Topological mapping antigenic sites on the influenza A/PR/8/34 virus hemagglutinin using monoclonal antibodies

We have previously constructed an antigenic map of the PR8 virus hemagglutinin (HA) using monoclonal antibodies and PR8 virus antigenic variants. Four different antigenic sites were operationally defined: two predominantly “strain-specific” antigenic sites (designated Sa and Sb) and two predominantly “cross-reactive” antigenic sites (designated Ca and Cb). In the present study, we have conducted competitive binding assays using monoclonal antibodies to investigate the topologic relationship among these antigenic sites. It was found that several antibodies directed against the site Sa did not compete with the binding of a radiolabeled antibody to the site Cb and, vice versa, antibodies directed against site Cb did not compete with the binding of a radiolabeled antibody to site Sa. This proved unequivocally that at least part of the “strain-specific” site Sa and of the “cross-reactive” site Cb are topologically distinct. Various degrees of competition were observed, however, between other antibodies directed against operationally defined antigenic sites. This may indicate that parts of these sites are either structurally overlapping or in close proximity. Alternatively, it is possible that the binding of antibody to any of these sites allosterically affects the binding of antibody at topologically distant sites.     

Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies

Competitive radioimmunoassays using monoclonal antibodies established that the neuraminidase of A/RI/5+/57 (H2N2) influenza can be divided into four overlapping antigenic regions. Antigenic regions 1 and 4 are sufficiently far apart so that there was no competition between antibodies for these sites. Region 1 is conserved in neuraminidases from N2 viruses over a 10-year period, while the other regions changed antigenically during this time. The antibodies belonging to groups 2 and 3 completely inhibited catalytic activity on fetuin substrate, whereas antibodies in groups 1 and 4 inhibited weakly or not at all. Antigenic region 2 can be further divided into four overlapping areas (2a, 2b, 2c, and 2d) based on the reactivity patterns of monoclonal antibodies with antigenic variants, chemically modified neuraminidase, and the ability of the antibodies to inhibit enzyme activity of different molecular weight substrates. Previous studies [R. G. Webster, V. S. Hinshaw , and W. G. Laver (1982) Virology 117, 93-104; D. C. Jackson and R. G. Webster (1982) Virology 123, 69-77] characterized only region 2 of the neuraminidase molecule. Each of the monoclonal antibodies inhibited virus release from MDCK cells when incorporated in an agar overlay, and some antibodies in each group inhibited hemagglutination by intact virus, but only antibodies in group 2 neutralized virus in embryonated eggs and permitted selection of antigenic variants. The results indicate that antibodies to some antigenic sites on the neuraminidase may inhibit virus release more efficiently than others, depending on their relation to the enzyme active center. None of the monoclonal antibodies inhibited the hemolytic activity of viruses possessing N2. Based on antigenic mapping and biological properties of the monoclonal antibodies, a topographical map of the neuraminidase can be constructed. It is proposed that antigenic regions 1 and 4 are spacially separated and, based on their failure to inhibit biological activity, may be located on the bottom surface of the molecule; region 3 may be on the top surface of the molecule but at some distance from the catalytic center. Antigenic region 2 probably encompasses most of the top surface of the molecule; region 2d being closest to the enzyme center, with subregions 2a and 2b adjacent to it on the top surface. Chemical treatment of the neuraminidase with trinitrobenzenesulfonic acid (TNBS) causes modification of the 2b region, confirming the antigenic mapping results.     

Identification by monoclonal antibodies of a new epitope in the glycoprotein complex of Sindbis virus

Monoclonal antibodies against a deletion mutant of Sindbis virus were produced and characterized in order to determine the fine mapping and functional activities of single viral epitopes. All monoclonal antibodies so far tested showed a certain degree of reciprocal competition and were directed against an antigenic determinant which was present only on the undissociated complex of the E1 and E2 glycoproteins. A biological assay measuring viral haemagglutination showed no decrease in the titre of viral samples preincubated with monoclonal antibodies. Conversely, a reduction in viral infectivity was demonstrated, particularly with two of these antibodies. The results suggest that the antibodies which we characterized seem to recognize a new epitope which is represented on both glycoproteins on the surface of this mutant of Sindbis virus.     

