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WO2023280303A1 - Avc-29作为疫苗佐剂的用途以及含有该佐剂的疫苗组合物 - Google Patents

Avc-29作为疫苗佐剂的用途以及含有该佐剂的疫苗组合物 Download PDF

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WO2023280303A1
WO2023280303A1 PCT/CN2022/104620 CN2022104620W WO2023280303A1 WO 2023280303 A1 WO2023280303 A1 WO 2023280303A1 CN 2022104620 W CN2022104620 W CN 2022104620W WO 2023280303 A1 WO2023280303 A1 WO 2023280303A1
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vaccine
antigen
avc
immune response
immunogenic
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李松
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海南大学
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    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P31/14—Antivirals for RNA 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/544—Mucosal route to the airways
    • 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
    • A61K2039/55511—Organic 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the application relates to the field of biomedicine, in particular to the use of the small molecule compound AVC-29 as a vaccine adjuvant or in the preparation of a vaccine adjuvant, an immunogenic or immunostimulatory composition containing AVC-29 and its preparation method and its use in the preparation of a vaccine use in .
  • Vaccination is the most economical and effective measure to prevent and control infectious diseases, and it is an effective means to deal with new and sudden infectious diseases (such as the new crown pneumonia epidemic). Relying on the rapid development of modern biotechnology and genetic engineering in recent years, great progress has been made in the development of vaccines.
  • Commonly used vaccines include inactivated vaccines, recombinant subunit vaccines, adenovirus vector vaccines, anti-idiotypic antibody vaccines, nucleic acid vaccines, and newly developed peptide vaccines in recent years.
  • inactivated vaccines, recombinant subunit vaccines and polypeptide vaccines all have defects such as weak immunogenicity of protein or polypeptide antigens and insufficient immune protection induced.
  • adjuvants to enhance the body's adaptive immune response to antigens, including increasing the level of antibodies and cellular immune responses, to induce effective immune protection.
  • adjuvants can also reshape the type of immune response to antigens in the body, making vaccines more effective against pathogens.
  • Aluminum salt adjuvant is the first vaccine adjuvant approved and commonly used in humans. Its mechanism of action is to form an antigen storage pool; produce particulate antigen to promote antigen presentation to immune cells; make antigen retention and slow release, thereby Attract active lymphocytes and activate the complement system.
  • the advantage of the adjuvant is that it is safe, but the disadvantage is that the stimulated immune response is relatively weak, especially the cellular immune response cannot be effectively induced.
  • Aluminum salt adjuvants are not very effective when developing against certain intracellular infectious pathogens such as Mycobacterium tuberculosis and herpes zoster virus. In addition, aluminum salt adjuvants may also lead to the occurrence of neurodegenerative diseases.
  • AVC-29 structure shown below
  • AVC-29 has a highly effective vaccine adjuvant effect.
  • AVC-29 has significant advantages in inducing antibody production and cellular immune response.
  • AVC-29 has good safety and can be applied to various types of vaccine preparations, for example, for recombinant protein vaccines against SARS-CoV-2 virus. Therefore, AVC-29 small molecule compound is a potential ideal vaccine adjuvant.
  • the present invention provides the use of AVC-29 or a pharmaceutically acceptable salt thereof as a vaccine adjuvant or in the preparation of a vaccine adjuvant and/or vaccine, wherein the AVC-29 structure is as follows :
  • AVC-29 stimulates an immune response in a subject, eg, elicits or enhances an immune response in a subject.
  • the immune response is a non-specific immune response.
  • the immune response is an antigen-specific immune response.
  • the immune response includes activation of B cells, activation of T cells, production of antibodies, and/or release of cytokines.
  • the present invention provides an immunogenic or immunostimulatory composition
  • AVC-29 or a pharmaceutically acceptable salt thereof comprising AVC-29 or a pharmaceutically acceptable salt thereof.
  • the immunogenic or immunostimulatory composition further comprises one or more antigens.
  • the antigen is a protein, recombinant protein, glycoprotein, peptide, polysaccharide, lipid, lipopolysaccharide, or nucleic acid (including DNA and mRNA) of a pathogen.
  • the antigen is derived from a cell (eg, a tumor cell), a bacterium, or a virus.
  • the antigen is a viral antigen of SARS-CoV-2, and the SARS-CoV-2 is a novel coronavirus named by the International Committee on Taxonomy of Viruses (ICTV).
  • the antigen is a receptor binding domain antigen of the SARS-CoV-2 virus.
  • the antigen has the amino acid sequence shown in SEQ ID NO:1.
  • the immunogenic or immunostimulatory composition is presented in unit dosage form.
  • the immunogenic or immunostimulatory composition contains 0.1-500 ⁇ g, preferably 1-100 ⁇ g, more preferably 5-50 ⁇ g of AVC-29 or a pharmaceutically acceptable salt thereof per unit dose.
  • the unit dosage is the reference amount of conventional single administration (injection) for clinical use.
  • the mass ratio of AVC-29 or a pharmaceutically acceptable salt thereof to the antigen in the immunogenic or immunostimulatory composition is (0.1-100):1, preferably (0.1-10):1 , more preferably (0.5-5): 1, for example (0.5-1): 1, (0.5-1.5): 1, (0.5-2): 1, (0.5-2.5): 1, (0.5-3): 1, (0.5-3.5): 1, (0.5-4): 1, (0.5-4.5): 1, (1-1.5): 1, (1-2): 1, (1-2.5): 1, (1-3):1, (1-3.5):1, (1-4):1, (1-4.5):1, or (1-5):1.
  • the immunogenic or immunostimulatory composition is in the form of injectable formulation, inhalable formulation or oral formulation, such as powder injection, suspension, aqueous injection, spray, aerosol, powder mist , paste tablet, sublingual tablet or film.
  • the immunogenic or immunostimulatory composition further contains one or more of buffers, isotonic agents, preservatives, stabilizers and solubilizers, such as sugars (lactose, sucrose), Amino acid (glycine), gelatin and protein (recombinant human albumin), etc.
  • buffers such as sugars (lactose, sucrose), Amino acid (glycine), gelatin and protein (recombinant human albumin), etc.
  • the present invention further provides a method for preparing the immunogenic or immunostimulatory composition, which comprises the following steps:
  • AVC-29 or a pharmaceutically acceptable salt thereof is mixed with one or more antigens described above and optionally other components, preferably in physiological saline, to obtain the immunogenicity or immune stimulation Composition; preferably, after the mixing, a drying step, such as freeze-drying, is also included.
  • the present invention also provides the use of the immunogenic or immunostimulatory composition as a vaccine or in the preparation of a vaccine.
  • the vaccine is an inactivated vaccine, a live attenuated vaccine or a gene recombinant vaccine.
  • the vaccine can be prophylactic (ie, protect the subject from the disease) or therapeutic (ie, help the subject fight the disease with which he has contracted it).
  • the vaccine is used to prevent and/or treat a disease associated with the antigen.
  • the disease is COVID-19 (Novel Coronavirus Pneumonia).
