Immunopotentiator and immunopotentiating method, and method for producing aggregate and / or assembly of protein

Abstract

The present invention provides: a new and safer immunopotentiator that exhibits an immunopotentiating effect comparable to that of conventional immunopotentiators; an immunopotentiating method using the same; and a method for producing an aggregate and / or an assembly of protein.　The immunopotentiator contains, as an active ingredient, an ion of at least one metal selected from the group consisting of nickel, cobalt, zinc, iron, copper, and manganese, or a salt of the metal.

Description

Immune enhancing agent, immune enhancing method, and method for producing protein aggregates and / or associations

[0001] The present invention relates to an immunopotentiating agent and an immunopotentiating method that are administered together with an antigen to enhance the immunogenicity of the antigen, and also to a method for producing aggregates and / or associations of proteins such as antigens.

[0002] An immunopotentiator, or adjuvant, is a substance administered together with a vaccine antigen to enhance immunogenicity. For example, vaccines that are administered using purified proteins from pathogens generally have low efficacy, so the addition of an adjuvant is usually required.

[0003] To date, the adjuvant effects of various substances have been investigated, and mineral oil, aluminum salts, and microbial-derived components have been found to be effective adjuvants (Non-Patent Documents 1 and 2). Among these, aluminum salts are used clinically as adjuvants. Aluminum salts are used as adjuvants in many vaccines against pertussis, tetanus, human papillomavirus (HPV), pneumococcus (PCV13), hepatitis B, and other diseases.

[0004] Recombinant protein vaccines (also known as subunit vaccines) have been attracting attention in recent years. A unique feature of viral vaccines is that they do not administer the virus itself, but rather use specific subunits (proteins) that make up the virus as antigens. The world's first recombinant protein vaccine was for hepatitis B virus. Hepatitis B virus is an enveloped virus with three envelope proteins. These proteins are produced in yeast and self-assemble into nanovirus particles with a diameter of 22 nm. 90-95% of healthy, immunocompetent adults who received this vaccine demonstrated an immune response to hepatitis B virus antigens, even in the presence of an adjuvant. These envelope proteins contain hepatitis B virus-specific T cell epitopes, which activate T cells. Activated T cells then release cytokines, which promote B cell differentiation and antibody production.

[0005] Coffman, RL, Sher, A. and Seder, RA (2010). Vaccine adjuvants: putting innate immunity to work. Immunity 33, 492-503. Moyle, PM and Toth, I. (2013). Modern subunit vaccines: development, components, and research opportunities. Chem. Med. Chem. 8, 360-376.

[0006] However, the above-mentioned conventionally known immunopotentiators and immunopotentiating methods using the same can sometimes cause various side effects, which is one of the major reasons for vaccine aversion in Japan. An object of the present invention is to provide a new, safer immunopotentiator that exhibits an immunopotentiating effect comparable to that of conventional immunopotentiators, an immunopotentiating method using the same, and a method for producing aggregates and / or associations of proteins such as antigens.

[0007] In order to achieve the above-mentioned object, the present inventors have conducted extensive research and found that certain metal ions have excellent immune enhancing effects and further promote the formation of aggregates and / or associations of proteins such as antigens, thereby completing the present invention.

[0008] (1) An immunopotentiator comprising, as an active ingredient, ions of at least one metal selected from the group consisting of nickel, cobalt, zinc, iron, copper, and manganese, or a salt of said metal. (2) The immunopotentiator according to (1), which is added to a solution containing an antigen so as to achieve a concentration of 3 to 100 μM. (3) The immunopotentiator according to (1), which is characterized in that the antigen is a recombinant protein having a histidine tag. (4) The immunopotentiator according to (1), which is characterized in that the antigen is a recombinant protein without a histidine tag. (5) A method for enhancing immunity, which comprises administering to a non-human animal a solution containing ions of at least one metal selected from the group consisting of nickel, cobalt, zinc, iron, copper, and manganese, and an antigen. (6) The method for enhancing immunity according to (5), which is characterized in that the concentration of the ions of the metal is 3 to 100 μM. (7) The method for enhancing immunity according to (5), which is characterized in that the antigen is a recombinant protein having a histidine tag. (8) The immune-enhancing method according to (5), wherein the antigen is a recombinant protein without a histidine tag. (9) A method for producing protein aggregates and / or assemblies, comprising the step of mixing, into a solution containing a recombinant protein with a histidine tag, ions of at least one metal selected from the group consisting of nickel, cobalt, zinc, iron, copper, and manganese, or a metal salt capable of generating ions of these metals in the solution, thereby aggregating and / or associating the recombinant protein with the ions of the metal. (10) The method for producing protein aggregates and / or assemblies according to (9), wherein ions of the metal are added to the solution to a concentration in the range of 3 to 100 μM.

[0009] This specification includes the disclosure of Japanese Patent Application No. 2023-127119, from which the present application claims priority.

[0010] The immunopotentiators and immunopotentiating methods of the present invention can enhance the immunogenicity of protein antigens. Therefore, application of the immunopotentiators and immunopotentiating methods of the present invention allows for lower antigen doses in vaccines. Furthermore, the methods for producing protein aggregates and / or associations of the present invention enable specific association and / or aggregation of proteins, such as histidine-tagged recombinant proteins used as antigens, by adding extremely low concentrations of metal ions. It is particularly important to emphasize that the metal ion concentrations used in the present invention are 10 to 10,000 times lower than those previously used for such applications (protein association and immunopotentiation). Therefore, the present invention is a significantly more advantageous technology than conventional techniques in terms of environmental pollution (wastewater treatment), safety (production facilities), and toxicity (in the body). Furthermore, the liquid properties, such as viscosity, of a liquid containing metal ions at a concentration of 100 μM are comparable to those of distilled water, making it easy to use during filtration or injection.

[0011] 1 is a characteristic diagram showing the results of measuring the particle radius of D3ED3 having a histidine tag associated with various metal ions. FIG. 1 is a characteristic diagram showing the relationship between the pH of a buffer solution and the particle radius of D3ED3 having a histidine tag. FIG. 2 is a characteristic diagram showing the relationship between the concentration of nickel ions and the particle radius of D3ED3 having a histidine tag. FIG. 3 is a characteristic diagram showing the results of measuring the IgG antibody titer (after initial immunization and after booster immunization) when D3ED3 having a histidine tag and nickel are administered, and when wild-type D3ED3 and nickel are administered. FIG. 4 is a characteristic diagram showing the distribution of CD markers expressed on the surface of helper T cells when D3ED3 having a histidine tag and nickel are administered, and when wild-type D3ED3 and nickel are administered. FIG. 5 is a table summarizing the experimental results of the examples. Ni of precipitates of histidine-tagged JEV-ED3 2+ The concentration dependence is shown as the percentage of JEV-ED3 in the precipitated fraction (Pellet) and the supernatant fraction (Sup) of proteins calculated from SDS-PAGE images. 2+Nearly 90% of JEV-ED3 was precipitated at a concentration of 100 μM. Analysis of the monomeric and aggregated states of JEV ED3 in the presence or absence of metal ions. Particle radius (Rh) measured by dynamic light scattering (DLS) at 25°C and 37°C is shown. Rh was calculated from the DLS number distribution measured immediately before administration. Data represent the mean and standard deviation of three independent measurements. Immune responses (absorbance and IgG titers by ELISA) to JEV Wt and Wt-His ED3 in the presence or absence of metal ions are shown. Average IgG titers were calculated from serum collected from the tails of mice after the third administration. n = 3 except for Wt-His+Co (n = 4). Long-term antibody titers to JEV ED3 in the presence or absence of metal ions are shown. "TB-(number)" indicates the number of weeks after the initial inoculation at which tail bleeding was performed. Regardless of the presence or absence of protein aggregation, mice treated with Co showed a stronger immune response, inducing immune enhancement.

