(19)
(11)EP 3 075 393 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
05.10.2016 Bulletin 2016/40

(21)Application number: 14865764.6

(22)Date of filing:  17.11.2014
(51)Int. Cl.: 
A61K 39/39  (2006.01)
A61K 39/00  (2006.01)
A61K 47/24  (2006.01)
A61K 9/127  (2006.01)
A61K 47/14  (2006.01)
A61K 47/28  (2006.01)
(86)International application number:
PCT/JP2014/080380
(87)International publication number:
WO 2015/079952 (04.06.2015 Gazette  2015/22)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME

(30)Priority: 29.11.2013 JP 2013248543

(71)Applicant: Terumo Kabushiki Kaisha
Tokyo 151-0072 (JP)

(72)Inventor:
  • SAKAGUCHI, Naoki
    Ashigarakami-gun Kanagawa 259-0151 (JP)

(74)Representative: Casalonga 
Casalonga & Partners Bayerstraße 71/73
80335 München
80335 München (DE)

  


(54)ADJUVANT COMPOSITION, VACCINE COMPOSITION COMPRISING SAME, AND METHOD FOR PRODUCING SAME


(57) Disclosed herein is an adjuvant composition which includes a pH-sensitive carrier and a substance with stimulus to activate innate immune system. The adjuvant composition serves as a carrier which is highly safe and capable of efficient induction of CTL.




Description

Technical Field



[0001] The present invention relates to an adjuvant composition, a vaccine composition containing the same, and a method for producing both of them.

Background Art



[0002] The immune system is defined as a protective mechanism that saves an organism from diseases by eliminating foreign matter, abnormal cells, etc. which might have invaded the organism.

[0003] The immune system usually functions through one of two mechanisms - humoral immunity and cell-mediated immunity.

[0004] The response of humoral immunity usually expresses itself in the following manner. First, an exogenous antigen such as bacteria which has invaded an organism is captured by antigen presenting cells through endocytosis. Next, it is digested and degraded into peptide fragments by protease in endosomes in the antigen presenting cells. The peptide fragments are loaded on major histocompatibility complex (MHC) Class II molecules. The resulting complex is presented to CD4 positive cells on the surface of the antigen presenting cells, so that the CD4 positive T cells are activated. Finally, the CD4 positive T cells activated in this manner release cytokine, eventually causing B cells to produce antibodies. Incidentally, the above-mentioned MHC Class II molecules express themselves in macrophages, dendritic cells, activated T cells, B cells, and the like.

[0005] By contrast, the response of cell-mediated immunity usually expresses itself in the following manner. First, an endogenous antigen, such as protein, which has been generated in virus-infected cells or cancer cells, is ubiquitinated and then degraded into peptide by proteasome. The peptide resulting from degradation combines with MHC Class I molecules. The resulting complex is presented to CD8 positive T cells on the surface of the antigen presenting cells, so that the CD8 positive T cells are activated. Finally, the activated CD8 positive T cells differentiate into cytotoxic T lymphocytes (CTL). Incidentally, the above-mentioned MHC Class I cells exist on the surface of almost all nucleated cells and blood platelets.

[0006] Meanwhile, the CTL responsible for cell-mediated immunity particularly attracts attention because of its ability to eliminate virus-infected cells and cancer cells. Recently, efforts are being made to investigate so-called cross-presentation which induces CTL by exogenous antigens. To be more specific, the exogenous antigen is degraded in endosomes in the antigen presenting cells and then loaded on MHC Class II molecules, as mentioned above. However, the cross-presentation causes even part amount of exogenous antigen to pass through the cell membrane or endosome membrane so that it transfers to the cytosol. The exogenous antigen which has thus been transferred to the cytosol functions as an endogenous antigen, thereby eventually is loaded on MHC Class I molecules to induce CTL.

[0007] The cross-presentation requires that the exogenous antigen passes through the endosome membrane. This is being investigated extensively. For example, Non-Patent Document 1 describes that pH-sensitive poly(propylacrylic acid) (PPAA) conjugates efficiently transport the antigen to the cytosol. Also, Non-Patent Document 2 describes the vaccine delivery by nano-particles for cancer immune therapy.

Prior Art Documents


Non-Patent Documents



[0008] 

Non-Patent Document 1: Bioconjug Chem. 2009 Feb. 20 (2) :241-248

Non-Patent Document 2: Immune Netw. 2013 Oct 13(5) 177-183


Summary of Invention



[0009] There is a strong desire to realize the induction of CTL responsible for cell-mediated immunity from the standpoint of developing new vaccines for therapy. From this point of view, efforts are being made toward the search and improvement of adjuvant molecules. However, no satisfactory effect has been achieved so far for the induction of CTL.

[0010] Some research has been conducted, with an attention paid to the cross-presentation, to realize the strong induction of CTL. However, such researches are apprehensive about safety because they often rely on new synthetic materials or virus-derived components.

[0011] Thus, the present invention was completed to provide a new carrier which is highly safe and capable of effectively inducing CTL.

[0012] With this object in mind, the present inventors conducted extensive research, which has led to a finding that the above-mentioned problems are solved if a pH-sensitive carrier is used in combination with a substance with stimulus to activate innate immune system. This finding is the basis of the present invention.

[0013] Thus, the present invention has solved the above-mentioned problems owing to the compositions and methods defined below.

[0014] 

(1) An adjuvant composition including a pH-sensitive carrier and a substance with stimulus to activate innate immune system.

(2) The adjuvant composition as defined in Paragraph (1) above, wherein the pH-sensitive carrier contains:

at least one species of pH-sensitive compound; and

at least one species of amphipathic substance, and

produces the membrane disruptive function promoting effect,

the pH-sensitive compound being selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof;

the amphipathic substance being selected from the group consisting of phosphatidylcholine having 10 to 12 carbon atoms, polyoxyethylene sorbitan monofatty acid ester having 12 to 18 carbon atoms, sorbitan fatty acid ester having 16 to 18 carbon atoms, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol.

(3) The adjuvant composition as defined in Paragraph (1) or (2) above, wherein the substance with stimulus to activate innate immune system is monophosphoryl lipid A.

(4) The adjuvant composition as defined in Paragraph (2) or (3) above, wherein the substance with stimulus to activate innate immune system is contained in an amount of 0.0227 to 22.7 mol for 100 mol of the amphipathic substance.

(5) A vaccine composition including the adjuvant composition defined in any one of Paragraphs (1) to (4) above and an antigen.

(6) The vaccine composition as defined in Paragraph (5) above, wherein the antigen is a peptide or protein.

(7) The vaccine composition as defined in Paragraph (5) or (6) above, wherein the antigen is contained in an amount of 3.2 to 400 µg for 100 nmol of the amphipathic substance.

(8) A method for producing an adjuvant composition, including:

the step of associating among

at least one species of pH-sensitive compound,

at least one species of amphipathic substance, and

a substance with stimulus to activate innate immune system;

the pH-sensitive compound being selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof;

the amphipathic substance being selected from the group consisting of phosphatidylcholine having 10 to 12 carbon atoms, polyoxyethylene sorbitan monofatty acid ester having 12 to 18 carbon atoms, sorbitan fatty acid ester having 16 to 18 carbon atoms, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol.

(9) A method for producing a vaccine composition, including:

the step of associating among at least one species of pH-sensitive compound, at least one species of amphipathic substance, and a substance with stimulus to activate innate immune system, the pH-sensitive compound being selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof, the amphipathic substance being selected from the group consisting of phosphatidylcholine having 10 to 12 carbon atoms, polyoxyethylene sorbitan monofatty acid ester having 12 to 18 carbon atoms, sorbitan fatty acid ester having 16 to 18 carbon atoms, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol;

the step of mixing the product of the association with an antigen;

the step of freeze-thawing the mixture obtained by the mixing; and

the step of freeze-drying the melt obtained by the freeze-thawing.


Brief Description of Drawings



[0015] 

[Fig. 1]
Fig. 1 is a schematic diagram depicting an adjuvant composition and a vaccine composition containing the same, which are concerned with one embodiment of the present invention.

[Fig. 2]
Fig. 2 depicts the result of evaluation of CTL induction by ELIspot method. Fig. 2(A) is a graph depicting the number of IFNγ spots, Fig. 2(B) depicts spot formation in the case where peptide is used alone, and Fig. 2(C) depicts spot formation in the case where peptide is used in combination with MPL-containing DLPC-deoxycholic acid complex (adjuvant composition). The condition of evaluation is 2 x 106 cells/well.

[Fig. 3]
Fig. 3 depicts the result obtained in the case where the CTL induction rate was evaluated by Intracellular Cytokine Staining (ICS) method. Fig. 3(A) depicts the result in the case where peptide is used alone, Fig. 3(B) depicts the result in the case where peptide and MPL-dispersion are used, and Fig. 3(C) depicts the result in the case where peptide is used in combination with monophosphoryl lipid A (MPL)-containing DLPC-deoxycholic acid complex (adjuvant composition).

[Fig. 4]
Fig. 4 depicts the result obtained in the case where the CTL induction rate was evaluated by using any one of the vaccine compositions prepared by various preparation methods. Fig. 4(A) depicts the result in the case where MPL-containing DLPC-deoxycholic acid complex (prepared by the preparation method by dispersion) was used, Fig. 4(B) depicts the result in the case where MPL-containing DLPC-deoxycholic acid complex (prepared by the preparation method by mixing) was used, and Fig. 4(C) depicts the result in the case where MPL-containing DLPC-deoxycholic acid complex (prepared by the preparation method by freeze-thawing and freeze-drying) was used.

[Fig. 5]
Fig. 5 depicts the result of evaluation of incorporation rate. Fig. 5(A) is a graph depicting absorbance determined by Lowry method before and after the peptide solution was filtered, Fig. 5(B) is a graph depicting absorbance determined by lipid test Wako before and after the adjuvant composition was filtered, and Fig. 5(C) depicts the result of evaluation of the incorporation rate determined for various vaccine compositions.

[Fig. 6]
Fig. 6 depicts the result of test on the stimulus to activate innate immune system. This test is intended to verify the evaluation system. Fig. 6(A) depicts the result of evaluation of flow cytometry, with the cultured mice spleen cells not stained, Fig. 6(B) depicts the enhanced expression of CD80 in the case where PBS is added alone, Fig. 6(C) depicts the enhanced expression of CD80 in the case where LPS is added, Fig. 6(D) is a graph depicting the result of flow cytometry in the case where LPS is added in various concentrations, Fig. 6(E) depicts the enhanced expression of CD86 as other costimulatory molecules, and Fig. 6(F) depicts the enhanced expression of CD40 as other costimulatory molecules.

[Fig. 7]
Fig. 7(A) depicts the result of evaluation of the enhanced expression of CD80. Fig. 7(B) depicts the result of evaluation of the enhanced expression of CD86 as another indicator. Fig. 7(C) depicts the result of evaluation of the enhanced expression of CD40 as another indicator.

[Fig. 8]
Fig. 8 depicts the result of examination for the presence or absence of the stimulus to activate innate immune system in various materials. Fig. 8(A) depicts the result of examination for the enhanced expression of CD80 in the case where MPL-free DLPC-deoxycholic acid complex is added in various amounts, and Fig. 8(B) depicts the result of examination for the enhanced expression of CD80 in the case where the sample solutions in various kinds are added.

[Fig. 9]
Fig. 9(A) depicts the leakage in the case where MPL is administered in an amount equivalent to the MPL content which is 0.227 mol. Fig. 9(B) depicts the leakage (at varied pH values) of the MPL-containing DLPC-deoxycholic acid complex. Fig. 9(C) depicts the result of the leakage (at pH 7.4 and pH 5.0) of the DLPC-deoxycholic acid complex, with the MPL content varied.

[Fig. 10]
Fig. 10 depicts the result of examination for the leakage (at pH 7.4 and pH 5.0) of the vaccine composition which was prepared by the preparation method by dispersion, preparation method by mixing, or preparation method by freeze-thawing and freeze-drying.

[Fig. 11]
Fig. 11 depicts the result of examination for the influence of MPL content on the adjuvant composition's membrane disruptive function promoting effect, with the amount of deoxycholic acid varied. Fig. 11(A) depicts the result in the case where the amount is 10, Fig. 11(B) depicts the result in the case where the amount is 20, and Fig. 11(C) depicts the result in the case where the amount is 640.

[Fig. 12]
Fig. 12 depicts the result of examination for the influence of the MPL inclusion on the adjuvant composition's membrane fusion function promoting effect.

[Fig. 13]
Fig. 13 depicts the result of examination for the influence of the antigen amount on the vaccine composition's ability to activate innate immune system. Fig. 13(A) depicts the result in the case where peptide is used, and Fig. 13(B) depicts the result in the case where OVA is used.

[Fig. 14]
Fig. 14 depicts the result of examination for the influence of the amphipathic substance on the adjuvant composition's stimulus to activate innate immune system.

[Fig. 15]
Fig. 15 depicts the result of examination for the influence of the complexing of the pH-sensitive compound on the strength of the adjuvant composition's and the vaccine composition's stimulus to activate innate immune system. Fig. 15(A) depicts the result in the case where the compositions are prepared by using deoxycholic acid varying in complex amount, with the MPL content fixed at 0.0227 mol, and Fig. 15(B) depicts the result in the case where the compositions are prepared by using deoxycholic acid varying in complex amount, with the MPL content fixed at 22.7 mol.

[Fig. 16]
Fig. 16 depicts the result of examination for the influence of the pH-sensitive compound varying in kind on the strength of the vaccine composition's stimulus to activate innate immune system. Fig. 16(A) depicts the result in the case where the vaccine composition is prepared by using the pH-sensitive compound varying in kind, with the MPL content fixed at 0.0227 mol, and Fig. 16(B) depicts the result in the case where the vaccine composition is prepared by using the pH-sensitive compound varying in kind, with the MPL content fixed at 22.7 mol.

[Fig. 17]
Fig. 17 depicts the result of evaluation by ELIspot method in the case where the vaccine composition is used. Fig. 17(A) depicts spot formation in the case where the peptide and the MPL dispersion are used under the condition of high MPL level, and Fig. 17(B) depicts spot formation in the case where the peptide and the adjuvant composition are used under the condition of high MPL level. The condition of evaluation is 1 × 106 cells/well.

[Fig. 18]
Fig. 18 depicts the result of evaluation by ELIspot method for the vaccine composition prepared with the pH-sensitive compound varying in kind. In this examination, the antigen is OVA, and the substance to stimulate innate immune system or the adjuvant composition is varied as follows. Fig. 18(A) depicts spot formation in the case where MPL dispersion is used, Fig. 18(B) depicts spot formation in the case where MPL-containing DLPC-deoxycholic acid is used, Fig. 18(C) depicts spot formation in the case where MPL-containing DLPC-cholic acid is used, Fig. 18(D) depicts spot formation in the case where MPL-containing DLPC-ursodeoxycholic acid is used, and Fig. 18(E) depicts spot formation in the case where MPL-containing DLPC-hyodeoxycholic acid is used. The condition of evaluation is 2 × 106 cells/well.

[Fig. 19]
Fig. 19 is a graph depicting the CTL induction rate of the vaccine composition prepared by various preparation methods.

[Fig. 20]
Fig. 20 depicts the result of evaluation for the antigen specificity of the CTL induced by the vaccine composition. Fig. 20(A) depicts the CTL induction rate in the case where the antigen is given restimulation, and Fig. 20(B) depicts the CTL induction rate in the case where the antigen is not given restimulation.

[Fig. 21]
Fig. 21 depicts the result of evaluation by ELIspot method for the antigen specificity of CTL. Figs. 21(A) to 21(F) depict spot formation in the case where there is restimulation, Figs. 21(G) to (L) depict spot formation in the case where there is no restimulation. Figs. 21(A) and 21(G) depict the result in the case where OVA (80 µg) and MPL dispersion are used, Figs. 21(B) and 21(H) depict the result in the case where OVA (80 µg) and the adjuvant composition which is prepared with deoxycholic acid (160 nmol) are used, Figs. 21(C) and 21(I) depict the result in the case where OVA (80 µg) and the adjuvant composition which is prepared with deoxycholic acid (640 nmol) are used, Figs. 21(D) and 21(J) depict the result in the case where peptide (80 µg) and MPL dispersion are used, Figs. 21(E) and 21(K) depict the result in the case where peptide (80 µg) and the adjuvant composition which is prepared with deoxycholic acid (160 nmol) are used, and Figs. 21(F) and 21(L) depict the result in the case where peptide (80 µg) and the adjuvant composition which is prepared with deoxycholic acid (640 nmol) are used for administration to mice. The condition of evaluation is 2 × 106 cells/well.

[Fig. 22]
Fig. 22 is a graph depicting the IgG antibody titer in the case where the vaccine compositions prepared by various preparation methods are used.

[Fig. 23]
Fig. 23 depicts the result of evaluation by ELIspot method in the case where CpG-ODN is used. Fig. 23(A) depicts spot formation in the case OVA (80 µg) and CpG-ODN alone are administered to the mice, Fig. 23(B) depicts spot formation in the case where OVA (80 µg) and the adjuvant composition containing CpG-ODN are used, Fig. 23(C) depicts spot formation in the case where OVA (80 µg) and CpG-ODN alone are administered to the mice, with the antigen given no restimulation, and Figs. 23(D) depicts spot formation in the case where OVA (80 µg) and the adjuvant composition containing CpG-ODN are used, with the antigen given no restimulation.


Modes for Carrying Out the Invention



[0016] According to one embodiment of the present invention, there is provided an adjuvant composition containing a pH-sensitive carrier and a substance with stimulus to activate innate immune system. The foregoing adjuvant composition serves as a carrier for effective induction of CTL. The adjuvant composition is also a safe carrier.

<Adjuvant composition>



[0017] The adjuvant composition pertaining to the embodiment contains a pH-sensitive carrier and a substance with stimulus to activate innate immune system. (The pH-sensitive carrier will be simply referred to as "carrier," "associated body," or "complex" in some instances hereinafter.)

[0018] The adjuvant composition pertaining to the embodiment is characterized in that the pH-sensitive carrier therein is composed of at least one species of pH-sensitive compound and at least one species of amphipathic substance, and the adjuvant composition produces the membrane disruptive function promoting effect. The pH-sensitive compound is selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof, and the amphipathic substance is selected from the group consisting of C10-12 phosphatidylcholine, C12-18 polyoxyethylene sorbitan monofatty acid ester, C16-18 sorbitan fatty acid ester, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol.

[0019] The embodiment will be described below in more detail with reference to the accompanying drawings. It is not intended to restrict the technical scope of the present invention, which may be defined according to the claims appended hereto. The attached drawings may not be on the correct scale, but may be exaggerated for the sake of explanation.

[0020] Fig. 1 is a schematic diagram depicting the adjuvant composition and the vaccine composition containing the same, both pertaining to one embodiment of the present invention.

[0021] It is noted from Fig. 1 that an adjuvant composition 4 contains an amphipathic substance 1, a pH-sensitive compound 2, and a substance 3 with stimulus to activate innate immune system. It is also noted from Fig. 1 that, according to one embodiment, both the substance 3 with stimulus to activate innate immune system and the pH-sensitive compound 2 associate with the hydrophobic moiety of the amphipathic substance 1. In this case, the adjuvant composition 4 may be regarded as an adjuvant complex. It is also noted that, according to another embodiment, the substance 3 with stimulus to activate innate immune system exists independently of the pH-sensitive carrier containing the amphipathic substance 1 and the pH-sensitive compound 2.

[0022] Moreover, a vaccine composition 6 contains the adjuvant composition 4 and an antigen 5. As depicted in Fig. 1, the antigen 5 may exist either inside or outside the adjuvant composition 4 pertaining to the above-mentioned two embodiments. The vaccine composition 6 in which the adjuvant composition 4 contains the antigen 5 may be regarded as a vaccine complex.

