Mucosal adjuvants

Abstract

To provide a mucosal adjuvant useful for preparation of a mucosal vaccine with high mucosal immunogenicity and high safety, and a mucosal vaccine composition containing the same.SOLUTION: A mucosal adjuvant is composed of TGDK.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a mucosal adjuvant that enhances the induction of mucosal immunity by an antigen. [Background technology]

[0002] Mucosal vaccines induce both local mucosal and systemic immune responses by administering antigens via mucosal routes such as intranasal mucosa, thereby providing a double line of defense against pathogens. However, mucosal vaccines in practical use are those that use live infectious vaccines or special toxins with mucosal affinity as antigens, while other inactivated antigens cannot induce sufficient immunity when administered alone and require the use of adjuvants or other agents.

[0003] Traditionally, attenuated infectious pathogens have been used to induce sufficient immunity in mucosal vaccines. However, because live attenuated vaccines are infectious, they can cause severe side effects. For example, polio vaccine-associated paralysis caused by oral live polio vaccines is a rare but unavoidable side effect, believed to occur due to reversion of the vaccine strain to neurovirulence. Therefore, safe inactivated antigens should be used in mucosal vaccines. However, with the exception of antigens such as special toxins, it is difficult to confer sufficient immunity through mucosal administration. To address this issue, the addition of mucosal adjuvants has been considered, and cholera toxin and the heat-labile toxin (LT) of enterotoxigenic Escherichia coli are known as representative mucosal adjuvants (Non-Patent Documents 1-2).

[0004] However, previous clinical trials have shown that intranasal administration of LT can cause facial nerve paralysis (Bell's palsy), and the use of the toxin itself as an adjuvant is considered to be a safety issue. Furthermore, double-stranded RNA (polyI:C) (Patent Document 1) also has mucosal adjuvant activity, but has not yet been put to practical use due to the induction of inflammation and cytokineemia.

[0005] In recent developments in Japan regarding intranasal influenza vaccines, clinical trials are being conducted using inactivated whole-particle antigens, which are more immunogenic than the split antigens used in commercially available influenza HA vaccines (Non-Patent Document 3). This is because split antigens are unable to induce a sufficient immune response when administered mucosally, and inactivated whole-particle antigens are not currently available on the market due to side effects (local reactions and fever at the administration site) when administered subcutaneously, particularly in children. Therefore, there is a desire to achieve immune induction to the same extent as inactivated whole-particle antigens using highly safe split antigens.

[0006] Tetragalloyl-D-lysine dendrimer (TGDK) is a molecule that specifically binds to microfold cells (M cells), antigen-uptake cells present in mucosal membranes (Non-Patent Document 4). Therefore, chemically conjugating TGDK to antigens or the like enables efficient delivery of vaccine antigens to M cells, thereby improving immune responses. For example, Patent Document 2 discloses that TGDK-CH2-CH2-NH2 can be used as an intestinal immunostimulant by binding to peptides, proteins, lipids, polyethylene glycols, or sugars via peptide bonds or Schiff bases. Furthermore, Patent Document 3 discloses that a covalent conjugate of a hub antigen, TGDK, and fetuin can serve as a molecular mimetic mucosal vaccine for HIV / AIDS.

[0007] However, it is not known that the TGDK molecule itself has any mucosal adjuvant activity. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-97267 [Patent Document 2] International Publication No. 2007 / 052641 [Patent Document 3] International Publication No. 2013 / 024859

Non-Patent Literature

[0009]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

[0010] The present invention relates to providing a mucosal adjuvant useful for preparing a mucosal vaccine that is highly mucosally immunogenic and highly safe, and a mucosal vaccine composition containing the same. [Means for solving the problem]

[0011] In light of the above-mentioned problems, the present inventors have conducted studies and have unexpectedly found that TGDK, which has conventionally been used to chemically bind vaccine antigens and deliver the antigens to M cells, has adjuvant activity in itself and can enhance the ability of antigens to induce mucosal immunity.

[0012] That is, the present invention relates to the following 1) to 5). 1) A mucosal adjuvant consisting of TGDK. 2) A mucosal adjuvant composition containing TGDK and a pharmaceutically acceptable carrier. 3) A mucosal vaccine composition comprising the mucosal adjuvant of 1) and an immunogen. 4) The mucosal vaccine composition of 3), wherein the immunogen is a whole particle or split antigen of influenza virus. 5) A method for preparing a mucosal vaccine composition according to 3) or 4), which comprises mixing TGDK with an immunogen. [Effects of the Invention]

