WO2017193535A1 - 氢氧化铝凝胶-氯化钠复合免疫佐剂及其制备方法和用途 - Google Patents

氢氧化铝凝胶-氯化钠复合免疫佐剂及其制备方法和用途 Download PDF

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WO2017193535A1
WO2017193535A1 PCT/CN2016/102131 CN2016102131W WO2017193535A1 WO 2017193535 A1 WO2017193535 A1 WO 2017193535A1 CN 2016102131 W CN2016102131 W CN 2016102131W WO 2017193535 A1 WO2017193535 A1 WO 2017193535A1
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antigen
sodium chloride
immunoadjuvant
aluminum hydroxide
vaccine
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PCT/CN2016/102131
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French (fr)
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魏霞蔚
罗敏
魏于全
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四川大学
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Priority to US16/300,295 priority Critical patent/US10869922B2/en
Priority to JP2018559377A priority patent/JP6796146B2/ja
Priority to EP16901490.9A priority patent/EP3456350A4/en
Publication of WO2017193535A1 publication Critical patent/WO2017193535A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/02—Bacterial antigens
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • 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
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to an aluminum hydroxide gel-sodium chloride composite immunoadjuvant, a preparation method thereof and use thereof.
  • Adjuvants are a non-specific immunopotentiator that has been used for decades to improve the body's immune response to vaccine antigens.
  • the purpose of adding adjuvant to the vaccine is to enhance the body's immune response to the antigen or to change the type of immune response, which is to promote specific humoral and/or cellular immunity of the antigen, thereby increasing the production of specific antibodies or/and specific cells.
  • Immune Function Injecting the antigen into the body together with the adjuvant can reduce the amount of antigen and the frequency of immunization required for effective immunization, improve the response success rate of the early immune response and immune dysfunction, and the like.
  • Aluminum adjuvants represented by aluminum hydroxide and aluminum phosphate are still the human vaccine adjuvants approved for legal use in China.
  • Aluminum hydroxide is also the only human adjuvant approved for use by the FDA.
  • the mechanism of action of the aluminum salt adjuvant is not fully understood. At present, there are two mechanisms of “inventory effect” and “immunostimulation effect”.
  • the aluminum adjuvant forms an antigen depot in the inoculated area after adsorbing the antigen, and on the one hand, non-specific immunostimulation at this point attracts antigen-presenting cells (APC) such as dendritic cells macrophages to recognize and endocytosis; Injecting a local slow release of antigen prolongs the interaction time between APC and T lymphocytes, thereby increasing antibody response and enhancing humoral immunity.
  • APC antigen-presenting cells
  • Sodium chloride is very important for life on Earth, and is widely used in life, industry, and medicine.
  • the main component of the salt required for daily diet and the physiological saline injection used in medicine is sodium chloride. It has the advantages of easy access, low price and high security.
  • the technical problem to be solved by the present invention is to provide a new and good performance immunoadjuvant, which provides a new and effective choice for the field.
  • the composite immunoadjuvant mainly contains an aluminum hydroxide gel and a physiologically higher level of sodium chloride.
  • the amount of sodium chloride in the immunoadjuvant is higher than the physiological level, which means that the mass fraction of sodium chloride in the final adjuvant-antigen complex solvent is higher than 0.9%.
  • the final mass fraction refers to the concentration of the complex of the immunoadjuvant and the antigen prior to injection.
  • the final mass fraction of the sodium chloride in the composite immunoadjuvant is 1.2% to 7.2%. Further, the final mass fraction of the sodium chloride in the above composite immunoadjuvant is 2.7% to 4.5%. Preferably, the final mass fraction of the sodium chloride in the above composite immunoadjuvant is 3.6%.
  • the above mass fraction means the mass (g) of sodium chloride per 100 ml of the adjuvant-antigen complex solvent.
  • the particle diameter of the aluminum hydroxide gel in the composite immunoadjuvant described above is in the range of 1 ⁇ m to 10 ⁇ m.
  • the aluminum hydroxide gel has a particle size ranging from 2 ⁇ m to 6 ⁇ m.
  • a particle size of about 3 ⁇ m is preferred.
  • the present invention further provides an immunoadjuvant-antigen complex formed by adding an antigen to the above composite immunoadjuvant.
  • Immunological adjuvant-antigen complexes are also known in the art as vaccines.
  • antigen in the above immunoadjuvant-antigen complex
  • antigen any substance capable of inducing an immune reaction.
  • the antigen is one or more of a tumor antigen, a viral antigen or a bacterial antigen.
  • the above tumor antigen may be selected from an OVA tumor model antigen (OVA tumor model antigen is internationally used as a modeled or standardized tumor specific antigen, and is often used as a standard tumor specific antigen to test the induction of an adjuvant.
  • OVA tumor model antigen is internationally used as a modeled or standardized tumor specific antigen, and is often used as a standard tumor specific antigen to test the induction of an adjuvant.
  • Anti-tumor immune response ability if the adjuvant induces an anti-tumor immune response, indicating that the adjuvant is representative of tumor immune response
  • NY-ESO-1 human melanoma-associated antigen gP100
  • melanin mage-1 or carcinoma One or several of commonly used tumor antigens such as embryo antigens.
  • the viral antigen is selected from the group consisting of hepatitis B virus antigen, hepatitis A virus antigen, hepatitis C virus antigen, poliovirus antigen, rabies virus antigen, yellow fever virus antigen, HIV antigen or measles, epidemic One or several common viral antigens such as mumps, rubella, varicella, rotavirus, Japanese encephalitis, papillomavirus, epidemic hemorrhagic fever virus, and plague virus antigen.
  • the bacterial antigen is selected from the group consisting of Staphylococcus aureus antigen, Pseudomonas aeruginosa antigen, pertussis antigen, diphtheria antigen, H. influenzae antigen, Neisseria meningitidis antigen, tetanus antigen, hemolytic streptococcus antigen, At least one of common bacterial antigens such as non-hemolytic streptococcus antigen, pneumococcal antigen, Mycobacterium tuberculosis, Bacillus anthracis, Vibrio cholerae, Leptospira or Helicobacter pylori.
  • the invention further provides a method of preparing the above-described immunoadjuvant/antigen complex.
  • the method includes the following steps:
  • the above immunoadjuvant/antigen complex can also be prepared by the following steps:
  • the antigen is one or more of a tumor antigen, a viral antigen or a bacterial antigen.
  • the tumor antigen may be selected from one or more of the commonly used tumor antigens such as OVA tumor model antigen, NY-ESO-1; human melanoma-associated antigen gP100; melanin mage-1 or carcinoembryonic antigen.
  • the above viral antigen is selected from the group consisting of hepatitis B virus antigen, hepatitis A virus antigen, hepatitis C virus antigen, poliovirus antigen, rabies virus antigen, yellow fever virus antigen, HIV antigen or measles, epidemic One or several common viral antigens such as mumps, rubella, chickenpox, rotavirus, Japanese encephalitis, papillomavirus, epidemic hemorrhagic fever virus, and plague virus antigen.
  • the above bacterial antigen is selected from the group consisting of Staphylococcus aureus antigen, Pseudomonas aeruginosa antigen, pertussis antigen, diphtheria antigen, H. influenzae antigen, Neisseria meningitidis antigen, tetanus antigen, hemolytic streptococcus antigen, non- At least one of a common bacterial antigen such as a hemolytic streptococcus antigen, a pneumococcal antigen, Mycobacterium tuberculosis, Bacillus anthracis, Vibrio cholerae, Leptospira or Helicobacter pylori.
  • the mass fraction in the above technical scheme refers to the mass (gram) of sodium chloride per 100 ml of the adjuvant-antigen complex solution.
  • the invention also provides a vaccine prepared from the above immunoadjuvant-antigen complex.
  • the vaccine can be a prophylactic and/or therapeutic vaccine.
  • the antigen when the antigen is a hepatitis antigen such as HBsAg, it can be prepared as a hepatitis vaccine; when the antigen is a pertussis, diphtheria toxoid or a bacterial antigen such as Staphylococcus aureus or Pseudomonas aeruginosa, it can be prepared as a bacterial vaccine; for example, when the antigen is an OVA tumor model In the case of antigens or tumor-specific antigens, prophylactic and/or therapeutic tumor vaccines can be prepared.
  • the vaccine of the present invention can be administered by subcutaneous, intraperitoneal or intramuscular injection to immunize an individual. It is of course also possible to use other means available in the art for immunization or a combination of means. And the vaccine of the invention can have different immunization intervals. Can be administered once or more Times. During the specific implementation process, the number of immunizations and the time of immunization may be changed or adjusted according to actual conditions.
  • the present invention has examined a variety of aluminum hydroxide-sodium chloride formulations.
  • the present invention screens sodium chloride of different mass fractions, and finds that the best effect is obtained when the mass fraction of sodium chloride in the vaccine solution prepared by using the complex of sodium chloride and aluminum hydroxide as an adjuvant is 3.6%.
  • the invention provides the use of sodium chloride in the preparation of an immunological adjuvant.
  • the immunoadjuvant described in the above application also has an aluminum hydroxide gel.
  • the amount of the sodium chloride used in the above-mentioned use is higher than the physiological level.
  • the sodium chloride in the above-mentioned use is used in the immunoadjuvant in an amount of 1.2% to 7.2% of the sodium chloride in the complex of the immunoadjuvant and the antigen prepared by the preparation.
  • This concentration is the concentration of the complex of the immunoadjuvant and the antigen prior to injection.
  • the invention also provides the use of sodium chloride in the preparation of an immunoadjuvant-antigen complex.
  • the immunoadjuvant-antigen complex further contains an aluminum hydroxide gel as an adjuvant.
  • the content of sodium chloride in the above application is 1.2% to 7.2% by mass. This concentration is the concentration of the complex of the immunoadjuvant and the antigen prior to injection.
  • the content of sodium chloride is from 2.7% to 4.5% by mass. The optimal is 3.6%.
  • the weight ratio of the antigen to the aluminum hydroxide in the above application is 1:1 to 100.
  • the weight ratio of the antigen in the above-mentioned application to the aluminum hydroxide in the composite immunoadjuvant is 1:5 to 50.
  • the antigen in the above use is one or more of a tumor antigen, a viral antigen or a bacterial antigen.
  • the invention also provides a method of formulating a vaccine. The method steps are to add sodium chloride and an appropriate amount of water for injection before the use of the vaccine, and to achieve a mass fraction of sodium chloride of 1.2% to 7.2%. The method can be implemented separately according to the actual situation as follows:
  • a hypertonic aluminum hydroxide gel stock solution is added, and the mass fraction of sodium chloride in the formulated vaccine is 1.2% to 7.2%;
  • the vaccine is prepared with the antigen and the aluminum hydroxide gel, the sodium chloride is directly added as an adjuvant during the preparation, so that the prepared vaccine contains 1.2% of the sodium chloride before the injection. ⁇ 7.2%.
