WO2007148924A1 - Use of colloidal gold as an adjuvant - Google Patents

Use of colloidal gold as an adjuvant Download PDF

Info

Publication number
WO2007148924A1
WO2007148924A1 PCT/KR2007/003002 KR2007003002W WO2007148924A1 WO 2007148924 A1 WO2007148924 A1 WO 2007148924A1 KR 2007003002 W KR2007003002 W KR 2007003002W WO 2007148924 A1 WO2007148924 A1 WO 2007148924A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
colloidal gold
vaccine
cell
adjuvant
Prior art date
Application number
PCT/KR2007/003002
Other languages
French (fr)
Inventor
Jung Sun Yum
Byung Cheol Ahn
Hong Mo Moon
Original Assignee
Dobeel Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dobeel Co., Ltd. filed Critical Dobeel Co., Ltd.
Publication of WO2007148924A1 publication Critical patent/WO2007148924A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/242—Gold; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nanotechnology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Inorganic Chemistry (AREA)
  • Medical Informatics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Ceramic Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Dispersion Chemistry (AREA)
  • Virology (AREA)

Abstract

The present invention relates to an adjuvant composition comprising colloidal gold. The administration of the colloidal gold with an antigen significantly increases cell-mediated immunity, thereby being used as an effective adjuvant for a therapeutic vaccine.

