JP5633152B2 - Adjuvant - Google Patents

Description

  The present invention relates to a novel adjuvant and a method for immunizing an animal using the same. In particular, the present invention is useful as an adjuvant for immunizing animals with DNA.

  In the conventional immunization method in which a protein or a partial peptide thereof is used as an antigen, humoral immunity can be induced but cellular immunity is often difficult to induce. For this reason, live vaccines with attenuated microorganisms and viruses are used for infectious diseases that need to induce cellular immunity. However, live vaccines have problems to be solved in terms of safety such as side effects. Many. On the other hand, as another immunization method, a DNA immunization method for inducing humoral immunity and cellular immunity against the target protein by administering an expression vector containing the target protein gene to an animal is known (Patent Document 1, Non-Patent Documents 1 to 4).

  Since DNA immunization can induce both humoral immunity and cellular immunity, clinical application research is being promoted as a safe immunization technique for infectious diseases in which induction of cellular immunity is difficult. Further, in recent years, it has been difficult to obtain animals by the conventional antibody production method (ie, the method of producing an antibody from the antibody-producing cells obtained by immunization using a protein or a partial peptide thereof as an antigen) because of the ability to induce humoral immunity. Create antibodies that specifically recognize antigenic proteins whose amino acid sequences are highly conserved among species, and antibodies that specifically recognize antigenic proteins that have a structure that exists naturally in the living body (so-called natural type) (Patent Documents 2 and 3, Non-Patent Document 5). In conventional immunization methods, there is a possibility that the structure changes in the process of preparing a protein or peptide as an immunogen, so that the antibody obtained by using the above method has high specificity and binding to the natural antigen. May not have power. On the other hand, in the DNA immunization method, the natural antigen protein is expressed in the body of the immunized animal by the expression vector containing the administered antigen protein gene, and thus has high specificity and binding power to the natural antigen. Antibody production is possible.

  In the DNA immunization method, an expression vector containing an antigen protein gene is taken into a cell in the vicinity of the immunization site to express the antigen protein in the cell, and immunity against the antigen protein is induced. It is important to efficiently introduce an expression vector and to effectively induce the immune mechanism of the cell itself. Further, in the DNA immunization method, the amount of antigen protein expressed in the body of the immunized animal is significantly smaller than the amount of antigen administered by the conventional immunization method, and the sensitizing effect is low as compared with the conventional immunization method. Therefore, it is necessary to enhance the sensitization effect by other methods.

JP 2000-582427 Gazette JP 2004-344118 A JP 2009-067678 A JP 2002-114708 A JP 2008-174466 A JP-A-1-252599

Proc. Natl. Acad. Sci. USA, 94, 14626-14631 (1997) Annu. Rev. Immunol. 18, 927-974 (2000) Nature, 356, 152-154 (1992) Immunol. Today, 19, 89-97 (1998) Nature Biotechnology, 21 (9), 1088-1092 (2003) Japanese Journal of Gout and Nucleic Acid Metabolism, 29 (2), 119-124 (2005)

  For the purpose of enhancing the sensitization effect in the DNA immunization method, methods for immunization and immunostimulatory substances (adjuvants) have been studied.

  As for the immunization method, the gene gun is said to have the highest sensitization effect, but it requires the preparation of antigen gold particles and the use of a dedicated immunity device. There are many problems to be solved for clinical application, such as a marked decrease in In addition, although research is underway to improve the expression vector stability and introduction efficiency by modifying the expression vector with ion polymers, liposomes, viral envelope vectors, etc., and sonoporation using ultrasound, etc. A method with a high sensitizing effect is required.

On the other hand, for adjuvants,
(1) Induction of the immune mechanism of the immunized cell itself due to inflammation, etc.
(2) Promotion of antigen uptake into immunized cells,
(3) Sustained release of antigen (so-called sustained release action),
For example, Ribi adjuvant (manufactured by Corixa), oil-containing adjuvant such as Titermax (manufactured by Titermax), α antigen, muramyl dipeptide (MDP), lipid A, lipopolysaccharide (LPS) ) And other bacterial-derived adjuvants, inorganic compounds such as aluminum phosphate, aluminum hydroxide and calcium phosphate, liposomes, saponins complexed with membrane protein antigens (immunostimulatory complexes), polyamines such as spermidine, various cytokine genes Alternatively, cytokine proteins are used alone or in combination (Patent Documents 4 and 5). However, the improvement of the sensitization effect by the exemplified adjuvant is not always sufficient.

