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Identification and characterization of an Onchocerca volvulus cDNA clone encoding a highly immunogenic calponin-like
M Irvine1, T Huima, A M Prince
1Lindsley F. Kimball Research Institute, New York Blood Center, NY 10021.
Abstract:
The identification and characterization of a recombinant cDNA clone, designated OV9M, expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Clone OV9M was identified by screening a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using pooled rabbit antisera raised against the third (L3) and fourth (L4) stage larvae of the parasite. The cDNA clone encodes an open reading frame of 238 amino acids corresponding to a 27-kDa polypeptide. This polypeptide contains a series of five highly conserved repeats of 25 amino acids that are similar to repeats found in calponin, a protein previously only identified in vertebrate smooth muscle. Extension of the 5' end of the cDNA clone revealed two additional repeats extending the sequence to 378 amino acids, encoding a 41.8-kDa protein. Affinity purified antibodies, which bound specifically to the glutathione S-transferase-OV9M fusion polypeptide, recognize a series of antigens in extracts of O.volvulus microfilariae, L3, L4 and adult stages. The apparent molecular weight of the native OV9M protein in the adult is 45 kDa. Similar proteins are present in extracts of other nematodes including Caenorhabditis elegans, and antibodies from other filarial infections are cross reactive with glutathione S-transferase-OV9M fusion polypeptide. Immunoelectron microscopy revealed that the antigen encoded by this clone is present in the longitudinal muscles of the various larval stages and adult worms. Antibodies to the OV9M protein are present in 40-60% of both patently infected and non-patent individuals residing in onchocerciasis endemic areas.
Insights
Researchers identified a new antigen, OV9M, from the parasite Onchocerca volvulus. This antigen is present across all parasitic stages and may be a target for diagnosing and treating onchocerciasis.
Area of Science:
- Parasitology
- Molecular Biology
- Immunology
Background:
- Onchocerca volvulus is the causative agent of onchocerciasis, a debilitating parasitic disease.
- Identifying parasite-specific antigens is crucial for developing diagnostic tools and therapeutic strategies.
Purpose of the Study:
- To identify and characterize a novel antigen expressed by Onchocerca volvulus.
- To investigate the potential of this antigen as a diagnostic marker for onchocerciasis.
Main Methods:
- Screening of a cDNA expression library from O. volvulus using specific antibodies.
- Characterization of the cDNA clone OV9M and its encoded polypeptide.
- Antibody production and validation using various parasitic stages and nematode extracts.
- Immunoelectron microscopy to determine antigen localization.
Main Results:
- A recombinant cDNA clone, OV9M, was identified, encoding a 27-kDa polypeptide with conserved repeats similar to calponin.
- The full-length clone encoded a 41.8-kDa protein; antibodies recognized antigens from microfilariae to adult stages.
- The native OV9M protein is approximately 45 kDa and found in longitudinal muscles.
- Cross-reactivity was observed with other nematodes and filarial infections.
- Antibodies to OV9M are present in a significant percentage of individuals in endemic areas.
Conclusions:
- The OV9M clone expresses a conserved antigen present throughout the Onchocerca volvulus lifecycle.
- This antigen shows potential as a diagnostic marker for onchocerciasis.
- Further research into OV9M could lead to improved diagnostic and therapeutic interventions for onchocerciasis.