Anemia and antibodies to the 19-kDa fragment of MSP1 during Plasmodium falciparum infection in Aotus monkeys

To determine whether antibodies to the 19-kDa fragment of merozoite surface protein 1 (MSP1(19)) help to control blood-stage Plasmodium falciparum infection, we performed a rechallenge experiment of previously infected Aotus monkeys. Monkeys previously exposed to the FVO strain of P. falciparum that did or did not develop high antibody titers to MSP1(19) and malaria-na?ve monkeys were challenged with erythrocytes infected with the same strain. Prepatent periods were prolonged in previously infected monkeys compared with malaria-na?ve monkeys. Previously infected monkeys with preexisting anti-MSP1(19) antibodies showed low peak parasitemias that cleared spontaneously. Previously infected monkeys that had no or low levels of pre-existing anti-MSP1(19) antibodies also showed low peak parasitemias, but because of low hematocrits, all of these animals required treatment with mefloquine. All previously malaria-na?ve animals were treated because of high parasitemias. The results of this study suggest that antibody to the 19-kDa carboxy-terminal fragment of MSP1 plays a role in preventing the development of anemia, an important complication often associated with malaria.     

The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.     

Cerebral malaria is associated with IgG2 and IgG4 antibody responses to recombinant Plasmodium falciparum RIFIN antigen

RIFIN proteins belong to the largest Plasmodium falciparum multicopy family of variant surface antigens (VSA) expressed by infected erythrocytes. VSA antibodies have been shown to be associated with protection against malaria. Here, antibody subclass responses to a recombinant RIFIN protein (RIF-29) in 116 Ghanaian children were determined by ELISA to investigate the relationship between severe malaria and anti-RIF-29 antibodies. The study group was composed of 23 children diagnosed exclusively for cerebral malaria and 35 children who had non-cerebral severe malaria. The remaining 58 individuals were age-, gender- and area-matched asymptomatic controls. The finding that IgG1 and IgG3 responses predominated in severe malaria patients compared to matched controls suggests that these antibodies are not protective, but are most probably induced by a current infection, an observation substantiated by the equally high reactivity to both recombinant RIF-29 protein and to P. falciparum crude lysate proteins. The exclusive detection of IgG2 and IgG4 antibodies to RIF-29 protein only in cerebral malaria children brings to mind the possibility that these antibodies are pathogenic. This is a new finding that may go some way towards explaining why these children are at risk of developing the life-threatening form of cerebral malaria.     

Immunogenicity of C-terminus of Plasmodium falciparum merozoite surface protein 1 expressed as a non-glycosylated polypeptide in yeast

The C-terminal region of the merozoite surface protein 1 (MSP1_(19)) is one of the mostpromising vaccine candidates against the erythrocytic forms of malaria.In the present study,a gene encodingPlasmodium falciparum MSP1_(19) was expressed in yeast Pichia pastoris.A non-glycosylated form of therecombinant protein MSP1_(19) was purified from culture medium.This recombinant protein maintains itsantigenicity.Significant immune responses were seen in C57BL/6 mice after the second immunization.Moreover,the specific antibodies recognized the native antigens of P.falciparum,The prevailing isotypesof immunoglobulin (Ig)G associated with immunization were IgG1,IgG2a and IgG2b.The antibodiesisolated from mouse sera immunized with MSP1_(19) can inhibit parasite growth in vitro.Based on theseimmunological studies,we concluded that MSP1_(19) deserves further evaluation in pre-clinical immunizationsagainst P.falciparum.     

Genetic polymorphism of the serine rich antigen N-terminal region in Plasmodium falciparum field isolates from Brazil

In this work we investigated the frequency of polymorphism in exon II of the gene encoding most of the amino-terminal region of the serine rich antigen (SERA) in Plasmodium falciparum field samples. The blood samples were collected from P. falciparum infected individuals in three areas of the Brazilian Amazon. Two fragments have been characterized by polymerase chain reaction: one of 175 bp corresponding to the repeat region with 5 octamer units and one other of 199 bp related to the 6 repeat octamer units of SERA protein. The 199 bp fragment was the predominant one in all the studied areas. The higher frequency of this fragment has not been described before and could be explained by an immunological selection of the plasmodial population in the infected individuals under study. Since repeat motifs in the amino-terminal region of SERA contain epitopes recognized by parasite-inhibitor antibodies, data reported here suggest that the analysis of the polymorphism of P. falciparum isolates in different geographical areas is a preliminary stage before the final drawing of an universal vaccine against malaria can be reached.     

A carboxyl-terminal fragment of Plasmodium falciparum gp195 expressed by a recombinant baculovirus induces antibodies that completely inhibit parasite growth.

