WO2014084333A1 - Protéine de protozoaire associée à une infection par un plasmodium au stade hépatique - Google Patents

Protéine de protozoaire associée à une infection par un plasmodium au stade hépatique Download PDF

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WO2014084333A1
WO2014084333A1 PCT/JP2013/082139 JP2013082139W WO2014084333A1 WO 2014084333 A1 WO2014084333 A1 WO 2014084333A1 JP 2013082139 W JP2013082139 W JP 2013082139W WO 2014084333 A1 WO2014084333 A1 WO 2014084333A1
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lisp2
infection
liver
stage
malaria
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油田　正夫
史朗 岩永
伊澄 金子
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国立大学法人三重大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
    • G01N2333/445—Plasmodium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a protozoan protein involved in infection of a malaria parasite liver stage.
  • LS liver stage
  • sporozoite After the sporozoite moves to the liver, it forms a parasite (parasitophorous vacuole (PV)) in the hepatocyte, enters, and proceeds to the liver infection stage.
  • the malaria parasite at the stage of liver infection eventually turns into thousands of merozoites and is released into the blood before infecting red blood cells. Hepatocytes are the first line of defense against malaria parasites.
  • Hepatocytes have an immune system against Plasmodium infection (Bongfen et al., 2007; Chakravarty et al., 2007; Cockburn et al., 2011: Prior art documents are summarized at the end), interferon and It is known to eliminate malaria parasites in cells by responding to cytokines such as TNF- ⁇ (Depinay et al., 2011). It is also known that parasitic hepatocytes undergo apoptosis to suppress the spread of red blood cell infection (van de Sand et al., 2005). It is thought that there is a dynamic interaction between protozoa and host hepatocytes during malaria infection. However, little is known about the interaction between Plasmodium and hepatocytes at the stage of liver infection. In particular, many remain unknown about the mechanism by which malaria parasites maintain infection in host cells and the genes that are important for it.
  • Circumsporozoite® (CS) ® protein the major surface protein of sporozoites, is known as the only malaria parasite protein that is transported to the cytoplasm of hepatocytes.
  • CS protein is observed in the cytoplasm of hepatocytes several hours after sporozoite invasion (Khan et al., 1992; Singh et al., 2007; Cockburn et al., 2011).
  • the present invention has been made in view of the above problems, and its purpose is to identify a protein essential for the malaria parasite in the liver infection stage to reach the next stage, and to search for a compound that inhibits this protein.
  • the present invention provides a test method for inhibiting malaria parasite infection by inhibiting the protein.
  • the inventors have also developed a technique for identifying a resistance gene by introducing an artificial chromosome into a malaria parasite, creating a gene library directly with the parasite, and screening. Such a method is unprecedented in all pathogenic eukaryotes including malaria parasites and is very original. If this method is used, it becomes possible to identify a resistance gene in only a few weeks, and it is expected to identify a resistance gene for the aforementioned antimalarial drug.
  • LISP2 LS-specific protein 2
  • LISP2 is one of the most abundant proteins in malaria parasites in the liver infection stage. LISP2 is produced in the middle to late liver infection stages, transported to PV by secretory vesicles, and then transported to the host cell cytoplasm and nucleus. According to our findings, this is the first reported protein that is produced and transported to the host cell by the Plasmodium parasite in the middle to late liver infection stages.
  • LISP2 is considered as an adhesion protein in the blood stage and is considered useful for diagnostic treatment. However, this protein is not actually expressed in the blood stage and has nothing to do with the present invention intended for use in the liver.
  • the LISP2-deficient type goes through a normal breeding stage and reaches merozoites. However, most LISP2-deficient types are inhibited when transformed into merozoites, resulting in abnormal merozoites that lack the ability to enter red blood cells.
  • exogenous LISP2 contributes to the growth of Plasmodium by acting directly with host hepatocytes. Based on this finding, a compound that inhibits LISP2 becomes a drug that inhibits infection with Plasmodium, and a vaccine that uses LISP2 as an antigen can be used as a malaria vaccine that inhibits infection with Plasmodium.
  • the method for testing a malaria parasite infection inhibitor according to the present invention is based on the LS-specific protein 2 (LS-specific protein 2 (LISP2)) that is specifically expressed in host cells from the middle stage to the late stage of the liver infection stage. )
  • LS-specific protein 2 LS-specific protein 2 (LISP2)
  • the test method is preferably an in vitro method using cultured cells derived from the liver. As such cultured cells, for example, primary cultured cells or cell lines can be used.