[0023] In this specification, the term "adjuvant composition" denotes a composition which contains the pH-sensitive carrier and the substance with stimulus to activate innate immune system. It may take on any form without specific restrictions. In other words, the "adjuvant composition" may be a mixture composed of the pH-sensitive carrier and the substance with stimulus to activate innate immune system. It may also be one (adjuvant complex) in which the pH-sensitive carrier supports thereon or contains therein the substance with stimulus to activate innate immune system. Both of them may be collectively referred to as "adjuvant composition" in this specification.

[0024] Also, in this specification, the term "vaccine composition" denotes any composition which contains the adjuvant composition and the antigen. It may take on any form without specific restrictions. In other words, the "vaccine composition" may be a mixture composed of two or more components selected from the group consisting of the constituents of the adjuvant composition and the antigen, or one (vaccine complex) in which the adjuvant complex supports thereon or contains therein the antigen. In this specification, both of them are collectively referred to as "vaccine composition."

[pH-sensitive carrier]



[0025] The pH-sensitive carrier is sensitive to pH, so that it transports the antigen in cells to the cytosol when its surrounding decreases in pH.

[0026] The pH-sensitive carrier is not specifically restricted. However, it may preferably be one which is composed of at least one species of pH-sensitive compound and at least one species of amphipathic substance, and is capable of producing the membrane disruptive function promoting effect. The pH-sensitive compound is selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof. The amphipathic substance is selected from the group consisting of C10-12 phosphatidylcholine, C12-18 polyoxyethylene sorbitan monofatty acid ester, C16-18 sorbitan fatty acid ester, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol. The "carbon number" for the amphipathic substance denotes the number of carbon atoms in the fatty acid (acyl group) constituting the hydrophobic moiety of the amphipathic substance.

[0027]  The following is a detailed description of the pH-sensitive carrier which is composed of the pH-sensitive compound and the amphipathic substance, and is capable of producing the membrane disruptive function promoting effect.

(Structure of pH-sensitive carrier)



[0028] It is considered that the pH-sensitive carrier is formed by association between the pH-sensitive compound and the amphipathic substance at a physiological pH or higher. To be more specific, it is considered that the pH-sensitive carrier is formed when the pH-sensitive compound associates with the hydrophobic moiety of the amphipathic substance. Incidentally, the form of association of the pH-sensitive carrier is hypothetical, and pH-sensitive carrier is not restricted by the form of association.

(Membrane disruptive function promoting effect)



[0029] The term "membrane disruptive function" denotes the function to bring about release in the release test. The release test described in this specification is a test consisting of the steps of adding liposomes (dispersion) encapsulating an aqueous solution containing a quenching substance and a fluorescent substance and a dispersion of a sample for evaluation to a properly pH-adjusted aqueous solution, incubating the resulting aqueous solution at 37°C for 90 minutes or 30 minutes, and measuring the fluorescence intensity of the incubated aqueous solution. This method permits the measurement of the amount of the fluorescent substance which has been released from the liposomes. In this way it is possible to verify that the pH-sensitive carrier functions to disrupt the membrane of the liposomes. Incidentally, the release test will be described in more detail in Working Examples to be described later.

[0030] The term "to produce the membrane disruptive function promoting effect" means that the following two conditions are satisfied simultaneously.
  1. (1) The release test indicates that the leakage is higher at a prescribed pH (lower than the physiological pH) than that at the physiological pH, and the amount of increase is larger than that observed in the experiment which is carried out with the pH-sensitive compound alone.
  2. (2) The release test at a prescribed pH lower than the physiological pH indicates that the leakage in the case where the pH-sensitive compound and the amphipathic substance form a complex (or the pH-sensitive carrier) is larger than the sum of the leakage due to the pH-sensitive compound used alone and the leakage due to the amphipathic substance used alone.


[0031] To be more specific, the term "to produce the membrane disruptive function promoting effect" means the following. The release test at pH 7.4 and pH 5.0 or pH 4.5 indicates that the relation among La, Lb, and Lc exists as indicated by the following two equations, where La represents the leakage for the pH-sensitive compound existing alone, Lb represents the leakage for the amphipathic substance existing alone, and Lc represents the leakage for the pH-sensitive carrier (which is a complex of the pH-sensitive compound and the amphipathic substance). In other words, what is mentioned in (1) and (2) above is expressed respectively in terms of the equations (1) and (2) below. In the following equations, La7.4, Lb7.4, and Lc7.4 denote the leakage at pH 7.4, and Lax, Lbx, and Lcx denote the leakage at pH 5.0 or pH 4.5.
[Math 1]





[0032]  In the equation (1) above, Δ should be larger than 0, preferably 5 or larger, more preferably 10 or larger, and most preferably be 30 or larger. Also, in the equation (2) above, Δ' should be larger than 0, preferably 5 or larger, more preferably 10 or larger than, and most preferably 15 or larger.

[0033] According to one embodiment of the present invention, it is desirable that the values of both Δ and Δ' in the equations (1) and (2) above should be 5 or larger, and that the pH-sensitive carrier should be one which contains bile acid and lipid. According to another embodiment of the present invention, it is desirable that the values of both Δ and Δ' in the equations (1) and (2) above should be 5 or larger and that the pH-sensitive carrier should be one which contains glycyrrhizinic acid or both glycyrrhetinic acid and lipid.

[0034] In this specification, the term "physiological pH" means a pH value in normal tissue or normal body fluid. The physiological pH is usually 7.4, but it may slightly vary (± 0.1) from one normal tissue or one normal body fluid to another. The term "prescribed pH lower than the physiological pH" means a pH lower than 7.4, preferably a pH 3.0 or more but less than 7.4, more preferably a pH 4.0 or more but less than 7.3, and most preferably a pH 4.5 or more but less than7.0.

[0035] No elucidation has been made yet on the mechanism by which the pH-sensitive carrier produces the membrane disruptive function promoting effect. However, the following conjecture may be made, although the conjecture does not restrict the scope of the present invention.

[0036] It is assumed that the pH-sensitive carrier changes the mode of association between the pH-sensitive compound and the amphipathic substance when the environment decreases in pH lower than the physiological pH, and this leads to the membrane disruptive function promoting effect. For example, if a system containing a pH-sensitive carrier and a biomembrane (such as cell membrane and vesicle membrane) changes such that its pH decreases below the physiological pH, the pH-sensitive carrier would change in the mode of association after contact with the biomembrane, thereby undergoing change in the structure of the biomembrane. In other words, the pH-sensitive carrier would bring about change in the structure of the biomembrane. A probable reason for this is that the environment of weakly acidic pH makes unstable the pH-sensitive compound in the pH-sensitive carrier in the structure of the carrier, with the result that the pH-sensitive carrier undergoes rearrangement with the biomembrane existing in the system, thereby producing the membrane disruptive function promoting effect. The foregoing may be put in another way as follows. The pH-sensitive compound consists of molecules which, when it becomes weakly acidic pH , undergo protonation and change in solubility for hydrophobic association. In other words, the hydrophobic association involving the pH-sensitive compound responds to the weak acid environment, thereby producing its function. Incidentally, the term "membrane disruption" means the change in membrane structure as mentioned above. It does not necessarily mean that the membrane-constituting components are entirely separated or decomposed. As the result of such "membrane disruption," any component that may be contained inside the biomembrane (such as endosome) is released to the outside (e.g., cytosol) of the biomembrane.

[0037] The pH-sensitive carrier may preferably be one which has a leakage lower than 20% at pH 7.4 and higher than 20% at pH 4.0 in the release test. The leakage in the release test may preferably be lower than 20% at pH 6.5 and higher than 20% at pH 4.0. The leakage at pH 7.4 or pH 6.5 may preferably be 15% or lower, more preferably 10% or lower. The leakage at pH 4.0 may preferably be 40% or higher, more preferably 50% or higher. With the leakage as defined above, the pH-sensitive carrier easily produces the effect of promoting the membrane-disruption effect at a slightly low pH.

[0038] In addition, the pH-sensitive carrier produces not only the membrane disruptive function promoting effect but also the membrane fusion function promoting effect.

[0039] The term "membrane-fusion function" as used in the present invention means the function that brings about membrane-fusion in the membrane-fusion test. The membrane-fusion test used in this specification denotes a test which consists of the steps of adding liposomes (dispersion) having two kinds of fluorescent substances built into the liposomalmenbrane and a dispersion of sample for evaluation into an aqueous solution adjusted to a prescribed pH, incubating the resulting aqueous solution at 37°C for 60 minutes, and measuring the fluorescence intensity of the incubated aqueous solution. This test permits the measurement of change in energy resonance transfer for the two kinds of fluorescent substances incorporated into the liposomes, thereby verifying the membrane-fusion function of the pH-sensitive carrier. Incidentally, a detailed description of the membrane-fusion test will be given later in Working Examples.

[0040] The term "to produce the membrane fusion function promoting effect" means a situation in which the membrane-fusion test indicates that the fusion rate at a prescribed pH lower than the physiological pH is higher than the fusion rate at the physiological pH and the amount of increase is larger than that observed when the pH-sensitive compound is tested alone. To be more specific, the term "to produce the membrane fusion function promoting effect" means that the membrane-fusion tests at pH 7.4 and pH 5.0 indicate that there exists the relation represented by the following equation (3) between the fusion rate Rc(%) of the pH-sensitive carrier (which is a complex of a pH-sensitive compound and an amphipathic substance) and the fusion rate Ra(%) of the pH-sensitive compound used alone. Incidentally, in the equation below, Rc7.4 and Ra7.4 represent the fusion rate at pH 7.4, and Rcx and Rax represent the fusion rate at pH 5.0.




[0041] The value of ΔR in equation (3) above should be larger than 0, preferably 2 or larger, more preferably 5 or larger, and most preferably 10 or larger.

[0042] The pH-sensitive carrier should preferably be one which has ΔR equal to or larger than 2 in equation (3) and contains bile acid and lipid.

[0043] The pH-sensitive carrier produces the membrane fusion function promoting effect at a slightly low pH (or a prescribed pH lower than the physiological pH). Although the mechanism of this phenomenon is not yet elucidated, the mechanism is probably the same as that in the membrane disruptive function promoting effect. This conjecture is not intended to restrict the scope of the present invention.

[0044]  It is assumed that the pH-sensitive carrier used in the present invention contributes to membrane-fusion as the result of rearrangement of biomembranes existing in the system when the environment decreases in pH lower than the physiological pH so as to change the mode of association between the pH-sensitive compound and the amphipathic substance. The membrane-fusion takes place as the result of rearrangement among components having affinity with each other, and hence those components (such as antigen) which have no or little affinity with biomembrane are excluded and released from the membrane which undergoes rearrangement.

[0045] As mentioned above, the antigen is usually surrounded by endosome, which is one kind of biomembrane, so that it is taken into cells (such as antigen presenting cells). After that, the endosome decreases in pH due to proton pumping action. The endosome further fuses with lysosome containing hydrolases. In this way, the antigen is degraded. (Subsequently, the degraded antigen forms a complex with MHC Class II molecules, thereby presenting the antigen to CD4 positive T cells.) Therefore, most of the antigen is not delivered into the cytosol.

[0046] By contrast, the pH-sensitive carrier pertaining to the embodiment of the present invention permits the antigen (such as exogenous antigen) to be delivered to the cytosol. To be more specific, an environment with a decreased pH similarly occurs when the antigen together with the pH-sensitive carrier is surrounded by endosomes and taken into cells. As the result of pH decrease (or acidification), the pH-sensitive compound makes unstable the pH-sensitive carrier, so that the rearrangement of membrane takes place between the endosome and the pH-sensitive carrier. This causes the pH-sensitive carrier to produce the membrane-disruption function (which produces itself together with the membrane-fusion function in some cases). This membrane-disruption function (or both the membrane-fusion function and the membrane-disruption function) causes the antigen to be delivered into the cytosol from the endosome. The foregoing mechanism suggests that, in principle, the antigen can be transported to the cytosol once it has been taken into the endosome together with the pH-sensitive carrier. This indicates the possibility of using the antigen and the pH-sensitive carrier in the form of their mixed composition or using the antigen in such a form that it is supported on or contained in the pH-sensitive carrier.

(pH-sensitive compound)



[0047] As mentioned above, the pH-sensitive compound is at least one species selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof. The pH-sensitive compounds in salt form unrestrictedly include alkali metal salts (such as lithium salt, sodium salt, and potassium salt), alkaline earth metal salts (such as magnesium salt, calcium salt, and barium salt), and ammonium salt. These pH-sensitive compounds may be used alone or in combination with one another.

[0048] According to one embodiment of the present invention, the pH-sensitive compound may preferably be at least one species selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, glycodeoxycholic acid, and glycyrrhizinic acid, and salts thereof.

[0049] According to another embodiment of the present invention, the pH-sensitive compound may preferably be at least one species selected from the group consisting of deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, glycodeoxycholic acid, glycyrrhizinic acid, and salts thereof. More preferably, it may be at least one species selected from the group consisting of deoxycholic acid, ursodeoxycholic acid, and glycyrrhizinic acid, and salts thereof.

[0050] The preferable examples of the pH-sensitive compounds, such as deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, and glycodeoxycholic acid, are generally called bile acid. Bile acid has been known as a typical steroid derivative for long time before the 1920s, and it has been used in the field of bacteriology. The bile acid in the living human body forms a complex with cholesterol, lipid, and fat-soluble vitamin, thereby assisting their absorption. Moreover, bile acid forms a complex with lipid, protein, or hydrophobic material owing to its physicochemical properties, and hence it has long been used for protein separation and purification and also as a solubilizer or emulsifier. Recently, it is attracting attention in the field of vaccine production and drug absorption promoter, which is enabled bile acid transporter. Typical examples of bile acid for such purposes are sodium deoxycholate (also known as sodium desoxycholate) and ursodeoxycholic acid (also known as ursodesoxycholic acid); they have found use as a pharmaceutical additive suitable for injection into human bodies owing to high safety. For this reason, the pH-sensitive compound may preferably be selected from deoxycholic acid, and ursodeoxycholic acid, and salts thereof (such as sodium salt).

[0051] The amount of the pH-sensitive compound for 100 mol of the amphipathic substance may be 10 mol or more, preferably 10 to 640 mol, more preferably 20 to 320 mol, and most preferably 20 to 160 mol. Incidentally, in this specification, the term "amount of the complex of the pH-sensitive compound" may be used to denote the content of the pH-sensitive compound for 100 mol of the amphipathic substance.

(Amphipathic substance)



[0052] As mentioned above, the amphipathic substance is at least one species selected from the group consisting of C10-12 phosphatidylcholine, C12-18 polyoxyethylene sorbitan monofatty acid ester, C16-18 sorbitan fatty acid ester, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and a-tocopherol. These amphipathic substances may be used alone or in combination with one another.

[0053] Incidentally, in this specification, the "carbon number" of the amphipathic substance denotes the number of carbon atoms in the fatty acid (acyl group) constituting the hydrophobic moiety of the amphipathic substance.

[0054] The C10-12 phosphatidylcholine may preferably be diacylphosphatidylcholine having saturated acyl groups. Examples of this compound include
didecanoylphosphatidylcholine (DDPC; 1,2-didecanoyl-sn-glycero-3-phosphatidylcholine) and
dilauroylphosphatidylcholine (DLPC; 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine). The phosphatidylcholine may be natural, synthetic prepared by any known method, or commercial.

[0055]  The C12-18 polyoxyethylene sorbitan monofatty acid ester includes, for example, polyoxyethylene sorbitan monolauric acid ester (polyoxyethylene sorbitan monolaurate), polyoxyethylene sorbitan myristic acid ester (polyoxyethylene sorbitan monomyristate), polyoxyethylene sorbitan monopalmitic acid ester (polyoxyethylene sorbitan palmitate), polyoxyethylene sorbitan monostearic acid ester (polyoxyethylene sorbitan monostearate), and polyoxyethylene sorbitan monooleic acid ester (polyoxyethylene sorbitan monooleate). Although the polyoxyethylene is not specifically restricted in the degree of polymerization, the polyoxyethylene chains (combined together) are preferably added to sorbitan have the degree of polymerization of 10 to 200, preferably 15 to 100, and more preferably 20 to 50. The polyoxyethylene sorbitan monofatty acid ester may be synthetic or commercial. The polyoxyethylene sorbitan monofatty acid ester may be commercially available under the trade name of Tween 20 (polyoxyethylene sorbitan monolaurate ester), Tween 40 (polyoxyethylene sorbitan monopalmitate ester), Tween 60 (polyoxyethylene sorbitan monostearate ester), and Tween 80 (polyoxyethylene sorbitan monooleate ester). Preferable among these products are C16-18 polyoxyethylene sorbitan monofatty acid ester (Tween 20, Tween 40, Tween 60, and Tween 80).

[0056] The C16-18 sorbitan fatty acid ester includes sorbitan mnofatty acid ester, such as sorbitan monopalmitic acid ester (sorbitan monopalmitate), sorbitan monostearic acid ester (sorbitan monostearate), and sorbitan monooleic acid ester (sorbitan monooleate). It also includes sorbitan trifatty acid ester, such as ester of sorbitan tripalmitic acid (sorbitan tripalmitate), ester of sorbitan tristearic acid (sorbitan tristearate), and ester of sorbitan trioleic acid (sorbitan trioleate). The sorbitan fatty acid ester may be synthetic or commercial. The sorbitan fatty acid ester may be commercially available under the trade name of SPAN 40 (sorbitan palmitic acid ester), SPAN 60 (sorbitan stearic acid ester), SPAN 80 (sorbitan oleic acid ester), SPAN 65 (sorbitan tristearic acid ester), and SPAN 85 (sorbitan trioleic acid ester). Preferable among them are SPAN 80, SPAN 65, and SPAN 85.

[0057] The glycerol monooleate (glyceryl monooleate), glycerol dilaurate (glyceryl dilaurate), glycerol distearate (glyceryl distearate), and glycerol dioleate (glyceryl dioleate) are acyl glycerol formed by ester linkage from glycerin and one or two molecules of fatty acid. The site of linkage for the fatty acid is not specifically restricted. In the case of glycerol monooleate which is monoacyl glycerol, for example, the ester linkage of the fatty acid may exist at the C1 or C2 position of glycerin. Also, in the case of glycerol dilaurate, glycerol distearate, or glycerol dioleate which is diacyl glycerol, the ester linkage of the fatty acid may exist at the C1 and C2 positions of glycerin or at the C1 and C3 positions of glycerin. A preferable example of glycerol dilaurate is α,α'-dilaurin, in which substitution occurs at the C1 and C3 positions. Preferable examples of the glycerol distearate and glycerol dioleate include diacyl glycerol in which substitution occurs at the C1 and C2 positions. These glycerol derivatives may be synthetic or commercial.

[0058] The polyoxyethylene castor oil is formed by addition of polyoxyethylene to castor oil. The polyoxyethylene may have a degree of polymerization (which is not specifically restricted) in the range of 3 to 200, preferably 5 to 100, more preferably 10 to 50. The polyoxyethylene castor oil may be synthetic or commercial.

[0059] The α-tocopherol may be natural, synthetic prepared by any known method, or commercial.

[0060] Preferable among the above-mentioned amphipathic substances are those selected from the group consisting of C10-12 phosphatidylcholine, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, sorbitan monooleate, polyoxyethylene castor oil, and α-tocopherol. More preferable examples of them are those selected from the group consisting of dilauroyl phosphatidylcholine, didecanoyl phosphatidylcholine, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, sorbitan monooleate, polyoxyethylene castor oil, and α-tocopherol.

