Each represents the sequential loss of a single histidine (minus 137.5 Da per histidine) from the C-terminal six-histidine affinity tag. both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage. Deployment of a viable malaria vaccine is regarded as the most cost-effective and practical method of reducing the high human and economic toll of this devastating disease. Raising the immunocompetence of Tafenoquine those individuals most Tafenoquine at risk for severe disease by vaccination could significantly lower the number of deaths due to clinically severe malaria. Two major requirements for producing a successful malaria vaccine are the ability to cheaply manufacture large amounts of high-quality antigen and a rapid, inexpensive way of analyzing the bioactivity of candidate antigens or combinations of antigens. In this paper we present data addressing both of these issues for apical membrane antigen 1 (AMA1). Antigenic polymorphism is an important mechanism by which malaria parasites evade host immune responses (17). Vaccine strategies involving a single target antigen may have their effectiveness limited by antigenic polymorphisms, which allow divergent parasites to circumvent a vaccine’s protective properties. Pursuing a strategy involving multiple allelic variants of a single antigen is one way to overcome this mechanism of immune evasion. Studies using gene substitution suggest that AMA1 is a critical component necessary for successful invasion of red blood cells (RBCs) by merozoites (24). Vaccination with AMA1 has been shown to elicit antibody responses that give good protection against homologous parasite challenges in a number of rodent and primate models (1, 3, 6-8, 14, 27). Additional support for the importance of AMA1-specific antibodies was provided by adoptive-transfer experiments where monoclonal Tafenoquine antibodies or purified hyperimmune rabbit immunoglobin protected mice against or challenge (3, 7). However, the protection provided in all these models was strain or species specific. This is probably also true of infections, for while AMA1 is a relatively conserved molecule, 64 single-amino-acid substitutions have been found to date in AMA1 sequenced from field isolates and laboratory strains (12). Analysis of the frequency and distribution of these substitutions has yielded evidence that this genetic diversity is maintained by selective pressures of the host immune response (16). Indirect evidence supporting this hypothesis has come from previous investigations, which have shown little effective cross-strain protection from immunization with TNFRSF10D a single allelic form of AMA1, even though the immunogen produced high levels of growth inhibition against homologous parasites (11). Here we have produced two divergent allelic forms of AMA1, one based on the sequence of the Vietnam Oak Knoll (FVO) parasite Tafenoquine clone, the other based on the sequence of the 3D7 clone. Using these, we show that the amino acid substitutions in the AMA1 genes of these two clones of with a codon used with the same frequency for that amino acid by expression plasmid pPIC9K (Invitrogen Corporation, Carlsbad, Calif.). The pPIC9K plasmid encodes a preprosecretory -factor sequence. The resulting recombinant proteins, after removal of the signal peptides by the yeast enzyme KEX2, have the sequences YVQNYWEHPYQKSDVYHPIN…TYDNMKTSHHHHHH (FVO) and YVQNYWEHPYQNSDVYRPIN…TYDKMKTSHHHHHH (3D7), where underlined sequences are AMA1 derived and nonunderlined sequences are vector derived. Gene expression is under the control of the alcohol oxidase I (and genes and metabolize methanol at the wild-type rate. The pPIC9K plasmid has a functional gene, so transformants are then selected.
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