Description

[DESCRIPTION] [Invention Title]
USE OF COLLOIDAL GOLD AS AN ADJUVANT
[Technical Field]
<i> The present invention relates to an adjuvant composition comprising colloidal gold.
<2>
[Background Art]
<3> There are two major branches of the adaptive immune responses; one is a humoral immune response and the other is a cell-mediated immune response. The cell-mediated immune response is believed to play an important role in the protection against cancer or virus infection and exhibited as the formation of interferon-g and TNF-a, immunogen-specific cytotoxic T lymphocyte and selective production of IgG2a, IgG2b, and IgG3. Therefore, in developing immuno-therapeutics for cancer or chronic virus infections, it is an important task having cell-mediated immune response induced strongly and effectively.
<4> Immune responses are generated by immune cells, which recognize foreign material as antigen. The foreign material is engulfed by antigen presenting cells (APC) such as dendritic cell, B lymphocyte, and macrophage, digested into smaller pieces, and then presented with MHC Class I or MHC Class II on the cell surface. The presented antigens (Epitope) are recognized by T cell receptors present in the immune cells, in particular, CD4 T cell and CD8 T cell. At this time, immune cells and APC are simultaneously activated to induce costimulatory molecules, which provide costimulatory signals, and produce specific cytokines according to characteristics of antigen. If self-antigens, not foreign materials, are displayed, the costimulatory molecules are not induced, and immune responses are not generated. Further, APC has pathogen-associated molecular pattern recognition receptors such as Toll-like receptors, which recognize molecular patterns of microorganism. APC recognition of the molecular pattern of antigen generates signals to produce specific cytokines, thereby determines the types of immune responses; that is, either humoral or cell-mediated immune response.
<5> Inducing what type of immune response is very important in the development of immuno-therapy, since the induction of cell-mediated immune response is essential for the treatment of cancer or chronic virus infections. The cell-mediated immune response is represented by the differentiation of antigen specific naive CD4 T cell lineage into ThI (T helper cell 1) cell, expansion of it, and production of ThI cell specific cytokines such as interferon-g.
<6> The naive CD4 T cell is a precursor cell having potential of differentiating into ThI, Th2, or other regulatory T cells. Accordingly, for the development of immuno-therapy, driving the differentiation of naive CD4 T cells into ThI cells is required. Differentiation of naive CD4 T cells into ThI cells helps the production of CD8 T cells (cytotoxic T lymphocyte, CTL) and determines the quality of induced antibody. Interferon-g secreted by ThI cells induces isotype switching toward IgG2a and IgG2b, which play an important role in the protection against virus infection (T.R. Mosmann, R.L. Coffman, Annu Rev Immunol, 1989, 7:145).
<7> In inducing antigen-specific immune response, it is important to understand that any antigen has two properties that determine the outcome of immune response. They include the epitope structure (protein antigen: a unit of 8 to 25 amino acids) that determines specificity of immune response, and the molecular pattern that determines quality of immune response. The epitope structure selects out a specific TCR bearing CD4 clone to be differentiated and expanded, whereas the moleculecular pattern determines the differentiation of naive CD4 T cell either to ThI cell or Th2 cell. For example, if a microorganism (antigen) recognized by a subset of toll-like receptors on APC induces to express IL-12 and IL-18, the outcome of the differentiated and proliferated naive CD4 T cells are ThI cells. Further, such cytokines influence the differentiation and proliferation of CD8 T cell. That is, the molecular pattern of microorganism (antigen) is a major factor of determining quality of immune responses. The expression of a transcription factor, T-bet (S. J. Szabo et al , Cell, 2000, 100:655. E. L. Pearce et al , Science, 2003, 302-'104I) plays a critical role in the differentiation into ThI cell and proliferation of CD8 T cell, in which IL-12 (A. C. Mullen et al , Science, 2001, 292:1907) functions as a pivotal cytokine.
<8> Therapeutic vaccine for chronic viral infection is an immuno-therapy that has to have two important fuctions; it must be able to break immune tolerance to the chronically producing viral antigen and induce strong immune responses against the viral antigen. Further, the therapeutic vaccine should induce strong enough humoral and cell-mediated immunity to resolve the chronic viral infection. Therefore, the immunogenicity of viral antigen must be improved as much as possible, and using an adjuvant that is capable of inducing both strong humoral and cell-mediated immunity is one way to accomplish the purpose.
<9> Currently, the adjuvant that has generally been used is an aluminum compound (aluminum sulfate, aluminum hydroxide, aluminum phosphate or the like). Aluminum compounds induce good humoral immune response leading to production of antigen specific antibodies for the protection against that antigen bearing pathogen and are widely used as an adjuvant for prophylactic vaccine. Even though aluminum compounds can induce well humoral immune response, there is limitation of inducing cell-mediated immune response. In order to overcome the problem, new adjuvants such as bacterial toxins (cholera toxin or the like), surfactants (saponins, higher fatty acid or the like), microorganisms or plant components (BCG, muramyl peptide), cytokines (interleukin or the like), stress induced molecules (heat shock protein or the like), or synthetic polyanion and polycation have been studied and utilized. In preparing a highly effective vaccine, in particular a therapeutic vaccine, an effective and safe adjuvant capable of inducing both strong humoral and cell-mediated immunity is still needed.
<io> There are examples that gold salts are used as a conventional treatment for Rheumatoid arthritis. Gold particles have been used to improve the antibody response of peptide antigen by efficiently delivering small peptides to immune cells, in that the peptide antigen is too small to be immunogenic (PCT WO 96/14855, US Patent Publication No.: US 2003/0180252 Al). It has also been used as DNA vaccine carrier (PCT W02004/028560 Al), or as a drug delivery material, on to which a biologically active material (e.g. TNF) is immobilized to deliver them to specific targets (US 2005/0267204, US 6989158). Further, PCT WO 94/21288 discloses the technology of using colloidal metals including colloidal gold, in order to reduce the toxicity of biologically-active factors such as cytokines or growth factors, but the colloidal metals used therein are not used as an adjuvant. Further, the use of colloidal gold as an adjuvant, in which antibody response was stimulated by conjugating antigen to colloidal gold, is mentioned in Izv Akad Nauk Ser Biol (Dykman L A, et al, 2004, (1):86-91). However, colloidal gold in inducing a cell-mediated immune response to antigens that are simply mixed with the particle is not mentioned.
<ii> The present inventors have surprisingly found out that colloidal gold, when it is mixed with antigen, has no effect or slightly inhibitory activity for the antigen specific antibody response, which is contradictory to the afore mentioned previous invention, while it can induce a strong cell- mediated immune response to the mixed antigen. And it is described that colloidal gold as an adjuvant for a therapeutic vaccine, which requires a cell-mediated immune response, thereby completing the present invention.
<12>
[Disclosure]
[Technical Problem] <13> It is an object of the present invention to provide an adjuvant composition comprising colloidal gold. <14> It is another object of the present invention to provide a vaccine composition comprising the colloidal gold and antigen.
<15> It is still another object of the present invention to provide a method for improving antigen-specific cell-mediated immunity by using the colloidal gold as an adjuvant.
<16>
[Technical Solution]
<17> Present inventors have made extensive studies on adjuvant inducing cell-mediated immunity, and have found that the administration of colloidal gold mixed with virus antigen can improve an immune response, in particular, cell-mediated immune response, as compared to a control without the colloidal gold, and demonstrated that colloidal gold can be used as an adjuvant to induce cell-mediated immune response.
