WO2023280303A1 - Use of avc-29 as vaccine adjuvant and vaccine composition containing adjuvant - Google Patents

Abstract

The present application relates to a use of AVC-29 as a vaccine adjuvant and a vaccine composition containing said adjuvant. Compared with conventional aluminum adjuvants., AVC-29 has significant advantages in inducing antibody production and cellular immune response. In addition, AVC-29 has good safety, can be applied in many types of vaccine preparations, and is a potentially ideal vaccine adjuvant.

Description

Use of AVC-29 as vaccine adjuvant and vaccine composition containing the adjuvant

This application is based on the application with CN application number 202110779345.1 and the application date is July 9, 2021, and claims its priority. The disclosure content of this CN application is hereby incorporated into this application as a whole.

technical field

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 .

Background technique

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. Among them, 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. Therefore, it is necessary to add 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. In addition, 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.

In the development of new adjuvants, developed countries in Europe and the United States have made great progress in recent decades. MF59 adjuvants, AS series adjuvants and CpG adjuvants have all been used in marketed vaccines. In comparison, our country is relatively backward in this area, and there is an urgent need to develop new and efficient vaccine adjuvants.

With the in-depth research on the mechanism of adjuvants and the development of molecular biology, people will be more targeted to choose appropriate vaccine adjuvants to prevent and treat diseases, and the application of adjuvants will be safer and more efficient , but there are not many human adjuvants currently available. Therefore, it is of great significance to develop new immune adjuvants.

Contents of the invention

The inventors found that the small molecule compound AVC-29 (structure shown below) 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. In addition, 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.

Therefore, in a first aspect, 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 :

In some embodiments, AVC-29, or a pharmaceutically acceptable salt thereof, stimulates an immune response in a subject, eg, elicits or enhances an immune response in a subject. In some embodiments, 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.

In another aspect, the present invention provides an immunogenic or immunostimulatory composition comprising AVC-29 or a pharmaceutically acceptable salt thereof.

In some embodiments, the immunogenic or immunostimulatory composition further comprises one or more antigens. In some embodiments, the antigen is a protein, recombinant protein, glycoprotein, peptide, polysaccharide, lipid, lipopolysaccharide, or nucleic acid (including DNA and mRNA) of a pathogen. In some embodiments, the antigen is derived from a cell (eg, a tumor cell), a bacterium, or a virus. In some embodiments, 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). In some embodiments, the antigen is a receptor binding domain antigen of the SARS-CoV-2 virus. In some embodiments, the antigen has the amino acid sequence shown in SEQ ID NO:1.

In some embodiments, the immunogenic or immunostimulatory composition is presented in unit dosage form. In some embodiments, the immunogenic or immunostimulatory composition contains 0.1-500 μg, preferably 1-100 μg, more preferably 5-50 μg 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In another aspect, 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.

In another aspect, the present invention also provides the use of the immunogenic or immunostimulatory composition as a vaccine or in the preparation of a vaccine.

In some embodiments, 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). In some embodiments, the vaccine is used to prevent and/or treat a disease associated with the antigen. In some embodiments, the disease is COVID-19 (Novel Coronavirus Pneumonia).

In some embodiments, the immunogenic or immunostimulatory composition stimulates an immune response in a subject, eg, elicits or enhances an immune response in a subject. In some embodiments, 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.

In another aspect, 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.

In some embodiments, 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.

In some embodiments, the immunogenic or immunostimulatory composition is administered orally, intravenously, intradermally, transdermally, nasally, subcutaneously, or anally.

In some embodiments, 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.

Definition of Terms

Unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the technical solutions of the present invention, definitions and explanations of relevant terms are provided below.

The term "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. In some embodiments, 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.

Thus, 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). In some embodiments, the compositions described herein can significantly stimulate CD4 T cell responses, particularly CD4 T cell responses positive for IFNγ, IL-2, and IL-5 cytokines.

The term "vaccine adjuvant", "adjuvant" refers to a substance capable of modifying or enhancing the immune response to an antigen. In other words, 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).

The term "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.

Examples of pathogens for which antigens may be used in immunogenic or immunostimulatory compositions include any pathogens of infectious diseases (viruses, bacteria, parasites, fungi).

For infectious diseases, 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. (P. falciparum, P. vivax .vivax), Pneumocystis carinii, Giardia duodenalis, Giardiose, Schistosoma, Bilharziose, Aspergillus , Cryptococcus, Candida albicans, Listeria monocytogenes or Toxoplasma gondii, etc.

Examples of diseases that may benefit from immunization with an appropriate antigen include, but are not limited to, cancer (benign or malignant), allergies, autoimmune diseases such as COVID-19.

The term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts of AVC-29. For example, sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrochloric acid salt, hydroiodic acid salt, nitrate salt, sulfate salt, hydrogen sulfate salt, phosphate salt, hydrogen phosphate salt, ethyl Acid, propionate, butyrate, oxalate, trimethylacetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate , fumarate, picrate, aspartate, gluconate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate or pamoate salt etc.

