Inhibition of Plasmodium falciparum growth by IgG antibody produced by human lymphocytes transformed with Epstein-Barr virus.

Supernatants from Epstein-Barr virus (EBV)--stimulated B lymphocytes obtained from two adult Gambians who were partially immune to malaria markedly inhibited the growth of Plasmodium falciparum in vitro (55-95% inhibition). When 22 separate colonies were derived by micromanipulation from one of these primary cultures and their supernatants assayed, the degree of inhibition correlated with levels of IgG fluorescent antibody and total IgG. The inhibitory anti-P. falciparum IgG immunoprecipitated an antigen of mol. wt 195,000, identified as the major schizont surface glycoprotein by dual biosynthetic labelling with 3H-glucosamine or 35S-methionine. Other studies on the analogous schizont surface protein of rodent malarias have shown that this antigen stimulates protective immunity. Production of this inhibitory antibody by adult Gambians may therefore contribute to their immunity to malaria. Human antibodies produced by EBV-stimulated B lymphocytes may be used to identify other important P. falciparum antigens and have potential applications for immunotherapy.     

Association between human serum-induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan.

Clinical histories with regard to falciparum malaria were collected from adults living in holo-, hyper-, and hypoendemic areas of Sudan and matched to serum samples which were assayed for antiparasitic activity in cultures of Plasmodium falciparum. The adult population of the endemic areas could be divided into three groups based on oral histories: those who never experience falciparum malaria; those with a childhood history of malaria, who experience only mild occasional malaria as adults; and those who suffer serious recurring malaria symptoms. In vitro parasite inhibition was greatest with sera from individuals with no clinical histories of malaria, and generally, more inhibition was noted in sera from holoendemic versus hyperendemic areas. Serum from hypoendemic urban Khartoum was not inhibitory. There was no relationship between serum indirect fluorescent antibody titers and parasite inhibition, but there was strong association between clinical immunity and intraerythrocytic parasite inhibition resulting in "crisis" forms. Purified immunoglobulin G was not strongly associated with crisis forms, which were consistently associated with fractions of immune serum remaining after immunoglobulin removal. Thus, it appears that clinical immunity to malaria in Sudan is based on nonantibody serum factors, possibly associated with cell-mediated immunity. Human leukocyte alpha-interferon had no inhibitory effects on cultured P. falciparum. Some umbilical cord sera were profoundly inhibitory, producing crisis forms, whereas others were not inhibitory, suggesting that factors that induce crisis forms may play a role in protecting neonates from falciparum malaria.     

Cell-mediated immune responses to Plasmodium falciparum antigens in adult Gambians.

Peripheral blood mononuclear cells from clinically immune Gambian adults were assayed for in vitro proliferation in response to crude and partially purified Plasmodium falciparum antigens. Lymphoproliferative responses to malaria antigens, lectin mitogens and Candida albicans were compared with those of control donors with no previous exposure to malaria. Cells of malaria-immune individuals were significantly more responsive to conconavalin A, and less responsive to phytohaemagglutinin, than cells from the control donors in both non-immune human serum and autologous serum. Cells from a proportion of immune donors proliferated in response to soluble malaria antigens but a substantial minority did not. Young adults and women were over-represented in the non-responding population. Responses to soluble malaria antigens were depressed in autologous serum compared with normal human serum. Both immune and control cells produced low levels of gamma-IFN when stimulated with crude P. falciparum schizont antigens. Approximately half the immune donors, and none of the controls, produced significant levels of gamma-IFN in response to purified soluble malaria antigen or malaria parasite culture supernatant. There was no direct correlation between lymphoproliferation and gamma-IFN production.     

Measurement of acute phase proteins for assessing severity of Plasmodium falciparum malaria.

Seventeen adult patients with acute Plasmodium falciparum malaria, admitted to the Hospital for Tropical Diseases, were studied. Serial measurements of the serum concentration of C-reactive protein, serum amyloid A protein, and percentage parasitaemia were determined, together with initial measurement of serum electrolytes, liver function, haemoglobin, white cell and platelet counts. Initial C-reactive protein and serum amyloid A concentrations were increased (C-reactive protein mean 49.0 mg/l serum amyloid A 28 mg/l) falling towards the normal range by the seventh day of treatment. There was a significant correlation between the pretreatment parasite count and clinical and laboratory markers of inflammation. C-reactive protein and serum amyloid A concentrations correlated inversely with the serum sodium. These results indicate that measurement of acute phase reactants such as C-reactive protein and serum amyloid A may prove valuable in assessing the severity of P falciparum malaria, and in following the response to antimalarial treatment.     

