JP2001253833A - Method for inducing cell-mediated immunity of live vaccine even in inactivated vaccine, and combined vaccine obtained by the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To overcome the shortcoming that the conventional inactivated vaccine does not induce cell-mediated immunity, and at the same time, to achieve the simplification, labor saving and cost reduction of preventive inoculation. SOLUTION: A method of inducing cell-mediated immunity of a live vaccine even in an inactivated vaccine, and a combined vaccine of the inactivated vaccine obtained by the method and a live vaccine. The inoculation of the combined vaccine induces not only humoral immunity but also cell-mediated immunity against an antigen of the inactivated vaccine accompanied by the induction of the cell-mediated immunity due to the live vaccine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inducing the cell-mediated immunity of a live vaccine to an inactivated vaccine, and to a mixed vaccine of an inactivated vaccine and a live vaccine obtained by such a method.

[0002]

2. Description of the Related Art Combination vaccine technology has been in practical use for about half a century, and can be roughly classified into the following three types: (1)
Combination vaccines prepared by complex mixing of heterologous antigens, such as diphtheria pertussis tetanus combination vaccine (DPT
(2) a multivalent vaccine prepared by simple mixing of different serotype antigens within the same species, such as an oral live polio vaccine (type I, type II) And III
A trivalent vaccine comprising a live attenuated poliovirus), an influenza HA vaccine (generally a trivalent vaccine comprising an inactivated influenza virus HA antigen derived from two A strains and one B strain), and (3) a heterologous vaccine , For example, two (the above-mentioned MMR and live attenuated varicella vaccine) and three (the above-mentioned DPT, hemophilis influenza b type (Hib) inactivated vaccine and hepatitis A inactivated vaccine) etc. (“New” Generatio
n Vacines ", pp. 619-629, MM.
Ed., Levine et al., Marcel Dekker, ln
c. , 1997). By the way, a live attenuated vaccine induces both humoral immunity and cellular immunity. However, it has been known that inactivated vaccines induce humoral immunity but usually do not induce cellular immunity. In order to solve such a defect, an attenuated recombinant virus constructed by inserting and linking a gene encoding an antigen for an inactivated vaccine to the genome of an attenuated virus for a live vaccine has been produced. For example, a recombinant attenuated VZV that simultaneously produces both varicella virus (VZV) and hepatitis B surface (HBs) antigens (Japanese Patent No. 3,026,029, US Pat. No. 5,653,976, European Patent No. 0) , 510,9
96), VZV antigen and human immunodeficiency virus (HI
V) Recombinant attenuated VZV simultaneously producing V3 antigen region of V)
(Japanese Patent Application H11-104337) and the like have already been created. All of these recombinant attenuated VZVs have 2
It has been confirmed by preclinical studies that the function and effect as an active ingredient of the mixed live vaccine, that is, both humoral immunity and cellular immunity against two different antigens are induced. However, safety for humans has not been confirmed.

[0003]

The present invention solves the above-mentioned deficiencies of inactivated vaccines that do not induce cell-mediated immunity, and implements the above-mentioned clinical tests on the safety of recombinant attenuated VZV. It was newly conceived and completed with the aim of avoiding difficult conditions. In addition, in vaccination, the recipient is geographically, temporally, and economically restricted, while the recipient needs to secure and deploy personnel and places to perform it. Therefore, simplification of vaccination, labor saving and cost reduction are important issues in public health, and the present invention also contributes to solving such issues.

