Prevention of tumors in rats by cross-protective immunization.

F344 inbred were repeatedly immunized (days 0, 28, and 42) with normal syngeneic or allogeneic rat tissues or transplantable syngeneic or allogeneic rat tumors (some of which were virus producing). Immunized rats were challenged by sc injection of 10(5) or 10(6) syngeneic rat tumor cells from either of two different tumor lines. Successful cross-protective immunization prevented tumor development in rats that were challenged at 100-1,000 times the 50% tumor dose. The protection was essentially lifelong and complete in that no tumors appeared up to 200 days post challenge in some experiments. To be successful, the tumor cell vaccines had to express a complement-fixing cross-reacting antigen detected with sera from rats bearing any of several different tumors and to be able to induce a spontaneously regressing tumor in the host.

Prevention of 3-methylcholanthrene-induced fibrosarcomas in rats pre-inoculated with endogenous rat retrovirus.

Weanling Fischer 344 rats received a single intraperitoneal injection of a 1000-fold concentrated preparation of endogenous nontransforming rat retrovirus. Ten days later, the rats were each given a single subcutaneous injection of 3-methylcholanthrene. The rats inoculated with the endogenous rat retrovirus were significantly protected against the development of cancer, whereas uninoculated rats and rats given one of several murine retroviruses or baboon retrovirus were not protected.

Existence of a reverse transcriptase-p30 complex in AKR mice with a high incidence of spontaneous lymphocytic leukaemia.

The virus proteins, reverse transcriptase (RT) and p30, were found to increase with time in the subcellular fractions of lymphocytic tissue from either the thymus or spleen of AKR mice with spontaneous lymphocytic leukaemia. Significant levels of RT activity were first detected in the microsomal fractions of the two tissues at 15 and 20 weeks old, respectively. Although low amounts of p30 could be found in both tissues within the first week of life, the overall increase in the amount of p30 within each tissue followed much the same course as that shown by RT. In addition, a protein complex consisting of p30 and RT was first found in thymus and spleen lymphocytes of 15 and 20 week-old animals, respectively. The complex increased in amount in both organs as the animals aged, reaching a maximum level in 30 week-old mice. Repeated attempts to detect other virus proteins such as gp70 in association with the complex by immunological means were unsuccessful. The complex could not be found in lymphocytic tissue taken from younger animals or in 'non-target' organs, such as liver or kidney, of animals with leukaemia. In animals treated with antiviral IgG, which delayed the development of spontaneous leukaemia, the complex did not appear until much later in life (45 weeks) and then in considerably smaller amounts.

Prevention of transplantable tumors by adoptive transfer of spleen cells from immunized rats.

Fischer 344 rats were specifically hyperimmunized with allogeneic, nonvirus-producing [Kirsten murine sarcoma virus (KiMSV)] or syngeneic, virus-producing [KiMSV (Rasheed)] rat tumors. Spleen cells taken from these rats adoptively transferred protection against a 100 to 1,000 X rat tumor dose50 cell challenge with several different transplantable rat tumors. Protection was obtained with spleen cells after removal of adherent cells and macrophages but not peritoneal cells. The spleen cells were not directly cytotoxic but required more than 3 days residence in the recipient before protecting the recipient against challenge. No protection against tumor cell challenge was observed when spleen cells were lethally x-ray irradiated before injection into nontreated rats. Spleen cells taken from rats immunized with normal histocompatibility antigens did not protect in this test system.

Induction of rat sarcomas in rats treated with antithymocyte sera after transplantation of human cancer cells.

Human cancer cells that had had high (greater than 160) tissue culture passages, when transplanted into antithymocyte-treated F344 newborn rats, caused induction of rat sarcomas in the rats within 2 or 3 subcultures, whereas human cancer cells with low (5-33) passages in vitro did not cause overt induction of rat sarcomas until after 5-10 subtransplantations. Because oncornavirus activity was not detected in either rat or human tumors, it is suggested that transforming sequences located on the human tumor cells may have been transferred to supporting rat reticulum cells in close contact with the human cancer cells.

Prevention of spontaneous leukemia in AKR mice by passive immunization and type specificity of this protection.

