Antibodies and DNA probes used to analyze variant populations of the Indochina-1 strain of Plasmodium falciparum

Abstract

Ten variant populations derived from the Indochina-l strain of Plasmodium fakciparum were analyzed by using (i) hyperimmune serum raised against some of these populations in squirrel monkeys and (ii) an oligonucleotide probe based on the rep-20 sequence, which had previously been shown to be a useful marker of diversity. Although all 10 subpopulations had an identical fingerprint pattern on Southern blots probed with the oligonucleotide, thus demonstrating a homogeneous genetic makeup, they all had a different phenotype for erythrocyte-associated antigens, thus confirming serological variant-specific differences. Antibodies to erythrocyte-associated antigens were measured with a new technique including immunogold and silver enhancement. The results of this study indicate that antigenic variation can occur without major genomic reorganization.The presence of new antigens on the surface of malariainfected erythrocytes is a well-established phenomenon (reviewed in references 14 and 15). In Plasmodiumfalciparum, these antigens are present only during the second half of the 48-h intraerythrocytic cycle, are trypsin sensitive, and are believed to be associated with the parasite molecule Pf-EMP1 (17). There is a considerable diversity of erythrocyteassociated antigens from one P. falciparum isolate to another (10), and the expression of these antigens can be modulated in a given parasite population either by immune pressure or transfer from intact to splenectomized animals (13). This modulation of antigen expression has been termed antigenic variation, by analogy with the phenomenon described in African trypanosomes (21) and in Plasmodium knowlesi (4). The genes encoding for the erythrocyte-associated antigens of P. falciparum have not been identified, and consequently it is not known whether antigenic variation is related to a change at genomic level (e.g., point mutation or deletion) or to a differential phenotypic expression.In this study, we compared 10 variant populations derived from the Indochina-1 strain of P. falciparum by using a combination of two different markers (antibodies and DNA probes). Variant-specific antibodies raised in hyperimmune squirrel monkeys were used to demonstrate diversity of erythrocyte-associated antigens, and DNA fingerprinting was used to examine the overall genetic background of the 10 populations studied. The rep-20 sequence, which had previously been shown to be of value in the identification of different P. falciparum isolates (16), was used for DNA fingerprinting after Southern blotting. The objective of the study was to establish whether the serological differences observed between variant populations were accompanied by detectable differences in gene organization.MATERIALS AND METHODS Parasites. The squirrel monkey-adapted strain of P. falciparum, Indochina-1, was used in this study. Variant populations of the original strain (INDO-A) were obtained by * Corresponding author. isolating recrudescence populations that emerged either spontaneously or after challenge infections in i...

Immune pressure selects for Plasmodium falciparum parasites presenting distinct red blood cell surface antigens and inducing strain-specific protection in Saimiri sciureus monkeys.

“…This was confirmed by DNA fingerprinting using the highly repetitive probe pPFrep20 (34) ( Fig. 3 B), previously shown to be highly sensitive in detecting genomic diversity (15,(30)(31)(32), as well as using the telomer-spedfic repeats (35) (Fig. 3 C) and Pf60.1, a repetitive probe recently isolated in our laboratory (15a) ( Fig.…”

“…The Palo Alto FUP/SP O parasites used in the experiments described here were not clonal. Genotyping using a panel of probes well-documented for their ability to detect polymorphism (15,(30)(31)(32)(33)(34), indicated that resistant and original parasites were isogenic for all genetic markers analyzed, except for a few differences in the restriction profiles of some subtelomeric and telomeric sequences. These regions are prone to frequent recombinations and rearrangements, mainly documented during in vitro cultivation (reviewed in reference 46), the significance of which is still unclear.…”

“…Strain-speciflc Protection in Plasmodium fakiparum Malaria Molecular Typing of Parasites. In P. fakiparum parasites, the strain-specific genetic background is readily visualized using repetitive probes (15,(30)(31)(32). Fig.…”

Antigenic variation during malaria infection—the contribution from the murine parasite Plasmodium chabaudi

“…We have, therefore, examined the in vivo switching rates of RC4, 7 & 10 during exponential growth of the parent population during the ascending primary parasitaemia in naive mice. An immunogold-silver staining technique (Hommel et al 1991) combined with the variant specific hyperimmune sera were used. The minimal estimates calculated for overall switching rates was 1.2-1.6% and, most interestingly, the rates varied depending on the VAT being switched on (Brannan, Turner & Phillips 1994).…”

Intravenous immunoglobulin in the treatment of paediatric cerebral malaria

“…The immunogiobulin preparation was used in an immunogold/ silver enhancement technique, developed and routinely used in our laboratory [19], to assay the binding of antibody to erythrocyte-associatcd malaria surface antigen in 22 isolates obtained from children with severe malaria in Malawi, Antibody which remained bound to the surface of infected erythrocytes was then eluted, and its specificity tested by immunogold/ silver enhancement against homologous and heterologous isolates.…”