Directory of Researchers

IRIKO Hideyuki
Graduate School of Health Sciences / Faculty of Health Sciences
Associate Professor
Medicine
Last Updated :2022/04/15

Researcher Profile and Settings

Affiliation

  • <Faculty / Graduate School / Others>

    Graduate School of Health Sciences / Faculty of Health Sciences
  • <Related Faculty / Graduate School / Others>

    School of Medicine / Faculty of Health Sciences, Center for Mathematical and Data Sciences

Teaching

Research Activities

Research Interests

  • Malaria
  • Parasitology (including Medical Zoology)
  • マラリア
  • 寄生虫学
  • 寄生虫
  • 電子顕微鏡

Research Areas

  • Life sciences / Parasitology
  • Life sciences / Cell biology

Published Papers

  • Mayumi Tachibana, Hideyuki Iriko, Minami Baba, Motomi Torii, Tomoko Ishino

    Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Activated gametocytes in the mosquito midgut egress from erythrocytes followed by fertilization and zygote formation. Zygotes differentiate into motile invasive ookinetes, which penetrate the midgut epithelium before forming oocysts beneath the basal lamina. Ookinete development and traversal across the mosquito midgut wall are major bottlenecks in the parasite life cycle. In ookinetes, surface proteins and proteins stored in apical organelles have been shown to be involved in parasite-host interactions. A group of ookinete proteins that are predicted to have such functions are named PSOPs (putative secreted ookinete protein). PSOP1 is possibly involved in migration through the midgut wall, and here its subcellular localization was examined in ookinetes by immunoelectron microscopy. PSOP1 localizes to the micronemes of Plasmodium yoelii and Plasmodium berghei ookinetes, indicating that it is stored and possibly apically secreted during ookinete penetration through the mosquito midgut wall.

    Oct. 2021, Parasitology international, 84, 102407 - 102407, English, International magazine

    Scientific journal

  • Tomoko Ishino, Mayumi Tachibana, Minami Baba, Hideyuki Iriko, Takafumi Tsuboi, Motomi Torii

    Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Gametocytes, which differentiate from asexual-stage parasites, are activated by environmental changes when ingested into the mosquito midgut, and are rapidly released from erythrocytes prior to fertilization. Secretory proteins localized to osmiophilic bodies (OBs), organelles unique to gametocytes, have been reported to be involved in female gametocyte egress. In this study, we investigate the dynamics of OBs in activated gametocytes of Plasmodium falciparum and Plasmodium yoelii using the female OB-specific marker protein, G377. After activation, female gametocyte OBs migrate to the parasite surface and fuse to form large vesicles beneath the parasite plasma membrane. At the marginal region of female gametocytes, fused vesicles secrete contents by exocytosis into the parasitophorous vacuole space, prior to parasite egress via the break-down of the erythrocyte membrane. This is the first detailed description of how proteins are transported through osmiophilic bodies.

    Mar. 2020, Molecular and biochemical parasitology, 236, 111261 - 111261, English, International magazine

    [Refereed]

    Scientific journal

  • Daisuke Ito, Eizo Takashima, Tsutomu Yamasaki, Shinya Hatano, Tomoyuki Hasegawa, Kazutoyo Miura, Masayuki Morita, Amporn Thongkukiatkul, Mahamadou Diakite, Carole A Long, Jetsumon Sattabongkot, Rachanee Udomsangpetch, Hideyuki Iriko, Tomoko Ishino, Takafumi Tsuboi

    Proteins coating Plasmodium merozoite surface and secreted from its apical organelles are considered as promising vaccine candidates for blood-stage malaria. The rhoptry neck protein 12 of Plasmodium falciparum (PfRON12) was recently reported as a protein specifically expressed in schizonts and localized to the rhoptry neck of merozoites. Here, we assessed its potential as a vaccine candidate. We expressed a recombinant PfRON12 protein by a wheat germ cell-free system to obtain anti-PfRON12 antibody. Immunoblot analysis of schizont lysates detected a single band at approximately 40 kDa under reducing conditions, consistent with the predicted molecular weight. Additionally, anti-PfRON12 antibody recognized a single band around 80 kDa under non-reducing conditions, suggesting native PfRON12 forms a disulfide-bond-mediated multimer. Immunofluorescence assay and immunoelectron microscopy revealed that PfRON12 localized to the rhoptry neck of merozoites in schizonts and to the surface of free merozoites. The biological activity of anti-PfRON12 antibody was tested by in vitro growth inhibition assay (GIA), and the rabbit antibodies significantly inhibited merozoite invasion of erythrocytes. We then investigated whether PfRON12 is immunogenic in P. falciparum-infected individuals. The sera from P. falciparum infected individuals in Thailand and Mali reacted with the recombinant PfRON12. Furthermore, human anti-PfRON12 antibodies affinity-purified from Malian serum samples inhibited merozoite invasion of erythrocytes in vitro. Moreover, pfron12 is highly conserved with only 4 non-synonymous mutations in the coding sequence from approximately 200 isolates deposited in PlasmoDB. These results suggest that PfRON12 might be a potential blood-stage vaccine candidate antigen against P. falciparum.

    Feb. 2019, Parasitology international, 68 (1), 87 - 91, English, International magazine

    [Refereed]

    Scientific journal

  • Yuki Oda-Yokouchi, Mayumi Tachibana, Hideyuki Iriko, Motomi Torii, Tomoko Ishino, Takafumi Tsuboi

    Invasion of host cells by apicomplexan parasites is mediated by proteins released from microneme, rhoptry, and dense granule secretory organelles located at the apical end of parasite invasive forms. Microneme secreted proteins establish interactions with host cell receptors and induce exocytosis of the rhoptry organelle. Rhoptry proteins are involved in target cell invasion as well as the formation of the parasitophorous vacuole in which parasites reside during development within the host cell. In Plasmodium merozoites, the rhoptry neck protein (RON) complex consists of RON2, RON4, and RON5, and interacts with apical membrane antigen 1 (AMA1) as a critical structure of the invasion moving junction. PfRON12 is known to localize to the rhoptry neck of merozoites, but its function remains obscure. The roles of RON proteins are largely unknown in sporozoites, the second invasive form of Plasmodium which possesses a conserved apical end secretory structure. Here, we confirm that RON12 is expressed in the rhoptry neck of merozoites in rodent malaria parasites, whereas in contrast we show that RON12 is localized to the rhoptry body in sporozoites. Phenotypic analysis of Plasmodium berghei ron12-disrupted mutants revealed that RON12 is dispensable for sporogony, invasion of mosquito salivary glands and mouse hepatocytes, and development in hepatocytes.

