The extent and nature of heterogeneity among representative Trichomonas vaginalis isolates were evaluated by flow cytofluorometry analysis. Monoclonal antibody and monospecific antiserum to an immunodominant trichomonad surface glycoprotein with a molecular weight of 267,000 (267K glycoprotein) were used to evaluate fresh isolates (JHH and RU375) and long-term grown isolates (NYH286, IR78, and JH31A) of T. vaginalis. Isolates NYH286, JH31A, and JHH were made up of heterogeneous staining (positive [pos] phenotype) and nonstaining (negative [neg] phenotype) populations of trichomonads, whereas RU375 and IR78 285 Vol. 53,No. 2

To produce monoclonal antibodies (MAbs) to highly immunogenic membrane proteins of Trichomonas vaginalis NYH286, the sera of subcutaneously infected BALB/c mice were first monitored for antibody to trichomonad surface proteins. The sera possessed antibody to one major surface protein by 7 days and antibody to numerous other trichomonad membrane proteins by 4 weeks postinfection. A hybridoma was then generated that synthesized an MAb, designated C20A3, which reacted to a parasite-derived glycoprotein possessing a molecular weight of 267,000 (267K glycoprotein). The immunogen corresponded to the single high-molecularweight immunogenic surface protein recognized by 7-day mouse antisera. The MAb differentiated T. vaginalis isolates by a whole-cell enzyme-linked immunosorbent assay and by indirect immunofluorescence, using either fixed or live organisms. All isolates, however, possessed C20A3-reactive material when tested by enzyme-linked immunosorbent assay, using detergent extracts of the isolates incubated with MAb-coated microtiter well plates. The epitope was accessible to antibody binding on live T. vaginalis organisms expressing the major immunogen, and the 267K glycoprotein was readily removed from the parasite membranes by trypsinizing the intact trichomonads. The antigen incorporated radiolabeled glucose, mannose, and acetate. Also, an unlabeled 267K glycoprotein on nitrocellulose blots was detected by '25I-concanavalin A and 1251-wheat germ agglutinin, confirming the glycoprotein nature of the immunogen. Finally, of seven isolates used in this study, one possessed a cross-reactive 170K, rather than 267K, antigen. The data reinforce the idea that antigenic heterogeneity among T. vaginalis isolates may be a function of the presence or absence of high-molecular-weight glycoprotein immunogens from trichomonal membranes.

The antibody response in trichomoniasis patients was examined with a variety of methodologies including enzyme-linked immunosorbent assays, indirect immunofluorescence, immunoblotting, and radioimmunoprecipitation-electrophoresis-autoradiography. Based on enzyme-linked immunosorbent assay recognition of trichomonal isolates, sera from patients with trichomoniasis were categorized into reactive class I (IA, IB, and IC) and nonreactive class II sera. A diminished ability to precipitate antibody-binding trichomonad membrane proteins by the whole cell radioimmunoprecipitation assay was noted from class IA to class II sera.

Fresh isolates of Trichomonas vaginalis were examined for reactions to a panel of five monoclonal antibodies (MAbs). Four MAbs (C20A3, DM126, DM116, and C55) were to distinct surface immunogens and one MAb (L64) was to a cytoplasmic component. The fresh isolates were evaluated by indirect immunofluorescence (IF), immunoblotting, and radioimmunoprecipitation. IF assay with C20A3 MAb gave isolates which were homogeneous nonstaining (negative [Neg] phenotype) and isolates which were heterogeneous staining and nonstaining (positive [Pos] and Neg phenotype, respectively) organisms. Immunoblotting and radioimmunoprecipitation assays revealed that surface phenotypic heterogeneity among isolates with C20A3 MAb was due to the presence or absence of the immunogen from the parasite surface. IF assay with DM126 MAb also gave Pos and Neg phenotypes among parasites of some isolates. All of the isolates were always Neg phenotype with DM116 and C55 MAbs. The occurrence of Neg phenotype organisms with DM126, DM116, and C55 was due to epitope inaccessibility to their respective MAbs and not to the absence of the immunogen from trichomonal membranes. All isolates possessed the cytoplasmic protein recognized by L64 MAb. Paired isolates (taken 5 to 6 days apart) from 24 women were also studied. Four of the 24 paired isolates (16%) had different phenotype distributions at the two timepoints for C20A3. Fresh isolates also underwent phenotypic variation during in vitro growth and multiplication, as determined with C20A3. Also, 7 of the 24 paired isolates demonstrated dramatic changes in the accessibility of DM126 MAb to epitope binding. Lastly, 55 (90%) of 60 serum samples from patients with trichomoniasis evaluated in this study possessed antibody to the C20A3 reactive molecule. The data show that the fresh T. vaginalis isolates were predominantly Neg phenotype and confirm the occurrence of protein and epitope phenotypic variation for major immunogens among fresh isolates of the pathogenic human trichomonads.

An immunoglobulin G type 2a (IgG2a) monoclonal antibody (MAb), designated C20A3, which reacts with a highly immunogenic trichomonad membrane glycoprotein (approximately 270,000 daltons), produced complement-independent cytolysis of Trichomonas vaginalis organisms. Time- and temperature-dependent lysis of parasites was observed following incubation of washed, live T. vaginalis with certain concentrations of C20A3 IgG. Differential killing of trichomonal isolates and clones of a given isolate by C20A3 was dependent on the presence of the glycoprotein antigen on the parasite surface.