JPWO2013011942A1 - Mutant rabies virus and vaccine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective and inexpensive rabies vaccine with high safety. [MEANS FOR SOLVING PROBLEMS] Among the structural proteins of rabies virus, a mutation in which at least position 273 of N protein is an amino acid other than phenylalanine, position 394 of N protein is an amino acid other than tyrosine, and position 333 of G protein is an amino acid other than arginine or lysine A rabies virus (for example, ERA-N273 / 394-G333 strain in FIG. 2), a rabies vaccine preparation containing these mutant rabies viruses, and the like are provided. In addition to the attenuation of rabies virus by introducing a mutation of amino acid at position 333 of G protein, immunogenicity and safety can be improved by introducing mutation to amino acids at positions 273 and 394 of N protein. In addition, by introducing mutations to the amino acids at positions 273 and 394 of the N protein, even if the 194 position of the G protein is mutated to lysine, the virulence can be restored by mutation. Can be reduced, and safety can be further increased.

Description

  The present invention relates to a rabies virus structural protein, wherein at least the 273 position of the N protein is an amino acid other than phenylalanine, the 394 position of the N protein is an amino acid other than tyrosine, and the 333 position of the G protein is an amino acid other than arginine or lysine It relates to rabies virus, rabies vaccine preparation containing this virus, rabies prevention / treatment method, rabies vaccine production method, use for rabies vaccine preparation production, and the like.

  Rabies is a zoonotic disease caused by infection from a rabies virus bite or the like. Treatment has not yet been established, and when it occurs, almost 100% die with severe neurological symptoms.

  Rabies virus has a very broad host range and is believed to infect all mammals including humans. In developing countries, dogs are the main source of infection, and in developed countries, wild animals such as foxes, raccoons, skunks, and bats are the main source of infection. Even today, more than 50,000 people die from rabies each year. ing.

  Rabies can be prevented by vaccination, and inactivated rabies vaccine is now widely used. However, in the case of an inactivated vaccine, a large amount of antigen is required to obtain a sufficient effect, and the vaccine is expensive. On the other hand, live attenuated vaccines can be manufactured at a lower cost than inactivated vaccines, but there are safety concerns. For this reason, vaccines are not sufficiently spread, particularly in developing countries.

  The rabies virus is classified into the genus Lissavirus of the Rhabdoviridae family. Viral particles have a bullet-like shape with a width of 60 to 110 nm and a length of 130 to 250 nm, and contain five structural proteins: G protein, M protein, N protein, P protein, and L protein. The viral genome is a single-stranded RNA with a total length of about 12,000 bases and contains genes encoding five structural proteins in the order of N, P, M, G, and L from the upstream (3 'end side). .

  In recent years, with the progress of research on the pathogenicity determinants of rabies virus, it has been clarified that the virus can be attenuated by substituting the amino acid at position 333 of the G protein with an amino acid other than arginine or lysine (non-patented). Reference 1 etc.). A virus in which the amino acid at position 333 of the G protein is substituted with an attenuated type is actually used as a live attenuated vaccine for oral immunization of wild animals in Europe and the like (see Patent Document 1, etc.).

  This attenuated mutant virus has been reported to be toxic by mutating position 194 of G protein to lysine even if position 333 of G protein is mutated to attenuated form (Non-patented) Reference 2). For this reason, there is concern about the return of pathogenicity due to mutation at position 194 of the G protein, and the attenuated mutant virus only at position 333 of the G protein is not necessarily sufficiently safe.

  In the Ni-CE strain, which is a rabies attenuated strain, the present inventors determined that the amino acids at positions 273 and 394 of the rabies N protein are antiviral responses and pathogenicity via RIG-I (Retinoic acid-inducible gene I). (See Non-Patent Document 3).

