WO2024242174A1 - Rotavirus infectieux à cycle unique et son utilisation - Google Patents

Rotavirus infectieux à cycle unique et son utilisation Download PDF

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WO2024242174A1
WO2024242174A1 PCT/JP2024/019050 JP2024019050W WO2024242174A1 WO 2024242174 A1 WO2024242174 A1 WO 2024242174A1 JP 2024019050 W JP2024019050 W JP 2024019050W WO 2024242174 A1 WO2024242174 A1 WO 2024242174A1
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rotavirus
cells
infectious
virus
mutation
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剛 小林
祐太 金井
将裕 小瀧
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University of Osaka NUC
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present invention relates to a single-infectious rotavirus, a rotavirus vaccine containing the single-infectious rotavirus, and a rotavirus neutralization test method using the single-infectious rotavirus.
  • the present invention includes the following inventions.
  • a monoinfectious rotavirus characterized by having a mutation in at least one viral protein selected from the group consisting of rotavirus VP1, VP2, VP3, VP4, VP6, VP7, NSP2, NSP3 and NSP4.
  • the mono-infectious rotavirus described in [1] above which has a mutation that induces dysfunction of a structural protein.
  • a vaccine comprising the monoinfectious rotavirus described in any one of [1] to [14].
  • the present invention can provide a single-infectious rotavirus.
  • the present invention can also provide a rotavirus vaccine containing a single-infectious rotavirus, and a rotavirus neutralization test method using a single-infectious rotavirus.
  • FIG. 1 shows the results of infecting cells that do not express VP6 with the artificial recombinant rotavirus ⁇ 169-174+linker and examining the proliferation ability of the virus after five repeated passages.
  • This figure shows the results of infecting cells persistently expressing VP6 with a green fluorescent protein-expressing single-infectious rotavirus, which was prepared by inserting a gene encoding green fluorescent protein (ZsG) into the segmented RNA genome encoding NSP1 in the single-infectious rotavirus ⁇ 169-174 + linker, and with a single-infectious rotavirus ⁇ 169-174 + linker that does not express green fluorescent protein, and observing the expression of ZsG and NSP4.
  • ZsG green fluorescent protein
  • This figure shows the results of infecting VP6 persistently expressing cells with a luciferase-expressing single-infectious rotavirus prepared by inserting a gene encoding luciferase (NLuc) into the segmented RNA genome encoding NSP1 in the single-infectious rotavirus ⁇ 169-174 + linker, and with a single-infectious rotavirus ⁇ 169-174 + linker that does not express luciferase, and measuring luciferase activity over time.
  • NLuc gene encoding luciferase
  • FIG. 1 shows the results of producing five types of single-infectious rotaviruses ⁇ 169-174+linker having a segmented RNA genome encoding human rotavirus VP7 and measuring the viral titer.
  • FIG. 1 shows the results of a neutralization test of single-infectious rotaviruses having segmented RNA genomes encoding four types of human rotavirus VP7, using mouse serum immunized with wild-type simian rotavirus SA11 strain.
  • FIG. 1 shows the results of examining the vaccine effect by orally administering single-infectious rotavirus ⁇ 169-174+linker to mice three times, and subjecting the blood samples collected two weeks after each administration to a neutralization test to obtain sera.
  • FIG. 1 shows the structure of rotavirus (A) and the structure of VP6 (B).
  • FIG. 1A is a diagram showing the structure of rotavirus VP7
  • FIG. 1B is a diagram showing the mutated region of the mutant VP7 used in Example 12.
  • FIG. 1A is a diagram showing the structure of rotavirus VP4, and FIG. 1B is a diagram showing the mutated region of the mutant VP4 used in Example 15.
  • FIG. 1 shows the results of infecting cells that do not express VP4 with an artificial recombinant rotavirus VP4-120bp, and examining the proliferation ability of the virus after five passages.