  • cell lines those derived from humans (eg, Alexander cells, HepG2, HLE, HLF, huH-1, HUH-6, HUH-7, JHH-1, JHH-2, JHH-4, JHH- 5, JHH-6, JHH-7, LI90, PLC / PRF / 5, etc., or rodent-derived (rat, mouse-derived (eg, Hepa1-6, 3'-mRLh- 2, 3'-mRLN-30, 3'-mRLN-31, Ac2F, AH601, AH66tc, AH70Btc, ARLJ301-3, BRL-3A, dRLa-74, dRLh-84, dRLN-4, dRLN-6, dRLN- 9, FAA-HTC1, FF101, FLS3, FLS5, IAR-20, JTC-16P3, M, M.P3, NCTC clone1469, RI-T, RL-34, RLC-10P3, RLN-8, RLN
  • the test method is preferably an in vivo method using rodents.
  • rodents include rats or mice.
  • LISP2 is expressed in the middle to late stages of the malaria parasite liver infection stage in host hepatocytes and is an essential protein for merozoite formation in the malaria parasite liver infection stage. For this reason, merozoite formation can be inhibited by suppressing the action of LISP2. Therefore, in the present invention, “suppressing the action of LISP2” means LISP2 transcription / translation, transport to the host cell, transport for localization distribution in the host cell, and interaction with other molecules. This means that any one or two or more points of action such as exerting the action of the protein of 1) are obstructed so that the action is not exhibited.
  • the function of such substances include, for example, control of LISP2 expression promoter, transcription inhibition, inhibition of post-translational modification, inhibition of transport to host cells, inhibition of transport for localization distribution in host cells, Examples include inhibition of interaction with other molecules.
  • the time during which an inhibitor should be present in order to suppress the action of LISP2 varies depending on any of the above action points. For this reason, the effect can be produced at any stage from before infection with malaria parasite to the host, at the same time as infection, from several hours to 60 hours after infection. In the present invention, it is possible to appropriately evaluate the time when the inhibitor is present.
  • a malaria vaccine according to another invention is characterized by containing LISP2 as an antigen.
  • various malaria parasites eg, P. falciparum, P. vivax
  • P. malariae P. malariae
  • Egg-shaped malaria parasites P. ovale
  • salmalaria parasites P. knowlesi
  • murine malaria parasites P. berghei, P. yoelii
  • All or part of LISP2 can be used as an antigen.
  • All or part of LISP2 can be used alone or as a malaria vaccine by adding an appropriate adjuvant (immuno-adjuvant).
  • a malaria parasite infection inhibitor according to another invention is detected by the test method described above, and suppresses the action of LISP2, thereby inhibiting merozoite formation in the malaria parasite liver infection stage.
  • a malaria parasite infection inhibitor related to another invention wherein the malaria parasite is specifically expressed in a host cell in the middle to late stage of liver infection (LS-specific protein 2 (LISP2)) )
  • the infection inhibitor include low molecular weight compounds, high molecular weight compounds, and nucleic acids (including DNA, RNA, and DNA-RNA hybrids.
  • nucleic acids including DNA, RNA, and DNA-RNA hybrids.
  • proteins including homologous, heterologous, or hybridized antibodies, including monoclonal antibodies and polyclonal antibodies, and the like.
  • LISP2 is expressed in the liver infection stage by the malaria parasite and transported into the hepatocyte, so that the malaria parasite plays an extremely important role in the proliferation in the hepatocyte. Proved to be.
  • the presence of LISP2 in the cytoplasm of hepatocytes means that it is likely to be a target for drugs and vaccines.
  • the compound which inhibits the infection of the malaria parasite can be searched by the test method for searching the compound which inhibits LISP2.
  • Such compounds can be provided as pharmaceuticals that inhibit malaria parasite infection.
  • LISP2 is a diagram illustrating a putative protein that is specifically expressed and secreted in the liver infection stage.
  • A Image diagram showing the structure of P. berghei LISP2 LISP2 is a protein consisting of 2172 amino acids, having a signal sequence at the N-terminus, and having a long repetitive portion of about 70 amino acids. Repeated sequences are indicated by rectangles in the figure.
  • B During the life cycle of the malaria parasite, the expression of LISP2 was examined by a promoter assay using GFP as a reporter gene.
  • the centromere plasmid Pcen rep-GFP was used, and the 1.2 kb upstream region of the LISP2 gene was incorporated upstream of the GFP gene.
  • FIG. 1 representative images of sporozoites in the salivary glands and liver infection stage are shown.
  • HepG2 cells were used for the liver infection stage. GFP expression was first observed 36 hours after infection. GFP expression was not observed at the blood stage or the intra-mosquito stage (see FIG. 1). The white bar is a 10 ⁇ m scale.
  • C The activity of the LISP2 promoter was evaluated using the luciferase gene as a reporter. At the stage of liver infection, HepG2 cells were used and collected at the time indicated on the horizontal axis.
