Association of Plasmodium berghei proteins with the host erythrocyte membrane: binding to inside-out vesicles

Two acidic phosphoproteins of Plasmodium berghei origin, of 65 and 46 kDa, are associated with the plasma membrane of the host mouse erythrocyte. The 65-kDa protein partitions between a soluble and particulate phase upon host cell lysis, whereas the 46-kDa protein is localized exclusively in the particulate fraction. Both proteins bind to inside-out vesicles derived from erythrocyte ghosts and the conditions of the reassociation reaction indicate that the binding is specific and that the proteins interact only with the cytoplasmic face of the erythrocyte membrane. The 65-kDa protein appears to exist in two membrane-associated states; one loosely bound, which readily dissociates from the membrane, and a more tightly associated state, which does not dissociate under non-denaturing conditions. The 46-kDa protein is tightly bound to the host erythrocyte membrane and does not dissociate. Cross-linking studies suggest that both of these parasite proteins interact with the submembrane cytoskeleton of the erythrocyte, and that the 65-kDa protein also appears to interact simultaneously with the lipid bilayer and erythrocyte membrane proteins. However, direct interaction between the malarial proteins and distinct erythrocyte membrane proteins could not be demonstrated. In summary, these findings indicate that the acidic phosphoproteins of the malarial parasite interact with the cytoplasmic face of the erythrocyte membrane both in vivo and in vitro.     

Glycoprotein recognition mediates attachment of Plasmodium chabaudi to mouse erythrocytes

The interaction between Plasmodium falciparum merozoites and human erythrocytes is mediated by specific parasite proteins and sialoglycoproteins (SGPs) on the surface of the host cell. To investigate whether a similar mechanism functions in rodent malaria, a series of experiments was performed to identify the proteins involved in the interaction of Plasmodium chabaudi parasites and mouse erythrocytes. Labeled parasite proteins incubated with purified mouse SGP bound specifically to glycoprotein 2.1. Two parasite proteins (72 and 126 kilodaltons [kDa]) were coprecipitated with antibody directed to mouse erythrocyte membrane proteins. The lower band (72 kDa) as well as a band of 105 kDa were also observed to bind to N-acetyl-D-galactosamine affinity columns, suggesting a carbohydrate component in the binding of these parasites to erythrocytes. These experiments indicate that P. chabaudi possesses specific proteins which recognized SGP on the surface of murine erythrocytes in a manner similar to that of the merozoites of P. falciparum. Thus P. chabaudi in mice may provide an in vivo model of the human parasite for testing ways to inhibit merozoite recognition and invasion of host cells.     

Antibody recognition of rodent malaria parasite antigens exposed at the infected erythrocyte surface: specificity of immunity generated in hyperimmune mice

In regions where malaria is endemic, inhabitants remain susceptible to repeated reinfection as they develop and maintain clinical immunity. This immunity includes responses to surface-exposed antigens on Plasmodium sp.-infected erythrocytes. Some of these parasite-encoded antigens may be diverse and phenotypically variable, and the ability to respond to this diversity and variability is an important component of acquired immunity. Characterizing the relative specificities of antibody responses during the acquisition of immunity and in hyperimmune individuals is thus an important adjunct to vaccine research. This is logistically difficult to do in the field but is relatively easily carried out in animal models. Infections in inbred mice with rodent malaria parasite Plasmodium chabaudi chabaudi AS represent a good model for Plasmodium falciparum in humans. This model has been used in the present study in a comparative analysis of cross-reactive and specific immune responses in rodent malaria. CBA/Ca mice were rendered hyperimmune to P. chabaudi chabaudi (AS or CB lines) or Plasmodium berghei (KSP-11 line) by repeated infection with homologous parasites. Serum from P. chabaudi chabaudi AS hyperimmune mice reacted with antigens released from disrupted P. chabaudi chabaudi AS-infected erythrocytes, but P. chabaudi chabaudi CB and P. berghei KSP-11 hyperimmune serum also contained cross-reactive antibodies to these antigens. However, antibody activity directed against antigens exposed at the surfaces of intact P. chabaudi chabaudi-infected erythrocytes was mainly parasite species specific and, to a lesser extent, parasite line specific. Importantly, this response included opsonizing antibodies, which bound to infected erythrocytes, leading to their phagocytosis and destruction by macrophages. The results are discussed in the context of the role that antibodies to both variable and invariant antigens may play in protective immunity in the face of continuous susceptibility to reinfection.     

