Adjuvant Composition

Abstract

The present invention pertains to an adjuvant composition that contains a vitamin D compound and that is not used in combination with an aluminum salt.

Description

[Technical Field]

[0001] The present invention relates to adjuvant compositions. [Background technology]

[0002] Vaccines are one of the greatest inventions in preventive medicine in the history of human health. The incidence and death rates of the top ten vaccine-preventable diseases, such as measles, mumps, whooping cough, poliomyelitis, rubella, smallpox, and tetanus, decreased by more than 92% and 98% in the United States before 1980 due to vaccine recommendations. The incidence and death rates of other infectious diseases, hepatitis A, acute hepatitis B, Haemophilus influenzae type b (HPV), and chickenpox, decreased by more than 87% and 81% in the United States between 1980 and 2005 due to vaccine recommendations. Adjuvants enhance immune responses and reduce antigen burden. Aluminum salts (Alum) are the only adjuvants approved for human use for 80 years.

[0003] Today, Alum remains the primary adjuvant used in many vaccines, including those for diphtheria, pertussis, tetanus, HPV, poliomyelitis, and acute hepatitis B. Although this long-lasting adjuvant has shown excellent results in vaccine efficacy, it does have some side effects: generalized joint pain, fatigue, fever, headache, GI and muscle pain, and localized pain, redness, and swelling.

[0004] Public concern about side effects from vaccinations has increased recently. Specifically, although HPV vaccination could theoretically prevent more than 500,000 cases of cervical, anal, vaginal, penile, or oropharyngeal cancers per year worldwide, parents of teenage children who have received the HPV vaccine frequently cite side effects, leading to a decline in vaccination uptake and hindering the public introduction of new HPV vaccines. These side effects are primarily due to inflammation caused by alum. The challenge is that an enhanced immune response is positively correlated with and inseparable from adverse side effects. Alum and new adjuvants under investigation cause inflammation and the aforementioned side effects. Reputational damage related to HPV vaccination refusal has led to a resurgence of measles, for example. Given the growing public concern about adjuvant side effects, there is an increasing need to understand how to minimize local and systemic side effects while maintaining vaccine efficacy.

[0005] Vitamin D3 is a biomolecule that plays an important role in calcium and phosphate homeostasis, and analogues such as oxalol are used to treat psoriasis.

[0006] Patent Document 1 describes that administration of 1,25(OH)2D3 after vaccination with an aluminum hydroxide adjuvant (Alum) enhances antibody responses, but does not describe or suggest the use of 1,25(OH)2D3 alone as an adjuvant in place of Alum. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO94 / 17823 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides novel adjuvant compositions. [Means for solving the problem]

[0009] The present inventors have demonstrated that vitamin D compounds have an adjuvant effect, i.e., they act by inhibiting epidermal CD11c + The present inventors have found for the first time that germinal center B cells, memory B cells, and TFH cells can be increased by targeting these cells, and have completed the present invention. [1] An adjuvant composition comprising a vitamin D compound, wherein the composition is not used in combination with an aluminum salt; [2] The composition according to [1], wherein the vitamin D compound is active vitamin D3, active vitamin D2, or a derivative thereof. [3] The composition of [1], wherein the vitamin D compound is maxacalcitol. [4] The composition of [1], which is for intradermal or nasal administration. [5] A vitamin D compound for use as an adjuvant, wherein the compound is not in combination with an aluminum salt. [6] The compound according to [5], wherein the vitamin D compound is active vitamin D3, active vitamin D2, or a derivative thereof. [7] The compound of [5], wherein the vitamin D compound is maxacalcitol. [8] the compound of [5], which is for transdermal administration; [9] A method for stimulating an immune response in a subject, comprising administering a vitamin D compound to a subject who has not received an aluminum salt as an adjuvant.

[10] The method according to [9], wherein the vitamin D compound is active vitamin D3, active vitamin D2, or a derivative thereof.

[11] The method of [9], wherein the vitamin D compound is maxacalcitol.

[12] The method of [9], wherein the vitamin D compound is administered transdermally or intranasally.