Sera from lupus patients inhibit growth of P. falciparum in culture.

A large number of malaria immune persons from Eastern India were found to possess antibodies to the ribosomal phosphoprotein P0 (PfP0) of the human malarial parasite Plasmodium falciparum. The characterization of PfP0 has been reported recently, and it has been shown that antibodies against PfP0 inhibit P. falciparum in vitro. About 10-15% of the patients suffering from the autoimmune disorder Systemic Lupus Erythematosus (SLE) possess autoantibodies to the human ribosomal P proteins. In order to test the cross-reactivity of the human and Plasmodium falciparum P0 proteins and to compare the SLE patients' and malaria immune persons' response, sera from 41 Indian SLE patients were tested against the P. falciparum PfP0 by Western blot analysis. Four of these samples (9.75%) were found to be cross-reactive to the carboxy-terminal domain of PfP0, but not to the amino-terminal domain of PfP0. The PfP0 reactive SLE sera inhibited the growth of Plasmodiumfalciparum in vitro. IgG purified from one such cross-reactive serum sample inhibited the growth of P. falciparum. Depletion of anti-P0 antibodies from this IgG preparation resulted in the removal of growth inhibition. Sera samples were collected from one of the PfP0 positive SLE patient from Mumbai, India, at different stages of the disease progression, and screened for the presence of anti-PfP0 antibodies. This patient's serum inhibited the parasite growth in vitro only during the phase in which anti-PfP0 antibodies were detected.     

Cell-Mediated Immunity to Plasmodium falciparum Infection: Evidence against the Involvement of Cytotoxic Lymphocytes

Blood mononuclear cells (PBMC) recognizing soluble malaria antigens (SPag) are present in the peripheral blood of individuals clinically immune to malaria, and they proliferate after exposure to such antigens. To test whether these cells have effector activity against Plasmodium falciparum, we stimulated PBMC from malaria-immune donors by SPag and purified protein derivative (PPD) in culture for 7 days. The PBMC were then co-incubated with P. falciparum for 48 h, and parasitaemia was determined by microscopy. Parasite growth was only significantly impaired after incubation with PBMC stimulated by either SPag or PPD in the presence of immune serum. Studies on subpopulations of PBMC indicated that the inhibitory cells resided among the adherent cell fraction. Furthermore we tested PBMC for cytotoxic activity against P. falciparum-infected autologous or heterologous erythrocytes. Experiments were done both in the absence and the presence of immune serum. Neither fresh PBMC nor PBMC activated by SPag or PPD for 7 days prior to assay were cytotoxic, indicating that cytotoxic T cells, natural killer (NK) cells, and K cells did not possess cytotoxic activity directed against parasitized erythrocytes. These data support the hypothesis that activated monocytes are the most important effector cells in the peripheral blood of malaria immune individuals.     

Detection of human antibodies against Plasmodium falciparum sporozoites using synthetic peptides.

A large peptide consisting of about 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was synthesized. It was recognized specifically by monoclonal antibodies produced against P. falciparum sporozoites. Moreover, this peptide strongly inhibited the binding of such monoclonal antibodies to antigens present in a sporozoite extract. The (NANP)40 peptide was employed without any carrier to develop an enzyme-linked immunosorbent assay to detect sporozoite-specific serum antibodies arising after natural malaria infections. Antibodies were detected in a high percentage (43.1%) of European patients suffering from acute P. falciparum malaria and in Africans living in an area of Gabon endemic for malaria. In the latter group, the frequency of antisporozoite antibodies increased with age, reaching 65.9% in individuals more than 40 years old. There was a significant correlation between the results obtained with an immunofluorescence assay with glutaraldehyde-fixed sporozoites and those obtained by enzyme-linked immunosorbent assay with (NANP)40. Therefore, such synthetic peptides representing the repetitive epitope of P. falciparum circumsporozoite protein can be used for the detection of antisporozoite antibodies and for the epidemiological studies required to obtain base-line data concerning the immune status of individuals before their participation in a sporozoite vaccine trial.     

Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes.

In vitro human monocytes from normal blood donors ingest red blood cells infected with Plasmodium falciparum more efficiently than normal red blood cells (NRBC). The phagocytic activity of human monocytes for infected red blood cells (IRBC) is greatly enhanced by the addition of immune sera obtained from individuals living in areas with endemic malaria. In contrast, the addition of sera obtained from individuals recovering from a first infection, or pooled normal sera, does not result in increased phagocytosis of IRBC. The phagocytosis enhancing activity of immune sera is associated with the IgG fraction and IgG depleted sera do not stimulate phagocytosis. Enhanced immune serum mediated phagocytosis occurs as a result of opsonization of IRBC. This was demonstrated by experiments in which monocytes or IRBC were preincubated with immune serum prior to the phagocytic assay. The opsonic activity could be absorbed by IRBC but not by NRBC. The opsonization of IRBC and subsequent phagocytosis were also dependent on the stage of development of the intracellular parasite. IRBC containing schizonts and trophozoites were preferentially phagocytosed as compared with ring forms. The role of malaria induced surface alterations and/or malaria surface antigens in the opsonization of IRBC by immune sera is discussed. These experiments suggest that phagocytosis of P. falciparum IRBC by monocytes may play a role in the immune elimination of malaria infection in humans.     

Inhibition of in Vitro Growth of Plasmodium falciparum by Purified Antimalarial Human IgG Antibodies

In the search for candidate molecules for a malaria vaccine the in vitro inhibition of Plasmodium falciparum cultures by polyclonal or monoclonal antibodies has become a major tool. In the present study antigens identical to antigens circulating in plasma during attacks of malaria have been isolated from supernatants of P. falciparum cultures and used for immunoadsorbent purification of IgG antibodies from a pool of human immune serum collected in Liberia. Approximately 50% growth inhibition of three different P. falciparum isolates from Africa was obtained with the affinity-purified antibodies at a concentration of 25 micrograms ml-1 culture medium after 48 h of incubation. The target antigen/antigens for the protective antibodies have been partly characterized by radiolabelling, polyacrylamide gel electrophoresis and autoradiography but have not yet been identified unequivocally. However, the results indicate that one or more of the easily isolated antigens from the supernatant of P. falciparum cultures could be used in a malaria vaccine. The results also indicate that antigenic differences between strains from geographically disparate areas may not constrain the development of such a vaccine.     

Characterization of peripheral blood lymphocyte subsets in patients with acute Plasmodium falciparum and P. vivax malaria infections at Wonji Sugar Estate, Ethiopia

We investigated the absolute counts of CD4+, CD8+, B, NK, and CD3+ cells and total lymphocytes in patients with acute Plasmodium falciparum and Plasmodium vivax malaria. Three-color flow cytometry was used for enumerating the immune cells. After slide smears were stained with 3% Giemsa stain, parasite species were detected using light microscopy. Data were analyzed using STATA and SPSS software. A total of 204 adults of both sexes (age, >15 years) were included in the study. One hundred fifty-eight were acute malaria patients, of whom 79 (50%) were infected with P. falciparum, 76 (48.1%) were infected with P. vivax, and 3 (1.9%) were infected with both malaria parasites. The remaining 46 subjects were healthy controls. The leukocyte count in P. falciparum patients was lower than that in controls (P=0.015). Absolute counts of CD4+, CD8+, B, and CD3+ cells and total lymphocytes were decreased very significantly during both P. falciparum (P<0.0001) and P. vivax (P<0.0001) infections. However, the NK cell count was an exception in that it was not affected by either P. falciparum or P. vivax malaria. No difference was found in the percentages of CD4, CD8, and CD3 cells in P. falciparum or P. vivax patients compared to controls. In summary, acute malaria infection causes a depletion of lymphocyte populations in the peripheral blood. Thus, special steps should be taken in dealing with malaria patients, including enumeration of peripheral lymphocyte cells for diagnostic purposes and research on peripheral blood to evaluate the immune status of patients.     