  • FIG. 7 is a diagram comparing the amino acid sequence of a site predicted to be a 6-cysteine domain between Plasmodium bergberg LISP2: SEQ ID NO: 22 and other P. berghei 6-cysteine family proteins. The upper side shows the result of comparison between LISP2 and p230 (PBANKA — 030610: SEQ ID NO: 60), and the lower side shows the result of comparison between LISP2 and p41 (PBANKA — 100260: SEQ ID NO: 61). “*” Indicates a conserved amino acid.
  • the malaria parasite that failed to progress to the Cytomere stage was not judged to be “abnormal” because it could fail to progress to the Cytomere stage even in general cultured cells.
  • D Wild blood (WT) or LISP2 mutant (LISP2 (-)) malaria parasite liver infection stage, and infection in rat blood when merozoite released into the medium is administered intravenously in rats It is a graph which shows a red blood cell rate. Sporozoites were inoculated into the cultured cells, and the medium was collected from each well 60 to 66 hours in total for a total of 4 times. The merozoites were recovered from the medium by centrifugation and injected into rats.
  • FIG. 2 is a photomicrograph showing that many LISP2 mutant malaria parasites could not be converted into merozoites. Malaria parasites at the liver infection stage were cultured in HepG2 cells, and merozoites released into the medium were collected from each medium and administered to mice. After this test, the liver infection stage remaining in the LISP2 mutant malaria parasite culture medium (69 hours later) was stained with DAPI and observed under an epifluorescence microscope.
  • Atypical nuclei were observed in many LISP2 mutant malaria parasites (arrows). It is a figure which shows the expression profile and intracellular localization of LISP2 which united mCherry.
  • A shows the gene structure of a malaria parasite that expresses mCherry fusion LISP2.
  • a targeting construct containing the mCherry gene and the human DHFR-TS gene (selection marker) was inserted into the 3 ′ portion of the LISP2 locus of the GFP-expressing malaria parasite by double crossover homologous recombination. The insert sequence into the construct was analyzed by PCR and Southern blot (right side).
  • B Micrograph showing the expression of mCherry fusion LISP2 in the liver infection stage.
  • LISP2 is a photomicrograph showing that mCherry fusion LISP2 is not expressed in the blood stage and the intra-mosquito stage.
  • the upper row shows a fluorescence image of GFP constitutively activated in the strain 507 under the control of the elongation factor promoter.
  • the lower row shows the red fluorescence image in the upper field of view. No signal of mCherry fusion LISP2 was observed at any stage.
  • White bar indicates 10 ⁇ m.
  • LISP2 is a photomicrograph showing that it is carried by secretory vesicles surrounded by LISP1.
  • FIG. 3 is a micrograph showing that an anti-LISP2 antibody does not react with a LISP2 mutant malaria parasite.
  • FIG. 2 is a photomicrograph showing that LISP2 is transported to host cells and distributed in the nucleus and cytoplasm.
  • A An immunofluorescent staining microscope image of a malaria parasite in the liver infection stage observed by epifluorescence microscopy at 48 hours after infection. The malaria parasite at the liver infection stage was cultured in HepG2 cells.
  • the left side is an image stained with a secondary antibody conjugated with FITC and an antibody against the LISP2 repeat region.
  • the image of staining the nucleus with DAPI is shown on the right.
  • Open arrows indicate malaria parasites at the stage of liver infection, and closed arrows indicate host cell nuclei.
  • White bar indicates 10 ⁇ m.
  • LISP2 is a photomicrograph showing that it is exported to the cytoplasm and nucleus of hepatocytes in vivo. Wild type sporozoites were injected into the veins of rats, and after 48 hours, phosphate buffered saline was perfused and the livers were collected and fixed with formaldehyde. A 20 ⁇ m frozen section was prepared from the fixed liver and immunostained with an anti-LISP2 antibody. Observation was performed using a confocal microscope. Nuclei were stained with DAPI. It is the photograph which western blotted the malaria parasite of a liver infection stage.
  • LISP2 mCherry type, wild type, and LISP2 mutant type [LISP2 (-)] malaria parasites were cultured in HepG2 cells and collected together with host cells by lysis in SDS sample buffer. 48 hours after infection, Western blot analysis was performed using an antibody against the LISP2 repeat region (left side) or an antibody against mCherry (right side).
  • the band indicated by a black circle is a 150 kDa band detected with antibodies against mCherry and LISP2 in LISP2 :: mCherry type malaria parasite.
  • the band indicated by a white circle is a 120 kDa band detected with an antibody against LISP2 in a wild-type malaria parasite.
  • the major band indicated by the arrow is that of 30 kDa detected with an antibody against the LISP2 repeat region in both wild-type and LISP2 :: mCherry-type malaria parasites.