[0013] The mucosal adjuvant of the present invention enables the preparation of mucosal vaccines using safe inactivated antigens. For example, mucosal vaccines using split antigens, which are safer than currently available influenza HA vaccines, can be provided, and these vaccines can make a significant contribution to the pharmaceutical industry as prophylactic drugs. [Brief explanation of the drawings]

[0014] [Figure 1] A / California / 07 / 2009 strain-specific IgG antibody titers in the split antigen administration group. [Figure 2] B / Texas / 2 / 2013 strain-specific IgG antibody titer in the split antigen administration group. [Figure 3] A / California / 07 / 2009 strain-specific IgG antibody titer in the inactivated whole particle antigen administration group. [Figure 4] B / Texas / 2 / 2013 strain-specific IgG antibody titer in the inactivated whole particle antigen administration group. [Figure 5] Geometric mean values ​​of B / Texas / 2 / 2013 strain-specific IgG1 and IgG2a antibody titers in each group. [Figure 6] A / California / 07 / 2009 strain-specific IgG antibody titer. [Figure 7] B / Texas / 2 / 2013 strain-specific IgG antibody titer. DETAILED DESCRIPTION OF THE INVENTION

[0015] In the present invention, "TGDK" is an abbreviation for Tetragalloyl-D-Lysine Dendrimer, which refers to N2,N6-bis[N2,N6-bis(3,4,5-trihydroxybenzoyl)-lysyl]-N-(2-aminoethyl)-lysine amide. TGDK is known as a target molecule for M cells, which are antigen-uptake cells present in mucous membranes. TGDK can be produced, for example, using gallic acid and D-lysine by the tetragalloyl-D-trilysinyl diethylamine solid phase method (see Non-Patent Document 4 mentioned above).

[0016] As shown in the Examples below, when a vaccine composition prepared by mixing influenza vaccine strains (split antigen / inactivated whole antigen) with TGDK is administered mucosally to mice, the titer of IgG that specifically binds to the antigen is significantly higher than that of a group to which TGDK is not added. Furthermore, when immunization is induced by adding TGDK to split antigen, the IgG titer is comparable to that obtained when immunization is induced using inactivated whole antigen alone. Furthermore, when the geometric mean antibody titers (GMT) of IgG1 and IgG2a are examined, there is a significant increase in IgG2a, which has excellent protective ability against influenza virus infection.

[0017] Specifically, TGDK has mucosal adjuvant activity that enhances antibody induction when an immunogen (antigen) is administered mucosally, and therefore TGDK can serve as a mucosal adjuvant, and a composition containing TGDK and a pharmaceutically acceptable carrier can serve as a mucosal adjuvant composition. TGDK can also be used to produce a mucosal adjuvant or a mucosal adjuvant composition.

[0018] In the present invention, the term "mucosal adjuvant" refers to a substance that enhances the immune response to an immunogen when the immunogen is administered mucosally. Here, "mucosal administration" refers to an administration form via a mucosa, and "mucosa" refers to the inner wall of an external hollow organ, particularly the digestive system, respiratory system, urogenital system, eye, etc. in vertebrates. Therefore, such mucosal administration includes, but is not limited to, nasal administration (intranasal administration), oral administration, intravaginal administration, upper respiratory tract administration, alveolar administration, and ophthalmic administration.

[0019] The mucosal adjuvant or mucosal adjuvant composition of the present invention can be administered mucosally in combination with an immunogen, and administration may be simultaneous with, before, or after administration of the immunogen. The dosage of the mucosal adjuvant or mucosal adjuvant composition of the present invention can be appropriately determined depending on the subject of administration, the administration method, the administration form, and the type of antigenic substance.

[0020] The mucosal adjuvant of the present invention can be combined with an immunogen to form a mucosal vaccine composition. The mucosal vaccine composition of the present invention can be prepared by mixing the immunogen with TGDK, and a pharmaceutically acceptable carrier can be added as needed to form an appropriate formulation. In the mucosal vaccine composition of the present invention, TGDK is not chemically bound to the immunogen or other components and exists in a free molecular state.

[0021] Examples of "immunogens" (antigens) include mucosally transmitted pathogens (e.g., viruses or pathogenic bacteria), natural products purified from such pathogens, and proteins, peptides, and polysaccharides artificially produced by techniques such as genetic recombination. Specifically, examples include virions, which are complete virus particles, incomplete virus particles, virion-constituting particles, viral nonstructural proteins, proteins or glycoproteins derived from pathogenic bacteria, protective antigens, and epitopes for neutralization reactions. These immunogens include those that are infectious and those that have lost their infectious ability (inactivated antigens). Examples of inactivated antigens include, but are not limited to, those inactivated by physical (e.g., X-ray irradiation, heat, ultrasound) or chemical (formalin, mercury, alcohol, chlorine) manipulations. From the viewpoint of safety, immunogens derived from mucosally transmitted pathogens are desirably inactivated antigens derived from the above viruses or pathogenic bacteria.

