JP5944210B2 - B5R gene-deficient recombinant vaccinia virus incorporating a hemagglutinin protein gene derived from a new influenza virus - Google Patents

Description

  The present invention relates to preventive and therapeutic agents for new influenza. Specifically, the vaccinia virus B5R protein gene (hereinafter sometimes referred to as “B5R gene”) region was recombined with a new influenza virus hemagglutinin protein gene (hereinafter also referred to as “HA gene”). The present invention relates to B5R gene-deficient recombinant vaccinia virus, and preventive and therapeutic agents for new influenza containing the recombinant vaccinia virus.

  The vaccinia virus LC16m8 strain is a safe and effective attenuated vaccine strain that was approved by the Ministry of Health and Welfare in 1975 as a smallpox vaccine (decubitus vaccine) and has been inoculated to more than 50,000 infants. The weak toxicity of the LC16m8 strain stems from the fact that the full length B5R protein is not expressed due to the single base deletion of the B5R protein gene of the LC16mO strain, which is thought to be involved in cell adsorption / infection (non-patented) Reference 1). On the other hand, mutations occurred while the LC16m8 strain repeated replication and growth, a single base insertion occurred in this B5R gene region, and the emergence of a reverted virus expressing the full-length B5R protein and the enhancement of its growth potential was There are concerns about ensuring the safety of However, it has been reported so far whether recombinants lacking the B5R gene by homologous recombination between the B5R protein gene region and the foreign gene can ensure safety and express the foreign gene well. Not.

  On the other hand, H1N1 pandemic influenza virus (H1N1 (2009) pdm), which is thought to have originated in Mexico in April 2009, is significantly different in antigenicity from conventional seasonal influenza viruses and has no immunity. Because there were many, the infection spread rapidly to the whole world. The H1N1 (2009) pdm vaccine is an inactivated split vaccine produced by the same method as the seasonal influenza vaccine. Since this split vaccine is also an inactivated vaccine, it is not always possible to exert a vaccine effect against epidemic strains.

Currently, there are Tamiflu and Relenza, which inhibits the action of neuraminidase (NA), which is important for influenza virus budding, as influenza drugs, but its effects are limited, such as medication within 48 hours after onset. ing. Other NA inhibitors and influenza virus polymerase inhibitors are under development, but are currently in clinical trials.
Under such circumstances, it is strongly desired to establish preventive and therapeutic drugs that can exert effects over a wide range and for a long time against influenza viruses that are difficult to predict.

Morikawa S. et al., An attenuated LC16m8 smallpox vaccine: analysis of full-genome sequence and induction of immune protection., J. Virol., 2005, vol. 79 (18), p. 11873-11891

  The problem to be solved by the present invention is to provide a recombinant vaccinia virus effective for preventing the onset due to infection with a new influenza virus, and a preventive and therapeutic agent for new influenza containing the recombinant vaccinia virus. is there.

  The present inventors conducted further earnest research based on the analysis and examination results regarding the new influenza virus infection. As a result, we found that the use of recombinant vaccinia virus in which the B5R protein gene region of vaccinia virus is replaced with a new influenza virus antigen gene as a vaccine leads to the activation of immunity, leading to a powerful new influenza virus infection prevention method. It was. The present inventors have confirmed that the above recombinant vaccinia virus is effective in preventing the onset of pandemic influenza virus belonging to the H1 subtype as a new influenza virus (eg, H1N1 (2009) pdm). did. In particular, it was confirmed that a single inoculation of the above recombinant vaccinia virus can be prevented by 3 days before challenge infection with H1N1 (2009) pdm, and the present invention was completed.

That is, the present invention is as follows.
(1) Recombination in which all or part of the B5R protein gene region of vaccinia virus is recombined with the expression promoter and all or part of the cDNA encoding the pandemic influenza virus-derived hemagglutinin protein belonging to the H1 subtype Vaccinia virus.

  Here, examples of the vaccinia virus include the LC16m8 strain. An example of a pandemic influenza virus belonging to the H1 subtype is H1N1 (2009) pdm.

Examples of the cDNA encoding the HA protein include the following DNAs (a) and (b).
(a) DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 which is SEQ ID NO: 1 (H1 subtype; California vaccine strain sequence) or a variant thereof
(b) DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 3 and encodes the HA protein of a virus strain belonging to the H1 subtype

Furthermore, examples of the expression promoter contained in the recombinant vaccinia virus of the present invention include a hybrid promoter. Specifically, examples of the hybrid promoter include those consisting of the following DNA (a) or (b).
(a) DNA consisting of the base sequence shown in SEQ ID NO: 2
(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2 and has promoter activity

(2) A pharmaceutical composition comprising the recombinant vaccinia virus of (1) above.
The pharmaceutical composition can be used as a preventive and / or therapeutic agent for new influenza, specifically as a prophylactic and / or therapeutic agent for influenza caused by pandemic influenza belonging to the H1 subtype.

  ADVANTAGE OF THE INVENTION According to this invention, it is effective in the prevention and treatment of new influenza (especially influenza by the pandemic influenza virus which belongs to H1 subtype), and the novel group with high safety | security which does not make the reversal virus which promotes proliferation increase appear. It is possible to provide a replacement vaccinia virus and a pharmaceutical composition containing the recombinant vaccinia virus, particularly a preventive and therapeutic agent for new influenza (a vaccine for preventing and treating new influenza).

