Plasmodium Falciparum Merozoite Surface Protein Research Articles

Shift in epitope dominance of IgM and IgG responses to Plasmodium falciparum MSP1 block 4

BackgroundPlasmodium falciparum merozoite surface protein-1 (MSP1) has been extensively studied as a blood-stage malaria vaccine candidate, with most work focused on the conserved 19 kDa and semi-conserved 42 kDa C-terminal regions (blocks 16-17) and the hypervariable N-terminal repeat region (block 2). However, recent genotyping studies suggest that additional regions of MSP1 may be under selective pressure, including a locus of intragenic recombination designated as block 4 within the 3' region of the gene.MethodsThe current study examined the antibody response to the two parental and two recombinant forms of block 4 and to blocks 16-17 (3D7) in study populations from Colombia, Papua New Guinea and Cameroon that differ in malaria transmission intensity and ethnic composition.ResultsIgM and IgG antibodies were detected against parental and recombinant MSP1 block 4 peptides in all three populations. Overall, 32-44% of the individuals produced IgM to one or more of the peptides, with most individuals having IgM antibodies reactive with both parental and recombinant forms. In contrast, IgG seropositivity to block 4 varied among populations (range 15-65%), with the majority of antibodies showing specificity for one or a pair of block 4 peptides. The IgG response to block 4 was significantly lower than that to blocks 16-17, indicating block 4 is subdominant. Antibodies to block 4 and blocks 16-17 displayed distinct IgG subclass biases, with block 4 responses biased toward IgG3 and blocks 16-17 toward IgG1. These patterns of responsiveness were consistently observed in the three study populations.ConclusionsProduction of antibodies specific for each parental and recombinant MSP1 block 4 allele in different populations exposed to P. falciparum is consistent with balancing selection of the MSP1 block 4 region by the immune response of individuals in areas of both low and high malaria transmission. MSP1 block 4 determinants may be important in isolate-specific immunity to P. falciparum.

The Plasmodium falciparum merozoite surface protein-1 19 KD antibody response in the Peruvian Amazon predominantly targets the non-allele specific, shared sites of this antigen

BackgroundPlasmodium falciparum re-emerged in Iquitos, Peru in 1994 and is now hypoendemic (< 0.5 infections/person/year). Purportedly non-immune individuals with discrete (non-overlapping) P. falciparum infections can be followed using this population dynamic. Previous work demonstrated a strong association between this population's antibody response to PfMSP1-19KD and protection against febrile illness and parasitaemia. Therefore, some selection for PfMSP1-19KD allelic diversity would be expected if the protection is to allele-specific sites of PfMSP1-19KD. Here, the potential for allele-specific polymorphisms in this population is investigated, and the allele-specificity of antibody responses to PfMSP1-19KD are determined.MethodsThe 42KD region in PfMSP1 was genotyped from 160 individual infections collected between 2003 and 2007. Additionally, the polymorphic block 2 region of Pfmsp1 (Pfmsp1-B2) was genotyped in 781 infection-months to provide a baseline for population-level diversity. To test whether PfMSP1-19KD genetic diversity had any impact on antibody responses, ELISAs testing IgG antibody response were performed on individuals using all four allele-types of PfMSP1-19KD. An antibody depletion ELISA was used to test the ability of antibodies to cross-react between allele-types.ResultsDespite increased diversity in Pfmsp1-B2, limited diversity within Pfmsp1-42KD was observed. All 160 infections genotyped were Mad20-like at the Pfmsp1-33KD locus. In the Pfmsp1-19KD locus, 159 (99.4%) were the Q-KSNG-F haplotype and 1 (0.6%) was the E-KSNG-L haplotype. Antibody responses in 105 individuals showed that Q-KNG and Q-TSR alleles generated the strongest immune responses, while Q-KNG and E-KNG responses were more concordant with each other than with those from Q-TSR and E-TSR, and vice versa. The immuno-depletion ELISAs showed all samples responded to the antigenic sites shared amongst all allelic forms of PfMSP1-19KD.ConclusionsA non-allele specific antibody response in PfMSP1-19KD may explain why other allelic forms have not been maintained or evolved in this population. This has important implications for the use of PfMSP1-19KD as a vaccine candidate. It is possible that Peruvians have increased antibody responses to the shared sites of PfMSP1-19KD, either due to exposure/parasite characteristics or due to a human-genetic predisposition. Alternatively, these allelic polymorphisms are not immune-specific even in other geographic regions, implying these polymorphisms may be less important in immune evasion that previous studies suggest.

