Dengue virus vaccine and dengue virus vaccine kit

Abstract

This dengue virus vaccine contains a recombinant vaccinia virus having both DNA that encodes a region other than NS1 in a non-structural protein of a dengue virus and a promoter that regulates the expression of a region other than NS1. The dengue virus vaccine is administered twice or more to a patient to be vaccinated.

Description

Dengue virus vaccine and dengue virus vaccine kit

[0001] The present invention relates to a dengue virus vaccine and a dengue virus vaccine kit.

[0002] Dengue virus (hereinafter referred to as "DENV") is endemic mainly in tropical regions, and is said to infect approximately 400 million people worldwide annually, with approximately 100 million developing dengue fever. Although infections in Japan ceased in 2014, DENV is still brought into Japan every year via travelers from endemic countries, leaving the country at risk of chronic infection. Since 2010, the number of imported cases reported annually has exceeded 200.

[0003] Currently, there are no clinically available therapeutic drugs, and symptomatic treatment is being carried out. Meanwhile, clinical trials of vaccines against DENV are underway. The tetravalent dengue / yellow fever chimeric vaccine (CYD-TDV) from Sanofi Pasteur, which has made the most progress in development, has been approved in 20 countries, including Mexico in December 2015. However, it has been reported that inoculation of CYD-TDV into uninfected individuals increases the risk of developing dengue fever and dengue hemorrhagic fever (see Non-Patent Document 1).

[0004] DENV belongs to the Flaviviridae family and exists in four serotypes (serotypes 1, 2, 3, and 4). If a person is infected with DENV followed by another DENV belonging to a different serotype, excessive production of DENV can be induced through a phenomenon known as antibody-dependent enhancement (ADE), in which antibodies against the first DENV protect against the second DENV infection, leading to the onset of severe diseases such as dengue hemorrhagic fever.

[0005] Antibodies against structural proteins may induce ADE. To avoid ADE, Patent Document 1 discloses a recombinant vaccinia virus having a gene encoding the NS1 or a region other than NS1 of the nonstructural protein of DENV.

[0006] Japanese Patent Application Laid-Open No. 2020-150940

[0007] The World Health Organization (WHO), "Dengue vaccine safety update", 2018 (URL: https: / / www.who.int / groups / global-advisory-committee-on-vaccine-safety / topics / dengue-vaccines / safety-update)

[0008] The recombinant vaccinia virus disclosed in Patent Document 1 is useful as a vaccine that does not induce ADE. It is desired to further improve the antiviral effect by investigating the use of the recombinant vaccinia virus.

[0009] The present invention has been made in view of the above circumstances, and has an object to provide a dengue virus vaccine and a dengue virus vaccine kit having a high antiviral effect.

[0010] (Dengue Virus Vaccine) The dengue virus vaccine described herein comprises a recombinant vaccinia virus having DNA encoding a region other than NS1 in the nonstructural proteins of the dengue virus and a promoter that controls the expression of the region other than NS1, and is administered to a subject two or more times.

[0011] The dengue virus vaccine described herein comprises a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of the region other than NS1, and is used in combination with a viral vector that expresses IL-15.

[0012] The dengue virus vaccine described herein comprises: a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of the region other than NS1; and a viral vector that expresses IL-15.

[0013] The dengue virus vaccine described herein comprises a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of the region other than NS1, and is administered to a subject who has been administered an expression vector that expresses a region other than NS1 in a nonstructural protein of the dengue virus.

[0014] (Dengue Virus Vaccine Kit) The dengue virus vaccine kit described in this specification comprises: a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of the region other than NS1; and a viral vector that expresses IL-15.

[0015] The dengue virus vaccine kit described in this specification comprises: a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of the region other than NS1; and an expression vector that expresses the region other than NS1 in a nonstructural protein of the dengue virus.

[0016] The region other than NS1 may be a region consisting of NS2A, NS2B, NS3, NS4A, NS4B, and NS5.

[0017] The dengue virus may be of serotype 2.

[0018] The gene encoding the NS5 in the DNA may have a mutation that reduces the RNA polymerase activity of the NS5.

[0019] The gene encoding the NS5 in the DNA may encode the following amino acid sequence (a) or (b): (a) the amino acid sequence shown in SEQ ID NO: 1; or (b) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1.

