Adjuvant and transmucosal vaccine

Abstract

The present invention provides an adjuvant and a transmucosal vaccine that are highly capable of inducing secretory IgA in addition to serum IgA and IgG when transmucosally administered.　The invention relates to the adjuvant that comprises, as an active ingredient, one or more polypeptides selected from: (1) a polypeptide having at least one or two amino acid sequences represented by SEQ ID NO: 1; and (2) a polypeptide having 93% or more identity with the aforesaid amino acid sequence.

Description

Adjuvants and mucosal vaccines

[0001] The present invention relates to adjuvants and mucosal vaccines.

[0002] Because the novel coronavirus (SARS-CoV-2) and influenza viruses typically invade through the upper respiratory tract, mucosal vaccines can not only induce IgG in serum but also secretory IgA in the mucosa of the upper respiratory tract, including the nasal cavity, thereby inhibiting viral invasion itself. On the other hand, vaccines administered via intramuscular injection, etc., produce IgG in serum, which then protects against the virus via the blood. Therefore, while they are highly effective in preventing the onset of severe illness, they are said to have a weak ability to protect against viral infection itself. Side effects from intramuscular injections are also a problem. From this perspective, the development of mucosal vaccines, such as nasal vaccines, has been desired, and a live attenuated influenza vaccine (trade name FluMist) that is sprayed into the nasal cavity has been developed (Non-Patent Document 1). Mucosal vaccines for SARS-CoV-2 are also being developed, but none have yet been commercialized.

[0003] http: / / www.slclinic.com / aboutus / ichikawaclinic / flumist / Vaccine, 30 Sep 2013, 31(48): 5729-5735

[0004] Aluminum salts, adjuvant MF59, etc. are used as adjuvants for injectable vaccines, but although there have been reports of research into adjuvants for mucosal vaccines, none have been put to practical use. The object of the present invention is to provide an adjuvant and mucosal vaccine that have a high ability to induce secretory IgA in addition to serum IgA and IgG upon mucosal administration.

[0005] The present inventors have found that intranasal inoculation of a peptide (MP3 (100 kDa)) derived from the PnxIIIA protein of the rodent opportunistic bacterium Pasteurella pneumoniae, which has been stripped of its cytotoxicity, induces antibodies (IgA and IgG) against MP3 alone, without the need for an adjuvant (Non-Patent Document 2). However, MP3 is a large polypeptide with a molecular weight of 100 kDa, and because it functions as an antigen itself, it cannot be used as an adjuvant. Therefore, the present inventors removed additional sites predicted to be cytotoxic and immunogenic from MP3 and redesigned it to be more easily expressed in Escherichia coli. They found polypeptides containing at least one or two specific 61 amino acid residues. Furthermore, when these polypeptides were conjugated to antigens such as viral antigens and administered transmucosally, high IgG and IgA antibody titers were obtained against the conjugated antigens, rather than antibodies against MP3. Furthermore, they found that these polypeptides had extremely low cytotoxicity, leading to the completion of the present invention.

[0006] That is, the present invention provides the following inventions [1] to

[15] . [1] An adjuvant comprising, as an active ingredient, one or more polypeptides selected from (1) a polypeptide having at least one or two amino acid sequences set forth in SEQ ID NO: 1 and (2) polypeptides having 93% or more identity to said amino acid sequence. [2] An adjuvant comprising, as an active ingredient, one or more polypeptides selected from (1) a polypeptide having at least one or two amino acid sequences set forth in SEQ ID NO: 1 and (2) polypeptides having 95% or more identity to said amino acid sequence. [3] The adjuvant according to [1] or [2], wherein the amino acid sequence set forth in SEQ ID NO: 1 is the amino acid sequence set forth in SEQ ID NO: 2 or 3. [4] An adjuvant comprising, as an active ingredient, a polypeptide having at least one or two amino acid sequences set forth in SEQ ID NO: 2 or 3. [5] The adjuvant according to [4], wherein the polypeptide having at least one amino acid sequence set forth in SEQ ID NO: 2 is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4. [6] The adjuvant according to [5], wherein the polypeptide having the amino acid sequence set forth in SEQ ID NO: 4 is a polypeptide having an amino acid sequence selected from SEQ ID NOs: 5 to 9. [7] The adjuvant according to [3], wherein the polypeptide having at least two copies of the amino acid sequence set forth in SEQ ID NO: 1 is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10. [8] The adjuvant according to [7], wherein the polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 is a polypeptide having an amino acid sequence selected from SEQ ID NOs: 11 to 15. [9] A polypeptide for producing an antibody against an antigen, the active ingredient of which is a conjugate of the adjuvant according to any of [1] to [8] above with the antigen.

