WO2019093347A1 - Pb1 mutant dans une arn polymérase du virus de la grippe - Google Patents

Pb1 mutant dans une arn polymérase du virus de la grippe Download PDF

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WO2019093347A1
WO2019093347A1 PCT/JP2018/041262 JP2018041262W WO2019093347A1 WO 2019093347 A1 WO2019093347 A1 WO 2019093347A1 JP 2018041262 W JP2018041262 W JP 2018041262W WO 2019093347 A1 WO2019093347 A1 WO 2019093347A1
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influenza virus
mutant
amino acid
strain
seq
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忠相 内藤
峰輝 齊藤
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学校法人 川崎学園
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

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  • the present invention relates to a mutant PB1 of influenza virus RNA polymerase, a ribonucleic acid containing a base sequence complementary to a base sequence encoding the mutant PB1, a mutant influenza virus containing the ribonucleic acid, a mutant type obtainable from the virus BACKGROUND OF THE INVENTION 1.
  • Field of the Invention relates to an influenza virus library, a method of screening for possible influenza viruses using the library in the future.
  • Influenza which causes a major epidemic each winter, is the nation's largest infectious disease that significantly affects the social life and health of all generations of people, from infants to the elderly.
  • the genome of influenza virus is an RNA with a total number of about 10,000 divided into eight, and is replicated by a low fidelity viral RNA polymerase, resulting in frequent gene mutation.
  • seasonal influenza viruses frequently undergo antigenic mutations, and there is a discrepancy between the antigenicity of the vaccine strain selected from the epidemic forecast and the actual epidemic strain, and the vaccine's onset and aggravation inhibition effect is significantly reduced. Is a problem.
  • Non-patent Document 1 The conventional technology at the vaccine development site can only predict the epidemic strain of the next season from mutation information introduced into the gene of the virus that has spread in the past (desktop analysis using a database), and there is a possibility that it will spread in the future Attempts have been made to artificially create certain virus candidate strains and to examine the effects of vaccines beforehand.
  • attempts have been made to actually produce a recombinant virus (antigen mutation virus) to predict an antigen mutation site, and the results have been reported (Non-patent Document 1).
  • HA hemagglutinin
  • NA neuraminidase
  • An object of the present invention is to provide a system (future epidemic strain prediction system) capable of predicting in advance the antigenic mutation site occurring in a pandemic strain after the next season using a mutant influenza virus having a recent pandemic strain as a source strain. .
  • the present inventors used the A / Puerto Rico / 8/1934 (H1N1) strain (PR8 strain), which is the maternal virus at the time of production of the current influenza vaccine, to reverse control the PB1 subunit of the viral RNA polymerase Were modified to try to develop a "low fidelity vaccine producing strain" that is susceptible to mutations during genome replication.
  • the present inventors produced a low-fidelity vaccine with a virus (PR8-PB1-K471H virus) having a genome encoding mutant PB1 in which the 471th Lys residue of PB1 strain PB1 was replaced with a His residue (PR8-PB1-K471H virus)
  • the strain was confirmed to be a strain to which a mutation was more easily introduced than the PR8 strain (mutation efficiency was improved by about 3.6 times).
  • the present inventors confirmed that there is no bias in the base substitution species introduced into the mutant influenza virus library, and the PB1-K471H virus is a very excellent "mutaable strain", and this function We considered that we could construct a prediction system for a pandemic strain with an antigen mutation by applying.
  • the present invention [1] Mutant polymerase basic protein 1 (PB1) of influenza virus RNA polymerase, which comprises the amino acid sequence represented by SEQ ID NO: 2; [2] The mutant PB1 according to [1], which comprises the following amino acid sequence (1) or (2): (1) the amino acid sequence represented by SEQ ID NO: 4, (2) A partial amino acid sequence (SEQ ID NO: 2) substantially identical to the amino acid sequence represented by SEQ ID NO: 4 and represented by amino acid numbers 471 to 486 of SEQ ID NO: 4 is left An amino acid sequence; [3] RNA comprising a nucleotide sequence complementary to the nucleotide sequence encoding the mutant PB1 described in [1] or [2]; [4] [3] a mutant influenza virus comprising a genomic RNA comprising the RNA according to [4] [3]; [5] The mutant influenza virus according to [4], which is derived from A / Puerto Rico / 8/1934 (H1N1) strain; [6] The hemagglutinin (HA), 5-
  • the mutant influenza virus having a genome encoding mutant PB1 in which the 471th Lys residue of PB1 of PR8 strain (the 1st Lys residue of polymerase motif D of PB1 of PR8 strain) is replaced with a His residue is Influenza viruses in which mutations are randomly introduced at "all amino acid sites" of "all influenza virus proteins" can be produced. Therefore, by propagating the mutant influenza virus containing the epidemic strain HA and NA, it is possible to efficiently obtain a mutated influenza virus library containing an influenza virus having HA and NA different from those of the epidemic strain. By isolating influenza virus which may be prevalent in the future from the library, it becomes possible to prepare and supply a vaccine against the influenza virus in advance.
  • FIG. 6 shows the hemagglutination activity of PR8-PB1-K471H virus. It is a figure which shows the mutagenesis efficiency of PR8-PB1-K471H virus. It is a figure which shows the kind and ratio of the conversion base of PR8-PB1-K471H virus.
  • the present invention relates to a mutated Polymerase basic protein 1 (PB1) of influenza virus RNA polymerase comprising the amino acid sequence represented by SEQ ID NO: 2 (hereinafter sometimes referred to as a mutated PB1 (1) of the present invention) I will provide a.
