WO1998055627A1 - Antigenes mutants du virus de la rougeole et genes les codant - Google Patents
Antigenes mutants du virus de la rougeole et genes les codant Download PDFInfo
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- WO1998055627A1 WO1998055627A1 PCT/JP1998/002481 JP9802481W WO9855627A1 WO 1998055627 A1 WO1998055627 A1 WO 1998055627A1 JP 9802481 W JP9802481 W JP 9802481W WO 9855627 A1 WO9855627 A1 WO 9855627A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- 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
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a mutant measles virus antigen and a gene encoding the same. More specifically, a mutant measles virus antigen containing at least one protein antigen selected from the H protein antigen and the F protein antigen of the mutant measles virus strain, and each of the mutant measles virus antigens About the gene to be loaded.
- a mutant measles virus antigen of the present invention or the mutant measles virus gene encoding the same is used, a live attenuated measles virus or a gene vaccine compatible with an epidemic strain measles virus, and a measles virus flowing out of the strain may be used. Diagnostic agents that can accurately detect infection can be provided efficiently and economically.
- Conventional technology Conventional technology
- Measles virus is a measles pathogen and is widely distributed throughout the world. The virus is highly contagious and, when splashed to humans, primarily affects the respiratory system and reticuloendothelial tissue, causing acute illness. When affected, it causes systemic symptoms such as high fever, catarrh, and rash, and in severe cases, bacterial pneumonia, otitis media, and acute encephalitis. In addition, measles in the world of 1996 The estimated number of patients is about 4.2 million, and the estimated number of deaths is about 110,000. ["The World Health Report 1997", p. 15, WHO (World Health Organization) ion) published in 1997].
- measles is a very important infectious disease, and eradication of measles by vaccines is now desired worldwide.
- WHO World Health Organization
- EPI Expanded Program on Immunization
- the measles virus belongs to the genus Morbilivirus, belonging to the order Paramyxoviridae, Mononegavirus.
- the virion has a substantially spherical shape with a diameter of about 150 nm and an envelope composed of a lipid bilayer membrane. H on the entire surface of the envelope
- the (hemagglutinin) protein and the F (fusion) protein protrude like spikes, and the base is supported by the Matrix membrane protein on the lower surface of the envelope.
- the nucleocapsid present inside the envelope consists of a measles virus genomic approximately 16 kb long linear single-stranded (one) -sense (so-called mononega) gene RNA and protein.
- the RNA of the gene is N (nucleocaps id-associated protein ins), PZC / V (phosphoprotein in / C protein / V protein in) around 3 ′ terminal force to 5 ′ terminal direction.
- tr ic ist ron ic gene M (ma tr ix prot e in), F (fus ion prot e in), H (hemagg lut in in prote in) and L (large put at ive polymerase prote in), respectively, and report them (i rus Taxonomy: Sixth Repor tof The International 1 Community of Taxonomy of Viruses ", Archives of Violology, Supplement 10, 268-270, and 271-227, 1995).
- the host cell used for subculturing the isolate wild measles virus
- the culture temperature were used.
- the virus of the host range mutant strain or the temperature mutant strain is attenuated. You.
- Measles prevention vaccines have been put into practical use since the early 1960s. In these early days, inactivated vaccines containing inactivated measles virus as an active ingredient (K; "ki 1- Led virus vacc ine ”) was mainly used. The vaccine had a poor immune effect and induced severe atypical measles, In the latter half of the year, live vaccines (L; abbreviation for "live virus vaccine”) using attenuated measles virus as an active ingredient began to be widely used, and KL was sometimes used during this period. , since 1 9 7 1990s, these further attenuated highly attenuated live vaccines. (FL: f ur ther at tenuated l ive vacc ine) force s are widely commercially available in the world, has been put to practical use in addition, All of these live vaccines are described above.
- the live attenuated measles vaccine strain virus of (3) is used as an active ingredient.
- the inventor of the present invention has been involved in research on clinical, epidemiological and vaccinological studies of measles for more than 30 years, as well as various measles viruses such as vaccine strains, epidemic strains and fresh isolates.
- the researchers confirmed the differences in antigenicity or immunogenicity between the conventional virulent strains and the mutant virulent strains and epidemic strains, and conducted intensive research to identify the factors of such differences.
- the mutant strain had a mutation involving amino acid substitution in a specific region of the gene encoding each of the H protein and the F protein.
- the present inventors have found that the mutated regions of the H protein and the F protein are useful as mutant measles virus antigens.
- the present invention has been completed based on this new finding.
- one of the main objects of the present invention is to provide at least one selected from a mutant measles virus H protein antigen and a mutant measles virus F protein antigen which are effective for preparing an epidemic measles virus vaccine and a diagnostic agent.
- Another object of the present invention is to provide a mutant measles virus antigen containing the same protein antigen.
- Another object of the present invention is to address the epidemic measles virus.
