[go: up one dir, main page]

WO2023157880A1 - Vaccin contre le paludisme et méthode de prévention/traitement du paludisme - Google Patents

Vaccin contre le paludisme et méthode de prévention/traitement du paludisme Download PDF

Info

Publication number
WO2023157880A1
WO2023157880A1 PCT/JP2023/005232 JP2023005232W WO2023157880A1 WO 2023157880 A1 WO2023157880 A1 WO 2023157880A1 JP 2023005232 W JP2023005232 W JP 2023005232W WO 2023157880 A1 WO2023157880 A1 WO 2023157880A1
Authority
WO
WIPO (PCT)
Prior art keywords
csp
malaria
vaccine
amino acid
acid sequence
Prior art date
Application number
PCT/JP2023/005232
Other languages
English (en)
Japanese (ja)
Inventor
壽利 志田
栄人 吉田
光洋 伊從
浩明 水上
Original Assignee
国立大学法人金沢大学
学校法人自治医科大学
壽利 志田
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人金沢大学, 学校法人自治医科大学, 壽利 志田 filed Critical 国立大学法人金沢大学
Priority to JP2024501407A priority Critical patent/JP7649504B2/ja
Publication of WO2023157880A1 publication Critical patent/WO2023157880A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/23—Parvoviridae, e.g. feline panleukopenia virus
    • 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
    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • A61K39/285—Vaccinia virus or variola virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/863—Poxviral vectors, e.g. entomopoxvirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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 malaria vaccine composition containing a recombinant vaccinia virus as a prime and a recombinant adeno-associated virus as a boost, and a method for preventing and treating malaria using the composition.
  • This application claims priority to Japanese Application No. 2022-24221 and Japanese Application No. 2022-131078, which are incorporated herein by reference.
  • Malaria is one of the world's three major infectious diseases, infecting more than 200 million people each year and killing 500,000 people. There is a cure, but it is far from a system that can treat all infected people. Furthermore, there is concern about the emergence of drug-resistant malaria parasites. Therefore, the development of an infection-protecting vaccine is awaited. Moreover, as a method of protecting society from malaria infection, the development of a method of blocking transmission routes from humans to mosquitoes and to humans is also expected. GSK's RTS,S vaccine (using a fusion protein of malaria antigen PfCSP and HBsAg and special adjuvant AS01) is an infection-protective vaccine that has been tested up to Phase III clinical trials, but it has an efficacy rate of only 30%.
  • Non-Patent Document 2 a vaccinia virus that cannot proliferate in mammals
  • Non-Patent Document 4 An infection prevention method combining the LC16m8 ⁇ strain (Non-Patent Document 4) and other viral vectors, which induces much stronger immunity in mice than the non-proliferative vaccinia strains (DIs, MVA). (Patent Document 1).
  • RTS,S is designed to prevent malaria parasite infection, maturation, and proliferation in the liver, reducing the risk of liver-multiplied parasites re-entering the bloodstream and infecting red blood cells, leading to malaria.
  • Non-Patent Document 6 the efficacy rate of the phase III clinical trial of RTS,S is only 30%, the development of new strategies/concepts for malaria is required (see: Non-Patent Document 9).
  • Patent Document 2 describes "A malaria vaccine containing a recombinant baculovirus containing: (1) a gene encoding an amino acid sequence of a malaria antigen and a gene encoding an amino acid sequence of DAF, or an amino acid sequence of a malaria antigen (2) a promoter capable of expressing the gene; and further, the recombinant baculovirus is a recombinant virus other than the baculovirus containing a gene encoding the amino acid sequence of the malaria antigen, the amino acid of the malaria antigen
  • a malaria vaccine containing the recombinant baculovirus characterized by administering to a patient a plasmid DNA into which a gene encoding the sequence or a protein having the amino acid sequence of the malaria antigen is administered to the patient.” is disclosed.
  • Patent Document 2 does not disclose or suggest "the composition of the malaria vaccine composition of the present invention”.
  • Patent Document 3 discloses that "at least one gene encoding a protein that can be a constituent of virus particles and at least one A method for producing a transfer vector comprising a construct incorporating a dual promoter and a fusion gene, characterized in that the fusion gene containing two immunogenic foreign genes is ligated.
  • Patent Document 3 does not disclose or suggest "the composition of the malaria vaccine composition of the present invention”.
  • Patent Document 4 describes "a pharmaceutical composition containing a recombinant Autographa nuclear polyhedrosis virus (AcNPV) as an active ingredient, wherein the recombinant AcNPV is a dual promoter linked to at least a polyhedrin promoter and a CAG promoter.
  • Patent Document 4 does not disclose or suggest "the composition of the malaria vaccine composition of the present invention”.
  • Patent Document 5 describes "(a) a nucleotide sequence encoding a foreign envelope protein of a virus, (b) a first promoter operably linked to said envelope coding sequence, and (c) a nucleotide sequence encoding an antigenic protein. and (d) a second promoter operably linked to said antigen coding sequence.” However, Patent Document 5 does not disclose or suggest "the composition of the malaria vaccine composition of the present invention.”
  • Patent Document 6 describes "a kit comprising the following first and second components, wherein said first and second components optionally further comprise a pharmaceutically acceptable carrier. a) 1 and b) a recombinant modified vaccinia virus Ankara (MVA), wherein said MVA is a fusion A second component comprising a nucleic acid sequence encoding a protein, said fusion protein comprising ubiquitin or a functional portion thereof and one or more foreign proteins or functional portions thereof.”
  • MVA modified vaccinia virus Ankara
  • Patent Document 6 does not disclose or suggest "the composition of the malaria vaccine composition of the present invention”.
  • Patent document 7 WO 2005/010200 discloses "An immunogenic composition comprising a viral vector comprising a nucleic acid sequence encoding a C4bp domain or a variant or fragment thereof and a nucleic acid sequence encoding an antigen of interest.” However, Patent Document 7 does not disclose or suggest "the composition of the malaria vaccine composition of the present invention.
  • Non-Patent Document 7 discloses that "by the priming-boosting method, the malaria infection protective effect reached 40% by inoculating adenovirus vaccine (ChAd63) followed by vaccinia virus vaccine (MVA)". However, Non-Patent Document 7 does not disclose or suggest "the composition of the malaria vaccine composition of the present invention".
  • Non-Patent Document 8 states, "Protection against malaria infection by inoculating MVA-CS (recombinant virus incorporating CSP into vaccinia virus) after inoculation with FP9-CS (recombinant virus incorporating CSP into fowlpox virus) The effect has reached 90%.” However, the method described in Non-Patent Document 8 uses an adjuvant, and when the adjuvant is not used, the malaria infection-preventing effect is reduced to 35%. Furthermore, Non-Patent Document 8 does not disclose or suggest “the composition of the malaria vaccine composition of the present invention”.
  • An object of the present invention is to provide a malaria vaccine and a malaria prophylactic/therapeutic method that are superior to conventional malaria vaccines in not only infection-preventing effects but also malaria transmission-blocking effects.
  • the present inventors as a result of research to solve the above problems, using a recombinant vaccinia virus containing a gene encoding the amino acid sequence of the gene and s25 encoding the amino acid sequence of CSP for prime, the amino acid sequence of CSP
  • a recombinant adeno-associated virus containing the encoding gene and the gene encoding the amino acid sequence of s25 for boosting it was found that the malaria vaccine composition was excellent not only in the effect of protecting against infection but also in the effect of inhibiting malaria transmission. , completed the present invention.
  • a malaria vaccine composition comprising: (1) Recombinant vaccinia virus containing the gene encoding the amino acid sequence of CSP for prime and the gene encoding the amino acid sequence of s25 (2) The gene encoding the amino acid sequence of CSP for boost and the amino acid sequence of s25 A recombinant adeno-associated virus containing a gene encoding for 2.
  • a malaria vaccine comprising a recombinant vaccinia virus comprising a gene encoding an amino acid sequence of CSP and a gene encoding an amino acid sequence of s25, Furthermore, the recombinant vaccinia virus is administered to the human or animal prior to administration of the recombinant adeno-associated virus containing the gene encoding the amino acid sequence of CSP and the gene encoding the amino acid sequence of s25 to the human or animal. characterized by malaria vaccine. 3.
  • a malaria vaccine comprising a recombinant adeno-associated virus comprising a gene encoding an amino acid sequence of CSP and a gene encoding an amino acid sequence of s25, Furthermore, the recombinant adeno-associated virus is administered to a human or animal after administering a recombinant vaccinia virus containing a gene encoding the amino acid sequence of CSP and a gene encoding the amino acid sequence of s25 to the human or animal. characterized by malaria vaccine. 4. 2. The malaria vaccine composition according to the preceding item 1, wherein the vaccinia virus is the LC16 strain lacking the B5R gene. 5. 4. 4.
  • a malaria vaccine composition that confers lifelong immunity to a recipient comprising: (1) Recombinant vaccinia virus containing the gene encoding the amino acid sequence of CSP for prime and the gene encoding the amino acid sequence of s25 (2) The gene encoding the amino acid sequence of CSP for boost and the amino acid sequence of s25 A recombinant adeno-associated virus comprising a gene that encodes the recombinant vaccinia virus for prime and the recombinant adeno-associated virus for boost, inoculated twice or more to the inoculated person. , Malaria vaccine composition. 9. 2. The malaria vaccine composition according to the preceding item 1, wherein the malaria is malaria vivax. 10.
  • a method of preventing and/or treating malaria comprising the steps of: 1) the step of administering to humans or animals a recombinant vaccinia virus containing a gene encoding the amino acid sequence of CSP for prime and the gene encoding the amino acid sequence of s25; and 2) the amino acid sequence of CSP for boost administering to said human or said animal a recombinant adeno-associated virus comprising the encoding gene and the gene encoding the amino acid sequence of s25.
  • 11. 11 The method according to 10 above, wherein the vaccinia virus is the LC16 strain lacking the B5R gene. 12. 11. The method according to 10 above, which is for protection against malaria infection and inhibition of malaria transmission. 13. 11.
  • a method of conferring lifetime immunity on a vaccinated person comprising the steps of: 1) administering to the inoculator a recombinant vaccinia virus containing a gene encoding the amino acid sequence of CSP for prime and a gene encoding the amino acid sequence of s25; and 2) encoding the amino acid sequence of CSP for boost administering to the recipient a recombinant adeno-associated virus comprising the gene encoding the amino acid sequence of s25 and the gene for