  • PB1 Polymerase basic protein 1
  • Influenza virus is a virus surrounded by an envelope with a particle size of about 100 nm in diameter belonging to the Orthomyxoviridae family.
  • the influenza virus genome is a single-stranded RNA (minus strand) segmented into eight strands, which binds to viral RNA polymerase and RNA binding protein (NP) to form a vRNP complex.
  • the viral RNA polymerase is composed of three subunits, PB1, PB2 (polymerase basic protein 2) and PA (polymerase acidic protein), among which PB1 functions as a catalytic subunit.
  • PB1 is a protein encoded by the second segment of the influenza virus genome and has a common polymerase motif A to D highly conserved among subtypes.
  • the Lys residue of amino acid No. 1 of polymerase motif D (SEQ ID NO: 1; A / Puerto Rico / 8/1934 (H1N1) strain (hereinafter, PR8 strain)) is a nucleotide approach required for RNA synthesis reaction
  • a Lys residue to His residue substitution (SEQ ID NO: 2), located in the vicinity of the mouth, is a function of wild type PB1 of the PR8 strain (a protein comprising the amino acid sequence represented by SEQ ID NO: 3).
  • a decrease in function refers to a decrease in viral RNA polymerase activity at 37 ° C., a decrease in the accuracy of viral genome replication, and the like. The degree of reduction is not particularly limited unless it is lost.
  • the mutant PB1 (1) of the present invention is preferably a mutant PB1 having the amino acid sequence shown by SEQ ID NO: 2 and having a function lower than that of a protein containing the amino acid sequence shown by SEQ ID NO: 3. .
  • the mutant PB1 (1) of the present invention preferably comprises the following amino acid sequence (1) or (2) (hereinafter sometimes referred to as the mutant PB1 (2) of the present invention).
  • a partial amino acid sequence (SEQ ID NO: 2) substantially identical to the amino acid sequence represented by SEQ ID NO: 4 and represented by amino acid numbers 471 to 486 of SEQ ID NO: 4 is left Amino acid sequence.
  • the mutant PB1 (2) of the present invention is an amino acid sequence obtained by replacing the Lys residue of amino acid number 471 of wild type PB1 (amino acid sequence represented by SEQ ID NO: 3) of PR8 strain with a His residue. That is, the amino acid sequence represented by SEQ ID NO: 4 is included.
  • the Lys residue (the Lys residue corresponds to the Lys residue of amino acid No. 1 of polymerase motif D (SEQ ID NO: 1)) of the amino acid No. 471 of the amino acid sequence represented by SEQ ID NO: 3 to His residue
  • substitution results in a decrease in the function of PB1 containing the amino acid sequence of SEQ ID NO: 3.
  • the "reduced function" may be the same as the mutant PB1 (1) of the present invention.
  • the mutant PB1 (2) of the present invention is also a partial amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 and represented by amino acid numbers 471 to 486 of SEQ ID NO: 4 SEQ ID NO: 2) contains the remaining amino acid sequence.
  • the partial amino acid sequence (SEQ ID NO: 2) represented by amino acid numbers 471 to 486 of SEQ ID NO: 4 is not changed, and in amino acid sequence parts other than the partial amino acid sequence, SEQ ID NO: 4
  • homology is an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is a sequence for optimal alignment. It means the ratio (%) of identical amino acid residues and similar amino acid residues to total overlapping amino acid residues in which the introduction of a gap in one or both may be considered.
  • Similar amino acids means amino acids similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), small side chain amino acids (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is predicted that such substitution with similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and described in various references (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
  • NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
  • algorithm for determining amino acid sequence homology for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [The algorithm is NBLAST and XBLAST. Described in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997)), Needleman et al., J. Mol.
  • the partial amino acid sequence (SEQ ID NO: 2) substantially identical to the amino acid sequence represented by SEQ ID NO: 4 and represented by amino acid numbers 471 to 486 of SEQ ID NO: 4 remains.
  • the partial amino acid sequence (SEQ ID NO: 2) represented by amino acid numbers 471 to 486 of SEQ ID NO: 4 does not change, and the amino acid sequence in the amino acid sequence part other than the partial amino acid sequence It is an amino acid sequence having an identity of about 85% or more, preferably about 90% or more, more preferably about 95% or more, and most preferably about 98% or more to the amino acid sequence represented by No. 4.
  • a partial amino acid sequence (SEQ ID NO: 4) substantially identical to the amino acid sequence represented by SEQ ID NO: 4 and represented by amino acid numbers 471 to 486 of SEQ ID NO: 4 as a mutant PB1 containing a sequence : 2) is retained, and a mutant PB1 having a function that is lower than that of the protein containing the amino acid sequence shown by SEQ ID NO: 3 is preferable.
  • the "reduced function" may be the same as the mutant PB1 (1) of the present invention.
  • mutant PB1 (2) of the present invention may be, for example, 1 or 2 or more (preferably about 1 to 30 or so, more preferably 1 to 2) of the amino acid sequence represented by SEQ ID NO: 4 Amino acid sequence from which about 10, more preferably 1 to 5) amino acids are deleted, (2) 1 or 2 or more (preferably about 1 to 30 or so, preferably about 1 to 30 amino acid sequences in the amino acid sequence represented by SEQ ID NO: 4 Preferably, about 1 to 10, more preferably 1 to 5) amino acids are added, (3) 1 or 2 or more (preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 4 1 or more (preferably 1 to 2 or more (preferably) of the amino acid sequence represented by SEQ ID NO: 4), more preferably 1 to 10 or so, more preferably 1 to 5 amino acids.