  • the immunogenic or immunostimulatory composition stimulates an immune response in a subject, eg, elicits or enhances an immune response in a subject.
  • the immune response is a non-specific immune response.
  • the immune response is an antigen-specific immune response.
  • the immune response includes activation of B cells, activation of T cells, production of antibodies, and/or release of cytokines.
  • the invention provides a method of stimulating (eg, eliciting or enhancing) an immune response in a subject, comprising administering to the subject an effective amount of the immunogenic or immunostimulatory composition.
  • the immune response is a non-specific immune response. In some embodiments, the immune response is an antigen-specific immune response. In some embodiments, the immune response includes activation of B cells, activation of T cells, production of antibodies, and/or release of cytokines.
  • the immunogenic or immunostimulatory composition is administered orally, intravenously, intradermally, transdermally, nasally, subcutaneously, or anally.
  • the subject is a mammal, such as a human or a non-human mammal (including but not limited to dogs, cows and horses), preferably a human.
  • vaccine is a composition administered to produce or artificially increase immunity to a particular antigen.
  • the terms “immunogenic composition”, “immunostimulatory composition” are compositions that are capable of generating an immune response in vivo when administered to an individual. Accordingly, it should be understood that the terms “immunogenic composition”, “immunostimulatory composition” and “vaccine” are synonymous terms.
  • the individual is preferably a mammal, more preferably a human, and of course other mammals, for example, the composition can be administered to a cow (cattle) (including a cow (cow)), a sheep, a goat or a horse. Induce immunity in, or pets, such as dogs or cats.
  • an immunogenic composition or immunostimulatory combination as described herein is a composition that contains an antigen and is capable of generating an immune response against such an antigen.
  • the immune response generated can be a cellular (T cell mediated) or humoral (B cell mediated, antibody producing) immune response. It can also induce cellular and humoral immune responses.
  • the cellular immune response can be CD8 T lymphocyte mediated response (i.e. cytotoxic response) or CD4 T lymphocyte mediated response (helper response). It can also combine cytotoxic and auxiliary cellular immune responses.
  • a helper response may involve Th1, Th2 or Th17 lymphocytes (such lymphocytes are capable of eliciting different cytokine responses).
  • the compositions described herein can significantly stimulate CD4 T cell responses, particularly CD4 T cell responses positive for IFN ⁇ , IL-2, and IL-5 cytokines.
  • vaccine adjuvant refers to a substance capable of modifying or enhancing the immune response to an antigen.
  • the immune response to the antigen in the presence of the adjuvant can be higher or different than that in the absence of the adjuvant (including when the response is modified, e.g., activated T cell subsets in the presence of the adjuvant versus those in the absence of the adjuvant different subsets of activation).
  • antigen is a molecule or combination of molecules that elicits an immune response in order for it to be recognized by an individual's immune system. Such antigens may be foreign to the body of the host seeking an immune response. In this case, the antigen may be a protein expressed by bacteria or viruses. Antigens can also be self-antigens, ie proteins expressed by host cells, such as tumor antigens.
  • Antigens can be produced from whole organisms (viruses or bacteria, fungi, protozoa, or even cancer cells), dead or not, cells (irradiated or not, genetically modified or not), or these Subfractions of organisms or cells such as cell extracts or cell lysates. Antigens may also consist of single molecules such as proteins, recombinant proteins, peptides, polysaccharides, lipids, glycolipids, glycopeptides or mixtures thereof. An antigen may also be one of the above-listed molecules modified by chemical modification or stabilization.
  • pathogens for which antigens may be used in immunogenic or immunostimulatory compositions include any pathogens of infectious diseases (viruses, bacteria, parasites, fungi).
  • preferred pathogens are selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A and B viruses, hepatitis C virus (HCV), Rous sarcoma virus (RSV), Ebola virus, giant Cytoviruses, herpes viruses, varicella-zoster virus, Epstein Barr virus (EBV), influenza viruses, coronaviruses (e.g., MERS, SARS, SARS-CoV-2, especially SARS- CoV-2), adenovirus, rotavirus, measles and rubella viruses, smallpox virus, Staphylococcus, Chlamydiae, Mycobacterium tuberculosis, Streptococcus pneumoniae, anthrax spores Bacillus anthracis, Vibrio cholerae, Helicobacter Pilorii, Salmonella, Plasmodium sp.
  • HCV human immunodeficiency virus
  • HCV hepatitis C virus
  • RSV Rou
  • P. falciparum P. vivax .vivax
  • Pneumocystis carinii Giardia duodenalis, Giardiose, Schistosoma, Bilharziose, Aspergillus , Cryptococcus, Candida albicans, Listeria monocytogenes or Toxoplasma gondii, etc.
  • pharmaceutically acceptable salt includes acid addition salts and base addition salts of AVC-29.
  • AVC-29 has a highly effective vaccine adjuvant effect. Compared with traditional aluminum adjuvants, AVC-29 has significant advantages in inducing antibody production and cellular immune response:
  • AVC-29 adjuvant can stimulate CD4 T cell response more strongly, especially CD4 T cell response positive for IFN ⁇ , IL-2 and IL-5 cytokines.
  • -29 adjuvant can potently stimulate antigen-specific T cell immune response.
  • AVC-29 has good safety and can be applied to various types of vaccine preparations, for example, for recombinant protein vaccines against SARS-CoV-2 virus.
  • AVC-29 small molecular compound is a potential ideal vaccine adjuvant.
  • FIG. 1 shows the OVA-specific IgG antibody titer of the OVA vaccine using AVC-29 adjuvant determined according to Example 2.
  • Figure 2 shows the SARS-CoV-2 RBD antigen-specific IgG antibody titer of the SARS-CoV-2 vaccine using AVC-29 adjuvant determined according to Example 5.
  • Figure 3 shows the true virus neutralizing antibody titer of the SARS-CoV-2 vaccine using AVC-29 adjuvant determined according to Example 6.
  • Figure 4 shows the cytokine-positive CD4 T cell immune response induced by the SARS-CoV-2 vaccine using the AVC-29 adjuvant of the present invention measured according to Example 7.
  • Embodiment 1 the vaccine preparation of model antigen OVA
  • Ovalbumin OVA purchased from Sigma
  • Aluminum (Al) adjuvant was purchased from U.S. Thermo Fisher Company;
  • AVC-29 was purchased from Ambow Company.
  • the OVA protein antigen was dissolved in physiological saline with AVC-29 adjuvant and aluminum adjuvant respectively, and vortexed to mix well to prepare vaccines.
  • the obtained vaccines were called OVA-AVC-29 and OVA-Al respectively.
  • mice were immunized according to the following groups:
  • Al alone adjuvant group (aluminum adjuvant, 50 ⁇ l);
  • AVC-29 adjuvant group alone (AVC-29, 5 ⁇ g);
  • OVA antigen panel 5 ⁇ g
  • OVA-Al group (OVA, 5 ⁇ g; aluminum adjuvant, 50 ⁇ l);
  • OVA-AVC-29 group (5 ⁇ g each of OVA and AVC-29 adjuvants);
  • each dose of vaccine is shown in brackets, and the volume of each dose of vaccine is 100 ⁇ l.