- the purpose of the present invention is to provide a mutant measles virus gene.
- the left end is the N-terminus and the right end is the C-terminus.
- SEQ ID NO: 1 shows the nucleotide sequence of cDNA corresponding to the RNA encoding the H protein of attenuated measles virus CAM-70 strain, and the full-length amino acid sequence of the H protein encoded by the sequence.
- SEQ ID NO: 2 is the full-length amino acid sequence of the H protein of attenuated measles virus CAM-70 strain
- SEQ ID NO: 3 is an amino acid sequence of a fragment peptide consisting of amino acids 93 to 61 of SEQ ID NO: 2;
- SEQ ID NO: 4 is the amino acid sequence of a fragment peptide consisting of amino acids 176 to 316 of SEQ ID NO: 2;
- SEQ ID NO: 5 is SEQ ID NO: 2
- SEQ ID NO: 6 is the amino acid sequence of a fragment peptide consisting of amino acids Nos. 172 to 178;
- SEQ ID NO: 6 is SEQ ID NO: 238 to 238 of SEQ ID NO: 2 Amino acid sequence of a fragment peptide consisting of amino acid No. 4;
- SEQ ID NO: 7 is composed of amino acids Nos.
- SEQ ID NO: 8 is the amino acid sequence of the fragment peptide consisting of amino acids Nos. 301-307 of SEQ ID NO: 2.
- SEQ ID NO: 9 is a nucleotide sequence of cDNA corresponding to gene RNA encoding H protein of virulent measles virus NA strain, and a full-length amino acid sequence of H protein encoded by the sequence.
- SEQ ID NO: 10 is the full-length amino acid sequence of H protein of the virulent measles virus NA strain,
- SEQ ID NO: 11 is the amino acid sequence of a peptide fragment consisting of amino acids 93 to 61 of SEQ ID NO: 10;
- SEQ ID NO: 12 is SEQ ID NO: 10
- SEQ ID NO: 13 is the amino acid sequence of the amino acid sequence of the 176th to 316th amino acids of SEQ ID NO: 10;
- 78 is the amino acid sequence of a fragment peptide consisting of the amino acid No. 8;
- SEQ ID NO: 14 is the amino acid Nos. 238 to 244 of SEQ ID NO.
- SEQ ID NO: 15 is the amino acid sequence of a peptide fragment consisting of amino acids 277 to 282 of SEQ ID NO: 10
- SEQ ID NO: 16 is SEQ ID NO:
- SEQ ID NO: 17 is the amino acid sequence of a peptide fragment consisting of amino acids Nos. 301 to 307 of No. 10
- SEQ ID NO: 17 is an attenuated measles virus CAM-70 strain
- SEQ ID NO: 18 is a full-length amino acid sequence of F protein of attenuated measles vinoles CAM-70 strain
- SEQ ID NO: 19 is the nucleotide sequence of cDNA corresponding to the gene RNA encoding the F protein of the virulent measles virus NA strain, and the full-length amino acid sequence of the F protein encoded by the sequence;
- SEQ ID NO: 20 is the full-length amino acid sequence of F protein of highly virulent measles virus NA strain.
- At least one mutant measles virus F protein antigen selected from the group consisting of fragments comprising at least six amino acid sequences adjacent to the amino acid.
- (h) Contains No. 11, No. 52, No. 107, No. 165, No. 398, No. 417, or No. 523 amino acid And at least one mutant measles virus F protein antigen selected from the group consisting of genes encoding fragments comprising at least six amino acid sequences adjacent to the amino acid. Code gene.
- a in the DNA base sequence indicates adenine
- C indicates cytosine
- G indicates guanine
- T indicates thymine
- a 1 a in the amino acid sequence is alanine, Arg is arginine, A sn is asno, ° argin, Asp is aspartic acid, Cys is cystine, G 1n is glutamine, G1u is glutamic acid, G1y is glycine, His is histidine, I1e is isoleucine, Leu is lysine, and Lys is lysine.
- Met is methionine
- Phe ferulanine
- Pro is proline
- Ser is serine
- Thr threonine
- Trp is triptophan
- Tyr Tyrosine and Val are balins.
- H and F protein genes involved in the adsorption and invasion of pirions into cells to establish viral infection. confirmed.
- a mutation in the H protein gene results in a substitution of 17 to 19 amino acids in a specific region of the sequence of all 6 17 amino acids constituting the protein.
- antigenic mutations antigenic variations have occurred due to changes in the three-dimensional structure of proteins. Such an antigenic mutation is determined to be as large and severe as an antigenic shift of the H protein.
- a recombinant virus is produced in which the H protein gene of the conventional live vaccine strain virus is replaced with the: H protein gene of the epidemic strain virus.
- an attenuated virus strain for a live vaccine suitable for the antigenicity of the epidemic strain can be quickly obtained.
