Lack of association between maternal antibody and protection of African infants from malaria infection

Maternally derived antibodies are believed to protect infants against infection, but there is little direct evidence for a protective role of passively acquired antibodies against malaria. A longitudinal study of malaria infection in 143 infants was conducted in a region of southern Ghana where Plasmodium falciparum is endemic. Infants born in the high-transmission season were less likely to become infected in the first 20 weeks of life than children born in the low-transmission season. Plasma, obtained at birth, was tested for immunoglobulin G (IgG) and IgG subclasses to P. falciparum schizonts and recombinant circumsporozoite antigen, MSP-1(19), MSP-2, AMA-1, and Pf155 (also called ring-infected erythrocyte surface antigen). Antibody levels at birth were not associated with resistance to malaria infection. On the contrary, antibodies at birth were positively associated with infection, indicating that high levels of maternally derived antibodies represent a marker for intensity of exposure to malaria infection in infants. However, all five children who experienced high-density infections (>100 parasites/microl of blood) were seronegative for MSP-1(19) at the time of infection.     

Identification of a novel antigenic domain of Plasmodium falciparum merozoite surface protein-1 that specifically binds to human erythrocytes and inhibits parasite invasion, in vitro

Merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a promising candidate for vaccine development against malaria. Identification of protective epitopes within MSP-1 is an important step towards the elucidation of mechanisms of parasitic invasion and for the creation of a multi-subunit vaccine. In this study, we show that a 115 amino acid region (p115MSP-1) within the p38 domain of MSP-1 can: (i) specifically bind to human erythrocytes, independent of glycophorin A; (ii) inhibit parasite invasion at significant levels, in vitro; and (iii) be recognized by human sera of individuals from malaria-endemic regions of Africa. More importantly, we also show that polyclonal antibodies specific to this region prevent parasite invasion at levels approaching 90%, in vitro. Our data illustrate that not only is p115MSP-1 involved in parasite recognition/invasion of human erythrocytes, but that this region is highly antigenic, producing high titer antibodies. The delineation of the role of MSP-1 in parasite invasion is an important component of the development of a multi-subunit malaria vaccine, and this study identifies a candidate antigen in this context.     

Allelic diversity and antibody recognition of Plasmodium falciparum merozoite surface protein 1 during hypoendemic malaria transmission in the Brazilian amazon region.

The polymorphic merozoite surface protein (MSP-1) of Plasmodium falciparum is a major asexual blood-stage malaria vaccine candidate. The impact of allelic diversity on recognition of MSP-1 during the immune response remains to be investigated in areas of hypoendemicity such as the Brazilian Amazon region. In this study, PCR was used to type variable regions, blocks 2, 4, and 10, of the msp-1 gene and to characterize major gene types (unique combinations of allelic types in variable blocks) in P. falciparum isolates collected across the Amazon basin over a period of 12 years. Twelve of the 24 possible gene types were found among 181 isolates, and 68 (38%) of them had more than one gene type. Temporal, but not spatial, variation was found in the distribution of MSP-1 gene types in the Amazon. Interestingly, some gene types occurred more frequently than expected from random assortment of allelic types in different blocks, as previously found in other areas of endemicity. We also compared the antibody recognition of polymorphic (block 2), dimorphic (block 6), and conserved (block 3) regions of MSP-1 in Amazonian malaria patients and clinically immune Africans, using a panel of recombinant peptides. Results were summarized as follows. (i) All blocks were targeted by naturally acquired cytophilic antibodies of the subclasses IgG1 and IgG3, but the balance between IgG1 and IgG3 depended on the subjects' cumulative exposure to malaria. (ii) The balance between IgG1 and IgG3 subclasses and the duration of antibody responses differed in relation to distinct MSP-1 peptides. (iii) Antibody responses to variable blocks 2 and 6 were predominantly type specific, but variant-specific antibodies that target isolate-specific repetitive motifs within block 2 were more frequent in Amazonian patients than in previously studied African populations.     