It is a figure which shows the gene structure of the plasmid vector (pBMSF7c / (DELTA) B5R-mIVR153 transfer vector etc.) for preparation of the recombinant vaccinia virus which has the HA protein gene of a novel influenza virus. The figure on the left shows the construction of a vector used to produce a recombinant vaccinia virus in which the HA protein gene derived from H1N1 (2009) pdm is recombined with the B5R protein gene of the attenuated vaccinia virus LC16m8 strain. FIG. 1 is a construction diagram of a vector for producing a recombinant vaccinia virus in which an H1N1 (2009) pdm-derived HA protein gene is inserted into the HA protein gene region of the attenuated vaccinia virus strain LC16m8. It is a photograph of a PVDF membrane showing the results of confirming the expression of H1N1 (2009) pdm-derived HA protein in infected cells such as rVVΔB5R-mIVR153 by Western blotting. It is a figure which shows the comparison result of the vaccine effect of rVV-mIVR153 and rVV (DELTA) B5R-mIVR153. Specifically, it is a figure showing changes in body weight due to challenge infection of H1N1 (2009) pdm (specifically H1N1 / 2619) to BALB / c mice inoculated once with rVVΔB5R-mIVR153 or the like. In the figure, `` rVV-Empty '' is inoculated with rVV-Empty at 1 × 10 <sup>7</sup> PFU / mouse, `` rVV-mIVR153 '' is inoculated with rVV-mIVR153 at 1 × 10 ⁷ PFU / mouse, `` rVVΔB5R- “mIVR153” shows the results when rVVΔB5R-mIVR153 was inoculated at 1 × 10 ⁷ PFU / mouse. It is a figure which shows the antibody titer by inoculation of rVV-mIVR153 and rVVΔB5R-mIVR153. Specifically, the figure shows the results of measuring the antibody titer against HA protein of H1N1 (2009) pdm in the obtained serum after single inoculation of BALB / c mice with rVVΔB5R-mIVR153 etc. It is. It is a figure which shows the reduction effect of the viral load in lung tissue by inoculation with rVV-mIVR153 and rVVΔB5R-mIVR153. Specifically, as in the experimental schedule shown in FIG. 3, BALB / c mice were inoculated once with rVVΔB5R-mIVR153, etc., and then infected with H1N1 (2009) pdm (specifically H1N1 / 2619), 3 days after infection. It is a figure which shows the result of having measured the viral load in lung tissue. It is a figure which shows the pneumonia reduction effect by inoculation of rVV-mIVR153 and rVV (DELTA) B5R-mIVR153. Specifically, similar to the experimental schedule shown in FIG. 3, BALB / c mice were inoculated once with rVVΔB5R-mIVR153, etc., and then infected with H1N1 (2009) pdm (specifically H1N1 / 2619), 9 days after infection. It is a figure which shows the result of the pathological analysis of a lung. It is a figure which shows the comparison of the vaccine effect by inoculating rVV-mIVR153 and rVVΔB5R-mIVR153 3 days before the attack infection by H1N1 (2009) pdm (specifically H1N1 / 2619). Specifically, as shown in the experimental schedule of FIG. 7, BALB / c mice were infected with H1N1 (2009) pdm 3 days after the single inoculation of rVVΔB5R-mIVR153 etc. and showed changes in body weight after 9 days from infection. It is a figure.

  Hereinafter, although the recombinant vaccinia virus concerning this invention and its use are demonstrated in detail, the range of this invention is not restrained by these description, The range which does not impair the meaning of this invention except the following illustrations. Can be implemented with appropriate changes. Note that patent documents, non-patent documents, and other publications cited in this specification are incorporated herein by reference.

1. Summary of the Invention Among various vaccines, live vaccines are one of particularly effective, but it is generally known that a very long period is required to develop an attenuated vaccine for an emerging virus. This is considered to be the same for the new influenza.
As one of the techniques employed in such a case, a genetic engineering technique for producing a recombinant vaccinia virus (hereinafter sometimes referred to as “rVV”) as a live vaccine is known. For example, a recombinant vaccinia virus for rabies virus or Lindapes developed by the present inventors has been demonstrated to exhibit an excellent infection prevention effect in field tests and the like (for example, Tsukiyama K. et al. al., Arch. Virol., 1989, vol. 107, p. 225-235).