  • the preferred sodium chloride has a mass fraction of 2.7% to 4.5% in the vaccine system to be injected.
  • the optimal is 3.6%.
  • the weight ratio of the antigen to the aluminum hydroxide in the vaccine is 1..1 to 100.
  • the weight ratio of the antigen to the aluminum hydroxide in the formulated vaccine is 1.. 5 to 50.
  • weight ratio of the antigen to aluminum hydroxide described in the present invention means the ratio of the antigen to the pure substance of aluminum hydroxide in the aluminum hydroxide gel.
  • the sodium chloride is sodium chloride which satisfies the purity required for vaccine preparation.
  • sodium chloride of chemical purity or chemical purity above is required.
  • the present invention provides the use of sodium chloride in the preparation of vaccine adjuvants and in the preparation of vaccines.
  • a new vaccine adjuvant aluminum hydroxide gel-sodium chloride complex immunoadjuvant, is available, which can be applied to the development of infectious diseases vaccines and tumor vaccines such as bacteria and viruses.
  • Preliminary analysis of its mechanism may be that higher than physiological levels of hypertonic sodium chloride can activate the activity of M ⁇ , NK, DC and other immune cells, activate MAPK signaling pathway, and promote DC maturation and antigen uptake or cross-presentation.
  • the aluminum salt combines with the antigen to form an antigen reservoir, so that the antigen can be slowly and stably released, and the aluminum salt adjuvant can also induce and stimulate humoral immunity. Therefore, the combination of the two can stimulate humoral immunity and stimulate cellular immunity to achieve better results.
  • the beneficial effects of the present invention are that the present invention creatively develops the use of sodium chloride in the preparation of vaccine adjuvants and in the preparation of vaccines, and further develops an aluminum hydroxide-sodium chloride complex immunoadjuvant. It has the advantages of strong immunological activity and high clinical safety. It has a specific cellular immune response compared with aluminum hydroxide alone. It is an excellent aluminum hydroxide-sodium chloride complex immunoadjuvant for various antigens. Tests have shown that the use of the aluminum hydroxide-sodium chloride of the present invention as an adjuvant for preparing a vaccine can have higher immune effects and anti-tumor effects, and provides new development and application for various preventive and/or therapeutic vaccines. select.
  • Figure 1 shows the results of an experiment in which an aluminum hydroxide gel-sodium chloride immunoadjuvant enhances the serum antibody titer against HBsAg in each group of mice in the HBsAg model.
  • Figure 2 is an experimental result of an aluminum hydroxide gel-sodium chloride immunoadjuvant to enhance the specific cellular immune response against HBsAg in each group of mice in the HBsAg model.
  • Figure 3 is an aluminum hydroxide gel-sodium chloride immunoadjuvant to enhance the serum against OVA in each group of mice in the OVA model. Experimental results of antibody titers.
  • Figure 4 is an experimental result of an aluminum hydroxide gel-sodium chloride immunoadjuvant to enhance the specific cellular immune response against HBsAg in each group of mice in the HBsAg model.
  • Figure 5 is an aluminum hydroxide gel-sodium chloride immunoadjuvant that enhances the anti-tumor effect of the vaccine in both prophylactic and therapeutic tumor models.
  • Figure 6 is an experimental result of the anti-tumor effect of the aluminum hydroxide gel-sodium chloride immunoadjuvant as a result of induction of specific killer CD8+ T cells (CTL response).
  • FIG. 7 shows the results of an experiment in which hypertonic sodium chloride can promote the maturation of DC cells in vitro.
  • Figure 8 is a graph showing the results of experiments in which hypertonic sodium chloride can promote antigen phagocytosis of DC cells in vitro.
  • Figure 9 is a graph showing the results of experiments in which hypertonic sodium chloride can promote the secretion of inflammatory cytokines in DC cells in vitro.
  • Figure 10 is a graph showing the results of experiments in which hypertonic sodium chloride can promote cross-antigen presentation of DC cells in vitro.
  • FIG 11 shows the results of an experiment in which an aluminum hydroxide gel-sodium chloride immunoadjuvant can increase memory T cells and improve the tumor microenvironment.
  • Figure 12 is an experimental result of an aluminum hydroxide gel-sodium chloride immunoadjuvant enhancing the serum antibody titer of each group of mice in the S. aureus model.
  • Figure 13 is an experimental result of an aluminum hydroxide gel-sodium chloride immunoadjuvant enhancing the serum antibody titer of each group of mice in the P. aeruginosa model.
  • the present inventors have creatively found that sodium chloride above physiological levels can be used as an immunological adjuvant and can be used to prepare a composite immunological adjuvant.
  • the use of aluminum hydroxide gel-sodium chloride combination as a composite adjuvant can effectively induce humoral immunity and effectively induce cellular immunity.
  • the composite adjuvant provided by the invention adopts two components: the aluminum hydroxide gel adjuvant is a commonly used inorganic salt adjuvant, has good protein adsorption, and can adsorb soluble antigen on the surface of the aluminum gel molecule. It is also possible to concentrate the antigen and reduce the injected dose. Aluminium hydroxide gel is the most widely used adjuvant because of its low cost, convenient use and non-toxicity. It is also the only adjuvant approved by the US FDA for human vaccine. Sodium chloride is widely used in life, industry and medicine, and it is safe for the human body and the cost is extremely low.
  • the amount of sodium chloride in the composite adjuvant of the present invention is the final quality of sodium chloride.
  • a score greater than the physiological concentration can achieve an improvement in the immune effect.
  • the increase in immune effect is more obvious in 1.2% to 7.2%.
  • Preferably, from 2.7% to 4.5% has a better immune adjuvant effect.
  • the best effect in the experiment of the invention was 3.6%.
  • the mass fraction of sodium chloride in the present invention means the mass (gram) of sodium chloride per 100 ml of the antigen-adjuvant complex solvent.
  • the vaccine can be prepared according to the standards in the above scheme, and the concentration of sodium chloride in the system can be changed by lyophilization, evaporation or dilution.
  • concentration of sodium chloride in the system can be changed by lyophilization, evaporation or dilution.
  • the technique of adjusting the concentration of sodium chloride in the system to within the above range before being used officially is also within the scope of protection of the present invention.
  • the above-mentioned aluminum hydroxide gel-sodium chloride new adjuvant of the present invention is a widely used and safely used immunological adjuvant, and various antigens known in the art can be added.
  • An immunoadjuvant-antigen complex is obtained and used as a vaccine.
  • the antigen (Ag) described in the present invention is referred to in the art as any substance which induces an immune response.
  • the antigen is one or more of a tumor antigen, a viral antigen or a bacterial antigen.
  • the tumor antigen refers to a tumor antigen form commonly known in the art including a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA) protein or a glycolipid component.
  • TSA tumor-specific antigen
  • TAA tumor-associated antigen
  • the above tumor antigen may be selected from one or more of commonly used tumor antigens such as NY-ESO-1; human melanoma-associated antigen gP100; melanin mage-1 or carcinoembryonic antigen.
  • the above viral antigen may be selected from the group consisting of hepatitis B virus antigen, hepatitis A virus antigen, hepatitis C virus antigen, poliovirus antigen, rabies virus antigen, yellow fever virus antigen, HIV antigen or measles, One or several common viral antigens such as mumps, rubella, varicella, rotavirus, Japanese encephalitis, papillomavirus, epidemic hemorrhagic fever virus, and plague virus antigen.
  • the above viral antigen may be in the form of a virus antigen, an inactivated virus, a viral subunit vaccine, a synthetic peptide vaccine, a nucleic acid vaccine, and the like, which are common in the art.
  • the bacterial antigen is selected from the group consisting of Staphylococcus aureus antigen, Pseudomonas aeruginosa antigen, pertussis antigen, diphtheria antigen, Haemophilus influenzae antigen, Neisseria meningitidis antigen, tetanus antigen, hemolytic streptococcus antigen, non-hemolytic At least one of a streptococcal antigen or a pneumococcal antigen or a common bacterial antigen such as Mycobacterium tuberculosis, Bacillus anthracis, Vibrio cholerae, Leptospira, or Helicobacter pylori.
  • the form of the bacterial antigen may be a dead bacterin of various bacteria, attenuated (attenuated) seedlings, a virulent seedling, or a pathogenic toxin of various bacteria, or various bacterial polysaccharides or bacterial proteins. Bacterial polysaccharides bind antigens and other forms of bacterial antigens commonly found in the art.
  • the invention adds sodium chloride adjuvant to the aluminum hydroxide type hepatitis B vaccine or the pertussis and diphtheria vaccine, and obtains higher antibody titer and IFN- ⁇ secretion increase; in the aluminum hydroxide type golden yellow Staphylococcus or green Adding sodium chloride adjuvant to the Pseudomonas vaccine to obtain higher antibody titer and protection against bacteria; also preparing an OVA tumor using aluminum hydroxide gel-sodium chloride new adjuvant A novel vaccine for model antigens and confirmed the effectiveness of this new adjuvant vaccine against tumors.
  • Aluminum hydroxide is generally used as aluminum hydroxide particles having a particle diameter ranging from 1 ⁇ m to 10 ⁇ m. Aluminum hydroxide particles having a particle diameter of 2 ⁇ m to 6 ⁇ m are preferred. Of course, it may be possible to have a reasonable change in the preferred particle size of the particles in the aluminum hydroxide when combined with different antigens. Aluminum hydroxide particles having a particle diameter of about 3 ⁇ m are preferred.
  • the weight ratio of the above antigen to the composite immunoadjuvant aluminum hydroxide is from 1..1 to 100, preferably from 1..5 to 50.
  • the preparation of the immunoadjuvant-antigen complex of the present invention can be carried out by the following two methods:
  • the amount of HBsAg or pertussis and diphtheria toxoid in the immunoadjuvant/antigen complex is 1 ⁇ g, and the weight of the antigen and the aluminum hydroxide gel.
  • the ratio may be 1..5 to 1..100, and most preferably 1..25.
  • the amount of OVA in the immunoadjuvant/antigen complex is 5 ⁇ g, and the weight ratio of the antigen to the aluminum hydroxide gel may be 1..5 to 1..50, and most preferably 1:25.
  • the present invention also develops a new vaccine preparation method.
  • the steps of the method are Before the vaccine is used, sodium chloride and an appropriate amount of water for injection are added, and the mass fraction of sodium chloride is 1.2% to 7.2%.
