Adjuvant containing zinc aluminum risedronate and use thereof

Abstract

An adjuvant containing zinc aluminum risedronate, an immunogenic composition comprising said adjuvant and an immunogen, and use of said adjuvant and said immunogenic composition.

Description

[Technical Field]

[0001] Technical Field The present application relates to the technical field of biopharmaceuticals. Specifically, the present application relates to an adjuvant containing zinc aluminum risedronate, an immunogenic composition comprising the adjuvant and an immunogen, and uses of the adjuvant and the immunogenic composition. [Background technology]

[0002] Background technology An adjuvant is a substance or mixture that can bind specifically or nonspecifically to an immunogen and stimulate and induce an organism to produce a long-lasting and effective specific immune response. The immunobiological effects of adjuvants include reducing the dose of an immunogen, enhancing the immunogenicity of an antigen, and changing the type of immune response. Currently approved adjuvants for human vaccines include aluminum adjuvant, MF59, AS04, and AS01. B , CpG1018, and several new adjuvants, including AS03, RC-529, Advax, and Matrix-M, are currently undergoing clinical trials. Aluminum adjuvants were the first adjuvants approved for human vaccines. They have been used for nearly 90 years and are recognized as the most widely used, safe, and effective adjuvants. However, as the number of vaccines studied increases, it has become clear that aluminum adjuvants stimulate the induction of Th2 immunodominant responses and have limited effectiveness in inducing Th1 immune responses, which also limits their use in therapeutic vaccines, such as varicella-zoster virus vaccines, hepatitis B therapeutic vaccines, and tumor vaccines. Furthermore, compared with many new vaccine adjuvants, aluminum adjuvants have weaker activity and their immunoenhancing effect on most genetically engineered antigens other than virus-like particle antigens is less than ideal, limiting their use in non-multimerized protein antigen vaccines.

[0003] Aluminum-containing adjuvant systems refer to adjuvant systems based on aluminum adjuvants and other adjuvant components or presentation systems (e.g., MPL, CPG, QS21, and liposomes), which can induce a relatively balanced Th1 and Th2 immune response. Currently, several aluminum-containing adjuvant systems are approved for sale or undergoing clinical trials (NPJ Vaccines. 2018, Vol. 10, No. 3, p. 51). For example, AS04, developed by GlaxoSmithKline (GSK), is based on an aluminum adjuvant supplemented with the TLR-4 (Toll-like receptor 4) agonist 3-O-deacyl-4'-monophosphoryl lipid A (MPL). Due to its glucosamine phosphorylation, the agonist is an aluminum adjuvant. 3+ It has a high affinity for the antigen and is adsorbed by aluminum adjuvant to form a complex adjuvant, which binds antigen-specific CD4 + It can improve the expression of gamma interferon (IFN-γ) in T cells (IFN-γ is an important indicator of T cell responses). Currently, AS04 has been successfully used in the hepatitis B vaccine (Fendrix, approved for sale in 2005) and the HPV16 / 18 bivalent cervical cancer vaccine (Cervarix, approved for sale in 2007). Compared with the use of aluminum adjuvant alone, AS04 significantly increases neutralizing antibody titers in the HPV16 / 18 bivalent cervical cancer vaccine (Cervarix), prolonging the duration of immune protection. When used for one or two immunizations, AS04 can produce a protective effect similar to that provided by three immunizations. CpG, a TLR-9 receptor agonist, is currently in clinical trials as an adjuvant for malaria and hookworm vaccines in combination with aluminum adjuvant.

[0004] The adsorption of aluminum adjuvants to antigens is one of the factors that contribute to their immunopotentiating effects. Ligand exchange is the strongest interaction between adjuvants and antigens, occurring through ligand exchange between the phosphate groups of the antigen and the hydroxyl groups of aluminum hydroxide or aluminum phosphate. This is the concept of "phosphophilicity" of the aluminum adjuvant surface, proposed by inventor Zhao in 2001 (Analytical Biochemistry, 2001, Vol. 295 (Issue 1): pp. 76-81). AS04 and MPL, mentioned above, are new types of composite adjuvants formed by adsorption of phosphate groups and aluminum adjuvants, and are widely used in clinical settings.

[0005] Bisphosphonates (BPs) are a type of artificially synthesized pyrophosphate analogues with a PCP core. As organic bisphosphonates, they have a high affinity for ions such as calcium, aluminum, zinc, and magnesium due to their phosphate groups. Therefore, they are used to treat bone and calcium metabolism disorders, such as osteoporosis, osteitis deformans, and hypercalcemia, as well as bone pain caused by bone metastasis from malignant tumors. Clinical studies have also shown that the use of BPs in the adjuvant treatment of multiple myeloma, breast cancer, kidney cancer, and prostate cancer can reduce the incidence of bone-related diseases and cancer recurrence, improving patient survival and clinical outcomes. Furthermore, bisphosphonates exhibit adjuvant activity due to their positive immunomodulatory effects, and patent inventions using bisphosphonates as immune-enhancing substances in vaccine formulations have been reported (Chinese Patent No. CN103768595B; US Patent No. US20170281759A1; Chinese Patent No. CN108289902A). Bisphosphonate drugs, which have strong mucosal stimulating effects, are usually administered orally or intravenously in clinical settings, whereas in vaccines, immunization is usually carried out by intramuscular injection. Furthermore, bisphosphonates have potential side effects, and further improvement is still needed.

[0006] Coronaviruses (CoVs) are enveloped, nonsegmented, single-stranded, positive-sense RNA viruses belonging to the Orthocoronavirinae subfamily, Coronaviridae family, and Nidovirales order. They are divided into four genera: α, β, γ, and δ. Seven coronaviruses have been identified to infect humans, including seasonal epidemic coronaviruses 229E and NL63 in the α genus, OC43 and HKU1 in the β genus, and epidemic coronaviruses: severe acute respiratory syndrome-related coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2). Currently, coronaviruses are known to be capable of infecting vertebrates, such as humans, mice, pigs, cats, dogs, mink, guinea pigs, hamsters, rhesus monkeys, and birds. At the same time, Dutch virologists have confirmed that SARS-CoV-2 can be transmitted from humans to mink and then back to humans, confirming the animal-to-human transmission chain of SARS-CoV-2. SARS-CoV, MERS-CoV, and SARS-CoV-2 are highly contagious and pathogenic. The current global SARS-CoV-2 pandemic poses a major threat to people's lives, health, safety, and social activities, and has caused huge economic losses. Vaccines are the most effective means of controlling and preventing viral infections.

[0007] The coronavirus spike protein (S protein) is a type I transmembrane protein composed of two protein subunits, S1 and S2, which mediate binding to host cell surface receptors and fusion of cell membranes, allowing the virus to enter target cells. Multiple lines of evidence suggest that antibodies against the spike protein may play an important role in the immunoprophylaxis and treatment of SARS-CoV-2-induced pneumonia. Therefore, the spike protein is currently an important target for the development and design of recombinant protein vaccines against SARS-CoV-2. Research has shown that acute SARS-CoV-2 infection can weaken long-lasting neutralizing antibody responses but still achieve immunological memory through virus-specific memory T cells (Cell, 2020, Vol. 183(1):13-15), demonstrating the importance of T cell responses in preventing SARS-CoV-2 infection. Subunit protein vaccines prepared by recombinant DNA technology have excellent safety profiles and can achieve multi-shot booster immunization, but their immunogenicity is weak. At the same time, the World Health Organization (WHO) established the target product characteristics for a coronavirus disease 2019 (COVID-19) vaccine in April 2020. In the event of an epidemic, the vaccine must be effective rapidly, providing protection within two weeks, conferring basic immunity with a single dose, not exceeding two doses, and providing protection for at least six months in terms of durability. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a need in the art to develop new adjuvants to improve the efficacy of vaccines (e.g., COVID-19 vaccines). [Means for solving the problem]

[0009] Contents of the present invention After thorough research, the inventors of the present application have developed a new type of adjuvant containing zinc aluminum risedronate, which can be used as a vaccine adjuvant or a drug delivery carrier, etc., and can be used to effectively improve the immunogenicity of antigens.

[0010] In particular, it has surprisingly been found that when the adjuvant of the present application is used in combination with an immunogen (e.g., SARS-CoV-2 S protein), it can effectively stimulate or induce high levels of functional antibodies and balanced cellular and humoral immune responses in various animals, such as Balb / c mice, mink, guinea pigs, Syrian golden hamsters, and cynomolgus monkeys. Thus, the adjuvant of the present application can improve the druggability of vaccines (e.g., SARS-CoV-2 S protein vaccines).

[0011] Furthermore, it has been found that when the adjuvant of the present application is used in combination with the SARS-CoV-2 S protein, it can effectively prevent infection with SARS-CoV-2. For example, a vaccine containing the adjuvant of the present application and the SARS-CoV-2 S protein can effectively prevent or reduce the replication of SARS-CoV-2 in the upper and lower respiratory tract of animals (e.g., mink), and prevent SARS-CoV-2 from infecting and damaging the lungs of animals (e.g., mink).

[0012] Adjuvants In one aspect, the present application provides an adjuvant comprising risedronate zinc aluminum (also referred to herein as a risedronate zinc aluminum adjuvant) having a zinc:risedronate molar ratio ranging from 1:1 to 16:1 (e.g., 2:1 to 16:1) and a zinc:aluminum molar ratio ranging from 1:1 to 50:1 (e.g., 5:1 to 50:1).

[0013] In certain embodiments, the risedronate zinc aluminum is present in the form of particles. In certain embodiments, the risedronate zinc aluminum is present in the form of nanoparticles or microparticles. In some embodiments, the particles have a particle size of 0.01 μm to 100 μm, e.g., 0.01 μm to 60 μm, 0.01 μm to 50 μm, 0.1 μm to 60 μm, 0.1 μm to 30 μm, 0.4 μm to 30 μm, or 0.4 μm to 20 μm.

[0014] In some embodiments, the adjuvant has a zinc:risedronic acid molar concentration ratio ranging from 1:1 to 2:1, 2:1 to 4:1, 4:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 14:1, or 14:1 to 16:1. In certain embodiments, the adjuvant has a zinc:risedronic acid molar concentration ratio of at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, or at least 15:1. In certain embodiments, the adjuvant has a zinc:risedronic acid molar concentration ratio of 16:1 or less, 14:1 or less, 12:1 or less, 10:1 or less, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less, 3:1 or less, or 2:1 or less.

[0015] In certain embodiments, the adjuvant has a zinc:risedronic acid molar ratio of 1:1, 2:1, 4:1, 4.5:1, 6:1, 8:1, 10:1, 12:1, 14:1, or 16:1, hi certain embodiments, the adjuvant has a zinc:risedronic acid molar ratio of 4:1 or 4.5:1.

[0016] In certain embodiments, the adjuvant has a zinc:aluminum molar concentration ratio ranging from 1:1 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 15:1, 15:1 to 20:1, 20:1 to 30:1, 30:1 to 40:1, or 40:1 to 50:1. In certain embodiments, the adjuvant has a zinc:aluminum molar concentration ratio of at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 30:1, or at least 40:1. In certain embodiments, the adjuvant has a zinc:aluminum molar concentration ratio of 50:1 or less, 40:1 or less, 30:1 or less, 20:1 or less, 15:1 or less, 12:1 or less, 10:1 or less, 8:1 or less, 6:1 or less, 5:1 or less, 4:1 or less, 3:1 or less, or 2:1 or less.

[0017] In some embodiments, the adjuvant has a zinc:aluminum molar ratio of 50:1, 40:1, 30:1, 20:1, 15:1, 12:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In certain embodiments, the adjuvant has a zinc:aluminum molar ratio of 10:1.

[0018] In certain embodiments, the adjuvant has a zinc:risedronic acid molar ratio in the range of 2:1 to 8:1, e.g., 2:1 to 4:1, 4:1 to 6:1, or 6:1 to 8:1, and the zinc:aluminum molar concentration ratio is in the range of 2:1 to 50:1 (e.g., in the range of 5:1 to 20:1), e.g., 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 15:1, or 15:1 to 20:1.

[0019] In certain embodiments, the adjuvant has a zinc:risedronic acid molar ratio of 4:1 and a zinc:aluminum molar ratio of 10:1.

[0020] In certain embodiments, the adjuvant has a zinc:risedronic acid molar ratio of 4.5:1 and a zinc:aluminum molar ratio of 10:1.

[0021] In certain embodiments, the adjuvant has a pH of 5.0 to 8.0, e.g., 5.0 to 7.0, 5.0 to 5.5, 5.5 to 6.0, 6.0 to 6.5, 6.5 to 7.0, 7.0 to 7.5, or 7.5 to 8.0. In certain embodiments, the adjuvant has a pH of 7.0 to 7.5. In certain embodiments, the adjuvant has a pH of 5.5 to 6.5. In certain embodiments, the adjuvant has a pH of 5.8 to 6.3.

[0022] In certain embodiments, the adjuvant has a point of zero charge between 3.0 and 8.0, e.g., between 4.0 and 8.0, between 3.0 and 4.0, between 4.0 and 5.0, between 5.0 and 6.0, between 6.0 and 7.0, or between 7.0 and 8.0. In some embodiments, the adjuvant has a point of zero charge between 4.0 and 6.0.

[0023] The adjuvants of the present application have good adsorption rates for immunogens (e.g., proteins). In certain embodiments, the adjuvants of the present invention have adsorption rates for immunogens (e.g., proteins) of at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0024] immunogenic composition Without being limited by theory, the adjuvants of the present invention can be used in combination with various immunogens to enhance the immunogenicity of the immunogen, including but not limited to proteins, nucleic acids, polysaccharides, or immunogenic portions thereof.

[0025] Thus, in one aspect, the present application provides an immunogenic composition comprising an immunogen and an adjuvant as described above.

