A composite adjuvant and its application in vaccine preparation

Abstract

The present invention provides a composite adjuvant preparation and application comprising cyclic dinucleotides and analogs thereof and / or oligodeoxynucleotides, aluminum salt adjuvants, etc. The composite adjuvant of the present invention is used for the preparation of a vaccine composition. The present invention determines the composite adjuvant preparation process through research on composite adjuvant preparations and animal experiments. The vaccine prepared based on the composite adjuvant of the present invention can effectively enhance the immunogenicity of the vaccine, and provides a new method for vaccine research and development under the existing production capacity and technology.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to a preparation method and application of a composite adjuvant comprising cyclic dinucleotide, oligodeoxynucleotide and aluminum salt. Background Art

[0002] Infectious diseases pose a serious threat to human health. In the face of these infectious diseases, vaccination has significantly reduced the morbidity and mortality of infectious diseases, and some infectious diseases are close to being cured or even eliminated. For example, after efforts to vaccinate, smallpox was declared eradicated worldwide in 1980. In the early stages of vaccine development, live vaccines or live attenuated vaccines dominated, but in the early 20th century, people found that traditional vaccines could not produce an effective immune response with purified diphtheria and tetanus toxoids, so the concept of adjuvants was proposed. Adjuvants are part of vaccines that are designed to enhance a stronger immune response. The composition of adjuvants has undergone a change from natural ingredients to artificially synthesized compounds, and the discovery of the adjuvant effect of aluminum salts in 1926 was a milestone. So far, adjuvants used in FDA-approved human vaccines include aluminum salts, oil-in-water emulsions (MF59, AS03, AF03), AS04 (MPL plus aluminum salts), CpG ODN, and AS01 (MPL and QS-21).

[0003] Toll-like receptor pathway (TLRs) belongs to the innate immune pathogen pattern recognition receptors, which can identify the proteins, nucleic acids, lipids of pathogenic microorganisms that invade the body and their intermediates and metabolites synthesized during the reaction process, such as LPS, peptidoglycan, double-stranded RNA, etc. TLRs can quickly activate intracellular signal cascades by recognizing PAMPs, ultimately leading to the body's production of pro-inflammatory cytokines, anti-inflammatory cytokines and chemokines, activating a variety of immune cells, initiating nonspecific immune responses and stimulating adaptive immune responses to eliminate pathogens.

[0004] CpG ODN is a TLR9 agonist. After the first report in 1995 that CpG motifs can enhance immune stimulation, people have conducted extensive research on bacterial DNA and its synthetic product CpG oligodeoxynucleotides (CpG ODN). People have found that CpG ODN is an adjuvant that can induce a strong Th1 immune response and enhance antigen-specific humoral and cellular immune responses. As a vaccine adjuvant, CPG ODN can work through intramuscular, subcutaneous, oral and intranasal routes.

[0005] Since its discovery in 2008, the STING (stimulator of interferon genes) pathway has been gradually recognized as a core and promising target for immunotherapy. The STING pathway can be stimulated by cyclic dinucleotides (CDNs) to produce type I interferons for immunotherapy of cancer or other diseases. The cyclic dinucleotide adjuvant, cyclic AMP-GMP (cGAMP), is an endogenous cyclic dinucleotide catalyst synthesized by the recently discovered cyclic GMP-AMP synthase (cGAS) and activated by pathogen-derived cytoplasmic double-stranded DNA. cGAMP can bind to the stimulator of interferon genes (STING), thereby activating IRF3 and inducing interferon-β, so cGAMP plays the role of an endogenous second messenger in innate immune signaling through cytoplasmic DNA. In vivo and in vitro studies have shown that cGAMP can be used as an effective adjuvant for antigens.

[0006] In order to improve the effectiveness of vaccines, composite adjuvants can be designed. Compared with single adjuvants, composite adjuvants can simultaneously improve the humoral immune response and cellular immune response of vaccines, and can enhance the effectiveness of vaccines. Providing guidance for the design of composite adjuvants, including the optimization of adjuvant formulas, the design of new adjuvants and their clinical transformation, will be conducive to the development of preventive vaccines. Summary of the invention

[0007] The purpose of the present invention is to screen and provide an effective composite adjuvant formula, and to provide a composite adjuvant preparation method and application capable of improving the immunogenicity of a vaccine.

[0008] In a first aspect of the present invention, a preparation or composition is provided, comprising an aluminum salt, a stimulator of interferon genes (STING) agonist and / or a TLR9 agonist, and the preparation or composition is used as a vaccine adjuvant to prepare a vaccine composition.

[0009] In another preferred embodiment, the aluminum salt includes aluminum hydroxide and aluminum phosphate, preferably aluminum hydroxide.

[0010] In another preferred embodiment, the aluminum salt is aluminum hydroxide hydrate gel.

[0011] In another preferred embodiment, the aluminum hydroxide hydrate gel particle size is 50-150 nm, preferably 80-120 nm, more preferably 90-110 nm, most preferably 95-105 nm.

[0012] In another preferred example, the concentration of the aluminum salt in the prepared vaccine composition is 1 mg / mL-1.5 mg / mL.

[0013] In another preferred embodiment, the STING agonist is an oligonucleotide.

[0014] In another preferred embodiment, the oligonucleotide comprises a cyclic dinucleotide (CDN).

[0015] In another preferred embodiment, the cyclic dinucleotide includes cyclic AMP-GMP (cGAMP).

