JP2005082581A - SECRETORY IgA ANTIBODY INDUCER - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preventing infection with a pathogen by administering a double chain or a single chain RNA which is a TLR (Toll-like receptor) ligand and inducing production of secretory IgA antibody specific to the virus or the pathogenic bacterium in respiratory mucosa tissues, and obtain a secretory IgA antibody inducer. <P>SOLUTION: The secretory IgA antibody inducer comprises the double chain or the single chain RNA as effective ingredient. The method induces the IgA antibody specific to the virus or the pathogen by administering the double chain or single chain RNA. Infections caused by the pathogen is protected by inducing the secretory IgA antibody by the method. Preferably, the double chain RNA is a Poly(I:C) secretory IgA antibody-inducing agent. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for protecting against infection by a pathogen comprising inducing production of an IgA antibody specific to a virus or pathogen by administering double-stranded or single-stranded RNA, and also to induce secretory IgA antibody It relates to the agent.

  Humans have a function to protect against external attacks such as viruses and bacteria, and such a biological defense function has been established by the cooperative action of innate immunity and acquired immunity. That is, the human immune mechanism can be broadly divided into innate immunity and acquired immunity. Acquired immunity is considered to deal with all foreign antigens by creating a myriad of receptors with different antigen specificities on B cells and T cells by the method of gene rearrangement.

  On the other hand, conventional innate immunity was considered to be a system for treating foreign substances and pathogens by nonspecific phagocytosis mainly by macrophages and leukocytes. However, it has recently been revealed that immune cells involved in this innate immunity also recognize a common pattern of pathogen components using specific receptors encoded on the genome without gene rearrangement. It is becoming. Among them, so-called antigen-presenting cells such as macrophages and dendritic cells responsible for innate immunity recognize pathogens through a group of membrane proteins called Toll-like receptors (TLRs). Receptors have been found to be essential molecules for pathogen recognition and subsequent immune responses. In mammals including humans, it has become clear that innate immunity dominates acquired immunity via TLR.

  By the way, the ligand of Toll-like receptor (TLR) does not exist in the host, and it is becoming clear that it is an essential component for microorganisms such as viruses and bacteria, and LPS (lipopolysaccharide: lipopolysaccharide specific to microorganisms). Polysaccharides), LP (lipoprotein: lipoprotein), peptide glucan (PGN) and the like have been identified as representative TLR ligands.

  The present inventors have been conducting various studies on double-stranded or single-stranded RNA, which is a ligand of TLR that recognizes microbial components of living organisms and stimulates the innate immune system, and in particular, inosinic acid and We have been studying the effect of Poly (I: C), a polynucleotide copolymer consisting of cytidylic acid, on the immune system. That is, since polylysine binds and stabilizes with Poly (I: C), Poly (I: C) is known as a potent adjuvant for immunity against influenza virus. (I: C) is considered to be capable of exhibiting the ability to acquire protective immunity against various pathogenic bacteria or viruses. Therefore, these double-stranded or single-stranded RNAs, in particular, Poly (I: C), which is a double-stranded RNA, serve as a TLR ligand that stimulates the innate immune system and protects against attack by microorganisms such as pathogenic bacteria or viruses. It is considered that the ability to acquire immunity can be exhibited.

  In fact, various reports have been made so far in which Poly (I: C) is used as an adjuvant and administered to a mucosa together with, for example, an influenza vaccine, and the effect thereof is examined. For example, Non-Patent Document 1 discloses that UV-inactivated whole (all) influenza virus using Poly (I: C) as an adjuvant was administered by the respiratory tract route and the effect was examined. However, FIG. 2 of this non-patent document 1 shows that there is no difference in the increase in IgG antibody from the case where Poly (I: C) is not added. Therefore, it was thought that Poly (I: C) was not so effective as an adjuvant.

  Non-Patent Document 2 and Non-Patent Document 3 describe nasal inoculation with an inactivated vaccinia virus using Poly (I: C) as an adjuvant. However, Non-Patent Document 2 describes that IgA antibody production in tears is increased, but does not mention the infection prevention effect of Poly (I: C).

  Furthermore, Patent Document 1 discloses that an antibody reaction of an influenza vaccine is enhanced by adding a polynucleotide (including Poly (I: C)) to an adsorptive adjuvant. However, patent document 1 does not mention the infection prevention effect.

  Non-Patent Document 4 reports a significant increase in IgG and IgM after intraperitoneal inoculation with an inactivated vaccine using Poly (I: C) as an adjuvant. Not mentioned.