  An object of the present invention is to provide a novel adjuvant capable of enhancing the sensitizing effect particularly in DNA immunization, and a method for immunizing an animal using the adjuvant.

  As a result of intensive studies to solve the above problems, the needle-like crystal of the inorganic compound promotes the uptake of cells in the vicinity of the antigen immunity site by physically damaging the cells, and also causes inflammation of the cells. And found that a sufficient sensitizing effect can be obtained.

  That is, the first invention is an adjuvant containing a needle crystal of an inorganic compound.

  Moreover, 2nd invention is an adjuvant as described in 1st invention whose acicular crystal | crystallization is a crystal body containing an acicular single crystal.

  The third invention is the first invention, wherein the inorganic compound is zinc oxide, and the acicular crystal is a crystal composed of a core and a needle-like single crystal extending from the core in different four-axis directions. The adjuvant described in 1. above.

  The fourth invention is a method for immunizing an animal, comprising a step of administering to the animal a mixture of the adjuvant and antigen according to any one of the first to third inventions.

  The fifth invention is the method according to the fourth invention, wherein the antigen is an expression vector containing a protein gene.

  The sixth invention is a method for producing an antibody using the antibody-producing cells obtained by the method described in the fourth or fifth invention.

  Hereinafter, the present invention will be described in more detail.

  The present invention is characterized in that a sharp and strong inorganic acicular crystal is used as an adjuvant, and the acicular crystal induces physical injury and inflammation of cells, thereby immunizing an animal. The sensitization effect of an antigen administered together with an adjuvant can be enhanced and humoral immunity can be induced. As long as the needle-like crystal of the inorganic compound used in the adjuvant of the present invention contains a sharp and strong needle-like crystal, it may be a needle-like crystal made of a polycrystal or a needle-like crystal made of a single crystal ( A so-called whisker) may be used, but a needle-like crystal including a needle-like crystal made of a single crystal, which has few structural defects such as crystal grain boundaries and has almost no impurities and high strength, is preferable. Examples of the preferred inorganic crystalline needle crystals include graphite whiskers, potassium titanate whiskers, alumina whiskers, silicon carbide whiskers, silicon nitride whiskers, mullite whiskers, magnesia whiskers, magnesium borate whiskers, zinc oxide whiskers, and boride. The whisker used as an additive for increasing the strength of resin, metal, ceramics, etc., such as titanium whisker, can be mentioned.

  As an example of a preferable needle-like crystal of an inorganic compound contained in the adjuvant of the present invention, a zinc oxide whisker having a three-dimensional shape consisting of a core and a needle-like single crystal extending from the core in different four axial directions ( For example, Panatetra (manufactured by Amtec), which is commercially available as an additive for improving the precision moldability, sliding, abrasion resistance, antistatic property, and conductivity of a resin, is described above. It can be used as a zinc whisker.

  The adjuvant of the present invention only needs to contain an acicular crystal of an inorganic compound, may be an acicular crystal of an inorganic compound, or a known substance used as an adjuvant (for example, polyamines such as spermidine, It may be mixed with uric acid crystals.

  The amount of adjuvant used when immunizing an animal by administering the adjuvant of the present invention and an antigen to the animal depends on the size of the animal to be immunized and the inorganic compound in the needle crystal of the inorganic compound contained in the adjuvant of the present invention. And the length of the acicular crystal (needle-shaped single crystal part), etc., may be appropriately determined. For animals that are normally used for immunization, such as mice and rats, one individual in one immunization The adjuvant of the present invention containing about 50 to 5000 μg of an inorganic compound needle-like crystal may be administered together with the antigen. For example, the animal to be immunized is a mouse, and the zinc oxide whisker having a three-dimensional shape consisting of a nucleus and an acicular single crystal portion extending in different four-axis directions from the nucleus (Patent Document 6). In this case, the adjuvant of the present invention containing 800 to 1000 μg (for example, 900 μg) of the zinc oxide whisker may be administered together with the antigen to one mouse in one immunization.

  The needle-like crystal of an inorganic compound contained in the adjuvant of the present invention enhances the sensitizing effect by inducing physical damage and inflammation of cells. Therefore, in the method of immunizing an animal using the adjuvant of the present invention, the antigen administered together with the adjuvant may be a protein or a partial peptide thereof or an expression vector containing a protein gene. The effect of the present invention is great when an expression vector containing a protein gene is used as an antigen.