(Combination of pH-sensitive compound and amphipathic substance)



[0061] The pH-sensitive carrier produces the membrane disruptive function promoting effect at a desired pH because it is composed of the pH-sensitive compound and the amphipathic substance which are combined together. The combination of the pH-sensitive compound and the amphipathic substance affects the pH at which the pH-sensitive carrier starts to produce the membrane disruptive function promoting effect. A probable reason for this is that the pH-sensitive compound varies in pKa and the mode of association with the amphipathic substance also varies depending on the combination of the pH-sensitive compound and the amphipathic substance. This implies that an adequate combination of the pH-sensitive compound and the amphipathic substance makes it possible to select any pH suitable for the desired effect and to meticulously set up the mode of delivery.

[0062] The following are preferable examples of the combination of the pH-sensitive compound and the amphipathic substance in the pH-sensitive carrier.

[0063] Cholic acid and DLPC; deoxycholic acid and DDPC; deoxycholic acid and DLPC; deoxycholic acid and Tween 20; deoxycholic acid and Tween 40; deoxycholic acid and Tween 60; deoxycholic acid and Tween 80; deoxycholic acid and SPAN 40; deoxycholic acid and SPAN 60; deoxycholic acid and SPAN 80; deoxycholic acid and SPAN 65; deoxycholic acid and SPAN 85; deoxycholic acid and α-tocopherol; deoxycholic acid and glycerol monooleate; deoxycholic acid and glycerol distearate; deoxycholic acid and glycerol dioleate; deoxycholic acid and glycerol dilaurate (α,α'-dilaurin); deoxycholic acid and polyoxyethylene castor oil; chenodeoxycholic acid and DLPC; hyodeoxycholic acid and DLPC; glycodeoxycholic acid and DLPC; ursodeoxycholic acid and DDPC; ursodeoxycholic acid and DLPC; ursodeoxycholic acid and Tween 20; ursodeoxycholic acid and Tween 40; ursodeoxycholic acid and Tween 60; ursodeoxycholic acid and Tween 80; ursodeoxycholic acid and SPAN 40; ursodeoxycholic acid and SPAN 60; ursodeoxycholic acid and SPAN 80; ursodeoxycholic acid and SPAN 65; ursodeoxycholic acid and SPAN 85; ursodeoxycholic acid and α-tocopherol; ursodeoxycholic acid and glycerol monooleate; ursodeoxycholic acid and glycerol distearate; ursodeoxycholic acid and glycerol dioleate; ursodeoxycholic acid and glycerol dilaurate (α,α'-dilaurin); ursodeoxycholic acid and polyoxyethylene castor oil; glycyrrhizinic acid and DDPC; glycyrrhizinic acid and DLPC; glycyrrhizinic acid and Tween 20; glycyrrhizinic acid and Tween 40; glycyrrhizinic acid and Tween 60; glycyrrhizinic acid and Tween 80; glycyrrhizinic acid and SPAN 40; glycyrrhizinic acid and SPAN 60; glycyrrhizinic acid and SPAN 80; glycyrrhizinic acid and SPAN 65; glycyrrhizinic acid and SPAN 85; glycyrrhizinic acid and α-tocopherol; glycyrrhizinic acid and glycerol monooleate; glycyrrhizinic acid and glycerol distearate; glycyrrhizinic acid and glycerol dioleate; glycyrrhizinic acid and glycerol dilaurate (α,α'-dilaurin); and glycyrrhizinic acid and polyoxyethylene castor oil.

[0064] More preferable examples are listed below.

[0065] Cholic acid and DLPC; deoxycholic acid and DDPC; deoxycholic acid and DLPC; deoxycholic acid and Tween 20; deoxycholic acid and Tween 40; deoxycholic acid and Tween 60; deoxycholic acid and Tween 80; deoxycholic acid and SPAN 40; deoxycholic acid and SPAN 65; deoxycholic acid and SPAN 80; deoxycholic acid and SPAN 85; deoxycholic acid and α-tocopherol; deoxycholic acid and glycerol monooleate; deoxycholic acid and polyoxyethylene castor oil; chenodeoxycholic acid and DLPC; hyodeoxycholic acid and DLPC; glycodeoxycholic acid and DLPC; ursodeoxycholic acid and DDPC; ursodeoxycholic acid and DLPC; ursodeoxycholic acid and Tween 40; ursodeoxycholic acid and Tween 60; ursodeoxycholic acid and Tween 80; ursodeoxycholic acid and SPAN 40; ursodeoxycholic acid and SPAN 65; ursodeoxycholic acid and SPAN 85; ursodeoxycholic acid and α-tocopherol; ursodeoxycholic acid and monooleate; ursodeoxycholic acid and polyoxyethylene castor oil; glycyrrhizinic acid and DDPC; glycyrrhizinic acid and DLPC; glycyrrhizinic acid and Tween 40; glycyrrhizinic acid and Tween 60; glycyrrhizinic acid and Tween 80; glycyrrhizinic acid and SPAN 40; glycyrrhizinic acid and SPAN 65; glycyrrhizinic acid and SPAN 85; glycyrrhizinic acid and α-tocopherol; glycyrrhizinic acid and glycerol monooleate; and glycyrrhizinic acid and polyoxyethylene castor oil.

[Substance with stimulus to activate innate immune system]



[0066] The substance with stimulus to activate innate immune system is defined as a substance which is recognized by the receptor for structural pattern recognition and activates immunocompetent cells.

[0067] The substance with stimulus to activate innate immune system is not specifically restricted; however, it may preferably be an agonist for Toll-like receptors.

[0068] The substance with stimulus to activate innate immune system includes the following examples which are not specifically restricted.

[0069] Mineral salts such as alum; gel-type adjuvants such as aluminum hydroxide, aluminum phosphate, and calcium phosphate; immunoregulatory DNA sequences including CpG motif; immunostimulatory RNA molecules; endotoxins (lipopolysaccharide (LPS: endotoxin)); monophosphoryl lipid A (MPL: registered trademark), exotoxins, such as cholera toxin, E. coli, thermolabile toxin, and pertussis toxin; microbial adjuvants, such as muramyl dipeptide and flagellin; oil adjuvants, such as incomplete Freund's adjuvant (IFA); oil adjuvants, such as liquid paraffin and lanolin; biodegradable microspheres; saponins (QS-21, Quil-A, etc.); nonionic block copolymers; muramyl peptide analogs; polyphosphazenes; synthetic polynucleotides, such as non-CpG synthetic polynucleotide; synthetic adjuvants, such as imidazoquinoline; cationic lipids, such as DOTAP, DC-Chol, and DDA; single-stranded RNA; and double-stranded RNA.

[0070] Preferable examples of the substance with stimulus to activate innate immune system among the foregoing are listed below. Mineral salts such as alum; gel-type adjuvants, such as aluminum hydroxide, aluminum phosphate, and calcium phosphate; immunoregulatory DNA sequences including CpG motif; immunostimulatory RNA molecules; monophosphoryl lipid A (MPL: registered trademark), exotoxins, such as cholera toxin, E. coli, thermolabile toxin, and pertussis toxin; microbial adjuvants, such as flagellin; saponins (QS-21, Quil-A, etc.); synthetic polynucleotides, such as non-CpG synthetic polynucleotide; synthetic adjuvants, such as imidazoquinoline; single-stranded RNA; and double-stranded RNA. Most preferable are monophosphoryl lipid A and immunoregulatory DNA sequences including CpG motif.

[0071] The substance with stimulus to activate innate immune system may be used alone or in combination with one another.

[0072] The substance with stimulus to activate innate immune system may vary in content depending on the kind of the substance with stimulus to activate innate immune system used. An adequate content for 100 mol of the amphipathic substance is 0.0227 to 22.7 mol, preferably 0.227 to 2.27 mol. A content of the substance with stimulus to activate innate immune system, which content is equal to or more than 0.0227 mol, is desirable because it is high enough to adequately induce the immune response. Also, a content of the substance with stimulus to activate innate immune system, which content is equal to or lower than 22.7 mol, is desirable for cost reduction.

[Aqueous solvent]



[0073] The adjuvant composition may contain an aqueous solvent.

[0074] In the case where the adjuvant composition contains an aqueous solvent, the pH-sensitive carrier and the substance with stimulus to activate innate immune system may form a dispersion in the aqueous solvent.

[0075] In this situation, the pH-sensitive carrier may preferably form in the aqueous medium a complex containing the pH-sensitive compound and the amphipathic substance. This complex may take on any form without specific restrictions. That is, the pH-sensitive compound and the amphipathic substance may form a membrane. Alternatively, the amphipathic substance may form a structure in which the pH-sensitive compound is partly or entirely embedded by association. It is desirable that the pH-sensitive compound and the amphipathic substance form micelle particles together. (Micelle particles result from hydrophobic interaction between the pH-sensitive compound and the amphipathic substance. They are typically particles of monomolecular film structure.) The micelle particles may have a diameter of 10 to 200 nm, preferably 10 to 100 nm, because they actively perform phagocytosis and endocytosis only when they have a particle diameter equal to or larger than a certain level. Incidentally the above-mentioned micelle particles do not include those which form lipid bimolecular membrane structure (such as liposome). In this specification, the particle diameter of the pH-sensitive carrier is measured by the dynamic light scattering method (with NanoZS90 made by MALVERN Instruments Co., Ltd.).

[0076] The adjuvant composition may preferably be one which forms in an aqueous medium a complex (adjuvant complex) which is composed of the pH-sensitive carrier in the form of complex and the substance with stimulus to activate innate immune system. Although the complex is not specifically restricted in its form, it may preferably take on the form of micelle particles composed of the pH-sensitive compound and the amphipathic substance composing the pH-sensitive carrier and the substance with stimulus to activate innate immune system. The micelle particles may have a particle diameter of 10 to 200 nm, preferably 10 to 100 nm.

[0077] Incidentally, in the aqueous solution containing the adjuvant composition, at least one of the pH-sensitive compound, the amphipathic substance, and the substance to activate innate immune system remains free without undergoing association.

[0078] The medium for the aqueous solution containing the pH-sensitive carrier according to the present invention may preferably be an aqueous solution containing a buffering agent, NaCl, sugar (such as glucose and sucrose), etc.

[0079] The buffering agent is not specifically restricted; any known one may be used so long as it can keep the pH of the adjuvant composition equal to or higher than the physiological pH. Examples of the buffering agent are listed below.

[0080] Phosphate buffers, citrate buffers, citrate-phosphate buffers, tris(hydroxymethyl)aminomethane-HCl buffers (tris-hydrochloric acid buffer), MES buffers (2-morpholinoethane sulfonate buffer), TES buffers (N-tris(hydroxymethyl)methyl-2-aminoethane sulfonate buffer), acetate buffers, MOPS buffers (3-morpholinopropane sulfonate buffer), MOPS-NaOH buffers, HEPES buffers (4-(2-hydroxyethyl)-1-piperazine ethanesulfonate buffer), HEPES-NaOH buffers and other GOOD buffers, amino acid buffers (such as glycine-hydrochloric acid buffer, glycine-NaOH buffer, glycylglycine-NaOH buffer, and glycylglycine-KOH buffer), boric acid-based buffers (such as Tris-borate buffer, borate-NaOH buffer, and borate buffer), and imidazole buffers. Preferable among these buffers are phosphate buffers, citrate buffers, citrate-phosphate buffers, Tris-hydrochloric acid buffers, MES buffers, acetate buffers, and HEPES-NaOH buffers. These buffers may be used in any concentration without specific restrictions. An adequate concentration is 0.1 to 200 mM, preferably 1 to 100 mM. Incidentally, the term "concentration of buffer" used in this specification denotes the concentration (mM) of the buffer contained in the aqueous solution.

[0081] The concentration of NaCl and sugar (such as glucose and sucrose) is not specifically restricted; it may preferably be 0.1 to 200 mM, more preferably 1 to 150 mM.

[0082] The concentration of the pH-sensitive carrier in the aqueous solution is not specifically restricted; it may be such that the total molar concentration of the pH-sensitive compound and the amphipathic substance is 0.73 µmol/L to 7.4 mmol/L, preferably 7.3 µmol/L to 6.5 mmol/L, and more preferably 8.0 µmol/L to 4.2 mmol/L.

[0083] Moreover, the aqueous solution may contain the substance with stimulus to activate innate immune system in any molar concentration without specific restrictions. An adequate concentration is 0.14 nmol/L to 0.227 mmol/L, preferably 1.4 nmol/L to 0.19 mmol/L, and more preferably 1.6 nmol/L to 0.12 mmol/L.

[Additional components]



[0084] The adjuvant composition may further contain additional components without specific restrictions. A typical example of them is a stabilizer.

[0085] The stabilizer is not specifically restricted and any known one may be used so long as it does not adversely affect the pH-sensitive carrier and the substance with stimulus to activate innate immune system. Examples of the stabilizer are listed below.

[0086] Saturated or unsaturated C4-20 alcohols, such as 1-octanol, 1-dodecanol, 1-hexadodecanol, and 1-eicosanol; saturated or unsaturated C12-18 fatty acids, such as lauric acid, myristic acid, palmitic acid, stearic acid, and oleic acid; C1-3 alkyl esters of saturated or unsaturated C8-18 fatty acids, such as methyl caprylate (methyl octanoate), ethyl caprylate (ethyl octanoate), methyl laurate, ethyl laurate, ethyl myristate, ethyl palmitate, ethyl stearate, methyl oleate, and ethyl oleate; D(L)-amino acids, such as D(L)-alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, leucine, isoleucine, lysine, methionine, proline, serine, threonine, triptophan, tyrosine, valine, and phenylalanine; amino acid triglycerides, such as tricaproin and tricaprylin; C12-18 polyoxyethylene sorbitan trifatty acid esters, such as polyoxyethylene sorbitan tripalmitic acid ester and polyoxyethylene sorbitan trioleic acid ester (known, for example, as Tween 65 and Tween 85); C12-18 polyoxyethylene alkyl esters, such as polyoxyethylene lauric acid ester, polyoxyethylene myristic acid ester, polyoxyethylene palmitic acid ester, and polyoxyethylene stearic acid ester (known, for example, as PEG20 stearyl ether and PEG23 lauryl ether); polyoxyalkylene hardened castor oil (known, for example, as PEG10 hardened castor oil, PEG40 hardened castor oil, and PEG60 hardened castor oil); saturated or unsaturated C8-18 monofatty acid glycerol esters, such as caprylin (glycerol octanoate), glycerol monocaprylate, glycerol monolaurate, glycerol monomyristate, glycerol monopalmitate, glycerol monostearate, and glycerol monooleate; glycerols of C8-16 difatty acids, such as glycerol dioctanoate, glycerol dicaprylate, glycerol dilaurate, glycerol dimyristate ester, and glycerol dipalmitate; α-tocopherol acetate ester, castor oil, soybean oil, cholesterol, squalene, squalane, lactose, ascorbyl palmitate, benzyl benzoate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate. Incidentally, the "carbon number" for the stabilizer denotes the number of carbon atoms in the fatty acid component (acyl group) constituting the hydrophobic moiety.

[0087] The content of the additional components is not specifically restricted so long as it does not adversely affect the pH-sensitive carrier and the substance with stimulus to activate innate immune system. An adequate content for 100 mol of the amphipathic substance is 150 mol or lower, preferably more than 0 mol and 66.4 mol or lower.

[0088] The adjuvant composition pertaining to the embodiment of the present invention effectively induces CTL when administered together with an antigen.

[0089] In other words, the adjuvant composition pertaining to the embodiment of the present invention permits the pH-sensitive carrier used in combination with the substance with stimulus to activate innate immune system to appropriately exhibit its function, for example, its membrane disruptive function promoting effect (and its membrane fusion function promoting effect). Moreover, the pH-sensitive carrier used in combination with the substance with stimulus to activate innate immune system allows the substance with stimulus to activate innate immune system to appropriately exhibit its function. This may be reasoned as follows although no complete elucidation has been made yet.

[0090] The pH-sensitive carrier in its desirable form contains the pH-sensitive compound and the amphipathic substance and also has the membrane disruptive function promoting effect (and the membrane disruptive function promoting effect as well as the membrane fusion function promoting effect in some cases). The membrane disruptive function promoting effect (and the membrane fusion function promoting effect) is attributable to the fact that the pH-sensitive carrier changes in the state of association owing to the pH-sensitive compound in an acidic environment and the rearrangement with the cell membrane (such as endosome) is brought about by the amphipathic substance in such a situation. The pH-sensitive compound changes the state of association of the pH-sensitive carrier because it does not change in pH sensitivity even though the pH-sensitive carrier is combined with the substance with stimulus to activate innate immune system. Moreover, the amphipathic substance does not affect the rearrangement with cell membrane even though the substance with stimulus to activate innate immune system is incorporated into the amphipathic substance or exists independently of the pH-sensitive carrier. Thus, the substance with stimulus to activate innate immune system does not impair the function of the pH-sensitive carrier even though it is used in combination with the pH-sensitive carrier. Further, the substance with stimulus to activate innate immune system keeps its function unimpaired because it is merely incorporated into the amphipathic substance of the pH-sensitive carrier by the hydrophobic mutual action and it also merely exists independently of the pH-sensitive carrier. Consequently, the adjuvant composition pertaining to the present invention causes the antigen to be introduced into a cytosol by the function of the pH-sensitive carrier and, at the same time, the substance with stimulus to activate innate immune system acts when it is administered together with the antigen. As the result, the adjuvant composition effectively induces cross-presentation due to the antigen introduced into the cytosol and hence effectively induces CTL.

[0091] The foregoing reason is a mere conjecture, and any other effects produced by other causes will be included in the scope of the present invention.

[0092] According to another preferred embodiment of the present invention, the adjuvant composition also effectively induces humoral immunity.

[0093] As mentioned above, any exogenous antigen is usually degraded into peptide fragments by the endosome in the antigen presenting cells and forms a complex with MHC class II molecules to be presented to the CD4 positive T cells.

[0094] The adjuvant composition according to this embodiment leads the antigen administered together with it to cross-presentation, thereby inducing CTL. Thus, when the pH-sensitive carrier brings about rearrangement of the cell membrane of endosome, the antigen and the substance with stimulus to activate innate immune system are introduced into the cytosol. In one embodiment, however, there may exist an instance in which the antigen and the substance with stimulus to activate innate immune system remain partly or entirely in the endosome even though the rearrangement is brought about. Also, in another embodiment, there may exist an instance in which only the antigen is taken up in part of endosome if the antigen and the adjuvant composition exist mutually independently. Thus, the antigen is degraded into peptide fragments by endosome and such peptide fragments form a complex with MHC class II molecules, thereby presenting antigen to the CD4 positive T cells and inducing humoral immunity. The induction of humoral immunity appropriately occurs because the dendritic cells which effectively induce cross-presentation are in the immunologically activated state. Alternatively, the dendritic cells which effectively induce cross-presentation actively generates cytokine (e.g., IFNγ), which activates immunity, thereby leading the surrounding environment to the environment suitable for immune induction.

[0095] Therefore, the adjuvant composition according to another embodiment of the present invention induces the cross-presentation or the humoral immunity in place of the cross-presentation.

<Vaccine composition>



[0096] The vaccine composition contains an adjuvant composition and an antigen.

[Adjuvant composition]



[0097] The adjuvant composition is the same one as mentioned; and hence its description is omitted here.

[Antigen]



[0098] The antigen is not specifically restricted so long as it produces immune response; it may preferably be a peptide or protein.

[0099] Examples of the peptide and protein include viral antigens, bacterial antigens, mycotic antigens, protozoan antigens or verminous antigens, cancer antigens, allergy-related antigens, disease-related antigens, and graft rejection-related antigens.