  • the combination inoculation of the recombinant vaccinia virus for prime and the recombinant adeno-associated virus for boost is performed on the inoculated person two or more times, Method.
  • the vaccinia virus is the LC16 strain lacking the B5R gene. 16. 15. The method according to 14 above, which is for protection against malaria infection and inhibition of malaria transmission. 17. 15. The method according to 14 above, wherein the malaria is malaria vivax. 18. Use of the following (1) and (2) for the production of a malaria vaccine composition. (1) Recombinant vaccinia virus containing the gene encoding the amino acid sequence of CSP for prime and the gene encoding the amino acid sequence of s25 (2) The gene encoding the amino acid sequence of CSP for boost and the amino acid sequence of s25 18.
  • a recombinant adeno-associated virus containing a gene encoding Use of the following (1) and (2) for the manufacture of a malaria vaccine composition that imparts lifelong immunity to inoculated persons.
  • the malaria vaccine composition according to the preceding item 1 or 4 which is for inhibiting oxinate conversion.
  • the malaria vaccine of the present invention has one or more of the following effects as compared with conventional malaria vaccines.
  • High malaria infection prevention effect (2) High malaria infection prevention effect and malaria transmission prevention effect (3) Malaria infection prevention effect in a short period after boost (4) Malaria infection prevention effect that lasts for a long time (5) Twice Effect of inoculation (6) Effect of conferring lifetime immunity to vaccinated persons
  • Figures 1 to 6 are m8 ⁇ /AAV1-Pf (s25-CSP) falciparum malaria vaccine
  • Figures 7 to 11 are m8 ⁇ /AAV1-Pv (s25-CSP-S/P) vivax malaria vaccine
  • Figures 12 to 11 Figure 15 shows the description of the m8 ⁇ /AAV5-Pf(s25-CSP) Plasmodium falciparum vaccine.
  • FIG. 2 shows m8 ⁇ /AAV1-Pf(s25-CSP) vaccine constructs and protection against infection in a low-dose PfCSP/Pb sporozoite challenge test performed after inoculation of the vaccine.
  • HA is hemagglutinin
  • p7.5 is p7.5 promoter
  • pCMVie cytomegalovirus immediate early promoter
  • S is signal sequence
  • F FLAG epitope tag
  • G6S is GGGGGGS hinge sequence
  • G is vesicular stomatitis virus G protein transmembrane domain
  • WPRE stands for woodchuck hepatitis virus posttranscriptional regulatory element.
  • mice 40 days after the final immunization, mice were challenged with 1,000 PfCSP/Pb sporozoites, blood smears were prepared, and the number of days until 1% parasitemia was evaluated.
  • P-values ( **** P ⁇ 0.0001) were calculated using the Log-Rank (Mantel-Cox) test.
  • P-values **** P ⁇ 0.0001 were calculated using the Mann-Whitney U test.
  • mice 29 days (a, short term) and 102 days (b, long term) after the final immunization, mice were challenged with 2,500 PfCSP/Pb sporozoites, blood smears were prepared, and 1% parasitemia was evaluated. It was evaluated by the number of days until the day when the disease was reached. P-values ( **** P ⁇ 0.0001) were calculated using the Log-Rank (Mantel-Cox) test.
  • (c) IgG antibody titers against PfCSP (c) and Pfs25 (d) were measured using peripheral blood collected periodically until 210 days after the final immunization of the mice protected against infection in Figure 2 (a) above. It was measured.
  • FIG. 4 shows the propagation blocking effect of m8 ⁇ /AAV1-Pf(s25-CSP) vaccine.
  • the propagation-blocking effect was determined by inoculating Pb-Pfs25DR3 (Pfs25 gene-introduced murine malaria parasite) infected erythrocytes to mice 37 days (a, short term) and 236 days (b, long term) after the final immunization. After that, the mosquitoes were allowed to suck blood. 10 to 12 days after the blood feeding, the midgut of the mosquito was dissected and the number of oocysts was measured. Each data point represents the number of oocysts from a single blood-sucking mosquito and the horizontal line indicates the average number.
  • Transmission inhibitory activity indicates a reduction in the number of oocysts in mosquitoes sucked from the vaccinated mice compared to mosquitoes sucked from non-vaccinated mice.
  • Propagation blocking activity indicates the oocyst-negative rate in mosquitoes sucked from the vaccinated mice compared to mosquitoes sucked from non-vaccinated mice. Effect of m8 ⁇ /AAV1-Pf(s25-CSP) vaccine against multiple sporozoite challenge tests against infection and propagation.
  • the propagation-blocking effect was evaluated by inoculating Pb-Pfs25DR3-infected erythrocytes 276 days after the final immunization (176 days after the sporozoite challenge in FIG. 4(c) above) to inoculate the mosquitoes, and then sucking the blood. 10 to 12 days after the blood feeding, the midgut of the mosquito was dissected and the number of oocysts was measured. Each data point represents the number of oocysts from a single blood-sucking mosquito and the horizontal line indicates the average number.
  • P-values for TRA **** P ⁇ 0.0001) were calculated using the Mann-Whitney U test.
  • P-values for TRA **** P ⁇ 0.0001 were calculated using Fisher's exact test.
  • IgG antibody titers against PfCSP and Pfs25 were measured using peripheral blood collected from the vaccine-immunized mice in FIG. 4(a)-(d) before each challenge and before the propagation blocking experiment.
  • P-values ( *** P ⁇ 0.001, ** P ⁇ 0.01, * P ⁇ 0.05) were one-way using Dunnett's multiple comparison test to compare each data point with data on day 25 post-last immunization. Calculated using a layout analysis of variance.
  • Figure 2 shows antigen gene expression on mammalian cell surface by m8 ⁇ malaria vaccine.
  • the transduced cells were fixed with methanol and then stained with Alexa Fluor 488-labeled 2A10 (anti-PfCSP antibody) and Alexa Fluor 594-labeled 4B7 (anti-Pfs25 antibody) to detect fluorescence signals. Detected by fluorescence microscopy.
  • FIG. 1 illustrates the experimental design of FIGS. 1 to 5 above.
  • the boost time point with AAV1-Pf(s25-CSP) was set at day 0. Serum for titer determination was performed the day before AAV1-Pf(s25-CSP) immunization, sporozoite challenge, or infected red blood cell infection, or periodically after infection control. Ts (tail scarification immunization) means inoculation with a bifurcated needle in the mouse tail, im for intramuscular inoculation, iv for intravenous administration and ip for intraperitoneal administration. The structure of Pv(s25-CSP-S/P) is shown (see SEQ ID NOS: 8, 9). (a) Pv(s25-CSP(S/P) artificial synthetic gene sequence.
  • the Pv(s25-CSP-S/P) gene is codon-optimized for efficient expression in mammalian cells.
  • b Recombinant m8 ⁇ construct with Pv(s25-CSP-S/P) gene inserted under the 7.5 promoter
  • c Recombinant AAV construct
  • the Pv(s25-CSP-S/P) gene was fused with the transmembrane domain (G) of the VSV-G protein, and WPRE was inserted downstream of it.
  • S gp64 signal sequence
  • pCMV CMV promoter
  • F flag epitope tag
  • G 6 S GGGGGGS hinge sequence
  • G VSV-GTM
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element.
  • CSP-PNG (SEQ ID NO: 11: CSP-PNG from Plasmodiumvivax) was derived from Plasmodium vivax .
  • Western blotting and indirect immunofluorescence of the m8 ⁇ /AAV1-Pv(s25-CSP-S/P) vaccine are shown.
  • (a) (b) Confirmation of the expression of PvCSP-Sal (a) and PvCSP-PNG (b) in mammalian cells transfected with m8 ⁇ /AAV1-Pv (s25-CSP-S/P) using Western blotting. .
  • Anti-PvCSP-Sal IgG antibody titer (a) and Pvs25 IgG antibody titer (b) after immunization with m8 ⁇ /AAV1-Pv(s25-CSP-S/P) vaccine are shown.
  • Serum was collected from each immunized mouse before the final immunization and before the infection experiment and measured by ELISA. Significant differences were assessed by the Kruskal Wallis test. **** p ⁇ 0.0001
  • the results of the infection protective effect of the m8 ⁇ /AAV1-Pv (s25-CSP-S/P) vaccine are shown.
  • DMFA was performed using serum 28 days after the final immunization. Each dot indicates the number of oocysts in the midgut of one mosquito sucked by DMFA, and the horizontal line indicates the average number. It also shows TRA and TBA with respect to serum dilution ratio.
  • AAV5-Pf(s25-CSP) vaccine constructs and Western blotting results of the vaccine are shown. (a) Shows the construction of AAV5-Pf(s25-CSP) expressing the Pfs25-PfCSP fusion protein [Pf(s25-CSP)].
  • pCMVie stands for cytomegalovirus immediate early promoter, S for signal sequence, F for FLAG epitope tag, H for GGGGGGS hinge sequence, G for vesicular stomatitis virus G protein transmembrane region, and WPRE for woodchuck hepatitis virus posttranscriptional regulatory element.
  • m8 ⁇ represents the same sample as m8 ⁇ -Pf(P7.5-s25-CSP)-HA (Fig. 1b).
  • the results of antibody titer of m8 ⁇ /AAV5-Pf(s25-CSP) vaccine are shown.
  • Prime is m8 ⁇ -Pf(P7.5-s25-CSP)-HA vaccine antibody titer 6 weeks after primary immunization
  • Boost is AAV5-Pf(s25-CSP) vaccine booster 4 weeks after anti-PfCSP antibody titer (a ), showing the anti-Pfs25 antibody titer (b).
  • the results of the infection protective effect of the m8 ⁇ /AAV5-Pf(s25-CSP) vaccine are shown.
  • mice 40 days after the final immunization, mice were challenged with 2,500 PfCSP-Tc/Pb sporozoites, blood smears were prepared, and the number of days until 1% parasitemia was evaluated. P-values ( **** P ⁇ 0.0001) were calculated using the Log-Rank (Mantel-Cox) test. The results of the propagation blocking effect of the m8 ⁇ /AAV5-Pf(s25-CSP) vaccine are shown.
  • DMFA was performed using serum 28 days after the final immunization. Each dot indicates the number of oocysts in the midgut of one mosquito sucked by DMFA, and the horizontal line indicates the average number. It also shows TRA and TBA with respect to serum dilution ratio.
  • Figure 2 shows the monkey blood sampling schedule. The results of 14 items of monkey blood test data are shown. (a) shows the results of anti-PfCSP antibody titers in monkeys. (b) Anti-Pfs25 antibody titer results at 12w. Figure 2 shows the results of the sporozoite neutralization test. The results of the propagation blocking test are shown. The results of the oxinate conversion inhibition test are shown.
  • the "malaria vaccine” of the present invention targets the following.
  • a prime recombinant vaccinia virus for a malaria vaccine comprising a gene encoding a CSP amino acid sequence and a gene encoding an s25 amino acid sequence, or a prime malaria vaccine comprising the virus.
  • a recombinant adeno-associated virus for boosting a malaria vaccine comprising a gene encoding an amino acid sequence of CSP and a gene encoding an amino acid sequence of s25, or a malaria vaccine for boost comprising the virus.
  • the "malaria vaccine composition” of the present invention includes: (1) Recombinant vaccinia virus containing the gene encoding the amino acid sequence of CSP for prime and the gene encoding the amino acid sequence of s25 (2) The gene encoding the amino acid sequence of CSP for boost and the amino acid sequence of s25
  • the malaria vaccine and malaria vaccine composition of the present invention are described below.
  • the term "gene (DNA molecule)" is intended to include not only double-stranded DNA, but also single-stranded DNA such as the sense strand and antisense strand that constitute it. not to be Therefore, unless otherwise specified, the gene (polynucleotide) encoding the amino acid sequence of the malaria antigen of the present invention includes double-stranded DNA containing genomic DNA, single-stranded DNA (sense strand) containing cDNA, and the sense strand. Both single-stranded DNA (antisense strand) and synthetic DNA having a sequence complementary to , and fragments thereof are included.