[Description of Drawings] <19> Fig. 1 shows an electron-microscopic photograph of a colloidal gold conjugate coated with a preS protein. <20> Fig. 2 shows a schematic diagram of an expression vector expressing an entire recombinant surface antigen (S protein, M protein, L protein). <2i> Fig. 3 shows a photograph of SDS-PAGE and western blotting of the purified entire recombinant surface antigen (S protein, M protein, L protein). <22> Fig. 4 shows the induction of humoral immune response by the colloidal gold. <23> Fig. 5 shows the induction of cell-mediated immune response by the colloidal gold. <24> Fig. 6 shows the induction of humoral immune response by a therapeutic vaccine consisting of L-form of HBV surface antigen and HBV core antigen
(HBcAg) adsorbed onto Alum and mixed with colloidal gold. <25> Fig. 7 shows the induction of cell-mediated immune response by the therapeutic vaccine. <26> Fig. 8 shows the induction of humoral immune response by the therapeutic vaccine in transgenic mice. <27> Fig. 9 shows the ratio of antibody isotype induced by the therapeutic vaccine in transgenic mice. <28> Fig. 10 shows the induction of interferon-g (ELISPOT assay) by the therapeutic vaccine in transgenic mice. <29> Fig. 11 shows the induction of interferon-g (ELISA) by the therapeutic vaccine in transgenic mice. <30> Fig. 12 shows the reduction of HBV surface antigen in the blood by the therapeutic vaccine. <3i> Fig. 13 shows the reduction of virus gene expression and increase of interferon-g gene expression by the therapeutic vaccine. <32> Fig. 14 shows the induction of humoral immune response by the therapeutic vaccine in transgenic mice. <33> Fig. 15 shows the ratio of antibody isotype induced by the therapeutic vaccine in transgenic mice. <34> Fig. 16 shows the induction of interferon-g (ELISPOT assay) by the therapeutic vaccine in transgenic mice. <35> Fig. 17 shows the induction of interferon-g (ELISA) by the therapeutic vaccine in transgenic mice. <36> Fig. 18 shows the reduction of HBV surface antigen in the blood by the therapeutic vaccine.
<37>
[Best Mode]
<38> In one embodiment, the present invention relates to an adjuvant composition comprising colloidal gold.
<39> As used herein, "adjuvant" refers to a substance or supplement to an antigen that cannot by itself induce specific immunity, but can stimulate the immune system to increase an immune response to specific antigen when it is mixed with the substance.
<40> Method of producing colloidal gold, which is used as an adjuvant, is not specifically limited to any method. In the present invention, the colloidal gold was prepared by a partial modification of sodium citrate procedure developed by Frens (Frens G, Controlled nucleation for the regulation of the particle size in monodisperse gold solutions. Nature Phys. Sci.241: 20, 1973).
<4i> The size and shape of the colloidal gold particle are preferably spherical and a diameter of 5 to 100 nm, more preferably a diameter of 10 to 40 nm, but are not limited thereto.
<42> The colloidal gold itself can be mixed with an antigen, and preferably in a form of colloidal gold conjugated to a protein can be used with antigen. That is, a colloidal gold conjugate, in which the surface of colloidal gold is coated with BSA or other proteins, can be mixed with an antigen. In order to prepare protein-colloidal gold conjugate, the protein used to coat the surface of colloidal gold is not limited thereto, preferably antigen used with a colloidal gold adjuvant is a protein, other immunogenic molecules, fragment of antigen, or a peptide. The colloidal gold adjuvant in the present invention can be referred as the colloidal gold itself and the colloidal gold conjugate of a protein.
<43> In one Example of the invention, a HBV surface antigen including S, M, L proteins and core antigen as a vaccine were administered to a mouse, and the degree of immune response was assessed. The degrees of immune response were compared between mice administered with the vaccine mixed with the colloidal gold and mice administered with the vaccine without the colloidal gold. From this, it was found out that, in the mouse administered with the antigen formulated with aluminum hydroxide (Alum) mixed with the colloidal gold, the humoral immunity (antibody response) was not affected much by the colloidal gold compare to the vaccine formulation without the colloidal gold, but the cell-mediated immunity was increased. That is, in particular, the cell-mediated immunity was prominently increased. Therefore, the colloidal gold seems to play a critical role in the augmentation of cell-mediated immunity when it is mixed with antigen.
<44> Further, one group of HBV envelop antigen gene transgenic mouse, which is an animal model of chronic hepatitis virus infection that continuously secrete the surface antigen into blood and shows immune tolerance against the secreted surface antigen, was administered with the mixture comprising Alum formulated HBV surface antigen including S, M, L proteins and core antigen and the colloidal gold (referred as "therapeutic vaccine"). And another transgenic mouse group was administered with only the HBV surface antigen including S, M, L proteins and core antigen without the colloidal gold. Then, the outcome of immune response was assessed. First, the "therapeutic vaccine" with or without colloidal gold broke immune tolerance, inducing strong HBV envelop antigen specific antibody response. Second, it was found that in the mice administered with the mixture of the colloidal gold and the "therapeutic vaccine" had strong antigen-specific cell-mediated immune response, showing that the expression of INF-r gene was increased 3 to 4 times and the percentage of IgG2a was increased from 48% to 82%. However, the addition of colloidal gold in the "therapeutic vaccine' decreased the antibody response (humoral immunity) by 35%, indicating that the colloidal gold effectively skewed immune response toward cell-mediated immunity.
Finally, it was found out that the least amount of blood surface antigen was measured in the mice administered with the mixture of the colloidal gold and the therapeutic vaccine, suggesting that the clearance of HBV antigen in the blood was dependent on both humoral and cell-mediated immunity.
<45> Based on these results, it was established that the colloidal gold has an adjuvant activity, and specifically an adjuvant activity that is capable of increasing cell-mediated immunity and improving the quality of antibody response by producing large amount of IgG2a required for strong vaccine formulation including therapeutic vaccine. Although the therapeutic vaccine with or without colloidal gold was good for breaking immune tolerance in the transgenic mice, clearance of HBV antigen in the transgenic mice was more efficient in the group with good cell-mediated immune response by using therapeutic vaccine with colloidal gold.
<46>
<47> In another embodiment, the present invention relates to a vaccine composition comprising the colloidal gold and antigen. The colloidal gold of the invention can be used as either naked gold particle or, preferably, a colloidal gold conjugate, in which the surface of colloidal gold is coated with BSA or other proteins.
<48> As described above, the colloidal gold can improve antigen-specific immune responses, thereby making it possible to be employed in the formulation of a variety of vaccines including therapeutic vaccine. In particular, the colloidal gold adjuvant provided in the present invention can improve the quality of humoral immunity and induce strong cell-mediated immunity. Therefore, the colloidal gold adjuvant of the invention can be administered with a conventional vaccine with Alum to improve immunogenicity, where cell mediated immunity is important.
<49> Examples of the vaccine antigen capable of being used with the colloidal gold of the invention as an adjuvant may include, but are not limited to, purified natural or recombinant antigen, a live attenuated pathogen (nonpathogenic) or killed pathogen including virus, bacteria, protozoa, and fungi, and protein, peptide, lipid, and carbohydrate antigens derived from a pathogen, preferably a protein antigen derived from a virus. Examples of the virus may include picorna virus, calici virus, corona virus, arena virus, parvo virus, toga virus, flavivirus, corona virus, rhabdovirus, filovirus, orthomyxo virus, paramyxo virus, bunya virus, retrovirus, papovavirus, adenovirus, herpes virus, pox virus, and hepadnavirus.
<50> The prepared colloidal gold can be used in an amount of 10 βg to 2000 βg, and the antigen can be used in an amount of 0.5 βg to 500 βg. The mixing ratio of the prepared colloidal gold and antigen can be determined by those skilled in the art. The colloidal gold and antigen can be mixed together in a ratio of 4000:1, preferably 10:1.
<5i> The vaccine comprising the colloidal gold as an adjuvant provided in the present invention can further comprise a conventional adjuvant, in addition to the colloidal gold, to synergically induce cell-mediated immune response. Examples of the conventional adjuvant include aluminum compounds (aluminum sulfate, aluminum hydroxide, aluminum phosphate or the like), bacterial toxins (cholera toxin or the like), surfactants (saponins, higher fatty acid or the like), microorganisms or plant components (BCG, muramyl peptide), cytokines (interleukin or the like), functional protein (heat shock protein or the like), and synthetic polyanion and polycation.