- such a recombinant virus can be used as an active ingredient of a qualifying vaccine that is excellent in preventing epidemic strain infection.
- a further advantage of this method is that It is economical. That is, the time and labor required for attenuating the virus can be significantly reduced and saved.
- Conventional methods for attenuating viruses are mainly based on the passage of the virus, since there is no fixed method as described above.Therefore, there are few methods for establishing an attenuated strain for live vaccines. It took several years to about 10 years.
- a non-propagating recombinant virus produced by inserting and linking the cDNAs of the H protein gene and the F protein gene into a non-proliferating virus genome, as an active ingredient of the gene vaccine or DNA protein Induces both humoral and cellular immunity, similar to conventional live measles vaccines. Notably, such vaccines can be given intranasally.
- naked DNA is prepared by inserting and linking a cDNA fragment containing a mutant region of the H protein gene of the epidemic virus, for example, into plasmid vector.
- DNA can be used as an active ingredient of a measles-preventing DNA vaccine or gene vaccine.
- PCR primers are synthesized in consideration of mutations in H protein gene and F protein gene of epidemic strain virus. Such a primer can be used as a genetic diagnostic agent for identifying an epidemic measles virus strain and for distinguishing a highly virulent strain from an attenuated strain.
- mutant antigen full-length protein or its fragment peptide encoded by the gene is prepared, and the epitope is chemically synthesized. These are provided as optimal diagnostic antigens for epidemic measles.
- Antigen analysis of various measles virus antigens For antigen analysis, neutralization test, HI (hemagglutinat ion inhi ion) test, PA (passive agglutinat ion) method, enzyme immunization using monoclonal antibody A measuring method or a fluorescent antibody method can be employed. Of these, analysis based on the measurement of antibody titer by neutralization reaction is particularly essential, and is performed in accordance with Ueda's modified method using microplates (Biken Journal, 14, 155-160, 1971). I can.
- serum for example, measles patient serum, mouse immune serum against measles virus shown below, and the like are used.
- Tanabe and Edmonston are representative of epidemic strains in the 1950s to 1960s (past virulent strains), etc., and the above-mentioned raw water was established by attenuating such strains.
- the Kuching strains include CAM_70 and Edmonston B, and the recent epidemic strains (strongly virulent strains) have been around the world since the 1990s. Can be used.
- F-t separated in 1991 from the throat swab of a re-infected person
- F-b blood of a re-infected person
- U—t isolated from throat swa in non-vaccinated patients
- U—b obtained from blood in non-vaccinated patients
- Tanabe Tanabe
- Edmons ton Edmo
- CAM CAM
- the analysis software DNASIS-Mac is used to analyze the hydrophobic pattern and to determine the secondary structure of the protein (Chou-Fasman analysis).
- Mutant measles virus antigen and gene encoding the same The present inventors have considered the recent measles virus outbreak based on the antigen analysis of (1) and the analysis of base and amino acid sequences of (2) above. In comparison with past highly virulent strains and conventional attenuated measles live vaccine strains, we found that amino acid-substituted regions were present in H proteins and proteins. Vaccinated strains and antigens useful for diagnosis were identified.
- the mutant measles virus antigen of the present invention is the full-length or fragment of H-protein and F-protein of CAM-70 which is an attenuated measles virus strain or NA strain which is an epidemic measles virus strain. All amino acid sequences were first disclosed by the present inventors.
- the measles virus nores antigen of the present invention is at least selected from the group consisting of the following (I) mutant measles virus H protein antigen and (II) mutant measles virus F protein antigen. It is an antigen containing a kind of protein antigen.
- (e) contains the No. 174, No. 176, No. 243, No. 279, or No. 302 amino acid and is adjacent to the amino acid A fragment consisting of a sequence of at least six amino acids;
- (h) Contains No. 11, No. 52, No. 107, No. 165, No. 398, No. 417, or No. 523 amino acid And at least one mutated measles virus F protein antigen selected from the group consisting of fragments comprising at least six amino acid sequences adjacent to the amino acid.
- the protein antigens contained in the mutant measles virus antigen of the present invention are H protein and protein, respectively, and (b) to (f) and (h) are It is a peptide (fragment).
- the four fragments described in (d), that is, the 17th fragment specified by the amino acid number of the amino acid sequence having a total length of 6 17 amino acids described in SEQ ID NO: 2 or 10 Nos. 2 to 178, 238 to 244, 277 to 282, and 301 to 307 are H proteins disclosed by the present inventors. It is an epitop.
- the mutant measles virus antigen of the present invention contains at least one protein antigen selected from the above-mentioned full-length protein and its fragment peptides, and the protein antigen is appropriately selected according to the purpose of use. Can be. Also, a plurality of antigens can be used in combination.
- a gene encoding the above mutant measles virus antigen Specifically, at least one selected from the group consisting of (I) a gene coding for the mutant measles virus H protein antigen and (II) a gene coding for the mutant measles virus F protein antigen shown below. Also provided is a mutant measles virus gene containing a gene.