Plasmodium falciparum Malaria: reduction of endothelial cell apoptosis in vitro

Organ failure in Plasmodium falciparum malaria is associated with neutrophil activation and endothelial damage. This study investigates whether neutrophil-induced endothelial damage involves apoptosis and whether it can be prevented by neutralization of neutrophil secretory products. Endothelial cells from human umbilical veins were coincubated with neutrophils from healthy donors and with sera from eight patients with P. falciparum malaria, three patients with P. vivax malaria, and three healthy controls. Endothelial apoptosis was demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V staining. The rate of apoptosis of cells was markedly increased after incubation with patient serum compared to that with control serum. Apoptosis was most pronounced after incubation with sera from two patients with fatal cases of P. falciparum malaria, followed by sera of survivors with severe P. falciparum malaria and, finally, by sera of patients with mild P. falciparum and P. vivax malaria. Ascorbic acid, tocopherol, and ulinastatin reduced the apoptosis rate, but gabexate mesilate and pentoxifylline did not. Furthermore, in fatal P. falciparum malaria, apoptotic endothelial cells were identified in renal and pulmonary tissue by TUNEL staining. These findings show that apoptosis caused by neutrophil secretory products plays a major role in endothelial cell damage in malaria. The antioxidants ascorbic acid and tocopherol and the protease inhibitor ulinastatin can reduce malaria-associated endothelial apoptosis in vitro.     

Plasmodium falciparum infection elicits both variant-specific and cross-reactive antibodies against variant surface antigens

Naturally acquired antibodies to Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1), the variant surface antigens expressed on the surface of infected erythrocytes, are thought to play a role in protection against P. falciparum malaria. Here, we have studied the development of antibodies to PfEMP-1 in adult malaria patients living in Rourkela, India, an area with a low malaria transmission rate, and prevalence of antibodies to PfEMP-1 in residents of San Dulakudar, India, a village in which P. falciparum malaria is hyperendemic. Convalescent-phase sera from adult malaria patients from Rourkela agglutinate homologous P. falciparum isolates as well as some heterologous isolates, suggesting that they develop partially cross-reactive antibodies to PfEMP-1 following infection. Adult sera from San Dulakudar agglutinate diverse P. falciparum isolates, suggesting that they have antibodies with wide recognition of diverse PfEMP-1. Mixed-agglutination assays using pairs of P. falciparum isolates confirm the presence of both variant-specific and partially cross-reactive antibodies in convalescent-phase sera from Rourkela and adult sera from San Dulakudar. Analysis of PfEMP-1 sequences suggests a molecular basis for the observed cross-reactivity.     

Immunity to blood stages of Plasmodium falciparum is dependent on a specific pattern of immunoglobulin subclass responses to multiple blood stage antigens

BACKGROUND: In view of the lack of effectiveness of vaccines against Plasmodium falciparum malaria new approaches in the search for antigens and formulations for a vaccine are required. Immunoglobulin responses against several blood stage antigens were only partially associated with protection from symptoms in prospective immuno-epidemiological studies. HYPOTHESIS: Immunity to blood stages of P. falciparum is dependent on a specific pattern of immunoglobulin subclass responses to multiple blood stage antigens. SUPPORTING EVIDENCE FOR THE HYPOTHESIS: This hypothesis results from previous studies showing that IgG1 and IgG3 responses against antigens like ring-infected erythrocyte surface antigens, merozoite surface proteins and variant surface antigens on schizonts were associated with clinical immunity against malaria. None of the specific responses against a single antigen was completely protective. IMPLICATIONS OF THE HYPOTHESIS: A confirmation of the hypothesis would support efforts to generate a P. falciparum vaccine containing a comprehensive set of blood stage antigens, which, using adjuvant technology, would lead to the appropriate immunoglobulin subclass response. MEANS TO TEST THE HYPOTHESIS: Detection of multiple immunorelevant P. falciparum blood stage antigens could be achieved by two-dimensional electrophoresis and blotting of the antigens onto nitrocellulose followed by exposure to the participant's serum. Bound immunoglobulins are visualized by isotype specific peroxidase conjugated anti-human immunoglobulin and quantified by transmission densitometry. The resulting pattern of responses for each immunoglobulin isotype could be compared between clinically immune participants remaining asymptomatic with subsequent P. falciparum infections and people who develop malaria. Immunorelevant antigens are identifiable by mass spectrometry with the fully decoded P. falciparum genome.     