  • mice 6-8 week-old female BALB/c mice were immunized respectively, with 6 mice in each group. Each mouse was immunized with two doses of vaccine on the 0th day and the 14th day respectively, the way of immunization was thigh muscle injection, and the volume of each vaccine injection was 100 ⁇ l. On the 28th day, mice were sacrificed, blood was collected, serum was separated at 4°C, inactivated at 56°C for 30 minutes, and then stored at -80°C until use.
  • Embodiment 2 Serum OVA-specific antibody titer determination
  • the serum OVA-specific antibody titer was determined by ELISA method. The specific steps are as follows: OVA protein was diluted to 1 ⁇ g/ml with ELISA coating solution, 100 ⁇ l was added to each well of a 96-well plate, and left overnight at 4°C.
  • the ELISA plate was closed, and the mouse serum was diluted in a 2-fold gradient, added to the ELISA plate and incubated at 37°C for 1 hour, then washed three times with PBS (PBST) containing 0.05% Tween 20, and then added goat anti-mouse HRP II Antibody (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), incubated at 37°C for 1 hour, washed three times with PBST, added TMB chromogenic solution for display, terminated with 2M hydrochloric acid, and read at OD450 on a microplate reader.
  • PBS PBS
  • HRP II Antibody purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
  • the results are shown in Figure 1.
  • the results in Figure 1 show that the antibody titer induced by the OVA antigen group alone was about 68; the antibody titers induced by the OVA-Al group and the OVA-AVC-29 group were higher than those of the OVA antigen group alone It can be seen that the antibody titer induced by the vaccine formulated with AVC-29 adjuvant is significantly higher than that of the vaccine using traditional aluminum adjuvant.
  • the inventor of the present application uses the RBD dimer construct described in the patent application number CN202010581414.3 as an antigen, the antigen has the amino acid sequence shown in SEQ ID NO: 1, and is represented by SEQ ID NO: 2
  • SEQ ID NO: 2 The nucleotide sequence code shown (corresponding to SEQ ID NO: 20 in CN202010581414.3).
  • nucleotide sequence (ATGATCCACT CAGTGTTCT CTTAATGTTT CTACTAACTC CCACGGAGTC G; SEQ ID NO:3 ) to the 5' end of the nucleotide sequence shown in SEQ ID NO:2. :4) , after adding a nucleotide sequence encoding 6 histidines at its 3' end, add a stop codon; insert the nucleotide sequence thus obtained into the EcoRI and XhoI restriction sites in the vector pCAGGS point, with the Kozak sequence gccacc upstream of its start codon.
  • the plasmid obtained above was transfected into 293T cells, and then the supernatant was harvested.
  • the cell supernatant was filtered through a 0.22 ⁇ m pore size filter to remove cell debris.
  • the cell culture supernatant was hung on a nickel affinity column (Histrap) at 4 overnight.
  • the column was eluted with buffer A (20mM Tris, 150mM NaCl, pH 8.0) to remove non-specifically bound proteins.
  • the target protein was eluted from the column with buffer solution (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole), and the eluate was concentrated to less than 5ml with a 10K cutoff (10cutoff) concentrator tube.
  • Embodiment 4 SARS-CoV-2 RBD antigen immunization mouse experiment
  • the SARS-CoV-2 RBD antigen obtained in Example 3 was dissolved in physiological saline with AVC-29 adjuvant and aluminum adjuvant, and vortexed to prepare the vaccine.
  • the resulting vaccines were respectively called RBD-AVC-29 and RBD-Al.
  • mice were immunized according to the following groups:
  • Al alone adjuvant group (aluminum adjuvant, 50 ⁇ l);
  • AVC-29 adjuvant group alone (AVC-29, 5 ⁇ g);
  • RBD-Al group (RBD antigen, 5 ⁇ g; aluminum adjuvant, 50 ⁇ l);
  • RBD-AVC-29 group (5 ⁇ g each of RBD antigen and AVC-29 adjuvant);
  • each dose of vaccine is shown in brackets, and the volume of each dose of vaccine is 100 ⁇ l.
  • mice Use normal saline or vaccine to immunize 6-8 week old female BALB/c mice, 6 mice in each group.
  • Each mouse was immunized with two doses of vaccine on the 0th day and the 14th day respectively, the way of immunization was thigh muscle injection, and the volume of each vaccine injection was 100 ⁇ l.
  • the mice were killed, and the blood and spleen were collected; for the blood, the serum was separated at 4°C, then inactivated at 56°C for 30 minutes, and then stored at -80°C for later use; the treatment method of the spleen is shown in Example 7 below .
  • Embodiment 5 Determination of serum SARS-CoV-2 RBD specific antibody titer
  • the serum RBD-specific antibody titer was determined by ELISA method. The specific steps are as follows: dilute the SARS-CoV-2 RBD dimer protein with ELISA coating solution to 1 ⁇ g/ml, add 100 ⁇ l to each well of a 96-well plate, and place it overnight at 4°C.
  • the ELISA plate was closed, and the mouse serum was diluted in a 2-fold gradient, added to the ELISA plate and incubated at 37°C for 1 hour, then washed three times with PBS (PBST) containing 0.05% Tween 20, and then added goat anti-mouse HRP II Antibody (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), incubated at 37°C for 1 hour, washed three times with PBST, added TMB chromogenic solution for display, terminated with 2M hydrochloric acid, and read at OD450 on a microplate reader.
  • PBS PBS
  • HRP II Antibody purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
  • the results are shown in Figure 2.
  • the results in Figure 2 show that the serum antigen-specific antibody titer of the RBD-Al immunized group is about 1.1*10 5 , while the serum antigen-specific antibody titer of the RBD-AVC-29 immunized group can reach 1.9* 10 6 ; This shows that the effect of AVC-29 as a vaccine adjuvant in inducing antibody levels is significantly higher than that of traditional aluminum adjuvants.
  • Embodiment 6 Determination of serum SARS-CoV-2 neutralizing antibody titer
  • Example 4 Using a microneutralization experiment based on cytopathic effect (CPE), the SARS-CoV-2 true virus neutralizing antibody titer of the immunized mouse serum obtained in Example 4 was determined. Specific steps are as follows:
  • the serum of the immunized mice obtained in Example 4 was serially diluted in a 2-fold ratio, and the diluted serum was mixed with 100 TCID 50 wild-type SARS-CoV-2 true virus (HB01 strain, stored in the P3 laboratory of the Institute of Microbiology, Chinese Academy of Sciences), etc. Mix by volume and incubate at 37°C for 1 hour, add 100 ⁇ l of Vero E6 cells at a density of 1.5 ⁇ 10 5 cells/mL to 100 ⁇ l of the mixture. After incubation at 37°C for 72 hours, the pathological changes of the cells were observed under a microscope. Finally, the serum dilution factor for protecting 50% of the cells from virus infection was calculated by the Karber method, which was the true virus neutralizing antibody titer NT 50 value.