Mapping of antigenic determinants within peptides of a variant surface glycoprotein of Trypanosoma brucei

The location of antigenic determinants in the primary amino acid sequence of the variant surface glycoprotein of Trypanosoma brucei MITat 1.6 was investigated using monoclonal antibodies in conjunction with the known cyanogen bromide and tryptic cleavage patterns of this antigen. The cyanogen bromide digestion fragments of the antigen were purified and used to raise polyclonal antisera, which were specific for the appropriate cyanogen bromide fragment and partial digestion products, as well as recognising the intact variant surface glycoprotein. Competition radioimmunoassays were carried out between these antisera and nine monoclonal antibodies specific for MITat 1.6 variant surface glycoprotein, which have previously been characterised and shown to recognise five antigenic determinants of which only one is exposed on the surface of the living trypanosome. The binding of the monoclonal antibodies to the major tryptic peptide of MITat 1.6 variant surface glycoprotein was investigated by immunoblotting and by competition radioimmunoassay, and revealed that the five antigenic determinants recognised by the nine monoclonal antibodies are all located in the N-terminal two thirds of the MITat 1.6 variant surface glycoprotein molecule. Three of the determinants are located in an immunodominant region apparently formed by the folding together of two of the cyanogen bromide peptides. The other two determinants appear to be more conformationally labile; one of these is the determinant which is exposed on the surface of the living trypanosome, which is located in the N-terminal one third of the molecule.     

Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceus and pemphigus vulgaris

Pemphigus foliaceus and pemphigus vulgaris are skin diseases in which antibodies against the cell surface of keratinocytes destroy the adhesion between epidermal cells, producing blisters. Patients with pemphigus foliaceus have antibodies to a complex of three polypeptides of 260, 160, and 85 kd (the foliaceus complex), whereas patients with pemphigus vulgaris have antibodies to a complex of 210-kd, 130-kd, and 85-kd polypeptides (the vulgaris complex). The 160-kd polypeptide of the foliaceus complex has been identified as desmoglein, a desmosomal glycoprotein. We suspected that the 85-kd component in both these antigenic complexes might be plakoglobin, another molecule in the adhering junctions of cells. To characterize these antigenic complexes, we used the serum of five patients with pemphigus foliaceus, that of four patients with pemphigus vulgaris, and monoclonal antiplakoglobin antibodies. We found that monoclonal antibodies to plakoglobin immunoprecipitated the 85-kd polypeptide from the dissociated foliaceus and vulgaris complexes and precipitated both complexes from epidermal extracts. Serum from patients with pemphigus foliaceus or pemphigus vulgaris (but not from four normal controls) bound desmoglein and the 130-kd polypeptide, respectively, showing that these peptides (and not plakoglobin) are the antigenic binding sites in these disorders. We conclude that plakoglobin, a protein of the adhering junctions of epidermal cells, is the 85-kd molecule in the antigenic complexes found in both pemphigus foliaceus and pemphigus vulgaris, although it is not the binding site in either disorder.     

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.     

Anti-idiotypic and anti-anti-idiotypic antibodies as highly specific tools in epitope and active site studies on human interferon-gamma.

Immunization of rabbits with F(ab')2 fragments of different monoclonal antibodies directed against human interferon-gamma yielded antisera with anti-idiotypic characteristics. Isolation of the anti-idiotypic fraction, resulting in a highly specific antiserum, allowed us to prove that out of six competing monoclonal antibodies directed against human interferon-gamma, only two really recognize the same epitope. The other monoclonal antibodies compete on the basis of steric hindrance, which is not surprising, because of the large difference in Mr between interferon-gamma and an immunoglobulin. The anti-idiotypes provided us also with a tool to study isolated epitopes on the human interferon-gamma molecule, a task which was previously not practicable. Exploration of the biological properties of these anti-idiotypes allows us to determine whether the investigated epitopes are involved in receptor binding. The production of an anti-anti-idiotypic antiserum not only proved that we generated real internal images, but also that these images conserved all of their properties, although with a decreased affinity in comparison with the original monoclonal antibody. As the former is a polyclonal antiserum, directed against a single epitope of the human interferon-gamma molecule, competition experiments yielded additional information on the relative position of three epitopes recognized by inhibiting monoclonal antibodies. These antisera will possibly open new ways for the affinity purification of interferon-gamma and perhaps for the treatment of autoimmune diseases.     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