<52> In one specific embodiment of the invention, the vaccine antigen adsorbed onto an aluminum compound (alum) was mixed with the colloidal gold conjugate to improve cell-mediated immunity. The vaccine antigen was adsorbed onto alum, which has been known as an adjuvant effectively inducing a humoral immune response and mixed with the colloidal gold before immunization. Consequently, it can be used for improving the quality of humoral immune response and for inducing cell-mediated immune response.
<53> Examples of the vaccine formulation prepared by using the colloidal gold of the invention as an adjuvant include a vaccine which is effective against virus, bacteria, fungus, protozoa, and any other microorganisms infecting a human or animal, specifically, influenza vaccine, pertussis vaccine, purified pertussis-diphtheria-tetanus mixed vaccine, Japanese Encephalitis vaccine, hepatitis A vaccine, hepatitis B vaccine, rota vaccine, measles vaccine, rubella vaccine, mumps vaccine, measles-rubel la-mumps 3-kind mixed vaccine, measles-rubella 2-kind mixed vaccine, and Haemophilus influenza vaccine. Further, examples of the vaccine include tuberculosis vaccine, methici 11 in-resistant Staphylococcus aureus (MRSA) vaccine, Helicobacter pylori vaccine, enterohemorrhagic E.coli (EHEC) vaccine, salmonella vaccine, chlamydia vaccine, mycoplasma vaccine, AIDS vaccine, malaria vaccine, coccidium vaccine, and schistosomiasis vaccine.
<54> The adjuvant composition and vaccine composition comprising the colloidal gold may contain a pharmaceutically acceptable carrier, and be formulated for human or veterinary use, and then be administered by various routes. The composition can be administered orally, intraperitoneal Iy, intravenously, intramuscularly, subcutaneousIy, intradermal Iy, or the like. It is preferable that the composition is formulated into an injectable preparation. The injectable preparation can be formulated using an aqueous solution such as saline solution or Ringer's solution, and a non-aqueous solution such as vegetable oil, higher fatty acid ester (for example, ethyl oleate), and alcohols (for example, ethanol , benzylalcoho1 , propyleneglycol , or glycerine). Further, the injectable preparation may contain a pharmaceutically acceptable carrier such as a stabilizer to prevent degradation (for example, ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, and EDTA), an emulsifier, a buffering agent to adjust pH, and an antimicrobial preservative (for example, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzylalcohol). <55> The composition of the invention is administered in a pharmaceutically effective amount. The phrase "pharmaceutically effective amount" refers to an amount enough to exert the vaccine effects, and further an amount that not to cause an adverse reaction, or serious or excessive immunity response. An exact concentration of administration varies depending on the antigen to be administered, and can be easily determined by those skilled in the art, depending on the factors well known in the medical field including patient's age, weight, health condition, sex, and sensitivity to drug, administration route, and administration method, and it can be administered once or multiple times.
<56> In still another embodiment, the present invention relates to a method for increasing antigen-specific cell-mediated immunity by using the colloidal gold as an adjuvant.
<57> The administration of an antigen and the colloidal gold as an adjuvant is able to increase cell-mediated immunity, thereby improving the efficiency of vaccine. Specifically, the colloidal gold is used as an adjuvant of therapeutic vaccine for virus infection, so as to treat infectious diseases. The colloidal gold can be administered to humans, as well as domestic animals such as cattle, horses, sheep, swine, goats, camels, antelopes, and dogs.
<58>
[Mode for Invention]
<59> Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
<60>
<6i> Example 1. Preparation of colloidal gold adjuvant
<62> 1.1. Preparation of colloidal gold
<63> Colloidal gold was prepared by a method based on sodium citrate procedure, which was developed by Frens (Frens G, Controlled nucleation for the regulation of the particle size in monodisperse gold solutions. Nature Phys. Sci. 241: 20, 1973). Two hundred mg of gold chloride (HAuCl43H20) was dissolved in 10 ml of distilled water to prepare 2% gold stock solution. One hundred ml of distilled water was heated to boiling with stirring, and 1 ml of the 2% gold stock solution was added thereto to make final concentration of 0.02% and maintained under heating and stirring for about 5 minutes. 10% sodium citrate solution was added to make final concentration of 0.032 to 0.036%, and maintained under heating and stirring for 5 to 10 minutes. At this time, the color of the solution was initially gray, gradually changed to violet, and after 1 to 3 minutes, changed to red. The solution was put in a water bath, and cooled. Then, ODs40 and OU6oo were measured. The number of gold particle or concentration was measured by reading OD540 value, which was 2 to 4. The OD6oo reading measures the size of particle or quality of particle, which was 0.55 to 0.75 values. The particle size of the prepared colloidal gold was about 10 to 40 nm.
<64>
<65> 1.2. Preparation of colloidal gold conjugate
<66> One hundred mM sodium carbonate monohydrate (or other buffer) was added to the prepared colloidal gold solution, and the pH of the solution was titrated to be 7.5. Then, while stirring the colloidal gold solution, 20 μg of Bovine Serum Albumin (BSA) or 10 μg of preS antigen per 1 ml of the solution (200 μg of colloidal gold contained) was added, and continuously stirred at room temperature for 15 minutes. After centrufugation, the supernatant was removed, and the precipitate was washed three times with a sterilized PBS buffer (Phosphate Buffered Saline) to remove unbound BSA or preS antigen. Then, the precipitate was resuspended in the PBS buffer, and stored at 4°C. An electron microscopic photograph of colloidal gold conjugate coated with preS protein is shown in Fig. 1 (JEMlOlO, 67.0k). The quantification of protein adsorption was confirmed by measuring the amount of proteins in the supernatant, which was obtained by centrufugation after the BSA or preS antigen adsorption. <67>
<68> Example 2. Preparation of vaccine antigen
<69> 2.1. Recombinant preS~S antigen
<70> (I)-I Cloning
<7i> The coding region for HBsAg (preSl-preS2-S) and the entire 3'-UTR containing polyadenylation site were amplified using a vector containing HBV genome (HBV315, Korean Biochem. J. 17: 70-79, 1984) as a template by PCR, and introduced into an expression vector. The PCR was performed using a Pfu DNA polymerase, and primers for amplifying the coding region of HBsAg and the entire 3'-UTR were prepared. They are the forward primer of 5'-GGA AGA TCT CAA TCT CGG GAA-3' (SEQ ID NO. 1) and the reverse primer of 5'-GGA AGA TCT CGA ATA GAA GGA AAG-31 (SEQ ID NO. 2). BgIII restriction enzyme sequence is underlined. A PCR product having a size of about 2.75 kbp was obtained, and ligated with a pMSG vector (Korean Patent Application No. 10-2000-0043996, PCT Application NO. PCT/KRO1/01285) linearized by a BgIU restriction enzyme. A schematic diagram of the prepared vector is shown in Fig. 2. The vector was transfected into CHO cells, resulting in a transformant . The expression of preS-S antigen (SEQ ID NO. 3) was determined by a western blotting assay, and the cell lines showing a high expression rate were screened. The nucleotide sequences encoding the protein of SEQ ID NO. 3 are represented by SEQ ID NO.4.
<72>
<73> (l)-2 Establishment of cell line in suspension culture
<74> The selected cell (5X10 cells) line was inoculated and cultured in T-
175 flasks with media containing 10% serum. Then, the cultured cells were detached by using 0.25% trypsin, and centrifuged at 1200 rpm for 5 minutes to remove the residual trypsin. The single cells were resuspended in protein- free media (HyQ SFM4CH0, Hyclone), and inoculated in 250 ml spinner flasks with 100 ml working volume. The cells were inoculated at the initial concentration of 5X10 cells/ml and cultured at 37°C and 80 rpm. When the concentration of the cells was approached to 1.5X10 cells/ml, the cells were continuously subcultured using the same initial concentration. Finally, the cell lines adapted to suspension culture were obtained.
<75>
<76> (2) Culture conditions
<77> Cell inoculation was prepared by subculturing from MCB (Master Cell Bank). At this time, serum-free media (HyQ SFM4CH0, Hyclone) were used as a