[0012] The immune enhancing agent and immune enhancing method according to the present invention are described in detail below. The immune enhancing agent and immune enhancing method according to the present invention are based on the finding that nickel, cobalt, zinc, iron, copper, and manganese ions enhance the immunogenicity of antigens. The immune enhancing agent according to the present invention may be these metal ions contained in a solution containing an antigen, or a metal salt capable of generating these metal ions in a solution containing an antigen. Furthermore, the immune enhancing agent according to the present invention may contain ions of at least one metal selected from the group consisting of nickel, cobalt, zinc, iron, copper, and manganese, or a salt of the metal.

[0013] Here, nickel, cobalt, and iron may be either divalent or trivalent ions. Copper may be either monovalent or divalent ions. Manganese may be either divalent, trivalent, tetravalent, hexavalent, or heptavalent ions. Zinc is a divalent ion.

[0014] In the immune enhancing agent and immune enhancing method of the present invention, one metal ion selected from nickel, cobalt, zinc, iron, copper, and manganese may be used, or two, three, four, five, or six metal ions may be used. Among these, nickel ions are preferred in the immune enhancing agent and immune enhancing method of the present invention because they have the most effective effect in improving immunogenicity. Furthermore, zinc or iron ions are preferred in the immune enhancing agent and immune enhancing method of the present invention from the viewpoint of safety.

[0015] Nickel salts are not particularly limited and include, but are not limited to, nickel chloride, nickel sulfate, nickel nitrate, nickel fluoride, nickel bromide, nickel acetate, nickel oxalate, basic nickel carbonate, nickel carbonate, nickel iodide, nickel phosphate, and hydrates thereof. Cobalt salts are not particularly limited and include, but are not limited to, cobalt chloride, cobalt sulfate, cobalt nitrate, cobalt fluoride, cobalt bromide, cobalt acetate, cobalt oxalate, basic cobalt carbonate, cobalt carbonate, cobalt iodide, cobalt phosphate, and hydrates thereof. Zinc salts are not particularly limited and include, but are not limited to, zinc chloride, zinc sulfate, zinc nitrate, zinc fluoride, zinc bromide, zinc acetate, zinc oxalate, basic zinc carbonate, zinc carbonate, zinc iodide, zinc phosphate, zinc pyrophosphate, and hydrates thereof. Iron salts are not particularly limited, and examples thereof include ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferrous nitrate, ferric nitrate, ferrous fluoride, ferric fluoride, ferrous bromide, ferric bromide, ferrous acetate, ferric acetate, ferrous oxalate, ferric oxalate, ferrous carbonate, ferric carbonate, ferrous iodide, ferric iodide, ferrous phosphate, ferric phosphate, ammonium ferrous sulfate, ammonium ferric sulfate, and hydrates thereof. Copper salts are not particularly limited, and examples thereof include cuprous chloride, cupric chloride, cuprous sulfate, cupric sulfate, cuprous nitrate, cupric nitrate, cuprous fluoride, cupric fluoride, cuprous bromide, cupric bromide, cuprous acetate, cupric acetate, cuprous oxalate, cupric oxalate, cuprous carbonate, cupric carbonate, cuprous iodide, cupric iodide, cuprous phosphate, cupric phosphate, ammonium copper sulfate, and hydrates thereof. The manganese salt is not particularly limited, and examples thereof include manganese chloride, manganese sulfate, manganese nitrate, manganese fluoride, manganese bromide, manganese acetate, manganese oxalate, basic manganese carbonate, manganese carbonate, manganese iodide, manganese phosphate, and hydrates thereof.

[0016] In the immune enhancing agent and immune enhancing method of the present invention, the term "antigen" refers to, but is not limited to, a foreign substance or a part thereof that enters a living body from the outside and induces an immune response in the living body. Antigens include foreign pathogens such as bacteria and viruses that cause various infectious diseases, as well as allergens such as pollen and food that induce allergic reactions. The immune enhancing agent and immune enhancing method of the present invention enhance the immune response after these antigens enter the body. Therefore, the immune enhancing agent and immune enhancing method of the present invention are suitable for vaccines using these antigens.

[0017] Antigens derived from viruses include at least one virus selected from the group consisting of influenza virus, norovirus, rotavirus, human papillomavirus, chickenpox virus, measles virus, mumps virus, poliovirus, adenovirus, herpes virus, human coronavirus, rubella virus, HIV, smallpox virus, Ebola virus, hepatitis virus, Japanese encephalitis virus, parvovirus, cowpox virus, and dengue virus, or a part or component thereof that has been inactivated or attenuated.

[0018] Antigens derived from bacteria include at least one bacterium selected from the group consisting of Haemophilus influenzae, Streptococcus pneumoniae, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Escherichia coli such as enterohemorrhagic Escherichia coli, Vibrio cholerae, Salmonella enterica, and methicillin-resistant Staphylococcus aureus, or a part or component thereof that has been inactivated or attenuated.

[0019] Allergens include pollen (cedar pollen, grass pollen, asteraceae pollen, etc.), fungi, insects, foods (soybean, egg, milk, etc.), or parts or components thereof.

[0020] The immunopotentiating agent and immunopotentiating method according to the present invention can particularly enhance the immunogenicity of a recombinant protein used as an antigen. Recombinant proteins can be prepared by applying genetic recombination techniques to various virus-derived proteins listed above as examples of virus-derived antigens, using a host such as Escherichia coli. Examples of the virus-derived protein include proteins exposed on the surface of the virus, or partial fragments of the protein containing a surface-exposed region of the protein.

[0021] Furthermore, the immunopotentiating agent and immunopotentiating method of the present invention can particularly enhance immunogenicity when a recombinant protein having a histidine tag added to the C-terminus and / or N-terminus is used as an antigen. A histidine tag typically consists of 5 to 11, preferably 5 to 9, histidine residues. The histidine tag also includes a sequence consisting of consecutive histidine residues, but may also contain amino acid residues other than histidine. Furthermore, the immunopotentiating agent and immunopotentiating method of the present invention can enhance immunogenicity even when a recombinant protein without a histidine tag is used as an antigen.