[0100] Examples of the foregoing viral antigen include the following without specific restrictions.

[0101] Human immunodeficiency virus (HIV) antigens, such as gene product of gag, pol, and env genes, Nef protein, reverse transcriptase, and other HIV components; hepatitis virus antigens, such as S, M, and L protein of hepatitis B virus, pre-S antigen of hepatitis B virus, hepatitis C virus RNA, and virus components of hepatitis A, B, and C; influenza virus antigens, such as hemagglutinin, neuraminidase, and other influenza virus components; measles virus antigens; rubella virus antigens; rotavirus antigens; cytomegalovirus antigens; respiratory syncytial virus antigens; herpes simplex virus antigens; varicella zoster virus antigens; Japanese encephalitis virus antigens; and rabies virus antigens. Additional examples include peptides derived from adenoviruses, retroviruses, picornaviruses; herpesviruses, rotaviruses, hantaviruses, coronaviruses, togaviruses, flaviviruses, rhabdoviruses, paramyxoviruses, orthomyxoviruses, bunyaviruses, arenaviruses, reoviruses, papillomaviruses, parvoviruses, poxviruses, hepadnaviruses, and spongy viruses.

[0102] Examples of the foregoing bacterial antigens include the following without specific restrictions. Bacterial antigens, such as pertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, cyclase adenylate, and other pertussis bacterial antigen components; diphteria bacterial antigens, such as diphteria toxin or toxoid, and other diphteria bacterial antigen components; bacterial antigens of tetanus bacillus and streptococcus, such as tetanus toxin or toxoid, and other tetanus bacterial antigen components; Gram-negative bacillary bacterial antigens, such as lipopolysaccharide, and other Gram-negative bacterial antigen components; tubercle bacillus bacterial antigens, such as mycolic acid, heat-shock protein 65 (HSP 65), 30 kDa major secretory protein, antigen 85A, and other mycobacteria antigen components; helicobacter and pylori bacterial antigen components; pneumococcus bacterial antigen; influenza bacillus bacterial antigen; anthrax bacillus bacterial antigen; and rikettsia bacterial antigen.

[0103]  Examples of the foregoing mycotic antigen include the following without specific restrictions. Candida mycotic antigen components; histoplasma mycotic antigens; cryptococcus mycotic antigens; coccidioides mycotic antigens; and ringworm mycotic antigens.

[0104] Examples of the foregoing protozoan antigen or verminous antigen include the following without specific restrictions. Plasmodium falciparum antigens; toxoplasma antigens; schistosoma antigens; Leishmania antigens; and trypanosome cruzi antigens.

[0105] Examples of the foregoing cancer antigen unrestrictedly include those cancer antigens derived from the cell surface of cells of tumor tissue, protoplasm, nuclei, and cell organelles. Examples of the cancer include the following. Leukemia, lymphoma, nervous tumor, melanoma, breast cancer, lung cancer, head and neck cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, cancer of the uterine cervix, uterine cancer, ovarian cancer, vaginal cancer, testis cancer, prostatic cancer, penile cancer, bone tumors, hemangioma, lip cancer, cancer of epipharynx, pharyngeal cancer, carcinoma of the esophagus, rectal cancer, carcinoma of the gallbladder, cancer of the bile duct, laryngeal cancer, lung cancer, bladder carcinoma, kidney cancer, brain tumor, thyroid carcinoma, Hodgkin's disease, and non-Hodgkin lymphoma. Incidentally, typical examples of the cancer antigen are listed below. HER2/neu (Human EGFR related 2), CEA (Carcinogenic Embryonic Antigen), MAGE (Melanoma-associated Antigen), XAGE (X antigen family member), NY-ESO-1, gp100, Melan/mart-1, Tyrosinase, PSA (Prostate Sepecific Antigen), PAP (Prostate Acid Phosphatase), p53, K-ras, N-ras, Bcr-Abl, MUC-1 (Mucin-1), PSMA (Prostate Specific Membrane Antigen), survivin, WT-1 (Wilms tumor suppressor gene 1), AFP (Alpha Fetoprotein), GPC (Glypican), and EGFR (Epidermal Growth Factor Receptor).

[0106] Examples of the foregoing allergy-related antigens include the following without specific restrictions. Pollen antigens, such as cedar pollen antigen, ragweed pollen antigen, and ryegrass pollen antigen; animal-derived antigens, such as rat mite antigen and cat antigen; histocompatible antigens; and therapeutic drugs such as penicillin.

[0107] The foregoing disease-related antigen (autoimmune disease and allergy) results from the diseases whose examples are listed below without specific restrictions. Diabetes, articular rheumatism, myasthenia gravis, systemic lupus erythematosus, atopic dermatitis, psoriasis, Sjögren's syndrome, alopecia areata, Crohn's disease, ulcerative colitis, conjunctivitis, keratoconjunctivitis, asthma, allergic asthma, proctitis, drug eruption, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, hypoplastic anemia, erythroblast anemia, idiopathic thrombocytsopenia, Wegener granulomatosis, Stevens-Johnson syndrome, and interstitial pulmonary fibrosis. Specific examples of the antigens involved in autoimmune disease include, for example, glutamic acid decarboxylase 65 (GAD 65), natural DNAs, myelin basic proteins, myelin proteolipid proteins, acetylcholine receptor components, thyroglobulins, and thyroid stimulating hormone (TSH) receptors.

[0108] Examples of the foregoing graft rejection-related antigens unrestrictedly includes the antigenic components of the graft to be transferred to the graft recipient, the graft including heart, lung, liver, pancreas, kidney, and nerve.

[0109]  The above-mentioned antigens may be used alone or in combination with one another.

[0110] The antigen may be contained in an amount of 3.2 to 400 µg, preferably 16 µg to 80 µg, for 100 nmol of the amphipathic substance as a constituent of the pH-sensitive carrier.

[0111] Although the antigen may exist independently of the adjuvant composition, the ratio of the antigen to be incorporated into the adjuvant composition may be equal to or higher than 3%, preferably 5 to 80%, more preferably 10 to 60%. The ratio equal to or higher than 3% is desirable because, when the vaccine composition is incorporated into cells by endocytosis, the antigen is readily introduced into the same endosome as that into which the adjuvant composition is introduced. This leads to the desirable effect of the present invention. Incidentally "the incorporation rate of the antigen" denotes the ratio of the antigen to the adjuvant composition which contains or supports the antigen. The value of the ratio is obtained by the methods described in the working example.

[Additives]



[0112] The vaccine composition may additionally contain pharmaceutical additives.

[0113] The suitable additives vary depending on the dosage form of the vaccine composition. The vaccine composition may be solid preparations, such as tablets, powders, and capsules or liquid preparations, such as parenteral solution, with the latter being preferable. Incidentally, the liquid preparations may be supplied in a dried form which can be restored with water or any other adequate diluent at the time of use.

[0114] The vaccine composition in the form of tablets or capsules may preferably be provided with enteric coating in the ordinary way. The enteric coating may be one which is ordinarily used in the field concerned. The vaccine composition may also contain any one of capsules, powders, and liquids.

[0115] The vaccine composition in the form of solid preparations may contain a diluent (such as sugar e.g., lactose and sucrose, starch e.g., corn starch, cellulose e.g., crystalline cellulose, acacia, magnesium metasilicate aluminate, and calcium phosphate), lubricant (such as magnesium stearate, talc, and polyethylene glycol), binder (such as sugar e.g., mannitol and sucrose, crystalline cellulose, polyvinylpyrrolidone, and hydroxypropylmethyl cellulose), disintegrator (such as starch e.g., potato starch, cellulose e.g., carboxymethyl cellulose, and crosslinked polyvinylpyrrolidone), coloring agent, and corrigent.

[0116] On the other hand, the vaccine composition in the form of liquid preparations may contain a solvent (such as physiological saline, sterilized water, and buffer solution), membrane stabilizer (such as cholesterol), tonicity adjusting agent (such as sodium chloride, glucose, and glycerin), antioxidant (such as tocopherol, ascorbic acid, and glutathione), and anticeptics (such as chlorobutanol and paraben). Incidentally, the solvent may be the same one as used for preparation of the vaccine composition.

[0117] According to one embodiment of the present invention, the vaccine composition may induce cell-mediated immunity through effective cross-presentation of the antigen. This permits the induction of CTL. The term "induce cell-mediated immunity" in this specification means that the CTL induction rate is higher than that in control which is not treated with the vaccine composition. Additionally, the term "CTL induction rate" means the ratio of IFNγ-secreting cells that accounts for in all the CD8 positive cells.

[0118] Incidentally, the adjuvant composition pertaining to this embodiment produces the effect of enhancing cell-mediated immunity in view of the fact that it gives a higher CTL induction rate than in control treated with the substance to activate innate immune system even though the same antigen is administered. In this specification, the term "produce the effect of enhancing CTL induction" means that a higher CTL induction rate is obtained than in the case where the substance to stimulate innate immune system is used alone. Thus, the effect of enhancing CTL induction is accompanied by the induction of cell-mediated immunity.

[0119] The vaccine composition pertaining to another embodiment of the present invention is able to induce immune response for humoral immunity. Thus, it is able to produce an antibody such as IgG. Here, the term "induce humoral immunity" means that the IgG antibody titer is higher than in that of control which is not treated with the vaccine composition.

[0120] Incidentally, the vaccine composition pertaining to this embodiment involves the effect of enhancing humoral immunity in view of the fact that it gives a higher IgG antibody titer than in control treated with the substance to activate innate immune system.

[Applications]



[0121] The vaccine composition of the present invention produces the membrane disruptive function promoting effect or the membrane disruptive function promoting effect and the membrane fusion function promoting effect, thereby allowing the antigen to efficiently release to the cytosol, when it is administered to a subject and the external environment of the vaccine composition decreases in pH below the physiological pH (for example, pH 6.5). Thus, it appropriately induces cell-mediated immune to develop immunity.

[0122] Thus, the vaccine composition pertaining to this embodiment provides a method for therapy or prevention of diseases by including administration of the vaccine composition in an effective amount to a subject who needs therapy or prevention of diseases.

[0123] The method for administration of the vaccine composition is not specifically restricted. Oral administration and parenteral administration such as intravenous injection, arterial injection, subcutaneous injection, endodermic injection, intramuscular injection, intrathecal injection, dermal administration, and percutaneous absorption are mentioned. The vaccine composition that contains peptide or protein as the antigen is suitable for parenteral administration, particularly subcutaneous injection, endodermic injection, intramuscular injection, and intravenous injection. Note that the vaccine composition in which the antigen is simply mixed with the adjuvant composition without being supported on or contained in it is suitable for local administration, specifically subcutaneous injection, endodermic injection, and intramuscular injection.

[0124] The above-mentioned subject may preferably be a mammal, especially human.

[0125]  The above-mentioned diseases include, for example, virus infectious diseases such as human immunodeficiency syndrome (HIV), hepatitis, and influenza; bacterial infections such as pertussis, diphtheria, tetanus, tuberculosis, Helicobactger pylori, and microbism caused by pneumococcus; mycosis such as candida; protozoiassis or vermiculous infections such as malaria; cancers such as leukemia, lymphoma, nervous tumor, melanoma, breast cancer, lung cancer, head and neck cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, cancer of the uterine cervix, uterine cancer, ovarian cancer, vaginal cancer, testis cancer, prostatic cancer, penile cancer, osteoncus, hemangioma, lip cancer, cancer of epipharynx, pharyngeal cancer, carcinoma of the esophagus, rectal cancer, carcinoma of the gallbladder, cancer of the bile duct, laryngeal cancer, lung cancer, bladder carcinoma, kidney cancer, brain tumor, thyroid carcinoma, Hodgkin's disease, and non-Hodgkin lymphoma; allergy caused by cedar pollen; autoimmune diseases such as diabetes, articular rheumatism, myasthenia gravis, and systemic lupus erythematosus; and graft rejections such as graft-versus-host disease (GVHD).

[0126] One preferable embodiment of the present invention provides a method for therapy or prevention of diseases. Such a method may be found in the common technical knowledge at the time of application of the present invention.

[0127] According to one embodiment of the present invention, the vaccine composition permits the antigen to be transported to cells by culture. In other words, the embodiment of the present invention provides a method for culture to transport the antigen to cells.

[0128] The above-mentioned method for culture includes a step of cultivating cells by using a culture medium containing the vaccine composition.

[0129] The culture medium is not specifically restricted; any known ones can be used. Their typical examples include MEM, DMEM, and RPMI.

[0130] No specific restrictions are imposed on the amount of the vaccine composition to be added to the culture medium. An adequate amount is such that the molar concentration of the amphipathic substance is 0.66 µmol/L to 1.0 mmol/L, preferably 6.6 µmol/L to 0.88 mmol/L, and more preferably 7.2 µmol/L to 0.56 mmol/L.

[0131] The above-mentioned culture medium may have a pH value equal to or higher than 7.0, preferably 7.2 to 7.8. Any culture medium with a pH value equal to or higher than 7.0 is desirable because it keeps stable the pH-sensitive compound constituting the pH-sensitive carrier in the culture medium.

[0132] The above-mentioned cells include, without specific restrictions, those cells collected from the subject and established cultured cells.

[0133] Typical examples of the cells collected from the subject or the cells collected from established cultured cells include dendritic cells, NK (Natural Killer) cells, T lymphocytes, B lymphocytes, and lymphoblasts.

[0134] Preferable among the cells mentioned above are cells collected from the subject, more preferably dendritic cells, NK cells, T lymphocytes, and lymphoblasts collected from the subject, and most desirably dendritic cells.

[0135] Collection of cells from the subject may be accomplished by blood collection or biopsy. The method of culture in one embodiment includes a step of collecting cells from the subject.

[0136] Incidentally, the cultured cells may be administered to the subject for therapy or prevention of the subject's disease. Thus, one embodiment of the present invention provides a method of treating and preventing diseases.

[0137] The method for treating and preventing disease, which is provided in one preferred embodiment of the present invention, includes a step of collecting cells from the subject, a step of cultivating the collected cells by using a culture medium containing the vaccine composition, and a step of administrating the cultured cells to the subject.

[0138] Thus, the foregoing method is used to treat and prevent diseases, whose examples were mentioned above.

<Method for producing the adjuvant composition>



[0139] The adjuvant composition pertaining to the present invention can be produced by various methods without specific restrictions.

[0140] The adjuvant composition in which the pH-sensitive carrier and the substance with stimulus to activate innate immune system exist independently can be produced by mixing together the pH-sensitive carrier and the substance with stimulus to activate innate immune system. In addition, the adjuvant composition in which the substance with stimulus to activate innate immune system is supported on or contained in the pH-sensitive carrier can be produced by associating the pH-sensitive carrier with the substance with stimulus to activate innate immune system.

[0141] The following is a detailed description of the preferred embodiment of the method for producing the adjuvant composition in which the pH-sensitive carrier is composed of a prescribed pH-sensitive compound and a prescribed amphipathic substance, the pH-sensitive carrier possessing the membrane disruptive function promoting effect.

[0142] According to this embodiment, the method for producing the adjuvant composition has a step of associating with one other at least one species of pH-sensitive compound, at least one species of amphipathic substance, and a substance with stimulus to activate innate immune system. The pH-sensitive compound is one which is selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof. The amphipathic substance is one which is selected from the group consisting of C10-12 phosphatidylcholines, C12-18 polyoxyethylene sorbitan monofatty acid esters, C16-18 sorbitan fatty acid esters, glycerol monooleates, glycerol dilaurates, glycerol distearates, glycerol dioleates, polyoxyethylene castor oils, and α-tocopherols.

[0143] The step of associating with one other the pH-sensitive compound, the amphipathic substance, and the substance with stimulus to activate innate immune system may be accomplished in such a way that they come into contact with one another in an aqueous solution. Therefore, the adjuvant composition according to this embodiment can be produced by causing the pH-sensitive compound, the amphipathic substance, and the substance with stimulus to activate innate immune system to come into contact with one another in an aqueous solution.

[0144] There are no specific restrictions as to the method for bringing the pH-sensitive compound, the amphipathic substance, and the substance with stimulus to activate innate immune system into contact with one another in an aqueous solution. Any method is acceptable so long as it forms an associated body. Thus the adjuvant composition may be produced by either of the following methods. (1) The first method consists of preparing separately an aqueous solution containing a pH-sensitive compound, an aqueous solution containing an amphipathic substance, and an aqueous solution containing a substance with stimulus to activate innate immune system, mixing these aqueous solutions, and vigorously stirring the mixed solution by means of an emulsifier, vortex mixer, ultrasonic wave, or the like to achieve their dispersion. (2) The second method known as Bangham method for producing liposome. The Bangham method typically consists of the following steps. First, the constituents of the adjuvant composition are dissolved in an organic solvent (e.g., methanol and chloroform) in a glass container. The resulting solution is freed of the organic solvent by means of a rotary evaporator or the like, so that a thin film is formed on the wall of the glass container. Then, the glass container, in which a thin film has been formed, is given an aqueous solution, so that the thin film is moistened at 5 to 35°C. The glass container is vigorously shaken at 5 to 35°C by means of an emulsifier, vortex mixer, ultrasonic wave, or the like, so that the thin film is thoroughly dispersed into the aqueous solution. The details of Bangham method will be found in known literature on the method for producing liposome, such as "Liposome" (compiled by Shoshichi Nojima, Junzo Sunamoto, and Keizo Inoue, published by Nankoudo), and "Liposome in Life Science" (compiled by Hiroshi Terada and Tetsuro Yoshimura, published by Springer-Verlag, Tokyo). The first method (1) mentioned above may be modified such that the aqueous solution containing the amphipathic substance is mixed with the pH-sensitive compound and the substance with stimulus to activate innate immune system. The solvent for the aqueous solution may be the same one as mentioned above. In the first method (1) mentioned above, no specific restrictions are imposed on the temperature at which the aqueous solutions are prepared and stirred. An adequate temperature is 5 to 35°C, preferably 15 to 25°C at normal temperature.

[0145] The adjuvant composition containing an aqueous solvent as a component thereof may be incorporated with additional components such as stabilizer in any way without specific restrictions. For example, it may be incorporated with an aqueous solution containing a pH-sensitive compound, an aqueous solution containing an amphipathic substance, and/or an aqueous solution containing a substance with stimulus to activate innate immune system. It is also possible to prepare an aqueous solution containing the adjuvant composition from the thin film prepared by Bangham method in which they are dissolved together with the constituents of the adjuvant composition.

<Method for producing vaccine composition>



[0146] The vaccine composition according to the present invention may be produced in various ways without specific restrictions. The method for producing the vaccine composition typically includes the preparation method by dispersion, the preparation method by mixing, and the preparation method by freeze-thawing and freeze-drying.

(Preparation method by dispersion)



[0147] The preparation method by dispersion includes the step of mixing together the pH-sensitive compound, the amphipathic substance, the substance with stimulus to activate innate immune system, and the antigen. That is, the constituents of the adjuvant composition are allowed to form a thin film on the wall of a glass container. Then, the glass container, in which a thin film has been formed, is given a solution containing the antigen, so that the thin film is moistened at 5 to 35°C. The glass container is vigorously shaken for dispersion by means of an emulsifier, vortex mixer, ultrasonic wave, or the like. In this way, the vaccine composition is prepared. Alternatively, a thin film containing the pH-sensitive compound and the amphipathic substance is formed on the wall of a glass container. Then, the glass container, in which a thin film has been formed, is given a solution containing the antigen and the substance with stimulus to activate innate immune system, so that the thin film is moistened at 5 to 35°C. The glass container is vigorously shaken by means of an emulsifier, vortex mixer, ultrasonic wave, or the like. In this way, the vaccine composition is prepared.

