Identification of a candidate vaccine peptide on the 37 kDa Schistosoma mansoni GAPDH.

A previous study performed in adolescents living in an area endemic for Schistosoma mansoni in Brazil has shown that a 37 kDa schistosome surface antigen is a selective target for antibodies in sera from those who were resistant to reinfection. This antigen was shown by molecular cloning to be the schistosome GAPDH. The aim of the present work was to assess whether peptides corresponding to GAPDH antigenic determinants could be used in a subunit vaccine. Five B cell and two T cell epitopic regions were identified on Sm37-GAPDH. One of the B cell determinants (Sm37-5, aa 268-289) is highly antigenic in human infections and antibody reactivity toward this determinant is associated with resistance to reinfection. Mice and rats immunized with Sm37-5 were partially protected against a challenge infection, indicating that this peptide can induce protective immunity. Analysis of Sm37-5 amino acid sequence indicated that this antigenic determinant is likely conserved among other pathogenic strains of schistosome (S. haematobium, S. intercalatum and S. japonicum), although it shows major amino acid differences with the corresponding human GAPDH sequence. All together these results indicate that Sm37-5 should be considered as a candidate component for an anti-schistosome subunit vaccine.

Characterization of a schistosome T cell-stimulating antigen (Sm10) associated with protective immunity in humans.

Parasite antigens that are strong T cell immunogens represent potential candidates for vaccines against pathogens susceptible to T cell-mediated immunity. We have previously shown that chromatographic fractions of schistosomula extracts contain components that are major T cell immunogen(s) in natural schistosome infections in humans and might contribute to the induction of human protective immunity against this parasite. In the present study, we report on the molecular cloning and on the biochemical characterization of the active components of these fractions. The screening of a schistosomula cDNA expression library with antibodies raised against the fractions allowed the cloning of a cDNA that hybridized to a 0.56-kb mRNA of schistosomula and adult worms. This cDNA contains an open reading frame of 267 base pairs (bp) which encodes a 10-kDa polypeptide. The analysis of the cDNA sequence revealed 70% homology with the sequences of previously reported proteins of unknown function. The native molecules in the active fractions were analyzed by mass spectrometry after additional purification by reverse phase high-performance liquid chromatography (HPLC). This procedure revealed two components in the fractions of molecular mass 10383 +/- 2 Da and 10401 +/- 9 Da. Both polypeptides stimulated immune T cells and yielded tryptic peptides whose sequences matched the sequence of the cloned molecule. These two polypeptides probably correspond to different post-translationally modified forms of the polypeptide encoded by the cloned cDNA.

Identification of a major T cell immunogen in the anti-schistosome response of adult residents in an area endemic for Schistosoma mansoni.

Vaccine-induced immunity to Schistosoma mansoni infection depends on the specific priming of certain T cell subsets and on the recall of this response by natural infections months or years after vaccine administration. Thus, those schistosome proteins that activate T cells in individuals stimulated by natural infections are potential candidate vaccine antigens. In the present study, we identified and purified one such T cell-stimulating antigen and evaluated its immunological properties in subjects living in an area endemic for Schistosoma mansoni. Chromatography fractions (gel filtration, followed by ion exchange chromatography) of soluble extracts of schistosomula were screened for their ability to stimulate schistosome-specific T cell clones derived from a subject sensitized by natural infection. A fraction stimulating most clones was identified and characterized. A few nanograms of this fraction, containing a major 9-10-kDa component, stimulated the T helper cells of most adults living in an endemic area of Brazil, and was able to trigger a strong cutaneous immediate hypersensitivity reaction. In contrast, children reacted weakly to this antigen preparation both in blastogenesis and in skin tests, although they mounted a significant reaction to crude larval antigen preparations. In conclusion, this work identifies a schistosomula antigen that induces a strong T cell response in adults sensitized by natural infections. This T cell response develops gradually in children and adolescents, is apparently not restricted by the HLA haplotypes common in the study area, and allows the production of parasite-specific IgE antibodies. Thus, this T cell response has some features of the immune response that is believed to protect chronically exposed humans from reinfection.

Monoclonal antibodies against antigens expressed by human sperm.

The production and characterization of 21 mouse monoclonal antibodies (TüS1-TüS21) with specificity predominantly for human spermatozoa antigens is described. Reactivity of cells from human ejaculates, peripheral blood and several organs was determined using the alkaline phosphatase-anti-alkaline phosphatase (APAAP)-technique as well as the indirect immunofluorescence test. 15 of the monoclonal antibodies reacted with various regions of human sperm and often also with their precursor cells in the testis. Cross-reactivity with animal spermatozoa was frequently observed.

Immunoelectron microscopy of human spermatozoa.

A number of immunocytochemical methods using various markers for electron microscopy have been developed in recent years. The immunogold technique has been especially effective in histotopochemical studies. The value of this technique for demonstrating sperm antigens results from the high electron density of gold, which makes it easily detectable under the electron microscope. The high resolution of the electron microscope permits precise localization of immunologic reactions in the sperm cell. Light microscopy findings can thus be elucidated. We tested a number of monoclonal antibodies that react with sperm antigens. Of three techniques for preparing the spermatozoa, the pre-embedding method and marking of cryoultra-microtome sections proved best.