[0016] In another preferred embodiment, the concentration of the cyclic dinucleotide in the prepared vaccine composition is 1.0 mg / mL-3.0 mg / mL, preferably 1.5 mg / mL-3.0 mg / mL.

[0017] In another preferred embodiment, the TLR9 agonist is an oligonucleotide.

[0018] In another preferred embodiment, the oligonucleotide comprises an oligodeoxynucleotide.

[0019] In another preferred embodiment, the oligodeoxynucleotide comprises CpG oligodeoxynucleotide (CpG ODN).

[0020] In another preferred embodiment, the CpG ODN includes the type B fully sulfhydryl-modified CpG sequence CpG ODN2006 (also known as CpG ODN7909).

[0021] In another preferred embodiment, the concentration of the oligodeoxynucleotide in the prepared vaccine composition is 1.0 mg / mL-3.0 mg / mL.

[0022] In another preferred embodiment, the preparation or composition can enhance the immunogenicity of the vaccine, thereby inducing the body to produce a strong immune response level.

[0023] The second aspect of the present invention provides a method for preparing a vaccine composition: using the preparation or composition as described in the first aspect of the present invention as a vaccine adjuvant to prepare a vaccine composition.

[0024] In another preferred embodiment, the method comprises the steps of:

[0025] (1) Providing an antigen solution for preparing a vaccine composition;

[0026] (2) adding aluminum salt and mixing it thoroughly with the antigen;

[0027] (3) adding the TL9 agonist and / or STING agonist and mixing thoroughly for t1 time to prepare a suspension.

[0028] In another preferred embodiment, the method comprises the steps of:

[0029] (1) Providing an antigen solution for preparing a vaccine composition;

[0030] (2) adding TL9 agonist and / or STING agonist and mixing them thoroughly with the antigen;

[0031] (3) Add aluminum salt and mix thoroughly for t1 time to form a suspension.

[0032] In another preferred embodiment, the method comprises the steps of:

[0033] (1) mixing a TL9 agonist and / or a STING agonist with an aluminum salt;

[0034] (2) The mixture in step (1) is added to the antigen solution used to prepare the vaccine composition, and after being fully mixed for t1 time, a suspension is prepared.

[0035] In another preferred embodiment, the aluminum salt includes aluminum hydroxide and aluminum phosphate, preferably aluminum hydroxide.

[0036] In another preferred embodiment, the aluminum salt is aluminum hydroxide hydrate gel.

[0037] In another preferred embodiment, the particle size of the aluminum hydroxide hydrate gel is 50-150 nm, preferably 80-120 nm, more preferably 90-110 nm, and most preferably 95-105 nm.

[0038] In another preferred example, the concentration of the aluminum salt in the vaccine composition is 1 mg / mL-1.5 mg / mL.

[0039] In another preferred embodiment, the STING agonist is an oligonucleotide.

[0040] In another preferred embodiment, the oligonucleotide comprises a cyclic dinucleotide (CDN).

[0041] In another preferred embodiment, the cyclic dinucleotide includes cyclic AMP-GMP (cGAMP).

[0042] In another preferred example, the concentration of the cyclic dinucleotide in the vaccine composition is 1.0 mg / mL-3.0 mg / mL, preferably 1.5 mg / mL-3.0 mg / mL.

[0043] In another preferred embodiment, the TLR9 agonist is an oligonucleotide.

[0044] In another preferred embodiment, the oligonucleotide comprises an oligodeoxynucleotide.

[0045] In another preferred embodiment, the oligodeoxynucleotide comprises CpG oligodeoxynucleotide (CpG ODN).

[0046] In another preferred embodiment, the CpG ODN includes the B-type fully thiolated CpG sequence CpG ODN2006 (CpGODN7909).

[0047] In another preferred embodiment, the concentration of the oligodeoxynucleotide in the vaccine composition is 1.0 mg / mL-3.0 mg / mL.

[0048] In another preferred embodiment, the buffer of the antigen solution is selected from the following group: phosphate buffer, physiological saline, histidine buffer, Tris-HCl buffer, or a combination thereof.

[0049] In another preferred embodiment, the concentration of the phosphate buffer is 0.01 mM-20 mM, preferably 0.05-10 mM, more preferably 0.05-1 mM.

[0050] In another preferred embodiment, the concentration of the phosphate buffer is 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or 0.05 mM.

[0051] In another preferred embodiment, the pH of the phosphate buffer is 5-10, preferably 6-9, more preferably 6-8.

[0052] In another preferred embodiment, the concentration of the Tris-HCl buffer is 5-15 mM, preferably 8-12 mM, and more preferably 10 mM.

[0053] In another preferred embodiment, the pH of the Tris-HCl buffer is 6-10, preferably 7-9, more preferably 7.5-8.5, and most preferably 8.0.

[0054] In another preferred embodiment, the concentration of the histidine buffer is 1-10 mM, preferably 4-6 mM, and more preferably 5 mM.

[0055] In another preferred embodiment, the buffer is a phosphate buffer with a concentration of 0.05-10 mM and a pH of 6-9.

[0056] In another preferred embodiment, the phosphate buffer is PBS.

[0057] In another preferred embodiment, the pH of the PBS buffer is 6-9, preferably 6-8, more preferably 7.0-7.5, and most preferably 7.2.

[0058] In another preferred embodiment, the concentration of the antigen in the vaccine composition is 50-300 μg / mL, preferably 50-100 μg / mL.