[0004]

According to the present invention, when an inactivated vaccine and a live vaccine are inoculated as a mixed vaccine,
Based on the surprising finding that inactivated vaccines induce both humoral and cellular immunity, as well as live vaccines, the composition is based on the infectious form of cell-mediated immunity produced by live vaccines or the induction of live vaccines Inactivated vaccines have the same activity as cell-mediated immunity. In other words, the active ingredient of the inactivated vaccine and that of the live vaccine coexist during the immune response, and the live vaccine is used as a kind of adjuvant to induce the inactivated vaccine to induce cellular immunity. The present invention solves the above-mentioned problems by providing the following (1) to (4): (1) The amount of each of the active components of the inactivated vaccine and the live vaccine to induce cellular immunity. , By mixing
Cell-mediated immunity caused by infection of the active ingredient of a live vaccine,
Alternatively, a method for causing the cell-mediated immunity activity of a live vaccine to occur in an inactivated vaccine; (2) a mixed vaccine containing the active components of the inactivated vaccine and the live vaccine in amounts that induce cellular immunity, respectively; (3) Inactivated antigens derived from hepatitis B, non-A non-B hepatitis, HIV, rota, Japanese encephalitis and influenza viruses and tetanus, pertussis and diphtheria bacteria as active ingredients of the inactivated vaccine. And (4) the combination vaccine of (2) or (3), wherein the active ingredient of the live vaccine is the attenuated varicella virus.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION [Points to keep in mind when mixing vaccines]
When mixing the inactivated vaccine and the live vaccine,
(A) It is the first decision to confirm the effect of mixing, that is, the induction of cellular immunity by the inactivated vaccine. However, it is important to further confirm the following (b) to (e), and to avoid mixing if these correspond. That is, such mixing or coexistence does not inhibit (b) the growth of the active ingredient of the live vaccine, for example, an attenuated virus or attenuated bacterium; (c) the immunogenicity of each active ingredient after mixing is lower than that before mixing. No; (d) the side reaction and frequency of inoculation of the combined vaccine are not enhanced and diversified as compared to before the mixing; and (e) the preservation of each active ingredient after the mixing is higher than that before the mixing. It is not spoiled.

[Inactivated Vaccine and Its Active Ingredient] Inactivated vaccines for human body, animal vegetation, fish and the like can be used. In addition, as an active ingredient of such an inactivated vaccine,
Known antigens or various antigens to be developed in the future can optionally be used. For example, hepatitis A, hepatitis B,
Antigens derived from viruses such as polio, rota, influenza, Japanese encephalitis, dengue, rabies, etc. Also, non-A non-B hepatitis virus (NANBV), HIV Hib, pneumococcus,
Antigens derived from meningococci, diphtheria, tetanus, pertussis, etc. More specifically, for example, all particles of HBs, Japanese encephalitis virus, all particles of rotavirus, VP4, VP
7 etc., NANBV E, C, M etc., HIV Gag, P
ol, Env, Nef, and other accessories, pneumococcal polysaccharide, DPT, tetanus toxin B-
Antigens such as C fragments can be raised. In the present invention, as an active ingredient of the inactivated vaccine, for example, an inactivating agent or denaturing agent such as formalin, ethylene oxide, β-propiolactone, and glutardialdehyde, and its proliferation and toxicity are determined by a conventional method using ultraviolet rays and the like. Not only antigens prepared by inactivating or detoxifying
An antigen prepared by fixing and stabilizing the three-dimensional structure is also used. However, in order to maintain the immunogenicity and antigenicity of such an antigen, selective setting of conditions, for example, the concentration, temperature, pH, etc. of the inactivating agent or denaturing agent used,
It is necessary to devise an inactivation relieving agent such as glycine or arginine.

[Live vaccines and their active ingredients] Live vaccines for humans, animal vegetation, fish and the like can be used.
As an active ingredient of such a live vaccine, a known attenuated virus, an attenuated bacterium, or the like, or various attenuated pathogens to be developed as an active ingredient of a live vaccine in the future can be optionally used. For example, chickenpox, vaccinia, Marek's disease, measles, rubella, polio, attenuated viruses such as influenza, and also attenuated bacteria such as Mycobacterium tuberculosis and anthrax, more specifically, for example, attenuated VZV Oka strain, attenuated measles virus Tanabe strain, Attenuated rubella virus Matsuura strain, Marek's disease virus C2 strain, 01 HVT strain, Calmette-Guerin bacteria, acapsular attenuated anthrax 34F2 strain and the like can be mentioned.