- the gene encoding the mutant measles virus antigen of the present invention is not particularly limited as long as it encodes a full-length protein or a fragment peptide thereof, which is a mutant measles virus antigen. It is not limited to the nucleotide sequence of the genomic RNA of the NA strain. As the mutant measles virus gene, the cDNAs shown in SEQ ID NOs: 1, 9, 17 and 19 can be used, or based on the amino acid sequence of the mutant measles virus antigen. Nucleotide sequences can also be synthesized.
- the mutant measles virus gene of the present invention contains at least one gene selected from the above-mentioned genes coding for the mutant measles virus antigen, and the gene contained is appropriately determined according to the purpose of use. You can choose.
- the mutant measles virus gene of the present invention includes attenuated strain genes and epidemic strain genes. Based on the disclosure of the present invention, for example, it becomes possible to prepare a live vaccine that is effective against epidemic strains (see [II] (1) below and Examples 2 and 3). When multiple genes are used in combination, the genes are linked together. (See [ ⁇ ] (2) below and Example 4).
- the antigen of the present invention comprising the above sequence and the gene encoding the same are useful as differential markers for virulence / attenuation, and are extremely useful for improving conventional vaccines and developing diagnostic agents. Important and useful.
- a recombinant virus in which the gene is replaced between a live vaccine strain virus and an epidemic strain virus is created.
- the raw vaccine strain various strains described in the prior art can be used, but at least for a long term of at least 10 years, and as an active ingredient of the raw vaccine. Preference is given to using strains that are used worldwide and whose safety and effectiveness are certified, for example, CA II-70.
- the epidemic strain is appropriately selected from a widespread strain virus in which the antigenic mutation based on the gene mutation is remarkable when compared with the antigenicity of the live-acting strain virus.
- the frequency of isolation is high, and the area of the epidemic is wide, and the antigenic mutation is not special but universal.
- MO or NA strains isolated in the year It is preferable to adopt MO or NA strains isolated in the year.
- the gene to be replaced can be appropriately selected from the genes encoding the antigens listed in the above [1] (3) and used alone or in combination.
- the H protein fragment of No considering prevention of reversion from attenuated to virulent by recombination, simplification of the recombination process, etc., the H protein fragment of No.
- a cDNA of the (+) sense RNA of the full-length genomic DNA of the CAM-70 virus was prepared, and the region encoding amino acid substitution described above was excised from the cDNA with a restriction enzyme.
- Corresponding region prepared from the viral genome of the strain MO or NA at the cleavage site was prepared, and the region encoding amino acid substitution described above was excised from the cDNA with a restriction enzyme.
- the resulting recombinant cDNA is linked to the busmids pBluescript SK and KS (manufactured by Stratagene Ltd., UK), and the recombinant cDNA is transferred to T7 fur Construct a vector (hereinafter referred to as "Vo") prepared to be transcribed by the diRNA polymerase. After co-transfection of Vo and VI into the helper cell described above, the cells are cultured, whereby a recombinant virus can be obtained.
- Propagation of the recombinant virus in infected cells can be determined by determining CPE (cytopathic effect) that produces syncytia, or by using a fluorescent antibody method using a monoclonal antibody to a protein epitope whose substitution region encodes. Can be confirmed with a microscope.
- CPE cytopathic effect
- the gene encoding amino acid number 176 to 316 (SEQ ID NO: 4) of the H-protein of CAM-70 can be expressed as MO or NA.
- a recombinant attenuated measles virus of the CAM-70 strain was obtained, which was replaced with a gene encoding amino acid number 176 to 316 (SEQ ID NO: 12) of the H protein of the strain.
- each of the N, P, and L protein genes of CAM-70 is individually inserted into pcDNA3.1 (-) and linked to the restriction site of pcDNA3.1 (-) to construct each expression vector.
- recMVA recomb inant MVA
- the three expression vectors and the above-mentioned expression vector Vo were co-transfected into MRC-5 or WI-38 cells transfected with recMVA in advance, and then about 35 Culture at ⁇ 38 ° C yields the desired recombinant attenuated measles virus.
- the propagation of such a virus can be confirmed by microscopy using CPE determination or the monoclonal immunofluorescence method as described above. The eligibility of the obtained virus for antigenicity or immunogenicity is
- This non-proliferative virus is derived from human adenovirus type 5 and lacks the genes E1A and E1B essential for its growth, so that it grows in cells other than the 293 cells that constantly produce these genes. Can not. Furthermore, this virus is also deficient in the gene encoding the E3 protein, which antagonizes the recognition of viral antigens by CTL (Cytotoxic T Lymphocyte), so that CTL can be expected to establish cell-mediated immunity. It is devised.
- the measles virus gene used can be appropriately selected from the genes encoding the antigens listed in the above [I] (3), and can be used alone or in combination.