In addition, Non-Patent Document 4 and Non-Patent Document 5 are documents relating to artificial synthesis of rabies virus mutants by the reverse genetics method described later.
US2011 / 0064764 A1 Dietzschold B et al, “Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus.” Proc. Natl. Acad. Sci. USA. 80: 70-74, 1983 Milosz Faber et al, “A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity.” Journal of Virology, vol.79, No.22 p14141-14148.2005 Tatsunori Masatani et al, “Amino acids at position 273 and 394 in rabies virus nucleoprotein are important for both evasion of host RIG-I-mediated antiviral response and pathogenicity.” Virus Research 155 (2011) 168-174. Naoto Ito et al, “Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: Glycoprotein gene is associated with virulence for adult mice.” Journal of Virology Vol. 75, No. 19 p9121-9128 2001. Naoto Ito et al, “Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system.” Microbiol Immunol. 2003; 47 (8): 613-7.

  As described above, a live attenuated vaccine for rabies can be produced at a lower cost than an inactivated vaccine, but there are safety concerns such as reversion to pathogenicity. Accordingly, an object of the present invention is to provide a safe, effective and inexpensive rabies vaccine.

  In the structural protein of the rabies virus, the present inventors, for the attenuated rabies virus in which the amino acid at position 333 of the G protein is substituted, in addition to the position at position 333 of the G protein, the amino acids at positions 273 and 394 of the N protein. It was newly found that by introducing mutations, immunogenicity and safety can be improved, and even when position 194 of the G protein is mutated to lysine, it is not toxic.

  Therefore, in the present invention, at least 273 of the N protein is an amino acid other than phenylalanine, 394 of the N protein is an amino acid other than tyrosine, and 333 of the G protein is an amino acid other than arginine or lysine. We provide certain mutant rabies viruses and rabies vaccine preparations containing these mutant rabies viruses.

  It is possible to attenuate the rabies virus by introducing a mutation into the amino acid at position 333 of the G protein, and to improve immunogenicity by introducing mutation into the amino acids at positions 273 and 394 of the N protein. In addition to the 333 position of the G protein, by introducing a mutation at the 273th and 394th amino acids of the N protein, a small inoculation amount is sufficient compared to the case where a mutation is added only at the 333th position of the G protein. In order to achieve a safe immune effect, the amount of inoculation can be reduced and the safety can be further increased.

  In addition, by introducing mutations to the amino acids at positions 273 and 394 of the N protein, even if the 194 position of the G protein is mutated to lysine, the virulence can be restored by mutation. Can be reduced, and safety can be further increased.

  Therefore, the present invention is useful as a rabies vaccine preparation containing a mutant rabies virus, and rabies can be effectively prevented and treated by administering this vaccine preparation.

  In addition, since this recombinant virus can be artificially synthesized using cultured cells, for example, when used as a live attenuated vaccine, there is an advantage that a rabies vaccine preparation can be produced at a relatively low cost.

  According to the present invention, immunogenicity can be increased. In addition, it is possible to provide an effective and inexpensive rabies vaccine with little concern about safety such as reversion of pathogenicity due to mutation.

<About Mutant Rabies Virus According to the Present Invention>
The present invention relates to a rabies virus structural protein, wherein at least the 273 position of the N protein is an amino acid other than phenylalanine, the 394 position of the N protein is an amino acid other than tyrosine, and the 333 position of the G protein is an amino acid other than arginine or lysine Includes all rabies viruses. That is, the present invention is not narrowly limited by virus production means and the like. For example, mutant viruses obtained by subculture in chicken embryos and / or cultured cells, mutant viruses artificially synthesized using clone cDNA, etc. And so on.

  Among the structural proteins of rabies virus, at least phenylalanine at position 273 of N protein is other amino acid, tyrosine at position 394 of N protein is other amino acid, and arginine or lysine at position 333 of G protein is other than that By substituting each with an amino acid, the rabies virus can be attenuated, the immunogenicity and safety can be improved, and the risk of pathogenicity reversion by mutation can be kept low.

