and therefore is of doubtful significance. This is particularly true when multiple-inoculation sites are used, with three different immune sera inoculated into the same host. Finally, Dmochowski (1948) has shown that there is extensive cross reaction in complement fixation between highly purified Rous and Fujinami viruses, but there is no cross reaction between these and normal tissue. This suggests that the results found in the present paper would be applicable to the Fujinami virus as well and poses a direct problem as to the interpretation of the early Fujinami work. The second work which merits consideration is that of Eckert et al. (1955). This extensive research establishes an apparent neutralization of the virus of avian erythromyeloblastic leukemia with antiserum to normal chick tissues and, to a lesser extent, with Forssmann antibody. Here, no complement was required in the in vitro mixture, but the inoculation was made intravenously, thus obviating the requirement. The dilution of the serum on intravenous inoculation would weaken the applicability of the interpretation that the effective serum action occurred against the cells. This objection would not be valid, however, if the serum was powerful and the assay highly accurate, as indeed they appeared to be. There were distinct qualitative and quantitative differences between the action of the antiviral and anticell sera which suggest that the two acted in different ways. Finally, just as in the case of the Rous sarcoma, the normal -chick tissues failed to absorb specific antiviral antibodies from the sera of convalescent chickens, but successfully accomplished this with antiserum to normal chick tissues made in rabbits (Eckert, personal communication). There is considerable evidence that antinormal tissue antibodies can localize and produce damage in a variety of organs (Pressman, 1951; Smadel, 1936; Seegal et al., 1951). It. is likely that the demyelinating encephalomyelitis that follows injection of normal brain tissue is an example of a specific tissue-damaging immunological condition. The
NEUTRALIZATION
OF
ROUS
SARCOMA
VIRUS
557
cell destruction in paroxysmal hemoglobinuria, and in hemolytic anemia probably is due to the action of anticell antibodies (Raffel, 1953). Since there is, in general, cross-reaction between normal and tumorous tissues (Landsteiner, 1945), there is ample precedent for the tumor-suppressing activity of antinormal cell serum. It is concluded that antichick serum inhibits tumor formation by CAM cells infected with the Rous sarcoma virus by damaging these cells rather than by neutralizing the virus. Some of the antigens involved are of the Forssmann heterogenetic type. This result calls for a re-examination of other similar systems to determine whether the same mechanism is at work. ACKNOWLEDGMENTS I greatly appreciate Dr. W. R. Bryan’s generous provision of concentrated Rous sarcoma virus and convalescent chicken serum. I also wish to acknowledge the role which Drs. J. W. Beard and E. A. Eckert’s prepublication discussion of their work played in provoking the thought which led to the present work. REFERENCES C. R., and CARR, J. G. (1939). Immunological experiments with a highly concentrated suspension of Rous 1 tumor producing agent. J. Patho2. Bacterio2. 49, 497-513. BARRETT, M. K. (1940). The antigenic nature of purified chicken tumor agent. Cancer Research 1, 543-4. DULBECC~, R., and VOGT, M. (1954). One step growth curve of Western equine encephalomyelitis on chicken embryo cells grown in oitro, and analysis of virus yields from single cells. J. Exptl. Med. 99, 183-199. DTTLBECCO, It., VOGT, M., and STRICKLAKD, A. G. R. (1956). A study of the basic aspects of neutralization of two animal viruses. Western equine encephalomyelitis virus and poliomyelitis virus. Virolog!/ 2. 162-205. DMOCHOWSKI, L. (1948). Investigations on the properties of agents causing fowl tumors. 1. Attempts at isolation of the fowl tumor agents by differential ccntrifugation. J. #all. Cancer Inst. 9, 57-67. &KERT, E. A., SHARP, 1). G., BEARD, I).. GREEN, I., and BEARD, J. W. (1955). Virus of avian erythromyeloblast,ic leukosis. 1X. Antigenic constitution and immunologic characterization. J. .%‘atZ. Cancer Inst. 16, 593-643. GYE, W. E., and PURDY, W. J. (1931). The Cause of&ncer. Cassell &Co., London. GYE, W. E., and PURDY, \I’. J. (1933). The infective agent in tumor filtrates: A flu%her investigation by means of antisera to normal tissues. Urit. J. Exptl. Path&. 14, 250-259. KAB~T, E. A., and FURTH, J. (1941). Neutralization of the agent causing leukosis and sarcoma of fowls by rabbit antisera. J. Exptl. Med. 74, 257-261. KALFAYAN, B., and KIDD, J. G. (1953). Structural changes produced in BrownAMES,