[0148] The foregoing solution containing the antigen and the foregoing solution containing the antigen and the substance with stimulus to activate innate immune system may be the same ones or the referenced ones as prepared by the preparation method by mixing which is mentioned below.

(Preparation method by mixing)



[0149] The preparation method by mixing includes the step of mixing solutions each containing the pH-sensitive compound, the amphipathic substance, the substance with stimulus to activate innate immune system, and the antigen. In other words, the preparation method by mixing includes the step of preparing the adjuvant composition by the above-mentioned methods (1) and (2). In the case where the adjuvant composition is prepared by the method (2), the vaccine composition is obtained by mixing together a dispersion of the adjuvant composition and the antigen or a solution containing the antigen. Alternatively, in the case where the adjuvant composition is obtained by the method (1), it is desirable to prepare the following solutions.

Solution containing pH-sensitive compound



[0150] The above-mentioned solution containing the pH-sensitive includes of the pH-sensitive compound and the solvent. It may also contain optional additives.

[0151] The above-mentioned pH-sensitive compound may be the same one as mentioned above. Therefore its description is not repeated here.

[0152] The above-mentioned solvent may be an aqueous solution containing buffer, NaCl, or sugar (such as glucose and sucrose), or sterilized water. It is desirable to use physiological saline, sterilized water, or buffer solution from the standpoint of administrating the vaccine composition into a living body.

[0153] The concentration of the pH-sensitive carrier in the solution containing the pH-sensitive compound is one which is attained when the molar concentration of the pH-sensitive compound is 0.066 µmol/L to 6.4 mmol/L, preferably 0.66 µmol/L to 5.6 mmol/L, and more preferably 0.72 µmol to 3.6 mmol/L.

Solution containing amphipathic substance



[0154] The solution containing the amphipathic substance mentioned above is composed of an amphipathic substance and a solvent. It may also contain optional additives.

[0155] The above-mentioned amphipathic substance and the above-mentioned solvent may be the same as mentioned earlier. Therefore their descriptions are not repeated here.

[0156] The concentration of the amphipathic substance in the solution containing the amphipathic substance may be such that the molar concentration of the amphipathic substance is 0.66 µmol/L to 1.0 mmol/L, preferably 6.6 µmol/L to 0.88 mmol/L, and more preferably 7.2 µmol to 0.56 mmol/L.

Solution containing substance with stimulus to activate innate immune system



[0157] The above-mentioned solution containing the substance with stimulus to activate innate immune system includes the substance with stimulus to activate innate immune system and a solvent. It may also contain optional additives.

[0158] The substance with stimulus to activate innate immune system and the solvent may be the same as mentioned earlier. Therefore their descriptions are not repeated here.

[0159] The concentration of the substance with stimulus to activate innate immune system in the solution containing the substance with stimulus to activate innate immune system is attained when the molar concentration of the substance with stimulus to activate innate immune system is 0.14 nmol/L to 0.227 mmol/L, preferably 1.4 nmol/L to 0.19 mmol, and more preferably 1.6 nmol/L to 0.12 mmol/L.

Solution containing antigen



[0160] The solution containing the antigen includes the antigen and a solvent.

[0161] The antigen and the solvent may be the same as mentioned earlier. Therefore their descriptions are not repeated here.

Mixing



[0162] No specific restrictions are imposed on the method of mixing together the solution containing the pH-sensitive compound, the solution containing the amphipathic substance, the solution containing the substance with stimulus to activate innate immune system, and the antigen.

[0163] The resulting mixed solution may preferably undergo dispersion. This dispersion may be accomplished with the help of emulsifier, vortex mixer, ultrasonic wave, or the like.

[0164] According to one embodiment, it is not necessarily need to prepare separately the solution containing the pH-sensitive compound, the solution containing the amphipathic substance, the solution containing the substance with stimulus to activate innate immune system, and the solution containing the antigen. Instead, it is possible to use equal to or not less than two solutions in the form of mixture. For example, a first solution is prepared which contains the pH-sensitive compound and the substance with stimulus to activate innate immune system, and a second solution is prepared which contains the amphipathic substance and the antigen. Then the two solutions are mixed together.

(Preparation method by freeze-thawing and freeze-drying)



[0165] The preparation method by freeze-thawing and freeze-drying includes a step of freeze-thawing a solution obtained by the preparation method by dispersion or mixing, thereby giving a molten solution, and a step of freeze-drying the molten solution.

Step of preparing molten solution



[0166] The molten solution is obtained by freeze-thawing the solution obtained by the preparation method by dispersion or mixing.

[0167] The term "freeze-thawing" means a step of freeze-drying a solution and melting the resulting dried product.

[0168] The method for freeze-drying is not specifically restricted. It should be achieved preferably by subliming water with the help of liquefied nitrogen or cooled methanol.

[0169] The dried product may be molten in any way without specific restrictions. It is desirable to heat the dried product resulting from cooling, or it is desirable to add a solvent to the dried product.

Step of freeze-drying



[0170] This step is intended to freeze-dry the molten solution obtained as mentioned above.

[0171] The method for freeze-drying is not specifically restricted as in the above-mentioned case, it is desirable to sublime water by means of liquefied nitrogen or cooled methanol.

[0172] Preferable among the above-mentioned methods for production of the vaccine composition is the preparation method by freeze-thawing and freeze-drying of the antigen. The preparation method by freeze-thawing and freeze-drying permits the antigen to be easily supported on or contained in the adjuvant composition. This leads to a high incorporation rate of the antigen. More specifically, the preparation method by freeze-thawing and freeze-drying proceeds in such a way that thawing takes a certain period of time when the dried product obtained by freeze-drying is molten in the stage of freeze-thawing. The result is that the antigen and the adjuvant composition approach each other in the early stage of thawing. Once the antigen and the adjuvant composition approach each other, this state remains steady, and hence the antigen and the adjuvant composition stay close to each other in the solution which has undergone thawing. Freeze-drying in this state permits the antigen to be easily supported on or contained in the adjuvant composition, this leads to a high incorporation rate with the antigen.

[0173] According to one preferable embodiment of the present invention, the pH-sensitive carrier contains a prescribed pH-sensitive compound and a prescribed amphipathic substance. Therefore, the method for producing the vaccine composition according to this preferred embodiment includes a step of associating with one another at least one species of pH-sensitive compound, which is selected form the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof, at least one species of amphipathic substance selected from the group consisting of C10-12 phosphatidylcholine, C12-18 polyoxyethylene sorbitan monofatty acid ester, C16-18 sorbitan fatty acid ester, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol, and a substance with stimulus to activate innate immune system; a step of mixing the product obtained by the association with an antigen; a step of freeze-thawing the thus obtained mixture; and a step of freeze-drying the molten product obtained in freeze-thawing mentioned above.

EXAMPLES



[0174] The present invention will be described below in more detail with reference to the examples to be given later. The scope of the present invention is not restricted by these examples.

<Raw materials>



[0175] The examples were carried out with the following compounds. In case where the reagent name is identical with the product name, the product name is omitted.

[0176] 
  1. (1) pH-sensitive compound
    • Sodium deoxycholate (from Nacalai Tesque Inc.)
    • Sodium cholate (from Nacalai Tesque Inc.)
    • Sodium ursodeoxycholate (from Tokyo Chemical Industry Co., Ltd.)
    • Chenodeoxycholic acid (from Tokyo Chemical Industry Co., Ltd.)
    • Hyodeoxycholic acid (from Tokyo Chemical Industry Co., Ltd.)
    • Sodium glycodeoxycholate (from Nacalai Tesque Inc.)
    • Monoammonium glycyrrhizinate (from Tokyo Chemical Industry Co., Ltd.)
  2. (2) Amphipathic substance
    • DDPC (1,2-didecanoyl-sn-glycero-3-phosphatidylcholine: from NOF Corporation, COATSOME MC-1010)
    • DLPC (1,2-dilauroyl-sn-glycero-3-phosphatidylcholine: from NOF Corporation, COATSOME MC-1212)
    • Polyoxyethylene sorbitan monofatty acid ester (Tween 20, 80: from Tokyo Chemical Industry Co., Ltd.)
    • Sorbitan fatty acid ester (SPAN 80: from Nacalai Tesque Inc. - sorbitan nomooleate)
    • Polyoxyethylene castor oil (from Wako Pure Chemical Industries, Ltd. Polyoxyethylene 10 castor oil)
    • α-tocopherol (from Nacalai Tesque Inc., DL-α-tocopherol)
  3. (3) Substance with stimulus to activate innate immune system
    • MPL (Monophosphoryl lipid A) (from Sigma Corporation, Lipid A, Monophosphory salmonella serotype / from InvivoGen, Monophosphoryl Lipid A (synthetic)
    • IFA (Freund's Incomplete Adjuvant) (from Santa Cruz Biotechnology, Inc.)
    • CpG-DNA (CpG-ODN: from InvivoGen, ODN-2395)
    • LPS (endotoxin) (from Wako Pure Chemical Industries, Ltd., derived from Escherichia coli 0111, extracted by phenol)
  4. (4) Solvent, etc.
    • Water for injection (from Otsuka Pharmaceutical Co., Ltd. )
    • MES-Na (from Merck Ltd.)
    • Sodium chloride (from Kanto Chemical Co., Inc.)
    • PBS Tablets (Phosphate buffered saline: from Takara Bio Inc.)
    • Methanol (from Nacalai Tesque Inc.)
    • Chloroform (from Wako Pure Chemical Industries, Ltd.)
    • Sodium hydroxide aqueous solution (0.1 mol/L: from Nacalai Tesque Inc.)
    • Hydrochloric acid (0.1 mol/L, 1 mol/L: from Nacalai Tesque Inc.)
    • OVA peptide: SIINFEKL, (outsourced for synthesis by PH Japan Co., Ltd.) (This may be simply referred to as "peptide" hereinafter.)
    • OVA protein: OVA (EndoFit Ovalbumin: from InvivoGen) (This may be simply referred to as "OVA" hereinafter.)
  5. (5) Culture medium, etc.
    • RPMI (from Nacalai Tesque Inc. RPMI 1640 culture medium (liquid))
    • Penicillin-Streptomycin Mixed Solution (from Nacalai Tesque Inc.)
    • FBS (Fetal Bovine Serum, certified, heat inactivated, US origin: from Gibco)
  6. (6) Reagents
    • EYPC (Egg yolk phosphatidylcholine with no water added: from NOF Corporation, COATSOME NC-50)
    • Phosphlipid C-test wako (from Wako Pure Chemical Industries, Ltd.): Reagent for phospholipid assay
    • Pyranine (from Tokyo Chemical Industry Co., Ltd.): Fluorescent substance
    • DPX (p-xylene-bis-pyridiniumn bromide: from Molecular Probes Inc.): Quencher
    • Triton-X100 (from Wako Pure Chemical Industries, Ltd.): Surface active agent
    • NBD-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)ammonium: from Avanti Polar Lipids, Inc.)
    • Rh-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)ammonium: from Avanti Polar Lipids, Inc.): fluorescence labeling lipid
    • Rh-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)ammonium: from Avanti Polar Lipids, Inc.): fluorescence labeling lipid
    • Bio-Rad DC Protein Assay Reagent A, B (from Bio-Rad Laboratories, Inc.): Protein assay kit
    • IsoFlow (from Beckman Coulter, Inc.): Sheath fluid for flow cytometry
    • RBC lysis buffer (from Santa Cruz Biotechnology, Inc.): Buffer for hematolysis of erythrocyte
    • Collagenase for cell dispersion (from Wako Pure Chemical Industries, Ltd.): Reagent for cell dispersion
    • Anti-CD11cFITC (from eBioscience Inc., Anti-Mouse CD11cFITC): Fluorescein isothiocyanate (FITC), labeled antibody
    • Anti-CD80PE (from eBioscience, Inc., Anti-Mouse CD80(B7-1) PE): R-phycoerythrin (PE), labeled antibody (This may be referred to as CD80PE hereinafter.)
    • Anti-CD86PE (from eBioscience, Inc., Anti-Mouse CD86(B7-2) PE): PE labeled antibody (This may be referred to as CD86PE hereinafter.)
    • Anti-CD40PE (from eBioscience, Inc., Anti-Mouse CD40PE): PE labeled antibody (This may be referred to as CD40PE hereinafter.)
    • Anti-mouse CD16/32 (from BD Biosciences, Inc.)
    • Cytofix/Cytoperm (from BD Biosciences, Inc.): cell immobilization-cell membrane permeation kit
    • BD Stain Buffer: Buffer for staining
    • BD GolgPlug: Cell stimulating kit
    • Anti-CD8aPE (from eBioscience, Inc.): PE labeled antibody
    • Mouse IFNγ ELISPOT Set (from BD Biosciences, Inc.)
    • AEC Substrate Set (from BD Biosciences, Inc.)
    • Sodium hydrogen carbonate (from Wako Pure Chemical Industries, Inc.)
    • Sodium carbonate (from Wako Pure Chemical Industries, Inc.)
    • Secondary antibody (anti-mouse IgG HRP conjugate, from R&D systems, Inc.)
    • Albumin (Albumin from bovine serum, from Sigma Corporation)
    • Color development kit for peroxidase (from Sumitomo Bakelite Co., Ltd.)

(7) Animals



[0177] C57BL/6N mice, female, (6 to 8 weeks old), were bought from Japan S.L.C, Inc. Experiments were carried out according to the guideline for animal experiments drafted by Terumo Corporation.

<Sample preparation, etc.>


• MES Buffer



[0178] Prepared from MES (25 mM) and NaCl (125 mM). The MES Buffer has a pH value of 7.4, unless otherwise specified.

• PBS



[0179] Prepared from PBS Tablets (from Takara Bio Inc.). To be more specific, PBS was prepared by dissolving 10 pieces of PBS Tablets in distilled water and diluting the solution to 1000 mL. It has a pH of 7.35 to 7.65.

• Preparation of MPL stock solution



[0180] Prepared by dissolution in a 7:3 mixed solvent of chloroform and methanol, so that the concentration was 100 ng/µL. It was further diluted according to need.

• RPMI medium



[0181] RPMI medium containing 10% serum was prepared by adding penicillin (100 unit/mL) and streptomycin (100 mg/mL), both as antibiotics. It was supplemented with additional FBS according to need.

<Apparatus used>



[0182] 

Ultrasonic irradiator: USC-J

Spectrophotometer: FP-6500

Flow cytometer: (FC500; software: CXP Software ver. 2) UV-vis spectrophotometer: UV-3600

Freeze-dryer: EYELA FREEZE DRYER FDU506

Vacuum pump: GCD 135XA

CO2 incubator: MCO20AIC

Separation filter: Amicon Ultra 30K


<Cell culture>



[0183] Cell culture was carried out at 37°C by using an incubator (MCO20AIC) containing 5% CO2.

(Preparation of adjuvant composition and vaccine composition)


Production by preparation method by dispersion



[0184] A mixture was prepared in a 10-mL eggplant-shaped flask from an amphipathic substance (1000 nmol dissolved in methanol (or chloroform), a pH-sensitive compound dissolved in methanol (or chloroform), and MPL stock solution. The resulting mixture was made into thin film by using a rotary evaporator.

[0185] With the help of an ultrasonic irradiator, the thus produced thin film was dispersed together with 1.0 mL of MES Buffer (for the release test and the membrane-fusion test) or 1.0 mL of PBS (for the measurement of incorporation rate, the evaluation of stimulus to activate innate immune system, and the immunization of mice) added thereto. In this way there was prepared a dispersion of the adjuvant composition. For preparation of the vaccine composition, the MES Buffer or the PBS was replaced by one which contains a prescribed amount of antigen dissolved therein.

[0186] Incidentally, the ratio between the amount of the amphipathic substance and the amount of the pH-sensitive compound was properly adjusted as desired. In the case where a plurality of amphipathic substances are used, adjustments were made so that their total amount is 1000 nmol (which is as desired molar concentration). Incidentally, the amount of the pH-sensitive compound used indicated in the Working Examples and the explanation of Figures is the amount for 100 nmol of the amphipathic substance used.

Production by preparation method by mixing



[0187] A mixture was prepared in a 10-mL eggplant-shaped flask from the amphipathic substance (1000 nmol dissolved in methanol (or chloroform), the pH-sensitive compound dissolved in methanol (or chloroform), and the MPL stock solution. The resulting mixture was made into thin film by means of a rotary evaporator. The thus produced thin film was dispersed at 5 to 35°C by means of an ultrasonic irradiator together with 0.5 mL of MES Buffer (for the release test and the membrane-fusion test) or 0.5 mL of PBS (for the measurement of incorporation rate, the evaluation of stimulus to activate innate immune system, and the immunization of mice) added thereto. In this way there was prepared a dispersion of the adjuvant composition.

[0188] The thus obtained adjuvant composition in dispersion form was incorporated with the same amount of the antigen solution in varied concentrations. In this way there was obtained the vaccine composition in dispersion form.

[0189] Production by the preparation method by freeze-thawing and freeze-drying

[0190] The adjuvant composition in dispersion form was prepared in the same way as the preparation method by mixing. The dispersion of the adjuvant composition was incorporated with the same amount of the antigen solution in varied concentrations. The resulting solution underwent sequentially freeze-thawing and freeze-drying. The thus obtained freeze-dried product was dispersed again into 1.0 mL of water for injection at 5 to 35°C. In this way there was prepared the dispersion of the vaccine composition.

[0191] Incidentally, the freeze-thawing was accomplished by dipping the 10-mL eggplant-shaped flask in cooled methanol, thereby freezing the dispersion, and then dipping the eggplant-shaped flask in distilled water at 5 to 35°C.

[0192] Also, the dispersion underwent freeze-drying, which was accomplished by using the freeze dryer (EYELA FREEZE DRYER FDU506) and the vacuum pump (GCD135XA).

[0193] In the case where CpG-DNA (CpG-ODN) was used, thin film was produced from a mixture of 1000 nmol of the amphipathic substance and a prescribed amount of the pH-sensitive compound, and the resulting thin film, together with 0.5 mL of PBS, was made into a dispersion by using an ultrasonic irradiator. The resulting dispersion was further incorporated with 0.5 mL of PBS containing a prescribed mount of CpG-DNA and a prescribed mount of antigen dissolved therein. The thus prepared dispersion was used for experiments.

(Preparation of samples for comparison)



[0194] Samples for comparison were prepared as follows from a dispersion of MPL alone (MPL dispersion), a dispersion of an amphipathic substance alone, and a dispersion of a pH-sensitive carrier alone. They additionally contain a prescribed amount of antigen in the case where they are used to immunize mice.

[0195] The first step started with providing a 10-mL eggplant-shaped flask with a prescribed amount of amphipathic substance or pH-sensitive carrier dissolved in MPL stock solution or methanol (or chloroform). Then, the content in the flask was made into thin film by means of a rotary evaporator. The thus obtained thin film was dispersed together with 1.0 mL of MES Buffer (for the release test and the membrane-fusion test) or 1.0 mL of PBS (for the measurement of incorporation rate, the evaluation of stimulus to activate innate immune system, and the immunization of mice) added thereto by means of an ultrasonic irradiator. Thus, there were obtained the dispersion of MPL alone, the dispersion of the amphipathic substance alone, or the dispersion of the pH-sensitive carrier alone. For evaluation of the CTL induction rate, PBS containing a prescribed amount of antigen dissolved therein was used.

<Method for measurement>


(Release test: Measurement of leakage)



[0196] The leakage was evaluated according to the method described in K. Kono et al., Bioconjugate Chem. 2008 19 1040-1048. The evaluation was performed with the help of EYPC liposomes which contain Pyranine (as a fluorescent substance) and DPX (as a quencher).

