Production of monoclonal antibody to bovine leukemia virus glycoprotein gp51 by in vitro immunization

The monoclonal antibody against glycoprotein gp51 of bovine leukemia virus (BLV) envelope antigen was produced by in vitro immunization. This monoclonal antibody reacted with viral antigen was observed at the 69 kilodalton (kDa) glycoprotein. However, this monoclonal antibody was not involved in neutralizing. It was shown that in comparison with in vivo immunization, in vitro immunization has some advantages, namely a short immunization period and a small antigen quantity.     

Iodoacetamide treatment of bovine leukemia virus glycoprotein gp51 enhances the western blotting reactivity of anti-peptide antibodies

Rabbit polyclonal antibodies were raised against synthetic peptides of the bovine leukemia virus envelope glycoprotein gp51 and tested against the full size protein by the Western blotting technique. We show that acetylation of gp51 by iodoacetamide either maintains or significantly increases the antigen-antibody reaction and conclude therefrom that the reactive potential of an anti-peptide antibody may require acetylation of the sulfhydryl groups of the blotted protein.     

Protection of cattle against bovine leukemia virus (BLV) infection could be attained by DNA vaccination

The bovine leukemia virus (BLV) envelope gene encoding extracellular glycoprotein gp51 and transmembrane glycoprotein gp30 was cloned into a vehicle expression vector under the human cytomegalovirus (CMV) intermediate early promoter. The intramuscular injection of this plasmid vector generated a cellular immune response. Seven out of ten cows vaccinated with the DNA construct resisted a drastic challenge (500 BLV-infected lymphocytes as an infectious dose).     

Fusion of bovine leukemia virus with target cells monitored by R18 fluorescence and PCR assays.

PCR and R18 fluorescence dequenching assays have been combined to monitor the kinetics of fusion of bovine leukemia virus with target cells (CC81, OVK, or Raji). Antibodies raised against gp51 allow us to demonstrate that not only the hydrophobic N-terminal domain of the transmembrane glycoprotein gp30 but also specific domains of gp51 (amino acids 39 to 103) are involved in bovine leukemia virus-cell fusion.     

Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems.

Bovine leukemia virus 60 to 70S RNA was heat denatured, the polyadenylic acid-containing species were separated by velocity sedimentation, and several size classes were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The major RNA species sedimented at 38S and migrated as a single component of molecular weight 2.95 x 10(6) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The predominant polypeptides of the in vitro translation of bovine leukemia virus 38S RNA were products with molecular weights of 70,000 and 45,000; minor components with molecular weights of 145,000 and 18,000 were also observed. Two lines of evidence indicate that the 70,000- and 45,000-molecular weight polypeptides represent translation products of the gag gene of the bovine leukemia virus genome (Pr70gag and Pr45gag). First, they are specifically precipitated by a monospecific antiserum to the major internal protein, p24, and second, they are synthesized and correctly processed into virion proteins p24, p15, and p10 in Xenopus laevis oocytes microinjected with bovine leukemia virus 38S RNA. The 145,000-molecular weight polypeptide was immunoprecipitated by the anti-p24 serum and not by an antiserum to the major envelope glycoprotein, gp60. It contained all the tryptic peptides of Pr70gag and additional peptides unique to it, and thus represents in elongation product of Pr70gag in an adjacent gene, presumably the pol gene. The 18,000-molecular weight product was antigenically unrelated to p24 and gp60 and shared no peptides in common with Pr70gag, Pr45gag, or the 145,000-molecular weight polypeptide. It was maximally synthesized on a polyadenylic acid-containing virion 16 to 18S RNA, and we present evidence that this RNA is a 3' end-derived subgenomic fragment of the bovine leukemia virus genome rather than a contaminating cellular RNA.     

