Identification, expression, localization and serological characterization of a tryptophan-rich antigen from the human malaria parasite Plasmodium vivax

Plasmodium vivax is most common but non-cultivable human malaria parasite which is poorly characterized at the molecular level. Here, we describe the identification and characterization of a P. vivax Tryptophan-Rich Antigen (PvTRAg) which contains unusually high (8.28%) tryptophan residues and is expressed by all blood stages of the parasite. The pvtrag gene comprises a 978bp open reading frame interrupted by two introns. The first intron is located in the 5'-untranslated region while the second one is positioned 174bp downstream to the ATG codon. The encoded approximately 40kDa protein contains a transmembrane domain near the N-terminus followed by a tryptophan-rich domain with significantly high surface probability and antigenic index. It is localized in the parasite cytoplasm as well as in the cytoplasm of the parasitized erythrocyte. The purified E. coli expressed recombinant PvTRAg protein showed a very high seropositivity rate for the presence of antibodies amongst the P. vivax patients, indicating that the antigen generates significant humoral immune response during the natural course of P. vivax infection. Analysis of various field isolates revealed that the tryptophan-rich domain is highly conserved except for three-point mutations. The PvTRAg could be a potential vaccine candidate since similar tryptophan-rich antigens of P. yoelii have shown protection against malaria in murine model.     

Complement in malaria immunity and vaccines

Developing efficacious vaccines for human malaria caused by Plasmodium falciparum is a major global health priority, although this has proven to be immensely challenging over the decades. One major hindrance is the incomplete understanding of specific immune responses that confer protection against disease and/or infection. While antibodies to play a crucial role in malaria immunity, the functional mechanisms of these antibodies remain unclear as most research has primarily focused on the direct inhibitory or neutralizing activity of antibodies. Recently, there is a growing body of evidence that antibodies can also mediate effector functions through activating the complement system against multiple developmental stages of the parasite life cycle. These antibody-complement interactions can have detrimental consequences to parasite function and viability, and have been significantly associated with protection against clinical malaria in naturally acquired immunity, and emerging findings suggest these mechanisms could contribute to vaccine-induced immunity. In order to develop highly efficacious vaccines, strategies are needed that prioritize the induction of antibodies with enhanced functional activity, including the ability to activate complement. Here we review the role of complement in acquired immunity to malaria, and provide insights into how this knowledge could be used to harness complement in malaria vaccine development.     

A prospective study of the association between the human humoral immune response to Plasmodium falciparum blood stage antigen gp190 and control of malarial infections.

The human humoral immune response to the Plasmodium falciparum merozoite surface antigen gp190 was analyzed to determine the rate of reinfection by the parasite and the ability to control parasite density. The prospective study was carried out in a West African village where malaria is hyperendemic. No correlation between the antibody titers and protection against infection was observed within the group of children. Positive and negative associations of antibody specificities with protection against and/or control of parasitemia were, however, found for adolescents and adults, respectively. Thus, in adolescents, the presence of antibodies to gp190 fragment M6 correlates with a 50% reduced risk of P. falciparum infection and an increased ability to control parasitemia, whereas in adults, the humoral response to some of the polymorphic regions of gp190 associates with an increased risk of infection.     

Humoral responses to Plasmodium falciparum blood-stage antigens and association with incidence of clinical malaria in children living in an area of seasonal malaria transmission in Burkina Faso, West Africa

