JP2004307477A - Method for enhancing neutralization antibody-inducing ability of transgenic vaccine and method for administering vaccine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for enhancing the neutralization antibody-inducing ability of DNA vaccines to heighten the effectiveness thereof. <P>SOLUTION: The method for enhancing the neutralization antibody-inducing ability of DNA vaccine comprises simultaneously administering transgenic and protein-based vaccines in organisms. By the simultaneous administration, the neutralization antibody-inducing ability of the vaccine in the organism is enhanced synergistically compared with the performance expressed by the independent administration of the transgenic or protein-based vaccine. The method is useful for heightening the effectiveness of DNA vaccine having low inducing ability. Further, it is preferable to administer a mixture of both vaccines using a needle-free injector. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for enhancing a neutralizing antibody-inducing ability of a gene-introduced vaccine, which comprises administering a protein-introduced vaccine to a living body simultaneously with administration of the gene-introduced vaccine. Furthermore, the present invention provides a method for administering a gene-introduced vaccine and a protein-introduced vaccine to a living body at the same time, whereby the neutralizing antibody of the vaccine in the living body is compared with when the gene-introduced vaccine or the protein-introduced vaccine is administered alone. The present invention relates to a method for administering a vaccine, wherein the inducibility is synergistically enhanced.

  DNA vaccines are a type of gene-based vaccine, in which an antigen-specific immune response is obtained by inoculating a target antigen gene (for example, a fragment of a viral gene) into an expression plasmid and inoculating it. It is characterized by guiding. DNA vaccines have the characteristic of producing antigens in the body of a recipient and inducing antibodies against the antigens, and are attracting attention as next-generation vaccines. Since DNA is a stable substance and can be provided in large quantities at low cost by using gene replication technology, the practicality of DNA vaccines is high. Also, unlike conventional inactivated vaccines, DNA vaccines can induce an immune response in the host similar to the original infection and have a sustained action. Furthermore, since DNA vaccines are not infectious and do not replicate in cells, they have the advantage of higher safety than conventional live vaccines using attenuated viruses.

  However, DNA vaccines are excellent in inducing cell-mediated immunity, but have the problem that humoral immunity cannot be effectively induced. Therefore, enhancement of neutralizing antibody-inducing ability has been an issue. In particular, it has been desired to enhance the immunogenicity of DNA vaccines for infectious diseases, such as Japanese encephalitis and dengue fever, in which protective immunity with neutralizing antibodies is the main component.

  One of the attempts to increase the ability of DNA vaccines to induce neutralizing antibodies is to use a different type of vaccine at different times to increase immunogenicity, prime-boost. Law is being attempted. The prime boost method involves first priming with a DNA vaccine and then administering another type of vaccine, such as a protein-based vaccine, to efficiently induce neutralizing antibodies. This is the characteristic method. The prime boost method is also used in, for example, clinical trials of AIDS, and is one of the methods that are currently receiving attention as an approach for increasing the effectiveness of DNA vaccines.

  In addition, the above-mentioned protein-introduced vaccines include inactivated vaccines using killed viruses or pathogenic bacteria, component vaccines using only some components of inactivated vaccines, and subunit vaccines using only the antigenic portion of the virus. Is included. Protein-introduced vaccines directly elicit a host immune response rather than through the process of host-expressing proteins as in the DNA vaccines described above. For this reason, protein-introduced vaccines have the advantage of high neutralizing antibody-inducing ability, but DNA vaccines are superior in terms of sustained action.

  As an example of trying the prime boost method, a report by Nam et al. (Non-patent Document 1) of administering a recombinant virus vaccine or attenuated vaccine of Japanese encephalitis after priming with a DNA vaccine, and DNA vaccine and protein against dengue fever Simmonns et al. (Non-Patent Document 2) reported administration of a combination of vaccines, but in these reports, no apparent enhancement of neutralizing antibody induction was observed.

