Antigenic Heterogeneity in the 115,000 Mr Major Surface Antigen of Trichomonas vaginalis1

ABSTRACT The 115,000-molecular-weight antigen of Trichomonas vaginalis was characterized using monoclonal antibodies developed to three different strains of T. vaginalis and one strain of Tritrichomonas foetus. The antigen was found to be present on all strains or isolates of T. vaginalis examined and was demonstrated to be located on the external surface plasma membrane by agglutination assays and complement-mediated lysis assays. Characteristics of the antigen were assessed with a proteolytic enzyme and periodate oxidation. Periodate treatment of whole T. vaginalis abrogated binding for eight antibodies while use of pronase-treated antigen resulted in loss of antibody binding for two different antibodies. Screening of 19 axenized clinical isolates of T. vaginalis and one strain each of T. foetus and Giardia lamblia with type-specific antibodies delineated three major groups of T. vaginalis based on antigenic specificities (epitope distributions) within the 115,000-molecular-weight antigen. In addition, one epitope of the 115,000-molecular-weight antigen was found only on the immunizing strain. Two epitopes were present on all T. vaginalis isolates as well as T. foetus and G. lamblia. One epitope was common to all T. vaginalis except one. A minimum of six different epitopes of the 115,000-molecular-weight antigen were identified. Antigens purified with type-specific or “common” monoclonal antibodies shared the same partial peptide maps demonstrating relatedness.     

Clonal and geographic distribution of a surface antigen of Tritrichomonas foetus

Geographically diverse strains and clones of Tritrichomonas foetus have been examined with respect to their expression of a major surface antigen of approximately 150,000 relative molecular weight (Mr), designated the 150 Ag. Radioiodination and 35S-methionine labeling of T. foetus followed by immunoprecipitation with monoclonal antibodies (MAbs), separation of polypeptides by SDS-PAGE, and autoradiography or fluorography confirmed the parasite origin of the 150 Ag. The results of flow cytometry analysis employing a panel of MAbs against live T. foetus parasites revealed that from 5 to 84% of individuals in a given population of T. foetus expressed a particular epitope of the 150 Ag. All strains and clones were positive for surface expression of epitopes of this antigen. These results show that the 150 Ag is widely distributed in populations of T. foetus, confirm the surface location of this antigen, and suggest its importance as a target for protective immune responses.     

Monoclonal antibodies to gonococcal outer membrane protein IB: use in investigation of the potential protective effect of antibodies directed against conserved and type-specific epitopes

Several monoclonal antibodies directed against gonococcal outer membrane protein IB have been used in in vitro assays to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, virulence of the variant P9-17 for epithelial cells in tissue culture was reduced in the presence of three of the four antibodies which recognized type-specific epitopes. Similarly, virulence of P9-17 as well as a recent isolate was reduced in the presence of the one antibody, SM24, which reacted with a conserved epitope. This antibody was also bactericidal in the presence of complement, and in addition was opsonic for several protein IB-expressing strains as determined by polymorphonuclear leucocyte chemiluminescence measurements. Similarly, all the type-specific antibodies were opsonic for P9 variants. However, only two of these antibodies mediated complement-dependent killing although those which were ineffective were nevertheless complement-fixing antibodies. These results indicate that antibodies to closely positioned epitopes on protein I vary in their biological activities and that the conserved epitope recognized by the antibody SM24 is potentially an effective target on the gonococcal surface for immunoprophylaxis.     

Reactivity of Monoclonal Antibodies to Species-Specific Antigens of Entamoeba histolytica

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica -like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica -like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia , and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis     

Detection of microtubules of the flagellate Trichomonas vaginalis by monoclonal antibodies specific for beta-tubulin