  Non-patent document 5 reports that when immunization is carried out by intravenously injecting sheep erythrocytes using Poly (I: C) as an adjuvant, the antibody in the blood rises. Is not mentioned.

Japanese Patent Publication No. 50-2009 J. Clinical Investigation, 110 (8), 1175-1184 (2002) Invest. Ophtalmol., 10 (10), 750-759 (1971) Invest. Ophtalmol., 10 (10), 760-769 (1971) Veterinary Microbiology, 88 (4), 325-338 (2002) Pro. Soc. Exp. Biol. & Med., 133, 334-338 (1970)

  In view of the above situation, the present inventors enhance the vaccine ability when double-stranded or single-stranded RNA, which is a ligand of TLR containing Poly (I: C), is administered together with a vaccine as an adjuvant. Instead, Poly (I: C), which is a double-stranded or single-stranded RNA, enhances the protective immunity against attack of various pathogens or pathogens such as viruses, in particular, virus or pathogen-specific IgA antibodies We thought that we could protect against infection by pathogens by inducing

  Based on this concept, when Poly (I: C), a double-stranded RNA, is administered together with an inactivated antigen that is a vaccine such as various pathogenic bacteria or viruses, it induces production of a virus or pathogen-specific IgA antibody. It was confirmed that the present invention was completed. Moreover, it was confirmed that single-stranded RNA also induces the production of similar virus or pathogen-specific IgA antibodies, and the present invention has been completed.

  Accordingly, the present invention is based on a pathogen comprising inducing the production of secretory IgA antibodies in respiratory mucosal tissue specific for viruses or pathogens by administering double-stranded or single-stranded RNA that is a ligand for TLR. It is an object of the present invention to provide a method for preventing infection, as well as a secretory IgA antibody inducer.

Specifically, the present invention for solving such problems is as follows:
(1) A secretory IgA antibody inducer comprising double-stranded or single-stranded RNA as an active ingredient;
(2) The secretory IgA antibody inducer according to (1) above, wherein the double-stranded RNA is Poly (I: C);
(3) The secretory IgA antibody inducer according to (2) above, wherein the molecular size of Poly (I: C) is 300 bp or more;
(4) The secretory IgA antibody inducer according to (1) to (3), which is administered to the respiratory mucosa;
(5) the secretory IgA antibody inducer according to (1) to (4), which is administered together with an inactivated antigen derived from a virus or a pathogen; and
(6) The virus is varicella virus, measles virus, mump virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, severe acute respiratory infection syndrome (SARS) virus (a type of coronavirus) and HIV virus, The secretory IgA antibody inducer according to (5) above, wherein the pathogenic bacteria are Bordetella pertussis, Neisseria meningitidis, influenza b type bacteria, pneumoniae, and cholera bacteria.

The present invention also relates to a method for inducing a pathogen-specific IgA antibody, specifically,
(7) A method of inducing a virus or pathogen-specific IgA antibody by administering double-stranded or single-stranded RNA;
(8) The method for inducing a virus or pathogen-specific IgA antibody according to (7) above, wherein the double-stranded RNA is Poly (I: C);
(9) The method for inducing a virus or pathogen-specific IgA antibody according to (7) or (8) above, wherein the molecular size of Poly (I: C) is 300 bp or more;
(10) The method for inducing the virus or pathogen-specific IgA antibody according to (7) to (9) above, wherein the administration of Poly (I: C) is to the respiratory mucosa;
(11) The method for inducing a virus or pathogen-specific IgA antibody according to (7) to (10), which is administered together with an inactivated antigen derived from a virus or pathogen; and
(12) The virus is varicella virus, measles virus, mump virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, severe acute respiratory infection syndrome (SARS) virus (a type of coronavirus) and HIV virus, A method for inducing the virus or pathogen-specific IgA antibody according to (11) above, wherein the pathogenic bacteria are Bordetella pertussis, Neisseria meningitidis, influenza b type bacteria, pneumococci and Vibrio cholerae;
It is.

