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All the known genes of the bovine leukemia proviral genome were found to be heavily methylated in both fresh and short-term-cultured leukemic cells, the latter of which expressed viral antigens. However, the pXBL region appeared to be mostly demethylated or at least hypomethylated in both cells, indicating that there is a non-uniform methylation pattern of the proviral genome.

Monoclonal antibody against tumor-associated antigen (TAA) expressed on bovine leukemia cells was conjugated to liposomes containing adriamycin (ADM), and the specificity and therapeutic effects of the conjugates were examined in vitro and in vivo using a TAA-positive bovine leukemia cell line as the target tumor. In vitro studies with the TAA-positive cell line clearly indicated that the antibody-conjugated liposomes containing ADM exerted selective effects on TAA-positive cells in the inhibition assay of 3H-thymidine incorporation. Three injections of liposomes containing ADM (4 mg/kg) into tumor-bearing nude mice significantly inhibited the tumor growth and the therapeutic effect of the antibody-conjugated liposomes was far greater than that of normal mouse IgG-conjugated liposomes as assessed in terms of tumor size.

Thirteen monoclonal antibodies directed against tumor cells from cattle from enzootic bovine leukosis (EBL) were obtained. They reacted with tumor cells but not with normal bovine cells or bovine leukemia virus antigens. According to the reactivities of these antibodies with 19 individual tumors, the 13 monoclonal antibodies can be divided into three groups. Antibodies of the first group reacted with all the EBLs tested; those of the second group reacted with several, but not all, of the EBLs tested; and those of the third group reacted only with homologous tumor cells. Therefore, tumor-associated antigen (TAA) on the EBL tumor may possess common TAA, partially common TAA, and individually distinct TAA. The TAAs were solubilized from the tumor cells by treatment with 0.2% sodium deoxycholate and partially purified by diethylaminoethyl-cellulose column chromatography. Eleven of the 13 monoclonal antibodies reacted with this soluble TAA. The monoclonal antibodies belonging to the first group inhibited in vitro the growth of the bovine lymphoid cell line derived from the EBL tumor.

Mouse monoclonal antibody c 143 was purified and F(ab')2 fragments were generated by pepsin digestion and then radiolabeled with 125I. The 125I-labeled c 143 F(ab')2 fragments were injected into athymic mice bearing bovine lymphoid tumor cells. The fragments became preferentially localized in tumor tissues, but not in normal tissues, as determined by differential counting of tissue radioactivity. The fragments became localized specifically in those tumors that were reactive with c 143 in vitro, but did not become localized in unrelated tumors. Localization of labeled F(ab')2 fragments of a monoclonal antibody of the same isotype directed against Taka virus (a variant of Newcastle disease virus) was not observed in athymic mice bearing bovine lymphoid tumor cells. Tumors were detectable by radioimmunoscintigraphy, using radiolabeled c 143 F(ab')2 fragments, without background subtraction, and by use of silver-grain scattering in light microscopic autoradiography.

An ELISA, using viable bovine lymphosarcoma cells, was developed to detect tumor-associated antigens (TAA) expressed on lymphosarcoma cells from cows with enzootic bovine leukosis (EBL). Monoclonal antibodies (MAB) against TAA were used. Using viable-cell ELISA, MAB reacted with tumor cells from 9 cows with EBL, but not with peripheral blood lymphocytes from 10 clinically normal cows. Titers of MAB against tumor cells from 1 cow with EBL were 2,048 (direct immunofluorescence assay), 8,192 (flow cytometry), 2,048 (fixed-cell ELISA), and 16,384 (viable-cell ELISA). Viable-cell ELISA was the most sensitive method for detection of TAA. Reactivities of 7 MAB to tumor cells from cows with EBL were compared between viable-cell ELISA and a complement-dependent antibody cytotoxicity test. Of 5 MAB with no cytotoxic activity against tumor cells, 3 were reactive against tumor cells from the same cow, as determined by viable-cell ELISA, with titers ranging from 128 to 2,048.

Hybrid cell lines were prepared by the fusion of BALB/c myeloma, P3X63Ag 8.653 cells with the spleen cells of BALB/c mice that were immunized with SF-28 cells, sheep fibroblasts transformed with BLV in vitro. Five clones of hybrid progeny produced cytotoxic IgG1 antibodies against SF-28 cells. The tested monoclonal antibodies showed cytotoxic activity against 4 out of 6 BLV-transformed sheep fibroblasts as well as SF-28 cells but failed to react with other cells, including BLV-producing FLK cells, bovine lymphosarcomas and normal sheep cells. These monoclonal antibodies showed suppressive effect on colony formation of SF-28 cells in soft agar culture.

