A principal target of human immunity to malaria identified by molecular population genetic and immunological analyses.

New strategies are required to identify the most important targets of protective immunity in complex eukaryotic pathogens. Natural selection maintains allelic variation in some antigens of the malaria parasite Plasmodium falciparum. Analysis of allele frequency distributions could identify the loci under most intense selection. The merozoite surface protein 1 (Msp1) is the most-abundant surface component on the erythrocyte-invading stage of P. falciparum. Immunization with whole Msp1 has protected monkeys completely against homologous and partially against non-homologous parasite strains. The single-copy msp1 gene, of about 5 kilobases, has highly divergent alleles with stable frequencies in endemic populations. To identify the region of msp1 under strongest selection to maintain alleles within populations, we studied multiple intragenic sequence loci in populations in different regions of Africa and Southeast Asia. On both continents, the locus with the lowest inter-population variance in allele frequencies was block 2, indicating selection in this part of the gene. To test the hypothesis of immune selection, we undertook a large prospective longitudinal cohort study. This demonstrated that serum IgG antibodies against each of the two most frequent allelic types of block 2 of the protein were strongly associated with protection from P. falciparum malaria.

Polymorphism of a high molecular weight schizont antigen of the human malaria parasite Plasmodium falciparum.

Intraspecies antigenic diversity in the blood stages of the human malaria parasite Plasmodium falciparum was investigated using a collection of murine monoclonal antibodies and clones of the parasite. The results were as follows: (a) The schizont and merozoite stages of the parasite express on their surface clonally restricted antigens detectable by strain-specific antibodies in indirect immunofluorescence tests. (b) These restricted antigens are phenotypically stable characteristics of clones grown in vitro. (c) The molecules carrying the specific antigens were isolated by immunoprecipitation and were found to be parasite proteins ranging in size from Mr 190,000 to 200,000 between clones. (d) Comparative immunoprecipitation and peptide mapping of these molecules showed that each parasite clone expresses a protein that is antigenically and structurally distinct from the equivalent products of several other clones. (e) The different clonal products are, however, immunologically interrelated, since they possess determinants in common with all tested isolates of the parasite. (f) These polymorphic molecules are closely related to a previously described schizont protein of P. falciparum that is posttranslationally cleaved into fragments located on the merozoite surface. These findings show the existence of a family of related polymorphic schizont antigens (PSA) of P. falciparum, whose expression is clonally restricted, and indicate that these proteins have regions of constant and variable antigenicity. We propose that a system of immunological classification of the parasite can be developed based on the polymorphism of these proteins.

A single fragment of a malaria merozoite surface protein remains on the parasite during red cell invasion and is the target of invasion-inhibiting antibodies.

A complex of polypeptides derived from a precursor is present on the surface of the malaria merozoite. During erythrocyte invasion only a small fragment from this complex is retained on the parasite surface and carried into the newly infected red cell. Antibodies to this fragment will interrupt invasion.

Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies.

Merozoite surface protein-1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti-MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies. Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

Allelic forms of gp195, a major blood-stage antigen of Plasmodium falciparum, are expressed in liver stages.

Mature exoerythrocytic (EE) forms of two cloned lines (3D7 and HB3) of Plasmodium falciparum were obtained in the livers of splenectomized chimpanzees. Sectioned preparations were examined by immunofluorescence (IFA) using mAbs that distinguished allelic variants of the blood-form antigen gp195 and mAbs that recognized multiple conserved epitopes of gp195. EE forms and blood schizonts exhibited identical IFA reactions for each respective clone, showing that the antigen was expressed identically in liver and blood-stage parasites. A third chimpanzee was infected with sporozoites derived from a mixture of 3D7 and HB3 gametocytes that had undergone cross-fertilization in the mosquitoes. IFAs on the EE forms in this animal showed that segregation of each gp195 allele had occurred earlier in the life cycle, providing evidence that the parasite is haploid for the whole of its mammalian development.