Monoclonal antibodies specific for mammalian beta-tubulin recognized the microtubule cytoskeleton of the flagellated protozoon Trichomonas vaginalis. Of seven antibodies, two demonstrated the axostyle, costa, recurrent flagellum, and anterior flagella by indirect immunofluorescence microscopy. The remaining five stained a hazy reticular pattern in the cytoplasm of formaldehydefixed, detergent-extracted organisms. Western immunoblots of whole T. vaginalis extracts treated with protease inhibitors and electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate showed a major band at molecular weight 50,000 when probed with only one of the antibodies which stained the axial cytoskeleton. The antibodies which stained only the cytoplasm showed a different western blot pattern with a major doublet band at MW 58,000-60,000. Another antibody, which stained both the axial cytoskeleton and the reticular cytoplasmic pattern showed major bands at MW 58,000-60,000 and also at MW 40,000-42,000. The recognition of microtubule populations in T. vaginalis by these monoclonal antibodies was different than we found earlier with Leishmania donovani and Toxoplasma gondii, where all seven antibodies recognize cytoskeletal microtubules and produce western blots characteristic of tubulin. Only one of these seven antibodies recognizes tubulin in T. vaginalis by immunoblot. The microtubules of T. vaginalis do not demonstrate all epitopes recognized by monoclonal antibodies specific for mammalian beta-tubulin; one of the antibodies appears to recognize an epitope which is morphologically associated with microtubules but does not have the characteristic MW of tubulin.     

Monoclonal antibodies against avian reticuloendotheliosis virus: identification of strain-specific and strain-common epitopes

We report the generation and partial characterization of a panel of 11 monoclonal antibodies (MCA) made against the nondefective strain T reticuloendotheliosis virus (REV). In an enzyme-linked immunosorbent assay (ELISA), nine MCA cross-reacted with both homologous strain T and heterologous strain chick syncytial virus (CS), whereas two MCA were strain specific and reacted with strain T but not with CS. Competitive antibody inhibitory ELISA tests demonstrated that the nine MCA recognized at least two distinct type-common epitopes. By using MCA-mediated immunoprecipitation and polyacrylamide gel electrophoresis analysis, we identified a 62,000 dalton glycoprotein that may contain both type-common epitopes and a 21,000 dalton glycoprotein that contains one of these epitopes. The competitive antibody inhibitory ELISA tests confirmed that the remaining two MCA recognize a strain T-specific epitope. We identified a 54,000 to 72,000 dalton glycoprotein that contains the type-specific epitope. All of the MCA reacted in an ELISA assay with cell-free virus preparations, suggesting that the polypeptides we identified are virion envelope glycoproteins. To identify the nonglycosylated precursor proteins, we treated infected cells with tunicamycin. We found a 48,000 polypeptide that was the nonglycosylated precursor to the 54,000 to 72,000 glycoprotein, and 48,000 and 20,000 dalton proteins that were the nonglycosylated precursors to the 62,000 and 21,000 dalton glycoproteins, respectively. These MCA may be of value in the field. They were able to distinguish in preliminary tests in ELISA between strains T and CS, which were otherwise undistinguishable in assays that made use of conventional polyvalent serum.     

Conservation of diagnostic antigen epitopes among biologically diverse isolates of Trichinella spiralis

Crude and immunoaffinity-purified excretory-secretory antigens derived from a domestic pig isolate of Trichinella spiralis were used in an enzyme-linked immunosorbent assay to test serum from mice infected with 25 different pig and wild animal isolates of T. spiralis sspp. All of the sera were found positive by ELISA using either of the antigen preparations, indicating all isolates shared certain antigen epitopes. Excretory-secretory antigens were prepared from 3 distinct isolates of T. spiralis sspp.--Trichinella spiralis spiralis (pig isolate), Trichinella spiralis nativa (polar bear isolate), and Trichinella spiralis pseudospiralis--and compared by electrophoresis and monoclonal antibody binding. While protein profiles varied among the isolates, a monoclonal antibody recognizing a major immunodiagnostic antigen epitope bound all 3 antigen preparations. However, this antigen epitope occurred on different molecular weight excretory-secretory proteins from the different isolates.     

Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica.

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa.     

Variable expression and geographic distribution of Mycoplasma agalactiae surface epitopes demonstrated with monoclonal antibodies

Abstract Species-speciGc monoclonal antibodies (MAbs) were developed against Mycoplasma agalactiae reference strain PG2 and French isolate P89 to study the in vitro expression of surface epitopes and to probe the antigenic profiles of 245 field isolates originating from 10 different countries. Colony immunostaining with MAbs on clonal lineage showed that 4 out of 9 species-specific epitopes exhibited a high rate of variation, demonstrating that M. agalactiae possesses a capacity for phenotypic diversification of its surface antigenicity. The emphasis was on dot immunobinding screening of the field isolates with MAbs recognizing permanently expressed epitopes. Eight different profiles could be defined. Great differences in epitope conservation were demonstrated with some area-specific strains completely lacking certain specific determinants. These results indicate that the antigenic variability of M. agalactiae relies not only upon surface switching mechanisms but also upon true epitope differences, partially related to the geographic origin of the isolates.     