[0059] In another preferred embodiment, the antigen is ovalbumin (OVA protein).

[0060] In another preferred embodiment, the antigen is a viral antigen.

[0061] In another preferred embodiment, the virus includes influenza virus, herpes zoster, coronavirus, or a combination thereof.

[0062] In another preferred embodiment, the coronavirus includes SARS-CoV, 2019 novel coronavirus (SARS-CoV-2), Middle East Respiratory Syndrome Virus (MERS-CoV), or a combination thereof.

[0063] In another preferred embodiment, the influenza virus includes seasonal influenza virus.

[0064] In another preferred embodiment, the seasonal influenza virus includes type A, type B, or a combination thereof.

[0065] In another preferred embodiment, the seasonal influenza virus includes H1N1, H3N2, B Victoria strain, B Yamagata strain, or a combination thereof.

[0066] In another preferred embodiment, the virus comprises a recombinant virus.

[0067] In another preferred embodiment, the virus includes recombinant influenza virus, recombinant herpes zoster virus, and recombinant novel coronavirus.

[0068] In another preferred embodiment, the mixing method includes stirring, shaking, or a combination thereof.

[0069] In another preferred embodiment, the stirring or shaking rate is 30-300 rpm, preferably 50-200 rpm, more preferably 150-200 rpm.

[0070] In another preferred embodiment, t1 is 1.-3 hours, preferably 1.5-2.5 hours, and more preferably 2 hours.

[0071] The third aspect of the present invention provides a vaccine composition, which comprises the preparation or composition as described in the first aspect of the present invention as a vaccine adjuvant, or is prepared by the method as described in the second aspect of the present invention.

[0072] In another preferred embodiment, the content of the antigen in the vaccine composition is 50-300 μg / mL, preferably 50-100 μg / mL.

[0073] In another preferred embodiment, the vaccine is an ovalbumin (OVA protein) vaccine.

[0074] In another preferred embodiment, the vaccine is a viral vaccine.

[0075] In another preferred embodiment, the virus includes influenza virus, herpes zoster, coronavirus, or a combination thereof.

[0076] In another preferred embodiment, the coronavirus includes SARS-CoV, 2019 novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome virus (MERS-CoV), or a combination thereof.

[0077] In another preferred embodiment, the influenza virus includes seasonal influenza virus.

[0078] In another preferred embodiment, the seasonal influenza virus includes type A, type B, or a combination thereof.

[0079] In another preferred embodiment, the seasonal influenza virus includes H1N1, H3N2, B Victoria strain, B Yamagata strain, or a combination thereof.

[0080] In another preferred embodiment, the virus comprises a recombinant virus.

[0081] In another preferred embodiment, the vaccine comprises an influenza virus split vaccine.

[0082] In another preferred embodiment, the vaccine comprises a recombinant herpes zoster vaccine.

[0083] In another preferred embodiment, the vaccine includes a recombinant new coronavirus vaccine.

[0084] In another preferred embodiment, the vaccine is administered to a subject in need thereof, thereby enabling the subject to acquire and / or enhance antigen-specific antibody levels.

[0085] In another preferred embodiment, the administration comprises intramuscular injection.

[0086] In another preferred embodiment, the subject includes an animal model.

[0087] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0088] In another preferred embodiment, the non-human mammals include primates, rodents, carnivores, felines, and caniformes.

[0089] In another preferred embodiment, the subject includes humans, mice, rats, rabbits, monkeys, dogs, cats, camels, and alpacas.

[0090] The fourth aspect of the present invention provides the use of the preparation or composition as described in the first aspect of the present invention for preparing a vaccine composition.

[0091] In another preferred embodiment, the vaccine is an ovalbumin (OVA protein) vaccine.

[0092] In another preferred embodiment, the vaccine is a viral vaccine.

[0093] In another preferred embodiment, the virus includes influenza virus, herpes zoster, coronavirus, or a combination thereof.

[0094] In another preferred embodiment, the coronavirus includes SARS-CoV, 2019 novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome virus (MERS-CoV), or a combination thereof.

[0095] In another preferred embodiment, the influenza virus includes seasonal influenza virus.

[0096] In another preferred embodiment, the seasonal influenza virus includes type A, type B, or a combination thereof.

[0097] In another preferred embodiment, the seasonal influenza virus includes H1N1, H3N2, B Victoria strain, B Yamagata strain, or a combination thereof.

[0098] In another preferred embodiment, the virus comprises a recombinant virus.

[0099] In another preferred embodiment, the vaccine comprises an influenza virus split vaccine.

[0100] In another preferred embodiment, the vaccine comprises a recombinant herpes zoster vaccine.

[0101] In another preferred embodiment, the vaccine includes a recombinant new coronavirus vaccine.

[0102] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 : Graph showing the results of CpG adsorption rate of composite adjuvant preparation methods.

[0104] Figure 2 : Graph showing protein adsorption rate results for composite adjuvant preparation methods.

[0105] Figure 3 : Graph showing the CpG adsorption rate results of composite adjuvant preparation buffer.

[0106] Figure 4 : Graph showing protein adsorption rate results of composite adjuvant preparation buffer.

[0107] Figure 5: The result diagram of the effect of the concentration of the composite adjuvant preparation buffer on the CpG adsorption rate.

[0108] Figure 6 : The result diagram of the effect of the concentration of composite adjuvant preparation buffer on protein adsorption rate.