[0022] The method of administering a vaccine to which the immune enhancing agent and immune enhancing method of the present invention are applied is not particularly limited, and can be, for example, a method selected from the group consisting of transdermal administration, subcutaneous administration, intradermal administration, and intramuscular administration. The vaccine dosage is determined taking into consideration the age, sex, weight, etc. of the subject, but when a recombinant protein derived from a virus is used as the antigen, the vaccine can usually be administered once or twice or more times. Multiple administrations are preferred, and in this case, administration is preferably performed at intervals of 1 to 4 weeks.

[0023] The immunopotentiator of the present invention is not particularly limited, but it is preferable that the metal ion or metal salt be present in a solution containing an antigen at a concentration ranging from 3 to 100 μM. If the concentration of the immunopotentiator of the present invention is lower than this range, the effect of enhancing the immunogenicity of the antigen may be reduced. On the other hand, if the concentration of the immunopotentiator of the present invention is higher than this range, the effect of enhancing the immunogenicity of the antigen may be reduced. In particular, when a histidine-tagged recombinant protein is used as the antigen, if the concentration of the immunopotentiator is higher than this range, the histidine-tagged recombinant protein may form large aggregates and precipitate in the solution.

[0024] In this way, an immune composition containing the immune enhancer of the present invention and an antigen and having enhanced immunogenicity of the antigen can be provided. The immune composition preferably contains the immune enhancer at a concentration ranging from 3 to 100 μM in a solution containing the antigen. The immune composition can be used in vaccines that contribute to the prevention, treatment, and prevention of aggravation of diseases caused by viruses, bacteria, etc., as described above.

[0025] The dosage form of the vaccine formulation to which the immune enhancing agent and immune enhancing method of the present invention are applied is not particularly limited, and examples include liquids, suspensions, sprays, powders, etc. Various additives commonly used in vaccine formulations, such as solubilizers, anti-aggregating agents, viscosity regulators, pH regulators, isotonicity agents, emulsifiers, antioxidants, bulking agents, surfactants, diluents, preservatives, stabilizers, moisture-proofing agents, and humectants, can be added to the vaccine formulation as needed. Furthermore, the immune enhancing agent of the present invention can also be used in combination with a conventionally known adjuvant as needed.

[0026] The subjects to which the vaccine is administered can be appropriately determined depending on the type of antigenic substance, and examples include humans and mammals other than humans (non-human animals), such as mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, horses, sheep, dogs, cats, monkeys, orangutans, and chimpanzees.

[0027] [Method for producing protein aggregates and / or assemblies] Next, a method for producing protein aggregates and / or assemblies according to the present invention will be described. Here, a protein aggregate refers to an aggregate of protein molecules that has formed a higher-order structure consisting of multiple protein molecules, or that does not form a higher-order structure. A protein aggregate refers to an aggregate of multiple protein molecules that does not form a higher-order structure, and is generally an aggregate that is larger than a protein aggregate. Furthermore, protein aggregates also include, for example, particles that are difficult to see with the naked eye (sub-visible aggregates) of 1 μm or less.

[0028] In the method of the present invention for producing protein aggregates and / or assemblies, nickel, cobalt, zinc, iron, copper, and manganese ions contribute to the association and aggregation of a histidine-tagged recombinant protein, thereby facilitating the purification of the protein, etc. The method of the present invention for producing protein aggregates and / or assemblies involves mixing these metal ions into a solution containing a histidine-tagged recombinant protein, or a metal salt capable of generating these metal ions into the solution.

[0029] Here, nickel, cobalt, and iron may be either divalent or trivalent ions. Copper may be either monovalent or divalent ions. Manganese may be either divalent, trivalent, tetravalent, hexavalent, or heptavalent ions. Zinc is a divalent ion.

[0030] In the method for producing protein aggregates and / or associations of the present invention, one metal ion selected from nickel, cobalt, zinc, iron, copper, and manganese may be used, or two, three, four, five, or six metal ions may be used. Among these, nickel ions are preferred in the method for producing protein aggregates and / or associations of the present invention, as they have the most effective protein aggregation effect. Furthermore, zinc or iron ions are preferred in the method for producing protein aggregates and / or associations of the present invention, from the viewpoint of safety.

[0031] Nickel salts are not particularly limited and include, but are not limited to, nickel chloride, nickel sulfate, nickel nitrate, nickel fluoride, nickel bromide, nickel acetate, nickel oxalate, basic nickel carbonate, nickel carbonate, nickel iodide, nickel phosphate, and hydrates thereof. Cobalt salts are not particularly limited and include, but are not limited to, cobalt chloride, cobalt sulfate, cobalt nitrate, cobalt fluoride, cobalt bromide, cobalt acetate, cobalt oxalate, basic cobalt carbonate, cobalt carbonate, cobalt iodide, cobalt phosphate, and hydrates thereof. Zinc salts are not particularly limited and include, but are not limited to, zinc chloride, zinc sulfate, zinc nitrate, zinc fluoride, zinc bromide, zinc acetate, zinc oxalate, basic zinc carbonate, zinc carbonate, zinc iodide, zinc phosphate, zinc pyrophosphate, and hydrates thereof. Iron salts are not particularly limited, and examples thereof include ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferrous nitrate, ferric nitrate, ferrous fluoride, ferric fluoride, ferrous bromide, ferric bromide, ferrous acetate, ferric acetate, ferrous oxalate, ferric oxalate, ferrous carbonate, ferric carbonate, ferrous iodide, ferric iodide, ferrous phosphate, ferric phosphate, ammonium ferrous sulfate, ammonium ferric sulfate, and hydrates thereof. Copper salts are not particularly limited, and examples thereof include cuprous chloride, cupric chloride, cuprous sulfate, cupric sulfate, cuprous nitrate, cupric nitrate, cuprous fluoride, cupric fluoride, cuprous bromide, cupric bromide, cuprous acetate, cupric acetate, cuprous oxalate, cupric oxalate, cuprous carbonate, cupric carbonate, cuprous iodide, cupric iodide, cuprous phosphate, cupric phosphate, ammonium copper sulfate, and hydrates thereof. The manganese salt is not particularly limited, and examples thereof include manganese chloride, manganese sulfate, manganese nitrate, manganese fluoride, manganese bromide, manganese acetate, manganese oxalate, basic manganese carbonate, manganese carbonate, manganese iodide, manganese phosphate, and hydrates thereof.

[0032] In the method for producing protein aggregates and / or assemblies according to the present invention, the target protein is not particularly limited as long as it has a histidine tag. The histidine tag is added to the C-terminus and / or N-terminus of the protein. The histidine tag can typically be a peptide containing 5 to 11, preferably 5 to 9, histidine residues. The histidine tag may include a sequence consisting of consecutive histidine residues, but may also contain amino acid residues other than histidine. The portion of the protein excluding the histidine tag is not particularly limited, and proteins with any amino acid sequence can be used.

