Differential Recognition of Plasmodium falciparum Merozoite Surface Protein 2 Variants by Antibodies from Malaria Patients in Brazil

Four variants of merozoite surface protein 2 (MSP-2) of Plasmodium falciparum were used in serology to examine whether changes in repeat units affect its recognition by antibodies during infection with parasites of known MSP-2 types. The results indicate that variation in MSP-2 repeats may represent a mechanism for parasite immune evasion.     

Antigenic Characterization of an Intrinsically Unstructured Protein,Plasmodium falciparum Merozoite Surface Protein 2

Merozoite surface protein 2 (MSP2) is an abundant glycosylphosphatidylinositol (GPI)-anchored protein of Plasmodium falciparum, which is a potential component of a malaria vaccine. As all forms of MSP2 can be categorized into two allelic families, a vaccine containing two representative forms of MSP2 may overcome the problem of diversity in this highly polymorphic protein. Monomeric recombinant MSP2 is an intrinsically unstructured protein, but its conformational properties on the merozoite surface are unknown. This question is addressed here by analyzing the 3D7 and FC27 forms of recombinant and parasite MSP2 using a panel of monoclonal antibodies raised against recombinant MSP2. The epitopes of all antibodies, mapped using both a peptide array and by nuclear magnetic resonance (NMR) spectroscopy on full-length recombinant MSP2, were shown to be linear. The antibodies revealed antigenic differences, which indicate that the conserved N- and C-terminal regions, but not the central variable region, are less accessible in the parasite antigen. This appears to be an intrinsic property of parasite MSP2 and is not dependent on interactions with other merozoite surface proteins as the loss of some conserved-region epitopes seen using the immunofluorescence assay (IFA) on parasite smears was also seen on Western blot analyses of parasite lysates. Further studies of the structural basis of these antigenic differences are required in order to optimize recombinant MSP2 constructs being evaluated as potential vaccine components.     

恶性疟原虫裂殖子表面蛋白3功能区片段的制备及其免疫原性分析

目的构建恶性疟原虫裂殖子表面蛋白3功能片段MSP3（69）与谷胱甘肽（GST）的融合蛋白，并在大肠杆菌中进行表达，分析其免疫原性。方法采用大肠杆菌系统表达融合蛋白GST—MSP3（69），以疟疾病人血清进行Western-blot，并以ISA720、佛氏、氢氧化铝与CpG联合3种佐剂与GST—MSP3（69）融合蛋白乳化，免疫Balb／ca小鼠．分析其免疫原性。结果在大肠杆菌系统中成功表达GST-MSP3（69）融合蛋白，疟疾病人血清能与融合蛋白反应。3种佐剂免疫Balb／ca小鼠均能诱发Balb／ca小鼠产生较高水平的特异抗体，3次免疫后的抗体滴度分别为4．5×10^5，4．1×10^5和3．5×10^5。3次免疫后血清抗体亚型分析显示该蛋白能够诱导Balb／ca小鼠产生较高滴度的亲细胞亚型IgG1和IgG2b．ISA720、佛氏、氢氧化铝与CpG联合佐剂组IgG1分别为8×104、7．8×10^4、6．8×10^4．IgG2b分别为1．2×10^4，1．25×10^4、1．5×10^4。统计学分析显示不同佐剂组的抗体滴度及抗体亚型滴度之间差异均无统计学意义（P〉0．05）。结论制备了在大肠杆菌表达的GST-MSP3（69）融合蛋白，该蛋白具有高免疫原性，并产生亲细胞亚型抗体，这些为研究MSP3蛋白功能区的免疫保护作用打下了基础。     

Plasmodium falciparum Merozoite Surface Protein 1 (MSP-1)-MSP-3 Chimeric Protein: Immunogenicity Determined with Human-Compatible Adjuvants and Induction of Protective Immune Response