  • This figure shows the results of infecting cells that do not express VP4 with a green fluorescent protein-expressing single-infectious rotavirus, which was created by inserting a gene encoding green fluorescent protein (ZsG) into the segmented RNA genome of a single-infectious rotavirus VP4-120bp in which most of the VP4 gene had been deleted, and a single-infectious rotavirus (VP4-120bp) that does not express green fluorescent protein, and observing the expression of ZsG and NSP4.
  • ZsG green fluorescent protein
  • This figure shows the results of infecting cells that do not express VP4 with a luciferase-expressing single-infectious rotavirus prepared by inserting a gene encoding luciferase (NLuc) into the segmented RNA genome of the single-infectious rotavirus VP4-120bp in which most of the VP4 gene has been deleted, and a single-infectious rotavirus (VP4-120bp) that does not express luciferase, and measuring luciferase activity over time.
  • NLuc gene encoding luciferase
  • This figure shows the results of infecting cells that do not express VP4 with an RBD-expressing single-infectious rotavirus (VP4-120bp-RBD) created by inserting a gene encoding the receptor binding domain (RBD) of the spike protein of the novel coronavirus (SARS-CoV-2) into the segmented RNA genome of the single-infectious rotavirus VP4-120bp in which most of the VP4 gene has been deleted, and a single-infectious rotavirus that does not express RBD (VP4-120bp), and observing the expression of RBD and NSP3 by Western blotting.
  • RBD receptor binding domain
  • the present invention provides a mono-infectious rotavirus.
  • mono-infectious rotavirus means a rotavirus that is unable to form infectious virus particles after a single infection, or whose production amount of infectious virus particles is suppressed after a single infection.
  • mono-infectious rotavirus means a rotavirus whose viral titer decreases by subculture.
  • Rotavirus is a non-enveloped virus belonging to the Reoviridae family.
  • the virus particle has a triple structure consisting of an outer coat, an inner coat, and a core protein, and contains a double-stranded RNA genome consisting of 11 segments (see Figure 13 (A)). These 11 gene segments code for six structural proteins (VPs) and six non-structural proteins (NSPs) (Table 1).
  • VPs structural proteins
  • NSPs non-structural proteins
  • the single-infectious rotavirus of the present invention may have a mutation in at least one viral protein selected from the group consisting of rotavirus VP1, VP2, VP3, VP4, VP6, VP7, NSP2, NSP3, and NSP4.
  • VP1, VP2, VP3, VP4, VP6, and VP7 are structural proteins of rotavirus
  • NSP2, NSP3, and NSP4 are non-structural proteins.
  • the single-infectious rotavirus of the present invention may have a mutation in a structural protein of rotavirus, or may have a mutation that induces dysfunction of a structural protein.
  • the number of viral proteins having a mutation may be two or more, or may be one, but is preferably one.
  • the structural protein having a mutation may be VP6 (inner coat protein). If the structural protein having a mutation is VP6, it is preferable that the mutation is in a region that interacts with VP7 (outer coat protein).
  • the region that interacts with VP7 may be domain H of VP6. Domain H contains three loop structures (A'-A'' loop, D'-D'' loop, and ⁇ A-I loop), and any one of these loops may have a mutation.
  • the mutated structural protein may be VP4 (spike protein).
  • the mutated structural protein may be VP4 (spike protein).
  • the mutated VP4 may be a deletion of the lectin domain, a deletion of the ⁇ -barrel domain, or a deletion of both the lectin domain and the ⁇ -barrel domain.
  • a mutant VP4 having deletions of both the lectin domain and the ⁇ -barrel domain of VP4 may be a mutant VP4 in which at least 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp or more remain on the N-terminus and at least 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp or more remain on the C-terminus in the base sequence of the segmented genome encoding VP4.
  • the number of bases remaining on the N-terminus and C-terminus is not particularly limited, and the number of bases on both sides does not need to be the same.
  • the monoinfectious rotavirus of the present invention can be produced using a known method for producing an artificial recombinant rotavirus.