Design and pre-clinical profiling of a Plasmodium falciparum MSP-3 derived component for a multi-valent virosomal malaria vaccine

BackgroundClinical profiling of two components for a synthetic peptide-based virosomal malaria vaccine has yielded promising results, encouraging the search for additional components for inclusion in a final multi-valent vaccine formulation. This report describes the immunological characterization of linear and cyclized synthetic peptides comprising amino acids 211-237 of Plasmodium falciparum merozoite surface protein (MSP-3).MethodsThese peptides were coupled to phosphatidylethanolamine (PE); the conjugates were intercalated into immunopotentiating reconstituted influenza virosomes (IRIVs) and then used for immunizations in mice to evaluate their capacity to elicit P. falciparum cross-reactive antibodies.ResultsWhile all MSP-3-derived peptides were able to elicit parasite-binding antibodies, stabilization of turn structures by cyclization had no immune-enhancing effect. Therefore, further pre-clinical profiling was focused on FB-12, a PE conjugate of the linear peptide. Consistent with the immunological results obtained in mice, all FB-12 immunized rabbits tested seroconverted and consistently elicited antibodies that interacted with blood stage parasites. It was observed that a dose of 50 μg was superior to a dose of 10 μg and that influenza pre-existing immunity improved the immunogenicity of FB-12 in rabbits. FB-12 production was successfully up-scaled and the immunogenicity of a vaccine formulation, produced according to the rules of Good Manufacturing Practice (GMP), was tested in mice and rabbits. All animals tested developed parasite-binding antibodies. Comparison of ELISA and IFA titers as well as the characterization of a panel of anti-FB-12 monoclonal antibodies indicated that at least the majority of antibodies specific for the virosomally formulated synthetic peptide were parasite cross-reactive.ConclusionThese results reconfirm the suitability of IRIVs as a carrier/adjuvant system for the induction of strong humoral immune responses against a wide range of synthetic peptide antigens. The virosomal formulation of the FB-12 peptidomimetic is suitable for use in humans and represents a candidate component for a virosomal multi-valent malaria subunit vaccine.

Antibody-Dependent Transplacental Transfer of Malaria Blood-Stage Antigen Using a Human Ex Vivo Placental Perfusion Model

Prenatal exposure to allergens or antigens released by infections during pregnancy can stimulate an immune response or induce immunoregulatory networks in the fetus affecting susceptibility to infection and disease later in life. How antigen crosses from the maternal to fetal environment is poorly understood. One hypothesis is that transplacental antigen transfer occurs as immune complexes, via receptor-mediated transport across the syncytiotrophoblastic membrane and endothelium of vessels in fetal villi. This hypothesis has never been directly tested. Here we studied Plasmodium falciparum merozoite surface protein 1 (MSP1) that is released upon erythrocyte invasion. We found MSP1 in cord blood from a third of newborns of malaria-infected women and in >90% following treatment with acid dissociation demonstrating MSP1 immune complexes. Using an ex vivo human placental model that dually perfuses a placental cotyledon with independent maternal and fetal circuits, immune-complexed MSP1 transferred from maternal to fetal circulation. MSP1 alone or with non-immune plasma did not transfer; pre-incubation with human plasma containing anti-MSP1 was required. MSP1 bound to IgG was detected in the fetal perfusate. Laser scanning confocal microscopy demonstrated MSP1 in the fetal villous stroma, predominantly in fetal endothelial cells. MSP1 co-localized with IgG in endothelial cells, but not with placental macrophages. Thus we show, for the first time, antibody-dependent transplacental transfer of an antigen in the form of immune complexes. These studies imply frequent exposure of the fetus to certain antigens with implications for management of maternal infections during pregnancy and novel approaches to deliver vaccines or drugs to the fetus.

Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection.

BackgroundMSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP119), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP119 had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP119 would affect critical T-cell responses to epitopes in this antigen.MethodsThe cellular responses to wild-type MSP119 and a panel of modified MSP119 antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults.ResultsInterestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP119. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins.ConclusionThis study suggests that specific MSP119 variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.

Plasmodium falciparumMerozoite 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-Fu(24), 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-1(19)]) and merozoite surface protein 3 (11-kDa conserved region [PfMSP-3(11)]). The recombinant MSP-Fu(24) protein was produced in Escherichia coli cells and purified to homogeneity by a two-step purification process with a yield of approximately 30 mg/liter. Analyses of conformational properties of MSP-Fu(24) using PfMSP-1(19)-specific monoclonal antibody showed that the conformational epitopes of PfMSP-1(19) that may be critical for the generation of the antiparasitic immune response remained intact in the fusion protein. Recombinant MSP-Fu(24) was highly immunogenic in mice and in rabbits when formulated with two different human-compatible adjuvants and induced an immune response against both PfMSP-1(19) and PfMSP-3(11). Purified anti-MSP-Fu(24) 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-1(19) component. The protective potential of MSP-Fu(24) was demonstrated by in vitro parasite growth inhibition using an antibody-dependent cell inhibition (ADCI) assay with anti-MSP-Fu(24) antibodies. Overall, the antiparasitic activity was mediated by a combination of growth-inhibitory antibodies generated by both the PfMSP-1(19) and PfMSP-3(11) components of the MSP-Fu(24) protein. The antiparasitic activities elicited by anti-MSP-Fu(24) antibodies were comparable to those elicited by antibodies generated with immunization with a physical mixture of two component antigens, PfMSP-1(19) and PfMSP-3(11). The fusion protein induces a protective immune response with human-compatible adjuvants and may form a part of a multicomponent malaria vaccine.

Identification of cultivation condition to produce correctly folded form of a malaria vaccine based on Plasmodium falciparum merozoite surface protein-1 in Escherichia coli

The C-terminal, 19-kDa domain of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1(19)) is among the leading vaccine candidate for malaria due to its essential role in erythrocyte invasion by the parasite. We designed a synthetic gene for optimal expression of recombinant PfMSP-1(19) in Escherichia coli and developed a scalable process to obtain high-quality PfMSP-1(19). The synthetic gene construct yielded a fourfold higher expression level of PfMSP-1(19) in comparison to the native gene construct. Optimization of cultivation conditions in the bioreactor indicated important role of yeast extract and substrate feeding strategy for obtaining enhanced expression of soluble and correctly folded PfMSP-1(19). It was observed that the higher expression level of PfMSP-1(19) was essentially associated with the generation of higher level of incorrectly folded PfMSP-1(19). A simple purification procedure comprising metal affinity and ion exchange chromatography was developed to purify correctly folded form of PfMSP-1(19) from cell lysate. Biochemical and biophysical characterization of purified PfMSP-1(19) suggested that it was highly pure, homogeneous, and correctly folded.

Human IgG subclass antibodies to the 19 kilodalton carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) and predominance of the MAD20 allelic type of MSP1 in Uganda.