  The amino acid sequences of portions other than the 273th position of the N protein, the 394th position of the N protein, and the 333th position of the G protein may be substantially the same as those in known rabies virus strains, and are not particularly limited. Examples of rabies virus strains with known amino acid sequences include CVS strain, ERA strain, Nishigahara strain, HEP-Flury strain, LEP-Flury strain, SAD Bern strain, and SAD B19 strain. Note that “substantially identical” means that the amino acid sequence of the corresponding region has 80 to 100% identity, more preferably 90 to 100% identity.

  The amino acid at position 273 of the N protein is not particularly limited as long as it is an amino acid other than phenylalanine. For example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, tyrosine, tryptophan, asparagine sun, glutamic acid, arginine, lysine, histidine are preferred, valine, Either leucine or isoleucine is more preferred, and leucine is most preferred.

  The amino acid at position 394 of the N protein is not particularly limited as long as it is an amino acid other than tyrosine. For example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, phenylalanine, tryptophan, asparagine sun, glutamic acid, arginine, lysine, histidine are preferred, arginine, Either lysine or histidine is more preferred, and histidine is most preferred.

  The amino acid at position 333 of the G protein is not particularly limited as long as it is an amino acid other than arginine or lysine. For example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, phenylalanine, tyrosine, tryptophan, asparagine sun, glutamic acid, histidine are preferred, glycine, leucine, Any one of isoleucine, serine, methionine, and glutamic acid is more preferable, and glutamic acid is most preferable.

  As a mutant rabies virus in which 273 of the N protein is leucine, 394 of the N protein is histidine, and 333 of the G protein is glutamic acid, for example, the N protein having the amino acid sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 described And a mutant rabies virus containing a G protein having the amino acid sequence as a structural protein.

<About the rabies vaccine preparation according to the present invention>
The present invention encompasses all rabies vaccine preparations containing at least the mutant rabies virus described above.

  The mutant rabies virus according to the present invention can be applied to both attenuated live vaccines and inactivated vaccines, as described above, because it can produce a rabies vaccine preparation at a relatively low cost and has a high effect as a vaccine. It is more preferable to use it as a live attenuated vaccine.

  A buffer, an isotonic agent, a soothing agent, a preservative, an antioxidant and the like may be appropriately added to the vaccine according to the purpose and use.

  As a suitable example of a buffering agent, buffer solutions, such as a phosphate, acetate, carbonate, citrate, etc. can be used, for example.

  As a suitable example of an isotonizing agent, sodium chloride, glycerin, D-mannitol etc. can be used, for example.

  As a suitable example of the soothing agent, for example, benzyl alcohol or the like can be used.

  Suitable examples of antiseptic agents include, for example, thimerosal, paraoxybenzoates, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, other antiseptics, antibiotics, and synthetic antibacterials. An agent or the like can be used.

  As a suitable example of an antioxidant, a sulfite, ascorbic acid, etc. can be used, for example.

  In addition, this drug includes auxiliary ingredients such as light-absorbing dyes (riboflavin, adenine, adenosine, etc.) that serve as storage and efficacy aids, and chelating / reducing agents (vitamin C, citric acid, etc.) for stabilization. , Carbohydrates (sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, etc.), casein digests, various vitamins, and the like may be included.

  About the dosage form etc. of a vaccine formulation, a well-known thing can be employ | adopted and it does not specifically limit. For example, it may be used as a liquid preparation, or may be mixed with food for oral administration after treatment such as lyophilization.

  On the other hand, when used as an inactivated vaccine preparation, known methods such as physical treatment (ultraviolet irradiation, X-ray irradiation, heat treatment, ultrasonic treatment, etc.), chemical treatment (organic by formalin, chloroform, etc.) Inactivation can be performed by solvent treatment, acid treatment with a weak acid such as acetic acid, treatment with alcohol, chlorine, mercury, or the like.

  For example, formalin is added to the culture solution at a volume concentration of 0.001 to 2.0%, more preferably 0.01 to 1.0%, and the culture solution is sensitized at 4 to 30 ° C. for 1 to 3 days to inactivate with formalin. It can be performed. For example, the inactivated virus may be washed with a buffer or the like to remove an inactivating agent such as formalin, or may be neutralized by adding a neutralizing agent to the inactivated virus. Further, the inactivated virus may be recovered by membrane filtration or centrifugation.