Furthermore, the present invention relates to a method for protecting against infection, more specifically,
(13) A method for protecting against infection by a pathogen, which comprises inducing a secretory IgA antibody by administering double-stranded or single-stranded RNA;
(14) The method for protecting against infection by a pathogen according to (13), wherein the double-stranded RNA is Poly (I: C);
(15) The method for protecting against infection by a pathogen according to the above (13) or (14), wherein the molecular size of Poly (I: C) is 300 bp or more;
(16) The pathogen is a virus or pathogen, and the virus is varicella virus, measles virus, mump virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, severe acute respiratory infection syndrome (SARS) virus (corona) A method for protecting against infection according to claim 11 or 12, which is a kind of virus) and an HIV virus, and the pathogenic bacteria are Bordetella pertussis, Neisseria meningitidis, Influenza b type bacteria, Streptococcus pneumoniae and Vibrio cholerae
(17) A method for preventing infection according to the above (13) to (16), comprising administering to the respiratory mucosa; and
(18) The method for protecting an infection according to any one of (13) to (17), which is administered together with an inactivated antigen derived from the pathogen according to (16) above;
It is.

  Administration of double-stranded or single-stranded RTNA, particularly Poly (I: C), which is a ligand of the TLR of the present invention, induces production of secretory IgA antibody in respiratory mucosal tissue specific to viruses or pathogens Is done. Secretory IgA is a major immunoglobulin in the exocrine fluid and is a pathogen-specific IgA antibody that is useful for defense against infection of the mucosal surface, and is secreted in saliva, nasal secretion, intestine, trachea, etc., or colostrum It is common in sera and is also present in serum. Therefore, double-stranded or single-stranded RTNA, particularly Poly (I: C), which is a ligand of TLR provided by the present invention, induces the production of this IgA antibody, thereby infecting by a pathogen such as virus or pathogen. And can be a secretory IgA antibody inducer for preventing infection.

The present invention will be described in detail below.
Double-stranded RNA (dsRNA (double-stranded RNA)) used in the present invention refers to any double-stranded RNA. Examples of such double-stranded RNA include Poly (I: C), Poly ( A: C), Poly (G: C), and the like. Among them, the present invention gave good results for the production of secretory IgA antibodies by using Poly (I: C).

  Regarding the double-stranded RNA used in the present invention, the molecular size of the base pair (bp) can be various low to high sizes, but it is more excellent for immune response. It was found that those exhibiting a good response need to have a molecular size of 300 bp or more. As what has such a molecular size, for example, 100-1000 bp of Poly (I: C) can be easily obtained from Toray Industries, Inc.

  Single-stranded RNA is RNA transcribed from double-stranded RNA that constitutes the genome. Among RNA viruses, genomes such as poliovirus, retrovirus, and influenza virus are single-stranded RNA. The RNA has a double-stranded structure only when it is a genome, and the RNA that performs other functions is based on the single-stranded structure. As a ligand, it induces the production of secretory IgA antibodies in respiratory mucosal tissue specific for viruses or pathogens. An example of such a single-stranded RNA is Poly (A, U).

  In the present invention, such a double-stranded or single-stranded RNA exhibits the ability to acquire protective immunity against the attack of microorganisms such as pathogenic bacteria or viruses as a ligand of Toll-like receptor (TLR) that stimulates the innate immune system. However, it has been confirmed that, when administered together with inactivating antigens of microorganisms such as these pathogenic bacteria or viruses, a better effect is exhibited.

  Such inactivated antigens of microorganisms such as pathogenic bacteria or viruses generally exhibit an action as a vaccine. Therefore, the double-stranded or single-stranded RNA that is the ligand of the TLR of the present invention is preferably administered together with the vaccine as an embodiment, in which case the double-stranded or single-stranded RNA of the present invention is used. Will also act as an adjuvant for vaccines.

  A vaccine to which a double-stranded or single-stranded RNA of the present invention is administered as an adjuvant is an antigenic suspension comprising an infectious agent or a portion of an infectious agent that is inoculated into the body and generates active immunity. Suspension or solution. The antigenic part constituting the vaccine may be a microorganism (eg, a virus or a bacterium) or a natural product purified from a microorganism, a synthetic product or a genetically engineered protein, peptide, polysaccharide or similar product. Good.

  For example, specific examples of live vaccines include BCG, seed varieties, polio, measles, rubella, mumps, cowpox, NDV, Marek's disease, and the like. Inactivated vaccines include pertussis, diphtheria (toxoid), tetanus (toxoid), influenza (HA), Japanese encephalitis, rickettsia and the like.

  The inactivated antigen referred to in the present invention refers to an antigen that has lost its infectivity, and is a complete virus particle virion, an incomplete virus particle, a virion constituent particle, a post-translational modification thereof, a virion non-constitutive protein, Examples include post-translational modifications, infection protective antigens, neutralization epitopes, and the like. Inactivation can be performed, for example, by physical (for example, X-ray irradiation, heat, ultrasonic) or chemical (formalin, mercury, alcohol, chlorine) operations.