  • a known method for producing an artificial recombinant rotavirus for example, the method described in Patent Document 1 (WO2018/062199) or Non-Patent Document 1 (Kanai et al., Proc Natl Acad Sci USA. 2017 Feb 28; 114(9):2349-2354), or a method partially modified from these methods, can be suitably used. It is preferable to introduce a normal protein expression vector corresponding to the mutated viral protein into the host cell.
  • the single-infectious rotavirus of the present invention can be used in a rotavirus neutralization test. That is, the present invention provides a method for a rotavirus neutralization test using the single-infectious rotavirus of the present invention.
  • the rotavirus neutralization test can be carried out by mixing serially diluted test serum and the single-infectious rotavirus of the present invention to carry out an antigen-antibody reaction, inoculating the mixture into culture cells on a microplate and culturing the mixture, and then measuring the number of virus-infected cells.
  • VP6 persistent expression cells The rotavirus VP6 gene was introduced into the pLVSIN-CMV-Neo vector (Takara Bio) and co-transfected with psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into 293T cells to produce lentivirus. The lentivirus was then infected into African green monkey MA104 cells (ATCC CRL-2378.1), and drug selection was performed with G418 (800 ⁇ g/mL) two days later. The cells expressing VP6 most strongly were cloned from the VP6-expressing MA104 cells obtained by drug selection using limiting dilution. Limiting dilution was performed four times to obtain MA104 cells that persistently express VP6.
  • transfection reagent 2 ⁇ L was used per ⁇ g of DNA.
  • BHK-T7/P5 cells were cultured in DMEM medium containing 5% FBS, 100 units/mL penicillin, and 100 ⁇ g/mL streptomycin at 37°C under 5% CO2 .
  • 24 hours after transfection the medium was replaced with FBS-free, trypsin-containing medium, and VP6-sustained MA104 cells were added at 2 ⁇ 105 cells/well. After co-culture for 5 days, the medium and cells were harvested. The harvested medium and cells were frozen and thawed three times to prepare a cell lysate.
  • Example 1 The nine types of viruses selected in Example 1 were infected at an MOI of 0.01, and 24 hours after infection, the medium and cells were frozen and thawed three times to prepare cell lysates, and the virus titer was measured. A wild-type virus without VP6 mutation was used as a control. The virus titer was measured in the same manner as in Example 1.
  • Example 3 Comparison of proliferation ability between normal MA104 cells and MA104 cells that continuously express VP6
  • the proliferation ability of the VP6 mutant rotavirus ⁇ 169-174+linker selected in Example 2 was compared between normal MA104 cells and MA104 cells that persistently express VP6.
  • Materials and Methods Normal MA104 cells and MA104 cells expressing VP6 were seeded in a 24-well culture plate at 7.5 x 104 cells/well and cultured overnight. ⁇ 169-174 + linker was infected at an MOI of 0.01, and the medium and cells were frozen and thawed three times at 0, 24, 48, and 72 hours after infection to prepare cell lysates and measure the virus titer.
  • a wild-type virus without VP6 mutation was used as a control. The virus titer was measured in the same manner as in Example 1.
  • Example 4 Examination of proliferation ability in normal MA104 cells To further confirm that the VP6 mutant rotavirus ⁇ 169-174 + linker was unable to grow in normal MA104 cells, it was subcultured in normal MA104 cells. Materials and Methods Normal MA104 cells were seeded at 3 x 105 cells/well in a 6-well culture plate and cultured overnight. ⁇ 169-174 + linker was infected at MOI 1.0, and after one week of culture, the medium and cells were collected and frozen and thawed to prepare a cell lysate. 10% of the obtained cell lysate was added to new normal MA104 cells and cultured for one week. The virus was passaged five times, and the virus titer was measured at each passage using the same method as in Example 1.
