Tropomyosin implicated in host protective responses to microfilariae in onchocerciasis

A cDNA from adult female Onchocerca volvulus encoding the C-terminal portion of a tropomyosin isoform (termed MOv-14) has been shown previously to confer protective immunity in rodent models of onchocerciasis. The full-length sequence (designated Ov-tmy-1) obtained by PCR amplification, codes for a protein of 33 kDa and shares 91% identity with tropomyosins from other nematodes, falling to 57% identity with human α-tropomyosin. Ov-TMY-1 migrates with an apparent molecular mass of 42 kDa on SDS/PAGE and is present in all life-cycle stages, as determined by immunoblotting. Immunogold electron microscopy identified antigenic sites within muscle blocks and the cuticle of microfilariae and infective larvae. Anti-MOv14 antibodies were abundant in mice exhibiting serum-transferable protection against microfilariae conferred by vaccination with a PBS-soluble parasite extract. In contrast, little or no MOv14-specific antibody was present in mice inoculated with live microfilariae, in which resistance is mediated by antibody-independent mechanisms. In human infections, there was an inverse correlation between anti-tropomyosin IgG levels and densities of microfilariae in the skin. Seropositivity varied with the relative endemicity of infection. An immunodominant B cell epitope within Ov-TMY-1 (AQLLAEEADRKYD) was mapped to the N terminus of the MOv14 protein by using sera from protectively vaccinated mice. Intriguingly, the sequence coincides with an IgE-binding epitope within shrimp tropomyosin, believed to be responsible for hypersensitivity in individuals exhibiting allergy to shellfish. IgG and IgE antibodies reacting with the O. volvulus epitope were detected in human infections. It is concluded that antibody responses to tropomyosin may be important in limiting microfilarial densities in a proportion of individuals with onchocerciasis and have the potential to mediate hypersensitivity reactions to dead microfilariae, raising the possibility of a link with the immunopathology of infection.

Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors.

A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta-galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans.