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.     

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.     

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。     

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.     

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.     

A Chimeric Plasmodium falciparum Merozoite Surface Protein Vaccine Induces High Titers of Parasite Growth Inhibitory Antibodies

The C-terminal 19-kDa domain of Plasmodium falciparum merozoite surface protein 1 (PfMSP1₁₉) is an established target of protective antibodies. However, clinical trials of PfMSP1₄₂, a leading blood-stage vaccine candidate which contains the protective epitopes of PfMSP1₁₉, revealed suboptimal immunogenicity and efficacy. Based on proof-of-concept studies in the Plasmodium yoelii murine model, we produced a chimeric vaccine antigen containing recombinant PfMSP1₁₉ (rPfMSP1₁₉) fused to the N terminus of P. falciparum merozoite surface protein 8 that lacked its low-complexity Asn/Asp-rich domain, rPfMSP8 (ΔAsn/Asp). Immunization of mice with the chimeric rPfMSP1/8 vaccine elicited strong T cell responses to conserved epitopes associated with the rPfMSP8 (ΔAsn/Asp) fusion partner. While specific for PfMSP8, this T cell response was adequate to provide help for the production of high titers of antibodies to both PfMSP1₁₉ and rPfMSP8 (ΔAsn/Asp) components. This occurred with formulations adjuvanted with either Quil A or with Montanide ISA 720 plus CpG oligodeoxynucleotide (ODN) and was observed in both inbred and outbred strains of mice. PfMSP1/8-induced antibodies were highly reactive with two major alleles of PfMSP1₁₉ (FVO and 3D7). Of particular interest, immunization with PfMSP1/8 elicited higher titers of PfMSP1₁₉-specific antibodies than a combined formulation of rPfMSP1₄₂ and rPfMSP8 (ΔAsn/Asp). As a measure of functionality, PfMSP1/8-specific rabbit IgG was shown to potently inhibit the in vitro growth of blood-stage parasites of the FVO and 3D7 strains of P. falciparum. These data support the further testing and evaluation of this chimeric PfMSP1/8 antigen as a component of a multivalent vaccine for P. falciparum malaria.     

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.     

Inhibitory Humoral Responses to the Plasmodium falciparum Vaccine Candidate EBA-175 Are Independent of the Erythrocyte Invasion Pathway

Plasmodium falciparum utilizes multiple ligand-receptor interactions for invasion. The invasion ligand EBA-175 is being developed as a major blood-stage vaccine candidate. EBA-175 mediates parasite invasion of host erythrocytes in a sialic acid-dependent manner through its binding to the erythrocyte receptor glycophorin A. In this study, we addressed the ability of naturally acquired human antibodies against the EBA-175 RII erythrocyte-binding domain to inhibit parasite invasion of ex vivo isolates, in relationship to the sialic acid dependence of these parasites. We have determined the presence of antibodies to the EBA-175 RII domain by enzyme-linked immunosorbent assay (ELISA) in individuals from areas of Senegal where malaria is endemic with high and low transmission. Using affinity-purified human antibodies to the EBA-175 RII domain from pooled patient plasma, we have measured the invasion pathway as well as the invasion inhibition of clinical isolates from Senegalese patients in ex vivo assays. Our results suggest that naturally acquired anti-EBA-175 RII antibodies significantly inhibit invasion of Senegalese parasites and that these responses can be significantly enhanced through limiting other ligand-receptor interactions. However, the extent of this functional inhibition by EBA-175 antibodies is not associated with the sialic acid dependence of the parasite strain, suggesting that erythrocyte invasion pathway usage by parasite strains is not driven by antibodies targeting the EBA-175/glycophorin A interaction. This work has implications for vaccine design based on the RII domain of EBA-175 in the context of alternative invasion pathways.     

Erythrocyte invasion phenotypes of Plasmodium falciparum in The Gambia

In vitro experimentation with Plasmodium falciparum has determined that a number of different receptor-ligand interactions are involved in the invasion of erythrocytes. Most culture-adapted parasite isolates use a mechanism of invasion that depends primarily on the erythrocyte sialoglycoprotein glycophorin A (GYPA) and erythrocyte-binding antigen 175 (EBA-175) of the parasite blood-stage merozoite. However, a minority of culture-adapted parasites and a majority of Indian field isolates can apparently invade by other means. Here, erythrocyte invasion phenotypes of P. falciparum field isolates in Africa were studied. For 38 Gambian isolates, invasion of neuraminidase-treated and trypsin-treated erythrocytes was inhibited, on average, by more than 60 and 85%, respectively, indicating a high level of dependence on sialic acid and trypsin-sensitive proteins on the erythrocyte surface. These results support the hypothesis that African P. falciparum parasites use GYPA as a primary receptor for invasion. However, the considerable variation among isolates confirms the idea that alternative receptors are also used by many parasites. Three amino acid polymorphisms in the GYPA-binding region of EBA-175 (region II) were not significantly associated with invasion phenotype. There was variation among isolates in the selectivity index (i.e., a statistical tendency toward aggregation or multiple invasions of host erythrocytes), but this variation did not correlate with enzyme-determined invasion phenotype or with eba-175 alleles. Overall, these invasion phenotypes in Africa support a vaccine strategy of inhibiting EBA-175 binding to GYPA but suggest that parasites with alternative phenotypes would be selected for if this strategy were used alone.     

Merozoite surface protein 8 of Plasmodium falciparum contains two epidermal growth factor-like domains

By motif searching of the unfinished sequences in the Malaria Genome Sequencing Project databases we have identified a novel EGF-like domain-containing protein of Plasmodium falciparum. The sequence lies within a single open reading frame of 1791 bp and is predicted to encode a polypeptide of 597 amino acids. There are hydrophobic regions at the extreme N- and C-termini, which could represent secretory signal peptide and GPI attachment sites, respectively. Similar to MSP1, there are two EGF-like domains located near the C-terminus. RT-PCR analysis of the novel gene shows that it is transcribed in asexual stages of the malaria parasite. We have expressed portions of the protein as recombinant GST fusions in Escherichia coli and raised antisera in rabbits. Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, MSP4 or MSP5 expressed as GST fusion proteins. Antiserum raised to the most C-terminal region of the protein reacts with four bands of 98, 50, 25 and 19 kDa in P. falciparum parasite lysates whereas antisera to the N-terminal fusion proteins recognise the 98 and 50 kDa bands, suggesting that the novel protein may undergo processing in a similar way to MSP1. Immunoblot analysis of stage-specific parasite samples reveals that the protein is present throughout the parasite asexual life cycle and in isolated merozoites, with the smaller fragments present in ring stage parasites. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites, schizonts and free merozoites by indirect immunofluorescence. Antisera to the C-terminus stain the surface of rings, whereas antisera to the N-terminus do not, suggesting that a fragment of the protein is carried into the developing ring stage parasite. Based on the accepted nomenclature in the field we designate this protein MSP8. We have shown that the MSP8 fusion proteins are in a conformation that can be recognised by human immune sera and that there is very limited diversity in the MSP8 gene sequences from various P. falciparum laboratory isolates. MSP8 shows significant similarity to the recently reported sequence of the protective P. yoelii merozoite surface protein pypAg-2 [Burns JM, Belk CC, Dunn PD. Infect Immun 2000;68:6189-95.] suggesting that the two proteins are homologues. Taken together, these findings suggest that MSP8/pypAg-2 may play an important role in the process of red cell invasion and is a potential malaria vaccine candidate.     

Plasmodium falciparum Field Isolates Commonly Use Erythrocyte Invasion Pathways That Are Independent of Sialic Acid Residues of Glycophorin A

Erythrocyte invasion by malaria parasites is mediated by specific molecular interactions. Sialic acid residues of glycophorin A are used as invasion receptors by Plasmodium falciparum. In vitro invasion studies have demonstrated that some cloned P. falciparum lines can use alternate receptors independent of sialic acid residues of glycophorin A. It is not known if invasion by alternate pathways occurs commonly in the field. In this study, we used in vitro growth assays and erythrocyte invasion assays to determine the invasion phenotypes of 15 P. falciparum field isolates. Of the 15 field isolates tested, 5 multiply in both neuraminidase and trypsin-treated erythrocytes, 3 multiply in neuraminidase-treated but not trypsin-treated erythrocytes, and 4 multiply in trypsin-treated but not neuraminidase-treated erythrocytes; 12 of the 15 field isolates tested use alternate invasion pathways that are not dependent on sialic acid residues of glycophorin A. Alternate invasion pathways are thus commonly used by P. falciparum field isolates. Typing based on two polymorphic markers, MSP-1 and MSP-2, and two microsatellite markers suggests that only 1 of the 15 field isolates tested contains multiple parasite genotypes. Individual P. falciparum lines can thus use multiple invasion pathways in the field. These observations have important implications for malaria vaccine development efforts based on EBA-175, the P. falciparum protein that binds sialic acid residues of glycophorin A during invasion. It may be necessary to target parasite ligands responsible for the alternate invasion pathways in addition to EBA-175 to effectively block erythrocyte invasion by P. falciparum.     

Antibody Recognition of Plasmodium falciparum Erythrocyte Surface Antigens in Kenya: Evidence for Rare and Prevalent Variants

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the name given to a family of parasite proteins that are inserted into the infected erythrocyte surface. Studies using agglutination assays have shown previously that PfEMP1 epitopes are extremely diverse. In a study in Kenya, 21 parasite isolates, including nine from children with severe malaria, were tested for agglutination by 33 pairs of plasma, 21 of which were from the corresponding children. Each plasma pair consisted of a sample taken at the time of disease (acute) and one taken 3 weeks later (convalescent). In agreement with previous studies, infection was generally followed by the induction of antibodies specific to the homologous parasite isolate. In addition however, the results show that (i) some isolates were agglutinated very frequently by heterologous plasma; (ii) unexpectedly, these frequently agglutinated isolates tended to be from individuals with severe malaria; (iii) an inverse relationship existed between the agglutination frequency of each parasite isolate in heterologous plasma and the agglutinating antibody repertoire of the homologous child at the time of disease; and (iv) A 3-month-old child apparently still carrying maternal antibodies was infected by a rarely agglutinated isolate. This child’s plasma agglutinated all isolates at the time of disease, apart from the homologous isolate. These results support the idea that preexisting anti-PfEMP1 antibodies can select the variants that are expressed during a new infection and may suggest the existence of a dominant subset of PfEMP1 variants.     

Invasion of Rh Null Cells by Plasmodium falciparum identifies a new invasion pathway

The malaria parasite, Plasmodium falciparum, invades the human erythrocyte through a complex interaction with erythrocyte receptors characterized by patterns of resistance to various enzymes. As invasion rates are influenced by blood group polymorphisms, we reasoned that the extremely rare rhesus null (Rh_null) erythrocytes could be informative in characterizing receptors. The aim was to test whether the complete absence of the Rh complex from the cell membrane impacted on parasite invasion. Enzyme treatment patterns for four P. falciparum isolates were first characterised for normal Rh cells. Two isolates showed an enzyme treatment pattern not hitherto described, with resistance to neuraminidase, trypsin and chymotrypsin. In contrast, all isolates had enhanced invasion rates for the Rh_null cell for all enzyme treatment regimens. The first finding suggests there is another pathway that P. falciparum can utilise to invade the host. We speculate that the Rh null cell membrane exposes a novel ligand defined as Receptor N. Wendy Y. Chung and Donald L. Gardiner contributed equally to this work.     

Invasion pathways and malaria severity in Kenyan Plasmodium falciparum clinical isolates

The invasion of erythrocytes by Plasmodium falciparum occurs through multiple pathways that can be studied in vitro by examining the invasion of erythrocytes treated with enzymes such as neuraminidase, trypsin, and chymotrypsin. We have studied the invasion pathways used by 31 Kenyan P. falciparum isolates from children with uncomplicated or severe malaria. Six distinct invasion profiles were detected, out of eight possible profiles. The majority of isolates (23 of 31) showed neuraminidase-resistant, trypsin-sensitive invasion, characteristic of the pathway mediated by an unknown parasite ligand and erythrocyte receptor "X." The neuraminidase-sensitive, trypsin-sensitive phenotype consistent with invasion mediated by the binding of parasite ligand erythrocyte binding antigen 175 to glycophorin A, the most common invasion profile in a previous study of Gambian field isolates, was seen in only 3 of 31 Kenyan isolates. No particular invasion profile was associated with severe P. falciparum malaria, and there was no significant difference in the levels of inhibition by the various enzyme treatments between isolates from children with severe malaria and those from children with uncomplicated malaria (P, >0.1 for all enzymes; Mann-Whitney U test). These results do not support the hypothesis that differences in invasion phenotypes play an important role in malaria virulence and indicate that considerable gaps remain in our knowledge of the molecular basis of invasion pathways in natural P. falciparum infections.     

Allele Specificity of Gamma Interferon Responses to the Carboxyl-Terminal Region of Plasmodium falciparum Merozoite Surface Protein 1 by Kenyan Adults with Naturally Acquired Immunity to Malaria

Cross-sectional seroepidemiological studies of populations naturally exposed to Plasmodium falciparum suggest an association between protection from malaria and circulating antibodies to the carboxyl terminus of merozoite surface protein 1 (MSP1). Questions remain regarding the significance of cell-mediated immunity to MSP1 in conferring protection and inducing immunologic memory. Vaccine constructs have been based on the 42-kDa recombinant MSP1 protein (MSP1₄₂), which includes the 19-kDa (MSP1₁₉) and 33-kDa (MSP1₃₃) fragments containing the major B- and T-cell epitopes, respectively. To evaluate T-cell responses to the MSP1₃₃ fragment, two libraries of overlapping 18-mer peptides from the 3D7 and FVO MSP1₃₃ regions were used to screen a cohort of asymptomatic Kenyan adults. Gamma interferon (IFN-γ) measured by enzyme-linked immunospot assay (ELISPOT) at multiple time points assessed the magnitude and stability of these responses. The percentage of individuals with IFN-γ responses to single MSP1₃₃ peptides ranged from nil to 24%, were clustered among a subset of peptides, and were not consistently recalled over time. In comparison to peptide responses, IFN-γ ELISPOT responses to recombinant MSP1₄₂ were more prevalent, more frequently elicited by the 3D7 as opposed to the FVO allele, and more stable over time. The prevailing MSP1₃₃ genotype infection was 3D7, with few mixed infections and no sole FVO infections. This study demonstrates that immunity against MSP1₃₃ after cumulative natural infections consists of low-magnitude and difficult-to-detect IFN-γ responses. Although immunity against MSP1 alone will not confer protection against malaria, demonstrating a relative and sustained increase in T-cell immunity to MSP1 after vaccination would be a reasonable measurement of vaccine responsiveness.Infection with Plasmodium spp., the protozoan parasites responsible for malaria, results in an estimated 350 to 500 million infections per year with an ensuing 1 to 2 million deaths, the majority of which are caused by Plasmodium falciparum (45). Adults who have maintained lifelong residence in areas where malaria transmission is stable and of high intensity gradually develop naturally acquired immunity, an age-related phenomenon marked by a reduced frequency and severity of clinical malaria and high-density blood stage parasitemia relative to that of infants and children. Maintaining naturally acquired immunity is thought to require continuing exposure to malaria blood stage antigens mediated through asymptomatic low-density blood stage infection. A malaria vaccine that would accelerate the development of naturally acquired immunity during infancy and childhood has been a high priority for many years. The recent success of vector interventions, such as insecticide-treated bed