  • Example 5 Examination of viral protein expression in normal cells In order to use a single-infectious virus as a vaccine, it is necessary to express viral proteins, even though infectious viral particles are not formed in normal cells that do not express VP6. Therefore, we infected normal MA104 cells with ⁇ 169-174+linker, which was determined to be a single-infectious rotavirus in Example 3, and observed the expression of viral proteins.
  • MA104 cells were seeded at 7.5 ⁇ 104 cells/well in a 24-well culture plate containing a coverslip and cultured overnight.
  • ⁇ 169-174 + linker and wild-type simian rotavirus (SA11 strain) were infected at an MOI of 1.0, and the cells were fixed with formalin 8 hours later.
  • the cells were permeabilized with 0.5% Triton-X and immunostained with rabbit anti-NSP4 and mouse anti-VP6 antibodies as primary antibodies, and CF488-labeled anti-rabbit IgG antibodies (Biotum) and CF594-labeled anti-mouse IgG antibodies (Biotum) as secondary antibodies. Nuclei were stained with Hoechst and observed under a fluorescent microscope.
  • Example 6 Construction of a single-infectious rotavirus containing a reporter gene 6-1 Single-infectious rotavirus expressing green fluorescent protein ⁇ Materials and methods> (1) Green fluorescent protein gene-inserted NSP1 expression plasmid
  • the ZsGreen (hereinafter referred to as "ZsG") gene was used as the green fluorescent protein gene.
  • the ZsG gene-inserted NSP1 gene expression plasmid was pT7-NSP1-ZsG- ⁇ 722 described in Kanai et al. (J Virol. 2019 Feb 15; 93(4): e01774-18.).
  • pT7-NSP1-ZsG- ⁇ 722 is a plasmid in which the ZsG gene is inserted between positions 111 and 112 of the NSP1 gene of pT7-NSP1SA11, and 722 nt (positions 134 to 855) are deleted from the NSP1 gene.
  • capping enzyme expression vectors pCAGD1R and pCAG-D12L
  • rotavirus NSP2 expression vector pCAG-NSP2SA11
  • rotavirus NSP5 expression vector pCAG-NSP5SA11
  • rotavirus VP6 expression vector pcDNA3-VP6 expression vector
  • Luciferase-expressing single-infectious rotavirus ⁇ Materials and methods>
  • luciferase gene-inserted NSP1 expression plasmid As the luciferase gene, the NanoLuc (hereinafter referred to as "NLuc") gene, which is a luciferase gene derived from Oplophorus gracilirostris, was used.
  • NLuc the NanoLuc gene-inserted NSP1 gene expression plasmid
  • pT7-NSP1-NLuc- ⁇ 722 described in Kanai et al. J Virol. 2019 Feb 15; 93(4): e01774-18.
  • pT7-NSP1-NLuc- ⁇ 722 is a plasmid in which the NLuc gene is inserted between positions 111 and 112 of the NSP1 gene of pT7-NSP1SA11, and 722 nt (positions 134 to 855) are deleted from the NSP1 gene.
  • capping enzyme expression vectors pCAGD1R and pCAG-D12L
  • rotavirus NSP2 and NSP5 expression vectors pCAG-NSP2SA11 and pCAG-NSP5SA11
  • rotavirus VP6 expression vector pcDNA3-VP6
  • Luciferase activity measurement MA104 cells expressing VP6 were seeded in a 96-well culture plate at 1 ⁇ 104 cells/well and cultured overnight. The cells were infected with the NLuc-expressing single-infective rotavirus prepared in (2) at an MOI of 0.01, and the medium and cells were collected over time and frozen and thawed to prepare cell lysates. The luciferase activity of the cell lysates was measured using the Nano-Glo Luciferase Assay System (trade name, Promega).
