Patent Application
20230174586
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “2021-11-20_4456-0277PUS1_ST25.txt” created on Nov. 20, 2021 and is 10,304 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates to a prophylactic medicine and therapeutic medicine for H7 subtype avian influenza virus infection that causes the onset of a new type influenza. Specifically, the present invention relates to a recombinant vaccinia virus DIs strain, which can express the hemagglutinin (HA) protein gene of H7 subtype avian influenza virus, and a prophylactic medicine and therapeutic medicine for a new type influenza comprising the recombinant vaccinia virus DIs strain.
Seasonal influenza epidemics occur every winter, where 10 to 20% of the population is reported to be affected. The pathogen of such influenza could be influenza virus type A/H1N1, A/H3N2 or B but their antigenicity slightly changes every year and thus a vaccine needs to be produced in accordance with the strain that is predicted to dominate. Meanwhile, there are new types of influenza such as highly pathogenic H5N1 avian influenza, H7N9 avian influenza and highly pathogenic H7N7 avian influenza. In particular, epidemics of infection with H7N9 avian influenza virus in humans have been reported from China since 2013. To date, at least 1564 infected cases and 612 deaths have been reported. Further, the infection scale is the largest in 2016-2017 season when compared to the previous four seasons, and there is concern for further epidemic expansion in future. Currently, in preparation for epidemic expansion of infection with a new type influenza virus in humans, candidate strains for vaccine production have been selected and stored in a plurality of countries including the United States and the United Kingdom. Preparation has been made to immediately start vaccine production in case of emergencies.
However, vaccines planned to be produced are inactivated whole virus particle vaccines, and for this reason, it is required to conduct inoculation with a vaccine a plurality of times, and accordingly, it takes time to obtain sufficient immunity induction. Moreover, it is reported that a sequence that causes activation of suppressor T cells (Treg) is contained in the hemagglutinin (HA) protein sequence of H7N9 avian influenza virus (Non-Patent Document 1), and there is a possibility that the ability to induce an antibody specific to H7 HA protein may be reduced. In consideration of temporal transition of H1N1 (2009) pandemic influenza virus, since the first wave of the epidemic of domestic infection reached each country 2 to 3 months after the epidemic in the country where it occurred first, it is required to develop a vaccine which can respond to H7 subtype influenza virus more quickly.
At present, therapeutic agents for influenza include Tamiflu and Relenza that inhibit the function of neuraminidase (NA) that is essential for the budding of the influenza virus, but their effectiveness is limited because, for example, it is critical to administer them within 48 hours following the onset. Beside them, an intravenous agent of a NA inhibitor has already been developed. Furthermore, although manufacturing and sales of Avigan, an influenza virus polymerase inhibitor, were approved in Japan in March 2014, it can only be manufactured and distributed under certain conditions such as upon a request from the Minister of Health, Labour and Welfare and cannot be sold in the general market.
Among the variety of vaccines, a live vaccine is one of the most effective vaccines. However, the development of an attenuated vaccine for an emerging virus is generally known to take a very long time, and it is considered that the same applies to H7 subtype influenza. The present inventors diligently made researches, and making full use of genetic engineering techniques, developed a recombinant vaccinia virus (RVV) that expresses the H7 subtype HA protein gene as an influenza live vaccine using a vaccinia virus as a viral vector. So far, recombinant vaccinia viruses (RVV) against rabies virus and rinderpest developed by the present inventors have been demonstrated to exert an excellent effect in preventing infection and onset in the field tests and else (Non-Patent Document 2). In addition, for severe acute respiratory syndrome (SARS), the production of a recombinant vaccinia virus that has cDNA of SARS-CoV known as a pathogen thereof has also been successful (Patent Document 1), and it has been confirmed to be a formulation that shows an excellent preventive effect and that can be repeatedly administered (Non-Patent Document 3). Furthermore, for H5N1 HPAIV, the production of a recombinant vaccinia virus having cDNA of H5N1 HPAIV HA has also been successful, and it has been confirmed to be a formulation that shows an excellent preventive effect (Patent Document 2 and Non-Patent Document 4).
Under such circumstances, it has been strongly desired to establish a therapeutic medicine and prophylactic medicine comprising a recombinant vaccinia virus (RVV) effective for preventing the onset of a disease caused by H7 subtype avian influenza virus infection.