  • the "protein having the amino acid sequence of CSP" used in the present invention has the same amino acid sequence or the same function as the amino acid sequence of CSP presented (presentable) by adeno-associated virus or vaccinia virus and has 90% homology. Above, 95% or more, 98% or more, 99% or more, there is no particular limitation. Furthermore, the amino acid sequence may be modified by a method known per se.
  • the "protein having the s25 amino acid sequence” used in the present invention has the same amino acid sequence or the same function as the s25 amino acid sequence presented (presentable) by adeno-associated virus or vaccinia virus and has 90% homology. Above, 95% or more, 98% or more, 99% or more, there is no particular limitation. Furthermore, the amino acid sequence may be modified by a method known per se.
  • CSP CercumsporozoiteProtein
  • CSP CercumsporozoiteProtein
  • sporozoiteProtein is the major membrane protein of sporozoite-stage protozoa that invade humans from mosquitoes. It has a molecular weight of about 50 kDa and has 10 to 40 repeats of 4 to 8 amino acid sequences in the central region with high antigenicity. CSPs are thought to play an important role in entry into hepatocytes, as they showed extremely high inhibition of entry in experiments in which sporozoites were mixed with monoclonal antibodies against repeat sequences and sprinkled on hepatocytes. Among many malaria antigens, CSP is particularly expected as a vaccine candidate antigen and has been studied for many years.
  • RTS,S which is the most developed vaccine for falciparum malaria
  • PfCSP Plasmodium falciparum CSP antigen
  • SEQ ID NO: 1 Plasmodium falciparum
  • Plasmodium falciparum a nucleotide sequence derived from Plasmodium falciparum, or a nucleotide sequence encoding an amino acid region of Leu19-Val377 lacking a signal sequence and a GPI anchor.
  • Plasmodium falciparum PvCSP gene is codon-optimized for expression in mammalian cells (SEQ ID NO: 2: sPfCSP2 derived from Plasmodium falciparum), but other Plasmodium falciparum CSP genes (e.g., Plasmodium falciparum PvCSP gene) can also be used. be.
  • sPfCSP2 derived from Plasmodium falciparum
  • Plasmodium falciparum PvCSP gene can also be used.
  • multiple types of CSP genes corresponding to multiple strains of malaria parasites may be linked.
  • CSPs for use in the present invention also include: (1) a protected derivative, glycosylated derivative, acylated derivative, or acetylated derivative of CSP; (2) A protein having 90% or more, 95% or more, 98% or more, or 99% or more homology with CSP and having substantially the same action as CSP. (3) In CSP, 100 to 10, 50 to 30, 40 to 20, 10 to 5, 5 to 1 amino acids are substituted, deleted, inserted and / or added, and CSP and substantial A protein with a similar effect.
  • CSP genes used in the present invention include the following. (1) A gene encoding a polypeptide consisting of the amino acid sequence of CSP.
  • s25 (sexual-stage 25-kDa protein) is a multi-step target antigen protein for the sexual-stage antigen of the okinate stage of Plasmodium falciparum ⁇ see: Mizutani M. et al. "Baculovirus-vectored multistage Plasmodium vivax vaccine induces both protective and transmission-blocking immunity against transgenicrodent malaria parasites.” Infection and Immunity (2014): IAI-02040. ⁇ .
  • s25 used in the present invention is preferably sPfs25 (SEQ ID NO: 3: sPfs25 derived from Plasmodium falciparum, amino acid sequence: SEQ ID NO: 4: sPfs25 derived from Plasmodium falciparum), but other Plasmodium s25 genes (eg, Plasmodium vivax Pvs25 gene) can also be used.
  • s25 as used in the present invention also includes: (1) a protected derivative, glycosylated derivative, acylated derivative, or acetylated derivative of s25; (2) A protein having 90% or more, 95% or more, 98% or more, or 99% or more homology with s25 and having substantially the same action as s25. (3) in s25, 100 to 10, 50 to 30, 40 to 20, 10 to 5, 5 to 1 amino acids are substituted, deleted, inserted and / or added, and s25 and substantial A protein with a similar effect.
  • the s25 gene used in the present invention includes the following. (1) A gene encoding a polypeptide consisting of the s25 amino acid sequence.
  • the mutated protein may be naturally occurring or may be obtained by introducing mutation based on a naturally occurring gene.
  • Means for introducing mutations are known per se, and for example, site-directed mutagenesis, homologous recombination, primer extension, or polymerase chain reaction (hereinafter abbreviated as PCR) can be used alone or in combination as appropriate. can.
  • PCR polymerase chain reaction
  • a method described in a book (Sambrook et al., "Molecular Cloning, Laboratory Manual 2nd Edition", 1989, Cold Spring Harbor Laboratory; Edited by Masami Muramatsu, "Lab Manual Genetic Engineering", 1988, Maruzen Co., Ltd.) or by modifying those methods, Ulmer's technology (Ulmer, KM, "Science", 1983, Vol.
  • peptides from the viewpoint that the introduction of mutations does not change the basic properties of the peptide (physical properties, functions, physiological activity or immunological activity, etc.), for example, homologous amino acids (polar amino acids, non-polar amino acids, Reciprocal substitutions between hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids, etc.) are readily envisioned.
  • homologous amino acids polar amino acids, non-polar amino acids, Reciprocal substitutions between hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids, etc.
  • the CSP gene and the s25 gene can preferably be introduced by linking via a hinge sequence.
  • a hinge sequence is not particularly limited as long as it does not affect the expression of CSP and s25 on the cell surface, but an amino acid sequence consisting of SEQ ID NO: 6 (Hinge sequence for Pfs25 and PfCSP) is preferred, Hinge sequences for Pfs25 and PfCSP) are preferred. Therefore, in the present invention, it is preferable to introduce a hinge sequence (eg, SEQ ID NO: 6) between s25 and CSP. Furthermore, in the present invention, it is preferable to fuse in the order of [N-terminus-s25-hinge-CSP-VSG membrane anchor] in consideration of the retention of the s25 conformation.
  • Adeno-associated virus is a virus that is incapable of self-replication, is classified into a genus of Parvoviridae, and consists of single-stranded DNA.
  • Adeno-associated viruses (vectors) used in the present invention include, for example, human adeno-associated virus type 1 (AAV1), AAV8, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9 ⁇ see: Nieto et al. AAV Vectors Vaccines against Infectious FrontImmunol.
  • Adeno-associated virus type 1 which expresses high and long term
  • AAV5 vector which is suitable for intradermal administration, are preferred. Since AAV vectors have low pathogenicity, they are highly safe and pose few problems for practical use. Malaria vaccine development requires long-term immunity persistence as an essential function. AAV is a non-pathogenic virus that can express foreign genes for a long period of time.
  • the gene encoding the amino acid sequence of CSP, the gene encoding the amino acid sequence of s25, and, if necessary, the gene encoding the amino acid sequence of a protein that can be a component of virus particles are used. It is introduced into a publicly known (commercially available) adeno-associated virus vector-producing plasmid, and the introduced plasmid is transduced into human embryonic kidney-derived cells by a known method and cultured. Next, a recombinant adeno-associated virus expressing the gene encoding the amino acid sequence of CSP and the gene encoding the amino acid sequence of s25 is obtained from the culture supernatant or cultured cells.
  • the vaccinia virus (vector) used in the present invention is superior in that it induces a suitable immune response in humans in addition to its safety.
  • examples include LC16 strain, LC16m8 strain, LC16mO strain, DIs strain, MVA strain, etc.
  • one or more nucleotides have been substituted, added, inserted and/or deleted in their B5R gene. It is preferred to use a vaccinia virus vector that does not produce a B5R gene product with normal function.
  • Vaccinia virus vectors that do not produce B5R gene products with such normal functions include the B5R gene-deleted LC16 strain, m8 ⁇ B5R (LC16m8 ⁇ strain, LC16m8 ⁇ 2 strain), mO ⁇ B5R (LC16mO ⁇ strain), m8proB5RdTM, mOproB5RdTM, and the like.
  • the LC16mO strain includes not only the LC16mO strain itself, but also the LC16m8 ⁇ 2 strain, the LC16m8 strain, the LC16mO ⁇ strain, the LC16m8 ⁇ strain, and the LC16m8 ⁇ VNC110 strain (reference: Vaccines (Basel). 2014 Dec. ;2(4): 755-771.), preferably the LC16m8 ⁇ 2 strain.
  • Vaccinia virus LC16m8 is a live smallpox vaccine that has been inoculated to more than 100,000 infants in Japan and is approved by the Ministry of Health and Welfare.
  • LC16m8 ⁇ was developed by the present inventor Dr. Shida and co-researcher Dr.
  • a method for producing a recombinant vaccinia virus containing a gene encoding an amino acid sequence of a malaria antigen used in the present invention comprises preparing a plasmid (transfer vector) in which a gene encoding a polypeptide of a malaria antigen to be introduced is linked, and producing the plasmid. can be made by introducing into cells infected with vaccinia virus and allowing homologous recombination to occur within the cells.
  • a gene fragment encoding a malaria antigen polypeptide to be introduced is digested with a restriction enzyme and directly linked to the vaccinia virus genome digested with the same enzyme, and the recombinant vaccinia virus genome is It can also be produced by introducing into virus-infected cells.
  • Plasmids that can be used in the preparation of vaccinia virus vectors include, for example, pSFJ1-10, pSFJ2-16, pMM4, pGS20, pSC11, pMJ601, p2001, pBCB01-3,06, pTKgpt-F1-3s, pTM1, pTM3, pPR34, pPR35, pgpt-ATA18-2, pHES1-3, pJW322, pVR1, pCA, pBHAR and the like.
  • the introduction region of the gene encoding the polypeptide of the malaria antigen is a gene region that is not essential for the life cycle of vaccinia virus, for example, hemagglutinin (HA) gene, thymidine kinase (TK) gene, B5R gene B6R gene), regions such as the F fragment.
  • HA hemagglutinin
  • TK thymidine kinase
  • B5R gene B6R gene regions such as the F fragment.
  • the gene to be introduced is desirably one that changes viral traits by substitution, addition, insertion and/or deletion of one or more nucleotides and facilitates the selection of recombinants.
  • Cells to be infected with vaccinia virus vectors include RK13 cells, Vero cells, HeLa cells, CV1 cells, COS cells, BHK cells, primary rabbit kidney cells, BSC-1 cells, HTK-143 cells, Hep2 cells, MDCK cells, etc. Examples can be given (see: International Publication No. 2012/053646).
  • an appropriate promoter can be operably linked upstream of the gene encoding the peptide.
  • Such promoters are not particularly limited, but for example, 7.5 promoter (sometimes referred to as P7.5), EL promoter (sometimes referred to as EL), AT1 promoter, SFJ1-10 promoter (sometimes referred to as SFJ ), SFJ2-16 promoter, improved 7.5 promoter (7.5E), mPH5 promoter, 11K promoter, T7.10 promoter, CPX promoter, HF promoter, H6 promoter, T7 hybrid promoter and the like.
  • 7.5 promoter sometimes referred to as P7.5
  • EL promoter sometimes referred to as EL
  • AT1 promoter AT1 promoter
  • SFJ1-10 promoter sometimes referred to as SFJ
  • SFJ2-16 promoter improved 7.5 promoter (7.5E)
  • mPH5 promoter 11K promoter
