Identification and characterization of the Plasmodium yoelii PyP140/RON4 protein, an orthologue of Toxoplasma gondii RON4, whose cysteine-rich domain does not protect against lethal parasite challenge infection

Previously, we identified a Plasmodium yoelii YM 140-kDa merozoite protein, designated PyP140, which formed a complex with apical membrane antigen 1 (AMA1). Furthermore, we produced a nonprotective monoclonal antibody (MAb), 48F8, that immunoprecipitated metabolically labeled PyP140 and localized the protein to the merozoite's apical end and, less frequently, to the merozoite surface, as observed by immunofluorescence assay (IFA). Here, using MAb 48F8, we have identified the pyp140 gene by screening a P. yoelii λ-Zap cDNA expression library. The pyp140 cDNA covers approximately 90% of the putative open reading frame (ORF) of PY02159 from the P. yoelii NL genome sequencing project. Analysis of the complete gene identified the presence of two introns. The ORF encodes a 102,407-Da protein with an amino-terminal signal sequence, a series of three unique types of repeats, and a cysteine-rich region. The binding site of MAb 48F8 was also identified. A BLAST search with the deduced amino acid sequence shows significant similarity with the Toxoplasma gondii RON4 protein and the Plasmodium falciparum RON4 protein, and the sequence is highly conserved in other Plasmodium species. We produced the cysteine-rich domain of PyP140/RON4 by using the Pichia pastoris expression system and characterized the recombinant protein biochemically and biophysically. BALB/c mice immunized with the protein formulated in oil-in-water adjuvants produced antibodies that recognize parasitized erythrocytes by IFA and native PyP140/RON4 by immunoblotting but failed to protect against a lethal P. yoelii YM infection. Our results show that PyP140/RON4 is located within the rhoptries or micronemes. It may associate in part with AMA1, but the conserved cysteine-rich domain does not appear to elicit inhibitory antibodies, a finding that is supported by the marked sequence conservation in this protein within Plasmodium spp., suggesting that it is not under immune pressure.     

Model for In Vivo Assessment of Humoral Protection against Malaria Sporozoite Challenge by Passive Transfer of Monoclonal Antibodies and Immune Serum

Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge.     

Falcipain-1, a Plasmodium falciparum cysteine protease with vaccine potential

Cysteine proteases (falcipains) of Plasmodium falciparum are potential targets for antimalarial chemotherapy, since they have been shown to be involved in important cellular functions such as hemoglobin degradation and invasion/rupture of red blood cells during parasite life cycle. The role of falcipain-1 at the asexual blood stages of the parasite still remains uncertain. This is mainly due to a lack of methods to prepare this protein in an active form. In order to obtain biologically active falcipain-1, a number of falcipain-1 constructs were designed and a systematic assessment of the refolding conditions was done. We describe here the expression, purification, and characterization of a falcipain-1 construct encoding mature falcipain-1 and 35 amino acids from the C-terminal of the pro domain. Recombinant falcipain-1 was overexpressed in the form of inclusion bodies, solubilized, and purified by Ni²⁺-nitrilotriacetic acid affinity chromatography under denaturing conditions. A systemic approach was then followed to optimize refolding parameters. An optimum refolding condition was obtained, and the yield of the purified refolded falcipain-1 was ~1 mg/liter. Activity of the protein was analyzed by fluorometric and gelatin degradation assays. Immunolocalization studies using anti-falcipain-1 sera revealed a distinct staining at the apical end of the P. falciparum merozoites. Previous studies using falcipain-1-specific inhibitors have suggested a role of falcipain-1 in merozoite invasion. Based on its localization and its role in invasion, we analyzed the immunogenicity of falcipain-1 in mice, followed by heterologous challenge with Plasmodium yoelii sporozoites. Our results suggest a possible role of falcipain-1 in merozoite invasion.     

Identification of a Potent Combination of Key Plasmodium falciparum Merozoite Antigens That Elicit Strain-Transcending Parasite-Neutralizing Antibodies

Blood-stage malaria vaccines that target single Plasmodium falciparum antigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine against P. falciparum that targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixed in vitro against a diverse set of six key merozoite ligands, including the novel ligands P. falciparum apical asparagine-rich protein (PfAARP), EBA-175 (PfF2), P. falciparum reticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, and Plasmodium thrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverse P. falciparum clones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.     

Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations.     