Furthermore, the present inventors have succeeded in producing a recombinant vaccinia virus having SARS-CoV cDNA known as a pathogen for the new type of pneumonia SARS (WO 2006/038742), and can be re-administered with an excellent preventive effect. (See, for example, Kitabatake M. et al., Vaccine, 2007, vol. 25, p. 630-637).
The recombinant vaccinia virus used for the production of rVV needs to be a vaccine strain with established safety, but such a vaccine strain may be a vaccinia virus strain LC16m8 (for example, clinical and viral, vol. 3, No. 3, p. 269, 1975) are known. The LC16m8 strain was isolated from the Lister strain, and has actually been administered as a preventive vaccine, and has been confirmed to be safe and effective, and is the only vaccine strain currently produced. However, a single-base deletion of the B5R protein caused by the weak toxicity of the LC16m8 strain caused the appearance of a reverting virus that expresses the full-length B5R protein that was mutated and inserted into the same gene region while the virus replicated. (See, for example, Morikawa S. et al., J. Virol., 2005, vol. 79, p. 11873-11891). In addition, the present inventors use a gene expression promoter that can greatly enhance the ability to produce antibodies and induce cellular immunity in the process of developing recombinant vaccinia viruses against Lindapesto, HIV, SARS-CoV, etc. Was found to be effective against the vaccinia virus of the present invention. Specifically, it was found that it is preferable to use pSFJ1-10 or pSFJ2-16 as a plasmid vector (for example, Jin NY et al., Arch. Virol., 1994, vol. 138, p. 315-330; Elmowalid GA. Et al., Pros. Natl. Acad. Sci., 2007, vol. 104, p. 8427-8432;

As a result, the present inventor recombined the gene encoding the hemagglutinin (HA) protein of H1N1 pandemic influenza virus (H1N1 (2009) pdm) with the B5R protein gene of vaccinia virus together with the expression promoter. ) A B5R protein gene-deficient recombinant vaccinia virus expressing pdm-derived HA protein was prepared, the safety of the LC16m8 strain was fixed, and a foreign gene was successfully expressed.
The virus serving as the parent of the recombinant vaccinia virus according to the present invention is vaccinia virus as described above. H1N1 (2009) cDNA encoding the HA protein of pdm (all or part of the cDNA) is recombined with the B5R protein gene region (all or part of the gene region) in the genome of the present invention. It is a recombinant vaccinia virus. H1N1 (2009) cDNA encoding the HA protein of pdm (all or a part of the cDNA) was used as an expression unit, and each was introduced into a vaccinia virus vector. This expression unit was homologously recombined with the B5R protein gene of vaccinia virus strain LC16m8. It is already known that deletion of the B5R protein gene does not affect the growth activity of the vaccinia virus strain LC16m8 (see PNAS, vol. 102, p. 4152-4157, 2005). Rather, a safe vaccine strain can be used as a recombinant maternal virus because it can eliminate the possibility of the appearance of a reverted virus that expresses the full-length B5R protein.

Recombinant vaccinia live vaccines have been tested in the field against rabies virus and Linda virus, and their superior infection prevention effect has been proven.
The present inventor inserted the H1N1 (2009) pdm HA protein gene downstream of the hybrid promoter, and recombined these genes with the B5R protein gene region of the attenuated vaccinia virus strain LC16m8, thereby producing a recombinant vaccinia virus (rVV). Was made. The hybrid promoter is composed of a poxvirus type A inclusion body (ATI) promoter and a multi-repeating vaccinia virus 7.5 kDa protein (p7.5) early expression promoter (Jin NY et al., Arch. Virol., 1994, vol. 138, p. 315-330). This promoter was developed by Dr. Satoshi Shida from Hokkaido University, and can also be provided by him.
The B5R protein gene-deficient LC16m8 strain-derived rVV was confirmed to express a large amount of HA protein of H1N1 (2009) pdm by infecting animal cells, and H1N1 (2009) by inoculating animal individuals It was also confirmed that a high-titer antibody against the HA protein of pdm was produced at an early stage, thereby completing the present invention.

2. Production of recombinant vaccinia virus carrying HA protein gene of new influenza virus
H1N1 (2009) The pdm HA protein gene is already available from the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) provided by the National Center for Biotechnology Information (NCBI). Are registered with a predetermined accession number (Accession No.). For example, a gene (cDNA: SEQ ID NO: 1) encoding an HA protein derived from H1N1 (2009) pdm (virus strain belonging to H1 subtype) is registered in GenBank accession number: FJ969540.

  As a gene (cDNA) encoding the HA protein contained in the recombinant vaccinia virus of the present invention, in addition to the above gene, a part thereof or a mutant sequence thereof can be used as long as it has HA aggregation activity. For example, as a gene (cDNA) encoding the HA protein of H1N1 (2009) pdm, a DNA obtained by mutating (substituting) a part of the base of the DNA consisting of the base sequence shown in SEQ ID NO: 1 (specifically, Further, DNA consisting of the base sequence shown in SEQ ID NO: 3) can also be used.

These HA protein genes (including partially deleted and mutated genes) have already been cloned and inserted into plasmids. Therefore, the gene contained in the recombinant vaccinia virus of the present invention, that is, the entire cDNA (including mutant sequences) encoding the HA protein of H1N1 (2009) pdm or a part thereof can be obtained by a normal genetic engineering technique. Can do. For example, a nucleic acid synthesis method using a DNA synthesizer generally used as a genetic engineering technique can be used. In addition, after isolating or synthesizing the gene sequence as a template, designing primers specific to each gene and amplifying the gene sequence using a PCR device, or gene amplification method using a cloning vector Can be used. The above method can be performed with reference to “Moleculer cloning, A Laboratory Manual 3rd ed.” (Cold Spring Harbor Laboratory Press (2001)). A known method can be used to purify the obtained PCR product. The mutant sequence, particularly the mutation-substituted DNA, is described in, for example, “Moleculer cloning, A Laboratory Manual 3rd ed.” And “Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997)”. It can be prepared according to the site-specific displacement induction method. Specifically, it can be prepared using a mutagenesis kit using site-directed mutagenesis by a known method such as the Kunkel method or the Gapped duplex method, and examples of the kit include QuickChange ^TM Site. -Directed Mutagenesis Kit (Stratagene), GeneTailor ^TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Prime STAR (registered trademark) Mutagenesis Basal kit, Mutan (registered trademark) -Super Express Km Etc .: manufactured by Takara Bio Inc.).