[0033] In the method for producing protein aggregates and / or assemblies of the present invention, the metal ions are preferably present in a solution containing a histidine-tagged protein at a concentration ranging from 3 to 100 μM, although this is not a particular limitation. If the metal ion concentration is lower than this range in the method for producing protein aggregates and / or assemblies of the present invention, the effect of causing protein aggregation or aggregation may not be observed. Furthermore, if the metal ion concentration is higher than this range in the method for producing protein aggregates and / or assemblies of the present invention, the aggregates or assemblies of the histidine-tagged recombinant protein may become too large, preventing the protein from exhibiting its inherent properties.

[0034] The method for producing protein aggregates and / or assemblies according to the present invention facilitates the association and aggregation of a histidine-tagged protein in a solution containing the protein. According to the method for producing protein aggregates and / or assemblies according to the present invention, the proteins associate or aggregate in solution, allowing the proteins to be recovered from the solution as a precipitate fraction, for example, by centrifugation. However, even when the method for producing protein aggregates and / or assemblies according to the present invention is applied, salting out, isoelectric precipitation, dialysis, and various types of chromatography, which are included in typical protein purification processes, may be combined. Furthermore, the size of the aggregates or assemblies produced by the method for producing protein aggregates and / or assemblies according to the present invention can be controlled by adjusting the temperature of the protein-containing solution, the protein concentration, the type and pH of the buffer, the salt concentration, and the incubation time.

[0035] Furthermore, a solution containing a histidine-tagged protein can be prepared by a conventional protein production process. That is, a histidine-tagged protein can be produced by transforming an appropriate host cell with an expression vector incorporating a cDNA encoding the histidine-tagged protein and growing the transformed cell under conditions suitable for its growth. Such a method is described in "Current Protocols in Molecular Biology," F. Ausubel et al., Publ. Wiley Interscience, New York, 1997, or Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, etc.

[0036] For example, to produce an expression vector incorporating a cDNA encoding a protein with a histidine tag, an expression plasmid incorporating a cDNA encoding a protein without a histidine tag can be used as a template. Then, for example, a nucleotide sequence encoding the amino acid sequence of the histidine tag can be added to the 5' or 3' end of the ORF encoding the protein. Kits for carrying out such methods are commercially available from Stratagene (USA) and other companies. Depending on the type of host cell used, the expression vector may be a plasmid, virus, phage, transposon, IS element, phasmid, cosmid, or vector consisting of linear or circular DNA, etc. For example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11, or pBdCI for E. coli; pUB110, pC194, or pBD214 for Bacillus; and pSA77 or pAJ667 for Corynebacterium. Suitable vectors for animal host cells include pCDM8 and pMT2PC, as well as adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, herpes virus vectors, and the like.

[0037] The expression vector can be introduced into host cells by conventional transfection methods such as coprecipitation, electroporation, retroviral transfection, etc. Bacteria such as E. coli and animal cells such as CHO cells can be used as host cells.

[0038] By incubating host cells transformed with the vector of the present invention under optimal culture conditions for the cells as described above, the histidine-tagged recombinant protein can be accumulated in the host cells. Then, by adding the above-mentioned metal ions, the histidine-tagged recombinant protein specifically associates or aggregates, allowing the preparation of a highly pure protein.

[0039] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0040] Example 1 In this example, nickel ions were used as an example of metal ions, and the effect of nickel ions on antigen proteins was investigated.

[0041] 1. Materials and Methods 1-1. Protein Design, Expression, Assembly, or Aggregation A plasmid was used in which a polynucleotide sequence encoding the amino acid sequence of dengue type 3 virus envelope protein domain 3 (D3ED3) (Table 1), which includes a His-tag region and a base sequence encoding a thrombin cleavage sequence at the N-terminus, was inserted between the NdeI and BamHI recognition sequences in the pET15b vector (Novagen). The product from which the His-tag region had been removed during purification was designated D3wt, and the product from which the His-tag region had been retained was designated D3His.

[0042]

[0043] In this example, E. coli was first transformed by the heat shock method. 1 μL of a plasmid containing the DNA sequence encoding D3wt was added to 100 μL of JM109(DE3)pLysS, and the mixture was left to stand on ice for 30 minutes, then placed on a hot plate at 42°C for 45 seconds, and then left to stand on ice for 3 minutes. 50 μL of this mixture was spread onto LB / Amp / Cp agar medium and incubated at 37°C for approximately 16 hours.

[0044] One of the colonies obtained on the agar medium by transformation was picked and transferred to 50 mL of LB / Amp / Cp liquid medium and pre-cultured for approximately 16 hours at 30°C and 250 rpm. The entire culture was transferred to 950 mL of LB / Amp / Cp liquid medium and cultured at 37°C and 125 rpm. When the OD value reached approximately 0.6, 1 mL of 1 M IPTG was added to a final concentration of 1 mM to induce protein expression, and the culture was again continued at 37°C and 125 rpm for 5 hours.

[0045] The culture medium was transferred to a 1 L centrifuge tube and centrifuged at 4,500 rpm at 4°C for 20 minutes, and the supernatant was removed. Approximately 30 mL of the removed supernatant was used to suspend the precipitate, which was then centrifuged in a 50 mL centrifuge tube at 8,000 rpm at 4°C for 10 minutes, and the supernatant was removed.

[0046] The resulting cells were suspended in 30 mL of 1x Lysis buffer and sonicated three times (one cycle consisted of 0.5 seconds of pulse irradiation and 0.5 seconds of pause, with 30 cycles constituting one disruption program). The cells were then centrifuged at 8000 rpm at 4°C for 20 minutes, and the supernatant was removed. 30 mL of 1x Lysis Wash buffer was added to the pellet, which was then suspended. The pellet was then sonicated, centrifuged, and the supernatant was removed under the same conditions. 30 mL of 1x Lysis Wash buffer was added to the pellet, which was then suspended. The pellet was then sonicated, centrifuged, and the supernatant was removed under the same conditions.

[0047] The precipitate obtained by sonication was added to 30 mL of 10 mM Tris HCl (pH 8.7) containing 6 M GuHCl and stirred at 30°C for approximately 36 hours. The air-oxidized sample was centrifuged at 8000 rpm for 20 minutes at 4°C, and the supernatant was filtered through a 0.2 μm pore size filter. This was added to 5 mL of resin equilibrated with 10 mM Tris HCl (pH 8.7) containing 6 M GuHCl and stirred at 4°C for 1 hour. The resin-adsorbed sample was loaded onto an open column, and the eluate was collected as Flow Through 1. Flow Through 1 was loaded onto the column again, and the eluate was collected as Flow Through 2. Next, 5 mL of 10 mM Tris HCl (pH 6.7) containing 6 M GuHCl was added to the column, and the resin was allowed to adsorb for 5 minutes before being collected as Wash. This cycle was repeated three times.