  • the method can be specifically carried out by adding an appropriate amount of water for injection and sodium chloride to the antigen, and then adding an aluminum hydroxide gel adjuvant, so that the mass fraction of sodium chloride in the formulated vaccine reaches 1.2%. 7.2%;
  • a hypertonic aluminum hydroxide gel stock solution is added, and the mass fraction of sodium chloride in the formulated vaccine is 1.2% to 7.2%;
  • the method comprises the steps of: adding a sodium chloride and an appropriate amount of water for injection to prepare a vaccine system to be injected before the vaccine is used, and making the sodium chloride and the sodium chloride The mass fraction in the vaccine system to be injected reaches 1.2% to 7.2%.
  • the mass fraction of sodium chloride in the vaccine system to be injected is 2.7% to 4.5%, which is a preferred solution.
  • the optimal is about 3.6%.
  • the vaccine contains an antigen and an aluminum hydroxide gel.
  • an antigen and an aluminum hydroxide gel may be a commercially available aluminum hydroxide immunoadjuvant, or may be an aluminum hydroxide colloid adjuvant currently formulated according to the Chinese Pharmacopoeia, or may be in accordance with the requirements of the US FDA or An aluminum hydroxide colloid that can be used for vaccine preparation in accordance with the relevant requirements of other countries.
  • Aluminum hydroxide adjuvant (Alhydrogel, aluminum hydroxide gel) was purchased from InvivoGen (white suspension, 10 mg/mL, particle size 3 ⁇ m).
  • Hepatitis B surface antigen was purchased from American Rearch Products, sodium chloride (NaCl) and ovalbumin (OVA) were purchased from Sigma, USA.
  • Whooping cough P7208) and diphtheria toxoid (D0564) were purchased from Sigma, USA.
  • the method for preparing the above immunoadjuvant-antigen complex can be carried out in the following two ways:
  • Method 1 a, take the desired antigen diluted or dissolved with water; b, add the required sodium chloride, mix; c, add the required amount of aluminum hydroxide gel, mix, that is.
  • Method 2 a, adding appropriate sodium chloride to the aluminum hydroxide gel adjuvant to prepare a hypertonic aluminum hydroxide gel composite adjuvant stock solution; b, taking the desired antigen diluted or dissolved with water; c, adding required A quantity of hypertonic aluminum hydroxide gel compound adjuvant, mixed, that is.
  • the final mass fraction of the sodium chloride is from 1.2% to 7.2%, preferably 3.6%. Have the best immune adjuvant effect.
  • the mass fraction of sodium chloride in the present invention means the mass (gram) of sodium chloride per 100 ml of the antigen adjuvant complex solvent.
  • the weight ratio of the above antigen to the composite immunoadjuvant aluminum hydroxide is 1:1 to 100.
  • a preferred weight ratio is 1:5 to 50.
  • the weight ratio of the antigen and the aluminum hydroxide gel may be 1..5 to 1 ..100. Most preferably it is 1..25.
  • the amount of OVA in the immunoadjuvant/antigen complex is 5 ⁇ g, and the weight ratio of the antigen to the aluminum hydroxide gel may be 1..5 to 1.50. Most preferred is 1..25.
  • Example 2 Animal immunization test using sodium chloride as adjuvant for hepatitis B vaccine of the present invention
  • mice aluminum hydroxide adjuvant, sodium chloride, HBsAg as in the first example; BALB/c mice were purchased from Beijing Weitong Lihua Company; hepatitis B virus surface antibody detection kit (Wantai Biopharmaceutical) ELISA kit for IFN- ⁇ (eBioscience, USA); lymphocyte separation solution (Daktronics, China); 70 ⁇ m nylon mesh filter (BD, USA); round bottom 24-well plate (NUNK).
  • hepatitis B virus surface antibody detection kit (Wantai Biopharmaceutical) ELISA kit for IFN- ⁇ (eBioscience, USA); lymphocyte separation solution (Daktronics, China); 70 ⁇ m nylon mesh filter (BD, USA); round bottom 24-well plate (NUNK).
  • the experimental animals were grouped as follows: 1. Control; 2, HbsAg; 3, HbsAg/Al(OH) 3 ; 4, HbsAg/Al(OH) 3 /1.8% NaCl; 5, HbsAg/Al(OH) 3 /3.6% NaCl;6, HbsAg/Al(OH) 3 /7.2% NaCl; HbsAg is 1 ⁇ g in all groups, Al(OH) 3 is 25 ⁇ g, and the weight ratio of HbsAg and Al(OH) 3 is 1:25, and the NaCl concentration is According to each group, they were prepared to 0, 1.8%, 3.6% and 7.2%. 5 per group. Muscle immunization was performed at 0, 2, and 3 weeks, and serum and spleen lymphocytes were collected at 4 weeks for the following experiment.
  • the measurement of IFN- ⁇ was performed by ELISA and flow cytometry. Specifically, the mice were sacrificed one week after the last immunization, and the spleen was taken out. The spleen lymphocytes were obtained and stimulated with HBsAg in vitro, and cultured in a CO 2 incubator for 72 hours, and the concentration of the culture supernatant IFN- ⁇ was measured by an ELISA test kit.
  • Example 3 Animal immunoassay using sodium chloride as an adjuvant for OVA tumor-specific antigen in the present invention
  • mice aluminum hydroxide adjuvant, sodium chloride, OVA as in the first example
  • mouse IFN- ⁇ ELISA kit
  • lymphocyte separation solution 70 ⁇ m nylon mesh filter, round bottom 24, 96-well plate
  • mouse lymphoma cell line EG.7 US ATCC
  • Na 2 51 CrO 4 PerkinElmer, USA.
  • mice were randomly divided into 4 groups: 1, Control 2, OVA 3, OVA/Al(OH) 3 4, OVA/Al(OH) 3 /3.6% NaCl; in all groups, OVA was used as the tumor.
  • the specific antigen was 5 ⁇ g
  • Al(OH) 3 was 125 ⁇ g
  • the weight ratio of OVA and Al(OH) 3 was 1:25, and the sodium chloride concentration was prepared according to the grouping requirements.
  • Ten mice in each group were subcutaneously injected into the left dorsal side of each group of mice at 0, 2, and 3 weeks.
  • mice lymphoma cells were inoculated subcutaneously on the right side of the mice by 3 ⁇ 10 6 /, and a mouse xenograft model was established, and the tumor volume of the mice was measured every 3 days.
  • mice were randomly divided into 4 groups as above, and the mouse lymphoma cells were inoculated subcutaneously on the right side of the mice 3 ⁇ 10 6 /piece, until the tumor was long enough to be touched (about 3 mm, 3 to 5 after inoculation). Days, mice began to undergo subcutaneous immunotherapy according to the 4-component dose schedule of the above prophylactic experiments, once a week, 3 consecutive treatments, and the tumor volume of the mice was measured every 3 days.
  • the experimental method and grouping are the same as those in the second embodiment, as follows: Control; 2, OVA; 3, OVA/Al(OH) 3 ; 4, OVA/Al(OH) 3 /1.8% NaCl; 5, OVA/Al(OH) 3 / 3.6% NaCl; 6, OVA / Al (OH) 3 / 7.2% NaCl.
  • OVA was 5 ⁇ g
  • Al(OH) 3 was 125 ⁇ g
  • OVA and Al(OH) 3 were 1:25, and sodium chloride concentration was prepared according to grouping requirements.
  • Five mice in each group were collected from the mice on the 7th day after the last immunization, and the antibody titer in the serum of each group was detected by self-labeled ELISA.
  • the detection results of antibody absorbance of each group are shown in Fig. 3.
  • the OVA group alone produced almost no antibodies.
  • the Al(OH) 3 group and the Al(OH) 3 + sodium chloride complex adjuvant group alone can effectively stimulate the production of antibodies.
  • the Al(OH) 3 + sodium chloride complex adjuvant produced higher IgG and IgG1 antibody titers than the Al(OH) 3 group alone, and reached the highest value when the sodium chloride mass fraction was 3.6%. .
  • the measurement of IFN- ⁇ was carried out using a self-coated ELISA kit (eBioscience) and flow cytometry. Specifically, the mice were sacrificed one week after the last immunization, and the spleen was taken out. After obtaining spleen lymphocytes, 5 ⁇ g of different OVA peptides were added and stimulated, and placed in a CO 2 incubator for 72 hours, and the concentration of IFN- ⁇ in the culture supernatant was measured by an ELISA test kit. Cells were used for INF- ⁇ and specific OVA-Tetramer experiments.
  • FIG 4c represents the proportion of positive specific OVA-Tetramer increased, indicating that Al (OH) 3 + NaCl Compound Adjuvant do induce specific cellular immunity.
  • novel adjuvant vaccine of the present invention stimulates tumor-specific cytotoxic T lymphocyte killing function
  • the detection of cytotoxic T lymphocyte killing function was detected by the 51 Cr release test.
  • mice were randomly selected from each group 7 days after the last immunization.
  • the mice were dissected under aseptic conditions, the spleen lymphocytes were isolated, and the cell concentration was adjusted to 1 ⁇ 10 7 /ml, and the obtained lymphocytes were effector cells.
  • 96-well plate horizontal bricks were centrifuged at 1500 rpm/min for 30 s, 37 ° C, 5% CO 2 . After incubation in the incubator, after 4 to 6 hours, 96-well plate horizontal bricks were centrifuged at 1500 rpm/min for 5 min, and 100 ⁇ l of supernatant was taken from each well.
  • Mouse EG.7 cells were injected into the right posterior 3 ⁇ 10 6 /only, and a mouse xenograft model was established and randomly divided into 4 groups, 5 in each group.
  • Cell Adoption One day before and after the establishment of the mouse xenograft model, the spleen lymphocytes obtained in the prophylactic experiment by the tail vein injection, 10 7 cells/only, were injected 3 times every 3 days. Changes in tumor volume were observed in mice.
  • Serum adoptive For the 3 weeks before and after the establishment of the mouse xenograft model, the serum obtained in the prophylactic test by tail vein injection, 250 ⁇ l/mouse, twice a week, observed the tumor volume of the mice every 3 days. The change.
  • Anti-CD4, CD8, NK monoclonal antibodies or control non-specific RAT antibodies were injected intraperitoneally on the day before and during immunization, twice a week for 3 weeks. After the immunization was completed, the mice were inoculated with EG.7 cells, and the tumor production curve of each group was monitored every 3 days.
  • mice in the prophylactic test or the therapeutic vaccine test were prepared, and after the single cell suspension was prepared, the changes of various immune cell populations of the mice were detected by flow cytometry.