The major merozoite surface Ag (gp195) of Plasmodium falciparum has been shown to protect monkeys against parasite infection, and gp195-based synthetic peptides and recombinant polypeptides have been evaluated as potential malaria vaccines. A major problem in developing a gp195-based recombinant vaccine has been the difficulty in obtaining a recombinant polypeptide that is immunologically equivalent to the native protein. In this study, the carboxyl-terminal processing fragment (p42) of gp195 was produced in yeast and in a baculovirus recombinant system. Immunologic analyses indicated that the secreted baculovirus p42 (BVp42) expressed native, disulfide-dependent conformational epitopes, whereas these epitopes were poorly represented in the intracellular yeast p42. BVp42, but not yeast p42, was also recognized by the majority of gp195-specific antibodies of animals immunized with purified native gp195, indicating that the anti-gp195 response of these animals was focused on conformational determinants of the p42 processing fragment. Sera against native gp195 of congenic mice of diverse H-2 haplotypes recognized the BVp42 polypeptide, demonstrating that a genetically heterogeneous population is capable of responding to p42 epitopes. BVp42 was highly immunogenic and induced high titers of antibodies that were cross-reactive with purified native gp195 in an ELISA and also reacted with schizonts and merozoites by immunofluorescence. Anti-BVp42 antibodies completely inhibited the in vitro growth of the malaria parasite, whereas anti-yeast p42 antibodies had no effect. These results indicate that native, conformational epitopes of p42 are critical for the induction of gp195-specific, parasite growth-inhibitory antibodies and that the BVp42 polypeptide efficiently induces antibodies specific for these native determinants.     

The Plasmodium vivax rhoptry-associated protein 1

Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.     

The merozoite surface protein 1 complex of human malaria parasite Plasmodium falciparum: interactions and arrangements of subunits

The major protein component at the surface of merozoites, the infectious form of blood stage malaria parasites, is the merozoite surface protein 1 (MSP-1) complex. In the human malaria parasite Plasmodium falciparum, this complex is generated by proteolytic cleavage of a 190-kDa glycosylphosphatidylinositol-anchored precursor into four major fragments, which remain non-covalently associated. Here, we describe the in vitro reconstitution of the MSP-1 complex of P. falciparum strain 3D7 from its heterologously produced subunits. We provide evidence for the arrangement of the subunits within the complex and show how they interact with each other. Our data indicate that the conformation assumed by the reassembled complex as well as by the heterologously produced 190-kDa precursor corresponds to the native one. Based on these results we propose a first structural model for the MSP-1 complex. Together with access to faithfully produced material, this information will advance further structure-function studies of MSP-1 that plays an essential role during invasion of erythrocytes by the parasite and that is considered a promising candidate for a malaria vaccine.     

Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity

In areas of endemic parasite transmission, protective immunity to Plasmodium falciparum malaria is acquired over several years with numerous disease episodes. Acquisition of Abs to parasite-encoded variant surface Ags (VSA) on the infected erythrocyte membrane is important in the development of immunity, as disease-causing parasites appear to be those not controlled by preexisting VSA-specific Abs. In this work we report that VSA expressed by parasites from young Ghanaian children with P. falciparum malaria were commonly and strongly recognized by plasma Abs from healthy children in the same area, whereas recognition of VSA expressed by parasites from older children was weaker and less frequent. Independent of this, parasites isolated from children with severe malaria (cerebral malaria and severe anemia) were better recognized by VSA-specific plasma Abs than parasites obtained from children with nonsevere disease. This was not due to a higher infection multiplicity in younger patients or in patients with severe disease. Our data suggest that acquisition of VSA-specific Ab responses gradually restricts the VSA repertoire that is compatible with parasite survival in the semi-immune host. This appears to limit the risk of severe disease by discriminating against the expression of VSA likely to cause life-threatening complications, such as cerebral malaria and severe anemia. Such VSA seem to be preferred by parasites infecting a nonimmune host, suggesting that VSA expression and switching are not random, and that the VSA expression pattern is modulated by immunity. This opens the possibility of developing morbidity-reducing vaccines targeting a limited subset of common and particularly virulent VSA.     

A co-ligand complex anchors Plasmodium falciparum merozoites to the erythrocyte invasion receptor band 3

In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP1(42)) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/Delta1 and MSP9/Delta2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/Delta1 and MSP9/Delta2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP1(42), the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.     