  • Example 7 Neutralization test using luciferase-expressing single-infectious rotavirus Materials and Methods (1) Luciferase-expressing, single-infectious rotavirus The NLuc-expressing, single-infectious rotavirus prepared in Example 6-2 was used. As a control, a NLuc-expressing, self-replicating rotavirus was used, which was prepared by replacing pT7-VP6SA11/ ⁇ 169-174+linker with pT7-VP6SA11, in which no mutation was introduced into VP6, in the NLuc-expressing, single-infectious rotavirus prepared in Example 6-2. The viral titers of both viruses were measured, and viruses with a titer of 100 FFU were prepared for each.
  • Serum Anti-rSA11 as described by Kanai et al. (J Virol. 2020 Dec 22;95(2):e01374-20.), was used. Anti-rSA11 was obtained by orally administering wild-type SA11 strain at 1.0 ⁇ 10 6 FFU/mouse to BALB/c mice (male, 3 weeks old) and repeating the administration 7 times at 2-week intervals, followed by blood sampling.
  • Example 8 Preparation of a single-infectious rotavirus having a human rotavirus coat protein
  • SA11 strain is a simian rotavirus
  • a single-infectious rotavirus is actually used as a vaccine or an antigen for neutralization tests, it is desirable to have the human rotavirus envelope protein. Therefore, Kanai et al. (J Virol. 2020 Dec 22;95(2):e01374-20.) attempted to create a single-infectious rotavirus carrying a segmented RNA genome encoding VP7 of human rotavirus isolated from the stool of nine children with acute gastroenteritis in Japan.
  • VP7 is an envelope protein located in the outermost layer of the virus particle and is a protein that determines the antigenicity and immunogenicity of the virus (see Figure 13 (A)).
  • pT7-VP6 ⁇ 169-174+linker encoding a mutated VP6 was used instead of pT7-VP6SA11
  • pT7-hVP7 a segmented RNA genome expression vector encoding VP7 of human rotavirus
  • capping enzyme expression vectors pCAGD1R and pCAG-D12L
  • rotavirus NSP2 rotavirus NSP2 expression vectors
  • pCAG-NSP2SA11 rotavirus NSP5 expression vectors
  • pcDNA3-VP6 expression vector rotavirus VP6 expression vector
  • the virus titers were calculated for the five types of single-infectious rotaviruses ( ⁇ 169-174 + linker/G1, ⁇ 169-174 + linker/G2, ⁇ 169-174 + linker/G3, ⁇ 169-174 + linker/G8, ⁇ 169-174 + linker/G9) with segmented RNA genomes encoding VP7 of the human rotaviruses produced, and the single-infectious rotavirus SA11 strain ( ⁇ 169-174 + linker) using the same method as in "Materials and Methods" (10) of Example 1.
  • Example 9 Neutralization test using single-infectious rotavirus expressing VP7 of human rotavirus To analyze whether single-infectious rotaviruses expressing VP7 from human rotaviruses of different genotypes (serotypes) have different antigenicities, neutralization tests were performed.
  • Single-episode infectious rotaviruses having human rotavirus VP7 As single-episode infectious rotaviruses expressing human rotavirus VP7, ⁇ 169-174+linker/G1, ⁇ 169-174+linker/G3, ⁇ 169-174+linker/G8, and ⁇ 169-174+linker/G9, which have different VP7 genotypes (serotypes) prepared in Example 8, were used.
  • ⁇ 169-174+linker which is a single-episode infectious rotavirus of the SA11 strain, and wild-type SA11 strain were used. The viral titer of each virus was measured, and a virus with a titer of 100 FFU was prepared for each virus.
  • Serum Anti-rSA11 described in Kanai et al. (J Virol. 2020 Dec 22;95(2):e01374-20.) was used. Anti-rSA11 was obtained by orally administering wild-type SA11 strain to BALB/c mice (male, 3 weeks old) at 1.0 ⁇ 10 6 FFU/mouse, repeating the administration 7 times at 2-week intervals, and then collecting blood serum.
  • the VP7 genotype (serotype) of the wild-type SA11 strain is G3.
  • the primary antibody for the immunofluorescence antibody technique was rabbit-derived anti-rotavirus NSP4 antibody, and the secondary antibody was CF488-labeled anti-rabbit IgG antibody (Biotum).