There is longstanding evidence that immunoglobulin G (IgG) has a role in protection against clinical malaria, and human antibodies of the cytophilic subclasses are thought to be particularly critical in this respect. In this cohort study, 286 Burkinabè children 6 months to 15 years old were kept under malaria surveillance in order to assess the protective role of antibody responses against four antigens which are currently being evaluated as vaccine candidates: apical membrane antigen 1 (AMA1), merozoite surface protein 1-19 (MSP1-19), MSP3, and glutamate-rich protein (GLURP). Total IgG, IgM, and IgG subclass responses were measured just before the malaria transmission season. The incidence of malaria was 2.4 episodes per child year of risk. After adjusting for the confounding effects of age, the level of total IgG to GLURP was strongly associated with reduced malaria incidence (incidence rate ratio associated with a doubling of total IgG, 0.79; 95% confidence interval, 0.66 to 0.94; P = 0.009.); there was a borderline statistically significant association between the level of total IgG to MSP3 and malaria incidence and no evidence of an association for total IgG to AMA1 and to MSP1-19. Of the IgG subclass responses studied, only IgG3 and IgG4 against GLURP and IgG1 against AMA1 were associated with reduced risk of clinical malaria. There was no evidence of an interaction between responses to AMA1 and baseline parasitemia in their effects on malaria incidence. Currently included in malaria vaccine formulations for clinical trials in humans, these blood-stage antigens, AMA1 and GLURP, offer good prospects for malaria vaccine development.     

Antibodies to the N-terminal block 2 of Plasmodium falciparum merozoite surface protein 1 are associated with protection against clinical malaria

This longitudinal prospective study shows that antibodies to the N-terminal block 2 region of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) are associated with protection against clinical malaria in an area of stable but seasonal malaria transmission of Ghana. Antibodies to the block 2 region of MSP-1 were measured in a cohort of 280 children before the beginning of the major malaria transmission season. The cohort was then actively monitored for malaria, clinically and parasitologically, over a period of 17 months. Evidence is presented for an association between antibody responses to block 2 and a significantly reduced risk of subsequent clinical malaria. Furthermore, statistical survival analysis provides new information on the duration of the effect over time. The results support a conclusion that the block 2 region of MSP-1 is a target of protective immunity against P. falciparum and, thus, a promising new candidate for the development of a malaria vaccine.     

Characterization of human T- and B-cell epitopes in the C terminus of Plasmodium falciparum merozoite surface protein 1: evidence for poor T-cell recognition of polypeptides with numerous disulfide bonds.

We have investigated the relationship between cellular and humoral immune responses to defined epitopes of the C terminus of merozoite surface protein 1 (MSP-1) of the human malaria parasite, Plasmodium falciparum, in immune blood donors. Sera from almost all donors contained antibodies to the 33-kDa processing product of the MAD20 allele of MSP-1 (MSP-1(33)), but these antibodies did not cross-react with the equivalent sequence of the Wellcome allele. In contrast, T-cell responses to MSP-1(33) are directed towards epitopes that are conserved between the two allelic families. Only 50% of adult blood donors possessed antibodies which recognized the 19-kDa processing product of MSP-1 (MSP-1(19)). These antibodies predominantly recognized conserved epitopes involving both of the constituent epidermal growth factor-like domains of MSP-1(19). T-cell responses were found in only 26% (for recombinant proteins) or 44% (for synthetic peptides) of donors and were directed mainly at dimorphic sequences of the protein. There was no obvious association, at an individual level, between the presence of antibodies and the detection of T-cell proliferative or gamma interferon responses, suggesting that the T cells identified in this manner are not providing significant levels of help to B cells. T-cell responses to reduced recombinant proteins and linear peptides were more prevalent than responses to disulfide-bonded proteins, suggesting that the complex disulfide-bonded structure of native MSP-1(19) may inhibit antigen processing or presentation.     

Malaria transmission-blocking immunity induced by natural infections of Plasmodium vivax in humans.

Immunity to malarial infections in human populations is known to affect the development of the asexual blood stages of the parasites in the human host and to be capable of conferring significant protection against morbidity and mortality due to the disease. In this study we show that during acute infection with Plasmodium vivax malaria, one of the two main malarial pathogens of humans, most individuals also develop immunity that suppresses the infectivity of the sexual stages of the parasite to mosquitoes. The immunity is antibody mediated and is directed against the parasites in the mosquito midgut shortly after ingestion of blood by a mosquito. This immunity could be expected to have significant effects on the natural transmission of P. vivax malaria.     