5 basic medium, and the cells were inoculated at the concentration of 5X10 cells/ml in 250 ml spinner flasks and cultured at 34°C and 80 rpm. Three days after culturing, basic components (Transferrin, Selenium, Glucose), growth factors (HyQ LSlOOO, Lipid), and fed batch supplements (Carbohydrate, Amino acid, Vitamin) were added. The cells were subcultured in IL Spinner flasks to expand the number of cells. Then, the cells were inoculated in a 7.5 L bioreactor, and cultured at pH 7.2, 34°C, and at the stirring speed of 80rpm. Three days after culturing, citric acid and HyQ LSlOOO were added, and the cells were further cultured for another three days.
<78>
<79> (3) Purification of the recombinant HBV surface protein <80> The culture media recovered from the bioreactor were centrifuged to remove cell debris and passed through a 0.45um filter to remove impurities. The expressed HBV surface antigen was purified by an equilibrated phenyl- sepharose chromatography, DEAE-sepharose chromatography, and sepharose 4 FF chromatography. The entire purified surface antigen consists of S protein, M protein, and L protein, and forms a particle consisting of six kinds of recombinant proteins (Fig. 3). Fig. 3A shows the result of SDS-PAGE of the entire purified surface antigen, and Fig. 3B shows the result of a western blotting assay by using anti-S antibody (Lane 1), anti-preSl antibody (Lane 2), and anti-preS2 antibody (Lane 3).
<81>
<82> 2.2 Recombinant core antigen (HBcAg) <83> (1) Cloning
<84> Amino acid sequence Nos. 1 to 149 except arginine cluster in c-terminus of core antigen were expressed as a recombinant protein (SEQ ID NO. 5). Nucleotide sequences encoding the protein are represented by SEQ ID NO. 6. A vector containing HBV genome (HBV315) was used as a template, and PCR was performed to amplify the corresponding region. The amplified gene was inserted into the Ndel, and BaaRl restriction sites of a pETlla vetor (Novagen). For the PCR amplification of the core gene, a forward primer: 5'- CCC CAT..ATG GAC ATT GAC CCG TA-3' (SEQ ID NO. 7) and a reverse primer: 5'-CGC GGA TCC AAC AAC AGT AGT TTC CGG-3' (SEQ ID NO. 8) were used. E. coli BL21 (DE3) was transformed with the pETlla-core expression vector. Its expression was confirmed, and high production clones were selected.
<85>
<86> (2) Culture conditions of the high production strain <87> Optimal production conditions were determined by using the production strain in a 5L fermentor. The media containing 2% Bacto Tryptone, 1% Yeast extract, 2% NaCl, 2% Glucose, 1.33% KH2PO4, 0.4% (NH4)2HP04, 0.17% Citric acid,
0.12% MgSO4, 0.01% Thiamine-HCl, and 0.0371% Ampicillin were used. The strain was cultured at 37°C for 11 hours, and then 0.05 mM/g cell of IPTG
(Isopropyl-β-D- thiogalactopyranoside) was added thereto. After adding
IPTG, the cells were subjected to induction by IPTG for 18 hours, and harvested.
<88>
<89> (3) Purification of the recombinant HBV core protein <90> The cells were harvested, and washed three times with a lysis buffer (50 mM Tris-Cl pH7.6, 150 mM NaCl, 5 mM EDTA, 10 mM 2-mercaptoethanol , 0.2 mM PMSF). Then, the lysis buffer was added, and the cells were disrupted by sonication. The supernatant was collected by centrufugation, and incubated at 65°C for 30 minutes. Ammonium sulfate was added thereto, and core antigens were precipitated. After centrufugation, the precipitate was dissolved in 50 mM Tris—Cl (pH 7.6), and passed through a butyl sepharose column to isolate the pure core antigens. The purified recombinant core antigens featuring virus-like particles were highly immunogenic and induced strong cell-mediated immunity.
<91>
<92> Example 3. Preparation of vaccine composition
<93> The entire HBV surface antigen and core antigen prepared in Example 2 were mixed to use as a vaccine antigen.
<94> The colloidal gold conjugate coated with BSA prepared in Example 1 was used as an adjuvant for therapeutic vaccine. Aluminum hydroxide previously known to induce strong humoral immune response was also used.
<95>
<96> 3.1 Alum adsorption
<97> The entire recombinant surface antigen or recombinant core antigen was adsorbed onto alum in a solution containing 150 mM sodium chloride at 4°C for 24 hours. In order to confirm that all of the used antigens were adsorbed onto alum, 200 μi of the solution containing the antigen adsorbed onto alum was put in an eppendorf tube, and centrifuged. The supernatant was separated, and an ELISA and western blotting assay were performed with the supernatant. Further, in order to separate the adsorbed antigen again, 20 μi of 1 M sodium phosphate was added to the precipitated alum, and vortexed at room temperature for 30 minutes. 30 μi of SDS-PAGE sample buffer was added thereto, and vortexed at room temperature for 20 minutes, and then heated for 5 minutes. A western blotting assay was performed to confirm the antigen adsorbed onto alum. The completely adsorbed antigen was stored at 4°C, and used for the efficacy test on a therapeutic vaccine by using an animal model.
<98>
<99> 3.2 Mixing with colloidal gold adjuvant
<ioo> The purified antigen itself was mixed with the colloidal gold conjugate, or the antigen was adsorbed onto alum first, and then mixed with the colloidal gold conjugate. 0.5 μg (immunization of normal mouse, C57BL/6) or 5 βg (immunization of transgenic mouse) of the antigen was mixed with 200 βg of the colloidal gold conjugate coated with BSA prepared in Example 1. Then, the each of the test vaccine was intramuscularly administered.
<101>
<iO2> Example 4. Effect of colloidal gold adjuvant on inducing immune response
<1O3> 4.1 Conditions for immunization experiment and analysis method
<iO4> (1) Purpose^ In order to confirm the efficacy of colloidal gold conjugate as an adjuvant, the immune response induced in immunized mouse was analyzed.
<iO5> (2) Materials and methods
<iO6> (a) Experimental animal; 6 week-old female C57BL/6 mouse was used. <iO7> (b) Test vaccine <1O8> - A test vaccine was prepared by adsorbing the purified recombinant core antigen and the entire recombinant surface antigen onto alum. 0.5 βg of each antigen per dose was used. In another group, 0.5 βg of the free antigen not being adsorbed onto alum was mixed with 200 βg of the colloidal gold conjugate to be used.
<1O9> (c) Immunization group and immunization condition <πo> - Immunization group <iii> Group 1: A negative control immunized with PBS (Phosphate buffered saline). <ii2> Group 2: A group immunized with the entire recombinant surface antigen and recombinant core antigen adsorbed onto alum. <ii3> Group 3: A group immunized with a mixture, in which the entire recombinant surface antigen and recombinant core antigen not being adsorbed onto alum was mixed with colloidal gold conjugate. <ii4> -Administration method: Each test vaccine was intramuscularly administered twice at two weeks intervals.
<115>
<ii6> (d) Analysis method of immune response <ii7> 1) Analysis of humoral immune response
<ii8> Pre-immune serum and sera taken after the second immunization were separated, and the antibodies produced in the sera were analyzed. In order to analyze the antibodies, 96 well-microplates were coated with each purified antigen at a concentration of 100 ng/well, and blocked with Bovine Serum Albumin (1%) for 1 hour. The microplates were washed. The sequentially diluted sera were added to each well, and reacted at 37°C for 2 hours. Then, an anti-mouse IgG-HRP was added as a secondary antibody, and reacted for 1 hour under the same condition. After washing, a developing agent was added, and reacted at room temperature for 20 minutes. Then, OD value was measured at 450 nm using an ELISA reader.
<119>
<12O> 2) Analysis of cell-mediated immune response
<i2i> Within 2 weeks after the secondary immunization, spleens were taken out from all of the mice, and total splenocytes were isolated and cultured to analyze the cell-mediated immune response. The spleens taken out from each mouse were put in a cell strainer, and crushed. Then, the red blood cells were completely removed using RBC lysis buffer, and only splenocytes were isolated. The isolated splenocytes were cultured in complete media (1 X glutamine and 1 X antibiotics added to RPMI1640 medium). To see the immune responses specific to each antigen, the core antigen or entire surface antigen was added to the culture media at a concentration of 1 μg/ml , so as to stimulate immune cells specific to each antigen. Then, the cytokine (interferon-g as an indication of cell-mediated immune response) secreted from the cell were analyzed by an ELISA kit (BD Biosciences) .
<122>
<i23> 4.2 Efficacy of colloidal gold conjugate as adjuvant
<124> (1) Effect on humoral immune response
<125> Anti-HBs antibody and anti-HBc antibody, which are antibodies specific to the antigen, were induced in the experimental groups (groups 2 and 3) administered with the vaccine antigen as compared to the negative control (group 1) which was not administered with the vaccine antigen. However, higher amount of antibody formation was found in the case of using alum as an adjuvant (group 2) than in the case of using colloidal gold conjugate as an adjuvant (group 3) (Fig. 4). Alum, as previously known, is an adjuvant capable of inducing strong humoral immune response. It appears that the colloidal gold conjugate used in the present invention seems to induce humoral immune response yet slightly less activity compared to Alum adjuvant.