[0109] Figure 7 : Elisa results of mice immunized with OVA protein and compound adjuvant once and twice 21 days after immunization.

[0110] Figure 8 : Elispot results of mice immunized with OVA protein and compound adjuvants 21 days after the second immunization.

[0111] Fig. 9 :21d Elisa results of mice immunized with influenza virus split vaccine adjuvanted with composite adjuvant.

[0112] Fig.10 : Elisa results of the second immunization of mice with recombinant herpes zoster vaccine adjuvanted with composite adjuvant 21 days later.

[0113] Fig.11 :Elispot results of mice immunized with recombinant herpes zoster vaccine adjuvanted with composite adjuvant at 21 days after the second immunization.

[0114] Fig.12 : 21d Elisa results of the first and second immunizations of mice with the recombinant new coronavirus vaccine adjuvanted with a composite adjuvant.

[0115] Fig.13 : The results of pseudovirus neutralization after two immunizations of mice with the recombinant new coronavirus vaccine adjuvanted with a composite adjuvant for 21 days. DETAILED DESCRIPTION

[0116] The inventors have conducted extensive and in-depth research and, after a large number of screenings, unexpectedly discovered for the first time a class of preparations or compositions that can be used as vaccine adjuvants to prepare vaccine combinations. Experimental results show that the vaccine adjuvants made from the preparations or compositions of the present invention can significantly enhance the immunogenicity of the vaccinated subjects to a variety of viral vaccines. The preparations or compositions of the present invention have simple formulations and are easy to obtain, and are expected to be applied in the field of vaccine production. On this basis, the present invention was completed.

[0117] the term

[0118] In order to better understand the present invention, the following terms are defined.

[0119] Unless otherwise specified, all singular terms also include plural, active and past tenses of the terms. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" refers to more than one compound.

[0120] Unless the context clearly dictates otherwise, the term "about" includes values ​​within a standard deviation of the stated value.

[0121] Where a numerical range is provided, it is to be understood that each intermediate integer of the value and each tenth of each intermediate integer of the value (unless the context clearly indicates otherwise), between the upper and lower limits of the range and any other specified or intermediate values ​​in the specified range are included in the present invention, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed in the present invention, subject to any explicitly excluded limitations within the specified ranges. When the specified range includes one or two of these limits, the range excluding (i) any one or (ii) two of those included limits is also included in the present invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0122] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0123] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0124] As used herein, the term "administering" refers to physically introducing a product of the disclosure into a subject, such as by injection or infusion, using any of a variety of methods and delivery systems, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration.

[0125] As used herein, the terms "disease" and "disorder" are used interchangeably to refer to a condition in a subject. In some embodiments, the condition is a viral infection, particularly an influenza virus infection, a herpes zoster virus infection, or a coronavirus infection. In specific embodiments, the term "disease" refers to a pathological state caused by the presence of a virus in a cell or subject, or by the invasion of a cell or subject by a virus. In certain embodiments, the condition is a disease in a subject, the severity of which is reduced by inducing an immune response in the subject through administration of an immunogenic composition.

[0126] As used herein, the term "effective amount" refers to the amount of a therapy with a preventive and / or therapeutic effect in the case of administering therapy to an individual. In certain embodiments, an "effective amount" refers to an amount of therapy sufficient to obtain the following effects in the case of administering therapy to an individual: reducing or improving the severity of a viral infection, a disease associated therewith, or a symptom, such as (but not limited to) reducing the duration of a viral infection, a disease associated therewith, or a symptom; preventing the development of a viral infection, a disease associated therewith, or a symptom; preventing the occurrence or onset or recurrence of a viral infection, a disease associated therewith, or a symptom; preventing or reducing the spread of a virus from one individual to another; reducing the hospitalization and / or duration of hospitalization of an individual; improving the survival rate of an individual with a viral infection or a disease associated therewith; eliminating a viral infection or a disease associated therewith; inhibiting or reducing viral replication; reducing viral titer; and / or enhancing and / or improving the preventive or therapeutic effect of another therapy. In certain embodiments, an effective amount does not cause full protection from a viral disease, but causes a reduction in viral titer or a reduction in number compared to an untreated individual. Benefits of a reduction in the titer, number, or total viral load include, but are not limited to, less severe symptoms of infection, fewer symptoms of infection, and a reduced duration of illness associated with infection.

[0127] As used herein, the term "infection" means invasion by multiplication and / or presence of a virus in a cell or individual. In one embodiment, the infection is an "active" infection, that is, an infection in which the virus replicates in a cell or individual. The infection is characterized by the spread of the virus from the cells, tissues and / or organs initially infected by the virus to other cells, tissues and / or organs. The infection may also be a latent infection, that is, an infection in which the virus does not replicate. In certain embodiments, infection refers to a pathological state caused by the presence of a virus in a cell or individual, or by the invasion of a cell or individual by a virus.

[0128] Preparations or compositions of the present invention

[0129] The preparation or composition of the present invention can be used as a vaccine adjuvant for preparing a vaccine composition. As used herein, the term "preparation or composition of the present invention" and "composite adjuvant" can be used interchangeably.

[0130] It has been determined that the preparation or composition of the present invention, compared with the prior art, can stimulate animals to produce a significantly stronger immune response to viral vaccines, thereby protecting animals (humans) from influenza virus infection.