558
HARRY
RUBIN
Pearce carcinoma cells by means of specific antibody
and complement. J. Exptl. Med. 97, 145-162. KNIGHT, C. A. (1946). Precipitin reactions of highly purified influenza viruses and related materials. J. Exptl. Med. 83,281-294. KEOGH, E. V. (1938). Ectodermal lesions produced by the virus of Rous sarcoma. Brit. J. Exptl. Pathol. 19, 1-8. LANDSTEINER, K. (1945). The Specificity of Serological Reactions, Harvard Univ. Press, Cambridge, Mass. G. R., and WABD, T. G. (1949). The effect of complement in the neutralization of mumps virus. J. Immunol. 61,95-105. MORGAN, I. M. (1955). Cytopathogenic effect of antiserum to human malignant epithelial cells (strain HeLa) on HeLa cell culture. J. ImmunoZ. 76, 478484. PRESSMAN, D. (1951). Radiolabelled antitissue antibodies. Federation Proc. 10, LEYMASTER,
568. RAFFEL, RUBIN,
S. (1953). Immunity. Appleton-Century-Crofts, New York. H. (1955). Quantitative relations between causative virus and cell in the Rous No. 1 chicken sarcoma. Virology 1, 445-473. SCHREK, R., and PRESTON, F. W. (1956). Toxicity of homologous immune serum to a transplantable tumor: Studies using phase microscopy and cinematography. J. N&Z. Cancer Inst. 16, 1021-1033. SEEGAL, B. C., HASSON, M. W., LOEB, E. N., and KNOWLTON, A. I. (1951). Production of cytotoxic nephritis in the dog by specific antiplacenta serum. Fedem-
tion Proc. SMADEL, J.
10, 418.
E. (1936). Experimental nephritis in rats induced by injection of antikidney serum. I. Preparation and immunological studies of nephrotoxin. J. ExplZ. Med. 64, 921-942.
2,
545-558
An Analysis
Biology
(1956)
of the Apparent Sarcoma Virus with Normal Chick
Division,
California
Neutralization Antiserum Tissues
HARRY
RUBIN’
Institute
of Technology,
Accepted
May
of Rous to
Pasadena,
California
21, 1956
The effect of antiserum to normal chick embryo tissues on the infectivity assay of the Rous sarcoma virus was investigated. It was found that such serum, when mixed with the virus and inoculated on the chorioallantoic membrane (CAM) of the developing chick embryo, caused a sharp reduction in the number of virus-induced tumors when compared with a control containing normal serum. In contrast to neutralization with specific antiviral serum, however, the reaction required complement and was completely reversible on dilution of the serum-virus mixture. Furthermore, the anticell serum suppressed tumor formation even when added after virus had adsorbed to susceptible cells, whereas the antiviral serum was effective only before adsorption. Most of the tumor-inhibiting activity of the antichick serum could be removed by absorption with normal chick cells and to a lesser degree by absorption with sheep red blood cells. Moreover, antiserum to boiled sheep red blood cell stroma was also capable of reducing the number of tumors. The antichick serum had no significant effect on the assay of vaccinia virus on the chorioallantoic membrane. It was concluded that the apparent neutralization of ROW sarcoma virus by antichick serum was due to impairment of the ability of the infected cells to multiply rather than to a direct neutralization of virus itself. This contradicts some earlier reports which indicated that unaltered normal cell protein forms an integral part of the functional surface of the Rous sarcoma virus. INTRODUCTION
There have been several contradictory reports dealing with the effect of antiserum to normal chick tissues on the infectivity of the Rous sarcoma virus. Gye and Purdy (1931) and Amies and Carr (1939) reported that such antiserum neutralized the virus, whereas Keogh ’ Research Fellow of the American Cancer Society on recommendation of the Committee on Growth, National Research Council. Aided in part by a grant from The American Cancer Society. 545
546
HARRY
RUBIN