Lipoproteins and malaria. I. Immunogenicity of serum lipoproteins in mice infected with Plasmodium chabaudi

Anti-lipoprotein antibodies (anti-Lp Ab) have been investigated during the course of acute infection with P. chabaudi in Swiss mice using a radio-immunoprecipitation assay with (125)-iodine radiolabelled lipoproteins from normal or infected mice. Antibodies were detected 11 days after the beginning of infection; however, the highest lipoprotein precipitations were observed with purified (125)-I labelled lipoproteins from day-5 or day-7 infected mice. P. chabaudi infected mice were treated with chloroquine at various intervals after the beginning of infection and anti-Lp Ab were assayed on day 13. Anti-Lp Ab were not observed in mice treated before day 7 but were present in all mice treated after day 7. Anti-Lp Ab were not detected in mice infected with P. yoelii 17 X. Injection of purified lipoproteins from day-7 P. chabaudi infected mice (Lp day-7) to normal uninfected mice did not induce an antibody response to lipoproteins but anti-Lp Ab were observed when the same injection was performed in P. yoelii infected mice. Moreover, anti-Lp Ab were detected in uninfected mice injected concomitantly with Lp day-7 and hematin extracted from malarial pigment. Our results suggest that anti-Lp Ab observed in P. chabaudi infected mice are mainly against modified lipoproteins produced during infection and that the induction of the antibody response against lipoproteins requires an adjuvant effect such as the hematin which is released during infection.     

Proteins with molecular masses of 25 to 40 kilodaltons elicit optimal protective responses against Plasmodium chabaudi adami infection.

The presence of the CD4+ T cell has been shown to be crucial for resolution of acute infection in the Plasmodium chabaudi adami murine malaria model. This model is, therefore, suitable for the isolation of malaria antigens that are capable of activating protective T cells. In light of this, we set out to identify P. chabaudi adami molecules that activate protective responses in this model. Denatured P. chabaudi adami proteins were isolated by continuous-flow electrophoresis on the basis of their apparent molecular masses and then sequentially assessed for the ability to protect mice in immunization experiments. We report here that low-molecular-mass P. chabaudi adami polypeptides in the range from 25 to 40 kDa are most effective at immunizing mice against a challenge infection with viable P. chabaudi adami. The method used to obtain these proteins could also be applied to identify molecules that activate protective cell-mediated responses in other infectious disease models.     

Inhibitory monoclonal antibodies recognise epitopes adjacent to a proteolytic cleavage site on the RAP-1 protein of Plasmodium falciparum.

The low-molecular-weight rhoptry-associated protein (RAP) complex of Plasmodium falciparum consists of at least two gene products, RAP-1 and RAP-2, and has the ability to immunise Saimiri monkeys against experimental P. falciparum infection. Several monoclonal antibodies specifically recognise this complex and in this study we show that purified immunoglobulin derived from these monoclonals is capable of inhibiting parasite growth in vitro. It has previously been shown that RAP-1 initially appears as an 80-kDa protein (p80) in early schizogony and is processed to a 65-kDa protein (p65) in late schizogony. Several of the inhibitory monoclonals recognise both the 80- and 65-kDa proteins by Western blot analysis suggesting that they recognise linear epitopes on RAP-1. We have mapped these epitopes by testing the reactivity of the monoclonals against fragments of the rap-1 gene expressed as beta-galactosidase fusion proteins and subsequently against synthetic peptides. All of the epitopes map to a region 10-20 amino acids C-terminal to the proteolytic cleavage site for the processing of p80 to p65 at amino acid 190. We also show that the 65-kDa protein is not present in purified merozoites, suggesting that its generation is associated with merozoite release rather than erythrocyte invasion. These results are discussed with respect to possible inhibitory mechanisms for the monoclonals.     

Serological cross-reaction between high molecular weight proteins synthesized in blood schizonts of Plasmodium yoelii, Plasmodium chabaudi and Plasmodium falciparum

A 230 000 molecular weight (MW) Plasmodium yoelii protein, a 250 000 MW P. chabaudi protein and a 195 000 MW P. falciparum protein, identified using monoclonal antibodies, have similar characteristics, and have been implicated as protective antigens. In this study the serological relationship between these proteins was investigated by Western transfer analysis. The monoclonal antibodies specific for each of the high molecular weight proteins did not cross-react with antigens of the other two parasites, but a polyvalent mouse serum raised against the purified 230 000 MW P. yoelii protein cross-reacted with the high molecular weight proteins of P. chabaudi and P. falciparum and also with the fragments derived from these proteins. This result indicates that these proteins belong to the same class of malaria parasite antigen.     

Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

The major outer membrane proteins (OMPs) of the human granulocytic ehrlichiosis (HGE) agent, with molecular sizes of 44 to 47 kDa, are immunodominant antigens in human infection. Monoclonal antibodies (MAbs) to the OMPs were made by immunizing BALB/c mice with the purified HGE agent and then by fusing spleen cells with myeloma cells. The immunologic specificities of three MAbs (3E65, 5C11, and 5D13) were examined with five human HGE agent isolates and one tick isolate. By Western blot analysis, all three MAbs recognized the HGE agent but not Ehrlichia chaffeensis, Ehrlichia sennetsu, Ehrlichia canis, or their host cells. MAb 3E65 reacted with a 44-kDa protein in the homologous human isolate but not in the remaining five isolates. The two remaining MAbs recognized proteins with molecular sizes of 44 to 47 kDa in all six isolates. Western blot results with the OMP fraction of the six isolates were consistent with results with the whole HGE agent. Immunofluorescent-antibody staining and immunogold labeling with these MAbs showed that these antigens were primarily present on the membrane of the HGE agent. MAbs 5C11 and 5D13 recognized the recombinant 44-kDa protein by Western immunoblot analysis, but MAb 3E65 did not. Passive immunization with MAb 3E65 was more effective in protecting mice from HGE agent infection than with MAbs 5C11 and 5D13. These MAbs would be useful for analyzing the role of the major OMP antigens in HGE agent infection and for serodiagnosis.     

Monoclonal antibodies that protect in vivo against Plasmodium chabaudi recognize a 250,000-dalton parasite polypeptide.

Twenty monoclonal antibodies have been prepared to the erythrocytes from CBA/Ca mice infected with the rodent malaria Plasmodium chabaudi. By immunofluorescence, 15 of these antibodies recognized parasite antigens expressed only during the development of mature trophozoites to schizonts and merozoites, 2 recognized parasite antigens that were expressed throughout most of the intraerythrocytic cycle, and 3 recognized the membranes of all infected and uninfected erythrocytes. By immunoprecipitation of [35S]methionine-labeled, parasitized erythrocytes, parasite antigens recognized by all of the antiparasite antibodies were characterized. Eleven precipitated a 250,000-dalton parasite polypeptide which was synthesized and expressed late in the intraerythrocytic cell cycle and which appeared to be the major coat protein of the merozoites. In passive protection experiments, transfer of hyperimmune serum before infection with the parasite resulted in a delay in the rise of parasitemia, reduction in peak parasitemias, and a delay in the clearance of the parasitemia. Two monoclonal antibodies to the 250,000-dalton polypeptide had a similar but not as marked effect on parasitemia when given as a single dose before infection. When mixed and administered throughout the course of infection, their effects were greater. They had no influence on the course of Plasmodium berghei KSP11 parasitemia. Monoclonal antibodies to other parasite antigens and normal erythrocyte antigens failed to have a significant and reproducible effect on P. chabaudi parasitemia. The results suggest that this 250,000-dalton malaria parasite antigen may be important in the induction and expression of antibody-mediated immunity to malaria.     

Analysis of the polypeptides synthesized in rinderpest virus-infected cells

We have identified, by [35S]methionine labeling, eight major induced proteins and a number of minor proteins in rinderpest virus-infected bovine kidney cells. The polypeptides ranged in molecular weight from 212 to 21.5 kDa. The majority of these polypeptides are virus specific, as demonstrated by immunoprecipitation with rabbit hyperimmune serum against rinderpest. Infected cells radiolabeled with glucosamine contained a 75-kDa polypeptide and a broad band migrating at 80 kDa, both identified as virus specific by immunoprecipitation. Phosphorylated virus-specific proteins of 65 kDa and a complex of polypeptides at 92.5 kDa were also identified. Monospecific and monoclonal antibodies against measles virus and canine distemper virus hemagglutinin, fusion protein, nucleocapsid protein, and phosphoproteins confirmed the identity of the corresponding rinderpest virus-specific polypeptides.     

