The development of transfection technology for malaria parasites holds significant promise for a more detailed characterization of molecules targeted by vaccines or drugs. One asexual blood stage vaccine candidate, apical membrane antigen-1 (AMA-1) of merozoite rhoptries has been shown to be the target of inhibitory, protective antibodies in both in vitro and in vivo studies. We have investigated heterologous (trans-species) expression of the human malaria Plasmodium falciparum AMA-1 (PF83/AMA-1) in the rodent parasite Plasmodium berghei. Transfected P. berghei expressed correctly folded and processed PF83/AMA-1 under control of both pb66/ama-1 and dhfr-ts promoters. Timing of expression was highly promoter-dependent and was critical for subsequent subcellular localization. Under control of pb66/ama-1, PF83/AMA-1 expression and localization in P. berghei was limited to the rhoptries of mature schizonts, similar to that observed for PF83/ AMA-1 in P. falciparum. In contrast the dhfr-ts promoter permitted PF83/AMA-1 expression throughout schizogony as well as in gametocytes and gametes. Localization was aberrant and included direct expression at the merozoite and gamete surface. Processing from the fulllength 83-kDa protein to a 66-kDa protein was observed not only in schizonts but also in gametocytes, indicating that processing could be mediated outside of rhoptries by a common protease. Trans-species expressed PF83/ AMA-1 was highly immunogenic in mice, resulting in a response against a functionally critical domain of the molecule.The protozoan parasite Plasmodium falciparum is a causative agent of malaria, one of the major human infectious diseases. In the search for new methods to combat the disease, the advent of transfection technology for Plasmodium species is critical, because it offers the opportunity to relate genotype to phenotype, and this will permit a more rational design of vaccines and drugs. To date, stable episomal maintenance of plasmid DNA introduced into Plasmodium has been reported (1-3) as well as site-directed integration of DNA into the parasite genome (4 -8). This technology also offers the possibility to dissect the thus far poorly characterized Plasmodium promoter function (9 -11) and study the relation between the tightly controlled timing of expression and the subcellular trafficking and localization of stage-specific proteins. Trans-species expression of malarial antigens will allow targeted development of attenuated parasite vaccines and opens possibilities for complementation of otherwise detrimental integration into essential genes. Apical membrane antigen-1 (AMA-1)1 is an attractive candidate for such studies, because it appears to be intimately involved in red cell invasion (12). Expression and post-translational N-terminal proteolytic cleavage of AMA-1 are restricted to the final stages of schizogony (13), during which the protein is localized within the neck of the rhoptry, an apical secretory organelle of the merozoite involved in red cell invasion (14). AMA-1 is a major cand...

Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression of Plasmodium falciparum FVO strain AMA-1 (PfAMA-1) in Pichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites in Plasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologous P. falciparum by immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologous P. falciparum.Accumulated data, including those from nonhuman primate (2, 5) and rodent (1, 3, 16) studies, have indicated that the apical membrane antigen 1 (AMA-1) family of molecules are targets for antibody-mediated protective immune responses. In all Plasmodium species reported to date, with the exception of Plasmodium falciparum (19) and P. reichenowi (13) (two parasites that form a phylogenetic clade distinct from other malaria parasites), AMA-1 is synthesized de novo as a 66-kDa transmembrane protein. The protein contains a predicted Nterminal signal sequence, an ectodomain, a predicted transmembrane region, and a C-terminal cytoplasmic domain. The ectodomain is further divided into three domains defined by disulfide bonds (10). In P. falciparum and P. reichenowi the protein is expressed as an 83-kDa protein, having an N-terminal extension compared to the 66-kDa forms that has been referred to as the prosequence (10). AMA-1 is processed by proteolytic cleavage between the different domains (11). Intraspecies sequence polymorphism due to point mutations (13,15,18,23) reveals clustering of mutations in particular domains of the molecule. Despite this, between species there is considerable conservation of primary and predicted secondary amino acid structures. Evidence to date indicates that protection invoked by AMA-1 is directed at epitopes dependent on the disulfide bonding (1-3, 6, 9, 16) located in the AMA-1 ectodomain. Immunization with reduced AMA-1 fails to induce parasite-inhibitory antibodies (1, 6, 9), and so far only those monoclonal antibodies (MAbs) that recognize reduction-sensitive AMA-1 epitopes have been shown elsewhere to inhibit parasite multiplication in vitro for P. knowlesi (4, 21) and P. falciparum (13,14). This indicates that for an AMA-1 vaccine the correct conformation will be critical.Recombinant expression of P. falciparum AMA-1 (PfAMA...

SUMMARYTwo mouse monoclonal antibodies, designated DB-1 and DB-2, were isolated and used for the purification and characterization of recombinant rat interferon gamma (rRIF-~,) derived from Chinese hamster ovary (CHO) cells. The two antibodies belong to different classes (DB-1 is an IgGl and DB-2 an IgA) and display similar epitope specificities as shown in competition binding experiments. Both antibodies, raised against rRIF-% exhibited high affinity for rat and mouse gamma interferon and efficiently neutralized the antiviral activity of both animal interferon species. Affinity chromatography analysis showed that a column with immobilized DB-1 was capable of complete binding of rat and mouse gamma interferon, both natural and recombinant DNA-derived. As visualized by SDS-polyacrylamide gel electrophoresis and Western blot analysis, the purified rRIF-~ preparation consisted of at least seven molecular forms with M~ values ranging between 14 000 and 25 000, with a relative abundance of a 18 000 Mr protein. Gel permeation chromatography of crude rRIF-~ gave coincident peaks of rRIF-~ proteins (all different forms) and interferon activity corresponding to a M~ value of 45000. The results suggest that the molecular heterogeneity was due to differential glycosylation and was not the consequence of a proteolytic degradation process.

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