Detection of cross-reactive antibody to BLV p24 in sera of human patients infected with HTLV

For detection of antibody to bovine leukemia virus (BLV) major core protein of p24 and cross-reactive antibody in human patients infected with human T cell leukemia virus type I (HTLV-I), monoclonal antibody, D432 against BLV p24 was used by competitive binding enzyme-linked immunoadsorbed assay (ELISA). In sera from cattle with enzootic bovine leukosis (EBL) which were positive for BLV antibodies by immunodiffusion test, 109 out of 112 (97.3%) were positive for BLV p24 antibody by competitive binding ELISA. By using the same procedures, 21 samples from adult T cell leukemia (ATL) patients and healthy carriers with HTLV-I were tested for cross-reactive antibody to BLV p24. All 21 samples were positive for HTLV-I antibodies by immunofluorescence test and/or ELISA. By competitive binding ELISA using non-treated BLV antigens, none of these 21 samples inhibited the binding of the D432. When the BLV antigen was treated by several different denaturation procedures, several HTLV-I positive samples showed the inhibition of the D432 binding and the most effective treatment was by 2-mercaptoethanol (2-ME). Sixteen out of 21 samples showed the presence of cross-reactive antibody against 2-ME-treated BLV antigens. The cross-reactivity of human sample to BLV p24 antigen was further confirmed by Western blotting of the 2-ME-treated BLV antigens. None of the 28 samples from leukemia patients other than ATL which were negative for HTLV-I antibodies showed inhibition of the D432 by the competitive binding ELISA.     

Sequence variability of bovine leukemia virus env gene and its relevance to the structure and antigenicity of the glycoproteins.

The nucleotide sequences of the env genes of seven bovine leukemia viruses and the encoded peptide sequence were compared, with the objective of (i) determining the genetic distance separating bovine leukemia virus isolates from different geographical regions, (ii) identifying particular amino acids that contribute to the sequential and conformational epitopes, and (iii) relating such epitopes to their projected position in a three-dimensional model of the structure of the gp51 surface glycoprotein. Two bovine leukemia virus subgroups were clearly identified, a Japanese-American subgroup represented by strains lambda BLV-1, VdM, and FLK-BLV and a European subgroup by strains T15-2, LB285, and LB59. It was possible to identify amino acids that were important in determining three of the epitopes (F, G, and H) recognized by neutralizing monoclonal and polyclonal antibodies. On the model, these epitopes were adjacent and located on the exposed region of the molecule. Amino acid sequences contributing to a fourth cryptic epitope were identified; as predicted by the model, they lay on the opposite side to the neutralizable epitopes in a region involved in glycoprotein subunit association. The fact that this region is not normally exposed on the virion surface provides further evidence for the validity of the model.     

Immunogenicity of a bovine rotavirus glycoprotein fragment.

Previous experiments demonstrated that an antigenic site responsible for virus neutralization and cell attachment was located on a 14,000-molecular-weight fragment of the major bovine rotavirus (BRV) glycoprotein (M. Sabara, J. E. Gilchrist, G. R. Hudson, and L. A. Babiuk, J. Virol. 53:58-66, 1985). However, it was necessary to investigate whether this fragment also had the ability to induce the production of neutralizing antibodies. Upon immunization of mice, the bovine serum albumin-conjugated 14,000-molecular-weight fragment, the unconjugated 14,000-molecular-weight fragment, and the native glycoprotein all induced a similar neutralizing antibody response, albeit to a lesser extent than did the infectious, whole virus. In addition, immuno-blot enzyme-linked immunosorbent assay analysis of the reactivity of anti-peptide serum versus anti-glycoprotein serum with the glycoprotein was very comparable. These results suggest that the 14,000-molecular-weight fragment may represent not only a biologically active region but also an immunodominant area of the glycoprotein.     

Sulfated components of enveloped viruses.