  • T7.10 promoter CPX promoter
  • CPX promoter HF promoter
  • H6 promoter H6 promoter
  • T7 hybrid promoter and the like.
  • the recombinant virus of the present invention may contain a gene encoding an amino acid of a protein that can serve as a membrane anchor component for the virus used in the present invention to express the vaccine antigen protein on the surface of infected cells.
  • Genes encoding amino acids of proteins that can be membrane anchor constituents for expressing vaccine antigens in infected cells in the virus used in the present invention include, for example, gp64 protein (GenBank Accession No. L22858), vesicular stomatitis virus sugar. glycoprotein G (VSV-G: GenBank Accession No.
  • herpes simplex virus glycoprotein (KOS: GenBank Accession No. K01760), human immunodeficiency virus type 1 gp120 (GenBank Accession No. U47783), human respiratory system Genes such as syncytial virus membrane glycoprotein (GenBank Accession No. M86651), influenza A virus hemagglutinin protein (GenBank Accession No. U38242) or parts thereof, or genes such as viral envelope proteins related to baculovirus, or A part of the gene can be exemplified, and the transmembrane region of VSV-G (VSV-G TM) (SEQ ID NO: 5: Vesicular stomatitis virus) is preferable.
  • VSV-G TM VSV-G TM
  • the combination and order of the amino acid sequences encoded by the genes contained in the recombinant adeno-associated virus containing the gene encoding the amino acid sequence of CSP and the gene encoding the amino acid sequence of s25 of the present invention are not particularly limited, but preferably N s25, hinge sequence, CSP and transmembrane region of vesicular stomatitis virus glycoprotein G protein (VSV-G TM) in order from the terminal side, more preferably s25, hinge sequence, CSP and VSV in order from the N-terminal side - Including GTM.
  • VSV-G TM vesicular stomatitis virus glycoprotein G protein
  • a priming-boosting method can be exemplified as a method of using the recombinant virus of the present invention as a vaccine (prophylactic/therapeutic method).
  • a recombinant vaccine of the following combination is administered intramuscularly to a recipient ⁇ human or animal (especially vertebrates including mammals, e.g., companion animals, horses, cattle, dogs, cats, rabbits, horse races) ⁇ . , nasally, or through the respiratory tract, etc., once or multiple times.
  • the method of administration to humans can employ already known administration methods, and the following can be exemplified, but are not particularly limited. Based on GSK's RTS,S/AS01 vaccine Phase 3 clinical trial (N Engl JMed 367:2284-95, 2012).
  • 1st immunization Subcutaneous inoculation to target human Recombinant vaccinia virus containing the gene encoding the amino acid sequence of CSP and the gene encoding the amino acid sequence of s25 (1 ⁇ 10 5 to 1 ⁇ 10 10 pfu) Second immunization: intramuscular injection 6 weeks later Recombinant adeno-associated virus (1 ⁇ 10 9 to 1 ⁇ 10 14 vg) containing the gene encoding the amino acid sequence of CSP and the gene encoding the amino acid sequence of s25
  • the malaria vaccine composition of the present invention is effective with only 2 inoculations in principle.
  • the combination of the malaria vaccine or malaria vaccine composition of the present invention can be exemplified below, but is not particularly limited.
  • the gene encoding the amino acid sequence of CSP is preferably PfCSP or PvCSP
  • the gene encoding the amino acid sequence of s25 is preferably Pfs25 or Pvs25
  • the recombinant vaccinia virus is m8 ⁇ B5R (LC16m8 ⁇ and LC16m8 ⁇ 2).
  • the recombinant adeno-associated virus is AAV1 or AAV5.
  • the malaria vaccine or malaria vaccine composition of the present invention has been confirmed to be suitable for the following uses according to the following examples.
  • the vaccinia virus containing the gene encoding the amino acid sequence of CSP and the gene encoding the amino acid sequence of s25 is used as prime, and the adeno-associated virus containing the gene encoding the amino acid sequence of CSP and the gene encoding the amino acid sequence of s25 is used as boost. It has an excellent infection-preventing effect and an excellent malaria transmission-inhibiting effect by using it.
  • the malaria vaccine composition of the present invention showed a high protective effect without using an adjuvant, as shown in the results of the examples below. Therefore, an adjuvant need not be an essential component, but may be included.
  • the malaria vaccine composition (including malaria vaccine) of the present invention is a recombinant virus suspension suspended in PBS (phosphate buffered saline) or physiological saline, etc., locally (for example, in lung tissue, liver). Intravenously, intramuscularly, and intracerebrally, etc.), inhaled transcutaneously, nasally, through the respiratory tract, or administered intravascularly (eg, intraarterially, intravenously, and intraportally).
  • the present invention is also directed to methods of preventing and treating malaria. More specifically, by the prime / boost immunization method, the recombinant vaccinia virus containing the gene encoding the amino acid sequence of CSP and the gene encoding the amino acid sequence of s25, the gene encoding the amino acid sequence of CSP and the amino acid sequence of s25 Before administering a recombinant adeno-associated virus containing the encoding gene to subjects (malaria patients (including subjects for prevention before the onset of malaria)) once or multiple times intramuscularly, nasally, or through the respiratory tract In the second, the patient is administered intramuscularly, intranasally, or intratracheally in one or more doses.
  • the CSP protein and s25 protein displayed on the surface of cells infected with recombinant adeno-associated virus and recombinant vaccinia virus function as component vaccines. Furthermore, the CSP protein and the s25 protein are produced in mammalian cells and function as prophylactic or therapeutic agents for malaria infections due to their immunopotentiating action.
  • the sporozoite-stage malaria parasites injected from mosquitoes reach the liver through the bloodstream, invade and multiply.
  • Humoral immune responses are effective against sporozoite stage parasites before they reach the liver, and cell-mediated immune responses are necessary for liver stage parasites after they invade the liver.
  • Gametocytes called gametocytes are born at a certain frequency from the erythrocyte stage, and when the mosquito sucks blood from these gametocytes, the mosquito stage begins anew.
  • the LC16m8 ⁇ /AAV vaccine of the present invention targets the sporozoite-liver stage and the mosquito stage, and effectively induces a hybrid response of both humoral and cellular immunity against sporozoite/liver stage surface antigen CSP and mosquito stage surface antigen s25. It is designed to be inducible, and is a bivalent vaccine that exerts an "infection-preventing effect” and a "propagation-blocking effect" according to the results of the following examples.
  • the malaria vaccine composition of the present invention was used by the inoculated person to inoculate a combination of the recombinant vaccinia virus for prime and the recombinant adeno-associated virus for boost two or more times (twice, 3 times). 3 or more times, 4 times or more, 4 times or more, 5 times or 5 times or more) can confer lifetime immunity to the inoculated person. More particularly, the vaccinated person can maintain the immune status for 1, 5, 10, 15, 20, 30 or 50 years.
  • the vaccine target antigens are the sporozoite stage PvCSP antigen as a "protective vaccine” and the mosquito okinate stage Pvs25 antigen as a "transmission blocking vaccine” to prevent transmission of mosquitoes from an infected person.
  • Plasmodium falciparum was targeted. Details are as follows. ⁇ Materials and methods> (Construction of pVR1-Pf(P7.5-s25-CSP)-HA-VV plasmid)
  • the transmembrane region of the vsv-g gene which is a cell membrane expression anchor, is arranged downstream of the pfcsp gene in pENTR-CAG-sPfCSP2-G2-sWPRE. (T 5 NT) sequences are present.
  • the vsv-g gene having Pst I and HindIII sites at both ends and the "TTCTTCT” sequence was artificially synthesized. was cleaved with PstI/HindIII and replaced with the vsv-g gene of pENTR-CAG-sPfCSP2-G2-sWPRE cleaved with PstI/HindIII to create pENTR-CAG-sPfCSP2-G2-sWPRE-Nhe.
  • pUC57-Simple-sPfCSP2-VV having Age I and Mun I sites at both ends was used. made. That is, pUC57-Simple-VV-WPRE was prepared by artificially synthesizing the wpre sequence having Age I and MunI sites at both ends and immediately preceding the Nco I site, and then pENTR-CAG-sPfCSP2- cleaved with Nco I.
  • the open reading frame of G2-sWPRE-Nhe and the wpre sequence were placed at the NcoI site of pUC57-Simple-VV-WPRE, and this was named pUC57-Simple-sPfCSP2-VV.
  • the m8 ⁇ transfer vector pVR1 was cleaved with XmaI/Eco RI, and the open reading frame and wpre sequence of pUC57-Simple-sPfCSP2-VV cleaved at the Age I/Mun I site were placed at the same site, and this was pVR1-Pf. (P7.5-CSP)-HA-VV.
  • the vsv-g gene having Xma I/Fse I sites at both ends was amplified by PCR.
  • pVSV-G-F1 CACCCGGGCGTTCGAACATCCTCACATTCAAGAC
  • pVSV-G-R7 TTTTGGCCGGCCTTACTTTCCAAGTCGGTTCATC
  • pVR1-Pf(P7.5-CSP)-WPRE(-)-HA-VV was constructed without the wpre sequence.
  • pVR1-Pf(P7.5-CSP)-WPRE(-)-HA-VV is m8 ⁇ -Pf(P7.5-CSP)-G-WPRE(-)-HA (hereafter m8 ⁇ -Pf(P7.5- CSP)-HA) was used as a transfer vector for virus production.
  • a DNA fragment obtained by cleaving pENTR-CAG-sPfs25-sPfCSP-G2-sWPRE with EcoRI/XmaI was inserted into the EcoRI/XmaI site of pVR1-Pf(P7.5-CSP)-WPRE(-)-HA-VV. Inserted to construct pVR1-Pf(P7.5-s25-CSP)-HA-VV. pVR1-Pf(P7.5-s25-CSP)-HA-VV was used as a transfer vector for generating m8 ⁇ -Pf(P7.5-s25-CSP)-HA virus.
  • the PVDF membrane on which the non-reduced sample was electrophoresed and transferred was incubated with the anti-Pfs25 antibody 4B7mAb diluted 1:10,000 with 5% skim milk. After washing with PBS-T, blots were probed with goat anti-mouse secondary antibody labeled with IRDye 800 (Rockland Immunochemicals) diluted 1:20,000 in 5% skimmed milk. The membrane was visualized using an Odyssey infrared imager (LI-COR). Molecular weight prediction was performed using the ExPASy server.
  • IRDye 800 Rockland Immunochemicals
  • NGS Normal Goat Serum
  • 4B7 labeled with Alexa Fluor 594 and 2A10 labeled with Alexa Fluor 488 were diluted 100-fold and 200-fold with 10% NGS/PBS, respectively, and added to each well and allowed to stand for 1 hour while shielding from light.
  • the cells were washed five times with PBS-T for 2 minutes and mounted with Vectashield containing 4,6'-diamidino2-phenylindole (DAPI; Vector Laboratories).
  • DAPI 4,6'-diamidino2-phenylindole
  • the slides were visualized with an LSM710 inverted laser scanning microscope (Carl Zeiss).
  • mice and protozoa used The mammals and protozoa used were as follows. Mice: 6-week-old BALB/c female mice and 6-week-old ICR female mice were obtained from Japan SLC, Inc. and used after 1 week.
  • Mouse malaria parasite salivary glands of Anopheles stephensi SDA500 strain infected with transgenic murine malaria ⁇ PfCSP-expressing P. berghei (Pb) PfCSP-Tc/Pb ⁇ (described in Insect Mol. Biol. 2013, 22:41) were dissected and transgenic murine malaria parasite sporozoites were isolated.
  • RPMI 1640 Medium Gibco, Life Technologies
  • homogenizing After adding RPMI 1640 Medium (Gibco, Life Technologies) and homogenizing, centrifugation was performed to collect the supernatant, and the number of sporozoites was counted using an optical microscope. They were prepared to have 1,000, 2,500 or 10,000 sporozoites/animal (body).