The amount of inactivated virus contained in the inactivated vaccine is not particularly limited. For example, the amount of virus before inactivation is preferably in the range of 10 ³ to 10 ¹¹ FFU, and in the range of 10 ⁴ to 10 ¹¹ FFU. More preferred.

  When used as an inactivated vaccine preparation, a known adjuvant may be added. Known adjuvants include, for example, animal oils (such as squalene) or hydrogenated oils thereof, vegetable oils (such as palm oil and castor oil) or hydrogenated oils thereof, mannitol / oleic anhydride, liquid paraffin, polybutene, caprylic acid, oleic acid , Oily adjuvants including higher fatty acid esters, PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymer, methacrylic acid copolymer, maleic anhydride copolymer, alkenyl derivative polymer, Oil-in-water emulsions, water-soluble adjuvants such as cationic lipids containing quaternary ammonium salts, precipitation adjuvants such as aluminum hydroxide (alum), sodium hydroxide, cholera toxin, E. coli Examples include microorganism-derived toxin components such as heat-labile toxin, bentonite, muramyl dipeptide derivatives, and interleukins. Moreover, what mixed these may be used.

  In addition, this vaccine formulation may be a mixed vaccine formulation with one or more vaccines against other diseases.

<About the rabies vaccine preparation method according to the present invention>
The present invention encompasses all methods for producing a rabies vaccine preparation comprising the step of artificially synthesizing the mutant rabies virus described above using cultured cells.

  This mutant rabies virus can also be artificially synthesized using cultured cells by the reverse genetics method, so that a vaccine preparation can be produced at a relatively low cost and with little lot difference.

  First, a recombinant plasmid for a rabies virus is prepared.

  As the parent strain of the rabies virus, for example, those described above can be used. Among the full-length cDNA of the parent strain of the rabies virus, mutations are introduced into the nucleotide sequences encoding the three amino acids of the N protein at positions 273 and 394 and the G protein at position 333. Mutagenesis can be performed by a known point mutation introduction method such as an overlap PCR method.

  The mutated cDNA fragment is inserted into the multicloning site of the plasmid vector by restriction enzyme digestion and ligation. In that case, for example, by arranging the T7 promoter sequence upstream (3 ′ end) of the cDNA fragment and the base sequence encoding hepatitis D virus ribozyme downstream (5 ′ end), T7 RNA polymerase The 3 ′ end of the artificial plus-strand genomic RNA transcribed by can be made the same sequence as the original virus.

  A known plasmid vector can be used and is not particularly limited.

  Subsequently, the cultured cells are transfected with the genomic plasmid of the recombinant rabies virus and the recombinant virus is expressed in the culture supernatant. For the transfection, a known method can be used, and it is not particularly limited. In addition, well-known cells such as BHK cells can be widely used as the cultured cells. For example, when expressing a virus with T7 RNA polymerase, a cultured cell that constantly expresses T7 RNA polymerase is used. May be.

  When transfecting a recombinant rabies virus genomic plasmid, a plasmid that also expresses the N protein of rabies virus, a plasmid that also expresses the P protein, and a plasmid that also expresses the L protein are simultaneously transfected as helper plasmids. By doing so, the mutant rabies virus can be efficiently synthesized.

  Subsequently, recombinant rabies virus expressed in the culture supernatant is added to the cultured cells to express the recombinant virus in large quantities. For example, mouse neuroblastoma-derived NA cells or Vero cells can be used as cultured cells.

  Virus recovery can be performed by a known method such as centrifugation or membrane filtration. Further, by adding the recovered virus to the cultured cells, the recombinant virus can be scaled up and mass-produced.

