Injection of high doses of KT-FeLV feline leukemia virus produced both viremia and leukemia in a portion of 1- to 2-day-old kittens but not in 17- to 24-week-old animals. Contact of susceptible cats with infected cagemates did not result in viremia or disease. Development of cytotoxic antibody was coordinated with protection against viremia and disease in kittens challenged with FeL V virus. The rapid evolution of resistance to KT-FeLV virus precluded the development of a practical means for conventional vaccination and challenge experiments in cats. Killed vaccine given to pregnant queens did, however, afford protection against FeLV virus on challenge in the progeny.

Envelope glycoprotein of feline leukemia virus (FeLV), strain FeLV-1, was extracted with 2% Triton X-100 and 0.43 M NaCI In 10 mM Tris (pH 7.5) and purified by ion-exchange chromatography on phosphocellulose. Purified FeLV envelope gp75 had a molecular weight of 75,000 daltons and induced antibodies of high titers in the goat. These antibodies specifically neutralized FeLV, precipitated radiolabeled FeLV envelope gp75, and lysed FL74 cells. The immunogenicity of purified FeLV envelope gp75 with and without adjuvant was tested and compared in guinea pigs and cats. Administration of 20 μg of FeLV envelope gp75 to guinea pigs in either alum or adjuvant 65 formulation induced neutralizing antibody [geometric mean titer (GMT), 8-25], radioimmune precipitating antibody (GMT, 1,382-6,450), and cytotoxic antibody (titer, ≥2) in all animals. Aqueous FeLV envelope gp75 vaccine induced neutralizing antibody (GMT, <4) in only 50% of the guinea pigs. In contrast to results with guinea pigs, immunization of speclflc-pathogen-free cats, 10-15 weeks old, with twice the guinea pig dose level of FeLV envelope gp75 (40 μg) induced significantly less immune response to FeLV when its envelope gp75 was administered in alum or Freund's adjuvant and little or no response when FeLV envelope gp75 was given as aqueous or adjuvant 65 formulation. Cats immunized with 40 μg FeLV envelope gp75 in alum or Freund's adjuvant developed radiolmmune antibody with GMT's of 127-207 [100% seroconverslon (SC)], but little or no neutralizing antibody (22-38% SC; GMT, <2) or cytotoxic antibody (0-44% SC) developed.

Chemical vaccine containing di-N-acetylchitobiosyl poly(L-asparagine) prepared as described by Shier was assayed for its ability to prevent XS63 5 myeloma transplant tumors and 3-methylcholanthrene-induced tumors in BALB/c mice. The vaccine was ineffective.

Adenovirus-7 hamster tumor cells propagated in cell culture varied markedly with respect to capability to immunize against homologous transplant tumor after treatment with formaldehyde or disruption by various mechanical means. Protective vaccines employed cells for vaccine preparation or for challenge that were at passage levels comparable to those of the nonprotective vaccines. The protective vaccines were made with highly oncogenic cells, while the cells used for nonprotective vaccines varied, ranging from weakly to highly oncogenic. The basis for the differences in immunogenicity of the vaccines is not known. However, all protective vaccines were prepared from the descendants of a single vial of frozen parent-cell seed stock, while the nonprotective vaccines came from descendants of other frozen vials of the same parent seed. Selection of qualitatively different progeny from the parent cell population appeared to have taken place. Possible difference in the kind of immune response e.g., cell-mediated compared with humoral antibody (tumor-enhancing), is being investigated.

Inbred LSH and MHA hamsters were compared with random bred LVG hamsters for susceptibility to induction of tumor by SV40, adenovirus 7, or adenovirus 12 viruses and for appearance of tumor after homologous transplantation. The LSH inbred strain proved superior to the MHA inbred line in trems of susceptibility to induction of tumor by SV40 and adenovirus 7 virus and showed a shorter latent period after transplant with homologous SV40 virus tumor. The LSH animals were also somewhat better than MHA hamsters from the point of view of husbandry. The susceptibilities of the inbred LSH and random bred LVG animals to induction of tumor by viruses were similar and the transplantation characteristics of tumors evolved in the two breeds of animals were approximately the same. The inbred LSH hamster provides a satisfactory model for induction of tumor by virus and for investigations of transplant tumor immunology employing a truly syngeneic system.

Thymectomy of newborn hamsters enhanced the tumorigenicity of polyoma virus given 1 week or 4 months after surgery, of SV40 virus given 1 week after surgery, and of adenovirus 3 and 7 injected immediately after thymectomy. There was no effect on tumor incidence following adenovirus 12 given in high or low dose immediately after surgery. Adenovirus types 1 and 4, influenza types A, A2, and B, ECHO 25, poliovirus 1, reovirus 1, vaccinia, and M. orale 1 failed to induce tumor when inoculated into newborn thymectomized and nonsurgically treated hamsters. The influence of age on host susceptibility to viral oncogenesis was studied by inoculating adenovirus 7 and SV40 virus into newborn, 1-, 3-, and 12.5-14-month-old hamsters. Adenovirus 7 induced tumors in the newborn group alone; by contrast, SV40 virus induced tumors in all age groups. The influence of virus strain and cell culture passage on oncogenicity was studied with the Pinckney and Gomen strains of adenovirus 7. These latter factors did not affect the induction of tumors in newborn hamsters; instead, the amount of virus given was the most important determinant. The significance of the findings in relation to detection of oncogenic viruses are discussed.