    Feb. 2019, Parasitology international, 68 (1), 17 - 23, English, International magazine

    [Refereed]

    Scientific journal

  • Skeleton binding protein 1 (SBP1) of Plasmodium falciparum accumulates in electron-dense material before passing through the parasitophorous vacuole membrane.

    Iriko H, Ishino T, Tachibana M, Omoda A, Torii M, Tsuboi T

    2019, 2019 Nov 13;75:102003. doi: 1, English

    [Refereed]

    Scientific journal

  • Dibothriocephalus nihonkaiensis infection identified by pathological and genetic analyses -a case report and a recent literature review of human diphyllobothriasis

    Yoshitane Tsukamoto, IRIKO HIDEYUKI, Shohei Matsuo, Yoshihiro Shimono, Junichi Miyazaki, Hironori Tanaka, Toshiya Komatsu, Seiji Azuma, Hiroko Oota, Yui Yamashita, Mizuka Ohkouchi, Yuka Hashikura, Seiichi Hirota

    2019, Human Pathology: Case Reports, 16, 1 - 5, English

    [Refereed]

    Scientific journal

  • Plasmodium falciparum Exported Protein 1 is localized to dense granules in merozoites.

    Iriko H, Ishino T, Otsuki H, Ito D, Tachibana M, Torii M, Tsuboi T

    Jun. 2018, Parasitol Int., 67 (5), 637 - 639, English

    [Refereed]

    Scientific journal

  • Imtiaj Hasan, Miharu Watanabe, Naoto Ishizaki, Yoshiko Sugita-Konishi, Yasushi Kawakami, Jun Suzuki, Chikaku Dogasaki, Sultana Rajia, Sarkar M.A. Kawsar, Yasuhiro Koide, Robert A. Kanaly, Shigeki Sugawara, Masahiro Hosono, Yukiko Ogawa, Yuki Fujii, Hideyuki Iriko, Jiharu Hamako, Taei Matsui, Yasuhiro Ozeki

    MDPI AG, 01 Jan. 2016, Molecules, 21 (1), English

    [Refereed]

    Scientific journal

  • Imtiaj Hasan, Miharu Watanabe, Naoto Ishizaki, Yoshiko Sugita-Konishi, Yasushi Kawakami, Jun Suzuki, Chikaku Dogasaki, Sultana Rajia, Sarkar M. A. Kawsar, Yasuhiro Koide, Robert A. Kanaly, Shigeki Sugawara, Masahiro Hosono, Yukiko Ogawa, Yuki Fujii, Hideyuki Iriko, Jiharu Hamako, Taei Matsui, Yasuhiro Ozeki

    MDPI AG, Jan. 2016, MOLECULES, 21 (1), 129, English

    [Refereed]

  • Joao C. Aguiar, Jessica Bolton, Joyce Wanga, John B. Sacci, Hideyuki Iriko, Julie K. Mazeika, Eun-Taek Han, Keith Limbach, Noelle B. Patterson, Martha Sedegah, Ann-Marie Cruz, Takafumi Tsuboi, Stephen L. Hoffman, Daniel Carucci, Michael R. Hollingdale, Eileen D. Villasante, Thomas L. Richie

    Background Nearly 100% protection against malaria infection can be achieved in humans by immunization with P. falciparum radiation-attenuated sporozoites (RAS). Although it is thought that protection is mediated by T cell and antibody responses, only a few of the many pre-erythrocytic (sporozoite and liver stage) antigens that are targeted by these responses have been identified. Methodology Twenty seven P. falciparum pre-erythrocytic antigens were selected using bioinformatics analysis and expression databases and were expressed in a wheat germ cell-free protein expression system. Recombinant proteins were recognized by plasma from RAS-immunized subjects, and 21 induced detectable antibody responses in mice and rabbit and sera from these immunized animals were used to characterize these antigens. All 21 proteins localized to the sporozoite: five localized to the surface, seven localized to the micronemes, cytoplasm, endoplasmic reticulum or nucleus, two localized to the surface and cytoplasm, and seven remain undetermined. PBMC from RAS-immunized volunteers elicited positive ex vivo or cultured ELISpot responses against peptides from 20 of the 21 antigens. Conclusions These T cell and antibody responses support our approach of using reagents from RAS-immunized subjects to screen potential vaccine antigens, and have led to the identification of a panel of novel P. falciparum antigens. These results provide evidence to further evaluate these antigens as vaccine candidates.

    PUBLIC LIBRARY SCIENCE, Aug. 2015, PLOS ONE, 10 (8), English

    [Refereed]

    Scientific journal

  • Mayumi Tachibana, Nantavadee Suwanabun, Osamu Kaneko, Hideyuki Iriko, Hitoshi Otsuki, Jetsumon Sattabongkot, Akira Kaneko, Socrates Herrera, Motomi Torii, Takafumi Tsuboi

    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-Ill 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. (C) 2015 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, Apr. 2015, VACCINE, 33 (16), 1901 - 1908, English

    [Refereed]

    Scientific journal

  • Imtiaj Hasan, Miharu Watanabe, Naoto Ishizaki, Yoshiko Sugita-Konishi, Yasushi Kawakami, Jun Suzuki, Chikaku Dogasaki, Sultana Rajia, Sarkar M. A. Kawsar, Yasuhiro Koide, Robert A. Kanaly, Shigeki Sugawara, Masahiro Hosono, Yukiko Ogawa, Yuki Fujii, Hideyuki Iriko, Jiharu Hamako, Taei Matsui, Yasuhiro Ozeki