  • mice (7-week-old females) purchased from SLC were used in groups of 10.
  • m8 ⁇ vaccine m8 ⁇ -Pf(P7.5-CSP)-HA and m8 ⁇ -Pf(P7.5-s25-CSP)-HA at 1 ⁇ 10 7 pfu/100 ⁇ L/mouse
  • AAV1 vaccine AAV-PfCSP and AAV -Pf(s25-CSP)
  • AdHu5 vaccine AdHu5-s25-CSP
  • the m8 ⁇ vaccine was immunized at the base of the tail of mice by vaccination with a bifurcated needle (ts), and the AAV1 vaccine or AdHu5 vaccine was immunized by left thigh muscle inoculation (im) using an insulin syringe with a 29G needle.
  • ts bifurcated needle
  • Boost booster immunization
  • ELISA Enzyme-linked immunosorbent assay
  • the Pfs25 antigen constructed using the same pfs25 gene used in m8 ⁇ and AAV1 vaccines was produced using a wheat germ cell-free (WGCF) protein expression system (CellFreeSciences).
  • WGCF wheat germ cell-free protein expression system
  • ABTS (2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (SIGMA) and H 2 O 2 in citrate phosphate buffer were detected at an absorption wavelength of 414 nm.
  • the endpoint titer was , expressed as the reciprocal of the final dilution that yielded an optical density at 414 nm that was 0.15 U above the value of the negative control ( ⁇ 0.1) All mice used in the experiment were seronegative prior to immunization.
  • ⁇ Challenge infection test ⁇ (Challenge test by administration of sporozoites) Sporozoites were isolated from the salivary glands of Anopheles stephensi mosquito infected with PfCSP-Tc/Pb. This was suspended in RPMI-1640 medium (Gibco, Life Technologies). Mice boosted with the AAV1-Pf(s25-CSP) vaccine were tail-veined 26 days later with 100 ⁇ L of medium containing 1,000 or 2,500 sporozoites. Infection was monitored from day 4 to day 14 by Giemsa staining of thin blood smears obtained from the tail. Protection against infection was calculated using the number of days after administration of sporozoites when parasitemia reached 1% (1% parasitemia) and complete protection against infection.
  • ⁇ Propagation blocking test ⁇ (Direct blood sucking test) Mice boosted with the AAV1-Pf(s25-CSP) vaccine were intraperitoneally administered 200 ⁇ L of 6 mg/ml phenylhydrazine solution (Sigma) 22 days later, and 3 days later, 1 ⁇ 10 6 Pb-Pfs25DR3 was infected. Erythrocytes were administered intraperitoneally. Three days later, the mice were sucked by approximately 60 starved Anopheles mosquitoes. The midguts of the blood-sucking mosquitoes were dissected 10 to 12 days after blood feeding, and the number of oocysts in the midgut was quantified. Propagation inhibitory activity (TRA) was calculated using the following formula.
  • TRA propagation inhibition activity
  • % non-oocyst formation rate [1 - (number of mosquitoes that sucked blood from vaccine group mice but did not form oocysts / total number of mosquitoes that sucked blood from vaccine group mice) / (blood-sucking mice from control group) (number of mosquitoes that did not form oocysts/total number of mosquitoes that sucked blood from control mice)] ⁇ 100.
  • TRA was calculated using Mann-Whitney U test and TBA was calculated using Fisher's exact test. All tests were considered significant when P value ⁇ 0.05. Prism version 8 (GraphPad Software Inc., La Jolla, Calif.) was used for statistical analysis.
  • both the m8 ⁇ /AAV1-Pf (s25-CSP) vaccine and the m8 ⁇ /AAV1-PfCSP vaccine induced significantly higher anti-PfCSP antibody responses as a booster effect compared to a single m8 ⁇ vaccine (Fig. 1d, p ⁇ 0.0001).
  • the m8 ⁇ /AAV1-Pf(s25-CSP) vaccine also induced an anti-Pfs25 antibody response with a boosting effect (FIG. 1d, p ⁇ 0.0001).
  • closed colony ICR mice express different MHC haplotypes among individuals.
  • the m8 ⁇ /AAV1-Pf(s25-CSP) vaccine is also effective in ICR mice with diverse MHC haplotypes, for which known vaccines (e.g. AdHu5/AAV1-Pf(s25-CSP)) have not been shown to be effective. A significant effect was confirmed.
  • AdHu5/AAV1-Pf(s25-CSP) an adenovirus-based vaccine previously demonstrated to be fully protective and to provide long-lasting antibody responses.
  • CSP 2,500 sporozoites (high dose) were used in the challenge test.
  • mice On days 86 and 100 after the last immunization, the same mice were intravenously injected with 2,500 and 10,000 sporozoites, respectively. All vaccinated mice (100%, p ⁇ 0.0001) were completely protected from infection, whereas all control mice were infected (Fig. 4b, 4c). Five random mice among the infection-protected mice were examined for propagation-blocking effect on day 276 after the final immunization. TRA was 99.43% (p ⁇ 0.0001) and TBA was 85.02% (p ⁇ 0.0001) (Fig. 4d).
  • vivax malaria was targeted. Details are as follows.
  • the structure of Pv(s25-CSP-S/P) was constructed by connecting a DNA sequence encoding a hinge (GlyGlyGlyGlyGlyGlySer) to the 3' end of the Pvs25 gene of Plasmodium vivax and connecting the PvCSP gene derived from the VK210 Salvador strain. . In addition, three repeat regions of the PvCSP gene from the VK247 Papua New Guinea strain were inserted.
  • the m8 ⁇ transfer vector pVR was cleaved with EcoR I/Xma I, and the open reading frame of pUC57-Simple-Pv (s25-CSP-S/P) cleaved at the EcoR I/Xma I site was placed at the same site, This was named pVR1-Pv(P7.5-s25-CSP-S/P)-HA-VV.
  • pVR1-Pv(P7.5-s25-CSP-S/P)-HA-VV was used as a transfer vector for generating m8 ⁇ -Pv(P7.5-s25-CSP-S/P)-HA virus.
  • a reduced sample in which 2-mercaptoethanol was added to the cell lysate and a non-reduced sample in which 2-mercaptoethanol was not added were prepared. Electrophoresis was performed on a 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel. Samples were electrophoretically developed on Immobilon FL® PVDF membranes (Merck Millipore). The membrane was blocked with 5% skimmed milk in PBS containing 0.1% Tween 20 (PBS-T) for 1 hour.
  • Anti-PvCSP-Sal antibody 2F2 monoclonal antibody (mAb) diluted 1:10,000 in 5% skim milk (Fig.
  • IFA Indirect fluorescent assay
  • NGS Normal Goat Serum
  • 2F2 labeled with R-Phycoerythrin LK23, 2E10E9 labeled with Fluorescein LK01, and anti-Pvs25 polyclonal antibody labeled with HiLyte Flour TM 647 LK13 were each diluted 1000-fold with 10% NGS/PBS and added to each well for 1 hour. It was allowed to stand while being shielded from light. The cells were washed five times with PBS-T for 2 minutes and mounted with Vectashield containing 4,6'-diamidino2-phenylindole (DAPI; Vector Laboratories). The slide was visualized with an LSM710 inverted laser scanning microscope (Carl Zeiss) (Fig. 8c).
  • mice and protozoa used The mammals and protozoa used were as follows. Mice: BALB/c female mice aged 6 weeks were obtained from Japan SLC, Inc. and used after 1 week.
  • Mouse malaria parasite Anopheles stephensi SDA500 strain infected with recombinant murine malaria ⁇ PvCSP-Sal expression P. berghei (Pb) PvCSP-Sal ⁇ (Sal/Pb; described in Infect Immun. 2014, 82. 10) Salivary glands were dissected and transgenic murine Plasmodium sporozoites were isolated.
  • mice Balb/c mice (7-week-old females) purchased from SLC were used in groups of 10.
  • m8 ⁇ vaccine [m8 ⁇ -Pv(P7.5-s25-CSP-S/P)-HA] was 1 ⁇ 10 7 pfu/10 ⁇ l/mouse
  • AAV1 vaccine [AAV1-Pv(s25-CSP-S/P)] was diluted with PBS to 1 ⁇ 10 10 vg/100 ⁇ L/mouse.
  • Each m8 ⁇ vaccine was immunized at the base of the tail of mice using a bifurcated pox injection technique (ts), and the AAV1 vaccine was immunized by left thigh muscle injection (im) using an insulin syringe with a 29G needle. Immunization was performed by the heterologous prime-boost method, with m8 ⁇ vaccine as primary immunization (Prime), followed by booster immunization (Boost) with AAV1 vaccine 6 weeks later.
  • Primary immunization Primary im
  • ELISA Enzyme-linked immunosorbent assay
  • Anti-PvCSP-Sal and Pvs25 antibody titers were quantified by ELISA as described in ⁇ Infect Immun. 2014, 82.10. doi: 10.1128/IAI.02040-14 ⁇ .
  • Costar® EIA/RIA polystyrene plates precoated with 200 ng/well PvCSP-Sal or 400 ng/well Pvs25 were blocked with 1% bovine serum albumin (BSA) in PBS and then Serially diluted serum samples, negative and positive controls (mAb 2A10 or mAb NHH10, respectively) were incubated.
  • BSA bovine serum albumin
  • ⁇ Challenge infection test ⁇ (Challenge test by administration of sporozoites) Sporozoites were isolated from the salivary glands of Anopheles stephensi mosquito infected with PvCSP-Sal/Pb. This was suspended in RPMI-1640 medium (Gibco, Life Technologies). Mice boosted with AAV1-Pv(s25-CSP-S/P) vaccine were tail-veined 28 days later with 100 ⁇ L of medium containing 1,000 sporozoites. Infection was monitored from day 4 to day 14 by Giemsa staining of thin blood smears obtained from the tail. Protection against infection was calculated using the number of days after administration of sporozoites when parasitemia reached 1% (1% parasitemia) and complete protection against infection.
  • ⁇ Propagation blocking test Blood was collected from vivax malaria patients, blood smears were prepared, and the presence of gametes (gametocytes) was confirmed. Blood was centrifuged at 2,000 rpm for 5 minutes and plasma removed with a Pasteur pipette. Add RPMI 1640 (equal volume to blood concentrate) and gently mix with blood. Five 1.5 mL tubes were taken and identified as follows (Control, 1:5, 1:10, 1:50). Three- to five-day-old female mosquitoes that had been fed sugar solution only were isolated (approximately 120 or 200) (sugar was removed approximately 24 hours prior to experimentation) and placed in containers.
  • the DirectMembrane Feeding Assay (DMF) infection experimental system consists of a 37°C water bath and a water circulation system connected to blood samples.
  • a well-stretched piece of parafilm (5 cm x 5 cm) was wrapped around a 500 ⁇ l glass feeder, and the glass feeder, hose, and water pump were placed in a water tank to construct a circuit. I looked at the last hose to make sure the water flow was continuous.
  • a feeder was taped to each plastic pot containing female mosquitoes (marked control or treatment group on each pot). Each sample of blood was added to each feeder with a glass pipette and allowed to fully feed for 30-120 minutes.
  • the female mosquito was removed from the infection room, transported to the dissecting room, removed from the cage using a trap, and placed in a freezer at -20°C for 3-5 minutes. After sleeping, the mosquitoes were placed in a Petri dish containing 70% alcohol and immediately washed with 1 ⁇ PBS. The midgut was removed from the mosquito abdomen using a microscope. After that, one drop of 2% mercurochrome dye was applied to the midgut, and after staining for 5-8 minutes, oocysts were counted under a microscope (Fig. 11).
  • TRA Transmission reducing activity
  • TBA Transmission blocking activity
  • TRA was calculated using Mann-Whitney U test and TBA was calculated using Fisher's exact test. All tests were considered significant when P value ⁇ 0.05.
  • Prismversion 8 GraphPad Software Inc., La Jolla, Calif. was used for statistical analysis.
  • Example 1 the effect of AAV5 as a booster vaccine was shown, and the effect with AAV1 used in Example 1 was verified. Details are as follows. ⁇ Materials and methods> (Preparation of vaccinia virus) A method for producing recombinant vaccinia virus m8 ⁇ -Pf(P7.5-s25-CSP)-HA virus is described in Example 1.
  • a method for producing recombinant adeno-associated virus AAV5-Pf(s25-CSP) virus is described in AAV1-Pf(s25-CSP) virus using AAV1 (Example 1). The only difference is the use of AAV5.
  • IFA Indirect fluorescent assay
  • mice (7-week-old females) purchased from SLC were used in groups of 10.
  • m8 ⁇ vaccine [m8 ⁇ -Pf(P7.5-s25-CSP)-HA] is 1 ⁇ 10 7 pfu/100 ⁇ L/mouse