  The administration of double-stranded or single-stranded RNA provided by the present invention is preferably carried out in the form of mucosal administration. The mucous membranes in vertebrates include the inner walls of particularly extrinsic luminal organs such as the digestive, respiratory, excretory, and genital organs. Therefore, examples of the mucosal administration which is a preferable administration form of the present invention include nasal administration (nasal administration), buccal administration, intravaginal administration, upper respiratory tract administration and alveolar administration. Of these, intranasal mucosal administration is preferred. In particular, the nasal cavity is the gate of the infection route of respiratory illness diseases caused by influenza virus and the like, causing a secretory IgA reaction by mucosal administration, and producing secretory IgA antibody in mucosal epithelial cells. It leads to defense against infectious diseases.

  The administration of the double-stranded or single-stranded RNA of the present invention is not limited to the above mucosal administration, and it goes without saying that various administration forms can be taken.

  A pathogen as a target in which production of secretory IgA antibody, which is the object of the present invention, is induced and exerts immune defense, is a microorganism that can cause a disease or disorder to a host. Specifically, examples of pathogens for humans include viruses, bacteria, and fungi. The virus may be of any kind, and examples thereof include DNA viruses and RNA viruses. More specifically, for example, varicella virus, measles virus, mump virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, severe acute respiratory infection syndrome (SARS) virus (a kind of coronavirus), HIV Examples include but are not limited to viruses. Such a virus is preferably an influenza virus.

  In addition, the bacteria targeted by the present invention may be any bacteria, and examples include gram positive bacteria and gram negative bacteria. Examples of such bacteria include pertussis bacteria and meningitis. Bacteria, influenza b-type bacteria, pneumoniae and cholera bacteria.

  The double-stranded or single-stranded RNA provided by the present invention can be administered together with various pharmaceutically acceptable carriers in the form of a composition, vaccine, or the like. Such carriers can include diluents, emulsifiers, suspending agents, fillers, extenders, buffers, excipients, antioxidants, preservatives, colorants, flavors and the like.

  In addition, the dose of the double-stranded or single-stranded RNA of the present invention cannot be generally limited by the age, body weight, and administration method of the subject to be administered, but usually in the case of oral administration per day for an adult, In the case of mucosal administration, particularly nasal administration, it is about 0.1 to 10 mg, preferably about 0.1 to 1 mg.

Hereinafter, the present invention will be described with reference to specific examples.
In addition, in the following Example with respect to humans, it was performed after obtaining informed consent from the subject patient in advance.
Example 1 : Poly (I: C), which is a synthetic double-stranded RNA IgA, IgG production ability Poly (I: C) as a synthetic double-stranded RNA as an adjuvant, an inactivated virus or sub as an inactivated antigen The ability of the unit antigen to induce neutralizing antibodies, and thus the anti-pathogen effect, was confirmed.
(material)
Mouse: BALB / c mouse (6 weeks old, female)
Virus: Influenza virus H1N1 (A / PR8) strain (obtained from National Institute of Infectious Diseases, Tokyo).
Vaccine: Influenza virus H1N1 (A / PR8) strain H1N1 (A / Beijing) strain (National Institute of Infectious Diseases); H1N1 ((A / Yamagata) strain (National Institute of Infectious Diseases); H3N2 (A / Guizou) strain ( National Institute of Infectious Diseases); ether-treated inactivated HA vaccine (Osaka University Microbiological Society, Kagawa)
Adjuvant: CTB * [contains CTB (cholera toxin B subunit), 0.1% CT (cholera toxin)] as a positive control, Poly (I: C)

(Method)
Six 6-week-old BALB / c mice (Japan SLC Co., Ltd., Tokyo) were used in each group. PR-8HA vaccine (National Institute of Infectious Diseases or Osaka University Microbial Disease Research Association) 1 μg of each adjuvant as an adjuvant, Poly (I: C), 0.1 μg, 1 μg, 3 μg, 10 μg and 5 μL of the mouse's nose were inoculated Three weeks later, the same amount of vaccine was injected intranasally with or without adjuvant, and further two weeks later, 100 pfu of PR8 influenza virus was inoculated into each nose in an amount of 1.2 μL.
As a control, only 10 μg and 1 μg of Poly (I: C), PR8HA vaccine only, and no treatment were placed.
Three days after the infection, nasal lavage fluid and serum were collected, IgA in nasal lavage fluid and IgG in serum were measured by ELISA, and virus titer in nasal lavage fluid was measured by plaque assay using MDCK cells.