  Biswas et al. Reported that two types of vaccines were simultaneously administered as a different approach from the prime boost method, in which different types of vaccines were administered at different times (Non-patent Document 3). is there. In their report, Biswas et al. Co-administered a combination of a rabies virus inactivated vaccine and a DNA vaccine. However, according to Biswas et al., Co-administration was performed 2 to 5 times, and the ability to induce neutralizing antibodies was not always satisfactory.

Nam JH, et al., Immunogenecity of a recombinant MVA and a DNA vaccine for Japanese encephalitis virus in swaine, Microbiol Immunol 2002; 46: 23-8 Simmons M, et al., Characterization of antibody respones to combinations of a dengue-2 DNA and dengue-2 recombinant subunit vaccine, Am J Trop Med Hyg 2001; 65: 420-6 Biswas S, et al., Preexposure efficacy of a novel combination DNA and inactivated rabies virus vaccine, Hum Gene Ther 2001; 12: 1917-22

  Accordingly, an object of the present invention is to provide a new method for enhancing the neutralizing antibody-inducing ability of a DNA vaccine having various advantages.

  In order to solve the above-mentioned problems, the present inventors have studied in detail the effects of simultaneous administration of a combination of a DNA vaccine and a protein-introduced vaccine. As a result, it was found that simultaneous administration of the DNA vaccine and the protein-introduced vaccine was effective for obtaining a high neutralizing antibody titer. In particular, when a subunit vaccine having the same immunogenicity as a co-administered DNA vaccine is used as a protein-introduced vaccine, the effect of inducing an immune response increases synergistically, and a very high neutralization is achieved by a combination of both vaccines. It was possible to obtain an antibody titer. Further, in the method of the present invention, a sufficient effect was recognized by performing simultaneous administration of the two vaccines once. Furthermore, by mixing a DNA vaccine and a protein-introduced vaccine and administering the mixture with a needleless syringe, it has become possible to synergistically induce neutralizing antibodies simply by inoculating the vaccine at one site.

  Therefore, the present invention is a method for enhancing the neutralizing antibody-inducing ability of a gene-introduced vaccine, which comprises simultaneously administering the gene-introduced vaccine and the protein-introduced vaccine to a living body. Furthermore, the present invention provides a method for administering a gene-introduced vaccine and a protein-introduced vaccine to a living body at the same time, whereby the neutralizing antibody of the vaccine in the living body is compared with when the gene-introduced vaccine or the protein-introduced vaccine is administered alone. This is a vaccine administration method characterized in that the inducing ability is synergistically enhanced.

  According to the present invention, there is provided a method for enhancing the neutralizing antibody-inducing ability of a gene-introduced vaccine, which comprises administering a protein-introduced vaccine to a living body at the same time as administering the gene-introduced vaccine. Further, according to the present invention, by simultaneously administering a gene-introduced vaccine and a protein-introduced vaccine to a living body, the neutralizing antibody of the vaccine in the living body is compared with when the gene-introduced vaccine or the protein-introduced vaccine is administered alone. A method of administering the vaccine was also provided, characterized in that the inducibility was synergistically enhanced. The method of the present invention is useful for the purpose of enhancing the effectiveness of a DNA vaccine having a low ability to induce antibody neutralization. In addition, since a high neutralizing antibody titer can be obtained by the method of the present invention, the amount of vaccine to be inoculated can be significantly reduced. In a preferred embodiment of the present invention, the two types of vaccines are mixed and administered using a needleless syringe.