  When the recombinant virus is used as a live attenuated vaccine, for example, a buffer, an isotonic agent, a soothing agent, a preservative, an antioxidant and the like are appropriately added to the collected virus to produce a product. When the recombinant virus is used as an inactivated vaccine, for example, after inactivating the virus by a known method, a buffer, an isotonic agent, a soothing agent, a preservative, an antioxidant, etc. are appropriately added. It may be commercialized.

<About the use of the virus for manufacturing the rabies vaccine preparation according to the present invention>
The present invention encompasses all uses of the mutant rabies virus described above for the production of a rabies vaccine formulation.

  By using this mutant rabies virus for the production of a rabies vaccine preparation, there are few safety concerns such as reversion to pathogenicity, and a rabies vaccine with a high vaccine effect can be produced relatively inexpensively and in large quantities.

<About the rabies prevention / treatment method according to the present invention>
The present invention includes all rabies prevention / treatment methods including at least a treatment for administering a rabies vaccine preparation containing at least the mutant rabies virus described above.

  The present invention can be applied to animals that can be infected with rabies, such as humans and non-human animals such as dogs, cats, foxes, raccoons, skunks, bats, and mice.

  For example, the rabies vaccine preparation may be administered to healthy individuals to immunize them to prevent the development of rabies. Rabies may be treated by eliciting an immune response.

This rabies vaccine preparation may be administered by subcutaneous, intracutaneous, intramuscular injection or the like, or in the case of wild animals or the like, and may be administered orally by mixing with food. When using a live attenuated vaccine preparation, for example, 10 ³ to 10 ¹¹ FFU is administered per subcutaneous injection, intradermal, intramuscular injection, and 10 ⁴ to 10 ¹¹ FFU is administered per oral administration. . In the case of using an inactivated vaccine preparation, for example, 10 ³ to 10 ¹¹ viruses at a time for subcutaneous, intradermal, intramuscular injection, and 10 ⁴ for virus by oral administration. Administer ~ 10 ¹¹ doses. The frequency of administration is not particularly limited, but is preferably once or several times at intervals of 1 week to 3 months. In addition, administration at least once a year is preferable.

  In Example 1, artificial synthesis of recombinant rabies virus was attempted by the reverse genetics method.

  Encodes the T7 promoter sequence in the upstream region (3 'end side) of the full-length cDNA of ERA strain, one of the highly toxic fixed strains of rabies virus, and the hepatitis D virus ribozyme directly under the downstream (5' end) CDNA fragments each having the nucleotide sequence to be amplified were amplified by PCR. The coding sequence for hepatitis D virus ribozyme was inserted so that the 3 ′ end of the artificial plus-strand genomic RNA transcribed by T7 RNA polymerase had the same sequence as the original virus.

  The cDNA fragment was inserted into the multicloning site of plasmid vector pUC19 by restriction enzyme cleavage and ligation. Through the above procedure, a genome plasmid carrying the ERA strain genome full-length cDNA (hereinafter referred to as “ERA strain genome plasmid”) was prepared.

  Next, a cDNA fragment in which the nucleotide sequence was mutated so that three amino acids at the N protein 273 and 394 positions and the G protein 333 position were replaced by leucine, histidine and glutamic acid, respectively, in the entire genome of the ERA strain. Was amplified by overlap PCR using the ERA strain genomic plasmid as a template (see Non-Patent Document 4).

  The genomic full length cDNA coding region on the ERA strain genomic plasmid was replaced with a mutated genomic full length cDNA fragment by restriction enzyme digestion and ligation. Through the above procedure, a genome plasmid (hereinafter referred to as “ERA-N”) having the genome full length cDNA of the mutant strain (ERA-N273 / 394-G333 strain) in which the amino acid sequence at three positions is mutated out of the genome length of the ERA strain. N273 / 394-G333 strain genomic plasmid ”) was prepared (see SEQ ID NO: 3 for the nucleotide sequence of the ERA-N273 / 394-G333 strain genome).

  In addition, among the full-length genome of the ERA strain, a genome plasmid containing the full-length genomic cDNA of the mutant strain (ERA-G333 strain) in which the amino acid at position 333 of G protein is substituted with glutamic acid (hereinafter referred to as “ERA-G333 strain genome ・The “plasmid” was prepared in the same procedure.