  • AAV5 vaccine [AAV5-Pf(s25-CSP)] is 1 ⁇ 10 10 vg/100 It was diluted with PBS so as to be ⁇ L/mouse.
  • the m8 ⁇ vaccine was immunized at the base of the tail of mice using a bifurcated vaccination technique (ts), and the AAV5 vaccine was immunized via a left thigh muscle injection (im) using an insulin syringe with a 29G needle. Immunization was performed by the heterogeneous prime-boost method, with m8 ⁇ vaccine as primary immunization (Prime), and after 6 weeks, booster immunization with AAV5 vaccine (Boost). A detailed method is described in Example 1.
  • ⁇ Challenge infection test ⁇ (Challenge test by administration of sporozoites) Sporozoites were isolated from the salivary glands of Anopheles stephensi mosquito infected with PfCSP-Tc/Pb. This was suspended in RPMI-1640 medium (Gibco, Life Technologies). Mice boosted with the AAV5-Pf(s25-CSP) vaccine were tail-veined 40 days later with 100 ⁇ L of medium containing 2,500 sporozoites. Infection was monitored from day 4 to day 14 by Giemsa staining of thin blood smears obtained from the tail. A detailed method is described in Example 1.
  • mice boosted with AAV5-Pf(s25-CSP) vaccine received 200 ⁇ L of 6 mg/ml phenylhydrazine solution (Sigma) intraperitoneally 29 days later, and 1 ⁇ 10 6 Pb-Pfs25 -DR3-infected erythrocytes were administered intraperitoneally. Three days later, the mice were sucked by approximately 60 starved Anopheles mosquitoes. The midguts of the blood-sucking mosquitoes were dissected 10 to 12 days after blood feeding, and the number of oocysts in the midgut was quantified. A detailed method is described in Example 1.
  • FIG. 12a an AAV5-based vaccine expressing Pfs25 and PfCSP as a fusion protein using a hinge G6S linker was constructed (Fig. 12a).
  • the structure is the same as in Figure 1a, but the AAV used is AAV5.
  • Western blotting confirmed that AAV5-Pf(s25-CSP) expressed a Pfs25-PfCSP fusion protein (Fig. 12b), and further confirmed cell surface expression of PfCSP and Pfs25 (Fig. 12b).
  • the protective effect of m8 ⁇ /AAV5-Pf(s25-CSP) vaccination was determined by complete protection against PfCSP/Pb sporozoites.
  • Balb/c mice all 10 control mice were infected after i.v. Complete infection protection was achieved (Fig. 14).
  • This effect was as complete protection as the m8 ⁇ /AAV1-Pf(s25-CSP) vaccine with AAV1 (100%).
  • the m8 ⁇ /AAV5-Pf(s25-CSP) vaccine induced high anti-PfCSP antibody responses as a booster effect with AAV5-Pf(s25-CSP) (Fig. 13a, 2.71 x 106 ).
  • the m8 ⁇ /AAV5-Pf(s25-CSP) vaccine also induced an anti-Pfs25 antibody response with a boosting effect (Fig. 13b, 7.17 x 105 ).
  • These anti-PfCSP antibody titers and anti-Pfs25 antibody titers were as high as the m8 ⁇ /AAV1-Pf (s25-CSP) vaccine using AAV1.
  • the m8 ⁇ /AAV5-Pf (s25-CSP) vaccine using AAV5 of the present invention has a complete infection protective effect equivalent to the m8 ⁇ /AAV1-Pf (s25-CSP) vaccine using AAV1 (Example 1) ( 100%) was confirmed.
  • TRA and TBA were 99.94% (p ⁇ 0.0001) and 95.27% (p ⁇ 0.0001), respectively ( Figure 15).
  • the TRA and TBA effects were as high as the m8 ⁇ /AAV1-Pf(s25-CSP) vaccine using AAV1.
  • the m8 ⁇ /AAV5-Pf(s25-CSP) vaccine using AAV5 of the present invention exhibits a high transmission blocking effect equivalent to the m8 ⁇ /AAV1-Pf(s25-CSP) vaccine using AAV1 (Example 1). It was confirmed.
  • Example 1 vaccinia virus and adeno-associated virus
  • the method for producing recombinant vaccinia virus m8 ⁇ -Pf(P7.5-s25-CSP)-HA is described in Example 1, and the method for producing recombinant adeno-associated virus type 1 AAV1-Pf(s25-CSP) is described in Example 1. It is described in Example 1.
  • the monkeys and protozoa used were as follows.
  • Rhesus monkeys Four Indian rhesus monkeys bred at the Institute of Biomedical Sciences, Kyoto University were used. Details are as follows.
  • Mouse malaria parasite genetically modified murine malaria ⁇ PfCSP-expressing P. berghei (Pb) PfCSP-Tc/Pb ⁇ (described in Insect Mol. Biol. 2013, 22:41) was used.
  • m8 ⁇ vaccine [m8 ⁇ -Pf(P7.5-s25-CSP)-HA] at 1 ⁇ 10 8 pfu/10 ⁇ L
  • AAV1 vaccine (AAV1-Pf(s25-CSP)) at 3.3 ⁇ 10 12 vg/100 ⁇ L diluted with PBS to The m8 ⁇ vaccine was primed into the depilated upper back by a bifurcated vaccination technique (ts). Eight weeks later, the AAV1 vaccine was boosted with a left thigh muscle inoculation (im) using a 29G needled insulin syringe.
  • the collected blood was stored at ⁇ 20° C. until it was used in the following blood tests or as plasma from which blood cell components were removed by centrifugation. Purification of IgG from monkey plasma was performed using HiTrap Protein G HP (cytiva).
  • the Pfs25 antigen constructed using the same pfs25 gene used in m8 ⁇ and AAV1 vaccines was produced using a wheat germ cell-free (WGCF) protein expression system (CellFree Sciences).
  • WGCF wheat germ cell-free protein expression system
  • Recombinant malaria parasite (PfCSP-Tc/Pb)-infected mosquitoes were dissected, salivary glands were added to a biomassher column (Biomasher III), homogenized, centrifuged at 8,000 rpm, 40 s, 4°C, and C-CHIP (NanoEnTek) was added. counted using 50 ⁇ L of RPMI (without serum) and 10,000 sporozoites/50 ⁇ L of immunized and pre-immunized monkey IgG final concentrations of 1,000 ⁇ g/mL, 500 ⁇ g/mL, and 100 ⁇ g/mL, respectively.
  • a solution diluted with RPMI (without serum) was prepared so that The IgG solution and spz solution were mixed in a 1.5 mL microtube and allowed to stand on ice for 40 minutes. After completion of the culture, a mixture of IgG and sporozoites was added to the wells, centrifuged at 1,000 rpm for 5 minutes at room temperature, and cultured in an incubator at 37°C for 24 hours. After the culture was completed, the cell supernatant was removed from the wells, 300 ⁇ L/well of RPMI (without serum) was added, and the cells were cultured in an incubator at 37°C for 24 hours.
  • RNA extraction and qRT-PCR were performed to measure the amount of protozoan-derived ribosomal RNA (rRNA).
  • Reaction data were analyzed using GraphPad Prism (GraphPad Software).
  • the neutralizing activity was determined as the ratio (percentage) of the amount of rRNA in sporozoite-infected cells containing each concentration of antibody to the amount of protozoan-derived rRNA in cells infected with sporozoites that do not react with antibody, which was defined as 100%.
  • the concentration of antibody that inhibits sporozoite invasion by 50% was calculated from the regression line.
  • mice were sucked by mosquitoes.
  • IgG purified from preimmune serum collected from the same individual was used. Twenty-four hours after sucking, non-sucking mosquitoes were removed, and the number of oocysts in the mosquito midgut was counted 10-12 days after sucking. The protozoan growth inhibitory effect of the antibody was calculated by comparison with the control antibody group.
  • Figure 19 shows the results of the in vitro sporozoite neutralization test. Infection ratios were significantly reduced in all four immunized monkey IgG compared to pre-immune monkey IgG. Specifically, at 1,000 ⁇ g/mL, it was 5.0% (monkey MM638), 0.03% (monkey MM641), 12.7% (monkey MM651), and 0.44% (monkey MM633). Immune monkey IgG was confirmed to have a high anti-invasion effect (neutralizing activity).
  • Fig. 20 shows the results of the propagation blocking test.
  • the malaria-positive rate in mosquitoes after feeding decreased to 67.9% (monkey MM651), 90.3% (monkey MM641), 53.9% (monkey MM633), and 23.4% (monkey MM638) in the immunized group compared to the control group, respectively. did.
  • the vaccine of the present invention not only provides 100% protective effect against PfCSP/Pb sporozoites, but also provides a transmission-blocking effect that inhibits migration of Pb-Pfs25DR3 protozoa to mosquitoes by 99% or more.
  • the vaccine of the present invention has a vaccine-induced immune response effect that lasts for 7 months or longer, and provides complete protection against multiple infections with parasites.
  • the vaccine of the present invention can express the genes encoding Pfs25 and PfCSP as a fusion protein via the hinge sequence. It was confirmed that it also functions as (TBV).
  • the vaccine of the present invention showed 90 to 100% complete infection protection against 1,000 sporozoite challenge, and 100% complete infection against 2,500 sporozoite challenge and blood-sucking challenge by infected mosquitoes. showed a protective effect.
  • the vaccine of the present invention can induce a rapid and strong humoral immune response, sustained antigen expression by AAV continuously enhances the response, and long-term anti-PfCSP antibody response and anti-Pfs25 antibody response. maintained.
  • the long-lasting antibody response with vaccination according to the invention was significantly superior to that with known AdHu5/AAV1 vaccination (Fig. 2c, 2d).
  • the vaccine of the present invention showed 100% complete protection against sporozoite challenge repeated 26 days, 86 days and 100 days after the final immunization ( Figure 4a- c). Furthermore, the same mice showed a high propagation-blocking effect 276 days after the final immunization (Fig. 4d). 7)
  • the vaccine [m8 ⁇ /AAV1-Pf(s25-CSP)] of the present invention which has a propagation-blocking effect of 99% or more, showed a higher infection-preventing effect than m8 ⁇ /AAV1-PfCSP, which does not have a propagation-blocking effect. Therefore, the synergistic effect of the vaccine was shown by the expression of both PfCSP and Pfs25 antigens.
  • the vaccine of the present invention has a significant prevalence suppressing effect of about 7 times (71% ⁇ 10%) compared to known vaccines (especially AdHu5/AAV1-Pf(s25-CSP)).
  • the vaccine of the present invention has the ability to confer lifelong immunity to humans by inoculating m8 ⁇ -Pf(P7.5-s25-CSP)-HA and AAV1-Pf(s25-CSP) twice or more in total. is considered to have More specifically, the results of the example (1) antibody response persists until 210 days after immunization (Figs. 2c and 2d), (2) antibody response increases with multiple exposures to protozoa (Fig.
  • the vaccine effect persists until 276 days after immunization; Infection with protozoa, which is prevalent in the past, provides a booster immunization effect, and considering the lifespan of mice, the vaccine of the present invention can confer lifetime immunity in humans as well as in mice.
  • the vaccine of the present invention is m8 ⁇ /AAV1-Pv (s25-CSP- S/P) vivax malaria vaccine. This vaccine expresses two PvCSP gene polymorphisms of Plasmodium vivax (Salvador strain VK210, Papua New Guinea strain VK247).
  • the vaccine of the present invention induced a very high anti-PfCSP antibody titer (more than 500,000 times) in all monkeys after booster immunization with AAV1 vaccine. The antibody titer was maintained even after half a year, demonstrating its usefulness as a two-dose malaria vaccine.
  • the anti-PfCSP antibody induced by the vaccine of the present invention showed very strong in vitro sporozoite neutralizing activity.
  • the vaccine of the present invention has demonstrated utility as a malaria-protective vaccine.
  • the vaccine of the present invention induced a very high anti-Pfs25 antibody titer (200,000- to 500,000-fold) in all monkeys after booster immunization with AAV1 vaccine. The antibody showed very strong propagation-blocking activity.
  • the vaccine of the present invention has demonstrated utility as a malaria transmission-blocking vaccine. 16) According to 12) to 15) above, the vaccine of the present invention provides life-long immunity to humans by inoculating m8 ⁇ -Pf(P7.5-s25-CSP)-HA and AAV1-Pf(s25-CSP) twice. is thought to have the ability to confer