One embodiment of the present invention is as described below.
<1> A recombinant vaccinia virus, wherein an expression promoter and the full length or a part of a cDNA encoding hemagglutinin protein derived from H7 subtype avian influenza virus are contained in the genome of vaccinia virus DIs strain.
<2> The recombinant vaccinia virus according to item <1>, wherein said H7 subtype avian influenza virus is H7N9 avian influenza virus or highly pathogenic H7N7 avian influenza virus.
<3> The recombinant vaccinia virus according to item <1> or <2>, wherein said cDNA encoding hemagglutinin protein is any one of DNAs of (a) to (h) below:
<4> A pharmaceutical composition comprising the recombinant vaccinia virus according to any one of items <1> to <3>.
<5> The pharmaceutical composition according to item <4>, which is a prophylactic medicine for H7 subtype avian influenza.
<6> The pharmaceutical composition according to item <4>, which is a therapeutic medicine for H7 subtype avian influenza.
<7> The pharmaceutical composition according to item <5> or <6>, wherein said H7 subtype avian influenza is H7N9 avian influenza or highly pathogenic H7N7 avian influenza.
<8> The pharmaceutical composition according to any one of items <4> to <7>, which is for intradermal administration.
<9> A formulation for intradermal administration comprising the recombinant vaccinia virus according to any one of items <1> to <3>.
<10> The formulation for intradermal administration according to item <9>, which is a prophylactic medicine for H7 subtype avian influenza.
<11> The formulation for intradermal administration according to item <9>, which is a therapeutic medicine for H7 subtype avian influenza.
<12> The formulation for intradermal administration according to item <10> or <11>, wherein said H7 subtype avian influenza is H7N9 avian influenza or highly pathogenic H7N7 avian influenza.
<13> An influenza vaccine comprising the recombinant vaccinia virus according to any one of items <1> to <3>.
<14> The influenza vaccine according to item <13>, wherein said influenza is H7 subtype avian influenza.
<15> The influenza vaccine according to item <14>, wherein said H7 subtype avian influenza is H7N9 avian influenza or highly pathogenic H7N7 avian influenza.
<16> The influenza vaccine according to any one of items <13> to <15>, which is for intradermal administration.
<17> A method for preventing and/or treating H7 subtype avian influenza, wherein the method comprises administering to a subject an effective amount of the recombinant vaccinia virus according to any one of items <1> to <3>.
<18> The method according to item <17>, wherein said H7 subtype avian influenza is H7N9 avian influenza or highly pathogenic H7N7 avian influenza.
<19> The method according to item <17> or <18>, wherein said administration is intradermal administration.
<20> The method according to any one of items <17> to <19>, wherein the method comprises administering in combination with a vaccine against another subtype influenza virus.
<21> A combination vaccine comprising the recombinant vaccinia virus according to any one of items <1> to <3> and one or more recombinant vaccinia viruses derived from other subtype influenza viruses.
<22> The combination vaccine according to item <21>, wherein said recombinant vaccinia viruses derived from other subtype influenza viruses are rDIs-H1, rDIs-H3 and rDIs-H5.
<23> Use of the recombinant vaccinia virus according to any one of items <1> to <3> in the manufacture of an influenza vaccine.
According to the present invention, it is possible to provide a novel recombinant vaccinia virus, which is effective for preventing or treating a new type influenza and has high safety, and a prophylactic medicine or therapeutic medicine for a new type influenza comprising the same.
Hereinafter, the recombinant vaccinia virus of the present invention and intended use thereof will be described in detail. The scope of the present invention should not be limited to the following description, and the present invention may suitably be modified and carried out in any way apart from the following illustration without departing from the gist of the present invention. The patent documents, the non-patent documents and other publications cited herein are incorporated herein by reference.
Under the above-described circumstances, in consideration of vaccination of many and unspecified subjects, the present inventors developed a DIs strain-derived recombinant vaccinia virus (RVV) having an H7 subtype influenza virus-derived hemagglutinin protein gene by using a vaccinia virus DIs strain, which is non-proliferative in mammalian cells and therefore is highly safe, and which has actually been used for vaccinating humans.