<126>
<127> (2) Effect of colloidal gold on cell-mediated immune response <128> To confirm whether a cell-mediated immune response was induced by the colloidal gold conjugate, after immunizations with the HBV antigens formulated with colloidal gold, splenocytes were prepared from each mouse and the production of interferon-g in response to the core antigen or surface antigen stimulation was analyzed by an ELISA assay. In the case of using the colloidal gold conjugate as an adjuvant, the production of interferon-g after stimulation with each antigen was induced (Fig. 5), specifically, interferon- g response to the surface antigen was increased 4 times more than the case of using alum. Therefore, the colloidal gold fits to be a good adjuvant that can be effectively utilized in the formulation of a therapeutic vaccine. Accordingly, the property of colloidal gold conjuge in inducing strong cell- mediated immune response can be exploited in the development of an effective therapeutic vaccine.
<129>
<i30> Example 5. Optimization of therapeutic vaccine
<i3i> 5.1 Conditions for immunization experiment and method of analysis
<i32> (1) Purpose: In order to induce both maximal humoral and cell-mediated immune response, the compositions of therapeutic vaccine were compared.
<i33> (2) Materials and methods
<134> (a) Experimental animal; 6 week-old female C57BL/6 mice were used.
<135> (b) Vaccine formulation
<136> - A test vaccine was prepared by adsorbing the purified recombinant HBV core antigen and the entire recombinant HBV surface antigen onto alum. 0.5 μg of each antigen per dose was used. Further, 0.5 μg of each antigen adsorbed onto alum was mixed with 200 μg of the colloidal gold conjugate coated with BSA, and the immunization was performed.
<137>
<138> (c) Immunization group and immunization condition
<139> - Immunization group
<14O> Group 1: A negative control immunized with PBS.
<i4i> Group 2: A group immunized with the entire recombinant surface antigen and recombinant core antigen adsorbed onto alum. <142> Group 3: A group immunized with a mixture, in which the entire recombinant surface antigen and recombinant core antigen adsorbed onto alum are mixed with colloidal gold conjugate. <143> -Administration method: Each test vaccine was intramuscularly administered twice at two week intervals.
<144>
<145> (d) Analytical methods of immune response
<i46> Humoral and cell-mediated immune response induced by the test vaccine were analyzed in the same manners as described in Example 4.
<147>
<i48> 5.2 Analysis of immune responses induced
<149> (1) Humoral immune response
<15O> Antigen-specific antibody formation was induced in the experimental groups administered with HBV antigens (groups 2 and 3), but there was no significant difference in the amount of the induced antibody between in the case of immunizing with only test vaccine adsorbed on alum (group 2) and in the case of immunizing with the test vaccine mixed with colloidal gold conjugate as an adjuvant (group 3) (Fig. 6). Therefore, the addition of colloidal gold conjugate to the Alum adorbed antigen had no significant effect on the antigen specific humoral immune response, showing only slight increase in the group with colloidal gold. <151>
<152> (2) Cell-mediated immune response
<153> To confirm whether a cell-mediated immune response was induced by the colloidal gold conjugate after secondary immunization, splenocytes were prepared from each mouse and the production of interferon-g was analyzed by ELISA assay.
<154> In the case of immunizing with the vaccine antigen adsorbed onto alum (group 2), the production of antigen stimulated interferon-g was increased about 2 times compared to the negative control group. In the case of immunizing with the colloidal gold conjugate and antigen adsorbed onto alum (group 3), however, the production of HBV surface antigen stimulated interferon-g was increased by 4.5 times (Fig. 7).
<155> Therefore, it has been conformed again that the colloidal gold conjugate is an adjuvant capable of strongly inducing cell-mediated immune response as shown in Example 4. Thus, the immunization with a vaccine antigen adsorbed onto alum and mixed with colloidal gold conjugate can realize the development of a therapeutic vaccine capable of optimizing both the humoral immune response and cell-mediated immune response.
<156>
<i57> Example 6. Efficacy of test vaccine by using transgenic mouse (1)
<i58> 6.1 Experimental conditions for immunization and analysis method
<159> (1) Purpose: To see the efficacy of the test vaccine in a transgenic mouse (HBsAg/HLA-A2).
<16O> (2) Materials and methods
<i6i> (a) Experimental animal; 6 week-old female HBsAg/HLA-A2 transgenic mice were used (Loirat D et . al, HBsAg/HLA-A2 transgenic mice: a model for T cell tolerance to hepatitis B surface antigen in chronic hepatitis B virus infection International Immunology 15: 1125-1136, 2003). The mouse model continuously expresses HBV surface antigen (HBsAg), and secrets virus-like particles consisting of surface antigens into blood. Further, the mouse model recognizes HBV surface antigen gene as a self gene, and does not induce the immune response against the gene, exhibiting immune tolerance. Therefore, this is a mouse model for HBV chronic carriers, in which there is no induction of sufficient immune response against HBV antigen to remove the virus antigen and maintains the condition of chronic infection.
<162>
<163> (b) Vaccine antigen and adjuvant
<164> The purified recombinant core antigen and the entire recombinant surface antigen were adsorbed onto alum, and used as a vaccine antigen. The colloidal gold conjugate coated with BSA was used as an adjuvant for therapeutic vaccine.
<165>
<166> (c) Immunized groups
<167> Group 1: A negative control immunized with PBS (Phosphate buffered saline) . <168> Group 2: A group immunized with the entire recombinant surface antigen and recombinant core antigen adsorbed onto alum. <169> Group 3: A group immunized with a mixture, in which the entire recombinant surface antigen and recombinant core antigen adsorbed onto alum are mixed with colloidal gold conjugate. <17O> -Administration method: Each test vaccine was intramuscularly administered three times at two week intervals.
<171>
<i72> (d) Analysis of the virus antigen in serum
<173> Pre-immune sera and sera after the third immunization were collected, and the amount of the surface antigen (HBsAg) in the serum was analyzed using a Genedia HBsAg ELISA kit 3.0 (GREEN CROSS).
<174>
<i75> (e) Analysis of humoral immune response
<i76> Pre-immune sera and sera after the third immunization were obtained, and the antigen-specific antibody formation was analyzed in the same way as described in Example 4. Further, IgG subtype of the induced antibody was determined to see the ratio of IgG2a and IgGl.
<177>
<178> (f) Analysis of cell-mediated immune response
<179> Splenocytes were prepared and cultured by the method as described in Example 4, and the induction of interferon-g as an indication of cell- mediated immune response was analyzed by commercially available ELISA kit and ELISPOT assay kit.
<180>
<i8i> (g) Analysis of interferon-g gene and surface antigen gene expressions in liver
<182> Total RNA was extracted from the mouse liver, and the gene expression was analyzed by a RT-PCR method. Total RNA was separated using an RNeasy Mini kit (Qiagen) and the expression of surface antigen gene (SEQ ID NOs. 9 and 10 were used) and interferon-g gene (SEQ ID NOs. 11 and 12 were used) were analyzed using the following primers and a one-step RT-PCR kit (Qiagen). The expression of β-actin gene (SEQ ID NOs. 13 and 14 used) was used as a control .
<183>
<i84> S-(F) 5 ' -ATG GAG AGC ACA ACA TCA GG-31 (SEQ ID NO. 9)
<185> S-(R) 5 ' -TTA AAT GTA TAC CCT AAG-3 ' (SEQ ID NO. 10)
<i86> INF- Y (F) 5 ' -AGC GGC TGA CTG AAC TCA GAT TGT AG-3 ' (SEQ ID NO. 11)
<i87> INF- Y (R) 5 ' -GTC ACA GTT TTC AGC TGT ATA GGG-31 (SEQ ID NO. 12)
<188> β -act in(F) 5 ' -TCC TGT GGC ATC CAT GAA AC-3 ' (SEQ ID NO. 13)
<i89> β -act in(R) 5 ' -CTT CGT GAA CGC CAC GTG C-31 (SEQ ID NO. 14)
<190>
<i9i> 6.2 Efficacy of colloidal gold conjugate as adjuvant for therapeutic vaccine
<192> 1) Breakage of immune tolerance
<193> The transgenic mouse model recognizes HBV surface antigen as a self antigen, and does not induce immune response against the antigen, exhibiting immune tolerance. However, administering the therapeutic vaccine to the mouse model, the immune tolerance against the surface antigen was broken, and humoral and cell-mediated immune responses were induced.
<194>
<i95> (a) Induction of humoral immune response
