WO2023280303A1 - Utilisation d'avc-29 en tant qu'adjuvant vaccinal et composition vaccinale contenant un adjuvant - Google Patents

Utilisation d'avc-29 en tant qu'adjuvant vaccinal et composition vaccinale contenant un adjuvant 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

La présente invention concerne une utilisation de l'AVC-29 en tant qu'adjuvant vaccinal et une composition vaccinale contenant ledit adjuvant. Par comparaison avec des adjuvants d'aluminium classiques. l'AVC-29 a des avantages significatifs dans l'induction de la production d'anticorps et de la réponse immunitaire cellulaire. De plus, l'AVC-29 présente de bonnes propriétés en termes de sécurité, peut être appliqué dans de nombreux types de préparations de vaccin, et est un adjuvant de vaccin potentiellement idéal.
PCT/CN2022/104620 2021-07-09 2022-07-08 Utilisation d'avc-29 en tant qu'adjuvant vaccinal et composition vaccinale contenant un adjuvant WO2023280303A1 (fr)

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