Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

Zona pellucida glycoproteins based immunocontraceptive vaccines: strategies for development and their applications.

The mammalian oocyte is surrounded by an extra-cellular matrix, the zona pellucida (ZP), composed of three major glycoproteins (ZP1, ZP2 and ZP3). The ZP glycoproteins, by virtue of their tissue specificity and critical role during mammalian fertilization, have emerged as potential candidate antigens for the development of an immunocontraceptive vaccine. Molecular characterization of ZP glycoproteins from several species, reveals a variable degree of homology among the deduced primary amino acid sequences, which provided an opportunity to undertake active immunization studies in heterologous animal models. Active immunization of various animal species with either native ZP glycoproteins or those obtained by recombinant DNA technology led to the inhibition of fertility. Thus ZP glycoproteins based immunocontraceptive vaccines offer an attractive proposition for controlling wild life population. To make it a practical proposition, additional research inputs are required to optimize and devise novel strategies for vaccine delivery. Observed ovarian dysfunction, often associated with immunization by ZP glycoproteins is one of the major stumbling blocks for their use in humans. Ongoing studies to delineate appropriate B cell epitopes of ZP glycoproteins that are devoid of oophoritogenic T-cell epitopes, which will inhibit fertility without concomitant oophoritis, will be critical to determine their feasibility for human use.

Active immunization of rats with chicken egg thiamin carrier protein results in early embryonic loss at periimplantation stages.

Active immunization with chicken egg white thiamin carrier protein (TCP) was performed to assess the functional importance of the vitamin carrier during gestation in rats. Towards this, fertile female rats were immunized with heterologous TCP and when the circulatory titres of anti-TCP IgG were high, these animals were mated with fertile male rats. Progression of pregnancy was monitored by measuring circulatory progesterone levels. A sudden fall in the steroid hormonal levels around day 8 post coitus was observed. Histological examination of the uterine tissue sections on day 7 revealed that active immunization with TCP affected the blastocyst viability resulting in unsuccessful implantation and hence pregnancy termination.

Establishment of the functional importance of thiamin carrier protein in pregnant rats by using monoclonal antibodies.

Monoclonal antibodies (mAbs) to chicken thiamin carrier protein (TCP) have been produced by hybridoma technology to identify the crucial epitopes involved in bioneutralization of the vitamin carrier. The monoclonality of these mAbs (A4C4, F3H6, H8H3, C8C1 and G7H10) was sought to be confirmed by sub-class isotyping; they all belong to IgG1, k type. The epitopes recognized by all the five mAbs are conserved in TCP from the chicken to the rat as assessed by liquid phase RIA and immunoprecipitation of 125I-labelled proteins from pregnant rat serum. Among these mAbs. passive immunization of pregnant rats with the mAb C8C1 only on three consecutive days (day 10, 11 and 12) resulted in embryonic resorption. These results demonstrate the importance of epitopic structure specified by the mAb C8C1 on TCP during pregnancy in rats.

Characterization of antigenic determinants on porcine zona pellucida-3 beta(ZP3 beta) glycoprotein by monoclonal antibodies.

Out of 11 monoclonal antibodies(MAbs) developed against porcine zona pellucida-3 beta(ZP3 beta) glycoprotein, 6 (MA-451, -454, -455, -462, -467 and -470) reacted with ZP2 beta, deglycosylated ZP3 beta(DGZP3 beta), and reduced and carboxyamidomethylated ZP3 beta(RCMZP3 beta) in ELISA and Western blot suggesting that these monoclonals recognise a linear protein epitope. Five MAbs(MA-452, -456, -459, -474 and -476) showed reduced binding with DGZP3 beta and RCMZP3 beta as compared to ZP3 beta in ELISA suggesting that these may recognize either carbohydrate moities or conformation dependent epitopes. Moreover, these MAbs failed to recognize these antigens on Western blot. Based on competitive inhibition studies in ELISA for the ability of MAbs to inhibit the binding of biotinylated ZP3 beta to solid phase antibody, 6 distinct domains were discernible. Domain V recognised by MA-467 partially overlaps with the domain corresponding to MA-454 and MA-455. All 11 MAbs reacted with zona pellucida of intact oocytes as revealed by indirect immunofluorescence but only 3 antibodies (MA-454, -455 and -467) delayed the zona lysis by trypsin. In immunoblots MA-467 recognised 18kDa fragment of RCMZP3 beta digested with endoproteinase lys-C and 37 and 30kDa bands of elastase digest of ZP3 beta. The results will help in delineation of functionally relevant domains and design of contraceptive vaccine based on zona pellucida aiming for pre-fertilization block.