- the full-length H protein (SEQ ID NO: 2 or 10) described in (a) and the The full-length F protein (SEQ ID NO: 18 or 20) was It is preferable to use a combination of the genes to be coded.
- the H protein gene and the F protein gene cDNA are prepared (the combination for linking in the 5 'to 3' direction is HF Or FH), which is inserted into the E1A.E1B-deficient site of the cosmid cassette pAdexl to obtain pAdexl / HF or pAdexl / FH.
- DNA-TPC was extracted from the parent adenovirus and digested with the restriction enzyme Eco T22I (Takara Shuzo Co., Ltd., Japan) to digest the DNA-TPC / Obtain EcoT22I. next,
- pAdexl / HF or pAdexl / FH and DNA-TPC / EcoT22I are subjected to c0-ans Iecti0 ⁇ in 2993 cells by the calcium phosphate method, homologous recombination occurs.
- a non-proliferating recombinant adenovirus having the measles virus ⁇ protein gene and the F protein gene can be obtained.
- the fact that this virus carries both the measles virus ⁇ and F protein genes means, for example, that the HeLa cells infected with this virus can be detected by a fluorescent antibody method using a monoclonal antibody against the protein. You can check it.
- the recombinant attenuated measles virus of [1] (1) can be used as a seed virus to produce a live attenuated measles vaccine.
- the recombinant virus is cultured in a susceptible cell, for example, a nitrile embryo cell, and the virus is suspended. After obtaining the liquid, centrifuge it at a low speed, and remove the cells by filtration to prepare a vaccine stock solution.
- a suitable medium for example, a BME medium (Eagle's
- a virus stabilizer can be added and mixed.
- the prepared vaccine solution is dispensed into a vial having an appropriate volume, for example, about 1 to 20 ml, and sealed and sealed, and then used as a vaccine.
- a vaccine can be used not only as a liquid but also as a dry preparation after freeze-drying after dispensing. In order to guarantee the quality of manufactured vaccine preparations, it is necessary to carry out various tests for safety and efficacy before use. This test is based on the Pharmaceutical Affairs Law (Act No. 1945 of 1958) and 1993 ⁇ Ministry of Health, Labor and Welfare No. 2117 “Biological Drug Standards” Perform according to the rules.
- Vaccines are, for example, doses of about 0.25 to
- 0.5 ml is inoculated subcutaneously.
- the non-proliferating recombinant virus of [H] (2) can be mass-produced in 2993 cells.
- PFU / ml PFU- Forming Unit
- the preparation can be used at a dose of about 0.25 to 0.5 ml subcutaneously, intramuscularly or intranasally, but in view of simplification of inoculation, nasal inoculation is particularly preferred. I like it.
- the diagnostic antigen As the diagnostic antigen, the antigens (full-length protein or its fragment peptide) listed in the above [1] (3) can be used alone or in combination. As a combination, it is preferable to combine antigens containing different epitopes in order to broaden the antigen spectrum.
- the diagnostic antigen of the present invention can be provided, for example, as an antigen such as a sedimentation reaction, an agglutination reaction, a neutralization reaction, a fluorescent antibody method, an enzyme immunoassay, and a radioimmunoassay.
- immune serum antibodies and the like.
- This antibody can be provided, for example, as an antibody for antigen detection in various diagnostic methods.
- the diagnostic antigen or antibody according to the present invention is used after being diluted so that the content in the diagnostic agent is an amount necessary for causing an antigen-antibody reaction.
- genes encoding the antigens listed in the above [I] (3) can be used alone or in combination, for example, as a probe reagent or an identification reagent in genetic diagnosis.
- H- and F-protein sequences of attenuated and endemic strains disclosed in this application (E.g., SEQ ID NOs: 2, 10, 18, and 20) or the gene encoding them (e.g., SEQ ID NOs: 1, 9, 17, and 19) to design PCR primers be able to.
- the obtained PCR primer can be provided as a reagent for diagnosis using PCR.
- Example 1
- Antigen analysis and gene analysis were performed as follows, to show past epidemic strains (virulent strains), conventional live vaccine strains (attenuated strains) and recent epidemic strains
- the difference between the base sequence and the amino acid sequence among the three (virulent strains) is determined.
- the amino acid sequence of the mutant antigen is specified, and its epitope is also determined.
- Measurement of neutralizing antibody titer (1) Measure by a modified Ueda method using microplate. Vaccin strain CAM-70 and epidemic strain Momo are used for the attacking virus, and B95a cells are used for virus culture. Specimens included sera from children who had been vaccinated with measles CAM-70 vaccine during the period of 1994 to 1996, before vaccination and 1 to 2 months after vaccination. One sample was obtained using 11 samples of serum (Group A) with 8 times higher HI antibody titer and 14 samples of serum (Group B) with 64 times higher HI antibody titer measured with HI antigen of Toshima strain (separated in 1995). Use two rows of gels each.