<i96> The production of antibody against the surface antigen was determined using the pre-immune sera and sera obtained after each immunization. As expected, the concentration of antigen in the blood was high in the negative control (experimental group 1) without showing any antibody response against the surface antigen. In the group received the vaccine (group 2, group 3), however, immune tolerance against the surface antigen was broken, and the antibody against the surface antigen was produced. In the case of using the colloidal gold conjugate coated with BSA as an adjuvant (group 3), the production of the induced antibody was significantly lower compared to the group received the vaccine antigen adsorbed onto alum (group 2) (Fig. 8). However, the ratio of IgG2a to IgGl in the experimental group 3 using the colloidal gold conjugate as an adjuvant is significantly higher compared to that in the group 2 used only alum (Fig. 9). It appears that the colloidal gold conjugate used in the present invention seems to have slightly suppressing activity for Alum adjuvant induced antibody response, but it is capable of inducing good quality humoral immune response, producing more IgG2a compare to the Alum.
<197> This is a good indication that, when the colloidal gold conjugate was used as an adjuvant, the immune response was skewed toward ThI cell response.
<198>
<i99> (b) Induction of cell-mediated immune response
<200> It has been known that stronge cell-mediated immune response is essential for removing virus harboring cells and control ing virus replication inside the infected cell. Therefore, confirming the efficacy of the therapeutic vaccine in terms of breaking the immune tolerance and inducing cell-mediated immune response specific to the surface antigen after immunization was an important part of this invention. And that was the ultimate test of utility of the colloidal gold for therapeutic vaccine fomulation. For this purpose, splenocytes were prepared and the production of interferon-g as an indication of the cell-mediated immune response was determined by ELISPOT and ELISA assays. Similar to the results shown in Example 5 using C57BL/6 normal mice, the group 3 using the colloidal gold conjugate as an adjuvant showed significantly higher induction of the cell- mediated immune response (Figs. 10 and 11) in the experiment with the transgenic mice.
<201>
<202> 2) Reduction of the surface antigen (HBsAg) in the serum <203> The amount of surface antigen (HBsAg) in the blood was analyzed using sera taken from the pre-immune mice and mice after each immunizations. As previously reported, in the negative control, the amount of virus antigen in the blood was naturally reduced about 40%. However, in the case of mice immunized with the therapeutic vaccine, the amount of virus antigen was rapidly reduced. Further, in the case of mice immunized with the vaccine antigen and the colloidal gold conjugate (group 3), the least amount of virus antigen was detected after three immunizations, as compared to the case of mice immunized with only the vaccine antigen adsorbed onto alum (group 2) (Fig. 12) or the control.
<204>
<205> 3) Analysis of virus gene expression.
<206> Cell-mediated immune response leads to expansion of CD 8 T cell (CTL) and production of interferon-g. And in chronic viral infection both of these are known to play pivotal role in removing virus infected cells and repressing virus replication inside the infected cells. Therefore, first to confirm whether the colloidal gold induced cell-mediated immune response specific to the antigen actually upregulated in the liver of the transgenic mouse, total RNA was extracted from the liver, and the expression of the interferon-g was analyzed by RT-PCR method. The expression of interferon-g was found to be low in the control group (group 1) and group 2, whereas the expression of the interferon-g was found to be 3 to 4 times higher in the group 3 that had the colloidal gold conjugate (Fig. 13). Further, to confirm whether the expression of the virus gene in the liver cells were suppressed by the interferon-g, the expression of HBV surface antigen gene was compared by the similar method described above. It was found that in the control group (group 1) and experimental group 2 immunized with only the antigen adsorbed onto alum, the expression of surface antigen was maintained high as for the control, and in the group 3, the expression of surface antigen was markedly decreased (Fig. 13).
<207> These experiments demonstrated that the colloidal gold is an adjuvant that can induce cell-mediated immune responses and can be utilized in the formulation of a therapeutic vaccine that might be good for resolving chronic infection.
<208>
<209> Example 7. Confirmation of efficacy of colloidal gold of therapeutic vaccine. (2)
<2io> 7.1 Conditions for immunization and analysis method
<2ii> The colloidal gold conjugate coated with a preS protein, which is a portion of an HBV surface antigen protein, was used as an adjuvant for therapeutic vaccine. Experimental animals, immunization groups, and analysis methods were similar to Example 6. <2i2> (1) Purpose: To see the efficacy of the test vaccine in a transgenic mouse (HBsAg/HLA-A2) . <2i3> (2) Materials and methods <2i4> (a) Experimental animal; 6 week-old female HBsAg/HLA-A2 transgenic mice were used.
<2i5> (b) Vaccine antigen and adjuvant <2i6> The purified recombinant core antigen and the entire recombinant surface antigen were adsorbed onto alum, and used as a vaccine antigen. The colloidal gold conjugate coated with preS was used as an adjuvant for therapeutic vaccine. <2i7> (c) Immunized groups
<2i8> Group l: A negative control immunized with PBS (Phosphate buffered saline) . <2i9> Group 2: A group immunized with the entire recombinant surface antigen and recombinant core antigen adsorbed onto alum. <220> Group 3: A group immunized with a mixture, in which the entire recombinant surface antigen and recombinant core antigen adsorbed onto alum are mixed with colloidal gold conjugate. <22i> -Administration method: Each test vaccine was intramuscularly administered three times at two week intervals.
<222>
<223> 7.2 Efficacy of colloidal gold conjugate coated with preS as adjuvant for therapeutic vaccine
<224> 1) Breakage of immune tolerance
<225> When the colloidal gold conjugate coated with preS was used as an adjuvant in the formulation of therapeutic vaccine, immune tolerance in the HBV transgenic mouse was broken, and humoral and cell-mediated immune responses were induced as shown in Example 6 using the colloidal gold conjugate coated with BSA.
<226> (a) Induction of humoral immune response
<227> The production of antibody against the surface antigen was confirmed using the pre-immune sera and sera obtained after the third immunization. In the case of mice administered with the vaccine antigen (group 2, group 3), the immune tolerance against the surface antigen was broken, and the antibody against the surface antigen was produced. In the case of using the colloidal gold conjugate coated with preS as an adjuvant (group 3), the production of the induced antibody was significantly lower than the mice immunized with only the vaccine antigen adsorbed onto alum (group 2) (Fig. 14). However, the ratio of IgG2a to IgGl was significantly higher compared to that in the experimental group 2 using only alum (Fig. 15). As for the previous experiment with the colloidal gold conjugate coated with BSA, colloidal gold conjugate coated with preS skewed the immune response toward ThI cell response, producing more IgG2a.
<228> (b) Induction of cell-mediated immune response
<229> In order to confirm that colloidal gold conjugate of pre S in the therapeutic vaccine formulation can break immune tolerance and induce a cell- mediated immune response specific to the surface antigen, splenocytes were prepared after the third immunization with the vaccine formuation and the production of interferon-g was determined by ELISPOT and ELISA assays. As shown in Example 5, the group 3 with colloidal gold conjugate of pre S had significantly higher amount of interferon-g induction (Figs. 16 and 17).
<230>
<23i> 2) Reduction of the virus surface antigen (HBsAg) in the blood.
<232> The amount of HBV surface antigen (HBsAg) in the blood was analyzed using the pre-immune sera and sera obtained after the third immunization. As previously reported, the amount of the virus antigen in blood was naturally reduced in some extent. However, in the group of mice immunized with the therapeutic vaccine containing colloidal gold conjugate of pre S, the amount of the virus antigen was rapidly reduced (group 3). As with previous experiment with colloidal gold conjugate of BSA, colloidal gold conjugate of pre S is much better compared to the case of immunizing with only the vaccine antigen adsorbed onto alum (group 2) (Fig. 18).
<233>
[Industrial Applicability]
<234> As demonstrated thus far, colloidal gold or its conjugate of protein can be efficiently utilized in the formulation of therapeutic vaccine for chronic viral infection to induce cell-mediated immune response, thereby resolving chronic infection.
<235>

