The complexity of the life cycle of the protozoan malaria parasite Plasmodium falciparum has hindered genetic analysis; even the number of chromosomes in P. falciparum is uncertain. The blood stages of rodent malaria parasites are haploid and hybridization with cloned complementary DNAs similarly suggests a haploid genome in P. falciparum blood stages (ref. 4 and our unpublished results). A novel approach to karyoptic and linkage analysis in P. falciparum has been provided recently by the technique of pulsed-field gradient (PFG) gel electrophoresis, which allows the fractionation of DNA molecules of 30-3,000 kilobases (kb), a range including the sizes of intact chromosomal DNA molecules from eukaryotes such as yeast and trypanosomatids. We describe here the fractionation by PFG electrophoresis of chromosomal DNA molecules from P. falciparum into at least seven discrete species which vary in size by up to 20% between different isolates. Several genes for P. faciparum antigens which contain repetitive sequences are located on different chromosomes. Surprisingly, two of the chromosomes seem to contain the same sequences.

The S antigen of a Papua New Guinean isolate of Plamodium falciparum was identified by immunoblotting as the dominant antigen in culture supernatants. An antigen identical in molecular weight (Mr 220,000), isoelectric point (pI 4.2), and immunoreactivity with sera from individuals exposed to malaria was expressed by four Papua New Guinean isolates and one isolate of unknown origin. The Mr 220,000 antigen was not detected in culture supernatants derived from two isolates from Thailand and one from Ghana. The Mr 220,000, pI 4.2 S antigen may characterize a subpopulation of parasites common to many isolates of P. falciparum, which is selected for by continuous culture in vitro A variant S antigen, 30 kilodaltons larger but with similar immunoreactivity, was expressed by 1 of 26 clonal populations derived by limit-dilution culture from one of the Papua New Guinean isolates of P. falciparum. The characteristics of the S antigen, defined by immunoblotting, allowed it to be identified in two-dimensional separations of [mS]methionine-labeled parasite proteins, thus confirming the parasite origin of the antigen.Plasmodiumfalciparum, the major cause of morbidity and mortality in malaria infections of man, exhibits considerable antigenic heterogeneity, which may be a major problem in developing an effective vaccine against malaria. The most extensive studies of antigenic heterogeneity in P. falciparum have concerned a system of soluble heat-stable antigens known as the S antigens. The S antigens were first demonstrated in extracts of infected placentas but are also found in the sera of some infected individuals. The S antigens, which have been the subject of a series of detailed studies by Wilson and colleagues (1-9), have the following characteristics: (i) stability to heating at 100°C for 5 min; (ii) serological complexity with individual isolates expressing more than one specificity; (iii) poor immunogenicityno heterologous antiserum has been produced and there is a marked age-dependent acquisition of antibodies in man; (iv) presence in the medium when P. falciparum is maintained in culture in vitro; and (v) molecular weight and charge heterogeneity.Techniques such as two-dimensional gel electrophoresis of biosynthetically labeled immunoprecipitates (10) and immunofluorescence with monoclonal antibodies (11) have provided new approaches to the direct demonstration of antigenic heterogeneity in P. falciparum. However, these techniques have not been directly applied to an analysis of the S antigens. A major limitation to the more detailed analysis of S antigens has been the failure to label the antigens with radioactive metabolites (12). Thus, to date, analyses of the S antigen have been dependent on relatively insensitive gel precipitation techniques and have been restricted to sources in which antibodies and antigens are sufficiently abundant to give visible precipitates.To circumvent the apparent problems of biosynthetic labeling S antigens, we have used the technique of "immunoblotting" to identify the pre...

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Summary After oral administration of cysts of the intestinal protozoan parasite, Giardia muris, young male C3H/He mice are chronically infected, whereas BALB/c mice demonstrate a rapidly resolving pattern of infection. Both strains of mice injected with trophozoites in adjuvant and challenged orally with cysts develop serum antibodies to numerous trophozoite proteins. A limited number of these protein antigens was differenliatly immunoprecipitated by sera from resistant BALB/c and susceptible C3H/He mice exposed to G. muris. 35S‐methionine‐labelled protein antigens better recognized by immune BALB/c sera included molecules of relative mobility (Mr) 82,000 and a series of proteins of Mr 25,000 to 32,000, Differential recognition extended to a subset of solubilized trophozoite antigens that bind to the Iectin. wheat germ agglutinin (WGA), and that can be radio‐iodinated. In particular, a complex of 4 acidic protein antigens of approximate Mr 32,000. and designated collectively as Gm32, was better recognized by immune BALB/c serum than C3H/He serum. Isolated WGA‐binding antigens were not able to consistently vaccinate BALB/c mice against subsequent G. muris infection. Moreover, preliminary evidence has been obtained that lack of antibody responsiveness to Gm32 does not segregate strictly with susceptibility to chronic infection in (BALB/c × C3H/He)F2 mice. These data, plus the observation that drug‐cured C3H/He mice are highly resistant to reinfection, has led to examination of whether mice differ in the capacity of the intestines to support inflammatory responses. Mast cell deficient Wf/Wf mice, unlike wild‐type litter‐mates, developed chronic giardiasis although no reconstitution of resistance has yet been achieved with inocula of bone marrow cells from +/+ mice. BALB/c mice injected with the antihistamine and antiserotonin drug, cyproheptadine, also showed prolonged infections with G. muris. The data suggest that analysis of specificity differences in immune responses of mice varying in susceptibility to intestinal parasites must be supplemented by examination of the capacity of the intestine to support induced immune responses.

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A prospective study in 207 children aged 0.5-15 years was carried out to examine the relationship between cellular responses to Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA) and malaria infection and morbidity. The prevalence of lymphoproliferative response to RESA was 13%, IFN-gamma prevalence was 40% and IL-4 prevalence was 22%. Only the IFN-gamma, response to RESA increased significantly with age. When proliferation or stimulation of either cytokine was used to assess T-cell activation the overall frequency of responders increased to 55%. The proliferative and IFN-gamma response to RESA were positively associated. Although there was no association between any of the CMI responses to RESA and concurrent morbidity the prevalence of IL-4 response to RESA was significantly lower in children who experienced clinical malaria in the following year. These results coupled with our earlier data showing a negative relationship between humoral responses to RESA and malaria morbidity support the inclusion of RESA in a subunit vaccine against malaria.

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