A linear double-stranded RNA in Trichomonas vaginalis

A "double-stranded" RNA was identified in the anaerobic, parasitic protozoan Trichomonas vaginalis. Electron microscopic evidence indicated linear double-stranded structure 1.5 micron in length, with no apparent hairpins or loops. Boiling in 30% dimethyl sulfoxide denatured it into single strands of 1.5 micron and shorter fragments. It consists of 23.4% G, 23.4% C, 23.0% A, and 30.3% U and melts at a transition temperature of 81.7 degrees C in 75 mM NaCl and 7.5 mM sodium citrate, pH 7.0, with 7-15% hyperchromicity. The 32P-labeled double-stranded RNA hybridized specifically with T. vaginalis DNA fragments in a single DNA band from EcoRI digest and two DNA bands from HindIII digest. Of the 33 different strains or isolates of T. vaginalis examined, all contained this double-stranded RNA. However, the only two metronidazole-resistant T. vaginalis strains examined thus far (IR78 and 85) contained no detectable double-stranded RNA, although the corresponding DNA sequence was present. DNA fragments of Escherichia coli and Giardia lamblia did not hybridize with the double-stranded RNA. But DNA fragments of a metronidazole-sensitive Tritrichomonas foetus hybridized specifically with the double-stranded RNA, even though this organism does not contain the double-stranded RNA itself.     

Analysis of antigenic profiles of inactivated poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method.

A new block-ELISA test for quantitative evaluation of relative reactivity of antigenic sites was developed and used to reveal the detailed epitope structure of inactivated poliovirus vaccines (IPV) and live poliovirus strains. Poliovirus was captured on ELISA plates coated with rabbit anti-poliovirus IgG and blocked by monoclonal antibodies (Mabs) specific to individual epitopes before the remaining reactive antigenic sites were quantified by polyclonal anti-poliovirus IgG conjugate. The decrease of conjugate binding by the pre-treatment with a Mab reflects its contribution to the overall reactivity of poliovirus antigen. The level of block activity of Mabs for a given antigen can be expressed as a percent of reduction of antigenic reactivity as determined by ELISA test. It can be normalized by expressing this value as a ratio to the block activity of a reference sample. The data on the blocking-activity of a panel of monoclonal antibodies specific to different antigenic sites represents the epitope composition (antigenic profile) of a sample. Quantitative differences in epitope composition were determined for nine samples of inactivated poliovirus vaccine (IPV) and compared with the International Reference Reagent. This method could be used for monitoring consistency of IPV production, comparison of vaccines made by different manufacturers, and for the analysis of antigenically modified strains of attenuated poliovirus. Antigenic structures of two isolates of type 1 vaccine-derived poliovirus (VDPV) were compared with the structures of parental Sabin 1 and wild-type Mahoney strains using 17 monoclonal antibodies and revealed significant differences, suggesting that the method can be used for screening of field isolates and rapid identification of antigenically divergent VDPV strains.     

Monoclonal antibodies to gonococcal outer membrane protein I: location of a conserved epitope on protein IB

Hybrid cell lines have been derived which produce monoclonal antibodies reacting with outer membrane protein I from Neisseria gonorrhoeae strain P9. The antibodies obtained showed variable reactivity with other strains but one antibody recognized an epitope present on all of the strains tested which expressed the protease sensitive protein IB. Purified IgG labelled with 125I was used in competitive radioimmunoassays with unlabelled antibody to investigate the spacial distribution of the epitopes recognized. Each pair of antibodies showed some degree of inhibition. The relative magnitude of inhibition suggested that the conserved epitope lies within a variable region containing other epitopes which determine the antigenic specificity of the protein. Western blotting of peptides derived by proteolytic digestion of protein IB revealed that the conserved epitope is located close to the chymotrypsin cleavage site within a 7000 Mr surface exposed region of the molecule.     