(1938), Kabat and Furth (1941), and Barrett (1941) failed to confirm this finding. This question has important implications, since a positive result would suggest a very close structural relationship between the protein of the virus and of the host. The problem was therefore reinvestigated with the aim of resolving the contradictory claims. The findings of the disagreeing groups were not strictly comparable, since their methods differed. Thus, Gye and Purdy added guinea pig complement to the serum-virus mixtures, as did Amies and Carr when unpurified virus suspensions were used. Furthermore, both these groups incubated their virus with strong concentrations of serum, which were then inoculated into the assay animal without dilution. By contrast, neither Kabat and Furth nor Barrett used complement in their mixtures, and the former workers diluted their serum-virus mixtures before inoculation. In all cases but one, the assay methods (tumor size or endpoint dilution) have large innate variability. Keogh employed the CAM but gave no details as to procedure. The disparate results obtained with such different techniques suggested a possible explanation for the apparent contradiction; i.e., the antiserum might act by suppressing multiplication of infected cells in the indicator host rather than by inactivating the virus. This hypothesis would be consistent with the suggestion that complement might be required and that dilution of the virus-serum mixture before inoculation may eliminate the effect. Complement is known to be necessary for anticell serum to exert some of its cell-damaging effects (Kalfayan and Kidd, 1953; Morgan, 1955, Schrek and Preston, 1956), whereas dilution of the serum before inoculation might dilute it beyond a concentration at which it could act effectively in the host. The phenomenon may therefore be an apparent rather than a true neutralization. This interpretation would also explain why this neutralization is restricted to tumor viruses, although other viruses containing normal tissue precipitins are known (Knight, 1946). The experiments reported in this paper were designed to clarify this point. They show that the neutralization when tested on the CAM is indeed apparent and that it derives from a marked effect of the antichick serum on the infected cells. MATERIALS
AND
METHODS
Virus. Frozen ampules of concentrated Rous sarcoma virus in 0.15 M Na citrate were kindly supplied by Dr. W. R. Bryan of the National
NEUTRALIZATION
OF
ROUS
SARCOMA
VIRUS
547
Institutes of Health. This virus titered about 8 X lo6 tumor-forming particles per milliliter and was used in varying dilutions in all the experiments. Antiviral serum. This was obtained from a chicken convalescing from an infection with the Lous sarcoma virus and had a high degree of neutralizing activity against the virus. It, too, was supplied by Dr. W. R. Bryan. Antichick serum. Six lo-day-old chick embryos were homogenized in 50 ml of phosphate-buffered saline in a Waring blendor. Two milliliters of (*rude homogenate was used to inoculate rabbits intramuscularly and subcutaneously twice a week for 2 months. The antichick serum was obtained 2 weeks after the final inoculation. Normal serum had been obtained from these rabbits before immunization. Owing to the requirement for concentrated serum in the experiments, it. was necessary to use three sera of varying activities in order to carry out all t,he required tests. CompEement. Complement was obtained from fresh guinea pig serum kept frozen at -20”. :7ledia. Phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954) was used as a diluent throughout all the experiments. Virus assay. The virus was assayed on the chorioallantoic membrane of the developing chick embryo, as described previously (Rubin, 1955). RESULTS
Apparent neutralization of Row sarcoma virus by antichick serum. The earlier reports of successful neutralization of the Rous sarcoma virus by antiserum to normal chick tissues arose from experiments assayed in adult chickens (Gye and Purdy, 1931; Amies and Carr, 1939). The first step in the present experiments was to determine whether analogous findings would be encountered when tumor counts on the CAM of the chick embryo were used for assay. Seven-tenths milliliter of virus containing about 2000 tumor-forming particles was mixed with 0.2 ml of undiluted antichick serum and 0.1 ml of guinea pig complement. A control WSLSset up in which normal prevaccination serum was substituted for the antichick serum. The mixtures were incubated for an hour at 5”, and 0.1 ml was inoculated directly on the CAM without further dilution. As shown in columns 1 and 2, Table 1, there were fewer than 10% as many tumors resulting from the mixture containing the antichick serum as from the control.
548
HARRY
RUBIN
TABLE APPARENT NORMAL
NEUTRALIZATION CHICK EMBRYOS,
1
OF Rous SARCOMA VIRUS WITH AND REVERSAL OF THE EFFECT
ANTISERUM ON DILUTION
TO
(Two-tenths milliliter of the appropriate serum (serum excess) was mixed with 0.7 ml of the indicated dilution of virus and 0.1 ml of guinea pig complement. The mixtures were incubated for 2 hours at 37”; 0.1 ml of each was then either inoculated directly into chick embryos or diluted 1:lOO before inoculation.)