Outcome of primary lethal and nonlethal Plasmodium yoelii malaria infection in BALB/c and IFN-γ receptor-deficient mice following chloroquine treatment

IFN-γ receptor-deficient (IFN-γR^?/?) mice and control wild-type (WT) mice, with or without chloroquine (CQ) treatment, were infected intraperitoneally with Plasmodium yoelii 17XL (lethal) and P. yoelii 17XNL (nonlethal), and then mouse survival, parasitemia, and antibody production were investigated during the course of infection. Without CQ treatment, both IFN-γR^?/? and WT mice were susceptible to infection showing 100 % mortality after infection with 1?×?10⁵ P. yoelii 17XL-parasitized erythrocytes. The P. yoelii 17XL-infected WT mice could survive by CQ treatment at a dose of 20 mg/kg for 3 days from day 3 postinfection (pi). Malaria parasites in their bloodstream could not be detected in the surviving mice after day 13 pi. CQ treatment, however, could not rescue IFN-γR^?/? mice infected with P. yoelii 17XL. Next, we examined the production of the parasite-specific antibodies in P. yoelii 17XL-infected, CQ-treated mice. Although the production of malaria-specific IgG1, IgG2a, IgG2b, and IgG3 antibodies was observed on days 14 and 28 pi in WT mouse sera, only IgG1 was detected on day 28 pi in IFN-γR^?/? mouse sera. On the other hand, in the nonlethal P. yoelii 17XNL infection, WT mice could control a primary infection with 1?×?10⁵ parasitized erythrocytes. Although IFN-γR^?/? mice could not control and died with increasing parasitemia, the mice could survive by CQ treatment. Both WT and IFN-γR^?/? mice with and without medication, which survived from P. yoelii 17XNL infection, showed the variable levels of malaria-specific IgG1, IgG2a, IgG2b, and IgG3 antibodies during the course of infection. The present data indicate that the IFN-γ receptors are needed to control the infection and parasite-specific IgG2a antibody plays an essential role in recovery from the infection of erythrocytic stages of P. yoelii 17XL or P. yoelii 17XNL parasite.     

Reduced antibody response to the repetitive sequence of the Plasmodium falciparum circumsporozoite protein in mice infected with Plasmodium yoelii blood forms

The immunogenicity of the carrier-free synthetic peptide, (NANP)₄₀, from the repetitive region of the Plasmodium falciparum circumsporozoite (CS) protein was investigated in genetically responder mice (C57BL/6, H-2^b) acutely infected with blood forms of the non-lethal murine malaria parasite, P. yoelii. As compared to non-infected mice, P. yoelii-infected C57BL/6 mice produced significantly lower titers of anti-(NANP)₄₀ IgG antibodies. This decrease in the anti-(NANP)₄₀ antibody response peaked with the peak of parasitemia, and involved all the IgG subclasses. Interestingly, this P. yoelii-mediated effect was evident both on the development of the antibody response to the (NANP)₄₀ peptide, and on an already established anti-(NANP)₄₀ antibody titer, as seen in mice immunized with the peptide 1 month before the infection. Since (NANP)_n-based constructs are strongly envisaged as potential vaccines against falciparum malaria, these results might be important in the evaluation of the efficacy of these vaccine candidates, when they will be used in individuals living in endemic areas.     

A Chimeric Plasmodium falciparum Merozoite Surface Protein Vaccine Induces High Titers of Parasite Growth Inhibitory Antibodies

The C-terminal 19-kDa domain of Plasmodium falciparum merozoite surface protein 1 (PfMSP1₁₉) is an established target of protective antibodies. However, clinical trials of PfMSP1₄₂, a leading blood-stage vaccine candidate which contains the protective epitopes of PfMSP1₁₉, revealed suboptimal immunogenicity and efficacy. Based on proof-of-concept studies in the Plasmodium yoelii murine model, we produced a chimeric vaccine antigen containing recombinant PfMSP1₁₉ (rPfMSP1₁₉) fused to the N terminus of P. falciparum merozoite surface protein 8 that lacked its low-complexity Asn/Asp-rich domain, rPfMSP8 (ΔAsn/Asp). Immunization of mice with the chimeric rPfMSP1/8 vaccine elicited strong T cell responses to conserved epitopes associated with the rPfMSP8 (ΔAsn/Asp) fusion partner. While specific for PfMSP8, this T cell response was adequate to provide help for the production of high titers of antibodies to both PfMSP1₁₉ and rPfMSP8 (ΔAsn/Asp) components. This occurred with formulations adjuvanted with either Quil A or with Montanide ISA 720 plus CpG oligodeoxynucleotide (ODN) and was observed in both inbred and outbred strains of mice. PfMSP1/8-induced antibodies were highly reactive with two major alleles of PfMSP1₁₉ (FVO and 3D7). Of particular interest, immunization with PfMSP1/8 elicited higher titers of PfMSP1₁₉-specific antibodies than a combined formulation of rPfMSP1₄₂ and rPfMSP8 (ΔAsn/Asp). As a measure of functionality, PfMSP1/8-specific rabbit IgG was shown to potently inhibit the in vitro growth of blood-stage parasites of the FVO and 3D7 strains of P. falciparum. These data support the further testing and evaluation of this chimeric PfMSP1/8 antigen as a component of a multivalent vaccine for P. falciparum malaria.     