AKR mice were given sc injections of goat antiviral immune globulin from birth through 14 or 31 days of age. Although the shorter immunization schedule greatly reduced expression of spontaneous leukemia, the longer immunization period was required for essentially complete prevention of leukemia. Immune globulin with a high neutralization titer for ecotropic virus provided this protection, whereas antibody with a high neutralization titer for murine xenotropic virus did not.

Prevention of spontaneous leukemia in mice by oncornavirus immunization.

C57L, NIH, and SWR mice were immunized with inactivated Gross leukemia virus (GLV) and then mated with AKR males. Their F1 offspring were then immunized with the murine sarcoma virus pseudotype of GLV, MSV(GLV). The concentrations of infectious ecotropic AKR virus in tail extracts of immunized mice were 100- to 1,000-fold lower than in non-immunized controls when tested at 30--40 days of age. Although viral titers increased slightly with time, the titers remained at least one log10 lower in the immunized mice than in non-immunized F1 control mice at all times tested. The reduction in the level of expression of endogenous ecotropic virus showed a highly significant positive correlation with the reduction in incidence of spontaneous leukemia in these mice. These data thus show that successful immunoprevention of leukemia in mice can be achieved with viral vaccines.

Prevention of spontaneous leukemia in AKR mice by type-specific immunosuppression of endogenous ecotropic virogenes.

AKR/J mice, 80-90% of which ordinarily die of spontaneous lymphocytic leukemias by 12 months of age, were significantly protected from developing leukemia in the initial experiment by a single course of treatment with AKR serotype-specific antibodies mad in goats and processed as immune gamma globulin (IgG). In experiment 1, IgG was given on the day of birth and on four additional days, and finished on day 14. This schedule resulted in suppression of over 4 logarithms of normal virogene expressions up to 25 days of age and led to partial viral suppression for over 200 days of age. At 365 days of age, 20 of 24 (83.3%) control animals were dead of leukemia whereas six of 30 (20%) treated animals had died of leukemia. In a second experiment, only four inoculations of IgG were given from birth to 20 days, after which they were given three inoculations of radiation-killed vaccine specific for AKR-Gross leukemia virus and one injection of murine sarcoma virus-Gross leukemia virus 10 days later. This combined immunization procedure provided significant virus suppression up to 288 days of age. At 300 days of age, 30 of the 50 (60%) controls had died of leukemia while only 1 of 24 (4.2%) of the immunized mice developed fatal leukemia; the significance of protection for each of the experiments was P LESS THAN 0.001. We conclude that these data establish in classical fashion with type-specific immunosuppression the determining role of type-C endogenous virogenes in leukemogenesis and, at the same time, also established the feasibility of nearly total prevention of leukemia in AKR mice.

Purification and properties of in vitro-produced anthrax toxin components.

The three components of the toxin of Bacillus anthracis, edema factor (EF), protective antigen (PA), and lethal factor (LF), were purified 197-, 156-, and 1,025- fold, with 38, 78, and 11% recovery, respectively. Each purified component was serologically active, distinct, and free from the other components. The purified EF produced edema when mixed with PA, and the purified PA was an active immunogen. The components did not appear to be simple proteins by spectrophotometric analysis. As they were purified, the pH range in which they were most stable narrowed, centering between pH 7.4 and 7.8. Heat readily destroyed the biological activity of the components but not their serological activity. The rat lethality test showed that, with a constant amount of LF and an increasing amount of PA, the time to death reached a minimum and then was extended. When an increasing amount of LF was added to a constant amount of PA, the time to death became shorter as more LF was added. The biological, immunological, and serological properties of the components were shown to vary independently with storage and extent of purification so that serological activity was not always directly correlated with biological activity. Evidence is presented that the components can exist in different molecular configurations or as aggregates, and that this property is influenced by the state of component purity and by the environment.

In vivo-produced anthrax toxin.

Specific anthrax antigens were demonstrated in the blood of animals dying from anthrax. These antigens, which appear in the blood at the time when organisms are first detected and whose concentration continues to increase as the number of organisms increases, do not elicit a strong antibody response. The in vivo-produced toxin differs from the in vitro in killing more rapidly and being more difficult to detect. The in vivo toxin exists as an aggregate whose biological and serological activity depends upon its particular composition or configuration, or both.