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Le problème décrit par la présente invention est de fournir un vaccin contre le paludisme qui a un excellent effet préventif contre l'infection et un excellent effet d'inhibition de la transmission du paludisme, par comparaison aux vaccins classiques contre le paludisme. Il a été découvert que l'utilisation, en tant que première immunisation, d'un virus de la vaccine recombinant qui comprend un gène qui code pour une séquence d'acides aminés CSP et un gène qui code pour une séquence d'acides aminés s25, et l'utilisation, en tant que rappel, d'un virus adéno-associé recombinant qui comprend un gène qui code pour une séquence d'acides aminés CSP et un gène qui code pour une séquence d'acides aminés s25 résulte en un vaccin contre le paludisme qui a un excellent effet préventif contre l'infection et un excellent effet d'inhibition de la transmission du paludisme.
PCT/JP2023/005232 2022-02-18 2023-02-15 Vaccin contre le paludisme et méthode de prévention/traitement du paludisme WO2023157880A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2024501407A JP7649504B2 (ja) 2022-02-18 2023-02-15 マラリアワクチン及びマラリア予防・治療方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2022-024221 2022-02-18
JP2022024221 2022-02-18
JP2022131078 2022-08-19
JP2022-131078 2022-08-19

Publications (1)

Publication Number Publication Date
WO2023157880A1 true WO2023157880A1 (fr) 2023-08-24