  • B Western blot analysis was performed using antibodies against the LISP2 repeat region 24, 36, and 48 hours after inoculation with wild-type sporozoites.
  • a white circle indicates a 30 kDa band, and an arrow indicates a 120 kDa band.
  • “LS” means liver infection stage
  • LS ( ⁇ )” means uninfected HepG2 cells (negative control). It is a graph which shows a result when the vaccine which used LISP2 as an antigen is administered.
  • mice immunized with the vaccine were 50% less susceptible to malaria parasite infection than the mice without the vaccine (Control-1). It is a graph which shows a result when administering the DNA vaccine which made LISP2 an antigen. Mice immunized with the vaccine (DNA-vaccine) were 70% less susceptible to malaria parasite infection than the mice without the vaccine (control).
  • malaria vector mosquitoes (Anopheles stephensi mosquitoes) were sucked from malaria infected mice (6-10 weeks old, female Balb / c mice, purchased from SLC), infected with malaria parasites and kept at 20 ° C. . Oocysts and sporozoites in salivary glands were collected on days 14 and 24, respectively, after the malaria vector mosquito sucked the infected blood. In order to infect rats, sporozoites in salivary glands after confirming gliding movement were intravenously administered. In order to prepare malaria parasites at the liver infection stage, sporozoites in salivary glands were collected and added to HepG2 cells seeded on 8-well chamber slides.
  • the malaria parasite at the liver infection stage was cultured at 37 ° C. in an atmosphere containing 5% CO 2 using RPMI1640 medium containing 10% FCS and 3 mg / ml glucose. The medium was changed twice a day.
  • a malaria parasite (P. berghei) using a rat as a host is used. This is because the test system using the malaria parasite with the human host cannot be performed, and the results observed in the test system using P. berghei are also recognized in human malaria. .
  • RT-PCR Sporozoites in salivary glands were intravenously administered to rats. After 24 hours, 36 hours, 48 hours, and 60 hours, the liver was perfused with PBS and then collected. Total RNA was extracted from each liver and subjected to RT-PCR analysis.
  • the primer sequences used in this study are shown in Table 1.
  • Recombinant GST fusion protein was prepared as follows. A genomic sequence containing the LISP2 repeat region was amplified by PCR, and the fragment was subcloned into a GEX vector (GE Healthcare, Piscataway, NJ). Recombinant GST fusion protein was produced in Escherichia coli DH5 ⁇ , purified on a glutathione-sepharose column, and used for rabbit immunization. After the anti-GST antibody was adsorbed by recombinant GST, the anti-LISP2 antibody was purified from rabbit antiserum using a Sepharose column to which the recombinant protein was bound. The primer sequences used for the preparation of the expression plasmid are shown in Table 1.
  • Promoter assay using PAC A promoter assay using GFP as a marker gene was performed based on a previously reported method using malaria centromere plasmid Pcen rep-GFP (Yuda et al., 2009). The 1.2-kb region upstream of LISP2 was used for the assay.
  • the malaria centromere plasmid Pcen rep-DL and the dual luciferase reporter assay system Were used. The constructs used for these assays are shown in FIG. The primers used for constructing the construct are shown in Table 1.
  • Plasmodium berghei (LISP2 :: mCherry parasites) expressing mCherry fusion LISP2 is a method used when preparing malaria parasites expressing GFP fusion proteins in the previous report (Yuda et al. al., 2009) was prepared using a transgenic malaria parasite strain (P. berghei 507 line) that always expresses GFP, using basically the same method.
  • the mCherry gene was fused to the terminal part of the LISP2 gene. 8).
  • Targeting of LISP2 Targeting in Plasmodium berghei is a method using a malaria parasite dihydrofolate reductase / thymidylate synthase (DHFR-TS) gene as a selection marker for pyrimethamine resistance (Yuda et al., 1999). And basically using the same method.
  • the primer sequences used for the preparation of the targeting construct are shown in Table 1.
  • LISP2 is a secreted protein-like molecule expressed in malaria parasites in the liver infection stage
  • a cDNA library was prepared from malaria parasites in the liver infection stage 31 hours after entry into the host (Ishino et al. , 2009).
  • This library was subjected to a random DNA sequence, and transcripts derived from rat hepatocytes were removed, and about 3000 P. berghei-derived sequence data were obtained (this data was obtained from PlasmoDB (http: // PlasmoDB. org)). These sequence data were compared with the P. berghe gene (PlasmoDB), and the genes were arranged in descending order of the corresponding number of sequence data.
  • PBANKA_100300 (SEQ ID NO: 21 (base sequence), SEQ ID NO: 22 (amino acid sequence)) is located second from the top (62 sequence data), and is the most transcribed in the liver infection stage. It was one of those that have been PBANKA_100300 is a protein encoding 2172 amino acids and has a long repeat region composed of about 1000 amino acids (FIG. 2 (A)).