As a technique to be employed in such a case, a genetic engineering technique for preparing a recombinant vaccinia virus (RVV) as a live vaccine is known. A vaccinia virus that serves as a recombinant parent for preparing a RVV needs to be a vaccine strain with established safety, and examples of such vaccine strains include vaccinia virus DIs strain and LC16m8 strain. The vaccinia virus DIs strain is a highly attenuated vaccinia virus strain separated by passaging vaccinia virus Dairen strain (DEI strain) in chicken egg embryos (Tagaya I, et al., A new mutant of dermovaccinia virus. Nature, 1961, vol. 192, pp. 1187-1188). This DIs strain can propagate in chicken embryonic fibroblasts (CEF) but is non-proliferative in other mammalian cells and thus is highly safe. Meanwhile, vaccinia virus LC16m8 strain was approved as a smallpox vaccine by the then Ministry of Health and Welfare in 1975, and is a safe and effective attenuated vaccine strain that has actually been used for vaccinating more than 50,000 infants. Attenuation of the LC16m8 strain owes to the fact that the full-length B5R protein is not expressed due to a single base deletion in the B5R protein gene of LC16mO strain that is considered to be involved in adhesion to cells and infection (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), pp. 11873-1189). When using such a vaccine strain with established safety, application to a subject undergoing immunodeficiency or immunosuppression can be realized, and a recombinant live influenza vaccine having advantages such as leaving no vaccine scar can be developed. Therefore, a new type of recombinant influenza vaccine was established using this DIs strain as a viral vector, and it was aimed to establish a prevention and treatment method for a rapidly spreading new type influenza using a novel vaccine showing effectiveness even in the case of a single inoculation and a short period of time.
As described in detail below, in individuals vaccinated with rDIs-H7 HA, the present invention showed satisfactory effects of protection against H7N9 avian influenza virus infection and H7N7 avian influenza virus infection. Moreover, in the case of infection with H7N9 avian influenza virus 1 week or 2 weeks after vaccination, symptoms were successfully attenuated significantly, and the rapid protection effects of rDIs-H7 HA were confirmed. According to the above-described results, rDIs-H7 HA can serve as a vaccine for H7 subtype influenza which can immediately induce the specific immune responses.
As described above, the present inventors established a new type of recombinant influenza vaccine (rDIs-H7 HA) using the DIs strain as a viral vector. In mice that had been given a single inoculation with rDIs-H7 HA, temporal weight loss was observed after challenge infection with A/Anhui/1/2013 (H7N9) influenza virus, but then they rapidly gained weight and showed 100% survival rate (
A method for preparing a H5N1 HPAIV vaccine is already-known (Japanese Patent No. 5884100), and the vaccine for H7 subtype influenza virus of the present invention (rDIs-H7 HA) can be prepared according to this already-known method. The outline of the preparation method will be described below.
The HA protein gene of H7 subtype avian influenza virus, for example, H7N9 avian influenza virus represented by SEQ ID NO: 1 has already been registered with a predetermined accession number (Accession No. EPI439507) in the influenza virus gene database (GISAID; Global Initiative on Sharing All Influenza Data).
Such HA protein genes (including partially mutated genes) have already been cloned and inserted into plasmids. Therefore, the whole (including mutated sequences) or a part of the gene contained in the recombinant vaccinia virus of the present invention, i.e., cDNA encoding the HA protein of H7 subtype avian influenza virus of H7N9 avian influenza virus can be obtained by a general genetic engineering technique. For example, it is possible to use a nucleic acid synthesis method using a DNA synthesis apparatus, which is generally used as a genetic engineering technique. Further, a PCR method, in which a gene sequence as a template is isolated or synthesized; then a primer specific to each gene is designed; and the gene sequence is amplified using a PCR device, or a gene amplification method using a cloning vector can be used. The above-described methods can be conducted with reference to “Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring Harbor Laboratory Press (2012)), etc. For purifying a PCR product obtained, an already-known method can be used. Further, a mutated sequence, in particular, a substitution mutation type DNA can be prepared, for example, in accordance with site-directed mutagenesis described in “Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring Harbor Laboratory Press (2012)) described above, “Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997)”, etc. Specifically, preparation can be carried out using a mutagenesis kit utilizing site-directed mutagenesis in accordance with an already-known technique such as the Kunkel method and the Gapped duplex method. Preferred examples of the kit include QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by Agilent Technologies), GeneTailorTM Site-Directed Mutagenesis System (manufactured by Invitrogen) and TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: manufactured by Takara Bio Inc.).