    A specific galactose-binding lectin was shown to inhibit the hemolytic effect of streptolysin O (SLO), an exotoxin produced by Streptococcus pyogenes. Commercially available lectins that recognize N-acetyllactosamine (ECA), T-antigen (PNA), and Tn-antigen (ABA) agglutinated rabbit erythrocytes, but had no effect on SLO-induced hemolysis. In contrast, SLO-induced hemolysis was inhibited by AKL, a lectin purified from sea hare (Aplysia kurodai) eggs that recognizes a-galactoside oligosaccharides. This inhibitory effect was blocked by the co-presence of D-galactose, which binds to AKL. A possible explanation for these findings is that cholesterol-enriched microdomains containing glycosphingolipids in the erythrocyte membrane become occupied by tightly stacked lectin molecules, blocking the interaction between cholesterol and SLO that would otherwise result in penetration of the membrane. Growth of S. pyogenes was inhibited by lectins from a marine invertebrate (AKL) and a mushroom (ABA), but was promoted by a plant lectin (ECA). Both these inhibitory and promoting effects were blocked by co-presence of galactose in the culture medium. Our findings demonstrate the importance of glycans and lectins in regulating mechanisms of toxicity, creation of pores in the target cell membrane, and bacterial growth.

    MDPI AG, Sep. 2014, MOLECULES, 19 (9), 13990 - 14003, English

    [Refereed]

    Scientific journal

  • Natural Infection of Plasmodium falciparum Induces Inhibitory Antibodies against Gametocyte Development in Human Hosts

    Natda Tonwong, Jetsumon Sattabongkot, Takafumi Tsuboi, Hideyuki Iriko, Satoru Takeo, Jeeraphat Sirichaisinthop, Rachanee Udomsangpetch

    We identified naturally induced antibodies from malaria patients in Thailand and clarified the effect of the antibodies on gametocyte development. Fifty-nine percent of the Plasmodium falciparum-infected blood samples (17 of 29) fed to female Anopheles mosquitoes showed no oocyst infection. Seventeen percent of the samples (5 of 29) distorted the morphology and hampered the maturity of the gametocytes. A possible mechanism for the gametocyte inhibitory activity was shown by the binding of the plasma antibodies to live, immature, intraerythrocytic gametocytes during the incubation period. One hundred fifty-seven proteins specific to different gametocyte stages were explored to find the targets of the antisera that bound to the live gametocytes. However, no additional gametocyte transmission- blocking vaccine candidate was detected. Therefore, the development of alternative transmission-blocking vaccines in high-transmission areas should focus on the identification of more gametocyte antigens-inducing inhibitory antibodies that reduce gametocytemia.

    NATL INST INFECTIOUS DISEASES, Mar. 2012, JAPANESE JOURNAL OF INFECTIOUS DISEASES, 65 (2), 152 - 156, English

    [Refereed]

    Scientific journal

  • Mayumi Tachibana, Yimin Wu, Hideyuki Iriko, Olga Muratova, Nicholas J. MacDonald, Jetsumon Sattabongkot, Satoru Takeo, Hitoshi Otsuki, Motomi Torii, Takafumi Tsuboi

    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.

    AMER SOC MICROBIOLOGY, Aug. 2011, CLINICAL AND VACCINE IMMUNOLOGY, 18 (8), 1343 - 1350, English

    [Refereed]

    Scientific journal

  • Helminth Infections Prevent Autoimmune Diseases through Th2-Type Immune Response

    Soji Fukumoto, Hideyuki Iriko, Hitoshi Otsuki

    Helminth parasites are known to elicit the immune response towards T helper 2 (Th2)type, characterized by Th2 related cytokines, that typically include interleukin-4 (IL4), IL-5 and IL-13. In this review we will describe the mechanisms involved in helminth induced Th2 immune response. Intestinal epithelial cells (IECs) produce thymic stromal lymphopoietin (TSLP), which is both necessary and sufficient for the initiation of Th2 cytokine-driven inflammation. IL-33 mRNA is expressed early during parasite infection and IL-33 binds ST2 receptor, both of which are associated with optimal CD4(+) Th2 polarization. Following innate immune cell recognition, basophils and mast cell can secrete Th2 type cytokines that are thought to contribute to CD4(+) Th2 differentiation. Additionaly, dendritic cell conditioned with some helminth products can promote CD4(+) Th2 differentiation. Alternatively activated macrophages, activated by the Th2 cytokines IL-4 and IL-13 in parasitic infections, contribute to the host protective response: control of Th1-type inflammation, wound healing and worm expulsion. Experimentally, helminths have been associated with protection against a number of autoimmune disorders, including inflammatory bowel diseases and type 1 diabetes. It may be a novel strategy to ameliorate autoimmune inflammation by expanding and activating the Th2 response originated from parasites.

    TOTTORI UNIV, FACULTY MEDICINE, Sep. 2009, YONAGO ACTA MEDICA, 52 (3), 95 - 104, English

    [Refereed]

    Scientific journal

  • Hideyuki Iriko, Ling Jin, Osamu Kaneko, Satoru Takeo, Eun-Taek Han, Mayumi Tachibana, Hitoshi Otsuki, Motomi Torii, Takafumi Tsuboi

    During the last decade transcriptome analyses demonstrated that alternative splicing plays an important role to generate a large number of mRNA and protein isoforms from a limited number of genes. However, the frequency of the alternative splicing dramatically Varies among living organisms. For example, 35-65% of human genes are involved in alternative splicing, whereas only a few are reported for unicellular organism yeast. Alternative splicing has been observed for several genes in the deadliest malaria parasite Plasmodium falciparum, but the frequency and the type were not systematically analyzed so far. In this study, we determined partial cDNA sequences for 88 open reading frames surrounding 246 introns in P falciparum which were transcribed at schizont and gametocyte stages, and observed 15 instances of alternative splicing within a total of 14 gene transcripts, 16% of the analyzed genes. Among 5 basic splicing patterns, alternative 5' and 3' splicing, and intron retention were detected. Alternative splicing in 7 open reading frames had effects on the domain architectures of the gene products, which might result in modifying the cellular localization and function of these products. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

    ELSEVIER IRELAND LTD, Jun. 2009, PARASITOLOGY INTERNATIONAL, 58 (2), 196 - 199, English

    [Refereed]

    Scientific journal

  • Otsuki H, Kaneko O, Thongkukiatkul A, Tachibana M, IRIKO HIDEYUKI, Takeo S, Tsuboi T, Torii M

    2009, Proc Natl Acad Sci U S A, 106 (17), 7167 - 7172, English

    [Refereed]

    Scientific journal

  • Takafumi Tsuboi, Satoru Takeo, Hideyuki Iriko, Ling Jin, Masateru Tsuchimochi, Shusaku Matsuda, Eun-Taek Han, Hitoshi Otsuki, Osamu Kaneko, Jetsumon Sattabongkot, Rachanee Udomsangpetch, Tatsuya Sawasaki, Motomi Torii, Yaeta Endo

    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 mu g/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.

