Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1.

Plasmodium vivax is one of the most widely distributed human malaria parasites and due to drug-resistant strains, its incidence and prevalence has increased, thus an effective vaccine against the parasites is urgently needed. One of the major constraints in developing P. vivax vaccine is the lack of suitable in vivo models for testing the protective efficacy of the vaccine. P. vivax and P. cynomolgi bastianelli are the two closely related malaria parasites and share a similar clinical course of infection in their respective hosts. The merozoite surface protein-1 (MSP-1) of these parasites has found to be protective in a wide range of host-parasite systems. P. vivax MSP-1 is synthesized as 200 kDa polypeptide and processed just prior to merozoite release from the erythrocytes into smaller fragments. The C- terminal 42 kDa cleavage product of MSP-1 (MSP-1(42)) is present on the surface of merozoites and a major candidate for blood stage malaria vaccine. In the present study, we have biochemically and immunologically characterized the soluble and refolded 42 kDa fragment of MSP-1 of P. vivax (PvMSP-1(42)) and P. cynomolgi B (PcMSP-1(42)). SDS-PAGE analysis showed that both soluble and refolded E. coli expressed P. vivax and P. cynomolgi B MSP-1(42) proteins were homogenous in nature. The soluble and refolded MSP-1(42) antigens of both parasites showed high reactivity with protective monkey sera and conformation-specific monoclonal antibodies against P. cynomolgi B and P. vivax MSP-1(42) antigens. Immunization of BALB/c mice with these antigens resulted in the production of high titres of cross-reactive antibodies primarily against the conformational epitopes of MSP-1(42) protein. The immune sera from rhesus monkeys. immunized with soluble and refolded MSP-1(42) antigens of both parasites also showed high titered cross-reactive antibodies against MSP-1(42) conformational epitopes. These results suggested that the soluble and refolded forms of E. coli expressed P. vivax MSP-1(42) antigens were highly immunogenic and thus a viable candidate for vaccine studies.

Partial purification and characterization of acetylcholinesterase isozymes from adult bovine filarial parasite Setaria cervi.

Filariasis is a major health problem, affecting millions of people in tropical and sub-tropical regions of the world. The isolation and characterization of parasite-specific enzyme targets is essential for developing effective control measures against filariasis. Acetylcholinesterase (AchE, E.C. 3.1.1.7), an important enzyme of neuromuscular transmission is found in a number of helminths including filarial parasites and may be playing a role in host-parasite interactions. Earlier, we demonstrated the presence of two isozymes of AchE, different from the host enzyme in the human (Brugia malayi) and bovine (Setaria cervi) filarial parasites. In the present study, two isozymes of AchE (pAchE1 and pAchE2) were isolated from S. cervi adults and characterized biochemically and immunochemically. The AchE was partially purified on Con-A Sepharose column and then subjected to preparative polyacrylamide gel electrophoresis (PAGE) for separation of the isozymes. The AchE activity was localized by the staining of gel and the isozymes were isolated from the PAGE strips by electroelution. Both isozymes preferentially utilized acetylcholine iodide as substrate and were strongly inhibited by the true AchE inhibitor (BW284c51), suggesting that they were true AchE. The polyclonal antibodies produced against the isozymes showed significant cross-reactivity with B. malayi AchE, but not against the host enzyme. These findings suggested that both the isozymes were biochemically (in terms of their substrate specificity and inhibitor sensitivity) and immunochemically similar, but different from the host enzyme.