- the antibody titer against the endemic strain is less than 0 (the numerical value is 10 g 2 ) and is not detected.
- the relative antibody titer of the remaining two cases is 1.
- Group B Out of a total of 14 specimens, the relative antibody titer (endemic strain Z vaccine strain) of 10 cases (10 Z14; 71.4%) was as low as 12 to 1-8, and the remaining In four cases it is about one. As a result, cases in which the relative antibody titer (epidemic vaccine strain) was 1 to 2 or less were frequently detected, and the antigens (H and F) involved in the neutralization or infection of recent epidemic measles virus were frequently detected. Both proteins are judged to have a mutation.
- the relative antibody titer endemic strain Z vaccine strain
- the neutralizing antibody titer against the mouse immune serum of the NA strain H protein is measured in the same manner as (1) above, except for the following items.
- Three strains of CAM-70, Tanabe, and NA are used as the attacking virus.
- a mouse immune serum is prepared as follows. NA for each of 10 4-week-old BALB / C mice The strain H protein expression vector pcDNA3.1 (-) / H (naked DNA) is intramuscularly inoculated in 100 ⁇ l portions, and two weeks later, the same inoculation is performed again for booster immunization. Five mice are inoculated with saline as control.
- each mouse is bled individually and used as immunized mouse serum.
- the naked DNA described above was attached to Example 3 described below, and the plasmid pcDNA3.1 (_) / H constructed as an expression vector for the NA strain H protein was amplified with Escherichia coli and the culture solution thereof was used. It is prepared by purification from
- Determination of the nucleotide sequences of both H protein and F protein genes The determination is performed according to the method of Isegawa et al. (Mol. Cell. Prob., 6, 467-475, 1992). After extracting the RNA from the 95a cells infected with each of the measles viruses listed in Tables 2 and 3 by the GTC / CsCl method of Chirgwin et al. (Biochemistry, 18, 5294-5299, 1979), Prepare cDNA using 6 mer random primer. Next, referring to the nucleotide sequence of the cDNA of the gene of Edmons n strain (Viology, vol. 173, no. 2, pp. 415-425, 1989).
- the nucleotide sequence is determined by the direct sequence method using PCR, and the amino acid sequence is read based on Universal Code, and the amino acid sequence is determined by gene mutation. Identify replacements for The results are shown in Tables 2 and 3, and SEQ ID NOS: 1, 2, 9, 10 and 17 to 20, respectively.
- Substituted amino acids are described based on the amino acid number and amino acid of Edmonston strain gene cDNA, and the portions not described are the same as the standard amino acids.
- H protein secondary structure The secondary structure of H protein was determined by performing a combinatorial analysis on the amino acid sequence obtained above.
- DNASIS-Mac version 3.6 [manufactured by S-Ritsu Software Engineering Co., Ltd., Japan] is used as the analysis software, and hydrophobic pattern analysis and Chou-Fasman analysis are performed.
- epitope Advisor Japan, manufactured by Fujitsu Kyushu System Engineering (FQS) Co., Ltd.
- FQS Fujitsu Kyushu System Engineering
- cDNA is prepared by RT-PCR (reverse transcript-PCR), and the primer is used from the cDNA by using a primer.
- Clones genes encoding the N, P and L proteins of the AM-70 strain virus. are referred to as pcDNA3.1 (-) / N, pcDNA3.1 (-) / P, and pcDNA3.1 (-) / L). These are amplified in E. coli, stored, and used for the next step.
- the NA strain corresponding to a part of the H protein gene in the cDNA of the CAM-70 strain full-length genomic RNA was obtained in the same manner as described above. Clones the gene cDNA that has been replaced with the above sequence. The obtained clone is hereinafter referred to as pB1uescript / MV, and is cultured and stored, and is provided for the next step.
- the sequence to be substituted is the nucleotide sequence of the CAM-70 strain H protein gene described in SEQ ID NO: 1 from No. 52 to No. 948 (432 bases; amino acid No. 1). (No. 76 to No. 316) and the corresponding region of the NA strain shown in SEQ ID NO: 9 (No. 526 to No. 948 bases).
- a non-replicating recombinant adenovirus is produced according to the method of Saito et al.
- the gene that is inserted and linked to the viral genome is used in combination with the gene cDNA that encodes each of the H and F proteins of the NA strain described in SEQ ID NO: 9 and SEQ ID NO: 19, respectively.
- Coordination was performed in the order of F-H in the direction from 5 'to 3' so that F and H were expressed as fusion proteins, and this cDNA was cleaved with the restriction enzyme SwaI to obtain a cosmid cassette. Insert and link to the E1A and E1B deletion sites of pAdexl to obtain pAdexl / FH.