[0197] EYPC (3000 nmol) dissolved in chloroform was measured and placed in a 10-mL eggplant-shaped flask, and the EYPC was made into thin film by means of a rotary evaporator. To the flask was added a Pyranine solution (500 µL), which is composed of Pyranine (35 mM), DPX (50 mM), and MES (25 mM), and has pH 7.4. The thin film in the flask was dispersed with the help of an ultrasonic irradiator (USC-J). The resulting dispersion was passed through a porous polycarbonate film having a pore diameter of 100 nm with the help of an extruder, so as to make the particle size uniform. The resulting dispersion underwent substitution of the outer aqueous layer with the help of MES Buffer and G100 column. Thus, there was obtained a dispersion of EYPC liposomes encapsulating the fluorescent substance. The concentration of the lipid was determined with the help of phospholipid C-test Wako, and then it was adjusted with MES Buffer so that phospholipid became 1.0 mmol/L.

[0198] The dispersion of EYPC liposomes (with its concentration adjusted) in an amount of 20 µL and the dispersion of a sample for evaluation in an amount of 20 µL were added to MES Buffer (with its pH adjusted) in an amount of 2960 µL. The resulting mixture underwent incubation at 37°C for 90 or 30 minutes. (Values resulting from incubation for 90 minutes are adopted in Working Examples, unless otherwise indicated.) Finally, fluorescence at Ex416 nm and Em512 nm was measured by means of the spectrometer FP-6500 to monitor the Leakage.

[0199] The leakage was calculated assuming that the sheer dispersion of EYPC liposomes has 0% and the dispersion of EYPC liposomes incorporated with 30 µL of 10-fold diluted Triton-X100 has 100%. Specifically, the leakage was calculated from the following formula. In the following formula, L denotes the intensity of fluorescence measured, L0 denotes the intensity of fluorescence of the sheer dispersion of EYPC liposomes containing the fluorescent substance, and L100 denotes the intensity of fluorescence of the dispersion of EYPC liposomes incorporated with Triton-X100.


(Membrane-fusion test: Measurement of Fusion (membrane-fusion))



[0200] The Fusion (membrane-fusion) was evaluated according to the method described in K. Kono et al. Biomaterials 2008 29 4029-4036, by means of FRET (Fluorescence Resonance Energy Transfer). The fluorescence labeling employed NBD-PE and Rh-PE.

[0201]  A dispersion of EYPC liposomes (with double fluorescence labeling) was prepared as follows. First, there was produced thin film from EYPC (1000 nmol) containing 0.6 mol% of NBD-PE and Rh-PE. Then, the thin film was dispersed together with MES Buffer (1.0 mL) added thereto by means of an ultrasonic irradiator (USC-J). Finally, the resulting dispersion was passed through a polycarbonate film having a pore diameter of 100 nm by means of an extruder.

[0202] The dispersion of EYPC liposomes with double fluorescence labeling (20 µL) and the dispersion of sample for evaluation (20 µL) were added to the pH-adjusted MES Buffer (2960 µL), followed by incubation at 37°C for 60 minutes. The resulting liquid was measured for fluorescent spectrum at 500 nm to 620 nm (with excitation light at 450 nm) by means of the spectrophotometer (FP-6500). There was obtained the ratio between the fluorescence intensity at 520 nm and the fluorescence intensity at 580 nm.

[0203] The fusion rate was calculated on the following assumptions. The fluorescence intensity ratio of the dispersion of double fluorescence labeled EYPC liposomes and the amphipathic substance, which have undergone incubation, is 0%. The fluorescence intensity ratio of the dispersion of double fluorescence labeled EYPC liposomes and the dispersion of sample for evaluation, which have undergone methanol treatment, is 100%. Incidentally, the methanol treatment consists of dissolving the dispersion of double fluorescence labeled EYPC liposomes and the dispersion of sample for evaluation in methanol, making the resulting solution into thin film by means of a rotary evaporator, and dispersing the thin film into MES Buffer (3.0 mL) by means of the ultrasonic irradiator.

[0204] To be more specific, the fusion rate was calculated from the following formula, wherein R denotes the intensity ratio of fluorescence measured, R0 denotes the intensity ratio of fluorescence of the dispersion of double fluorescence labeled EYPC liposomes and the amphipathic substance, both of which underwent incubation, and R100 denotes the intensity ratio of fluorescence of the dispersion of double fluorescence labeled EYPC liposomes and the dispersion of sample for evaluation, both of which underwent methanol treatment.


(Measurement of incorporation rate)



[0205] The incorporation rate was evaluated as follows on the basis of the fact that the dispersion in which the antigen exists alone passes through the filter and the dispersion in which the antigen is supported on or contained in the adjuvant composition does not pass through the filter.

[0206] The dispersion containing the adjuvant and the antigen was passed through the Amicon Ultra 30K Filter at room temperature, at 7000 rpm, and for 10 minutes.

[0207] The incorporation rate was calculated by determining the antigen before and after filtration. Before and after filtration, the antigen underwent color reaction by Lowry method and was tested for absorbance at 750 nm by means of the spectrometer UV-vis. The incorporation rate was calculated from the following formula. Incidentally, the test for color reaction was performed by using 200 µL. In the following formula, Abefore denotes absorbance observed before filtration, due to color reaction caused by the antigen in the dispersion; Aafter denotes absorbance observed after filtration, due to color reaction caused by the antigen in the dispersion; and Abuffer denotes absorbance in the case where PBS was used. In other words, the numerator represents the antigen which did not pass through the filter or the antigen which was incorporated into (supported on or contained in) the adjuvant composition.


(Evaluation of stimulus to activate innate immune system)



[0208] The evaluation of stimulus to activate innate immune system was performed as follows.

[0209] A spleen was harvested from C57BL/6N mice. The harvested spleen was injected with 500 µL of collagenase solution, 2 mg/mL (prepared with RPMI medium) followed by incubation at 37°C for 30 minutes. The spleen was made into cell suspension by treatment with BD Falcon cell strainer. The cells underwent hemolysis with RBC lysis buffer, followed by washing with RPMI medium. The cells were dispersed into RPMI medium, and the number of cells was counted. The cells were used for the subsequent procedure.

[0210] The cells were inoculated into a 96-well dish such that 1.0 × 106 cells/100 µL. The cell dispersion was given 100 µL of RPMI medium containing a dispersion of different kind, followed by incubation overnight. These procedures were performed by using serum-free RPMI medium.

[0211] The cells were recovered and washed with BD stain buffer. Then, the cells underwent incubation together with Anti-CD11cFITC, 0.25 µg/100 µL (4°C for 30 minutes), so that the cells were stained. After washing, the cells underwent again incubation with Anti-CD80PE, Anti-CD86PE, or Anti-CD40PE, 0.25 µg/100 µL (4°C for 30 minutes), so that the cells were stained. After washing at least more than three times, the cells were evaluated by using the flow cytometer (Cytomics FC500, Software: CXP software ver. 2).

(Immunization of mice)



[0212] Administration (100 µL/head) was performed by subcutaneous injection at one position on the back under anesthesia. The dose of the amphipathic substance was 100 nmol/head; the dose of the pH-sensitive compound was 10 to 640 nmol/head; MPL content was 0.0227 to 22.7 nmol/head; and the amount of the antigen was 3.2 to 400 µg/head. For evaluation of cell-mediated immunity, administration was performed once and the assay was performed seven days after administration. For evaluation of humoral immunity, administration was performed twice. The second administration was performed 14 days after the first administration, and assay was performed seven days after the second administration.

(Preparation of dispersion of cells from mice's spleen)



[0213] The mice were sacrificed on the seventh day after the last administration and had its spleen harvested. The spleen was given RPMI medium (3.0 mL) containing 10% serum and then treated by the BD Falcon cell strainer. Thus there was obtained a suspension of cells. The cells underwent hemolysis with RBC lysis buffer, followed by washing with RPMI medium containing 10% serum. The cells were dispersed into RPMI medium containing 10% serum. The number of cells was counted. Thus there was obtained a dispersion of spleen cells.

(Evaluation of CTL induction rate -- ICS method)



[0214] The dispersion of spleen cells was inoculated into the medium containing 10% serum such that 1.0 × 106 cells/100 µL. For restimulation of the antigen, 10% serum-containing RPMI medium (100 µL) containing OVA peptide (40 µg/mL) was added, followed by incubation for three hours. Then, BD GolgiPlug was added such that its concentration was 0.2 µL/100 µL. Cell culture was performed overnight. In the case where the restimulation was not given, a 10% serum-containing RPMI medium not containing OVA peptide was used.

[0215] The cells were recovered and washed with BD stain buffer. Then, the cells underwent incubation together with Anti-mouse CD16/32 at 4°C for 10 minutes. After washing, the cells were stained with Anti-CD8αPE (at 4°C for 30 minutes), followed by washing again. Then the cells were permeabilized with Cytofix/Cytoperm. After washing, the cells were stained with Anti-IFNγFITC (at 4°C for 30 minutes). After washing equal to or more than three times, the cells were evaluated by using the flow cytometer (Cytomics FC500, Software: CXP software ver. 2). CTL induction rate was calculated in terms of the ratio between the IFNγ secreting cells and the total CD8 positive cells.

(ELIspot method)



[0216] The ELIspot method was practiced by using the Mouse IFNγ ELISPOT Set. One day before cell inoculation, the 96-well ELIspot plate was allowed to adsorb the detection antibody attached to the kit, so that the plate was made ready for use. The plate made in this manner was washed with 10% serum-containing RPMI medium and then given 10% serum-containing RPMI medium in an amount of 200 µL. The plate was allowed to stand at 37°C for two hours for blocking. The plate was washed with 10% serum-containing RPMI medium. In the case where the antigen undergoes restimulation, the plate was given 10% serum-containing RPMI medium (100 µL) containing OVA peptide (40 µg/mL). In the case where the antigen does not undergo restimulation, the plate was given 10% serum-containing RPMI medium (100 µL). The foregoing plate was inoculated with the dispersion of spleen cells so that there existed a prescribed number of cells. Finally, the 10% serum-containing RPMI medium was added so that the total amount per well was adjusted to 200 µL. After that, cell culture was performed for two nights so that the plate assumed a color.

[0217] The procedure for the plate to assume a color conformed to the protocol described in the Mouse IFNγ ELISPOT Set and the AEC Substrate Set.

(Measurement of antibody titer)



[0218] Serum was obtained by blood collection which was performed seven days after the second administration. Block buffer was prepared by dissolving albumin (5 g) in PBS (500 mL). Coating buffer was prepared by dissolving sodium hydrogen carbonate (1.47 g) and sodium carbonate (0.80 g) in water (500 mL). Washing of the plate was performed with PBS (500 mL) added with Tween 20 (2.5 mL).

[0219] In the coating buffer was dissolved OVA protein, and the resulting solution as added to the 96-well plate such that the amount was 0.1 µg/well (100 µL). After standing at 37°C for two hours, the coating buffer was replaced by the Block buffer. After standing overnight at 4°C, the plate was washed, and then each well was given 100 µL of serum diluted at a prescribed ratio. After standing at 37°C for two hours, the plate was washed, and each well was given 100 µL of secondary antibody solution diluted 1000 fold. After standing at 37°C for two hours, the plate was washed and allowed to develop color with the help of the coloring kit for peroxidase. In this way there was obtained the antibody titer.

[Evaluation]



[0220] Various evaluations were performed by using the dispersions prepared according to the above-mentioned methods.

(1) Induction of immune response



[0221] The induction of cell-mediated immunity was reviewed. The amphipathic substance in the adjuvant composition is DLPC, and the pH-sensitive compound in the adjuvant composition is deoxycholic acid (160 nmol). The MPL content is 0.227, and the model antigen is OVA peptide (occasionally referred to as "peptide" hereinafter). Incidentally, the MPL content denotes the amount (in nmol) of MPL relative to the amount (100 nmol) of the amphipathic substance. The amount of the pH-sensitive compound denotes the amount relative to the amount (100 nmol) of the amphipathic substance. The adjuvant composition was prepared by the preparation method by dispersion.

[0222]  Samples used for comparison include a solution of the peptide alone, a dispersion of the pH-sensitive carrier (DLPC-deoxycholic acid) and the peptide, and a solution of incomplete Freund's adjuvant (IFA) and peptide. The above-mentioned solution or dispersion was subcutaneously administered once to the back of C57BL/6N mice. The number of IFNγ-secreting cells in the spleen was counted, and the CTL induction was evaluated.

[0223] Evaluation was typically accomplished by the ELIspot method. The result of evaluation is depicted in Fig. 2. This result was obtained under the condition of 2 × 106 cells/well. Fig. 2(A) represents a graph depicting the number of IFNγ spots, Fig. 2(B) represents the spots formed in the case where peptide is used alone, and Fig. 2(C) represents the spots formed in the case where peptide is used in combination with MPL-containing DLPC-deoxycholic acid complex (adjuvant composition). The sample with peptide alone gave the equivalent number of spots as the untreated sample, with no CTL induction (Fig. 2(A)). The sample with MPL-free DLPC-deoxycholic acid complex also gave the equivalent number of spots as the untreated sample, with no CTL induction (Fig. 2(A)). This is a very interesting result. It suggests that the CTL induction needs stimulus to activate innate immune system.

[0224] By contrast, the sample with a complex of MPL-containing DLPC-deoxycholic acid, which is the adjuvant composition) gave a larger number of spots than the untreated sample or the sample containing peptide alone (Figs. 2(B) and 2(C)). The fact that the number of spots is almost equal to that in the positive control (or IFA) suggests that the adjuvant composition induced CTL as strongly as IFA. In view of the fact that IFA is widely used in clinical studies as the adjuvant to induce CTL, the adjuvant of the present invention, which is as active as IFA, is expected to be effective for clinical use.

(2) Comparison with MPL



[0225] It is known that MPL activates innate immune system and functions as an adjuvant even when used alone. It is also known that MPL induces cell-mediated immunity. Thus, a comparison was made as to the induction of cell-mediated immunity between MPL used alone and MPL used in combination with a pH-sensitive carrier in the form of the adjuvant composition. Each of the MPL and the adjuvant composition is administered together with an antigen. Incidentally, the antigen is peptide (80 µg), and the MPL is that which contains MPL in an amount of 0.227 mol or in an amount equivalent to that mentioned above. The adjuvant composition is a dispersion of the MPL-containing DLPC-deoxycholic acid complex, as mentioned in (1) above. The above-mentioned solution or dispersion was subcutaneously administered once to the back of C57BL/6N mice, and the CTL induction rate was obtained according to the ICS method. The CTL induction rate is expressed in terms of the ratio of the IFNγ-secreting cells to the total CD8 positive cells in a specific area which is hardly affected by dead cells (IFNγ-secreting cells / total CD8 positive cells).

[0226] The results are depicted in Fig. 3. Fig. 3(A) represents the result in the case where a solution of peptide alone was use. Fig. 3(B) represents the result in the case where peptide and a dispersion of MPL alone were used. Fig. 3(C) represents the result in the case where an MPL-containing DLPC-deoxycholic acid complex (as the adjuvant composition) was used.

[0227] In the case where peptide and MPL dispersion were used, the CTL induction rate is 0.27%, which is slightly higher than that (0.14%) in the case where peptide was used alone, as depicted in Figs. 3(A) and 3(B). Also, in the case where peptide and MPL-containing DLPC-deoxycholic acid complex were used, the CTL induction rate is 0.53%, which is higher than that in the case where MPL dispersion was used, as depicted in Figs. 3(B) and 3(C). In spite of the fact that the same antigen was used, the pH-sensitive carrier containing MPL (as the adjuvant composition) gave a higher CTL induction rate than the MPL dispersion. This result suggests that the adjuvant composition produces the effect of enhancing the CTL induction.

[0228] The foregoing results demonstrate that the effect of CTL induction by MPL is enhanced by the pH-sensitive carrier. A probable reason for this is that the pH-sensitive carrier promotes the membrane-disruption function, thereby enabling the delivery of the antigen to the cytosol, and enhances the effect of CTL induction through promotion of cross-presentation.

(3) Discussion on method for preparation



[0229] The vaccine composition is composed of the antigen and the adjuvant composition. The discussion on the method for the preparation of the vaccine composition will help to improve the adjuvant composition in its effect of inducing CTL. This idea led to the following discussion.

[0230] Several vaccine compositions in dispersion form were prepared by the preparation method by dispersion, the preparation method by mixing, and the preparation method by freeze-thawing and freeze-drying. The resulting vaccine compositions were examined for the CTL induction rate in the similar way as mentioned in (2) above. The amphipathic substance is DLPC, and the pH-sensitive compound is deoxycholic acid (160 nmol). The content of MPL is 0.227, and the antigen is peptide (80 µg).

[0231] The results are depicted in Fig. 4. Fig. 4(A) depicts the result in the case where an MPL-containing DLPC-deoxycholic acid complex was used (prepared by the preparation method by dispersion). Fig. 4(B) depicts the result in the case where an MPL-containing DLPC-deoxycholic acid complex was used (prepared by the preparation method by mixing). Fig. 4(C) depicts the result in the case where an MPL-containing DLPC-deoxycholic acid complex was used (prepared by preparation method by freeze-thawing and freeze-drying).

[0232] All of the vaccine compositions prepared by the preparation method by dispersion, the preparation method by mixing, and the preparation method by freeze-thawing and freeze-drying gave a higher CTL induction rate than the vaccine composition containing MPL alone (0.53%, 0.54%, 1.47%; Figs. 4(A) to 4(C)). These results suggest that the adjuvant composition in each vaccine composition produces the effect of enhancing the CTL induction regardless of the method for preparation. Particularly, the vaccine composition prepared by the preparation method by freeze-thawing and freeze-drying gave a very high CTL induction rate. This result apparently suggests that the preparation method by freeze-thawing and freeze-drying yields the adjuvant composition (in the vaccine composition) which produces the effect of greatly enhancing the CTL induction rate (Fig. 4(C)).

(4) Evaluation of incorporation rate



[0233] It has been depicted in (3) above that the vaccine composition prepared by the preparation method by freeze-thawing and freeze-drying gave a very high CTL induction rate.

[0234]  A probable reason for this is that the incorporation of the antigen into the adjuvant composition improves the efficiency of delivery to the cytosol, which in turn produces the effect of greatly enhancing the CTL induction rate. Thus, the incorporation rate of the antigen into the adjuvant composition was evaluated. First, the system for evaluation was verified. In the case of solution containing peptide alone, there was no change in absorbance measured by Lowry method before and after filtration, and the peptide was not caught by the filter (Fig. 5(A)). On the other hand, in the case of solution containing peptide test Wako, absorbance completely disappeared after filtration (Fig. 5(B)). This suggests that the adjuvant composition is caught by the filter. The foregoing suggests that the evaluation system mentioned herein permits the evaluation of the incorporation rate of the antigen into the adjuvant composition.

[0235] Fig. 5(C) depicts the result of evaluation of the incorporation rate in the case where each of the vaccine compositions were prepared by the preparation method by dispersion, the preparation method by mixing, or the preparation method by freeze-thawing and freeze-drying. The preparation method by dispersion resulted in a low incorporation rate (approximately 5%), and the preparation method by mixing also resulted in a low incorporation rate. By contrast, the preparation method by freeze-thawing and freeze-drying resulted in a high incorporation rate (approximately 60% at the maximum) (Fig. 5(C)). This suggests that the vaccine composition prepared by the preparation method by freeze-thawing and freeze-drying is composed of the adjuvant composition and the antigen which are integrated with each other in a high ratio. Incidentally, the amphipathic substance is DLPC, and the pH-sensitive compound is deoxycholic acid (160 nmol). The content of MPL is 0.227.