A chimeric gene, MSP-Fu₂₄, was constructed by genetically coupling immunodominant, conserved regions of the two leading malaria vaccine candidates, Plasmodium falciparum merozoite surface protein 1 (C-terminal 19-kDa region [PfMSP-1₁₉]) and merozoite surface protein 3 (11-kDa conserved region [PfMSP-3₁₁]). The recombinant MSP-Fu₂₄ protein was produced in Escherichia coli cells and purified to homogeneity by a two-step purification process with a yield of ∼30 mg/liter. Analyses of conformational properties of MSP-Fu₂₄ using PfMSP-1₁₉-specific monoclonal antibody showed that the conformational epitopes of PfMSP-1₁₉ that may be critical for the generation of the antiparasitic immune response remained intact in the fusion protein. Recombinant MSP-Fu₂₄ was highly immunogenic in mice and in rabbits when formulated with two different human-compatible adjuvants and induced an immune response against both PfMSP-1₁₉ and PfMSP-3₁₁. Purified anti-MSP-Fu₂₄ antibodies showed invasion inhibition of P. falciparum 3D7 and FCR parasites, and this effect was found to be dependent on antibodies specific for the PfMSP-1₁₉ component. The protective potential of MSP-Fu₂₄ was demonstrated by in vitro parasite growth inhibition using an antibody-dependent cell inhibition (ADCI) assay with anti-MSP-Fu₂₄ antibodies. Overall, the antiparasitic activity was mediated by a combination of growth-inhibitory antibodies generated by both the PfMSP-1₁₉ and PfMSP-3₁₁ components of the MSP-Fu₂₄ protein. The antiparasitic activities elicited by anti-MSP-Fu₂₄ antibodies were comparable to those elicited by antibodies generated with immunization with a physical mixture of two component antigens, PfMSP-1₁₉ and PfMSP-3₁₁. The fusion protein induces a protective immune response with human-compatible adjuvants and may form a part of a multicomponent malaria vaccine.Malaria is among the major parasitic diseases in tropical and subtropical countries. With as many as 300 to 500 million new cases each year, malaria accounts for the death of over 2 million people globally each year, and most are children (41). Among the four species of Plasmodium that infect humans, the most threatening is Plasmodium falciparum. The extensive spread of drug-resistant P. falciparum strains as well as the insecticide-resistant mosquito necessitates the development of a malaria vaccine on an urgent basis. Collectively, the major objective of the ongoing vaccine effort in this field is to develop a multistage, multivalent vaccine against P. falciparum (34).The blood-stage cycle of the parasite is responsible for malaria pathogenesis. Intervention at this stage of the parasite''s development through vaccination is likely to reduce malaria-related clinical symptoms. As a major interface between host and pathogen, the merozoite surface is an obvious target for the development of a malaria vaccine. A number of potential vaccine candidate antigens identified so far are located on or associated with the surface of the merozoite or in apical organelles. These include merozoite surface protein 1 (MSP-1), MSP-2, MSP-3, MSP-4, MSP-5, MSP-8, RAP1/2, AMA-1, and EBA-175, which are implicated in the process of merozoite invasion of the erythrocyte (23).MSP-1 is one of the most extensively studied proteins of P. falciparum (18). It is synthesized as a ∼200-kDa precursor and then processed in two steps: the primary processing step produces a complex of four fragments that are present on the merozoite surface, and the secondary processing step at invasion results in the shedding of the complex from the surface, except for the C-terminal 19-kDa domain (MSP-1₁₉), which remains anchored to the parasite surface by a glycosylphosphatidylinositol (GPI) moiety (2). The C-terminal 19-kDa fragment of MSP-1 is well conserved among P. falciparum isolates and contains two epidermal growth factor (EGF)-like domains that play a role in merozoite invasion. Substantial data from studies with P. falciparum MSP-1 and in vivo immunization studies of mice with Plasmodium yoelii and Plasmodium chabaudi indicate that the protective immune responses are directed against the C-terminal 19-kDa domain (10, 12, 15, 20, 27, 35). The inhibition of MSP-1 processing by conformation-specific antibodies (Abs) was previously proposed to be one of the possible mechanism for the inhibition of merozoite invasion (1).Another merozoite surface protein, MSP-3, was also shown to be the target of the protective immune responses in humans (29). The PfMSP-3 protein contains three blocks of four tandem heptad repeats based on the AXXAXX motif at the N terminus, a glutamic acid-rich domain, and a putative leucine zipper sequence at the C terminus (25). Although a clear surface localization of PfMSP-3 is known, it lacks any transmembrane domain or glycosylphosphatidylinositol (GPI) anchor site (24, 25) and is therefore considered to be loosely associated with the merozoite surface by interactions with other merozoite surface proteins. PfMSP-3 was identified as a candidate vaccine antigen by an antibody-dependent cellular inhibition (ADCI) assay using human immune sera (28). The potential of PfMSP-3 as a vaccine candidate was further illustrated by ADCI using mice antibodies and was further confirmed by the suppression of P. falciparum growth in an immunocompromised mouse after the passive transfer of human antibodies purified on MSP-3 peptides together with human monocytes (28, 40, 42). The immunization of Aotus and Saimiri monkeys with recombinant PfMSP-3 or its fragments provided protection against parasite challenge (6, 16). A 70-amino-acid-long conserved domain of PfMSP-3, referred to here as the PfMSP-3₁₁ region, was identified as the target of protective antibodies in human immune responses (40). The presence of high titers of cytophilic antibodies, IgG3, against this conserved region of MSP-3 has been correlated with protection against the parasite. In addition, immunization of humans with a synthetic peptide corresponding to this region was previously shown to induce antiparasitic antibodies that suppress parasite growth in an ADCI assay (11).It is generally believed that a combination vaccine for malaria is likely to be more effective than vaccines based on a single antigen, and attempts are being made to develop a malaria vaccine by using a mixture of more than one antigen or by combining immunologically relevant proteins of the target antigens as fusion proteins (31, 43, 45). In the present study, we have constructed a fusion chimera (MSP-Fu₂₄) consisting of PfMSP-1₁₉ and PfMSP-3₁₁ and produced the corresponding recombinant MSP-Fu₂₄ protein in Escherichia coli cells. The two individual components, PfMSP-1₁₉ and PfMSP-3₁₁, were also expressed and purified separately; the immunological properties of MSP-Fu₂₄ were compared with a physical mixture of the two individual components. MSP-Fu₂₄ retained the native conformation of the PfMSP-1₁₉ component and was highly immunogenic in small animals. The anti-MSP-Fu₂₄ antibodies inhibited parasite invasion into host red blood cells (RBCs) and also inhibited parasite growth in a monocyte-dependent manner, suggesting the potential of the fusion protein as a malaria vaccine candidate.     

Pathways for Potentiation of Immunogenicity during Adjuvant-Assisted Immunizations with Plasmodium falciparum Major Merozoite Surface Protein 1

Vaccine adjuvants exert critical and unique influences on the quality of immune responses induced during active immunizations. We investigated the mechanisms of action of immunological adjuvants in terms of their requirements for cytokine-mediated pathways for adjuvanticity. Antibody responses potentiated by several adjuvants to a Plasmodium falciparum MSP1-19 (C-terminal 19-kDa processing fragment of MSP1) vaccine were studied in gamma interferon (IFN-γ) or interleukin (IL-4) knockout mice. The levels of anti-MSP1-19 antibodies and the induction of Th1- and Th2-type antibodies were analyzed. Results revealed a spectrum of requirements for cytokine-mediated pathways in the potentiation of immunogenicity, and such requirements were influenced by interactions among individual components of the adjuvant formulations. One adjuvant strictly depended on IFN-γ to induce appreciable levels of anti-MSP1-19 antibodies, while some formulations required IFN-γ only for the induction of Th1-type antibodies. Other formulations induced exclusively Th2-type antibodies and were not affected by IFN-γ knockout. There were three patterns of requirements for IL-4 by various adjuvants in the induction of Th2-type anti-MSP1-19 antibodies. Moreover, the induction of Th1-type anti-MSP1-19 antibodies by adjuvants showed two distinct patterns of regulation by IL-4. The utilization of an IL-4 regulated pathway(s) for the induction of Th2-type antibodies by the same adjuvant differed between mouse strains, suggesting that animal species variability in responses to vaccine adjuvants may be due, at least in part, to differences in the utilization of immune system pathways by an adjuvant among animal hosts.     

Inhibitory Antibodies Specific for the 19-Kilodalton Fragment of Merozoite Surface Protein 1 Do Not Correlate with Delayed Appearance of Infection with Plasmodium falciparum in Semi-Immune Individuals in Vietnam

Inhibitory antibodies specific for the 19-kDa fragment of merozoite surface protein 1 (MSP1₁₉) are a significant component of inhibitory responses in individuals immune to malaria. Nevertheless, conflicting results have been obtained in determining whether this antibody specificity correlates with protection in residents of areas where malaria is endemic. In this study, we examined sera collected from a population of semi-immune individuals living in an area of Vietnam with meso-endemicity during a 6-month period. We used two Plasmodium falciparum parasite lines that express either endogenous MSP1₁₉ or the homologous region from Plasmodium yoelii to measure the MSP1₁₉-specific inhibitory activity. We showed that (i) the level of MSP1₁₉-specific inhibitory antibodies was not associated with a delay in P. falciparum infection, (ii) MSP1₁₉-specific inhibitory antibodies declined significantly during the convalescent period after infection, and (iii) there was no significant correlation between the MSP1₁₉-specific inhibitory antibodies and the total antibodies measured by enzyme-linked immunosorbent assay. These results have implications for understanding naturally acquired immunity to malaria and for the development and evaluation of MSP1₁₉-based vaccines.Infection of humans by Plasmodium falciparum remains one of the most deadly infectious diseases worldwide, leading to approximately 1 million deaths annually, predominantly in children under 5 years of age. It is the infection of red blood cells by asexual parasites that is associated with all clinical signs and symptoms and is responsible for malaria morbidity and mortality. In areas where malaria is endemic, immunity to this stage develops after repeated exposure and acts to prevent symptomatic illness and severe complications and to limit parasitemia (19). This immunity can be transferred passively among humans (6, 29), suggesting that antibodies are an important component of protective immunity. Attention has been devoted to mechanisms by which antibodies act to protect humans and to the identification of P. falciparum proteins that may be the targets of such protective antibodies and that may in turn induce such protective antibodies when administered in a vaccine.The C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP1₁₉) is a major target of protective antibodies against blood-stage infection and a leading candidate for inclusion in a subunit malaria vaccine. Studies with rodent and nonhuman primate models have shown that passive transfer of anti-MSP1₁₉ antibodies or immunization with recombinant MSP1₁₉ can provide significant protection against lethal challenge (8, 17, 18). Antibodies to MSP1₁₉, affinity purified from either immune human sera or monoclonal or polyclonal experimental sera, are capable of inhibiting parasite growth in vitro (3, 12, 27). However, the association between levels of MSP1₁₉-specific antibodies in humans and clinical immunity remains unclear. Using approaches such as enzyme-linked immunosorbent assay (ELISA), the levels of MSP1₁₉-specific antibodies have been quantified in many field studies, and correlations with protection have not been observed consistently (1, 11, 13, 16, 28, 30). ELISAs do not account for antibody affinity and fine specificity, which may be critical for functional activity. Monoclonal antibodies directed against MSP1₁₉ have been shown to have various effects on parasite growth, ranging from inhibition to enhancement. These specificities, as well as the presence of antibodies that block the action of inhibitory antibodies, have been detected in naturally acquired responses (15, 23). Thus, it remains unclear how antibody levels relate to inhibitory function in immune humans.Relatively few field studies have examined the association between the subset of growth inhibitory antibodies and protective immunity due to methodological constraints on performing these assays in a reproducible and reliable manner (10, 20, 25). The recent development of paired transgenic P. falciparum lines that differ only in their MSP1₁₉ region has provided a tool with which to measure MSP1₁₉-specific inhibitory antibodies (24). By calculating the difference in the levels of inhibition of the two parasite lines in the presence of a particular serum, the inhibitory effect attributable to MSP1₁₉-specific antibodies can be determined. Using this assay, O''Donnell et al. demonstrated that MSP1₁₉-specific antibodies capable of inhibiting parasite growth were a major component of inhibitory responses in serum samples from individuals living in Papua New Guinea (24). However, such a finding does not establish if individuals with these particular inhibitory antibodies are immune and whether detection of these antibodies is an accurate correlate of protection. Two field studies using this functional assay reached conflicting conclusions: one study in western Kenya during a malaria epidemic revealed a correlation between MSP1₁₉-specific inhibitory antibodies and protection from P. falciparum infection (16), whereas a study conducted in Gambia showed that the MSP1₁₉-specific inhibitory antibodies were not associated with protection (7). It is important to resolve this discrepancy and to determine the generality of the observation and whether it applies outside Africa to different ethnic groups and differing levels of malaria transmission. It is also important to determine the kinetics of acquisition and maintenance of these specificities during infection.We previously examined a group of transmigrants who experienced sequential infections during settlement in an area where malaria is highly endemic and showed that the acquisition of the MSP1₁₉-specific inhibitory antibodies required two or more infections (22). This contrasts with the case for Australian travelers returning with P. falciparum infection, as nearly half of the individuals showed significant inhibitory antibodies after their first infection (14).In this study, we examined a population of semi-immune individuals from southern central Vietnam who were drug cured and then intensively monitored over a 6-month period for infection. We measured the MSP1₁₉-specific inhibitory antibodies in the serum samples from these individuals and showed that they did not correlate with a delay in infection. We also investigated the kinetics of MSP1₁₉-specific inhibitory antibodies during infection, drug treatment, and convalescence, as well as their correlation with antibody levels measured by ELISA.     

Temporal Variation of the Merozoite Surface Protein-2 Gene of Plasmodium falciparum

Extensive polymorphism of key parasite antigens is likely to hamper the effectiveness of subunit vaccines against Plasmodium falciparum infection. However, little is known about the extent of the antigenic repertoire of naturally circulating strains in different areas where malaria is endemic. To address this question, we conducted a study in which blood samples were collected from parasitemic individuals living within a small hamlet in Western Irian Jaya and subjected to PCR amplification using primers that would allow amplification of the gene encoding merozoite surface protein-2 (MSP2). We determined the nucleotide sequence of the amplified product and compared the deduced amino acid sequences to sequences obtained from samples collected in the same hamlet 29 months previously. MSP2 genes belonging to both major allelic families were observed at both time points. In the case of the FC27 MSP2 family, we observed that the majority of individuals were infected by parasites expressing the same form of MSP2. Infections with parasites expressing 3D7 MSP2 family alleles were more heterogeneous. No MSP2 alleles observed at the earlier time point were detectable at the later time point, either for the population as a whole or for individuals who were assayed at both time points. We examined a subset of the infected patients by using blood samples taken between the two major surveys. In no patients could we detect reinfection by a parasite expressing a previously encountered form of MSP2. Our results are consistent with the possibility that infection induces a form of strain-specific immune response against the MSP2 antigen that biases against reinfection by parasites bearing identical forms of MSP2.The development of a host-protective immune response against Plasmodium falciparum takes several years and many episodes of infection, at least for children living in areas where malaria is endemic. One of the reasons for this is believed to be the large number of distinct parasite strains circulating within an area of endemicity and the assumption that exposure must occur to a sufficiently large sample of these before lasting immunity is induced. However, the detailed epidemiology of endemic malaria infection remains poorly understood at the molecular level, and there is surprisingly little nucleotide sequence data to support the concept of a large repertoire of antigenically distinct strains.There are at least six antigenically diverse proteins of the asexual stage that are known to be the target of potentially protective host responses. The definition of antigenically distinct strains involves identification of the allelic form expressed at all antigenically diverse loci—the extended antigenic haplotype. The loci would include merozoite surface protein-1 to -3 (3), apical membrane antigen-1 (17), S-antigen (6), and P. falciparum erythrocyte membrane protein-1 (PfEMP-1) (5). Such a complete molecular definition of infecting parasites is a highly ambitious task, particularly in the case of blood samples collected from patients harboring mixed infections. Accordingly, most studies focus on one or other of the antigenically diverse antigens. We have elected to study merozoite surface protein-2 (MSP2) (27), a 45- to 50-kDa glycoprotein anchored in the merozoite surface by a glycosylphosphatidylinositol anchor. This surface protein is a promising candidate for inclusion in a malaria subunit vaccine, as both in vitro and in vivo studies have demonstrated the ability of immune responses to MSP2 to inhibit parasite multiplication (23, 25). However, the efficacy of any subunit vaccine containing a single form of MSP2 may be limited by the presence of antigenically distinct parasite strains within an area of endemicity. We will adopt the recently proposed convention for parasite genes and gene products of denoting the gene sequence as MSP2 and the protein as MSP2.Sequence polymorphism has been described for MSP2 genes of both laboratory-maintained isolates (29) and field isolates (14, 16, 19, 30). Comparison of MSP2 gene sequences from these isolates reveals highly conserved 5′ and 3′ sequences that flank a central variable region. This central region is composed of repeats flanked by nonrepetitive sequences. The nonrepetitive sequences are one or other of two distinct forms that define two allelic families, FC27 and IC-1/3D7 (29). The central repeats are more variable and define the individual alleles of MSP2. There is a correlation between the general form of the central repeat sequence and the allelic family. For example, FC27 family members have variants of a central 96-bp pair sequence that may be present in one to four copies followed by a 21-bp partial repeat and a variably represented 36-bp sequence that may be present in one to five copies. In contrast, alleles belonging to the 3D7 family show a central repeat region made up of variable numbers of 12- to 24-bp repeats separated by repeating 6-bp sequences.Field studies aimed at defining the antigenic diversity of MSP2 have approached the problem by determining MSP2 gene structure by various forms of PCR. The rationale for this is that P. falciparum is haploid and MSP2 has been shown to be present in all laboratory and field isolates examined (8–10, 15). Most studies examining the distribution and frequency of different allelic forms of MSP2 have enumerated the presence of the allelic families (11, 12, 14). Whereas a skewed distribution of predominantly 3D7 family alleles exists among laboratory-adapted strains, in the field a more even distribution of FC27 and 3D7 alleles occurs. Often FC27 family alleles are more prevalent than 3D7 alleles, and novel FC27 and 3D7 family alleles have been found in field malaria strains (14, 16). Some field studies examining recurrent MSP2 infections have been performed, but these have classified MSP2 alleles on the basis of family and length of the central repeat region (7, 11, 14). This makes it difficult to form conclusions about the repertoire of repeat sequences in the circulating pool of parasites and to infer the possible action of immune responses to MSP2 repeats. We were interested to examine the sequences of MSP2 alleles circulating in an area of endemicity over time and to determine the persistence of various MSP2 alleles within a localized area. This study describes MSP2 genotypes from malaria-infected inhabitants of the Oksibil region of Irian Jaya and allows comparison of the variation in MSP2 sequences seen over a 2.5-year period within the region as a whole and in particular individuals.     

Passive Immunization with Antibodies against Three Distinct Epitopes on Plasmodium yoelii Merozoite Surface Protein 1 Suppresses Parasitemia

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an ~42-kDa fragment (MSP-1₄₂) that is derived from the C terminus of MSP-1. MSP-1₄₂ is further cleaved to an N-terminal ~33-kDa polypeptide (MSP-1₃₃) and a C-terminal ~19-kDa polypeptide (MSP-1₁₉) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-1₄₂ but not with either of the constituents MSP-1₃₃ and MSP-1₁₉, B4 recognized an epitope within the N terminus of MSP-1₃₃, and B6, B10, F5, and G3 bound to MSP-1₁₉. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.     

Identification of a Potent Combination of Key Plasmodium falciparum Merozoite Antigens That Elicit Strain-Transcending Parasite-Neutralizing Antibodies

Blood-stage malaria vaccines that target single Plasmodium falciparum antigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine against P. falciparum that targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixed in vitro against a diverse set of six key merozoite ligands, including the novel ligands P. falciparum apical asparagine-rich protein (PfAARP), EBA-175 (PfF2), P. falciparum reticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, and Plasmodium thrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverse P. falciparum clones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.     

Sequential Processing of Merozoite Surface Proteins during and after Erythrocyte Invasion by Plasmodium falciparum

Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.     

Acquired Antibodies to Merozoite Antigens in Children from Uganda with Uncomplicated or Severe Plasmodium falciparum Malaria

Malaria can present itself as an uncomplicated or severe disease. We have here studied the quantity and quality of antibody responses against merozoite antigens, as well as multiplicity of infection (MOI), in children from Uganda. We found higher levels of IgG antibodies toward erythrocyte-binding antigen EBA181, MSP2 of Plasmodium falciparum 3D7 and FC27 (MSP2-3D7/FC27), and apical membrane antigen 1 (AMA1) in patients with uncomplicated malaria by enzyme-linked immunosorbent assay (ELISA) but no differences against EBA140, EBA175, MSP1, and reticulocyte-binding protein homologues Rh2 and Rh4 or for IgM against MSP2-3D7/FC27.Patients with uncomplicated malaria were also shown to have higher antibody affinities for AMA1 by surface plasmon resonance (SPR). Decreased invasion of two clinical P. falciparum isolates in the presence of patient plasma correlated with lower initial parasitemia in the patients, in contrast to comparisons of parasitemia to ELISA values or antibody affinities, which did not show any correlations. Analysis of the heterogeneity of the infections revealed a higher MOI in patients with uncomplicated disease, with the P. falciparum K1 MSP1 (MSP1-K1) and MSP2-3D7 being the most discriminative allelic markers. Higher MOIs also correlated positively with higher antibody levels in several of the ELISAs. In conclusion, certain antibody responses and MOIs were associated with differences between uncomplicated and severe malaria. When different assays were combined, some antibodies, like those against AMA1, seemed particularly discriminative. However, only decreased invasion correlated with initial parasitemia in the patient, signaling the importance of functional assays in understanding development of immunity against malaria and in evaluating vaccine candidates.     

Development and pre-clinical analysis of a Plasmodium falciparum Merozoite Surface Protein-1(42) malaria vaccine

Merozoite Surface Protein-1(42) (MSP-1(42)) is a leading vaccine candidate against erythrocytic malaria parasites. We cloned and expressed Plasmodium falciparum MSP-1(42) (3D7 clone) in Escherichia coli. The antigen was purified to greater than 95% homogeneity by using nickel-, Q- and carboxy-methyl (CM)-substituted resins. The final product, designated Falciparum Merozoite Protein-1 (FMP1), had endotoxin levels significantly lower than FDA standards. It was structurally correct based on binding conformation-dependent mAbs, and was stable. Functional antibodies from rabbits vaccinated with FMP1 in Freund's adjuvant inhibited parasite growth in vitro and also inhibited secondary processing of MSP-1(42). FMP1 formulated with GlaxoSmithKline Biologicals (GSK) adjuvant, AS02A or alum was safe and immunogenic in rhesus (Macaca mulatta) monkeys.     

Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene–oil-in-water emulsion, and GLA plus squalene–oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene–oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene–oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration.     

The Glutamate-Rich Protein (GLURP) of Plasmodium falciparum Is a Target for Antibody-Dependent Monocyte-Mediated Inhibition of Parasite Growth In Vitro

Monocyte-dependent as well as direct inhibitory effects of antimalarial antibodies point toward antigens accessible at the time of merozoite release as targets for biologically active antibodies capable of mediating protection against Plasmodium falciparum. The glutamate-rich protein (GLURP), being an antigen associated with mature schizont-infected erythrocytes, was therefore the object of the present investigation, in which we analyzed whether anti-GLURP antibodies can either interfere directly with merozoite invasion or act indirectly by promoting a monocyte-dependent growth inhibition, antibody-dependent cellular inhibition. GLURP-specific human immunoglobulin G (IgG) antibodies, from pooled IgG of healthy Liberian adults who were clinically immune to malaria, were purified by affinity chromatography on columns containing R0 (N-terminal nonrepetitive region of GLURP) or R2 (C-terminal repetitive region of GLURP) recombinant protein or synthetic peptides as ligands. Analysis of the pattern of reactivity of highly purified anti-GLURP antibodies led to the definition of at least four B-cell epitopes. One epitope was specific for R0, two were specific for R2, and the fourth displayed cross-reactivity between R0 and R2. None of the purified IgG antibodies had direct invasion-inhibitory effects, even at high concentrations. In contrast, when allowed to cooperate with monocytes, all anti-GLURP IgG preparations mediated a strong monocyte-dependent parasite growth inhibition in a dose-dependent manner.Epidemiological surveys performed in areas of intense malaria transmission have consistently shown that individuals who are continuously exposed to repeated malaria infection gradually develop clinical immunity (14, 20, 29). This acquired immunity is strong, although incomplete, and is nonsterilizing (3, 25, 26). Experiments with antibodies purified from the sera of African adults who were clinically immune to malaria and given by passive transfer to susceptible children have established that immunoglobulin G (IgG) is at least a main component of defense against the asexual blood stage of Plasmodium falciparum (5, 9, 11). Recent passive transfer experiments have enabled us to acquire clinically demonstrated protective antibodies from the donor and nonprotective antibodies from the recipients. These sets of antibodies were used to assess the extent to which the in vitro data correlated with the in vivo results for each recipient isolate (5). Results from these in vitro studies suggested that clinically protective antibodies had little direct effect on merozoite invasion, but that they could act in conjunction with blood monocytes to contain parasite multiplication. This mechanism was called antibody-dependent cellular inhibition (ADCI) (5, 17, 19).The assay provides a screen to select molecules which may be targeted by clinically effective antibodies. Further experiments have indicated that antibody-monocyte cooperation in parasite inhibition is mediated not through parasite opsonization but rather through indirect effects. These activities were mediated by soluble monocyte-derived substances whose release was triggered through monocyte interaction with cytophilic antibodies bound to merozoite antigens (7). A critical role for merozoite surface molecules in this mechanism is also supported by the identification of Msp3, a new molecule from the merozoite surface, when an expression library was screened by ADCI (23).Based on the published immunoepidemiological data for the glutamate-rich protein (GLURP) (4, 12, 13, 15) and the report that this molecule is located on the surface of the merozoite (2), we chose to investigate the potential of affinity purified anti-GLURP human IgG in assays of direct parasite inhibition and to compare this with activity in ADCI assays.     

Detection of Plasmodium falciparum,P. vivax,P. ovale,and P. malariae Merozoite Surface Protein 1-p19 Antibodies in Human Malaria Patients and Experimentally Infected Nonhuman Primates

Approximately 3.2 billion people live in areas where malaria is endemic, and WHO estimates that 350 to 500 million malaria cases occur each year worldwide. This high prevalence, and the high frequency of international travel, creates significant risk for the exportation of malaria to countries where malaria is not endemic and for the introduction of malaria organisms into the blood supply. Since all four human infectious Plasmodium species have been transmitted by blood transfusion, we sought to develop an enzyme-linked immunosorbent assay (ELISA) capable of detecting antibodies elicited by infection with any of these species. The merozoite surface protein 1 (MSP1), a P. falciparum and P. vivax vaccine candidate with a well-characterized immune response, was selected for use in the assay. The MSP1 genes from P. ovale and P. malariae were cloned and sequenced (L. Birkenmeyer, A. S. Muerhoff, G. Dawson, and S. M. Desai, Am. J. Trop. Med. Hyg. 82:996-1003, 2010), and the carboxyl-terminal p19 regions of all four species were expressed in Escherichia coli. Performance results from individual p19 ELISAs were compared to those of a commercial test (Lab 21 Healthcare Malaria enzyme immunoassay [EIA]). The commercial ELISA detected all malaria patients with P. falciparum or P. vivax infections, as did the corresponding species-specific p19 ELISAs. However, the commercial ELISA detected antibodies in 0/2 and 5/8 individuals with P. malariae and P. ovale infections, respectively, while the p19 assays detected 100% of individuals with confirmed P. malariae or P. ovale infections. In experimentally infected nonhuman primates, the use of MSP1-p19 antigens from all four species resulted in the detection of antibodies within 2 to 10 weeks postinfection. Use of MSP1-p19 antigens from all four Plasmodium species in a single immunoassay would provide significantly improved efficacy compared to existing tests.More than 3.2 billion people in the world today live in areas where malaria is endemic. The World Health Organization estimates that more than 350 to 500 million malaria clinical disease episodes occur each year worldwide, with more than 1 million deaths occurring annually in sub-Saharan Africa, mostly among children under the age of 5 years (50). The combination of high disease prevalence and high frequency of international travel creates a significant risk for the exportation of malaria to countries where the disease is nonendemic. This risk is accompanied by the potential for introduction of malaria-causing organisms into the blood supplies used for transfusions. All four principal species of Plasmodium that infect humans have been transmitted via blood transfusion in the United States (36), France (4), the United Kingdom (23), and Switzerland (19). This has resulted in the implementation of donor deferral policies in many countries that restrict blood donation by those with a history of recent travel to or emigration from regions of endemicity and by those with recent cases of clinical malaria. Recent publications indicate that the prevalence of Plasmodium knowlesi, a pathogen of simian origin, in human populations in Southeast Asia (11, 12), Singapore (37), the Philippines (31), and Thailand (21) is much higher than previously believed. However, P. knowlesi malaria appears to be a zoonotic disease and to our knowledge has been not implicated in cases of transfusion-transmitted malaria in humans.The effectiveness of donor deferral programs has previously been questioned (29), and there is concern that many donors are needlessly deferred, since the rates of imported malaria are much lower than the rates of travel to areas of endemicity (17, 35). To prevent erosion of qualified donor populations, some countries have implemented antibody screening such that only individuals who are known to have been exposed to organisms causing malaria are subject to deferral of donations rather than all donors who have traveled to or lived in regions where malaria is endemic. Commercial antibody enzyme-linked immunosorbent assays (ELISAs) are currently in use (in the United Kingdom, France, and Australia), and reinstatement of questionnaire-deferred donors is being discussed in Canada and the United States (16, 24, 42). In these cases, potential donors are tested for antibodies to Plasmodium-derived antigens within several months of deferral; when the tested individuals show negative antibody results, donation is allowed.Antibodies to asexual malaria parasites (i.e., merozoites) appear within days to weeks after the invasion of erythrocytes and can persist for months or even years (14, 49). Historically, antibodies to parasite antigens have been detected using the immunofluorescence assay (IFA). This assay is not particularly sensitive or specific and is labor-intensive, requiring careful preparation of reagents. Commercially available ELISAs have been developed that use recombinant antigens or P. falciparum whole-organism lysates for detection of immunoglobulins (IgG and/or IgM, IgA) in human serum or plasma (Lab 21 Healthcare Laboratories, United Kingdom; Cellabs, Australia; DiaMed AG, Switzerland; LG Chemical Inc., Iksan, South Korea; Green Cross, Inc., Youngin, South Korea [Genedia Malaria Ab Rapid]; and Standard Diagnostics, Suwon, South Korea). These assays are typically easier to perform and exhibit higher throughput and better sensitivity and specificity than IFA (25, 42, 47), though this is not always the case (32). Some ELISAs may be better than others for detection of antibodies against all four Plasmodium species that cause malaria in humans (44). However, none of the available commercial assays currently include P. ovale- or P. malariae-derived antigens. Because these organisms have been implicated in transfusion-transmitted malaria (TTM), it would be advantageous to include antigens from these organisms in an antibody detection assay.Antigens used in some commercial ELISAs for the capture of antibodies have included vaccine candidates, since their ability to elicit antibody responses in animals and human vaccine recipients has been predetermined and naturally occurring antibodies are measured prior to vaccination. Examples of such antigens include circumsporozoite protein (CSP), apical membrane antigen 1 (AMA-1), merozoite surface protein 1 (MSP1), and, in particular, a 19-kDa C-terminal fragment of MSP1 (MSP1-p19) (22, 25, 40). Plasmodium falciparum MSP1 has been extensively studied and was one of the very earliest vaccine candidates; it elicits a protective antibody response against severe malaria, and the presence of MSP1 antibodies correlates with protective immunity (45). MSP1 is expressed as an ∼200-kDa precursor molecule linked by a glycosyl phosphatidylinositol anchor to the merozoite surface membrane. MSP1 is processed into a complex of polypeptides on the merozoite surface, including N-terminal and central regions of 82, 30, and 38 kDa, as well as the C-terminal region of 42 kDa. At the time of invasion of red blood cells, MSP1-p42 is further processed by proteolytic cleavage into a 33-kDa fragment (MSP1-p33), which is shed with the rest of the complex, and a C-terminal 19-kDa fragment (MSP1-p19). Only the C-terminal MSP1-p19 fragment remains anchored on the merozoite surface and is carried into parasitized red blood cells (RBC) (10). In monkeys, immunization with recombinant P. falciparum MSP1-p42 and P. falciparum MSP1-p19 has been shown to elicit various degrees of protection against P. falciparum challenge (15, 26). MSP1-p19 proteins from both P. falciparum and P. vivax have been proposed as vaccine candidates (18, 41, 48).By analogy to P. falciparum and P. vivax findings, one would predict that the MSP1 genes of P. ovale and P. malariae would be useful as reagents for vaccination or antibody detection. We recently cloned and expressed the MSP1-p19 proteins of P. malariae and P. ovale as recombinant antigens in Escherichia coli (2). We report here the independent evaluation of these proteins as reagents for antibody detection using sera from human malaria patients and experimentally infected nonhuman primates. In addition, a prototype immunoassay combining MSP1-p19 antigens from all four Plasmodium species was evaluated and its performance compared to a commercially available antibody test.     

Analysis of Antibodies to Newly Described Plasmodium falciparum Merozoite Antigens Supports MSPDBL2 as a Predicted Target of Naturally Acquired Immunity

Prospective studies continue to identify malaria parasite genes with particular patterns of polymorphism which indicate they may be under immune selection, and the encoded proteins require investigation. Sixteen new recombinant protein reagents were designed to characterize three such polymorphic proteins expressed in Plasmodium falciparum schizonts and merozoites: MSPDBL1 (also termed MSP3.4) and MSPDBL2 (MSP3.8), which possess Duffy binding-like (DBL) domains, and SURFIN4.2, encoded by a member of the surface-associated interspersed (surf) multigene family. After testing the antigenicities of these reagents by murine immunization and parasite immunofluorescence, we analyzed naturally acquired antibody responses to the antigens in two cohorts in coastal Kenya in which the parasite was endemic (Chonyi [n = 497] and Ngerenya [n = 461]). As expected, the prevalence and levels of serum antibodies increased with age. We then investigated correlations with subsequent risk of clinical malaria among children <11 years of age during 6 months follow-up surveillance. Antibodies to the polymorphic central region of MSPDBL2 were associated with reduced risk of malaria in both cohorts, with statistical significance remaining for the 3D7 allelic type after adjustment for individuals'' ages in years and antibody reactivity to whole-schizont extract (Chonyi, risk ratio, 0.51, and 95% confidence interval [CI], 0.28 to 0.93; Ngerenya, risk ratio, 0.38, and 95% CI, 0.18 to 0.82). For the MSPDBL1 Palo Alto allelic-type antigen, there was a protective association in one cohort (Ngerenya, risk ratio, 0.53, and 95% CI, 0.32 to 0.89), whereas the other antigens showed no protective associations after adjustment. These findings support the prediction that antibodies to the polymorphic region of MSPDBL2 contribute to protective immunity.     