For a review of suitable salts see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts are known to those skilled in the art.

Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations thereof such as "includes" or "includes" and the like will be understood to include the stated elements or constituents, and not Other elements or other components are not excluded.

Beneficial Effects of the Invention

The study found that the small molecule compound 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:

(1) The serum SARS-CoV-2 RBD-specific antibody titer experiment showed that the effect of AVC-29 as a vaccine adjuvant to induce antibody levels was nearly 20 times higher than that of Al adjuvant;

(2) The serum SARS-CoV-2 neutralizing antibody titer experiment showed that the level of neutralizing antibody induced by AVC-29 as a vaccine adjuvant was nearly 10 times higher than that of Al adjuvant;

(3) Compared with traditional Al adjuvant, AVC-29 adjuvant can stimulate CD4 T cell response more strongly, especially CD4 T cell response positive for IFNγ, IL-2 and IL-5 cytokines. -29 adjuvant can potently stimulate antigen-specific T cell immune response.

At the same time, 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.

Description of drawings

The accompanying drawings described here are used to provide a further understanding of the present invention and constitute a part of the application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention. In the attached picture:

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.

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative in nature and in no way taken as any limitation of the invention, its application or uses. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

In addition, in order to better illustrate the present invention, numerous specific experimental details are given in the following examples. It will be understood by those skilled in the art that the present invention may be practiced without certain of the specific details. In the embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the gist of the present invention.

Embodiment 1: the vaccine preparation of model antigen OVA

Reagent source:

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:

Normal saline group;

Al alone adjuvant group (aluminum adjuvant, 50 μl);

AVC-29 adjuvant group alone (AVC-29, 5 μg);

Individual OVA antigen panel (OVA, 5 μg);

OVA-Al group (OVA, 5 μg; aluminum adjuvant, 50 μl); and

OVA-AVC-29 group (5 μg each of OVA and AVC-29 adjuvants);

In each of the above groups, the antigen or adjuvant content of each dose of vaccine is shown in brackets, and the volume of each dose of vaccine is 100 μl.

The procedure for immunizing mice was as follows:

According to the above-mentioned vaccine groups, 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 μl. 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

For the immunized mouse serum obtained in Example 1, the serum OVA-specific antibody titer was determined by ELISA method. The specific steps are as follows: OVA protein was diluted to 1 μg/ml with ELISA coating solution, 100 μl was added to each well of a 96-well plate, and left overnight at 4°C. The next day, 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.

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.

Example 3: Expression and purification of SARS-CoV-2 RBD antigen

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 The nucleotide sequence code shown (corresponding to SEQ ID NO: 20 in CN202010581414.3).

SEQ ID NO: 1:

SEQ ID NO: 2:

Add the 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 μm 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. Finally, 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. Molecular sieve chromatography was carried out through Superdex 200 Hi load 16/60 column (GE) for further purification of the target protein. Molecular sieve chromatography buffer was PBS. After molecular sieve chromatography, the only main peak appeared at the elution volume of 80ml, which was collected and concentrated, and stored at -80°C.

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:

Normal saline group;

Al alone adjuvant group (aluminum adjuvant, 50 μl);

AVC-29 adjuvant group alone (AVC-29, 5 μg);

RBD-Al group (RBD antigen, 5 μg; aluminum adjuvant, 50 μl); and

RBD-AVC-29 group (5 μg each of RBD antigen and AVC-29 adjuvant);

In each of the above groups, the antigen or adjuvant content of each dose of vaccine is shown in brackets, and the volume of each dose of vaccine is 100 μl.

The procedure for immunizing mice was as follows:

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 μl. On the 28th day, 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

For the immunized mouse serum obtained in Example 4, 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 μg/ml, add 100 μl to each well of a 96-well plate, and place it overnight at 4°C. The next day, 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.

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

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 μl of Vero E6 cells at a density of 1.5×10 ⁵ cells/mL to 100 μl 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.

The results are shown in Figure 3, and the results in Figure 3 show that in the normal saline group, the Al adjuvant group alone and the AVC-29 adjuvant group immunized mouse serum, no neutralization against SARS-CoV-2 true virus was observed. and antibodies; and for RBD-Al group and RBD-AVC-29 group immunized mice, it was shown that the neutralizing antibody titers induced by them reached 498 and 4469 respectively, that is, the RBD-AVC-29 group was higher than the RBD-Al group Nearly 10-fold higher levels of neutralizing antibodies were induced.

