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1.
The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed, and the 19-kDa C-terminal fragment (MSP119) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth, MSP119 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based on BiGGER is employed on MSP119-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP119 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria treatment and provide valuable information for drug design.  相似文献   

2.
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.  相似文献   

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.  相似文献   

4.
P30P2MSP1(19) is a recombinant subunit vaccine derived from merozoite surface protein 1 (MSP1) of Plasmodium falciparum, the causative agent of malaria. P30P2MSP1(19) consists of two universal T-cell epitopes fused to the most C-terminal 19-kDa portion of MSP1, and this protein has previously shown promising potential as a vaccine for malaria. However, previous attempts at producing this molecule in Saccharomyces cerevisiae resulted in the production of a truncated form of the molecule missing most of the universal T-cell epitopes. Here, we report the production of full-length P30P2MSP1(19) in Pichia pastoris. As salt precipitation is a common problem during P. pastoris high-density fermentation, we utilized an alternative low-salt, fully defined medium that did not reduce growth rates or biomass yields to avoid precipitation. A total of 500 mg/L of secreted purified protein was produced in high cell density fermentation and the protein was purified in one step utilizing nickel-chelate chromatography. P30P2MSP1(19) produced in Pichia was reactive with monoclonal antibodies that recognize only conformational epitopes on correctly folded MSP1. Rabbits immunized with this molecule generated higher and more uniform antibody titers than rabbits immunized with the protein produced in Saccharomyces. P30P2MSP1(19) produced in Pichia may prove to be a more efficacious vaccine than that produced in Saccharomyces and Pichia would provide a system for the cost-effective production of such a vaccine.  相似文献   

5.
Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.  相似文献   

6.
The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in ‘priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.  相似文献   

7.
Antibodies from malaria-exposed individuals can agglutinate merozoites released from Plasmodium schizonts, thereby preventing them from invading new erythrocytes. Merozoite coat proteins attached to the plasma membrane are major targets for host antibodies and are therefore considered important malaria vaccine candidates. Prominent among these is the abundant glycosylphosphatidylinositol (GPI)-anchored merozoite surface protein 1 (MSP1) and particularly its C-terminal fragment (MSP119) comprised of two epidermal growth factor (EGF)-like modules. In this paper, we revisit the role of agglutination and immunity using transgenic fluorescent marker proteins. We describe expression of heterologous MSP119'miniproteins' on the surface of Plasmodium   falciparum merozoites. To correctly express these proteins, we determined that GPI-anchoring and the presence of a signal sequence do not allow default export of proteins from the endoplasmic reticulum to merozoite surface and that extra sequence elements are required. The EGFs are insufficient for correct trafficking unless they are fused to additional residues that normally reside upstream of this fragment. Antibodies specifically targeting the surface-expressed miniprotein can inhibit erythrocyte invasion in vitro despite the presence of endogenous MSP1. Using a line expressing a green fluorescent protein–MSP1 fusion protein, we demonstrate that one mode of inhibition by antibodies targeting the MSP119 domain is the rapid agglutinating of merozoites prior to erythrocyte attachment.  相似文献   

8.
Merozoite surface protein 3 (MSP3), an important vaccine candidate, is a soluble polymorphic antigen associated with the surface of Plasmodium falciparum merozoites. The MSP3 sequence contains three blocks of heptad repeats that are consistent with the formation of an intramolecular coiled-coil. MSP3 also contains a glutamic acid-rich region and a putative leucine zipper sequence at the C-terminus. We have disrupted the msp3 gene by homologous recombination, resulting in the expression of a truncated form of MSP3 that lacks the putative leucine zipper sequence but retains the glutamic acid-rich region and the heptad repeats. Here, we show that truncated MSP3, lacking the putative leucine zipper region, does not localize to the parasitophorous vacuole or interact with the merozoite surface. Furthermore, the acidic-basic repeat antigen (ABRA), which is present on the merozoite surface, also was not localized to the merozoite surface in parasites expressing the truncated form of MSP3. The P. falciparum merozoites lacking MSP3 and ABRA on the surface show reduced invasion into erythrocytes. These results suggest that MSP3 is not absolutely essential for blood stage growth and that the putative leucine zipper region is required for the trafficking of both MSP3 and ABRA to the parasitophorous vacuole.  相似文献   

9.
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.  相似文献   

10.
Immunizing pregnant women with a malaria vaccine is one approach to protecting the mother and her offspring from malaria infection. However, specific maternal Abs generated in response to vaccination and transferred to the fetus may interfere with the infant's ability to respond to the same vaccine. Using a murine model of malaria, we examined the effect of maternal 19-kDa C-terminal region of merozoite surface protein-1 (MSP1(19)) and Plasmodium yoelii Abs on the pups' ability to respond to immunization with MSP1(19). Maternal MSP1(19)-specific Abs but not P. yoelii-specific Abs inhibited Ab production following MSP1(19) immunization in 2-wk-old pups. This inhibition was correlated with the amount of maternal MSP1(19) Ab present in the pup at the time of immunization and was due to fewer specific B cells. Passively acquired Ab most likely inhibited the development of an Ab response by blocking access to critical B cell epitopes. If a neonate's ability to respond to MSP1(19) vaccination depends on the level of maternal Abs present at the time of vaccination, it may be necessary to delay immunization until Abs specific for the vaccinating Ag have decreased.  相似文献   