Identification of VAR2CSA Domain-Specific Inhibitory Antibodies of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Using a Novel Flow Cytometry Assay

VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, is a leading candidate for use in vaccines to protect first-time mothers from placental malaria (PM). VAR2CSA, which is comprised of a series of six Duffy binding-like (DBL) domains, binds chondroitin sulfate A (CSA) on placental syncytiotrophoblast. Several recombinant DBL domains have been shown to bind CSA. In order to identify and develop recombinant proteins suitable for clinical development, DBL2X and DBL3X, as well as their respective third subdomain (S3) from the FCR3 parasite clone, were expressed in Escherichia coli, refolded, and purified. All but DBL3X-S3 recombinant proteins bound to CSA expressed on Chinese hamster ovary (CHO)-K1 cells but not to CHO-pgsA745 cells, which are CSA negative as determined by flow cytometry. All but DBL3X-S3 bound to CSA on chondroitin sulfate proteoglycan (CSPG) as determined by surface plasmon resonance (SPR) analysis. Purified IgG from rats and rabbits immunized with these four recombinant proteins bound homologous and some heterologous parasite-infected erythrocytes (IE). Using a novel flow cytometry inhibition-of-binding assay (flow-IBA), antibodies against DBL3X-S3 inhibited 35% and 45% of IE binding to CSA on CHO-K1 cells compared to results for soluble CSA (sCSA) and purified multigravida (MG) IgG, respectively, from areas in Tanzania to which malaria is endemic. Antibodies generated against the other domains provided little or no inhibition of IE binding to CSA on CHO-K1 cells as determined by the flow cytometry inhibition-of-binding assay. These results demonstrate for the first time the ability to identify antibodies to VAR2CSA DBL domains and subdomains capable of inhibiting VAR2CSA parasite-IE binding to CSA by flow cytometry. The flow cytometry inhibition-of-binding assay was robust and provided an accurate, reproducible, and reliable means to identify blocking of IE binding to CSA and promises to be significant in the development of a vaccine to protect pregnant women.     

Molecular Genetic Characterization of the Merozoite Surface Protein 1 Gene of Plasmodium vivax from Reemerging Korean Isolates

Plasmodium vivax merozoite surface protein 1 (PvMSP-1) has been considered a major candidate for the development of an antimalaria vaccine, but the molecule exhibits antigenic diversity among isolates. The extent of genetic polymorphism in the region between interspecies conserved blocks 4 and 5 (ICB4 and ICB5) of the PvMSP-1 gene was analyzed for 30 Korean isolates. Two genotypes, SK-A and SK-B, were identified on the basis of amino acid substitution. Almost all the amino acid sequences of the Korean isolates were nearly identical to those of the Solomon Island isolate Solo-83 (97.8 to 99.9% similarity) and Philippine isolates Ph-79, Ph-52-2, and Ph-49 (97.3 to 99.8% similarity). Also, we report two sequences in the isolates that were characterized on the basis of restriction fragment length polymorphism (RFLP). The RFLP profiles following digestion with the DraI restriction enzyme produced two distinguishable patterns. This study might be the first report of the region between ICB4 and ICB5 of the MSP-1 gene of P. vivax in South Korea.Plasmodium vivax malaria represents a major public health problem for many tropical and subtropical countries, which has been exacerbated by the expansion of drug-resistant strains (2, 10, 29, 30). The enormous toll of mortality caused by Plasmodium falciparum has tended to overshadow the public health importance of P. vivax. For this reason and on account of technical difficulties, relatively little investigation has been done toward the development of a vaccine against P. vivax (27). One of the major problems in vaccine development is the antigenic diversity of the vaccine candidates. The critical emerging problem is that the host response to one allele is not very effective against parasites expressing different allelic forms (8, 25). Therefore, genetic variation studies for the antigens of vaccine candidates are very important for P. falciparum and P. vivax. The polymorphism of potential malaria vaccine targets is rather greater for P. vivax than for P. falciparum. Also, the growing resistance of P. vivax strains to chloroquine is spurring the development of a vaccine against P. vivax malaria.The study of polymorphism is important not only for establishing the antigenic repertoire of isolates from regions where malaria is endemic but also for elucidating the mechanisms by which antigenic diversity is generated. The WHO declared in 1979 that malaria had been eradicated in Korea, but in 1993 (4), a case of malaria in a soldier working in the Demilitarized Zone (the border area between North and South Korea) of the Republic of Korea was reported. After 1993, the number of malaria cases expanded exponentially each year, with 3,932 patients diagnosed in 1998 (15). Current epidemiological results suggest that the malaria that has reemerged did not originate from overseas. All indigenous cases of malaria are due to P. vivax, with the occasional imported case of P. falciparum. However, the genetic characteristics of the reemergent Korean strain are not known at present.In previous studies, P. vivax circumsporozoite protein (13), P. vivax Duffy binding protein (12), and P. vivax apical membrane antigen 1 (6) showed genotypes with at least two new phenotypes among Korean isolates. However, the extent of genetic diversity of Korean P. vivax isolates is not accurately known at present, due to the fact that very few polymorphic markers are available for studying P. vivax.P. vivax merozoite surface protein 1 (PvMSP-1) is a well-characterized antigen whose diversity is maintained by host immune selection pressure (20). There is extensive allelic diversity of MSP-1 among isolates (5, 11, 19, 21), and this polymorphism may hamper the development of an effective vaccine against malaria. The polymorphism of PvMSP-1 has been considered to result from interallelic recombination in nature (20). Although the polymorphism of P. falciparum MSP-1 is well characterized, little is known about P. vivax MSP-1. To contribute useful information regarding genetic diversity and to facilitate rational vaccine design, the polymorphism of PvMSP-1 in Korean isolates was investigated in this study. In addition, we also describe the molecular phylogenetic characteristics of Korean P. vivax isolates.     

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.