[Preparation of a Combination Vaccine of an Inactivated Vaccine and a Live Vaccine] The preparation and manufacture of a vaccine, and the quality assurance and control of its safety and efficacy are described in “Biological Product Standards” [Pharmaceutical Affairs Law (Showa 35th Law No. 145) No. 4
Notification of the Ministry of Health and Welfare based on Article 2, Paragraph 1 (October 1, 1993) No. 217] or WH
O (World Health Organization) recommendation "Requirements
for Biological Substances
and othersets of recommendation
ndations "(WHO Technical
Reports Series, No. 889, p.
105-111, 1999). For example, in the preparation of an inactivated vaccine, "precipitated hepatitis B vaccine"
Manufacture and various tests are performed in accordance with the above-mentioned standards for "influenza HA vaccine", "Japanese encephalitis vaccine", "precipitated purified pertussis vaccine", etc., to ensure eligibility as an inactivated vaccine. Also, in the preparation of live vaccines,
Manufacture and various tests are performed in accordance with the above-mentioned standards for "dry attenuated live varicella vaccine", "dry cell culture varicella vaccine", "dry attenuated measles vaccine", etc., to ensure eligibility as a live vaccine. Furthermore, in the preparation of a mixed vaccine, for example, production and various tests are carried out in accordance with the above-mentioned standards relating to, for example, `` pertussis diphtheria tetanus '', `` dried attenuated live shin shinobi mumps measles vaccine '', and the qualification as a mixed vaccine is ensured. can do. Mixed vaccine 1
The final amount of antigen contained in each ml is 0.01 μg to 10 mg, preferably 0.1 μg to
100 μg, for live vaccines, for example, for an attenuated virus, 10 ² to 10 ⁷ infectious particles, preferably 10 ³ to 1
0 ⁵ is an infectious particle. Regarding the preparation of a mixed vaccine, for example, a mixed vaccine in which the final amount of antigen in one dose (0.5 ml) is composed of 20 μg of inactivated antigen and 10 ⁴ PFU (plaque forming unit) of the attenuated virus is obtained by diluting the vaccine stock solution, Inactivated vaccine (antigen amount 80μg / ml)
And live vaccine (attenuated virus 4 × 10 ⁴ PFU / m
Each vaccine containing 1) can be prepared by separately preparing each vaccine in advance, and then mixing these two vaccines in equal amounts. Regarding the type of vaccine to be mixed, in the present invention, at least one inactivated vaccine and at least one live vaccine can be combined and mixed, and at least two mixed vaccines, and further, three or more A multi-combination vaccine obtained by combining vaccines can be obtained. As the stabilizer for the mixed vaccine, commonly used substances such as amino acids such as arginine and sodium glutamate, sugars such as lactose and saccharose, plasma albumin, vitamins and the like can be used. The mixed vaccine preparation is dispensed into vials or ampoules of, for example, 0.5 to 10 ml, and then sealed or sealed, and provided in a liquid or dry form. A dry preparation can be produced by freeze-drying after dispensing and filling the inside of the container with nitrogen gas. Such vaccine formulations are usually stored at 1-5 ° C,
For use, the liquid preparation is directly opened after opening, the dried preparation is opened, and then dissolved in the attached dissolving solution, and usually 0.5 to 1.0 ml per inoculated patient is inoculated subcutaneously or intramuscularly. Used.