[0022] Examples of viruses include chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, severe acute respiratory syndrome (SARS) virus, human immunodeficiency virus (HIV), human papillomavirus, rubella virus, etc., and are preferably influenza virus or human immunodeficiency virus, more preferably influenza virus. Although whole influenza virus particles can be used, in the present invention, split antigens obtained by splitting virus particles and removing lipids in the envelope can be used.

[0023] Pathogenic bacteria include Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholerae, and Corynebacterium diphtheriae.

[0024] The dosage form of the mucosal vaccine composition includes, for example, a liquid, a suspension, a powder, and the like. Examples of liquid preparations include those dissolved in purified water, buffer solutions, etc. Examples of suspensions include those suspended in purified water, buffer solutions, etc. together with methylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, gelatin, casein, etc. Examples of powder preparations include those thoroughly mixed with methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, etc. These preparations may contain commonly used agents such as absorption enhancers, surfactants, preservatives, stabilizers, moisture-proofing agents, moisturizing agents, solubilizers, etc., as needed.

[0025] The mucosal vaccine composition of the present invention may also contain adjuvants other than TGDK, as long as they do not impair the immunogenicity and safety of the vaccine.

[0026] The amount of immunogen contained in the mucosal vaccine composition of the present invention is not particularly limited, as long as it is sufficient to produce antigen-specific IgG, and can be appropriately determined taking into account the ratio with the TGDK used in combination. For example, when an influenza virus split antigen is used as the antigen, the content should be within the range of 1 to 60 μg HA (equivalent to HA), which is the single dose administered, with 9 to 15 μg HA (equivalent to HA) being more preferable. The above concentration is a value obtained by measuring the concentration of HA protein using a test method specified by WHO or national standards, such as a single radial immunodiffusion test or an HA content method.

[0027] The content of TGDK in the mucosal vaccine composition can be adjusted appropriately taking into consideration the antibody titer, but may be, for example, within the range of 0.03 to 30 μg, which is the single administration dose, and more preferably 0.03 to 0.3 μg.

[0028] The route of administration of the vaccine composition of the present invention is not particularly limited, and may be oral or parenteral (e.g., nasal or ocular administration), and may be administered, for example, by dropping, atomizing, or spraying into the nasal or oral cavity.

[0029] The adjuvant composition or vaccine composition of the present invention can be administered to humans and non-human mammals, preferably humans, including mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, horses, sheep, dogs, cats, monkeys, orangutans, and chimpanzees. [Example]

[0030] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0031] Example 1 (1) Influenza HA vaccine "Seiken" A / H1N1 subtype (A / California / 07 / 2009 strain) and B / Yamagata lineage (B / Texas / 2 / 2013 strain) stock solutions were used as split antigens. Split antigens were mixed so that each strain's hemagglutinin was 1 μg per 10 μL, and TGDK was added to a final concentration of 0.03-30 μg / 10 μL. As controls, a dosing solution without adjuvant was also prepared. Because TGDK is a primary amine skeleton consisting of three lysine molecules to which four molecules of gallic acid are linked, a dosing solution containing gallic acid at 30 μg / 10 μL was also prepared (Table 1). In addition, as with the split antigens, inactivated whole particle antigens of the A / H1N1 subtype (A / California / 07 / 2009 strain) and B / Yamagata lineage (B / Texas / 2 / 2013 strain) were mixed so that the hemagglutinin of each strain was 1 μg per 10 μL, and administration solutions of each inactivated whole particle antigen were also prepared so that the TGDK was 0.03 or 0.3 μg and the gallic acid was 30 μg (Table 1).

[0032] The inactivated whole particle antigen used in this example was prepared as follows. The virus was inoculated into the chorioallantoic cavity of 12-day-old embryonated chicken eggs, and after two days of incubation, the chorioallantoic fluid was collected. The collected chorioallantoic fluid was clarified by filtration, adsorbed onto barium sulfate, and eluted with 12% sodium citrate solution to recover influenza virus. The recovered virus was further purified by ultrafiltration into 6.7 mM phosphate-buffered saline (pH 7.2) after buffer replacement, and then by sucrose density gradient centrifugation to recover the influenza virus-containing fraction. The inactivating agent beta-propiolactone was added to this purified influenza virus to a final concentration of 0.05%, and the influenza virus infectivity was inactivated by incubation at 4°C for 24 hours. After this inactivation reaction, the buffer was replaced by 6.7 mM phosphate-buffered saline (pH 7.2) containing 1 w / w% sucrose by ultrafiltration (MWCO: 100,000), and this was used as an inactivated whole particle vaccine.