[0048] The column was then loaded with 5 mL of 10% acetic acid containing 6 M GuHCl, and the resin was allowed to adsorb for 5 minutes before collecting the elute. This procedure was repeated three times (Elutes 1–3). The collected elute volumes (approximately 15 mL total) were transferred to an 8 cm dialysis membrane with a pore size of 3500 Da and dialyzed at 4°C. The RO water was replaced with 3 L of 50 mM Tris HCl (pH 8.0) four times, every 30 minutes. The solution was then replaced with 3 L of 50 mM Tris HCl (pH 8.0) overnight. The next day, the solution was replaced with 3 L of 50 mM Tris HCl (pH 8.0) for 2 hours.

[0049] After dialysis, the sample was centrifuged at 8000 rpm for 10 minutes at 4°C, and the supernatant was filtered through a 0.2 μm pore size filter. Protein concentration was measured using a nano dropper, and the protein amount was estimated. The required amount of Wako thrombin was added and stirred at 30°C for 3 hours. This step was not performed when purifying D3His.

[0050] Acetic acid was added to the sample to a final concentration of 10% and filtered through a 0.2 μm filter. Four mL of this mixture was injected into a reverse-phase HPLC system, and the peak of the target protein was collected. The acetonitrile, which was approximately 40% by volume in the collected sample, was evaporated using a speed vac for approximately 2 hours. After removing the acetonitrile, the sample was flash-frozen using liquid nitrogen, freeze-dried over one day, and stored at -30°C as a powder. Finally, the molecular weight of the purified protein was measured using MALDI-TOF / MS.

[0051] All samples for physical property measurements and immune response experiments were prepared by diluting stock protein prepared as described below with the specified buffer. MilliQ solution was added to powdered protein to a concentration of 1 mg / mL, and the protein was dissolved overnight on ice or in a refrigerator. This was centrifuged at 4°C and 20,000 x g for 20 minutes, and the supernatant was passed through a 0.2 μm filter to prepare stock protein. Unless otherwise specified, measurement samples had a protein concentration of 0.3 mg / mL and were prepared in phosphate-buffered saline (PBS, pH 7.4) as the buffer.

[0052] 1-2. DLS (Dynamic Light Scattering) Measurements DLS (Dynamic Light Scattering) measurements were performed using a Zetasizer Nano ZS (Malvern) to estimate the particle radius of the sample. Measurements were performed 5 to 6 times, and the integrated values ​​were obtained as measurement data. The hydrodynamic radius (Rh) was calculated from the obtained number-based distribution (Numbers) using the Einstein-Stokes equation.

[0053] 1-3. Immune Response Experiments. Immune response experiments were performed using 4-week-old female Jcl:ICR mice (CLEA Japan). Stock protein was diluted with PBS to a protein concentration of 0.3 mg / mL, and nickel sulfate solution was added to a final concentration of 10 μM. The resulting mixture was incubated at 37°C for 40 minutes. Each mouse received 30 μg of antigen protein (total volume: 100 μL) subcutaneously at weekly intervals for a total of five doses (Dose 1–5) (n=4). Six weeks after Dose 5, a booster dose of 30 μg of 0.3 mg / mL D3wt was administered subcutaneously (Dose 6). DLS was used to monitor the particle size and aggregation state of the samples immediately before each dose. Control mice received 0.3 mg / mL D3wt supplemented with 10 μM nickel (n=2) and the same concentration and volume of D3wt (n=1).

[0054] Four days after administration, blood samples were collected from the tail vein (TB: Tail Bleeding) (TB1-5 and TB11). Tail vein blood was diluted 50-fold with PBS and centrifuged at 25°C and 10,000 rpm for 5 minutes to remove cellular components such as red blood cells. The plasma fraction was then stored at -30°C. Blood samples were collected weekly, even during the non-administration period (TB6-10, 12, 13). Three weeks after Dose 6, whole blood was collected from the heart and the spleen was removed. As with the tail vein blood, the plasma fraction from cardiac blood was diluted 5-fold with PBS and a final concentration of 20% glycerol and stored at -30°C.

[0055] 1-4. ELISA Method The amount of anti-D3wt IgG antibody in mouse plasma was measured by indirect ELISA. Antigen (D3wt) was added to PBS at a concentration of 25 μg / mL, and 100 μL of the antigen was added to each well of a 96-well microtiter plate (TPP) and incubated overnight at 25°C (coating). Each well of the plate was washed four times with 200 μL of PBS, blotted, and then 200 μL of PBS containing 1% BSA was added and incubated at 37°C for 45 minutes (blocking). Each well of the plate was washed four times with 300 μL of PBS, blotted, and then 100 μL of mouse plasma was added. Mouse plasma was serially diluted 3-fold with PBS containing 0.1% BSA to 50x, 150x, 450x, 1350x, 4050x, 12150x, and 36450x concentrations. 100 μL of PBS (negative control) or positive control was added to empty wells and incubated at 37°C for 2 hours (primary antibody addition). The plate was then washed six times with 300 μL each of PBS containing 0.05% Tween 20 and five times with 300 μL each of PBS, blotted, and dried. 100 μL of IgG-HRP diluted 3000-fold in PBS containing 0.1% BSA and 0.05% Tween 20 was added to each well and incubated at 37°C for 90 minutes (secondary antibody addition). The plate was washed six times with 300 μL each of PBS containing 0.05% Tween 20 and five times with 300 μL each of PBS, blotted, and dried. 100 μL of OPD substrate (4 mg OPD, 4 μL H2O2 per 10 mL of citrate buffer (pH 5.0)) was added to each well and incubated at room temperature for 20 minutes (fluorescent substrate addition). After 20 minutes, 50 μL of 0.5 M sulfuric acid was added to each well as a reaction stopper, and the absorbance at 492 nm was measured using a corona multigrating microplate reader SH-9000Lab (Hitachi High-Tech Science Corporation).

[0056] 1-5. Flow cytometry The cells in the spleen were fluorescently stained and then counted and selected by flow cytometry. The removed spleen was ground in FACS buffer (PBS + 2% FBS + 1 mM EDTA (pH 8.0)) and centrifuged at 4°C and 4000 xg for 5 minutes. RBC (Red Blood Cell) Lysis buffer was added to the precipitate, and the mixture was centrifuged at 4°C and 4000 rpm for 5 minutes. This process was repeated twice to lyse red blood cells and remove them from the cell suspension. The cells were then resuspended in FACS buffer and observed under a microscope to determine the number of red blood cells in the spleen at 1000 xg. 6 The cells were diluted to 100 μL. Fluorescently labeled antibodies (anti-CD3 antibody PE / Cyanine5, anti-CD4 or anti-CD8 antibody PE / Cyanine7, anti-CD44 antibody FITC, anti-CD62L antibody PE: BioLegend) were added to the cell suspension and incubated at room temperature in the dark for 30 minutes. Unbound fluorescent antibodies were removed by washing with FACS buffer and centrifuging at 4°C and 4000 rpm for 5 minutes. The precipitate was resuspended in FACS buffer and analyzed using a CytoFLEX (Beckman Coulter).