Association between protection against clinical malaria and antibodies to merozoite surface antigens in an area of hyperendemicity in Myanmar: complementarity between responses to merozoite surface protein 3 and the 220-kilodalton glutamate-rich protein

We performed a longitudinal clinical and parasitological follow-up study in OoDo, a village in southeast Asia in which malaria is hyperendemic, in order to assess the association between protection against malaria attacks and antibodies to three currently evaluated vaccine candidates, merozoite surface protein 1 (MSP1), MSP3, and the 220-kDa glutamate-rich protein (GLURP) from Plasmodium falciparum. Our results showed that the levels of cytophilic immunoglobulin G3 (IgG3) antibodies against conserved regions of MSP3 and GLURP were significantly correlated with protection against clinical P. falciparum malaria. In contrast, the levels of noncytophilic IgG4 antibodies against GLURP increased with the number of malaria attacks. Furthermore, we observed a complementary effect of the MSP3- and GLURP-specific IgG3 antibodies in relation to malaria protection. In the individuals that did not respond to one of the antigens, a strong response to the other antigen was consistently detected and was associated with protection, suggesting that induction of antibodies against both MSP3 and GLURP could be important for the development of protective immunity. The complementarity of the responses to the two main targets of antibody-dependent cellular inhibition identified to date provides the first rational basis for combining these two antigens in a hybrid vaccine formulation.     

Acquisition of Antibodies against Plasmodium falciparum Merozoites and Malaria Immunity in Young Children and the Influence of Age,Force of Infection,and Magnitude of Response

Individuals in areas of Plasmodium falciparum endemicity develop immunity to malaria after repeated exposure. Knowledge of the acquisition and nature of protective immune responses to P. falciparum is presently limited, particularly for young children. We examined antibodies (IgM, IgG, and IgG subclasses) to merozoite antigens and their relationship to the prospective risk of malaria in children 1 to 4 years of age in a region of malaria endemicity in Papua New Guinea. IgG, IgG1, and IgG3 responses generally increased with age, were higher in children with active infection, and reflected geographic heterogeneity in malaria transmission. Antigenic properties, rather than host factors, appeared to be the main determinant of the type of IgG subclass produced. High antibody levels were not associated with protection from malaria; in contrast, they were typically associated with an increased risk of malaria. Adjustment for malaria exposure, using a novel molecular measure of the force of infection by P. falciparum, accounted for much of the increased risk, suggesting that the antibodies were markers of higher exposure to P. falciparum. Comparisons between antibodies in this cohort of young children and in a longitudinal cohort of older children suggested that the lack of protective association was explained by lower antibody levels among young children and that there is a threshold level of antibodies required for protection from malaria. Our results suggest that in populations with low immunity, such as young children, antibodies to merozoite antigens may act as biomarkers of malaria exposure and that, with increasing exposure and responses of higher magnitude, antibodies may act as biomarkers of protective immunity.     

Plasmodium vivax promiscuous T-helper epitopes defined and evaluated as linear peptide chimera immunogens

Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1* alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development.     

Immunization of Aotus nancymai with recombinant C terminus of Plasmodium falciparum merozoite surface protein 1 in liposomes and alum adjuvant does not induce protection against a challenge infection.

Merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is an antimalarial vaccine candidate. The highly conserved 19-kDa C-terminal processing fragment of MSP-1 (MSP-1(19)) is of particular interest since it contains epitopes recognized by monoclonal antibodies which inhibit the invasion of erythrocytes in vitro. The presence of naturally acquired anti-MSP-1(19) antibodies in individuals exposed to malaria has been correlated with reduced morbidity, and immunization with an equivalent recombinant P. yoelii antigen induces substantial protection against this parasite in mice. We have expressed P. falciparum MSP-1(19) in Escherichia coli as a correctly folded protein and immunized Aotus nancymai monkeys by using the protein incorporated into liposomes and adsorbed to alum. After vaccination, the sera from these animals contained anti-MSP-1(19) antibodies, some of which competed for binding to MSP-1(19) with monoclonal antibodies that inhibit parasite invasion of erythrocytes in vitro. However, after challenge with either a homologous or a heterologous strain of parasite, all animals became parasitemic and required treatment. The immunization did not induce protection in this animal model.     