[13] A vaccine composition comprising maxacalcitol as an adjuvant, and not comprising maxacalcitol as an adjuvant.

[14] The composition,

[13] wherein the vaccine is an influenza vaccine. [Effects of the Invention]

[0010] The adjuvant compositions of the present invention can enhance the immunogenicity of vaccines. [Brief explanation of the drawings]

[0011] [Figure 1] Transdermal application of oxarol protects against death due to virus challenge. After vaccination under different conditions, mice vaccinated with the H1N1 A / California / 04 / 2009 strain were infected with the same virus. Survival rates are shown. There were five mice per group. A representative example of the results of two independent experiments is shown. [Figure 2] Transdermal application of oxarol protects against infectious diseases. After vaccination under different conditions, mice vaccinated with the H1N1 A / California / 04 / 2009 strain were challenged with the same virus. Weight changes were shown. There were five mice per group. A representative example of the results of two independent experiments is shown. [Figure 3] Transdermal application of oxalorenhanced recall humoral responses. One month after vaccination with influenza vaccine antigen without adjuvant, or with Alum or oxaloren, the influenza vaccine antigen was injected intraperitoneally without adjuvant for booster. Ten days after the booster, serum was collected and subjected to ELISA to determine serum levels of anti-NP IgG1. The number of animals was five per group. A representative example of the results of two independent experiments is shown. [Figure 4] Antigen-specific memory B cells expand after oxalol-adjuvanted immunization. After hapten immunization under different conditions, the number of hapten-positive IgG1+CD38+GL7- B cells in the auricular lymph nodes was determined by total lymphocyte count and flow cytometry analysis in the auricular lymph nodes. [Figure 5]Follicular T cells expand after oxalol-adjuvanted immunization. After hapten immunization under different conditions, the number of CD4+CXCR5+PD-1+FoxP- T cells in auricular lymph nodes was determined by total lymphocyte count and flow cytometry analysis in auricular lymph nodes. [Figure 6] Antigen-specific GCB cells expanded after oxalol adjuvant immunization. After hapten immunization under different conditions, total lymphocytes in the auricular lymph nodes were counted and the number of hapten-positive CD38-GL7+ B cells in the auricular lymph nodes was determined by flow cytometry analysis. [Figure 7] Requirement of Vdr expression in keratinocytes for germinal center B cell proliferation. hLangerin-Cre, CD11c-Cre, K5-Cre-driven conditional Vdr knockout mice or Cre- littermates were immunized in the ear skin with NP-CGG / Oxarol. The total number of NIP+CD38-GL7+B220+ cells in the auricular lymph nodes is shown. [Figure 8] Reduced serum anti-NP antibody titers in K5-Vdr cKO mice. K5-Vdr cKO mice (n = 9) or Cre- littermates (n = 13) were vaccinated and boosted with NP-CGG without oxalol adjuvant 1 month postvaccination. Anti-NP IgG1 titers were measured by ELISA at the indicated time points. NP30 and NP0.43 as hapten antigens were used to detect low- and high-affinity anti-NP antibodies, respectively. [Figure 9] Cyp24a1 induction by oxalol adjuvant. Ear skin samples were collected from naive mice (n = 3), PBS-injected mice (n = 3), oxalol-injected wild-type (n = 3), and K5-VDR mice (n = 3) on day 1. Cyp24a1 expression levels were expressed as FKPM.

[0012] In the present invention, the term "vitamin D compound" refers to a type of vitamin, a fat-soluble steroid derivative secosteroid, which includes natural vitamin D2 and natural vitamin D3 with different side chain structures, as well as synthetic vitamin analogs that can be converted into activated vitamins. The vitamin D compound of the present invention is preferably activated vitamin D3, activated vitamin D2, or a derivative thereof, and more preferably oxalol.

[0013] In the present invention, an adjuvant refers to a substance used to enhance the immunogenicity of a vaccine, more preferably a substance injected together with a vaccine to enhance its immunogenicity.

[0014] The adjuvant composition of the present invention can be administered using any conventionally known administration system, preferably intradermal or nasal administration.