Mice immunized intranasally by the same method were infected with 20 μL of a 40LD ₅₀ lethal dose (10 ^4.7 EIO ⁵⁰ (about 10000 times the amount of virus immunity in 50% embryonated chicken eggs) of influenza virus, The survival was observed.

  In addition, in order to examine the cross protection of nasal influenza vaccine using Poly (I: C) as an adjuvant, influenza virus H1N1 (A / PR8) strain H1N1 (Beijing) strain, H1N1 (A / Yamagata) with different subtypes H3N2 (A / Guizou) strain vaccine with 3 μg of Poly (I: C) nasally, 3 weeks later with the vaccine alone, and 2 weeks later with 100 μfu PR8 influenza virus 1.2 μL per nose Inoculated and infected. Three days after the infection, nasal lavage fluid and serum were collected, IgA in nasal lavage fluid and IgG in serum were measured by ELISA, and virus titer in nasal lavage fluid was measured by plaque assay using MDCK cells.

Furthermore, in order to examine the safety of Poly (I: C) to the central nervous system, 0.25 μg, 2.5 μg, and 25 μg of Poly (I: C) were dissolved in 25 μL of PBS, and a two-stage needle was used. A brain inoculation was performed. Changes in body weight after inoculation were measured and survival was observed.
As a control group, a group in which 2.5 μg, 10 μg, and 25 μg of CTB * (including CTB and 0.1% CT) were similarly dissolved in 25 μL of PBS was inoculated into the brain.

(result)
(1) Antibody induction and infection protection by nasal influenza vaccine using Poly (I: C) as an adjuvant Poly (I: C) mucosal adjuvant ability was evaluated. Six weeks ago, 1 μg of PR8 vaccine was intranasally inoculated with Poly (I: C) dosed to 0.1 μg to 10 μg, and two weeks ago, the same amount of vaccine was intranasally inoculated with the vaccine alone or with an adjuvant. did. Table 1 summarizes the IgA antibody response in the nasal mucosa and the blood IgG response.

In order to see the dose-dependent adjuvant effect of Poly (I: C), the amount of Poly (I: C) was gradually increased from 0.1 μg to 10 μg, and the adjuvant action was observed.
As a result, as can be seen from the results shown in Table 1, for IgA response to the nasal mucosa, at least 0.1 μg of Poly (I: C) should be used at the time of the first immunization. There was found.

The amount of IgA induced in the nasal mucosa was dependent on the amount of Poly (I: C), and the adjuvant effect was recognized as the amount of Poly (I: C) was increased.
When Poly (I: C) is used for both immunizations, secretion of IgA of 100 ng / mL or more was observed in the nasal lavage fluid in an amount of 1 μg of Poly (I: C). When 3 μg of Poly (I: C) was added, induction of a specific IgA antibody of 100 ng / mL or more was observed.
The production of IgG in serum was also examined at the same time. The production correlates with the secretion of IgA, and 1.6 μg was obtained when 1 μg of PR8 vaccine was immunized twice with Poly (I: C) at intervals of 4 weeks. / ML blood IgG was obtained.

In addition, when 100 pfu of PR8 virus was infected at a rate of 1.2 μL per nose two weeks after the second immunization under the same immunization conditions, 10 ² pfu in nasal lavage fluid was obtained in the control group without vaccination. Virus growth was observed at a virus titer of / mL or more.
On the other hand, in the group in which nasal vaccination was performed twice using Poly (I: C) in combination, virus growth was completely suppressed, and the group immunized twice with 1 μg or more of the vaccine alone, In addition, in the group in which 3 μg or more of Poly (I: C) was used only at the time of the first immunization, no virus suppressing effect was observed.

In addition, even in the group inoculated with 1 μg and 0.1 μg of Poly (I: C) only for the first time, significant virus growth suppression was observed at 10 ^0.8 pfu / mL and 10 ^1.6 pfu / mL. It was. In addition, no virus growth inhibition was observed in the two-dose administration group of vaccine alone.
These results are summarized in Table 2.

＊：ｐ＜０．００１

*: P <0.001

(2) Confirmation that the structure of double-stranded RNA is important for the expression of action of Poly (I: C) The structure of double-stranded RNA is important for the expression of action of Poly (I: C) To confirm this, Poly (I: C) was heated at 100 ° C. for 5 minutes, immediately cooled on ice to denature, and 1 μg thereof was inoculated nasally with the vaccine.
As a result, the IgA response, which was seen at 121 μg / mL when nasally inoculated with Poly (I: C) that was not heat-denatured, was obtained by nasally inoculating Poly (I: C) with heat denaturation, The IgG response in the blood decreased from 1.5 μg / mL to 0.7 μg / mL.
Further, inoculation with heat-denatured Poly (I: C) no longer showed the effect of suppressing virus growth.
From the above facts, it was confirmed that the structure of double-stranded RNA is important for the expression of the action of Poly (I: C).

