Peptide nucleic acid-based adjuvants

Abstract

Provided are: an adjuvant useful for the preparation of a vaccine having high efficacy and a high level of safety; and a vaccine composition containing the same.　This adjuvant consists of a peptide nucleic acid to which a cell-penetrating peptide is bound.

Description

[Technical Field]

[0001] The present invention relates to peptide nucleic acids modified with cell membrane-permeable peptides having immunopotentiating activity. [Background technology]

[0002] The adaptive immune system has the ability to form immunological memory that allows it to quickly respond to a foreign substance that it has encountered once, and vaccines utilize this immunological memory, allowing it to obtain immunity that can neutralize a pathogen before it is exposed to the pathogen.

[0003] Approved vaccines include live attenuated vaccines, inactivated vaccines, and subunit vaccines such as purified recombinant proteins. Live attenuated vaccines, in particular, are highly effective, but their infectivity makes them difficult to ensure safety and raises concerns about unexpected side effects. Inactivated and subunit vaccines, on the other hand, are safer than live attenuated vaccines. However, because they lack infectivity, they lack the ability to elicit natural immunity, and therefore antigens alone cannot confer sufficient acquired immunity. For this reason, many approved inactivated and subunit vaccines are formulated with antigens supplemented with immunostimulating adjuvants. Therefore, the development of safe vaccines should use safe inactivated and subunit vaccine antigens, but the addition of immunostimulating adjuvants is necessary to achieve sufficient efficacy.

[0004] Furthermore, to enhance the effectiveness of pathogen infection prevention, it is also important to induce immunity in the mucosa, which is the gateway for pathogen invasion. Mucosal immunity can be induced by administering a mucosal vaccine. However, currently approved mucosal vaccines are live attenuated vaccines or vaccines derived from toxins with mucosal affinity. Inactivated vaccines and subunit vaccines lacking mucosal affinity are difficult to induce sufficient immunity for infection prevention. Therefore, adjuvants that enhance mucosal immunity are required even when administering such antigens mucosally.

[0005] Adjuvants used in approved vaccines include aluminum salts and squalene-based emulsions, but there are few approved adjuvants, and aluminum salts are known to promote IgE induction (Non-Patent Document 1). Furthermore, the above adjuvants are for injectable use, making them difficult to combine with mucosal vaccines.

[0006] On the other hand, peptide nucleic acids (PNAs), which are modified nucleic acids in which the sugar phosphate backbone of nucleic acids has been converted to an N-(2-aminoethyl)glycine backbone, have high double-strand formation ability and are not degraded by in vivo nucleases, and are therefore expected to be used as antisense molecules, etc. Furthermore, in fields such as drug delivery, a technique for introducing proteins, peptides, or low molecular weight compounds into cells and enabling them to function is to bind these molecules to cell penetrating peptides (CPPs). However, it is not known that PNA or CPP-conjugated PNA can be used as an adjuvant. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Marichal T, Ohata K, Bedoret D, Mesnil C, Sabatel C, Kobiyama K, Lekeux P,Coban C, Akira S, Ishii KJ, Bureau F, Desmet CJ. DNA released from dying host cells mediates aluminum adjuvant activity. Nat Med. 2011 Jul 17;17(8):996-1002. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to providing an adjuvant useful for preparing a highly effective and safe vaccine, and a vaccine composition containing the same. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have modified a cell membrane-penetrating peptide (CPP) such as TAT ​​(Trans-Activator of Transcription Protein) of HIV (Human Immunodeficiency Virus) into a peptide nucleic acid and found that this CPP-bound peptide nucleic acid has adjuvant activity in both injection and transmucosal administration.

[0010] That is, the present invention relates to the following 1) to 6). 1) An adjuvant consisting of a peptide nucleic acid bound to a cell membrane-permeable peptide. 2) An adjuvant composition comprising a peptide nucleic acid bound to a cell membrane-permeable peptide and a pharmaceutically acceptable carrier. 3) A vaccine composition containing the adjuvant of 1) and an antigen. 4) Use of peptide nucleic acids bound to cell membrane-permeable peptides to produce adjuvants. 5) Use of peptide nucleic acids bound to cell membrane-permeable peptides as adjuvants. 6) A method in which a peptide nucleic acid bound to a cell membrane-permeable peptide is used as an adjuvant. [Effects of the Invention]

[0011] According to the present invention, it is possible to enhance the effectiveness of highly safe inactivated vaccines, and the invention can make a significant contribution to the pharmaceutical industry by creating high-quality prophylactic drugs. [Brief explanation of the drawings]