- the cDNAs of the H and F genes are each prepared from the NA strain genomic RNA by RT_PCR using primers corresponding to each gene. Also, pAdexl / FH is packaged with ⁇ phage, amplified and stored in E. coli, and used for the next step. Next, the parental adenovirus DNA-TPC is extracted and purified from the infected cells by CsC1 ultracentrifugation, and then digested with the restriction enzyme Eco T22I to obtain a cleavage product DNA-TPC / Eco T22I.
- the pAdexl / FH and DNA-TPC / Eco T22I were cultured by the calcium phosphate method in co-transfection with 2293 cells, and cultured at 37 ° C for 18 hours. Cause homologous recombination.
- a non-recombinant recombinant adenovirus carrying both the H and F protein genes of the NA strain is produced. The virus and its growth were confirmed by the NA strain H And F. Select and confirm by fluorescent antibody method using monoclonal antibody against each protein.
- mutant measles virus antigen of the present invention or the mutant measles virus gene encoding the same is used, a live attenuated measles vaccine or a gene vaccine compatible with the epidemic measles virus, and further, infection by the epidemic measles virus can be detected accurately. Diagnostic agents can be provided efficiently and economically.
- Sequence Listing SEQ ID NO: 1 Sequence length: 18 5 4 Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear type of linear sequence: cDNA to genomic RNA encoding H Protein Origin Organism name: Meas les vi rus Strain name: Attenuated measles virus CAM—70 shares
- AAA ATC ATC GGT GAT GAA GTG GGC CTG AGG ACA CCT CAG AGA TTC ACT 336 Lys lie He Gly Asp Glu Val Gly Leu Arg Thr Pro Gin Arg Phe Thi
- GAG CTC ATG AAT GCA TTG GTG AAC TCA ACT CTA CTG GAG ACC AAA ACA 528 Glu Leu Met Asn Ala Leu Val Asn Ser Thr Leu Leu Glu Thr Lys Thr
- AGC AAA AGG TCA GAG TTG TCA CAA CTG AGC ATG TAC CGA GTG TTT GAA 768 Ser Lys Arg Ser Glu Leu Ser Gin Leu Ser Met Tyr Arg Val Phe Glu
- GTC TCC TTA TCA ACG GAT GAT CCA GTG ATA GAC AGG CTT TAC CTC TCA 1056
- GCG TGT AAG GGT AAA ATC CAA GCA CTC TGC GAG AAT CCC GAG TGG GCA 1200
- Pro Leu Lys Asp Asn Arg lie Pro Ser Tyr Gly Val Leu Ser Val Asp
- Organism name Goenjin winores (measles virus)
- Strain name Attenuated measles virus CAM—70 strain Sequence
- Lys lie He Gly Asp Glu Val Gly Leu Arg Thr Pro Gin Arg Phe Thr
- Arg lie Lys Leu Asp Tyr Asp Gin Tyr Cys Ala Asp Ala Ala Ala Glu 145 150 155 160
- Organism name Measles virus (measles virus)
- Strain name Attenuated measles vinoles CAM—70 strain Sequence
- Pro Glu Trp Ala Pro Leu Lys Asp Asn Arg lie Pro Ser Tyr Gly Val 305 310 315 320
- Organism name measles vis virus
- Strain name Attenuated measles vineless CAM—70 strain Sequence
- Organism name measles vinoles
- Strain name attenuated measles virus CAM—70 strains Array
- Organism name Gonochin ⁇ inoresu (meas les vi rus)
- Strain name Attenuated measles virus CAM—70 strain Sequence
- Organism name measles virus
- Strain name Attenuated measles virus CAM—70 strain Sequence is Gly Glu Asp Serlie Thr
- Sequence length 18 5 4
- Sequence type Nucleic acid number of strands: Single strand Topology: Linear arrangement
- Lj class cDNA to genomic RNA encoding H rotein Origin Organism name: Measles Vinoles (meas les vi rus)
- Strain name Poisonous measles virus NA strain Sequence
- AGA ATC AAA TTG GAT TAT GAT CAA TAC TGT GCA GAT GTG GCT GCT GAA 480 Arg lie Lys Leu Asp Tyr Asp Gin Tyr Cys Ala Asp Ala Ala Ala Glu 145 150 155 160
- GAA CTC ATG AAT GCA TTG GTG AAC GCA ACT CTA CTG GAG GCC AGG GCA 528 Glu Leu Met Asn Ala Leu Val Asn Ala Thr Leu Leu Glu Ala Lys Ala
- GGG GTC CCC ATC GAA TTA CAA GTG GAA TGC TTC ACA TGG GAC CAA AAA 1728
- Organism name gonorrhea (measles virus)
- Strain name Poisonous measles virus NA strain
- Lys lie lie lie Gly Asp Glu Val Gly Leu Arg Thr Pro Gin Arg Phe Thr
- Arg lie Lys Leu Asp Tyr Asp Gin Tyr Cys Ala Asp Val Ala Ala Glu 145 150 155 160