Diagnosis of vivax malaria using an IgM capture ELISA is a sensitive method,even for low levels of parasitemia

Although diagnosis of Plasmodium vivax malaria has been difficult when it is present at a low parasite density, it was recently revealed that an antibody assay was a good method of screening for malaria in blood banks. However, the use of this method for the diagnosis of malaria is limited due to the persistence of specific immunoglobulin (Ig) G. Therefore, we evaluated specific IgM antibody responses against the C-terminal region of the merozoite surface protein 1 of P. vivax (PvMSP1c) in sera obtained from patients with vivax malaria using various assays. The IgM capture enzyme-linked immunosorbent assay showed good sensitivity (97.7%; 308/315) and specificity (99.1%, 446/450). In addition, the results of this assay were not related to parasite density, and a high reactivity was observed when there was a low level of parasitemia. Furthermore, we found that patients with cases of malaria that had relapsed still had the IgM titers against PvMSP1c. Therefore, the use of IgM ELISA for the detection of specific IgM that was not involved in memorial immune activity could be an alternative tool for the diagnosis of malaria and blood screening, even in areas in which malaria is endemic.     

Parity and placental infection affect antibody responses against Plasmodium falciparum during pregnancy

Women are at higher risk of Plasmodium falciparum infection when pregnant. The decreasing risk of malaria with subsequent pregnancies is attributed to parity-dependent acquisition of antibodies against placental parasites expressing variant surface antigens, VAR2CSA, that mediate placental sequestration through adhesion to chondroitin sulfate A (CSA). However, modulation of immunity during pregnancy may also contribute to increase the risk of malaria. We compared antibody responses among 30 Mozambican primigravidae and 60 multigravidae at delivery, 40 men, and 40 children. IgG levels were measured against the surface antigens of erythrocytes infected with P. falciparum isolated from 12 pregnant women (4 placental and 8 peripheral blood isolates) and 26 nonpregnant hosts. We also measured IgG levels against merozoite recombinant antigens and total IgG. Placental P. falciparum infection was associated with increased levels of total IgG as well as IgG levels against merozoite antigens and parasite isolates from pregnant and nonpregnant hosts. We therefore stratified comparisons of antibody levels by placental infection. Compared to multigravidae, uninfected primigravidae had lower total IgG as well as lower levels of IgGs against peripheral blood isolates from both pregnant and nonpregnant hosts. These differences were not explained by use of bed nets, season at delivery, neighborhood of residence, or age. Compared to men, infected primigravidae had higher levels of IgGs against isolates from pregnant women and CSA-binding lines but not against other isolates, supporting the concept of a pregnancy-specific development of immunity to these parasite variants. Results of this study show that parity and placental infection can modulate immune responses during pregnancy against malaria parasites.     

Cytophilic immunoglobulin responses to Plasmodium falciparum glutamate-rich protein are correlated with protection against clinical malaria in Dielmo, Senegal

The goal of this study was to analyze antibody responses to Plasmodium falciparum glutamate-rich protein (GLURP) using clinical data and plasma samples obtained from villagers of Dielmo, Senegal. This molecule was chosen because it is targeted by human antibodies which induce parasite growth inhibition in antibody-dependent cellular inhibition (ADCI) assays. The results showed a strong correlation between protection against malaria attacks and levels of immunoglobulin G2 (IgG2) and IgG3 against GLURP(94-489) (R0) and IgG3 against GLURP(705-1178) (R2) when corrected for the confounding effect of age-related exposure to malaria. Thus, GLURP may play a role in the induction of protective immunity against P. falciparum malaria.     

Acquired immune responses to the N- and C-terminal regions of Plasmodium vivax merozoite surface protein 1 in individuals exposed to malaria.