[0020] The dengue virus vaccine and dengue virus vaccine kit according to the present invention have a high antiviral effect.

[0021] Fig. 1 is a diagram schematically showing the structure of the DENV genome. Fig. 2 is a diagram showing the change over time in the copy number of DENV type 2 RNA present in 1 mL of blood in a test example. Fig. 3 is a diagram showing the change over time in the copy number of DENV type 2 RNA present in 1 mL of blood in a test example. Fig. 4 is a diagram showing the result of ELISpot of splenocytes cultured in the presence of NS3 peptide in a test example. Fig. 5 is a diagram showing the result of ELISpot of splenocytes cultured in the presence of NS4A peptide in a test example.

[0022] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0023] [DENV Vaccine] The DENV genome is a single-stranded RNA. As shown in Figure 1, the DENV genome encodes three structural proteins and seven nonstructural proteins between the 5'-end untranslated region and the 3'-end untranslated region. The structural proteins are C, prM, and E. The nonstructural proteins are NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The DENV vaccine of this embodiment comprises a recombinant vaccinia virus having DNA encoding a region other than NS1 in the DENV nonstructural proteins and a promoter that controls the expression of the DNA, i.e., the expression of the region other than NS1. "Encoding a region other than NS1 in the DENV nonstructural proteins" means encoding a region other than NS1 that is produced in cells when the virus replicates. More specifically, the region other than NS1 in the nonstructural protein is a region consisting of NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (hereinafter collectively referred to as the "NS2-5 region").

[0024] The serotype of DENV from which the DNA encoding the region other than NS1 in the nonstructural protein of DENV according to this embodiment is derived is not particularly limited, but is preferably type 2. Type 2 is further divided into Asian and cosmopolitan strains, and DNA derived from a type 2 cosmopolitan strain is particularly preferred as the DNA encoding the region other than NS1. For example, when the gene encoding the NS2-5 region is derived from a type 2 cosmopolitan strain, the base sequence of the DNA is, for example, as shown in SEQ ID NO: 2.

[0025] DNA encoding regions other than NS1 in the nonstructural proteins of DENV can be obtained by conventional genetic engineering techniques. For example, nucleic acid synthesis using a DNA synthesizer commonly used in genetic engineering techniques may be used. Alternatively, PCR may be used in which the nucleotide sequence of a template gene is isolated, primers specific to each gene are designed, and the nucleotide sequence is amplified using a PCR device, or a gene amplification method using a cloning vector. Preferably, DNA encoding regions other than NS1 in the nonstructural proteins can be obtained by performing PCR using the DENV gene inserted into a plasmid as a template and primers specific to the nucleotide sequence encoding regions other than NS1 in the nonstructural proteins of the DENV gene.

[0026] In order not to inhibit the proliferation of the recombinant vaccinia virus, the gene encoding NS5 in the DNA may have a mutation that reduces the RNA polymerase activity of NS5. The reduction in RNA polymerase activity may be such that the RNA polymerase activity of the encoded NS5 is lower than that of wild-type NS5, and preferably the RNA polymerase activity of NS5 is inactivated. In this case, the gene encoding NS5 in the DNA encodes, for example, the following amino acid sequence (a) or (b): (a) the amino acid sequence shown in SEQ ID NO: 1; (b) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1.

[0027] In this embodiment, "sequence identity" refers to the ratio of the number of positions where identical amino acids exist in both amino acid sequences when the two amino acid sequences are aligned to maximize their degree of identity to the total number of amino acids in the entire sequence.

[0028] "90% or more sequence identity" with respect to an amino acid sequence means at least 90% sequence identity. The amino acid sequence of NS5 having a mutation may have 90% or more, preferably 92% or more, more preferably 95% or more, even more preferably 97% or more, still more preferably 98% or more, and particularly preferably 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1.

[0029] When the gene encoding NS5 in the DNA has a mutation that reduces the RNA polymerase activity of NS5, the base sequence of the DNA is, for example, as shown in SEQ ID NO:3.

[0030] The promoter controlling the expression of the region other than NS1 is not particularly limited, and the expression promoter contained in the vaccinia virus can be any of various expression promoters used for gene expression in vaccinia viruses, such as the Pm2H5, PsynII, and mH5 promoters.