Example 7: Evaluation of Cellular Immune Response Induced by SARS-CoV-2 Vaccine

Spleen treatment: the spleen of the immunized mice obtained in Example 4 was placed in pre-cooled 1640 medium. Put the 40μm 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. Add 4ml of erythrocyte lysate to the collected cell mass, suspend the cells, let stand at room temperature for 5-10 minutes, then add 8ml of 1640 medium, centrifuge, and collect the cells; add 12ml of 1640 medium to wash again, centrifuge again, and collect cell. Add 10ml of 1640 medium to suspend the cells, and count the total number of cells using a cell counter. Centrifuge again to collect the cells; add a certain volume of 10% FBS1640 medium to the collected cell mass to make the cell concentration 1×10 ⁷ cells/ml.

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 μg/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. Then, use PE-labeled anti-mouse CD3 antibody, FITC-labeled anti-mouse CD4 antibody, APC-Cy7-labeled anti-mouse CD8 antibody, BV605-labeled anti-mouse IL-2 antibody, BB700-labeled anti-mouse TNFα antibody, BV421-labeled Anti-mouse IFNγ antibody, BV786-labeled anti-mouse IL-4 antibody and APC-labeled anti-mouse IL-5 antibody (all antibodies were purchased from Biolegend Company), and the cells were stained with antibodies, and the operation steps were completely in accordance with BD Company Cytofix/Cytoperm ^TM Fixation/Permeabilization kit manual and antibody manual operation. Then, cell fluorescence was detected on a FACSCanton flow cytometer.

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 IFNγ, 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.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims (11)

1. An immunogenic or immunostimulatory composition containing AVC-29 or a pharmaceutically acceptable salt thereof, wherein the structure of AVC-29 is as follows:

   
2. The immunogenic or immunostimulatory composition of claim 1, further comprising one or more antigens;

   Preferably, the antigen is a protein, recombinant protein, glycoprotein, peptide, polysaccharide, lipid, lipopolysaccharide or nucleic acid (including DNA and mRNA) of a pathogen;

   Preferably, the antigen is derived from a cell (eg, tumor cell), bacterium or virus;

   Preferably, the antigen is a SARS-CoV-2 virus antigen;

   Preferably, the antigen is a receptor binding domain antigen of SARS-CoV-2 virus;

   Preferably, the antigen has the amino acid sequence shown in SEQ ID NO:1.
3. The immunogenic or immunostimulatory composition of claim 1 or 2, in unit dosage form;

   Preferably, each unit dose of the immunogenic or immunostimulatory composition contains 0.1-500 μg, preferably 1-100 μg, more preferably 5-50 μg of AVC-29 or a pharmaceutically acceptable salt thereof.
4. The immunogenic or immunostimulatory composition according to any one of claims 1-3, wherein the mass ratio of AVC-29 or its pharmaceutically acceptable salt to the antigen is (0.1-100): 1, preferably (0.1 -10):1, more preferably (0.5-5):1.
5. The immunogenic or immunostimulatory composition according to any one of claims 1-4, which is in the form of injectable preparations, inhalable preparations or oral preparations, such as powder injections, suspensions, aqueous injections, sprays, aerosols, etc. Spray, powder spray, paste tablet, sublingual tablet or film;

   Preferably, the immunogenic or immunostimulatory composition further contains one or more of buffers, isotonic agents, preservatives, stabilizers and solubilizers.
6. The preparation method of the immunogenicity or immunostimulatory composition according to any one of claims 1-5, which comprises the following steps:

   AVC-29 or a pharmaceutically acceptable salt thereof is mixed with antigen and optionally other ingredients, preferably in physiological saline, to obtain said immunogenic or immunostimulatory composition; preferably, said After mixing, a drying step, such as freeze drying, is also included.
7. Use of AVC-29 or a pharmaceutically acceptable salt thereof as a vaccine adjuvant or in the preparation of a vaccine adjuvant and/or a vaccine.
8. Use of the immunogenic or immunostimulatory composition of any one of claims 1-5 as a vaccine or in the preparation of a vaccine.
9. The purposes of claim 7 or 8, wherein said vaccine is an inactivated vaccine, an attenuated live vaccine or a gene recombinant vaccine;

   Preferably, the vaccine is a prophylactic or therapeutic vaccine;

   Preferably, said vaccine is used to prevent and/or treat diseases associated with said antigen;

   Preferably, the disease is COVID-19 (new coronavirus pneumonia).
10. The use of any one of claims 7-9, wherein the AVC-29 or a pharmaceutically acceptable salt thereof, or an immunogenic or immunostimulatory composition stimulates (for example, elicits or enhances) the immunity of a subject answer;

    Preferably, the immune response is a non-specific immune response;

    Preferably, said immune response is an antigen-specific immune response;

    Preferably, the immune response includes activation of B cells, activation of T cells, production of antibodies and/or release of cytokines.
11. A method of stimulating (e.g., eliciting or enhancing) an immune response in a subject comprising the step of administering to the subject an effective amount of the immunogenic or immunostimulatory composition of any one of claims 1-5 ;

    Preferably, the immune response is a non-specific immune response;

    Preferably, said immune response is an antigen-specific immune response;

    Preferably, the immune response includes activation of B cells, activation of T cells, production of antibodies and/or release of cytokines.

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