co
(1) Normal rabbit serum undiluted + virus 1:670, incubated and inoculated directly
Apparent virus
1.5 x
106
(3)
Antichick serum undiluted + virus 1: 670, incubated and inoculated directly
1.3
x
106
Antichick serum undiluted + virus 1:6.7, dilutxl;; 100
(4)
I
Antivirus serum 1: 10 virus + 1:670
inoculation
(5) Antivirus serum 1: 10 + virus 1:6.7, dilut;;;; 100 inoculation
5 x
104
1.7
x
106
titer
E$ect of dilution on mixtures of the ROW sarcoma virus with antichick and antiviral sera. Careful kinetic study of neutralization of several viruses (poliomyelitis, Western equine encephalomyelitis, Newcastle disease virus) by specific antiviral sera has shown the inactivation to be largely irreversible (Dulbecco et al., 1956; Rubin and Franklin, unpublished observations). Consequently, dilution of a serum-virus mixture leads to no appreciable dissociation of serum-virus complex. The following experiment was carried out to determine the effect of dilution on mixtures of Rous sarcoma virus, and either antichick serum or antiviral sera. In each case, complement was added, the mixture incubated, and then diluted. The dilution was designed to bring the serum well beyond the concentration at which it would inhibit tumor formation on the CAM as previously determined. The original incubation was made in a region of great antibody excess, so the antibody concentration would not be limiting when mixed with the higher virus concentrations. It may be seen from Table 1 that all evidence of neutralization with the antichick sertim disappeared on dilution, the diluted mixture producing as many tumors as the mixture containing only normal serum. In contrast, about 90% of the virus mixed with specific antiviral serum was irreversibly neutralized, since this fraction remained inactive after dilution. There wax a suggestion of reactivation of about 8 % of the inactivated virus by dilution in the latter case. This may be due to the
NEUTRALIZATION
OF
ROWS
SARCOMA
549
VIRUS
continued action of the concentrated serum in the host until the virus is adsorbed, rather than to the reversible inactivation of this small fraction (Dulbeceo et al., 1956). Eflect of serum concentration in vitro and in viva on the degree of’ apparent neutralization. Results of the previous section suggested that the effect of the antichick serum was independent of the serum concentration during in vitro incubation, depending only on the concentration inoculated into the host. The following experiment was carried out to learn more about the quantitative aspects of this dependence. Two series of four tubes each were set up. In the first series the undiluted serum was mixed with serial fourfold dilutions of the virus and incubated for 1 hour at, 5”. Each of the tubes was then diluted to give an equal concentration of virus in all tubes before inoculation. Thus, the virus of all the tubes was incubated with the same concentration of serum but was inoculated on the CAM with successive fourfold dilutions of this serum. The second series was incubated with concentrations of serum and virus which were identical to the first series after its final dilution. The second series was then inoculated without further dilution. Thus both series were identical in serum concentration on inoculation but differed during incubation. The results are present,ed in Table 2, and t’hey show t,hat the degree of apparent neutralization was strictly dependent, on the concentration TABLE EFFECT
6~
VARYING
SERUM
2
CONCENTRATIONS
APPARENT
I;itro
in.
AND
in
Viva
ON
NEUTRALIZATIOS
(Series A consisted of four tubes with successive fourfold dilutions of virus and undiluted serum, which were incubated at 37” for 2 hours, in t,he presence of excess complement. The contents of each was then diluted to give equal concentrations of virus, with successive fourfold dilutions of serum, and inoculated onto the CAM. Series B consisted of four tubes which were &o&ted with the same concentrations of virus and serum as w-ere present in series A after final dilution. These were then inoculated without further dilution. The results are expressed as number of tumors on the CAM.) Series
A B
Apparent
virus
Undiluted
5.7 5.7
x x
titer
at preliminary
1:4
104 104
5.6 4.2
X lo5 x 105
serum concentration
of:
1:16
7..5 x 8.3 x
1:64
105 105
1.3 9.6
x 10” X IO5
550
HARRY
RUBIN
of serum inoculated into the host, being completely independent of the concentration during incubation. Role of complement. Although complement may enhance the neutralization of certain viruses by their specific antisera (Leymaster and Ward, 1949), it is not an essential component (Dulbecco et al., 1956). It is, however, required to produce certain types of cell damage (Kalfayan and Kidd, 1953; Morgan, 1955; Schrek and Preston, 1956). The earliest report of neutralization by antichick serum explicitly stated a requirement for complement (Gye and Purdy, 1931), whereas a later report indicated that it was required with one type of virus preparation but not with another (Amies and Carr, 1939). Requirement for complement would lend weight to the theory that host cell damage is responsible for apparent neutralization. To learn whether complement is necessary to obtain this effect on the CAM, the antichick serum was heated to destroy complement (56” for 30 minutes) and the serum used in the experiments described in Table 3 to determine the role of complement. A parallel study was made with antiviral serum. The results in the table show that complement is necessary in order to obtain any evidence of apparent neutralization with antichick serum. On the other hand, it did not prove to be essential for neutralization with the antiviral serum, although perhaps it exerted some slight enhancing effect. E$ect of adding serum after the virus has adsorbed to the host cell. The experiments already reported suggested that the antichick serum reduced the number of tumors either by damaging the cells or by neutralizing the virus in a completely reversible way. To distinguish between these alternatives, the action of the antiserum on previously infected cells was determined. Once a virus particle has irreversibly adsorbed TABLE ROLE
OF COMPLEMENT
IN
(Both rabbit antichick serum and 30 minutes to destroy complement. 0.2 ml was mixed with 0.7 ml of virus. of 0.1 ml of complement or 0.1 ml of
7.8 titer
x
106
4.4
x
NEUTRALIZATION
chick antiviral serum were heated at 56” for These were then appropriately diluted, and The mixtures were incubated in the presence buffer andtheninoculated onto the CAM.)