In this study, we evaluated the naturally acquired immune response to Plasmodium vivax merozoite surface protein 1 (PvMSP1) in individuals with recent clinical episodes of malaria from the state of Para, Brazil. Ten recombinant proteins representing the first 682 amino acids (aa) of the N-terminal region and one representing the final 111 aa of the C-terminal region were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Both of these regions have been suggested as candidates for development of a vaccine against Plasmodium sp. The total frequencies of individuals with antibodies and cellular immune responses to PvMSP1 were high (83.8 and 75%, respectively). The recombinant proteins representing the N- and C-terminal regions were recognized by 51.4 and 64.1% of sera, respectively. The frequency of responders to the C-terminal region increased according to the number of previous malaria episodes, reaching 83.3% after four episodes. Cellular immune response was measured by in vitro proliferation and gamma interferon production. Peripheral blood mononuclear cells of 75 and 47.2% of individuals proliferated in response to stimulation by the N- and C-terminal regions, respectively. Also, we found that one protein representing the N terminus and a second representing the C terminus of PvMSP1 stimulated 54.5% of individuals to secrete gamma interferon. We concluded that PvMSP1 is immunogenic to a large proportion of individuals exposed to malaria. Our results also suggested that the C-terminal region of PvMSP1 containing the two epidermal growth factor-like domains is particularly immunogenic to antibodies and T cells during natural infection in humans.     

Vaccine efficacy of recombinant Plasmodium falciparum merozoite surface protein 1 in malaria-naive, -exposed, and/or -rechallenged Aotus vociferans monkeys

Protection against a lethal challenge infection of Plasmodium falciparum was elicited in malaria-naive Aotus vociferans monkeys by vaccination with the C terminus 19-kDa protein of the major merozoite surface protein (MSP-1(19)) fused to tetanus toxoid universal T-cell epitopes P30 and P2. Three of four monkeys were protected against a 10(4)-parasite challenge. Four monkeys were challenged with 10(5) parasites; one self-cured the infection, two were protected against high parasitemia (<2%) but were treated for severe anemia (hematocrit of <25%), and the fourth was not protected. In this model system, anemia appears to be a manifestation of incomplete protection (prolonged low-level parasitemia). Enzyme-linked immunosorbent assay (ELISA) antibody titers correlated with protection. Antibodies from some protected monkeys inhibited secondary processing of MSP-1(42) to MSP-1(33) and MSP-1(19). To mimic the repeated reinfections seen in regions where malaria is endemic, a second malaria parasite challenge was administered 4 months later. All P30P2MSP-1(19)-vaccinated monkeys were protected; thus, a single challenge infection may underestimate vaccine efficacy. ELISA antibody titers correlated with protection against a second infection but had decreased compared to the first challenge. As most target populations for asexual blood-stage malaria vaccines will have been exposed to malaria parasites, a malaria parasite-exposed monkey was vaccinated with P30P2MSP-1(19). This monkey was completely protected, while a malaria parasite-naive P30P2MSP-1(19)-vaccinated monkey self-cured a low-grade parasitemia. Prior malaria parasite infection primed the production of anti-native MSP-1(19) antibodies, which were boosted by vaccination with recombinant P30P2MSP-1(19). Preliminary data suggest that immunogenicity studies of vaccines designed for malaria parasite-exposed populations should also be conducted in malaria parasite-exposed subjects.     

Expression, purification, and characterization of the immunological response to a 40-kilodalton Plasmodium vivax tryptophan-rich antigen

We describe here an approximately 40-kDa Plasmodium vivax tryptophan-rich antigen (PvTRAg40) which contains 321 amino acids and 11.4% tryptophan residues. This protein shows 65% homology (35% identity) with the previously described PvTRAg, besides sharing 23 of 27 positionally conserved tryptophan residues and similar genomic organization. The nucleotide sequence of the entire tryptophan-rich domain of PvTRAg40 was identical among 35 P. vivax clinical isolates. The protein is expressed by ring, trophozoite, and schizont stages of the parasite. The cDNA covering exon 2 of PvTRAg40 was cloned and expressed in the pPROEXHTa vector, and recombinant protein was purified. A high humoral immune response (90.7% seropositivity; n = 43) against this recombinant protein was detected in humans during the course of natural P. vivax infection. Eighty percent of the total of 20 P. vivax-exposed individuals exhibited lymphoproliferative responses against this antigen. The T cells of these individuals produced larger amounts of interleukin-12 (IL-12), IL-4, and IL-10 than gamma interferon and tumor necrosis factor alpha cytokines in response to the recombinant protein. Production of Th2-biased cytokines, conserved T- and B-cell epitopes, and an enhanced humoral immune response indicate that PvTRAg40 could possibly induce antibody-mediated immune protection against infection.     