    AMER SOC MICROBIOLOGY, Apr. 2008, INFECTION AND IMMUNITY, 76 (4), 1702 - 1708, English

    [Refereed]

    Scientific journal

  • Hideyuki Iriko, Osamu Kaneko, Hitoshi Otsuki, Takafumi Tsuboi, Xin-zhuan Su, Kazuyuki Tanabe, Motomi Torii

    complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Understanding the level of polymorphism and the evolutionary processes is important for advancements in both vaccine design and knowledge of the evolution of cell invasion in this parasite. In the present study, we sequenced the entire open reading frames of seven genes encoding the proteins of the PfRhopH complex (rhoph2, rhoph3, and five rhoph1/clag gene paralogs). We found that four rhoph1/clag genes (clag2, 3.1, 3.2, and 8) were highly polymorphic. Amino acid substitutions and indels are predominantly clustered around amino acid positions 1000-1200 of these four rhoph1/clag genes. An excess of nonsynonymous substitutions over synonymous substitutions was detected for clag8 and 9, indicating positive selection. The McDonald-Kreitman test with a Plasmodium reichenowi orthologous sequence also supports positive selection on clag8. Based on the ratio of interspecific genetic distance to intraspecific distance, the time to the most recent common ancestor of the clag2 and 8 polymorphisms was estimated to be 1.89 and 0.87 million years ago, respectively, assuming divergence of P. falciparum and P. reichenowi 6 million years ago. In addition to a copy number polymorphism, gene conversion events were detected for the rhoph1/clag genes on chromosome 3, which likely play a role in increasing the diversity of each locus. Our results indicate that a high diversity of the PfRhopH1/Clag multigene family is maintained by diversifying selection forces over a considerably long period. (c) 2007 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, Mar. 2008, MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 158 (1), 11 - 21, English

    [Refereed]

    Scientific journal

  • Expression of malaria vaccine candidates using a wheat germ cell-free protein synthesis system without codon optimisation.

    Takafumi Tsuboi, Satoru Takeo, Hideyuki Iriko, Ling Jin, Masateru Tsuchimochi, Shusaku Matsuda, Eun-Taek Han, Hitoshi Otsuki, Osamu Kaneko, Jetsumon Sattabongkot, Rachanee Udomsangpetch, Tatsuya Sawasaki, Motomi Torii, Yaeta Endo

    PERGAMON-ELSEVIER SCIENCE LTD, Jan. 2008, INTERNATIONAL JOURNAL FOR PARASITOLOGY, 38, S77 - S77, English

    [Refereed]

  • Eun-Taek Han, Risa Watanabe, Jetsumon Sattabongkot, Benjawan Khuntirat, Jeeraphat Sirichaisinthop, Hideyuki Iriko, Ling Jin, Satoru Takeo, Takafumi Tsuboi

    Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovate. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovate and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovate. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.

    AMER SOC MICROBIOLOGY, Aug. 2007, JOURNAL OF CLINICAL MICROBIOLOGY, 45 (8), 2521 - 2528, English

    [Refereed]

    Scientific journal

  • コムギ胚芽無細胞タンパク質合成法:マラリアワクチン研究への応用

    坪井 敬文, 竹尾 暁, IRIKO HIDEYUKI, 金子 修, 鳥居 本美, 遠藤 弥重太

    2007, 愛媛医学, 26, 8 - 11, Japanese

    [Refereed]

    Research institution

  • Osamu Kaneko, Thomas J. Templeton, Hideyuki Iriko, Mayumi Tachibana, Hitoshi Otsuki, Satoru Takeo, Jetsumon Sattabongkot, Motomi Torii, Takafumi Tsuboi

    The Plasmodium circumsporozoite protein/thrombospondin-related anonymous protein-related protein (CTRP) is expressed at the mosquito midgut ookinete stage and is considered to be a transmission-blocking vaccine candidate. CTRP is composed of multiple von Willebrand factor A (vWA) and thrombospondin type I domains in the extracellular portion of the molecule, and a short acidic cytoplasmic domain that interacts with the actomyosin machinery. As a means to predict functionally relevant domains within CTRP we determined the nucleotide sequences of CTRP from the Plasmodium vivax Sall and the Plasmodium yoelii 17XL strains and characterized the conservation of domain architectures and motifs across Plasmodium genera. Sequence alignments indicate that the CTRP 1st to 4th vWA domains exhibit greater conservation, and thereby are perhaps functionally more important than the 5th and 6th domains. This point should be considered for the development of a transmission-blocking vaccine that includes CTRP recombinant subunit. To complement previous cellular studies on CTRP, we further determined the expression and cellular localization of CTRP protein in R vivax and P. yoelii. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

    ELSEVIER IRELAND LTD, Sep. 2006, PARASITOLOGY INTERNATIONAL, 55 (3), 227 - 231, English

    [Refereed]

    Scientific journal

  • Screening of novel malaria transmission-blocking vaccine candidates using wheat germ cell-free protein synthesis system

    Hideyuki Iriko, Satoru Takeo, Ling Jin, Osamu Kaneko, Motomi Torii, Jetsumon Sattabongkot, Sanjay Singh, Tatsuya Sawasaki, Yaeta Endo, Takafumi Tsuboi