To determine the natural human humoral immune responses to the 19 kilodalton carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)), a malaria candidate vaccine antigen and to determine the prevalence of MAD20 and K1 alleles of P. falciparum MSP1. Community based cross-sectional study. Atopi Parish, Apac District, Uganda, 1995. Three hundred and seventy four Ugandans between <1 and 70 years old provided serum samples. IgG subclass antibodies by ELISA; MAD20 and K1 allelic types of MSP1 by PCR. Both the prevalence and the mean concentration of serum IgG1, and to a lesser extent IgG3, antibodies increased with age. IgG2 or IgG4 antibodies were virtually nonexistent. The cross-reactivity between the 4 sequence variants (E-KNG, E-TSR, Q-KNG and Q-TSR) of MSP1(19) was confirmed; however, a minority of sera preferentially recognised the KNG but not the TSR variants. All 33 P. falciparum isolates from different parts of Uganda carried the E-TSR (Mad20) allelic type and 3 isolates were mixed infections with E-TSR (MAD20) and Q-KNG (K1) allelic types, confirming the rarity of the K1 allele in Uganda. There is a robust IgG1 antibody response to the malaria vaccine candidate antigen MSP1(19) which begins at an early age. Future cohort studies are necessary to estblish the impact of these antibodies on clinical immunity to malaria. The MAD20 allelic type of MSP1 id predominant in Ugandan P. falciparum isolates.

Inhibition of Erythrocyte Invasion andPlasmodium falciparumMerozoite Surface Protein 1 Processing by Human Immunoglobulin G1 (IgG1) and IgG3 Antibodies

Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP1(19) can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab')(2) fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human gamma1 and gamma3 constant regions, retain the ability to bind to both parasites and recombinant MSP1(19), and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcgamma receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy.

Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)

BackgroundGenotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported.MethodsThe original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis.ResultsThe CE-based method showed high precision and reproducibility in determining fragment size (< 1 bp). More genotypes were detected in mixtures of laboratory lines and blood samples from malaria infected children, compared to gel electrophoresis. The capacity to distinguish recrudescent parasites from new infections in an anti-malarial drug trial was similar by both methods, resulting in the same outcome classification, however with more precise determination by CE.ConclusionThe improved resolution and reproducibility of CE in fragment sizing allows for comparison of alleles between separate runs and determination of allele frequencies in a population. The more detailed characterization of individual msp1 and msp2 genotypes may contribute to improved assessments in anti-malarial drug trials and to a further understanding of the molecular epidemiology of these polymorphic P. falciparum antigens.

Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies has confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying β-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular β-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.

Genetic Diversity of the Malaria Vaccine Candidate Plasmodium falciparum Merozoite Surface Protein-3 in a Hypoendemic Transmission Environment

The N-terminal domain of Plasmodium falciparum merozoite surface protein-3 (PfMSP3) has been excluded from malaria vaccine development largely because of genetic diversity concerns. However, no study to date has followed N-terminal diversity over time. This study describes PfMSP3 variation in a hypoendemic longitudinal cohort in the Peruvian Amazon over the 2003-2006 transmission seasons. Polymerase chain reaction was used to amplify the N-terminal domain in 630 distinct P. falciparum infections, which were allele-typed by size and also screened for sequence variation using a new high-throughput technique, denaturing high performance liquid chromatography. PfMSP3 allele frequencies fluctuated significantly over the 4-year period, but sequence variation was very limited, with only 10 mutations being identified of 630 infections screened. The sequence of the PfMSP3 N-terminal domain is relatively stable over time in this setting, and further studies of its status as a vaccine candidate are therefore warranted.

AFCo1, a meningococcal B-derived cochleate adjuvant, strongly enhances antibody and T-cell immunity against Plasmodium falciparum merozoite surface protein 4 and 5

BackgroundWhilst a large number of malaria antigens are being tested as candidate malaria vaccines, a major barrier to the development of an effective vaccine is the lack of a suitable human adjuvant capable of inducing a strong and long lasting immune response. In this study, the ability of AFCo1, a potent T and B cell adjuvant based on cochleate structures derived from meningococcal B outer membrane proteoliposomes (MBOMP), to boost the immune response against two Plasmodium falciparum antigens, merozoite surface protein 4 (MSP4) and 5 (MSP5), was evaluated.MethodsComplete Freund's adjuvant (CFA), which is able to confer protection against malaria in animal MSP4/5 vaccine challenge models, was used as positive control adjuvant. MSP4 and 5-specific IgG, delayed-type hypersensitivity (DTH), T-cell proliferation, and cytokine production were evaluated in parallel in mice immunized three times intramuscularly with MSP4 or MSP5 incorporated into AFCo1, synthetic cochleate structures, CFA or phosphate buffered saline.ResultsAFCo1 significantly enhanced the IgG and T-cell response against MSP4 and MSP5, with a potency equivalent to CFA, with the response being characterized by both IgG1 and IgG2a isotypes, increased interferon gamma production and a strong DTH response, consistent with the ability of AFCo1 to induce Th1-like immune responses.ConclusionGiven the proven safety of MBOMP, which is already in use in a licensed human vaccine, AFCo1 could assist the development of human malaria vaccines that require a potent and safe adjuvant.