[0031] The recombinant vaccinia virus according to this embodiment can be produced, for example, by inserting DNA encoding a region other than NS1 in the nonstructural protein of DENV and a promoter that controls the expression of the region other than NS1 into a vector (plasmid), and then introducing the vector into vaccinia virus. Examples of the vector that can be used include the pSMART™ vector.

[0032] Although not particularly limited, an attenuated strain of vaccinia virus is preferred. A preferred example of the vaccinia virus is the DIs strain, which was isolated by subculturing the Dalian strain (DIE strain) of vaccinia virus in chicken egg embryos. The DIs strain grows in primary chicken fibroblasts but hardly grows in other mammalian cells. Therefore, safety is ensured even when the recombinant vaccinia virus is inoculated into immunodeficient or immunosuppressed subjects.

[0033] Any known method can be used to introduce the vector into a vaccinia virus. For example, by introducing the above-mentioned vector into an animal cell infected with the vaccinia virus, homologous recombination can be induced within the vaccinia virus genome to produce a recombinant vaccinia virus that expresses a region other than NS1 in the nonstructural protein of DENV.

[0034] To confirm the introduction of DNA encoding a region other than NS1 in the DENV nonstructural protein into a recombinant vaccinia virus, PCR can be performed using the genome of the recombinant vaccinia virus as a template and primers specific to the DNA. Expression of a region other than NS1 in the DENV nonstructural protein can also be confirmed by Western blotting using animal cells infected with the recombinant vaccinia virus as a sample. Examples of antibodies that can be used in Western blotting include commercially available antibodies that specifically recognize regions other than NS1, and IgG purified with Protein G from antisera prepared by immunization with a DENV polypeptide.

[0035] The DENV vaccine according to this embodiment can be used as a preventive and therapeutic drug for DENV infections, such as dengue fever and dengue hemorrhagic fever.

[0036] The DENV vaccine according to this embodiment may be administered parenterally or orally. Depending on the mode of administration, the DENV vaccine may be in the form of tablets, capsules, powders, granules, pills, liquids, syrups, injections, topical preparations, suppositories, eye drops, or the like. The DENV vaccine is preferably administered by local injection, such as subcutaneous, intradermal, intramuscular, or abdominal injection. Alternatively, the DENV vaccine may be administered to a recipient by inhalation, for example, via the nasal cavity, oral cavity, or lungs. Preferably, the DENV vaccine is administered subcutaneously to stimulate subcutaneous immune cells. Subcutaneous administration may be by abrasion or a non-invasive method, such as a microneedle.

[0037] The DENV vaccine of this embodiment may contain, in addition to the recombinant vaccinia virus as an active ingredient, pharmaceutically acceptable carriers such as excipients, bulking agents, binders, lubricants, as well as buffers, isotonicity agents, chelating agents, colorants, preservatives, fragrances, flavorings, sweeteners, etc.

[0038] The DENV vaccine of this embodiment is administered to humans and non-human animals, preferably mammals, including primates such as humans and chimpanzees, laboratory animals such as rats, mice, and rabbits, livestock animals such as pigs, cows, horses, sheep, and goats, and pet animals such as dogs and cats.

[0039] The dose for inoculation of the DENV vaccine according to this embodiment is appropriately determined depending on the route of inoculation, the recipient, the recipient's age, weight, sex, symptoms, and other conditions. Examples of the daily dose of the recombinant vaccinia virus include, but are not limited to, 1,000 to 100,000,000 PFU (plaque forming units), preferably 100,000 to 100,000,000 PFU for oral administration, and 100 to 100,000,000 PFU, preferably 1,000 to 100,000,000 PFU for parenteral administration.

[0040] The DENV vaccine of this embodiment is preferably administered to a recipient two or more times. The number of inoculations is not particularly limited as long as it sufficiently induces cellular immunity. The number of inoculations may be, for example, two or three. The dose of DENV vaccine administered in the first inoculation may be the same as or different from the dose administered in the second or subsequent inoculations to the same recipient. The interval between inoculations to the same recipient may be set appropriately depending on the induction of cellular immunity. The second inoculation may be administered, for example, one day, three days, five days, seven days, ten days, two weeks, three weeks, four weeks, six weeks, or two months after the first inoculation. The interval between the second and third inoculations to the same recipient is the same as the interval between the first and second inoculations. The interval between the first and second inoculations and the interval between the second and third inoculations may be the same or different. Furthermore, the second and subsequent inoculations may be administered via the same route as the first inoculation or via a different route.