(3) About the protective effect of pneumonia caused by lethal dose of influenza virus infection by combined nasal vaccination with Poly (I: C) Combined with 10 μg, 3 μg and 1 μg of 1 μg PR8 vaccine and Poly (I: C) 6 weeks ago Nasal inoculation and booster immunization with the vaccine alone two weeks ago, infected with 20 μL of 40LD ₅₀ PR8 virus, and examined the protective ability of pneumonia.
In the non-vaccinated group, the mice died within 1 week (5/5), and the viral titer of the lung 3 days after viral infection was 10 ⁶ pfu or more.
In contrast, in the vaccinated group, all mice survived by inoculating 1 μg or more of Poly (I: C) together. The results are shown in Table 3.

  From the above facts, it was found that Poly (I: C) produces secretory IgA antibodies sufficient for infection protection and elicits mucosal IgA antibody responses.

Example 2 : Cross protection by poly (I: C) combined nasal vaccine The cross protection ability of influenza (N: C) combined nasal influenza vaccine induced by nasal influenza vaccine was examined.
Immunized with 3 μg of Poly (I: C) for the first time with the vaccines of influenza virus strains H1N1 (A / Beijing), H1N1 (Yamagata), and H3N2 (A / Guzhou), which are different in subtype from PR8. 2 weeks later, 100 pfu of H1N1 (A / PR8) strain was infected with 100 pfu, and 3 days later, IgA cross-reacting with PR8 in nasal lavage, IgG in serum were measured, and We examined the cross protection of PR8 virus.
The results are shown in Tables 4 and 5.

注：二次は、ワクチンのみ。
＊：Ｐ＜０．００１

Note: Secondary is vaccine only.
*: P <0.001

As can be seen from the results in each table, both the IgA and IgG responses were observed against the same subtype of H1N1 (A / beijing) strain and H1N1 (A / Yamagata), and virus infection was completely suppressed. It was.
Small amounts of cross-reacting IgA and IgG were observed against H3N2 (A / Guzhou) strains with different subtypes, and partial protection against viral infection was observed.
Based on the above facts, cross protection was confirmed by Poly (I: C) combined nasal influenza vaccine.

Example 3 : Preventive effect when using inactivated virus particles as a nasal influenza vaccine in combination with Poly (I: C) (material)
Vaccine: ether-treated HA vaccine (Osaka Univ.); Formalin inactivated whole particle vaccine (A / New Caledonia / 20/99 (H1N1) virus) (Osaka Univ.)
Mouse: BALB / c mouse (6 weeks old, female)
(Method)
A / New Caledonia / 20/99 (H1N1) virus formalin-inactivated whole particle vaccine 0.1 μg combined with Poly (I: C) [100-1000 bp: Toray] 0.1 μg As a vaccine component of the nasal influenza vaccine, mice were administered to BALB / c mice (6 weeks old, female), and the same vaccine was administered a second time after 3 weeks.
One week later, antibody responses to HA and NA in mice's nasal lavage fluid and serum were measured as indicators of mucosal and systemic protective immunity, respectively.

(result)
The results are shown in Table 6.

As can be seen from the results in the table, mucosal protective immunity and systemic protective immunity were also enhanced when inactivated whole virus particles were used as a vaccine component of a Poly (I: C) combined influenza vaccine.
Moreover, even when 0.1 μg each of vaccine and Poly (I: C) is used, a positive control for adjuvant activity is predicted in which split-product vaccine is used in combination with CTB * to prevent safe viral infection. The response was equivalent to the group.
Furthermore, these responses were higher than when split-product vaccine was used with Poly (I: C).
From the above facts, the usefulness of the nasal vaccine combined with Poly (I: C) was confirmed not only when using the spirit-product vaccine but also when using other forms of vaccine.