  • FIG. 11a The results of memory T lymphocyte experiments are shown in Figure 11a. Immunization with Al(OH) 3 + sodium chloride complex adjuvant can significantly increase central and effector CD4+CD8+ T lymphocytes. For the tumor microenvironment, the results are shown in Figures 11b and c. Al(OH) 3 + sodium chloride complex adjuvant not only increases the invasiveness of CD8+ T lymphocytes in tumor tissues, but also reduces the immunosuppressive effect. The number of MDSC and M2 macrophages.
  • novel sodium chloride adjuvant of the invention can promote various functions of DC in vitro
  • DC plays an indispensable role in the vaccine immune response. so I We studied the effects of high salt on DC function in vitro.
  • C57BL/6 mouse primary bone marrow DCs were prepared by classical methods. After in vitro stimulation with cytokine differentiation such as GM-CSF for 6 days, the cells were collected and continued to be cultured for 48 hours in medium containing different mass fractions of sodium chloride, and the cells were detected by flow cytometry to indicate the ratio of mature molecules CD80, CD86, etc. Simultaneous ELISA was used to detect the secretion of inflammatory cytokines in the supernatant. At the same time, the lysed part of the cells were collected to prepare RNA, and the mRNA level of the relevant inflammatory molecule was detected by RT-PCR.
  • cytokine differentiation such as GM-CSF
  • Simultaneous ELISA was used to detect the secretion of inflammatory cytokines in the supernatant.
  • the lysed part of the cells were collected to prepare RNA, and the mRNA level of the relevant inflammatory molecule was detected by RT-PCR.
  • DCs were cultured for 1 h in medium containing different mass fractions of sodium chloride and fluorescently labeled OVA model tumor antigen or dextran, and relevant antigen phagocytosis was detected by fluorescence microscopy or flow cytometry.
  • high salt stimulates DC in vitro and promotes its maturation and increases the expression of CD80, CD86, MHCII and other molecules on its molecular surface. It can be seen from Fig. 8 that high salt can also promote the phagocytosis of antigen by DC. It is also known that the release of the inflammatory cytokines IL6 and IL1b can be increased at the mRNA and protein levels.
  • DC/T lymphocyte co-culture experiment The experimental grouping is shown in Figure 10.
  • DC cells stimulated with high salt and OVA antigen in vitro were co-cultured with CFSE-labeled CD8+ T lymphocytes derived from OT-1 transgenic mice. After 3 days, the formation of CLUSTER was observed under microscope white light, and the proliferation of CFSE-labeled CD8+ T lymphocytes was detected by flow cytometry. In addition, the culture supernatant was collected for detection of IL2 secretion by ELISA.
  • Immunofluorescence colocalization experiment The experimental group is the same as above. DC cells stimulated with high-salt and fluorescent-labeled OVA in vitro were fixed, perforated, stained with proteasome and lysosomal organelle fluorescent protein markers, and observed under a confocal microscope.
  • Example 4 Animal immunoassay using sodium chloride as a bacterial vaccine adjuvant in the present invention
  • the experimental animals were immunized as follows: 1. Control; 2, Sa or Pa; 3, Sa or Pa/Al(OH) 3 ; 4, Sa or Pa/Al(OH) 3/3.6% NaCl; the specific immunization protocol is as follows: The cultured bacteria were expanded, washed, 1% paraformaldehyde fixed inactivated, washed, resuspended in PBS, 5 mice in each group, subcutaneously immunized 3 times at 0, 2, 3 weeks, the first bacterial solution 0.05 OD (1 ⁇ 10 6 CFU / 200 ⁇ L), the second bacterial liquid 0.5 OD (1 ⁇ 10 7 CFU / 200 ⁇ L), the third bacterial liquid 2.5 OD (5 ⁇ 10 7 CFU / 200 ⁇ L); after each immunization Serum was collected for antibody detection.
  • Detection of antibody titer 96-well plates were self-coated with 1 ⁇ 10 7 CFU/100 ⁇ L whole bacterial antigen, and specific IgG antibody titers against different bacteria in the serum of each group of mice were detected by ELISA.
  • the experimental results are shown in Fig. 12, and antibodies against S. aureus can be detected; Al(OH)3 adjuvant helps to increase antibody levels; high salt Al(OH)3 adjuvant has a better effect.