Antichick St!llUD + buffer
Apparent virus
3 APPARENT
106
Antichick SWUIII + complement
2.5
X lo4
Antiviral S~KUlll + buffer
1.5
x
Antiviral StTlUll + complement
10’
1.2
x
104
NEUTRlLIZATION
OF
ROW
TABLE ~';FFE('T
OF ADDISC:
SERUM
S’:ARCOMA
1
AFTER
I.VFECTION
(Virus was diluted to give about 200 tumors on the CAM of 36 embryos. At, appropriate serum mixture (5 parts serum, 2 parts buffer, in groups of 6 embryos.) Hours after infection
1 ‘l 48
Nomal rabbit serum
~~~-~~
8.1
x
10”
531
VIRI:S
HAS
(kCIIRRED
per 0.1 ml of inoculum and placed intervals, 0.1 ml of the indicated 1 part complement.) was inoculated
Rabbit
antichick Serum
-2.3 7.8 5.0
X IO” x 106 x 105
Chirken
antiviral
1:lO serum -
4.5 x X.8 x
IO” IO”
to a cell, it is no longer susceptible to neutralization by a specific antiviral serum (Rubin and Franklin, unpublished observations). The cells themselves, however, may be just as sensitive to damage with anticell antibody after infection as they had been before infection. If this serum decreased the number of tumors by damaging t’he cells, then it might be expected to act just as effectively soon after infect,ion as before. Accordingly, either antiviral or antichick serum was added to the CAM at appropriate intervals after virus inoculation. The results in Table 4 show that the antiviral serum, capable of neutralizing over 90% of t’he virus in z&o, had no effect on the number of tumors when added 4 hours after the virus. The ant’ichick serum retained its effectiveness, even if added as late as 2 days after infection. This result favored the interpretation that the effect of the serum was on t’he cells. It should be noted that there was about 50 % reduction in the number of tumors when the antiviral serum was added 1 hour after infection. This suggests that virus adsorption was only half-complete at this t,ime. Absorptio?z of sera with chick embryo and sheep red blood cells. Roth antichick and antiviral sera were incubated with chick embryo cells to remove antibodies to normal chick cells. The neutralizing capacities of the sera were assayed before and after this Absorption, and the results are presented in Table 5. The antiviral serum was unaffected by this treatment, but the antichick serum lost almost all it,s capacity for apparent neutralization, clearly indicating t)hat two unrelated antibodies were involved in direct neutralization and apparent neutraliaation. Thus, it appears that no anticell antibody was active in the direct neutralization with antiviral serum, whereas the effective component in apparent neutralization with antichick serum was antibody against
552
HARRY
RUBIN
.TABLE ABSORPTION
OF SERA
WITH
CHICK
5
EMBRYO
BLOOD
TISSIJES
AND
WITH
SHEEP
RED
CELLS
(A suspension of lo-day-old chick embryo cells at a concentration of 108 cells/ml was made (Dulbecco, 1954) in phosphate buffer. A 10% suspension of sheep red blood cells was also prepared (6 X lo8 cells/ml). One-milliliter cell aliquots were centrifuged and resuspended in the appropriate serum. The mixtures were incubated at 37” for 2 hours, and the cells were again centrifuged. The superantant serum was absorbed once more and then used to test for neutralizing behavior.)