[0026] In some embodiments, immunogens include, but are not limited to, proteins, nucleic acids, polysaccharides, or immunogenic portions thereof, etc. In certain embodiments, immunogens are derived from pathogens such as viruses, bacteria, and fungi.

[0027] In certain embodiments, the immunogen is a protein or immunogenic fragment thereof, for example, a protein or immunogenic fragment thereof derived from a pathogen such as a virus, bacteria, or fungus. In certain embodiments, the virus includes, but is not limited to, a respiratory virus (e.g., influenza, parainfluenza, rhinovirus, coronavirus, respiratory syncytial virus), an enterovirus (e.g., EV71 virus, rotavirus), or a varicella-zoster virus (VZV).

[0028] In certain embodiments, the immunogen is a coronavirus protein (e.g., spike protein) or an immunogenic fragment thereof. In some embodiments, the coronavirus is selected from the group consisting of orthocoronavirus alphaviruses (e.g., 229E and NL63), orthocoronavirus betaviruses (e.g., OC43 and HKU1), severe acute respiratory syndrome-associated coronavirus (SARS-CoV), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and SARS-CoV-2. In certain embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the immunogen is a structural protein or an immunogenic fragment thereof, such as the S protein or an immunogenic fragment thereof, of SARS-CoV-2.

[0029] In certain embodiments, the immunogen is a varicella-zoster virus protein or an immunogenic fragment thereof, such as the gE protein of varicella-zoster virus or an immunogenic fragment thereof.

[0030] In certain embodiments, the immunogen is an influenza virus protein or immunogenic fragment thereof, such as the HA protein of an influenza virus or an immunogenic fragment thereof.

[0031] In certain embodiments, the immunogen is a rotavirus protein or an immunogenic fragment thereof, such as the VP4 protein of rotavirus or an immunogenic fragment thereof.

[0032] In certain embodiments, the immunogenic composition further comprises pharmaceutically acceptable auxiliary materials, such as excipients, preservatives, antimicrobial agents, buffers, and / or additional immunoadjuvants, hi certain embodiments, the additional immunoadjuvants are selected from the group consisting of aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium pumilus, lipopolysaccharides, cytokines, or any combination thereof.

[0033] In certain embodiments, the immunogenic composition further comprises a second immunogen, including, but not limited to, a protein, a nucleic acid, a polysaccharide, or an immunogenic portion thereof.

[0034] In certain embodiments, the immunogenic composition is a vaccine. Methods for Preparing Adjuvants In one aspect, the present application provides a method for producing a pharmaceutical composition comprising the steps of: 1) providing a soluble salt solution containing zinc ions and aluminum ions; 2) mixing the soluble salt solution of step (1) with an alkaline risedronate solution to obtain an adjuvant; The present invention provides a method for preparing an adjuvant as described above, comprising:

[0035] In some embodiments, the method further comprises sterilizing the adjuvant obtained in step (2). In certain embodiments, the adjuvant is sterilized by filter sterilization or high temperature and high pressure sterilization. In certain embodiments, the adjuvant is sterilized by sterilization at 121°C for at least 15 minutes (e.g., at least 30 minutes, e.g., 30 to 60 minutes).

[0036] In certain embodiments, the soluble salt solution has a zinc:aluminum molar ratio in the range of 1:1 to 50:1 (e.g., in the range of 5:1 to 50:1). In certain embodiments, the zinc:aluminum molar ratio is in the range of 1:1 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 15:1, 15:1 to 20:1, 20:1 to 30:1, 30:1 to 40:1, or 40:1 to 50:1. In certain embodiments, the zinc:aluminum molar concentration ratio is at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 30:1, or at least 40:1. In certain embodiments, the zinc:aluminum molar concentration ratio is 50:1 or less, 40:1 or less, 30:1 or less, 20:1 or less, 15:1 or less, 12:1 or less, 10:1 or less, 8:1 or less, 6:1 or less, 5:1 or less, 4:1 or less, 3:1 or less, or 2:1 or less.

[0037] In certain embodiments, the soluble salt solution has a zinc:aluminum molar concentration ratio of 50:1, 40:1, 30:1, 20:1, 15:1, 12:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In certain embodiments, the soluble salt solution has a zinc:aluminum molar concentration ratio of 10:1.

[0038] In certain embodiments, in step (2), the soluble salt solution is mixed with an alkaline risedronate solution at a zinc:risedronate molar ratio ranging from 1:1 to 16:1 (e.g., ranging from 2:1 to 16:1). In some embodiments, the zinc:risedronate molar ratio is in the range of 1:1 to 2:1, 2:1 to 4:1, 4:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 14:1, or 14:1 to 16:1. In certain embodiments, the zinc:risedronate molar ratio is at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, or at least 15:1. In certain embodiments, the zinc:risedronic acid molar ratio is 16:1 or less, 14:1 or less, 12:1 or less, 10:1 or less, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less, 3:1 or less, or 2:1 or less.

[0039] In certain embodiments, the zinc:risedronic acid molar ratio is 1:1, 2:1, 4:1, 4.5:1, 6:1, 8:1, 10:1, 12:1, 14:1, or 16:1. In certain embodiments, the zinc:risedronic acid molar ratio is 4:1 or 4.5:1.

[0040] In certain embodiments, in step (2), the soluble salt solution is mixed with the alkaline risedronate solution in a manner that allows for the coprecipitation of zinc ions, aluminum ions, and risedronate. In some embodiments, in step (2), the alkaline risedronate solution is added dropwise to the soluble salt solution to allow for the coprecipitation of zinc ions, aluminum ions, and risedronate. In certain embodiments, during the process of mixing the soluble salt solution with the alkaline risedronate solution in step (2), coprecipitation of zinc ions, aluminum ions, and risedronate occurs, resulting in risedronate zinc aluminum particles. In certain embodiments, the resulting risedronate zinc aluminum particles have a particle size of 0.01 μm to 100 μm, for example, 0.01 μm to 60 μm, 0.01 μm to 50 μm, 0.1 μm to 60 μm, 0.1 μm to 30 μm, 0.4 μm to 30 μm, or 0.4 μm to 20 μm.

[0041] In some embodiments, the alkaline risedronate solution is selected from the group consisting of a risedronate and sodium hydroxide solution, a risedronate and phosphate solution (e.g., a risedronate and disodium hydrogen phosphate solution, a risedronate and sodium dihydrogen phosphate solution), or any combination thereof. In some embodiments, the alkaline risedronate solution is a risedronate and sodium hydroxide solution or a risedronate and disodium hydrogen phosphate solution.

[0042] In some embodiments, the soluble salt solution in step (1) is selected from, for example, a sulfate solution, a chlorate solution, an acetate solution, or any combination thereof. In certain embodiments, the soluble salt solution is a chlorate solution or an acetate solution.

[0043] Use of adjuvants In one embodiment, the present application further provides the use of an adjuvant in the manufacture of an immunogenic composition, or as a carrier for delivering an immunogen, or as an immunostimulant for an immunogen. In one embodiment, the present application also provides the use of an adjuvant to enhance the immunogenicity of an immunogen. In one embodiment, the present application also provides the use of an adjuvant to enhance a subject's immune response to an immunogen. In one embodiment, the present application also provides the use of an adjuvant in the manufacture of a formulation for enhancing a subject's immune response to an immunogen. It is easy to understand that the various descriptions above regarding adjuvants, immunogens, etc. can also be applied to these embodiments.

[0044] In certain embodiments, the immune response is a cellular immune response and / or a humoral immune response. In certain embodiments, the cellular immune response is a T cell immune response. In certain embodiments, the T cell immune response is a Th1 immune response and / or a Th2 immune response.

[0045] Method for preparing an immunogenic composition / method for enhancing the immunogenicity of an immunogen In one embodiment, the present application also provides a method for preparing an immunogenic composition, comprising mixing an adjuvant with an immunogen. In one embodiment, the present application also provides a method for enhancing the immunogenicity of an immunogen, comprising mixing an immunogen with an adjuvant. It is easy to understand that the adjuvants of the present invention can be used in combination with various possible immunogens to enhance the immunogenicity of the immunogen. Therefore, the various descriptions above regarding adjuvants, immunogens, etc. can also be applied to these embodiments.

[0046] In certain embodiments, immunogens include, but are not limited to, proteins, nucleic acids, polysaccharides, or immunogenic portions thereof, etc. In certain embodiments, immunogens are derived from pathogens such as viruses, bacteria, and fungi.

[0047] In certain embodiments, the immunogen is a protein or immunogenic fragment thereof, for example, a protein or immunogenic fragment thereof derived from a pathogen such as a virus, bacteria, or fungus. In certain embodiments, the virus includes, but is not limited to, a respiratory virus (e.g., influenza, parainfluenza, rhinovirus, coronavirus, respiratory syncytial virus), an enterovirus (e.g., EV71 virus, rotavirus), or a varicella-zoster virus (VZV).

[0048] In certain embodiments, the immunogen is a coronavirus protein (e.g., spike protein) or an immunogenic fragment thereof. In some embodiments, the coronavirus is selected from the group consisting of an orthocoronavirus alphavirus (e.g., 229E and NL63), an orthocoronavirus betavirus (e.g., OC43 and HKU1), a severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and SARS-CoV-2. In certain embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the immunogen is a structural protein or an immunogenic fragment thereof, such as the S protein or an immunogenic fragment thereof, of SARS-CoV-2.

[0049] In certain embodiments, the immunogen is a varicella-zoster virus protein or an immunogenic fragment thereof, such as the gE protein of varicella-zoster virus or an immunogenic fragment thereof.

[0050] In certain embodiments, the immunogen is an influenza virus protein or immunogenic fragment thereof, such as an influenza virus HA protein or immunogenic fragment thereof.

[0051] In certain embodiments, the immunogen is a rotavirus protein or immunogenic fragment thereof, such as the rotavirus VP4 protein or immunogenic fragment thereof.

[0052] In some embodiments, the method further comprises adding a pharmaceutically acceptable auxiliary material. In certain embodiments, the auxiliary material is selected from, for example, an excipient, a preservative, an antibacterial agent, a buffer, and / or an additional immunological adjuvant. In certain embodiments, the additional immunological adjuvant is selected from the group consisting of aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium pumilus, lipopolysaccharide, cytokines, or any combination thereof.

[0053] In certain embodiments, the method further comprises adding a second immunogen, such as, but not limited to, a protein, a nucleic acid, a polysaccharide, or an immunogenic portion thereof.

[0054] In certain embodiments, the immunogenic composition is a vaccine. Uses of immunogenic compositions In one aspect, the application provides the use of an immunogenic composition in the manufacture of a medicament for preventing and / or treating a disease in a subject, the disease being a disease to be prevented or treated by an immune response induced by the immunogen.

[0055] It is easy to understand that the various descriptions above regarding adjuvants, immunogens, immunogenic compositions, etc. are also applicable to this embodiment. It is also easy to understand that various immunogens can be included in the immunogenic composition, and therefore, the prepared medicament can be used to prevent or treat various corresponding diseases. For example, if the immunogenic composition includes an immunogen derived from a pathogen (e.g., a virus), the immunogenic composition of the present application can be used to prepare a medicament for preventing and / or treating a pathogen infection (e.g., a viral infection) or a disease associated with a pathogen infection (e.g., a viral infection).

[0056] In certain embodiments, the immunogen is derived from a coronavirus (e.g., SARS-CoV-2) and the disease is a coronavirus (e.g., SARS-CoV-2) infection or a disease associated with a coronavirus (e.g., SARS-CoV-2) infection. In certain embodiments, the immunogen is a coronavirus (e.g., SARS-CoV-2) protein (e.g., spike protein) or an immunogenic fragment thereof, and the disease is a coronavirus (e.g., SARS-CoV-2) infection or a disease associated with a coronavirus (e.g., SARS-CoV-2) infection.

[0057] In some embodiments, the immunogen is a structural protein of SARS-CoV-2 (e.g., S protein) or an immunogenic fragment thereof, and the disease is SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection (e.g., pneumonia caused by SARS-CoV-2 (COVID-19)).

[0058] In certain embodiments, the immunogen is derived from varicella zoster virus and the disease is varicella zoster virus infection or a disease associated with varicella zoster virus infection.

[0059] In certain embodiments, the immunogen is a varicella-zoster virus protein (eg, gE protein) or an immunogenic fragment thereof, and the disease is a disease associated with varicella-zoster virus infection.

[0060] In certain embodiments, the immunogen is derived from an influenza virus and the disease is an influenza virus infection or a disease associated with an influenza virus infection.

[0061] In certain embodiments, the immunogen is an influenza virus protein (eg, an HA protein) or an immunogenic fragment thereof, and the disease is influenza virus infection or a disease associated with influenza virus infection.

[0062] In certain embodiments, the immunogen is derived from a rotavirus and the disease is a rotavirus infection or a disease associated with a rotavirus infection.

[0063] In certain embodiments, the immunogen is a rotavirus protein (e.g., the VP4 protein) or an immunogenic fragment thereof, and the disease is a rotavirus infection or a disease associated with a rotavirus infection.

[0064] In certain embodiments, the subject is an animal, such as a bird or a mammal. In certain embodiments, the subject is a rodent, a pig, a cat, a dog, a horse, a primate, or a bird. In certain embodiments, the subject is a non-human subject. In certain embodiments, the subject is a mouse, a mink, a guinea pig, a Syrian golden hamster, or a cynomolgus monkey. In certain embodiments, the subject is a human.

[0065] Methods for stimulating / boosting the immune response In one embodiment, the present application also provides a method for stimulating or enhancing an immune response to an immunogen in a subject, comprising administering to the subject an immunogenic composition comprising an effective amount of an immunogen and an adjuvant of the present invention. It is readily understood that the various descriptions above of adjuvant, immunogen, immunogenic composition, subject, etc. are also applicable to this embodiment.