Differential patterns of human immunoglobulin G subclass responses to distinct regions of a single protein, the merozoite surface protein 1 of Plasmodium falciparum

Comparisons of immunoglobulin G (IgG) subclass responses to the major polymorphic region and to a conserved region of MSP-1 in three cohorts of African villagers exposed to Plasmodium falciparum revealed that responses to Block 2 are predominantly IgG3 whereas antibodies to MSP-1(19) are mainly IgG1. The striking dominance of IgG3 to Block 2 may explain the short duration of this response and also the requirement for continuous stimulation by malaria infection to maintain clinical immunity.     

Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria

Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n = 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-1(19) and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P = 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n = 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.     

Familial correlation of immunoglobulin G subclass responses to Plasmodium falciparum antigens in Burkina Faso

Host genes are thought to determine the immune response to malaria infection and the outcome. Cytophilic antibodies have been associated with protection, whereas noncytophilic antibodies against the same epitopes may block the protective activity of the protective ones. To assess the contribution of genetic factors to immunoglobulin G (IgG) subclass responses against conserved epitopes and Plasmodium falciparum blood-stage extracts, we analyzed the isotypic distribution of the IgG responses in 366 individuals living in two differently exposed areas in Burkina Faso. We used one-way analysis of variance and pairwise estimators to calculate sib-sib and parent-offspring correlation coefficients, respectively. Familial patterns of inheritance of IgG subclass responses to defined antigens and P. falciparum extracts appear to be similar in the two areas. We observed a sibling correlation for the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against ring-infected-erythrocyte surface antigen, merozoite surface protein 1 (MSP-1), MSP-2, and P. falciparum extract. Moreover, a parent-offspring correlation was found for several IgG subclass responses, including the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against conserved MSP-2 epitopes. Our results indicated that the IgG subclass responses against P. falciparum blood-stage antigens are partly influenced by host genetic factors. The localization and identification of these genes may have implications for immunoepidemiology and vaccine development.     

Mass spectrometric identification of an HLA-A*0201 epitope from Plasmodium falciparum MSP-1

Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum-derived antigens were shown to play an important role for the protection against malaria. Although several CTL epitopes have been identified from P. falciparum sporozoite-derived antigens, none has been described for the merozoite form. Since the merozoite surface protein (MSP)-1 is a known target of the immune response, we focused on this protein to identify HLA-A*0201-associated epitopes. Using our mass spectrometry-based method [the 'predict-calibrate-detect' (PCD) approach], we were able to identify an MSP-1-derived epitope in the peptide mixture naturally associated with HLA-A*0201 molecules purified from an MSP-1-expressing cell line. CTLs against this epitope were generated from HLA-A*0201 monochain transgenic mice (HHD). They specifically killed MSP-1-expressing HLA-A2-positive target cells. Thus, we describe here the first MHC class I epitope from the merozoite form of P. falciparum. This epitope can be used as a tool for the immunomonitoring of natural or vaccine-induced CTL immune responses against malaria and could eventually be proposed as a component of an anti-malaria peptide-based vaccine.     

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

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

Distinct Th1- and Th2-Type prenatal cytokine responses to Plasmodium falciparum erythrocyte invasion ligands