    AMER SOC TROP MED & HYGIENE, Dec. 2005, AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 73 (6), 292 - 292, English

    [Refereed]

  • Wheat germ cell-free system: A powerful tool to identify novel vaccine candidates based on the Plasmodium Falciparum genome database

    Takafumi Tsuboi, Satoru Takeo, Hideyuki Iriko, Ling Jin, Eun-Taek Han, Osamu Kaneko, Jetsumon Sattabongkot, Rachanee Udomsangpetch, Tatsuya Sawasaki, Motomi Torii, Yaeta Endo

    AMER SOC TROP MED & HYGIENE, Dec. 2005, AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 73 (6), 338 - 338, English

    [Refereed]

  • O Kaneko, BYSY Lim, H Iriko, IT Ling, H Otsuki, M Grainger, T Tsuboi, JH Adams, D Mattei, AA Holder, M Torii

    The Plasmodium falciparum high molecular mass rhoptry protein (`PfRhopH') complex is important for parasite growth and comprises three distinct gene products: RhopH1, RhopH2 and RhopH3. We have previously shown that P. falciparum RhopH1 is encoded by either PFC0110w (clag3.2) or PFC0120w (clag3.1), members of the previously-named clag (cytoadherence-linked asexual gene) multigene family. In this report, we have further characterized rhoph1/clag members in terms of gene structure, transcription and protein expression. The cDNA sequences for all five rhoph1/clag members were determined, confirming previous in silico predictions of intron-exon boundaries. All member genes were transcribed in HB3 and 3D7 parasite lines, but clag3.2 was not transcribed in Dd2 parasites. The peak abundance of transcripts for all genes was observed during the late schizont stage. Antisera specific to Clag2 and Clag3.1 localized these proteins to the apical end of merozoites in segmented schizonts, and both proteins are found to be components of the PfRhopH complex. PfRhopH complex that was immunoprecipitated with anti-Clag9 antibody contained neither Clag2 nor Clag3.1, thereby suggesting that PfRhopH complexes contain only individual rhoph1/clag gene products. Since the PfRhopH complex binds the erythrocyte surface, and RhopH2 and RhopH3 are encoded by single copy genes, the RhopH1/Clag proteins may serve to confer some degree of specificity to the roles of the individual complexes. (c) 2005 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, Sep. 2005, MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 143 (1), 20 - 28, English

    [Refereed]

    Scientific journal

  • マラリア原虫の赤血球侵入に関わる多重遺伝子族

    金子 修, 大槻 均, IRIKO HIDEYUKI, 坪井 敬文, 鳥居 本美

    2003, 愛媛医学, 22, 102 - 109, Japanese

    [Refereed]

    Research institution

  • H Iriko, K Nakamura, H Kojima, N Iida-Tanaka, T Kasama, Y Kawakami, Ishizuka, I, A Uchida, Y Murata, Y Tamai

    Glycosphingolipids (GSLs) were purified from adults and plerocercoids of the tapeworm Diphyllobothrium hottai , and their chemical structures were determined. Total lipid fractions prepared from chloroform/methanol extracts of whole tissues were fractionated successively on ion-exchange chromatography, silicic acid column chromatography, and preparative TLC. The purified GSLs were characterized by methylation analysis, TLC-immunostaining, liquid secondary ion MS, MALDI-TOF MS, and (1) H-NMR. Ten GSLs were isolated from adult worms and four from plerocercoids, comprising mono-, di-, tri-, tetra-, and pentasaccharides. The GSL Galbeta1-4(Fucalpha1-3)Glcbeta1-3Galbeta1-Cer was found in adult worms but not in plerocercoids, whereas Galbeta1-4 (Fucalpha1-3)Glcbeta1-3(Galbeta1-6)Galbeta1-Cer was found in both adult worms and plerocercoids. We previously found a similar series of GSLs in plerocercoids of the cestode Spirometra erinaceieuropaei , and termed them 'spirometosides'[Kawakami, Y. et al . (1996) Eur J. Biochem . 239 , 905-911]. The core structure of spirometosides, Galbeta1-4Glcbeta1-3 Galbeta1-Cer, may have taxonomic significance, being characteristic of pseudophyllidean tapeworms. In the present study, GSL compositions were significantly different between adults and plerocercoids, and growth-dependent changes in composition were documented. We found a novel dihexosylceramide, Glcbeta1-3Galbeta1-Cer, which is a possible precursor for spirometosides. Immunohistochemical examination showed that spirometoside GSLs are highly enriched in the inner surface of bothria, the major point of contact between the adult worm and the host's intestine. Our findings indicate that spirometosides are involved in host-parasite interaction.

    BLACKWELL PUBLISHING LTD, Jul. 2002, EUROPEAN JOURNAL OF BIOCHEMISTRY, 269 (14), 3549 - 3559, English

    [Refereed]

    Scientific journal

  • M Yanagisawa, H Kojima, Y Kawakami, H Iriko, T Nakamura, K Nakamura, A Uchida, Y Murata, Y Tamai

    A mouse monoclonal antibody AK97 (IgM) was established against a new type of glycosphingolipid, SEGLx, isolated from plerocercoids of tapeworm, Spirometra erinaceieuropaei. The chemical structure of SEGLx (Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta 1-ceramide) had been previously characterized. The specificity of AK97 was determined by thin-layer chromatography-immunostaining and enzyme-linked immunosorbent assay. AK97 was found to be directed to SEGLx and GalSEGLx (Gal beta 1-4(Fuc alpha 1-3)G1c beta 1-3(Gal beta 1-6)Gal beta 1-ceramide) and also showed cross-reactivity with the stage specific embryonic antigen-1 (SSEA-I), the epitope being defined to be the non-reducing terminal trisaccharide sequence. On immunohistochemical examination, AK97 predominantly stained the tegument, the external surfaces of worms which have a brush border-like organization. Based on the immunohistochemical findings for the staining liability as to organic solvents and the results of Western blot analysis of the plerocercoid glycoproteins, it was proved that the antigens in the tapeworm were glycolipids. Considering that the tapeworm is in direct contact with its host's tissue through the tegument, the membrane surface of which is exposed to the external environment, it is suspected that SEGLx and GalSEGLx on the tegument play functionally important roles in the host-parasite interaction. (C) 1999 Elsevier Science B.V. All rights reserved.