[0066] In certain embodiments, the immune response is a cellular immune response and / or a humoral immune response. In certain embodiments, the cellular immune response is a T cell immune response. In certain embodiments, the T cell immune response is a Th1 immune response and / or a Th2 immune response.

[0067] In certain embodiments, the immunogenic composition is administered by a route selected from the group consisting of intramuscular injection, subcutaneous injection, intradermal administration, intranasal administration, oral administration, transdermal administration, or intravenous injection, hi certain embodiments, the immunogenic composition is administered by intramuscular injection.

[0068] Disease treatment / prevention methods In one aspect, the present application provides a method of preventing or treating a disease, comprising administering to a subject an effective amount of an immunogenic composition, wherein the disease is a disease that can be prevented or treated by an immune response induced by the immunogen.

[0069] It is easy to understand that the various descriptions above regarding adjuvants, immunogens, immunogenic compositions, subjects, etc., are also applicable to this embodiment. It is also easy to understand that various immunogens can be included in the immunogenic composition, and therefore, the immunogenic composition can be used to prevent or treat various corresponding diseases. For example, if the immunogenic composition contains an immunogen derived from a pathogen (e.g., a virus), the immunogenic composition of the present application can be used to prevent and / or treat pathogen infection (e.g., viral infection) or a disease associated with pathogen infection (e.g., viral infection) in a subject.

[0070] In some embodiments, the disease is a coronavirus (e.g., SARS-CoV-2) infection or a disease associated with a coronavirus (e.g., SARS-CoV-2) infection. Accordingly, in such embodiments, the immunogen is derived from a coronavirus (e.g., SARS-CoV-2). In certain embodiments, the immunogen is a coronavirus (e.g., SARS-CoV-2) protein (e.g., spike protein) or an immunogenic fragment thereof.

[0071] In some embodiments, the disease is SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection, such as pneumonia caused by SARS-CoV-2 (COVID-19). Thus, in such embodiments, the immunogen is derived from SARS-CoV-2. In certain embodiments, the immunogen is a structural protein (e.g., S protein) of SARS-CoV-2 or an immunogenic fragment thereof.

[0072] In certain embodiments, the disease is varicella-zoster virus infection or a varicella-zoster virus infection-related disease. Thus, in such embodiments, the immunogen is derived from varicella-zoster virus. In certain embodiments, the immunogen is a varicella-zoster virus protein (e.g., gE protein) or an immunogenic fragment thereof.

[0073] In certain embodiments, the disease is influenza virus infection or an influenza virus infection-related disease. Thus, in such embodiments, the immunogen is derived from an influenza virus. In certain embodiments, the immunogen is an influenza virus protein (e.g., HA protein) or an immunogenic fragment thereof.

[0074] In certain embodiments, the disease is a rotavirus infection or a rotavirus infection-related disease. Thus, in such embodiments, the immunogen is derived from a rotavirus. In certain embodiments, the immunogen is a rotavirus protein (e.g., the VP4 protein) or an immunogenic fragment thereof.

[0075] In certain embodiments, the subject is an animal, such as a bird or a mammal. In certain embodiments, the subject is a rodent, a pig, a cat, a dog, a horse, a primate, or a bird. In certain embodiments, the subject is a non-human subject. In certain embodiments, the subject is a mouse, a mink, a guinea pig, a Syrian golden hamster, or a cynomolgus monkey. In certain embodiments, the subject is a human.

[0076] Description and explanation of relevant terms in this application In this application, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art.

[0077] According to the present invention, the term "S protein" refers to the SARS-CoV-2 spike protein, which belongs to the type I transmembrane protein and consists of two parts, S1 and S2 protein subunits, and mediates binding to host cell surface receptors and fusion with the cell membrane to allow the virus to enter target cells. The amino acid sequence of the wild-type S protein is known to those skilled in the art, and its exemplary amino acid sequence can be found in Genbank: QHD43416.1. In some embodiments, the amino acid sequence of the wild-type S protein is set forth in SEQ ID NO: 1.

[0078] Those skilled in the art will readily appreciate that the wild-type S protein can be modified, for example, by removing the furin cleavage site, increasing the protein multimerization domain (e.g., increasing the trimerization domain of the T4 bacteriophage fibritin protein), and / or adding tags to achieve desired performance. In certain embodiments, the amino acid sequence of the modified S protein is set forth in SEQ ID NO:4.

[0079] Unless otherwise stated herein or clearly contradicted by the context, the term "S protein" is to be construed as encompassing wild-type S protein as well as modified S proteins.

[0080] According to the present invention, a "gE protein" is an envelope glycoprotein of varicella-zoster virus (VZV). The amino acid sequence of wild-type gE protein is known to those skilled in the art, and its exemplary amino acid sequence can be found in Genbank: DQ008355.1. In some embodiments, the amino acid sequence of the gE protein is set forth in SEQ ID NO:2.

[0081] According to the present invention, the term "HA protein" refers to influenza virus hemagglutinin (HA), which is an important protein mediating viral invasion into host cells and is also the main target of anti-influenza virus neutralizing antibodies. The amino acid sequence of the HA protein of each influenza virus subtype is well known to those skilled in the art. For example, a typical amino acid sequence of type B Victoria lineage influenza virus can be found in Genbank: AIU46088. In a specific embodiment, the amino acid sequence of the HA protein is set forth in SEQ ID NO: 3.

[0082] According to the present invention, the "VP4 protein" is an outer capsid protein of rotavirus. The VP4 protein is an important neutralizing antigen that can stimulate the organism to produce neutralizing antibodies. The amino acid sequence of the wild-type VP4 protein is known to those skilled in the art, and its exemplary amino acid sequence can be found in GenBank: KP752474 or GenBank: MG729832.

[0083] Unless otherwise specified herein or clearly contrary to the context, the gE, HA, and VP4 proteins described herein should be construed to encompass their corresponding wild-type proteins as well as their corresponding modified proteins.

[0084] According to the present invention, the term "adjuvant" refers to a nonspecific immunopotentiating substance that, when delivered to a subject together with or prior to an antigen, can enhance or alter the type of immune response of the subject to the antigen. There are many types of adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium pumilus, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is widely used in clinical trials.

[0085] According to the present invention, the term "pharmaceutically acceptable auxiliary material" may be, for example, an inert diluent such as water or other solvent, a solubilizer and emulsifier, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (specifically cottonseed oil, peanut oil, corn oil, malt oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions can also contain auxiliary materials such as wetting agents, emulsifying agents and suspending agents, sweeteners, flavoring agents, coloring agents, fragrances and preservatives; suspension preparations can also contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, crystalline cellulose, aluminum metahydride, bentonite, agar and tragacanth and their mixtures; these compositions can also contain auxiliary materials such as wetting agents, emulsifying agents and dispersing agents.It can also be desirable to include isotonic agents such as sugar, sodium chloride, etc. in the composition.In addition, prolonged absorption of injectable pharmaceutical forms can be caused by incorporating substances that can delay absorption, such as aluminum monostearate and gelatin.

[0086] As used herein, the term "immunogenicity" refers to the ability to stimulate a subject to form specific antibodies or sensitize lymphocytes.The term "immunogenicity" not only refers to the characteristic of an antigen to stimulate specific immune cells, activate, proliferate and differentiate immune cells, and ultimately produce substances with immune effects, such as antibodies and sensitized lymphocytes, but also refers to the ability of an antigen to stimulate a subject, and induce a specific immune response in the subject, causing the subject's immune system to produce antibodies or sensitized T lymphocytes.Immunogenicity is the most important characteristic of an antigen.Whether an antigen can successfully induce an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the immunization method.

[0087] According to the present invention, the term "immunogenic fragment" refers to a polypeptide fragment that at least partially retains the immunogenicity of the protein from which it is derived. For example, an immunogenic fragment of a coronavirus spike protein (S protein) refers to a fragment of the coronavirus spike protein that at least partially retains the immunogenicity of the S protein.

[0088] According to the present invention, the term "further immunoadjuvants" includes stabilizers; emulsifiers; pH adjusting substances, such as sodium hydroxide, hydrochloric acid, etc.; liposomes; iscom adjuvants; synthetic glycopeptides, such as muramyl dipeptide; bulking agents, such as dextran; carbopol; bacterial cell walls, such as mycobacterial cell wall extracts; derivatives thereof, such as Corynebacterium parvum; Propionibacterium acnes; Mycobacterium bovis, such as Bovine Calmette-Guerin; Examples of suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ-amine or NZ-amine AS, and other standard, metabolizable, non-toxic oils useful in injectable or intranasal vaccine compositions.

[0089] As used herein, the term "subject" can be any animal, such as a bird or a mammal. In certain embodiments, the subject is a rodent, pig, cat, dog, horse, primate, or bird. In certain embodiments, the subject is a non-human subject. In certain embodiments, the subject is a mouse, mink, guinea pig, Syrian golden hamster, or cynomolgus monkey. In certain embodiments, the subject is a human.

[0090] As used herein, the terms "pneumonia caused by SARS-CoV-2" and "COVID-19" refer to pneumonia caused by SARS-CoV-2 infection and have the same meaning.

[0091] According to the present invention, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, an amount effective for preventing a disease (e.g., COVID-19) refers to an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., COVID-19), and an amount effective for treating a disease refers to an amount sufficient to cure or at least partially prevent the onset of the disease and its complications in patients suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the mode of drug administration, and other concurrently administered therapies.

[0092] Beneficial Effects of the Invention Compared with the prior art, the present invention provides an adjuvant containing risedronate zinc aluminum, which can be used as a vaccine adjuvant or a drug delivery carrier, etc., and can be used to effectively improve the immunogenicity of antigens, and the effect is better than that of aluminum adjuvants.

[0093] In particular, when the adjuvants of the present application are used in combination with an immunogen (e.g., SARS-CoV-2 S protein), the adjuvants of the present application can stimulate or induce high levels of functional antibodies and balanced cellular and humoral immune responses in various animals, such as Balb / c mice, mink, guinea pigs, Syrian golden hamsters, and cynomolgus monkeys. Thus, the adjuvants of the present application can improve the drug potential of vaccines (e.g., SARS-CoV-2 S protein vaccines). [Brief explanation of the drawings]

[0094] [Figure 1] Figure 1 shows photographs of aluminum adjuvant (Al001) (Figure 1A) and zinc risedronate aluminum adjuvant (FH002C) (Figure 1B) for suspension appearance and particle morphology under light and electron microscopes. [Figure 2] FIG. 2 shows the particle size measurement results of Al001 (FIG. 2A) and FH002C (FIG. 2B); n=3, mean ± SD. [Figure 3] FIG. 1 shows the results of measuring the zeta potential of Al001 and FH002C at various pH values; n=3, mean±SEM. [Figure 4A] FIG. 1 shows photographs of FH002C adjuvants with various ratios of zinc, aluminum and risedronate in terms of suspension appearance and particle morphology under an electron microscope. [Figure 4B] FIG. 1 shows photographs of FH002C adjuvants with various ratios of zinc, aluminum and risedronate in terms of suspension appearance and particle morphology under an electron microscope. [Figure 4C] FIG. 1 shows photographs of FH002C adjuvants with various ratios of zinc, aluminum and risedronate in terms of suspension appearance and particle morphology under an electron microscope. [Figure 4D] FIG. 1 shows photographs of FH002C adjuvants with various ratios of zinc, aluminum and risedronate in terms of suspension appearance and particle morphology under an electron microscope. [Figure 4E] FIG. 1 shows photographs of FH002C adjuvants with various ratios of zinc, aluminum and risedronate in terms of suspension appearance and particle morphology under an electron microscope. [Figure 4F] FIG. 1 shows photographs of FH002C adjuvants with various ratios of zinc, aluminum and risedronate in terms of suspension appearance and particle morphology under an electron microscope. [Figure 5]Figure 1 shows the antigenicity detection results of recombinant SARS-CoV-2 S protein without adjuvant and dissociated vaccine formulation (FH002C combined with recombinant S protein), where ELISA detection results based on specific binding of ACE2 receptor at various protein concentrations are shown. [Figure 6] Figure 1 shows the results of detecting antibody binding titers and neutralization titers in mouse sera from mice immunized with various immunogen components (recombinant S protein 1 μg, recombinant S protein 10 μg, recombinant S protein + Al001 1 μg, recombinant S protein + Al001 10 μg, recombinant S protein + FH002C 1 μg, and recombinant S protein + FH002C 10 μg); *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=5, mean±SEM. [Figure 7] Figure 1 shows the results of specific antibody binding titers in Balb / C mice immunized with FH002C adjuvant containing various ratios of zinc, aluminum, and risedronate combined with recombinant SARS-CoV-2 S protein; *p<0.05; **p<0.01; ***p<0.001; n=5, mean ± SEM. Note: The abscissa represents the molar concentration ratio of zinc:aluminum:risedronate. [Figure 8] Figure 1 shows the results of specific antibody binding titers in Syrian golden hamsters immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=22 or 16, geometric mean ± geometric mean SD. [Figure 9] Figure 1 shows the results of specific antibody binding titers in cynomolgus monkeys immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, n=2, geometric mean ± geometric mean SD. [Figure 10]Figure showing the results of serum neutralizing antibody titers in Syrian golden hamsters immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=22 or 16, geometric mean ± geometric mean SD. [Figure 11] Figure 1 shows the results of neutralizing antibody titers in cynomolgus monkeys immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, n=2, geometric mean ± geometric mean SD. [Figure 12] Figure showing the results of serum blocking titers from Syrian golden hamsters immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=22 or 16, geometric mean ± geometric mean SD. [Figure 13] Figure 1 shows the results of serum blocking titers from cynomolgus monkeys immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, n=2, geometric mean ± geometric mean SD. [Figure 14] Serum antibody avidity of Balb / c mice immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, n=4 or 5, mean±SEM. [Figure 15] Serum antibody avidity in Syrian golden hamsters immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, n=16, mean±SEM. [Figure 16] Serum antibody subtypes of Balb / c mice immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=4 or 5, mean±SEM. [Figure 17]Figure 17 shows the results of T cell responses in Balb / c mice immunized with FH002C or Al001 in combination with recombinant SARS-CoV-2 S protein, as detected by enzyme-linked immunospot assay. Figure 17A shows the captured images, and Figure 17B shows the counting results of the captured images. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=8, mean±SD. [Figure 18] Figure showing the results of specific antibody binding titers in Balb / C mice immunized with FH002C or Al001 in combination with varicella-zoster virus gE protein, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=5, mean±SEM. [Figure 19] This figure shows the results of specific antibody binding titers in Balb / C mice immunized with FH002C or Freund's adjuvant combined with influenza virus HA protein. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=10, mean ± SEM. Note: "Victoria / WT" refers to the wild-type HA protein of Victoria strain influenza virus that was not treated with deglycosylase (Pn gauze), and "Victoria / Pn gauze" refers to the wild-type HA protein of Victoria strain influenza virus that was treated with Pn gauze. [Figure 20] FIG. 1 shows the results of neutralizing antibody titers in Balb / C mice immunized with FH002C or Al001 in combination with rotavirus VP4 protein, n=5, mean±SEM. [Figure 21] FIG. 1 shows the results of neutralizing antibody titers in guinea pigs immunized with FH002C or Al001 in combination with rotavirus VP4 protein, n=5, mean±SEM. DETAILED DESCRIPTION OF THE INVENTION

[0095] Sequence information The sequences referred to in this application are set forth in the table below.