  In the following examples, pcJEME prepared using the gene of the Japanese encephalitis virus Nakayama strain and pcD2ME prepared from the dengue virus New Guinea C strain were used as gene-introduced vaccines. As a protein-introduced vaccine, JEEP, a Japanese encephalitis virus extracellular particle (EP), and D2EP, a protein, a dengue virus extracellular particle (EP) were used. JEEP and D2EP are subunit vaccines composed of an envelope similar to the original particles.

  pcJEME and pcD2ME are DNA vaccines designed so that viral proteins expressed in cells are secreted extracellularly.The secreted proteins have a virion-like particle structure, so extracellular particles Called. Extracellular particles are advantageous in handling because they are easily purified. In addition, since the co-administered protein-introduced vaccines JEEP and D2EP are also extracellular particles produced in vitro, the protein expressed in the host body by the administration of the DNA vaccine is administered as a subunit vaccine. Become. In an embodiment of the present invention, the co-administered protein-introduced vaccine is preferably a subunit vaccine having the same immunogenicity as the protein encoded by the DNA vaccine, thereby synergistically enhancing the ability to induce neutralizing antibodies. It is possible to achieve the effect of enhancing.

  As used herein, the term "neutralizing antibody-inducing ability" refers to the ability of a vaccine to induce an immune response to an antigen such as a virus, a bacterium, or a parasite, thereby inducing the production of a protective antibody against the antigen. . And, "neutralizing antibody inducing ability is synergistically enhanced" means that neutralizing antibody inducing ability when co-administering gene-introduced vaccine and protein-introduced vaccine is the result of administration of both vaccines independently. Means more than the sum of the neutralizing antibody-inducing abilities obtained at this time.

  In the following examples, the vaccine against Japanese encephalitis and dengue fever was examined for the effectiveness of simultaneous administration of a combination of a gene-introduced vaccine and a protein-introduced vaccine. Was confirmed. Such effects are expected to be achieved not only with vaccines against Japanese encephalitis and dengue fever, but also with vaccines against other diseases.

  The vaccine administration method of the present invention is useful not only when administering vaccines against Japanese encephalitis and dengue fever, but also when administering vaccines against infectious diseases caused by various viruses. Vaccine administration method of the present invention, adult T cell leukemia virus, EB virus, hepatitis A virus, hepatitis B virus, herpes simplex virus, yellow fever virus, tick-borne encephalitis virus, RS virus, polio virus, human coronavirus, Human enterovirus, human immunodeficiency virus, human papillomavirus, human parvovirus, human reovirus, influenza virus, Lassa virus, human latinovirus, varicella-zoster virus, mumps virus, measles virus, hantavirus, rotavirus, swine cholera virus It is useful when administering a vaccine against an infection caused by a virus such as rabies virus. It should be noted that these listed viruses are merely examples, and do not exclude the aspect of using the method of the present invention when administering a vaccine against infectious diseases caused by other viruses.

  Furthermore, the method of the present invention is considered to be useful not only when administering a vaccine against an infection caused by a virus, but also when administering a vaccine against an infection caused by a bacterium or a parasite. The vaccine administration method of the present invention includes the following methods: It is also useful when administering vaccines against infectious diseases caused by bacteria such as pneumococci, gonococci, tetanus, Treponema pallidum, Borrelia burgdorferi, Salmonella typhi and Legionella. The vaccine administration method of the present invention is also useful when administering a vaccine against infectious diseases caused by parasites such as leishmania, malaria parasite, toxoplasma parasite, trypanoma and dysentery amoeba. Note that these listed bacteria and parasites are merely examples, and exclude the aspect of using the method of the present invention when administering a vaccine against an infection caused by other bacteria and parasites. It does not do.

  Therefore, the method of the present invention can be carried out by administering a gene-introduced vaccine against infectious diseases caused by various viruses, bacteria and parasites simultaneously with a protein-introduced vaccine. The method of the present invention provides a new method for remarkably enhancing the protective antibodies of a gene transfer vaccine such as a DNA vaccine against a wide range of infectious diseases. It provides a new possibility to increase and its significance is great. Further, the method of the present invention is considered to contribute not only to prevention but also to treatment for infectious diseases.