[0015] In the present invention, the aluminum salt refers to a mixture containing trivalent aluminum salts such as AlOOH, Al(OH)3, AlPO4, and AlK(SO4)2·12H2O. The aluminum salt of the present invention is preferably an aluminum salt containing Al(OH)3, and more preferably Imject® Alum.

[0016] The compositions of the present invention may be formulated by further comprising a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" has the meaning commonly used in the pharmaceutical industry, and in some cases refers to the use of a molecular entity or composition that does not cause an allergic or similar adverse reaction when administered to humans. The preparation of an aqueous composition containing a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injections, either as liquid solutions or suspensions, and solid dosage forms suitable for dissolution or suspension in liquid prior to injection can also be prepared. The preparations can also be emulsified.

[0017] "Carriers" can include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. Carriers can include buffers of phosphate, citrate, and other organic acid salts; antioxidants including ascorbic acid; low molecular weight (fewer than about 10 amino acid residues) polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophobic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants (e.g., polyoxyalkylenes).

[0018] The composition of the present invention may contain various surfactants used in formulations. The type of surfactant is not particularly limited, and examples thereof include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. [Example]

[0019] The effects of the present invention are illustrated in the following examples.

[0020] antigen The influenza H1N1 A / California / 04 / 2009 split vaccine was a gift from the Research Foundation for Microbial Diseases of Osaka University (BIKEN). NP-CGG was produced by conjugating 4-hydroxy-3-nitrophenylacetic acid active ester (LGC Biosearch Technology) to chicken gamma globulin (Rockland).

[0021] Adjuvants Oxarol ointment and Imject® Alum adjuvant were purchased from Chugai Pharmaceutical and ThermoFisher Scientific, respectively. Ointment containing 2.5 μg of maxacalcitol or a solution containing 2.5 μg of aluminum hydroxide was administered to the ears of mice.

[0022] immunization For oxalor adjuvant immunizations, 7.5 ng of H1N1 A / California / 04 / 2009 split vaccine antigen or 1 μg of NP-CGG antigen in 10–25 μL was injected intradermally into the ear skin, followed by administration of 100 μg of oxalor ointment (2.5 μg of maxacalcitol as the active ingredient) over the injection site. For antigen alone, 10–25 μL of a solution containing the above amount of antigen was injected intradermally into the ear skin. For alum adjuvant immunizations, 10–25 μL of a solution containing the above amount of antigen and alum equivalent to 2.5 μg of aluminum hydroxide was injected intradermally into the ear skin.

[0023] Influenza infection research 50 μL (2 × LD 50 ) influenza H1N1 A / California / 04 / 2009 virus solution was administered intranasally to mice under anesthesia, and then body weight and survival rate were monitored for 14 days.

[0024] Flow cytometry Single-cell suspensions of ear lymphocytes were incubated with Mouse BD Fc Block® anti-CD16 / CD32 (2.4G2, from BD), followed by immunoblotting with B220 (clone RA3-6B2, APC-Cy7, from BioLegend), GL7 (GL7, FITC and PE, from BD), CD38 (clone 90, PE-Cy7, from BioLegend), IgG1 (clone X56, BV510, from BD), CD4-FITC (clone RM4-5, from BD), CXCR5-BV421 (clone L138D7, from BioLegend), PD-1-PE (clone J43, from eBioscience), PD-1-PerCP-eFluor 710 (clone RMP1-30, from eBioscience), and Foxp3-APC (clone FJK-16s, from eBioscience). The Foxp3 intracellular staining kit (catalog 88-8824-00) was purchased from eBioscience. Various antibodies, including NIP (4-hydroxy-3-iodo-5-nitrophenylacetic acid)-APC (self-made), were used for staining. Dead cells were excluded by staining with 7-aminoactinomycin D (7-AAD). Stained cells were analyzed using a FACSCanto® II (BD Bioscience). Data were analyzed using FlowJo® software (FlowJo, LLC).