[0012] [Figure 1A] IgG antibody titer against the A / Singapore / GP1908 / 2015 strain after subcutaneous administration. [Figure 1B]IgG antibody titer against the B / Maryland / 15 / 2015 strain after subcutaneous administration. [Figure 2] Neutralizing antibody titers against the A / Singapore / GP1908 / 2015 strain on Day 35 after subcutaneous administration. [Figure 3A] IgG antibody titer against the A / Singapore / GP1908 / 2015 strain after nasal administration. [Figure 3B] IgG antibody titer against the B / Maryland / 15 / 2015 strain following nasal administration. [Figure 4A] IgA antibody titer against the A / Singapore / GP1908 / 2015 strain in nasal wash fluid after nasal administration. [Figure 4B] IgA antibody titers against the B / Maryland / 15 / 2015 strain in nasal washes after nasal administration. [Figure 5] NFκB activation effect of MIP03. [Figure 6] Cytokine (IL-1α) induction effect of MIP03. [Figure 7] Cytokine (IL-1β) induction effect of MIP03. [Figure 8] Cytokine (IL-13) induction effect of MIP03. [Figure 9] Cytokine (IFN-γ) induction effect of MIP03. DETAILED DESCRIPTION OF THE INVENTION

[0013] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments. In the present invention, "peptide nucleic acid bound to a cell membrane-permeable peptide" refers to a molecule in which a peptide nucleic acid is bound to the N- or C-terminus of a cell membrane-permeable peptide, and is also referred to herein as Mitsumata-Immunostimulatory Peptide nucleic acid (MIP).

[0014] "Cell membrane-penetrating peptides (CPPs)" refer to peptides that cross the cell membrane and enter cells when coexisting with cells. Examples of CPPs include, but are not limited to, peptides derived from HIV TAT, Penetratin derived from Drosophila antennapedia, polyarginine, Transportan derived from the neuropeptide galanin, Pep-1 derived from the SV40 Nuclear Localization Signal (NLS), and the antimicrobial peptide LL37, as shown in Table 1. A preferred example is a TAT-derived peptide (a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1).

[0015] [Table 1]

[0016] "Peptide nucleic acid (PNA)" is a nucleic acid analogue in which the sugar-phosphate backbone of nucleic acid is replaced with N-(2-aminoethyl)glycine, and bases are bound via methylenecarbonyl bonds. The bases in the peptide nucleic acid used in the present invention may be any of adenine, guanine, thymine, cytosine, and uracil, and the chain length is generally 20 mer or less, but from the viewpoint of adjuvant activity, it is preferably 3 to 12 mer, and more preferably 3 to 10 mer. The structure of a 3- to 12-mer peptide nucleic acid is shown in the following formula (1).

[0017] [ka]

[0018] [In the formula, Base may be the same or different and represents adenine, guanine, thymine, cytosine, or uracil, R represents —COOH or —CONH2, and n represents an integer of 1 to 10.]

[0019] In the present invention, suitable peptide nucleic acids include, for example, 3-mer or 10-mer peptide nucleic acids having at least one guanine or cytosine base, 3-mer peptide nucleic acids having three guanine bases (G3PNA), 3-mer peptide nucleic acids having three cytosine bases (C3PNA), and 10-mer peptide nucleic acids having ten cytosine bases (C10PNA).

[0020] The MIPs of the present invention can be produced by covalently bonding the N-terminus of a cell-penetrating peptide to the N- or C-terminus of the cell-penetrating peptide using a chemical method, for example, by using a crosslinker having N-hydroxysuccinimide esters at both ends to form a link between the cell-penetrating peptide and the N-terminus of a peptide nucleic acid. Peptide nucleic acids can be synthesized using, for example, Fmoc-type PNA monomer units and the like, by utilizing solid-phase peptide synthesis methods known in the art.

[0021] Suitable MIPs for use in the present invention include MIP01 (formula (2)), in which a 13-amino acid peptide derived from HIV TAT (SEQ ID NO: 1) shown below is covalently bound to G3PNA at its C-terminus, and MIP03 (formula (3)), in which a C10PNA is covalently bound to G3PNA at its C-terminus.