Studies were carried out to measure the effect of a synthetic double-stranded RNA, poly I:C, on SV40 transplant tumor growth and SV40 viral oncogenesis in hamsters. Single or multiple doses of poly I:C given intraperitoneally before and/or after subcutaneous inoculation of SV40 virus into newborn hamsters had little or no effect on the percentage of animals developing subcutaneous tumors later in life. Likewise, single or multiple doses of varying amounts of poly I:C administered intraperitoneally before and /or after subcutaneous inoculation of 1000 to 500,000 virus-free SV40 tumor cells had little or no effect on the rate of tumor appearance in adult male hamsters.

THE SUCCESSFUL propagation in cell culture of rubella virus by Parkman et al 1 and by Weller and Neva 2 opened the way to development of a vaccine. A prime factor in acceptability of a live virus vaccine is the cell in which it is prepared, especially if the vaccine is given parenterally. Though rubella virus propagates readily in cell cultures of monkey, dog, rabbit, bovine, or human origin, our principal effort was given to finding an avian source. Having demonstrated that only limited propagation occurred in cell cultures of chick embryo, we directed our attention to domestic fowl with longer periods of gestation, and successful propagation was achieved on the first attempt made on April 8, 1963. Successful growth of the virus in duck cell culture was later confirmed by Fuccillo et al. 3 The virus was rapidly attenuated for monkey and man in the Pekin duck embryo cell

Synthetic double-stranded ribonucleic acid (Poly I:C) and replicative form RNA from MU9 coliphage reduced the occurrence of internal but not subcutaneous tumor after injection of adenovirus 12 subcutaneously into newborn hamsters. The effect was more evident in female than in male animals. Poly I:C treatment was consistently effective only when started prior to virus. The single homopolymers Poly I and Poly C were also protective even though they exert little, if any, interferon-inducing capacity. The action of the ribonucleotides might have been related both to interferon and to ordinary immune mechanisms. The greater activity in females as compared with male animals and the fact that the single-stranded Poly I and Poly C homopolymers were active favors the latter possibility. Pyran copolymer and endotoxin showed a minor reduction in internal but not subcutaneous tumor when given prior to and after adenovirus.

Studies were carried out to measure the effect on Friend leukemia in BALB/c mice of synthetic double-stranded RNA, Poly I:C (rI:rC), given in different prophylactic and therapeutic regimens. The effect of Poly I:C was strongly influenced by the time of initiating treatment, the amount of drug given, and the number and frequency of doses administered. Necrosis and hemorrhage of the spleen were among the principal pathologic effects of Friend leukemia in mice and these were markedly reduced in those instances in which drug treatment decreased splenomegaly. When administered prior to virus, cortisone greatly increased splenomegaly in Friend leukemia both alone and in combination with Poly I:C. The effect of Poly I:C administration before virus was strongly dose-related and a significant beneficial effect was obtained when the amount of drug was reduced to 4 μg. In toto, Poly I:C was not strikingly beneficial in altering the events in experimental Friend leukemia. The complex and multifaceted relationships between virus and drug and their administration will require detailed examination before an optimal prophylactic or therapeutic regimen can be derived.

Division of Virus and Cell Biology Research Merck Institute for Therapeutic Research West Point, Pennsylvania

Studies were continued in SV40 and adenovirus 7 hamster tumor systems to develop means for isolating tumor-specific transplant antigen, essentially free of normal tissue antigens, preparatory to consideration for vaccination experiments employing autochthonous tumor vaccines in man. Consistent with previous experiments, SV40 virus-induced tumor cells lost their immunizing ability for the host when treated with formalin or disrupted by a French pressure cell or nitrogen decompression. By contrast, adenovirus 7 tumor cells treated with formalin or disrupted by freeze-thaw, by use of a French pressure cell, or by nitrogen decompression retained the ability to immunize against the same cell tumor challenge. The latter findings are regarded as a significant step toward eventual development of tumor cell vaccines employing fractionated and purified antigens.

Studies of cryogenic destruction of virus-induced SV40 tumor and of adenovirus 7 transplant tumor were carried out in hamsters. The effect on the treated tumors as well as on other tumors which were present at the time of cryosurgery or which developed subsequently was noted. The effect on the treated SV40 virus-induced tumor was, at best, temporary and palliative and there was no apparent suppression of untreated tumor. The same general effect was noted with adenovirus 7 transplant tumor except that complete regression of treated tumor occurred in 2 animals and similar regression of an untreated tumor was observed in 1 animal. In the overall, immunodestructive effect subsequent to cryosurgery was weak if present at all. This was consistent with the prior demonstration of destruction of tumor-immunizing quality of tumor antigens by the freeze-thaw procedure and with the commonly observed development of immunologic tolerance in cancer.