The C-terminal domain of Plasmodium falciparum merozoite surface protein 3 self-assembles into α-helical coiled coil tetramer

Proteins located on the surface of the pathogenic malaria parasite Plasmodium falciparum are objects of intensive studies due to their important role in the invasion of human cells and the accessibility to host antibodies thus making these proteins attractive vaccine candidates. One of these proteins, merozoite surface protein 3 (MSP3) represents a leading component among vaccine candidates; however, little is known about its structure and function. Our biophysical studies suggest that the 40 residue C-terminal domain of MSP3 protein self-assembles into a four-stranded α-helical coiled coil structure where α-helices are packed “side-by-side”. A bioinformatics analysis provides an extended list of known and putative proteins from different species of Plasmodium which have such MSP3-like C-terminal domains. This finding allowed us to extend some conclusions of our studies to a larger group of the malaria surface proteins. Possible structural and functional roles of these highly conserved oligomerization domains in the intact merozoite surface proteins are discussed.

Recombinant Viral Vaccines Expressing Merozoite Surface Protein-1 Induce Antibody- and T Cell-Mediated Multistage Protection against Malaria

SummaryProtecting against both liver and blood stages of infection is a long-sought goal of malaria vaccine design. Recently, we described the use of replication-defective viral vaccine vectors expressing the malaria antigen merozoite surface protein-1 (MSP-1) as an antimalarial vaccine strategy that elicits potent and protective antibody responses against blood-stage parasites. Here, we show that vaccine-induced MSP-1-specific CD4+ T cells provide essential help for protective B cell responses, and CD8+ T cells mediate significant antiparasitic activity against liver-stage parasites. Enhanced survival is subsequently seen in immunized mice following challenge with sporozoites, which mimics the natural route of infection more closely than when using infected red blood cells. This effect is evident both in the presence and absence of protective antibodies and is associated with decreased parasite burden in the liver followed by enhanced induction of the cytokine IFN-γ in the serum. Multistage immunity against malaria can thus be achieved by using viral vectors recombinant for MSP-1.

Characterization of a protective Escherichia coli-expressed Plasmodium falciparum merozoite surface protein 3 indicates a non-linear, multi-domain structure

Immunization with a recombinant yeast-expressed Plasmodium falciparum merozoite surface protein 3 (MSP3) protected Aotus nancymai monkeys against a virulent challenge infection. Unfortunately, the production process for this yeast-expressed material was not optimal for human trials. In an effort to produce a recombinant MSP3 protein in a scaleable manner, we expressed and purified near-full-length MSP3 in Escherichia coli (EcMSP3). Purified EcMSP3 formed non-globular dimers as determined by analytical size-exclusion HPLC with in-line multi-angle light scatter and quasi-elastic light scatter detection and velocity sedimentation ( R h 7.6 ± 0.2 nm and 6.9 nm, respectively). Evaluation by high-resolution atomic force microscopy revealed non-linear asymmetric structures, with beaded domains and flexible loops that were recognized predominantly as dimers, although monomers and larger multimers were observed. The beaded substructure corresponds to predicted structural domains, which explains the velocity sedimentation results and improves the conceptual model of the protein. Vaccination with EcMSP3 in Freund's adjuvant-induced antibodies that recognized native MSP3 in parasitized erythrocytes by an immunofluorescence assay and gave delayed time to treatment in a group of Aotus monkeys in a virulent challenge infection with the FVO strain of P. falciparum. Three of the seven monkeys vaccinated with EcMSP3 had low peak parasitemias. EcMSP3, which likely mimics the native MSP3 structure located on the merozoite surface, is a viable candidate for inclusion in a multi-component malaria vaccine.