Example 4 : Confirmation of molecular size of Poly (I: C) (material)
Virus: A / New caledonia / 20/99 (H1N1) virus Poly (I: C):
Size (L): 1 to 300 bp (Fluka)
Size (M): 100-1000bp (Toray)
Size (H):> 3.3 × 10 ⁶ bp (Fluka)
Poly (A: U)
Mouse: BALB / c mouse (6 weeks old, female)
(Method)
A / New Caledonia / 20/99 (H1N1) virus split-product vaccine (0.4 μg), 0.1 μg of Poly (I: C) of various sizes, and BALB / c mice (6 weeks old, The same vaccine was administered a second time three weeks later.
One week later, antibody responses to HA and NA in mice's nasal lavage fluid and serum were measured as indicators of mucosal and systemic protective immunity, respectively.

(result)
The results are shown in Table 7.

  As can be seen from the results shown in the table, a mucosal response lower than that of the other groups was observed in the experimental group using a Poly (I: C) molecular size of 10 to 300 bp. Therefore, the molecular size of Poly (I: C) useful as an adjuvant was considered to be about 300 bp or more.

Example 5 : Action of double-stranded RNA other than Poly (I: C) The action of Poly (I: C) was compared with other double-stranded RNAs Poly (A: U) and single-stranded Poly (A, U). ).
(material)
Subunit: Purified HA
Adjuvant: Poly (I: C), Poly (A: U), Poly (A, U)
(Method)
The HA molecule was purified from A / New Caledonia / 20/99 (H1N1) virus using a nasal anti-HA monoclonal antibody-binding column, and 1 μg of that was purified using Poly (A: U) (Toray) and single-chain Poly (A , U) (Toray) together with 1 μg of nasal administration to BALB / c mice (6 weeks old, female), and 3 weeks later, only HA was administered a second time. One week later, antibody responses to HA in nasal lavage fluid and serum of mice were measured as indicators of mucosal and systemic protective immunity, respectively.

(result)
The results are shown in Table 8.

As can be seen from the results shown in the table, adjuvant activity was also observed in double-stranded Poly (A: U) and single-chain Poly (A, U). Compared to the adjuvant activity of Poly (I: C), the order was double-stranded Poly (A: U) and single-chain Poly (A, U) in this order.
Accordingly, it was found that adjuvant activity was also observed when double-stranded RNA or single-stranded RNA other than Poly (I: C) was used in combination with a nasal influenza vaccine.

Example 6 : Examination of induction of protective immunity by several influenza virus subunits under Poly (I: C) combined nasal administration conditions Several flu virus infections under Poly (I: C) combined nasal administration conditions The subunit was confirmed to induce protective immunity.
(material)
Subunit: HA, NA and NP
Adjuvant: Poly (I: C) [100-1000 bp: Toray]
Mouse: BALB / c mouse (6 weeks old, female)
(Method)
The ability of the influenza virus subunits, HA, NA and NP, administered intranasally with Poly (I: C) to induce protective effects was compared. That is, HA, NA, and NP molecules were purified from A / New Caledonia / 20/99 (H1N1) virus using a specific anti-monoclonal binding column, and 1 μg of the purified poly (I: C) (100-1000 bp: Toray) Was administered intranasally to BALB / c mice (6 weeks old, female) together with 1 μg of the above, and each molecule was administered a second time after 3 weeks. One week later, antibody response to each molecule in nasal lavage fluid and serum of mice was measured as an index of mucosal and systemic protective immunity.

(result)
Poly (I: C) has been shown to enhance mucosal and systemic immune responses to any subunit. However, although the preventive effect was enhanced by enhancing humoral immunity against HA and NA, the preventive effect was not enhanced by enhancing humoral immunity against NP. Therefore, it was found that HA and NA are strong protective antigens, and the ability to induce preventive effects varies depending on the subunit.

Example 7 : Examination of administration frequency and administration interval (material) to enhance the effectiveness of poly (I: C) combined nasal influenza vaccine
Vaccine: A / New Caledonia / 20/99 (H1N1) virus Split-product vaccine (1 μg)
Adjuvant: Poly (I: C) [100-1000 bp: Toray]
Mouse: BALB / c mouse (6 weeks old, female)
(Method)
In order to examine the number of administrations and the administration interval to enhance the effectiveness of the Poly (I: C) combined influenza vaccine, the Split-product vaccine (1 μg) of the A / New Caledonia / 20/99 (H1N1) virus was C) NaLB was administered to BALB / c mice (6 weeks old, female) together with 1 μg of (100-1000 bp: Toray), and the same vaccine was administered a second time after 1, 3, 4, and 6 weeks. One or two weeks later, antibody responses to HA and NA in mice's nasal lavage fluid and serum were measured as indicators of mucosal and systemic protective immunity, respectively. At this time, there were some experimental groups 1 and 8 weeks after only one administration.