  • the partial amino acid sequence (SEQ ID NO: 2) containing the amino acid sequence in which the acid is substituted with another amino acid, or the amino acid sequence combining them (5) and represented by amino acid numbers 471 to 486 of SEQ ID NO: 4 remains.
  • amino acids used for substitution similar amino acids are preferably used.
  • the mutant PB1 (1) and the mutant PB1 (2) of the present invention can be produced according to known peptide synthesis methods.
  • the peptide synthesis method may be, for example, either solid phase synthesis or liquid phase synthesis.
  • a partial peptide or amino acid capable of constituting the mutant PB1 of the present invention and the remaining portion are condensed, and when the product has a protective group, the target mutant PB1 can be produced by removing the protective group. it can.
  • the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the methods described in the following (1) and (2).
  • the mutant PB1 of the present invention thus obtained can be purified and isolated by known purification methods.
  • the purification method for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, a combination thereof, and the like can be mentioned.
  • the mutant PB1 of the present invention obtained by the above method is a free form
  • the free form can be converted to a suitable salt by a known method or a method analogous thereto
  • the mutant of the present invention When PB1 is obtained as a salt, the salt can be converted to a free form or another salt by a known method or a method analogous thereto.
  • the mutant PB1 of the present invention is cultured in a transformant containing deoxyribonucleic acid (DNA) containing a nucleotide sequence encoding the same (hereinafter sometimes referred to as a DNA encoding the mutant PB1 of the present invention).
  • DNA deoxyribonucleic acid
  • they can be produced by separating and purifying mutant PB1 from the resulting culture.
  • the DNA encoding the mutant PB1 of the present invention may be double stranded or single stranded. When double stranded, it may be double stranded DNA, double stranded RNA or a hybrid of DNA: RNA.
  • Examples of DNA encoding the mutant PB1 of the present invention include synthetic DNA.
  • cDNA plus strand
  • RT-PCR method Reverse Transcriptase-PCR
  • a single-stranded genomic RNA minus strand
  • kits for example, Mutan TM -super Express Km (TAKARA BIO INC.), using Mutan TM -K (TAKARA BIO INC. ) or the like, ODA-LA PCR method, Gapped duplex method, known per se, such as Kunkel method It can be obtained by conversion according to a method or a method according to them.
  • influenza virus cDNA library prepared by inserting a single-stranded genomic RNA (minus strand) -derived cDNA (plus strand) fragment prepared from the above-mentioned influenza virus into a suitable vector, colonies or plaques may be generated.
  • the cloned cDNA can also be obtained by conversion according to the method described above, such as by hybridization or PCR.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • the present invention also provides a ribonucleic acid (RNA) containing a nucleotide sequence complementary to the nucleotide sequence encoding the mutant PB1 of the present invention (hereinafter sometimes referred to as RNA encoding the mutant PB1 of the present invention).
  • RNA ribonucleic acid
  • the RNA encoding mutant PB1 of the present invention is an antisense strand (ie, non-coding strand or minus strand).
  • RNA containing a nucleotide sequence complementary to the nucleotide sequence encoding the mutant PB1 of the present invention include, for example, nucleotides complementary to the nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 5
  • examples include RNA containing a sequence, preferably RNA containing a base sequence complementary to a base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 6.
  • the base sequence substantially identical to the base sequence represented by SEQ ID NO: 5 (or SEQ ID NO: 6) is, for example, a sequence in the base sequence represented by SEQ ID NO: 5 (or SEQ ID NO: 6)
  • a nucleotide sequence having a silent mutation which does not change the amino acid sequence represented by SEQ ID NO: 2 (or SEQ ID NO: 4) is included.
  • RNA encoding the mutant PB1 of the present invention is, as described above, a cDNA (plus strand) directly amplified by RT-PCR using single-stranded RNA (minus strand) prepared from influenza virus as a template, Using a synthetic DNA primer having a desired mutation, which is a part of the base sequence of cDNA, cloning by amplification according to the above-mentioned method, incorporating into an expression vector, and introducing the expression vector into cells for expression Can be obtained by
  • the present invention also provides a mutant influenza virus (hereinafter sometimes referred to as a mutant influenza virus of the present invention), which comprises a genomic RNA comprising an RNA encoding a mutant PB1 of the present invention.
  • a mutant influenza virus of the present invention has the property of causing mutations throughout the grown influenza virus genome at a high frequency as compared to wild-type influenza virus, since the function of PB1 is reduced.
  • influenza virus of the present invention may be any of A-type, B-type and C-type, but A-type having a diversity in viral genome is more preferable.
  • influenza A virus has many amino acid sequence differences between hemagglutinin (HA) and neuraminidase (NA), which are molecules on the surface of the envelope, and is represented by a combination of 16 types of HA and 9 types of NA. It may be any of the types (H1N1 to H16N9).
  • the mutant influenza virus of the present invention may be derived from any known strain, for example, a mutant influenza virus derived from A / Puerto Rico / 8/1934 (H1N1) strain. You may
  • the mutant influenza virus of the present invention can be produced by known methods. For example, it is possible to synthesize eight segmented influenza virus genomic RNAs, including an RNA encoding a mutant PB1 of the present invention, and PB1, PB2 isolated or recombinantly from each influenza virus genomic RNA and influenza virus
  • the eight vRNP complexes reconstituted in a test tube can be obtained by mixing PA, NP and NP. By introducing the obtained eight vRNP complexes into cells and culturing it, it is possible to isolate and purify the mutant influenza virus of the present invention from the cells (in vitro vRNP reconstitution method: Luytjes , W et al. Cell, 59, 1107-1113, 1989, etc.).
  • the mutant influenza virus of the present invention can be easily recovered.