Antigenic diversity in the human malaria parasite Plasmodium falciparum.

Monoclonal antibodies against blood forms of Plasmodium falciparum were used to demonstrate considerable antigenic diversity in this species. Different isolates were distinguished by their ability to react with certain antibodies, and most of the antibodies reacted specifically with merozoites, schizonts, or both. The distribution of different antigenic types appeared not to be related to geographic origin. Serological typing with monoclonal antibodies extends the range of methods for identification of different strains of this malaria parasite.

Structural and antigenic polymorphism of the 35- to 48-kilodalton merozoite surface antigen (MSA-2) of the malaria parasite Plasmodium falciparum.

Merozoite surface antigen MSA-2 of the human parasite Plasmodium falciparum is being considered for the development of a malaria vaccine. The antigen is polymorphic, and specific monoclonal antibodies differentiate five serological variants of MSA-2 among 25 parasite isolates. The variants are grouped into two major serogroups, A and B. Genes encoding two different variants from serogroup A have been sequenced, and their DNA together with deduced amino acid sequences were compared with sequences encoded by other alleles. The comparison shows that the serological classification reflects differences in DNA sequences and deduced primary structure of MSA-2 variants and serogroups. Thus, the overall homologies of DNA and amino acid sequences are over 95% among variants in the same serogroup. In contrast, similarities between the group A variants and a group B variant are only 70 and 64% for DNA and amino acid sequences, respectively. We propose that the MSA-2 protein is encoded by two highly divergent groups of alleles, with limited additional polymorphism displayed within each group.

Fragments of the polymorphic Mr 185,000 glycoprotein from the surface of isolated Plasmodium falciparum merozoites form an antigenic complex.

Merozoites of the human malaria parasite Plasmodium falciparum express on their surface several antigens derived from a polymorphic glycoprotein precursor of Mr 185,000 synthesised earlier on by trophozoites and schizonts. A panel of 18 monoclonal antibodies against a range of different specificities of the precursor was used to characterise its mature products in spontaneously released merozoites. Merozoites released by [35S]methionine or [14C]glucosamine-labelled schizonts, or surface 125I-labelled purified merozoites, were extracted in detergents, and the antigens were detected by immunoprecipitation or Western blotting. We show that a nonglycosylated peptide of Mr 80,000 and two glycosylated fragments of Mr 40,000 and Mr 16,000, all derived from the precursor, are exposed on the surface of the mature merozoite. Precipitations from extracts in different detergents indicate that the 80 and 40 kDa fragments can form a non-covalent complex with each other and two additional major surface antigens of 36 and 22 kDa. Several antibodies react strongly with the complex but not with its dissociated subunits, thus indicating presence of conformational epitopes. Other epitopes are positively mapped on different dissociated subunits by immunoprecipitation and Western blotting. The 80 and 40 kDa antigens each carry a different polymorphic marker epitope, and both of these markers are absent on the 16 kDa fragment. The 40 and 16 kDa glycoproteins share common epitopes, and the latter may be derived from the former fragments. Only epitopes present on the 16 kDa antigen, but not those specific for the larger fragments, are detectable by immunofluorescence in the ring-stage. This indicates that the whole or a part of the 16 kDa antigen remains on the parasite through the invasion process.

Antigenicity of recombinant proteins derived from Plasmodium falciparum merozoite surface protein 1.

We have expressed seven recombinant antigens representing two N-terminal regions of the polymorphic merozoite surface protein 1 (MSP-1) of Plasmodium falciparum. The antigens include the MAD20 and Palo Alto forms of the relatively conserved Block 1 region, and variants of the Block 2 region from isolates 3D7, Palo Alto FUP, MAD20, Wellcome and RO33, that are representative of a range or amino acid sequence diversity in this most polymorphic section of MSP-1. All recombinant antigens have been able to immunise mice to produce polyclonal antibodies which specifically recognise parasite MSP-1 in indirect immunofluorescence assays and in Western blots. The recombinant antigens also react appropriately in ELISA with murine monoclonal antibodies specific for variant epitopes in Block 2 of MSP-1. These results show that the antigenic structure of the recombinant proteins is similar to that of the native MSP-1 product from parasites. Importantly, human sera from malaria-exposed individuals contain IgG antibodies that recognise very specifically one or another of the Block 2 types, showing that different Block 2 types are immunogenic, antigenically distinct and distinguishable when presented during natural infections. In contrast, the conserved Block 1 is rarely recognised by human antibodies.