Plasmodium falciparum antigens associated with membrane structures in the host erythrocyte cytoplasm

Hybridomas were made from mice immunized with plasma membranes from erythrocytes infected with Plasmodium falciparum. Among the monoclonal antibodies produced, a series reacted with antigens in the host cell cytoplasm. Immunoelectron microscopy, along with indirect fluorescent antibody double labeling experiments, were used to further localize the antigens to membrane structures (presumably Maurer's clefts) in the erythrocyte cytoplasm. The epitopes thus localized are found on three parasite proteins (20 kDa, 29 kDa, and 45 kDa) and one parasite glycoprotein (45 kDa). They are likely to be part of a transport system for the parasite.     

Purification and characterization of 37-kilodalton proteases from Plasmodium falciparum and Plasmodium berghei which cleave erythrocyte cytoskeletal components

Cytosoluble 100,000 X g extracts from Plasmodium berghei or Plasmodium falciparum infected red blood cells were shown to hydrolyze erythrocyte spectrin. By Fast Protein Liquid Chromatography (FPLC), these enzymes were purified and exhibited a pI of 4.5 and Mr of 37,000 using SDS-PAGE under reducing conditions. An immunochemical enzyme assay using anti-spectrin antibodies was developed. The optimal activity using spectrin as substrate was at pH 5.0, and the enzymes were strongly inhibited by HgCl2, ZnCl2, chymostatin, leupeptin and aprotinin, and moderately by pepstatin. These properties of the Pf37 and Pb37 proteases differ from the Plasmodium lophurae and P. falciparum 'cathepsin D-like' enzymes and from the serine or cysteine neutral proteases previously described in P. falciparum and P. berghei infected red blood cells. While the Pf37 and Pb37 enzymes cleaved spectrin preferentially, degradation of band 4.1 was also observed with high concentration of enzyme. The parasite origin of the Pf37 protease was clearly demonstrated, since purified radiolabeled enzyme was active on spectrin. A high-molecular-weight polymer (greater than 240 kDa) was often observed on incubating purified spectrin and Pf37 protease. The breakdown of erythrocyte cytoskeletal components could be of interest in the release of merozoites from segmented schizonts or during the process of invasion of erythrocytes by merozoites.     

Lipoproteins and malaria. II. Immunoregulatory role of immunoglobulin-lipoprotein complexes in mice infected with Plasmodium chabaudi on antibody producing cells

Injection of lipoproteins from Plasmodium chabaudi infected mice into mice previously immunized with either human serum transferrin, bovine serum albumin, polyvinyl-pyrrolidone or tetanus toxoid, was followed by a decrease in the levels of antibodies directed against these antigens, suggesting a blockade of antibody secreting cells (Goumard et al., 1982). However, lipoproteins in P. chabaudi infected mice are complexed with immunoglobulins during the second week of infection (Demonchy et al., 1982). In this study, the effects of lipoproteins and Ig-lipoprotein complexes (Ig-Lp) on antibody secreting cells was investigated in vitro. Spleen cells from mice immunized with tetanus toxoid were cultured in microplates and the antitetanus antibodies (anti-TT Ab) were measured in the culture supernatants using a radioimmunoassay (Goumard et al., 1984). Ig-Lp purified from day-11 or day-13 P. chabaudi infected mice inhibited the secretion of anti-TT Ab when introduced into microcultures. On the contrary, lipoproteins purified from either day-5, day-7, day-21 or day-28 infected mice as well as lipoproteins from uninfected mice did not inhibit anti-TT Ab secreting cells. Ig-Lp formed in vitro with lipoproteins purified from day-7 infected mice (Lp J-7) and day-20 infected mice sera, inhibited anti-TT Ab secreting cells. IgG purified from day-20 sera and incubated with Lp J-7, inhibited anti-TT Ab secreting cells but no inhibitory effect was observed with the F(ab')2 fragments of these Ig. Pre-incubation of anti-TT Ab secreting cells with Fc fragments of mouse IgG blocked the inhibitory effect of Ig-Lp purified from infected mouse sera or formed in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gamma interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical

We have examined the roles of gamma interferon (IFN-gamma), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1(+) cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-gamma production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS-/-) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS-/- mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-gamma comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-gamma antibody led to the progression of parasitemia and fatal outcome. CD4(-/-) mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-gamma production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-gamma plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

The ring-infected erythrocyte surface antigen of Plasmodium falciparum associates with spectrin in the erythrocyte membrane.

The malaria parasite Plasmodium falciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P. falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton.     