  • Pseudomonas aeruginosa-specific antibodies can be detected, the experimental results are shown in Figure 13; Al(OH)3 adjuvants help to increase antibody levels, and high-salt Al(OH)3 adjuvants have better effects.
  • the aluminum hydroxide gel-sodium chloride immunoadjuvant can induce specific antibody production of diphtheria toxin and increase the production of about 48% of antibodies.
  • an aluminum hydroxide gel-sodium chloride immunoadjuvant can increase the induction of about 45% antibody production by pertussis toxin.

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Abstract

一种氢氧化铝凝胶-氯化钠复合免疫佐剂及其制备方法和用途。所要解决的技术问题是提供一种性能良好的免疫佐剂。解决该技术问题的技术方案是提供氯化钠在制备免疫佐剂中用途以及以之为基础得到的氢氧化铝凝胶-氯化钠复合免疫佐剂。该复合免疫佐剂主要含有氢氧化铝凝胶和氯化钠，具有方法简便、成本低、免疫活性强、临床安全性高等优点，是一种优秀的可用于各种抗原的复合免疫佐剂，为疫苗的开发和应用提供了有效选择。

Description

氢氧化铝凝胶-氯化钠复合免疫佐剂及其制备方法和用途 技术领域
本发明属于生物医药领域，具体涉及一种氢氧化铝凝胶-氯化钠复合免疫佐剂及其制备方法和用途。
背景技术
佐剂是一种非特异性免疫增强剂，作为改善机体对疫苗抗原的免疫应答已经有几十年应用的历史了。疫苗中加入佐剂目的是为了增强机体对抗原的免疫应答或改变免疫应答类型，表现为促进抗原的特异性体液免疫和/或细胞免疫，从而可以提高特异性抗体的产生或/和特异性细胞免疫功能。将抗原与佐剂一起注入机体，可以减少有效免疫接种所需的抗原量和免疫接种频率，提高早期免疫应答和免疫功能不全者的应答成功率等等。
以氢氧化铝和磷酸铝为代表的铝佐剂至今仍然是我国被批准合法使用的人用疫苗佐剂。氢氧化铝还是唯一被美国FDA批准使用的人用佐剂。虽然应用悠久，但是铝盐佐剂的作用机理目前尚不完全清楚。目前认为有“库存效应”和“免疫刺激效应”两种机制。铝佐剂吸附抗原后在接种区域形成抗原贮存库，一方面在该处的非特异免疫刺激吸引树突状细胞巨噬细胞等抗原提呈细胞(APC)识别与内吞；另一方面，在注射局部缓慢释放抗原，延长了APC与T淋巴细胞相互作用的时间，从而提高抗体应答，增强体液免疫。
氯化钠对于地球上的生命非常重要，无论是在生活，工业还是医学上都广泛应用。生活中饮食所需的食盐和医学上使用的生理盐水注射液的主要成分都是氯化钠。它具有获取方便，价格便宜和安全性高等优点。
随着新型佐剂的飞速发展，传统佐剂已经越来越无法满足疫苗的需求。大量的临床免疫试验认为氢氧化铝胶体佐剂对体液免疫反应具有很强的激发作用，而对细胞免疫反应的作用较弱。如何提高铝佐剂介导的细胞免疫，既能有效地诱导机体产生体液免疫，又能有效地诱导细胞免疫则是目前免疫佐剂研究的重点与难点。
目前还没有人使用氯化钠与氢氧化铝一起作为复合免疫佐剂，而本领域目前需要提供新的、性能良好的免疫佐剂，为疫苗的制备提供新的有效选择。
发明内容
本发明所要解决的技术问题是提供一种新的、性能良好的免疫佐剂，为本领域提供一种新的有效选择。
解决该技术问题的技术方案是提供一种复合免疫佐剂。该复合免疫佐剂主要含有氢氧化铝凝胶和高于生理水平的氯化钠。
其中，氯化钠在免疫佐剂中的用量为高于生理水平是指在最终佐剂-抗原复合物溶剂中氯化钠的质量分数高于0.9％。该最终质量分数指免疫佐剂与抗原的复合物在注射使用前的浓度。
其中，上述复合免疫佐剂中所述氯化钠的最终质量分数为1.2％～7.2％。进一步的，上述复合免疫佐剂中所述氯化钠的最终质量分数为2.7％～4.5％。优选的，上述复合免疫佐剂中所述氯化钠的最终质量分数为3.6％。
其中，上述的质量分数是指在每100毫升佐剂-抗原复合物溶剂中氯化钠的质量(克)。
其中，上述复合免疫佐剂中所述氢氧化铝凝胶的颗粒粒径范围为1μm～10μm。优选的，所述氢氧化铝凝胶的颗粒粒径范围为2μm～6μm。粒径3μm左右为佳。
本发明另外还提供了上述复合免疫佐剂添加抗原形成的免疫佐剂-抗原复合物。免疫佐剂-抗原复合物在本领域也可称为疫苗。
其中，上述免疫佐剂-抗原复合物中所述抗原(antigen，Ag)在本领域中指为任何可诱发免疫反应的物质。
进一步的，所述抗原为为肿瘤抗原，病毒抗原或者细菌抗原中的一种或者数种。
其中，上述的肿瘤抗原可选自OVA肿瘤模式抗原(OVA肿瘤模式抗原是国际上作为一种模式化的或标准化的肿瘤特异抗原，常用作标准的肿瘤特异性抗原来测试一种佐剂的诱导抗肿瘤的免疫反应能力，如果该佐剂诱导出抗肿瘤免疫反应，说明该佐剂对抗肿瘤免疫反应具有代表性)、NY-ESO-1；人黑色素瘤相关抗原gP100；黑色素mage-1或癌胚抗原等常用的肿瘤抗原中的一种或者数种。
其中，所述的病毒抗原选自乙型肝炎病毒抗原、甲型肝炎病毒抗原、丙型肝炎病毒抗原、脊髓灰质炎病毒抗原、狂犬病毒抗原、黄热病病毒抗原、艾滋病毒抗原或麻疹、流行性腮腺炎、风疹、水痘、轮状病毒、日本脑炎、乳头瘤病毒、流行性出血热病毒、鼠疫病毒抗原等常见病毒类抗原中的一种或者数种。
其中，所述的细菌抗原选自金色葡萄球菌抗原、绿脓杆菌抗原、百日咳菌抗原、白喉菌抗原、流感嗜血菌抗原、脑膜炎奈瑟球菌抗原、破伤风菌抗原、溶血链球菌抗原、非溶血链球菌抗原、肺炎球菌抗原、结核杆菌、炭疽桿菌、霍乱弧菌、钩端螺旋体或幽门螺杆菌等常用细菌抗原中的至少一种。
本发明另外还提供了制备上述的免疫佐剂/抗原复合物的方法。该方法包括以下步骤：
a、取所需抗原用水稀释或溶解；
b、加入所需的氯化钠，混匀；
c、加入所需量的氢氧化铝凝胶，混匀，即得。
同时，上述的免疫佐剂/抗原复合物也可以采用以下步骤制备：
a、向氢氧化铝凝胶佐剂中加入适当氯化钠配制成高渗氢氧化铝凝胶复合佐剂储备液；
b、取所需抗原用水稀释或溶解；
c、加入所需量的高渗氢氧化铝凝胶复合佐剂，混匀，即得。
进一步的，所述抗原为肿瘤抗原，病毒抗原或者细菌抗原中的一种或者数种。
其中，上述的肿瘤抗原可选自OVA肿瘤模式抗原、NY-ESO-1；人黑色素瘤相关抗原gP100；黑色素mage-1或癌胚抗原等常用的肿瘤抗原中的一种或者数种。
其中，上述的病毒抗原选自乙型肝炎病毒抗原、甲型肝炎病毒抗原、丙型肝炎病毒抗原、脊髓灰质炎病毒抗原、狂犬病毒抗原、黄热病病毒抗原、艾滋病毒抗原或麻疹、流行性腮腺炎、风疹、水痘、轮状病毒、日本脑炎、乳头瘤病毒、流行性出血热病毒、鼠疫病毒抗原等常见病毒类抗原中的一种或者数种。
其中，上述的细菌抗原选自金色葡萄球菌抗原、绿脓杆菌抗原、百日咳菌抗原、白喉菌抗原、流感嗜血菌抗原、脑膜炎奈瑟球菌抗原、破伤风菌抗原、溶血链球菌抗原、非溶血链球菌抗原、肺炎球菌抗原、结核杆菌、炭疽桿菌、霍乱弧菌、钩端螺旋体或幽门螺杆菌等常用细菌抗原中的至少一种。
上述技术方案中的质量分数是指在每100毫升佐剂-抗原复合物溶液中氯化钠的质量(克)。
可以理解的是，本发明同时提供了上述免疫佐剂-抗原复合物制备的疫苗。所述疫苗可为预防和/或治疗性疫苗。比如当抗原为HBsAg等肝炎抗原时，可以制备成为肝炎疫苗；当抗原为百日咳、白喉类毒素或者金色葡萄球菌、绿脓杆菌等细菌抗原时，可以制备成为细菌疫苗；比如当抗原为OVA肿瘤模式抗原或者肿瘤特异性抗原时，可以制备预防和/或治疗性肿瘤疫苗。本发明所述疫苗可以通过皮下、腹腔或肌肉等方式注射给药，对个体进行免疫。当然也可以采用本领域的其他可用方式进行免疫，或者是多种方式的组合。并且本发明所述疫苗可以有不同的免疫时间间隔。可以给药一次，也可以多 次。具体实施过程中可以根据实际情况改变或者调整免疫次数和免疫时间点。
为了构建好的可用于免疫佐剂的氢氧化铝-氯化钠复合佐剂，本发明考察了多种氢氧化铝-氯化钠配方。本发明对不同质量分数的氯化钠进行筛选，发现使用的氯化钠与氢氧化铝的复合物作为佐剂制成的疫苗溶液中氯化钠的质量分数为3.6％时效果最好。在肝炎疫苗模型中，体内产生了针对HBsAg的高滴度的抗体和IFN-γ；在细菌性疫苗中，产生了针对百日咳、白喉类毒素的高滴度的抗体和IFN-γ；产生了针对金色葡萄球菌、绿脓杆菌的高滴度的抗体和攻毒保护作用；在肿瘤疫苗模型中，分别通过预防性免疫和治疗性免疫实验证明，体内产生了针对OVA的抗体和激活了特异性CD8杀伤性T细胞，增加了IFN-γ的分泌，能有效抑制肿瘤生长。
同时，本发明提供了氯化钠在制备免疫佐剂中的用途。
其中，上述用途中所述的免疫佐剂里还有氢氧化铝凝胶。
其中，上述用途中所述氯化钠在免疫佐剂中的用量为高于生理水平。
其中，上述用途中所述氯化钠在免疫佐剂中的用量为使其制备得到的免疫佐剂与抗原的复合物中的氯化钠质量分数为1.2％～7.2％。该浓度为免疫佐剂与抗原的复合物在注射使用前的浓度。
同时，本发明也提供了氯化钠在制备免疫佐剂-抗原复合物中的用途。
其中，上述用途中免疫佐剂-抗原复合物中还含有氢氧化铝凝胶作为佐剂。
其中，上述用途中氯化钠的含量为质量分数1.2％～7.2％。该浓度为免疫佐剂与抗原的复合物在注射使用前的浓度。优选的，氯化钠的含量为质量分数达到2.7％～4.5％。最优的为3.6％。
其中，上述用途中抗原与氢氧化铝的重量配比为1:1～100。
其中，上述用途中抗原与复合免疫佐剂中氢氧化铝的重量配比为1:5～50。
其中，上述用途中所述抗原为肿瘤抗原，病毒抗原或者细菌抗原中的一种或者数种。本发明还提供了疫苗的配制方法。该方法的步骤为疫苗使用前，加入氯化钠和适量注射用水，并使氯化钠的质量分数达到1.2％～7.2％。该方法可根据实际情况按以下方式分别具体实施：
在疫苗使用前，先向抗原中加入适量注射用水和氯化钠，再加入氢氧化铝凝胶佐剂，使配得的疫苗中氯化钠的质量分数达到1.2％～7.2％；
