Monoclonal antibodies define eight independent antigenic regions on the bovine leukemia virus (BLV) envelope glycoprotein gp51

Fifteen monoclonal anti-BLV gp51 antibodies are characterized. Competition antibody binding assays show that they are directed against eight independent antigenic regions on the BLV gp51 molecule. Conformation or accessibility of some of these gp51 epitopes change with the test system used, namely the liquid phase radioimmunoassay with radiolabeled antigen or the solid phase enzyme immunoassay with plastic bound gp51 or BLV particles. A two-site immunometric assay using monoclonal antibodies directed against two independent epitopes allows detection of isolated gp51 molecules at a minimal concentration of 0.4 ng/ml and is also suitable for the detection of BLV particles.     

Antigenic variants of bovine leukemia virus (BLV) are defined by amino acid substitutions in the NH2 part of the envelope glycoprotein gp51

Previous studies with monoclonal antibodies of the antigenic structure of bovine leukemia virus (BLV) envelope glycoprotein (gp51) have identified three epitopes (F, G, H) directly involved in the infectivity of BLV, F, G, and H lost their reactivity with the respective monoclonal antibodies after treatment with a reducing agent, indicating that these epitopes were conformational. Sequence comparisons between BLV mutants and differential reactivities of urokinase or proteinase K gp51 fragments with monoclonal antibodies indicated that the NH2 moiety of the env protein harbored the three architectural determinants F, G, and H. ELISA tests demonstrated that anti-F, -G, and -H monoclonal antibodies were maximally reactive toward intact virions whereas they showed much poorer affinities for their respective epitopes when presented on a purified protein. Accordingly, an efficient vaccine against BLV infection will include at least the identified gp51 region presented in its native architectural configuration.     

Biologically active epitopes of bovine leukemia virus glycoprotein gp51: their dependence on protein glycosylation and genetic variability

A panel of monoclonal antibodies to the bovine leukemia virus envelope glycoprotein (BLV gp51) has previously demonstrated the association of the biological activities of the virus (infectivity, syncytia induction) with three out of eight epitopes of gp51. In BLV-infected cells, the unglycosylated homolog of the precursor to the BLV envelope glycoproteins (gPr72env) is a 47,000-MW polypeptide. Immunoprecipitation studies with monoclonal antibodies show that the neutralizing antibody-inducing sites, although present in gPr72env, are not conserved in the 47,000-MW unglycosylated homolog. Finally, it is demonstrated that the neutralizing antibody-inducing sites of gp51 are subject to antigenic variation among BLV isolates of the same or different geographical origins.     

Topographical analysis by monoclonal antibodies of BLV-gp51 epitopes involved in viral functions

Mouse monoclonal antibodies, directed against eight independent antigenic sites on the bovine leukemia virus (BLV) envelope gp51 (sites A, B, C, D, E, F, G, H), were tested for their capacity to interfere with the biological activities of BLV which are associated with the gp51 molecule, as follows: the pseudotype inhibition test (PI) measures the ability of antibodies to inhibit infectivity of BLV-VSV pseudotypes; the early polykaryocytosis inhibition test (EPI) measures inhibition of syncytia-inducing activity of BLV-producing cells in the presence of these antibodies; and the complement-dependent cell lysis measures the cytoxic activity of antibodies toward BLV-producing cells in the presence of complement. Three of the eight antigenic sites (sites F, G, H) were shown to be involved in the biological activities tested for in infectivity and syncytia neutralization tests. At least one of these three regions is exposed on the surface of BLV-producing cells (site G) and can be the target for complement-dependent cytotoxicity. Limited protease digestion of gp51 showed that a peptide fragment of MW 15,000 contained at least a part of sites FGH involved in the biological activities analyzed and of site E which is inactive. The major carbohydrate residues are localized on a fragment of apparent molecular weight 35,000, also containing the antigenic sites ABCD which are not involved in the biological activities tested for.     

Expression of env gene of bovine leukemia virus in rodent cells

Summary The BamHI-BamHI fragment of the env gene of bovine leukemia virus (BLV) cloned in pMMEx expression vector was transfected into Chinese hamster cells. Monoclonal antibodies (MAbs) directed against both conformational and sequential epitopes of gp51 of BLV recognized viral polypeptides expressed in hamster cells in Western blotting and enzyme-linked immunosorbent assay.     