Construction and immunogenicity in mice of attenuated Salmonella typhi expressing Plasmodium falciparum merozoite surface protein 1 (MSP-1) fused to tetanus toxin fragment C

One strategy to develop a multi-antigen malaria vaccine is to employ live vectors to carry putative protective Plasmodium falciparum antigens to the immune system. The 19 kDa carboxyl terminus of P. falciparum merozoite surface protein 1 (MSP-1), which is essential for erythrocyte invasion and is a leading antigen for inclusion in a multivalent malaria vaccine, was genetically fused to fragment C of tetanus toxin and expressed within attenuated Salmonella typhi CVD 908. Under conditions in the bacterial cytoplasm, the fragment C-MSP-1 fusion did not form the epidermal growth factor (EGF)-like domains of MSP-1; monoclonal antibodies failed to recognize these conformational domains in immunoblots of non-denatured protein extracted from live vector sonicates. The MSP-1 was nevertheless immunogenic. One month following intranasal immunization of BALB/c mice with the live vector construct, four out of five mice exhibited > or =four-fold rises in anti-MSP-1 by ELISA (GMT=211); a single intranasal booster raised titers further (GMT=1280). Post-immunization sera recognized native MSP-1 on merozoites as determined by indirect immunofluorescence. These data encourage efforts to optimize MSP-1 expression in S. typhi (e.g. as a secreted protein), so that the EGF-like epitopes, presumably necessary for stimulating protective antibodies, can form.     

Naturally occurring anti-band 3 autoantibodies recognize a high molecular weight protein on the surface of Plasmodium falciparum infected erythrocytes

Naturally occurring anti-band 3 autoantibodies bind to erythrocytes infected with a knobby variant of the human malaria Plasmodium falciparum (FCR-3 strain). The autoantibodies recognized a greater than 240 kDa protein in SDS extracts made from surface iodinated infected erythrocytes. The antigen was associated only with erythrocytes infected with a knobby variant, and was removed by trypsin treatment of intact infected cells. By two-dimensional peptide map analysis the antigen was shown to be structurally related to the human erythrocyte anion transporter, band 3.     

The stability and complexity of antibody responses to the major surface antigen of Plasmodium falciparum are associated with age in a malaria endemic area

Individuals that are exposed to malaria eventually develop immunity to the disease with one possible mechanism being the gradual acquisition of antibodies to the range of parasite variant surface antigens in their local area. Major antibody targets include the large and highly polymorphic Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of proteins. Here, we use a protein microarray containing 123 recombinant PfEMP1-DBLα domains (VAR) from Papua New Guinea to seroprofile 38 nonimmune children (<4 years) and 29 hyperimmune adults (≥15 years) from the same local area. The overall magnitude, prevalence and breadth of antibody response to VAR was limited at <2 years and 2-2.9 years, peaked at 3-4 years and decreased for adults compared with the oldest children. An increasing proportion of individuals recognized large numbers of VAR proteins (>20) with age, consistent with the breadth of response stabilizing with age. In addition, the antibody response was limited in uninfected children compared with infected children but was similar in adults irrespective of infection status. Analysis of the variant-specific response confirmed that the antibody signature expands with age and infection. This also revealed that the antibody signatures of the youngest children overlapped substantially, suggesting that they are exposed to the same subset of PfEMP1 variants. VAR proteins were either seroprevalent from early in life, (<3 years), from later in childhood (≥3 years) or rarely recognized. Group 2 VAR proteins (Cys2/MFK-REY+) were serodominant in infants (<1-year-old) and all other sequence subgroups became more seroprevalent with age. The results confirm that the anti-PfEMP1-DBLα antibody responses increase in magnitude and prevalence with age and further demonstrate that they increase in stability and complexity. The protein microarray approach provides a unique platform to rapidly profile variant-specific antibodies to malaria and suggests novel insights into the acquisition of immunity to malaria.     

Differential antibody responses to Plasmodium falciparum glycosylphosphatidylinositol anchors in patients with cerebral and mild malaria

Glycosylphosphatidylinositol (GPI) membrane anchors of Plasmodium falciparum surface proteins are thought to be important factors contributing to malaria pathogenesis, and anti-GPI antibodies have been suggested to provide protection by neutralizing the toxic activity of GPIs. In this study, IgG responses against P. falciparum GPIs and a baculovirus recombinant MSP1p19 antigen were evaluated in two distinct groups of 70 patients each, who were hospitalized with malaria. Anti-GPI IgGs were significantly lower in patients hospitalized with confirmed cerebral malaria compared to those with mild malaria (P < 0.01) but did not discriminate for fatal outcome. In contrast, a specific marker of the anti-parasite immunity, as monitored by the anti-MSP1p19 IgG response, was similar in both cerebral and mild malaria individuals, although it was significantly lower in a subgroup with fatal outcomes. These results are consistent with a potential anti-toxin role for anti-GPI antibodies associated with protection against cerebral malaria.     