[0057] Lymphocytes were first sorted based on forward scatter (FSC) and side scatter (SSC) signals, and then CD3-expressing T cells were selected from the selected group based on the fluorescence intensity of PE / Cyanine 5. Similarly, anti-CD4-expressing helper T cells and anti-CD8-expressing cytotoxic T cells were identified. The number of CD44-positive cells and CD62L-positive cells was counted within 10,000 events of CD4- or CD8-positive cells, counted as one measurement.

[0058] 2. Results and Discussion 2-1. Optimization of D3His for Metal Ion-Based Aggregation. First, we evaluated the effect of metal ions interacting with histidine on protein aggregation using DLS measurements. Six metals were used: divalent nickel ions (Ni: nickel sulfate), divalent cobalt ions (Co: cobalt chloride), divalent zinc ions (Zn: zinc sulfate), trivalent ferric ions (Fe: iron(III) chloride), divalent copper ions (Cu: copper sulfate), and divalent manganese ions (Mn: manganese chloride). DLS measurements revealed that all samples exhibited a single peak in the Number parameter, demonstrating that the protein existed in a single particle size group. In the absence of metal ions, D3His particle radii were 1.3–2 nm, and, like D3wt, existed in solution as monomers. This indicates that the histidine tag itself is not a factor inducing protein aggregation.

[0059] D3His was shown to form aggregates in the presence of the divalent or trivalent metal ions tested (Figure 1). In particular, D3His formed aggregates with radii of over 200 nm upon addition of nickel and cobalt. Nickel and cobalt have higher affinity than other metals, likely resulting in tighter binding to the histidine tag and growing the aggregates. Addition of zinc also led to the formation of aggregates, but the particle radius increased with temperature and time. Addition of iron resulted in the formation of small aggregates with radii of approximately 10 nm. Comparing nickel and cobalt, which formed large aggregates, the particle radius increased with increasing temperature from 25°C to 37°C and decreased with cooling back to 25°C when nickel was added and continued to grow the aggregates even when cooled from 37°C to 25°C.

[0060] Next, we investigated the buffer most suitable for nickel-D3His complex formation. The pH values ​​measured were 4.1, 7.1, 7.4, and 8.8. Samples were prepared by diluting stock protein with MilliQ (MQ), 10 mM phosphate buffer (PB), PBS, and 10 mM Tris-HCl buffer (Tris-HCl), respectively. Because the stock protein had a low pH due to residual trifluoroacetic acid remaining after lyophilization, dilution with MQ resulted in a low-pH sample.

[0061] The measurement results are shown in Figure 2. As shown in Figure 2, the particle radius of D3His at pH 7.4 and 7.1 increased significantly with the addition of nickel. Next, the particle radius of the sample at pH 8.8 was large, and at pH 4.1, D3His did not form aggregates even when nickel was added. The dissociation pH of the histidine side chain is 6.0, and at lower pHs, the nitrogen atom on the imidazole group is protonated. Nickel ions Ni 2+ compensates for its missing electrons by forming a coordinate bond with the non-bonding electron pair of histidine, but when the pH is low and the histidine is protonated, this coordinate bond does not occur. This is thought to be why aggregates do not form. Under buffer conditions with a pH of 6.0 or higher, aggregates were formed in all cases, but differences in particle radius were observed depending on the pH.

[0062] In particular, at pH 8.8, the particle radius was 10-20 nm, smaller than the aggregates formed at other pH levels. At this pH, protonation of proteins and buffer components other than histidine had not occurred, and there were many unshared electron pairs other than histidine, suggesting that nonspecific nickel coordination bonds occurred. In both pH 7.4 and pH 7.1 buffers, large aggregates with a maximum radius of approximately 1000 nm were formed. Among these, PBS, a physiological saline solution with pH buffering properties that matches the osmotic pressure of body fluids, was selected as the buffer, considering the possibility of future administration to mice.

[0063] 2-2. Particle size of D3His-nickel aggregates Next, we investigated the effect of the nickel concentration added to D3His on aggregate formation. When nickel was added to D3wt, the particle radius was 1.7-2.3 nm, regardless of the nickel concentration, 5 μM or 10 μM, and there was no change in size compared to D3wt without nickel. This indicates that nickel-mediated aggregate formation is due to an interaction with the histidine tag, and not an interaction with the dengue protein itself.

[0064] Furthermore, when nickel was added to D3His, the particle radius increased depending on the nickel concentration. The particle radius increased significantly between 3 μM and 5 μM (Fig. 3). The dengue protein concentration used in the measurements was 0.3 mg / mL, which, when converted to molar concentrations, was 25.8 μM for D3wt and 22.4 μM for D3His, respectively. This means that the addition of nickel at a molar concentration ratio of 4 to 5 times lower promoted the formation of large aggregates.

[0065] Without the addition of nickel, D3His existed as a monomer. The number of monomers in the aggregate could be estimated by calculating the volume from the particle radius of the aggregate. It was estimated that D3His formed a nonamer in the presence of 3 μM nickel, and 1.07 million in the presence of 5 μM nickel. This is thought to be due to hydrophobic interactions between aggregates, which did not form up to about 10-mer aggregates, beginning to function as the nickel concentration increased, eventually resulting in stable aggregates with large particle radii. However, when the aggregates were present in solution, there was an upper limit to the nickel concentration. Addition of 50 μM nickel resulted in precipitation of the measurement sample, making it impossible to evaluate aggregate formation using DLS.

[0066] From the above experiments, it can be seen that recombinant proteins having histidine tags can be associated with metal ions, and that aggregates and / or associations of the proteins can be produced extremely easily.

[0067] 2-3. Immune response experiment Although the results are not shown, when the particle diameter of the samples was measured immediately before each administration, there was little difference in particle diameter between administrations for all recombinant proteins, and stable aggregates could be administered. The particle diameter of the D3His aggregate was particularly stable at 37°C, suggesting that the aggregate would be maintained in vivo.

[0068] After Dose 5, the mice had received a total of approximately 750 ng of nickel sulfate, but all survived and did not develop sarcomas at the exposed site, a commonly reported effect of nickel on the body. Furthermore, the mice grew and survived as healthy as mice that had not been exposed to nickel until sacrifice by cardiac blood collection.