nets, in decreasing transmission of P. falciparum in sub-Saharan Africa suggests that a blood stage vaccine would also be desirable in order to maintain the strength and duration of naturally acquired immunity among adults (17).Merozoite surface protein 1 (MSP1) is one of several candidates that have been considered for a blood stage vaccine. The primary ∼195-kDa MSP1 protein is expressed during schizogony and is processed initially to form a 42-kDa carboxyl-terminal fragment (MSP1₄₂) that remains attached to the merozoite surface. A second proteolytic cleavage results in the shedding of a 33-kDa fragment (MSP1₃₃) with formation of a 19-kDa carboxyl-terminal fragment (MSP1₁₉) anchored to the merozoite surface during invasion of the red blood cell (RBC) (4, 5). The 19-kDa fragment of MSP1 contains the major B-cell epitopes (18), the recognition of which is enhanced by T-helper epitopes in the 33-kDa fragment (1). Antibodies to MSP1₁₉ that interfere with RBC invasion by merozoites are one of several possible mechanisms by which naturally acquired immunity and experimental MSP1 vaccines may mediate protection against blood stage infection (27, 34). Studies of primates and mice immunized with MSP1 and more limited observations of residents of areas in which P. falciparum is endemic who have naturally acquired immunity and malaria-naïve human volunteers vaccinated with MSP1₄₂ suggest that T-cell responses to MSP1 are driven primarily by epitopes within the shed MSP1₃₃ fragment and that gamma interferon (IFN-γ) is important for optimal protective immunity (20, 23, 44, 46).A challenge to developing malaria vaccines that enhance protective immunity against blood stage parasitemia has been antigenic polymorphism that arises through selection by the human immune response; i.e., inclusion of several blood stage alleles might be required in order to mitigate selection of alleles not included in the vaccine. The P. falciparum msp1 gene has been divided into 17 blocks based on conserved, semiconserved, and variant DNA sequences (30). The C-terminal MSP1₄₂ protein is encoded by blocks 15 to 17; MSP1₃₃ is encoded by block 15 and 16. MSP1₄₂ alleles expressed by most clinical isolates and laboratory cultures of P. falciparum exist as either the MAD20 (3D7) or FVO (K1 or Wellcome) allele of MSP1₁₉ and MSP1₃₃ (although genetic recombination between the two [and other] alleles encoded by block 17 and block 15 to 16 also occurs). Whereas most studies from sub-Saharan Africa and other areas where malaria is endemic have characterized msp1 block 17 haplotypes and cross-reactivity of IgG antibodies to the corresponding MSP1₁₉ polypeptides (14, 43), block 15 to 16 polymorphisms underlying the variation in MSP1₃₃ and its impact on naturally acquired T-cell immunity are less well understood. The objectives of the current study were to evaluate MSP1₃₃ polymorphism in an area of western Kenya in which malaria is holoendemic and the corresponding allele specificity of IFN-γ responses by adults with naturally acquired immunity to malaria.     

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.     

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.     

Invasion profiles of Brazilian field isolates of Plasmodium falciparum: phenotypic and genotypic analyses

The invasion of red blood cells (RBCs) by Plasmodium falciparum is dependent on multiple molecular interactions between erythrocyte receptors and parasite ligands. Invasion studies using culture-adapted parasite strains have indicated significant receptor heterogeneity. It is not known whether this heterogeneity reflects the parasite invasion arsenal in the field. We have studied the invasion phenotypes of 14 distinct field isolates from the Legal Amazon areas of Brazil by using erythrocyte invasion assays to investigate invasion into normal, enzyme-treated, and clinical-mutant RBCs. Analysis of these isolates revealed four distinct invasion profiles. Using En(a-) cells to get an unequivocal estimate of the use of glycophorin A (GPA) as a receptor, we found that the 175-kDa erythrocyte-binding antigen (EBA-175)/GPA pathway was used by a minority of the parasite isolates studied. Although polymorphism of region II domains at specific amino acid positions in both EBA-140 and EBA-181 was found in these field isolates, this did not correlate with invasion profiles and thus receptor selectivity. These studies have further confirmed the existence of a significant diversity of invasion pathways in nature and suggest that additional parasite ligands will have to be targeted to devise global vaccines that will work in the field.