[0041] Furthermore, the recombinant vaccinia virus according to this embodiment may be used in combination with a viral vector that expresses interleukin 15 (IL-15). By using the recombinant vaccinia virus in combination with IL-15, highly active DENV-specific CD8 ＋ The viral vector is not particularly limited, and examples of the viral vector include retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus. Preferably, a vaccinia virus vector can be used as the viral vector. The viral vector has DNA encoding IL-15 and a promoter that controls the expression of IL-15. The method for producing the viral vector is the same as the method for producing the recombinant vaccinia virus described above, and can be referenced by replacing the DNA encoding the region other than NS1 in the nonstructural protein of DENV in the method for producing the recombinant vaccinia virus described above with DNA encoding IL-15.

[0042] The mode of combined use of the recombinant vaccinia virus and the viral vector expressing IL-15 according to this embodiment is not particularly limited, as long as the viral vector is inoculated into a subject to be inoculated with the recombinant vaccinia virus. The recombinant vaccinia virus and the viral vector may be inoculated simultaneously, or one may be inoculated after a certain time has elapsed since the other was inoculated. The dosage of the viral vector in inoculation is appropriately determined depending on the route of inoculation, the subject, the subject's age, weight, sex, symptoms, and other conditions. Examples of the daily dosage of the viral vector include, but are not limited to, 1,000 to 100,000,000 PFU for oral administration, and 100 to 100,000,000 PFU for parenteral administration, and 100 to 100,000,000 PFU for parenteral administration, but are not limited to, for example.

[0043] Furthermore, the recombinant vaccinia virus according to this embodiment may be inoculated into a recipient that has been administered an expression vector that expresses a region other than NS1 in the nonstructural protein of DENV. The expression vector is not particularly limited, and known expression vectors can be used. Commercially available expression vectors may also be used. Examples of expression vectors include the pCAGGS-Neo virus vector. To prepare such an expression vector, DNA encoding a region other than NS1 in the nonstructural protein of DENV may be amplified by PCR, and the resulting PCR product may be inserted into a site in the expression vector under expression control by a promoter.

[0044] The expression vector may be administered as a pharmaceutical composition containing pharmaceutically acceptable carriers such as excipients, bulking agents, binders, lubricants, as well as buffers, isotonicity agents, chelating agents, colorants, preservatives, fragrances, flavorings, sweeteners, etc.

[0045] The mode of administration of the expression vector is not particularly limited, and may be parenteral or oral administration. Depending on the mode of administration, the expression vector may be in the form of tablets, capsules, powders, granules, pills, liquids, syrups, injections, topical preparations, suppositories, eye drops, etc. The mode of administration of the expression vector is preferably local injection into the subcutaneous, intradermal, muscle, or abdominal cavity, etc. Alternatively, the expression vector may be administered to the subject by inhalation, for example, from the nasal cavity, oral cavity, or lungs. Preferably, the expression vector is administered subcutaneously to the subject.

[0046] The interval between inoculations of the recombinant vaccinia virus of this embodiment after administration of the expression vector may be set appropriately depending on the state of induction of cellular immunity, and may be, for example, 1 day, 3 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, or 2 months after administration of the expression vector. The dose of the expression vector is set appropriately depending on the route of inoculation, the recipient, the age, weight, sex, symptoms, and other conditions of the recipient.

[0047] As shown in the examples below, the DENV vaccine according to this embodiment exhibits an antiviral effect at least equivalent to that of CYD-TDV when administered two or more times. Furthermore, when administered in combination with the expression vector, the DENV vaccine exhibits an antiviral effect at least equivalent to that of CYD-TDV even with a single administration. Furthermore, when the DENV vaccine is administered in combination with the viral vector, a significant antiviral effect can be achieved.

[0048] The DENV vaccine according to this embodiment has antiviral and protective effects against the onset of DENV not only against the serotype from which the DNA encoding the region other than NS1 is derived, but also against DENV of serotypes other than the serotype from which the DNA is derived. The DENV vaccine according to this embodiment can suppress the induction of severe disease caused by ADE, which is a particular problem associated with DENV infection.