Apparent virus titer
I
I
5.72
&xlO’
Antichick
Antichick serum absorbed with shee red bloo B calls
4.9 x 106 2.1 x
Antiviral se*um 1:20
Antiviral serum 1: 20 absorbed with chick tissues
105
normal cell antigens. A similar finding was made by Amies and Carr (1939). The antichick serum was similarly treated with sheep red blood cells to remove heterogenetic Forssmann-type antibody. Serum treated in this way permitted the formation of eight times as many tumors as the unabsorbed serum. The sheep cell-absorbed serum retained the capacity to reduce the number of tumors by more than 60% when compared with normal serum, however, indicating that not all the apparent neutralizing antibody was of the Forssmann type. Effect of antiserum to boiled sheepred cell stroma. Since absorption of the antichick serum with sheep red blood cells results in some loss of its capacity for apparent neutralization, then antiserum to sheep cells may simulate to some degree the action of the antichick serum. Antisera to sheep red blood cells made by subcutaneous injection of rabbits in this laboratory exhibited only a low order of activity. A potent antiserum to boiled sheep red blood cell stroma was provided by Dr. Jose Vinas of Johns Hopkins University, however. A 1: 19,500 dilution of this serum gave 50% lysis of a suspensioncontaining 5 X lo* sheep red blood cells per milliliter. At a final dilution of 1:5 it reduced the number of tumors on the CAM to about 4%; a powerful antichick serum at the same concentration reduced it to 1%. (Table 6). Action of anticell serum on pock production by vaccinia virus. The results presented up to this point indicate that the phenomenon of
NEUTRALIZATION
OF
ROUS
TABLE EFFECT
OF ANTISERUM
TO SHEEP
RED
SARCOMA
riS3
VIRUS
6
BLOOD SARCOMA
CELL
STROMA
ON THE
ASSAY
OF THE
Rous VIRUS (Two-tenths milliliter of the appropriate serum was mixed with 0.7 ml of diluted ROW sarcoma virus plus 0.1 ml of complement and incubated for 1 hour at 5”. Then 0.1 ml of each mixture was inoculated onto the CAM of 6 embryos.) Antichick SCNm
Apparent virus
infectivity
4 x 106
of Rous sarcoma
TABLE EFFECT
OF ANTICHICK
SERUM
ON THE
4 x 104
Antisheep stroma
RBC Serum
1.7 x 105
7 ASSAY
OF VACCINIA
VIRUS
(Two-tenths milliliter of the appropriate serum was mixed with 0.7 ml of diluted vaccinia virus and 0.1 ml of complement and incubated for 1 hour at 5”. Then 0.1 ml of each mixture was inoculated onto the CAM of 6 embryos.) Normal
serum
-__--
Pock titer of vaccinia
virus
2.5 X lo6
Antichick - ~-.-l__l_-~.
S~TUIII
1.9 x 106
apparent neutralization of the ROW sarcoma virus is mediated through action of the antichick serum on the infected cell rather than on the virus. Does the mechanism of this action consist in int’erference with the ability of the infected cell to multiply or, alternatively, in impairment, of its ability to produce virus? If the latter explanation were correct, it would seem likely that impairment of the cell’s ability to produce nontumor viruses as well would result. For this reason, the effect of the antichick serum on the production of pocks on the CAM by vaccinia virus was tested. The results are presented in Table 7. The data show that there was no significant decrease in the number of vaccinia pocks, nor was there any apparent difference in t,heir size. This indicates that antiserum-treated cells are capable of producing vaccinia virus and favors the hypothesis that the serum *acts by interfering with cell multiplication. The over-all thickened appearance of antiserum-treated membranes, when compared to the controls, indicates some damage to normal as well as to infected cells. It is evident that the cells are not totally destroyed, however, since they can produce virus. In addition, the pocks made up of cells destroyed by vaccinia infection can be clearly distiu-
554
HARRY
guished against the background tions suggest that the anticell but does not destroy them.
RUBIN
of serum-damaged cells. These observaserum probably damages normal cells
DISCUSSION
The original finding of Gye and Purdy that antiserum to normal chick cells, when mixed with the Rous sarcoma virus in the presence of complement, reduces the apparent infectivity of the virus has been confirmed. The effect of the antichick serum depended only on the concentration of that serum at the moment of inoculation into the host; the effect was independent of the serum concentration during the period of incubation in vi&o with the virus. This contrasts with neutralization by a convalescent antiviral serum, which is strongly dependent on the in v$ro serum concentration. It corresponds to Amies and Carr’s finding (1939) that the virus could be centrifuged from an antichick serumvirus mixture and its full infectivity recovered. Antiviral serum could no longer neutralize virus which had already infected cells of the CAM. However, the antichick serum was just as effective in reducing the number of tumors if applied shortly after infection as it had been when incubated with the virus in vitro before simultaneous inoculation of both components. This showed that the antichick serum exerted its effect through damage inflicted on the cells. The possible occurrence of a completely reversible neutralization as an explanation for the dilution-reactivation phenomenon