Monoclonal antibody analysis of bovine herpesvirus-1 glycoprotein antigenic areas relevant to natural infection

Neutralizing antigenic areas on the glycoproteins of bovine herpesvirus-1 (BHV-1) were identified by reciprocal competition radioimmunoassays using monoclonal antibodies. Three interrelated and two independent antigenic areas were identified on the 77-kDa (K) gIV envelope glycoprotein. Antigenic analysis of this protein has not been previously described. Four interrelated and one independent antigenic areas were found on the 97K gIII envelope glycoprotein. A third group of monoclonal antibodies reacting in Western blot with the 74K subunit of gI, a 130K disulfide-linked 74K/55K heterodimer, revealed four interrelated antigenic areas. All of the antigenic areas on all three glycoproteins were reactive with neutralizing monoclonal antibodies and all were targets for antibody-complement lysis. However, antibodies against gIV were the most efficient at neutralizing the virus and rendering infected cells susceptible to antibody-complement lysis. Convalescent sera from experimentally infected calves were used in a competitive radioimmunoassay to confirm that each antigenic area on the gI, gIII, or gIV glycoproteins was a target for bovine antibodies during primary infection with BHV-1.     

Antigenic sites of human growth hormone and related molecules detected by monoclonal antibodies to human growth hormone

Four major antigenic sites for human growth hormone (hGH) were identified by 27 mouse monoclonal antibodies to hGH. Sites 1 and 2 are spatially close whereas sites 3 and 4 are located in other parts of the molecule. There also appears to be a subdivision of antigenic sites. A panel of 10 monoclonal antibodies, which included representatives from each antigenic site group, were used to determine cross-reactivities between hGH and human placental lactogen (hPL), human prolactin (hPRL), the 20,000 mol. wt variant of hGH (hGH20K) and a disulfide-linked dimer of hGH (diS-dimer). The data suggest a high conformational dependence of antigenic sites in hGH. DiS-dimer retains all four antigenic sites of hGH, although all have been altered. hGH20K retains sites 2-4 but site 1 has been dramatically altered. hPL retains site 3, whereas sites 1 and 4 have been dramatically altered and site 2 may be lacking. The extremely low cross-reactivity observed for hPRL is consistent with the dissimilarity between hGH and hPRL. Antigenic site 3 is the most conserved of all sites. The lack of structural similarity compared with hGH of site 1 in hGH20K and of a portion of site 3 in diS-dimer suggests that it may be possible to develop specific radioimmunoassays for these structural variants of hGH.     

Targeting behavior of rat monoclonal IgG antibodies in vivo: role of antibody isotype, specificity and the target cell antigen density

The studies described in this report were designed to investigate factors that could influence the behavior of erythrocytes following their interaction with monoclonal antibodies (mAb) in a fully homologous experimental opsonization system in vivo. The clearance profiles and tissue distribution of target erythrocytes were examined in both normal and decomplemented rats preinjected with rat IgG2a or IgG2b mAb directed against the same or different sites on RT1Aa, the classical class I major histocompatibility complex antigen of the DA rat. Complement played a major role in augmenting the clearance and promoting hepatic sequestration of target erythrocytes in rats preinjected with IgG2a mAb directed against the S site. In contrast, an intact complement system was not an essential requirement for erythrocyte clearance when S site-specific IgG2b mAb were used. With each antibody tested, (DA x PVG)F1 cells, expressing about half as much antigen, were removed significantly slower than DA erythrocytes, this finding being more pronounced when the animals had been preinjected with mAb of the IgG2a isotype. A comparison of the tissue distribution of DA and (DA x PVG)F1 erythrocytes indicated that hepatic uptake was greater for target cells expressing higher antigen density. A considerable degree of heterogeneity was observed in the in vivo behavior of the target erythrocytes with three groups of IgG2b mAb that recognized different sites on the class I molecule. The S site-specific IgG2b mAb were much more efficient in the hepatic Fc receptor-mediated clearance system than were the P site-directed mAb of the same subclass. Our results suggest that antibody specificity may also be a contributory factor, in addition to antibody isotype and target cell antigen density, in determining the fate of target cells in vivo.     