[10] A mucosal vaccine, the active ingredient of which is a conjugate of the adjuvant according to any of [1] to [8] above with a pathogen or a substance produced by a pathogen, or a part thereof.

[11] The mucosal vaccine according to

[10] , wherein the pathogen or a substance produced by a pathogen, or a part thereof, is a pathogen or a substance produced by a pathogen, or a part thereof, selected from influenza virus, coronavirus, human papillomavirus, measles virus, tetanus toxin, and pertussis toxin.

[12] Use of a conjugate of the adjuvant according to any of [1] to [8] above with a pathogen or a substance produced by a pathogen, or a portion thereof, for producing a mucosal vaccine.

[13] A conjugate of the adjuvant according to any of [1] to [8] above with a pathogen or a substance produced by a pathogen, or a portion thereof, for use in mucosal vaccine therapy.

[14] Mucosal vaccine therapy, characterized by transmucosally administering a conjugate of the adjuvant according to any of [1] to [5] above with a pathogen or a substance produced by a pathogen, or a portion thereof.

[15] An antigen for producing a mucosal vaccine against coronavirus or SARS-CoV-2, consisting of a polypeptide (OC43ag) having the amino acid sequence shown in SEQ ID NO: 16 or a polypeptide having an amino acid sequence having 95% or more identity to said amino acid sequence.

[0007] Mucosal administration of a conjugate of the adjuvant and an antigen of the present invention not only induces high antibody titers of IgA and IgG in serum, but also induces high antibody titers of secretory IgA against the antigen in the mucosa, thereby enabling the adjuvant of the present invention to produce specific antibodies against polypeptides with low antigenicity. Mucosal administration of a conjugate of the adjuvant of the present invention and a pathogen, or a substance produced by a pathogen, or a part thereof, can induce high antibody titers of secretory IgA in the mucosa, thereby preventing invasion of pathogens containing the antigen through the mucosa and also inducing high antibody titers of IgA and IgG in serum, thereby preventing infectious diseases caused by pathogens or substances produced by pathogens that have entered the body. Furthermore, the mucosal vaccine of the present invention, when administered mucosally, such as via nasal administration, can induce high antibody titers of IgA and IgG in serum, as well as high antibody titers of secretory IgA in the mucosa, thereby reducing side effects of attenuated live vaccines and nucleic acid vaccines and pain during injectable vaccination.

[0008] 1 is a diagram showing the flow of the adjuvant of the present invention (NAIS291-1) and a portion of the spike protein of human coronavirus OC43 (OC43 vaccine antigen: OC43ag).

[0034] FIG. 1 is a diagram showing anti-HCoV-OC43 spike protein serum antibody titers when the mucosal vaccine of the present invention is administered intranasally.

[0035] FIG. 2 is a diagram showing the cytotoxicity of the adjuvant of the present invention (NAIS291-1) against L929 cells.

[0036] FIG. 3 is a diagram showing anti-thioredoxin antibody titers and anti-NAIS antibody titers when a conjugate of the adjuvant of the present invention (NAIS61-2, NAIS230-1, NAIS291-1) with thioredoxin (antigen) is administered intranasally.

[0037] FIG. 4 is a diagram showing anti-HA antibody titers when a conjugate of the adjuvant of the present invention (NAIS61-2, NAIS230-1) with hemagglutinin (HA) is administered intranasally.