[Assay for Immunogenicity of Mixed Vaccine] In the assay for humoral immunity, the antibody titer in the blood after vaccination is determined by a method commonly used in immunology and serodiagnosis, for example, neutralization reaction,
Fluorescent antibody reaction, ELISA (enzyme-link)
immunosorbent assay), PH
A (passive hemagglutinatio)
n) and the like. The immunity can be confirmed by comparing the antibody titer before inoculation with time as a control. For example, after the mixed vaccine is inoculated subcutaneously into small experimental animals such as monkeys, rabbits, guinea pigs, and mice, the immunized animals are bred and managed. During the breeding, after inoculation of the virus, about 3 ml of blood is partially collected from the femoral vein of the guinea pig at regular intervals, for example, weekly or monthly, and the antibody titer in the blood is measured. The antibody titer may be measured, for example, by using PHA using erythrocytes to which the antigen used for immunization is attached, ELISA using the antigen used for immunization and an anti-IgG antibody labeled with an enzyme, a known amount of infectious virus and its After antigen-antibody reaction with antiserum,
A neutralization test or the like in which the highest dilution factor of the antiserum that reduces the amount of the infectious virus by 50% by neutralization is measured by a CPE method or a plaque assay can be employed. On the other hand, cell-mediated immunity can be assayed as follows.

[Assay for Induction of Cellular Immunity by Mixed Vaccine and Inactivated Antigen] Cellular immunity is assayed by a measurement method using as an index the functional activity of sensitized T lymphocytes (T cells) induced by vaccination. it can. The cells involved in cell-mediated immunity include helper T cells 1 (Th
1), helper T cells 2 (Th2), cytotoxic T lymphocytes (CTL: cytotoxic T lymphocyte)
cyte or Tc), memory T cell (Tm), suppressor T cell (Ts), and the like are known. Specific examples of the measurement method thereof are described in “Current Protocol”.
ls in Immunology "[vol. 1, p
p. 3.0.1 to 4.8.17; E. FIG. Coliga
n et al., 1991-present (addition and subtraction), John Wil
ey & Sons (USA)]. For example, the presence of Th1 indicates delayed type hypersensitivity (DTH:
delayed-type hypersensitivity
ii) reaction or a method using a delayed-type skin hypersensitivity reaction (“Same as above”, pp. 4.5.1-4.5.5). For this, for example, an intradermal reaction in a mouse, a guinea pig, or the like is used. The intradermal response was determined by the T
When h1 is increased (cellular immunity is established), if the same antigen is intradermally inoculated, redness or induration occurs at the inoculated area. Therefore, the size and degree are measured over time. The CTL activity is, for example, ⁵¹ Cr
Chromium release method using target cells (infected cells) labeled with, a DNA fragment method using [ ³ H] thymidine-labeled target cells, a limiting dilution method for knowing the frequency of CTLs, etc. 3.11.1 to 3.11.20). Hereinafter, specific examples of the present invention will be described with reference to Reference Examples and Examples. However, the present invention is not limited only to these Reference Examples and Examples.

[0011]

[Reference Example] Reference Example 1 [PBS Preparation of (phosphate buffered saline) and NaCl 8.0 g, the _{KCl 0.2g, Na 2 HPO 4 ·} 12H
2.9 g of ₂ O, 0.2 g of KH ₂ PO ₄ , CaCl ₂
0.1 g and 0.1 g of MgCl ₂ .6H ₂ O were respectively dissolved in distilled water to make 1,000 ml to prepare PBS. In addition, divalent ions, CaCl ₂ and MgCl
₂ · 6H ₂ O together not containing PBS (-) was separately prepared.

Reference Example 2 [Preparation of Inactivated Vaccine and Live Vaccine] For preparation of inactivated hepatitis B vaccine and live attenuated varicella vaccine, the "precipitated hepatitis B vaccine" according to the "Biological Standards" described above. , And undiluted live attenuated varicella vaccine according to the regulations of each vaccine. The amount of antigen in the vaccine was adjusted by diluting these stock solutions with PBS (-).