  The transgenic vaccine is preferably administered by the intramuscular or intradermal route, whereas the protein-transduced vaccine is preferably administered by the subcutaneous route. However, the administration route is not limited to these, and any other administration route can be used as long as the gene transfer vaccine and the protein transfer vaccine can exert their effects. Therefore, if the effect of the vaccine can be obtained, it is considered that administration routes such as oral administration, transdermal administration, and nasal administration can be used, and the method of the present invention is carried out using various administration routes. be able to. As for the administration method, various methods such as a method using a normal syringe, a method using a needleless syringe, a method of applying to the skin, and a method of spraying with a spray can be used. The composition of the diluent for diluting the gene-introduced vaccine and the adjuvant added to the protein-introduced vaccine also affect the effectiveness of the vaccine.However, those skilled in the art may also vary these factors in light of the common general knowledge in the art. The present invention can be implemented by making modifications so as to make the most effect.

  The ratio of the dose of the gene-introduced vaccine to the dose of the protein-introduced vaccine is not particularly limited, and is most suitable for achieving the effect of the present invention of inducing an immune response by co-administering both. Ratio can be set as appropriate. In the following examples, it has been found that the combination of 50 μg of pcJEME (DNA vaccine), a vaccine for Japanese encephalitis virus, and 10 μg of JEEP (subunit vaccine) is most effective when administered simultaneously. The ratio of the two vaccines to be administered is not limited thereto, and those skilled in the art can determine the most effective ratio based on the common general knowledge in the art.

  In the method of the present invention, the effect of simultaneous administration of the gene-introduced vaccine and the protein-introduced vaccine can be sufficiently obtained by a single administration. However, a more remarkable effect can be expected by administering the vaccine in a plurality of times. When administering the vaccine in multiple doses, the interval between administrations is generally about 1 to 5 weeks, but is not limited to that interval, and the most appropriate number of administrations and administration intervals Can be set arbitrarily. After administration of the vaccine, the highest neutralizing antibody titer can be generally obtained in about 3 to 15 weeks.

  The synergistic effect in the present invention is based on the initial immune response caused by a relatively large amount of a protein-introduced vaccine having antigenicity and the small amount of protein secreted from host cells by administration of the gene-introduced vaccine. It is presumed that there is a prolonged immune response caused by This mutually promotes the neutralizing antibody-inducing ability of the two, and a single administration induces the same strong immunological application as when a so-called “boost” is applied. When a subunit vaccine, which is a protein-introduced vaccine, and a DNA vaccine, which is a gene-introduced vaccine, have the same immunogenicity, such a mechanism works most effectively, so that significant neutralizing antibody induction is required. It is thought that performance can be obtained. In the above-mentioned Biswas et al. (Non-Patent Document 3), the immunogenicity of the two vaccines administered cannot be said to be the same because the vaccine administered with the DNA vaccine is an inactivated vaccine for rabies virus. . Therefore, the present invention newly provides an excellent method for efficiently inducing the neutralizing antibody-inducing ability of a DNA vaccine.

  In Example 1 described below, two administration routes, i.e., intramuscular administration for a gene-introduced vaccine and subcutaneous administration for a protein-introduced vaccine, are administered simultaneously. However, if the number of inoculation sites can be reduced to one, its significance in clinical applications would be significant. The current vaccination requires multiple boosters.

  In recent years, by jetting a drug solution with strong pressure from a jet nozzle provided at the tip of the tip of a syringe, it is possible to inject the drug solution into the body from the skin without using a syringe needle. Syringes have been developed. The jet injection needleless syringe is almost painless and can easily inject a drug solution, so that it is used, for example, for a diabetic patient to inject insulin himself (self-injection) on a daily basis. It is also said that administration of a DNA vaccine using a needleless syringe increases the DNA introduction efficiency, which is presumably because vaccine DNA is introduced into more cells than a normal needle syringe.