  • a promoter sequence to which host cell RNA polymerase I (Pol I) can bind, a DNA complementary to eight segmented influenza virus genomic RNAs including RNA encoding mutant PB1 of the present invention
  • cells are transfected with eight influenza virus genome RNA synthesis plasmids containing a and a terminator sequence, and four influenza virus protein expression plasmids expressing PB1, PB2, PA and NP.
  • RNA polymerase I (Pol I) of the host cell non-modified RNA to which Cap structure or Poly A is not added can be synthesized from the plasmid for synthesizing influenza virus genome RNA, and can be functioned as influenza virus genome.
  • mRNA of each influenza virus protein can be transcribed by using RNA polymerase II (Pol II) of the host cell.
  • RNA polymerase II Poly II
  • the eight influenza virus genomic RNAs synthesized in the cell and each influenza virus protein form a vRNP complex, and by culturing the cells, the mutant influenza virus of the present invention is isolated and purified from the cells. It is possible.
  • the plasmid for synthesizing influenza virus genomic RNA for example, excises a DNA fragment having a base sequence complementary to eight segmented influenza virus genomic RNAs including the above-mentioned mutant PB1 encoding RNA of the present invention, Each DNA fragment can be produced by ligating downstream of a promoter in an appropriate plasmid.
  • the plasmid for synthesizing influenza virus genomic RNA of the present invention can be obtained by replacing the wild-type PB1 contained in and directly introducing the gene into cells.
  • the plasmid for influenza virus protein expression of the present invention may be prepared, for example, by excising the above-mentioned PB1, PB2, PA or NP-encoding DNA fragment and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured.
  • a plasmid for animal cells eg, pCAGGS, pSR ⁇ , pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo
  • pCAGGS plasmid for animal cells
  • pSR ⁇ plasmid for animal cells
  • pA1-11 plasmid for animal cells
  • pXT1 plasmid for animal cells
  • an RNA polymerase I promoter is used for a plasmid for influenza virus genomic RNA synthesis.
  • a chicken ⁇ actin-derived promoter eg, CAG promoter
  • CMV cytomegalovirus
  • HSV human immunodeficiency virus
  • HSV human immunodeficiency virus
  • RSV Rous sarcoma virus
  • MMTV mouse mammary tumor virus
  • MoMLV Moloney murine leukemia virus
  • HSV Herpes simplex virus
  • HSV HSV thymidine kinase
  • SV40 derived promoter eg SV40 early promoter
  • Epstein bar virus EBV
  • SV40 ori an SV40 origin of replication
  • selection markers include dihydrofolate reductase gene (hereinafter sometimes abbreviated as dhfr, methotrexate (MTX) resistance), neomycin resistant gene (hereinafter sometimes abbreviated as neo r , G418 resistance), etc. It can be mentioned.
  • the target gene can also be selected by a medium containing no thymidine.
  • a base sequence (signal codon) encoding a signal sequence suitable for the host is added to the 5 'end of the DNA encoding the influenza virus protein of the present invention (or replaced with a native signal codon) You may For example, an insulin signal sequence, an ⁇ -interferon signal sequence, an antibody molecule signal sequence, etc. may be used.
  • the mutant influenza virus of the present invention can be produced by introducing the above plasmid into cells and culturing it. Animal cells are used as cells.
  • animal cells for example, human-derived cells (eg, HEK293, Per. C6), canine-derived cells (eg, MDCK), monkey-derived cells (eg, Vero), duck-derived cells (eg, EB66), etc. are used .
  • human-derived cells eg, HEK293, Per. C6
  • canine-derived cells eg, MDCK
  • monkey-derived cells eg, Vero
  • duck-derived cells eg, EB66
  • Gene transfer can be carried out according to a known method, for example, described in Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology 52, 456 (1973)
  • the gene can be introduced according to the method of
  • Culturing of the cells can be carried out according to known methods, and examples of the medium include minimal essential medium (MEM) containing about 5 to about 20% fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 Medium, 199 medium, etc. are used.
  • the pH of the culture medium is preferably about 6 to about 8.
  • Culturing is usually performed at about 30 to about 40 ° C., preferably about 34 ° C., for about 72 hours. Aeration and / or stirring may be performed as necessary.
  • the mutant influenza virus of the present invention can be produced in cells.
  • the mutant influenza virus of the present invention budding extracellularly can be isolated and purified according to a method known per se.
  • influenza virus is known to adsorb to sulfated cellulose, sulfated cellulose beads are packed in a 1 ml column, the mutant influenza virus of the present invention is adsorbed from the culture supernatant to the beads, and washed with a washing buffer Then, the mutant influenza virus of the present invention can be eluted with elution buffer and recovered.
  • the sulfate ester content on the bead surface and the salt concentration of the elution buffer can be appropriately determined by those skilled in the art.
  • the mutant influenza virus of the present invention in the culture supernatant can be separated and concentrated also by sucrose concentration gradient centrifugation. These methods can also be combined as appropriate.
  • the present invention further comprises a mutant influenza virus virus of the present invention (hereinafter referred to as a pandemic strain-derived mutant influenza virus virus of the present invention), which comprises a genomic RNA further comprising the following (1) and / or (2) RNAs: Also provide).
  • a mutant influenza virus virus of the present invention hereinafter referred to as a pandemic strain-derived mutant influenza virus virus of the present invention
  • a genomic RNA further comprising the following (1) and / or (2) RNAs: Also provide.
  • RNA containing a nucleotide sequence complementary to the nucleotide sequence encoding neuraminidase (NA) derived from a pandemic strain a mutant influenza virus virus of the present invention
  • the endemic strain is not particularly limited as long as it is a known endemic strain, and examples thereof include A / New York / 4747/2009 (H1N1) and A / Texas / 50/2012 (H3N2).