[0009] Unless otherwise specified, the terms used herein are used in the sense commonly used in the relevant field. The present invention is described in more detail below. "Adjuvant" refers to an auxiliary agent for a main agent. In the field of immunology, it is also called an antigenicity enhancer and refers to a substance injected together with an antigen to enhance its antigenicity. In the field of preventive medicine, it refers to a substance used in combination with a vaccine to enhance its effectiveness. In the present invention, adjuvant has the meanings of both the fields of immunology and preventive medicine. "Antigen" refers to a substance that invades the body, induces antibody production, and binds and reacts only with the antibody. Examples of this include bacterial toxins, bacterial components, viruses, and many heterologous proteins. "Pathogen" refers to organisms such as protozoa, bacteria, and viruses that parasitize the body and cause disease, particularly those with pathogenicity. These pathogens can be considered a type of antigen because they have antigenicity. Substances produced by pathogens include bacterial toxins. Examples of pathogenic components include bacterial components, viral membrane proteins, viral spike proteins, and parts of these. "Antibodies" generally refer to proteins produced in the body in response to an antigen and specifically react with that antigen. Specifically, they are glycoproteins produced by B cells, a type of lymphocyte, a subtype of white blood cell, and are also called immunoglobulins. Immunoglobulins include IgG, IgE, IgA, IgM, and IgD. "Vaccines" are pharmaceuticals used to acquire immunity against infectious diseases and prevent them. Specifically, they stimulate the production of antibodies against pathogens in the body by administering detoxified or attenuated antigens themselves, chemically synthesized mRNA or DNA gene sequences designed based on pathogens (gene vaccines), or proteins mass-expressed using genetic engineering (recombinant vaccines). "Mucous membranes" refer to the soft tissues lining hollow organs such as the digestive, respiratory, and genitourinary tracts. Therefore, transmucosal administration refers to administration directly to these mucous membranes.Mucous membranes are present in the nasal cavity and oral cavity, and in the present invention, direct administration to these mucous membranes is preferred. Amino acid sequence identity refers to the percentage (%) of the number of positions at which identical amino acid residues exist in two amino acid sequences when the two sequences are aligned, relative to the total number of amino acid residues in both sequences. Specifically, it can be calculated, for example, by the Lipman-Pearson method (Science, 227, 1435, (1985)), and can be calculated by performing analysis using the Search homology program in the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with a unit size to compare (ktup) of 2.

[0010] One embodiment of the adjuvant of the present invention is an adjuvant containing as an active ingredient one or more polypeptides selected from (1) a polypeptide having at least one or two amino acid sequences set forth in SEQ ID NO: 1 and (2) a polypeptide having 93% or more identity to said amino acid sequence. The amino acid sequence set forth in SEQ ID NO: 1 is shown in Table 1.

[0011] In the above amino acid sequence, X １ represents S or Q, and X ２ represents I or V, and X ３ represents G or S, and X ４ represents A or K, and X represents ５ represents T or Q, and X ６ represents P or D, and X ７ represents A or E, and X represents ８ represents D or Q, and X ９ represents A or K, and X represents １０ indicates D or I.

[0012] The amino acid sequence shown in SEQ ID NO: 1 has 61 amino acid residues. １ ~X １０) is one of the two types of amino acids described above. This polypeptide having 61 amino acid residues is not known to function as an adjuvant. The polypeptide having the amino acid sequence shown in SEQ ID NO: 1 may be referred to as Nasal Antigen-Inducible Sequence (NAIS) 61.

[0013] The polypeptides used in the present invention include (2) polypeptides having an amino acid sequence that is 93% or more identical to the amino acid sequence of SEQ ID NO: 1. Preferably, it is a polypeptide having an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 1, more preferably a polypeptide having an amino acid sequence that is 97% or more identical to the amino acid sequence of SEQ ID NO: 1, even more preferably a polypeptide having an amino acid sequence that is 98% or more identical to the amino acid sequence of SEQ ID NO: 1, and even more preferably a polypeptide having an amino acid sequence that is 99% or more identical to the amino acid sequence of SEQ ID NO: 1.

[0014] Specific examples of the 61 amino acid polypeptide having the amino acid sequence shown in SEQ ID NO: 1 include the above-mentioned X １ From X １０ The amino acid sequence may be either of the two types of amino acid residues shown below, but particularly preferred is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 3 below.

[0015]

[0016]

[0017] The polypeptide used in the adjuvant of the present invention may have at least one or two copies of the amino acid sequence shown in SEQ ID NO: 1 or a polypeptide having 93% or more identity to said amino acid sequence, but a polypeptide having one or two copies of the amino acid sequence shown in SEQ ID NO: 1 is more preferred. Of these, a more preferred example of a polypeptide having one copy of the amino acid sequence shown in SEQ ID NO: 1 is a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 below, from the viewpoint of obtaining a higher adjuvant effect. Furthermore, it is preferable that the 61 amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 4 are the NAIS61-1.

[0018] In the above amino acid sequence, X １ represents S or Q, and X ２ represents I or V, and X ３ represents G or S, and X １１ represents V or A, and X １２ represents N or K, and X １３ represents Q or T, and X １４ represents I or V, and X １５ represents N or Q, and X １６ represents D or N, and X １７ indicates S or N.