  • Spleen treatment the spleen of the immunized mice obtained in Example 4 was placed in pre-cooled 1640 medium. Put the 40 ⁇ m sieve on the 50ml centrifuge tube, use a 5ml syringe to grind the spleen, then add 1640 medium to drop the cells into the 50ml tube, filter out impurities and large cell clusters. Transfer all the cells to a 15ml centrifuge tube, collect the cells by centrifugation at 2000rpm at room temperature, add 12ml of 1640 medium to wash again, and centrifuge again to collect the cells.
  • Intracellular cytokine staining experiment In a 96-well round-bottom plate, add 1 ⁇ 106 mouse spleen cells obtained above to each well, and then add RBD polypeptide library (the final concentration of each polypeptide is 2 ⁇ g/ml) to stimulate the cells , respectively set the group added with phytohemagglutinin (PHA) stimulation as the positive control group, and the group without any stimulus as the negative control group. After 4 hours of stimulation, monamycin GolgiStop (purchased from BD Bioscience) was added, followed by culturing in a cell culture incubator for 12 hours, and the cells were collected by centrifugation.
  • PHA phytohemagglutinin
  • FIG. 4 The statistical results of fluorescent detection of CD4 T cells positive for each cytokine are shown in Figure 4.
  • the results in Figure 4 show that compared with traditional Al adjuvant, AVC-29 adjuvant can stimulate CD4 T cell response more strongly, especially IFN ⁇ , CD4 T cell responses positive for IL-2 and IL-5 cytokines suggest that AVC-29 adjuvant can potently stimulate antigen-specific T cell immune responses.

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Abstract

本申请涉及AVC-29作为疫苗佐剂的用途以及含有该佐剂的疫苗组合物。与传统的铝佐剂相比，AVC-29在诱导抗体产生和细胞免疫应答方面，均具有显著优势。另外，AVC-29安全性良好，可应用于多种类型的疫苗制剂，是潜在的理想的疫苗佐剂。

Description

AVC-29作为疫苗佐剂的用途以及含有该佐剂的疫苗组合物
本申请是以CN申请号为202110779345.1，申请日为2021年7月9日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本申请涉及生物医药领域，具体涉及小分子化合物AVC-29作为疫苗佐剂或者在制备疫苗佐剂中的用途，含有AVC-29的免疫原性或免疫刺激组合物及其制备方法和在制备疫苗中的用途。
背景技术
疫苗接种是预防和控制传染性疾病最经济和最有效的措施，是应对新发、突发传染性疾病(如，新冠肺炎疫情)的有效手段。近年来依托现代生物技术和基因工程的迅猛发展，疫苗的研制取得了重大的进展。常用的疫苗种类包括灭活疫苗、重组亚单位疫苗、腺病毒载体疫苗、抗独特型抗体疫苗、核酸疫苗以及近年来新发展起来的多肽疫苗等。其中，灭活疫苗、重组亚单位疫苗以及多肽疫苗都存在蛋白或多肽抗原免疫原性弱，诱导的免疫保护力不足等缺陷。因此，需要加入佐剂来增强人体对抗原的适应性免疫应答强度，包含提升抗体和细胞免疫应答水平等，以诱导有效的免疫保护。另外，佐剂还可重塑人体对抗原的免疫应答类型，使疫苗能更有效地抵抗病原体。
铝盐佐剂是第一个被批准并普遍用于人类的疫苗佐剂，其作用机理是形成抗原贮藏库；产生颗粒性抗原来促进抗原提呈给免疫细胞；使抗原滞留，缓慢释放，从而吸引活性淋巴细胞，激活补体系统。该佐剂的优点是，安全性好，但缺陷是刺激的免疫反应较弱，特别是无法有效诱导细胞免疫应答。在开发针对某些胞内感染型病原体如结核分枝杆菌和带状疱疹病毒等时，铝盐佐剂效果很不理想。此外，铝盐佐剂还可能导致神经退行性疾病的发生。
在新型佐剂开发方面，欧美发达国家近几十年已取得较大发展，MF59佐剂、AS系列佐剂和CpG佐剂等都已在上市疫苗中使用。相比较而言，我国在这方面比较落后，亟需发展新型高效的疫苗佐剂。
随着研究者对于佐剂作用机制的深入研究和分子生物学的发展，人们将会更有针对性的选择合适的疫苗佐剂来预防和治疗疾病，而佐剂的应用也将会更加安全高效，但目前可 供使用的人用佐剂并不多。因此开发研制新型的免疫佐剂有着重要的意义。
发明内容
发明人发现，小分子化合物AVC-29(结构如下文中所示)具有高效的疫苗佐剂效应。与传统的铝佐剂相比，AVC-29在诱导抗体产生和细胞免疫应答方面，均具有显著优势。另外，AVC-29安全性良好，可应用于多种类型的疫苗制剂，例如，用于针对SARS-CoV-2病毒的重组蛋白疫苗。因此，AVC-29小分子化合物是潜在的理想的疫苗佐剂。
因此，在第一个方面，本发明提供AVC-29或其药学上可接受的盐作为疫苗佐剂或者在制备疫苗佐剂和/或疫苗中的用途，其中所述AVC-29结构如下所示：
Figure PCTCN2022104620-appb-000001