Rhop-3 protein conservation among Plasmodium species and induced protection against lethal P. yoelii and P. berghei challenge

In the present study, Rhop-3 polymorphism among Plasmodium falciparum field and laboratory isolates and among rodent Plasmodium species was investigated and identified. The Rhop-3 gene was found in all Plasmodium species so far tested. The overall structure of the Rhop-3 protein was found conserved among P. falciparum, Plasmodium yoelii, and Plasmodium berghei. However, it was more conserved among rodent Plasmodium species than between P. falciparum and Plasmodium vivax. The most conserved regions of Rhop-3 are the second half of exon 6 (amino acid #548 to #665) and the beginning of exon 3 (amino acid #59 to #210). Recombinant C-terminal partial and full-length Rhop-3 proteins of P. yoelii and P. berghei were expressed in Escherichia coli and purified. Immunization-challenge experiments in mice using recombinant Rhop-3 proteins led to a delay in parasite development and protected mice from a homologous lethal challenge infection. In a group of eight outbred Carworth Farm White (CFW) mice immunized with P. yoelii C-terminal recombinant His-Y1412 protein, three mice (37.5%) were protected from a lethal P. yoelii challenge. In BALB/cJ mice one mouse (20%) survived the infection. Immunization of mice with P. berghei recombinant full-length Rhop-3 protein in BALB/cJ mice led to a 40% survival from lethal P. berghei challenge. CFW mice immunized with P. berghei recombinant full-length Rhop-3 protein showed a significant delay in parasite development with a heterologous P. yoelii challenge. The Rhop-3 protein is a promising candidate for an asexual stage malaria vaccine.Nucleotide sequence data reported in this paper have been submitted to GeneBank with the Accession numbers: AY044907-AY044914.     

Plasmodium falciparum Merozoite Surface Protein 1 (MSP-1)-MSP-3 Chimeric Protein: Immunogenicity Determined with Human-Compatible Adjuvants and Induction of Protective Immune Response