Claims

[CLAIMS] [Claim 1]
<237> An adjuvant composition for therapeutic vaccine, comprising colloidal gold.
<238>
[Claim 2]
<239> The adjuvant composition according to claim 1, wherein the colloidal gold is in the form of colloidal gold conjugate coated with a protein.
<240>
[Claim 3]
<24i> The adjuvant composition according to claim 2, wherein the colloidal gold is in the form of colloidal gold conjugate coated with BSA or an antigen protein.
<242>
[Claim 4]
<243> The adjuvant composition according to claim 1, wherein the composition increases cell-mediated immunity.
<244>
[Claim 5]
<245> The adjuvant composition according to claim 1, wherein a diameter of the colloidal gold is 5 to 100 nm.
<246>
[Claim 6]
<247> The adjuvant composition according to claim 5, wherein the diameter of the colloidal gold is 10 to 40 nm.
<248>
[Claim 7]
<249> A vaccine composition comprising the composition of claim 1 and an antigen.
<250>
[Claim 8] <25i> The vaccine composition according to claim 7, further comprising an adjuvant in addition to the colloidal gold.
<252>
[Claim 9]
<253> The vaccine composition according to claim 8, further comprising aluminum hydroxide.
<254>
[Claim 10]
<255> The vaccine composition according to claim 7, wherein the antigen is derived from virus.
<256>
[Claim 11]
<257> The vaccine composition according to claim 7, wherein the composition increases cell-mediated immunity.
<258>
[Claim 12]
<259> The vaccine composition according to claim 7, wherein the vaccine composition is a therapeutic vaccine composition.
<260>
[Claim 13]
<26i> A method of using colloidal gold as an adjuvant, in order to prepare a vaccine capable of increasing a cell-mediated immune response.
<262>
[Claim 14]
<263> A method of improving antigen-induced cell-mediated immunity by using colloidal gold as an adjuvant.
<264>
PCT/KR2007/003002 2006-06-23 2007-06-21 Use of colloidal gold as an adjuvant WO2007148924A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2006-0057040 2006-06-23
KR1020060057040A KR100836893B1 (en) 2006-06-23 2006-06-23 Use of colloidal gold as an adjuvant

Publications (1)

Publication Number Publication Date
WO2007148924A1 true WO2007148924A1 (en) 2007-12-27

Family

ID=38833622

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2007/003002 WO2007148924A1 (en) 2006-06-23 2007-06-21 Use of colloidal gold as an adjuvant

Country Status (2)

Country Link
KR (1) KR100836893B1 (en)
WO (1) WO2007148924A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2114450A1 (en) * 2007-01-31 2009-11-11 Dobeel Co., Ltd. An hbv vaccine and a process of preparing the same
WO2019013361A1 (en) * 2017-07-14 2019-01-17 公益財団法人東京都医学総合研究所 Hepatitis b vaccine
US11324819B2 (en) * 2016-04-06 2022-05-10 University Of Washington Therapeutic vaccine for Hepatitis b virus (HBV) using the HBV core antigen

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030180252A1 (en) * 1997-11-10 2003-09-25 Lawrence Tamarkin Methods and compositions for enhancing immune response and for the production of in vitro Mabs
US20050267204A1 (en) * 1996-11-04 2005-12-01 Medical Innovations Limited Synergistic gold-containing compositions

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050267204A1 (en) * 1996-11-04 2005-12-01 Medical Innovations Limited Synergistic gold-containing compositions
US20030180252A1 (en) * 1997-11-10 2003-09-25 Lawrence Tamarkin Methods and compositions for enhancing immune response and for the production of in vitro Mabs

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2114450A1 (en) * 2007-01-31 2009-11-11 Dobeel Co., Ltd. An hbv vaccine and a process of preparing the same
EP2114450A4 (en) * 2007-01-31 2012-06-27 Dobeel Co Ltd An hbv vaccine and a process of preparing the same
US11324819B2 (en) * 2016-04-06 2022-05-10 University Of Washington Therapeutic vaccine for Hepatitis b virus (HBV) using the HBV core antigen
US11883488B2 (en) 2016-04-06 2024-01-30 Abacus Bioscience, Inc. Therapeutic vaccine for Hepatitis B virus (HBV) using the HBV core antigen
WO2019013361A1 (en) * 2017-07-14 2019-01-17 公益財団法人東京都医学総合研究所 Hepatitis b vaccine
JPWO2019013361A1 (en) * 2017-07-14 2020-07-16 公益財団法人東京都医学総合研究所 Hepatitis B vaccine
JP7295536B2 (en) 2017-07-14 2023-06-21 公益財団法人東京都医学総合研究所 Hepatitis B vaccine

Also Published As

Publication number Publication date
KR20070122056A (en) 2007-12-28
KR100836893B1 (en) 2008-06-11

Similar Documents

Publication Publication Date Title
EP2114450B1 (en) An hbv vaccine and a process of preparing the same
KR100517114B1 (en) Composition for adjuvant containing poly-gamma-glutamic acid
EP2125012B1 (en) Compositions comprising a b subunit of shiga toxin and a means stimulating nkt cells
EP3957312A1 (en) Manganese combination for immunological enhancement
JP2009298806A (en) Pharmaceutical composition comprising polynucleotide and optionally antigen especially for vaccination
JP2011026328A (en) Oligonucleotide composition and use thereof for modulation of immune response
JP2021006567A (en) Nanoemulsion compositions for preventing, suppressing and eliminating allergic and inflammatory diseases
KR20200134255A (en) Composition
CA2594911A1 (en) Peptides for delivery of mucosal vaccines
JP5429821B2 (en) Vaccine preparation against Neospora protozoa infection
WO2007148924A1 (en) Use of colloidal gold as an adjuvant
JP2022527206A (en) Optimized cell-free synthesis of invading plasmid antigen B, and related compositions, and methods of use.
US20040014661A1 (en) Periplasmic domain of an enterobacterium omp protein and its use as carrier or adjuvant
KR101632962B1 (en) Anti-infective immunostimulatory composition comprising modified outer membrane vesicle
US10232036B2 (en) Vaccine formulation, preparation method therefor and use thereof
RU2796973C2 (en) Intranasal vaccine inducing cell-mediated immunity
JP7445897B2 (en) Nasal vaccines that induce cellular immunity
AU783851B2 (en) Method for preparing a polypeptide soluble in an aqueous solvent in the absence of detergent
Apostolopoulos et al. Subunit protein-based vaccines
WO2023047129A1 (en) Immunogenic fusion peptides including fc fragment and a non toxic b subunit of ab5 toxin
TWI358302B (en) Fusion proteins for inhibiting or preventing hpv i
WO2017189448A1 (en) Bivalent immunogenic conjugate for malaria and typhoid
JP2003277292A (en) Mucous membrane vaccine for inhibiting plasmodium propagation
KR20120021854A (en) Preparation for recombinant istf(immunostimulating factor) protein to increase anti tumor effect as vaccine adjuvant

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07747036

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07747036

Country of ref document: EP

Kind code of ref document: A1