或者为直接用注射用水溶解抗原后，加入高渗氢氧化铝凝胶储备液，并使配得的疫苗中氯化钠的质量分数达到1.2％～7.2％；
或者为若带配制的为已含有抗原和氢氧化铝凝胶的疫苗，在配制时直接加入氯化钠作为佐剂，使配得的疫苗在注射前含有的氯化钠的质量分数达到1.2％～7.2％。
优选的氯化钠在待注射的疫苗体系中的质量分数达到2.7％～4.5％。最优的为3.6％。
其中，上述方法中，所述疫苗中抗原与氢氧化铝的重量配比为1︰1～100。优选的，配得的疫苗中抗原与氢氧化铝的重量配比为1︰5～50。
需要说明的是，本发明中所述的抗原与氢氧化铝的重量配比是指抗原与氢氧化铝凝胶中的氢氧化铝纯物质的比例。
显然，上述技术方案中，所述的氯化钠为满足疫苗制备要求纯度的氯化钠。一般来说，要求化学纯或者化学纯以上纯度的氯化钠。
本发明提供了氯化钠在制备疫苗佐剂以及制备疫苗中的用途。此外还提供了新型的疫苗佐剂，氢氧化铝凝胶-氯化钠复合免疫佐剂，可适用于细菌、病毒等传染性疾病疫苗和肿瘤疫苗的研发。其作用机制初步分析可能为高于生理水平的高渗氯化钠能激活MΦ、NK、DC等免疫细胞活性，激活MAPK信号通路，促进DC的成熟与抗原摄取或者交叉递呈。而氢氧化铝凝胶中铝盐与抗原结合形成抗原贮存库，使抗原得以缓慢稳定地释放，铝盐佐剂也能诱导、激发体液免疫。故两者配合使用则既可以激发体液免疫，又能够激发细胞免疫，达到更好的效果。
本发明的有益效果为：本发明创造性地开发出了氯化钠在制备疫苗佐剂以及制备疫苗中的用途，并进一步开发出了氢氧化铝-氯化钠复合免疫佐剂。具有免疫活性强、临床安全性高等优点，和单独的氢氧化铝相比引起了特异性的细胞免疫反应，是一种优秀的针对各种抗原的氢氧化铝-氯化钠复合免疫佐剂。试验表明，使用本发明的氢氧化铝-氯化钠作为佐剂制备疫苗，可具有更高的免疫效果以及抗肿瘤效果，为各种预防和/或治疗性疫苗的开发和应用提供了新的选择。
附图说明
图1为氢氧化铝凝胶-氯化钠免疫佐剂可以增强HBsAg模型中各组小鼠针对HBsAg的血清抗体滴度的实验结果。
图2为氢氧化铝凝胶-氯化钠免疫佐剂可以增强HBsAg模型中各组小鼠针对HBsAg的特异性细胞免疫反应的实验结果。
图3为氢氧化铝凝胶-氯化钠免疫佐剂可以增强OVA模型中各组小鼠针对OVA的血清 抗体滴度的实验结果。
图4为氢氧化铝凝胶-氯化钠免疫佐剂可以增强HBsAg模型中各组小鼠针对HBsAg的特异性细胞免疫反应的实验结果。
图5为氢氧化铝凝胶-氯化钠免疫佐剂在预防性与治疗性肿瘤模型中可以增强疫苗的抗肿瘤效果。
图6为氢氧化铝凝胶-氯化钠免疫佐剂的抗肿瘤效果是由于诱导了特异性的杀伤性CD8+T细胞(CTL反应)的实验结果。
图7为高渗氯化钠在体外可以促进DC细胞的成熟的实验结果。
图8为高渗氯化钠在体外可以促进DC细胞的抗原吞噬的实验结果。
图9为高渗氯化钠在体外可以促进DC细胞炎性细胞因子的分泌的实验结果。
图10为高渗氯化钠在体外可以促进DC细胞的交叉抗原递呈的实验结果。
图11为氢氧化铝凝胶-氯化钠免疫佐剂可以增加记忆性T细胞和改善肿瘤微环境的实验结果。
图12为氢氧化铝凝胶-氯化钠免疫佐剂可以增强金黄色葡萄球菌模型中各组小鼠的血清抗体滴度的实验结果。
图13为氢氧化铝凝胶-氯化钠免疫佐剂可以增强绿脓杆菌模型中各组小鼠的血清抗体滴度的实验结果。
具体实施方式
下面结合附图对本发明作进一步的描述说明。以下实施中将进一步说明该疫苗的制备和使用方法。本发明包括但不限于以下实施中所列举的具体方法步骤。
本发明创造性地发现高于生理水平的氯化钠能够用作免疫佐剂，并能用于制备复合免疫佐剂。使用氢氧化铝凝胶-氯化钠组合作为复合佐剂，既能有效地诱导机体产生体液免疫，又能有效地诱导细胞免疫。
本发明提供的复合佐剂采用了两种成分：其中的氢氧化铝凝胶佐剂，是一种常用的无机盐佐剂，具有良好的蛋白吸附作用，能将可溶性抗原吸附于铝胶分子表面；也可以浓缩抗原，减少注射剂量。氢氧化铝凝胶成本低廉、使用方便、无毒，是应用最广的一种佐剂，也是至今唯一被美国FDA批准可用于人类疫苗的佐剂。而氯化钠在生活，工业及医学上广泛应用，对人体也很安全，成本也极低。
经过进一步的筛选试验，本发明复合佐剂中所述氯化钠用量为使氯化钠的最后质量 分数大于生理浓度就能取得免疫效果的提高。如在1.2％～7.2％免疫效果的提高较为明显。优选的，2.7％～4.5％能有更好的免疫佐剂的效果。本发明实验中最好的效果是3.6％。本发明中氯化钠的质量分数是指在每100毫升抗原-佐剂复合物溶剂中氯化钠的质量(克)。显然本领域技术人员可以根据本发明公开的内容知晓，可按上述方案中的标准制得疫苗的过程中，还可以对疫苗采用冻干、蒸发或稀释等方式处理改变体系中氯化钠的浓度，但是只要在是在正式使用前，需调整体系中的氯化钠的浓度至上述范围之内的技术，也显然应在本发明的保护范围之内。
本领域技术人员从上述记载可以得知，本发明上述氢氧化铝凝胶-氯化钠新型佐剂是一种适用范围广，使用安全的免疫佐剂，可以添加本领域各种已知的抗原得到免疫佐剂-抗原复合物，进而作为疫苗使用。本发明所述的中所述抗原(antigen，Ag)在本领域中指为任何可诱发免疫反应的物质。
进一步的，所述抗原为肿瘤抗原，病毒抗原或者细菌抗原中的一种或者数种。
其中，肿瘤抗原是指包括肿瘤特异抗原(tumor-specific antigen，TSA)或肿瘤相关抗原(tumor-associated antigen,TAA)的蛋白或糖脂成分等本领域常见的肿瘤抗原形式。上述的肿瘤抗原可选自NY-ESO-1；人黑色素瘤相关抗原gP100；黑色素mage-1或癌胚抗原等常用的肿瘤抗原中的一种或者数种。
其中，上述的病毒抗原可以是选自乙型肝炎病毒抗原、甲型肝炎病毒抗原、丙型肝炎病毒抗原、脊髓灰质炎病毒抗原、狂犬病毒抗原、黄热病病毒抗原、艾滋病毒抗原或麻疹、流行性腮腺炎、风疹、水痘、轮状病毒、日本脑炎、乳头瘤病毒、流行性出血热病毒、鼠疫病毒抗原等常见病毒类抗原中的一种或者数种。而上述病毒抗原的采用的形式可以是减毒或者灭活病毒、病毒亚单位疫苗、合成肽疫苗、核酸疫苗等本领域常见的病毒抗原形式。
其中，上述细菌抗原选自金色葡萄球菌抗原、绿脓杆菌抗原、百日咳菌抗原、白喉菌抗原、流感嗜血菌抗原、脑膜炎奈瑟球菌抗原、破伤风菌抗原、溶血链球菌抗原、非溶血链球菌抗原或肺炎球菌抗原或结核杆菌、炭疽桿菌、霍乱弧菌、钩端螺旋体、幽门螺杆菌等常用细菌抗原中的至少一种。细菌抗原的形式可以是各种细菌的死菌苗、弱毒(减毒)苗、强毒苗，也可以是各种细菌中致病成分类毒素，或各种细菌多糖或以细菌蛋白为载体的细菌多糖类结合抗原等本领域常见的细菌抗原形式。
本发明在氢氧化铝型乙型肝炎疫苗或者百日咳、白喉类疫苗的基础上添加氯化钠佐剂，获得了更高的抗体滴度和IFN-γ的分泌增加；在氢氧化铝型金黄色葡萄球菌或者绿 脓杆菌疫苗的基础上添加氯化钠佐剂，获得了更高的抗体滴度和对细菌的攻毒保护作用；还采用氢氧化铝凝胶-氯化钠新型佐剂制备了一种OVA肿瘤模式抗原的新型疫苗，并证实了这一新型佐剂疫苗抗肿瘤的有效性。
氢氧化铝一般使用为粒径范围为1μm～10μm的氢氧化铝颗粒。优选为粒径2μm～6μm的氢氧化铝颗粒。当然，可能在与不同的抗原配合时，氢氧化铝中颗粒的粒径的优选值会有一定合理的变化。优选为粒径3μm左右的氢氧化铝颗粒。
而经过筛选，上述抗原与复合免疫佐剂中氢氧化铝的重量配比为1︰1～100，优选1︰5～50。
显然，本领域技术人员在使用不同抗原制备疫苗时，可以在上述范围内合理调整氯化钠的质量分数。也可以根据需要合理调整复合免疫佐剂中抗原用量及氢氧化铝凝胶和抗原的重量配比。
而制备本发明免疫佐剂-抗原复合物使用以下两种方法进行均可：
方法1：
a、取所需抗原用水稀释或溶解；
b、加入所需的氯化钠，混匀；
c、加入所需量的氢氧化铝凝胶，混匀，即得。
方法2：
a、向氢氧化铝凝胶佐剂中加入适当氯化钠配制成高渗氢氧化铝凝胶复合佐剂储备液；
b、取所需抗原用水稀释或溶解；
c、加入所需量的高渗氢氧化铝凝胶复合佐剂，混匀，即得。
比如，本发明的实施例中，当使用HBsAg或者百日咳、白喉类毒素作为抗原时，免疫佐剂/抗原复合物中HBsAg或者百日咳、白喉类毒素用量为1μg，抗原和氢氧化铝凝胶的重量配比可为1︰5～1︰100，最优选为1︰25。
使用OVA时，免疫佐剂/抗原复合物中OVA用量为5μg，抗原和氢氧化铝凝胶的重量配比可为1︰5～1︰50，最优选为1∶25。
在上述技术的基础上，本发明还发展出一种新的疫苗配制方法。该方法的步骤为在 疫苗使用前加入氯化钠和适量注射用水，并使氯化钠的质量分数达到1.2％～7.2％。
具体的，该方法可以以下步骤具体进行：先向抗原中加入适量注射用水和氯化钠，再加入氢氧化铝凝胶佐剂，使配得的疫苗中氯化钠的质量分数达到1.2％～7.2％；
或者为直接用注射用水溶解抗原后，加入高渗氢氧化铝凝胶储备液，并使配得的疫苗中氯化钠的质量分数达到1.2％～7.2％；
而当待配制的为已含有抗原和氢氧化铝凝胶的疫苗时，该方法的步骤为在疫苗使用前，加入氯化钠和适量注射用水配制成待注射的疫苗体系，并使氯化钠在待注射的疫苗体系中的质量分数达到1.2％～7.2％。
上述方法中氯化钠在待注射的疫苗体系中的质量分数达到2.7％～4.5％为优选的方案。最优的为3.6％左右。
其中，所述疫苗含有抗原和氢氧化铝凝胶。本领域技术人员有能力根据选择的抗原的不同，以及疫苗的具体使用方式的区别，适当的调整抗原和氢氧化铝之间的相对用量。一般地，抗原与氢氧化铝的重量配比为1︰1～100。本发明中的氢氧化铝凝胶可以是目前市售的氢氧化铝免疫佐剂，也可以是目前临床根据《中国药典》配制的氢氧化铝胶体佐剂，也可以是根据符合美国FDA要求或符合其他国家相关要求配置的可用于疫苗制备的氢氧化铝胶体。
实施例一：复合佐剂的筛选及制备
实验材料及试剂：氢氧化铝佐剂(Alhydrogel，氢氧化铝凝胶)购自美国InvivoGen(白色悬浊液，10mg/mL，粒径3μm)。
乙型肝炎表面抗原(HBsAg)购自美国ARP公司(American Rearch Products)，氯化钠(NaCl)与卵白蛋白(OVA)购自美国Sigma公司。百日咳(P7208)和白喉类毒素(D0564)购买自美国Sigma公司。金黄色葡萄球菌(S.a)或绿脓杆菌株(P.a)购买自ATCC公司(33591和27853).