was thus ruled out. The absence of neutralization with antiviral serum after the virus had adsorbed to susceptible cells, when considered in the light of the observations presented below, suggested that cell-to-cell transmission of the virus was a minor factor in formation of the tumor. It implies that the tumor is formed chiefly through the increased multiplication rate of the originally infected cell and its progeny. This is consistent with the finding that vir.us is produced by sarcoma cells at a very slow rate (Rubin, 1955). It is also consistent with the fact that the height of the CAM tumors is ap.proximately equal to their width. If cell-tocell transmission of the virus occurred at a significant rate, the lateral spread of the virus to adjacent ectodermal cells would be much more rapid than the multiplication of the cells, and the lesion, as in the early proliferative stage of vaccinia infection, would be much wider than it is high. That some limited reinfection does occur, however, is clear from
PU’EUTRALIZATIOK
OF
ROUS
SARCOMA
VIRUS
503
the fact that mesodermal involvement occurs in the late stages of tumor development. Experiments with a nontumor virus, vaccinia, showed that serumtreated cells could produce virus. Therefore the most likely mechanism of action of anticell serum in apparent neutralization is to impair multiplication of the infected cells themselves, rather than to inhibit their ability to produce virus. This would explain why this phenomenon has been uniquely described with tumor viruses and not with necrotizing viruses (Knight, 1946). Antiserum to boiled sheep red blood cells simulates the tumor-suppressing activity of the antichick cell serum to a limited degree. Moreover, a portion of the activity of the antichick serum can be absorbed out with sheep red blood cells. This indicates that some of the cell antigens involved may be of the Forssmann heterogenetic type. Before these findings can be generalized to cover tumor viruses as a class, some observations made by other workers with other tumor viruses have to be considered. Gye and Purdy (1933) reported that antichick serum, although it apparently neutralized Fujinami myxosarcoma virus of chicken origin, failed to do so with that of duck origin, whereas antiduck serum gave the reverse effect. This relationship held true whether the assay was done in chicks or ducklings. This would seem to establish a strong case for true neutralization by the homologous tissue antiserum. The following observations cast doubt on this interpret,ation, however. The neutralization with antiserum to normal tissues always required complement and was species-specific. Antiserum against> the myxosarcoma itself did not require complement for neutralization and was not, species-specific. The latter antiserum always resulted in complete absence of t,umors; the former very rarely did. Normal tissues failed t,o absorb the antibodies from the antimyxosarcoma serum, but they were capable of absorbing homologous antibodies. Taken at face value, these findings suggest that the virus can be inactivated by t’wo unrelated antibodies, one virus-specific and the other species-specific, the latter having an absolute requirement for complement, a unique condition iu virus neutralization. The work also implies that only the species-specific antigens are uniquely thus involved, and not the many antigens which are common to both ducks and chickens. In the present work, by way of contrast, it is shown that even the ubiquitous Forssmann antigens are concerned in apparent neutralization of Rous sarcoma virus. In addition, the possibility of aggregation or coprecipitation, particu-
556
HARRY
RUBIN
larly when crude filtrates are used, cannot be excluded. Knight (1946) has found that antiserum to normal tissues can precipitate purified influenza virus derived from the homologous tissues without neutralizing the virus. Aggregation of a tumor virus, however, might easily result in an apparent reduction in infectivity, if infectivity is measured by tumor size, as it was in the aforementioned experiments, since the number of tumor-initiating foci would then be decreased. The assay method of tumor size which was used is subject to large variation and therefore is of doubtful significance. This is particularly true when multiple-inoculation sites are used, with three different immune sera inoculated into the same host. Finally, Dmochowski (1948) has shown that there is extensive cross reaction in complement fixation between highly purified Rous and Fujinami viruses, but there is no cross reaction between these and normal tissue. This suggests that the results found in the present paper would be applicable to the Fujinami virus as well and poses a direct problem as to the interpretation of the early Fujinami work. The second work which merits consideration is that of Eckert et al. (1955). This extensive research establishes an apparent neutralization of the virus of avian erythromyeloblastic leukemia with antiserum to normal chick tissues and, to a lesser extent, with Forssmann antibody. Here, no complement was required in the in vitro mixture, but the inoculation was made intravenously, thus obviating the requirement. The dilution of the serum on intravenous inoculation would weaken