    ELSEVIER SCIENCE BV, Aug. 1999, MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 102 (2), 225 - 235, English

    [Refereed]

    Scientific journal

MISC

  • 原虫・寄生虫検査に強くなろう- II 吸虫類 肺寄生、肝・胆道寄生、消化管寄生、門脈寄生

    大槻 均, IRIKO HIDEYUKI, 蓼本 早百合, 福本 宗嗣

    2013, 臨床と微生物, 40 (3), 327 - 333, Japanese

    Introduction scientific journal

  • 感染症症候群(第2版)-症候群から感染性単一疾患までを含めてー 上 病原体感染症編 VI. 原虫症, 吸虫症 <日本海裂頭条虫症, 広節裂頭条虫症>

    福本 宗嗣, 大槻 均, IRIKO HIDEYUKI, 蓼本 早百合

    2013, 別冊日本臨床 新領域別症候群シリーズ, 24, 733 - 737, Japanese

    Introduction scientific journal

  • IMMUNIZATION WITH N-TERMINAL REGION OF A GAMETOCYTE PROTEIN PFS230 SUCCESSFULLY INDUCE TRANSMISSION-BLOCKING ANTIBODIES AGAINST PLASMODIUM FALCIPARUM

    Mayumi Tachibana, Hideyuki Iriko, Olga Muratova, Guanhong Song, Yimin Wu, Jetsumon Sattabongkot, Satoru Takeo, Hitoshi Otsuki, Motomi Torii, Takafumi Tsuboi

    AMER SOC TROP MED & HYGIENE, Nov. 2009, AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 81 (5), 160 - 160, English

    Summary international conference

  • IMMUNIZATION WITH RECOMBINANT PROTEINS OF A GAMETOCYTE PROTEIN PFS230 EXPRESSED USING WHEAT GERM CELL-FREE SYSTEM SUCCESSFULLY INDUCE TRANSMISSION-BLOCKING ANTIBODIES AGAINST PLASMODIUM FALCIPARUM

    Mayumi Tachibana, Hideyuki Iriko, Olga Muratova, Guanhong Song, Yimin Wu, Jetsumon Sattabongkot, Satoru Takeo, Hitoshi Otsuki, Motomi Torii, Takafumi Tsuboi

    AMER SOC TROP MED & HYGIENE, Dec. 2008, AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 79 (6), 307 - 307, English

    Summary international conference

  • Novel sporozoite antigen discovery of Plasmodium falciparum screened using human immunesera

    Ling Jin, Satoru Takeo, Hideyuki Iriko, Osamu Kaneko, Jetsumon Sattabongkot, Motomi Torii, Joao Carlos Aguiar, Takafumi Tsuboi

    AMER SOC TROP MED & HYGIENE, Nov. 2007, AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 77 (5), 165 - 165, English

    Summary international conference

  • Discovering novel malaria pre-erythrocytic antigens

    Joao Carlos Aguiar, Hideyuki Iriko, Fengying Huang, John B. Sacci, Laure Juompan, Ling Jin, Eun-Taek Han, Satoru Takeo, Urszula Krzych, Yaeta Endo, Thomas Richie, Takafumi Tsuboi

    AMER SOC TROP MED & HYGIENE, Nov. 2006, AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), 303 - 303, English

    Summary international conference

  • Discovering novel blood stage malaria vaccine candidates: Screening with immune sera from falciparum malaria patients and asymptomatic parasite carriers

    Satoru Takeo, Ling Jin, Hirokazu Sakamoto, Eun-Taek Han, Hideyuki Iriko, Osamu Kaneko, Motomi Torii, Jetsumon Sattabongkot, Rachanee Udomsangpetch, Tatsuya Sawasaki, Yaeta Endo, Takafumi Tsuboi

    AMER SOC TROP MED & HYGIENE, Nov. 2006, AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), 302 - 302, English

    [Refereed]

    Summary international conference

Books etc

  • Glycosphingolipids in prendophyllidean tapeworms (共著)

    9th International Congress of Parasitology, 1998

Presentations

  • Protein trafficking and parasite-derived membrane structures in P. falciparum-infected erythrocytes

    Hideyuki Iriko

    The 4th Asian Congress of Protistology -internet, 20 Nov. 2021, English

    [Invited]

    Nominated symposium

  • Expression profile and immuno-localization of Skeleton Binding Protein 1 (SBP1) during gametocyte stage in Plasmodium falciparum.

    Ayaka Omoda, Jerusha Kulamahan, Nozomi Yamasaki, Mayumi Tachibana, Tomoko Ishino, Motomi Torii, Hideyuki Iriko

    The 4th Asian Congress of Protistology -internet, 20 Nov. 2021, English

    Poster presentation

  • 熱帯熱マラリア原虫生殖母体感染赤血球のMaurer's cleft局在タンパク質の解析

    面田彩馨, 岡田小夏, 橘真由美, 石野智子, 鳥居本美, 入子英幸

    第76回日本寄生虫学会西日本支部大会, 25 Sep. 2021

    Oral presentation

  • 熱帯熱マラリア原虫生殖母体期におけるPTEX複合体構成タンパク質の発現プロファイル解析

    岡田小夏, 伊坂恵里奈, 面田彩馨, 橘真由美, 石野智子, 鳥居本美, 高島英造, 坪井敬文, 入子英幸

    第 76 回日本寄生虫学会 西日本支部大会, 25 Sep. 2021

    Oral presentation

  • 熱帯熱マラリア原虫の赤血球寄生ステージに形成される膜構造

    入子 英幸

    第30回 原生生物・寄生虫・進化(PPE)セミナー, 16 Jul. 2021

  • 熱帯熱マラリア原⾍の⽣殖⺟体期における原⾍タンパク質輸送機構の解析

    面田彩馨, 橘真由美, 石野智子, 鳥居本美, 坪井敬文, 入子英幸

    第90回日本寄生虫学会 第32回日本臨床寄生虫学会 合同大会, 16 Apr. 2021

    Poster presentation

  • Skeleton binding protein 1 (SBP1) of Plasmodium falciparum is localized in Maurer’s cleft of gametocyte infected erythrocyte