  • This region consists of several repeat motifs with 12 copies of 56 amino acid motifs as a unit.
  • This amino acid sequence was analyzed using various structural analysis software such as signal P and TMpred programs. However, although it has an N-terminal signal sequence, other membrane-related motifs (for example, transmembrane domains and GPI anchor motifs) This protein was judged to be a secreted protein (FIG. 2 (A)).
  • Homologous molecules of the PBANKA_100300 gene were found in all malaria parasite species with known gene sequences. These homologous molecules were all secreted proteins with a long repeat region.
  • SNPs single nucleotide polymorphisms
  • liver-specific protein 1 (LISP1), a protein related to parasites in hepatocytes, was expressed specifically in the liver infection stage, and the liver infection stage (Ishino et al., 2009). LISP1 gene expression was not observed in sporozoites, but in early mitotic bodies 24 hours after infection. As shown below, PBANKA_100300 expression was observed in the malaria parasite at the liver infection stage, and its expression pattern was similar to that of LISP1. Therefore, this protein was named LISP2.
  • LISP1 liver-specific protein 1
  • LISP2 is specifically detected in the middle or late stage of liver infection.
  • a promoter assay was performed using the centromere plasmid Pcen rep-GFP containing GFP as a reporter gene.
  • GFP is located in the regulatory region 1.2-kbp upstream of the LISP2 coding region.
  • the GFP fluorescence signal was not observed in the blood stage of protozoan parasites or in the mosquito body (including zygotes, zygosacs, sporozoites in zygosac and sporozoites in salivary glands)
  • a GFP signal was observed 36 hours after infection (hpi) and was enhanced up to 48 hours (hpi) (FIG. 2 (B)). From this result, it was found that LISP2 is specifically expressed in the malaria parasite at the liver infection stage.
  • the expression profile of LISP2 in the liver infection stage was quantitatively examined by a promoter assay using a centromere plasmid Pcen rep-Luc containing firefly luciferase as a reporter gene (FIG. 2 (C)). Malaria parasites incorporating this plasmid were cultured with HepG2 cells. Cells were collected after 12 hours and luciferase assay was performed.
  • LISP2 is important for liver infection
  • a pyrimesamine resistance gene (PbDHFR-ts) was inserted between the 5 ′ end and 3 ′ end of the LISP2 gene (FIG. 5 (A)).
  • PbDHFR-ts pyrimesamine resistance gene
  • This construct was introduced into wild-type merozoites by electroporation. Plasmodium that incorporated this construct into the LISP2 locus was selected by pyrimethamine by homologous recombination. The protozoa selected in this manner were further separated from the wild-type protozoa by limiting dilution, and it was confirmed by Southern blotting (FIG.
  • the percentage of sporozoites showing gliding movement was normal even in the mutant type (over 90%).
  • sporozoites in the salivary glands were administered into the rat vein, and incubation period (from sporozoite administration to blood stage) by Giemsa staining (Period) was examined (FIG. 5B).
  • the growth rate in the blood was similar between the mutant type and the wild type (about 10 times a day), but the incubation period was about 1.5 days longer in the mutant type than in the wild type.
  • both wild type and mutant types were in the exponential growth phase, but the number of parasites in mice infected with wild type was about 30 times higher than that of the mutant type.
  • the mutant malaria parasite In the mutant malaria parasite, merozoite formation is impaired in the liver infection stage.
  • the phenotype of the mutant malaria parasite in the liver infection stage was examined in HepG2 cells (FIGS. 6 (A) to (C)). ).
  • the number of malaria parasites in the liver infection stage was examined every predetermined time (FIG. 6 (A)). Up to 48 hours after infection, no significant difference was observed between the wild type and the mutant type, and the nuclear morphology was the same. However, 60 hours after infection, the number of wild-type malaria parasites was reduced to 1/2 to 1/3 compared to the number of mutants. Furthermore, the cell nucleus infested with the mutant type showed irregular or irregular shape, and it seemed that fission was suppressed (FIG.
  • FIG. 6 (C) shows the proportion of malaria parasites in the liver infection stage having abnormal nuclei of cultured cells after 77 hours.
  • the proportion of malaria parasites with abnormal nuclei reached nearly half of the malaria parasites in the LISP2 mutant liver infection stage.
  • Such protozoa were hardly observed in the wild type.
  • mice administered with the wild type were cultured with HepG2 cells, and merozoite released in the culture solution was collected and then administered intravenously to mice.
  • the state of parasitemia in mice after administration was evaluated daily by Giemsa staining. In mice given the wild type, parasitemia was observed on the second day after administration, whereas in mice receiving the mutant type, parasitemia was observed only on days 5-6. . On day 5 after administration, the parasitemia of mice administered with the wild type was about 100 times as high as that administered with the mutant type (FIG. 6 (D)).