Anti-malaria antibody-producing B cell frequencies in adults after a Plasmodium falciparum outbreak in Madagascar.

The central highlands of Madagascar offer a unique opportunity to explore the malaria immune memory, as the last murderous epidemic in the study area occurred 8 years ago. Quantification of the circulating memory B lymphocytes reacting to Plasmodium falciparum was assessed among 14 Madagascans by using a limiting dilution assay, applied to the EL4 culture system, which leads to activation, proliferation and differentiation into antibody-secreting cells (ASC) of most peripheral B cells. This system allowed us to observe, without any malaria-specific restimulation, a geometric mean frequency of one anti-P. falciparum ASC among 2992 circulating B cells, except for one Madagascan who did not have any detectable ASC. A geometric mean frequency of one anti-P. falciparum ASC among 1403 was obtained for six malaria hyperimmune Cameroonians, but conversely, no anti-malaria ASC was detected in the blood of six malaria non-immune French control subjects. Anti-P. falciparum ASC frequencies and serum specific antibodies were strongly related. Our results indicate that anti-malaria ASC are still present in peripheral blood of Madagascan subjects, who have not been exposed to P. falciparum for several years. These responder B cells reflect the malaria B cell memory acquired during the last epidemic.     

Inhibitory immunoglobulin M antibodies to tumor necrosis factor-inducing toxins in patients with malaria.

Cytokines such as tumor necrosis factor alpha (TNF) appear to play an important role in the pathogenesis of malaria. We have previously shown that TNF is produced in response to substances released at schizont rupture, which we have called malaria toxins. In mice these toxins stimulate a T cell-independent antibody response, generating short-lived immunoglobulin M (IgM) antibodies that inhibit the TNF-inducing activity of the toxins. We report here that a similar antibody response is seen in humans. Serum from a European adult infected with Plasmodium falciparum inhibited the induction of TNF by malaria toxins derived from P. falciparum-infected erythrocytes. We found that IgM antibodies were responsible for the inhibitory activity. These inhibitory antibodies could not be detected in convalescent-phase serum collected from the same patient 6 weeks later or in sera from healthy European and African controls. The antibodies appeared to be malaria specific in that they inhibited TNF induction by a variety of P. falciparum isolates but failed to inhibit TNF induction by bacterial lipopolysaccharide or lipoteichoic acid. The inhibitory antibodies bound to liposomes containing phosphatidylinositol but not other phospholipids. Serum from a European adult infected with P. vivax also inhibited the activity of toxins derived from P. falciparum-infected erythrocytes, and this too was mediated by IgM antibodies which were malaria specific and bound to phosphatidylinositol liposomes.     

Direct Coombs antiglobulin reactions in Gambian children with Plasmodium falciparum malaria. II. Specificity of erythrocyte-bound IgG.

The cause of direct Coombs (DAT) positivity in West African (Gambian) children has been investigated. Results obtained from a comparison of two groups of primary school children, one from an area hyperendemic for P. falciparum malaria and the other partially protected from malaria, indicate an association between a high incidence of DAT positivity and P. falciparum parasitaemia with raised serum antibody titres to falciparum schizonts. Erythrocytes taken from children with a high IgG DAT titre were subjected to acid elution and the eluates examined for IgG content and antigen specificity. Eluted IgG had specific antibody activity against P. falciparum schizont antigen as demonstrated by means of the indirect fluorescent antibody technique. Cross-reaction was not observed with either P. falciparum gametocytes or P. malariae schizonts. No blood group specificity could be demonstrated in either the red cell eluates or serum taken from children with DAT positive red cells. The results provide convincing evidence which implicates falciparum malaria in the aetiology of Coombs positivity in Gambian children. It is probable that erythrocyte sensitization results from passive attachment of circulating complement-fixing malaria antigen-antibody complexes.     

Immunoglobulin E, a pathogenic factor in Plasmodium falciparum malaria.