In a preferred embodiment of the present invention, the HA protein gene of H7 subtype avian influenza virus inserted in the above-described plasmid can be used as the above-described PCR template. In a more preferred embodiment of the present invention, the HA protein gene of H7N9 avian influenza virus can be used as the above-described PCR template. Further, by using a cDNA of the HA protein gene of highly pathogenic H7 subtype avian influenza virus, more preferably H7N9 avian influenza virus as a template and conducting PCR using a primer specific to each gene, the HA protein gene region of highly pathogenic H7 subtype avian influenza virus, more preferably H7N9 avian influenza virus can be prepared.
Each mutant DNA described above can be obtained by chemical synthesis, or can be obtained from a cDNA library and a genome library according to an already-known hybridization method such as colony hybridization, plaque hybridization and Southern blotting, using a DNA consisting of a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a fragment thereof as a probe. Examples of stringent conditions for the above-described hybridization include 0.1×SSC to 10×SSC, 0.1% to 1.0% SDS and 20° C. to 80° C. More specifically, for example, prehybridization is performed at 37° C. to 56° C. for 30 minutes or more, and then washing is performed in 0.1 ×SSC, 0.1% SDS at room temperature for 10 to 20 minutes from once to three times. For detailed procedures of the hybridization method, “Molecular Cloning, A Laboratory Manual 4th ed.” (Cold Spring Harbor Press (2012)), etc. can be referred to.
Further, it is possible to use a DNA (mutant DNA), which has at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% homology to the nucleotide sequence represented by SEQ ID NO: 1, and which encodes the HA protein derived from H7 subtype avian influenza virus; and a DNA (mutant DNA), which has at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% homology to the nucleotide sequence represented by SEQ ID NO: 2, and which encodes the HA protein derived from H7N9 avian influenza virus.
The expression promoter contained in the recombinant vaccinia virus of the present invention is not limited as long as it can be inserted into a gene deletion region of the vaccinia virus DIs strain, and it may, for example, be vaccinia virus promoter mH5. As said expression promoter, for example, a hybrid promoter consisting of a poxvirus A-type inclusion (ATI) promoter and repeats of a vaccinia virus 7.5 kDa protein (p7.5) early expression promoter may be used. This promoter can be linked to a suitable plasmid. For example, pBMSF7C and pUC/DIs are known (see Arch. Virol. vol. 138, pp. 315-330, 1994; and Japanese Laid-Open Patent Publication No. H06-237773).
The nucleotide sequence of the vaccinia virus promoter mH5 which can be used in the present invention is represented by SEQ ID NO: 3. In the present invention, in addition to a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3, a DNA, which hybridizes to a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 under stringent conditions, and which has promoter activity, can also be used as the expression promoter (particularly hybrid promoter). The “stringent conditions” are the same as those described above. The expression “has promoter activity” means having transcriptional activity of a gene encoding a structural protein or non-structural protein. A protein expressed by the above-described hybrid promoter can be expressed in a large amount in a form that has undergone complete sugar modification from the early stage to the late stage of vaccinia virus infection.
A plasmid vector obtained by inserting the DNA encoding the expression promoter and the HA protein is introduced into a vaccinia virus as a host, thereby preparing a recombinant vaccinia virus. The plasmid vector can be introduced into the host by employing any known technique. For example, by introducing a plasmid vector obtained into an animal cell infected with an attenuated vaccinia virus DIs strain, homologous recombination is caused in a sequence abutting the gene deletion region of the vaccinia virus DIs strain, and accordingly, a recombinant vaccinia virus (rDIs-H7 HA), by which the HA protein gene of H7 subtype avian influenza virus (e.g., H7N9 avian influenza virus or highly pathogenic H7N7 avian influenza virus) is expressed, can be prepared.
Methods for producing an inactivated vaccine are already known. As mentioned above, an influenza virus used for the vaccine production is propagated by inoculating embryonated chicken eggs with the virus, then the virus purified/concentrated from the chorioallantoic fluid is partially degraded with ether, and the resultant is further inactivated with formalin.