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novel anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.Key words: Plasmodium falciparum, malaria, histidine-rich protein, monoclonal antibodies, recombinant Fab, rapid diagnostic test     

Characterization of a modified red cell membrane protein expressed on erythrocytes infected with the human malaria parasite Plasmodium falciparum: possible role as a cytoadherent mediating protein

Infections with the human malaria Plasmodium falciparum are characterized by the retention of parasitized erythrocytes in tissue capillaries and venules. Erythrocytes containing trophozoites and schizonts attach to the endothelial cells that line these vessels by means of structurally identifiable excrescences present on the surface of the infected cell. Such excrescences, commonly called knobs, are visible by means of scanning or transmission electron microscopy. The biochemical mechanisms responsible for erythrocyte adherence to the endothelial cell are still undefined. In an attempt to identify the cytoadhesive molecule on the surface of the infected cell, we have prepared monoclonal antibodies to knob-bearing erythrocytes infected with the FCR-3 strain of P. falciparum. One of these monoclonal antibodies, designed 4A3, is an IgM that reacts (by means of immunofluorescence) with the surface of unfixed erythrocytes bearing mature parasites of the knobby line; it does not react with knobless lines or uninfected erythrocytes. By immunoelectron microscopy the monoclonal antibody 4A3 was localized to the knob region. In an in vitro cytoadherence assay, the monoclonal antibody partially blocked the binding of knob-bearing cells (FCR-3 strain) to formalin-fixed amelanotic melanoma cells. The monoclonal antibody was used to immunoprecipitate a protein from extracts of knobby erythrocytes that had been previously surface iodinated. By a two-dimensional peptide mapping technique, the antigen recognized by the monoclonal antibody was found to be structurally related to band 3 protein, the human erythrocyte anion transporter.     

Cellular and humoral immune responses to Plasmodium falciparum gametocyte antigens in malaria-immune individuals. Limited response to the 48/45-kilodalton surface antigen does not appear to be due to MHC restriction

We have examined immune responses to a cultured Plasmodium falciparum gametocyte lysate and to an affinity-purified preparation of the 48/45-kDa gamete surface Ag in a group of 30 malaria immune individuals and in 24 Europeans with no previous exposure to malaria. Cellular responses were assessed in vitro by lymphoproliferation and production of IFN-gamma; antigamete antibodies were detected by immunofluorescence, Western blotting, and competitive ELISA. Cells from all the malaria immune donors responded to the gametocyte lysate in both assays while cells from nonimmune donors gave only weak proliferative responses. Antigamete antibodies were detected in the serum of all the immune donors but not in serum from nonimmunes. Nonimmune donors were completely unresponsive to the purified 48/45-kDa surface Ag while cells from 40% of immune donors responded by either proliferation or IFN-gamma production. Only 3 of 30 immune donors had detectable antibodies to the 48/45-kDa Ag. Class II HLA type was determined for 27 of the immune donors but no relationship between HLA-DR or -DQ and responsiveness to the 48/45-kDa Ag was discerned. The possible reasons for limited recognition of this gamete surface Ag are discussed.     

Suramin and suramin analogues inhibit merozoite surface protein-1 secondary processing and erythrocyte invasion by the malaria parasite Plasmodium falciparum

Malarial merozoites invade erythrocytes; and as an essential step in this invasion process, the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP142) is further cleaved to a 33-kDa N-terminal polypeptide (MSP133) and an 19-kDa C-terminal fragment (MSP119) in a secondary processing step. Suramin was shown to inhibit both merozoite invasion and MSP142 proteolytic cleavage. This polysulfonated naphthylurea bound directly to recombinant P. falciparum MSP142 (Kd = 0.2 microM) and to Plasmodium vivax MSP142 (Kd = 0.3 microM) as measured by fluorescence enhancement in the presence of the protein and by isothermal titration calorimetry. Suramin bound only slightly less tightly to the P. vivax MSP133 (Kd = 1.5 microM) secondary processing product (fluorescence measurements), but very weakly to MSP119 (Kd approximately 15 mM) (NMR measurements). Several residues in MSP119 were implicated in the interaction with suramin using NMR measurements. A series of symmetrical suramin analogues that differ in the number of aromatic rings and substitution patterns of the terminal naphthylamine groups was examined in invasion and processing assays. Two classes of analogue with either two or four bridging rings were found to be active in both assays, whereas two other classes without bridging rings were inactive. We propose that suramin and related compounds inhibit erythrocyte invasion by binding to MSP1 and by preventing its cleavage by the secondary processing protease. The results indicate that enzymatic events during invasion are suitable targets for drug development and validate the novel concept of an inhibitor binding to a macromolecular substrate to prevent its proteolysis by a protease.