Inhibitory monoclonal antibodies recognise epitopes adjacent to a proteolytic cleavage site on the RAP-1 protein of Plasmodium falciparum.

The low-molecular-weight rhoptry-associated protein (RAP) complex of Plasmodium falciparum consists of at least two gene products, RAP-1 and RAP-2, and has the ability to immunise Saimiri monkeys against experimental P. falciparum infection. Several monoclonal antibodies specifically recognise this complex and in this study we show that purified immunoglobulin derived from these monoclonals is capable of inhibiting parasite growth in vitro. It has previously been shown that RAP-1 initially appears as an 80-kDa protein (p80) in early schizogony and is processed to a 65-kDa protein (p65) in late schizogony. Several of the inhibitory monoclonals recognise both the 80- and 65-kDa proteins by Western blot analysis suggesting that they recognise linear epitopes on RAP-1. We have mapped these epitopes by testing the reactivity of the monoclonals against fragments of the rap-1 gene expressed as beta-galactosidase fusion proteins and subsequently against synthetic peptides. All of the epitopes map to a region 10-20 amino acids C-terminal to the proteolytic cleavage site for the processing of p80 to p65 at amino acid 190. We also show that the 65-kDa protein is not present in purified merozoites, suggesting that its generation is associated with merozoite release rather than erythrocyte invasion. These results are discussed with respect to possible inhibitory mechanisms for the monoclonals.

Polymorphism of a 35-48 kDa Plasmodium falciparum merozoite surface antigen.

Glycoproteins of the asexual blood stages of Plasmodium falciparum were labelled with radioactive glucosamine and analysed by two-dimensional electrophoresis. Four major glycoproteins were detected in all eight parasite isolates studied. Two of the glycoproteins, designated GP2 and GP4, were invariant among the isolates, while the other two GP1 and GP3 were found to be polymorphic in both their biochemical and antigenic properties. By immunoblotting and immunoprecipitation with specific monoclonal antibodies, the two polymorphic glycoproteins were identified as surface antigens of merozoites.

46-53 kilodalton glycoprotein from the surface of Plasmodium falciparum merozoites.

A 46-53 kDa glycoprotein antigen of Plasmodium falciparum merozoites has been identified using a murine monoclonal antibody that inhibits infection of human erythrocytes in vitro. Immunofluorescence screening with the antibody of greater than 250 isolates of the parasite finds the inhibitory epitope expressed by only 18% of strains. The glycoprotein is metabolically labelled with methionine, cysteine, histidine and glucosamine but incorporates little lysine or leucine. It is synthesized early in schizogony and remains, without any apparent processing, on the surface of released merozoites where it is demonstrated by immuno-electronmicroscopy and also by vectorial radio-iodination.

A possible molecular basis for strain specific immunity to malaria.

A 250 kDa antigen implicated in the induction of protective immunity to Plasmodium chabaudi was examined with a panel of 11 monoclonal antibodies in cloned parasite lines. 2 antibodies cross-reacted with the different parasite lines while 9 were specific for one line. This antigenic diversity was correlated with major differences in one dimensional peptide maps between the purified antigen from different lines of parasites. The peptide maps also revealed some apparently conserved structure which may have been responsible for the antigenic cross reactivity. Using cloned lines of P. falciparum and a second series of monoclonal antibodies, similar antigenic and structural diversity was evident in the equivalent antigen from the important human pathogen. These findings are discussed with relationship to the induction of protective immunity to malaria.

