Hitoshi Otsuki

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

ABSTRACT One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 μg/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.

Rhoptry neck protein RON2 forms a complex with microneme protein AMA1 in Plasmodium falciparum merozoites.

Erythrocyte invasion is an essential step in the establishment of host infection by malaria parasites, and is a major target of intervention strategies that attempt to control the disease. Recent proteome analysis of the closely-related apicomplexan parasite, Toxoplasma gondii, revealed a panel of novel proteins (RONs) located at the neck portion of the rhoptries. Three of these proteins, RON2, RON4, and RON5 have been shown to form a complex with the microneme protein Apical Membrane Protein 1 (AMA1). This complex, termed the Moving Junction complex, localizes at the interface of the parasite and the host cell during the invasion process. Here we characterized a RON2 ortholog in Plasmodium falciparum. PfRON2 transcription peaked at the mature schizont stage and was expressed at the neck portion of the rhoptry in the merozoite. Co-immunoprecipitation of PfRON2, PfRON4 and PfAMA1 indicated that the complex formation is conserved between T. gondii and P. falciparum, suggesting that co-operative function of the rhoptry and microneme proteins is a common mechanism in apicomplexan parasites during host cell invasion. PfRON2 possesses a region displaying homology with the rhoptry body protein PfRhopH1/Clag, a component of the RhopH complex. However, here we present co-immunoprecipitation studies which suggest that PfRON2 is not a component of the RhopH complex and has an independent role. Nucleotide polymorphism analysis suggested that PfRON2 was under diversifying selective pressure. This evidence suggests that RON2 appears to have a fundamental role in host cell invasion by apicomplexan parasites, and is a potential target for malaria intervention strategies.

Nasal Immunization with a Malaria Transmission-Blocking Vaccine Candidate, Pfs25, Induces Complete Protective Immunity in Mice against Field Isolates of Plasmodium falciparum

ABSTRACT Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.

Single amino acid substitution in Plasmodium yoelii erythrocyte ligand determines its localization and controls parasite virulence.

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host–pathogen interactions and malaria virulence.

The wheat germ cell-free protein synthesis system: a key tool for novel malaria vaccine candidate discovery.

Malaria kills more than a million people a year, causes malady in about three hundred million people and poses risk to approximately 40% of the worlds population living in malarious countries. This disease is re-emerging mainly due to the development of drug-resistant parasites and insecticide-resistant mosquitoes. Therefore, we are now forced to resort to remedy through vaccination. Until now, not even a single licensed malaria vaccine has been developed despite intensive efforts. Even the efficacy of RTS,S, the most advanced and promising vaccine candidate in the pipeline of malaria vaccine development, was only around 50% based on a number of clinical trials. These facts urge malaria researchers to urgently enrich this pipeline, as much as possible, with potential vaccine candidates. With the availability of malaria genome database, the enrichment of this pipeline is possible if we could now employ an efficient protein expression technology to decode the malaria genomic data, without any codon optimization, into quality recombinant proteins. Then, these synthesized recombinant proteins can be characterized and screened for discovering novel potential vaccine targets. The wheat germ cell-free protein synthesis system will be a promising tool to this end. This review highlights the recent successes in synthesizing quality malaria proteins using this tool.

Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen.

ABSTRACT One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.

N-Terminal Prodomain of Pfs230 Synthesized Using a Cell-Free System Is Sufficient To Induce Complement-Dependent Malaria Transmission-Blocking Activity

ABSTRACT The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum.

Plasmodium vivax gametocyte protein Pvs230 is a transmission-blocking vaccine candidate.