[0131] In a preferred embodiment of the present invention, the preparation or combination comprises the following components: an aluminum salt (preferably aluminum hydroxide hydrate gel) and CpG ODN, wherein the concentration of aluminum hydroxide hydrate gel in the prepared vaccine composition is 1 mg / mL-1.5 mg / mL, and the concentration of CpG ODN in the prepared vaccine composition is 1.5 mg / mL-3.0 mg / mL.

[0132] In a preferred embodiment of the present invention, the preparation or combination comprises the following components: an aluminum salt (preferably aluminum hydroxide hydrate gel) and cGAMP, wherein the concentration of aluminum hydroxide hydrate gel in the prepared vaccine composition is 1 mg / mL-1.5 mg / mL, and the concentration of cGAMP in the prepared vaccine composition is 1.5 mg / mL-3.0 mg / mL.

[0133] In some embodiments, the preparation or composition of the present invention is used as a vaccine adjuvant and is suitable for the preparation of vaccine compositions against a variety of viruses. The virus can be selected from, but not limited to: positive-sense single-stranded RNA virus ((+)-ss-envRNAV), negative-sense single-stranded RNA virus ((-)-ss-envRNAV), double-stranded DNA virus (ds-DNAV) or positive-sense RNA derived from DNA virus.

[0134] In some embodiments, the virus is an influenza virus. Influenza viruses are classified into influenza virus types: A, B, and C. As used herein, the term "influenza virus subtype" refers to a type A influenza virus variant characterized by a combination of hemagglutinin (H) and neuraminidase (N) viral surface proteins. According to the present invention, an influenza virus subtype may be referred to as "an influenza virus comprising an HA of the H3 subtype," "an influenza virus of the H3 subtype," or "H3 influenza" using its H number, or as "an influenza virus subtype H3N2" or "H3N2" using a combination of the H number and the N number. The term "subtype" specifically includes all individual "strains" within each subtype, which are typically generated by mutations and display different pathogenic profiles, including natural isolates and artificial mutants or reassortants and the like. These strains may also be referred to as various "isolates" of virus subtypes. Therefore, as used herein, the terms "strain" and "isolate" may be used interchangeably. Current nomenclature for human influenza virus strains or isolates includes the virus type (genus) (i.e., A, B, or C), the geographic location of first isolation, the strain number, and the year of isolation, usually with the antigenic description of HA and NA given in parentheses, e.g., A / Moscow / 10 / 00 (H3N2). Non-human strains also include the host of origin in the nomenclature. Influenza A virus subtypes can additionally be classified by reference to their phylogenetic group. Phylogenetic analysis has shown that hemagglutinins are subdivided into two major groups: particularly the H1, H2, H5, H9 subtypes in phylogenetic group 1 ("group 1" influenza viruses), and particularly the H3, H4, H7, and H10 subtypes in phylogenetic group 2 ("group 2" influenza viruses).

[0135] In some embodiments, the virus is a coronavirus (CoV) that is pathogenic to humans, for example, can be selected from but not limited to: SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU20.

[0136] In some embodiments, the virus is herpes zoster virus (also known as varicella-zoster virus).

[0137] Preparation method of the present invention

[0138] The present invention provides a method for preparing a vaccine composition, wherein the vaccine composition is prepared by using the preparation or composition of the present invention as a vaccine adjuvant.

[0139] In a preferred embodiment of the present invention, the method comprises the following steps: adding aluminum salt to a buffer containing an antigen and mixing thoroughly, then adding CpG ODN or cGAMP and mixing thoroughly for 1-3 hours (optimally 2 hours) to prepare a suspension. The buffer is a phosphate buffer with a concentration of 0.05mM-1.0mM and a pH of 6-9; the mixing method is stirring or shaking at a rate of 50-200rpm. In the final vaccine composition, the antigen content is 50-100μg / mL, the aluminum salt concentration is 1mg / mL-1.5mg / mL, and the CpG ODN or cGAMP content is 1.5mg / mL-3.0mg / mL.

[0140] Vaccine composition of the present invention

[0141] The present invention also provides a vaccine composition prepared using the preparation method of the present invention. A preferred vaccine composition is a preventive vaccine composition. The vaccine composition of the present invention can be a monovalent or multivalent vaccine composition.

[0142] These vaccines contain high-purity, high-titer viral particles and are usually combined with a "pharmaceutically acceptable carrier", which includes any carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition. Suitable carriers are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive viral particles. These carriers are well known to those of ordinary skill in the art. In addition, these carriers can act as immunostimulants ("adjuvants").

[0143] In the present invention, the vaccine uses the preparation or composition described in the first aspect of the present invention as an adjuvant.

[0144] The vaccine composition of the present invention (including virus, pharmaceutically acceptable carrier and / or adjuvant) usually contains a diluent, such as water, saline, glycerol, ethanol, etc. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc. may be present in the vaccine composition.

[0145] More specifically, vaccines, including immunogenic compositions, contain an immunologically effective amount of the virus, as well as the other required components described above. "Immunologically effective amount" refers to an amount that is effective for treatment or prevention when administered to an individual in a single or continuous dose. The amount is determined based on the physiological condition of the animal (such as a human), the ability of the immune system to synthesize antibodies, the degree of protection required, and other relevant factors.

[0146] In the present invention, the vaccine composition or immunogenic composition can be prepared into an injectable preparation, such as a liquid solution or emulsion; it can also be prepared into a solid form suitable for being mixed with a solution or suspension or a liquid excipient before injection. The preparation can also be emulsified or encapsulated in a liposome to enhance the adjuvant effect under the above-mentioned pharmaceutically acceptable carrier.