  • the HA and NA derived from the A / New York / 4747/2009 (H1N1) strain include the amino acid sequence represented by SEQ ID NO: 7 and the amino acid sequence represented by SEQ ID NO: 8, respectively.
  • examples of HA and NA derived from the A / Texas / 50/2012 (H3N2) strain include the amino acid sequence represented by SEQ ID NO: 9 and the amino acid sequence represented by SEQ ID NO: 10.
  • the pandemic strain-derived mutant influenza virus of the present invention can be produced in the same manner as the mutant influenza virus of the present invention.
  • the RNA comprising a nucleotide sequence complementary to the nucleotide sequence encoding HA is the fourth segment
  • the RNA comprising a nucleotide sequence complementary to the nucleotide sequence encoding NA is Each exists in the sixth segment.
  • the fourth segment of influenza virus genomic RNA is derived from a pandemic strain Replace with RNA containing a nucleotide sequence complementary to the HA-encoding nucleotide sequence, and replace the sixth segment of influenza virus genomic RNA with RNA containing a nucleotide sequence complementary to the nucleotide sequence encoding NA from a pandemic strain
  • the pandemic strain-derived mutant influenza virus of the present invention can be obtained.
  • a plasmid containing a DNA containing a nucleotide sequence complementary to the nucleotide sequence encoding the mutant PB1 of the present invention In addition to substitution by, a plasmid containing a DNA containing a nucleotide sequence complementary to a nucleotide sequence encoding HA from a pandemic strain, Complementary to an RNA containing a nucleotide sequence complementary to a nucleotide sequence encoding NA from a pandemic strain
  • the pandemic strain-derived mutant influenza virus of the present invention can be obtained by replacing it with a plasmid containing the relevant DNA.
  • the pandemic strain-derived mutant influenza virus of the present invention like the mutant influenza virus of the present invention, has a reduced function of the mutant PB1 of the present invention as compared to wild-type PB1, and thus the entire influenza virus genome that has grown. And have the property of causing mutations more frequently than the epidemic strain. Therefore, the present invention provides a mutant influenza virus library (hereinafter sometimes referred to as a mutant influenza virus library of the present invention) obtained by propagating the pandemic strain-derived mutant influenza virus of the present invention. provide.
  • a mutant influenza virus library hereinafter sometimes referred to as a mutant influenza virus library of the present invention
  • the mutant influenza virus library of the present invention can be produced by known methods.
  • the allantoic cavity of embryonated chicken eggs hatched for about 11 days at 38 ° to 39 ° C. is inoculated with the pandemic strain-derived mutant influenza virus of the present invention.
  • the embryonated chicken eggs after virus inoculation are cultured at 32 ° C. to 36 ° C., 60 to 80% humidity for 48 to 72 hours to propagate the virus. After the end of the culture, it is cooled at 4 ° C for about 12 hours to stop the growth of the virus. Thereafter, the air chamber of the embryonated chicken egg is divided and the allantoic fluid is collected.
  • the mutant influenza virus library of the present invention can be purified by sucrose concentration gradient centrifugation, followed by concentration by ultrafiltration, chemical method or the like.
  • the mutant influenza virus library of the present invention is obtained by ultracentrifugation (35,000 rpm) of the virus in a density gradient of 0 to 60% sucrose and collecting fractions having a sucrose density of about 40%. can do.
  • influenza virus genome many mutations are introduced into HA and / or NA every year to cause antigenic drift (alteration of antigenicity), which is considered to be the cause of epidemic epidemic. Therefore, when the vaccine which has HA and NA which do not correspond with HA and NA of the influenza which spreads is vaccinated, the preventive effect can not be expected. Conventionally, prediction of antigenic drift against influenza virus has been attempted, but comprehensively predicting influenza virus that has maintained infectivity has many technical problems.
  • the mutant influenza virus library of the present invention is more frequently mutated in the entire amino acid sequence of all viral proteins including HA and NA than the influenza virus library obtained by propagating a pandemic strain Because it is a population of influenza virus, it can cover all possible pandemic strains in the future.
  • the mutant influenza virus library of the present invention it is possible to screen new mutant influenza viruses that may be prevalent in the future. For this reason, the present invention is also expected to spread in the future, including contacting a neutralizing antibody against a pandemic strain with the mutant influenza virus library of the present invention, and selecting a mutant influenza virus whose infectivity is not neutralized.
  • the present invention provides a screening method for a possible mutant influenza virus (hereinafter sometimes referred to as the screening method of the present invention).
  • the screening method of the present invention comprises the step of contacting a neutralizing antibody against a pandemic strain with the mutant influenza virus library of the present invention.
  • the neutralizing antibody against a pandemic strain may be any of an antiserum, a polyclonal antibody and a monoclonal antibody. These antibodies can be produced according to a method for producing antisera or antibodies known per se.
  • any of the pandemic strain itself described above or HA, NA or a partial peptide thereof derived from the pandemic strain may be used. can do.
  • the most preferred antigen among them is the pandemic strain itself.
  • a pandemic strain can be obtained from a known storage facility of influenza virus.
  • the HA, NA or its partial peptide derived from the pandemic strain is chemically synthesized by a known peptide synthesis method using, for example, a peptide synthesizer etc., and the DNA encoding the HA, NA or its partial peptide derived from the pandemic strain is used. It is produced by culturing the transformant containing the same or by biochemically synthesizing a nucleic acid encoding HA, NA or its partial peptide derived from the epidemic strain as a template using a cell-free transcription / translation system.