In the prepared DIs strain-derived recombinant vaccinia virus (rDIs-H7 HA), the HA protein gene of H7 subtype avian influenza virus (e.g., H7N9 avian influenza virus) is inserted in the gene deletion region of the vaccinia virus DIs strain. While the influenza virus-derived HA protein is involved in agglutination reaction of guinea pig red blood cells, the vaccinia virus-derived HA protein does not cause agglutination reaction of guinea pig red blood cells. Therefore, RVV is screened by infecting animal cells with rDIs-H7 HA and using agglutination reaction of guinea pig red blood cells to the resulting plaque as an indicator. For intended RVV, red plaques having hemagglutination activity may be selected.
Regarding the virus obtained from the red plaques, PCR is conducted with a primer specific to the HA protein gene of H7 subtype avian influenza virus such as H7N9 avian influenza virus, using the virus genome as a template, and thus introduction of the HA protein gene of H7 subtype avian influenza virus can be confirmed.
The expression of the HA protein of H7 subtype avian influenza virus such as H7N9 avian influenza virus can be confirmed by Western blotting, using an animal cell after infected with rDIs-H7 HA as a sample. As an antibody, a rabbit antiserum prepared by immunization with an HA peptide (SEQ ID NO: 4 or the like) derived from the HA protein of H7N9 avian influenza virus can be used.
As an insertion site of a target gene in the preparation of RVV, the thymidine kinase (TK) gene region is generally used as the gene deletion region of the vaccinia virus DIs strain. However, it is known that proliferation property of RVV is reduced by defective expression of TK due to insertion of a target gene into the TK gene region. Accordingly, in the present invention, as an insertion site of a target gene, the gene deletion region of the vaccinia virus DIs strain is preferred.
The present invention provides a prophylactic/therapeutic medicine (pharmaceutical composition for preventing and treating) for a new type influenza (i.e., H7N9 avian influenza and highly pathogenic H7N7 avian influenza), which comprises the above-described recombinant vaccinia virus derived from the DIs strain. Further, the present invention can also provide: a method for preventing and treating the new type influenza, which includes administering the recombinant vaccinia virus to a patient (subject); use of the recombinant vaccinia virus for preventing and treating the new type influenza; use of the recombinant vaccinia virus for producing a prophylactic/therapeutic medicine for the new type influenza; etc.
The pharmaceutical composition of the present invention may be introduced into a living body by any already-known method, for example, an intramuscular, intraperitoneal, intradermal or subcutaneous injection, a nasal, oral or pulmonary inhalation, or an oral administration. Moreover, the recombinant vaccinia virus contained in the pharmaceutical composition of the present invention may be used in combination with an existing antiviral drug (e.g., Tamiflu and Relenza). The mode of combined use is not particularly limited, and the recombinant vaccinia virus of the present invention can be administered simultaneously with the existing antiviral drug. Alternatively, they can be introduced into a living body by administering one of them and then administering the other after a certain lapse of time.
The pharmaceutical composition of the present invention may be mixed with an already-known pharmaceutically acceptable carrier such as an excipient, a filler, a binder or a lubricant, a buffer, a tonicity agent, a chelating agent, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetener or the like.
The pharmaceutical composition of the present invention can be administered orally or parenterally according to the dosage form, for example, as an oral administration agent such as tablets, capsules, powder, granules, pills, a liquid agent or a syrup, or as a parenteral administration agent such as an injection, a topical medication, a suppository or eye drops. Preferable examples include local injections such as intradermal, intramuscular and intraperitoneal injections. In a preferred embodiment of the present invention, the above-described pharmaceutical composition may be for intradermal administration.
In one embodiment of the present invention, the pharmaceutical composition of the present invention may be provided as a formulation for intradermal administration comprising the recombinant vaccinia virus of the present invention. In one embodiment of the present invention, the formulation for intradermal administration may be a prophylactic medicine for H7 subtype avian influenza. In one embodiment of the present invention, the formulation for intradermal administration may be a therapeutic medicine for H7 subtype avian influenza. In such embodiments, H7 subtype avian influenza may be H7N9 avian influenza or highly pathogenic H7N7 avian influenza.