在一些实施方案中，AVC-29或其药学上可接受的盐刺激受试者的免疫应答，例如，引发或增强受试者的免疫应答。在一些实施方案中，所述免疫应答为非特异性免疫应答。在一些实施方案中，所述免疫应答是抗原特异性免疫应答。在一些实施方案中，所述免疫应答包括B细胞的活化、T细胞的活化、抗体的产生和/或细胞因子的释放。
在另一个发面，本发明提供一种含有AVC-29或其药学上可接受的盐的免疫原性或免疫刺激组合物。
在一些实施方案中，所述免疫原性或免疫刺激组合物中进一步含有一种或多种抗原。在一些实施方案中，所述抗原为病原体的蛋白质、重组蛋白、糖蛋白、肽、多糖、脂质、脂多糖或核酸(包括DNA和mRNA)。在一些实施方案中，所述抗原衍生自细胞(例如，肿瘤细胞)、细菌或病毒。在一些实施方案中，所述抗原为SARS-CoV-2病毒抗原，所述SARS-CoV-2为新型冠状病毒，由国际病毒分类学委员会(ICTV)命名。在一些实施方案中，所述抗原为SARS-CoV-2病毒的受体结合结构域抗原。在一些实施方案中，所述抗原具有如SEQ ID NO:1所示的氨基酸序列。
在一些实施方案中，所述免疫原性或免疫刺激组合物以单位剂量形式存在。在一些实施方案中，每单位剂量的免疫原性或免疫刺激组合物中含有0.1-500μg，优选1-100μg，更优选5-50μg的AVC-29或其药学上可接受的盐。所述单位剂量为临床使用的常规单次施用(注射)参考量。
在一些实施方案中，所述免疫原性或免疫刺激组合物中AVC-29或其药学上可接受的盐与抗原的质量比为(0.1-100)：1、优选(0.1-10)：1、更优选(0.5-5)：1，例如(0.5-1)：1、(0.5-1.5)：1、(0.5-2)：1、(0.5-2.5)：1、(0.5-3)：1、(0.5-3.5)：1、(0.5-4)：1、(0.5-4.5)：1、(1-1.5)：1、(1-2)：1、(1-2.5)：1、(1-3)：1、(1-3.5)：1、(1-4)：1、(1-4.5)：1、或(1-5)：1。
在一些实施方案中，所述免疫原性或免疫刺激组合物为可注射制剂、可吸入制剂或口服制剂形式，例如粉针剂、混悬剂、水针剂、喷雾剂、气雾剂、粉雾剂、粘贴片剂、舌下片剂或膜剂。
在一些实施方案中，所述免疫原性或免疫刺激组合物中还含有缓冲剂、等渗剂、防腐剂、稳定剂和增溶剂中的一种或多种，如糖(乳糖、蔗糖)、氨基酸(甘氨酸)、明胶和蛋白质(重组人血白蛋白)等。
在另一个方面，本发明进一步提供所述免疫原性或免疫刺激组合物的制备方法，其包括以下步骤：
将AVC-29或其药学上可接受的盐与前文所述的一种或多种抗原和任选的其它成分进行混合，优选地，在生理盐水中混合，得到所述免疫原性或免疫刺激组合物；优选地，进行所述混合后，还包括干燥的步骤，例如冷冻干燥。
在另一个方面，本发明还提供所述的免疫原性或免疫刺激组合物用作疫苗或在制备疫苗中的用途。
在一些实施方案中，所述疫苗为灭活疫苗、减毒活疫苗或基因重组疫苗。所述疫苗可以是预防性的(即保护受试者免受所述疾病)或治疗性的(即帮助受试者抗击已感染的疾病)。在一些实施方案中，所述疫苗用于预防和/或治疗与所述抗原相关的疾病。在一些实施方案中，所述疾病为COVID-19(新型冠状病毒肺炎)。
在一些实施方案中，所述免疫原性或免疫刺激组合物刺激受试者的免疫应答，例如，引发或增强受试者的免疫应答。在一些实施方案中，所述免疫应答为非特异性免疫应答。在一些实施方案中，所述免疫应答是抗原特异性免疫应答。在一些实施方案中，所述免疫应答包括B细胞的活化、T细胞的活化、抗体的产生和/或细胞因子的释放。
在另一个方面，本发明提供一种刺激(例如，引发或增强)受试者免疫应答的方法，其包括向所述受试者施用有效量的所述免疫原性或免疫刺激组合物。
在一些实施方案中，所述免疫应答为非特异性免疫应答。在一些实施方案中，所述免疫应答是抗原特异性免疫应答。在一些实施方案中，所述免疫应答包括B细胞的活化、 T细胞的活化、抗体的产生和/或细胞因子的释放。
在一些实施方案中，所述免疫原性或免疫刺激组合物的施用途径为口服、静脉内、皮内、经皮、经鼻、皮下或肛门。
在一些实施方案中，所述受试者为哺乳动物，例如人或非人类哺乳动物(包括但不限于狗、牛和马)，优选为人。
术语定义
除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学等实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明的技术方案，下面提供相关术语的定义和解释。
术语“疫苗”是被施用以产生或人工增加对特定抗原的免疫的组合物。
术语“免疫原性组合物”、“免疫刺激组合物”是当施用于个体时能够在体内产生免疫应答的组合物。因此，应理解术语“免疫原性组合物”、“免疫刺激组合物”和“疫苗”是同义术语。在一些实施方案中，所述个体优选为哺乳动物，更优选为人，当然也可以是其它哺乳动物，例如所述组合物可在牛(cattle)(包括乳牛(cow))、绵羊、山羊或马中诱导免疫，或者宠物，如狗或猫。
因此，本文所述免疫原性组合物或免疫刺激组合为是这样一种组合物，其含有抗原并且能够产生针对这种抗原的免疫应答。产生的免疫应答可以是细胞(T细胞介导的)或体液(B细胞介导的，产生抗体)免疫应答。其也可以诱导细胞免疫应答和体液免疫应答。细胞免疫应答可以是CD8T淋巴细胞介导的应答(即细胞毒性应答)或CD4 T淋巴细胞介导的应答(辅助性应答)。它也可以组合细胞毒性和辅助性细胞免疫应答。辅助性应答可涉及Th1、Th2或Th17淋巴细胞(这种淋巴细胞能够引发不同的细胞因子应答)。在一些实施方案中，本文所述组合物可显著刺激CD4 T细胞相应，特别是IFNγ、IL-2和IL-5细胞因子阳性的CD4 T细胞响应。
术语“疫苗佐剂”、“佐剂”是指能够修饰或增强对抗原的免疫应答的物质。换言之，针对抗原的免疫应答在佐剂存在时可以高于或不同于当佐剂不存在时(包括当应答被修饰时，例如，佐剂存在时活化的T细胞子集与不存在佐剂时活化的子集不同)。
术语“抗原”是为了使个体的免疫系统识别它而引发免疫应答的分子或分子的组合。这种抗原对于寻求免疫应答的宿主的机体而言可能是外来的。在这种情况下，抗原可以是由细菌或病毒表达的蛋白。抗原还可以是自身抗原，即由宿主细胞表达的蛋白，如肿瘤抗原。
抗原可以由死亡或未死亡的完整生物体(病毒或细菌、真菌、原生动物甚至癌细胞)、细胞(经辐射的或未经辐射的，经遗传修饰的或未经遗传修饰的)、或这些生物体或细胞的亚组分如细胞提取物或细胞裂解物所组成。抗原还可以由单分子如蛋白、重组蛋白、肽、多糖、脂质、糖脂、糖肽或其混合物组成。抗原也可以是通过化学修饰或稳定化进行修饰的以上列举的分子之一。
作为免疫原性或免疫刺激组合物中可以使用抗原的病原体的示例，包括感染性疾病的任何病原体(病毒、细菌、寄生虫、霉菌)。