[0019] The amino acid sequence shown in SEQ ID NO: 4 has 230 amino acid residues. １ ~X １７ ) is one of the two amino acids described above. This polypeptide having 230 amino acid residues is not known to function as an adjuvant. The polypeptide having the amino acid sequence shown in SEQ ID NO:4 is sometimes referred to as NAIS230.

[0020] Specific examples of polypeptides having the amino acid residues shown in SEQ ID NO:4 include polypeptides having the amino acid sequences shown in the following SEQ ID NOs:5 to 9.

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] Among these, an example of a polypeptide having two copies of the amino acid sequence shown in SEQ ID NO: 1 is, from the viewpoint of obtaining a higher adjuvant effect, more preferably a polypeptide having the amino acid sequence shown in the following SEQ ID NO: 10. Furthermore, in the amino acid sequence of SEQ ID NO: 10, the 61 amino acids from the N-terminus are preferably the NAIS61-1.

[0027] In the above amino acid sequence, X １ represents S or Q, and X ２ represents I or V, and X ３ represents G or S, and X １１ represents V or A, and X １２ represents N or K, and X １３ represents Q or T, and X １４ represents I or V, and X １５ represents N or Q, and X １６ represents D or N, and X １７ indicates S or N.

[0028] The amino acid sequence shown in SEQ ID NO: 10 has 291 amino acid residues. １ ~X １７ ) is either of the two types of amino acids described above. The N-terminal 61 amino acids of the amino acid sequence shown in SEQ ID NO:10 are preferably the amino acid sequence shown in SEQ ID NO:2 (NAIS61-1), and the C-terminal 61 amino acids are the amino acid sequence shown in SEQ ID NO:3 (NAIS61-2). A polypeptide having this 291 amino acid residues is not known to function as an adjuvant. The polypeptide having the amino acid sequence shown in SEQ ID NO:10 may be referred to as NAIS291.

[0029] Preferred examples of the NAIS291 used in the present invention include polypeptides having the amino acid sequences selected from the following SEQ ID NOS: 11 to 15. The amino acid sequence represented by SEQ ID NOS: 11 is a polypeptide in which NAIS61-2 represented by SEQ ID NOS: 3 is bound to the C-terminus of the amino acid sequence represented by SEQ ID NOS: 5. The polypeptide having the amino acid sequence represented by SEQ ID NOS: 11 may also be referred to as NAIS291-1. Similarly, examples include a polypeptide in which NAIS61-2 represented by SEQ ID NOS: 3 is bound to the C-terminus of the amino acid sequence represented by SEQ ID NOS: 6 (SEQ ID NOS: 12), a polypeptide in which NAIS61-2 represented by SEQ ID NOS: 3 is bound to the C-terminus of the amino acid sequence represented by SEQ ID NOS: 7 (SEQ ID NOS: 13), a polypeptide in which NAIS61-2 represented by SEQ ID NOS: 3 is bound to the C-terminus of the amino acid sequence represented by SEQ ID NOS: 8 (SEQ ID NOS: 14), and a polypeptide in which NAIS61-2 represented by SEQ ID NOS: 3 is bound to the C-terminus of the amino acid sequence represented by SEQ ID NOS: 9 (SEQ ID NOS: 15).

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] Among these polypeptides, NAIS61, NAIS230, and NAIS291 are all polypeptides derived from a protein (30 kDa protein designated NAIS by the inventors) obtained by further deleting a region believed to be immunogenic from the protein MP3 (100 kDa), which is derived from the outer membrane protein PnxIIIA (250 kDa) of Pasteurella pneumoniae by removing a region believed to be cytotoxic. Therefore, as shown in Example 1 below, a gene encoding the PnxIIIA protein is amplified by PCR from the genome of Pasteurella pneumoniae, and the gene product encoding the region from amino acid residues 1,393 to 1,683 of the PnxIIIA protein is amplified using the resulting gene product as a template. This gene product is then inserted into a pBAD vector containing an araBAD promoter and a 6x histidine tag, and then introduced into an Escherichia coli strain in which the araB gene of the araBAD promoter has been replaced with T7 RNA polymerase. The transformed E. coli can be produced by culturing the transformed E. coli in LB medium.