A chimeric gene, MSP-Fu₂₄, was constructed by genetically coupling immunodominant, conserved regions of the two leading malaria vaccine candidates, Plasmodium falciparum merozoite surface protein 1 (C-terminal 19-kDa region [PfMSP-1₁₉]) and merozoite surface protein 3 (11-kDa conserved region [PfMSP-3₁₁]). The recombinant MSP-Fu₂₄ protein was produced in Escherichia coli cells and purified to homogeneity by a two-step purification process with a yield of ∼30 mg/liter. Analyses of conformational properties of MSP-Fu₂₄ using PfMSP-1₁₉-specific monoclonal antibody showed that the conformational epitopes of PfMSP-1₁₉ that may be critical for the generation of the antiparasitic immune response remained intact in the fusion protein. Recombinant MSP-Fu₂₄ was highly immunogenic in mice and in rabbits when formulated with two different human-compatible adjuvants and induced an immune response against both PfMSP-1₁₉ and PfMSP-3₁₁. Purified anti-MSP-Fu₂₄ antibodies showed invasion inhibition of P. falciparum 3D7 and FCR parasites, and this effect was found to be dependent on antibodies specific for the PfMSP-1₁₉ component. The protective potential of MSP-Fu₂₄ was demonstrated by in vitro parasite growth inhibition using an antibody-dependent cell inhibition (ADCI) assay with anti-MSP-Fu₂₄ antibodies. Overall, the antiparasitic activity was mediated by a combination of growth-inhibitory antibodies generated by both the PfMSP-1₁₉ and PfMSP-3₁₁ components of the MSP-Fu₂₄ protein. The antiparasitic activities elicited by anti-MSP-Fu₂₄ antibodies were comparable to those elicited by antibodies generated with immunization with a physical mixture of two component antigens, PfMSP-1₁₉ and PfMSP-3₁₁. The fusion protein induces a protective immune response with human-compatible adjuvants and may form a part of a multicomponent malaria vaccine.Malaria is among the major parasitic diseases in tropical and subtropical countries. With as many as 300 to 500 million new cases each year, malaria accounts for the death of over 2 million people globally each year, and most are children (41). Among the four species of Plasmodium that infect humans, the most threatening is Plasmodium falciparum. The extensive spread of drug-resistant P. falciparum strains as well as the insecticide-resistant mosquito necessitates the development of a malaria vaccine on an urgent basis. Collectively, the major objective of the ongoing vaccine effort in this field is to develop a multistage, multivalent vaccine against P. falciparum (34).The blood-stage cycle of the parasite is responsible for malaria pathogenesis. Intervention at this stage of the parasite''s development through vaccination is likely to reduce malaria-related clinical symptoms. As a major interface between host and pathogen, the merozoite surface is an obvious target for the development of a malaria vaccine. A number of potential vaccine candidate antigens identified so far are located on or associated with the surface of the merozoite or in apical organelles. These include merozoite surface protein 1 (MSP-1), MSP-2, MSP-3, MSP-4, MSP-5, MSP-8, RAP1/2, AMA-1, and EBA-175, which are implicated in the process of merozoite invasion of the erythrocyte (23).MSP-1 is one of the most extensively studied proteins of P. falciparum (18). It is synthesized as a ∼200-kDa precursor and then processed in two steps: the primary processing step produces a complex of four fragments that are present on the merozoite surface, and the secondary processing step at invasion results in the shedding of the complex from the surface, except for the C-terminal 19-kDa domain (MSP-1₁₉), which remains anchored to the parasite surface by a glycosylphosphatidylinositol (GPI) moiety (2). The C-terminal 19-kDa fragment of MSP-1 is well conserved among P. falciparum isolates and contains two epidermal growth factor (EGF)-like domains that play a role in merozoite invasion. Substantial data from studies with P. falciparum MSP-1 and in vivo immunization studies of mice with Plasmodium yoelii and Plasmodium chabaudi indicate that the protective immune responses are directed against the C-terminal 19-kDa domain (10, 12, 15, 20, 27, 35). The inhibition of MSP-1 processing by conformation-specific antibodies (Abs) was previously proposed to be one of the possible mechanism for the inhibition of merozoite invasion (1).Another merozoite surface protein, MSP-3, was also shown to be the target of the protective immune responses in humans (29). The PfMSP-3 protein contains three blocks of four tandem heptad repeats based on the AXXAXX motif at the N terminus, a glutamic acid-rich domain, and a putative leucine zipper sequence at the C terminus (25). Although a clear surface localization of PfMSP-3 is known, it lacks any transmembrane domain or glycosylphosphatidylinositol (GPI) anchor site (24, 25) and is therefore considered to be loosely associated with the merozoite surface by interactions with other merozoite surface proteins. PfMSP-3 was identified as a candidate vaccine antigen by an antibody-dependent cellular inhibition (ADCI) assay using human immune sera (28). The potential of PfMSP-3 as a vaccine candidate was further illustrated by ADCI using mice antibodies and was further confirmed by the suppression of P. falciparum growth in an immunocompromised mouse after the passive transfer of human antibodies purified on MSP-3 peptides together with human monocytes (28, 40, 42). The immunization of Aotus and Saimiri monkeys with recombinant PfMSP-3 or its fragments provided protection against parasite challenge (6, 16). A 70-amino-acid-long conserved domain of PfMSP-3, referred to here as the PfMSP-3₁₁ region, was identified as the target of protective antibodies in human immune responses (40). The presence of high titers of cytophilic antibodies, IgG3, against this conserved region of MSP-3 has been correlated with protection against the parasite. In addition, immunization of humans with a synthetic peptide corresponding to this region was previously shown to induce antiparasitic antibodies that suppress parasite growth in an ADCI assay (11).It is generally believed that a combination vaccine for malaria is likely to be more effective than vaccines based on a single antigen, and attempts are being made to develop a malaria vaccine by using a mixture of more than one antigen or by combining immunologically relevant proteins of the target antigens as fusion proteins (31, 43, 45). In the present study, we have constructed a fusion chimera (MSP-Fu₂₄) consisting of PfMSP-1₁₉ and PfMSP-3₁₁ and produced the corresponding recombinant MSP-Fu₂₄ protein in Escherichia coli cells. The two individual components, PfMSP-1₁₉ and PfMSP-3₁₁, were also expressed and purified separately; the immunological properties of MSP-Fu₂₄ were compared with a physical mixture of the two individual components. MSP-Fu₂₄ retained the native conformation of the PfMSP-1₁₉ component and was highly immunogenic in small animals. The anti-MSP-Fu₂₄ antibodies inhibited parasite invasion into host red blood cells (RBCs) and also inhibited parasite growth in a monocyte-dependent manner, suggesting the potential of the fusion protein as a malaria vaccine candidate.     