A gene-family encoding small exported proteins is conserved across Plasmodium genus

A gene-family, named sep, encoding small exported proteins conserved across Plasmodium species has been identified. SEP proteins (13-16 kDa) contain a predicted signal peptide at the NH(2)-terminus, an internal hydrophobic region and a polymorphic, low-complexity region at the carboxy-terminus. One member of the Plasmodium berghei family, Pbsep1, encodes an integral membrane protein expressed along the entire erythrocytic cycle. Immunolocalisation results indicated that PbSEP1 is targeted to the membrane of the parasitophorous vacuole up to the early phases of schizogony, while, in late schizonts, it re-locates in structures within the syncitium. After erythrocyte rupture, PbSEP1 is still detectable in free merozoites thus suggesting its involvement in the early steps of parasite invasion. Seven members of the sep-family in Plasmodium falciparum have been identified. Two of them correspond to previously reported gene sequences included in a family of early transcribed membrane proteins (etramp). Structural, functional and phylogenetic features of the sep family, shown in the present work, supercede this previous classification. PfSEP proteins are exported beyond the parasite membrane and translocated, early after invasion, to the host cell compartment in association with vesicle-like structures. Colocalisation results indicated that PfSEP-specific fluorescence overlaps, at the stage of trophozoite, with that of Pf332, a protein associated with Maurer's clefts, membranous structures in the cytosol of parasitised red blood cells, most probably involved in trafficking of parasite proteins. The specific signals necessary to direct SEP proteins to the vacuolar membrane in P. berghei or to the host cell compartment in P. falciparum remain to be determined.     

Evidence for major differences in ribosomal subunit proteins from Plasmodium berghei and rat liver

Purified polysomes were isolated in high yield from the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei, and from rat liver. Proteins extracted from the ribosomal subunits derived from these polysomes were fractionated and their number and molecular weights were estimated by two-dimensional polyacrylamide gel electrophoresis. Plasmodial small ribosomal subunits contained 30 proteins ranging in apparent molecular size from 11.7 to 40.7 kDa, while large subunits contained 35-36 proteins ranging from 12.1 to 42.6 kDa. None of these parasite proteins was shared by the two subunits nor altered in electrophoretic mobility by radioiodination. Rat liver 40 S ribosomal subunit proteins numbered 30 and ranged from 9.2 to 37.5 kDa, while liver 60 S subunits contained 41-43 proteins with apparent molecular sizes of 10.3-45.2 kDa. Coelectrophoresis of trace amounts of radioiodinated P. berghei ribosomal subunit proteins and stainable quantities of liver proteins demonstrated that most of these 139 parasite and host ribosomal proteins possessed different two-dimensional electrophoretic mobilities under the conditions of this study. Based upon a comparative analysis of P. berghei and rodent ribosomal RNA and these data, it was concluded that parasite and host ribosomes contain distinct ribosomal RNAs and ribosomal proteins.     

Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion

Erythrocyte invasion by malaria parasites requires specific receptor-ligand interactions. Plasmodium vivax and Plasmodium knowlesi are completely dependent on binding the Duffy blood group antigen to invade human erythrocytes. P. knowlesi invades rhesus erythrocytes by multiple pathways using the Duffy antigen as well as alternative receptors. Plasmodium falciparum binds sialic acid residues on glycophorin A as well as other sialic acid-independent receptors to invade human erythrocytes. Parasite proteins that mediate these interactions belong to a family of erythrocyte binding proteins, which includes the P. vivax Duffy binding protein, 175 kDa P. falciparum erythrocyte binding antigen (EBA-175), P. knowlesi alpha protein, which binds human and rhesus Duffy antigens, and P. knowlesi beta and gamma proteins, which bind Duffy-independent receptors on rhesus erythrocytes. The receptor-binding domains of these proteins lie in conserved, N-terminal, cysteine-rich regions that are referred to as region II. Here, we have examined the feasibility of inhibiting erythrocyte invasion with antibodies directed against receptor-binding domains of erythrocyte binding proteins. Region II of P. knowelsi alpha protein (Pk(alpha)RII), which binds the Duffy antigen, was expressed as a secreted protein in insect cells and purified from culture supernatants. Rabbit antibodies raised against recombinant Pk(alpha)RII were tested for inhibition of erythrocyte binding and invasion. Antibodies raised against Pk(alpha)RII inhibit P. knowlesi invasion of both human and rhesus erythrocytes. These data provide support for the development of recombinant vaccines based on the homologous binding domains of P. vivax Duffy binding protein and P. falciparum EBA-175.     

Two monoclonal antibodies with defined epitopes of P44 major surface proteins neutralize Anaplasma phagocytophilum by distinct mechanisms

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.