[0036] Furthermore, since the amino acid sequences of the above polypeptides, including NAIS61, NAIS230, and NAIS291, are known, they can be produced by standard genetic engineering peptide synthesis methods. Specifically, they can be produced by preparing a DNA fragment encoding the desired amino acid sequence, incorporating the DNA fragment into a vector, introducing the vector into cells capable of producing the polypeptide, selecting only cells that have incorporated the plasmid, growing the selected cells, and recovering the produced polypeptide. When producing a polypeptide in E. coli, a vector can be used in which the gene of interest is placed downstream of a T7lac promoter and the host is an E. coli strain incorporating a T7 RNA polymerase gene; or a vector in which the gene of interest is placed downstream of an araBAD promoter that controls L-arabinose metabolism and the host is an E. coli strain lacking the L-arabinose metabolic pathway or an E. coli strain in which araB in the araBAD promoter has been replaced with T7 RNA polymerase. Methods that can be used to introduce a vector into E. coli include the heat shock method, in which E. coli is treated with calcium chloride and the temperature is suddenly raised to allow the vector to be introduced, or the electroporation method, in which contaminants are removed from E. coli, the cells are suspended in a 10% glycerol solution, and then a high-voltage pulse is applied to introduce the vector into the E. coli cells. Cells that have taken up the plasmid can be selected using a medium containing ampicillin, kanamycin, or chloramphenicol.

[0037] When the polypeptide is conjugated to an antigen and administered transmucosally, it is possible to significantly produce antibodies specific to the antigen. In this case, antibodies against the polypeptide are hardly produced. Therefore, the polypeptide is useful as an adjuvant for producing antibodies against various antigens. Furthermore, a conjugate of the polypeptide and an antigen is useful as a polypeptide for producing antibodies against the antigen. Here, various antigens include pathogens, substances produced by pathogens, or parts thereof.

[0038] By conjugating the above polypeptide with a pathogen, a substance produced by a pathogen, or a portion thereof and administering it transmucosally, it is possible to significantly produce specific antibodies against the pathogen. More specifically, by conjugating the above polypeptide with a pathogen, a substance produced by a pathogen, or a portion thereof and administering it transmucosally, it is possible to induce secretory IgA at a high antibody titer, thereby providing protection against the invasion of the pathogen at the mucosa, and also to induce high IgA and IgG in the serum, thereby providing protection against infectious diseases caused by the pathogen or a substance produced by the pathogen that has entered the body. In this case, almost no antibodies against the above polypeptide are produced. Furthermore, the above polypeptide itself exhibits almost no cytotoxicity. Therefore, conjugates of the above polypeptide with a pathogen, a substance produced by a pathogen, or a portion thereof are useful as mucosal vaccines.

[0039] Another aspect of the present invention is a mucosal vaccine containing, as an active ingredient, a conjugate of the above-described polypeptide with a pathogen or a substance produced by a pathogen, or a portion thereof. Examples of pathogens include viruses or bacteria that infect humans. Examples of substances produced by pathogens include proteins (including envelope proteins, membrane proteins, and spike proteins) and toxins produced by pathogens that infect humans. Specific examples of pathogens include pathogens or substances produced by pathogens, or portions thereof, selected from influenza virus, coronavirus, human papillomavirus, measles virus, tetanus toxin, and pertussis toxin. Examples of coronaviruses include SARS-CoV, MERS-CoV, SARS-CoV-2, and coronaviruses that cause the common cold. Examples of mucosal vaccines of the present invention include those used as inactivated vaccines, VLP vaccines, and recombinant protein vaccines.

[0040] Specific examples of the pathogen or a substance produced by a pathogen, or a portion thereof, include a portion of the spike protein of coronavirus OC43, such as a polypeptide (OC43ag) having the amino acid sequence shown in SEQ ID NO: 16 (described below), and a polypeptide having an amino acid sequence having 95% or more identity to said amino acid sequence. The polypeptide (OC43ag) having the amino acid sequence shown in SEQ ID NO: 16 is the S2 subunit, which is highly conserved among coronavirus spike proteins, and shows 50% or more homology to the same protein of SARS-CoV-2.

[0041] When rabbits were intranasally inoculated with a conjugate of the adjuvant of the present invention (the above-described polypeptide) and OC43ag, not only high anti-coronavirus antibody titers but also high anti-SARS-CoV-2 spike protein antibody titers were obtained. Therefore, a polypeptide having the amino acid sequence set forth in SEQ ID NO: 16 (OC43ag) or a polypeptide having an amino acid sequence with 95% or greater identity to said amino acid sequence is useful as an antigen for producing mucosal vaccines against not only coronaviruses but also SARS-CoV-2. Here, the polypeptide having an amino acid sequence with 95% or greater identity to said amino acid sequence is preferably a polypeptide having an amino acid sequence with 97% or greater identity to said amino acid sequence, more preferably a polypeptide having an amino acid sequence with 98% or greater identity to said amino acid sequence, and even more preferably a polypeptide having an amino acid sequence with 99% or greater identity to said amino acid sequence.