Reference Example 3 [Measurement of antibody titer in blood]
An HA test [manufactured by Kokusai Reagent Co., Ltd.] and ELISA were performed. In the ELISA, a 96-well ELISA plate was used, and an anti-guinea pig IgG sheep IgG antibody labeled with peroxidase [Cappe] was used as the enzyme-labeled antibody.
1 (all molecules); ICN (USA)]. As antigens, both HBs and VZV glycoprotein (gE: gl) antigens were used. As the HBs antigen, a sugar chain-added HBs antigen derived from a hepatitis B carrier serum and a sugar-free HBs antigen produced by a recombinant yeast were used. Also, the gE: gl antigen is
Purified and prepared from crushed cells of infected cells of Oka strain virus, which is the active ingredient of the live attenuated varicella vaccine, using an affinity column using an anti-gE monoclonal antibody (Arc
hives of Virology, 142, 22
95-2301, 1997; and Vaccine, 1
6, 1263-1269, 1998).

Reference Example 4 [Assay of Cellular Immunity by Delayed Type Hypersensitivity (DHT) Reaction] After vaccination, the test was performed by intradermal reaction in guinea pigs kept and maintained. 0.1 ml of the antigen described below in Example 1 was intradermally inoculated into 3 backs / antigen (total of 6 per guinea pig for 2 antigens) of the back of the guinea pig whose back hair had been removed with a depilatory agent. , 24 and 48 hours respectively, the generated elliptic redness was measured, and each area was calculated by the formula [area (m
m ² ) = π × major axis (mm) × minor axis (mm) / 4], and the average value was determined.

[0015]

EXAMPLES Example 1 [Preparation of vaccine] Inactivated hepatitis B vaccine (H): As described in Reference Example 2, the vaccine stock solution was diluted with PBS (-) to give 20 μg.
An inactivated vaccine containing /0.1 ml of inactivated HBs antigen was prepared. Live attenuated varicella vaccine (V): As described in Reference Example 2, the vaccine stock solution was diluted with PBS (-), and 10 ⁴ PFU
A live vaccine containing (plaque forming units) /0.5 ml of attenuated VZV Oka strain virus was prepared. Combined vaccine (HV): Both H and V stock solutions were added to PBS
The mixture was diluted and mixed with (-) to prepare a mixed vaccine containing both 20 μg of inactivated HBs antigen and 10 ⁴ PFU of attenuated VZV Oka strain virus in 0.5 ml. [Preparation of Comparative Control] Comparative Control (M): M, which is a culture host of VZV, conforming to the various rules of “Dry attenuated live varicella vaccine” described in Reference Example 2.
After preparing a stock solution of a mock vaccine containing no attenuated Oka strain virus (pseudo-control) from an uninfected cell culture of RC-5, the stock solution was diluted with PBS (-) in the same manner as the stock solution of V above, and a control ( M) was obtained. Comparative control (heated antigen): Take a part of the stock solution of V above,
Heat at 0 ° C. for 30 minutes and compare with [K: Heat V (10
⁴ PFU equivalent heated VZV)]. [Immunization schedule] For immunization, guinea pigs (Hartley species, 4 weeks old, body weight 230 to 270 g) of 5 animals per group, 7 groups in total, were used to inoculate a sample subcutaneously on the back of each guinea pig. In addition, the amount of inoculation / guinea pig
0 μg, and V was 10 ⁴ PFU, and the immunization schedule was as follows: After completion of immunization for a total of seven groups as described above, an intradermal reaction described below was performed on the third week. After the intradermal reaction, the serum antibody titer of each serum collected from each guinea pig was measured. [Measurement of Blood Antibody Titer] The serum antibody titers for both HBs and VZV glycoprotein (gE: gl) antigens derived from yeast were determined for each of the above G1 to G7 sera of 35 guinea pigs by E.
Each was measured by LISA. First, 96-hole ELI
Each well of the SA plate was coated with 1 μg of HBs antigen or 0.5 μg of gE: gl antigen. Then 10% with PBS containing 2% (W / V) skim milk
The serum diluted 0-fold and a peroxidase-labeled anti-guinea pig IgG sheep IgG antibody [Cappel (all molecules)] were sequentially reacted, and then color-developed by HAT-EIA [Denka Seiken Co., Ltd.]. The absorbance at a wavelength of 490 nm was measured with a plate absorptiometer. Table 1 shows the results. Compared with the control group (G5 to G7), a marked increase in blood antibodies to both HBs and VZV antigens was observed, particularly in the mixed vaccination group (G1). In addition, a comparison between G1 to G4 and G5 showed that mixing or simultaneous inoculation of both antigens enhanced humoral immunity with the inactivated antigen HBs. Furthermore, the difference in each antibody titer in blood with respect to these two antigens (G1 >> G5 to G7)
Were significant at the significance level p <0.0001 by the t-test.