[0022] [ka]

[0023] As shown in the Examples below, when a vaccine composition prepared by mixing MIP01 represented by formula (2) with an influenza antigen (split antigen) was injected or administered transmucosally to mice, the antibody titer in the blood against the antigen was higher than that in a group not treated with MIP. Furthermore, when administered transmucosally, the mucosal IgA titer also increased. Furthermore, MIP03 represented by formula (3) was observed to have the ability to activate NFκB and induce IL-1β, which likely promotes activation of the innate immune system. Therefore, MIP is considered to be an adjuvant capable of activating innate immunity. Specifically, MIPs have adjuvant activity that enhances the antibody induction ability in the blood or mucosa when an antigen is injected or administered mucosally, and therefore MIPs can serve as adjuvants, and compositions containing MIPs and pharmaceutically acceptable carriers can serve as adjuvant compositions. Furthermore, MIPs can be used to produce adjuvants or adjuvant compositions.

[0024] In the present invention, the term "adjuvant" refers to a substance that enhances the immune response to an antigen when the antigen is injected or administered transmucosally. Here, "mucosa" refers to the inner walls of external cavity organs in vertebrates, such as the digestive system, respiratory system, urogenital system, eyes, etc. Therefore, examples of transmucosal administration include, but are not limited to, oral administration, nasal administration (intranasal administration), oral administration, intravaginal administration, upper respiratory tract administration, alveolar administration, and ophthalmic administration.

[0025] The adjuvant or adjuvant composition of the present invention can be administered in combination with an antigen, and may be administered simultaneously with, before, or after administration of the antigen. The dosage of the adjuvant or adjuvant composition of the present invention can be determined appropriately depending on the subject, administration method, administration form, and type of antigen.

[0026] The adjuvants of the present invention can be combined with antigens to form vaccine compositions. The vaccine compositions of the present invention can be prepared by mixing an antigen with an MIP, and a pharmaceutically acceptable carrier can be added as needed to form a suitable formulation. In the vaccine compositions of the present invention, the MIP may be chemically bound to the antigen or other components, or may exist in a free molecular state.

[0027] Antigens include natural products purified from pathogens, or proteins or peptides artificially produced by techniques such as genetic recombination. Specific examples include virions (complete virus particles), incomplete virus particles, viral structural proteins, viral nonstructural proteins, whole pathogen cells, proteins or glycoproteins derived from pathogens, protective antigens, and neutralizing epitopes. These antigens include both infectious and non-infectious antigens (inactivated antigens). Inactivated antigens include, but are not limited to, antigens inactivated by physical (X-ray irradiation, UV irradiation, heat, ultrasound) or chemical (formalin, beta-propiolactone, binary ethyleneimine, mercury, alcohol, chlorine) procedures. From the perspective of safety, pathogen-derived antigens are preferably inactivated antigens derived from the viruses or pathogens listed above.

[0028] Examples of viruses include measles virus, rubella virus, mumps virus, poliovirus, rotavirus, norovirus, adenovirus, enterovirus, herpes virus, varicella virus, severe acute respiratory syndrome (SARS) virus, human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV-1), human papillomavirus, Japanese encephalitis virus, West Nile virus, yellow fever virus, dengue virus, Zika virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, respiratory syncytial virus, and influenza virus, with influenza virus being preferred.

[0029] Pathogenic bacteria include Corynebacterium diphtheriae, Corynebacterium tetani, Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Streptococcus pneumoniae, Vibrio cholerae, Mycobacterium tuberculosis, and periodontal disease bacteria.

[0030] Examples of dosage forms of the vaccine composition include liquids, suspensions, freeze-dried preparations, etc. Liquids include those dissolved in purified water or buffer solutions, etc. Suspensions include those suspended in purified water or buffer solutions together with methylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, gelatin, casein, etc. For these formulations, commonly used absorption enhancers, surfactants, preservatives, stabilizers, anti-humectants, solubilizing agents, etc. can be added as necessary.

[0031] In addition, the vaccine composition of the present invention can also contain adjuvants other than MIP, as long as the immunogenicity and safety of the vaccine are not impaired.

[0032] The amount of antigen contained in the vaccine composition of the present invention is not particularly limited as long as it is sufficient to induce a specific antibody response, and can be appropriately set in consideration of the ratio with the combined MIP. For example, when using a split antigen of influenza virus as the antigen, it may be contained within the range of 1 to 60 μg HA (in terms of HA) per single-dose administration, and 9 to 15 μg HA (in terms of HA) is more preferable. The above concentration is a value obtained by measuring the concentration of HA protein by a test method defined by standards of the WHO or a country, such as the single-radial immunodiffusion test method.