Monoclonal antibodies to the circumsporozoite protein repeats of a Plasmodium vivax-like human malaria parasite and Plasmodium simiovale.

We have recently described a Plasmodium vivax-like human malaria parasite. The circumsporozoite protein of this parasite is identical to that of a simian malaria parasite, P. simiovale, but different from two known types of P. vivax. Here, we describe the production of two monoclonal antibodies, Pam 172 and Pam 135, specific for the circumsporozoite protein repeat sequence APGANQEGGAA of the P. vivax-like malaria parasite. These two monoclonal antibodies recognized air-dried sporozoites of P. simiovale but not other human, simian, or rodent malaria parasites tested.     

Neonatal and maternal immunological responses to conserved epitopes within the DBL-gamma3 chondroitin sulfate A-binding domain of Plasmodium falciparum erythrocyte membrane protein 1

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the adherence of P. falciparum-infected erythrocytes to placental syncytiotrophoblasts via interactions with chondroitin sulfate A (CSA), a characteristic of pregnancy-associated malaria. Pregnancy-associated malaria predicts increased susceptibility of newborns to malaria, and it is postulated that transplacental passage of parasite antigen induces immune regulatory activity in the neonate. We wished to examine the immune responsiveness to a CSA-binding domain of PfEMP1, the DBL-gamma3 domain, in cord and maternal venous blood obtained from pregnancies with various histories of P. falciparum infection. We assessed in vitro T-cell cytokine and plasma immunoglobulin G (IgG) and IgM responses to four peptides corresponding to highly conserved regions of a DBL-gamma3 domain common to central African parasite isolates. The presence of placental P. falciparum infection at delivery was associated with elevated frequencies of DBL-gamma3 peptide-specific CD3+ interleukin-10-positive T cells in cord blood, while treatment and clearance of infection prior to delivery was associated with elevated frequencies of CD3+ gamma interferon-positive T cells. DBL-gamma3 peptide-specific IgM antibodies were detected in 12 of 60 (20%) cord plasma samples from those born to mothers with P. falciparum infection during pregnancy. Consistent with polyclonal anti-PfEMP1 antibody responses that are associated with protection against pregnancy-associated malaria, the presence of maternal IgG antibodies with specificity for one of the DBL-gamma3 peptides showed a parity-dependent profile. These data demonstrate that peptides corresponding to conserved regions of the DBL-gamma3 domain of PfEMP1 are immunogenic in P. falciparum-infected mothers and their offspring.     

Naturally acquired human antibodies which recognize the first epidermal growth factor-like module in the Plasmodium falciparum merozoite surface protein 1 do not inhibit parasite growth in vitro.

Merozoite surface protein 1, one of the major surface proteins of the invasive blood stage of the malaria parasite, is a prime candidate for the development of a vaccine against the human disease. Previously, monoclonal antibodies which both inhibited the growth of Plasmodium falciparum in vitro and bound to the first of two epidermal growth factor-like modules located near the carboxy terminus of the protein had been identified. In this study, we have used affinity chromatography on a recombinant fusion protein corresponding to the first epidermal growth factor-like module in P. falciparum merozoite surface protein 1 to prepare antibody induced by natural infection. The antibody was purified from the total immunoglobulin G fraction of adult West African donors, shown to passively confer immunity against falciparum malaria. Such affinity-purified antibodies were shown to recognize the native protein by a number of separate criteria and to block the binding of an inhibitory monoclonal antibody, but they failed to inhibit parasite invasion in an in vitro growth assay. These results indicate that antibody alone is not sufficient to interfere with erythrocyte invasion.