In another embodiment of the present invention, a method for preventing and/or treating H7 subtype avian influenza, which includes administration of an effective amount of the recombinant vaccinia virus of the present invention to a subject, is provided. In such an embodiment, H7 subtype avian influenza may be H7N9 avian influenza or highly pathogenic H7N7 avian influenza. In a preferred embodiment of the present invention, administration of the recombinant vaccinia virus of the present invention may be carried out by means of intradermal administration.
The dosage may appropriately be selected according to the type of the active component, the administration route, the administration target, the age, weight, sex and symptoms of the patient, and other conditions. The daily dosage of the virus is about 1,000 to 1,000 ,000,000 PFU (plaque forming units), and preferably about 100,000 to 100,000 ,000 PFU for oral administration, and about 100 to 1,000 ,000,000 PFU, and preferably about 1,000 to 100,000 ,000 PFU for parenteral administration. The virus may be given once or in several times a day.
The recombinant vaccinia virus of the present invention can be used as a vaccine for preventing and treating a new type influenza. Further, in the development of a vaccine against H7 subtype avian influenza virus, researches have been made with attention being focused on an antibody against H7N9 avian influenza virus or highly pathogenic H7N7 avian influenza virus and a cytotoxic T cell (CTL). Accordingly, it is preferred to measure the antibody titer or cell-mediated immune activity as a vaccine in advance. In a preferred embodiment of the present invention, the above-described vaccine for preventing and treating influenza may be for intradermal administration.
For example, the antibody titer against a new type of influenza virus in subjects inoculated with the prepared recombinant vaccinia virus (rDIs-H7 HA) or the DIs strain as the parent strain can be obtained by a procedure in which: a mouse is inoculated with the virus strain; after that, serum is collected over time; and the ELISA value against the HA protein of H7N9 avian influenza virus or highly pathogenic H7N7 avian influenza virus in the serum is measured.
Thus, the recombinant vaccinia virus prepared by the present inventors can induce humoral immunity against a new type influenza.
The recombinant vaccinia virus of the present invention can be used in the production of the influenza vaccine of the present invention.
In one embodiment of the present invention, the vaccine comprising the recombinant vaccinia virus of the present invention can be administered in combination with a vaccine against another subtype influenza virus. In one embodiment of the present invention, the above-described administration in combination may be, for example, administering a combination vaccine which comprises the recombinant vaccinia virus of the present invention and a recombinant vaccinia virus derived from another subtype influenza virus.
In one embodiment of the present invention, a combination vaccine comprising the recombinant vaccinia virus of the present invention and one or more recombinant vaccinia viruses derived from other subtype influenza viruses is provided. In one embodiment of the present invention, one or more recombinant vaccinia viruses derived from other subtype influenza viruses may be contained in the combination vaccine of the present invention. In one embodiment of the present invention, the combination vaccine of the present invention may be a double vaccine. In another embodiment of the present invention, the combination vaccine of the present invention may be a triple vaccine. In yet another embodiment of the present invention, the combination vaccine of the present invention may be a quadruple vaccine. In one embodiment of the present invention, the recombinant vaccinia viruses derived from other subtype influenza viruses may be, for example, rDIs-H1, rDIs H3 and rDIs-H5, but are not limited thereto. In a preferred embodiment of the present invention, the combination vaccine of the present invention may be a quadruple vaccine (rDIs-H1, -H3, -H5 -H7 HAs). The combination vaccine of the present invention can be prepared using a method known in the art.
Hereinafter, the present invention will be described more specifically by way of examples. These examples are provided merely for illustration and are not intended to limit the scope of the present invention.
A hemagglutinin protein (HA) gene, namely, an antigen of H7N9 influenza virus (A/Anhui/1/2013) artificially synthesized, was linked downstream from a vaccinia virus promoter mH5 sequence, and the resultant was inserted into a plasmid vector for homologous recombination, thereby preparing RVV (
CEF was infected with a DIs strain-derived recombinant vaccinia virus prepared using SEQ ID NO: 1 (rDIs-H7 HA) with viral infectivity (multiplicity of infection) of 10. 24 hours after the infection, the medium was removed and washing was performed twice using PBS(-). A lysate was added to prepare a cell lysate. SDS-polyacrylamide electrophoresis was carried out with the amount of protein contained in the cell lysate of 10 µg, and transfer to a PVDF membrane was carried out. For confirmation of the influenza virus protein expression by RVV infection, with respect to H7N9 avian influenza virus, a mouse anti-H7N9-HA protein monoclonal antibody was used as the primary antibody, and a sheep anti-mouse IgG antibody-HRP was used as the secondary antibody. A coloring reagent manufactured by Millipore (Millipore; Immobilon western) was used.