[0049] In another aspect of the present embodiment, there is provided a DENV vaccine comprising a recombinant vaccinia virus having DNA encoding a region other than NS1 in a DENV nonstructural protein and a promoter that controls the expression of the DNA, and a viral vector that expresses IL-15.

[0050] Another aspect of this embodiment provides a DENV vaccine kit. The DENV vaccine kit comprises a recombinant vaccinia virus having DNA encoding a region other than NS1 in a DENV nonstructural protein and a promoter controlling the expression of the DNA, and a viral vector expressing IL-15. Yet another aspect of this embodiment provides a DENV vaccine kit comprising a recombinant vaccinia virus having DNA encoding a region other than NS1 in a DENV nonstructural protein and a promoter controlling the expression of the DNA, and an expression vector expressing a region other than NS1 in a DENV nonstructural protein. These DENV vaccine kits comprise the viral vector and expression vector, each separated from the recombinant vaccinia virus. For example, in this DENV vaccine kit, the recombinant vaccinia virus is held in a first container, and the viral vector or expression vector is held in a second container different from the first container. In this case, the DENV vaccine can be prepared immediately upon use by the user mixing the recombinant vaccinia virus held in the first container with the viral vector or expression vector held in the second container at the time of use.

[0051] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0052] Example 1: Preparation of DENV nonstructural protein gene recombinant vaccinia vaccine A vaccine was prepared based on the genome of the DENV type 2 cosmopolitan strain (Dengue-2 Cosmopolitan: olSa-054, hereinafter referred to as "DENV2"). The nucleotide sequence of the gene encoding the NS2-5 region of the DENV type 2 cosmopolitan strain is shown in SEQ ID NO: 2.

[0053] Using cDNA encoding the nonstructural protein of DENV2 as a template, the NS2-5 region gene was amplified by PCR using NS2-5 region primers (a forward primer whose nucleotide sequence is shown in SEQ ID NO: 4 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO: 5). The resulting PCR product was digested with Sbf-I enzyme and AsiSI enzyme, and then inserted into the transfer vector pUC / DIs (Koji Ishii et al., Virology, 2001, 302, 433-444), a DIs strain recombinant plasmid. The resulting plasmid is referred to as "DENV 2C NS25 plasmid."

[0054] The composition of the reaction solution and reaction conditions for PCR using the NS2-5 region primers are as follows:

[0055] [Reaction solution composition] Template cDNA (DENV 2C cDNA): 1.0 μL, 5×Q5 DNA polymerase buffer: 5 μL, 2.5 mM dNTP: 2.5 μL, Q5 DNA polymerase (2.0 U / μL): 0.5 μL, forward primer (10 μM): 1.25 μL, reverse primer (10 μM): 1.25 μL, sterilized water: 13.5 μL, total: 25 μL

[0056] [Reaction conditions] A total of 35 cycles were performed, each cycle consisting of "thermal denaturation and dissociation: 10 seconds at 98°C → annealing: 15 seconds at 72°C → synthesis and extension: 40 seconds at 72°C."

[0057] Subsequently, in order to reduce the RNA polymerase activity of NS5, the gene encoding NS5 was modified by PCR using the QuickChange™ Lightning Site-Directed Mutagenesis kit (Stratagene, #210518) so that the amino acid sequence "GDD" in NS5 was changed to "GND." NS2-5 region mutagenesis primers (a forward primer whose nucleotide sequence is shown in SEQ ID NO: 6 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO: 7) were used in the PCR. The reaction solution composition and reaction conditions for PCR using the NS2-5 region mutagenesis primers are as follows:

[0058] [Reaction solution composition] Template DNA (DENV 2C NS25 plasmid): 1.0 μL 10× reaction buffer: 2.0 μL 2.5 mM dNTP: 0.4 μL F primer (50 ng / ul): 1.0 μL R primer (50 ng / ul): 1.0 μL QuickSolution reagent: 0.6 μL QuickChange Lightning Enzyme: 0.4 μL Sterile water: 13.6 μL Total: 20 μL

[0059] [Reaction conditions] A total of 18 cycles were performed, each cycle consisting of "thermal denaturation and dissociation: 20 seconds at 95°C → annealing: 10 seconds at 60°C → synthesis and extension: 6 minutes at 68°C."

