An improved microassay for Plasmodium falciparum cytoadherence using stable transformants of Chinese hamster ovary cells expressing CD36 or intercellular adhesion molecule-1.

Stable transformants of Chinese hamster ovary (CHO) cell lines expressing high levels of human CD36 or intercellular adhesion molecule-1 (ICAM-1) have been produced as target cells for cytoadherence of Plasmodium falciparum-infected erythrocytes. An improved adherence microassay has been designed using small sample volumes and allowing convenient and reliable measurements on a large number of samples. The assay can be used both with purified proteins spotted on plastic and with the stably transformed CHO cell lines. The same assay plate can be evaluated either microscopically or by scintillation counting after use of 3H-hypoxanthine-labeled parasites. Using the microassay, functional expression of the transfected receptor molecules on CHO-CD36 and CHO-ICAM was confirmed using parasites with different cytoadherence phenotypes and cytoadherence inhibition experiments with a panel of anti-CD36 antibodies. The use of isolates from The Gambia confirmed the applicability of these assays for laboratory studies of these isolates.

Agglutination of Plasmodium falciparum-infected erythrocytes from east and west African isolates by human sera from distant geographic regions.

Plasmodium falciparum-infected erythrocytes (PfE) were collected from acutely infected children in The Gambia and Tanzania and cultured for more than 30 hr until the parasites were mature trophozoites. Sera collected from these countries, other African countries, Asia, and South America were used in the PfE microagglutination test to determine whether PfE from East and West Africa share surface antigens. From the patterns of agglutination reactivity, we identified extensive antigenic diversity in surface antigens, but obtained no evidence for greater differences between isolates from East or West Africa and those within one region. The majority of sera from immune adults from The Gambia, Tanzania, Sudan, Nigeria, or Ghana were pan-agglutinating, and agglutinated all PfE isolates from The Gambia and Tanzania. Some sera from immune adults of Irian Jaya also agglutinated each of the seven African isolates, while others agglutinated many but not all of the isolates, similar to sera from immune adults of Flores, Indonesia. In contrast, sera from nonimmune adults from Colombia agglutinated few of the African isolates. It was remarkable, however, that sera from nonimmune Colombians agglutinated any African isolates. Our results are consistent with the following conclusions: some PfE surface antigen(s) are very diverse; this diversity is a feature of the parasite worldwide; the repertoire of isolate-specific surface antigens, although large, includes antigens that are either identical or antigenically cross-reactive in geographically very distant parasite populations; and African adults have pan-agglutinating antibodies that may contribute to protective immunity. Such pan-agglutinating antibodies could reflect the accumulation of a large repertoire of isolate-specific antibodies. The contribution of antibody against any shared PfE surface antigen to the pan-agglutinating reactivities is unknown and awaits development of the appropriate reagents.

Surface molecules on Plasmodium falciparum-infected erythrocytes involved in adherence.

The identity of cell surface receptor molecules on Plasmodium falciparum-infected erythrocytes is of great interest since the functional sites involved in attachment to endothelial cells may be structurally conserved in wild isolates. Such conserved sites may represent suitable antigenic targets for a vaccine-induced immune response that would block or reverse infected cell sequestration in vivo. Identification of the infected cell receptor sites may also lead to novel methods for treatment of acute cerebral malaria. We review the likely roles, either direct or indirect, for the participation of knob protrusions, malarial proteins expressed at the cell surface, and modified host membrane proteins in the specific receptor properties acquired by infected erythrocytes.

Genes for Plasmodium falciparum surface antigens cloned by expression in COS cells.

Two genes encoding membrane antigens of Plasmodium falciparum were isolated by transient expression in mammalian cells and selection with human immune sera from African adults exposed to P. falciparum malaria. COS-7 cells were transfected with a plasmid expression library constructed from P. falciparum genomic DNA, and cells expressing reactive malaria antigens on their surface were enriched by adherence to antibody-coated dishes. One of the genes isolated is distinctive in that it does not contain repeat sequences typical of many malarial genes cloned by immunoscreening of bacterial expression libraries. The second gene apparently encodes a polymorphic version of the P. falciparum merozoite surface antigen Ag513, since the two sequences are identical in the 5' and 3' coding regions but diverge completely in the center. The COS-7 expression system provides an alternate means for cloning genes encoding malarial membrane antigens by using those antibodies in complex immune sera that bind membrane-associated, nondenatured molecules.

Detection of specific protein binding to the SV40 early promoter in vivo.

The interactions in vivo between cellular proteins and the Simian Virus (SV40) early promoter region, contained in a plasmid capable of replicating in Cos cells, have been characterized by DNaseI and dimethyl sulfate (DMS) footprinting. The relative contribution of each GC-motif within the 21 bp repeat upstream element to transcription was first determined after transfection of Cos cells with either the wild type 21 bp repeats or recombinants where the GC-motifs were mutated either individually or in neighboring pairs. Mutation of GC-motifs III and VI was the most detrimental, mutation of GC-I, -IV and -V also decreased promoter activity but to a lesser extent, while mutation of GC-II had little effect on transcription. All six GC-motifs of the wild type 21 bp repeats were found protected from DNaseI nuclease attack in vivo though to varying degrees. Motifs GC-III, -V and -VI were more strongly protected than GC-I, -II and -IV. In vivo DNaseI footprinting of recombinants bearing mutations in the GC-motifs demonstrated the specificity of factor interaction and further indicated that, in agreement with the previously published in vitro results, the binding of factor(s) to each of the GC-motifs was independent. DMS protection experiments identified specific guanine (G) contacts characteristic of Sp1 binding to the GC-motifs and this in vivo pattern was compared to that obtained in vitro using a crude nuclear extract. These results indicate that the transcription factor Sp1 interacts in vivo with the GC-motifs of the SV40 early promoter.