BALB/c mice were intradermally inoculated with 1 × 107 PFU of the recombinant vaccinia virus (rDIs-H7 HA) on the back. Challenge infection with H7N9 avian influenza virus (A/Anhui/1/2013) was conducted 5 weeks after the inoculation, and the weight and the survival rate were determined daily. Further, the protective effect on the onset against highly pathogenic H7N7 avian influenza virus (A/chicken/Netherlands/2586/2003), which is also the H7 subtype but a different HA subtype virus, was also examined. In addition, for the purpose of examining the immediate effect of the vaccine, BALB/c mice that were subjected to single inoculation with the recombinant vaccinia virus (rDIs-H7 HA) in the same manner were subjected to challenge infection with H7N9 avian influenza virus (A/Anhui/1/2013) (50x MLD50/individual) 1 week or 2 weeks after the inoculation with the vaccine, and thus the protective effect on the onset was examined.
Using a bifurcated needle, cynomolgus monkeys were inoculated with 1 × 107 PFU of the recombinant vaccinia virus (rDIs-H7 HA) on both the upper arms (skin scarification method using bifurcated needle). 3 weeks after the initial inoculation, the same amount of the recombinant vaccinia virus (rDIs-H7 HA) was additionally inoculated, and 1 week after the additional inoculation, challenge infection with H7N9 avian influenza virus (A/Anhui/1/2013) (3×106 TCID50/individual) was conducted. In addition, for the purpose of examining the immediate effect of the vaccine, cynomolgus monkeys that were subjected to single inoculation with the recombinant vaccinia virus (rDIs-H7 HA) in the same manner were subjected to challenge infection with H7N9 avian influenza virus (A/Anhui/1/2013) (3×106 TCID50/individual) 2 weeks after the inoculation with the vaccine, and thus the protective effect on the onset was examined.
Using the above-described cynomolgus monkeys, with respect to the unvaccinated group (group of inoculation with DIs), the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) twice and the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) once (rapid), the body temperature was measured over time from immediately prior to challenge infection, and the lung weight 7 days after the challenge infection was measured.
Further, the collected lung tissue was immersed in a 10% buffered formalin solution to perform virus inactivation and tissue fixation, and after that, a paraffin block of the tissue was prepared. A thin slice of 4 µm was prepared, attached to a slide glass, and then stained with hematoxylin and eosin, and pathological findings of the lung was analyzed.
Although temporal weight loss was observed after the challenge infection with H7N9 influenza virus in the mice that had been given a single inoculation with the prepared RVV (rDIs-H7 HA), they rapidly gained weight and showed 100% survival rate (
In the test of the onset protection of the recombinant vaccinia virus (rDIs-H7 HA) against H7N9 avian influenza virus infection using cynomolgus monkeys, a biphasic fever pattern was shown in the case of the unvaccinated group (group of inoculation with DIs), but the effect of quickly bringing down fever was obtained in the case of the vaccinated group with the recombinant vaccinia virus (rDIs-H7 HA) twice (
Using the above-described cynomolgus monkeys, with respect to the group of no inoculation with the vaccine (group of inoculation with DIs), the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) twice and the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) once (rapid), the effect of virus elimination in swab samples collected from the conjunctiva, nasal cavity, oral cavity and respiratory tract was examined. A biphasic pattern was shown in the case of the group of no inoculation with the vaccine (group of inoculation with DIs), but the effect of early virus elimination was confirmed in the case of the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) twice and the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) once (rapid) (
Using the above-described cynomolgus monkeys, with respect to the group of no inoculation with the vaccine (group of inoculation with DIs), the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) twice and the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) once (rapid), pathological findings of the lung 7 days after the challenge infection was analyzed. In the case of the group of no inoculation with the vaccine (group of inoculation with DIs), pneumonia was locally recognized. Meanwhile, in the case of both the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) twice and the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) once (rapid), the alveolar spaces were firmly maintained, and it was shown that pneumonia was remarkably alleviated (
Further, the lung weight was measured. In the case of the group of no inoculation with the vaccine (group of inoculation with DIs), increase in the lung wet weight was recognized. Meanwhile, in the case of both the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) twice and the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) once (rapid), increase in the lung wet weight was suppressed (
Single inoculation with 1 × 107 PFU of the DIs strain-derived RVV expressing the influenza virus HA protein gene as a vaccine for H7 subtype influenza virus (rDIs-H7 HA) showed, against the challenge infection with lethal H7N9 influenza virus and H7N7 influenza virus, the rapid effect of suppressing weight loss and 100% survival rate (administration of the vaccine 2 weeks before the challenge infection). In addition, 80% survival rate was shown even in the case of the administration of the vaccine 1 week before the challenge infection. Note that in the case of the control group (group of inoculation with DIs), all the individuals died. Accordingly, the DIs strain-derived RVV also has sufficient effects, in particular, immediate effects as a vaccine for H7 subtype influenza virus.