- Pro Leu Lys Asp Asn Arg lie Pro Ser Tyr Gly Val Leu Ser Val Asn
- Pro Glu Trp Ala Pro Leu Lys Asp Asn Arg lie Pro Ser Tyr Gly Val 305 310 315 320
- Type of sequence Number of amino acid chains: Single-chain Topology: Linear Sequence type: Peptide Origin Organism name: Machina vinoles. (Measles vi rus) Strain name: NA strain of virulent measles virus Array
- Organism name Goenzin Winores (measles virus)
- Strain name Poisonous measles virus NA strain strain IJ
- Organism name measles virus
- Strain name NA strain of virulent measles virus
- Organism name measles virus (measles virus)
- Strain name Virulent measles virus NA sequence
- Gly Pro Pro lie Ser Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly
- Organism name measles virus (measles virus)
- Strain name attenuated measles virus CAM—70 strain Sequence
- Tyr Tyr Thr Glu lie Leu Ser Leu Phe Gly Pro Ser Leu Arg Asp Pro
- Asn Leu lie Ala Asn Cys Ala Ser He Leu Cys Lys Cys His Thr Thr 385 390 395 400
- Gly Thr lie lie Asn Gin Asp Pro Asp Lys lie Leu Thr Tyr lie Ala
- Gly Pro Pro lie Ser Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly
- Gly Thr lie lie Asn Gin Asp Pro Asp Lys lie Leu Thr Tyr lie Ala
- Organism name Hemp Winores (measles virus)
- Strain name Poisonous measles virus NA sequence
- Gin Ala lie Asp Asn Leu Arg Ala Ser Leu Glu Thr Thr Asn Gin Ala 145 150 155 160 lie Glu Ala He Arg Gin Ala Gly Gin Glu Met He Leu Ala Val Gin
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JP50206999A JP4043054B2 (ja) | 1997-06-04 | 1998-06-04 | 変異麻疹ウイルス抗原及びそれをコードする遺伝子 |
CA002262500A CA2262500C (en) | 1997-06-04 | 1998-06-04 | Measles virus mutant antigen and gene coding for the same |
EP98923127A EP0949333B1 (en) | 1997-06-04 | 1998-06-04 | Measles virus mutant antigen and gene encoding the same |
US09/230,944 US6277380B1 (en) | 1997-06-04 | 1998-06-04 | Measles virus mutant antigen |
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US09/230,944 A-371-Of-International US6277380B1 (en) | 1997-06-04 | 1998-06-04 | Measles virus mutant antigen |
US09/873,233 Division US6605284B2 (en) | 1997-06-04 | 2001-06-05 | Gene coding for the measles virus mutant antigen |
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US8529884B2 (en) * | 1999-12-22 | 2013-09-10 | The Trustees Of The University Of Pennsylvania | Cosmid DNA constructs and methods of making and using same |
WO2002072776A2 (en) * | 2001-03-09 | 2002-09-19 | The Regents Of The University Of California | Cell culture system for synthesis of infectious hepatitis c virus |
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JPH07509122A (ja) * | 1992-04-08 | 1995-10-12 | ザ ガバメント オブ ザ ユナイテッド ステイツ オブ アメリカ,アズ レプリゼンテッド バイ ザ セクレター,デパートメント オブ ヘルス アンド ヒューマン サービシーズ | 野生型麻疹ウイルス糖タンパク質:ワクチンおよびその検出方法 |
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JP3770333B2 (ja) | 1995-03-15 | 2006-04-26 | 大日本住友製薬株式会社 | 組換えdnaウイルスおよびその製造方法 |
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JPH07509122A (ja) * | 1992-04-08 | 1995-10-12 | ザ ガバメント オブ ザ ユナイテッド ステイツ オブ アメリカ,アズ レプリゼンテッド バイ ザ セクレター,デパートメント オブ ヘルス アンド ヒューマン サービシーズ | 野生型麻疹ウイルス糖タンパク質:ワクチンおよびその検出方法 |
Non-Patent Citations (2)
Title |
---|
ROTA J. S., ET AL.: "COMPARISON OF SEQUENCES OF THE H, F, AND N CODING GENES OF MEASLES VIRUS VACCINE STRAINS.", VIRUS RESEARCH, AMSTERDAM, NL, vol. 31., 1 January 1994 (1994-01-01), NL, pages 317 - 330., XP002913150, ISSN: 0168-1702, DOI: 10.1016/0168-1702(94)90025-6 * |
See also references of EP0949333A4 * |
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EP0949333A1 (en) | 1999-10-13 |
CA2262500A1 (en) | 1998-12-10 |
JP4043054B2 (ja) | 2008-02-06 |
EP0949333A4 (en) | 2001-09-12 |
EP0949333B1 (en) | 2006-05-17 |
CA2262500C (en) | 2004-03-16 |
US20020146434A1 (en) | 2002-10-10 |
US6605284B2 (en) | 2003-08-12 |
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