Family

ID=87578353

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2023/005232 WO2023157880A1 (fr) 2022-02-18 2023-02-15 Vaccin contre le paludisme et méthode de prévention/traitement du paludisme

Country Status (2)

Country Link
JP (1) JP7649504B2 (fr)
WO (1) WO2023157880A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007091624A1 (fr) * 2006-02-09 2007-08-16 Educational Foundation Jichi Medical University Nouveau vecteur viral
JP2010535466A (ja) * 2007-08-07 2010-11-25 学校法人自治医科大学 新規ウイルスベクター
WO2016059911A1 (fr) * 2014-10-17 2016-04-21 国立大学法人金沢大学 Vaccin antipaludéen
JP2017502674A (ja) * 2013-12-30 2017-01-26 ヴイ−コア テクノロジーズ インコーポレイテッド ポックスウィルス−プラスモジウム組換え体、こうした組換え体を含む組成物、該組換え体の使用、および該組換え体を作製し、および用いる方法。
JP2017512498A (ja) * 2014-03-28 2017-05-25 フラウンホーファーゲゼルシャフト ツール フォルデルング デル アンゲヴァンテン フォルシユング エー．フアー． 複数成分複数段階マラリアワクチン
JP2017527293A (ja) * 2014-09-08 2017-09-21 フラウンホーファーゲゼルシャフト ツール フォルデルング デル アンゲヴァンテン フォルシユング エー．フアー． 三成分多段階マラリアワクチン
JP2019151617A (ja) * 2018-03-02 2019-09-12 国立大学法人金沢大学 マラリアワクチン

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007091624A1 (fr) * 2006-02-09 2007-08-16 Educational Foundation Jichi Medical University Nouveau vecteur viral
JP2010535466A (ja) * 2007-08-07 2010-11-25 学校法人自治医科大学 新規ウイルスベクター
JP2017502674A (ja) * 2013-12-30 2017-01-26 ヴイ−コア テクノロジーズ インコーポレイテッド ポックスウィルス−プラスモジウム組換え体、こうした組換え体を含む組成物、該組換え体の使用、および該組換え体を作製し、および用いる方法。
JP2017512498A (ja) * 2014-03-28 2017-05-25 フラウンホーファーゲゼルシャフト ツール フォルデルング デル アンゲヴァンテン フォルシユング エー．フアー． 複数成分複数段階マラリアワクチン
JP2017527293A (ja) * 2014-09-08 2017-09-21 フラウンホーファーゲゼルシャフト ツール フォルデルング デル アンゲヴァンテン フォルシユング エー．フアー． 三成分多段階マラリアワクチン
WO2016059911A1 (fr) * 2014-10-17 2016-04-21 国立大学法人金沢大学 Vaccin antipaludéen
JP2019151617A (ja) * 2018-03-02 2019-09-12 国立大学法人金沢大学 マラリアワクチン

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAUZA KAROLIS, MALINAUSKAS TOMAS, PFANDER CLAUDIA, ANAR BURCU, JONES E. YVONNE, BILLKER OLIVER, HILL ADRIAN V. S., REYES-SANDOVAL : "Efficacy of a Plasmodium vivax Malaria Vaccine Using ChAd63 and Modified Vaccinia Ankara Expressing Thrombospondin-Related Anonymous Protein as Assessed with Transgenic Plasmodium berghei Parasites", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 82, no. 3, 1 March 2014 (2014-03-01), US , pages 1277 - 1286, XP093084830, ISSN: 0019-9567, DOI: 10.1128/IAI.01187-13 *
HILL ADRIAN V S , REYES-SANDOVAL A, O'HARA G, ET AL: "Prime-boost vectored malaria vaccines: progress and prospects.", HUMAN VACCINES, LANDES BIOSCIENCE, GEORGETOWN, TX, US, vol. 6, no. 1, 1 January 2010 (2010-01-01), US , pages 78 - 83, XP002636116, ISSN: 1554-8600, DOI: 10.4161/hv.6.1.10116 *

Also Published As

Publication number Publication date
JPWO2023157880A1 (fr) 2023-08-24
JP7649504B2 (ja) 2025-03-21

Similar Documents

Publication Publication Date Title
Goodman et al. The utility of Plasmodium berghei as a rodent model for anti-merozoite malaria vaccine assessment
US9603916B2 (en) Treatment and prevention of malaria
WO2011163628A2 (fr) Séquences polypeptidiques du virus de la dengue (dv), épitopes de lymphocytes t et leurs procédés et leurs utilisations
KR20180108754A (ko) 말라리아 백신에 사용하기 위한 신규 항원
Mlambo et al. Functional immunogenicity of baculovirus expressing Pfs25, a human malaria transmission-blocking vaccine candidate antigen
US10213501B2 (en) Three-component-multistage malaria vaccine
Hota-Mitchell et al. Recombinant vaccinia viruses and gene gun vectors expressing the large subunit of Schistosoma mansoni calpain used in a murine immunization-challenge model
JP5980810B2 (ja) イヌバベシア症ワクチン抗原
US11197920B2 (en) Vaccines
JP7649504B2 (ja) マラリアワクチン及びマラリア予防・治療方法
WO2016059911A1 (fr) Vaccin antipaludéen
Iyori et al. Sterile protection and transmission blockade by a multistage anti-malarial vaccine in the pre-clinical study
US6855316B1 (en) Baculovirus produced Plasmodium falciparum vaccine
JP2019151617A (ja) マラリアワクチン
Yamamoto et al. A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study
US7737262B2 (en) Plasmodium falciparum polypeptides and methods of using same
Sun et al. Vesicular stomatitis virus-based vaccine targeting plasmodium blood-stage antigens elicits immune response and protects against malaria with protein booster strategy
Meerstein-Kessel et al. Monoclonal antibodies block transmission of genetically diverse Plasmodium falciparum strains to mosquitoes
WO2014188212A2 (fr) Traitement et prévention du paludisme
WO2024192465A1 (fr) Vaccins et leurs procédés d'utilisation
Namkung ADENO-ASSOCIATED VIRUS VECTORED IMMUNOPROPHYLAXIS OF HUMAN MALARIA PLASMODIUM FALCIPARUM: MURINE AND NON-HUMAN PRIMATE MODEL
WO2022098656A1 (fr) Vaccin contre les lyssavirus à gène réarrangé
US20190255167A1 (en) Fusion polypeptides
Hirota et al. A Baculovirus Dual Expression
Kei Expression of a Hexa-histidine TaggedPlasmodium falciparum Merozoite Surface Protein-1 C-terminal Processing Fragment (C-HisPfMSP-l42) in Silkworm Larvae usingBombyx mori Nuclear Polyhedrosis Virus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23756405

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2024501407

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23756405

Country of ref document: EP

Kind code of ref document: A1