Prenatal immunity to Plasmodium falciparum merozoite proteins involved in erythrocyte invasion may contribute to the partial protection against malaria that is acquired during infancy in areas of stable malaria transmission. We examined newborn and maternal cytokine and antibody responses to merozoite surface protein-1 (MSP-1), ribosomal phosphoprotein P0 (PfP0), and region II of erythrocyte binding antigen-175 (EBA-175) in infant-mother pairs in Kenya. Overall, 82 of 167 (50%), 106 of 176 (60%), and 38 of 84 (45%) cord blood lymphocytes (CBL) from newborns produced one or more cytokines in response to MSP-1, PfP0, and EBA-175, respectively. Newborns of primigravid and/or malaria-infected women were more likely to have antigen-responsive CBL than were newborns of multigravid and/or uninfected women at delivery. Newborn cytokine responses did not match those of their mothers and fell into three distinct categories, Th1 (21 of 55 CBL donors produced only gamma interferon and/or interleukin 2 [IL-2]), Th2 (21 of 55 produced only IL-5 and/or IL-13), and mixed Th1/Th2 (13 of 55). Newborns produced more IL-10 than adults. High and low levels of cord blood IL-12 p70 production induced by anti-CD40 activation were associated with malaria-specific Th1 and Th2 responses, respectively. Antigen-responsive CBL in some newborns were detected only after depletion of IL-10-secreting CD8 cells with enrichment for CD4 cells. These data indicate that prenatal sensitization to blood-stage Plasmodium falciparum occurs frequently in areas where malaria is holoendemic. Modulation of this immunity, possibly by maternal parity and malaria, may affect the acquisition of protective immunity against malaria during infancy.     

Enhanced protection against malaria by a chimeric merozoite surface protein vaccine

The 42-kDa processed fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is a prime candidate for a blood-stage malaria vaccine. Merozoite surface protein 8 contains two C-terminal epidermal growth factor (EGF)-like domains that may function similarly to those of MSP-1(42). Immunization with either MSP-1 or MSP-8 induces protection that is mediated primarily by antibodies against conformation-dependent epitopes. In a series of comparative immunogenicity and efficacy studies using the Plasmodium yoelii rodent model, we tested the ability of recombinant P. yoelii MSP-8 (rPyMSP-8) to complement rPyMSP-1-based vaccines. Unlike MSP-1, PyMSP-8-dependent protection required immunization with the full-length protein and was not induced with recombinant antigens that contained only the C-terminal EGF-like domains. Unlike PyMSP-8, the immunogenicity of the PyMSP-1 EGF-like domains was low when present as part of the rPyMSP-1(42) antigen. Immunization with a mixture of rPyMSP-1(42) and rPyMSP-8 further inhibited the antibody response to protective epitopes of rPyMSP-1(42) and did not improve vaccine efficacy. To improve PyMSP-1 immunogenicity, we produced a chimeric antigen containing the EGF-like domains of PyMSP-1 fused to the N terminus of PyMSP-8. Immunization with the chimeric rPyMSP-1/8 antigen induced high and comparable antibody responses against the EGF-like domains of both PyMSP-1 and PyMSP-8. This enhanced MSP-1-specific antibody response and the concurrent targeting of MSP-1 and MSP-8 resulted in improved, nearly complete protection against lethal P. yoelii 17XL malaria. Unexpectedly, immunization with rPyMSP-1/8 failed to protect against challenge infection with reticulocyte-restricted P. yoelii 17X parasites. Overall, these data establish an effective strategy to improve the efficacy of P. falciparum MSP-based vaccines.     

Analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite Plasmodium falciparum

The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.     

A recombinant baculovirus 42-kilodalton C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 protects Aotus monkeys against malaria.

The immunogenicity and protective efficacy of baculovirus recombinant polypeptide based on the Plasmodium falciparum merozoite surface protein 1 (MSP-1) has been evaluated in Aotus lemurinus griseimembra monkeys. The MSP-1-based polypeptide, BVp42, corresponds to the 42-kDa C-terminal processing fragment of the precursor molecule. Immunization of Aotus monkeys with BVp42 in complete Freund's adjuvant resulted in high antibody titers against the immunogen as well as parasite MSP-1. Fine specificity studies indicated that major epitopes recognized by these antibodies localize to conserved determinants of the 19-kDa C-terminal fragment derived from cleavage of the 42-kDa processing fragment. Effective priming of MSP-1-specific T cells was also demonstrated in lymphocyte proliferation assays. All three Aotus monkeys immunized with BVp42 in complete Freund's adjuvant showed evidence of protection of protection against blood-stage challenge with P. falciparum. Two animals were completely protected, with only one parasite being detected in thick blood films on a single days after injection. The third animal had a modified course of infection, controlling its parasite infection to levels below detection by thick blood smears for an extended period in comparison with adjuvant control animals. All vaccinated, protected Aotus monkeys produced antibodies which inhibited in vitro parasite growth, indicating that this assay may be a useful correlate of protective immunity and that immunity induced by BVp42 immunization is mediated, at least in part, by a direct effect of antibodies against the MSP-1 C-terminal region. The high level of protection obtained in these studies supports further development of BVp42 as a candidate malaria vaccine.     