[0042] Examples of means for binding the adjuvant and the antigen (including a pathogen or a substance produced by a pathogen, or a part thereof) include a means for directly binding the adjuvant and the antigen, a means for producing a polypeptide in which the adjuvant and the antigen are bound by genetic engineering, a means for binding the adjuvant and the antigen via a linker, etc. Furthermore, the binding form between the adjuvant and the antigen may be a form in which the antigen is bound to either the N-terminal side or the C-terminal side of the adjuvant, but a form in which the antigen is bound to the C-terminal side of the adjuvant is preferred.

[0043] The mucosal vaccine of the present invention may contain a conjugate of the adjuvant (the polypeptide) and an antigen, and may further contain a pharmaceutically acceptable carrier, such as purified water, physiological saline, various buffer solutions, pH adjusters, stabilizers such as sugars, and polyhydric alcohols.

[0044] The mucosal vaccine of the present invention is a vaccine administered mucosally. Among mucosal administrations, nasal administration is preferred from the viewpoints of irritation and ease of administration. The form of nasal administration may be any form that allows the vaccine to adhere from the nasal mucosa to the upper respiratory tract mucosa. It may be administered directly using a cotton swab, but a form that allows powder or spray droplets to reach the upper respiratory tract from the nasal cavity is preferred. Specific examples include inhalable powders, inhalable liquids, and inhalable aerosols. The dose of the mucosal vaccine of the present invention varies depending on the type of pathogen, but because of its strong adjuvant effect, a smaller dose is sufficient compared to administering a pathogen alone. The number of administrations also varies depending on the type of pathogen and can be selected appropriately.

[0045] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0046] Example 1 (Production of NAIS291-1 (Polypeptide of SEQ ID NO: 11)) The gene encoding the PnxIIIA protein was amplified by PCR from the genome of Pasteurella pneumoniae. Using the resulting gene product as a template, the gene product of the region encoding amino acid residues 1,393 to 1,683 of the PnxIIIA protein was amplified and inserted into a pBAD vector having an araBAD promoter and a 6x histidine tag. The amplified gene product was then introduced into an Escherichia coli strain in which araB in the araBAD promoter had been replaced with T7 RNA polymerase. The transformed Escherichia coli was cultured in LB medium for 3 hours, after which 0.2% L-arabinose was added to a final concentration and the culture was continued for an additional 3 hours. The cultured cells were disrupted by sonication, and NAIS291-1 (a polypeptide having the amino acid sequence of SEQ ID NO: 11) was isolated and purified using magnetic beads for histidine tag purification. Polypeptides having the amino acid sequences of SEQ ID NOs: 2, 3, 5-9, and 12-15 were obtained in a similar manner.

[0047] Example 2: NAIS291-OC43ag was prepared by ligating a portion of the spike protein of human coronavirus OC43 (OC43ag) to the NAIS291-1 obtained in Example 1. Using the vector obtained in Example 1 as a template, the NAIS291-1 expression vector was linearized using PCR, starting from the 3' end of the gene encoding NAIS291-1. A portion of the S2 subunit of the gene encoding the spike protein derived from human coronavirus OC43 was amplified by PCR and ligated into the linear NAIS291-1 expression vector using PCR cloning. This vector was introduced into an E. coli strain in which araB in the araBAD promoter had been replaced with T7 RNA polymerase. The transformed E. coli was cultured in LB medium for 3 hours, after which a final concentration of 0.2% L-arabinose was added and the culture was continued for an additional 3 hours. The cultured cells were disrupted by sonication, and NAIS291-OC43ag was isolated and purified using magnetic beads for histidine tag purification.