[0236] It is assumed that the preparation method by freeze-thawing and freeze-drying realized a high incorporation rate for the antigen and this led to a high probability that the adjuvant composition and the antigen are integrated into the same endosome. The result of this assumption is that the membrane disruptive function promoting effect efficiently increases the delivery to the cytosol, which further leads to the high CTL induction rate as depicted in the result mentioned in (3) above.

(5) Regarding amount of MPL



[0237] The result mentioned in (1) above depicts that the instance in which the MPL-free DLPC-deoxycholic acid complex was used did not bring about the CTL induction. This suggests that a substance to activate innate immune system is necessary if the pH-sensitive carrier is to be utilized as the adjuvant composition. In order to test the intensity of stimulus to activate innate immune system, ex vivo experiments (which use mice spleen cells) were conducted according to the reports in Nature Materials 2011 vol. 10(3) 243-251 and Cancer Res. 2011 71 2858-2870. In this way the amount of MPL required was sought.

[0238] First, the system for evaluation was verified.

[0239] Fig. 6 depicts the result of verification of the system for evaluation. Fig. 6(A) depicts the result of flow cytometry in which the cultured mice spleen cells were evaluated without staining. This indicates the region of dead cells. Figs. 6(B) and 6(C) depict respectively how much the expression of CD80 is enhanced when the cultured mice spleen cells are incorporated with PBS alone and LPS in a concentration of 400 ng/mL. Fig. 6(D) depicts the result obtained when LPS is added in varied concentrations. Figs. 6(E) and 6(F) depict the result of the increase in expression of CD86 and CD40 which are other costimulatory molecules.

[0240] It is noted that the fluorescence intensity of CD80PE increases in proportion to the amount of LPS in the region monitored (Fig. 6(B) to 6(D)). It is also noted that the fluorescence intensity of CD86 and CD40, which are another costimulatory molecules, increases in the same way as above (Fig. 6(E) and 6(F)). The foregoing verifies that the evaluation system mentioned herein is capable of evaluating the intensity of stimulus to activate innate immune system.

[0241] Next, an evaluation was made on the stimulus to activate innate immune system by the adjuvant composition, with the amount of MPL varied appropriately. The results are depicted in Fig. 7. The amphipathic substance and the pH-sensitive compound in the adjuvant composition are respectively DLPC and deoxycholic acid (160 nmol). The sample for comparison was prepared from PBS, pH-sensitive carrier (DLPC-deoxycholic acid) in dispersion form, and lipopolysaccharide (LPS) in solution form.

[0242] Fig. 7(A) depicts the result of evaluation for increase in expression of CD80, and Figs. 7(B) and 7(C) depict the result of evaluation for increase in expression of CD86 and CD40 as other indicators. The MPL-free DLPC-deoxycholic acid complex (added in an amount of 0.1 µL or 10 µL) gave the fluorescence intensity equivalent to that in the case where PBS was added alone. This indicates the absence of stimulus to activate innate immune system (Fig. 7(A)). On the other hand, it was found that the fluorescence intensity increases and the stimulus to activate innate immune system also increases in proportion to the MPL content (Fig. 7(A)). In the case where the MPL content is 0.00227, the value of MFI (fluorescence intensity) is 41.6. This value is larger than the MFI (39.1) in the case where PBS is used alone and the MFI (38.8) of the pH-sensitive carrier without MPL. In the case where the MPL content is 2.27 or 22.7, the value of MFI is 69.1 or 70.9, respectively. This indicates that the stimulus to activate innate immune system is close to the point of saturation (The value depicted in Fig. 7(A) is that in the case where the amount added is 10 µL.).

[0243] Incidentally, the increase of expression of CD40 and CD86, which are other costimulatory molecules, suggests that these adjuvant compositions certainly possess the stimulus to activate innate immune system (Figs. 7(B) and 7(C)).

(6) Regarding enhancement of cell-mediated immunity and activation of innate immune system



[0244] This section is intended to verify the relationship between the enhancement of cell-mediated immunity and the activation of innate immune system.

[0245] First, the MPL-free DLPC-deoxycholic acid complex which did not induce CTL as mentioned in (1) above was found to have no stimulus to activate innate immune system as mentioned in (5) above. This is depicted in more detail in Fig. 8(A). It is to be noted that the fluorescence intensity of CD80PE does not exceed that of PBS even though the MPL-free DLPC-deoxycholic acid complex increases in amount from 0.5 µL to 50 µL. This suggests that the MPL-free DLPC-deoxycholic acid complex does not have stimulus to activate innate immune system (Fig. 8(A)).

[0246] On the other hand, the adjuvant composition, which produced the effect of enhancing CTL induction as mentioned in (2) above, has the MPL content of 0.227, and it apparently has stimulus to activate innate immune system as mentioned in (5) above. This suggests a relationship between the enhancement of cell-mediated immunity and the activation of innate immune system.

[0247] Incidentally, Fig. 8(B) depicts the result obtained when cultured mice spleen cells were given a solution (5 µL) of various samples and examined for the enhanced expression of CD80 (activation of innate immune system). Each solution contains 800 µg/mL each of peptide and OVA, 1000 nmol/mL of DLPC alone, and 1600 nmol/mL of deoxycholic acid alone.

[0248] In the case where peptide, OVA, DLPC, and deoxycholic acid were added alone, the fluorescence intensity of CD80PE remained unchanged from that in the case where PBS was added. This suggests the absence of stimulus to activate innate immune system (Fig. 8(B)).

(7) Influence on membrane disruptive function promoting effect


(7-1) Influence of MPL



[0249] Investigations were made as to the influence of MPL content on the membrane disruptive function promoting effect possessed by the adjuvant composition. The results are depicted in Fig. 9. Fig. 9(A) depicts the leakage in the case where MPL (in an amount equivalent to the MPL content of 0.227 mol) is added to the evaluation system for the release test. Fig. 9(B) depicts the leakage for MPL-containing DLPC-deoxycholic acid complex at varied pH values. Fig. 9(C) depicts the leakage for DLPC-deoxycholic acid complex containing MPL in varied amount at pH 7.4 and pH 5.0.

[0250] An investigation was performed to see whether the leakage increases at a low pH value when MPL (dispersed in PBS or DMSO) is added to cuvette. In all the cases of investigation, the result in all the cases was equivalent to that in the case where PBS was used alone (Fig. 9(A)). This suggests that MPL has no ability to bring about the release.

[0251] Several adjuvant compositions were prepared in which the pH-sensitive carrier contains MPL in varied amount, and they were examined for the leakage at various pH values. Even in the case where the MPL content is 0.227 or 2.27, the leakage at each pH value coincided with that in the case where the normal pH-sensitive carrier (DLPC-deoxycholic acid complex) was used (Fig. 9(B)). This suggests that the MPL content has only little influence on the pH value suitable for the membrane disruptive function promoting effect. The influence of MPL content over a broad range was investigated by monitoring the leakage at pH 5.0. It was found that, even in the case where the MPL content is 22.7, the observed leakage remained unchanged from that in the case where an ordinary pH-sensitive carrier (with MPL content = 0) is used (Fig. 9(C)). It was confirmed again that the MPL content influences only little on the membrane disruptive function promoting effect. A probable reason for this is that the amount of MPL is so small relative to the amphipathic substance in the pH-sensitive carrier that MPL has only little influence on the membrane disruptive function promoting effect.

(7-2) Influence of method for preparation



[0252] An investigation was made as to how the method for preparing the vaccine composition (composed of the antigen and the adjuvant composition) influences the membrane disruptive function promoting effect. Fig. 10 depicts the leakage (at pH 7.4 and pH 5.0) of the vaccine composition prepared by the preparation method by dispersion, the preparation method by mixing, or the preparation method by freeze-thawing and freeze-drying. In this vaccine composition, the amphipathic substance is DLPC and the pH-sensitive compound is deoxycholic acid. The content of MPL is 0.227 and the amount of complex product is 160 nmol. The amount of peptide is 15 µg.

[0253] All the vaccine compositions, regardless of the method for preparation, gave the similar leakage at pH 7.4 and pH 5.0. This suggests that the method for preparing the vaccine composition does not influence the membrane disruptive function promoting effect (Fig. 10).

(7-3) Influence by amount of complex product in pH-sensitive compound



[0254] Fig. 11 is the results of an investigation which was made as to how much the MPL content influences the adjuvant composition's membrane disruptive function promoting effect which varies depending on the amount of complex product of deoxycholic acid. The amphipathic substance in the adjuvant composition is DLPC, and the amount of complex product of deoxycholic acid is 10 nmol, 20 nmol, and 640 nmol in Figs. 11(A), 11(B), and 11(C), respectively. The content of MPL is 0.0227 and 22.7. The samples for comparison are DLPC alone, deoxycholic acid alone, and pH-sensitive carrier (not containing MPL). The leakage was measured at pH 7.4 and pH 5.0.

[0255] First, the adjuvant composition not containing MPL (the pH-sensitive carrier; indicated as MPL-free in the figure) produced the membrane disruptive function promoting effect regardless of the amount of complex product (Figs. 11(A) to 11(C)). Next, the adjuvant compositions in which the MPL content is 0.0227 and 22.7 gave the leakages similar to each other or equivalent to those of the MPL-free adjuvant composition at both pH 7.4 and pH 5.0 (Fig. 11(A)). Also, similar results were found for the adjuvant composition in which the amount of complex product wss 20 and 640 (Figs. 11(B) and 11(C)). These results suggest that the content of MPL does not influence the adjuvant composition's membrane disruptive function promoting effect regardless of the amount of complex product.

[0256]  It was confirmed that the membrane disruptive function promoting effect is not influenced by the MPL content regardless of difference in the amount of complex product of the pH-sensitive compound (Fig. 11).

(7-4) Influence by kind of amphipathic substance or pH-sensitive compound



[0257] An investigation was made as to the influence of the amphipathic substance of different kind and the pH-sensitive compound of different kind on the adjuvant composition's membrane disruptive function promoting effect.

[0258] Tables 1 and 2 depict the result of evaluation of the membrane-disruption function of the adjuvant composition which was prepared from the amphipathic substance of different kind, and the result of evaluation of the membrane-disruption function of the adjuvant composition which was prepared from the pH-sensitive compound of different kind.

[0259] [Table 1]
Table 1
The membrane disruptive function promoting effect of adjuvant composition prepared from amphipathic substance of different kind
pH-sensitive substance or amphipathic substanceAloneComplex with deoxycholic acid
MPL: 0MPL: 0.227
pH 7.4pH 5.0pH 7.4pH 5.0pH 7.4pH 5.0
Deoxycholic acid 1.0 ± 0.1 12.9 ± 1.6 - - - -
DLPC 1.8 ± 1.2 5.5 ± 0.9 0.6 ± 0.2 58.6 ± 0.5 0.6 ± 0.1 63.6 ± 6.4
DDPC 17.4 ± 0.1 37.1 ± 1.3 24.3 ± 4.5 92.7 ± 5.3 27.7 ± 3.1 92.6 ± 1.9
Tween 20 70.4 ± 3.9 74.0 ± 2.0 63.7 ± 1.9 87.7 ± 2.8 75.3 ± 2.2 93.9 ± 1.6
Tween 80 5.0 ± 1.4 9.8 ± 1.3 5.5 ± 1.3 34.5 ± 5.6 3.7 ± 1.3 34.2 ± 4.5
Peg10 castor oil 1.0 ± 0.1 4.6 ± 0.7 2.1 ± 0.3 24.9 ± 1.7 1.4 ± 0.5 30.0 ± 0.9
Span 80 6.1 ± 0.6 18.1 ± 1.0 1.6 ± 0.4 36.3 ± 1.2 1.7 ± 0.3 34.9 ± 1.8
α-tocopherol 5.5 ± 0.3 10.6± 1.3 10.6 ± 0.5 51.3 ± 2.6 2.4 ± 0.5 43.5 ± 10.9
DLPC : EYPC (100 : 150) 1.5 ± 0.2 2.4 ± 0.1 0.8 ± 0.1 35.8 ± 2.1 0.5 ± 0.2 31.0 ± 2.2
DLPC : EYPC (100 : 25) 2.1 ± 0.3 4.6 ± 1.2 2.3 ± 0.3 63.8 ± 1.0 2.3 ± 0.2 62.9 ± 2.8
Values represent the leakage (%), and ± stands for SD.

[0260] [Table 2]
Table 2
The membrane disruptive function promoting effect of adjuvant composition prepared from pH-sensitive compound of different kind
pH-sensitive substanceAloneComplex with DLPC
MPL: 0MPL: 0.227
pH 7.4pH 5.0pH 7.4pH 5.0pH 7.4pH 5.0
Cholic acid 2.4 ± 0.1 9.8 ± 0.3 2.7 ± 0.3 34.5 ± 2.0 1.6 ± 0.3 34.1 ± 2.2
pH-sensitive substanceAloneComplex with DLPC
MPL: 0MPL: 0.227
pH 7.4pH 5.0pH 7.4pH 5.0pH 7.4pH 5.0
Ursodeoxycholic acid 3.6 ± 0.5 6.8 ± 0.3 1.4 ± 1.0 23.9 ± 2.5 2.2 ± 1.0 28.1 ± 2.8
Chenodeoxycholic acid 0.7 ± 0.5 14.1 ± 0.9 3.0 ± 1.4 52.8 ± 8.8 1.4 ± 0.1 48.5 ± 8.5
Hyodeoxycholic acid 4.2 ± 0.9 12.6 ± 0.4 2.1 ± 0.8 62.1 ± 8.3 2.2 ± 0.6 63.5 ± 6.4
Glycodeoxycholic acid 1.9 ± 1.2 9.8 ± 0.8 2.1 ± 0.2 36.3 ± 4.1 1.6 ± 0.3 35.4 ± 1.6
Glycyrrhizinic acid 4.2 ± 1.2 16.7 ± 2.9 1.8 ± 0.3 59.8 ± 3.3 1.3 ± 0.1 63.9 ± 6.8
Values represent the leakage (%), and ± stands for SD.

[0261] First, all the pH-sensitive carriers (represented as MPL: 0 in the table) produced the membrane disruptive function promoting effect regardless of the kind of the amphipathic substances used and the kind of the pH-sensitive compounds used (Tables 1 and 2). Next, the MPL-containing adjuvant composition (represented as MPL: 0.227 in the table) produced, at both pH 7.4 and pH 5.0, the similar membrane disruptive function promoting effect as that of the MPL-free adjuvant composition (or the pH-sensitive carrier) which was prepared with either of the amphipathic substance and the pH-sensitive compound (Tables 1 and 2). These results suggest that the inclusion of MPL has no influence on the adjuvant composition's membrane disruptive function promoting effect regardless of the kind of the amphipathic substance used and the kind of the pH-sensitive compound used.

[0262] It was confirmed that the kind of the amphipathic substance or the kind of the pH-sensitive compound has no connection with the membrane disruptive function promoting effect which is produced by the inclusion of MPL.

[0263] It is considered from the foregoing discussions (7-1) to (7-4) that the adjuvant composition produces the membrane disruptive function promoting effect irrespective of the method for preparation, the amount of complex product, the amphipathic substance, and the pH-sensitive compound.

(8) Influence on the membrane fusion function promoting effect



[0264] Fig. 12 depicts the result of an investigation made to examine the influence of the MPL inclusion on the adjuvant composition's membrane fusion function promoting effect. The amphipathic substance is DLPC, and the pH-sensitive compound is deoxycholic acid (160 nmol). The MPL content ranges from 0 to 0.227, and the adjuvant composition is prepared by the preparation method by dispersion. It is apparent from Fig. 12 that the MPL inclusion has no influence on the membrane-fusion promoting function.

(9) Influence on stimulus to activate innate immune system


(9-1) Influence of the amount of antigen



[0265] An investigation was made to examine how the amount of antigen influences the stimulus to activate innate immune system in the case of the vaccine composition composed of the antigen and the adjuvant composition. The amphipathic substance is DLPC and the pH-sensitive compound is deoxycholic acid (160 nmol). Two kinds of the adjuvant compositions were used, in one of which the MPL content is 0.0227 (referred to as "low MPL" hereinafter) and in the other of which the MPL content is 22.7 (referred to as "high MPL" hereinafter). The vaccine composition was prepared by the preparation method by dispersion.

[0266] The result is depicted in Fig. 13. Fig. 13(A) depicts the fluorescence intensity of CD80PE which was observed in the case where the vaccine composition containing peptide in varied amounts was added to the cultured mice spleen cells. Fig. 13(B) depicts the fluorescence intensity of CD80PE which was observed in the case where the vaccine composition containing OVA protein (simply referred to as "OVA" hereinafter) in varied amounts was added to the cultured mice spleen cells.

[0267] In both low MPL and high MPL, the fluorescence intensity of CD80PE remained constant regardless of the amount of OVA (Figs. 13(A) and 13(B)). This suggests that the amount of the antigen does not influence the strength of stimulus to activate innate immune system.

(9-2) Influence of the kind of amphipathic substance



[0268] An investigation was made to examine how the amphipathic substance influences the adjuvant composition's stimulus to activate innate immune system. Table 3 and Fig. 14 depicts the result of the investigation in which the adjuvant composition was prepared from a variety of amphipathic substances. The pH-sensitive compound is deoxycholic acid (160 nmol) and the MPL content ranges from 0.0227 to 22.7. The preparation method by dispersion was employed for sample preparation.

[0269] [Table 3]
Table 3
Influence of amphipathic substance on stimulus to activate innate immune system
Amphipathic substanceCD80 MFI
Amphipathic substanceMPL content: 0.0227MPL content: 22.7
DLPC 38.5 ± 2.9 44.3 ± 0.7 65.5 ± 2.6
DDPC 36.5 ± 1.1 43.8 ± 3.1 57.0 ± 2.7
Tween 20 38.0 ± 3.2 46.8 ± 2.9 54.8 ± 4.0
Tween 80 36.0 ± 3.2 45.6 ± 2.7 60.9 ± 3.5
Peg10 castor oil 42.1 ± 2.9 48.9 ± 4.6 67.0 ± 9.2
Span 80 39.6 ± 1.8 48.8 ± 4.7 65.9 ± 4.4
α-Tocopherol 43.8 ± 4.9 54.7 ± 1.8 64.0 ± 4.8


[0270] The adjuvant composition containing the amphipathic substance, no matter what their kind might be, gave a higher fluorescence intensity of CD80PE than that containing the amphipathic substance alone. This implies the addition of stimulus to activate innate immune system (Table 3 and Fig. 14). This result suggests that the adjuvant composition functions satisfactorily irrespective of the kind of the amphipathic substance used.

(9-3) Influence of amount of complex product of pH-sensitive compound



[0271] Fig. 15 depicts the result of an investigation to examine how the complex product of the pH-sensitive compound influences the strength of the adjuvant composition's and the vaccine composition's stimulus to activate innate immune system. The amphipathic substance is DLPC, the pH-sensitive compound is deoxycholic acid, and MPL content is 0.0227 or 22.7, and the amount of the antigen is 0 µg (in the adjuvant composition) and 400 µg (in the vaccine composition). All the samples were prepared by the preparation method by dispersion. Incidentally, evaluation was performed by using dispersion (5 µL).

[0272] Fig. 15(A) depicts the fluorescence intensity of CD80PE which is observed in the case where the MPL content is 0.0227 and DLPC is complexed with deoxycholic acid in varied amounts. Fig. 15(B) depicts the fluorescence intensity of CD80PE which is observed in the case where the MPL content is 22.7 and DLPC is complexed with deoxycholic acid in varied amounts.

[0273]  In the case where the MPL content is 0.0227 and 22.7, the fluorescence intensity of CD80PE remained constant regardless of the complexing of the deoxycholic acid and the amount of the complex product. This indicates that the complexing of the deoxycholic acid does not influence the adjuvant composition's and the vaccine composition's stimulus to activate innate immune system (Figs. 15(A) and 15(B)).