A simple micropore system for experimental studies on trichomonad parasites

A simple leak-free micropore chamber containing protozoan parasite species was implanted subcutaneously on the back of hamsters and evaluated for viability and multiplication of protozoan parasites. Trophozoites of defined strains of Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and Tritrichomonas foetus were used; their survival and multiplication in the chambers formed the basis of evaluation. Entamoeba histolytica and G. lamblia did not survive more than 6 hr and succumbed due to cellular adhesion. Trichomonas vaginalis and T. foetus survived 3 and 6 days and multiplied a maximum of 3.6 and 26 times, respectively. This indicated that exchange of body fluids and cells needed for the survival and multiplication of trichomonads readily occurs. This preliminary observation showed that micropore chambers may be useful for chemotherapeutic and immunological studies on trichomonads in ectopic sites.     

Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.     

Comparative study of epitopes recognized by two monoclonal antibodies that protects mice against Trichomonas vaginalis challenge

Trichomonas vaginalis is a flagellated protozoan which infects the urogenital tract of humans. Previous studies have demonstrated that monoclonal antibodies (MAbs) against a 62 kDa proteinase (4D8 and 1A8) decreased cytoadherence of the parasite to epithelial cells in vitro and passive inoculation of mice with two MAbs 24 h before the intraperitoneal challenge resulted in different grade of protections to T. vaginalis infection. In the present paper we describe the characterization of the epitopes recognized by MAbs 4D8 and 1A8. The epitopes were characterized by heat treatment, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, enzymes proteolysis and periodate oxidation. The results showed that the two MAbs 4D8 and 1A8 each react with a different protein epitope of repetitive nature found on the same excretory-secretory molecules of T. vaginalis and it could explain the variation in the protection grade obtained in the challenge experiments.     

Monoclonal antibody analysis of p53 expression in normal and transformed cells.

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.     

Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. We found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of the three epitopes are located in nonoverlapping domains on the matrix protein molecule.     

Monoclonal antibody characterization of the 195-kilodalton major surface glycoprotein of Plasmodium falciparum malaria schizonts and merozoites: identification of additional processed products and a serotype-restricted repetitive epitope

The gp195 from Camp strain parasites was characterized with eight monoclonal antibodies (MAb) that recognize different epitopes on gp195 and three of its merozoite-associated processed products. Four MAb (3H7, 3B10, 7F1, and 4G12) reacted with different epitopes on the 45-kDa glycosylated product (gp45), shown by differences in their reactivities with soluble and immunoblotted gp45. One MAb (7H10) reacted with a conformational epitope probably formed as a result of the interaction of gp45 with a nonglycosylated 45-kDa product (p45). Three other MAb (3D3, 7B11, and 7B2) reacted with different epitopes on a nonglycosylated 83-kDa product (p83), shown by differences in their reactivities against various parasite isolates in immunofluorescent antibody assays. Immunoprecipitation of antigens that were pulse-labeled with [3H]isoleucine and chased with cold isoleucine showed that p45 and gp45 were processed products of gp195 and p83 was sequentially processed into smaller fragments of 73 and 67 kDa (p73 and p67). Immunoblots showed that the 7B11 and 7B2 epitopes were present on p83, p73, and p67, but that the 3D3 epitope was present only on p83 and p73. A two-site immunoassay showed the 3D3 epitope to be repetitive. The 3D3 and 7B11 epitopes were serotype restricted (present in seven and 24 of 33 isolates, respectively), but the other five epitopes were common to all isolates tested. The gp195 and its processed products have Mr that are consistent with the Mr of a number of antigens shown previously to be associated with the immune complexes that are formed when merozoites are agglutinated by antibodies contained in some growth inhibitory immune sera.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

用标记分析法对我国海南地区登革2型病毒株的抗原表位分析

用标记分析(signature analvsis)法了解我国海南地区3年(1985—1987)来流行的登革2型(DEN-2)病毒株的抗原性变化。用3种单克隆抗体(McAb),包括黄病毒属特异、亚属特异和登革2型型特异,分析了8个DEN-2病毒流行株,并与标准株新几内亚B林进行比较．通过统计分析和微机处理,发现8株中有5株与株准株类似,有3株显示出明显差异。株记分析法为病毒抗原性分析提供了一个高度敏感的方法,并可用于监测一个地区病毒株群的变化及新株的引人。