[0060] The resulting PCR product was inserted into a DIs strain recombinant plasmid in the same manner as the PCR product from the above-mentioned PCR using the NS2-5 region primers. The DIs strain recombinant plasmid was then inserted downstream of the mH5 promoter in pSMART-DIs-L3-GPTF (GenScript), a recombinant vector for producing a DIs strain recombinant vaccine. This recombinant vector has the E. coli gpt gene under the control of the VACV p7.5 early promoter.

[0061] The resulting recombinant vector was linearized by digestion with ApaI restriction enzyme. The linearized vector was purified and transfected into primary chicken embryo fibroblasts that had been infected with attenuated vaccinia virus strain DIs at a multiplicity of infection of 10 for 1 hour. 20 hours after transfection, the cell layer was peeled off to collect the mixture containing the virus and cells, and the resulting suspension was frozen at -80°C until use.

[0062] Introduction of the vector induced homologous recombination within the genome of the DIs strain, resulting in a recombinant vaccinia virus (rDIs-DENV2C-NS25-GND) expressing the NS2-5 region with a mutation in NS5. The resulting virus was purified using the selection reagent mycophenolic acid (a purine metabolism inhibitor). rDIs-DENV2C-NS25-GND carrying the E. coli gpt gene could be cultivated in the presence of xanthine and hypoxanthine, even when the GMP synthesis pathway was inhibited by mycophenolic acid.

[0063] Example 2: Preparation of IL-15 recombinant vaccinia vaccine DNA (SEQ ID NO: 8) having an mH5 promoter and an IL-15 gene between Hind III recognition sites was artificially synthesized and inserted into a DIs strain recombinant plasmid using Hind III. Furthermore, the DIs strain recombinant plasmid was inserted into pSMART-DIs-L3-GPTF (GenScript) to prepare a recombinant vaccinia virus (rDIs-IL-15) expressing IL-15 in the same manner as the above rDIs-DENV2C-NS25-GND.

[0064] Example 3: Preparation of DENV nonstructural protein gene expression vector A PCR product obtained by PCR using the NS2-5 region mutation introduction primers in Example 1 was inserted into the pCAGGS-Neo vector to prepare a vector (pCAG-NS25) that expresses the NS2-5 region having a mutation in NS5.

[0065] Test Example: DENV Infection Experiment A129 mice (10 weeks old, manufactured by Marshall Bioresources) were infected with dengue fever vaccine CYD-TDV (manufactured by Sanofi Pasteur) at a dose of 1 / 10 of the human dose or 1 x 10 ８ PFU of rDIs-DENV2C-NS25-GND was subcutaneously inoculated by the scraping method. After 4 weeks, each individual was inoculated with the same vaccine as the first inoculation as the second inoculation. After another 4 weeks, DENV2 (10 ４Mice were infected intraperitoneally with DENV2 (PFU / mouse). Blood was collected from the mice on days 0, 2, 4, 7, 11, and 14 after infection. Mice in the control group (None) were infected with DENV2 without vaccination.

[0066] In addition, a DENV infection experiment was similarly performed by changing the number or type of vaccine administered as shown in Table 1. Blood was collected from the mice on days 0, 2, 4, and 7 after infection. Group D received 1 x 10 ８ PFU of rDIs-DENV2C-NS25-GND and 1 × 10 ８ The mice were inoculated with a combination vaccine containing rDIs-IL-15 (PFU) and pCAG-NS25 (100 μg) using Actranza (trademark) Lab (manufactured by Daicel Corporation).

[0067]

[0068] DENV2 in blood was quantified by quantitative reverse transcription PCR (qRT-PCR). RNA was extracted from blood using Isogen (Nippon Gene Co., Ltd.), and DENV2 RNA was quantified using a sense primer whose base sequence is shown in SEQ ID NO: 9, an antisense primer whose base sequence is shown in SEQ ID NO: 10, and a probe whose base sequence is shown in SEQ ID NO: 11 (all manufactured by Thermo Fisher Scientific). The 5' end of the probe was labeled with FAM.