the applicability of the interpretation that the effective serum action occurred against the cells. This objection would not be valid, however, if the serum was powerful and the assay highly accurate, as indeed they appeared to be. There were distinct qualitative and quantitative differences between the action of the antiviral and anticell sera which suggest that the two acted in different ways. Finally, just as in the case of the Rous sarcoma, the normal -chick tissues failed to absorb specific antiviral antibodies from the sera of convalescent chickens, but successfully accomplished this with antiserum to normal chick tissues made in rabbits (Eckert, personal communication). There is considerable evidence that antinormal tissue antibodies can localize and produce damage in a variety of organs (Pressman, 1951; Smadel, 1936; Seegal et al., 1951). It. is likely that the demyelinating encephalomyelitis that follows injection of normal brain tissue is an example of a specific tissue-damaging immunological condition. The
NEUTRALIZATION
OF
ROUS
SARCOMA
VIRUS
557
cell destruction in paroxysmal hemoglobinuria, and in hemolytic anemia probably is due to the action of anticell antibodies (Raffel, 1953). Since there is, in general, cross-reaction between normal and tumorous tissues (Landsteiner, 1945), there is ample precedent for the tumor-suppressing activity of antinormal cell serum. It is concluded that antichick serum inhibits tumor formation by CAM cells infected with the Rous sarcoma virus by damaging these cells rather than by neutralizing the virus. Some of the antigens involved are of the Forssmann heterogenetic type. This result calls for a re-examination of other similar systems to determine whether the same mechanism is at work. ACKNOWLEDGMENTS I greatly appreciate Dr. W. R. Bryan’s generous provision of concentrated Rous sarcoma virus and convalescent chicken serum. I also wish to acknowledge the role which Drs. J. W. Beard and E. A. Eckert’s prepublication discussion of their work played in provoking the thought which led to the present work. REFERENCES C. R., and CARR, J. G. (1939). Immunological experiments with a highly concentrated suspension of Rous 1 tumor producing agent. J. Patho2. Bacterio2. 49, 497-513. BARRETT, M. K. (1940). The antigenic nature of purified chicken tumor agent. Cancer Research 1, 543-4. DULBECC~, R., and VOGT, M. (1954). One step growth curve of Western equine encephalomyelitis on chicken embryo cells grown in oitro, and analysis of virus yields from single cells. J. Exptl. Med. 99, 183-199. DTTLBECCO, It., VOGT, M., and STRICKLAKD, A. G. R. (1956). A study of the basic aspects of neutralization of two animal viruses. Western equine encephalomyelitis virus and poliomyelitis virus. Virolog!/ 2. 162-205. DMOCHOWSKI, L. (1948). Investigations on the properties of agents causing fowl tumors. 1. Attempts at isolation of the fowl tumor agents by differential ccntrifugation. J. #all. Cancer Inst. 9, 57-67. &KERT, E. A., SHARP, 1). G., BEARD, I).. GREEN, I., and BEARD, J. W. (1955). Virus of avian erythromyeloblast,ic leukosis. 1X. Antigenic constitution and immunologic characterization. J. .%‘atZ. Cancer Inst. 16, 593-643. GYE, W. E., and PURDY, W. J. (1931). The Cause of&ncer. Cassell &Co., London. GYE, W. E., and PURDY, \I’. J. (1933). The infective agent in tumor filtrates: A flu%her investigation by means of antisera to normal tissues. Urit. J. Exptl. Path&. 14, 250-259. KAB~T, E. A., and FURTH, J. (1941). Neutralization of the agent causing leukosis and sarcoma of fowls by rabbit antisera. J. Exptl. Med. 74, 257-261. KALFAYAN, B., and KIDD, J. G. (1953). Structural changes produced in BrownAMES,
558
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RUBIN
Pearce carcinoma cells by means of specific antibody
and complement. J. Exptl. Med. 97, 145-162. KNIGHT, C. A. (1946). Precipitin reactions of highly purified influenza viruses and related materials. J. Exptl. Med. 83,281-294. KEOGH, E. V. (1938). Ectodermal lesions produced by the virus of Rous sarcoma. Brit. J. Exptl. Pathol. 19, 1-8. LANDSTEINER, K. (1945). The Specificity of Serological Reactions, Harvard Univ. Press, Cambridge, Mass. G. R., and WABD, T. G. (1949). The effect of complement in the neutralization of mumps virus. J. Immunol. 61,95-105. MORGAN, I. M. (1955). Cytopathogenic effect of antiserum to human malignant epithelial cells (strain HeLa) on HeLa cell culture. J. ImmunoZ. 76, 478484. PRESSMAN, D. (1951). Radiolabelled antitissue antibodies. Federation Proc. 10, LEYMASTER,
568. RAFFEL, RUBIN,
S. (1953). Immunity. Appleton-Century-Crofts, New York. H. (1955). Quantitative relations between causative virus and cell in the Rous No. 1 chicken sarcoma. Virology 1, 445-473. SCHREK, R., and PRESTON, F. W. (1956). Toxicity of homologous immune serum to a transplantable tumor: Studies using phase microscopy and cinematography. J. N&Z. Cancer Inst. 16, 1021-1033. SEEGAL, B. C., HASSON, M. W., LOEB, E. N., and KNOWLTON, A. I. (1951). Production of cytotoxic nephritis in the dog by specific antiplacenta serum. Fedem-
tion Proc. SMADEL, J.
10, 418.
E. (1936). Experimental nephritis in rats induced by injection of antikidney serum. I. Preparation and immunological studies of nephrotoxin. J. ExplZ. Med. 64, 921-942.