Conservation of epitopes in the oligosaccharide portion of the lipooligosaccharide of Haemophilus influenzae type b.

The antigenic characteristics of the lipooligosaccharide (LOS) of Haemophilus influenzae type b (Hib) were examined in strains obtained over an extended period of time. These Hib strains were isolated from patients with systemic Hib disease in Dallas, Tex., over a 20-year period and in New York City between 1941 and 1956. The antigenic characteristics of the LOS of these Hib strains were examined by using a set of four murine monoclonal antibodies directed against epitopes present in the oligosaccharide portion of the LOS molecule. The same basic set of LOS antigenic determinants that is expressed by recent Hib isolates was also found to be present in this collection of Hib strains spanning a 40-year period. Some variation with time was detected in the distribution of the systemic disease isolates among four Hib LOS antigenic groups; however, only 2 of 188 Hib isolates failed to react with a set of two LOS-specific monoclonal antibodies. Therefore, little variation has occurred among Hib strains with regard to the LOS epitopes defined by these monoclonal antibodies over a considerable period of time.     

A monoclonal antibody (3A33) that reacts with a mouse-specific epitope of Mac-1 antigen

The 3A33 monoclonal antibody, obtained by fusing rat immune lymphocytes with mouse plasmacytoma cells, was directed against mouse macrophages. Antibody 3A33, a rat IgG2a, reacted with macrophages from all the mouse strains tested, with mouse blood monocytes and with 56% of bone marrow cells, but not with T lymphocytes. It immunoprecipitated an antigen with alpha and beta subunits, found to be identical to Mac-1 antigen after cross-absorption experiments with M1/70 monoclonal antibody. The two antigenic determinants of the Mac-1 molecule identified by the 3A33 and M1/70 antibodies both displayed reduced expression on inflammatory macrophages and comparable resistance to trypsin digestion. The sites of the determinants on this molecule seemed close together judging from the ability of both the 3A33 and M1/70 antibodies to block C3bi receptor sites and compete for cell binding. However, unlike antibody M1/70, 3A33 never reacted with human cells bearing Mac-1 antigen. Therefore, two closely related epitopes of the Mac-1 molecule - one specific for mouse and one common to mouse and man, were recognized by these monoclonal antibodies.     

Monoclonal antibodies produced in BALB.B10 mice define new antigenic determinants in culture filtrate preparations of Mycobacterium tuberculosis.

A panel of monoclonal antibodies were derived from BALB.B10 mice immunized with a culture filtrate from Mycobacterium tuberculosis H37Rv. Of these antibodies, 10 were examined more closely for antigen specificity and interspecies reactivity. Six antibodies were used as immunosorbents for affinity purification of their corresponding antigens. Two monoclonal antibodies (HBT 2 and HBT 11) reacted with a 17-kilodalton antigen, and a competition assay showed that these antibodies are directed against the same epitope or against epitopes that are sterically very close to each other. Monoclonal antibody HBT 12 reacted with the same molecule with which a previously described 38-kilodalton reactive antibody reacted but was directed against a different epitope. Antibody HBT 10 reacted with a culture filtrate of M. tuberculosis but not of Mycobacterium bovis BCG. This latter finding was further studied by testing different preparations of M. tuberculosis H37Rv antigens and, additionally, culture filtrates of four M. tuberculosis and two BCG strains. Interspecies reactivity was assayed by immunoblotting and revealed that the majority of the monoclonal antibodies were specific to M. tuberculosis complex.     

Antigenic topography of bluetongue virus 17.