  In a preferred embodiment of the present invention, the HA protein gene of H1N1 (2009) pdm inserted into the plasmid can be used as the PCR template. Then, the HA protein gene region of H1N1 (2009) pdm can be prepared by PCR using H1N1 (2009) pdm HA protein gene cDNA as a template and using each gene-specific primer. In the present invention, the gene encoded by the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 is referred to as “mIVR153” as the gene encoding the HA protein derived from H1N1 (2009) pdm (virus strain belonging to H1 subtype). .

In the present invention, in addition to the DNA consisting of the nucleotide sequences shown in SEQ ID NOS: 1 and 3, the following DNAs can also be used as DNAs for the above HA protein genes (for example, mIVR153).
A DNA (mutant DNA) that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and encodes an HA protein derived from H1N1 (2009) pdm.
A DNA (mIVR153 mutant DNA) that hybridizes with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3 under stringent conditions and encodes an HA protein derived from H1N1 (2009) pdm.

  Each of the above mutant DNAs can be obtained by chemical synthesis, or colony hybridization, plaque hybridization, Southern blot, etc. using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 3 or fragments thereof as probes It can also be obtained from cDNA libraries and genomic libraries by known hybridization methods. Examples of stringent conditions in the above hybridization include 0.1 × SSC to 10 × SSC, 0.1% to 1.0% SDS, and 20 ° C. to 80 ° C., and more specifically, 37 ° C. to 56 ° C. An example is a condition in which prehybridization is performed for 30 minutes or more and then washing is performed 1 to 3 times in 0.1 × SSC and 0.1% SDS at room temperature for 10 to 20 minutes. For the detailed procedure of the hybridization method, “Molecular Cloning, A Laboratory Manual 3rd ed.” (Cold Spring Harbor Press (2001)) and the like can be referred to.

  Further, it has 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more homology with the base sequence shown in SEQ ID NO: 1, and H1N1 (2009) DNA encoding HA protein derived from pdm (mutant DNA) and the nucleotide sequence shown in SEQ ID NO: 3, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% As described above, DNA (mIVR153 mutant DNA) having a homology of 98% or more or 99% or more and encoding an HA protein derived from H1N1 (2009) pdm can be used.

The expression promoter contained in the recombinant vaccinia virus of the present invention is inserted downstream of the B4R protein gene of vaccinia virus, and includes a poxvirus type A inclusion body (ATI) promoter and a multi-repeat vaccinia virus 7.5 kDa protein ( p7.5) A hybrid promoter (ATI / p7.5 hybrid promoter) composed of early expression promoters. This promoter can be ligated to an appropriate plasmid. For example, pBMSF7cΔB5R is known (Arch. Virol., 1994, vol. 138, p. 315-330; see JP-A-6-337773).
The base sequence of a hybrid promoter that can be used in the present invention is shown in SEQ ID NO: 2. However, in the present invention, in addition to DNA consisting of the base sequence shown in SEQ ID NO: 2, it hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2, and a promoter DNA having activity can also be used as an expression promoter (particularly a hybrid promoter). “Stringent conditions” are the same as described above. “Having promoter activity” means having the transcriptional activity of a gene encoding a structural protein or a nonstructural protein.

The protein expressed by this hybrid promoter can be expressed in a large amount in the form of complete sugar modification from the early stage to the late stage of vaccinia virus infection. In the present invention, pBMSF7cΔB5R-Flu HA is a generic term for a plasmid vector in which a cDNA encoding a pandemic influenza virus belonging to the H1 subtype or a mutant HA protein thereof or a part thereof is inserted downstream of the hybrid promoter of pBMSF7cΔB5R. In particular, a plasmid vector into which the HA protein gene of H1N1 (2009) pdm encoded by the nucleotide sequence of SEQ ID NO: 3 is inserted is called pBMSF7cΔB5R-mIVR153.
A recombinant vaccinia virus (rVV) can be produced by introducing these plasmid vectors into the host vaccinia virus. rVV obtained using pBMSF7cΔB5R-Flu HA is referred to as rVVΔB5R-Flu HA, and rVV obtained using pBMSF7cΔB5R-mIVR153 is sometimes referred to as rVVΔB5R-mIVR153.
The plasmid vector can be introduced into the host by any known method. For example, by introducing the plasmid vector pBMSF7cΔB5R-mIVR153 into an animal cell infected with the attenuated vaccinia virus strain LC16m8, the B5R of vaccinia virus is introduced. A recombinant vaccinia virus (rVVΔB5R-mIVR153) that causes homologous recombination in the gene region and expresses the HA protein gene of H1N1 (2009) pdm can be produced.