对于感染性疾病，优选的病原体选自人免疫缺陷病毒(HIV)、甲型肝炎和乙型肝炎病毒、丙型肝炎病毒(HCV)、劳斯氏肉瘤病毒(RSV)、埃博拉病毒、巨细胞病毒、疱疹病毒、水痘带状疱疹病毒，爱泼斯坦-巴尔二氏病毒(Epstein Barr virus，EBV)、流感病毒、冠状病毒(例如，MERS、SARS、SARS-CoV-2，尤其是SARS-CoV-2)、腺病毒、轮状病毒、麻疹和风疹病毒、天花病毒、葡萄球菌(Staphylococcus)、衣原体(Chlamydiae)、结核分枝杆菌(Mycobacterium tuberculosis)、肺炎链球菌(Streptococcus pneumoniae)、炭疽芽孢杆菌(Bacillus anthracis)、霍乱弧菌(Vibrio cholerae)、幽门螺杆菌(Helicobacter Pilorii)、沙门氏菌(Salmonella)、疟原虫(Plasmodium sp.)(恶性疟原虫(P.falciparum)、间日疟原虫(P.vivax)、卡氏肺囊虫(Pneumocystis carinii)、十二指肠贾第鞭毛虫(Giardiaduodenalis)、梨形鞭毛虫(Giardiose)、血吸虫(Schistosoma)、体吸虫病(Bilharziose)、曲霉(Aspergillus)、隐球菌(Cryptococcus)、白色念珠菌(Candida albicans)、单核细胞增多性李斯特氏菌(Listeria monocytogenes)或弓形虫(Toxoplasma gondii)等。
作为可以受益于采用合适的抗原免疫的疾病的示例，包括但不限于癌症(良性或恶性肿瘤)、过敏、自身免疫疾病，如新型冠状病毒肺炎。
术语“药学上可接受的盐”包括AVC-29的酸加成盐及碱加成盐。例如钠盐、钾盐、钙盐、锂盐、葡甲胺盐、盐酸盐，氢澳酸盐，氢腆酸盐，硝酸盐，硫酸盐，硫酸氢盐，磷酸盐，磷酸氢盐，乙酸盐，丙酸盐，丁酸盐，草酸盐，三甲基乙酸盐，己二酸盐，藻酸盐，乳酸盐，柠檬酸盐，酒石酸盐，琥珀酸盐，马来酸盐，富马酸盐，苦味酸盐，天冬氨酸盐，葡糖酸盐，苯甲酸盐，甲磺酸盐，乙磺酸盐，苯磺酸盐，对甲苯磺酸盐或双羟萘酸盐等。
适合的盐的综述参见Stahl及Wermuth的“Handbook of Pharmaceutical Salts:Properties,Selection,and Use”(Wiley-VCH,2002)。用于制备可药用盐的方法为本领域技术人员已知的。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
发明的有益效果
研究发现，小分子化合物AVC-29具有高效的疫苗佐剂效应。与传统的铝佐剂相比，AVC-29在诱导抗体产生和细胞免疫应答方面，均具有显著优势：
(1)血清SARS-CoV-2 RBD特异性抗体滴度实验显示，AVC-29作为疫苗佐剂诱导抗体水平效应相较于Al佐剂高出近20倍；
(2)血清SARS-CoV-2中和抗体滴度实验显示，AVC-29作为疫苗佐剂诱导中和抗体水平相较于Al佐剂高出近10倍；
(3)相比传统Al佐剂，AVC-29佐剂可更强地刺激CD4 T细胞响应，特别是IFNγ、IL-2和IL-5细胞因子阳性的CD4 T细胞响应，由此可知，AVC-29佐剂可强效刺激抗原特异性T细胞免疫应答。
同时，AVC-29安全性良好，可应用于多种类型的疫苗制剂，例如，用于针对SARS-CoV-2病毒的重组蛋白疫苗。AVC-29小分子化合物是潜在的理想的疫苗佐剂。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1显示依据实施例2测定的使用AVC-29佐剂的OVA疫苗的OVA特异性IgG抗体滴度。
图2显示依据实施例5测定的使用AVC-29佐剂的SARS-CoV-2疫苗的SARS-CoV-2 RBD抗原特异性IgG抗体滴度。
图3显示依据实施例6测定的使用AVC-29佐剂的SARS-CoV-2疫苗的真病毒中和抗体滴度。
图4显示依据实施例7测定的使用本发明AVC-29佐剂的SARS-CoV-2疫苗诱导的细胞因子阳性的CD4 T细胞免疫应答。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，绝不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
另外，为了更好地说明本发明，在下文的实施例中给出了众多的具体实验细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本发明的主旨。
实施例1：模式抗原OVA的疫苗制备
试剂来源：
卵白蛋白OVA，购自Sigma；
铝(Al)佐剂，购自美国Thermo Fisher公司；
AVC-29购自安博公司。
将OVA蛋白抗原分别与AVC-29佐剂、铝佐剂一起溶于生理盐水，涡旋混匀，以配制疫苗，所得疫苗分别称为OVA-AVC-29和OVA-Al。
按照如下组别分别免疫小鼠：
生理盐水组；
单独的Al佐剂组(铝佐剂，50μl)；
单独的AVC-29佐剂组(AVC-29，5μg)；
单独的OVA抗原组(OVA，5μg)；
OVA-Al组(OVA，5μg；铝佐剂，50μl)；和
OVA-AVC-29组(OVA和AVC-29佐剂各5μg)；
上述各组中，括号中显示了每剂疫苗的抗原或佐剂含量，每剂疫苗体积为100μl。
免疫小鼠的程序如下：
按照上述疫苗组别，分别对6-8周龄雌性BALB/c小鼠进行免疫，每组6只小鼠。每只小鼠分别在第0天和第14天进行两针疫苗免疫，免疫方式为大腿肌肉注射，每次疫苗注射体积为100μl。第28天，对小鼠处死，采血，4℃分离血清，并于56℃灭活30分钟，然后保存于-80℃待用。
实施例2：血清OVA特异性抗体滴度测定
对于实施例1所得免疫小鼠血清，通过ELISA方法测定其血清OVA特异性抗体滴度。具体步骤如下：将OVA蛋白用ELISA包被液稀释至1μg/ml，96孔板每孔加入100μl，4℃放置过夜。第二天封闭ELISA板，小鼠血清按照2倍梯度稀释，加入到ELISA板中37℃孵育1小时，之后用含0.05％吐温20的PBS(PBST)清洗三次后，加入羊抗鼠HRP二抗(购自北京中杉金桥生物技术公司)，37℃孵育1小时后，PBST清洗三次，加入TMB显色液显示，并用2M盐酸终止，在酶标仪上于OD450处读值。
结果如图1所示，图1结果显示：单独使用OVA抗原组诱导的抗体滴度为约68；OVA-Al组和OVA-AVC-29组诱导的抗体滴度比单独使用OVA抗原组分别提升了约13倍和160倍；由此可见，用AVC-29佐剂配制的疫苗所诱导的抗体滴度显著高于使用传统铝佐剂的疫苗。
实施例3：SARS-CoV-2 RBD抗原的表达纯化
本申请的发明人采用发明专利申请号CN202010581414.3中记载的RBD二聚体构建物作为抗原，所述抗原具有如SEQ ID NO:1所示的氨基酸序列，并且由如SEQ ID NO:2所示(对应于CN202010581414.3中的SEQ ID NO:20)的核苷酸序列编码。
SEQ ID NO:1：
Figure PCTCN2022104620-appb-000002
SEQ ID NO:2：
Figure PCTCN2022104620-appb-000003
Figure PCTCN2022104620-appb-000004