Most children and adults living in areas where the endemicity of Plasmodium falciparum malaria is high have significantly elevated levels of both total immunoglobulin E (IgE) and IgE antimalarial antibodies in blood. This elevation is highest in patients with cerebral malaria, suggesting a pathogenic role for this immunoglobulin isotype. In this study, we show that IgE elevation may also be seen in severe malaria without cerebral involvement and parallels an elevation of tumor necrosis factor alpha (TNF). IgE-containing serum from malaria immune donors was added to tissue culture plates coated with rabbit anti-human IgE antibodies or with P. falciparum antigen. IgE-anti-IgE complexes as well as antigen-binding IgE antibodies induced TNF release from peripheral blood mononuclear cells (PBMC). Nonmalaria control sera with no IgE elevation induced significantly less of this cytokine, and the TNF-inducing capacity of malaria sera was also strongly reduced by passing them over anti-IgE Sepharose columns. The cells giving rise to TNF were adherent PBMC. The release of this cytokine probably reflects cross-linking of their low-affinity receptors for IgE (CD23) by IgE-containing immune complexes known to give rise to monocyte activation via the NO transduction pathway. In line with this, adherent monocytic cells exposed to IgE complexes displayed increased expression of CD23. As the malaria sera contained IgG anti-IgE antibodies, such complexes probably also play a role in the induction of TNF in vivo. Overproduction of TNF is considered a major pathogenic mechanism responsible for fever and tissue lesions in P. falciparum malaria. This overproduction is generally assumed to reflect a direct stimulation of effector cells by certain parasite-derived toxins. Our results suggest that IgE elevation constitutes yet another important mechanism involved in excessive TNF induction in this disease.     

Protective properties and surface localization of Plasmodium falciparum enolase

The enolase protein of the human malarial parasite Plasmodium falciparum has recently been characterized. Apart from its glycolytic function, enolase has also been shown to possess antigenic properties and to be present on the cell wall of certain invasive organisms, such as Candida albicans. In order to assess whether enolase of P. falciparum is also antigenic, sera from residents of a region of Eastern India where malaria is endemic were tested against the recombinant P. falciparum enolase (r-Pfen) protein. About 96% of immune adult sera samples reacted with r-Pfen over and above the seronegative controls. Rabbit anti-r-Pfen antibodies inhibited the growth of in vitro cultures of P. falciparum. Mice immunized with r-Pfen showed protection against a challenge with the 17XL lethal strain of the mouse malarial parasite Plasmodium yoelii. The antibodies raised against r-Pfen were specific for Plasmodium and did not react to the host tissues. Immunofluorescence as well as electron microscopic examinations revealed localization of the enolase protein on the merozoite cell surface. These observations establish malaria enolase to be a potential protective antigen.     

Naturally acquired immunity and reduced susceptibility to falciparum malaria in two subpopulations of endemic eastern India

This study was aimed to assess the prevalence of naturally acquired humoral immune responses and their association with reduced susceptibility to malaria in children and adults with differential clinical conditions from an Indian zone where malaria is endemic. The study was undertaken in an eastern province of India (Keonjhar, Orissa) in a group of 341 children (both younger and older) and 98 adults living in two different areas, Town area and Forest area. They were studied for their parasitological and immunological profiles. Sera from different age-matched groups were screened by ELISA to measure IgG reactivities for characterizing humoral immune responses to the B-cell epitopes of Plasmodium falciparum MSP1, AMA1, RAP1 and EBA175 peptides and P. falciparum -infected erythrocyte lysate. In Town area, overall P. falciparum cases were 5.5%, whereas those in Forest area were 26.7%. We observed an age-wise increasing trend of immunity in these two populations. It was also noticed that the frequency of responders to stage-specific antigens was higher in individuals from the Town area where the frequency of malaria was lower. The naturally acquired humoral immune responses to different stage-specific antigens of P. falciparum reflect the reduced risk of malaria in the study groups. The higher frequency of seroresponders showed correlation with lower risk of developing malaria.     

Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein.

This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactosidase fusion protein and used as antigen after purification and biotinylation. Specific IgM antibodies were found in 51% (39/77) of sera from adult Liberians immune to malaria. The binding of IgM antibodies was specific for the malaria portion of the fusion protein and no cross-reactivity was found in sera from patients with IgM antibodies due to other diseases. Inhibition studies with a fusion protein containing amino acid residues 816-1134 (GLURP816-1134) representing the carboxy-terminal repeat region suggested a different use of epitopes for IgM antibodies in different individuals.