The glycoproteins of several enveloped viruses, grown in a variety of cell types, are labeled with 35SO4(-2), whereas the nonglycosylated proteins are not. This was shown for the HN and F glycoproteins of SV5 and Sendai virus, the E1 and E2 glycoproteins of Sindbis virus, and for the major glycoprotein, gp69, as well as for a minor glycoprotein, gp52, of Rauscher leukemia virus. The minor glycoprotein of Rauscher leukemia virus is more highly sulfated, with a ratio of 35SO4- [3H]glucosamine about threefold greater than that of gp69. The G protein of vesicular stomatitis virus was labeled when virions were grown in the MDBK line of bovine kidney cells, although no significant incorporation of 35SO4(-2) into this protein was observed in virions grown in BHK21-F line of baby hamster kidney cells. In addition to the viral glycoproteins, sulfate was also incorporated into a heterogenous component with an electrophoretic mobility lower than that of any labeled with 35SO4(-2) and [3H]leucine, this component had a much greater 35S-3H ratio than any of the viral polypeptides and thus could not represent aggregated viral proteins. This material is believed to be a cell-derived mucopolysaccharide and can be removed from virions by treatment with hyaluronidase without affecting the amount of sulfate present on the glycoproteins.     

Role of the 3'' long open reading frame region of bovine leukemia virus in the maintenance of cell transformation.

Viral RNA expression was studied by dot blot hybridization with polyadenylated RNAs extracted from a bovine (YR-1) and an ovine (YR-2) tumor cell clone. Both clones were derived from in vivo bovine leukemia virus-induced tumors. The probes used were either the bovine leukemia virus information or only the long open reading frame sequences. No viral RNA corresponding to the bovine leukemia virus long open reading frame region was detected in YR-2, and a very limited amount of bovine leukemia virus messages was unraveled in YR-1. These results strongly suggest that viral expression, even in the long open reading frame region, is not required to maintain transformation of at least some tumor cells.     

Isolation of tumor-associated antigen from sera of bovine leukemia virus-infected cattle

Tumor-associated antigens (TAA) expressed on the surface of enzootic bovine leukemia (EBL) cells were detected and separated from sera of bovine leukemia virus (BLV)-positive cattle using monoclonal antibody-conjugated immunoaffinity matrix. Eluted fraction from these sera showed 3 polypeptides with molecular weights of 70K, 52K, and 30K daltons, and these polypeptides reacted with a monoclonal antibody against TAA. However, only 70K peptide was isolated from culture supernatant of EBL B-cell line. We also tried to examine a reversed passive hemagglutination test to develop a rapid screening system of serum TAA level, but its sensitivity was below the level of detection when EBL sera was applied directly. This is the first report on the existence of tumor antigens in sera from leukemic cattle.     

Glycoprotein (gp-51) antigen not associated with the bovine leukemia virus in FLK culture supernatant liquid]

After elimination of the virus fraction non-associated with bovine leukemia virus (free) glycoprotein gp-51 antigen has been preparatively isolated from the supernatant of the FLK cell culture. For this purpose ultrafiltration through PM-30 membrane, thrice-repeated isoelectric focusing with different (pH 3.0-10.0, pH 4.0-6.0) ampholine intervals and affinity chromatography with ConA-sepharose are used. The SDS-polyacrylamide gel electrophoresis has determined it to be a homogeneous protein with molecular weight 51,000 daltons. The results of the enzyme-linked immunosorbent assay, immunoelectrophoresis and agar-gel immunodiffusion test confirm the specific activity of isolated gp-51 antigen. The gp-51 antigen may be used for identification of antibodies in the blood serum of leukemia virus of infected cattle and for monoclonal screening of antibodies.     

A new monoclonal antibody recognizing an antigen of human lymphocytes similar or identical to Tac antigen

A new mouse monoclonal antibody (HIEI, IgG1 type) that reacts with a cell surface glycoprotein of human lymphocytes was isolated. Membrane immunofluorescence assay showed that HIEI, like the anti-Tac monoclonal antibody, reacted preferentially with activated normal human T-cells and adult T-cell leukemia (ATL) virus (ATLV)-carrying human T- and B-cell lines. However, an interesting difference between HIEI and anti-Tac antibody was that HIEI did not react with ATLV-transformed simian cell lines or those cultured with interleukin-2 (IL-2), whereas the anti-Tac antibody did. The immunoprecipitation assay showed that both HIEI and anti-Tac antibody precipitated a glycoprotein with a molecular weight of 60,000 daltons (gp60) from activated normal T-cells and ATLV-positive T- and B-cells, and also gp53 from MT-2 and MT-2-related T-cell lines transformed with ATLV in vitro by the MT-2 cocultivation method. HIEI inhibited the IL-2-dependent proliferation of normal T-cells, but its inhibitory effect was much weaker than that of the anti-Tac antibody. The anti-Tac antibody interfered with the binding of HIEI to target cells, but HIEI did not block binding of the anti-Tac antibody to the cells. These observations indicate that HIEI antibody recognizes a new antigenic determinant of the human Tac antigen.     

