In theSaimiri sciureusmonkey, erythrocytes infected with the varO antigenic variant of thePlasmodium falciparumPalo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express avargene having the characteristics of group Avargenes, and we show that the varO Duffy binding-like 1α1(DBL1α1) domain is implicated in the rosetting of bothS. sciureusand human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1α1recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the samevarOgene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1α1-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1α1domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology.

Why severe Plasmodium falciparum malaria occurs in only a small percentage of patients is unclear. The possibility that specific parasite characteristics contribute to severity has been investigated in French Guiana, a hypoendemic area, where parasite diversity is low and all patients with severe cases are referred to a single intensive care unit. Parasite genotyping in geographically and temporally matched patients with mild and severe disease showed that the association of a specific msp-1 allele (B-K1) with a specific var gene (var-D) was overrepresented among patients with severe versus mild disease (47% vs. 3%, respectively; P<.001). Moreover, this genotype combination was consistently observed in the most severe clinical cases. Reverse-transcription polymerase chain reaction demonstrated programmed expression of var-D in vivo, which is consistent with its potential implication in severe disease. These results provide field evidence of an association of severe malaria with specific genetic characteristics of parasites and open the way for intervention strategies targeting key virulence factors of parasites.

SummaryDuring erythrocyte invasion, the Plasmodium falciparum Ring-infected erythrocyte surface antigen (RESA) establishes specific interactions with spectrin. Based on analysis of strains with a large chromosome 1 deletion, RESA has been assigned several functions, none of which is firmly established. Analysis of parasites with a disrupted resa1 gene and isogenic parental or resa3 -disrupted controls confirmed the critical role of RESA in the surface reactivity of immune adult sera on glutaraldehyde-fixed ring stages. Absence of RESA did not influence merozoite invasion or erythrocyte membrane rigidity, was associated with a modest increase of cytoadhesion to CD36 under conditions of flow, but resulted in marked susceptibility to heat shock. resa1 -KO-infected erythrocytes were prone to heat-induced vesiculation like uninfected erythrocytes, whereas parental or resa3 -KO infected erythrocytes remained undamaged. Furthermore, a 6 h exposure of ring stages at 41 ∞ ∞ ∞ ∞ C resulted in 33% culture inhibition of resa1 -KO parasites while marginally impacting parental and resa3 -KO parasite growth. This points to a role for RESA in protecting the infected erythrocyte cytoskeleton during febrile episodes. Infection patterns of resa1 -KO and parental parasites in Saimiri sciureus indicated that RESA does not, at least on its own, modulate virulence in the squirrel monkey, as had been previously suggested.

SummaryThe passive transfer of specific antibodies to a naive splenectomized Saimiri sciureus monkey infected with the Palo Alto FUP/SP strain of Plasmodiumfakiparum resulted in the emergence of parasites resistant to the transferred antibodies. Molecular typing indicated that the original and resistant parasites were isogenic. Saimiri monkeys primed with original parasites were fully susceptible to a challenge by the resistant ones, and vice versa. This absence of crossprotection indicates that strain-specific determinants would be the major targets of protective immunity developed in these monkeys. Phenotypic analysis showed that the surface of the infected red blood cells differed in both lines. Original parasites formed rosettes, autoagglutinated, presented characteristic knobs at the surface of the infected red blood cell, and did not agglutinate in the presence of a pool of human immune sera. In contrast, the resistant parasites did not form rosettes, did not spontaneously autoagglutinate, presented abnormal flattened knobs, and formed large aggregates in the presence of a pool of human immune sera. The presence of strain-specific determinants at the surface of the resistant parasites was confirmed by surface immunofluorescence and agglutination using homologous Saimiri serum. Neither the original nor the resistant parasites cytoadhered to an amelanotic melanoma cell line, suggesting that cytoadherence and agglutination can be dissociated. These results indicate that parasites that differ by the antigens exposed at the surface of the red blood cell induce strain-specific immunity. Furthermore they show that rosetting and nonrosetting parasites differ in their antigenic properties and do not crossprotect.

Abstract. The recombinant R23, PfEB200, and GST-5 antigens derive from conserved antigens associated with the Plasmodium falciparum-infected erythrocyte membrane. They were identified as targets of protective antibodies in the Saimiri sciureus model. We have assessed here the humoral response to these antigens in humans. Crosssectional surveys were conducted in two Senegalese villages with different levels of endemicity. The prevalence of specific IgG and IgM was similar and influenced by age in both localities. The anti-R23 antibodies decreased after the rainy season, particularly in the children less than ten years old. The anti-PfEB200 response did not show significant seasonal variation. The anti-GST-5 response increased in both the less-than 10-year-old and the greaterthan 10-year-old groups after the rainy season in Dielmo, but only in the Ndiop villagers who were more than 10-years-old. Thus, antigen-specific seasonal variations of antibody levels were influenced differently by age in both villages. The isotype distribution was antigen-specific and differed for both seasons.

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