The method for inoculation with the recombinant vaccinia virus (rDIs-H7 HA) was changed from the skin scarification method to the intradermal inoculation method using a two-stage needle, the protective effect of the vaccine against H7N9 avian influenza virus infection when using the intradermal inoculation method was evaluated in cynomolgus monkeys.
Firstly, inoculation with a monovalent vaccine of the recombinant vaccinia virus (rDIs-H7 HA) using the intradermal inoculation method or skin scarification method (1×107 PFU), or intradermal inoculation with a quadruple vaccine including vaccines against H1, H3 and H5 subtype viruses in addition to H7 influenza virus (rDIs-H1, -H3, -H5 -H7 HAs) (1×107 PFU for each), or intradermal inoculation with DIs (control) were carried out (on both the upper arms of each cynomolgus monkey, single inoculation in each case). 2 weeks after the inoculation, challenge infection with H7N9 avian influenza virus (A/Anhui/1/2013) (3×106 TCID50/individual) was conducted, and the protective effects on the onset were examined.
Using the above-described cynomolgus monkeys, with respect to the group of no inoculation with the vaccine (group of inoculation with DIs; control group), the group of inoculation with the recombinant vaccinia virus (rDIs-H7 HA) by skin scraping, the group of intradermal inoculation with the recombinant vaccinia virus (rDIs-H7 HA) and the group of intradermal inoculation with the quadruple vaccine (rDIs-H1, -H3, -H5 -H7 HAs), the body temperature was measured over time from immediately prior to challenge infection.
As shown in
Moreover, also in the case of the group of intradermal inoculation with the quadruple vaccine (rDIs-H1, -H3, -H5 -H7 HAs), suppression of fever and the effect of bringing down fever equivalent to those of the other groups of inoculation with the vaccine were observed. The results indicate a possibility that a vaccine that can simultaneously provide immunities against viruses with 4 serotypes with which humans may be infected can be developed.
Further, regarding the above-described cynomolgus monkeys inoculated or not inoculated with the vaccine, the viral infectivity in the lung tissue was measured and pathological findings of the lung was analyzed. The collected lung tissue was immersed in a 10% buffered formalin solution to perform virus inactivation and tissue fixation, and after that, a paraffin block of the tissue was prepared. A thin slice of 4 µm was prepared, attached to a slide glass, and then stained with hematoxylin and eosin, and pathological findings of the lung was analyzed.
In the case of the group of no inoculation with the vaccine, the infectious virus was detected in a plurality of lobes of the lung and severe pneumonia with pulmonary edema was caused. Meanwhile, in each case of the group of intradermal inoculation with the monovalent vaccine, the group of skin scraping and the group of inoculation with the quadruple vaccine, the titer of infectious virus was equal to or lower than the detection limit, and pneumonia was successfully alleviated remarkably (
According to the above-described results, it was understood that the DIs strain-derived RVV expressing the influenza virus HA protein gene of the present invention serves as a vaccine that is safe and effective against H7 subtype influenza virus. Further, it was shown that the DIs strain-derived RVV expressing the influenza virus HA protein gene of the present invention can respond to rapid spread of infection as a prophylactic vaccine.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-213990 | Nov 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/044737 | 11/14/2019 | WO |