Administration of live SV40 virus vaccine to adult female hamsters prior to conception afforded protection of the progeny against tumorigenesis by the homologous virus given shortly following birth. The mechanism appeared to be passive transfer of maternal antibody which prevented infection. There was no apparent infection of the fetus with SV40 virus in utero and there was no detectable CF antibody in the mothers or the progeny against SV40 tumor antigen. Maternal immunization holds promise as a means for preventing tumor induction in man caused by hypothetical viral neoplastic agents.

Cell mediated immunity against homologous SV40 tumor cells was demonstrable in the homologous in vivo hamster system but not in cell culture. The effectiveness of exudate cells from immunized hamsters was clearly influenced by the ratio of target cells to immune cells and by the age of test hamsters used with protective effect demonstrable in adult but not in 1 week-old hamsters. High levels of immunity against homologous tumor were also demonstrable by direct subcutaneous challenge. The latter system was desirable for assay of vaccine potency because of simplicity and rapidity of obtaining results.

Summary A total of 123 extracts of tissues from 111 cases of human cancer of diverse type and 22 extracts of tissues from 20 cases of non-neoplastic disease of man were examined for presence of antigens fixing complement with sera from hamsters bearing adenovirus 7, 12 and 18, SV40, or Rous sarcoma tumor. Thirteen tissues from cases of cancer and 4 tissues from non-neoplastic disease gave positive CF reactions with adenovirus type 12 and sometimes with type 7 tumor antisera but not with the other tumor antisera. Interpretation was complicated in some instances by apparent isoantigen-anti-body reactions between human and hamster tissues. No evidence for etiologic role of adenovirus 18, SV40 or Rous sarcoma virus in human tumor was obtained and if a virus related to 7 and 12 plays a role in human neoplasia, it would appear to be relatively infrequent. Without substantial improvement in specificity and sensitivity, tests for T antigen in human neoplastic tissues would not appear worthy of further emphasis.

Hamster SV40 tumor cells rendered nonproliferative by exposure to gamma rays or by propagation in the presence of iododeoxyuridine were highly effective when used as immunizing antigens for preventing the appearance of tumors in hamsters which had received SV40 virus when newborn. Only a single injection of immunizing antigen was employed. Disruption of tumor cell preparations by freeze-thaw or by treatment in a French pressure cell for purpose of fractionation resulted in total or near total loss of immunizing capability, even when incorporated into alum adjuvant. Possible mechanisms for loss of potency are discussed.

Summary A total of 38 neoplastic tissue preparations from a variety of human cancers were tested for presence of a tumor-inducing principle in newborn ICR/Ha mice. The mice were sex-segregated to prevent pregnancy and to limit occurrence of spontaneous mammary carcinoma. The malignant tissue specimens were tested as crude extracts, after fluorocarbon extraction, or following treatment by the Burton-Friedman method which was reported to make possible tumor induction in mice. There was no evidence for a tumor-inducing principle in any of the specimens tested. Numerous spontaneous neoplasias were observed and mammary carcinoma and malignant lymphoma were most commonly present.

Cell culture lines of virus-free hamster tumors which were initiated by ade-novirus 7 (Pinckney and Grider) or by adenovirus 18 and which synthesized “T” antigen were tested exhaustively for release of infectious adenovirus following addition of candidate “helper” viruses including SV40 respiratory syncytial virus and reovirus type 1. Attempts to detect adenovirus were by copropagation with susceptible indicator cells and by subculture of supernatant fluid or of disrupted cells in a sensitive assay cell system. In no case was infectious adenovirus recovered. The data permit speculation that the adenovirus genetic material retained in the tumor cells which was sufficient to code for “T” antigen might have been too highly integrated in the cell or might have been insufficient in amount to produce infectious virus. Alternatively, the “helper” viruses chosen might not have been appropriate to demonstrate the effect. Whatever the factors involved, it appeared quite clear that SV40, RS and reovirus 1 agents failed to afford sufficient or proper complementation or facilitation to effect release of infectious adenovirus from adenovirus 7 and 18 tumor cells of hamsters.

Cell culture lines of virus-free hamster tumors initiated by SV40 virus or by the Pinckney and Grider strains of adenovirus 7 were tested exhaustively by procedures which might be hoped to induce the virus or to increase its chance for detection. This included treatment with actinomycin D, mito-mycin C, puromycin, proflavine, hydrocorti-sone, ACTH, thyroxine, insulin, H2O2, increased O2, γ-radiation and copropagation with indicator cells. In no case was infectious virus recovered. Superinfection of SV40 hamster tumor with SV40 virus could not be established and hypothetical inapparent super-infection was not demonstrated following treatment with mitomycin C, proflavine or H2O2. No virus was detected in the cell cultures of any of 3 human tumors tested. The findings supported the concept that viral genetic materials in the animal tumors which are sufficient to code for “T” antigen may be insufficient to replicate the complete infectious virus.