[0048] The amino acid sequence of a portion of the spike protein of OC43 (OC43ag) used as an antigen is as follows:

[0049]

[0050] Example 3 A TBS solution of NAIS291-OC43ag (2 μM, 200 μl) was intranasally inoculated into rabbits three times at weekly intervals, and the serum antibody titer was measured one week after the third inoculation. Specifically, 100 μL of serially diluted rabbit serum was added to a 96-well plate precoated with 100 μL of Human Coronavirus (HCoV-OC43) Spike S2 ​​Protein solution (Sino Biological, China, 1 μg / ml) and treated with a blocking buffer (Pierce Protein-Free (PBS) Blocking Buffer). The plate was incubated overnight at 4°C and then washed five times with 100 μL of 0.1% Tween 20 in PBS (PBST). Next, 100 μL of Anti-Rabbit IgG (H+L), HRP Conjugate (Promega, USA) or Anti-Rabbit IgA alpha chain (HRP) (Abcam, UK) diluted 3000-fold with Blocking buffer was added and reacted, followed by washing five times with 100 μL of PBST. 100 μL of TMB microwell peroxidase substrate (SeraCare Life Sciences, USA) was added and reacted at 25°C for 10 minutes, after which the absorbance at 650 nm was measured. As a result, as shown in Figure 2, the anti-HCoV-OC43 spike protein serum antibody titer for IgG exceeded the upper limit of measurement by ELISA. Both IgG and IgA showed antibody titers sufficient for protection against infection.

[0051] Example 4 HCT-8 cells (2.0 x 10^5 cells / 200 μl) were mixed with control serum or serum from a rabbit nasally inoculated with NAIS291-OC43ag (100 μl each), and then mixed with HCoV-OC43 strain (10,000 pfu / 100 μl), which was then used to infect HCT-8 cells and cultured. After 3 weeks of culture, cell degeneration was observed in the cells to which the control serum was added, and the number of virus copies in the medium also increased. However, no degeneration of HCT-8 cells was observed in the cells to which serum from rabbits nasally inoculated with NAIS291-OC43ag was added, which is consistent with the method of Vijgen et al. (Reference: Vijgen L, Keyaerts E, Moes E, Maes P, Duson G, Van Ranst M. Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E. J Clin Microbiol. 2005 43(11):5452-5456. When the virus copy number was measured by real-time PCR, it was below the lower limit of detection.

[0052] Example 5: OC43ag, having the amino acid sequence shown in SEQ ID NO: 16, is a highly conserved S2 subunit among coronavirus spike proteins and shows greater than 50% homology with the same protein in SARS-CoV-2. The antibody titer against the SARS-CoV-2 spike protein S2 subunit was measured for serum from rabbits nasally inoculated with NAIS291-OC43ag. Specifically, 100 μL of Recombinant SARS-CoV-2 S2 Subunit Protein with C-terminal His-tag, Transfected HEK293 Cell Culture Supernatant solution (RayBiotech Life, USA, 1 μg / ml) was precoated, and the antibody titer was measured in the same manner as in Example 3. As a result, the antibody titers for IgG were over 1,024 and for IgA were 64, both of which were sufficient for protection against infection.

[0053] Example 6: BSA and NAIS291-1 were diluted in medium to final concentrations of 10 μM, 1 μM, and 0.1 μM, respectively, and added to L929 cells (3.0 x 10^3 cells / 100 μl). After 24 hours of culture, % cytotoxicity was calculated using an LDH cytotoxicity assay kit (Nacalai Tesque). Four replicates were measured twice, and the average is shown. % cytotoxicity was calculated as (sample abs - low control abs) / (high control abs - low control abs) x 100. As a result, as shown in Figure 3, NAIS291-1 (NAIS in Figure 3) had lower cytotoxicity than BSA, which was used as a control.

[0054] Example 7 A thioredoxin gene-NAIS291-1 conjugate (Trx-NAIS-His_291: SEQ ID NO: 17), a thioredoxin gene-NAIS230-1 conjugate (Trx-NAIS-His_230: SEQ ID NO: 18), and a thioredoxin gene-NAIS61-2 conjugate (Trx-NAIS-His_61: SEQ ID NO: 19) were produced in the same manner as in Example 2, except that thioredoxin gene 6xHisTag (Trx-His: SEQ ID NO: 20) was used as an antigen.