  Focusing on such a needleless syringe, for the purpose of reducing the route of administration and increasing the efficiency of DNA introduction, the present inventors mixed two vaccines and administered them using a needleless syringe (implementation Example 2). As a result, a synergistic increase in neutralizing antibody-inducing ability was observed when the combined administration of the gene-introduced vaccine and the protein-introduced vaccine was performed using a needleless syringe. Therefore, the mixed administration using a needleless syringe made it possible to maintain a long-lasting neutralizing antibody titer by a single inoculation.

  When a DNA vaccine is administered in a mixed administration system using a needleless syringe, the efficiency with which DNA is introduced into host animal cells increases. In addition, as shown in the examples below, the CpG motif contained in DNA has an adjuvant effect, and these effects contribute to a synergistic increase in immunogenicity when combined administration with a needleless syringe. It is considered to be.

  The needleless syringe used in the present invention is not particularly limited, and mainly the one generally used for medical purposes, etc., is adjusted according to the amount of the DNA vaccine to be injected and the tissue to be injected with the DNA vaccine. Various types can be adopted. It is a particularly preferred embodiment of the present invention to use a needle-free jet injector. Examples of needleless syringes suitable for use for the purposes of the present invention include ShimaJet, available from Shimadzu Corporation, BioInjector 2000, available from Bioinject, Medicator Choice, available from Mediject, and Activa. Examples include, but are not limited to, AdvantaJet Activa provided by Brand Products, MardaJet XL provided by Marda International. Although an administration method using a gene gun (gene gun) is used for the purpose of introducing a foreign gene, the needleless syringe of the present invention is different from a so-called "gene gun" and does not mean that.

The route for administering the DNA vaccine using a needleless syringe is not particularly limited. Since a DNA vaccine is generally administered by an intramuscular or intradermal administration route, it is preferable in the present invention to employ such an administration route, and it is particularly preferable to administer a DNA vaccine intramuscularly. However, if the effect as a DNA vaccine can be obtained, it is theoretically possible to administer it by another route as needed.
The present invention will be described in more detail with reference to the following examples and drawings, but the description does not limit the scope of the present invention in any way.

(Study on simultaneous administration of DNA vaccine and subunit vaccine)
As a DNA vaccine, pcJEME prepared by incorporating the Japanese encephalitis virus prM / E gene into a pcDNA3 vector and pcD2ME produced by incorporating the DEN2 virus New Guinea C (NGC) strain prM / E gene into a pcDNA3 vector were used. As the subunit vaccine, JEEP, a Japanese encephalitis virus extracellular particle (EP), and D2EP, a dengue virus extracellular particle (EP), were used. The cells were transfected with the plasmid pcJEEP to prepare a subunit vaccine of JEEP. Cells were also transfected with the plasmid pcD2EP to produce a D2EP subunit vaccine. The extracellular particles were prepared from the culture supernatant of a continuous expression cell of the extracellular particles by polyethylene glycol precipitation and sucrose density gradient centrifugation. The nucleotide sequence of pcJEME is shown in SEQ ID NO: 1 in the Sequence Listing, the nucleotide sequence of pcJEEP is shown in SEQ ID NO: 2 in the Sequence Listing, the nucleotide sequence of pcD2ME is shown in SEQ ID NO: 3 in the Sequence Listing, and the nucleotide sequence of pcD2EP is shown in SEQ ID NO: 4 in the Sequence Listing. Respectively.

  Four week old ICR or ddY mice (5 per group) were vaccinated with DNA and subunit vaccines. For pcJEME and pcD2ME, 50 μg was inoculated intramuscularly. For JEEP and D2EP, 0.1 μg to 10 μg were inoculated subcutaneously in combination with Freund's complete adjuvant. After inoculation of these two immunogens singly or simultaneously at one time, blood was collected from the inoculated mice every three weeks for 15 weeks, and the neutralizing antibody titer in the serum was measured.