[0069] The degree of cellular immune activation was assessed by ELISpot assay using single splenocytes isolated from spleens harvested from mice in the control group and groups A to D 14 days after infection, according to standard procedures. Splenocytes were cultured for 24 hours in medium containing 1 μg / mL of DENV NS3 peptide or NS4A peptide, according to the manufacturer's instructions, in each well of an ELISpot plate coated with anti-mouse IFN-γ antibody (#3321-2H, Mabtech). The medium consisted of RPMI 1640 (Shimadzu Diagnostics) containing 10% fetal bovine serum, L-glutamine (4 mM), penicillin-streptomycin (100 U / mL), HEPES (10 mM), 2-mercaptoethanol (50 μM), non-essential amino acids, and 0.2% sodium carbonate. After the ELISpot plate was dried, the number of spots in each well was counted using an automated ELISpot plate reader (Advanced Imaging Devices GmbH).

[0070] (Results) Figure 2 shows the copy number of DENV2 RNA present in 1 mL of blood after infection. Two inoculations of rDIs-DENV2C-NS25-GND produced an antiviral effect equivalent to that of CYD-TDV.

[0071] Figure 3 shows the copy number of DENV2 RNA present in 1 mL of blood after infection for groups A to D. Inoculation of rDIs-DENV2C-NS25-GND after pre-immunization with pCAG-NS25 (group B) yielded an antiviral effect equivalent to that of two inoculations of rDIs-DENV2C-NS25-GND (group C). Inoculation of a mixture of rDIs-DENV2C-NS25-GND and rDIs-IL-15 (group D) demonstrated that DENV in the blood was suppressed to approximately 1 / 10,000 of that in the control group (none).

[0072] The results of ELISpot analysis of splenocytes cultured in the presence of NS3 peptide are shown in Figure 4A. The results of ELISpot analysis of splenocytes cultured in the presence of NS4A peptide are shown in Figure 4B. Stimulation with either NS3 peptide or NS4A peptide increased the amount of IFN-γ secreted compared to the control group, indicating activation of cellular immunity. Particularly, significant activation was observed in Group D stimulated with NS3 peptide.

[0073] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. Furthermore, the above-described embodiments are intended to illustrate the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0074] This application is based on Japanese Patent Application No. 2023-144930, filed on September 7, 2023. The entire specification, claims, and drawings of Japanese Patent Application No. 2023-144930 are incorporated herein by reference.

[0075] The present invention is useful in vaccines against DENV.

Claims

1. A dengue virus vaccine comprising a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of said region other than NS1, which is administered to a subject two or more times.

2. A dengue virus vaccine comprising a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of said region other than NS1, which is used in combination with a viral vector that expresses IL-15.

3. A dengue virus vaccine comprising: a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of the region other than NS1; and a viral vector that expresses IL-15.

4. A dengue virus vaccine comprising a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of said region other than NS1, which is administered to a subject who has been administered an expression vector that expresses a region other than NS1 in a nonstructural protein of the dengue virus.

5. A dengue virus vaccine kit comprising: a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of a dengue virus and a promoter that controls the expression of the region other than NS1; and a viral vector that expresses IL-15.

6. A dengue virus vaccine kit comprising: a recombinant vaccinia virus having DNA encoding a region other than NS1 in a nonstructural protein of the dengue virus and a promoter that controls the expression of the region other than NS1; and an expression vector that expresses a region other than NS1 in a nonstructural protein of the dengue virus.

7. The dengue virus vaccine according to claim 1, 2, 3 or 4, or the dengue virus vaccine kit according to claim 5 or 6, wherein the region other than NS1 is a region consisting of NS2A, NS2B, NS3, NS4A, NS4B and NS5.

8. A dengue virus vaccine according to claim 1, 2, 3 or 4, or a dengue virus vaccine kit according to claim 5 or 6, wherein the dengue virus is of serotype 2.

9. The dengue virus vaccine or dengue virus vaccine kit according to claim 7, wherein the gene in the DNA encoding the NS5 has a mutation that reduces the RNA polymerase activity of the NS5.

10. The dengue virus vaccine or dengue virus vaccine kit according to claim 9, wherein the gene encoding the NS5 in the DNA encodes the following amino acid sequence (a) or (b): (a) the amino acid sequence shown in SEQ ID NO: 1; (b) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1.

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