Neutralizing monoclonal antibodies (mAbs) have been produced and used to map the topographical relationship of the surface antigenic determinants of bluetongue virus (BTV) 17 that mediate neutralization. Eight monoclonal antibodies, at least five of which were directed to the major outer coat protein of BTV 17, P2, were studied in neutralization assays using variant BTV 17 and in competition binding experiments. Five different epitopes were identified that are involved in neutralization of viral infectivity. Three of the five epitopes are clearly associated with P2, while the location of the other two epitopes is not known. The potential association of these two epitopes with one or both outer coat proteins of BTV is discussed.     

Idiotypic characterisation of monoclonal antibodies with restricted epitope specificity for retinal S-antigen.

This paper describes the idiotypic specificities of eight murine monoclonal antibodies directed to three independent epitopes on retinal S-antigen. The antigenic sites recognised by these monoclonal antibodies have previously been localised to a small region near the C-terminal of bovine S-antigen. Xenogeneic, site-related anti-idiotypes prepared against each of the monoclonal antibodies recognised common idiotypes only amongst those monoclonal antibodies which reacted with the same epitope on S-antigen. Two of the three idiotypes were detected in the sera of BALB/c mice but not in two strains of rat immunised with xenogeneic S-antigen and none could be detected in the sera of patients with anti-photoreceptor autoantibodies. Our results demonstrate that the idiotypes of murine monoclonal antibodies to retinal S-antigen exhibit restricted epitope specificity but are species-restricted and imply that the S-antigen lacks a dominant antigenic epitope.     

Identification of conserved epitopes on a hog cholera virus protein

Summary Eight monoclonal antibodies directed against the hog cholera virus (HCV) strain Alfort/187 and displaying broad cross-reactivity with other HCV strains were characterized. An enzyme immunoassay on fixed monolayers of porcine or bovine cells infected with 14 different strains and isolates of HCV and 12 bovine viral diarrhea viruses (BVDV), respectively, showed that all antibodies reacted with HCV only. Seven antibodies recognized all HCV tested, thus indicating that they were directed against conserved epitopes. All antibodies neutralized the homologous strain and different patterns of the other HCV tested. Radioimmunoprecipitation analysis showed that the monoclonal antibodies were directed against a doublet of 56–60 kDa, presumably representing the major envelope glycoprotein of HCV. The results of reciprocal antibody blocking assays allowed the mapping of two distinct conserved antigenic domains on this protein.     

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.     

Binding characteristics of monoclonal antibodies raised against bovine growth hormone

Monoclonal antibodies have been raised against pituitary bovine growth hormone using the hybridoma procedure. The binding characteristics of the seven selected monoclonal antibodies toward the antigen molecule in its native, chemically or enzymatically treated form have been studied. The reactivities of the monoclonal antibodies with growth hormones from other species and bovine prolactin have also been investigated. The epitopes recognized by four of the produced monoclonal antibodies are conformational, whereas two other monoclonal antibodies bind to sequential determinants. Three antibodies define immunological sites located between residues 6-124 of the bovine growth hormone molecule, and one of this antibody shows higher affinity to human than bovine growth hormone. The immunoreactivity of one monoclonal antibody is enhanced by the previous binding of the antigen to polyclonal antibodies, probably because of a localized conformational change of the bovine growth hormone molecule. This antibody also shows cross-reactivity with all the homologous hormones tested, indicating to recognize a highly conserved antigenic determinant.     

Studies of Monoclonal Antibodies Specific for Major Histocompatibility Complex Products of the Rat

Sixteen monoclonal antibodies against antigens coded for by the RT1 complex of the rat have been produced. Fourteen are specific for the a haplotype: six recognize class II and eight class I antigens. Two are specific for the 1 haplotype, one reacting with class I and the other with class II antigens. By means of these monoclonal antibodies four independent clusters of antigens for class I antigens of the a haplotype and three for class II antigens could be defined. The three antigenic sites of class II antigens reside on the same heterodimer. The monoclonals described here are characterized with regard to Ig class and subclass, pI, and complement-activating capacity.