Lise Sibilli

Plasmodium falciparum: Molecular analysis of a putative protective antigen, the thermostable 96-kDa protein

A group of three Plasmodium falciparum antigens of distinct pI, migrating with an apparent MW of 96 kDa has been previously identified as a target of protective immunity both in humans and in monkeys (Jouin et al. 1987, Dubois et al. 1987). These antigens are produced during the late stages of asexual intraerythrocytic development. One of these 96-kDa proteins, the 96 tR, has a pI of 5.25, is thermostable, and is released in the culture supernatant (Jouin et al. 1987). We report here the cloning and expression in Escherichia coli of the gene coding for this antigen. Antibodies raised to the recombinant 96 tR immunoprecipitated exclusively the 96 tR, indicating that the other two antigens of 96 kDa are the product(s) of distinct gene(s). Northern and Southern blots as well as DNA sequencing of the gene showed that the 96 tR antigen is identical to proteins identified in other laboratories as the glycophorin binding protein GBP 130 (Perkins 1984, Ravetch et al. 1985) and Ag 78 (Bianco et al. 1987). The 96-kDa antigen is produced at the trophozoite stage and more actively in the schizonts. It is released in the culture supernatant at the time of schizont rupture, together with two minor products, forming a characteristic triplet. This triplet was also detected in immunoblots of merozoites. An approximate quantification on immunoblots indicated that the largest proportion of the protein is found in the culture supernatant, a minor fraction being loosely associated with merozoites. By immunofluorescence and immunoelectron microscopy, intense signals were observed in the erythrocyte cytoplasm. The 50-amino acid repeats were found in all strains examined, the protein showing some size polymorphism. The antigen was detected in the serum of infected monkeys as well as in that of infected humans.

The polymorphic 11.1 locus of Plasmodium falciparum

Clone pPF11.1 encodes a Plasmodium falciparum antigen expressed during the intraerythrocytic cycle and containing tandem repeats of a 9 amino acid unit. We report here an analysis of the genomic region specific for 11.1, which extends over 30 kb. It contains two blocks of repeats, spanning 13 kb and 9 kb. The restriction map suggests that the locus may result from a gene duplication. The 11.1 region is present in all P. falciparum strains examined so far. Southern analysis of 8 distinct isolates indicates that the locus is highly polymorphic. Thus the pPF11.1 repeats constitute a sensitive and discriminating probe to type P. falciparum strains.

Cloning of cDNAs that code for Plasmodium chabaudi antigens of the erythrocytic forms and cross-hybridize with P. falciparum

Several Plasmodium chabaudi antigens are synthesized and the corresponding mRNAs are detected by cell-free translation only during specific stages of the erythrocytic cycle. Ring stage mRNA was used to construct a cDNA library in the plasmid vector pBR 322. The library was screened by differential hybridization with cDNA specific for ring stage parasites or schizonts. Two clones which showed some stage specificity and seven which did not were retained. Three clones were characterized by hybrid-selected translation. Clones 451, 148 and 443 contain sequences homologous to the messages for a 27 kDa stage-specific antigen, a 37 kDa early antigen and a 24 kDa antigen, respectively. The nine clones cross-hybridize to various degrees with cDNA from P. falciparum but cross hybridization intensities do not reflect the strength of immunological cross-reactivity.