Comparison of the entire genomes of bovine leukemia virus and human T-cell leukemia virus and characterization of their unidentified open reading frames.

We have compared the sequence of the entire genomes of bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I). Both the gag and pol genes show overall strong homologies indicating the close evolutionary relationship of the two retroviruses. However, a surface glycoprotein portion of the env gene shows no appreciable homology, which probably reflects a difference in their host ranges. The 3' end portion of the BLV genome (designated as pXBL) contains an unidentified long open reading frame that has a typical protein-coding property. The potential product of this open reading frame may be a glycoprotein of approximately 40 000 daltons. We note that its amino acid sequence shows low but appreciable homology, especially in its N-terminal quarter, to that of the HTLV-I counterpart (pX product), and we thus suggest that BLV pXBL and HTLV-I pX have diverged from a common ancestral gene. It is tentatively concluded that both the putative pXBL and pX products are respectively produced from a spliced mRNA.     

Proteins of bovine leukemia virus. I. Characterization and reactions with natural antibodies.

The bovine leukemia virus (BLV) was purified from a chronically infected fetal lamb kidney cell line. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this virus revealed the presence of eight distinguishable viral components with molecular weights ranging from 80,000 to 11,000. The major component is a non-glycosylated protein having a molecular weight of 24,000 (p24). At least three heavier polypeptides were found, one of them representing a glycoprotein (gp 60). In addition, four minor polypeptides with respective molecular weights of 19,000, 16,000, 13,000, and 11,000 were identified. In a complement fixation assay using naturally occurring antibodies of a leukemic cow, four polypeptides, which included gp 60, p35, p24, and p16, were found to be reactive.     

Carbohydrate side chains of Rauscher leukemia virus envelope glycoproteins are not required to elicit a neutralizing antibody response.

Antisera raised against Rauscher leukemia virus (R-MuLV) contain a preponderance of antibodies against glycoprotein gp70 that are dependent on the presence of carbohydrate side chains for reactivity, as judged by immunoprecipitation or Western blotting. However, the majority of neutralizing antibodies were not dependent on the presence of carbohydrate, as indicated by (i) the ability of deglycosylated R-MuLV to adsorb neutralizing antibody from sera as efficiently as glycosylated R-MuLV and (ii) the ability of deglycosylated R-MuLV to induce neutralizing antibody responses when injected into rabbits. Moreover, a faster response was obtained with deglycosylated R-MuLV than with untreated control virus in the latter experiments. The results indicate that the neutralizing antibodies are a discrete subpopulation of the total antibody response. Furthermore, the carbohydrate moieties appear to afford protection to the virion during infection, rather than serve as a target for neutralization.     

Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus.

A detailed restriction map was deduced for the genome of the exogenous bovine leukemia virus. The cleavage sites for nine restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with bovine leukemia virus was isolated. The linear viral DNA had a unique restriction map, indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-enriched probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. Restriction enzyme analysis of integrated bovine leukemia virus information present in animals with enzootic bovine leukosis provided evidence for the existence of genetic variants of the virus.     

Experiments with cloned complete tumor-derived bovine leukemia virus information prove that the virus is totally exogenous to its target animal species.

Taking advantage of the existence of a unique SacI restriction site in the long terminal repeats of the integrated bovine leukemia virus proviral DNA isolated from a bovine tumor, the total viral information (about 9.2 kilobases) was cloned in the lambdoid vector lambda WES. lambda B. Use of this cloned bovine leukemia virus DNA allowed us, for the first time, to definitely rule out the existence of any endogenous bovine leukemia virus sequence in the bovine, ovine, caprine, murine, feline, chicken, or human genomes. These data prove the absence of acquired cellular information in the provirus that has given rise to a tumor.