Synthetic peptides approach to identification of epitopes on bovine leukemia virus envelope glycoprotein gp51

Peptides corresponding to residues 21-28, 39-48, 57-67, 59-69, 78-92, 144-155, 144-157, 195-205, 255-268, and 260-268 of envelope glycoprotein gp51 of bovine leukemia virus (BLV) were chemically synthesized and coupled to keyhole limpet hemocyanin or tetanus toxoid. All peptides were immunogenic in rabbits and induced production of antipeptide antibodies. Enzyme-linked immunosorbent assays using Tween 80-purified gp51 or BLV particles showed that antibodies against peptides 78-92, 255-268, and to a lesser extent 39-48 and 144-157 were able to react with the parent glycoprotein, purified or as an integer part of BLV particles. Antisera against peptides 39-48, 78-92, and 144-157 neutralized VSV (BLV) pseudotypes in vitro, the highest neutralization titer being obtained with the 78-92 cyclized peptide. These observations confirm that the NH2 moiety of gp51 carries epitopes involved in virus infectivity.     

Identification of an immunodominant region on the isolated bovine leukaemia virus (BLV) major envelope protein gp51 by monoclonal antibodies presumably not exposed during natural BLV infection

A panel of newly isolated monoclonal antibodies (MoAbs) is described which are specific for bovine leukaemia virus (BLV) envelope protein gp51. Epitope mapping using competition antibody binding assays and binding studies with gp51-related fusion proteins and synthetic peptides show that they are directed against seven independent antigenic determinants. Four of them are unrelated to the epitopes described earlier (Bruck et al., 1982a). We define three binding regions for the MoAbs on the gp51 molecule. The region between amino acids 170 and 217 is highly immunogenic when the isolated protein is used for immunization, and seems to be inaccessible for immune recognition when gp51 is associated with the virus envelope as it occurs during natural BLV infection.     

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.     

Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH₂-terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.     

Expression of the bovine leukemia virus envelope glycoprotein (gp51) by recombinant baculovirus and its use in an enzyme-linked immunosorbent assay

The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH2-terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.     

Use of two monoclonal antibodies in an ELISA test for the detection of antibodies to bovine leukaemia virus envelope protein gp51

A competition ELISA technique involving two monoclonal anti-gp51 antibodies has been developed for the detection of bovine leukaemia virus (BLV) antibodies. Precoated gp51 antigen-microtitre plates were obtained by incubation of plastic adsorbed monoclonal antibody with a non-purified BLV preparation. Samples to be tested were incubated in the wells of the gp51-coated plates; the presence of anti-gp51 antibodies was indicated by competition for antigen binding with an enzyme linked monoclonal antibody directed to an important epitope on gp51. This test is as sensitive as a routinely used indirect ELISA test; it is highly specific, reliable and easy to perform.     

Immunoaffinity purification of two major proteins of bovine leukemia virus (gp51 and p24) and their use for discrimination between vaccinated and infected animals.

The outer envelope glycoprotein gp51 and the core protein p24 of bovine leukemia virus (BLV), were purified from culture media of FLK-BLV cells by a single-step procedure, using immunoaffinity chromatography based on monoclonal antibodies to the respective proteins. About 90% of the envelope glycoprotein in the culture medium was recovered as a highly purified product. Both purified protein (gp51 and p24) preparations, were found to be highly specific antigens by ELISA, and did not cross-react with sera raised against the other antigen. The conformational epitopes on the purified gp51 were preserved as judged by their reactions with the corresponding monoclonal antibodies. The p24 ELISA reacted only with sera from naturally infected animals and not with sera from animals immunized with an experimental gp51-iscom vaccine. The p24 antigen is therefore useful for discriminating between BLV-infected animals and those immunized with a gp51 subunit vaccine.     

Serological analysis of antigenic determinants on the env gene products of AKR dualtropic (MCF) murine leukemia viruses

A panel of eight monoclonal antibodies recognizing individual antigenic determinan (epitopes) on the envelope proteins (gp70 and p15(E)) of murine leukemia virus (MuLV) has been used to probe the serological properties of recombinant dualtropic (MCF) viruses. Since six of these monoclonal antibodies showed specificity for epitopes that were exclusive to endogenous N-ecotropic MuLV, it was reasoned that they might provide a means to determine the relative genetic contribution of ecotropic information in the env gene of dualtropic isolates. It was anticipated that the loss of ecotropic-specific sequences in these viruses would be reflected by a loss of reactivity with the ecotropic-specific monoclonal antibodies. This expectation proved to be correct, as several of the monoclonal antibodies that reacted with endogenous ecotropic MuLV of AKR mice failed to react with isolates of recombinant dualtropic MuLV from the same strain. In addition, it was noted that the “nonreactivity” of the monoclonal antibodies with different dualtropic viruses occurred in characteristic patterns. A common feature of the patterns was coordinate loss of expression of the gp70^a, gp70^d, and gp70^e epitopes from all AKR dualtropic MuLV. In contrast to the common deletion of ecotropic-specific epitopes, the gp70^b, gp70^c, and p15(E)^a epitopes were variably deleted in different recombinant viruses.     