[0147] Conventional methods are to administer the immunogenic composition by injection from a parenteral (subcutaneous or intramuscular) route. Other formulations suitable for other modes of administration include oral and transdermal applications, etc. The therapeutic dose can be a single dose regimen or a multiple dose regimen. The vaccine composition of the present invention can be administered in combination with other immunomodulators.

[0148] The main advantages of the present invention include:

[0149] 1. Composite adjuvants can enhance the immunogenicity of various vaccines (such as influenza virus split vaccine, recombinant shingles vaccine and recombinant new coronavirus vaccine) and induce the body to produce a strong immune response level.

[0150] 2. Under the existing production capacity and technology, the combination of compound adjuvants and vaccines can effectively expand the immune population and the duration of immunity, and improve economic benefits while saving the amount of antigen used. Therefore, it has important social significance and use value during a viral pandemic.

[0151] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0152] Example 1 Comparison of compound adjuvant preparation methods

[0153] The composite adjuvant was prepared in the following three preparation methods:

[0154] (1) The preparation order of the composite adjuvant is to add 1500 μg of aluminum salt adjuvant to 200 μg of OVA antigen, and then add 2000 μg of CpG ODN 2006 (hereinafter referred to as CpG) or cGAMP adjuvant and mix for 2 hours to prepare 1 mL of suspension.

[0155] (2) The preparation order of the composite adjuvant can be: add 2000 μg CpG or cGAMP to 200 μg OVA antigen, then add 1500 μg aluminum salt adjuvant and mix for 2 hours to prepare 1 mL suspension.

[0156] (3) The preparation order of the composite adjuvant can be as follows: 2000 μg of CpG or cGAMP is mixed with 1500 μg of aluminum salt adjuvant, and then 200 μg of OVA antigen is added and mixed for 2 h to prepare 1 mL of suspension.

[0157] The samples prepared in the above three ways were taken out at 0 min, 2 h, 1 d, 2 d, and 7 d to detect the adsorption amount of OVA antigen and CpG.

[0158] From the results of CpG and protein adsorption rate ( Figure 1 , Figure 2 ), after 7 days of adsorption, the adsorption rate of aluminum salt adjuvant to CpG was between 75% and 80%, and the preparation method (1) was slightly higher than the other two preparation methods; while the adsorption rate of aluminum adjuvant to protein was 100%, which was completely adsorbed, and there was no significant difference among the three. The adsorption efficiency of aluminum salt adjuvant to cGAMP was similar to that of CpG. The above results show that the adsorption rate of aluminum salt adjuvant is different due to different preparation methods.

[0159] Example 2 Comparison of composite adjuvant preparation buffer types

[0160] (1) According to Example 1, the composite adjuvant preparation order is to add 0.75 mg aluminum salt adjuvant to 200 μg OVA antigen (prepared in buffer) and mix, then add 2000 μg CpG adjuvant and mix for 2 h to prepare 1 mL suspension.

[0161] (2) The buffers were 10 mM Tris-HCl (pH 8.0), 10 mM phosphate-buffered saline (PBS) buffer (pH 7.2), normal saline, and 5 mM histidine buffer.

[0162] (3) The samples prepared in the above manner were taken out at time points such as 0 min, 2 h, 1 d, 2 d, 3 d, and 7 d to detect the amount of antigen adsorption.

[0163] From the results of CpG and protein adsorption rate ( Figure 3 , Figure 4 ), except for phosphate buffer, the adsorption rate of aluminum salt adjuvant to CpG after 7 days of adsorption of the other three buffers was higher than 80%, and the adsorption rate of phosphate buffer was only about 40%; except for phosphate buffer, the adsorption rate of aluminum salt adjuvant to protein in the other three buffers was 100%, which was completely adsorbed, and there was no obvious difference among the three, while the protein adsorption rate of phosphate buffer was between 50-60%. This shows that the adsorption rate of aluminum salt adjuvant is different when the buffer is prepared differently.

[0164] Example 3 Comparison of composite adjuvants prepared with different aluminum salts

[0165] (1) According to Example 2, the aluminum salt adjuvants are aluminum hydroxide gel and aluminum phosphate gel adjuvant, respectively. The dosage of the aluminum salt adjuvant is 0.5 mg / mL, the dosage of CpG is 100 μg / mL, the dosage of protein is 100 μg / mL, and the buffer is phosphate saline (PBS) buffer.

[0166] (2) The samples prepared in the above manner were taken out at time points such as 0 min, 2 h, 1 d, 2 d, 3 d, and 7 d to detect the adsorption of antigen and CpG.

[0167] From the results of CpG and protein adsorption rates (Table 1 and Table 2), the adsorption rates of aluminum hydroxide gel to CpG and protein are better than those of aluminum phosphate adjuvant, and the adsorption rates are both greater than 95%.

[0168] Table 1 CpG adsorption rate results of different aluminum salt composite adjuvants.

[0169]

[0170] Table 2 Protein adsorption rate results of different aluminum salt composite adjuvants

[0171]

[0172] Example 4 Determination of the concentration and pH value of the preparation buffer of the composite adjuvant

[0173] Phosphate buffer was selected as the preparation buffer for the composite adjuvant, and the concentration and pH value of the buffer were explored.

