Monolayers of human erythrocytes (E) infected with Plasmodium falciparum were briefly fixed with 1% glutaraldehyde and air dried. They were then exposed to sera from patients with P. falciparum malaria or from donors immune to this parasite and tested in an indirect immunofluorescence assay (IFA). Parasites in infected E were made visible by counterstaining with ethidium bromide. Immunofluorescence (IF) was restricted to the surface of infected E. No antibody binding was detected unless the E were dried, suggesting that the relevant antigens were not available on the outer layers of the E surface. Staining over large parts of the E surface was seen already when the merozoite penetrated noninfected cells and was strong in E containing early stages of the parasite (rings, trophozoites). It was weak or absent from E containing schizonts. Antibodies in sera from different parts of Africa, Colombia, or Sweden reacted similarly with E infected with a Tanzanian P. falciparum strain kept in culture for many years and with parasitized E freshly drawn from African, Swedish, or Colombian patients. All sera from residents of a holoendemic area (Liberia) were IFA positive. In contrast, some sera from Colombian or Swedish patients with primary infection gave negative results. The results of the IFA and of an enzyme-linked immunosorbent assay in which fixed and dried E were the targets were well-correlated, suggesting that the same antibodies were detected by these assays. The antigens involved in the IFA were susceptible to pronase but not to trypsin or neuraminidase. E surface IF was inhibited by lysates of infected E, merozoite extracts, or soluble antigens present in P. falciparum culture supernatants but not by lysates of normal E or ghost extracts. The inhibitory antigens were heat stable (100 degrees C, 5 min). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting of either antigen-enriched preparations from culture supernatants or merozoite extracts showed that antibodies eluted from monolayers of infected E reacted consistently with a predominant polypeptide of Mr 155,000 and two to four minor polypeptides of lower molecular weights. Metabolic labeling of the parasites with 75Se-methionine indicated that these antigens were parasite derived. We conclude that the antigens involved in these reactions are released from bursting schizonts or merozoites and are deposited in the E membrane in the course of invasion.(ABSTRACT TRUNCATED AT 400 WORDS)

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IgG from a donor clinically immune to Plasmodiumfalciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, <1 ,ug/ml) than the original IgG preparation. falciparum (1, 2). These antigens are released from bursting schizonts or merozoites and are deposited in the RBC membrane in the course of invasion. Antibodies recognizing these antigens are of high prevalence in sera from residents of a holoendemic area of Africa (Liberia) but are also present in many sera of acutely infected patients from different parts of the world. A polypeptide of Mr 155,000 (Pf 155) appears to be primarily responsible for this RBC membrane IF. A similar antigen of the same apparent molecular weight has also been found recently by two other groups (3, 4). To further investigate the function of these antigens and the possible significance of the immune response against them, we have now tested the antibodies reacting with Pf 155 and related minor components for their capacity to inhibit RBC invasion by merozoites in vitro. MATERIAL AND METHODSParasites. Parasites were from a Tanzanian strain of P. falciparum (F 32) isolated in 1978 (5) and cultured in vitro in blood group O RBCs (6).Immune Sera. Five sera (Kinon, IS-6 to -8, X-12) were from Liberians, >15 years old (except X-12, from a 12-yearold boy), living in a P. falciparum holoendemic area in which in adult life clinical illness is rare and parasitemia is kept at a low-grade, mostly subpatent, level despite high sporozoite inoculation rates (7). These donors, designated as clinically immune, had not taken any antimalarial drug for the last 6 weeks (X-12) or longer. The 6 remaining donors had acute P. falciparum infections. Four donors (ASF, MV, JOR, FG) were South Americans from an endemic area of Colombia. Two Swedish patients (YC, HP) were suffering from a first infection acquired in Kenya. IgG was prepared by ammonium sulfate precipitation and ion-exchange chromatography on DEAE-Sephadex (Pharmacia). Immunoglobulin concentrations were determined by ELISA.In Vitro Growth Inhibition Assay. Infected RBCs from P. falciparum cultures (5-10% parasitemia, -70% schizonts) were diluted with normal O RBCs to a parasitemia of 0.5%. They were adjusted to 2% hematocrit with Hepes-buffered (20 mM) RPMI-1640 medium (GIBCO) containing 15% normal human serum, 2 mM glutamine, 25 pg of gentamycin per ml, and 0.2% NaHCO3 [complete tissue culture medium (TC medium)]. In some experiments, synchronized parasite cultures were used. In this case, the original cultures (5-10% parasitemia) were adjusted to 10% hematocrit and layered on top of 2.5 ml of 60% Percoll (Pharmacia) diluted in com...