在SEQ ID NO:2所示核苷酸序列的5’端加上编码MERS S蛋白自身信号肽(MIHSVFLLMFLLTPTES； SEQ ID NO:3)的核苷酸序列(ATGATCCACT CAGTGTTTCT CTTAATGTTT CTACTAACTC CCACGGAGTC G； SEQ ID NO:4)，在其3’端加上编码6个组氨酸的核苷酸序列后，再加上终止密码子；将如此所得核苷酸序列插入到载体pCAGGS中的EcoRI和XhoI酶切位点，其起始密码子上游有Kozak序列gccacc。
将如上所得质粒体转染293T细胞，之后收获上清。将细胞上清通过0.22μm孔径的滤膜过滤，除去细胞碎片。将细胞培养上清挂镍亲和柱(Histrap)，4度过夜。以缓冲液A(20mM Tris，150mM NaCl，pH 8.0)洗脱柱子，以去除非特异结合蛋白。最后，目的蛋白以缓冲液(20mM Tris，150mM NaCl，pH 8.0，300mM咪唑)从柱子洗脱下来，并以10K截留(10cutoff)浓缩管将洗脱液浓缩至5ml以内。再通过Superdex 200 Hi load 16/60柱子(GE)进行分子筛层析，以进行进一步的目的蛋白纯化。分子筛层析缓冲液为PBS。经过分子筛层析，在洗脱体积80ml处出现唯一主峰，将其收集浓缩后，-80℃保存。
实施例4：SARS-CoV-2 RBD抗原免疫小鼠实验
将实施例3中获得的SARS-CoV-2 RBD抗原分别与AVC-29佐剂、铝佐剂一起溶于生理盐水，涡旋混匀，以配制疫苗，所得疫苗分别称为RBD-AVC-29和RBD-铝。
按照如下组别分别免疫小鼠：
生理盐水组；
单独的Al佐剂组(铝佐剂，50μl)；
单独的AVC-29佐剂组(AVC-29，5μg)；
RBD-Al组(RBD抗原，5μg；铝佐剂，50μl)；和
RBD-AVC-29组(RBD抗原和AVC-29佐剂各5μg)；
上述各组中，括号中显示了每剂疫苗的抗原或佐剂含量，每剂疫苗体积为100μl。
免疫小鼠的程序如下：
使用生理盐水或疫苗，对6-8周龄雌性BALB/c小鼠进行免疫，每组6只小鼠。每只小鼠分别在第0天和第14天进行两针疫苗免疫，免疫方式为大腿肌肉注射，每次疫苗注射体积为100μl。第28天，对小鼠处死，采集血液和脾脏；对于血液，于4℃分离血清，然后于56℃灭活30分钟，之后保存于-80℃待用；脾脏的处理方法见以下实施例7。
实施例5：血清SARS-CoV-2 RBD特异性抗体滴度测定
对于实施例4所得免疫小鼠血清，通过ELISA方法测定其血清RBD特异性抗体滴度。具体步骤如下：将SARS-CoV-2 RBD二聚体蛋白用ELISA包被液稀释至1μg/ml，96孔板每孔加入100μl，4℃放置过夜。第二天封闭ELISA板，小鼠血清按照2倍梯度稀释，加入到ELISA板中37℃孵育1小时，之后用含0.05％吐温20的PBS(PBST)清洗三次后，加入羊抗鼠HRP二抗(购自北京中杉金桥生物技术公司)，37℃孵育1小时后，PBST清洗三次，加入TMB显色液显示，并用2M盐酸终止，在酶标仪上于OD450处读值。
结果如图2显示，图2结果显示：RBD-Al免疫组的血清抗原特异性抗体滴度约1.1*10 5，而RBD-AVC-29疫组的血清抗原特异性抗体滴度可达1.9*10 6；这表明，AVC-29作为疫苗佐剂诱导抗体水平的效应显著高于传统的铝佐剂。
实施例6：血清SARS-CoV-2中和抗体滴度测定
使用基于细胞病变效应(CPE)的微量中和实验，测定实施例4所得免疫小鼠血清的SARS-CoV-2真病毒中和抗体滴度。具体步骤如下：
将实施例4所得免疫小鼠的血清按2倍比进行梯度稀释，将稀释后血清与100TCID 50 野生型SARS-CoV-2真病毒(HB01株，保存于中国科学院微生物研究所P3实验室)等体积混合，37℃孵育1小时后，向100μl混合液中加入100μl密度为1.5×10 5个/mL的Vero E6细胞。37℃孵育72小时后，显微镜下观察细胞的病变情况。最后，通过Karber法计算保护50％的细胞免受病毒感染时的血清稀释倍数，即为真病毒中和抗体滴度NT 50值。
结果如图3所示，图3结果显示：在生理盐水组、单独Al佐剂组和单独AVC-29佐剂组免疫小鼠血清中，均未观察到针对SARS-CoV-2真病毒的中和抗体；而对于RBD-Al组和RBD-AVC-29组免疫小鼠，显示它们所诱导的中和抗体滴度分别达498和4469，即，RBD-AVC-29组比RBD-Al组所诱导的中和抗体水平高近10倍。
实施例7：SARS-CoV-2疫苗诱导的细胞免疫反应评价
脾脏处理：将实施例4所得免疫小鼠的脾脏放置于预冷的1640培养基中。将40μm筛网放在50ml离心管上，使用5ml注射器推头研磨脾脏，之后加1640培养基使细胞滴进50ml管中，过滤掉杂质及大的细胞团。把所有细胞转移到15ml离心管中，室温2000rpm离心收集细胞，再加入12ml的1640培养基清洗一遍，再次离心，收集细胞。向收集的细胞团块中，加入4ml的红细胞裂解液，悬浮细胞，室温放置5-10分钟，再加入8ml的1640培养基，离心，收集细胞；加入12ml 1640培养基清洗一遍，再次离心，收集细胞。加入10ml的1640培养基，悬浮细胞，使用细胞计数板计算细胞总数。再次离心，收集细胞；向收集的细胞团块中，加入一定体积的10％FBS1640培养基，使细胞浓度为1×10 7个/ml。
胞内细胞因子染色实验：在圆底96孔板中，向每孔加入1×10 6个上述所得小鼠脾脏细胞，然后加入RBD多肽库(每条多肽的终浓度为2μg/ml)刺激细胞，分别设置加入植物血凝素(PHA)刺激的组为阳性对照组，不加任何刺激物的组为阴性对照组。刺激4小时后，加入莫奈霉素GolgiStop(购自BD Bioscience)，接着在细胞培养箱中培养12小时后，离心收集细胞。然后，使用PE标记的抗鼠CD3抗体，FITC标记的抗鼠CD4抗体，APC-Cy7标记的抗鼠CD8抗体，BV605标记的抗鼠IL-2抗体，BB700标记的抗鼠TNFα抗体，BV421标记的抗鼠IFNγ抗体，BV786标记的抗鼠IL-4抗体和APC标记的抗鼠IL-5抗体(所有抗体全部购自Biolegend公司)，对细胞进行抗体染色，其中操作步骤完全按照BD公司Cytofix/Cytoperm TM Fixation/Permeabilization试剂盒说明书和抗体使用说明书操作。然后，在FACSCanton流式细胞仪上检测细胞荧光。
各细胞因子阳性的CD4 T细胞的荧光检测统计结果如图4所示，图4结果表明：相 比传统Al佐剂，AVC-29佐剂可更强地刺激CD4 T细胞响应，特别是IFNγ、IL-2和IL-5细胞因子阳性的CD4 T细胞响应，由此可知，AVC-29佐剂可强效刺激抗原特异性T细胞免疫应答。
除本文中描述的那些外，根据前述描述，本发明的各种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。

Claims (11)

  1. 一种含有AVC-29或其药学上可接受的盐的免疫原性或免疫刺激组合物，其中所述AVC-29结构如下所示：
    Figure PCTCN2022104620-appb-100001
  2. 权利要求1所述的免疫原性或免疫刺激组合物，其进一步含有一种或多种抗原；
    优选地，所述抗原为病原体的蛋白质、重组蛋白、糖蛋白、肽、多糖、脂质、脂多糖或核酸(包括DNA和mRNA)；