  FIG. 1 shows the results of examining the effect of simultaneous administration of vaccines against Japanese encephalitis virus, in which 50 μg of pcJEME (indicated by □) or 10 μg of JEEP (indicated by ▲) were used once, and both vaccines were simultaneously administered. The time course of the neutralizing antibody titer in the pooled sera of the administered group (shown by ●) is shown. Groups receiving 50 μg pcJEME or 10 μg JEEP had a neutralizing antibody titer of 1:10 or 1:20, whereas the groups receiving both vaccines simultaneously had a neutralizing antibody titer of up to 1: 160, and the effect of simultaneous administration was remarkable. The variation among individuals in each group at week 9 after administration was within 2 times the neutralizing antibody titer obtained with pooled sera, and the increase in neutralizing antibody titer in the group administered simultaneously was statistically It was shown to be significant.

  In addition, the neutralizing antibody titers induced by immunizing ICR mice at different concentrations of pcJEME and JEEP were compared. The results are shown in Table 1 below. Five 4-week-old mice in each group were inoculated with pcJEME and JEEP at the indicated doses (Table 1a). As a control, phosphate buffered saline mixed with 50 μg of pcDNA3 and an adjuvant was administered (Table 1b). After administration of the immunogen, neutralizing antibody titers were measured at weeks 3, 6, and 9 (Table 1c). In Table 1, simultaneous administration of 1.7 μg of pcJEME and 0.3 μg of JEEP showed the same neutralizing antibody inducing ability as administration of 50 μg of pcJEME or 10 μg of JEEP alone, and was compared with DNA vaccine or subunit vaccine alone. Thus, a high neutralizing antibody titer could be obtained by administering a small amount of vaccine.

  FIG. 2 shows the results of examining the effect of simultaneous administration of vaccines against dengue virus. The group in which 50 μg of pcD2ME (indicated by □) or 150 ng of D2EP (indicated by ▲) was administered once, and both vaccines were simultaneously administered. The change with time of the neutralizing antibody titer in the pooled serum (indicated by ●) is shown. As a control, a group to which 50 μg of pcDNA3 and a phosphate buffered saline prepared with an adjuvant were administered is also shown (indicated by x: control). In the vaccine against dengue virus, simultaneous administration induces a neutralizing antibody titer 4 to 16 times or more higher than that of the single administration in mice of both ICR (upper) and ddY (lower) strains. It was shown that neutralizing antibodies were induced.

(Study on administration using a needleless syringe)
Furthermore, we tried to administer the DNA vaccine pcJEME and the subunit vaccine JEEP in combination with a needleless syringe. Four weeks old ddY mice were administered a mixture of pcJEME and JEEP using a needleless syringe, and mice immunized with pcJEME or JEEP alone were compared for 9 weeks with neutralizing antibody titers (FIG. 3). The dose of pcJEME was kept constant (1 μg) and the dose of JEEP was varied between 0.001-1 μg.

  When 1 μg of pcJEME and 1 μg of JEEP were combined, the induced neutralizing antibody titers ranged from 1:20 to 1:80 (FIG. 3A). On the other hand, neutralizing antibodies induced by pcJEME and JEEP alone were below the detection limit (1:10 or lower) or low (1:10). Similarly, when 1 μg of pcJEME and 0.1 μg of JEEP were combined, neutralizing antibodies were also induced (FIG. 3B). No induction of neutralizing antibodies was observed by single administration. Neutralizing antibody levels induced by the mixture of DNA and subunit vaccines were dependent on the dose of JEEP (FIG. 3C). These results indicate that immunization with pcJEME and JEEP with a needleless syringe synergistically increases neutralizing antibody titers.