制备上述的免疫佐剂-抗原复合物的方法可采用以下两种方法：
方法1：a、取所需抗原用水稀释或溶解；b、加入所需的氯化钠，混匀；c、加入所需量的氢氧化铝凝胶，混匀，即得。
方法2：a、向氢氧化铝凝胶佐剂中加入适当氯化钠配制成高渗氢氧化铝凝胶复合佐剂储备液；b、取所需抗原用水稀释或溶解；c、加入所需量的高渗氢氧化铝凝胶复合佐剂，混匀，即得。
经过进一步的筛选试验，所述氯化钠的最后质量分数为1.2％～7.2％，优选3.6％能 有最好的免疫佐剂的效果。本发明中氯化钠的质量分数是指在每100毫升抗原佐剂复合物溶剂中氯化钠的质量(克)。
而经过筛选，上述抗原与复合免疫佐剂中氢氧化铝的重量配比为1:1～100。优选的重量配比为1:5～50。
当使用HBsAg或百日咳和白喉毒素作为抗原时，免疫佐剂/抗原复合物中HBsAg或百日咳、白喉类毒素用量为1μg时，抗原和氢氧化铝凝胶的重量配比可为1︰5～1︰100。最优选为1︰25。
当使用OVA作为模式肿瘤特异抗原时，免疫佐剂/抗原复合物中OVA用量为5μg，抗原和氢氧化铝凝胶的重量配比可为1︰5～1︰50。最优选的为1︰25。
实施例二：本发明使用氯化钠作为乙肝疫苗佐剂的动物免疫试验
实验材料及试剂：氢氧化铝佐剂，氯化钠，HBsAg同实施例一；BALB/c小鼠购自北京维通利华公司；乙型肝炎病毒表面抗体检测试剂盒(万泰生物药业)；IFN-γ的ELISA试剂盒(美国eBioscience公司)；淋巴细胞分离液(中国达科为试剂公司)；70μm尼龙网过滤器(美国BD公司)；圆底24孔板(NUNK)。
实验动物按如下分组：1、Control；2、HbsAg；3、HbsAg/Al(OH)3；4、HbsAg/Al(OH)3/1.8％NaCl；5、HbsAg/Al(OH)3/3.6％NaCl；6、HbsAg/Al(OH)3/7.2％NaCl；所有分组中HbsAg为1μg，Al(OH)3为25μg，HbsAg和Al(OH)3的重量配比1:25，其NaCl浓度则按各组分别为配制至0、1.8％、3.6％及7.2％。每组5只。于第0,2,3周肌肉免疫，第4周收集血清及脾脏淋巴细胞用于如下实验。
(一)抗体滴度的检测
通过ELISA试剂盒(万泰生物药业)检测各组小鼠血清中的总IgG和IgG分型抗体滴度。各组血清抗体稀释后的吸光度检测结果见图1。单独HBsAg组几乎不能产生抗体。单独使用Al(OH)3组和Al(OH)3+氯化钠复合佐剂组均能有效刺激抗体的产生。而Al(OH)3+氯化钠复合佐剂相比于单独使用Al(OH)3组能产生更高的IgG和IgG1抗体滴度，并且在氯化钠质量分数为3.6％时达到最高值。
(二)细胞因子IFN-γ的测定
IFN-γ的测定采用ELISA和流式细胞术进行。具体如下：小鼠末次免疫后一周处死，取出脾脏。得到脾脏淋巴细胞后在体外用HBsAg刺激，置于CO2孵箱培养72小时，用ELISA检测试剂盒测定培养上清IFN-γ的浓度。
实验结果见图2。我们发现在HBsAg模型中，高盐/铝佐剂组，CD8+INF-γ分泌显著增加，细胞免疫明显增强，流式与ELISA结果相符合。
实施例三：本发明使用氯化钠作为OVA肿瘤特异抗原佐剂的动物免疫试验
实验材料及试剂：氢氧化铝佐剂，氯化钠，OVA同实施例一；小鼠，IFN-γ的ELISA试剂盒，淋巴细胞分离液，70μm尼龙网过滤器，圆底24，96孔板同实施例二；小鼠淋巴癌细胞EG.7(美国ATCC)；Na2 51CrO4(美国PerkinElmer公司)。
(一)本发明新型佐剂对小鼠移植瘤生长的预防及治疗性实验
预防性实验：C57小鼠随机分为4组：1、Control 2、OVA 3、OVA/Al(OH)3 4、OVA/Al(OH)3/3.6％NaCl；其中所有分组中以OVA作为肿瘤特异抗原为5μg，Al(OH)3为125μg，OVA和Al(OH)3的重量配比1:25，氯化钠浓度按分组要求配制。每组10只,分别在第0，2，3周在各组小鼠的左背侧皮下注射疫苗。在最后一次免疫后第7天，在小鼠的右侧皮下接种小鼠淋巴瘤细胞3×106/只，建立小鼠移植瘤模型，每3天测量一次小鼠的肿瘤体积。
治疗性实验：小鼠按如上随机分为4组，在小鼠的右侧皮下接种小鼠淋巴瘤细胞3×106/只，待肿瘤长至可触及时(约3mm，接种后3～5天)，小鼠开始按上述预防性实验的4组分组剂量设计进行皮下免疫治疗，每周1次，连续治疗3次，每3天测量一次小鼠的肿瘤体积。
预防性和治疗性实验结果分别见图5，与对照组(Control，OVA)和单独Al(OH)3佐剂疫苗组相比，Al(OH)3+氯化钠复合佐剂组的肿瘤生长明显得到抑制。
(二)抗体滴度的测定
实验方法及分组同实施例二，如下1、Control；2、OVA；3、OVA/Al(OH)3；4、OVA/Al(OH)3/1.8％NaCl；5、OVA/Al(OH)3/3.6％NaCl；6、OVA/Al(OH)3/7.2％NaCl。其中所有分组中OVA为5μg，Al(OH)3为125μg，OVA和Al(OH)3的重量配比1:25，氯化钠浓度按分组要求配制。每组5只，小鼠最后一次免疫后第7天收集血清，通过自包被ELISA检测各组小鼠血清中的抗体滴度。各组抗体吸光度的检测结果见图3。单独OVA组几乎不能产生抗体。单独使用Al(OH)3组和Al(OH)3+氯化钠复合佐剂组均能有效刺激抗体的产生。而Al(OH)3+氯化钠复合佐剂相比于单独使用Al(OH)3组能产生更高的IgG和IgG1抗体滴度，并且在氯化钠质量分数为3.6％时达到最高值。
(三)细胞因子IFN-γ的测定
IFN-γ的测定采用自包被ELISA试剂盒(eBioscience公司)和流式细胞术进行。具体如下：小鼠末次免疫后一周处死，取出脾脏。得到脾脏淋巴细胞之后，再加入5μg不同的OVA肽段刺激，置于CO2孵箱培养72小时，用ELISA检测试剂盒测定培养上清IFN-γ的浓度。细胞用于INF-γ和特异性的OVA-Tetramer实验。
实验结果见图4。我们发现在OVA模型中，高盐/铝佐剂组，CD8+INF-γ分泌显著增加，流式与ELISA结果相符合。此外，图4c表示特异性的OVA-Tetramer阳性比例增高，说明Al(OH)3+氯化钠复合佐剂确实诱导了特异性的细胞免疫。
(四)本发明新型佐剂疫苗刺激肿瘤特异性的细胞毒性T淋巴细胞杀伤功能
细胞毒性T淋巴细胞杀伤功能检测采用51Cr释放试验检测。
小鼠免疫同预防性模型中所述，最后一次免疫后7天每组随机取3只小鼠。无菌条件下解剖小鼠，分离脾脏淋巴细胞，调整细胞浓度至1×107/ml,获得的淋巴细胞即为效应细胞。
收集体外培养的肿瘤细胞E.G7，细胞计数，用培养基将肿瘤细胞的浓度调整至1×107/ml，取200μl细胞，加入100μci Na2 51CrO4，37℃孵育2h进行标记，清洗3次后，细胞计数，将细胞浓度调整至0.5×105/ml，获得的51Cr标记的肿瘤细胞即为靶细胞。将靶细胞悬液加入圆底96孔板中，每孔100μl，设3个副孔，按照表1的不同比例加入效应细胞：
表1
Figure PCTCN2016102131-appb-000001
每孔加入100μl的51Cr标记的肿瘤细胞，自然释放组加入100μl培养基，最大释放组加入100μl 1％Triton X-100。96孔板水平砖头1500rpm/min离心30s，37℃，5％CO2 孵箱中培养，4～6小时后，96孔板水平砖头1500rpm/min离心5min，每孔吸取100μl上清，Backmen550B型γ计数仪检测cpm值CTL杀伤率计算公式为：杀伤率％＝〔(实验组cpm-自然释放组cpm)/(对照组cpm-自然释放组cpm)〕×100％。
实验结果显示见图6d,Al(OH)3+氯化钠复合佐剂组(OVA/Al/3.6％NaCl)能有效地诱导的针对肿瘤细胞的特异性CTL反应，并且作用明显优于对照组(Control,OVA)和单独Al(OH)3佐剂组(OVA/Al)。
(五)本发明新型佐剂疫苗免疫诱导的小鼠过继治疗实验和抗体阻断实验
小鼠右后方注射EG.7细胞3×106/只，建立小鼠移植瘤模型，随机分为4组，每组5只。细胞过继：在建立小鼠移植瘤模型前一天，后一天和后3天，经尾静脉注射预防性实验中所述获取的脾脏淋巴细胞，107细胞/只，共注射3次，每3天观察小鼠肿瘤体积的变化。
血清过继：在建立小鼠移植瘤模型前一天和之后的连续3周，经尾静脉注射预防性实验中所述获取的血清，250μl/只，每周2次，每3天观察小鼠肿瘤体积的变化。
抗体阻断：在小鼠免疫前一天和免疫期间，腹腔注射抗CD4，CD8，NK单克隆抗体或者对照非特异性的RAT抗体，每周2次，连续3周。免疫完成后，小鼠接种EG.7细胞后，每3天一次监测每组小鼠肿瘤生产曲线。
实验结果显示见图6a,b和c，血清过继治疗几乎没有抗肿瘤作用，而细胞过继治疗Al(OH)3+氯化钠复合佐剂组(OVA/Al/3.6％NaCl)具有显著的抗肿瘤作用。此外，阻断CD4+T细胞对抗肿瘤效果无影响，而阻断CD8+T几乎完全废除了复合免疫佐剂的抗肿瘤效果，表明高盐/铝复合佐剂的抗肿瘤作用主要归功于其诱导的CD8+杀伤性T淋巴细胞，而与CD4+T淋巴细胞无关。
(六)本发明新型佐剂疫苗免疫对记忆性T淋巴细胞和小鼠肿瘤微环境的改变
取预防性试验或者治疗性疫苗试验中小鼠的脾脏淋巴细胞或者肿瘤，制备得到单细胞混悬液后，用流式细胞术检测小鼠各种免疫细胞群的改变。
记忆性T淋巴细胞实验结果见图11a,Al(OH)3+氯化钠复合佐剂免疫可以显著增加中心及效应性CD4+CD8+T淋巴细胞。对于肿瘤微环境，结果见图11b和c，Al(OH)3+氯化钠复合佐剂不仅增加了肿瘤组织中杀伤性的CD8+T淋巴细胞的侵润，同时还减少了起免疫抑制作用的MDSC和M2型巨噬细胞的数量。
(七)本发明新型氯化钠佐剂在体外能够促进DC的各项功能
DC作为主要的抗原递呈细胞，在疫苗免疫反应中扮演者不可或缺的作用。因此我 们在体外研究了高盐对DC功能的影响。
实验分组见图7，C57BL/6小鼠原代骨髓DC通过经典的方法制备得到。在体外用GM-CSF等细胞因子分化刺激培养6天后，收集并继续在含不同质量分数氯化钠的培养基中培养48h，用流式细胞术检测细胞表明成熟分子CD80，CD86等的比例，同时ELISA检测上清炎性细胞因子的分泌。同时收集裂解部分细胞制备RNA，用RT-PCR检测相关炎性分子的mRNA水平。此外，将DC在含不同质量分数氯化钠和荧光标记的OVA模式肿瘤抗原或者葡聚糖的培养基中培养1h后，用荧光显微镜或者流式细胞术检测相关抗原吞噬。