[0174] The concentration of the preparation buffer of the composite adjuvant is 10mM, 5mM, 1mM, 0.5mM, 0.1mM and 0.05mM. The pH value of the composite adjuvant buffer is between 6-9. The adsorption of aluminum salts on antigens and CpG or cGAMP adjuvants is related to the isoelectric point of the target protein, but the adsorption effect is not much different. The concentration of the preparation buffer of the composite adjuvant has a great influence on the adsorption efficiency of aluminum salts. The lower the buffer concentration, the better the adsorption effect; when the buffer concentration is between 0.05mM-1mM, the adsorption effect is the best, and the antigen adsorption rate is>95% ( Figure 5 , Figure 6 ).

[0175] Example 5 Comparison of stirring or shaking to prepare composite adjuvant

[0176] The effects of stirring or shaking methods on the preparation of composite adjuvants were compared.

[0177] When the preparation speed is 50rpm, the adsorption effect of the composite adjuvant preparation method on antigen or CpG and cGAMP is not much different when it is stirred or shaken, and the antigen adsorption rate is >95%. In the stirring preparation method, when the preparation speed is 150-200rpm, the adsorption effect of aluminum salt adjuvant is better than 50rpm.

[0178] The compound adjuvant preparation method was determined to be stirring or shaking, and the preparation speed was 50-200 rpm.

[0179] Example 6 Determination of the dosage of the composite adjuvant

[0180] The composite adjuvant was prepared according to the optimal preparation method of the above embodiment, and the appropriate content of aluminum salt adjuvant, CpG or cGAMP and antigen in the composite adjuvant was explored.

[0181] It was finally determined that the aluminum salt adjuvant content was between 1mg / mL-1.5mg / mL, the CpG or cGAMP content was between 1.5mg / mL-3.0mg / mL, and the antigen content was 50-100μg / mL. The higher the aluminum salt content in the composite adjuvant buffer, the more antigen and CpG or cGAMP were adsorbed. In 1mL buffer, 1.5mg aluminum salt adjuvant can completely adsorb 50-100μg of antigen and 1.5-1.75mg of CpG or cGAMP.

[0182] Example 7 Composite adjuvant can enhance the immune effect of OVA protein

[0183] The composite adjuvant vaccine was prepared according to the formula determined in Example 6. The composite adjuvant vaccine was immunized with BALB / C mice by intramuscular injection, 100 μl / mouse, 10 mice in each group. Group A: OVA (10 μg); Group B: OVA (10 μg) + Al; Group C: OVA (10 μg) + CpG; Group D: OVA (10 μg) + CpG + Al; Group E: OVA (10 μg) + cGAMP + Al; Group F: OVA (10 μg) + cGAMP; Group G: PBS. On the 21st day of the first immunization and the 21st day of the second immunization, blood was collected from the tail vein, mouse serum was separated, and the level of humoral immune response in the immunized mice was determined by the Elisa method. Splenocytes were collected 21 days after the second immunization, and the level of IFN-γ secretion by splenocytes in the immunized mice was determined by the Elispot method. 21 days after the second immunization, the CpG and aluminum salt adjuvant and protein combination group produced a higher level of antigen-specific antibodies, with the antibody titer reaching 2 20 ; The cGAMP and aluminum adjuvant and protein combination group produced a higher level of antigen-specific antibodies, with the antibody titer reaching 2 18 ; Compared with the single protein group, the secretion level of IFN-γ increased by 5.1 and 1.9 times, respectively, indicating that a better humoral and cellular immune response was generated ( Figure 7 , Figure 8 ).

[0184] Table 3 Figure 7 The corresponding data table.

[0185]

[0186] Table 4 Figure 8 The corresponding data table.

[0187]

[0188] Example 8 Composite adjuvant can enhance the immune effect of influenza split vaccine

[0189] The composite adjuvant vaccine was injected intramuscularly into BALB / C mice, 100 μl / mouse, 10 mice in each group. Group A: 0.015 μg H1N1 influenza virus split vaccine + Al; Group B: 0.015 μg H1N1 influenza virus split vaccine + CpG; Group C: 0.015 μg H1N1 influenza virus split vaccine + CpG + Al; Group D: 0.015 μg H1N1 influenza virus split vaccine; Group E: PBS. On the 14th, 28th, 90th and 180th day of the first immunization, blood was collected from the tail vein, and the mouse serum was separated. The level of humoral immune response in the immunized mice was determined by the Elisa method. 14 days after the first immunization, compared with the single vaccine group, the antibody titers of each adjuvant combination group increased significantly, reaching the highest value at 28 days, and the antibody titer of the CpG and aluminum salt adjuvant and influenza virus split vaccine combination group reached 2 18 , the antibody titer remained at a high level for 180 days ( Fig. 9 ). This indicates that the combination of adjuvant and vaccine can not only improve the immunogenicity of the vaccine, but also prolong the duration of the immune response.

[0190] Table 5 Fig. 9 The corresponding data table.