[0096] [Table 1-1]

[0097] [Table 1-2]

[0098] Specific Models for Implementing the Invention The present invention will now be described with reference to the following examples which are intended to illustrate, but not limit, the present invention; however, those skilled in the art will understand that the following figures and examples are used merely to illustrate the invention, rather than to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

[0099] The reagents used in the preparative examples were as follows: Risedronate sodium (C7H) was purchased from Hunan Huateng Pharmaceutical Co., Ltd. 10 NNaO7P2); Anhydrous zinc chloride (ZnCl2) purchased from Xilong Chemical Industry; Aluminum chloride hexahydrate (AlCl3·6H2O) purchased from Xilong Chemical Industry; disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) purchased from Xilong Chemical Industry; Sodium hydroxide (NaOH) purchased from Xilong Chemical Industry.

[0100] Preparation Example 1: Preparation of Risedronate Zinc Aluminum Adjuvant (FH002C) (1) Preparation of solutions: According to the Zn / Al / risedronic acid molar ratio of 1:0.1:0.22, 0.5 L of (47 mM zinc chloride + 4.7 mM aluminum chloride) solution was prepared, which was defined as solution A, and 0.5 L of (10.3 mM risedronic acid + 55 mM sodium hydroxide + 63 mM disodium hydrogen phosphate) solution was prepared, which was defined as solution B. Solutions A and B were filtered through a 0.22 μm filter membrane for later use.

[0101] (2) Preparation of risedronate zinc aluminum adjuvant FH002C suspension: Risedronate zinc aluminum adjuvant was prepared by coprecipitation using a 1:1 volume ratio of solution A and solution B. That is, prepared solution B was added dropwise to solution A in a 1:1 volume ratio until solution B was completely added to form a suspension. The resulting suspension was sterilized once at 121°C for 60 minutes, and the post-sterilization physical and chemical properties, such as pH, particle size, and particle shape, were measured.

[0102] Preparative Example 2: Preparation of Risedronate Zinc Aluminum Adjuvant (FH002C) with Varying Ratios of Zinc, Aluminum, and Risedronate (1) Preparation of solutions: Using the molar concentration of Zn as 1, the designed Zn / Al / risedronate molar concentration ratios were 1:0.02:0.1, 1:0.02:0.15, 1:0.02:0.22, 1:0.02:0.33, 1:0.02:0.5, 1:0.02:1 and 1:0.05:0.1, 1:0.05:0.15, 1:0.05:0.22, 1:0.05:0.33, 1:0.05:0.5, 1:0.02:1. .05:1 and 1:0.1:0.1, 1:0.1:0.15, 1:0.1:0.22, 1:0.1:0.33, 1:0.1:0.5, 1:0.1:1 and 1:0.15:0.1, 1:0.15:0.15, 1:0.15:0.22, 1:0.15:0.33, 1:0.15:0.5, 1:0.15:1 and 1:0.25:0.1, 1:0.25:0.15, 1:0.25: 0.22, 1:0.25:0.33, 1:0.25:0.5, 1:0.25:1 and 1:0.5:0.1, 1:0.5:0.15, 1:0.5:0.22, 1:0.5:0.33, 1:0.5:0.5, 1:0.5:1, (47 mM zinc chloride + 0.94 mM, 2.35 mM, 4.7 mM, 7.05 mM, 11.75 mM, or 23.5 mM aluminum chloride), respectively. ) solution, 0.5 L, was prepared and defined as Solution A, and 0.5 L of (4.7 mM, 7.05 mM, 10.3 mM, 15.51 mM, 23.5 mM, or 47 mM risedronate + 35 mM to 250 mM sodium hydroxide + 63 mM disodium hydrogen phosphate) solution, 0.5 L, was prepared and defined as Solution B. Solutions A and B were filtered through a 0.22 μm filter membrane for later use.

[0103] 2) Preparation of the suspension: Risedronate zinc aluminum adjuvant was prepared by coprecipitation using a 1:1 volume ratio of solution A and solution B. That is, prepared solution B was added dropwise to solution A in a 1:1 volume ratio until solution B was completely added to form a suspension. The resulting suspension was sterilized once at 121°C for 60 minutes.

[0104] Preparation Example 3: Preparation of aluminum adjuvant Al001 According to a phosphate / Al molar ratio of 0.15:1, 0.5 L of 124 mM aluminum chloride solution was prepared, which was defined as solution A, and 0.5 L of 18.6 mM disodium hydrogen phosphate solution was prepared, which was defined as solution B, which contained 140 mM sodium hydroxide and was filtered through a 0.22 μm filter membrane for later use.

[0105] The preparation process of aluminum adjuvant Al001 suspension can refer to the preparation of FH002C suspension.

[0106] Example 1: Determination of the physical and chemical properties of aluminum zinc risedronate (FH002C) and aluminum adjuvant (Al001) The resulting FH002C suspension (Preparation Example 1) and Al001 suspension (Preparation Example 3) were sterilized once at 121°C for 60 minutes, and their physical and chemical properties after sterilization, such as pH, particle size, particle shape, and point of zero charge, were measured.

[0107] (1) Observation of the appearance and particle morphology of the adjuvant Risedronate zinc aluminum adjuvant FH002C was diluted 1x with 100mM NaAc buffer solution, and aluminum adjuvant Al001 was diluted 1x with saline. Photographs were taken using a China Huawei Mate30 5G mobile phone camera to record the appearance of the adjuvants, while a China Motic AE31E inverted biological microscope was used to observe and photograph their particle morphology, with the eyepiece and objective lenses both at 10x magnification.

[0108] Aluminum adjuvant Al001 and zinc risedronate aluminum adjuvant FH002C were diluted 50-fold and 100-fold with deionized water, respectively, and then observed using a Japan Electronics JEM-2100 transmission electron microscope (TEM). The specific steps were as follows: the adjuvant sample was dropped onto a copper grid and allowed to absorb for 10 minutes. After wiping off the remaining liquid with filter paper, the sample was transferred to the sample chamber of the transmission electron microscope, where the morphology was observed and photographed.

[0109] Experimental Results: As shown in Figure 1, both risedronate zinc aluminum adjuvant FH002C and aluminum adjuvant Al001 were milky white suspensions, and under an optical microscope, both appeared as amorphous clusters. Under an electron microscope, FH002C exhibited an irregular sheet-like structure, while Al001 adjuvant exhibited a transparent fibrous structure.

[0110] (2)pH measurement Test samples were taken, allowed to stand at room temperature for at least 30 minutes and measured using a Sartorius acidity meter.

[0111] Standard buffer solution (pH 7.00), standard buffer solution (pH 4.01), and standard buffer solution (pH 10.01) were selected and used to calibrate the instrument according to the instruction manual.

[0112] The "Mode" (switch) key could be pressed to switch between pH mode and mV mode. Usually, the mode was set to pH mode, which determines the solution pH value.

[0113] Press the "SETUP" key, the display will show "Clear buffer", and press the "ENTER" key to confirm and clear previous calibration data.

[0114] The "SETUP" key was pressed until the display showed the buffer solution group "4.01, 7.00, 10.01" followed by confirmation by pressing the "ENTER" key.

[0115] The electrodes were removed from the electrode storage solution and rinsed thoroughly with deionized water, and after rinsing, the surface water was dried with filter paper (note: the electrodes should not be wiped).

[0116] The electrode was immersed in the first buffer solution (pH 7.00) until the value stabilized and "S" appeared, and the "STANDARDIZE" key was pressed to automatically calibrate the instrument. After successful calibration, "7.00" and the electrode slope were displayed.

[0117] The electrode was removed from the first buffer solution, rinsed thoroughly with deionized water, and then immersed in the second buffer solution (pH 4.01) until the value stabilized and then "S" appeared. The "Standardize" key was pressed to automatically calibrate the instrument. After successful calibration, "4.01 7.00" and the message "Slope" were displayed, where "Slope" indicated the measured electrode slope value, which was acceptable within the range of 90% to 105%.

[0118] If there is a relatively large deviation from the theoretical value, an error message (Err) is displayed, the electrode should be washed, and the above steps should be repeated for calibration.

[0119] The above procedure was repeated to complete the third point calibration (pH 10.01). After calibration, the electrode was rinsed thoroughly with deionized water, then gently blotted and dried with filter paper. The test solution was shaken thoroughly, the glass electrode was immersed in the test solution, and a reading was taken when the pH value did not change by more than ±0.05 within 1 minute.

[0120] The test solution was shaken and measured again; the difference between the two pH values ​​should not exceed 0.1. The average of the two readings was taken as the pH value of the test solution.

[0121] Experimental results: The pH of Al001 adjuvant was 8.00-8.40 before sterilization and 6.50-6.90 after sterilization. The pH of FH002C adjuvant was 7.00-7.50 before sterilization and 5.80-6.30 after sterilization.

[0122] (3) Determination of particle size The Beckman LS 13320 laser particle sizer was turned on and allowed to preheat for 15 minutes.

[0123] The instrument control software and the closed compartment of the sample cell were opened, the sample cell was removed from the sample tank, and 12 mL of purified water was added.

[0124] The sample cell was placed in the sample holder and the door was closed. After opening the LS13320 software, the instrument self-test was performed by clicking "OK" in the pop-up window. The detection module was opened by clicking "run" on the menu bar and selecting "Use Optical Module." The automatic stirrer built into the sample cell was started by clicking "control" on the menu bar and selecting "stirrer on." The blank background calibration was initiated by clicking "start cycle," selecting "Measure Offsets," "Align," and "Measure Background" in that order, and finally clicking "start." The blank background calibration was initiated by clicking "OK" in the pop-up dialog box.

[0125] Remove the sample cell, add a certain amount of standard sample (provided with the instrument), click "Start Cycle", select "Measure Loading", "Enter Sample Info", "Enter run setting", "Start runs", and finally click "Start". Enter the standard sample name in the pop-up dialog box, and click "OK" when the software's "Obscuration" parameter is between 8% and 12%. The standard sample was then tested.

[0126] To ensure the accuracy and reliability of the experimental data, the blank background and test standard samples needed to be calibrated in size before each start of measurement.

[0127] The closed compartment of the sample cell was opened and the sample cell was removed. The aqueous solution containing the standard sample in the sample cell was discarded, and deionized water was poured into the sample cell to wash the sample cell three times.

[0128] After cleaning, 12 mL of deionized water was added, the sample cell was placed in the sample tank, and the door was closed.

[0129] Click "Start Cycle," select "Measure Offset," "Align," and "Measure Background" in sequence, and finally click "Start" and "OK" in the pop-up dialog box to begin blank background calibration.

[0130] The sample cell was removed, a certain amount of test sample was added, the sample test door was opened, the sample cell was placed in the sample tank, and the door was closed.

[0131] Click "Start Cycle," select "Enter Sample Information," "Enter Run Settings," and "Start Run," and finally click "Start." Enter the sample name in the pop-up dialog box. Click "OK" when the software's "Opacity" parameter was between 8% and 12%. The particle size of the measured sample was recorded. Three replicate measurements were performed for aluminum adjuvant Al001 and aluminum risedronate zinc FH002C, respectively.

[0132] Experimental Results: As shown in Figure 2, Al001 showed a unimodal particle distribution, with sizes ranging from 0.4 μm to 60 μm, with the main peak at 15 μm to 16 μm (Figure 2A); risedronate zinc aluminum adjuvant FH002C showed a bimodal particle distribution, with sizes ranging from 0.4 μm to 25 μm, with the main peak particle diameter at approximately 5.0 μm (Figure 2B).

[0133] (4) PZC (point of zero charge) detection Detection equipment: Nanobrook Omni (Brookhaven) Experimental Procedure: The power was turned on and the multi-angle particle size and high sensitivity zeta potential meter was preheated for 30 minutes.

[0134] By using 36% to 38% hydrochloric acid and 10 M NaOH, the Al001 adjuvant was adjusted to have a pH of 6.5, 7.0, 7.5, 8.0, or 8.5, and the FH002C adjuvant was adjusted to have a pH of 3.0, 4.0, 5.0, 6.0, or 7.0.