  As a result of sequence analysis, it was confirmed that the target mutation was present on each genome / plasmid.

  Subsequently, artificial synthesis of each recombinant virus was attempted using these genomic plasmids.

  First, as a helper plasmid, a plasmid expressing the N protein of rabies virus, a plasmid expressing the P protein, and a plasmid expressing the L protein were also prepared (see Non-Patent Document 5).

  The day before, T7 RNA polymerase constitutively expressing BHK cells (hereinafter referred to as “BHK / T7-9 cells”) were seeded in 24-well tissue culture plates. On the day, BHK / T7-9 cells were co-transfected with genomic plasmid (2 μg / well) and 3 types of helper plasmids (0.4, 0.1, 0.2 μg / well each) and cultured for 4-5 days Thereafter, the culture supernatant was collected and stored at −80 ° C.

  In addition, the transfected BHK / T7-9 cells were fixed and then stained with an anti-rabies virus N protein monoclonal antibody. Then, the presence of the virus mutant was confirmed using the expansion of the fluorescence signal as an index.

  Subsequently, an attempt was made to produce a recombinant virus stock.

  The collected culture supernatant was added to culture mouse neuroblastoma-derived NA cells, and when about 50% of the cells were lost due to cytopathic effect, the culture supernatant was recovered. The culture supernatant was centrifuged, and the supernatant was stored at −80 ° C. as a virus mutant stock.

  The virus titer of the recombinant virus artificially synthesized by the above procedure was measured.

  NA cells on a 24-well tissue culture plate were inoculated with 100 μL / well of each virus solution diluted 10-fold. After allowing to stand at 37 ° C. for 1 hour to adsorb the virus to the cells, the cells were washed. A culture solution containing 0.5% methylcellulose was added to the cells and cultured at 37 ° C. for 2 days. After fixing the infected cells, the cells were fluorescently stained with an anti-rabies virus N protein monoclonal antibody, and the number of fluorescent focus per well was counted. As the virus titer, the focus forming unit (FFU) / mL was calculated based on the average number of focus appearing in 3 holes of the same dilution.

  In Example 2, the proliferation of the recombinant virus (ERA-N273 / 394-G333 strain) was examined.

  Vero cells sheeted in a T-25 culture flask were inoculated with ERA strain or ERA-N273 / 394-G333 strain under the condition of multiplicity of infection = 0.01. After allowing to stand at 37 ° C. for 1 hour to adsorb the virus to the cells, the cells were washed. 7 mL of culture solution per flask was added to the cells and cultured at 37 ° C. On the third, fifth, seventh, ninth and eleventh days of infection, 100 μL of the culture solution was collected, and the virus titer was measured in the same manner as in Example 1.

As a result, the virus titer of ERA-N273 / 394-G333 strain in Vero cells was 1 × 10 ⁶ FFU / mL or more on the 7th day after infection and 1 × 10 ⁸ FFU / mL or more on the 11th day after infection. Yes, it was almost equivalent to the virus titer of ERA strain. From these results, it was shown that the virus growth ability was not impaired even if the amino acids at the three positions of N protein 273 and 394 and G protein 333 were simultaneously mutated in the structural protein of rabies virus.

  In Example 3, the immunogenicity of the recombinant virus (ERA-N273 / 394-G333 strain) was examined.

10 ⁴ FFU ERA-G333 strain or ERA-N273 / 394-G333 strain was immunized by intramuscular inoculation of 5 ddY mice (6 weeks old, female) per group.

  Two weeks after the inoculation, blood was collected from the immunized mouse, the serum was diluted 2-fold, and then immobilized at 56 ° C for 30 minutes. Next, 2-fold serially diluted serum (25 μL) was mixed with a virus solution (25 μL) containing a CVS strain corresponding to 200 times the 50% tissue culture infectious dose. This mixture was sensitized overnight at 4 ° C. to sensitize the neutralizing antibody in the diluted serum to the virus.