[0191]

[0192] Example 9 Composite adjuvant can enhance the immune effect of recombinant herpes zoster vaccine

[0193] The composite adjuvant vaccine was injected intramuscularly into BALB / C mice, 100 μl / mouse, 10 mice in each group. Group A: 5 μg recombinant herpes zoster vaccine; Group B: 5 μg recombinant herpes zoster vaccine + Al; Group C: 5 μg recombinant herpes zoster vaccine + CpG; Group D: 5 μg recombinant herpes zoster vaccine + CpG + Al. Group E: 5 μg recombinant herpes zoster vaccine + cGAMP; Group F: 5 μg recombinant herpes zoster vaccine + Al + cGAMP; Group G: PBS. On the 21st day of the second immunization, blood was collected from the tail vein, mouse serum was separated, and the humoral immune response level in the immunized mice was determined by the Elisa method; spleen cells were collected under sterile conditions, and the IFN-γ secretion level of spleen cells in the immunized mice was determined by the Elispot method. 21 days after the second immunization, compared with the vaccine alone group, the antibody titers of the adjuvant combination groups were significantly increased, and the antibody titer of the CpG and aluminum salt adjuvant and recombinant herpes zoster vaccine combination group reached 2 15 The antibody titer of the cGAMP and aluminum salt adjuvant combined with recombinant herpes zoster vaccine reached 2 15 ; Compared with the protein group alone, the secretion of IFN-γ levels increased by 18.7 and 14.7 times, respectively, indicating that the adjuvant combined with the recombinant herpes zoster vaccine produced a better humoral and cellular immune response ( Fig.10 , Fig.11 ).

[0194] Table 6 Fig.10 The corresponding data table.

[0195]

[0196] Table 7 Fig.11 The corresponding data table.

[0197]

[0198] Example 10 Composite adjuvant can enhance the immune effect of recombinant new coronavirus vaccine

[0199] The composite adjuvant vaccine was immunized with BALB / C mice by intramuscular injection, with 10 mice in each group. They were Group A: 3μg recombinant new coronavirus vaccine; Group B: 3μg recombinant new coronavirus vaccine + Al; Group C: 3μg recombinant new coronavirus vaccine + cGAMP; Group D: 3μg recombinant new coronavirus vaccine + cGAMP + Al; Group E: 3μg recombinant new coronavirus vaccine + CpG; Group F: 3μg recombinant new coronavirus vaccine + CpG + Al; Group G: PBS. On the 21st day of the first and second immunizations, blood was collected from the tail vein, mouse serum was separated, and the serum IgG antibody level in the immunized mice was determined by Elisa method. The serum was taken on the 21st day of the second immunization, and the serum neutralizing antibody level was detected by pseudovirus neutralization test. 21 days after the first immunization, compared with the vaccine group alone, the antibody titers of each adjuvant combination group were increased to varying degrees. 21 days after the second immunization, compared with the vaccine alone group, the antibody titers of each adjuvant combination group increased significantly, and the antibody titer of the cGAMP and aluminum salt adjuvant and recombinant new coronavirus vaccine combination group reached 2 20 , serum neutralizing antibody level NT 50 The antibody titer of the combination of CpG and aluminum salt adjuvant and recombinant new coronavirus vaccine reached 2 20 , serum neutralizing antibody level NT 50 was 8262; compared with the protein group alone, the serum neutralizing antibody levels increased by 13.2 and 11.8 times, respectively, indicating that the combination of adjuvant and recombinant new coronavirus vaccine enhanced the immunogenicity of the vaccine ( Fig.12 , Fig.13 ).

[0200] Table 8 Fig.12 The corresponding data table.

[0201]

[0202] Table 9 Fig.13 The corresponding data table.

[0203]

[0204] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A vaccine composition, characterized in that: The invention comprises a preparation or a composition as a vaccine adjuvant, wherein the preparation or the composition comprises an aluminum salt and an interferon gene stimulating factor agonist; or the preparation or the composition comprises an aluminum salt and a TLR9 agonist; wherein the aluminum salt is aluminum hydroxide hydrated gel, the interferon gene stimulating factor agonist is cyclic adeno-guanosine monophosphate, the TLR9 agonist is a CpG oligodeoxynucleotide, and the CpG oligodeoxynucleotide is a B-type fully thiolated CpG sequence CpG ODN2006; The concentration of the aluminum salt in the vaccine composition is 1 mg / mL-1.5 mg / mL; the concentration of the cyclic adeno-guanosine monophosphate in the vaccine composition is 1.5 mg / mL-3.0 mg / mL; the concentration of the CpG oligodeoxynucleotide in the vaccine composition is 1.0 mg / mL-3.0 mg / mL; the content of the antigen in the vaccine composition is 50-100 μg / mL, and the antigen is a herpes zoster virus antigen; The vaccine composition uses phosphate buffer as the preparation buffer, the concentration of the phosphate buffer is 0.05-10mM, and the pH of the phosphate buffer is 6-9.

2. The vaccine composition according to claim 1, characterized in that The particle size of the aluminum hydroxide hydrate gel is 80-120 nm.

3. The vaccine composition according to claim 1, characterized in that The vaccine composition is injectable.

4. A method for preparing the vaccine composition according to claim 1, characterized in that: The vaccine composition is prepared using the preparation or composition as claimed in claim 1 as a vaccine adjuvant.

5. The method according to claim 4, characterized in that The method comprises the following steps, (1) Providing an antigen solution for preparing a vaccine composition; (2) adding the aluminum salt and mixing it thoroughly with the antigen; (3) Add the TL9 agonist or interferon gene stimulator agonist and mix thoroughly for 1-3 hours to prepare a suspension.

6. The method according to claim 5, characterized in that The buffer of the antigen solution is a phosphate buffer, the concentration of the phosphate buffer is 0.05-10 mM, and the pH of the phosphate buffer is 6-9.

7. The method according to claim 5, characterized in that The mixing method includes stirring, shaking, or a combination thereof.

8. The method according to claim 7, characterized in that The stirring or shaking speed is 50-200 rpm.

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