  • the number of hepatic merozoites produced at the mutant liver infection stage was about 1/100 that of the wild type. it was thought.
  • the number of malaria parasites at the stage of liver infection remaining in the cultured cells infected with the mutant was about four times that of the wild type infected cells.
  • the cell nucleus was atypical (FIG. 7). Similar to the above result (FIG. 6 (A)), it was considered that the infectivity was reduced in the mutant malaria parasite by suppressing the change to merozoite in the liver infection stage. .
  • LISP2 is transported to parasites
  • a transgenic malaria parasite (LISP2 :: mCherry) that expresses mCherry fusion LISP2 and a P berghei 507cl1 line that constitutively expresses GFP ( Janse et al., 2006).
  • the mCherry gene was introduced into the 3 ′ end of the LISP2 coding sequence by homologous recombination.
  • the LISP2 :: mCherry mutant was infected with rats and mosquitoes, and it was found that the LISP2 function was not affected even when mCherry was fused (Table 3, FIG. 9).
  • a weak mCherry signal was observed around the GFP signal (FIG. 8 (B)).
  • mCherry signal was observed around the PVM of the Plasmodium parasite at the whole liver infection stage. The increased vesicle-like signal at the mitotic stage indicates that LISP2 was produced in large quantities at this stage.
  • LISP2 is carried by secretory vesicles that surround LISP1.
  • LISP1 is a PVM protein specific to the Plasmodium parasite in the middle to late liver infection stages (Ishino et al., 2009).
  • LISP1 is produced by mitotic bodies, secreted from Plasmodium, and then transported to PVM.
  • the LISP2 In order to confirm whether both proteins are transported through the same route, the LISP2 :: mCherry mutant malaria parasite was stained with both mCherry and LISP1 (FIGS. 11A to 11C).
  • FIGS. 11A to 11C granules of secretory vesicles were detected in Plasmodium by anti-LISP1 antibody (FIG. 11 (A)).
  • LISP2 is transported to the host cytoplasm. Furthermore, LISP2 expression and subcellular localization were examined by immunocytochemistry using anti-LISP2 antibodies.
  • a recombinant GST fusion protein protein corresponding to amino acids 496 to 665 of LISP2 was used as an antigen, and this was administered to rabbits to obtain antibodies.
  • Specific antibodies against LISP2 were purified by affinity column from rabbit antiserum using recombinant GST fusion protein. Using this antibody, malaria parasites in the liver infection stage were immunostained 48 hours after infection. Strong fluorescence signals were observed for the infected HepG2 cells and the malaria parasite at the liver infection stage (FIG. 13 (A)).
  • This fluorescence signal was significantly different from the image of the fluorescence signal when LISP2 :: mCherry was used. Although no fluorescence was seen in uninfected HepG2 cells, the complex shape was revealed by antibodies in infected host cells. This indicates that LISP2 was transported to the host cell and distributed throughout the cytoplasm. LISP2 was also localized in the nucleus of all infected HepG2 cells, and its signal was stronger than that from the cytoplasm. Such localization of LISP2 indicates that LISP2 was actively transferred from the cytoplasm of the host cell into the nucleus (FIG. 13 (A)).
  • LISP2 The expression state of LISP2 at another time after infection was examined. At 12 hours after infection, no fluorescent signal was observed in the host cells. At 24 hours after infection, LISP2 was localized in the malaria parasite at the liver infection stage and in the parasite around the parasite (FIG. 13). At 36 hours post infection, LISP2 was found in host cells, but was still observed in many malaria parasites and parasites. It is important that the cytoplasm and nucleus show stained images for all cells 36 hours after infection. After 48 hours of infection, although there was variation among cells depending on the stage of development of the malaria parasite, the number of granules decreased and the signal of the host cell became stronger than the signal in the parasite.
  • LISP2 Liver slice samples were stained with LISP2 antibody and DAPI (4 ′, 6-diamidino-2-phenylindole) and observed with a confocal microscope. LISP2 was found in the cytoplasm and nucleus of infected hepatocytes, similar to the observation data using HepG2 cells (FIG. 14).
  • LISP2 mCherry malaria parasite
  • the mCherry fluorescence signal was observed only inside the parasitophorous vacuole (PV).
  • LISP2 was observed in host cells by immunofluorescence microscopy. This finding indicates that LISP2 is secreted into the host cell after undergoing C-terminal processing.
  • Western blot analysis was performed on wild-type malaria parasites and LISP2 :: mCherry malaria parasites (FIGS. 15A and 15B).