11.
One of the most promising vaccine candidates against the erythrocytic forms of malaria is the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)). As part of our studies aimed at the development of a Plasmodium vivax malaria vaccine, we characterized the immunogenic properties of a new bacterial recombinant protein containing the P. vivax MSP1(19) and two helper T-cell epitopes, the synthetic universal pan allelic DR epitope (PADRE) and a new internal MSP1 P. vivax epitope (DYDVVYLKPLAGMYK). We found that the recognition of His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE was as good as the recognition of His6MSP1(19) indicating that the presence of the T-cell epitopes PADRE and DYDVVYLKPLAGMYK did not modify the MSP1(19) epitopes recognized by human IgG. The recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE proved to be highly immunogenic in marmosets (Callithrix jacchus jacchus) when administered in incomplete Freund's adjuvant. However, when administered in other adjuvant formulations such as Quil A, CpG ODN 2006 or MPL/TDM, antibody titers to MSP1(19) were significantly lower. Among these three adjuvants, Quil A proved to be the most efficient one generating antibody titers significantly higher than the others. These results indicated that under the circumstances evaluated, adjuvants were key for the immunogenicity of the recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE.  相似文献   

12.
Immunity induced by the 19-kDa fragment of merozoite surface protein 1 is dependent on CD4+ Th cells. However, we found that adoptively transferred CFSE-labeled Th cells specific for an epitope on Plasmodium yoelii 19-kDa fragment of merozoite surface protein 1 (peptide (p)24), but not OVA-specific T cells, were deleted as a result of P. yoelii infection. As a result of infection, spleen cells recovered from infected p24-specific T cell-transfused mice demonstrated reduced response to specific Ag. A higher percentage of CFSE-labeled p24-specific T cells stained positive with annexin and anti-active caspase-3 in infected compared with uninfected mice, suggesting that apoptosis contributed to deletion of p24-specific T cells during infection. Apoptosis correlated with increased percentages of p24-specific T cells that stained positive for Fas from infected mice, suggesting that P. yoelii-induced apoptosis is, at least in part, mediated by Fas. However, bystander cells of other specificities also showed increased Fas expression during infection, suggesting that Fas expression alone is not sufficient for apoptosis. These data have implications for the development of immunity in the face of endemic parasite exposure.  相似文献   

13.
Merozoite surface protein 1 (MSP1) of malaria parasites undergoes proteolytic processing at least twice before invasion into a new RBC. The 42-kDa fragment, a product of primary processing, is cleaved by proteolytic enzymes giving rise to MSP1(33), which is shed from the merozoite surface, and MSP1(19), which is the only fragment carried into a new RBC. In this study, we have identified T cell epitopes on MSP1(33) of Plasmodium yoelii and have examined their function in immunity to blood stage malaria. Peptides 20 aa in length, spanning the length of MSP1(33) and overlapping each other by 10 aa, were analyzed for their ability to induce T cell proliferation in immunized BALB/c and C57BL/6 mice. Multiple epitopes were recognized by these two strains of mice. Effector functions of the dominant epitopes were then investigated. Peptides Cm15 and Cm21 were of particular interest as they were able to induce effector T cells capable of delaying growth of lethal P. yoelii YM following adoptive transfer into immunodeficient mice without inducing detectable Ab responses. Homologs of these epitopes could be candidates for inclusion in a subunit vaccine.  相似文献   

14.
We have identified a Plasmodium vivax merozoite surface protein (MSP) that migrates on SDS-polyacrylamide gels at a Mr of about 185 kDa. This protein was recognized by a P. vivax monoclonal antibody (mAb) that localizes the protein by immunofluorescence to the surface of merozoites and also immunoprecipitates this protein from NP-40 detergent extracts of [35S]methionine metabolically radiolabeled P. vivax schizonts. The P. vivax MSP does not become biosynthetically radiolabeled with [3H]glucoamine, [3H]myristate, [3H]palmitate, or [3H]mannose, indicating that this P. vivax MSP is not posttranslationally modified and bound to the merozoite membrane by a glycosylphosphatidylinositol (GPI) lipid anchor. Thus, in this respect, this protein is different from members of the MSP-1 protein family and from MSP-2 and MSP-4 of P. falciparum. The mAb cross-reacts with and outlines the surface of P. cynomolgi merozoites and immunoprecipitates a 150-kDa P. cynomolgi homologue. The mAb was used as an affinity reagent to purify the native homologous MSP from NP-40 extracts of P. cynomolgi mature schizonts in order to develop a specific polyclonal antiserum. The resulting anti-PcyMSP rabbit antiserum cross-reacts strongly with the P. vivax 185-kDa MSP and also recognizes an analogous 110-kDa protein from P. knowlesi. We have determined via an immunodepletion experiment that the 110-kDa P. knowlesi MSP corresponds to the PK 110 protein partially characterized earlier (Perler et al. 1987). The potential of P. vivax MSP as a vaccine candidate was addressed by conducting in vitro inhibition of erythrocyte invasion assays, and the IgG fraction of both the P. vivax MSP mAb and the P. cynomolgi MSP rabbit antiserum significantly inhibited entry of P. vivax merozoites. We denote, on a preliminary basis, these antigenically related merozite surface proteins PvMSP-185, PcyMSP-150, and PkMSP-110.  相似文献   