Further characterization and subcellular localization of Sm and U1 ribonucleoprotein antigens

Sera from patients with systemic autoimmune diseases often contain antibodies against small nuclear ribonucleoprotein (snRNP) particles. Anti-Sm antibodies react with the entire set of U1, U2, U4, U5 and U6 (U1-U6) RNP particles whereas anti-(U1)RNP sera specifically recognize particles containing U1 RNA. Here we performed semi-quantitative immunoblotting using 16 human anti-Sm, 15 human anti-(U1)RNP sera and two mouse monoclonal antibodies to establish which snRNA-associated proteins carry antigenic determinants. Almost every (15/16) human anti-Sm sera recognized epitopes present on a 28-kDa (B/B') protein doublet and on a 16-kDa (D) polypeptide. Nine anti-(U1)RNP sera also recognized the B/B' doublet, but in all cases a much stronger reaction was observed with one or more of the specifically U1 RNA-associated 70 kDa, A or C antigens. With affinity-purified antibody fractions eluted from individual antigen bands on nitrocellulose blots it is shown that the anti-Sm-reactive polypeptides B/B' and D contain common epitopes. We also report the finding of one human anti-Sm serum with exclusive specificity for the B/B' doublet and a mouse monoclonal anti-Sm antibody recognizing only the D protein, indicating that these antigens also carry unique epitopes. In immunoprecipitation assays, purified anti-B/B' and -D antibodies react with (U1-U6) RNP while purified anti-70 kDa, anti-A and anti-C antibodies precipitate exclusively U1 RNP particles. Finally, we established the subcellular localization of Sm and U1 RNP antigens using a biochemical cell fractionation procedure. Part of the 70 kDa and B/B' antigens were found in a nuclease and high salt-resistant nuclear substructure, usually referred to as nuclear matrix, while the A and D antigens could be extracted completely from HeLa nuclei by ribonuclease treatment and subsequent high salt extraction.     

Selective small antigenic structures are capable of inducing widespread autoimmunity which closely mimics the humoral fine specificity of human SLE

Recent data have suggested that autoantibodies in lupus can progress from simple immunity against a few antigenic structures to a complex response against multiple autoantigens. Our aim was to determine whether these diverse epitope patterns can indeed be generated by antigenic challenge with a single, small structure. Rabbits were immunized with either a 60 kDa Ro peptide commonly antigenic in human systemic lupus erythematosus (SLE) (Ro 274-289) or one which is rarely a humoral target (Ro 500-515). Rabbits immunized with the antigenic peptide (Ro 274-289) not only developed antibodies to multiple epitopes of 60 kDa Ro and La, as has been described, but also produced non-cross-reactive antibodies to the common spliceosomal proteins Sm B' and D1, and nRNP A and C. Rabbits immunized with the Ro 274-289 peptide also mount a progressive, diversified immune response to the sequential antigenic regions of these proteins (60 kDa Ro, Sm B' and D1, nRNP A and C), which is nearly identical to that seen in human SLE. Animals immunized with the nonantigenic peptide Ro 500-515 develop antibodies only to 60 kDa Ro. These results demonstrate that loss of tolerance to select single, small antigenic structures can begin a cascade which virtually recreates, at the epitope level, the humoral autoimmune specificity seen in human SLE.     

Shiga toxin 1 targets bovine leukemia virus-expressing cells

Direct evidence that Escherichia coli Shiga toxin (Stx) acts against bovine leukemia virus (BLV)-expressing cells was obtained. The active A subunit of Stx type 1 (StxA1) targeted a selected population of permeable cells expressing BLV and inhibited BLV replication in a culture of bovine peripheral blood mononuclear cells. Cells were cultured with and without StxA1, and at various times cells expressing BLV were identified by being stained with MW1 monoclonal antibody specific for the BLV protein gp51. Before culture, permeable cells were tagged by uptake of one of the following: acetoxymethyl of 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF), BCECF conjugated to 70-kDa dextran, or 70-kDa dextran conjugated to fluorescein. The tagged cells costaining with anti-gp51 were selectively eliminated in StxA1-treated cultures. Electron microscopy analysis of purified B lymphocytes showed sharply reduced numbers of BLV particles in StxA1-treated cultures.     