  Further, instead of JEEP, the test was conducted using JEVAX (purchased from Takeda Pharmaceutical Co., Ltd.), a Japanese brain inactivated vaccine for encephalitis derived from mouse brain. Using a needleless syringe, the neutralizing antibody titers were compared between the case where pcJEME and JEVAX were mixedly administered to mice and the case where they were administered alone (FIG. 4). 0.1 μg to 10 μg for pcJEME and 1/1000 to 1/10 of the dose used for humans were used for JEVAX. When 10 μg of pcJEME and 1/10 dose of JEVAX were administered in combination, the induced neutralizing antibody titer was 1:80 to 1: 160 (FIG. 4A). On the other hand, the neutralizing antibody titer when pcJEME or JEVAX was administered alone was 1:40 for pcJEME and 1:10 for JEVAX. The neutralizing antibody titer of the group administered with 10 μg of pcJEME and 1/10 dose of JEVAX was examined up to 21 weeks after administration. As a result, a neutralizing antibody titer of 1: 160 was maintained.

  When 1 μg of pcJEME and 1/100 dose of JEVAX were combined, the effect of the mixed administration was more pronounced (FIG. 4B). When administered alone, the neutralizing antibody titer was below the limit of detection, whereas the combined administration of pcJEME and JEVAX induced neutralizing antibodies with neutralizing antibody titers of 1:20 to 1:80. Neutralizing antibody levels induced by the combined administration of pcJEME and JEVAX decreased as the dose of these vaccines decreased (FIG. 4C). These results indicate that the combination of pcJEME and JEVAX administered in a needleless syringe system has a synergistic effect.

  Furthermore, it was examined whether the adjuvant effect of the CpG motif contained in DNA had a synergistic effect on the increase in neutralizing antibody titer during mixed administration using a needleless syringe (FIG. 5). A 1/10 dose of JEVAX and an empty vector pcDNA3 (7.3 μg or 0.73 μg) or pcJEME (10 μg or 1 μg) were administered using a needleless syringe. In terms of molecular weight, the number of moles of the CpG motif contained in 10 μg and 1 μg of pcJEME corresponds to the number of moles of the CpG motif contained in 7.3 μg and 0.73 μg of pcDNA3, respectively.

  The neutralizing antibody titer induced by the combined administration of 10 μg of pcJEME and 1/10 dose of JEVAX was compared with that of 1/10 dose of JEVAX alone (1:10 or less or 1:10, indicated by ● in FIG. 5C). 4 times or more (1:40 to 1:80, FIG. 5A, indicated by ●). Under these conditions, when 7.3 μg of pcDNA3 and 1/10 dose of JEVAX were mixed and administered, the neutralizing antibody titer was 1:20 (shown by ○ in FIG. 5A). This result suggests that the effect of enhancing immunogenicity when pcJEME and JEVAX are administered in combination is derived to some extent from the vector. FIG. 5B shows the results of the mixed administration of 1 μg of pcJEME and JEVAX (1/10 dose) (FIG. 5B ●) and the mixed administration of 0.73 μg of pcDNA3 and JEVAX (FIG. 5B ○). This result also indicates that the effect of the CpG motif is partial.

  By administering a protein-introduced vaccine to a living body at the same time as administering a gene-introduced vaccine, it became possible to synergistically enhance the neutralizing antibody-inducing ability of the vaccine. In addition, by mixing and administering these two vaccines with a needleless syringe, it became possible to achieve an enhancement of neutralizing antibody ability only by inoculating the vaccine at one site. Given that conventional techniques require multiple administrations and that the method of the present invention can reduce the dose of the vaccine, the present invention is considered to be highly useful in the pharmaceutical field.