[0135] Passivation of the electrode: 3-4 mL of adjuvant was placed in the sample tube. After inserting the electrode, the instrument was run with the SOP set to 50 cycles to passivate the electrode.

[0136] Sample detection: Remove the electrode, rinse the bottom end with deionized water, add the corresponding sample, click "instrument parameters" in the SOP, select "square polystyrene cell" for "cell type," select "BI-SREL (1250 μL)" for "electrode assembly," click "advanced settings," set the "equilibration time" to 120 seconds, set the "inter-cycle delay" to 1 second, set "time dependent" measurements to 3, set the "time interval" to 0, click "liquid" to set the pH to the pH corresponding to each sample, click "model" in "Data Analysis," select "Smoluchowski," finally click "save," and then click "OK" to complete the parameter settings.

[0137] Instrument execution: After the adjuvants with different pH values ​​were thoroughly shaken, 2 mL was taken with a pipette and transferred into a plastic sample tank in order, the electrode was inserted and then placed in the sample tank, the instrument was connected to the sample tank, data was collected, and the measurement was started by clicking "Start". Then, the next sample test was carried out.

[0138] Data processing: The corresponding zeta potentials at various pH values ​​were obtained, and the software provided with the instrument was run to obtain PZC values, which were then plotted using GraphPad Prism 8.0.2 (manufacturer: GraphPad Software, LLC).

[0139] Experimental Results: As shown in Figure 3, the PZC of aluminum adjuvant Al001 was 7.52, and the PZC of zinc risedronate-aluminum adjuvant FH002C was 4.75.

[0140] (5) Precipitation rate of Zn / Al / risedronate in FH002C Experimental method: The contents of zinc and aluminum in the free supernatant were detected by an atomic absorption spectrometer (Shimadzu Corporation, AA6300C (P / N 206-52430)), and risedronate was detected by an ultraviolet spectrophotometer (Beckman, DU800). The details were as follows: Determination of zinc element content by flame photometry using atomic absorption spectrophotometer: Preparation of standard curve: Zinc single element standard solution (Beijing General Research Institute for Nonferrous Metals, GSB 04-1761-2004) was diluted with 0.2% nitric acid to obtain zinc standard solutions of 0 ng / mL, 500 ng / mL, 1000 ng / mL, 1500 ng / mL, and 2000 ng / mL.

[0141] Preparation of test solutions: Samples were diluted with 0.2% nitric acid solution until the concentration was within the range of the standard curve (Abs reading of 0.2-0.8), with shaking and mixing using a vortex mixer before each dilution.

[0142] Login to WizAArd: Double-click the WizzAArd icon to open the software, click the "Measurement" icon in the "Operation" column, enter "Admin" in the "Login ID" column, and enter the password without password. <ok>I clicked.

[0143] Wizard Selection: Select "Element Selection" <ok>I clicked.

[0144] Element Selection: Select the "Select Element" option on the "Element Selection" page, select "Zn" on the "Loading Parameters" page, select the "Flame Continuous" measurement method, uncheck the "Use ASC" option, and select "Common Lamp". <ok>I clicked. I didn't set anything in the "Calibration Curve Settings" and "Edit Parameters" pages, <ok>On the "Not Connected Instrument / Send Parameters" page, clicked <Connect / Send Parameters>.

[0145] On the "Instrument Initialization" page, the ASC and GFA options for the graphite furnace method do not need to be checked, and the "Check Gas" dialog box appears, <no>Select "Check safety device now, will the burner pressure monitor be checked?" <yes>Select "Please supply air, click the OK button after supplying air, air discharge" and a dialog box will appear. <ok>Select "Check NO2" and "Check NO2" will be displayed. <no>If you select "NO2-C2H2 cannot be used", a dialog box will appear. <ok>Select "Elevate the waste liquid probe above the liquid level" and follow the prompts to add enough ultrapure water to the waste liquid tank until water flows out of the waste pipe to prevent backfire, then follow the prompts to "Move the waste liquid probe below the liquid surface" and close the opening of the waste liquid tank. After checking all the items, <ok>I clicked.

[0146] Check all items on the "Instrument Inspection List for Flame Analysis" page and tick them. <ok>I clicked.

[0147] On the "Optical Parameters" page, set the wavelength to [213.86nm], set the slit width to [0.7], set the illumination method to [emission], set the [lamp position setting] to ensure that the actual position of the Zn hollow cathode lamp is the same as the set position, select [Lighting], and when "Ready" appears at the bottom of the screen, <ok>I clicked.

[0148] On the "Spectral Line Search" page, when both "Spectral Line Search" and "Beam Balance" displayed [OK], click [Close], return to the "Optical Parameters" page, and click <Next>. On the "Atomizer / Gas Flow Settings" page, do not change anything and click <Finish>.

[0149] Burner Origin Position Adjustment: Select [Instrument] → [Maintenance] → [Burner Origin Position Adjustment] and adjust the burner position using the forward / backward position knob and the wrench on the burner. Using the AA-6300C standard card, observe the position of the emitted light and use the scale on the card to ensure that the light passing through the entire burner slit is on the same horizontal line. After adjustment, the Abs value stabilizes. Click <move up> or <move down> to adjust the vertical height of the burner via the computer, first coarsely and then finely, until the Abs value is half the maximum reading (ensuring that half of the maximum light intensity passes through the burner). Select <Origin Memory>, close the current page, and place the safety cover on the burner.

[0150] Select [Parameter] → [Edit Parameter] on the menu bar and change the lighting method. <bgc-d2>I performed "Line Search" again, and when both "Line Search" and "Beam Balance" displayed [OK], I clicked <Close>.

[0151] Ignition: After ensuring that the C2H2 is turned on and the pressure meets the requirements, press the PURGE and IGNITE keys on the host simultaneously until ignition occurs.

[0152] Auto zero adjustment: Before the formal detection of the sample, select auto zero adjustment to remove residual impurities in the burner.

[0153] The blank group (BLK), standard substance (STD), and sample to be tested (UNK) were set on the MRT worksheet, the theoretical concentration of the standard substance and the sample name were entered, and the sample was manually loaded through the sample loading tube extending from the atomizer. The sample volume for each run was at least 1 mL, and detection was performed by selecting [Start].

[0154] Data processing: Create a scatter plot in Excel based on the Abs and concentrations of the standards, add trend lines, and calculate the equation and correlation coefficient R 2 This will display R 2 A correlation of ≥99% was demonstrated, demonstrating good correlation and could be used to calculate sample concentrations.

[0155] Determination of aluminum element content by graphite method using atomic absorption spectrometer: Preparation of standard curve: Aluminum single element standard solution (National Nonferrous Metals and Electronic Materials Analysis and Testing Center, GNM-SAl-002-2013) was diluted with 2% nitric acid to obtain aluminum standard solutions of 0 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, and 200 ng / mL.

[0156] Preparation of test solutions: Samples were diluted at 0.2% until the concentration was within the range of the standard curve (Abs reading of 0.2-0.8).

[0157] Login to WizAArd: Double-click the WizzAArd icon to open the software, click the "Measurement" icon in the "Operation" column, enter "Admin" in the "Login ID" column, and enter the password. <ok>Click Wizard Selection: Select "Element Selection" <ok>I clicked.

[0158] Element selection: Select the "Element Selection" option on the "Element Selection" page, select "Al" on the "Loading Parameters" page, check the "Use ASC" option, select [Common Light], and click <Next>. No special settings are required on the "Calibration Curve Settings" and "Parameter Editing" pages. <ok>I turned on the power switch wrench (HEAT) of the GFA-EX7i and clicked <Connect / Send Parameters> on the "Connect to Host / Send Parameters" page. When the dialog box "The instrument is not connected, will it be connected?" appears, <yes>I clicked.

[0159] On the "Instrument Initialization" page, the options for flame C2H2 values, burner, gas, flame monitor, burner, and waste probe do not need to be checked. <no>After checking all the items, <ok>Bypass flame analysis: When the "Will flame analysis be performed?" dialog box appears, <no>I clicked.

[0160] On the "Optical Parameters" tab page, set the wavelength to [396.2nm], set the slit width to [0.7nm], set the illumination method to [Emission], set the [Set Lamp Position] to ensure the aluminum lamp is in the set position, check the [Illumination] option, and when "Ready" appears in the bottom right corner of the screen, <ok>I clicked.

[0161] "Spectral Line Search" page: When "Spectral Line Search" and "Beam Balance" both displayed [OK], click [Close], return to the "Optical Parameters" page, and then click <Next>.

[0162] On the "Graphite Furnace Program" page, I selected [7] for "Maximum Phase Number," [6] for "Sampling Phase Number," and [Unknown] for "Graphite Tube Type," and clicked <Finish>.

[0163] Graphite furnace atomizer origin position adjustment: Select "Instrument", "Maintenance", "Graphite furnace origin position adjustment", and after the atomizer position reading stabilizes, move the graphite furnace atomizer by using the manual forward / backward position knob until the Abs reading reaches its maximum, and use the Wizard software to adjust the atomizer's up / down position, first with "Fast" adjustment and then with "Slow" adjustment, until the reading reaches its maximum value, and click <Origin Memorization>.

[0164] Checking the ASC nozzle position: Select [Instrument] → [Graphite Furnace Nozzle Position] from the menu bar and follow the computer prompts to move the sample aspiration nozzle first to the ASC rotating plate and then to the graphite furnace atomizer. Following the WizAArd software prompts, first loosen the arm guide screw and click <Move Down> to move the sample aspiration nozzle down. Once it approached the graphite furnace inlet, adjust the injection position adjustment knob on the ASC workbench to horizontally adjust the position of the sample aspiration nozzle back and forth and left and right until it was centered in the inlet. Using <Move Up> and <Move Down>, adjust the sample loading position first coarsely and then finely. Using the observation mirror on the ASC, observe the position of the sample aspiration nozzle at the inlet. Its position was optimized at 1 / 3 of the distance between the sample aspiration nozzle and the bottom of the inlet. <ok>Click to save your current location and close the current page.

[0165] Repeatedly check the ASC nozzle position: Tighten the knob, select [Instrument] → [Graphite Furnace Nozzle Position] from the menu bar, move the sample aspiration nozzle to the atomizer, and then select [Direction Moving Tube]. If the sample aspiration nozzle was in the center of the inlet, no adjustment was necessary. If the sample aspiration nozzle position was offset, adjust it to the center of the inlet and select <Cancel> until the sample aspiration nozzle was in the center of the inlet.

[0166] On the menu bar, select [Parameters] → [Edit Parameters] → and select the lighting method. <bgc-d2>I performed "Line Search" again, and when both "Line Search" and "Beam Balance" displayed [OK], I clicked <Close>. Cleaning: Before officially testing the sample, I selected <Cleaning> to remove any remaining impurities in the graphite tube.

[0167] On the MRT worksheet, the positions and sample volumes (10 μL) of the blank group (BLK), standard substance (STD), and sample to be tested (UNK) were set in order, the theoretical concentration of the standard substance and the sample name of the sample to be tested were entered, and <Start> was selected to perform detection.

[0168] Processing the results: According to the Abs values ​​and theoretical concentrations of the standards, create a scatter plot in Excel, add a trend line, and calculate the formula and correlation coefficient R 2 where R 2 A correlation of ≧99% was demonstrated to be good and could be used to calculate sample concentrations.

[0169] The risedronate content of FH002C supernatant was determined by UV spectrophotometer: Power on and preheating: The main power switch on the right rear of the device was turned on, and general preheating was carried out for 10 to 20 minutes.

[0170] Optional measurements were performed according to the requirements: Select "Fixed Wavelength" in the options column on the first line in the upper left corner of the screen and perform the measurements.

[0171] Fixed wavelength (fixed wavelength measurement): Click the "Edit Method" icon to display the wavelength setting page, select "262 nm wavelength" in the "Number of wavelengths" field, and click "OK" after setting. Load the sample from the blank tube into a clean cuvette, place it in the colorimetric rack, click the "BLK" icon to subtract the blank, then add the sample individually and click "Read."

[0172] Experimental results: Based on the obtained standard curve, the formula: (total content of each component - content of each component in the supernatant) / total concentration of each component * The concentrations of zinc ions, aluminum ions, and risedronate in the supernatant of FH002C were calculated based on the precipitation rate of each component, i.e., 100%. The precipitation rates of zinc and aluminum in the risedronate zinc-aluminum adjuvant FH002C were calculated to be greater than 99.0%, and those of aluminum and risedronate were greater than 99.9% (Table 2).

[0173] [Table 2]

[0174] (6) Determination of adsorption rate The recombinant SARS-CoV-2 S protein was designed and prepared as follows: Briefly, the trimerization domain of the T4 phage fibritin protein was fused to the C-terminus of the full-length extracellular segment of the wild-type S protein of SARS-CoV-2 (SEQ ID NO: 1), removing the furin cleavage site, and then 6 * A recombinant SARS-CoV-2 S protein (recombinant S protein, SEQ ID NO: 4) was engineered with a His-tag fused to the C-terminus. The recombinant S protein was expressed in a Chinese hamster ovary carcinoma cell (CHO) expression system (purchased from Thermo Scientific, A29133) and purified using a nickel-agarose column (Cytiva, 17-5318-03) to obtain recombinant S protein with a purity of over 95%. The purified recombinant S protein was quantified using the BCA method (Pierce™ BCA Protein Assay Kit, Thermo Scientific, 23227). The recombinant S protein was diluted to 400 μg / mL and mixed with FH002C adjuvant at a recombinant S protein:adjuvant ratio of 1:1 (volume ratio). The mixed sample preparation was shaken and allowed to stand at 4°C for adsorption. Sampling was performed individually at each time point. After shaking the wells, 500 μL of each sample was taken, and the sample was centrifuged at 13,000 rpm / min for 5 minutes, and then the supernatant was collected. The sample supernatant was then diluted to a corresponding ratio according to the results of the preliminary experiment for later use.

