[PMC free article] [PubMed] [Google Scholar] 20. demonstrated by immunofluorescence microscopy, flow cytometry, and immunoblotting. Furthermore, the antiparasitic effect of RAM1 has been tested in vitro in an antibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 and the IgG3 versions of the antibody show an inhibitory effect on parasite growth. Clinical immunity to malaria is gradually acquired over a dozen years of intense exposure to the parasite (12). Acquired immunity to malaria has been termed premunition and is characterized as being nonsterile and incomplete (43). The exact mechanism responsible for premunition is not known with certainty. However, a number of clinical studies carried out in the early sixties (9, 16, 23)and subsequently confirmed and extended in the nineties (2, 33)showed an unambiguous antiparasitic effect of antibodies transferred from adults with immunity to malaria to malaria-infected infants. Clinical effects observed in one of these studies correlated with the effect measured in the in vitro assay termed antibody-dependent cellular inhibition (ADCI) (2, 4). In the ADCI assay, immune antibody cooperates with monocytes in an in GPR120 modulator 2 vitro malaria culture, and the antiparasitic effect is demonstrated by parasite growth inhibition. It has been shown that the antibody-merozoite complex by a contact-dependent mechanism stimulates the monocyte to secrete substances toxic to the asexual blood stages. The specific substances responsible for the subsequent, non-contact-dependent parasite growth inhibition include tumor necrosis factor alpha together with other molecules that are yet to be identified (4). The ADCI assay has been used for identification and characterization of the merozoite surface protein 3 (MSP-3) (27). An invariable structural feature of all reported MSP-3 sequences is the presence of three regions GPR120 modulator 2 each of which contains three, four, or five conserved heptad repeat units. Previously published structural analyses suggest that the heptad repeat regions have an amphipathic alpha-helical secondary structure. A coiled-coil bundle conformation including these regions is a theoretical possibility supported by experimental data (24). The C-terminal part of MSP-3 contains a leucine zipper-like domain possibly implicated in dimerization and the formation of tetramers in vivo (5). MSP-3 contains a 96-amino-acid predicted globular region of high amino acid complexity. The region comprises amino acid residues 166 to 261 numbered according to the D10 sequence (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”L07944″,”term_id”:”507790″,”term_text”:”L07944″L07944) positioned C-terminal to the second putative -helix (18). Naturally occurring antibodies affinity purified on the C-terminal part of this globular region (MSP-3 amino acid residues 194 to 257 [MSP-3194-257]) have been shown to exert a strong inhibition in ADCI assays (27). A recent phase I vaccine trial using a long synthetic peptide spanning this region of MSP-3 has shown promising results in terms of raising both humoral and cellular responses (1). Functional studies of the long synthetic peptide-induced responses GPR120 modulator 2 have shown encouraging results in vitro in ADCI assays and in vivo in an immunocompromised BXN mouse model (13). Furthermore, an exceptional degree GPR120 modulator 2 of conservation in this region makes it a prominent vaccine candidate (39). Additionally, this region shows complete homology with a sequence of 11 NEDD4L amino acids (MSP-3220-230) from the MSP-6 antigen (MSP-6182-192), except for a valine-to-alanine substitution at position 229 of the MSP-3 sequence (49) as well as high homology with two additional antigens. The latter antigens and their resemblance to MSP-3 and MSP-6 have been described and designated H101 and H103 recently (31). It has been shown that naturally occurring antibodies affinity purified on MSP-6-derived peptides cross-react with MSP-3-derived homologous peptides and exert an ADCI effect in vitro, thereby confirming the biological relevance of these homologies (40). Recombinant antibodies would be excellent tools to elucidate the role of isotype and fine specificity of anti-MSP-3 antibodies. All previous work has relied on either polyclonal bulk antibody or affinity-purified antiparasitic antibody. In contrast to the polyclonal antibody preparations used so far, specificity and isotype of recombinant antibodies can be controlled at will by use of DNA techniques. To clarify the role of anti-MSP-3 antibodies, cross-reactivity with MSP-6, and functional properties in naturally occurring malaria immunity, we decided to clone antibodies directed against the target antigen fragment MSP-3194-257 (27). The present paper describes the isolation and characterization of three distinct antibodies directed to MSP-3194-257 as well as functional in vitro studies in the ADCI assay of one of these antibodies. The amino acid residues of the antigen fragments used in this study are numbered.