The development of vaccines is presently receiving major attention in malaria research. As it is not possible to base malaria vaccines on the use of killed or attenuated organisms, the vaccines which are being developed are subunit vaccines in which the immunogens consist of defined parasite antigens or antigenic fragments. Since protective immunity to malaria involves both antibody-dependent and antibody-independent mechanisms, the immunogens in a subunit vaccine must have the capacity to induce relevant B- and T-cell responses in the majority of vaccinees. In turn, this requires good knowledge of these responses in humans who have acquired immunity through natural infection. In this paper we have summarized our recent work on the dissection into epitope-specific components of the human antibody response to the Plasmodium falciparum antigen Pf155/RESA, a recognized candidate for a vaccine against the asexual blood stages of this parasite. Epitope mapping of the antigen by means of short synthetic peptides led to the identification in several molecular regions of short amino acid sequences constituting linear and probably immunodominant B-cell epitopes. The antigenically most active region was located in the C-terminus of the molecule. This region, which consists of approximately 40 related, 4- or 8-amino acid long repeats, induced higher antibody concentrations in a larger number of malaria-immune donors than any of the other regions. A large fraction of these antibodies bound to short synthetic peptides representing the major repeat motifs of Pf155/RESA. Although these repeats are made up of closely related amino acid sequences, the antibody response to them was highly polyclonal, indicating the presence of several linear and probably also conformational epitopes which gave rise to a variety of cross-reacting as well as monospecific antibodies. Further analysis revealed that the levels of antibodies differing in specificity and/or avidity for different peptides varied independently of each other in individual donors. In an area (Liberia) where malaria transmission is holoendemic and perennial, these antibody profiles remained constant when individual donors were followed over several years. Since the C-terminal repeat region of Pf155/RESA is conserved in different P. falciparum strains, the results reflect differences in the genetic regulation of epitope-specific host responses rather than antigenic differences between infecting parasites. In donors living in an area with high but seasonal malaria transmission, antibody levels usually drop to lower levels when there is no transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract. In this article we present methods for the purification and fractionation of human blood lymphocytes, which have been used in our laboratory to characterize antibody‐dependent cytotoxic effector cells (K cells). The assay system consists of highly purified lymphocytes, 51Cr‐labelled chicken erythrocytes (Ec) and IgG rabbit anti‐Ec in high dilutions. Various ways of comparing K‐cell potentials of different lymphocyte preparations in this system are discussed. When purified lymphocytes are partially depleted (60‐85% depletion) of cells forming rosettes with sheep erythrocytes (E+ cells), the K‐cell activity of the depleted fraction is increased, indicating that the majority of the E+ cells are inactive in this assay. Depletion of EAC‐rosette‐forming cells shows that most or all K cells have complement receptors. For depletion of B cells, the lymphocytes may be passed through glass bead columns, charged with F(ab')2 fragments of human IgG and F(ab')2 fragments of rabbit antibodies to the F(ab')2 part of human IgG. These columns give high yields of B‐cell depleted fractions. These preparations are rich in E+ cells and contain ˜80% of the Fc‐receptor lymphocytes which form rosettes with bovine erythrocytes, coated with IgG antibodies. Their K‐cell activity is unchanged or slightly elevated, indicating that mature B cells, i.e. SIg+ cells, have little or no K‐cell activity. In contrast, passage of the lymphocytes through immune complex columns (ovalbumin/anti‐ovalbumin) leads to ˜ 70% depletion of Fc receptor‐bearing cells, while most of the B cells (SIg+ cells) pass through the columns. The relative frequency of E+ cells in the passed fraction frequently shows a slight reduction. These preparations have a very low K‐cell activity, indicating that K cells are lymphocytes with Fc receptors of relatively strong avidity.

T cells play a crucial role in antibodymediated and antibody-independent immunity against Plasmodium falciparum malaria. Therefore, a vaccine immunogen should include parasite-derived B-and T-cell epitopes capable of giving rise to protective responses in both systems. The P.falciparum antigen PfI55/ring-infected erythrocyte surface antigen (RESA), a vaccine candidate, contains immunodominant T-and B-cell epitopes located in the central (5') and C-terminal (3') invariant repeat regions of the molecule. To relate Pfl55/RESA-peptide-specific responses of T cells to function, T cells from P. fakciparum immune donors were activated with peptides corresponding to these immunodominant regions. Activation was measured as induction of interferon-y secretion, T-cell proliferation (DNA synthesis), or transcription and translation of interleukin 4 (IL-4) mRNA.Peptides from both regions were shown to induce interferon-y, IL-4, proliferation, or any combination. In individual donors, there was no correlation between these different activities. Rather, they were negatively correlated, demonstrating the importance of examining multiple parameters of T-cell activation when estimating the proportion of individuals responding to a given epitope. However, IL-4 mRNA and intracellular IL-4 could be induced in T cells of donors who had elevated concentrations ofserum antibodies to the same peptide that was used for T-cell activation. These results suggest that a causal relationship exists between the activation of IL-4-producing T-cell subsets and production of the anti-Pf155/RESA-specific antibodies in individuals in which immunity has been induced by natural infection. This finding has implications that should be considered for the selection of immunogens to be included in a future P. falciparum subunit vaccine and for vaccine development in general.

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