[0069] Furthermore, although the results are not shown, mice that had received the D3His-nickel complex after Dose 5 and the initial immunization had finished showed higher antibody titers than mice that had received D3wt alone. Furthermore, the rise in IgG antibody titers occurred at the end of Dose 3, and antibody production was most active in three out of four mice at TB8, suggesting that the immunogenicity induced by this complex requires approximately two weeks after antigen administration.

[0070] Surprisingly, mice administered a mixture of D3wt and nickel, which should be a monomer, also showed enhanced immunogenicity. Antibody titers began to increase from the end of Dose 2, and while some individuals had the highest antibody production at TB6, others showed an increase in antibody titers about two weeks after antigen administration, demonstrating a tendency for immune responses similar to those seen in mice administered the D3His-nickel complex. Furthermore, individuals whose antibody titers increased early also showed a rapid decline in antibody titers, indicating that the duration of immunity does not depend on the speed at which an immune response occurs after encountering the antigen.

[0071] As an example, the results of measuring IgG antibody titers by ELISA at weeks 7 and 13 are shown in Figure 4. Although not shown in Figure 4, the specific increase in antibody titer was 529-fold higher in mice administered the D3His+Ni conjugate at TB8 compared with that in mice administered D3wt, and 656-fold higher in mice administered the D3wt+Ni monomer. These results suggest that the D3His+Ni conjugate sufficiently enhanced immunogenicity, and that D3wt+Ni also enhanced immunogenicity, suggesting the possibility that nickel may function as an immune enhancer.

[0072] Furthermore, when D3wt alone was given as a booster immunization after TB10, antibody titers increased in all mice, regardless of whether they were D3wt or D3His, exceeding the maximum titers observed after the initial immunization. While both the D3His+Ni aggregate and the D3wt+Ni monomer immunization induced immunological memory, the duration of the subsequent immune response differed. Mice administered with D3wt+Ni monomer exhibited high antibody titers 2 weeks after the booster immunization (TB12), but this decreased 1 week later, suggesting that the immunological memory induced by a single booster immunization only lasted 1 week. In contrast, mice administered with D3His+Ni aggregates initially showed an increase in antibody titers at TB12, similar to those administered with D3wt+Ni, but antibody titers remained constant or even increased the following week, demonstrating the induction of long-lasting immunological memory. Based on the above results, it is possible to select the antigen to be administered according to the purpose. For example, if a short-term but strong immune response or immune memory is desired, D3wt+Ni monomer should be administered, whereas if a long-term maintenance of immune memory or immune response is desired despite a lower antibody titer than D3wt+Ni, D3His+Ni aggregate should be administered.

[0073] ELISA assays of IgG antibody titers revealed that D3His+Ni aggregates and D3wt+Ni monomers enhanced B cell-dependent immunogenicity and induced immunological memory. T cell-derived immunological memory was then assessed by fluorescent detection of cell surface CD (Cluster of Differentiation) markers using a flow cytometer. The presence or absence of CD44 or CD62L on CD4- or CD8-expressing helper or cytotoxic T cells can determine whether they are memory cells or classify them as memory cells. CD44+CD62L+ (co-expressing CD44 and CD62L) cells are central memory T cells, CD44+CD62- cells are effector memory T cells, and CD44-CD62+ cells are naive T cells.

[0074] The results of fluorescence detection using a flow cytometer when D3His+Ni aggregate, D3wt+Ni monomer, or D3wt alone was administered are shown in FIG.

[0075] As shown in Figure 5, in mice that were administered only D3wt as a negative control, in which antibody titers did not increase sufficiently, 84% of the helper T cells were naive T cells, 6.31% were central memory T cells, and 3.98% were effector memory T cells, indicating that differentiation into memory cells did not occur in most of the helper T cells.

[0076] In contrast, as shown in Figure 5, the differentiation of helper T cells in mice administered the D3His+Ni conjugate showed that 55.53% were naive T cells, 19.32% were central memory T cells, and 15.57% were effector T cells, with approximately 35% of the total differentiating into memory T cells. Similarly, in mice administered the D3wt+Ni monomer, which also had high antibody titers, 26% of the helper T cells differentiated into memory cells (central memory T cells: 5.61%, effector memory T cells: 20.87%), while only 50.82% were naive T cells, as shown in Figure 5.

[0077] Compared with administration of D3wt alone, it can be said that both administration of the D3His+Ni aggregate and administration of the D3wt+Ni monomer were able to sufficiently induce immunological memory.

[0078] While the total production of memory T cells was increased by both D3His+Ni aggregates and D3wt+Ni monomers, the proportion of each type of memory T cell varied. Mice treated with D3His+Ni aggregates produced central memory T cells and effector memory T cells at roughly equal rates. In contrast, mice treated with D3wt+Ni monomers had a higher proportion of effector memory T cells. Central memory T cells are generally abundant in secondary lymphoid tissues, where they respond with high sensitivity upon re-exposure to the same antigen and play a key role in T cell proliferation. Effector memory T cells circulate in peripheral tissues and secondary lymphoid tissues, and upon antigen exposure, they rapidly differentiate into effector T cells and initiate an immune response. Therefore, the higher proportion of central memory T cells in mice treated with D3His+Ni aggregates may have contributed to the efficient proliferation of T cells during the immune response induced after booster administration, maintaining high antibody titers.

[0079] On the other hand, the proportion of central memory T cells in D3wt+Ni-treated mice was low, so the duration of the immune response that occurred after the booster immunization was shorter than that in D3His+Ni-treated mice. However, due to the large number of effector memory T cells, differentiation into effector memory T cells occurred quickly, which is thought to have led to a rapid increase in antibody titers.

[0080] The results of the immune response experiments above demonstrated that the His-tagged D3ED3 / nickel complex enhanced immunogenicity and induced immunological memory (Fig. 6). Furthermore, the control D3wt / nickel complex also enhanced immunogenicity and induced immunological memory, suggesting that very low concentrations of nickel ions act as an adjuvant (Fig. 6).

[0081] [Example 2] In this example, we investigated the production of a histidine-tagged recombinant protein aggregate using nickel ions and the purification of proteins using the aggregate. Histidine-tagged Japanese encephalitis virus envelope protein (JEV-ED3), BSA (commercially available), and histidine-tagged JEV-ED3 and BSA at a concentration of 0.3 mg / mL were purified with 100 μM Ni ions. 2+ The cells were incubated in 1× phosphate buffered saline (PBS, pH: 7.4) for 60 minutes at 37°C in the presence or absence of .

[0082] As a result, only histidine-tagged JEV-ED3 precipitated. 2+ These results suggest that Ni induces the precipitation of histidine-tagged JEV-ED3 through aggregate formation. 2+ Precipitation of histidine-tagged JEV-ED3 induced by Ni 2+ The results depend on the concentration and incubation time of Ni. 2+ At this concentration, more than 90% of JEV-ED3 was precipitated by incubation at 37°C for 60-75 minutes (Fig. 7).