[0016]

[Table 1] [Guinea pig intradermal reaction] 0.1 g was added to the back of each immunized guinea pig (3 sites / antigen; 6 sites / 2 antigen / guinea pig in total).
1 ml of HBs antigen (2 μg) and VZV glycoprotein (gE: gl) antigen (1 μg) were intradermally inoculated, respectively.
At 8, 24 and 48 hours later, the major axis and minor axis of each elliptic redness generated by DTH were measured, and the area of each redness was calculated by the formula described in Reference Example 4, and the average value of each immune group was calculated. The significant differences between these immunized groups were estimated by the t-test. Table 2 shows the results. In addition, the numerical value in a table | surface shows the redness area (mm < ² >) of each immunity group in 24 hours after an intracutaneous inoculation: [(pi) x major axis (mm) x average value of minor axis (mm) / 4 standard error] Show.

[0017]

[Table 2] The difference between the mixed vaccination group (G1) and each of the G2 to G7 groups, that is, G1 >> G2 to G7, is all significant at the significance level p
= 0.0001 or <0.0001, which was significant, and it was determined that cell-mediated immunity was induced by the HBs antigen (inactivated antigen). In addition, regarding VZV, G1 and G5
7, there is a significant difference at p <0.0001, and the combined vaccination group (G1) and the simultaneous vaccination group (G3)
It was determined that there was no significant difference between p and 0.4703. These results indicate that cell immunity against the inactivated vaccine antigen is induced by the mixture of the inactivated vaccine and the live vaccine.

[0018]

EFFECT OF THE INVENTION The method provided by the present invention for inducing the cell-mediated immunity of a live vaccine to an inactivated vaccine, and the combined vaccine obtained by such a method are suitable for infectious cell-mediated immunity produced by the live vaccine, Alternatively, it provides an epoch-making effect that an inactivated vaccine produces an activity equivalent to the cellular immunity induced by a live vaccine, and also contributes to simplification of vaccination, labor saving and cost reduction.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61K 39/145 A61K 39/145 39/21 39/21 39/25 39/25 39/29 39/29 // C12N 7 / 04 C12N 7/04 (C12N 7/04 (C12N 7/04 C12R 1:92) C12R 1:92) F term (reference) 4B065 AA95X BA14 BD10 BD13 CA45 4C085 AA04 BA10 BA12 BA17 BA55 BA62 BA69 BA79 BA89 BA92 CC07 CC08 DD88 EE03 GG04

Claims (4)

[Claims] 1. A method for causing an inactivated vaccine to have a cellular immune activity of a live vaccine, comprising mixing the active ingredients of the inactivated vaccine and the live vaccine in amounts that induce cellular immunity. 2. A combination vaccine comprising the active ingredients of an inactivated vaccine and a live vaccine in amounts that induce cell-mediated immunity. 3. The active ingredient of the inactivated vaccine is derived from hepatitis B, non-A non-B hepatitis, HIV, rota, Japanese encephalitis and influenza viruses and tetanus, pertussis and diphtheria bacteria. The combination vaccine according to claim 2, which is at least one kind of antigen selected from the group of activated antigens. 4. The combination vaccine according to claim 2, wherein the active ingredient of the live vaccine is attenuated varicella virus.