A 96-well tissue culture plate was inoculated with NA cell suspension (3 × 10 ⁵ cells / mL, 0.1 mL / hole) and inoculated with a serum-virus mixture. The cells were cultured at 37 ° C. for 3 days. After fixing the infected cells, the cells were fluorescently stained with an anti-rabies virus N protein monoclonal antibody, and the virus neutralizing antibody titer was calculated by the Reed and Munch method. At the same time, the same measurement was performed on standard serum (0.5 international units [IU / mL]) obtained from the International Organization for Animal Health (OIE), and neutralizing antibody titers were converted to international units [IU / mL].

  The results are shown in FIG. FIG. 1 is a graph showing the neutralizing antibody titer of mouse serum when mice were immunized with ERA-N273 / 394-G333 strain. In FIG. 1, "ERA-G333" on the horizontal axis shows the results when mice were immunized with ERA-G333 strain, and "ERA-N273 / 394-G333" immunized mice with ERA-N273 / 394-G333 strain In each case, the vertical axis represents the neutralizing antibody titer of mouse serum (unit: IU / mL).

As shown in FIG. 1, when immunized by intramuscular inoculation with 10 ⁴ FFU of recombinant rabies virus, the ERA-N273 / 394-G333 strain showed a neutralizing antibody titer of mouse serum of 0.5 IU / It was more than mL, and was higher than ERA-G333 strain. From these results, it was shown that the ERA-N273 / 394-G333 strain has higher immunogenicity than the ERA-G333 strain.

In Example 4, the ED ₅₀ (50% effective dose) of the recombinant virus (ERA-N273 / 394-G333 strain) was examined.

Immunization was performed by intramuscular inoculation of 5 ddY mice (6 weeks old, female) with 10 ² to 10 ⁵ FFU of ERA-G333 strain or ERA-N273 / 394-G333 strain. Six weeks after the inoculation, immunized mice were inoculated in the brain with a standard challenge strain for vaccine test (CVS strain) 25 times the 50% lethal dose. Each mouse was observed for 14 days, and the ED ₅₀ (50% effective dose) of each strain was calculated from the survival rate.

The results are shown in FIG. FIG. 2 is a graph showing the ED ₅₀ (50% effective dose) of the ERA-N273 / 394-G333 strain. In FIG. 2, “ERA-G333” on the horizontal axis represents the results for the ERA-G333 strain, “ERA-N273 / 394-G333” represents the results for the ERA-N273 / 394-G333 strain, and the vertical axis represents the ED ₅₀ (50% effective dose, unit: FFU).

As shown in Fig. 2, ED- ₅₀ (50% effective dose) of ERA-N273 / 394-G333 strain is 5,000 FFU or less, and the equivalent effect is obtained with about 1/3 dose compared to ERA-G333 strain. It was.

  This result shows that in the strains with mutations at position 333 of the G protein and positions 273 and 394 of the N protein, the immunogenicity is improved compared to the case where the mutation is added only at position 333 of the G protein. To suggest. In addition, due to the improved immunogenicity, the same immune effect can be expected even if the inoculation amount is reduced, so this result suggests that the safety can be further improved by reducing the inoculation amount. To do.

  As described above, the results of Example 3 and Example 4 indicate that the ERA-N273 / 394-G333 strain has high immunogenicity and safety, that is, is useful as a live attenuated vaccine against rabies.

  As described above, even when a mutation is added to the amino acid at position 333 of G protein and the rabies virus is attenuated, if the amino acid at position 194 of G protein is substituted with a mutation for lysine, the virus becomes virulent and the pathogenicity is restored. It is known to do. Therefore, in Example 5, it was examined whether virus virulence occurs when the amino acid at position 194 of G protein of ERA-N273 / 394-G333 strain is changed to lysine.