[0055]

[0056]

[0057]

[0058]

[0059] Rabbits were intranasally inoculated with TBS solutions (5 μM, 200 μl) of these polypeptides three times at weekly intervals, and serum antibody titers were measured one week after the third inoculation. Specifically, 100 μL of serially diluted rabbit serum was added to a 96-well plate precoated with 100 μL of Trx-His solution (SEQ ID NO: 20, 1 μg / ml) or NAIS291 solution (SEQ ID NO: 13, 1 μg / ml) and treated with blocking buffer (Pierce Protein-Free (PBS) Blocking Buffer). The plate was incubated overnight at 4°C and then washed five times with 100 μL of PBST. Next, 100 μL of Anti-Rabbit IgG (H+L) HRP Conjugate (Promega, USA) or Anti-Rabbit IgA alpha chain (HRP) (Abcam, UK) diluted 3000-fold with Blocking buffer was added and reacted, followed by washing five times with 100 μL of PBST. 100 μL of TMB microwell peroxidase substrate (SeraCare Life Sciences, USA) was added and reacted at 25°C for 10 minutes, after which the absorbance at 650 nm was measured. As a result, as shown in Figure 4, the thioredoxin gene-NAIS61-2 conjugate (Trx-NAIS-His_61: SEQ ID NO: 19) enhanced the anti-thioredoxin antibody titer (IgG), while the thioredoxin gene-NAIS291-1 conjugate (Trx-NAIS-His_291: SEQ ID NO: 17) and the thioredoxin gene-NAIS230-1 conjugate (Trx-NAIS-His_230: SEQ ID NO: 18) both exhibited extremely high anti-thioredoxin antibody titers (IgG). On the other hand, the thioredoxin gene 6xHisTag (Trx-His: SEQ ID NO: 20) hardly induced the anti-thioredoxin antibody titer (IgG). Furthermore, neither polypeptide induced the anti-NAIS antibody titer.

[0060] Example 8 NAIS230-1 and NAIS61-2 were cross-linked with influenza virus (Influenza A H1N1 (A / Puerto Rico / 8 / 1934)) hemagglutinin 6xHis (HA, Sino Biological, China) using N-Hydroxysuccinimide (Thermo Fisher Scientific, USA) according to the protocol of Thermo Fisher Scientific to prepare NAIS230-HA and NAIS61-HA. Mice were intranasally inoculated three times weekly with NAIS230-HA, NAIS61-HA, or HA in TBS (0.1 μM, 20 μl). One week after the third inoculation, serum anti-HA IgG antibodies were measured using enzyme-linked immunosorbent assay. Specifically, 100 μL of mouse serum was added to a 96-well plate precoated with 100 μL of HA solution (1 μg / ml) and treated with blocking buffer (Pierce protein-free T20). The plate was incubated overnight at 4°C and then washed four times with 200 μL of PBST. Next, 100 μL of Goat anti-mouse IgG-Fc Fragment HRP Conjugated (Bethyl Laboratories, USA) diluted 5000-fold with 10% Blocking buffer was added and reacted, followed by washing seven times with 200 μL of PBST. 100 μL of TMB microwell peroxidase Substrate (SeraCare Life Sciences, USA) was added and reacted at 25°C for 10 minutes, after which 100 μL of 1 mol / L sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured. As a result, as shown in FIG. 5, the absorbance (450 nm) of NAIS230-HA and NAIS61-HA was higher than that of HA alone, indicating that anti-HA IgG antibodies were induced.

Claims

1. An adjuvant comprising a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 4, or 10 as an active ingredient.

2. An adjuvant comprising a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 3, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15 as an active ingredient.

3. An adjuvant comprising a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15 as an active ingredient.

4. (Deleted)

5. (Deleted)

6. (Deleted)

7. (Deleted)

8. (Deleted)

9. A polypeptide for antibody production against an antigen, comprising a conjugate of the adjuvant according to any one of Claims 1 to 3 and the antigen as an active ingredient.

10. A transmucosal vaccine comprising a conjugate of the adjuvant according to any one of Claims 1 to 3 and a pathogen or a substance produced by the pathogen or a part thereof as an active ingredient.

11. The transmucosal vaccine according to Claim 10, wherein the pathogen or the substance produced by the pathogen or a part thereof is a pathogen or a substance produced by the pathogen or a part thereof selected from influenza virus, coronavirus, human papillomavirus, measles virus, tetanus toxin, and pertussis toxin.

12. Use of a conjugate of the adjuvant according to any one of Claims 1 to 3 and a pathogen or a substance produced by the pathogen or a part thereof for the manufacture of a transmucosal vaccine.

13. A conjugate of the adjuvant according to any one of Claims 1 to 3 and a pathogen or a substance produced by the pathogen or a part thereof for use in transmucosal vaccine therapy.

14. A transmucosal vaccine therapy, characterized by transmucosally administering a conjugate of the adjuvant according to any one of Claims 1 to 3 and a pathogen or a substance produced by the pathogen or a part thereof.