  The vaccinia virus LC16m8 strain used for the production of rVV is an attenuated strain that can be propagated in individual animals but has extremely low proliferation ability in nerve cells. It has been approved as a pressure ulcer vaccine in Japan, and no serious side effects have occurred as a result of inoculation to about 50,000 children (Ministry of Health and Welfare Vaccine Research Group Report: Clinical and Virus, vol. 3, No. 3, p. 269, 1975). On the other hand, it is reported that the immunity induction ability is equivalent to the parent Lister strain, and the LC16m8 strain is a safe and effective vaccine.

  The produced recombinant vaccinia virus (rVV) exhibits an agglutination reaction on guinea pig erythrocytes because the B5R protein gene region of vaccinia virus is recombined with the HA protein gene derived from influenza virus. Therefore, rVV screening is performed using as an index the agglutination reaction of guinea pig erythrocytes on plaques formed by infecting animal cells with rVVΔB5R-Flu HA. The target rVV may be selected from red plaques having hemagglutination activity.

The virus obtained from the red plaque can be subjected to PCR using the virus genome as a template with the influenza virus HA protein gene-specific primer used in the present invention to confirm the introduction of the HA virus gene of the influenza virus.
The expression of influenza virus-derived HA protein in rVV-infected cells can be confirmed, for example, by Western blotting using animal cells after infection with rVVΔB5R-mIVR153 as a sample. As the antibody, for example, rabbit antiserum prepared by immunization with an HA peptide derived from the HA protein of H1N1 (2009) pdm (aa 223-234 (SEQ ID NO: 4: YSKKFKPEIAIR) or the like) can be used.

3. TECHNICAL FIELD The present invention relates to preventive and therapeutic agents for influenza (new influenza) caused by pandemic influenza virus belonging to the H1 subtype, including the recombinant vaccinia virus. A composition). The present invention also provides a method for preventing and treating the above-mentioned new influenza, including the administration of the above-mentioned recombinant vaccinia virus to a patient (subject), use of the above-mentioned recombinant vaccinia virus for the prevention and treatment of the above-mentioned novel influenza, In addition, the use of the above recombinant vaccinia virus for producing the above-mentioned novel influenza preventive and therapeutic agents can be provided.

The pharmaceutical composition of the present invention can be introduced into a living body by any known method, for example, intramuscular, intraperitoneal, intradermal or subcutaneous injection, inhalation from the nasal cavity, oral cavity or lung, or oral administration. Furthermore, it is also possible to use a recombinant vaccinia virus contained in the pharmaceutical composition of the present invention in combination with an existing antiviral drug (eg, Tamiflu, Relenza). The mode of combined use is not particularly limited, and the recombinant vaccinia virus of the present invention and an existing antiviral agent can be administered simultaneously, and after administration of one, a method of administering the other after a certain period of time has elapsed. Can also be introduced into the living body.
In addition, the pharmaceutical composition of the present invention comprises known pharmaceutically acceptable carriers such as excipients, extenders, binders, lubricants, buffers, tonicity agents, chelating agents, coloring agents, preservatives. , Fragrances, flavors, sweeteners and the like.

The pharmaceutical composition of the present invention includes tablets, capsules, powders, granules, pills, solutions, syrups and other oral administration agents, injections, external preparations, suppositories, ophthalmic preparations and the like. Depending on the form, it can be administered orally or parenterally. Preferable examples include intradermal, intramuscular, abdominal injection and the like.
The dosage is appropriately selected depending on the type of active ingredient, administration route, administration subject, patient age, body weight, sex, symptom and other conditions, but the daily dose of virus is 1,000 to 1,000,000,000 for oral administration. About PFU (plaque forming units), preferably about 100,000 to 100,000,000 PFU. In the case of parenteral, about 100 to 1,000,000,000 PFU, preferably about 1,000 to 100,000,000 PFU. The virus can be administered once a day or in several divided doses.

  The recombinant vaccinia virus of the present invention is used as a vaccine for preventing and treating new influenza. In addition, so far, in the development of vaccines against H1N1 (2009) pdm, studies focusing on antibodies against H1N1 (2009) pdm and cytotoxic T cells (CTL) have been conducted. Therefore, it is preferable to previously measure the antibody titer or cellular immune activity as a vaccine.

For example, the antibody titer against the prepared recombinant vaccinia virus (rVVΔB5R-mIVR153) or the parent strain, LC16m8, was obtained by inoculating mice with these virus strains and collecting serum over time, and the serum H1N1 (2009) pdm It can be obtained by measuring the ELISA value for HA protein. The serum of mice inoculated with rVVΔB5R-mIVR153 shows an increase in antibody titer against HA protein of H1N1 (2009) pdm from 2 weeks after inoculation.
As described above, the recombinant vaccinia virus (rVVΔB5R-mIVR153) prepared by the present inventors can induce humoral immunity against H1N1 (2009) pdm, which is a pandemic influenza virus belonging to the H1 subtype.

  Hereinafter, the present invention will be described more specifically with reference to examples. These examples are illustrative only and are not intended to limit the scope of the invention.