[0033] The administration route of the vaccine composition of the present invention is not particularly limited, and can be any of administration by injection (subcutaneous administration, intramuscular administration, intradermal administration, intravenous administration), oral administration, parenteral administration (for example, nasal administration, eye drop administration, vaginal administration, sublingual administration, transdermal administration). For example, it is administered by dropping, spraying or spraying into the nasal cavity.

[0034] The administration targets of the adjuvant composition or vaccine composition of the present invention include humans and mammals other than humans, but humans are preferred. Examples of mammals other than humans include mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, sheep, dogs, cats, rhesus monkeys, cynomolgus monkeys, orangutans, chimpanzees, etc.

Examples

[0035] Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto. <CPP and MIP> The synthesis of CPP01 (TAT-derived peptide (SEQ ID NO: 1)) as a CPP, and MIP01 shown in formula (2) and MIP03 shown in formula (3) as MIPs was entrusted to Peptide Institute, Inc.

[0036] Example 1 Evaluation of adjuvant activity of subcutaneously administered MIP01 The stock solutions of the influenza HA vaccine "Seiken" A / H1N1 subtype (A / Singapore / GP1908 / 2015 strain) and B / Victoria lineage (B / Maryland / 15 / 2016 strain) were used as split antigens (SVs). Each split antigen was mixed so that 1 μg of hemagglutinin from each strain was obtained per 0.3 mL of the vaccine. To this was added 1 μg of a cell membrane-penetrating peptide consisting of 13 amino acids derived from HIV TAT or MIP01. A control solution containing only the antigen without adjuvant was also prepared.

[0037] The dosing solution (Table 2) prepared as described above was administered twice, 3 weeks apart, to BALB / c mice (female, 5 weeks old) subcutaneously in the dorsal region at 0.3 mL (five mice per group). Two weeks after each administration, exsanguination was performed. The collected blood was centrifuged to prepare serum, and the serum IgG (total IgG) titers that specifically bind to the A / Singapore / GP1908 / 2015 and B / Maryland / 15 / 2016 strains were measured. The serum after the second administration was also measured for neutralizing antibody titers against the A / Singapore / GP1908 / 2015 strain.

[0038] [Table 2]

[0039] The IgG titers for each administration group are shown in Figures 1A and 1B. Adding 1 μg of MIP01 per dose to the split antigen resulted in higher antigen-specific IgG titers in the blood against each strain after both the first and second administrations compared with the non-adjuvant group. Furthermore, the MIP01-administered group also demonstrated higher antigen-specific IgG titers against each strain after both the first and second administrations compared with the CPP01-administered group. Figure 2 shows the neutralizing antibody titers against the A / Singapore / GP1908 / 2015 strain after two administrations. The MIP01-administered group also demonstrated the highest neutralizing antibody titers, followed by the non-adjuvant group and the CPP01-administered group, with the lowest. Therefore, it was confirmed that administration of MIP01 not only increases antigen-specific IgG titers in the blood, but also neutralizing antibody titers, which are important for eliminating viruses, and it was suggested that the structure in which peptide nucleic acids are linked to cell membrane-permeable peptides is important for the adjuvant activity of MIP01.

[0040] Example 2 Evaluation of the adjuvant activity of MIP01 upon nasal administration As in Example 1, stock solutions of the influenza HA vaccine "Seiken" A / H1N1 subtype (A / Singapore / GP1908 / 2015 strain) and B / Victoria lineage (B / Maryland / 15 / 2016 strain) were used as split antigens, and the split antigens were mixed so that each strain's hemagglutinin was 1 μg per 10 μL, to which CPP01 or MIP01 was added so that each was 10 μg. A control solution containing only the antigen without added adjuvant was also prepared.

[0041] The dosing solution prepared as described above (Table 3) was administered twice, 3 weeks apart, to BALB / c mice (female, 5 weeks old) in 10 μL (5 μL per nostril) nasally (five mice per group). Two weeks after each administration, whole blood was collected and centrifuged to prepare serum. Two weeks after the second administration, blood was also collected and the nasal cavities were lavaged with 400 μL of D-PBS containing protease inhibitors per mouse. This lavage fluid was collected as nasal wash. Serum was used to measure IgG (total IgG) titers specifically binding to the A / Singapore / GP1908 / 2015 and B / Maryland / 15 / 2016 strains, and nasal washes were used to measure IgA titers specifically binding to the A / Singapore / GP1908 / 2015 and B / Maryland / 15 / 2016 strains.