When various indicator cells, including virus transformed and nontransformed cells, were cocultivated with bovine leukemia virus-producing cells, strong positive syncytia formation was found in transformed cells one day after cocultivation. The results of comparison of bovine leukemia virus antibody titers and the detection of bovine leukemia by the syncytium assay showed 89% of serologically positive cows were positive for bovine leukemia virus, whereas no reactors were found in serologically negative cows. However, the frequency of bovine leukemia virus detection differed according to the difference of incubation periods in the syncytium assay. Therefore, it is important to choose the appropriate indicator cell and culture conditions for the detection of bovine leukemia virus in the syncytium assay.

Tumor-associated antigens (TAAs) expressed on tumor cells from cattle with enzootic bovine leukosis were divided into three groups by using 13 monoclonal antibodies: common TAA; partially common TAA; and individually distinct TAA. TAA was extracted from tumor cells and purified by ion exchange chromatography on diethylaminoethyl-cellulose and isoelectric focusing. The common TAA, which was detected on all tumors tested, was eluted with 0.6 M KCl in ion exchange chromatography on diethylaminoethyl-cellulose, and the isoelectric point of the antigen was 6.8. The partially common TAA, which was detected on some (but not all) of the tumors tested, was eluted with 0.4 to 0.8 M KCl, and the isoelectric points of the antigen were 5.3, 5.8, and 6.4. The individually distinct TAA was present in the fractions eluted with 0.4 to 0.8 M KCl, and the isoelectric point of the antigen was 5.5. Results of competitive binding assay and Western blot analysis showed that the common TAA was a polypeptide with a molecular weight of 74,000; that it has at least two independent antigenic regions; that the partially common and individually distinct TAAs were a polypeptide with a molecular weight of 64,000; and that the antigenic determinants on the common TAA, partially common TAA, and individually distinct TAA existed independently from each other.

Four preparations were tested as potential vaccines to protect sheep against bovine leukemia virus (BLV) infection. Purified glycoprotein (gp) 51 and protein (p) 24 antigens from the virus and glutaraldehyde-fixed fetal lamb kidney (FLK) cells chronically infected with BLV or sheep fibroblasts transformed with BLV (SF-28 cells) were used to inoculate 12 sheep. Each vaccine was given 3 times 2 and 4 weeks apart to 3 sheep. Six sheep vaccinated with gp51 antigen or fixed FLK cells developed complement-fixing antibody against gp51; the titers ranged from 1:8 to 1:128 at the time of virus challenge exposure at postinoculation week 9. Although the 6 sheep inoculated with p24 antigen or fixed SF-28 cells developed antibody against the respective inoculum, none of these sheep had gp51 antibody at the time of challenge exposure. All 12 vaccinated and 2 control sheep were challenge exposed with BLV-infected lymphocytes, and cells from the sheep subsequently were tested for infection by syncytium assay. Sheep inoculated with gp51 antigen or FLK cells were protected, but sheep inoculated with p24 antigen or SF-28 cells became infected. The cytotoxic activity of lymphocytes from protected and nonprotected sheep was not different from that of normal sheep. Seemingly, purified gp51 antigen or fixed FLK cells were capable of preventing BLV infection in sheep. Humoral immune responses to gp51 appears to have an important role in protection against BLV infection, whereas cytotoxic activity of lymphocytes does not.

FLK cells are fetal lamb kidney cells persistently infected with bovine leukemia virus (BLV). 3178 cells, originating from calf-form bovine lymphosarcoma, also showed persistent production of BLV and alteration of cell morphology, after treatment with 5'-iodo-2'-deoxyuridine. In the present paper, the first in vitro transformation of sheep fibroblasts by inoculation with BLV materials from these two cell lines is described. In a few passages after inoculation with these viral materials, morphological alteration occurred. The morphologically altered cells were grown as stable cultures and showed such transformed phenotypes as growth in soft agar medium, increased uptake of 2-deoxy-D-glucose and tumorigenicity in athymic nude mice. This result, together with our previous observation of simultaneous induction of BLV expression and morphological alteration of 3178 cells, suggests the presence of some transforming capacity in these BLV materials similar to that in, for example, murine or avian acute leukemia viruses. The possible acquisition of such capacity during the prolonged passage is discussed.