Production of recombinant vaccinia virus (rVV)
By incorporating the H1N1 (2009) pdm HA protein gene between the SbfI-SgfI sites of the pBMSF7c / ΔB5R plasmid, the ATI / p7.5 hybrid promoter was inserted downstream of the B4R protein gene and the HA protein gene was inserted downstream thereof. Plasmid vector pBMSF7c / ΔB5R-mIVR153 was prepared (FIG. 1). Specifically, in pBMSF7c / ΔB5R-mIVR153, DNA consisting of the base sequence shown in SEQ ID NO: 3 is inserted as the HA protein gene of H1N1 (2009) pdm, and DNA consisting of the base sequence shown in SEQ ID NO: 2 is inserted. Inserted as an ATI · p7.5 hybrid promoter. Similarly, by inserting the HA protein gene of H1N1 (2009) pdm between the SbfI-SgfI sites of the pBMSF7c plasmid, the ATI / p7.5 hybrid promoter is inserted into the vaccinia virus protein gene region and the HA protein gene is inserted downstream thereof. The prepared plasmid vector pBMSF7c-mIVR153 was prepared and used as a control (FIG. 1).

  Primary kidney cultured cells were seeded in a 35 mm dish, and when they reached confluence, vaccinia virus attenuated strain LC16m8 was infected with moi = 10 at 30 ° C. for 1 hour. Note that moi (multiplicity of infection) refers to PFU (plaque forming units) per cell. After infection, the virus solution was removed by suction, and the cells were washed with PBS (−). Next, treat with 5-fold diluted TrypLE / 0.5 mM EDTA / PBS (-), wash with 10% FCS / DMEM medium antibiotics (-), PBS (-), HeBS buffer, then suspend cells in 600 μl of HeBS buffer did. 1.5 μg of the plasmid vector pBMSF7c / ΔB5R-mIVR153 was diluted with HeBS buffer to a total volume of 30 μl, mixed with the cell suspension, and allowed to stand on ice for 10 minutes. The cell suspension added with the plasmid vector was sucked up with a 10 μl chip-type gold electrode and electroporated (1200 V, pulse width 40 ms, number of pulses once) using an electroporator (Invitrogen). Immediately after electroporation, 10% FCS / DMEM medium was added to RK13 cells or primary kidney cultured cells previously seeded in a 35 mm dish with 2 ml of antibiotic (−). Two portions of electroporation were added to a 35 mm dish and cultured at 30 ° C. for 24 hours. A plasmid vector pBMSF7c-mIVR153 in which the HA protein gene of H1N1 (2009) pdm was incorporated into the HA gene region of LC16m8 was similarly treated and used as a control.

  After culturing for 24 hours, remove the cells from the dish using a scraper, collect the cell suspension, perform sonication in cold water (30 sec x 4 times), and then centrifuge (2000 rpm, 10 min) did. The supernatant was used as a virus solution. The virus solution was diluted in 5% FCS / MEM medium, and infected with primary kidney cultured cells that had been seeded in a 150 mm dish at 30 ° C. for 1 hour. After the virus solution was removed by suction, the cells were washed with PBS (−). 5% FCS / 0.5% methylcellulose / MEM medium was added and cultured at 30 ° C. for 96 hours. After 96 hours of culture, the supernatant was removed by aspiration and washed with PBS (-). Guinea pig erythrocyte solution diluted with PBS (+) was added to a 150 mm dish and incubated at 30 ° C. for 30 min. After removing the erythrocyte solution by suction, the cells were washed twice with PBS (+). Plaques adsorbed with guinea pig erythrocytes were collected using a Pipetman. About the collected plaques, introduction of each HA protein gene was confirmed by PCR and determination of gene sequence. Plaque purification was repeated three times for the plaques confirmed to have been transfected.

  Small scale cultures were performed on the virus purified plaque three times. The colony obtained in the third round was suspended in 500 μl of 5% FCS / MEM medium and sonicated in cold water. After centrifugation (2000 rpm, 10 min), 250 μl of the supernatant was added to primary kidney cultured cells that had been seeded in a 60 mm dish, and infected at 30 ° C. for 1 hour. After infection, 2.5 ml of 5% FCS / MEM medium was added and cultured at 30 ° C. for 96 hours. After 96 hours, the cells were detached from the flask using a scraper, and the cell suspension was recovered. The collected cell suspension was sonicated in cold water (30 sec, 4 times) and then centrifuged, and the supernatant was collected as a virus solution. The collected virus solution was added to RK13 cells or primary kidney cultured cells that had been serially diluted and seeded in a 6-well plate, and was infected at 30 ° C. for 1 hour. After removing the virus solution by suction, the cells were washed twice with PBS (−), 5% FCS / 0.5% methylcellulose / MEM medium was added, and the cells were cultured at 30 ° C. for 96 hours. After 96 hours, titer was calculated by counting the number of plaques formed in the well.