[0175] Drawing of standard curve: Recombinant S protein stock solution was used as the standard and subjected to gradient dilution, using sample diluent SD-1 as the dilution buffer, to dilute the standard to a series of concentrations specified in EP tubes for later use: 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.12 ng / mL, 1.56 ng / mL.

[0176] Testing Procedure: (1) Plate coating: 36H6 monoclonal antibody (self-produced in the laboratory; 36H6 monoclonal antibody recognizes the RBD epitope on the SARS-CoV-2 S protein and is prepared using hybridoma technology; the method was adapted from Li et al., Emerging Microbes Infection. 2020) was diluted to 1 μg / mL using 1x CB9.6 coating buffer, and then added at 100 μL / well to a polystyrene 96-well plate. Coating was carried out overnight at 4°C.

[0177] (2) Blocking: The coating solution in the wells was discarded, the plate was washed once with PBST washing solution, and centrifuged to dryness. Blocking solution-1 (200 μL / well) was added, and blocking was carried out at 25° C. for 4 hours.

[0178] (3) The blocking solution in the wells was discarded, the plate was washed once with PBST, and spun dry. The diluted standard and sample supernatants were added to the corresponding 96-well plate and incubated at 25°C for 1 hour.

[0179] (4) Addition of enzyme-labeled antibody (85F7-HRP) (prepared in-house): The liquid in the wells was discarded, the plate was washed five times with PBST, and spun dry. 100 μL / well of enzyme-labeled antibody (85F7-HRP, diluted 1:5000 (V:V) with ED-11 as enzyme diluent) was added, and the plate was incubated at 25°C for 1 hour.

[0180] (5) Color development: The enzyme-labeled antibody in the wells was discarded, the plate was washed five times with PBST, and centrifuged to dryness. An equal volume of mixed color development solutions A and B was added to each well at 100 μL / well, and the reaction was carried out at 25°C for 10 minutes.

[0181] (6) Stopping: 50 μL / well of 2 M sulfuric acid stopping solution was added to stop the reaction. (7) Plate reading: The detection wavelengths were set at 450 nm and 630 nm on a microplate reader, and the OD value of each reaction well was measured.

[0182] Calculation of FH002C adjuvant adsorption rate: The OD readings of the diluted sample wells were inserted into the standard curve to obtain the corresponding protein concentration of the sample wells, which was then multiplied by the corresponding dilution factor to obtain the protein concentration of the supernatant of the original sample. Adjuvant adsorption rate = (total protein concentration of the original sample - protein concentration of the supernatant) / total protein concentration of the original sample. * 100%.

[0183] The experimental results are shown in Table 3. According to the adsorption rate calculation formula, when the FH002C vaccine formulation combined with recombinant S protein was adsorbed for 30 hours, the adjuvant adsorption rate reached over 95%. When it was left standing at 4°C for 70 days, the protein adsorption rate of the vaccine formulation was always above 95% (96.5%-99.9%).

[0184] [Table 3]

[0185] Example 2: Determination of the physical and chemical properties of risedronate zinc aluminum adjuvant (FH002C) with various ratios of zinc, aluminum, and risedronate The resulting FH002C suspension (Preparation Example 2) and Al001 suspension (Preparation Example 3) were sterilized once at 121°C for 60 minutes, and their physical and chemical properties, such as pH, adsorption rate, metal precipitation rate, appearance and particle morphology, were determined.

[0186] (1) Determination of pH The pH determination method was the same as the pH determination process in Example 1.

[0187] Experimental Results: The pH values ​​of FH002C adjuvants with different ratios of zinc, aluminum and risedronate were 7.20-7.90 before sterilization and 6.00-6.60 after sterilization.

[0188] (2) Determination of adsorption rate Drawing a standard curve for BSA standard: BSA standard (2 mg / mL) was serially diluted using 100 mM NaAc as a dilution buffer, and the absorbance at 280 nm was measured using a UV spectrophotometer to draw a standard curve.

[0189] Preparation of BSA: Using 100 mM NaAc as the dilution buffer, a certain amount of BSA sample was weighed into an EP tube and diluted to a final concentration of 1 mg / mL.

[0190] Mixing of BSA and adjuvant: After shaking the adjuvant, mixing was carried out according to 1 mg / mL BSA:adjuvant = 1:1 (volume ratio), and adsorption was carried out at room temperature for 1 hour, during which time shaking was carried out 5 times, and after centrifugation (13000 rpm / min, 3 min), the supernatant was collected for later use.

[0191] Determination of adsorption rate: The absorbance value of the supernatant at 280 nm was measured directly using a UV spectrophotometer, resulting in a reading of 0.2-0.8. Calculation of adsorption rate: The OD reading of the diluted sample well was introduced into the standard curve to obtain the corresponding protein concentration of the sample well, and then multiplied by the corresponding dilution factor to obtain the protein concentration of the supernatant of the original sample. Adjuvant adsorption rate = (total protein concentration of the original sample - protein concentration of the supernatant) / total protein concentration of the original sample. * 100%.

[0192] The experimental results are shown in Table 4. According to the adsorption calculation formula, the adsorption rates of FH002C adjuvants with various ratios of zinc, aluminum, and risedronate ranged from 39% to 96%. Using 75% as the standard, adjuvants with adsorption rates above 75% were selected to observe the metal precipitation rate.

[0193] [Table 4]

[0194] (3) Determination of metal precipitation rate Preparation of mixed metal element standard solutions: In a 12 mL centrifuge tube, the standard solution with four single metal elements was diluted with 5% nitric acid to the highest concentration point of the calibration curve to obtain STD7 solution, which was then serially diluted with 1% nitric acid (6 mL of hydrochloric acid was added to 6 mL of the previous standard working solution) to obtain STD6 to STD1 solutions, with the final volume of each mixed standard solution being 6 mL to ensure complete sample loading.

[0195] [Table 5]

[0196] Preparation of test solution (at least 4 mL): Take the test sample, equilibrate it to room temperature, mix thoroughly with a vortex mixer, accurately sample the test sample, dilute it to an appropriate ratio with 5% nitric acid solution (200 times is recommended for determining calcium, magnesium and zinc content, and 50 times is recommended for determining aluminum content), mix well, and let it stand for 1 hour to prepare the test sample solution.

[0197] Preparation of blank control solution: 5% nitric acid solution was used as the blank control. Measurement: The argon gas bottle (adjusted to have an air pressure of approximately 0.6 MPa) was opened. Blowing gas equilibration: The ICP-OES instrument and software were turned on, and "Purge Gas Flow" was selected in the "Dashboard" of the software interface, followed by the "Normal" model in the drop-down options. The instrument was allowed to equilibrate in this state for at least 1 hour. Upon equilibration, the "Torch Compartment," "Plasma Gas Pressure," "Purge Gas Pressure," "Drain Flow," "Exhaust Flow," and "Optics Temperature" on the software interface should be observed and turn green. After opening the water tank and waiting approximately 2 minutes, the "Detector Water Flow" and "Detector Temperature" in the instrument status bar should turn green. The equipment pipeline was connected and cleaned: the catheter inlet was placed in ultrapure water, the catheter was correctly connected to the peristaltic pump, the "Pump Speed" in the selection interface was changed to 50, and "Apply" was clicked to observe whether the liquid could flow normally from the catheter outlet. After 1-2 minutes of cleaning, ignition could be performed. Ignition: Select "Dashboard" in the software interface, click the blue "Get Ready" icon in the center at the top of the operation interface, click "OK" in the pop-up window, and wait approximately 2-5 minutes for ignition to be successful. If the initial ignition was not recognized as normal, the ignition operation was performed again after the equipment had been completely cleaned automatically. The x and y values ​​of the "Carbon line" in the log were observed. Both absolute values ​​should be less than 10.Creating a LabBook: Click "LabBooks" on the right menu bar, enter the name of the experiment to be performed in the "Name" field (experiment date + experiment + operator), select "Create a new LabBook from an existing LabBook," open the "Risedronate Sodium-Zinc-Aluminum Adjuvant Method Template," and click "Create LabBook" at the bottom of the interface. A LabBook for the current experiment in the risedronate zinc aluminum adjuvant template was successfully created. Parameter selection: In the newly created template interface, the "Analytes" field on the right was checked for the elements to be measured and their detection wavelengths: Zn (213.856 nm), Al (396.152 nm), Ca (396.847 nm), and Mg (279.553 nm). In the "Measure Modes" column, check whether the observation direction is "Radial." Creating a sample loading sequence: Select "Sample list" in the right column, enter the sample information and loading position into the sequence, save it, and click the green triangle icon "Start" in the upper left corner, followed by the green triangle icon in the lower left corner to start the sequence. When measuring each sample, the catheter was placed in a centrifuge tube. After each injection measurement, residual droplets on the catheter should be wiped off with dust-free paper (or filter paper) before the system instructs the user to enter "wash" to clean the catheter. When moving the catheter from "wash" to the next sample, droplets on the catheter should also be wiped off. Calculating the results: Four standard curves were plotted with the concentration of the mixed standard solution as the abscissa and the corresponding peak area as the ordinate, and a linear regression equation was created. The peak area of ​​each single element corresponding to the diluted test sample solution was substituted into the regression equation and multiplied by the dilution factor to obtain the metal element concentration (ppm) of the test sample solution.

[0198] Experimental results: As shown in Tables 6, 7 and 8, Zn was mainly present in the precipitate, and Mg and Ca were partially present in the precipitate and partially present in the supernatant. According to the results of the precipitation rates of Mg and Ca, the grayed areas were selected to observe the appearance and particle morphology of the adjuvants.

[0199] [Table 6]

[0200] [Table 7]

[0201] [Table 8]

[0202] (4) Observation of the appearance and particle morphology of the adjuvant By referring to the detection process in Example 1, observation of the appearance and particle morphology of the adjuvant was carried out.

[0203] Experimental Results: As shown in Figures 4A-4F, the risedronate zinc aluminum adjuvants with various ratios of zinc, aluminum, and risedronate were all milky white suspensions with varying degrees of turbidity, and under electron microscope, most of them had irregular sheet-like structures, and some of them had fibrous structures.

[0204] Example 3: Determination of antigenic activity of recombinant SARS-CoV-2 S protein preparations containing FH002C (based on ACE2 receptor binding) FH002C (prepared as in Preparation Example 1) and recombinant SARS-CoV-2 S protein were mixed in a 1:1 volume ratio to form a vaccine formulation, which was then placed at 4°C for 1 week (fully adsorbed) to test its antigenicity. A non-adsorbed recombinant SARS-CoV-2 S protein stock solution was used as a control to compare antigenicity after adsorption and dissociation of the adjuvant.

[0205] The antigenicity of the protein was detected by an in vitro relative potency test based on ACE2 receptor binding: (1) Antibody coating solution: 1x CB9.6 buffer (15mM Na2CO3; 35mM NaHCO3).

[0206] (2) Washing solution: PBST, ELISA kit from INNOVAX Company. (3) Blocking solution: Blocking solution-1, ELISA kit from INNOVAX Company.

[0207] (4) Coloring solution A: ELISA kit from INNOVAX Company. (5) Coloring solution B: ELISA kit from INNOVAX Company.

[0208] (6) Stop solution: ELISA kit from INNOVAX Company. Testing Procedure: (1) Plate coating: SARS-CoV-2 S protein antibody 45C3 (self-prepared in the laboratory) was diluted to 1 μg / mL with 1× CB9.6 coating buffer and added to a polystyrene 96-well plate at 100 μL / well. Coating was carried out overnight at 4°C.

[0209] (2) Blocking: The coating solution in the wells was discarded, the plate was washed once with PBST washing solution, and spun dry. 200 μL / well of blocking solution was added, and blocking was carried out at room temperature for 4 hours.

[0210] (3) Sample preparation: recombinant S protein antigen stock solution (before adsorption) and antigen formed by dissociation of recombinant S protein vaccine formulation (the concentration of recombinant S protein antigen in the formulation was 100 μg / mL, and the adjuvant was FH002C). The antigen concentration in the first well was 4 μg / mL. The blocking solution in the wells was discarded, the plate was washed once with PBST, and spun dry. The sample to be tested was added to the first well at 200 μL / well, and 100 μL of sample diluent solution was added to each subsequent well to perform two-fold serial dilutions. The plate was then incubated at 25°C for 1 hour.

[0211] (4) Addition of ACE2-hFc (purchased from SinoBiological, Cat. No.: 10108-H02H): The liquid in the wells was discarded, the plate was washed five times with PBST, and spun dry. ACE2-hFc (1 μg / mL, 100 μL / well) was added, and the incubation reaction was carried out at 25°C for 1 hour.

[0212] (5) Addition of enzyme-labeled antibody (MAH-HRP) (purchased from SouthernBiotech, Cat. No.: 9042-05): The liquid in the wells was discarded, the plate was washed five times with PBST, and centrifuged to dryness. 100 μL / well of enzyme-labeled antibody (MAH-HRP, V:V=1:5000) was added, and the incubation reaction was carried out at 25°C for 1 hour.

[0213] (5) Color development: The enzyme-labeled antibody in the wells was discarded, the plate was washed five times with PBST, and centrifuged to dryness. An equal volume of mixed color development solution A and B (100 μL / well) was added, and the incubation reaction was carried out at 25°C for 10 minutes.

[0214] (6) Stopping: 50 μL / well of 2 M sulfuric acid stopping solution was added to stop the reaction. (7) Plate reading: The OD value of each reaction well was measured by setting the detection wavelengths at 450 nm and 630 nm on a microplate reader.