15.
Maternal Abs generated as a result of prior exposure to infectious agents such as the malaria parasite are transferred from the mother through the placenta to the fetus. Numerous studies have attributed the resistance to malaria infection observed in neonates and infants up to 6 mo of age to the presence of maternally derived Abs. However, recent studies have produced conflicting results suggesting that alternative protective mechanisms may be responsible. Although the presence of maternally derived Abs in the infant is not disputed, their exact role in the infant is unknown. Even less clear is the effect that maternally derived Abs, if generated in response to vaccination, may have on the infant's ability to respond to malaria infection. Studies on mouse pups were performed to determine the role of the 19-kDa region of merozoite surface protein 1 (MSP1(19)) and Plasmodium yoelii-specific Abs in neonatal malaria infection and to examine their effect on the development of a specific immune response in the pup. It was shown that P. yoelii- and MSP1(19)-specific Abs transferred to the pup from the mother act to suppress the growth of the parasite in the pup. However, the maternally derived Abs interfered with the development of the pups' own Ab response to the parasite by altering the fine specificity of the response. These results suggest that immunizing women of child-bearing age with a malaria vaccine candidate such as MSP1(19) would not prevent the infant from producing Abs in response to malaria infection, but it may affect the region of the Ag to which it responds.  相似文献   

16.
Vaccination of mice with the leading malaria vaccine candidate homologue, the 19-kDa carboxyl terminus of merozoite surface protein-1 (MSP119), results in sterile immunity to Plasmodium yoelii, with no parasites detected in blood. Although such immunity depends upon high titer Abs at challenge, high doses of immune sera transferred into naive mice reduce parasitemia (and protect from death) but do not result in a similar degree of protection (with most mice experiencing high peak parasitemias); this finding suggests that ongoing parasite-specific immune responses postchallenge are essential. We analyzed this postchallenge response by transferring Abs into manipulated but malaria-naive mice and observed that Abs cannot protect SCID, nude, CD4+ T cell-depleted, or B cell knockout mice, with all mice dying. Thus, in addition to the Abs that develop following MSP119 vaccination, a continuing active immune response postchallenge is required for protection. MSP119-specific Abs can adoptively transfer protection to strains of mice that are not protected following vaccination with MSP119, suggesting that the Ags targeted by the immune response postchallenge include Ags apart from MSP119. These data have important implications for the development of a human malaria vaccine.  相似文献   

17.
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.  相似文献   

18.
Murine immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. We still know relatively little about which IgG subclasses protect against this disease in mouse models, although IgG2a and IgG2b are considered to be the most potent and dominate in successful passive transfer experiments in rodent malarias. To explore the mechanism(s) by which the different mouse IgG subclasses may mediate a protective effect, we generated mouse IgG1, IgG2a, IgG2b and IgG3 specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1(19)), and to the homologous antigen from Plasmodium yoelii (P. yoelii), both major targets of protective immune responses. This panel of eight IgGs bound antigen with an affinity comparable to that seen for their epitope-matched parental monoclonal antibodies (mAbs) from which they were derived, although for reasons of yield, we were only able to explore the function of mouse IgG1 recognizing PfMSP1(19) in detail, both in vitro and in vivo. Murine IgG1 was as effective as the parental human IgG from which it was derived at inducing NADPH-mediated oxidative bursts and degranulation from neutrophils. Despite showing efficacy in in vitro functional assays with neutrophils, the mouse IgG1 failed to protect against parasite challenge in vivo. The lack of protection afforded by MSP1(19)-specific IgG1 against parasite challenge in wild type mice suggests that this Ab class does not play a major role in the control of infection with mouse malaria in the Plasmodium berghei transgenic model.  相似文献   

19.
The first interaction between the malaria merozoite and the red blood cell it will invade is mediated by molecules on the surface of the two cells. The Plasmodium falciparum merozoite surface protein (MSP)1 complex that contains MSP1 and two other parasite proteins, MSP6 and MSP7, is likely to be an important component in this process. This article reviews the role of the MSP1 complex in the biology of the host parasite interface with a focus on MSP7 and related proteins that are coded by gene families in each of the different Plasmodium spp.  相似文献   

20.
Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2–4 × 10−7 m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.  相似文献   

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