  • partial amino acid sequences include those consisting of 3 or more consecutive amino acid residues, preferably 4 or more, more preferably 5 or more, still more preferably 6 or more consecutive amino acid residues.
  • the amino acid sequence is composed of, for example, 20 or less consecutive amino acid residues, preferably 18 or less, more preferably 15 or less, still more preferably 12 or less consecutive amino acid residues Can be mentioned. Some of these amino acid residues (eg, 1 to several) may be substituted by substitutable groups (eg, Cys, hydroxyl group etc.).
  • the peptide used as an antigen has an amino acid sequence containing one to several such partial amino acid sequences.
  • Antiserum and polyclonal antibody can be produced according to a method known per se or a method analogous thereto.
  • the antigen is administered to a warm-blooded animal, for example, by intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, or the like, alone or in combination with a carrier or diluent at the site where antibody production is possible.
  • Ru Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance antibody production ability upon administration. The administration is usually performed once every 1 to 6 weeks, for a total of about 2 to 10 times.
  • warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, hamsters, sheep, goats, donkeys and chickens.
  • An antibody-containing product against a pandemic strain can be collected from the warm-blooded animal administered and used as an antiserum as it is.
  • the antibody titer in serum can be measured, for example, by reacting a labeled antigen with an antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • Polyclonal antibodies can be collected from the blood, ascites fluid and the like of warm-blooded animals immunized by the above method, preferably from blood.
  • the measurement of polyclonal antibody titer in antiserum can be measured in the same manner as the measurement of antibody titer in antiserum described above.
  • Separation and purification of polyclonal antibodies can be carried out by methods known per se, such as separation and purification of immunoglobulins [eg salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg DEAE, QEAE Specific purification method to obtain antibody by collecting only the antibody by adsorption / desorption method, ultracentrifugation method, gel filtration method, active adsorbent such as antigen-bound solid phase or protein A or protein G, etc. It can be done according to.
  • immunoglobulins eg salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg DEAE, QEAE Specific purification method to obtain antibody by collecting only the antibody by adsorption / desorption method, ultracentrifugation method, gel filtration method, active adsorbent such as antigen-bound solid phase or protein A or protein G, etc. It can be done according to.
  • animal cells used for extracorporeal immunization include lymphocytes isolated from human and the above-mentioned warm-blooded animals (preferably mice and rats), peripheral blood, spleen, lymph nodes and the like, preferably B lymphocytes and the like. .
  • individuals or cell populations with elevated antibody titers are selected from warm-blooded animals (eg, mice, rats) or animal cells (eg, humans, mice, rats) immunized with an antigen. After harvesting the spleen or lymph node 2 to 5 days after the final immunization, or culturing it for 4 to 10 days after extracorporeal immunization, recover the cells, isolate the antibody-producing cells, and fuse this with myeloma cells. Production hybridomas can be prepared. The antibody titer in serum can be measured by the same method as described above.
  • the fusion operation can be carried out according to known methods, for example, the method of Koehler and Milstein (Nature, vol. 256, p. 495 (1975)).
  • a fusion accelerator polyethylene glycol (PEG), Sendai virus and the like can be mentioned, and preferably PEG and the like are used.
  • Antibody-producing cell lines can also be obtained by infecting antibody-producing cells with a virus capable of transforming lymphocytes to immortalize the cells.
  • viruses include, for example, Epstein-Barr (EB) virus.
  • Antibody-producing cells which have acquired infinite proliferation ability by transformation can be back-fused with mouse or human myeloma cells in order to stably maintain the antibody production ability.
  • mouse or human myeloma cells in order to stably maintain the antibody production ability.
  • the same myeloma cells as those described above can be used.
  • Hybridoma Screening and breeding of hybridomas is usually performed by adding HAT (hypoxanthine, aminopterin, thymidine) and culturing in animal cell culture medium containing 5-20% FCS (eg RPMI 1640) or serum-free culture medium supplemented with cell growth factor. It will be.
  • the concentrations of hypoxanthine, aminopterin and thymidine include, for example, about 0.1 mM, about 0.4 ⁇ M and about 0.016 mM, respectively.
  • ouabain resistance can be used. Since human cell lines are more sensitive to ouabain than mouse cell lines, unfused human cells can be eliminated by adding to the medium at about 10 -7 to 10 -3 M.
  • the neutralizing antibody against the pandemic strain obtained as described above should be one that neutralizes the infectivity of the pandemic strain.
  • the neutralizing activity of the obtained neutralizing antibody can be measured, for example, by plaque assay of a pandemic strain in the presence and absence of the neutralizing antibody.
  • the contacting of a neutralizing antibody against a pandemic strain with the mutant influenza virus library of the present invention can be performed either in vitro or in vivo.
  • a neutralizing antibody against a pandemic strain can be contacted with the mutant influenza virus library of the present invention.
  • the buffer include various buffers such as phosphate buffered saline (PBS) and Tris buffer.
  • PBS phosphate buffered saline
  • Tris buffer Tris buffer
  • the epidemic strain is administered intraperitoneally, intravenously, subcutaneously or intracutaneously of a warm-blooded animal to induce the neutralizing antibody against the epidemic strain.
  • the mutant influenza virus library of the present invention can be administered to the warm-blooded animal, and the neutralizing antibody against a pandemic strain can be contacted with the mutant influenza virus library of the present invention in vivo.
  • the screening method of the present invention comprises the step of selecting a mutant influenza virus whose infectivity is not neutralized.
  • a mutant influenza virus whose infectivity is not neutralized can be obtained by contacting a neutralizing antibody against a pandemic strain and selecting a mutant influenza virus that has not shown an antigen-antibody reaction.