  Large-scale culture was performed based on the calculated titer. RK13 cells or primary kidney cultured cells were seeded on 10 150 mm dishes, and when they reached confluence, a recombinant vaccinia virus solution (2 ml) was infected at 30 ° C. for 1 hour at moi = 0.1. After infection, the virus solution was removed by suction, 18 ml of 5% FCS / MEM medium was added, and the mixture was cultured at 30 ° C. for 96 hours. After 96 hours, the cells were removed from the flask using a scraper, and the cell suspension was collected and stored frozen at -80 ° C. This cell suspension was freeze-thawed once, then sonicated in cold water (30 sec, 4 times), centrifuged, and the supernatant was recovered as a virus solution. This virus solution was serially diluted and infected with RK13 cells or primary kidney cultured cells that had been seeded in a 6-well plate, and the virus titer in the solution was calculated by the same method as described above. Each of these virus solutions (recombinant vaccinia virus: rVVΔB5R-mIVR153 and control: rVV-mIVR153) for which titer was calculated were used in various experiments shown in the following examples.

Confirmation of introduction of HA protein gene by PCR Using the following primers specific for the HA protein gene (cDNA: SEQ ID NO: 3) shown in Example 1 (primer for insert confirmation PCR), the genome of the obtained recombinant vaccinia virus By performing PCR using as a template, it was confirmed whether each HA protein gene was introduced into the viral genome.

Base sequence of primer for PCR for insert confirmation <F primer>
Fw: mIVR153-1889-S20
5'- AATGCGAACTGTTGGTTCT-3 '(SEQ ID NO: 5)
(For H1N1 (2009) pdm HA protein gene confirmation)

<R primer>
Rv: HA-6-R
5'- CTAGTTCTGAGAAACCAGAGG -3 '(SEQ ID NO: 6)
(For H1N1 (2009) pdm HA protein gene confirmation)

  Specifically, the composition of the reaction solution was DNA polymerase 1 U, dNTP 0.3 mM, F primer 1 μM, R primer 1 μM with respect to 50 μl of the buffer solution attached to the commercially available polymerase, and the cycle conditions were melting at 95 ° C. for 0.5 minutes. The cycle in which annealing was performed at 58 ° C. for 0.5 minutes and elongation was performed at 72 ° C. for 2 minutes was a total of 25 cycles. The obtained PCR product was electrophoresed using an agarose gel, and the band was confirmed. As a result, if a single band of the expected length is recognized by the primer design, it can be seen that the HA protein gene of H1N1 (2009) pdm has been introduced into the recombinant virus genome, while the band is If it is not recognized, it will be understood that the HA protein gene of H1N1 (2009) pdm has not been introduced.

Confirmation of H1N1 (2009) pdm HA protein expression by Western blotting Recombinant vaccinia virus was inoculated into RK13 cells previously seeded in a 6-well plate at moi = 10 at 30 ° C. for 1 hour. After infection, the virus solution was removed by suction, and the cells were washed once with PBS (−). 2 ml of 5% FCS / MEM medium was added to each well and cultured at 30 ° C. for 18 hours. After 18 hours, the medium was removed by aspiration, and the cells were washed twice with PBS (−). 100 μl lysis buffer (1% SDS, 0.5% NP-40, 0.15 M NaCl, 10 mM Tris-HCl (pH 7.4)) was added to lyse the cells, and the solution was transferred to a 1.5 ml Eppendorf tube. . The collected solution was sonicated in cold water until it became viscous. The amount of protein in the prepared solution was quantified by the Lowry method.

Electrophoresis was performed on 20 μg of protein using a 10% acrylamide gel. After the completion of electrophoresis, the gel was taken out and energized for 60 minutes at 2 mA / cm ² using a Semi-dry blotter to transfer the protein in the gel to a PVDF membrane. The membrane was washed with a TBS-T solution, and then blocked by applying it to a 5% skim milk-TBS-T solution. After blocking, the membrane was washed 3 times with TBS-T solution. The primary antibody used was a rabbit polyclonal antibody against the HA peptide derived from H1N1 (2009) pdm HA protein (aa 223-234 (SEQ ID NO: 4: YSKKFKPEIAIR). After the reaction with the primary antibody, the membrane was treated with TBS-T solution. The secondary antibody was anti-rabbit IgG-linked HRPO (from Donkey, manufactured by Amersham) After the reaction with the secondary antibody, the membrane was washed again 3 times with TBS-T solution. Immobilon western detection reagent (Millipore) was added to the membrane, and fluorescence was captured with a CCD camera and image analysis was performed.

  As a result, as shown in FIG. 2, it was found that the HA protein of H1N1 (2009) pdm was expressed in the recombinant virus genome of rVVΔB5R-mIVR153.

Mouse inoculation experiment with recombinant vaccinia virus (Figure 3)
FIG. 3 (upper figure) is also a diagram showing a method for confirming the ability to induce humoral immunity of a recombinant vaccinia virus (such as rVVΔB5R-mIVR153).
Recombinant vaccinia virus (rVVΔB5R-mIVR153, rVV-mIVR153) obtained in Example 1 or recombinant vaccinia virus (rVV-Empty) inserted with an empty vector was transferred to BALB / c mice (female) at 1 × 10 ⁷ PFU. After transendothelial inoculation (intradermal inoculation (id)), blood was collected from the orbital vein 1, 2, 3 and 4 weeks later. All the collected blood was collected in a 0.5 ml tube containing a serum separating agent and centrifuged (15000 rpm, 10 minutes) to separate and collect the serum. The serum was stored frozen at −80 ° C. until the ELISA test described below was performed.