Identification of an Erythrocyte Binding Peptide from the Erythrocyte Binding Antigen, EBA-175, Which Blocks Parasite Multiplication and Induces Peptide-Blocking Antibodies

A biotinylated peptide covering a sequence of 21 amino acids (aa) from the erythrocyte binding antigen (EBA-175) of Plasmodium falciparum bound to human glycophorin A, an erythrocyte receptor for merozoites, as demonstrated by enzyme-linked immunosorbent assay (ELISA) and to erythrocytes as demonstrated by flow cytometry analysis. The peptide, EBA(aa1076–96), also bound to desialylated glycophorin A and glycophorin B when tested by ELISA. The peptide blocked parasite multiplication in vitro. The glycophorin A binding sequence was further delineated to a 12-aa sequence, EBA(aa1085–96), by testing the binding of a range of truncated peptides to immobilized glycophorin A. Our data indicate that EBA(aa1085–96) is part of a ligand on the merozoite for binding to erythrocyte receptors. This binding suggests that the EBA(aa1085–96) peptide is involved in a second binding step, independent of sialic acid. Antibody recognition of this peptide sequence may protect against merozoite invasion, but only a small proportion of sera from adults from different areas of malaria transmission showed antibody reactivities to the EBA(aa1076–96) peptide, indicating that this sequence is only weakly immunogenic during P. falciparum infections in humans. However, Tanzanian children with acute clinical malaria showed high immunoglobulin G reactivity to the EBA(aa1076–96) peptide compared to children with asymptomatic P. falciparum infections. The EBA(aa1076–96) peptide sequence from EBA-175 induced antibody formation in mice after conjugation of the peptide with purified protein derivative. These murine sera inhibited EBA(aa1076–96) peptide binding to glycophorin A.     

Identification and characterization of the Plasmodium falciparum RhopH2 ortholog in Plasmodium vivax

Plasmodium vivax is one of the most important human malaria species that is geographically widely endemic and potentially affects a larger number of people than its more notorious cousin, Plasmodium falciparum. During invasion of red blood cells, the parasite requires the intervention of high molecular weight complex rhoptry proteins (RhopH) that are also essential for cytoadherence. PfRhopH2, a member of the RhopH multigene family, has been characterized as being crucial during P. falciparum infection. This study describes identifying and characterizing the pfrhoph2 orthologous gene in P. vivax (hereinafter named pvrhoph2). The PvRhopH2 is a 1,369-amino acid polypeptide encoded by PVX_099930 gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Both P. falciparum and P. vivax genes contain nine introns, and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. Moreover, PvRhopH2 contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. PvRhopH2 is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to that of PfRhopH2 in P. falciparum. The recombinant PvRhopH2 protein is recognized by serum antibodies of patients naturally exposed to P. vivax, suggesting that PvRhopH2 is immunogenic in humans.     

Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera

Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4⁺ and CD8⁺ PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development.     

A Replicating Adenovirus Capsid Display Recombinant Elicits Antibodies against Plasmodium falciparum Sporozoites in Aotus nancymaae Monkeys

Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization.     

Fine mapping of Plasmodium falciparum ribosomal phosphoprotein PfP0 revealed sequences with highly specific binding activity to human red blood cells