  • Mutant influenza viruses which have not shown an antigen-antibody reaction to a neutralizing antibody against a pandemic strain can be selected by a known means, and can be selected, for example, by a plaque assay. Specifically, a plaque assay is performed by infecting monolayer culture cells (generally using MDCK cells) with a mutant influenza virus library of the present invention contacted with a neutralizing antibody against a pandemic strain, and then adding agar.
  • the culture medium By overlaying the culture medium, it is possible to infect only the adjacent cells with progeny virus particles that budd from the first infected cells. As a result, only the periphery of the initially infected cells has a cytopathic effect, and the dead cells are concentrated and spotted. Since the spots (also called plaques) contain infectious cells as well as dead cells, a sterile chip or the like is used to isolate and recover a non-infectious mutant influenza virus from each plaque. be able to.
  • the mutant influenza virus whose infectivity is not neutralized which is selected by the screening method of the present invention, can not be neutralized by the neutralizing antibody against a pandemic strain, and thus can become a mutant influenza virus that may become prevalent in the future.
  • Viral protein expression plasmids pCAGGS-PB1, pCAGGS-PB2, pCAGGS-PA and pCAGGS-NP
  • model virus genome expression plasmids pHH-vNS-Luc
  • the pRL-SV40 plasmid was also cotransfected at the same time, and the plasmid transfer efficiency between samples was normalized by measuring the luciferase activity by the Renilla Luciferase protein expressed from pRL-SV40 (Promega). Binding of PB1, PB2, PA and NP proteins respectively expressed from pCAGGS-PB1, pCAGGS-PB2, pCAGGS-PA and pCAGGS-NP to a model virus genome encoding Firefly Luciferase gene expressed from pHH-vNS-Luc Induced a transcription / replication reaction of the genome in cells.
  • the Firefly Luciferase protein which is a transcript from the amplified model virus genome, was expressed by the Firefly Luciferase protein, and the viral polymerase activity was quantified by measuring its luciferase activity (Reference: Nuclear MxA proteins form a complex with influenza virus Nucleic acid Acids Res. 2004, vol 32, p643-652) NP and inhibitory the transcription of the engineered influenza virus genome.
  • Example 1 wild-type PB1 (PB 1 wt) of A / Puerto Rico / 8/1934 (H1N1) strain (PR8 strain) or each mutant PB1 (K471R, K471H, K479R, K479H, K480R, K480H, K481R or PCAGGS-PB1 expressing K481H) was used.
  • PR8-PB1-K471H Virus A method for preparing a recombinant influenza virus by reverse genetics has been established, and PR8-PB1-K471H virus was generated according to a standard method (Reference: Generation of influenza A viruses entirely) from cloned cDNAs. Proc Natl Acad Sci US A. 1999, vol 96, p 9345-9350).
  • the virus genome expression plasmid pPol-1R-PB1-K471H which is necessary for the production of recombinant virus, is based on pPol-1R-PB1-wild type, and is the 471st of wild type PB1 (SEQ ID NO: 3) of PR8 strain.
  • the codon sequence AAG corresponding to the Lys residue was generated by substituting the codon sequence CAC corresponding to the His residue.
  • the introduction of mutations from AAG to CAC was performed by overlapping PCR.
  • two sets of primer sets (Set1: 5'- GTGTGTCCCTGGGGTTGACCAGA-3 '(SEQ ID NO: 11) (pPol-For) and 5'- TTATCGAACCTGTCACCTACTTGGAATCA-3 '(SEQ ID NO: 12);
  • Set 2 5'- CATCGGTGATGTCGGCGATATAG-3 '(SEQ ID NO: 13) (pPol-Rev) and After synthesizing two DNA fragments using 5'- TTGATTCCAAGTAGGTGACAGGTTCGATAA-3 '(SEQ ID NO: 14), these DNA fragments are mixed, and pPol-For and pPol-Rev primers are further added, and PCR is performed again.
  • PR8-PB1-K471H The full-length sequence of the PR8-PB1-K471H gene was amplified.
  • the PR8-PB1-K471H full-length DNA fragment (insert) and pPol-1R-empty (vector) were digested with restriction enzymes ApaI and XhoI, and then the insert was coupled to the vector by a ligation reaction.
  • PR8-PB1-K471H virus by replacing the PB1 genome expression plasmid (pPol-1R-PB1-wild type: alias pPol-1R-Seg2) with pPol-1R-PB1-K471H in the plasmid set for recombinant influenza virus production was produced.
  • Viral protein expression plasmids pCAGGS-PB1, pCAGGS-PB2, pCAGGS-PA and pCAGGS-NP
  • viral genome expression plasmids pPol-1R-Seg1, pPol-1R-PB1-K471H, pPol-1R-Seg3, pPol-1R -Seg4, pPol-1R-Seg5, pPol-1R-Seg6, pPol-1R-Seg7 and pPol-1R-Seg8 were introduced into cells using DNA transfection reagent TransIT-LT1 (Mirus).
  • the medium was changed to Opti-MEM (Thermo Fisher Scientific) 24 hours after plasmid introduction, and TPCK-treated trypsin (Sigma) was added to a final concentration of 3.5 ⁇ g / ml.
  • 200 ul of culture supernatant was inoculated into the serum of growth chicken eggs (11th egg) to amplify seed virus sprouted from 293T cells, and cultured at 34 degrees for 3 days.
  • the shell of the embryonated chicken egg was broken, and the urine was collected using a syringe.
  • the amount of virus contained in the urine was determined by hemagglutination test using 0.5% chicken erythrocytes (Reference: WHO Global Influenza Surveillance Network. 2011. Manual for the laboratory diagnosis and virological surveillance of influenza. World Health Organization, Geneva).