  The ERA-N273 / 394-G333 strain genomic plasmid or ERA-G333 strain genomic plasmid was mutated by the same method as in Example 1 so that the amino acid at position 194 of the G protein was replaced with lysine. Using the genomic plasmid, a mutant virus (ERA-N273 / 394-) in which the above-mentioned three amino acid sequences and the G protein position 194 were mutated out of the full-length genome of the ERA strain in the same manner as in Example 1. G194 / 333 strain) and a mutant virus (ERA-G194 / 333 strain) in which amino acids at positions 333 and 194 were mutated were artificially synthesized.

10 ² to 10 ⁴ FFU of ERA-N273 / 394-G194 / 333 strain or ERA-G194 / 333 strain was inoculated in the brain into 5 groups of ddY mice (6 weeks old, female). Each mouse was observed for 18 days, and the transition of its survival rate was examined.

The results are shown in FIGS. 3A and 3B. 3A is a graph showing the survival rate when the ERA-G194 / 333 strain is inoculated in the brain, and FIG. 3B is a graph showing the survival rate when the ERA-N273 / 394-G194 / 333 strain is inoculated in the brain. is there. In both figures, the horizontal axis shows the number of days after virus inoculation, the vertical axis shows the survival rate (%), and each graph shows the case when no virus was administered (non-infected) or when 10 ² to 10 ⁴ FFU was inoculated with virus. Each result is represented.

When ERA-G194 / 333 strain was inoculated in the brain, as shown in FIG. 3A, the survival rate at 17 days after inoculation with 10 ² FFU was 20%, and at 17 days after inoculation with 10 ³ FFU When the viability was 40% and virus intensification was occurring, when ERA-N273 / 394-G194 / 333 strain was inoculated in the brain, as shown in FIG. However, no virus poisoning was observed at 100%.

  This result shows that, in the ERA-N273 / 394-G333 strain, even when the amino acid at position 194 of G protein is substituted with lysine by mutation, the virus is not toxic, and in addition to position 333 of G protein, N Mutation of amino acids at positions 273 and 394 of the protein suggests that even if the 194th position of the G protein is replaced with lysine, it is not toxic, that is, the risk of reverting to virulence due to mutation is low. To do.

In Example 3, the graph which shows the neutralizing antibody titer of a mouse | mouth serum at the time of immunizing a mouse | mouth with ERA-N273 / 394-G333 strain | stump | stock. Graph in Example 4, ED ₅₀ of ERA-N273 / 394-G333 strain (50% effective dose). In Example 5, the graph showing the survival rate at the time of inoculating ERA-G194 / 333 strain in the brain. In Example 5, the graph showing the survival rate when ERA-N273 / 394-G194 / 333 strain is inoculated in the brain.

Claims (8)

  Among the structural proteins of rabies virus, a mutant rabies virus in which at least 273 of the N protein is an amino acid other than phenylalanine, 394 of the N protein is an amino acid other than tyrosine, and 333 of the G protein is an amino acid other than arginine or lysine.   The mutant rabies virus according to claim 1, which is artificially synthesized using cultured cells.   The mutant rabies virus according to claim 1, wherein position 273 of the N protein is leucine, position 394 of the N protein is histidine, and position 333 of the G protein is glutamic acid.   The mutant rabies virus according to claim 1, comprising an N protein having the amino acid sequence of SEQ ID NO: 1 and a G protein having the amino acid sequence of SEQ ID NO: 2 as structural proteins.   A rabies vaccine preparation containing the mutant rabies virus according to claim 1.   A method for preventing and treating rabies, comprising administering the vaccine preparation according to claim 5.   Using cultured cells, artificially synthesize a mutant rabies virus in which at least 273 of the N protein is an amino acid other than phenylalanine, 394 of the N protein is an amino acid other than tyrosine, and 333 of the G protein is an amino acid other than arginine or lysine A method for producing a rabies vaccine preparation, comprising the step of:   For the production of a rabies vaccine preparation of a mutant rabies virus in which at least 273 of the N protein is an amino acid other than phenylalanine, 394 of the N protein is an amino acid other than tyrosine, and 333 of the G protein is an amino acid other than arginine or lysine use.

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