Immunoglobulin G antibodies to merozoite surface antigens are associated with recovery from chloroquine-resistant Plasmodium falciparum in Gambian children

We examined the hypothesis that recovery from uncomplicated malaria in patients carrying drug-resistant Plasmodium falciparum is a measure of acquired functional immunity and may therefore be associated with humoral responses to candidate vaccine antigens. Gambian children with malaria were treated with chloroquine in 28-day trials, and recovery was defined primarily as the absence of severe clinical malaria at any time and absence of parasitemia with fever after 3 days. Plasma samples from these children were assayed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) to recombinant merozoite antigens: apical membrane antigen 1 (AMA-1) and the 19-kDa C-terminal region of merozoite surface protein 1 (MSP-1(19)), including antigenic variants of MSP-1(19) with double and triple substitutions. Antigen-specific IgG was more frequent in children who recovered, particularly that for MSP-1(19) (age-adjusted odds ratios: 0.32 [95% confidence interval, 0.05, 1.87; P = 0.168] for AMA-1, 0.19 [0.03, 1.11; P = 0.019] for recombinant MSP-1(19), 0.24 [0.04, 1.31; P = 0.032] for the recombinant MSP-1(19) double variant, and 0.18 [0.03, 0.97; P = 0.013] for the triple variant). IgG titers to MSP-1(19) and to the triple variant were higher in plasma samples taken 7 days after chloroquine treatment from children who carried resistant parasites but recovered and remained parasite free. Moreover, in children who were parasitemic on day 14 or day 28, there was an age-independent relationship between parasite density and IgG to both MSP-1(19) and the triple variant (coefficients of -0.550 and -0.590 and P values of 0.002 and 0.001, respectively). The results validate the use of this approach to identify antigens that are associated with protection from malaria.     

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

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

A plant-produced Pfs230 vaccine candidate blocks transmission of Plasmodium falciparum

Plasmodium falciparum is transmitted to a new host after completing its sexual cycle within a mosquito. Developing vaccines against the parasite sexual stages is a critical component in the fight against malaria. We are targeting multiple proteins of P. falciparum which are found only on the surfaces of the sexual forms of the parasite and where antibodies against these proteins have been shown to block the progression of the parasite's life cycle in the mosquito and thus block transmission to the next human host. We have successfully produced a region of the Pfs230 antigen in our plant-based transient-expression system and evaluated this vaccine candidate in an animal model. This plant-produced protein, 230CMB, is expressed at approximately 800 mg/kg in fresh whole leaf tissue and is 100% soluble. Administration of 230CMB with >90% purity induces strong immune responses in rabbits with high titers of transmission-blocking antibodies, resulting in a greater than 99% reduction in oocyst counts in the presence of complement, as determined by a standard membrane feeding assay. Our data provide a clear perspective on the clinical development of a Pfs230-based transmission-blocking malaria vaccine.     