[0025] Enzyme-linked immunosorbent assay (ELISA) A 96-well ELISA plate (Thermo Scientific) was coated with an antigen solution in PBS at a concentration of 1 μg / mL for the H1N1 A / California / 04 / 2009 split vaccine. After antigen binding, nonspecific binding was blocked using BlockingOne (Nacalai Tesque). Serially diluted serum samples and standard sera were then added, followed by a goat anti-mouse IgG1 antibody conjugated with HRP (SouthernBiotech). The antibody titer of each serum sample was calculated based on the standard curve.

[0026] statistics Two-tailed Student's t-test and one-way analysis of variance were used for statistical analysis using GraphPadPrism software.

[0027] mouse C57BL / 6JJcl mice were purchased from CLEA Japan, Inc. Vdr-floxed mice, hLangerin-Cre BAC transgenic mice, K5-Cre transgenic mice, and B6.Cg-Tg(Itgax-Cre)1-1Reiz / J(Cd11c-Cre) transgenic mice were used in this study. All animal experiments were performed using protocols approved by the Institutional Animal Care at RIKEN IMS.

[0028] RNA-sequencing RNA was extracted from mouse ear skin using TRIzol™ reagent. cDNA libraries were generated using the NEBNext Ultra RNA Library Preparation Kit for Illumina (NEB). Sequencing was performed on a HiSeq2500 sequencer (Illumina) in 50-bp single-end read mode. Sequencing reads were trimmed for adapter sequences, masked for low-complexity or low-quality sequences, and mapped to the reference genome (mm9) using Bowtie2 v2.1.0 and TopHat2 v2.0.8. Transcript abundance was estimated as FPKM (fragments per kilobase of mapped exon million) values ​​using Cufflinks v2.1.1.

[0029] result Intradermal injection of influenza virus vaccine antigen followed by topical application of Oxarol ointment as an adjuvant increased survival rates compared with those receiving antigen alone (Figure 1). Body weight loss associated with infection was observed in all treatment groups up to day 7, as shown in Figure 2. However, in the Oxarol ointment-treated group, the weight loss reversed to an increase from day 8 onward. The humoral immune memory response was examined one month after vaccination. To mimic the infection situation, antigen alone was administered one month after vaccination. Following the second booster immunization with antigen alone, serum IgG1 antibody titers against the vaccine antigen were significantly higher in the Oxarol-treated group than in the Alum-vaccinated group (Figure 3). Furthermore, to investigate whether this humoral immune memory response was due to the number of memory B cells, we examined the number of antigen-specific IgG1 memory B cells produced in the auricular lymph node closest to the vaccine injection site (Figure 4). The results showed that the number of antigen-specific IgG1 memory B cells was significantly higher in the oxalor-treated group. We also examined germinal center B cells, antibody-producing cells, and TFH cells, which play an important role in the generation of memory B cells. The results showed that the proportions of these cells were significantly higher in the oxalor-treated group (Figure 5). Furthermore, we examined the number of antigen-specific germinal center B cells. The results showed a significant increase in the oxalor-treated group (Figure 6). It is noteworthy that oxalor-treated mice did not exhibit skin tissue hardening or rashes at the injection site, which are typically seen with alum administration. Further findings were obtained: Vitamin D3 targeted Vdr in keratinocytes (Figure 7); keratinocyte-specific Vdr deletion reduced humoral responses (Figure 8); and oxalol adjuvant induced Cyp24a1, a known target gene of Vdr (Figure 8). [Industrial Applicability]

[0030] The adjuvant compositions of the present invention are useful for enhancing the immunogenicity of vaccines.

Claims

1. An adjuvant composition comprising maxacalcitol, wherein the adjuvant composition is not used in combination with an aluminum salt.

2. The adjuvant composition according to claim 1, which is used for transdermal or intranasal administration.

3. 3. The adjuvant composition according to claim 1 or 2, which is used to stimulate an immune response in a subject that has not been administered an aluminum salt as an adjuvant.

4. A vaccine composition comprising maxacalcitol as an adjuvant and not comprising an aluminum salt as an adjuvant.

5. The vaccine composition of claim 4, wherein the vaccine is an influenza vaccine.

6. 6. The vaccine composition according to claim 4 or 5, for transdermal or nasal administration.

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