(9-4) Influence of the kind of pH-sensitive compound



[0274] Fig. 16 depicts the result of an investigation to examine the influence of the kind of the pH-sensitive compound on the strength of the vaccine composition's stimulus to activate innate immune system. Figs. 16(A) and 16(B) depict the fluorescence intensity of CD80 in the case where the pH-sensitive compound of different kinds was added to the cultured mice spleen cells. The amphipathic substance used is DLPC, and the amount of complex product of the pH-sensitive compound is 160 nmol. The MPL content is 0.0227 and 22.7, and the antigen is peptide (400 µg). The preparation method by mixing was used for preparation.

[0275] It is noted that the fluorescence intensity of CD80PE for all the pH-sensitive compounds is higher than in the case of the antigen and PBS (PBS alone in the figure), and that the stimulus to activate innate immune system was added. This suggests that the stimulus to activate innate immune system is not influenced by the kind of the pH-sensitive compound used (Figs. 16(A) and 16(B)).

[0276] It is considered from (9-1) to (9-4) above that the adjuvant composition functions satisfactorily independently of the amount of antigen, the amphipathic substance, the complex product, and the pH-sensitive compound.

(10) Verification of immune response induction


(10-1) Influence of the amount of MPL



[0277] The adjuvant composition, in which the MPL content is 0.0227 (low MPL) or 22.7 (high MPL), was examined for the effect of enhancing the induction of CTL. The amphipathic substance is DLPC, and the pH-sensitive compound is deoxycholic acid (160 nmol). The antigen is OVA peptide in an amount of 3.2 µg to 400 µg per head. The vaccine composition was subcutaneously administered once to the back of C57BL/6N mice. The preparation method by dispersion was used. For each group, n=3. The results are depicted in Table 4 below. Incidentally, "MPL alone" depicts the results in the case where the antigen and the MPL dispersion were used.

[0278] [Table 4]
Table 4
Verification of effect of enhancing CTL induction in the case of low MPL and high MPL
Amount of antigen (µg)Low MPLHigh MPL
MPL aloneAdjuvant compositionMPL aloneAdjuvant composition
3.2 0.14 0.57 0.45 1.11
16 0.31 0.66 0.36 1.00
80 0.26 0.74 0.37 2.61
400 0.46 0.50 0.58 2.10

Amphipathic substance: DLPC

pH-sensitive compound: deoxycholic acid (160 nmol)

MPL content: low MPL (0.0227), high MPL (22.7)

Antigen: peptide in an amount of 3.2 to 400 µg

Method of preparation: preparation method by dispersion

Each group: n = 3 (evaluation was made for three heads altogether)



[0279] The result in each experiment with the antigen in a fixed amount depicts that the adjuvant composition with either a high MPL content or a low MPL content gave a higher CTL induction rate than that containing MPL alone (Table 4). It was depicted that the adjuvant composition produces the effect of enhancing the induction of CTL under the above-mentioned conditions. Fig. 17 depicts the result of evaluation by ELIspot method in the case where the vaccine composition was used. The antigen used is OVA (80 µg). Fig. 17(A) depicts the spot formation in the case where the peptide and the MPL dispersion (MPL alone) were used under the condition of high MPL. Fig. 17(B) depicts the spot formation in the case where the vaccine composition composed of the peptide and the adjuvant composition was used under the condition of high MPL. The condition for evaluation is 1 × 106 cells/well.

[0280] Evaluation by ELIspot method also supports the foregoing result by the fact that the adjuvant composition induces more IFNγ-related spots than MPL used alone (Figs. 17(A) and 17(B)).

(10-2) Influence of amount of complex product of pH-sensitive compound



[0281] An investigation was made as to the influence of the amount of complex product of the pH-sensitive compound on the adjuvant composition's effect of enhancing the induction of CTL. To be more specific, the adjuvant composition was prepared in which the DLPC is complexed with deoxycholic acid in varied amounts, and the resulting adjuvant composition was examined for the effect of enhancing the induction of CTL. The content of MPL is 0.227. The vaccine composition containing peptide (80 µg) or OVA (80 µg) as the antigen was subcutaneously administered to C57BL/6N mice. It was prepared by the preparation method by dispersion. For each group, n=1. The result is depicted in Table 5.

[0282] [Table 5]
Table 5
Effect of enhancing CTL induction that varies depending on amount of complex product
Amount of complex product of deoxycholic acidCTL induction rate by adjuvant composition (%)
Antigen: peptideAntigen: OVA
MPL alone 0.22 0.18
Deoxycholic acid: 10 0.38 0.37
Deoxycholic acid: 20 0.44 0.42
Deoxycholic acid: 160 0.68 0.66
Deoxycholic acid: 640 0.40 0.57

Amphipathic substance: DLPC

pH-sensitive compound: deoxycholic acid (10 to 640 nmol)

MPL content: 0.227

Antigen: peptide (80 µg) or OVA (80 µg)

Method of preparation: preparation method by dispersion

Each group: n = 1



[0283] The CTL induction rate in the case where the adjuvant composition with peptide or OVA was used is higher than that in the case where MPL was used alone (antigen and MPL dispersion) regardless of the amount of complex product (Table 5). It was depicted that the effect of enhancing the induction of CTL is produced in the case where the pH-sensitive compound (10 to 640 nmol) is used for the amphipathic substance (100 nmol).

(10-3) Influence attributable to the kind of amphipathic substances or pH-sensitive compounds



[0284] Next, an investigation was made as to how the adjuvant composition's effect of enhancing the induction of CTL varies depending on the kind of the amphipathic substance and the kind of the pH-sensitive compound. To be concrete, samples of the adjuvant composition were prepared from various amphipathic substances or pH-sensitive compounds, and they were examined for the presence or absence of the effect of enhancing the induction of CTL.

[0285] In this investigation, the content of MPL is 0.227 and the antigen is OVA (80 µg). The amount of complex product of the pH-sensitive compound is 160 nmol. The preparation method by dispersion was employed. For each group, n=1.

[0286] Table 6 depicts the results obtained in the case where the adjuvant composition was prepared from various amphipathic substances. Table 7 depicts the results obtained in the case where the adjuvant composition was prepared from various pH-sensitive compounds.

[0287] [Table 6]
Table 6
Effect of enhancing CTL induction by adjuvant composition in the case where various kinds of amphipathic substance are used
 CTL induction rate (%)
Amphipathic substance-MPLAdjuvant composition
No treatment 0.11 --
MPL alone 0.20 --
DLPC 0.23 0.62
 CTL induction rate (%)
Amphipathic substance-MPLAdjuvant composition
DDPC 0.19 0.27
Tween 20 0.20 0.54
Tween 80 0.23 0.37
Peg 10 castor oil 0.16 0.32
Span 80 0.23 0.40
α-tocopherol 0.31 0.50

Amphipathic substance: 100 nmol each

pH-sensitive compound: deoxycholic acid (160 nmol)

MPL content: 0.227

Antigen: OVA (80 µg)

Method of preparation: preparation method by dispersion Each group: n = 1

Amphipathic substance-MPL : Prepared by dispersing a mixed thin film of amphipathic substance and MPL in an antigen solution



[0288] [Table 7]
Table 7
Effect of enhancing CTL induction by adjuvant composition and antigen specificity due to adjuvant composition in the case where various kinds of pH-sensitive compound are used
 CTL induction rate (%)
Adjuvant compositionAdjuvant composition (with antigen not restimulated)
No treatment 0.17 0.15
MPL alone 0.25 0.14
Cholic acid 0.43 0.21
Ursodeoxycholic acid 0.55 0.14
Chenodeoxycholic acid 0.50 0.20
Hyodeoxycholic acid 0.51 0.10
Glycodeoxycholic acid 0.35 0.09
Glycyrrhizinic acid 0.57 0.12

Amphipathic substance: DLPC

pH-sensitive compound: 160 nmol each

MPL content: 0.227

Antigen: OVA (80 µg)

Method of preparation: preparation method by dispersion

Each group: n = 1



[0289] The adjuvant composition prepared from the amphipathic substance of any kind and the pH-sensitive compound of any kind gave a higher CTL induction rate than that of the adjuvant composition prepared from MPL alone. This indicates that the adjuvant composition produces the effect of enhancing the induction of CTL (Tables 6 and 7).

[0290]  Also, Fig. 18 depicts the results of evaluation by ELIspot method for the vaccine composition in which the pH-sensitive compound was varied. The condition for evaluation is 2 × 106 cells/well, and the antigen used was OVA (80 µg). The MPL content is 0.227. Fig. 18 depicts spots which differ in appearance depending on the substance to stimulate innate immune system or the adjuvant composition. Fig. 18(A) depicts spots observed when the MPL dispersion was used. Fig. 18(B) depicts spots observed when the MPL-containing DLPC-deoxycholic acid was used. Fig. 18(C) depicts spots observed when the MPL-containing DLPC-cholic acid was used. Fig. 18(D) depicts spots observed when the MPL-containing DLPC-ursodeoxycholic acid was used. Fig. 18(E) depicts spots observed when the MPL-containing DLPC-hyodeoxycholic acid was used.

[0291] The adjuvant composition prepared from any one of the pH-sensitive compound induces more IFNγ-related spots than that prepared from MPL alone. This suggests that the adjuvant composition prepared from whatever pH-sensitive compound produces the effect of enhancing the induction of CTL (Figs. 18(A) to 18(E)). These results coincide with those obtained when the ICS method is employed.

(11) Regarding method for preparation



[0292] An investigation was made to examine how the vaccine composition varies depending on the method of preparation in the case where protein is used. Fig. 19 is a graph depicting the CTL induction rate due to various vaccine compositions prepared by different methods. The vaccine compositions were prepared by the preparation method by dispersion, preparation method by mixing, and preparation method by freeze-thawing and freeze-drying. The amphipathic substance is DLPC and the pH-sensitive compound is deoxycholic acid (160 nmol). The content of MPL is 0.0227 to 22.7. The antigen is OVA (80 µg). For each group, n=1.

[0293] It is noted from Fig. 19 that the vaccine composition, regardless of its method of preparation, gave a higher CTL induction rate that that prepared from MPL alone. Particularly, the one prepared by the preparation method by freeze-thawing and freeze-drying gave a high CTL induction rate. These results suggest that no matter how the vaccine composition is prepared, the adjuvant composition produces the effect of enhancing the induction of CTL (Fig. 19). Even in the case where protein is used as the antigen, the vaccine composition prepared by the preparation method by freeze-thawing and freeze-drying gave a higher CTL induction rate than those prepared by other methods (Fig. 19).

(12) Regarding antigen specificity



[0294] An investigation was made to confirm whether or not the CTL induced by the vaccine composition has antigen specificity. To be concrete, this object was achieved by checking the difference in the CTL induction rate between two methods of cell culture. Culture by the first method involves OVA peptide as an antigen added to the suspension of mice spleen cells (with restimulation); and culture by the second method relies on a medium alone containing no OVA peptide (without restimulation). The vaccine composition contains DLPC as the amphipathic substance and deoxycholic acid (160 nmol) as the pH-sensitive compound. The content of MPL is 22.7. OVA (80 µg) was administered to C57BL/6N mice. The dispersion of the vaccine composition was prepared by preparation method by mixing. For each group, n=1.

[0295] The results obtained are depicted in Fig. 20. Fig. 20(A) depicts the CTL induction rate in the case of culture with restimulation. Fig. 20(B) depicts the CTL induction rate in the case of culture without restimulation. In the case with restimulation, the CTL induction rate is 1.08%, (this value suggests the presence of CTL induction); in the case without restimulation, the CTL induction rate is 0.31%, (this value is smaller than that in the case with restimulation of the antigen) (Fig. 20). Similar results were observed also in the case where various pH-sensitive compounds were used (Table 7). The foregoing results suggest that the CTL induced by the vaccine composition is antigen-specific.

[0296] An additional investigation was made to confirm antigen specificity by the ELIspot method. The vaccine composition contains DLPC as the amphipathic substance and deoxycholic acid (160 nmol and 640 nmol) as the pH-sensitive compound. The MPL content is 0.227. OVA (80 µg) or peptide (80 µ) was administered to a C57BNL/6N mice. The dispersion of the vaccine composition was prepared by preparation method by dispersion. Evaluation was performed under the condition of 2 × 106 cells/well.

[0297] The results obtained are depicted in Fig. 21. Figs. 21(A) to 21(F) depict spot formation in culture with OVA peptide as antigen (with restimulation). Figs. 21(G) to 21(L) depict spot formation in culture with medium alone (without restimulation). In the case where the vaccine composition was used, spot formation without restimulation is much less insignificant than spot formation with restimulation. This indicates that the CTL induced by the vaccine composition has antigen specificity (Fig. 21). The result obtained by ELIspot method was also similar to that obtained by ICS method.

(13) Regarding enhancement of humoral immunity



[0298] An investigation was made to verify the induction of humoral immunity by the vaccine composition and the effect of enhancing the induction of humoral immunity by the adjuvant composition. To be concrete, in this investigation, one of the vaccine compositions prepared by various methods was subcutaneously administered twice into the back of C57BL/6N mice, and the IgG antibody titer in blood was measured. The amphipathic substance is DLPC, and the pH-sensitive compound is deoxycholic acid (160 nmol). The content of MPL is 0.227 and the antigen is OVA (80 µg). For comparison, one group was not given the antigen and one group was given the antigen-containing MPL alone. For each group, n=3.

[0299] The results obtained are depicted in Fig. 22. The subject which was given the vaccine composition depicted a higher IgG antibody titer than the untreated subject regardless of the method (preparation method by dispersion, preparation method by mixing, and preparation method by freeze-thawing and freeze-drying) by which the vaccine composition was prepared. This apparently suggests that the vaccine composition induced humoral immunity (Fig. 22).

[0300] The antibody titer is higher in the case of the vaccine compositions (each composed of the antigen and the adjuvant composition), regardless of the method of their preparation, than that containing MPL alone (or composed of antigen and MPL dispersion) (Fig. 22). The fact that the vaccine composition containing the adjuvant composition gives a higher antibody titer than that containing MPL alone suggests that the adjuvant composition produces the effect of enhancing the induction of humoral immunity (Fig. 22).

(14) Regarding adjuvant composition containing CpG-DNA



[0301] An investigation was made to verify the adjuvant composition's effect of enhancing the induction of CTL in the case where CpG-DNA (or CpG-ODN) is used as the substance (other than MPL) to activate innate immune system. To be concrete, the adjuvant composition was prepared from a mixture of CpG-ODN (5 µg per head) and DLPC-deoxycholic acid complex, and this adjuvant composition was mixed with OVA (80 µg) to give the desired vaccine composition. The amount of deoxycholic acid is 160 nmol and the total amount of the vaccine composition is 100 µL. This vaccine composition was administered to C57BL/6N mice (one head per group). Evaluation by ELIspot method was performed under the condition of 2 × 106 cells/well.

[0302] Table 8 depicts the CTL induction rate in the case where the restimulation of antigen was given or not given. These results were obtained when the antigen and CpG-ODN (CpG-ODN alone) were administered or the adjuvant composition containing CpG-ODN (vaccine composition) was administered. In the case where the adjuvant composition containing CpG-ODN is used, the CTL induction rate is higher than in the case of CpG-ODN alone (Table 8). It was found that even in the case where the substance other than MPL which activates innate immune system is used, the adjuvant composition produces the effect of enhancing CTL induction. The CTL induction rate in the absence of restimulation is lower than that in the presence of restimulation. This apparently suggests that the CTL induced by the vaccine composition containing CpG-ODN is antigen specific as in the case of MPL (Table 8). The same result as above was obtained in evaluation by ELIspot method (Fig. 23).

[0303] Incidentally, Fig. 23(A) depicts spot formation resulting from administration of OVA (80 µg) and CpG-ODN alone. Fig. 23(B) depicts spot formation resulting from administration of OVA (80 µg) and the adjuvant composition containing CpG-ODN. Fig. 23(C) depicts spot formation resulting from administration of OVA (80 µg) and CpG-ODN alone, without restimulation of the antigen. Fig. 23(D) depicts spot formation resulting from administration of OVA (80 µg) and the adjuvant containing CpG-ODN, without restimulation of the antigen.

[0304] [Table 8]
Table 8
Effect of enhancing CTL induction by adjuvant composition containing CpG-DNA, and antigen specificity
 CTL induction rate (%)
With restimulationWithout restimulation
CpG-ODN alone 0.27 0.12
Adjuvant composition (CpG-ODN) 0.80 0.09

Amphipathic substance: DLPC

pH-sensitive compound: deoxycholic acid (160 nmol)

CpG-ODN: 5 µg

Antigen: OVA (80 µg)

Each group: n = 1



[0305] This application is based on Japanese Patent Application No. 2013-248543 filed November 29, 2013, and the disclosure therein is referenced and integrated as a whole.


Claims

1. An adjuvant composition comprising a pH-sensitive carrier and a substance with stimulus to activate innate immune system.
 
2. The adjuvant composition according to claim 1, wherein the pH-sensitive carrier comprises:

at least one species of pH-sensitive compound; and

at least one species of amphipathic substance, and

produces the membrane disruptive function promoting effect,

the pH-sensitive compound being selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C(higher) bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof;

the amphipathic substance being selected from the group consisting of phosphatidylcholine having 10 to 12 carbon atoms, polyoxyethylene sorbitan monofatty acid ester having 12 to 18 carbon atoms, sorbitan fatty acid ester having 16 to 18 carbon atoms, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol.


 
3. The adjuvant composition according to claim 1 or 2, wherein the substance with stimulus to activate innate immune system is monophosphoryl lipid A.
 
4. The adjuvant composition according to claim 2 or 3, wherein the substance with stimulus to activate innate immune system is contained in an amount of 0.0227 to 22.7 mol for 100 mol of the amphipathic substance.
 
5. A vaccine composition comprising the adjuvant composition according to any one of claims 1 to 4 and an antigen.
 
6. The vaccine composition according to claim 5, wherein the antigen is a peptide or protein.
 
7. The vaccine composition according to claim 5 or 6, wherein the antigen is contained in an amount of 3.2 to 400 µg for 100 nmol of the amphipathic substance.
 
8. A method for producing an adjuvant composition, comprising:

the step of associating among

at least one species of pH-sensitive compound,

at least one species of amphipathic substance, and

a substance with stimulus to activate innate immune system;

the pH-sensitive compound being selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C bile acid, glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof;

the amphipathic substance being selected from the group consisting of phosphatidylcholine having 10 to 12 carbon atoms, polyoxyethylene sorbitan monofatty acid ester having 12 to 18 carbon atoms, sorbitan fatty acid ester having 16 to 18 carbon atoms, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol.


 
9. A method for producing a vaccine composition, comprising:

the step of associating among at least one species of pH-sensitive compound, at least one species of amphipathic substance, and a substance with stimulus to activate innate immune system, the pH-sensitive compound being selected from the group consisting of deoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, 27C bile acid,
glycodeoxycholic acid, glycyrrhizinic acid, and glycyrrhetinic acid, and salts thereof, the amphipathic substance being selected from the group consisting of phosphatidylcholine having 10 to 12 carbon atoms, polyoxyethylene sorbitan monofatty acid ester having 12 to 18 carbon atoms, sorbitan fatty acid ester having 16 to 18 carbon atoms, glycerol monooleate, glycerol dilaurate, glycerol distearate, glycerol dioleate, polyoxyethylene castor oil, and α-tocopherol;

the step of mixing the product of the association with an antigen;

the step of freeze-thawing the mixture obtained by the mixing; and

the step of freeze-drying the melt obtained by the freeze-thawing.


 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description