  In order to confirm whether the immunized animal obtained by the method of immunizing an animal using the adjuvant of the present invention produces an antibody, an immunoassay commonly used by those skilled in the art may be used. For example, liquid phase or solid phase EIA (enzyme immunoassay) using activated antigen, Western blotting, flow cytometry using a transient expression cell line into which an antigen gene has been introduced, or cell ELISA (Enzyme-Linked Immunosorbent Assay).

  The method of immunizing an animal using the adjuvant of the present invention is not limited to application to a vaccine for the purpose of disease treatment or disease protection in animals such as humans, but a polyclonal antibody of the immunized animal against the administered antigen. Alternatively, it can be applied to the production of monoclonal antibodies. In order to produce the antibody, an animal is immunized by administering a mixture of the adjuvant of the present invention and an antigen, and spleen, lymph nodes, peripheral blood, etc. are collected from the immunized animal and antibody-producing B cells are obtained. An antibody specific to the administered antigen can be produced by fusing the B cell with an appropriate immortal cell such as myeloma by a method known to those skilled in the art to produce a hybridoma.

  The present invention is characterized in that an acicular crystal of an inorganic compound is used as an adjuvant, and the acicular crystal accelerates the uptake of antigen into the cell by physically damaging the cells around the immune site. Moreover, humoral immunity can be effectively induced by activating the immune response reaction of the immunized object itself by inducing a strong inflammatory action in the periphery of the immunized site.

  The adjuvant of the present invention is a DNA immunization method in which an animal is immunized with an expression vector containing an antigen protein gene to immunize the animal, and can enhance the sensitizing effect on the expressed antigen protein in the immunized animal. Therefore, antigen proteins with highly conserved amino acid sequences between animal species and natural antigen proteins, which were difficult with conventional immunization methods (methods of immunizing animals by administering proteins or partial peptides thereof) Immunity to can be easily given to animals.

  The method for immunizing an animal using the adjuvant of the present invention is not limited to merely immunizing an animal with an antigen administered together with the adjuvant of the present invention, and an antibody-producing cell is obtained from the immunized animal and appropriately immortalized. An antibody specific to the administered antigen can be produced by producing a hybridoma by fusing with a modified cell.

The figure which showed the result of having performed the FACS analysis by the anti-FLAG antibody about the cell which introduce | transduced SND1 full length protein gene (SND1 full length section) or SND1 Region 3 gene (Region 3 section). The FACS assay format is shown on the right side of the figure. The more the fluorescence intensity (Count) is shifted to the right, the greater the protein expression level. The figure which showed the mouse | mouth serum antibody titer measurement result of each immunity group by CELISA analysis. The vertical axis represents the fluorescence intensity. The figure which showed the mouse serum antibody titer measurement result of each immunity group by FACS analysis. The vertical axis represents the fluorescence intensity. The figure which showed the average value (bar graph) of each immunity group obtained by CELISA analysis and FACS analysis, and a standard error (error bar).

  Examples will be shown below to describe the present invention in more detail. However, these examples are only examples of the present invention, and the present invention is not limited to the examples.