FIG. 1 is a graph showing the time course of the neutralizing antibody titer when a DNA vaccine against Japanese encephalitis virus (pcJEME) and a subunit vaccine (JEEP) are co-administered. FIG. 2 is a graph showing the time course of the neutralizing antibody titer when a DNA vaccine against dengue virus (pcD2ME) and a subunit vaccine (D2EP) are co-administered. FIG. 3 is a graph showing the time course of the neutralizing antibody titer when a DNA vaccine against the Japanese encephalitis virus (pcJEME) and a subunit vaccine (JEEP) are mixed and administered with a needleless syringe. FIG. 4 is a graph showing the time course of the neutralizing antibody titer when a DNA vaccine against the Japanese encephalitis virus (pcJEME) and a commercially available inactivated vaccine (JEVAX) are mixed and administered using a needleless syringe. FIG. 5 is a graph showing the effect of an empty vector, pcDNA3, on the synergistic induction of neutralizing antibody titer when mixed and administered using a needleless syringe.

Claims (21)

  A method for enhancing the neutralizing antibody-inducing ability of a gene-introduced vaccine, which comprises administering a protein-introduced vaccine to a living body simultaneously with administration of the gene-introduced vaccine.   The method according to claim 1, wherein the gene-introduced vaccine is a DNA vaccine, and the protein-introduced vaccine is a subunit vaccine.   The method according to claim 2, wherein the protein encoded by the DNA vaccine and the protein contained in the subunit vaccine have the same immunogenicity.   The method according to claim 1, wherein the gene-introduced and protein-introduced vaccines are vaccines against infectious diseases caused by viruses, bacteria or parasites.   The method according to claim 4, wherein the gene transfer vaccine and the protein transfer vaccine are vaccines against infectious diseases caused by viruses.   The method according to claim 5, wherein the gene transfer vaccine and the protein transfer vaccine are vaccines against Japanese encephalitis.   The method according to claim 6, wherein the transgenic vaccine is pcJEME, and the protein-transduced vaccine is JEEP.   The method according to claim 5, wherein the gene-introduced vaccine and the protein-introduced vaccine are vaccines against dengue fever, dengue hemorrhagic fever, or dengue shock syndrome.   9. The method according to claim 8, wherein the transgenic vaccine is pcD2ME and the protein-transduced vaccine is D2EP.   The method according to any one of claims 1 to 9, wherein the gene-introduced vaccine and the protein-introduced vaccine are mixed and administered with a needleless syringe.   Simultaneous administration of the transgenic vaccine and the protein-introduced vaccine to the living body, as compared to when the transgenic vaccine or the protein-introduced vaccine is administered alone, to induce neutralizing antibodies of the vaccine in the living body A method for administering a vaccine, wherein the ability is synergistically enhanced.   The method according to claim 11, wherein the gene-introduced vaccine is a DNA vaccine, and the protein-introduced vaccine is a subunit vaccine.   13. The method according to claim 12, wherein the protein encoded by the DNA vaccine and the protein contained in the subunit vaccine have the same immunogenicity.   By synergistically enhancing the neutralizing antibody-inducing ability, compared to when the gene-introduced vaccine or the protein-introduced vaccine is administered alone, it is possible to demonstrate its efficacy as a vaccine in a low dose in a living body. The method of claim 11, wherein the method is characterized by:   The method according to claim 11, wherein the gene-introduced vaccine and the protein-introduced vaccine are vaccines against infectious diseases caused by viruses, bacteria or parasites.   The method according to claim 15, wherein the gene-introduced vaccine and the protein-introduced vaccine are vaccines against infectious diseases caused by viruses.   17. The method according to claim 16, wherein the gene-introduced vaccine and the protein-introduced vaccine are vaccines against Japanese encephalitis.   18. The method according to claim 17, wherein the transgenic vaccine is pcJEME and the protein-transduced vaccine is JEEP.   17. The method according to claim 16, wherein the gene-introduced vaccine and the protein-introduced vaccine are vaccines against dengue fever, dengue hemorrhagic fever, or dengue shock syndrome.   20. The method according to claim 19, wherein said transgenic vaccine is pcD2ME and said protein-introduced vaccine is D2EP. The method according to any one of claims 11 to 20, wherein the gene-introduced vaccine and the protein-introduced vaccine are mixed and administered with a needleless syringe.

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