[0215] The experimental results are shown in Figure 5. The results of antigenicity detection based on ACE2 receptor binding showed that after FH002C combined with the recombinant SARS-CoV-2 S protein vaccine formulation was dissociated in the dissociation solution, its antigenicity was comparable to that of the recombinant S protein stock solution (rEC 50 =EC 50 , stock solution / EC 50 , after dissociation = 0.9), indicating that the recombinant SARS-CoV-2 S protein vaccine formulation containing FH002C has good stability.

[0216] Example 4: Dose-Effect Correlation Study of Recombinant SARS-CoV-2 S Protein with Risedronate Zinc Aluminum Adjuvant The FH002C adjuvant prepared according to Preparation Example 1 and the Al001 adjuvant prepared according to Preparation Example 3 were used as adjuvants and mixed with SARS-CoV-2 recombinant S protein at a volume ratio of 1:1 to form vaccines, which were then intramuscularly injected into mice to detect specific antibody binding titers and neutralization titers in the serum. The specific methods were as follows: Experimental animals: Balb / C mice (purchased from Shanghai Slack Experimental Animal Co., Ltd., Cat. No.: 2017000502480), 6 to 8 weeks old, 5 mice per group, female.

[0217] Experimental groups: (1) low-dose recombinant S protein aqueous solution group (1 μg); (2) high-dose recombinant S protein aqueous solution group (10 μg); (3) low-dose recombinant S protein + Al001 group (1 μg); (4) high-dose recombinant S protein + Al001 group (10 μg); (5) low-dose recombinant S protein + FH002C group (1 μg); (6) high-dose recombinant S protein + FH002C group (10 μg).

[0218] Immunization protocol: 1 μg or 10 μg of recombinant S protein per animal was mixed with adjuvant at a volume ratio of 1:1 to form a vaccine, which was then intramuscularly injected into mice at 100 μL per mouse, 50 μL into each hind leg of the mouse. Immunizations were performed at weeks 0 and 3. Three weeks after the initial immunization of the animals according to the immunization group, blood samples were collected from the orbit to determine the specific antibody titer in the serum. After a booster immunization at week 3, blood samples were collected from the orbit every week, and the specific antibody binding titer in the serum was determined by ELISA.

[0219] Detection of antibody binding titer by enzyme-linked immunosorbent assay (ELISA): (1) Antibody coating solution: 1x CB9.6 buffer (15mM Na2CO3; 35mM NaHCO3).

[0220] (2) Washing solution: PBST, ELISA kit from INNOVAX Company. (3) Blocking solution: Blocking solution-1, ELISA kit from INNOVAX Company.

[0221] (4) Coloring solution A: ELISA kit from INNOVAX Company. (5) Coloring solution B: ELISA kit from INNOVAX Company.

[0222] (6) Stop solution: ELISA kit from INNOVAX Company. Testing Procedure: (1) Plate coating: The antigen recombinant S protein was diluted to a certain concentration using CB9.6 coating buffer, and added to a polystyrene 96-well plate at 100 μL / well, followed by coating overnight at 4°C.

[0223] (2) Blocking: The coating solution in the wells was discarded, the plate was washed once with PBST washing solution, and dried by centrifugation. 200 μL / well of blocking solution was added, and blocking was carried out at room temperature for 4 hours.

[0224] (3) Addition of serum at a fixed dilution: Discard the blocking solution in the wells, wash the plate once with PBST, and centrifuge dry. Add 200 μL / well of the serum to be tested to the first well, add 100 μL of sample diluent to each subsequent well, perform two-fold serial dilutions, and perform the incubation reaction at 25°C for 1 hour.

[0225] (4) Addition of enzyme-labeled antibody (GAM-HRP) (purchased from Bio-Rad Laboratories Inc., Cat. No.: 1706516): The serum dilutions in the wells were discarded, and the plate was washed five times with PBST and centrifuged to dryness. 100 μL / well of enzyme-labeled antibody (GAM-HRP, V:V = 1:5000) was added, and the incubation reaction was carried out at 25°C for 1 hour.

[0226] (5) Color development: The enzyme-labeled antibody in the wells was discarded, the plate was washed five times with PBST, and dried by centrifugation. An equal volume of mixed color development solutions A and B was added to each well at 100 μL / well, and the reaction was carried out at 25°C for 10 minutes.

[0227] (6) Stopping: 50 μL / well of 2 M sulfuric acid stopping solution was added to stop the reaction. (7) Plate reading: The OD value of each reaction well was measured by setting the detection wavelengths at 450 nm and 630 nm on a microplate reader.

[0228] The detection method for specific antibody neutralization titers in serum was as described in HL Xiong et al., Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells. Emerg Microbes Infect, pp. 1-38 (2020).

[0229] The experimental results are shown in Figure 6: Three weeks after immunization, mice in the FH002C adjuvant group showed higher antibody titers than those in the aqueous solution and Al001 adjuvant groups, and 1 to 1.5 orders of magnitude higher than those in the aluminum adjuvant group, demonstrating rapid response. After two rounds of immunization, the humoral immune-boosting benefits of the FH002C adjuvant were still evident. At week 4, in the low-dose group, antibody titers in the FH002C adjuvant group were 1.5 orders of magnitude higher than those in the aluminum adjuvant group and 3 orders of magnitude higher than those in the aqueous solution group. In the high-dose group, antibody levels in the FH002C adjuvant and Al001 adjuvant groups were comparable, and both were 2 orders of magnitude higher than those in the aqueous solution group. At weeks 5 and 6, in both the high-dose and low-dose groups, antibody titers in the FH002C adjuvant group were still significantly higher than those in the Al001 group (0.5 to 1 order of magnitude higher) and than those in the aqueous solution group (2.5 to 3 orders of magnitude higher).

[0230] After one shot, only mice in the FH002C adjuvant group produced neutralizing antibodies at week 3, whereas mice in the aqueous solution and Al001 adjuvant groups did not. After the second shot of immunization, at weeks 4, 5, and 6, the neutralizing titers of mice in the FH002C adjuvant group, whether in the high-dose or low-dose group, were higher than those in the Al001 adjuvant group (0.5 to 1.5 orders of magnitude higher) and higher than those in the aqueous solution group (1.5 to 2.5 orders of magnitude higher).

[0231] Example 5: Specific antibody binding titers induced by FH002C adjuvant with various ratios of zinc, aluminum, and risedronate combined with recombinant SARS-CoV-2 S protein The prepared FH002C (see Preparation Example 2 for the preparation method) was used as an adjuvant and mixed with recombinant SARS-CoV-2 S protein at a volume ratio of 1:1 to form a vaccine, which was then intramuscularly injected into Balb / C mice to measure the titer of the specific antibodies produced. The specific method was as follows: Experimental animals: Balb / C mice, 6-7 weeks old, female, 5 mice per group Immunization protocol: 1 μg of antigen per mouse, i.e., 100 μL per mouse (half in the left limb and half in the right limb). Two immunization shots were performed at week 0 and week 3, and orbital blood was collected once a week from week 0 to week 6 to detect antibody titers in the serum.

[0232] Enzyme-linked immunosorbent assay (ELISA) was used to detect antibody binding titers, which was illustrated as follows: The reagents used in the ELISA were those described in Example 4.

[0233] Testing Procedure: (1) Coating and blocking: Recombinant S protein was diluted to 1 μg / mL in CB9.6 and added to a 96-well microtiter plate at 100 μL / well, placed, and incubated at 37° C. for 2 hours. After that, the plate was washed once with PBST buffer (i.e., PBS containing 0.05% Tween-20), and 200 μL of PBS containing 20% ​​NBS was added to each well, followed by incubation at 37° C. for 2 hours to perform blocking. After removing excess liquid, it was centrifuged and dried for later use.

[0234] (2) Serum dilution: 100 μL of 300-fold diluted serum was added to the first well, followed by 3-fold dilution with a 10-fold gradient, and incubated at 37° C. for 1 hour.

[0235] (3) Addition of enzyme-labeled antibody: After washing the plate five times with PBST buffer, 100 μL of goat anti-mouse horseradish peroxidase enzyme-labeled antibody GAM-HRP (self-prepared in the laboratory) diluted at a certain ratio was added and incubated at 37°C for 0.5 hours.

[0236] (4) Develop, stop, and read. Experimental Results: As shown in Figure 7, the FH002C adjuvants with various ratios of zinc, aluminum, and risedronate combined with recombinant SARS-CoV-2 S protein were all able to induce specific binding antibodies.

[0237] Example 6: Specific antibody binding titers induced by risedronate zinc aluminum adjuvant in combination with recombinant SARS-CoV-2 S protein The prepared Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were used as adjuvants and mixed with recombinant SARS-CoV-2 S protein in a volume ratio of 1:1 to form a vaccine, which was then intramuscularly injected into Syrian golden hamsters (also referred to herein as "Syrian hamsters") and cynomolgus monkeys to determine the titer of specific antibodies produced. The specific method was as follows: Experimental animals: Syrian golden hamsters (purchased from Shanghai Slack Experimental Animal Co., Ltd.), 6-7 weeks old, 22 animals per group for FH002C group, 16 animals per group for Al001 group, half male and half male; adult cynomolgus monkeys (purchased from Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.), 2 animals per group, half male and half male.

[0238] Experimental grouping: (1) recombinant S protein + Al001; (2) recombinant S protein + FH002C Immunization protocol for Syrian golden hamsters: 10 μg of antigen per animal was individually mixed with the recombinant SARS-CoV-2 S protein in a 1:1 volume ratio with the adjuvant to form a vaccine, which was then injected intramuscularly into Syrian golden hamsters at 200 μL per animal. Three immunization shots were administered at weeks 0, 2, and 6, and retro-orbital blood was collected weekly from weeks 0 to 7 for detection of serum antibody titers.

[0239] Immunization protocol for cynomolgus monkeys: 20 μg of antigen per monkey was individually mixed with the recombinant SARS-CoV-2 S protein in a 1:1 volume ratio with the adjuvant to form a vaccine, which was then injected intramuscularly into cynomolgus monkeys at 500 μL per animal. Two immunization shots were administered at weeks 0 and 2, and blood was collected weekly from weeks 0 to 6 for detection of antibody binding titers.

[0240] Enzyme-linked immunosorbent assay (ELISA) was used to detect antibody binding titers, which was illustrated as follows: The reagents used in the ELISA were those described in Example 4.

[0241] Testing Procedure: (1) Coating and blocking: Recombinant S protein was diluted to 2 μg / mL in CB9.6 and added to a 96-well microtiter plate at 100 μL / well, placed, and incubated at 37° C. for 2 hours. After that, the plate was washed once with PBST buffer (i.e., PBS containing 0.05% Tween-20), and 200 μL of PBS containing 20% ​​NBS was added to each well, followed by incubation for 2 hours at 37° C. for blocking. After removing excess liquid, it was centrifuged and dried for later use.

[0242] (2) Serum dilution: 100 μL of serum diluted 50-fold or 100-fold was added to each well and incubated at 37° C. for 1 hour.

[0243] (3) Addition of enzyme-labeled antibodies: After washing the plate five times with PBST buffer, 100 μL of HRP-conjugated goat anti-human IgG (H+L) (BEYOTIME, A0201) or goat anti-Syrian golden hamster IgG H&L (Abcam, ab6892) diluted at a certain ratio was added and incubated at 37°C for 0.5 hours.

[0244] (4) Develop, stop, and read. Experimental Results: As shown in Figure 8, two weeks after Syrian golden hamsters were immunized with a single shot, the antibody titer in the FH002C group was higher than that in the Al001 group, i.e., 15 times higher than that in the aluminum adjuvant group. FH002C demonstrated rapid response. After two immunizations, its advantage in enhancing humoral immunity remained evident. At week 4, the antibody titer in the FH002C group was 8 times higher than that in the aluminum adjuvant group. After three injections, the antibody titer in the FH002C group was 11 times higher than that of the control group at week 7.

[0245] Two weeks after immunization of the cynomolgus monkeys, the antibody titers in the FH002C group were higher than those in the Al001 group, i.e., six times higher than those in the aluminum adjuvant group, demonstrating that FH002C has the property of rapidly exerting its effect. After two shots of immunization, at week 4, the antibody titers in the FH002C group were equivalent to those in the aluminum adjuvant group (Figure 9).

[0246] Example 7: Neutralizing antibody titers induced by risedronate zinc aluminum adjuvant in combination with recombinant SARS-CoV-2 S protein Using the experimental procedures described in Example 6, adjuvants Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were combined with recombinant SARS-CoV-2 S protein and used to immunize Syrian golden hamsters and cynomolgus monkeys by intramuscular injection, respectively, and neutralizing antibody titers in the serum were detected. The immunization procedures for Syrian golden hamsters and cynomolgus monkeys were the same as those in Example 6.