Passive transfer of Plasmodium falciparum MSP-2 pseudopeptide-induced antibodies efficiently controlled parasitemia in Plasmodium berghei-infected mice

We have developed monoclonal antibodies directed against the pseudopeptide ψ-130, derived from the highly conserved malarial antigen Plasmodium falciparum merozoite surface protein 2 (MSP-2), for obtaining novel molecular tools with potential applications in the control of malaria. Following isotype switching, these antibodies were tested for their ability to suppress blood-stage parasitemia through passive immunization in malaria-infected mice. Some proved totally effective in suppressing a lethal blood-stage challenge infection and others reduced malarial parasitemia. Protection against P. berghei malaria following Ig passive immunization can be associated with specific immunoglobulins induced by a site-directed designed MSP-2 reduced amide pseudopeptide.

Formation of the Food Vacuole in Plasmodium falciparum: A Potential Role for the 19 kDa Fragment of Merozoite Surface Protein 1 (MSP119)

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

Effect of GPI anchor moiety on the immunogenicity of DNA plasmids encoding the 19‐kDa C‐terminal portion of Plasmodium falciparum MSP‐1

The glycosylphosphatidylinositol (GPI)-anchored Plasmodium falciparum merozoite surface protein 1 (MSP-1) is a widely studied malaria vaccine candidate. The C-terminal 19-kDa portion of MSP-1 (MSP-1(19)) is of particular interest because this polypeptide moiety remains bound to the parasite even after erythrocyte invasion, while the remainder of MSP-1 is shed during invasion. Studies have shown that antibodies against MSP-1(19) inhibit merozoite invasion of erythrocytes efficiently, and that MSP-1(19) produces protective immunity in mice and monkeys. To investigate the efficacy of MSP-1(19 )DNA vaccine and role of GPI anchor moiety in the immunogenicity of MSP-1(19), we constructed expression vectors that produce MSP-1(19) as either secretory or GPI-anchored polypeptide. Both constructs efficiently expressed MSP-1(19) in transfected HEK-293 cells. While the recombinant plasmid lacking GPI anchor signal sequence expressed MSP-1(19) mainly as secreted polypeptide, that containing GPI anchor signal sequence produced GPI-anchored MSP-1(19 )on cell surface. In immunized mice, both constructs produced substantial levels of MSP-1(19)-specific IgG1, IgG2a, IgG2b, IgG3, IgA and IgM antibodies. In both cases, the IgG1 level was significantly higher than other isotypes. Interestingly, the plasmid containing GPI anchor signal sequence produced significantly higher levels of IgG2a and IgG2b than the plasmid that lacks GPI signal sequence.

Identifying Merozoite Surface Protein 4 and Merozoite Surface Protein 7 Plasmodium falciparum Protein Family Members Specifically Binding to Human Erythrocytes Suggests a New Malarial Parasite-Redundant Survival Mechanism

Plasmodium falciparum merozoite surface proteins (MSP-1 to -11) have been involved in merozoite interaction with the red blood cell (RBC) surface. Peptides covering complete MSP-4 and MSP-7 amino acid sequences were synthesized and tested in RBC binding assays. One MSP-4 high activity binding peptide (HABP) and five MSP-7 HABPs were found having specific binding to RBC surface. MSP-4 and MSP-7 HABP binding was sensitive to enzymatic treatment; they recognized a 52 kDa erythrocyte membrane protein. MSP-4 HABP had low invasion inhibition, suggesting it might bind to RBCs and also be involved in physiological mechanisms, while MSP-7 HABPs displayed different invasion inhibition activity (83-24%) in in vitro tests, suggesting different roles for both proteins during invasion. Structural characteristics found when comparing the MSP-4 HABP with MSP-HABPs displaying epidermal growth factor-like sequences suggested that these redundant MSP-family proteins could be a new parasite strategy for evading host genetic variability and immune pressure.