  • the reduction rate of the number of infected cells compared to the number of infected cells in the control without serum was calculated as the neutralization rate.
  • results are shown in FIG. 11.
  • the wild-type SA11 strain (genotype G3), the single-infectious rotavirus ⁇ 169-174+linker (genotype G3) expressing VP7 of the SA11 strain, and the single-infectious rotavirus ⁇ 169-174+linker/G3 expressing human VP7 of genotype G3 were efficiently neutralized by serum immunized with the wild-type SA11 strain (genotype G3).
  • the single-infectious rotavirus expressing human VP7 of a genotype other than G3 had low neutralizing activity.
  • Trizol reagent Invitrogen
  • a Thunderbird probe One-step qRT-PCR kit (Toyobo) was used. The following primer-probe set was used to detect the VP1 segment of rotavirus. Forward: AGGCAAACCATTGGAGGCAGAC (SEQ ID NO: 14) Reverse: CCAACCAGAACATGACTGCATT (SEQ ID NO: 15) Probe: FAM-TCCAACAGCGGAGGAATATACGGAC-TAMRA (SEQ ID NO: 16)
  • cecal samples were added to the culture medium of VP6-expressing MA104 cells to infect them with the virus, and viral titers were measured.
  • a t-test was performed for the number of viral RNA copies, and a Mann-Whitney U test was performed for the viral titers.
  • a P value of 0.05 or less was considered to indicate a significant difference.
  • Example 12 Creation of a single-infectious rotavirus by VP7 mutation introduction Materials and Methods In the method for producing a single-infectious rotavirus in Example 1, the mutated gene was changed from VP6 to VP7, and the VP6 expression vector was changed to a VP7 expression vector to produce a single-infectious rotavirus. The materials not used in Example 1 are shown below.
  • Rotavirus VP7 expression vector The VP7 expression vector was prepared by inserting the protein coding region DNA of the VP7 gene (SEQ ID NO: 9) of SA11 strain into the BamHI and EcoRI cleavage sites of the pcDNA3.1(+) plasmid to prepare pcDNA3-VP7.
  • the coding region DNA was synthesized by an artificial gene synthesis service (GenScript).
  • VP7 persistent expression cells The rotavirus VP7 gene was introduced into the pLVSIN-CMV-Neo vector (Takara Bio) and co-transfected with psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into 293T cells to produce lentivirus. The lentivirus was then infected into African green monkey MA104 cells (ATCC CRL-2378.1), and after 2 days, drug selection was performed with G418 (800 ⁇ g/mL). From the VP7-expressing MA104 cells obtained by drug selection, cells that persistently and strongly expressed VP7 were cloned by limiting dilution.
  • Example 13 Comparison of proliferation ability between normal MA104 cells and MA104 cells continuously expressing VP7
  • the VP7 mutant rotavirus VP7- ⁇ Domain II prepared in Example 12 was compared for its proliferation ability in normal MA104 cells and in MA104 cells that persistently express VP7.
  • Materials and Methods Normal MA104 cells and MA104 cells expressing VP7 were seeded at 7.5 x 104 cells/well in a 24-well culture plate and cultured overnight.
  • VP7- ⁇ Domain II was infected at an MOI of 0.01, and the medium and cells were frozen and thawed three times at 0, 24, 48, and 72 hours after infection to prepare cell lysates and measure the virus titer.
  • a wild-type virus without VP7 mutation was used as a control.
  • the virus titer was measured in the same manner as in Example 1.
  • Example 19 Construction of VP4-deleted, single-infectious rotavirus having human rotavirus coat protein
  • Kanai et al. J Virol. 2020 Dec 22;95(2):e01374-20.
  • Kanai et al. produced a single-infectious rotavirus having a segmented RNA genome encoding VP7 of human rotavirus isolated from the stool of nine children with acute gastroenteritis in Japan.