由图7可知，高盐在体外刺激DC后，可以促进其成熟，增加其分子表面CD80，CD86，MHCII等分子的表达；由图8可知高盐还能促进DC对抗原的吞噬，由图9可知还能在mRNA和蛋白水平增加炎性细胞因子IL6和IL1b等的释放。
接下来我们继续探讨了高盐所诱导的细胞特异性免疫反应是否与DC的交叉抗原递呈有关。
DC/T淋巴细胞共培养实验：实验分组见图10，将体外用高盐和OVA抗原刺激后的DC细胞与CFSE标记的来源于OT-1转基因小鼠的CD8+T淋巴细胞共培养2-3天后，显微镜白光下观察CLUSTER的形成，以及用流式细胞术检测CFSE标记CD8+T淋巴细胞的增殖，此外收集培养上清用ELISA检测IL2的分泌。
免疫荧光共定位实验：实验分组同上。将体外用高盐和荧光标记OVA刺激后的DC细胞固定，打孔，染上蛋白酶体和溶酶体的细胞器荧光蛋白标志，在共聚焦显微镜下观察。
由图10a，b和c可知通过DC/T淋巴细胞共培养实验，发现高盐处理后的DC能将OVA抗原分子摄取递呈给特异性的CD8+T淋巴细胞，诱导其增殖并分泌IL2。由图10d可知免疫荧光共定位实验也表明在高盐处理的DC中，OVA更多的被蛋白酶体而非溶酶体降解。因此，高盐可显著提高DC的交叉抗原递呈。
实施例四：本发明使用氯化钠作为细菌疫苗佐剂的动物免疫试验
实验材料及试剂：氢氧化铝佐剂，氯化钠，金黄色葡萄球菌(S.a)或绿脓杆菌株(P.a)，百日咳和白喉毒素同实施例一；C57BL/6小鼠购自北京维通利华公司；IFN-γ的ELISA试剂盒(美国eBioscience公司)；淋巴细胞分离液(中国达科为试剂公司)；70μm尼龙网过滤器(美国BD公司)；圆底24孔板(NUNK)。
实验动物按如下分组免疫：1、Control；2、S.a或P.a；3、S.a或P.a/Al(OH)3；4、S.a或P.a/Al(OH)3/3.6％NaCl；具体免疫方案如下：扩大培养的菌液离心，洗涤，1％多聚甲醛固定灭活后，洗涤，PBS重悬，每组小鼠5只，于第0,2,3周皮下免疫3次，第一次菌液0.05OD(1×106CFU/200μL)、第二次菌液0.5OD(1×107CFU/200μL)、第三次菌液2.5OD(5×107CFU/200μL)；每次免疫后收集血清用于抗体检测。
抗体效价的检测：通过1×107CFU/100μL全菌抗原自包被96孔板，使用ELISA检测各组小鼠血清中针对不同细菌的特异IgG抗体滴度。实验结果如图12所示，可检测出对金葡菌特异性抗体；Al(OH)3佐剂有助于增加抗体水平；高盐Al(OH)3佐剂具有更好的效果。同样，可检测出对绿脓杆菌特异性抗体，实验结果如图13所示；Al(OH)3佐剂有助于增加抗体水平，高盐Al(OH)3佐剂具有更好的效果。百日咳或白喉毒素实验动物分组同金葡菌和绿脓杆菌，所有分组中百日咳或白喉毒素为1μg，Al(OH)3为25μg，百日咳或白喉毒素和Al(OH)3的重量配比1:25，氯化钠按分组要求配制相应浓度。每组5只。于第0,2,3周肌肉免疫，每周收集血清用于血清抗体ELISA实验。实验结果与金葡菌和绿脓杆菌类似，氢氧化铝凝胶-氯化钠免疫佐剂可以诱导白喉毒素的特异抗体产生，增加约48％抗体的产生。同样氢氧化铝凝胶-氯化钠免疫佐剂可以增加诱导百日咳毒素约45％抗体产生。

Claims (35)

  1. 复合免疫佐剂，其特征在于：含有氢氧化铝凝胶和氯化钠。
  2. 根据权利要求1所述的复合免疫佐剂，其特征在于：所述氯化钠在复合免疫佐剂中含量高于生理水平。
  3. 根据权利要求1～2任一项所述的复合免疫佐剂，其特征在于：所述氯化钠的最终质量分数为1.2％～7.2％。
  4. 根据权利要求1～3任一项所述的复合免疫佐剂，其特征在于：所述氯化钠的最终质量分数为2.7％～4.5％。
  5. 根据权利要求1～4任一项所述的复合免疫佐剂，其特征在于：所述氢氧化铝凝胶的粒径范围为1μm～10μm。
  6. 免疫佐剂-抗原复合物，其特征在于：含有抗原和权利要求1～5任一项所述的复合免疫佐剂。
  7. 根据权利要求6所述的免疫佐剂-抗原复合物，其特征在于：所述抗原与复合免疫佐剂中氢氧化铝的重量配比为1︰1～100。
  8. 根据权利要求7所述免疫佐剂-抗原复合物，其特征在于：所述抗原与氢氧化铝的重量配比为1︰5～50。
  9. 根据权利要求6～8任一项所述的免疫佐剂-抗原复合物，其特征在于：所述抗原为肿瘤抗原,病毒抗原或细菌抗原中的至少一种。
  10. 根据权利要求9所述的免疫佐剂-抗原复合物，其特征在于：所述的肿瘤抗原选自OVA肿瘤模式抗原、NY-ESO-1、人黑色素瘤相关抗原gP100、黑色素瘤抗原mage-1或癌胚抗原中的至少一种。
  11. 根据权利要求9所述的免疫佐剂-抗原复合物，其特征在于：上述的病毒抗原可以是选自乙型肝炎病毒抗原、甲型肝炎病毒抗原、丙型肝炎病毒抗原、脊髓灰质炎病毒抗原、狂犬病毒抗原、黄热病病毒抗原、艾滋病毒抗原或麻疹、流行性腮腺炎、风疹、水痘、轮状病毒、日本脑炎、乳头瘤病毒、流行性出血热病毒、鼠疫病毒抗原等常见病毒类抗原中的至少一种。
  12. 根据权利要求9所述的免疫佐剂-抗原复合物，其特征在于：所述的细菌抗原选自金色葡萄球菌抗原、绿脓杆菌抗原、百日咳菌抗原、白喉菌抗原、流感嗜血菌抗原、脑膜炎奈瑟球菌抗原、破伤风菌抗原、溶血链球菌抗原、非溶血链球菌抗原、肺炎球菌抗原结核杆菌、炭疽桿菌、霍乱弧菌、钩端螺旋体或幽门螺杆 菌等常用细菌抗原中的至少一种。
  13. 制备权利要求6～12任一项所述的免疫佐剂-抗原复合物的方法，其特征在于包括以下步骤：
    a、取所需抗原用水稀释或溶解；
    b、加入所需的氯化钠，混匀；
    c、加入所需量的氢氧化铝凝胶，混匀，即得。
  14. 制备权利要求6～12任一项所述的免疫佐剂-抗原复合物的方法，其特征在于包括以下步骤：
    a、向氢氧化铝凝胶佐剂中加入适当氯化钠配制成高渗氢氧化铝凝胶复合佐剂储备液；
    b、取所需抗原用水稀释或溶解；
    c、加入所需量的高渗氢氧化铝凝胶复合佐剂，混匀，即得。
  15. 权利要求1～5任一项所述的复合免疫佐剂在制备疫苗中的用途。
  16. 氯化钠在制备免疫佐剂中的用途。
  17. 根据权利要求16所述的用途，其特征在于：所述免疫佐剂中还有氢氧化铝凝胶。
  18. 根据权利要求17所述的用途，其特征在于：所述氯化钠在免疫佐剂中的用量为高于生理水平。
  19. 根据权利要求18所述的用途，其特征在于：所述氯化钠在免疫佐剂中的用量为使其制备得到的免疫佐剂与抗原的复合物中的氯化钠质量分数为1.2％～7.2％。
  20. 根据权利要求19所述的用途，其特征在于：所述氯化钠在免疫佐剂中的用量为使其制备得到的免疫佐剂与抗原的复合物中的氯化钠质量分数为2.7％～4.5％。
  21. 氯化钠在制备免疫佐剂-抗原复合物中的用途。
  22. 根据权利要求21所述的用途，其特征在于：所述免疫佐剂-抗原复合物中还含有氢氧化铝凝胶作为佐剂。
  23. 根据权利要求22所述的用途，其特征在于：所述氯化钠的含量为质量分数为1.2％～7.2％。
  24. 根据权利要求23所述的用途，其特征在于：所述氯化钠在免疫佐剂中的用量为使其制备得到的免疫佐剂与抗原的复合物中的氯化钠质量分数为2.7％～4.5％。
  25. 根据权利要求22所述的用途，其特征在于：所述抗原与氢氧化铝的重量配比为1︰1～100。
  26. 根据权利要求25所述的用途，其特征在于：所述抗原与复合免疫佐剂中氢氧化铝的重量配比为1︰5～50。
  27. 根据权利要求21～26任一项所述的用途，其特征在于：所述抗原为肿瘤抗原,病毒抗原或细菌抗原中的至少一种。
  28. 根据权利要求27所述的用途，其特征在于，所述的肿瘤抗原选自OVA肿瘤模式抗原、NY-ESO-1、人黑色素瘤相关抗原gP100、黑色素瘤抗原mage-1或癌胚抗原中的至少一种。
  29. 根据权利要求27所述所述的用途，其特征在于，所述的病毒抗原选自乙型肝炎病毒抗原、甲型肝炎病毒抗原、丙型肝炎病毒抗原、脊髓灰质炎病毒抗原、狂犬病毒抗原、黄热病病毒抗原、艾滋病毒抗原或麻疹、流行性腮腺炎、风疹、水痘、轮状病毒、日本脑炎、乳头瘤病毒、流行性出血热病毒、鼠疫病毒抗原等常见病毒类抗原中的至少一种。
  30. 根据权利要求27所述的用途，其特征在于，所述的细菌抗原选自金色葡萄球菌抗原、绿脓杆菌抗原、百日咳菌抗原、白喉菌抗原、流感嗜血菌抗原、脑膜炎奈瑟球菌抗原、破伤风菌抗原、溶血链球菌抗原、非溶血链球菌抗原、肺炎球菌抗原、结核杆菌、炭疽桿菌、霍乱弧菌、钩端螺旋体或幽门螺杆菌等常用细菌抗原中的至少一种。
  31. 疫苗的配制方法，其特征在于：在疫苗使用前，加入氯化钠和适量注射用水，并使氯化钠的质量分数达到1.2％～7.2％。
  32. 根据权利要求31所述的疫苗配制方法，其特征在于按以下步骤进行：
    在疫苗使用前，先向抗原中加入适量注射用水和氯化钠，再加入氢氧化铝凝胶佐剂，使配得的疫苗中氯化钠的质量分数达到1.2％～7.2％；
    或者直接用水溶解抗原后，加入高渗氢氧化铝凝胶储备液，并使配得的疫苗中氯化钠的质量分数达到1.2％～7.2％；
    或者若待配制的为已含有抗原和氢氧化铝凝胶的疫苗，在配制时则直接加入氯化钠作为佐剂，使配得的疫苗在注射前含有的氯化钠的质量分数达到1.2％～7.2％。
  33. 根据权利要求31所述的疫苗配制方法，其特征在于：配得的疫苗中所述氯化钠的最终质量分数为2.7％～4.5％。
  34. 根据权利要求32所述的疫苗配制方法，其特征在于：配得的疫苗中抗原与氢氧化铝的重量配比为1︰1～100。
  35. 根据权利要求34所述的疫苗配制方法，其特征在于：配得的疫苗中抗原与氢氧化铝的重量配比为1︰5～50。
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