Antigenic diversity found in isolates of Plasmodium falciparum from Papua New Guinea by using monoclonal antibodies.

Monoclonal antibodies were used to demonstrate antigenic diversity in over 100 primary isolates of Plasmodium falciparum collected from one area of Papua New Guinea. The frequencies of several parasite antigens in our sample is calculated. One particular antigen which had previously been shown to be absent in one-third of established isolates collected from several countries appeared ubiquitous in our sample from Papua New Guinea. A method is presented that can be used to test for associations between antigens; the analysis also reveals whether other variables affect these associations. Several associations between antigens were revealed. In one instance, the association between two particular parasite antigens was affected by the donor's age. The importance of characterizing the antigenic structure of P. falciparum populations, and its relevance to the introduction of an antimalarial vaccine are discussed.

Genetic evidence for the importance of interrupted feeding by mosquitoes in the transmission of malaria.

Plasmodium falciparum isolates were obtained from 17 pairs of Gambian children, each pair living in the same house and presenting with malaria at the same time. Frequencies of allelic serotypes of 3 polymorphic blood stage proteins (MSP1, MSP2, and Exp-1) were previously determined from a large number of isolates from patients in the local area, and the probability of a random pair of isolates containing an identical genotype was calculated to be less than 0.01. However, 3 of 8 household pairs in one year, and 6 of 9 in the next year, contained identical P. falciparum genotypes, a much higher frequency than expected randomly (P less than 0.00005, for each year). This finding is discussed in terms of the probable contribution of single mosquitoes infecting more than one person.

Serotyping Plasmodium falciparum from acute human infections using monoclonal antibodies.

Monoclonal antibodies specific for the schizont and merozoite stages of Plasmodium falciparum have been used to demonstrate antigenic differences between parasites obtained from individual patients with acute malaria. Parasites from East Africa are shown to share some strain-specific antigenic determinants with culture-adapted isolates from Africa as well as from Asia and Papua New Guinea.

EDI and imaging automate the business office.

By implementing electronic remittance posting and imaging in patient accounting, New York's Memorial Sloan-Kettering Cancer Center was able to eliminate 22 full-time equivalent supervisory and staff positions and save $365,000 in physical space, microfilm and media costs the first year the new system was operational. Memorial Sloan-Kettering currently is participating in a pilot program with its Medicare fiscal intermediary to determine how EDI can be used to manage claim-status information between payer and provider. By combining EDI, fax, and imaging technologies, Memorial Sloan-Kettering is making its patient accounting process more efficient and providing better service to its patients.

Memorial Sloan-Kettering cures paperwork problems with document imaging.

The world's largest cancer center, New York's Memorial Sloan-Kettering, is finding concrete efficiencies--and real savings--by implementing "OSCAR" the Optical System Controlling Administrative Records. The system now handles more than 2.7 million documents, including patient accounts and medical records.

Population genetics of Plasmodium falciparum within a malaria hyperendemic area.

Serotyping with monoclonal antibodies was used to estimate the number and frequencies of allelic variants of two merozoite surface proteins, MSP1 and MSP2, and an exported protein Exp-1, in a sample of 344 clinical isolates of Plasmodium falciparum from an urban region of The Gambia. Represented among the isolates were 36, 8 and 2 alleles of the MSP1, MSP2 and Exp-1 loci respectively. Relative frequencies of these alleles remained stable in the parasite population over the 2 years of the study. A computer program was used to calculate from the frequencies of individual alleles at the three loci, the probable number of different genotypes in samples from the population, assuming random assortment among the loci. No significant difference was found between the expected and the observed genotype diversity. It is concluded that recombination among unlinked loci is a common consequence of sexual reproduction of P. falciparum in The Gambia. Slightly lower genotype diversity was observed in each of two villages, which may be a consequence of smaller population size compared with the urban region.