Genetic polymorphism in Plasmodium falciparum vaccine candidate antigens

Malaria is still a major public health problem in many tropical and subtropical countries. Malaria vaccine is highly desirable as an adjunct to existing malaria control measures. The polymorphisms in malaria vaccine candidates antigens might be a hurdle in developing an effective vaccine. The present article reviews the genetic polymorphism in several antigens expressed on the parasite surface, which are targets for immunological responses of the host and are good candidates for vaccine development against P. falciparum. Variable regions of most genes are generally dimorphic probably as a result of intragenic recombinations. Each allele in turn shows polymorphism resulting from point mutations, or other mechanisms. Several antigens like merozoite surface protein-1 and 2 (MSP-1 and MSP-2) and S antigen show high polymorphism while in others like circumsporozoite protein (CSP), apical membrane antigen-1 (AMA-1) and erythrocyte binding antigen-175 (EBA-175) functional constraints limit the degree of polymorphism. Polymorphism reported in these genes is discussed.     

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.     

Selection of glutamate-rich protein long synthetic peptides for vaccine development: antigenicity and relationship with clinical protection and immunogenicity

Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to each LSP was observed in all three areas of endemic infection. Levels of cytophilic immunoglobulin G (IgG) antibodies against both GLURP regions were significantly correlated with protection from clinical P. falciparum malaria. Protected children from the Ghana cohort possessed predominantly IgG1 antibodies against the nonrepeat epitope and IgG3 antibodies against the repeat epitope. T-cell proliferation responses, studied in the cohort from Senegal, revealed that T-helper-cell epitopes were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of different H-2 haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination against P. falciparum malaria.     

Human recombinant antibodies against Plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties

Immunoglobulins from individuals with immunity to malaria have a strong antiparasitic effect when transferred to Plasmodium falciparum malaria infected patients. One prominent target of antiparasitic antibodies is the merozoite surface antigen 3 (MSP-3). We have investigated the antibody response against MSP-3 residues 194 to 257 (MSP-3(194-257)) on the molecular level. mRNA from peripheral blood leukocytes from clinically immune individuals was used as a source of Fab (fragment antibody) genes. A Fab-phage display library was made, and three distinct antibodies designated RAM1, RAM2, and RAM3 were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3 full-length antibodies have been produced in CHO cells. Reactivity with the native parasite protein was demonstrated by immunofluorescence microscopy, flow cytometry, and immunoblotting. Furthermore, the antiparasitic effect of RAM1 has been tested in vitro in an antibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 and the IgG3 versions of the antibody show an inhibitory effect on parasite growth.     

Amino acid dimorphism and parasite immune evasion: cellular immune responses to a promiscuous epitope of Plasmodium falciparum merozoite surface protein 1 displaying dimorphic amino acid polymorphism are highly constrained

Like most other surface-exposed antigens of Plasmodium falciparum, the leading malaria vaccine candidate merozoite surface protein (MSP)-1 contains a large number of dimorphic amino acid positions. This type of diversity is presumed to be associated with parasite immune evasion and represents one major obstacle to malaria subunit vaccine development. To understand the precise role of antigen dimorphism in immune evasion, we have analyzed the flexibility of CD4 T cell immune responses against a semi-conserved sequence stretch of the N-terminal block of MSP-1. While this sequence contains overlapping promiscuous T cell epitopes and is a target for growth inhibitory antibodies, three dimorphic amino acid positions may limit its suitability as component of a multi-epitope malaria vaccine. We have analyzed the CD4 T cell responses in a group of human volunteers immunized with a synthetic malaria peptide vaccine containing a single MSP-143-53 sequence variant. All human T cell lines and HLA-DR- or -DP-restricted T cell clones studied were exclusively specific for the sequence variant used for immunization. Competition peptide binding assays with affinity-purified HLA-DR molecules indicated that dimorphism does not primarily affect HLA binding. Modeling studies of the dominant restricting HLA-DRB1*0801 molecule showed that the dimorphic amino acids represent potential TCR contact residues. Lack of productive triggering of the TCR by MHC/variant peptide ligand complexes thus seems to be the characteristic feature of parasite immune evasion associated with antigen dimorphism.