    优选地，所述抗原衍生自细胞(例如，肿瘤细胞)、细菌或病毒；
    优选地，所述抗原为SARS-CoV-2病毒抗原；
    优选地，所述抗原为SARS-CoV-2病毒的受体结合结构域抗原；
    优选地，所述抗原具有如SEQ ID NO:1所示的氨基酸序列。
  3. 权利要求1或2所述的免疫原性或免疫刺激组合物，其以单位剂量形式存在；
    优选地，每单位剂量的免疫原性或免疫刺激组合物中含有0.1-500μg，优选1-100μg，更优选5-50μg的AVC-29或其药学上可接受的盐。
  4. 根据权利要求1-3任一项所述的免疫原性或免疫刺激组合物，其中AVC-29或其药学上可接受的盐与抗原的质量比为(0.1-100)：1、优选(0.1-10)：1、更优选(0.5-5)：1。
  5. 根据权利要求1-4任一项所述的免疫原性或免疫刺激组合物，其为可注射制剂、可吸入制剂或口服制剂形式，例如粉针剂、混悬剂、水针剂、喷雾剂、气雾剂、粉雾剂、粘贴片剂、舌下片剂或膜剂；
    优选地，所述免疫原性或免疫刺激组合物中还含有缓冲剂、等渗剂、防腐剂、稳定剂和增溶剂中的一种或多种。
  6. 根据权利要求1-5任一项所述的免疫原性或免疫刺激组合物的制备方法，其包括以下步骤：
    将AVC-29或其药学上可接受的盐与抗原和任选地其它成分进行混合，优选地，在生理盐水中混合，得到所述免疫原性或免疫刺激组合物；优选地，进行所述混合后，还包括干燥的步骤，例如冷冻干燥。
  7. AVC-29或其药学上可接受的盐作为疫苗佐剂或者在制备疫苗佐剂和/或疫苗中的用途。
  8. 权利要求1-5任一项所述的免疫原性或免疫刺激组合物用作疫苗或在制备疫苗中的用途。
  9. 权利要求7或8的用途，其中所述疫苗为灭活疫苗、减毒活疫苗或基因重组疫苗；
    优选地，所述疫苗为预防性或治疗性疫苗；
    优选地，所述疫苗用于预防和/或治疗与所述抗原相关的疾病；
    优选地，所述疾病为COVID-19(新型冠状病毒肺炎)。
  10. 权利要求7-9任一项所述的用途，其中所述AVC-29或其药学上可接受的盐、或免疫原性或免疫刺激组合物刺激(例如，引发或增强)受试者的免疫应答；
    优选地，所述免疫应答为非特异性免疫应答；
    优选地，所述免疫应答是抗原特异性免疫应答；
    优选地，所述免疫应答包括B细胞的活化、T细胞的活化、抗体的产生和/或细胞因子的释放。
  11. 一种刺激(例如，引发或增强)受试者的免疫应答的方法，其包括向受试者施用有效量的权利要求1-5任一项所述的免疫原性或免疫刺激组合物的步骤；
    优选地，所述免疫应答为非特异性免疫应答；
    优选地，所述免疫应答是抗原特异性免疫应答；
    优选地，所述免疫应答包括B细胞的活化、T细胞的活化、抗体的产生和/或细胞因子的释放。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459058A (zh) * 2014-05-01 2017-02-22 诺华股份有限公司 作为toll‑样受体7激动剂的化合物和组合物
CN111592602A (zh) * 2020-02-10 2020-08-28 中国科学院微生物研究所 一种β冠状病毒抗原、其制备方法和应用
CN111892648A (zh) * 2020-06-08 2020-11-06 中国科学院上海药物研究所 偶联tlr7激动剂的新型冠状病毒多肽疫苗及其应用
WO2021130195A1 (en) * 2019-12-24 2021-07-01 F. Hoffmann-La Roche Ag Method of treating virus infection using a tlr7 agonist
CN113476600A (zh) * 2021-07-09 2021-10-08 海南大学 Avc-29作为疫苗佐剂的用途以及含有该佐剂的疫苗组合物

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105903009A (zh) * 2016-04-05 2016-08-31 中国科学院过程工程研究所 一种基于阿拉伯半乳聚糖-聚肌苷酸胞苷酸修饰的结核分枝杆菌亚单位疫苗及其制备方法
CN111217917B (zh) * 2020-02-26 2020-10-23 康希诺生物股份公司 一种新型冠状病毒SARS-CoV-2疫苗及其制备方法
BR112023004799A2 (pt) * 2020-09-15 2023-04-18 Bharat Biotech Int Ltd Formulação de vacina de agonista de receptor do tipo toll (tlr)
CN112220920B (zh) * 2020-10-30 2023-06-13 上海泽润生物科技有限公司 一种重组新型冠状病毒疫苗组合物

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459058A (zh) * 2014-05-01 2017-02-22 诺华股份有限公司 作为toll‑样受体7激动剂的化合物和组合物
WO2021130195A1 (en) * 2019-12-24 2021-07-01 F. Hoffmann-La Roche Ag Method of treating virus infection using a tlr7 agonist
CN111592602A (zh) * 2020-02-10 2020-08-28 中国科学院微生物研究所 一种β冠状病毒抗原、其制备方法和应用
CN111892648A (zh) * 2020-06-08 2020-11-06 中国科学院上海药物研究所 偶联tlr7激动剂的新型冠状病毒多肽疫苗及其应用
CN113476600A (zh) * 2021-07-09 2021-10-08 海南大学 Avc-29作为疫苗佐剂的用途以及含有该佐剂的疫苗组合物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PARTLOW HALEY A, DAVISON CLARA J., LATHROP STEPHANIE K, EVANS JAY T.: "The comparison of two SARS-CoV-2 spike protein antigens in TLR-adjuvanted subunit vaccines.", THE JOURNAL OF IMMUNOLOGY, WILLIAMS & WILKINS CO., US, vol. 206, no. 1 Suppl., 1 May 2021 (2021-05-01), US , pages 30.06, XP009542556, ISSN: 0022-1767, DOI: 10.4049/jimmunol.206.Supp.30.06 *

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