[0033] (2) BALB / c mice (female, 5 weeks old) were given 5 μL of the solution (Table 1) in each nostril (10 μL total) twice at 3-week intervals (eight mice per group). Two weeks after the second administration, whole blood was collected. Serum was prepared by centrifugation, and the IgG (total IgG) titers specific for the A / California / 07 / 2009 and B / Texas / 2 / 2013 strains were measured. The IgG subclasses IgG1 and IgG2a were also measured for antibody titers specific for the B / Texas / 2 / 2013 strain in the sera from the split antigen and inactivated whole antigen groups without adjuvant and with 0.3 μg of TGDK.

[0034] [Table 1]

[0035] (3) IgG titers in the split antigen-administered groups (A–F) and the inactivated whole-particle antigen-administered group (G) without adjuvant are shown in Figures 1 and 2. Adding 0.03–30 μg of TGDK per dose to split antigen increased antigen-specific IgG titers in the blood for all strains compared with those administered without adjuvant, and reached levels comparable to those for inactivated whole-particle antigen. In particular, IgG titers against the A / California / 07 / 2009 strain were significantly higher in the group administered with 0.03–0.3 μg of TGDK, and IgG titers against the B / Texas / 2 / 2013 strain were significantly higher in the group administered with 0.03–3 μg of TGDK compared with those administered without adjuvant (Mann-Whitney U test, p<0.05). Furthermore, when gallic acid was added to the split antigen, the IgG titer against the A / California / 07 / 2009 strain was similar to that without the addition of an adjuvant, but the IgG titer against the B / Texas / 2 / 2013 strain was significantly lower than that without the addition of an adjuvant. Polyphenols are said to have antioxidant and immune-activating effects, but in this example, a single molecule of gallic acid did not have the adjuvant activity to enhance antibody induction, and the structure of TGDK, in which four molecules of gallic acid are bound to a primary amine in the backbone formed by three molecules of lysine, was thought to be important for exerting adjuvant activity.

[0036] Next, the results for the inactivated whole antigen administration groups (H–J) are shown in Figures 3 and 4. Similar to split antigen, the addition of 0.03 or 0.3 μg of TGDK per administration to inactivated whole antigen increased IgG titers against both strains. The addition of gallic acid reduced IgG titers against the A / California / 07 / 2009 strain to the same level as in the non-adjuvanted group, but reduced IgG titers against the B / Texas / 2 / 2013 strain. Therefore, similar results were obtained with inactivated whole antigen, but the effect of adding TGDK on IgG titer enhancement was smaller with inactivated whole antigen than with split antigen. This is due to the high immunogenicity of inactivated whole antigen itself. However, TGDK demonstrated adjuvant activity when administered intranasally with both types of antigens (split antigen and inactivated whole antigen) that differed in immunogenicity.

[0037] Figure 5 shows the geometric mean titers (GMT) of IgG1 and IgG2a in each group. It can be seen that IgG2a was significantly increased by the addition of TGDK for both split antigens and inactivated whole antigens. IgG2a, induced by a Th1-type response, is more effective at protecting against influenza virus infection than IgG1, induced by a Th2-type response, and therefore, the addition of TGDK is expected to further improve efficacy.

[0038] Reference example 1 (1) Using the same antigen as in Example 1, the adjuvant activity of TGDK when administered subcutaneously was evaluated in the test groups shown in Table 2. In this evaluation, Alm (Imject Alum, manufactured by Thermo Fisher Scientific), which has a proven track record as an adjuvant for subcutaneous administration, was added as a control.

[0039] [Table 2]

[0040] (2) Figure 6 shows the IgG titers against the A / California / 07 / 2009 strain, and Figure 7 shows the IgG titers against the B / Texas / 2 / 2013 strain. For both strains, the TGDK-treated group showed similar IgG titers compared to the split antigen-treated group, demonstrating no adjuvant activity. On the other hand, the Alm and inactivated whole particle-treated groups (WV) showed higher IgG titers than the split antigen-treated group. Furthermore, for both strains, the gallic acid-treated group showed similar IgG titers to the split antigen-treated group, demonstrating that neither the gallic acid derivatives TGDK nor gallic acid had adjuvant activity when administered subcutaneously.

Claims

1. A mucosal adjuvant composition for mucosal administration, comprising TGDK and a pharmaceutically acceptable carrier.

2. A mucosal vaccine composition comprising TGDK and an influenza virus, which composition enhances influenza virus-specific IgG production.

3. The mucosal vaccine composition according to claim 2, wherein the influenza virus is a whole particle or split antigen of the influenza virus.

Images