  • pT7-VP4SA11-120bp encoding the mutated VP4 was used instead of pT4-VP6SA11
  • pT7-hVP7 a segmented RNA genome expression vector encoding VP7 of human rotavirus
  • the viral titers were calculated in the same manner as in Example 1 for the four types of VP4-deleted single-infectious rotaviruses (VP4-120bp/G1, VP4-120bp/G2, VP4-120bp/G8, VP4-120bp/G9) having segmented RNA genomes encoding the VP7 of the human rotaviruses produced.
  • Example 20 Preparation of a single-infectious rotavirus carrying the spike protein gene of the new coronavirus in the VP4 segment
  • Materials and Methods (1) Preparation of a single-infectious rotavirus expressing the spike protein of the new coronavirus
  • a gene encoding the receptor binding domain (RBD) of the spike protein of the new coronavirus (SARS-CoV-2) was used as a foreign gene.
  • the new coronavirus RBD gene was amplified from the spike protein expression plasmid described in Minami et al. (Microbiol Spectr. 2024 Apr 2;12(4):e0285923.).
  • HRP-labeled anti-rabbit IgG antibody Nacalai Tesque
  • HRP-labeled anti-mouse antibody Nacalai Tesque
  • Chemi-Lumi-One Ultra trade name, Nacalai Tesque

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Abstract

La présente invention propose un rotavirus infectieux à cycle unique caractérisé en ce qu'il présente une mutation dans au moins une protéine virale du rotavirus, qui est choisie dans le groupe constitué par VP1, VP2, VP3, VP4, VP6, VP7, NSP2, NSP3 et NSP4. Le rotavirus infectieux à cycle unique selon la présente invention peut être utilisé dans un vaccin contre le rotavirus, un procédé test de neutralisation du rotavirus, et similaires.
PCT/JP2024/019050 2023-05-24 2024-05-23 Rotavirus infectieux à cycle unique et son utilisation Ceased WO2024242174A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003507040A (ja) * 1999-08-17 2003-02-25 スミスクライン ビーチャム バイオロジカルズ ソシエテ アノニム ワクチン
WO2018062199A1 (fr) * 2016-09-27 2018-04-05 国立大学法人大阪大学 Procédé de préparation de rotavirus recombinant artificiel
JP2022521217A (ja) * 2019-02-19 2022-04-06 メディカゴ インコーポレイテッド ロタウイルスｖｐ７融合タンパク質およびそれらを含むロタウイルス様粒子

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003507040A (ja) * 1999-08-17 2003-02-25 スミスクライン ビーチャム バイオロジカルズ ソシエテ アノニム ワクチン
WO2018062199A1 (fr) * 2016-09-27 2018-04-05 国立大学法人大阪大学 Procédé de préparation de rotavirus recombinant artificiel
JP2022521217A (ja) * 2019-02-19 2022-04-06 メディカゴ インコーポレイテッド ロタウイルスｖｐ７融合タンパク質およびそれらを含むロタウイルス様粒子

Non-Patent Citations (2)

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Title
MATHIEU MAGALI, PETITPAS ISABELLE, NAVAZA JORGE, LEPAULT JEAN, KOHLI EVELYNE, POTHIER PIERRE, VENKATARAM PRASAD B V, COHEN JEAN, R: "Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion", THE EMBO JOURNAL, vol. 20, no. 7, 1 January 2001 (2001-01-01), pages 1485 - 1497, XP093243051 *
SETTEMBRE ETHAN C, CHEN JAMES Z, DORMITZER PHILIP R, GRIGORIEFF NIKOLAUS, HARRISON STEPHEN C: "Atomic model of an infectious rotavirus particle", THE EMBO JOURNAL / EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, IRL PRESS, OXFORD, vol. 30, no. 2, 19 January 2011 (2011-01-19), Oxford , pages 408 - 416, XP093243054, ISSN: 0261-4189, DOI: 10.1038/emboj.2010.322 *

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