A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense.

Infectivity of Trypanosoma brucei rhodesiense to humans is due to its resistance to a lytic factor present in human serum. In the ETat 1 strain this character was associated with antigenic variation, since expression of the ETat 1.10 variant surface glycoprotein was required to generate resistant (R) clones. In addition, in this strain transcription of a gene termed SRA was detected in R clones only. We show that the ETat 1.10 expression site is the one selectively transcribed in R variants. This expression site contains SRA as an expression site-associated gene (ESAG) and is characterized by the deletion of several ESAGs. Transfection of SRA into T.b. brucei was sufficient to confer resistance to human serum, identifying this gene as one of those responsible for T.b. rhodesiense adaptation to humans.

Diagnostic value of a synthetic peptide derived from Echinococcus granulosus recombinant protein.

A specific monoclonal antibody (MAb; EG 02 154/12) directed against a protein epitope of Echinococcus granulosus antigen 5 was used to screen a cDNA library constructed from E. granulosus protoscoleces RNA. One clone designated Eg14 was selected and shown to code for an amino acid sequence partially homologous to that of the clone Eg6 previously identified with the same MAb. Hydrophobic cluster analysis showed that both recombinant antigens may adopt a similar alpha-helical organization and share a common conformational epitope. A synthetic peptide (89-122) mimicking the conformational site of Eg6 and Eg14 was constructed and demonstrated to be able to inhibit binding of the MAb and human hydatid sera to the Eg6 fusion protein (FP6) or to native hydatid antigens. To assess the diagnostic value of the peptide 89-122, we tested sera from patients infected with different parasites for their antibody reactivity with this peptide in ELISA. A high binding sensitivity and specificity of IgG-A-M antibodies were obtained with E. granulosus-infected patient sera. Moreover, the peptide 89-122 was found to be specifically recognized by IgE antibodies from patients with hydatid disease. These results indicate the particular interest of this synthetic peptide as a standardized antigen in diagnosis and treatment surveillance of hydatidosis.

Molecular cloning of an Echinococcus granulosus protein expressing an immunogenic epitope of antigen 5.

cDNA was synthesized from RNA extracted from Echinococcus granulosus protoscoleces and cloned in the lambda gt11 expression vector. A pool of 5 E. granulosus patient sera was used to screen the library and allowed the selection of 13 clones. Ten of these were shown to be identical, among which clone 6 (Eg6) was chosen for further analysis. The nucleotide sequence (456-bp) presented an entire open reading frame coding for 152 amino acids. The fusion protein (FP6) was recognized by a mouse monoclonal antibody (EG 02 154/12) specific for E. granulosus antigen 5. Moreover, the presence of antibodies to FP6 seemed to be correlated to the ability of sera from hydatidosis patients to immunoprecipitate antigen 5. These results indicate that the cloned protein could be used as a standardized antigen for the diagnosis of hydatidosis.

Use of a monoclonal antibody specific for a protein epitope of Echinococcus granulosus antigen 5 in a competitive antibody radioimmunoassay for diagnosis of hydatid disease.

A monoclonal antibody (mAb) designated as EG 02 154/12, specific for the major antigen (antigen 5) of Echinococcus granulosus was produced, and used to study the binding sites recognized by anti-antigen 5 antibodies from patients with hydatid disease. The nature of the target epitope was partially characterized. The antibody reactivity was analyzed towards sheep hydatid fluid antigens (SHF Ag) using ELISA, immunoelectrophoresis (IEP), Western blotting (WB), and immunoprecipitation (IP). In IEP, EG 02 154/12 mAb gave a single precipitin of Ag 5. The mAb and human hydatid patient sera recognized a major antigen of 64 kDa, in SHF Ag analyzed in non-reducing conditions. Both types of antibodies revealed two components of 37 and 22 kDa in reducing conditions. Deglycosylation and delipidation of SHF Ag did not affect the mAb binding. These results, together with the observation of mAb binding to in vitro translation products from protoscoleces messenger RNA, suggest the protein nature of the epitope recognized on the antigen 5. Using competitive antibody radioimmunoassay (CRIA), a competition between this mAb and hydatid patient sera, for the same epitope or closely related sites on antigen 5, was observed. No such competition was detected with the sera from other helminthiasis. The sensitivity and specificity of CRIA was compared to that of ELISA and CRIA found to be an improved diagnostic test for hydatid disease.

A monoclonal antibody to Ly-6 gene product inhibits generation of functionally active T cells and recognizes single antigenic specificity whose expression is up-regulated in virus-transformed rat fibroblast.

In order to elucidate the relationship between the structure and function of proteins encoded for by the Ly-6 gene complex, a cDNA was constructed for a Ly-6.2 specificity and then monoclonal antibodies (mAb) generated to bacterially synthesized protein. The addition of one of these mAb, designated Pb-19, inhibited the proliferative response of T cells to concanavalin A (Con A) or major histocompatability complex (MHC) alloantigens. Reactivity of Pb-19 to the Ly-6 specificity was blocked by a known anti-Ly-6.A.2 mAb but not by an anti-Ly-6.E.1 mAb. This mAb detected a Ly-6.A.2 specificity (a 33,000 MW antigen) whose expression was increased in a transformed rat fibroblast containing the entire genome of bovine papillomavirus.