The malaria transmission-blocking vaccine (TBV) aims to interfere the development of malaria parasite in the mosquito and prevent further transmission in the community. So far only two TBV candidates have been identified in Plasmodium vivax; ookinete surface proteins Pvs25 and Pvs28. The pvs230 (PVX_003905) is reported as an ortholog of Pfs230, a gametocyte/gamete stage TBV candidate in Plasmodium falciparum, however its candidacy for TBV has never been tested. Therefore here, we have investigated whether Pvs230 can be a TBV candidate using P. vivax samples obtained from Thailand. The mouse antiserum raised against the plasmid expressing CRDs I-IV of Pvs230 detected Pvs230 protein in the lysate of P. vivax gametocyte in western blot analysis under non-reducing condition. From the localization of Pvs230 on the outer most regions of gametocyte in the immunofluorescence assay, it appears that Pvs230 is localized on the surface of gametes. Importantly, the anti-Pvs230 mouse serum significantly reduced the number of P. vivax oocysts developed in the mosquito midgut. Moreover, the polymorphism in Pvs230 CRDs I-IV is limited suggesting that it may not be an impediment for the utilization of Pvs230 as an effective TBV candidate. In conclusion, our results show that Pvs230 is a transmission-blocking vaccine candidate of P. vivax.

Plasmodium vivax gametocyte proteins, Pvs48/45 and Pvs47, induce transmission-reducing antibodies by DNA immunization

Malaria transmission-blocking vaccines (TBV) aim to interfere with the development of the malaria parasite in the mosquito vector, and thus prevent spread of transmission in a community. To date three TBV candidates have been identified in Plasmodium vivax; namely, the gametocyte/gamete protein Pvs230, and the ookinete surface proteins Pvs25 and Pvs28. The Plasmodium falciparum gametocyte/gamete stage proteins Pfs48/45 and Pfs47 have been studied as TBV candidates, and Pfs48/45 shown to induce transmission-blocking antibodies, but the candidacy of their orthologs in P. vivax, Pvs48/45 (PVX_083235) and Pvs47 (PVX_083240), for vivax TBV have not been tested. Herein we investigated whether targeting Pvs48/45 and Pvs47 can inhibit parasite transmission to mosquitoes, using P. vivax isolates obtained in Thailand. Mouse antisera directed against the products from plasmids expressing Pvs48/45 and Pvs47 detected proteins of approximately 45- and 40-kDa, respectively, in the P. vivax gametocyte lysate, by Western blot analysis under non-reducing conditions. In immunofluorescence assays Pvs48/45 was detected predominantly on the surface and Pvs47 was detected in the cytoplasm of gametocytes. Membrane feeding transmission assays demonstrated that anti-Pvs48/45 and -Pvs47 mouse sera significantly reduced the number of P. vivax oocysts developing in the mosquito midgut. Limited amino acid polymorphism of these proteins was observed among 27 P. vivax isolates obtained from Thailand, Vanuatu, and Colombia; suggesting that polymorphism may not be an impediment for the utilization of Pvs48/45 and Pvs47 as TBV antigens. In one Thai isolate we found that the fourth cysteine residue in the Pvs47 cysteine-rich domain (CRD) III (amino acid position 337) is substituted to phenylalanine. However, antibodies targeting Pvs47 CRDI-III showed a significant transmission-reducing activity against this isolate, suggesting that this substitution in Pvs47 was not critical for recognition by the generated antibodies. In conclusion, our results indicate that Pvs48/45 and Pvs47 are potential transmission-blocking vaccine candidates of P. vivax.

Disruption of the Plasmodium berghei 2-Cys peroxiredoxin TPx-1 gene hinders the sporozoite development in the vector mosquito.

To investigate the physiologic role of cytosolic 2-Cys peroxiredoxin of Plasmodium berghei (PbTPx-1), we infected the vector mosquito Anopheles stephensi with a parasite carrying a targeted knockout of pbtpx-1 (Prx-KO). The number of Prx-KO midgut oocysts at 14-15 days post-feeding (pf) was comparable to that of the parent strain (WT); however, the numbers of sporozoites that formed in midgut oocysts and accumulated in the salivary gland of Prx-KO-infected mosquitoes by 21 days pf were decreased to 10-20% and 3-10%, respectively, of those values in WT-infected mosquitoes. A higher frequency of DNA strand breaks was detected in Prx-KO oocysts than in WT oocysts. Sporozoites carrying the targeted disruption had reduced infectivity in mice; however, the knockout did not affect the ability of the sporozoite to reach the liver parenchyma and initiate exo-erythrocytic form (EEF) development. TPx-1 may be involved in development during exponentially multiplying stages, such as sporozoites and EEF.