Antibody titer measurement by ELISA for H1N1 (2009) pdm HA protein in sera of mice immunized with rVVΔB5R-mIVR153
A 96-well plate was coated with H1N1 (2009) pdm HA protein, and a frozen serum sample was diluted 100 to 1000 times and added, and allowed to stand overnight at 4 degrees. After the 96-well plate was washed with a TBS-T solution, anti-mouse IgG-linked HRPO (from Sheep, manufactured by Amersham) was added as a secondary antibody to the 96-well plate. After reacting at room temperature for 2 hours, the 96-well plate was washed again with TBS-T solution three times. Coloring solution was added at 100 μl / well. After standing at room temperature for 30 minutes, 2M sulfuric acid solution was added at 50 μl / well to stop the reaction. Absorbance at 490 nm was measured with a microplate reader.
As a result, induction of a high antibody titer against the HA protein of H1N1 (2009) pdm was observed in the serum of mice inoculated with rVVΔB5R-mIVR153 (FIG. 4).

Examination of the protective effect of rVVΔB5R-mIVR153 against H1N1 (2009) pdm influenza as shown in FIG. 4, because the immune induction effect by rVVΔB5R-mIVR153 vaccination was confirmed, these vaccinated mice were 5 weeks after vaccination, H1N1 (2009) pdm (specifically, H1N1 / 2619/2009 strain) was attack-infected from the nose (FIG. 3 (upper figure)).
As a result of examining the viral load in the lung 3 days after the above-mentioned attack infection, the viral load in the lung tissue was greater in the rVVΔB5R-mIVR153 inoculated mice than in the uninoculated mice (rVV-Empty inoculated mice; the same applies hereinafter). Decreased to about 1/1000 (Figure 5).

Moreover, the daily body weight change after the said attack infection was measured, the autopsy was carried out 9 days after the infection, and the pathological analysis of the lung was performed.
In mice not vaccinated, the body weight decreased daily after the above-mentioned challenge infection, and after about 9 days a decrease of about 30% was observed, whereas the vaccinated group (rVV-mIVR153 Inoculated mice, rVVΔB5R-mIVR153-inoculated mice; the same applies below), although transient weight loss was observed in the early stage after the above-mentioned challenge infection, it was found that the body weight increased rapidly and recovered to near normal values thereafter. (Fig. 3 (bottom)).
Moreover, as a result of the pathological analysis of the lung, it was found that pneumonia caused by pandemic influenza virus infection can be remarkably reduced in the vaccinated group as shown in FIG.

Examination of Immediate Vaccine Effect of rVVΔB5R-mIVR153 against H1N1 (2009) pdm Influenza As shown in the experimental schedule of FIG. 7, the recombinant vaccinia virus (rVVΔB5R-mIVR153, rVV-mIVR153) or empty vector obtained in Example 1 the inserted recombinant vaccinia virus (rVV-Empty), since the transendothelial inoculated with 1 × 10 ⁷ PFU (intradermal inoculation (id)) after 3 days BALB / c mice (female), from the nose H1N1 (2009 ) pdm (specifically H1N1 / 2619/2009 strain) was attacked. The daily body weight change until 9 days after the attack was measured.
In mice not vaccinated, the body weight decreased daily after the challenge infection, and a decrease of about 25% was observed after 9 days. Although the body weight decreased temporarily in the early days after infection, it was found that the body weight increased rapidly and recovered to near normal values (Fig. 7).

Sequence number 3: Recombinant DNA
Sequence number 5: Synthetic DNA
Sequence number 6: Synthetic DNA

Claims (7)

Recombinant vaccinia virus in which all or substantially all of the B5R protein gene region of vaccinia virus is recombined with an expression promoter and all or part of the cDNA encoding the pandemic influenza virus-derived hemagglutinin protein belonging to the H1 subtype A pharmaceutical composition for preventing or treating influenza, comprising: The pharmaceutical composition according to claim 1, wherein the vaccinia virus is the LC16m8 strain. The pharmaceutical composition according to claim 1 or 2, wherein the pandemic influenza virus belonging to the H1 subtype is H1N1 (2009) pdm. The pharmaceutical composition according to any one of claims 1 to 3, wherein the cDNA encoding the hemagglutinin protein is the following DNAs (a) to (d):
(a) DNA consisting of the base sequence shown in SEQ ID NO: 3
(b) DNA encoding a hemagglutinin protein derived from a virus strain having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 3 and belonging to the H1 subtype
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1
(d) a DNA encoding a hemagglutinin protein derived from a virus strain having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and belonging to the H1 subtype The pharmaceutical composition according to any one of claims 1 to 4, wherein the expression promoter is a hybrid promoter. The pharmaceutical composition according to claim 5, wherein the hybrid promoter is composed of the following DNA (a) or (b).
(a) DNA consisting of the base sequence shown in SEQ ID NO: 2
(b) DNA having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 2 and having promoter activity The pharmaceutical composition according to any one of claims 1 to 6 , which is a pharmaceutical composition for preventing or treating influenza caused by pandemic influenza virus belonging to the H1 subtype.