(result)
Two or more administrations at intervals of one week or more were effective in enhancing the effectiveness of the Poly (I: C) flat y nasal influenza vaccine.

Example 8 : Preventive effect of poly (I: C) combined nasal influenza vaccine in humans Confirmation of safety and efficacy of poly (I: C) combined nasal influenza vaccine in humans from antibody response against influenza infection preventive effect did.
(material)
Vaccine: HA vaccine (Split-product vaccine)
Adjuvant: Poly (I: C) [100-1000 bp: Toray]
(subject)
Healthy people: 2 to several people (method)
For healthy adults, 300 μL of vaccine solution containing 400 μg / mL of the current HA vaccine (Split-product vaccine) and 700 μg / mL of Poly (I: C) [100-1000 bp: Toray] (150 μL each in the left and right nasal cavities) Nebulized. Re-administration was performed 4 weeks later, and the prophylactic effect was confirmed by measuring antibody responses to HA and NA of the serum material before administration, once after administration, and after twice administration.

According to the present invention, administration of double-stranded or single-stranded RTNA, particularly Poly (I: C), which is a ligand of TLR, enables production of secretory IgA antibody in respiratory mucosal tissue specific to viruses or pathogens. Be guided. Secretory IgA is the main immunoglobulin in the exocrine fluid, and therefore double-stranded or single-stranded RNA, particularly Poly (I: C), which is a ligand of the TLR provided by the present invention, is a component of this IgA antibody. By inducing production, a secretory IgA antibody inducer is provided that protects against infection by a pathogen such as a virus or pathogen, and also prevents infection.

Claims (18)

  A secretory IgA antibody inducer comprising double-stranded or single-stranded RNA as an active ingredient.   The secretory IgA antibody inducer according to claim 1, wherein the double-stranded RNA is Poly (I: C).   The secretory IgA antibody inducer according to claim 2, wherein the molecular size of Poly (I: C) is 300 bp or more.   The secretory IgA antibody inducer according to claim 1, 2 or 3, which is administered to the respiratory mucosa.   The secretory IgA antibody inducer according to any one of claims 1 to 4, which is administered together with an inactivated antigen derived from a virus or a pathogenic bacterium.   The virus is varicella virus, measles virus, mump virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, severe acute respiratory infection syndrome (SARS) virus (a type of coronavirus) and HIV virus, 6. The secretory IgA antibody inducer according to claim 5, which is Bordetella pertussis, Neisseria meningitidis, influenza b type bacteria, pneumoniae and cholera bacteria.   A method of inducing a virus or pathogen-specific IgA antibody by administering double-stranded or single-stranded RNA.   The method for inducing a virus or pathogen-specific IgA antibody according to claim 7, wherein the double-stranded RNA is Poly (I: C).   The method for inducing a virus or pathogen-specific IgA antibody according to claim 7 or 8, wherein the molecular size of Poly (I: C) is 300 bp or more.   The method for inducing a virus or pathogen-specific IgA antibody according to any one of claims 7 to 9, wherein the administration of Poly (I: C) is to the respiratory mucosa.   The method for inducing a virus or pathogen-specific IgA antibody according to any one of claims 7 to 9, which is administered together with an inactivated antigen derived from a virus or pathogen.   The virus is varicella virus, measles virus, mump virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, severe acute respiratory infection syndrome (SARS) virus (a type of coronavirus) and HIV virus, The method for inducing a virus or pathogen-specific IgA antibody according to claim 11, which is Bordetella pertussis, Neisseria meningitidis, influenza b type bacteria, pneumoniae, and cholera bacteria.   A method for protecting against infection by a pathogen, which comprises inducing a secretory IgA antibody by administering double-stranded or single-stranded RNA.   The method for protecting against infection by a pathogen according to claim 13, wherein the double-stranded RNA is Poly (I: C).   The method for protecting against infection by a pathogen according to claim 13 or 14, wherein the molecular size of Poly (I: C) is 300 bp or more.   The pathogen is a virus or pathogen, and the virus is varicella virus, measles virus, mump virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, severe acute respiratory infection syndrome (SARS) virus (a kind of coronavirus) ) And HIV viruses, and the pathogen is Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Streptococcus pneumoniae, and Vibrio cholerae, The method for protecting an infection according to claim 11 or 12.   The method for protecting against infection according to any one of claims 13 to 16, comprising administering to the respiratory mucosa. The method for preventing infection according to any one of claims 13 to 17, which is administered together with an inactivated antigen derived from a pathogen according to claim 16.