    Ayaka Omoda, Jerusha Kulamahan, Nozomi Yamasaki, Mayumi Tachibana, Tomoko Ishino, Motomi Torii, Hideyuki Iriko

    Joint meeting of the Japan Society of Protistology and Korean Society of Protistologists (Kobe2020), 22 Nov. 2020, English

    Poster presentation

Association Memberships

  • 日本生化学会

  • 日本寄生虫学会

Research Projects

  • Development of novel transmission blocking vaccine targeting microgamete surface antigen of Plasmodium falciparum

    鳥居 本美, 橘 真由美, 石野 智子, 入子 英幸, 伊藤 大輔, カレトン リチャード

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Ehime University, 01 Apr. 2020 - 31 Mar. 2023

  • 三日熱マラリア伝搬阻止効果のある患者血漿を用いた新規ワクチン候補抗原の探索

    石野 智子, 橘 真由美, 馬場 みなみ, 鳥居 本美, 入子 英幸

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)), Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)), Ehime University, 07 Oct. 2019 - 31 Mar. 2023

  • 入子 英幸

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 01 Apr. 2016 - 31 Mar. 2019, Principal investigator

    マラリア原虫は、赤血球侵入時に「寄生胞膜」と呼ばれる膜を形成する。この寄生胞膜は、宿主免疫応答・環境ストレスに対する障壁として機能するが、その一方で、栄養源であるヘモグロビンから原虫を隔離する。そのため、マラリア原虫は、サイトストームと呼ばれる特徴的な膜構造を形成し、ヘモグロビンを取り込み、代謝の場である食胞へと輸送する。本研究では、熱帯熱マラリア原虫の生殖母体期に着目し、ヘモグロビン輸送に関連する膜構造に局在する分子群を同定し、それらのヘモグロビン輸送における機能を明らかにすることを目的としている。本年度は、生殖母体期における寄生胞膜分子のプロファイリングを目的として、生殖母体期に発現するPfs16を指標として、間接蛍光抗体法により熱帯熱マラリア原虫の寄生胞膜分子4種類(ETRAMP4, 5, 10.2, 10.3)の発現及び局在を、無性生殖期と生殖母体間で比較した。これらの結果から、熱帯熱マラリア原虫の寄生胞膜を構成する分子は、生殖母体期に特有のもの(ETRAMP4, ETRAMP10.3)、無性生殖期と生殖母体期に共通のもの(ETRAMP10.2)、無性生殖期に特有のもの(ETRAMP5)に分類できることが明らかになった。マラリア原虫の無性生殖期に発現する赤血球侵入関連分子は、遺伝子欠損原虫の表現型が致死となるため解析が困難である。これに対し、生殖母体期に特異的に発現する分子群は、逆遺伝学的手法を用いた解析が可能である。現在、生殖母体期特異的に寄生胞膜に発現するETRAMP4, ETRAMP10.3に着目し、CRISPR-Cas9ゲノム編集システムを用いた遺伝子改変を試みている。

    Competitive research funding

  • IRIKO HIDEYUKI

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), 01 Apr. 2013 - 31 Mar. 2015, Principal investigator

    To elucidate the molecular mechanism and membrane dynamics of hemoglobin transport, I focused in ETRAMP4 (Early Transcribed Protein 4), the membrane proteins expressed in trophozoite stage of Plasmodium falciparum. By immunoelectron microscopy, ETRAMP4 localized in parasitophorous vacuole membrane and uptake by cytostomes, hemoglobin transport vesicles.

    Competitive research funding

  • A study of the effect of a recombinant immunosuppressive factor from parasites and immune-inhibitory mechanisms

    FUKUMOTO Soji, IRIKO Hideyuki, OTSUKI Hitoshi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Tottori University, 2009 - 2011

    An immunosuppressive factor(ES90) from Spirometra erinaceieuropaei plerocercoids was cloned and the recombinant ES90 was synthesized using wheat germ cell free expression system. However, the recombinant ES90 did not inhibit nitric oxide production in macrophages. Messenger RNA of Ym1, FIZZ1, and arginase 1 were expressed in peritoneal macrophages from Trichinella spiralis-infected mice. In connection with this, 2 types of peroxiredoxin(Prx) from T. spiralis were cloned, Prx-1 mRNA expression was stage-specific

  • Analysis of menbrane dynamics and identification of target signal to parasitophorous vacuole menbrane of malaria parasite.

    IRIKO Hideyuki

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), Tottori University, 2009 - 2010

    During the process of invasion, maralia parasite initiates the formation of a membrane, the so-called parasitophorous vacuole membrane (PVM). PVM is associated with host-parasite interaction. In this study, we analysed membrane dynamics and target signal to PVM in malaria parasite. We succeed in visualization of PVM and deternimation of forming period of tubulovesicular membrane network and circular clefts.

  • Functional analysis of an immunosuppressive factor secreted from a parasite and its application to rheumatoid arthritis model

    FUKUMOTO Soji, IRIKO Hideyuki

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Tottori University, 2007 - 2008

    マンソン裂頭条虫擬充尾虫(幼虫)由来の免疫抑制因子(ES90)のN末端と内部のアミノ酸配列に基づいてRT-PCR法でcDNAが増幅でき、クローニングの基礎試料が得られた。また、同じ幼虫由来の130kDaの免疫抑制因子(ES130)を精製した。ES130は活性化マクロファージが産生する3つのケモカイン(RANTES, MIP-2, IP-10)の遺伝子発現を抑制し、炎症や免疫応答の抑制への関与が推察された。

  • Comprehensive screening and identification of the novel malaria transmission-blocking vaccine candidate antigens

    TSUBOI Takafumi, TAKEO Satoru

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Ehime University, 2006 - 2007

