Characterization of Schistosoma mansoni dynein light chains: The potential for immunoprophylactic therapy

he majority of this dissertation involves the characterization of two developmentally regulated Schistosoma mansoni tegumental antigens which, by sequence similarity, can be classified as new members of the dynein light chain (DLC) protein family (Chapters two and three). Additionally, studies were designed to develop and optiminize schistosome naked DNA vaccines (Chapters four and five). Finally, the immunolocalization of a prominent schistosome vaccine candidate (Sm 23) to the membranous bodies within the tegument is described (Chapter six).;S. mansoni DLC (SmDLC) was identified utilizing a monoclonal antibody (mAb 709A2/2) that was raised in mice immunized with adult worm tegumental extracts. SmDLC was found to be developmentally regulated; the highest abundance was observed in cercaria with decreasing levels of expression detected in schistosomula and adults. Expression of SmDLC was specific for the tegument in these life-stages. A novel characteristic associated with SmDLC is its strong hydrophobicity, despite lacking a traditional membrane-spanning motif or lipid attachment consensus sequence. SmDLC also demonstrated a strong interaction with a 20.8-kDa polypeptide from adult worm tegumental extracts.;S. mansoni 20.8-kDa DLC (Sm 20.8) was found to be expressed in the same developmental stages and relative abundances as that observed for SmDLC. The cDNA (748 bp) encoding this polypeptide contained an open reading frame of 181 amino acids which displayed sequence similarity to an 18-kDa calcium binding DLC from C. reinhardtii as well as SmDLC. In addition, Sm 20.8 showed a high degree of sequence similarity to a group of schistosome antigens termed tegumental associated antigens (TAA). SmDLC and Sm 20.8 were found associated during velocity sedimentation in a fraction corresponding to 4.4 Svedberg units (S). Sm 20.8 was isolated from the aqueous fractions of adult worm extracts. The 20.8-kDa polypeptide displays ideal characteristics of a potential vaccine candidate, including (i) expression in the tegument, (ii) significant divergence from mammalian brain cytoplasmic dynein, and (iii) a conserved homolog in S. japonicum.;I also investigated the immunoprophylactic efficacy of IrV-5 (a 62-kDa Schistosoma mansoni antigen with sequence similarity to myosin II) as a naked DNA vaccine. Intramuscular immunization of mice with pcDNA1/IrV-5 led to a 61-62% reduction in worm burden, whereas no statistically significant reduction in worm burden occurred in mice immunized intramuscularly with VR1012/IrV-5. Mice vaccinated epidermally (gene gun) with VR1012/IrV-5-coated gold microspheres showed an intermediate level of protection that was statistically significant. In the animals immunized intramuscularly, there appeared to be a positive correlation between protection and generation of anti-IrV-5 specific IgG;Finally, immunoprophylactic studies of SmDLC were initiated. It was first determined that chronic schistosome infected mouse sera and sera from mice vaccinated two times with irradiated cercaria both recognize SmDLC. Secondly, in two passive transfer experiments, mAb 709A2/2 was capable of partially protecting naive mice against schistosome challenge. Lastly, mice actively immunized IP with recombinant SmDLC/Freund's incomplete adjuvant emulsions developed anti-SmDLC Abs of the...