The Plasmodium falciparum P0 ribosomal phosphoprotein (PfP0) was identified for the first time by screening a cDNA expression library of P. falciparum parasites with sera from malaria-immune individuals. Due to its localization on the surface of different parasite life-cycle stages (merozoites and gametocytes) and its recognition by invasion-blocking antibodies, PfP0 has been considered a potential malaria-vaccine component. In this study, 16 20-mer-long synthetic peptides spanning the entire PfP0 sequence were evaluated by means of receptor–ligand assays with human red blood cells (RBCs) in order to determine the role played by these peptides in the invasion process. Four RBC high-activity binding peptides (HABPs), located mostly toward the N-terminal region, were identified: HABP 33898 (¹MAKLSKQQKKQMYIEKLSSL²⁰), HABP 33900 (⁴¹ASVRKSLRGKATILMGKNTRY⁶⁰), HABP 33901 (⁶¹IRTALKKNLQAVPQIEKLLPY ⁸⁰), and HABP 33906 (¹⁶¹LIKQGEKVTASSATLLRKFNY¹⁸⁰). The binding pattern of HABPs 33898 and 33906 to enzyme-treated RBCs suggests receptors of protein nature for these two HABPs, one of which could correspond to a common 58-kDa RBC membrane protein, as indicated by results of cross-linking assays. Both HABPs exhibited high content of α-helical features and prevented P. falciparum merozoite invasion to RBCs in vitro by up to 91%. The invasion-blocking ability reported here for these PfP0 HABPs supports their inclusion in immunological studies with the aim of assessing their potential as candidates for a vaccine against P. falciparum malaria.     

Immunogenicity and in vitro protective efficacy of recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa merozoite surface protein-1 (MSP-1(19)) antigen of Plasmodium falciparum

Vaccine development against the blood-stage malaria parasite is aimed at reducing the pathology of the disease. We constructed a recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa C-terminus of Plasmodium falciparum merozoite surface protein-1 (MSP-1₁₉) to evaluate its protective ability against merozoite invasion of red blood cells in vitro. A mutated version of MSP-1₁₉, previously shown to induce the production of inhibitory but not blocking antibodies, was cloned into a suitable shuttle plasmid and transformed into BCG Japan (designated rBCG016). A native version of the molecule was also cloned into BCG (rBCG026). Recombinant BCG expressing the mutated version of MSP-1₁₉ (rBCG016) elicited enhanced specific immune response against the epitope in BALB/c mice as compared to rBCG expressing the native version of the epitope (rBCG026). Sera from rBCG016-immunized mice contained significant levels of specific IgG, especially of the IgG2a subclass, against MSP-1₁₉ as determined by enzyme-linked immunosorbent assay. The sera was reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA) and inhibited merozoite invasion of erythrocytes in vitro. Furthermore, lymphocytes from rBCG016-immunized mice demonstrated higher proliferative response against the MSP-1₁₉ antigen as compared to those of rBCG026- and BCG-immunized animals. rBCG expressing the mutated version of MSP-1₁₉ of P. falciparum induced enhanced humoral and cellular responses against the parasites paving the way for the rational use of rBCG as a blood-stage malaria vaccine candidate.     

Immunization with the MAEBL M2 Domain Protects against Lethal Plasmodium yoelii Infection

Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4⁺, but not CD8⁺, T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.     

Blackwater fever like in murine malaria

Blackwater fever (BWF) is the term used to designate the occurrence of hemoglobin pigments in the urine of patients infected with malaria parasites. BWF is more often associated with Plasmodium falciparum infection in man. The pathogenesis of BWF has not been explained satisfactorily. In the present study, the clinical and pathological observations made upon CD1 mice infected with Plasmodium yoelii yoelii lethal strain with clinical signs of hemoglobinuria and acute renal failure were evaluated. From the 40 P. yoelii yoelii-infected mice, 14 presented hemoglobinuria. In the observations, it was emphasized that hemoglobinuria occurred in the animals 1–2 days before they die. At 6 days post-infection, infected hemoglobinuric mice (HM) exhibited clinical signs such as dark red urine, apnea, and evident oliguria and hematuria; urine microscopical examination showed very few red blood cells. The entire non hemoglobinuric infected mice had a high parasitemia preceding the time of death, while the HM parasitemia was just detectable. In HM, marked hepatosplenomegaly, anemia, and renal and hepatic dysfunction were observed with the blood chemistry analysis at 6 days post-infection. Severe renal lesions were demonstrated in histopathological and scanning electron microscopy samples. Occlusion and necrosis of convoluted tubules were the main lesions found. The conditions required for the experimental production of hemoglobinuria in CD1 mouse infected by P. yoelii yoelii is still unknown. The clinical picture of a BWF, like in our rodents, was produced exclusively by the interaction between the parasite and its host. Results showed that hemoglobinuria in CD1 mice infected with P. yoelii yoelii and BWF in man infected with P. falciparum are similar in their pathogenesis.     

Immunogenicity of a Prime-Boost Vaccine Containing the Circumsporozoite Proteins of Plasmodium vivax in Rodents

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund''s adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.