[0083] Interestingly, at very low concentrations of Ni 2+ Incubation of histidine-tagged JEV-ED3 in the presence of α-glucan (1-50 μM) resulted in the formation of non-visible soluble aggregates of various sizes (Ni 2+ The aggregate size increased with increasing concentration), and finally precipitated at high Ni2+ concentrations. 2+ Circular dichroism spectroscopy in the presence of HCl demonstrated retention of near-native secondary structure in the non-visible aggregates.

[0084] These observations suggest that Ni 2+ These results clearly suggest that the Ni-induced protein precipitation is due to oligomerization of histidine-tagged JEV-ED3. Furthermore, very low to high concentrations (0.05 to 0.35 mg / mL) of JEV-ED3 inhibit Ni 2+ These findings suggest that Ni 2+This suggests that inducible precipitation may be useful for purifying proteins with very low expression levels, and the method of the present invention allows for the production of proteins with nearly 100% purity in analytical HPLC chromatograms in a single step.

[0085] Example 3 In this example, using a histidine-tagged Japanese encephalitis virus envelope protein as a model, we attempted to improve immunogenicity by forming an aggregate through the addition of an extremely low concentration (1 to 100 μM) of metal ions.

[0086] The amino acid sequences of the Japanese encephalitis virus envelope protein (JEV Wt ED3) used in this example and the histidine-tagged protein (JEV Wt-HIS ED3) are shown in Table 2.

[0087]

[0088] First, we analyzed the state of monomers and aggregates of JEV ED3 in the presence or absence of metal ions (Co, Cu, Mn, Ni, and Zn). Figure 8 shows the particle radius (Rh) measured by dynamic light scattering (DLS) at 25°C and 37°C.

[0089] Next, immunization experiments were performed as follows. JEV Wt ED3 and JEV Wt-HIS ED3 were prepared in phosphate-buffered saline (1X PBS, pH 7.4) at 15 μg / 100 μL in the presence or absence of 5 μM metal ions. At the start of the experiment, 4-week-old female Jcl:ICR (CLEA Shizuoka Japan) mice were divided into 13 groups containing at least three mice. Specifically, six groups contained JEV Wt ED3 with added metal ions (Wt, Wt + Co, Wt + Cu, Wt + Mn, Wt + Ni, and Wt + Zn), six groups contained histidine-tagged JEV Wt-His ED3 with added metal ions (Wt-His, Wt-His + Co, Wt-His + Cu, Wt-His + Mn, Wt-His + Ni, and Wt-His + Zn), and a control group.

[0090] Mice were subcutaneously injected at 14-day intervals. The JEV Wt ED3 group (Wt + Co) received three doses because of the strong immune response from the beginning, while the other groups received four doses (post-TB11). The control group received PBS alone. Anti-JEV ED3 IgG responses were monitored weekly by ELISA.

[0091] The results showed that the addition of metal ions improved the immunogenicity of the antigen, the Japanese encephalitis virus envelope protein, regardless of the presence or absence of a histidine tag. In other words, when the histidine tag was not added, no aggregates were formed even with the addition of metal ions, but immunogenicity was dramatically improved. In this example, Co enhanced the immune response regardless of the presence or absence of a His tag (Figures 9 and 10).

[0092] In this example, Ni(II) was added to a histidine-tagged anti-EGFR antibody to form an aggregate, and the binding affinity to EGFR was examined depending on whether the aggregate was formed. The sequence of VHH-C-His, in which a histidine tag was added to the C-terminus of the anti-EGFR antibody VHH-7D12 used as a model protein, is shown in Table 3.

[0093]

[0094] VHH-C-His protein was adjusted to 0.30 mg / ml in phosphate-buffered saline (1x PBS, pH 7.4) with or without 50 μM Ni ions. It was then incubated at 25°C for 30 minutes and diluted 1:10 with 1x PBS. This sample was immobilized on a chip (Octet® NTA Biosensors). Binding affinity was assessed by BLI using a SARTORIUS Octet N1.

[0095] The analyte used was EGFR protein from MedChem Express. Measurements were performed at five serially diluted concentrations (559.8, 279.9, 140, 69.98, and 34.99 nM), and the dissociation constants were calculated using global fitting (1:1) (in the presence of Ni ions). Samples without Ni ions were measured at four concentrations, excluding 140 nM. A sample containing only PBS was used as a control. DLS measurements were also performed on the same samples simultaneously with the BLI measurements.

[0096] In the presence of Ni ions, VHH-C-His proteins were immobilized in an aggregated state, and the particle radius (Rh) remained in the range of 400–600 nm regardless of the concentration of EGFR added. In the absence of Ni ions, VHH-C-His proteins were immobilized in a monomeric form.

[0097] K obtained by BLI measurement of immobilized aggregates (in the presence of Ni ions) and monomers (in the absence of Ni ions) D , k a , k ｄ The results are shown in Table 4. These results indicate that the EGFR protein binds with approximately 14-fold higher affinity when the VHH-C-His assemblies are immobilized compared to when the VHH-C-His monomers are immobilized.

[0098]

[0099] The results of this example demonstrate that immobilizing proteins that can serve as ligands on a chip in an aggregated state enables the binding of proteins that bind to the ligand with significantly higher affinity than when immobilized as monomers. This indicates that protein aggregate formation in the presence of metal ions not only facilitates the purification of the protein, but also helps improve methods for detecting and purifying binding partners that utilize the immobilization of the protein.

[0100] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. An immune enhancing agent comprising as an active ingredient an ion of at least one metal selected from the group consisting of nickel, cobalt, zinc, iron, copper and manganese, or a salt of said metal.

2. The immune enhancing agent according to claim 1, which is added to a solution containing an antigen so as to have a concentration in the range of 3 to 100 μM.

3. The immune enhancing agent according to claim 1, wherein the antigen is a recombinant protein having a histidine tag.

4. The immune enhancing agent according to claim 1, characterized in that the antigen is a recombinant protein having no histidine tag.

5. A method for enhancing immunity, which comprises administering to a non-human animal a solution containing an antigen and ions of at least one metal selected from the group consisting of nickel, cobalt, zinc, iron, copper and manganese.

6. The immune enhancing method according to claim 5, wherein the concentration of said metal ions is 3 to 100 μM.

7. The immune enhancing method according to claim 5, wherein the antigen is a recombinant protein having a histidine tag.

8. The immune enhancing method according to claim 5, wherein the antigen is a recombinant protein having no histidine tag.

9. A method for producing protein aggregates and / or associations, comprising the step of mixing, into a solution containing a recombinant protein having a histidine tag, ions of at least one metal selected from the group consisting of nickel, cobalt, zinc, iron, copper and manganese, or a metal salt capable of generating ions of such a metal in the solution, thereby aggregating and / or associating the recombinant protein with the ions of the metal.

10. The method for producing protein aggregates and / or associations according to claim 9, characterized in that the metal ions are added to the solution to have a concentration in the range of 3 to 100 μM.

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