Naturally occurring leukemia viruses in H-2 congenic C57BL mice. III. Characterization of C-type viruses isolated from lymphomas induced by milk transmission of B-ecotropic virus

The prevalence of different host-range classes of murine leukemia virus (MuLV) was studied in C57BL mice with (V+) and without (V-) milk transmission of a naturally occurring B-tropic ecotropic MuLV. Virus isolates were studied with respect to growth properties, XC-plaque formation, antigen profiles of their envelope proteins (gp70 and P15(E)), gp70 tryptic-peptide maps, and their potential to induce lymphomas after inoculation into newborn mice. B-tropic ecotropic MuLV with the capacity to cause plaques in XC cells was isolated from almost all lymphomas of both V+ and V- sublines. The reaction patterns of these ecotropic isolates with monoclonal antibodies reactive with MuLV-env proteins and the tryptic-peptide maps of the gp70 molecule indicate that they are similar to each other and differ only slightly from the ecotropic MuLV in the spleens of young V+ animals, which is identical to the milk-transmitted virus. XC-, B-tropic dualtropic mink cell focus-inducing (MCF) viruses were isolated from the majority of different types of lymphoma (B cell, T cell, or neither B nor T cell derived), but not from the spleens or milk of young V+ or V- animals. The env proteins of the MCF isolates are highly heterogeneous, but most isolates originating from B10.AV + T-cell lymphomas share MCF-related epitopes in their gp85 envelope precursor with AKR MCF-247 virus. Most MCF viruses isolated from non-T lymphomas do not possess these epitopes. The results indicate that also in this model the generation of dualtropic MCF viruses might be important in lymphoma induction, although only some of the cloned MCF viruses show enhanced oncogenic properties in comparison with ecotropic isolates. A cloned oncogenic MCF virus induced different lymphoma types in C57BL/10 (= B10, H-2b) and B10.A (H-2a) mice, similar to what was found earlier with the milk-transmitted virus. Hence, the lymphoma-type differences are not due to differences in the B-tropic ecotropic viruses transmitted through the milk in these strains, but reflect an influence of the H-2 complex on the phenotype of the virus-induced lymphomas.     

Monoclonal antibodies derived during graft-versus-host reaction. II. Antibodies detect unique determinants common to many MCF viruses

Hybridoma cell lines were recovered from the spleens of 6-week-old (B6 X D2)F1 mice undergoing graft-versus-host reaction induced by the transfer of 5-week-old B6 parental spleen cells. These cell lines produced antibodies reactive with envelope polypeptides of a variety of MuLV. The viral specificity assessed by membrane immunofluorescence and virus-binding radioimmunoassay indicated that the reactivity of these antibodies was distinctly different from monoclonal antibodies recovered from (B6 X D2)F1 recipients of D2 spleen cells reported previously (Portis et al., Virology 118, 181-190, 1982). Ten out of 17 monoclonal antibodies in the current study reacted exclusively with MCF viruses and three of these antibodies detected envelope determinants which were shared by a broad panel of MCF viruses of diverse origin. These common MCF determinants were expressed by the gp70 molecule as determined by Western blot analysis. The production of these antibodies by young mice in the absence of exogenous virus inoculation suggests that these antigens may be encoded by endogenous MCF-like sequences.     

Autoantibody production against the U small nuclear ribonucleoprotein particle proteins E, F and G in patients with connective tissue diseases

The nucleoplasmic U small nuclear ribonucleoprotein particles (snRNP) have a set of seven proteins in common which are designated B', B, D, D', E, F and G. Patients suffering from rheumatoid autoimmune diseases such as systemic lupus erythematosus often develop autoantibodies against the proteins B', B, and D. Here we describe a sensitive immunoassay which allows the specific detection of autoantibodies reacting with the E, F or G snRNP proteins. We were able to identify several patient sera containing autoantibodies against one or more of these proteins. This demonstrates that all snRNP proteins described so far are potentially antigenic in systemic rheumatoid diseases. The characterization of the antibodies showed an immunological cross-reactivity between the snRNP protein G and the 70-kDa protein of U1 snRNP. Several sera contained autoantibodies which were specific for the F snRNP protein.     

Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus.

Current serological assays using whole rubella virus (RV) as a target antigen for detecting RV-specific antibodies fail to define specific RV proteins and antigenic determinants such as hemagglutinin (HA) and virus-neutralizing (VN) epitopes of rubella virus. A panel of E1 deletion mutants and a subset of E1-specific monoclonal antibodies (MAb) were used for the initial analysis of HA and VN epitopes of E1 glycoprotein. A peptide region (E1(193) to E1(269)) was found to contain HA and VN epitopes. Using both overlapping synthetic peptides and truncated fusion proteins within this region, the HA epitope defined by MAb 3D9F mapped to amino acid residues E1(214) to E1(240), while two VN epitopes defined by MAb 21B9H and MAb 16A10E mapped to amino acid residues E1(214) to E1(233) and E1(219) to E1(233), respectively. The epitopes defined in this study are recognized by antibody whether or not the epitopes are glycosylated.