    Malaria transmission-blocking vaccines (TBVs) prevent the transmission of malaria by inducing antibodies against antigens specifically expressed on the sexual stage parasites. Since well-characterized TBV candidates are only four, it would be necessary to identify novel TBV candidates for a successful TBV development. In order to identify the novel TBV candidates, we searched a combined dataset from genome and transcriptome databases and selected 192 genes, which are expected to be expressed only in gametocyte stage of P. falciparum. These genes were cloned into plasmids and 170 of the template cDNA clones were prepared for transcription through PCR-based procedures, followed by high throughput recombinant protein synthesis by wheat germ cell-free system. Using this approach, we succeeded in obtaining 148 recombinant proteins. After the screening of these recombinant proteins to identify novel TBV candidates with malaria patient sera harboring parasite transmission-blocking antibodies, we have identified 27 novel antigens. After subclone these candidate genes into pEU plasmid for the large-scale expression using the wheat germ cell-free system, we have expressed and affinity purified 22 targets. In order to obtain antisera, we immunized these proteins into mice. After the confirmation of the immunoreactivity of these antisera against parasites, we examined the transmission-blocking efficacy of the sera. Finally we identified the two novel parasite antigens, which induced transmission-blocking antibodies in mice. Accordingly, this approach will be useful for the novel transmission-blocking vaccine candidate discovery.

  • 病原微生物が発現する宿主細胞認識分子のハイスループットな同定法の開発

    坪井 敬文, 竹尾 暁, 入子 英幸

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Exploratory Research, Grant-in-Aid for Exploratory Research, Ehime University, 2005 - 2006

    近年、多くの病原微生物のゲノム塩基配列が決定されているが、タンパク質の機能に関しては未だに得られる情報は少ない。核酸代謝・アミノ酸代謝といった生命現象に普遍的な分子は、生物全般で保存されており解析も比較的容易であるが、病原微生物の病原性に関与する分子、中でも感染の成立時に働く宿主細胞認識分子は病原微生物に固有のものが多く、ワクチンや創薬の標的であるにもかかわらず研究が遅れている。そこで我々は、病原微生物の宿主細胞認識分子をゲノムワイドに同定する手法の開発を目的として本研究を開始した。昨年度は、複雑な生活環を持ち多くの宿主細胞認識分子を持つと考えられる熱帯熱マラリア原虫の遺伝子約400個をモデルとして、コムギ無細胞タンパク質合成法を応用することにより、それらの約70%をGFP融合タンパク質として発現できた。本年度は、その原虫組換えタンパク質のレセプターとなるヒト赤血球膜ベジクルの作製を試みたが、均一なベジクルの作製は困難であった。そこで一分子蛍光分析システムを応用したレセプター・リガンドの同定の一例として、マラリア患者血清を用いた抗原抗体反応の測定を試みた。その結果、上記GFP融合原虫タンパク質の内約100種類と、患者血清を用いて検討した結果、3種類の原虫タンパク質がヒト血清中の抗体と反応し、抗原性を有していることが明らかとなった。したがって、本法は病原微生物と宿主分子間の結合をハイスループットに同定できる手法となりうることが示唆された。

  • High-throughput screening of novel malaria vaccine candidates using human immune sera

    TSUBOI Takafumi, TAKEO Satoru, IRIKO Hideyuki

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Ehime University, 2004 - 2006

    Malaria remains one of the leading causes of both morbidity and mortality in humans residing in the tropical countries. The evidence of increasing resistance of the Plasmodium falciparum parasite to chemotherapeutic agents highlights the critical need for an effective vaccine. After the establishment of malaria genome database, we have now free access to the genome data to search for novel vaccine candidates. However, one of the bottlenecks for the malaria vaccine research is the difficulty of the recombinant protein expression using conventional methods. We applied a high-throughput protein production method based on the wheat germ cell-free system to the malaria vaccine research. To identify novel malaria vaccine candidates, we selected and cloned more than 180 genes which are expected to be expressed in merozoite stage, then prepared transcription templates through PCR-based high throughput procedures, followed by protein synthesis by wheat germ cell-free system. Using this approach, we succeeded in obtaining 149 recombinant merozoite proteins. At the same time, we successfully obtained approximately 30 human immune sera from asymptomatic villagers living along the Thai-Myanmar border, and approximately 30 symptomatic malaria patient sera from hospital in Bangkok, Thailand. All human serum samples used in this study were reviewed and approved by the Institutional Ethics Committee of the Thai Ministry of Public Health and of the Ehime University, Japan. Finally, we tried to screen these recombinant proteins for identifying novel malaria vaccine candidates with five cases of the malaria immune sera by ELISA. As a result of this preliminary screening, we identified ten merozoite proteins as antigens. Accordingly, this strategy using wheat germ cell-free system will be a major breakthrough in the novel malaria vaccine candidate discovery research.

  • Expression of erythrocyte binding protein in Plasmodium falciparum merozoites isolated from endemic area

    TORII Motomi, MATSUDA Syouji, KANEKO Osamu, TSUBOI Takafumi, IRIKO Hideyuki

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Ehime University, 2001 - 2002

    Mutant erythrocyte that lacked certain proteins on the surface were frequently observed in the malaria endemic areas, and some of them were believed to be generated by the selective advantage against malaria infection. However, Plasmodium falciparum have an ability to invade many types of erythrocyte including these mutant erythrocytes. The molecular base of this ability of P.falciparum could be resulted by the erythrocyte-binding proteins encoded in the multi-gene family involved in the invasion steps. In this study, we explored the possibility if P.falciparum regulated the expression of these multi-gene family in order to overcome the erythrocyte polymorphism by quantitate the transcription and protein expression level of each members of the multi-gene family, PfRhopH1. Firstly, we designed a panel of oligonucleotide primers for real-time PCR method with SYBR green. Complementary DNA was made from the 3D7 clone of P.falciparum and used to make plasmids containing the real-time PCR targets for each member. These plasmids were used to create the standard curve. P.falciparum field samples were collected in the malaria endemic area in Thailand and total RNA were extracted. Simultaneously blood smear were made on the glass slides for the IFA analysis for the protein expression. In order to check the protein expression level, we successfully generated anti-PfRhopHl_2, 3.1, 9 specific sera. Difference in the protein expression was observed among the culture-adapted P. falciparum clones.