  • HepG2 cells infected with malaria parasites in the liver infection stage 48 hours after infection were dissolved in SDS sample buffer together with uninfected HepG2 cells, and Western blot analysis using an antibody against LISP2 repeat or an antibody against mCherry was performed.
  • anti-LISP2 repeat antibody When anti-LISP2 repeat antibody is used, two specific bands of 120-kDa (minor band) and 30-kDa (major band) are detected in the wild type, while minor bands are detected in the LISP2 :: mCherry type. It moved to 150-kDa (FIG. 15 (A)). The above band was not observed in uninfected HepG2 cells and HepG2 cells infected with LISP2 mutant malaria parasites.
  • the band size of 120 ⁇ ⁇ ⁇ kDa is only half of the molecular weight estimated from the amino acid sequence of LISP2 (excluding the signal sequence, it is predicted to be 240 kDa). This is because the N-terminal region is processed while LISP2 is transferred to PV, or because LISP2 has a highly biased amino acid composition in a large repetitive region, it has unusual movement characteristics in SDS-PAGE (Fig. 2) shows that In contrast, the 30-kDa major band observed in both wild-type and LISP2 :: mCherry types corresponds to the LISP2 product detected in host cells by the fluorescent antibody method using anti-LISP2 antibody. . In addition, Western blot analysis was performed at various times after sporozoite infection.
  • a weak 30-kDa band was observed 24 hours after infection. 36 hours after infection, two bands of 30-kDa and 120 kDa were detected. Since several bands were observed above 120 kDa, it was considered that the N-terminal part of this protein was processed. No full-length protein was detected by this processing. Since LISP2 is mainly observed in secretory vesicles during this time zone (Fig. 13 (B)), LISP2 modification is thought to be performed in secretory vesicles, and 120 kDa is expected 48 hours after infection. Since no larger bands were detected, LISP2 processing was considered complete. This finding is consistent with the result that almost no export vesicles are observed inside the malaria parasite by the fluorescent antibody method at the same time zone.
  • LISP2 knockout protozoa Analysis of the phenotype of the LISP2 knockout protozoa revealed that this protein plays an extremely important role in the growth of plasmodium in liver cells. Therefore, it is considered that a drug that inhibits the function of LISP2 can prevent the proliferation of Plasmodium parasite in hepatocytes.
  • LISP2 is produced by early mitotic bodies, it is interesting to note that the abnormal phenotype is seen in the knockout protozoa at the end of the liver stage where merozoites are formed. This time difference between the time of expression and the appearance of the phenotype suggests that the transported LISP2 first acts on the host cell and that the effect is subsequently exerted on the protozoa that grow in the parasite.
  • LISP2 may act on the nuclei of hepatocytes to inhibit the expression of genes involved in early immunity and help protozoa grow. Alternatively, it may inhibit host cell protein production by suppressing the expression of various host genes, thereby increasing the import of host cell nutrients into protozoa (Mikolajczak et al., 2007; Labaied et al., 2011).
  • the liver infection stage is the only stage where anti-infection immunity against malaria infection is established (Nussenzweig et al., 1967) and is one of the most important vaccine target stages.
  • CD8 + T cells play an important role in this immunity (Schofield et al., 1987; Weiss et al., 1988; Romero et al., 1989; Doolan and Hoffman, 2000; Morrot et al., 2005; Overstreet et al., 2008), proteins transported to the cytoplasm of host hepatocytes are presented to and activated by the host immune system, ultimately leading to removal of infected cells (Doolan et al. 1996; Bongfen et al., 2007; Chakravarty et al., 2007; Cockburn et al., 2011).
  • SNPs single nucleotide substitutions
  • Liver-derived cultured cells eg, HepG2
  • an appropriate culture plate eg, 8-well chamber plate
  • an appropriate medium eg, RPMI1640 medium containing 10% FCS and 3 mg / ml glucose.
  • sporozoites in the salivary glands are added to and infect the cultured cells, and a test substance for evaluating whether or not the action of LISP2 is suppressed is added to the culture solution.
  • the state of the malaria parasite in the cultured cells is observed 24 to 60 hours after infection with malaria parasite (preferably after 36 to 50 hours).
  • test substance suppresses the action of LISP2
  • merozoite formation is inhibited.
  • substances that inhibit malaria parasite infection can be tested by suppressing the action of LISP2.
  • such a substance can be used as a pharmaceutical against malaria parasite infection.
  • a sporozoite in the salivary gland is administered intravenously to a rodent (rat or mouse), and a test substance for evaluating whether or not the action of LISP2 is suppressed is administered.
  • Collect rodent liver 24 to 60 hours after malaria parasite administration (preferably 36 to 50 hours later), make a microscopic slice of the specimen, and use the microscope to find intracellular malaria parasite Observe the situation.