Example 1 Construction of Expression Vector In order to effectively induce humoral immunity by DNA immunization, it is desirable to localize the target antigen protein as a membrane-bound protein on the cell surface. In this study, Homo sapiens Staphylococcal Nuclease Domain Containing 1 (hereinafter referred to as SND1), which is an intracellular protein, was selected as a target antigen protein, so that a plasmid vector capable of expressing a protein having a GPI anchor added to SND1 was constructed.
(1) Gene of full length (genbank accession No. NM — 014390 region 230 to 2956) or region 3 (GenBank No. NM — 014390 region 1793 to 2956) excluding the start and stop codons of SND1 cDNA according to a conventional method The fragment was amplified by the RT-PCR method and then inserted into the BamHI-EcoRI site of pVAC1 (Invivogen). The primer combinations used for amplification are shown below.
(A) Primer for full-length amplification of SND1 Forward: 5′-gcatccaggggtcctccgcgcagagggg-3 ′ (SEQ ID NO: 1, 6 ′ base on the 5 ′ terminal side is BamHI recognition sequence, 3 bases on the 20 ′ terminal side are GenBank No. Equivalent to an array)
Reverse: 5'-gaattcgcggctgtcccaaaattcgt-3 '(SEQ ID NO: 3, 5' terminal 6 bases are EcoRI recognition sequences, 3 'terminal 20 bases correspond to GenBank No. NM_014390 from 2937 to 2956)
(B) SND1 Region 3 amplification primer Forward: 5′-ggatccagcgggcagggtcgttctgaagc-3 ′ (SEQ ID NO: 2, 5 ′ terminal 6 bases are BamHI recognition sequences, 3 ′ terminal 20 bases are GenBank No. NM — 014318th 1793 to 179318 Equivalent to
Reverse: 5′-gaattcgcggctgtcccaaaattcgt-3 ′ (SEQ ID NO: 3)
(2) After amplification of each gene fragment containing the full length of SND1 or the GPI anchor region of Region 3 and PVAC1-derived PAL1 derived Alkaline phosphatase by the PCR method, the gene fragment containing the full length of SND1 and the GPI anchor is pFLAG-Myc-CMV-21 (Invitrogen) The gene fragment containing SND1 Region 3 and the GPI anchor was inserted into the HindIII-BamHI site of pFLAG (manufactured by Invitrogen). The primer combinations used for amplification are shown below.
(C) (SND1 full length + GPI anchor) Amplification primer Forward: 5′-aagctttgcgtcctccgcgcaggcgg-3 ′ (SEQ ID NO: 4, 5 ′ terminal side 6 base is HindIII recognition sequence, 3 ′ terminal side 20 base is GenBank No. NM — 014390 230 To the 249th sequence from
Reverse: 5'-ggaccctcaggtttaggggagcagggg-3 '(SEQ ID NO: 6, 5' terminal 6 bases are EcoRI recognition sequences)
(D) Primer for amplification (SND1 Region 3 + GPI anchor) Forward: 5′-aagctttggggcagtcgttctgaagc-3 ′ (SEQ ID NO: 5, 5′-end 6 bases are HindIII recognition sequences, 3′-end 20 bases are GenBank No. NM — 014390 Corresponds to the 1812th sequence from
Reverse: 5′-ggatcctcaggtttagggggaggtgg-3 ′ (SEQ ID NO: 6)
(3) In order to confirm that the protein expressed by the gene inserted into each plasmid vector is localized on the cell surface as expected, using Chinese hamster ovary-derived mammalian cells (CHO-K1 cell line) Verified.
(3-1) The plasmid vector constructed in (2) was introduced into the CHO-K1 cell line according to a conventional method and transiently expressed.
(3-2) The introduced CHO-K1 cell line was cultured at 37 ° C. for 24 hours using a 10% FCS-added Hams F12 medium (manufactured by Wako Pure Chemical Industries, Ltd.) in a 5% CO ₂ incubator.
(3-3) A mouse anti-FLAG M2 antibody (manufactured by SIGMA) against the FLAG tag inserted in the introduced plasmid vector is added to the culture solution of (3-2) and left for 30 minutes, and then fluorescently labeled The anti-mouse IgG antibody (manufactured by BECKMAN COULTER) was added and allowed to stand for 30 minutes, and then FACS (Fluorescence Activated Cell Sorting) analysis was performed. In addition, the same analysis was performed using the CHO-K1 cell line culture solution which has not carried out gene transfer as a target section.

  The result of FACS analysis is shown in FIG. As a result of the analysis, no response was observed in the control group, but any of the cells into which the SND1 full-length gene (SND1 full-length group) or the SND1 Region 3 gene (Region 3 group) had been introduced showed a reaction. From this result, it was shown that the SND1 full length protein or SND1 Region 3 protein expressed was localized on the cell surface. In addition, since the reactivity of the FLAG antibody is higher in the Region 3 section than in the SND1 full-length section, it is presumed that the expression level on the cell surface is increased by fragmenting the full length.

Example 2 Immunization and Blood Collection (1) A plasmid vector into which SND1 Region 3 gene was inserted was prepared in 100 μL of a 5% sucrose solution so as to contain 40 μg of DNA and administered to A / J mice. / J mice were immunized. As an immunization group, a control group (administered only 40 μg of plasmid vector), a zinc oxide whisker group (administered 40 μg of plasmid vector and 900 μg of zinc oxide whisker (trade name: Panatetra, manufactured by Amtec)), a uric acid crystal group (40 μg of plasmid and 100 μL of a uric acid crystal saturated solution was administered, and uric acid crystals were prepared by the method described in Non-Patent Document 6), and 4 A / J mice were used for each immunization group.
(2) 3 days, 5 days, 7 days, 9 days, 12 days, 14 days, 21 days, 28 days, and 35 days after the first immunization, additional immunization is performed, and blood is collected on the 42nd day after the start of the first immunization. Antiserum was collected. Only in the zinc oxide whisker group, a strong inflammatory reaction accompanied by congestion was observed around the immunized site 7 days after the start of immunization.