  • virus-infected plaques were detected by staining the cells with an amide black 10B solution, and the number was counted to quantify the number of infectious particles (Reference: Plaque) Med microbiol Immunol. 1975, vol 162, p 9-14; Replication and plaque assay of influenza virus in an established line. Canine kidney cells. Appl Microbiol. 1968, vol 16, p 588-594).
  • amplicon DNA was produced by performing PCR using Seg8 for and Seg8 rev primers by using Seg8 cDNA as a template. Sequencing analysis of amplicon DNA was performed using the next-generation sequencer GSJunior (454 Life Sciences, Roche), and mutation introduction efficiency was calculated from the obtained sequence information (Reference: Oseltamivir expand quasispecies of influenza virus through cell- to-cell transmission. Sci Rep. 2015, vol 5: 9163).
  • Example 1 Analysis of Mutant PB1 Focusing on Lys Residue in Polymerase Motif D of PB1 Similar to influenza virus, RNA polymerase from the past research report using poliovirus that performs genome replication by viral RNA-dependent RNA polymerase, It has been revealed that the Lys residue in the polymerase motif D present in ⁇ 1> is involved in fidelity control (control of error introduction frequency) during the RNA synthesis reaction.
  • a similar motif present in PB1, which constitutes the RNA polymerase of influenza virus contains 4 Lys residues (Fig. 1, amino acid numbers 471, 479, 480 and 481 of SEQ ID NO: 3), PR8 The functional significance of these Lys residues was examined by variant analysis of strain PB1.
  • the measurement of the polymerase activity of influenza virus was performed by a minireplicon system in which cultured cells were transfected with a PB1 expression plasmid together with a reporter virus genome expression plasmid.
  • the mutant PB1 in which the 471st Lys residue was substituted with a His residue had significantly reduced polymerase activity at 37 ° C culture temperature conditions, but was as active as wild-type PB1 at 34 ° C culture temperature conditions was held ( Figure 2). From this result, it was considered that the virus encoding the PB1-K471H mutant grew as a temperature sensitive strain, and that some modification occurred in the polymerase function.
  • Example 2 Preparation of Recombinant PR8-PB1-K471H Virus
  • the Seed viruses of wild type PB1 and strain PB1-K471H were prepared by transfecting cultured cells with a virus genome expression plasmid, and then amplification of virus was performed using embryonated chicken eggs. For these viruses, measurements of plaque forming activity, virus titer, proliferation ability and hemagglutination activity were performed (FIG. 4, FIG. 5, FIG. 6 and FIG. 7).
  • the PB1-K471H strain had lower plaque forming activity as compared to the PB1 wild strain and had a 50 to 100-fold reduction in the ability to produce infectious particles.
  • the PB1 wild strain and the PB1-K471H strain had almost the same value.
  • Hemagglutination test is an experimental system that can visually evaluate the sialic acid binding activity of HA protein present on the particle surface of influenza virus particles and simply measure the amount of virus regardless of infectivity and noninfectiousness. . From the above results, it was considered that the number of infectious particles in the PB1-K471H virus was 50 to 100 times smaller than that in the PB1 wild strain.
  • Example 3 Examination of the mutation introduction efficiency of PR8-PB1-K471H virus
  • the mutation introduction efficiency of PR8-PB1-K471H virus was examined using a next-generation sequencer (FIG. 8).
  • the “base insertion / deletion frequency” and the “base substitution frequency” introduced into the viral genome in the amplification process were calculated, targeting the NS gene, which is the eighth segment of the influenza virus genome.
  • the frequency of base insertion / deletion was improved by about 8 times and the frequency of base substitution was improved by about 3.6 times as compared with the PB1 wild strain.
  • Example 4 Verification of whether or not it can become a pandemic strain
  • a pandemic strain which is the original strain of a mutant influenza virus library to produce an antiserum (neutralizing antibody)
  • the following experiment is carried out Isolate viruses that will be prevalent in the future.
  • I) In vitro experiment After reacting the antiserum and the "mutant influenza virus library” in vitro, isolate "the escape mutant from humoral immunity” by plaque assay which has not been neutralized .
  • II In vivo experiment: After being developed and recovered by infecting a mouse with a current pandemic strain, challenge infection with a "mutant influenza virus library" is performed.
  • recombinant viruses are generated from genomic sequence information, and attempts are made to identify viral proteins and amino acid mutations involved in immune escape mutation. Perform characterization of the isolated immune escape mutant. Antigen mutation levels are quantified by a hemagglutination inhibition test using a neutralizing antibody against a pandemic strain, and viral proteins and amino acid sites involved in antigen mutations are identified along with the results of genome sequence analysis. In addition, target antigens are prepared based on the result of genome sequence analysis and ELISPOT method is performed to identify CTL epitopes (CTL: Cytotoxic T Lymphocyte, cytotoxic T cells) involved in immune escape. Subsequently, the proliferative ability of the isolated immune escape mutant is compared with a pandemic strain to verify whether it can be a pandemic strain.
  • CTL Cytotoxic T Lymphocyte, cytotoxic T cells
  • RNA comprising a nucleotide sequence complementary to a nucleotide sequence encoding a mutant PB1 of the present invention
  • RNA comprising a nucleotide sequence complementary to a nucleotide sequence encoding HA from a pandemic strain
  • NA from a pandemic strain

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Abstract

La présente invention concerne une protéine 1 de base de polymérase mutante (PB1) dans l'ARN polymérase du virus de la grippe, la mutation comprenant la séquence d'acides aminés représentée par SEQ ID NO: 2.
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