  • the test substance suppresses the action of LISP2
  • merozoite formation is inhibited.
  • substances that inhibit malaria parasite infection can be tested by suppressing the action of LISP2.
  • such a substance can be used as a pharmaceutical against malaria parasite infection.
  • LISP2-like protein a protein corresponding to LISP2 of these malaria parasites (hereinafter referred to as “LISP2-like protein”) exhibits the same function as the above experimental result in the liver infection stage. For this reason, a malaria vaccine can be obtained by providing a vaccine using LISP2 as an antigen. All or part of the malaria parasite LISP2 is used as an antigen.
  • LISP2-like protein antigen conventionally known methods (for example, various expression vectors (plasmids, cosmids, viruses, artificial chromosomes) and various cells (E. coli, Bacillus, insect cells, animal cell lines, etc.) are used. Protein expression systems, cell-free protein synthesis systems, chemical synthesis, etc.).
  • a LISP2-like protein antigen can be used alone or with an appropriate adjuvant (immuno-adjuvant) added.
  • the malaria vaccine can be inoculated by a method such as subcutaneous injection, intramuscular injection, nasal inoculation, or oral administration.
  • Immunity against LISP2 can be acquired by administering a malaria vaccine containing a LISP2-like protein.
  • LISP2 action can be suppressed through cell immunity against LISP2 or anti-LISP2 antibody, so that infection with Plasmodium can be inhibited.
  • ⁇ Test method> 1 Effect confirmation test of malaria vaccine using LISP2 as antigen Recombinant adenovirus was prepared using Clontech (Takara Bio) Adeno-X Adenoviral System 3 (CMV, green) (Code: 632267). The adenovirus vector was E1 E3 deficient. A region corresponding to LISP2 amino acids 1692-2030 (SEQ ID NO: 65) of Plasmodium Yoelii was incorporated into a cosmid vector for virus vector preparation and cloned in E. coli.
  • SEQ ID NO: 62 gtaactataacggtcATGGAATACCTCTCATCACTCTCC
  • SEQ ID NO: 63 attacctctttctccttaTCCTGAATAGATGATTTTTCCATTG
  • SEQ ID NO: 65 The amino acid sequence expressed at this time is shown in SEQ ID NO: 65.
  • mice (Balb / c 7 weeks female) were immunized by subcutaneous injection of 1 ⁇ 10 9 pfu per mouse (5 mice each). Two weeks after subcutaneous injection, 100 P. yoelii sporozoites were injected intravenously per mouse and challenged. Blood was collected from the second day after intravenous injection, and infection was evaluated by the erythrocyte infection rate.
  • ⁇ Test results> 1 The results of a test for confirming the effect of a malaria vaccine using LISP2 as an antigen are shown in FIG. When mice were immunized with a recombinant adenovirus containing LISP2 as an antigen, infection with malaria parasites was reduced by 50% compared to mice not administered with the vaccine. 2. The results of the LISP2 DNA vaccine effect confirmation test are shown in FIG. When LISP2 expression DNA was immunized to mice as a vaccine, infection with malaria parasites was suppressed by 70% compared to mice that did not receive the vaccine.
  • LISP2 is expressed in the liver infection stage by the malaria parasite and transported into the hepatocyte, so that the malaria parasite plays an extremely important role in the proliferation in the hepatocyte. Proved to be.
  • the compound which inhibits the infection of the malaria parasite can be searched by the test method for searching the compound which inhibits LISP2.
  • Such compounds can be provided as pharmaceuticals that inhibit malaria parasite infection.
  • a malaria vaccine using LISP2 as an antigen can be provided.

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Abstract

La présente invention traite le problème de l'identification d'une protéine essentielle pour le plasmodium au stade d'infection hépatique pour passer au stade suivant et concerne un procédé de test pour rechercher un composé faisant obstruction à cette protéine, et concerne un composé permettant d'entraver l'infection par le plasmodium en faisant obstruction à la protéine. L'objectif est atteint par un procédé de test d'une substance permettant d'entraver l'infection par le plasmodium, le procédé de test se caractérisant en ce qu'une évaluation est faite pour déterminer si la formation de mérozoïte au stade hépatique d'une infection par le plasmodium est entravée ou non en restreignant l'action de la protéine 2 spécifique de LS (LISP2) par laquelle le plasmodium est spécifiquement exprimé dans des cellules hôtes de la période médiane à la période plus tardive du stade hépatique.
PCT/JP2013/082139 2012-11-30 2013-11-29 Protéine de protozoaire associée à une infection par un plasmodium au stade hépatique WO2014084333A1 (fr)

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WO2007041216A2 (fr) * 2005-09-30 2007-04-12 Seattle Biomedical Research Institute Antigenes du plasmodium en phase hepatique
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