Example 3 Production of SND1 full-length protein-expressing cells A CHO-K1 cell line that constantly expresses SND1 full-length protein was prepared for antiserum evaluation.
(1) After introducing the plasmid having the full-length SND1 gene into a CHO-K1 cell line according to a conventional method, using a Hams F12 medium (manufactured by Wako Pure Chemical Industries, Ltd.) with 10% FCS in a 5% CO ₂ incubator, The cells were cultured for 24 hours at 37 ° C.
(2) After culturing, an antibiotic Geneticin solution (manufactured by Invitrogen) was added to a concentration of 250 μg / mL and cultured for 3 weeks.
(3) CHO-K1 cells that constantly express the full-length SND1 protein were collected using a cell sorter using an anti-FLAG antibody.

Example 4 Evaluation of Mouse Antisera Each mouse antiserum prepared in Example 2 was analyzed by cell enzyme immunoassay (CELISA) and FACS using the SND1 full-length protein-expressing cells prepared in Example 3. The antiserum used was diluted 1000-fold with PBS containing 3% fetal bovine serum.
(1) CELISA analysis (1-1) SND1 full-length protein-expressing cells prepared in Example 3 were coated on a 96-well plate at 5 × 10 ⁴ cells per well, and 10% FCS-added Hams in a 5% CO ₂ incubator. The cells were cultured at 37 ° C. for 24 hours using F12 medium (manufactured by Wako Pure Chemical Industries, Ltd.).
(1-2) After reacting for 1 hour at room temperature with antiserum diluted 1000 times as the first antibody, the plate was washed and horseradish peroxidase (HRP) -labeled anti-mouse immunoglobulin G-Fc antibody (manufactured by SIGMA) was Reaction was performed as a secondary antibody.
(1-3) After reaction at room temperature for 1 hour, the plate was washed, an Amplex Red solution (manufactured by Invitrogen) was added, and the fluorescence intensity was calculated with a fluorescence measurement plate reader.
(2) FACS analysis The fluorescence intensity was calculated in the same manner as in Example 1.

  The result of CELISA analysis is shown in FIG. 2, and the result of FACS analysis is shown in FIG. In any analysis, the zinc oxide whisker group showed higher values than the control group and the uric acid crystal group. FIG. 4 shows the mean value of the fluorescence intensity in each immunization group and the statistical processing result by Welch T test between the control group and the zinc oxide whisker group, calculated by CELISA analysis and FACS analysis. Since the CELISA analysis (p = 0.0036) and the FACS analysis (p = 0.024) showed a statistically significant difference between the subject group and the zinc oxide whisker group, humoral immunity was induced by the zinc oxide whisker. It was shown that the antibody titer increased. On the other hand, no increase in antibody titer was observed in the group (uric acid crystal group) administered with uric acid crystals as compared with the control group even in the acicular crystals. From the above, it was shown that zinc oxide whiskers, which are inorganic compounds having a needle-like single crystal part, have an adjuvant effect (an effect of enhancing liquid immunity).

  By using an acicular crystal of an inorganic compound as an adjuvant when immunizing an animal, it is possible to enhance the humoral immunity of the immunized animal. In addition, an antibody with high specificity for the administered antigen can be produced from antibody-producing cells obtained by immunizing an animal by administering the adjuvant of the present invention and the antigen.

Claims (4)

Needle crystal of an inorganic compound A including A adjuvant, an inorganic compound is zinc oxide, consisting of needle-shaped crystals are needle-shaped single crystal portions extending in four different axial directions from said core portion and the core portion An adjuvant that is crystalline. A method for immunizing an animal (but not including a human) comprising the step of administering a mixture of the adjuvant and antigen according to claim 1 to the animal (but not including a human) . The method according to claim 2 , wherein the antigen is an expression vector containing a protein gene. A method for producing an antibody using the antibody-producing cells obtained by the method according to claim 2 or 3 .

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