[0042] [Table 3]

[0043] The IgG titer results for each strain are shown in Figures 3A and 3B. After both intranasal administration and one or two doses, the antigen-specific IgG titers in the blood against each strain were higher than those in the non-adjuvanted group. In particular, the IgG titers against the B / Maryland / 15 / 2016 strain were significantly higher than those in the non-adjuvanted group (Mann-Whitney U test, p<0.05). Furthermore, the antigen-specific IgA titers in the nasal washes against each strain were highest in the MIP01-treated group at both time points, followed by the non-adjuvanted group and lowest in the CPP01-treated group (Figures 4A and 4B). Therefore, MIPs, in which cell membrane-penetrating peptides are modified with peptide nucleic acids, have adjuvant activity not only when administered by injection but also when administered mucosally, suggesting that mucosal administration promotes not only IgG in the blood but also IgA induction in the mucosa.

[0044] Example 3: Innate immune activation ability of MIP03 The innate immune activation ability of MIP03 was evaluated in vitro.

[0045] 1×10 6 MIP03 was added to cells / mL of the Raw264.7 Reporter Cell Line (Novus biologicals, NBP2-26261) at final concentrations of 10, 20, 40, and 80 μM. Cultures were incubated at 37°C in 5% CO2 for 2 days, and the culture medium was harvested. The harvested culture medium was centrifuged at 12,000 × g for 1 minute, and the supernatant was assayed for alkaline phosphatase and cytokine levels. The SEAP Reporter Gene Assay kit Luminescence (Cayman Chemical, 600260) was used for alkaline phosphatase assays, and Bio-Plex Pro Mouse Cytokine GI23-Plex (Bio-Rad, M60009RDPDB03) was used for cytokine assays. Medium was used as a negative control (NC), and imiquimod (0.02 μM), a Toll-like receptor 7 agonist, was used as a positive control for cytokine assays.

[0046] The alkaline phosphatase assay results are shown in Figure 5, and demonstrate an increase in the alkaline phosphatase luminescence signal dependent on the addition of MIP03. Therefore, MIP03 was found to promote NFκB activation, suggesting its activation of innate immunity. Furthermore, cytokine assay results are shown in Figures 6 to 9, demonstrating that MIP03 concentration-dependently enhanced IL-1α and IL-1β induction (Figures 6 and 7). IL-1β, in particular, was induced significantly more significantly than imiquimod (Figure 7). Furthermore, the results of measurements of Th2- and Th1-associated cytokines, IL-13 (Figure 8) and IFN-γ (Figure 9), demonstrate comparable induction of both cytokines to that observed with the positive control, imiquimod. As mentioned above, MIP03 is highly capable of activating NFκB and inducing IL-1β, and therefore likely promotes activation of the innate immune system via C-type lectin receptors and Nod-like receptors, which may result in the induction of Th1 / Th2 cytokines related to antibody responses. Therefore, MIP is an adjuvant capable of activating innate immunity.

Claims

1. An adjuvant comprising a peptide nucleic acid bound to a cell membrane-permeable peptide, the cell membrane-permeable peptide being a peptide having an amino acid sequence represented by SEQ ID NO: 1, and the peptide nucleic acid being a peptide represented by the following formula (1): [Formula 1] (In the formula, Base may be the same or different and represents guanine or cytosine, R represents --COOH or --CONH 2 , and n represents an integer of 1 to 10.) Represented by, adjuvant.

2. The adjuvant according to claim 1, wherein in formula (1), n ​​is an integer of 1 to 8.

3. In formula (1), R is -CONH ２ The adjuvant according to claim 1 or 2,

4. The adjuvant according to any one of claims 1 to 3, wherein in formula (1), each Base is guanine or cytosine.

5. The adjuvant described in any one of claims 1 to 4, wherein the peptide nucleic acid is a 3mer peptide nucleic acid having three guanines as bases or a 10mer peptide nucleic acid having ten cytosines as bases.

6. An adjuvant composition comprising a peptide nucleic acid bound to a cell membrane-permeable peptide and a pharma- ceutically acceptable carrier, wherein the cell membrane-permeable peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and the peptide nucleic acid is a peptide having the following formula (1): [chemical 2] (In the formula, Base may be the same or different and represents guanine or cytosine, R represents --COOH or --CONH 2 , and n represents an integer of 1 to 10.) The composition represented by:

7. A vaccine composition comprising the adjuvant according to any one of claims 1 to 5 and an antigen.

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