[0247] Antibody neutralization titers were detected by pseudovirus neutralization (HL Xiong et al., Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells. Emerg Microbes Infect, pp. 1-38 (2020)), which was specifically described as follows: A vesicular stomatitis virus (VSV) pseudovirus (rVSV-SARS-CoV-2) carrying the SARS-CoV-2 S protein (Wuhan-Hu-1 strain, gene bank: QHD43416.1) was packaged using the recombinant plasmid pCAG-nCoVSdel18 and VSVdG-EGFP-G virus (Addgene, 31842). hACE2-expressing BHK21 (BHK21-hACE2) cells were seeded in a 96-well plate for 24 h. The serum was then diluted 50-fold and serially diluted 3-fold in high-glucose DMEM medium (Sigma-Aldrich, D6429) containing 10% FBS (GIBCO, 10099141) and penicillin-streptomycin (GIBCO, 15140122). Diluted rVSV-SARS-CoV-2 virus (MOI = 0.05) was mixed with diluted serum and incubated at 37°C for 1 hour. Immediately thereafter, the mixture was transferred to a plate coated with BHK21-hACE2 cells, followed by incubation at 37°C for 12 hours in an incubator containing 5% CO2. Fluorescence image data were captured using an Opera Phenix or Operetta CLS high-content analysis system (PerkinElmer), and the count of GFP-positive cells per well was analyzed using a Columbus system (PerkinElmer). Each plate contained eight serum-free wells as virus controls. Serum neutralization titers were calculated as the serum dilution factor (ID ) corresponding to a 50% reduction in the number of GFP-positive cells compared to positive wells. 50 ) was defined as

[0248] Experimental Results: As shown in Figure 10, two weeks after Syrian golden hamsters were immunized with a single shot, the antibody titer in the FH002C group was higher than that in the Al001 group, i.e., 1.8 times that of the aluminum adjuvant group. FH002C possessed rapid-acting properties. After two shots of immunization, its advantage in enhancing humoral immunity remained evident. At week 4, the antibody titer in the FH002C group was 2.6 times that of the aluminum adjuvant group. After three shots, the antibody titer in the FH002C group was 6.5 times that of the control group at week 7.

[0249] Two weeks after immunization of cynomolgus monkeys, the antibody titer of the FH002C group was higher than that of the Al001 group, i.e., 2.6 times that of the aluminum adjuvant group. After two shots of immunization, the antibody titer of the FH002C group was 3.5 times that of the aluminum adjuvant group at week 4 (Figure 11).

[0250] Example 8: Cell-based spike-blocking experiments of immune sera induced by risedronate zinc aluminum adjuvant combined with recombinant SARS-CoV-2 S protein Using the immunization procedure described in Example 6, Syrian golden hamsters and cynomolgus monkeys were immunized intramuscularly with adjuvants Al001 (Preparation Example 3) and FH002C (Preparation Example 1) in combination with recombinant SARS-CoV-2 S protein, and cell-based spike serum blocking experiments (Y. Zhang et al., Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibody and compound inhibitors. bioRxiv, (2020)) were performed as follows: 293T cells expressing hACE2-mRuby3 (293T-ACE2iRb3) were inoculated into 2 × 10 cells for 24 hours in advance. 4 Cells were seeded onto a 96-well plate pretreated with poly-D-lysine at 1000p / well. Syrian golden hamster and cynomolgus monkey sera were serially diluted 2-fold with DMEM medium containing 10% FBS. 11 μL of a SARS-CoV-2 spike trimer (STG) probe fused with Gamillus was mixed with 44 μL of diluted serum to a final probe concentration of 2.5 nM. Half of the culture medium was removed from the well containing 293T-ACE2iRb3 cells, and 50 μL of the mixture was pipetted and added to the cells. The cells were then incubated at 37°C and 5% CO2 for 1 hour. Cell images were then captured in confocal mode using an Opera Phenix high-content analysis system.

[0251] Data analysis: A Columbus system was used for quantitative analysis of imaging data. First, all inoculated cells were identified using the near-infrared channel (Ex: 640 / Em: 670) based on the principle that all 293T-ACE2iRb3 cells express H2B-fused iRFP670 localized in the nucleus. Based on the principle that hACE2 fused to mRuby3 is located on the membrane, the cell boundary was defined using the red channel (Ex: 561 / Em: 590). Next, the MFI of the STG probe channel (Ex: 488 / Em: 525) in the cytoplasmic region was calculated (cMFI, outer boundary: 20%, inner boundary: 45%) to obtain the inhibition ratio: [(cMFIpc-cMFItst) / (cMFIpc-cMFIblk)] × 100%. where cMFItst refers to the cMFI value of the test wells, and cMFIpc and cMFIblk refer to the cMFI values ​​of the probe-only (positive control) and cell-only wells, respectively. Serum blocking levels were calculated using ID 50 It is expressed as:

[0252] Experimental results: As shown in Figure 12, the serum blocking rate of the Syrian golden hamster FH002C group was higher than that of the Al001 group, and as shown in Figure 13, the serum blocking rates of the two adjuvant groups in cynomolgus monkeys were equivalent.

[0253] Example 9: Effect of Risedronate Zinc Aluminum Adjuvant in Combination with Recombinant SARS-CoV-2 S Protein on Antibody Affinity Using the experimental procedures described in Example 6, mice and Syrian golden hamsters were immunized intramuscularly with adjuvants A1001 (Preparation Example 3) and FH002C (Preparation Example 1) mixed with recombinant SARS-CoV-2 S protein to detect serum antibody affinity. The immunization procedures for Syrian golden hamsters were the same as those in Example 6, except that the FH002C and A1001 groups each contained 16 animals, half of which were male and half of which were female. The immunization procedures for mice were as follows: Experimental animals: Balb / C mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.), 6-8 weeks old, 4 or 5 mice per group, female.

[0254] Experimental grouping: (1) recombinant S protein + Al001; (2) recombinant S protein + FH002C.

[0255] Mouse immunization protocol: Vaccines were formed by individually mixing 1 μg of antigen per animal with the recombinant SARS-CoV-2 S protein and adjuvant at a volume ratio of 1:1, followed by intramuscular injection of 150 μL per animal into Balb / C mice. Three immunization shots were performed at weeks 0, 2, and 4, and retro-orbital blood was collected once a week from weeks 0 to 6 for detection of antibody affinity in the serum.

[0256] Antibody affinity was detected by enzyme-linked immunosorbent assay (ELISA), which is detailed as follows: The reagents used in the ELISA were those described in Example 4.

[0257] Testing Procedure: Coating and blocking: Purified recombinant S protein was diluted to 2 μg / mL in CB9.6 and then used for coating. 100 μL / well of the plate was added to an ELISA plate, placed in the well, and incubated at 37° C. for 2 hours. After that, the plate was washed once with PBST buffer (i.e., PBS containing 0.05% Tween-20), and 200 μL of PBS containing 20% ​​NBS was added to each well, placed, and incubated for 2 hours at 37° C. for blocking. After removing excess liquid, it was spun dry.

[0258] Serum antibody affinity detection: 100 μL of serum was added to each well, and each sample was incubated in duplicate at 37°C for 1 hour. After washing the plate once with PBST buffer, 100 μL of PBS was added to one well of the duplicate wells, and 100 μL of 4 M urea (prepared in PBS) was added to the other well. The plate was incubated at 37°C for 20 minutes and then washed four times with PBST buffer. Next, a fixed-fold dilution of HRP-conjugated goat anti-mouse IgG (H+L) (Proteintech, SA00001-1) or goat anti-Syrian hamster IgG H&L (Abcam, ab6892) was added and incubated at 37°C for 0.5 hours. The plate was washed five times with wash buffer. 100 μL of color-developing substrate mix was added to each well, and the absorbance at 450 nm was read using a PHOMO microplate reader. Antibody avidity was calculated as the antibody ratio, i.e., the EC 50 EC vs. untreated group 50 It could be expressed as a ratio of

[0259] Experimental Results: As shown in Figures 14 and 15, whether in mouse model or Syrian golden hamster model, the FH002C group was able to produce a higher proportion of high affinity antibodies than the Al001 group.

[0260] Example 10: Production of specific antibody subtypes by immunizing mice with risedronate zinc aluminum adjuvant combined with recombinant SARS-CoV-2 S protein The prepared Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were used as adjuvants and mixed with recombinant SARS-CoV-2 S protein in a volume ratio of 1:1 to form vaccines, which were then intramuscularly injected into mice. The immunization procedure was the same as in Example 9.

[0261] Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibody subtypes, as detailed below: The reagents used in the ELISA were those described in Example 4.

[0262] Testing Procedure: Coating and blocking: Purified recombinant S protein was diluted to 2 μg / mL with carbonate buffer and added to the ELISA plate at 100 μL / well and incubated at 37° C. for 2 hours. After that, the plate was washed once with PBST buffer (i.e., PBS containing 0.05% Tween-20), and 200 μL of PBS containing 20% ​​NBS was added to each well and incubated for 2 hours at 37° C. for blocking. After removing excess liquid, it was centrifuged and dried.

[0263] Detection of serum antibody subtype levels: 100 μL of serum was added to each well and incubated at 37°C for 1 hour. After washing the plate five times with PBST buffer, 100 μL of HRP-conjugated goat anti-mouse IgG1 (AbD Serotec, STAR132P), goat anti-mouse IgG2a (AbD Serotec, STAR133P), or goat anti-mouse IgG2b (AbD Serotec, STAR134P) was added and incubated at 37°C for 0.5 hours. The plate was washed five times with wash buffer. 100 μL of 3-fold diluted color-developing substrate mix was added to each well, and the absorbance at 450 nm was read using a PHOMO microplate reader. Each plate included five wells of negative serum as negative controls.

[0264] Experimental results: As shown in Figure 16, compared with the aluminum adjuvant group, the FH002C adjuvant group was able to induce higher levels of IgG2a and IgG2b subtype antibodies, and the ratio of IgG1 to IgG2a and IgG2b was lower than that of the aluminum adjuvant group, indicating that it has a certain stimulating effect on the Th1 immune pathway.

[0265] Example 11: Effect of Risedronate Zinc Aluminum Adjuvant in Combination with Recombinant SARS-CoV-2 S Protein on T Cell Responses The prepared Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were used as adjuvants and mixed with recombinant SARS-CoV-2 S protein in a volume ratio of 1:1 to form vaccines, which were then intramuscularly injected into mice to measure the induced T cell response. The specific method was as follows: Experimental animals: C57BL / 6, 6-8 weeks old, 8 animals / group, female.

[0266] Experimental groups: (1) blank group; (2) recombinant S protein + Al001; (3) recombinant S protein + FH002C.

[0267] Immunization protocol for C57BL / 6 mice: 10 μg of antigen per animal was mixed with the recombinant SARS-CoV-2 S protein in a volume ratio of 1:1, followed by intramuscular injection of 150 μL per animal. Two immunization shots were performed at weeks 0 and 3, and the mice were sacrificed at week 4, after which the spleens and lymph nodes were isolated for assay of T cell immune responses.

[0268] Enzyme-linked immunospot assay (ELISPOT) was used to detect T cell responses.

[0269] Spleens and lymph nodes of mice were harvested and prepared into single cell suspensions, with 10 cells per well. 6 (spleen) cells or 4 x 10 per well 5 Lymph node cells were seeded onto ELISPOT plates (DAKEWEI, 2210005) coated with mouse IFN-γ. They were then stimulated and cultured for 20 hours with PBS or a 15-unit SARS-CoV-2 S peptide library (Genscript, RP30020) with 11 amino acid overlaps. Detection was then performed according to the kit's instructions. Image capture and spot counting were performed using a CTL-ImmunoSpot® S5 (Cellular Technology Limited). The number of IFN-γ-secreting cells was calculated by subtracting the number of wells stimulated with PBS from the number of wells stimulated with the spiked peptide library.

[0270] Experimental Results: As shown in Figure 17, the number of IFN-γ-secreting cells in the FH002C and Al001 groups increased by 28.9-fold and 5.8-fold in the spleen, and by 14.0-fold and 2.3-fold in the lymph nodes, respectively. Compared with the Al001 group, the FH002C group showed a higher level of induced T cell responses.

[0271] Example 12: Specific antibody binding titers induced by aluminum adjuvant (Al001) and FH002C adjuvant in combination with varicella-zoster virus gE protein (VZV gE) The prepared Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were used as adjuvants and mixed with varicella-zoster virus gE protein (VZV gE, SEQ ID NO: 2) at a volume ratio of 1:1 to form vaccines, which were then intramuscularly injected into Balb / C mice to detect the titer of specific antibodies produced, where the gE protein was obtained by expression using an Escherichia coli expression system (purchased from Shanghai Weidi Biotechnology Co., Ltd., EC1060).

[0272] The specific method was as follows: Experimental animals: Balb / C mice, 6-7 weeks old, 5 females in each group.

[0273] Experimental grouping: (1) VZV gE+Al001; (2) VZV gE+FH002C. Immunization protocol for Balb / C mice: 5 μg of antigen per mouse was mixed with adjuvant at a volume ratio of 1:1 to form a vaccine, which was then intramuscularly injected into Balb / C mice at 100 μL per animal. Two immunization shots were performed at weeks 0, 2, and 4, and retro-orbital blood was collected once a week from week 0 to week 4 for detection of serum antibody titers.

[0274] Enzyme-linked immunosorbent assay (ELISA) was used to detect antibody binding titers, which is detailed as follows: The reagents used in the ELISA were those described in Example 4.

[0275] Testing Procedure: (1) Coating and blocking: VZV gE was diluted to 1 μg / mL in PB7.4+NaCl and added to a 96-well microplate at 100 μL / well. After overnight incubation at 4°C, the plate was washed once with PBST buffer (i.e., PBS containing 0.05% Tween-20), and 200 μL of PBS containing 20% ​​NBS was added to each well, followed by incubation for 2 hours at 37°C for blocking. After removing excess liquid, it was centrifuged and dried for later use.

[0276] (2) Serum dilution: 100 μL of serum diluted 50-fold or 100-fold was added to each well and incubated at 25° C. for 1 hour.

[0277] (3) Addition of enzyme-labeled antibody: After washing the plate five times with PBST buffer, 100 μL of enzyme-labeled antibody (GAM-HRP) (purchased from Bio-Rad Laboratories Inc., Cat. No.: 1706516) was added and incubated at 25°C for 1 hour.

[0278] (4) Develop, stop, and read. Experimental Results: As shown in Figure 18, after two shots of immunization, the binding titer of the FH002C adjuvant group was significantly higher than that of the Al001 group, about 10 times.

[0279] Example 13: Specific antibody binding titers induced by Freund's adjuvant and FH002C adjuvant in combination with influenza virus HA protein A commercially available Freund's adjuvant (purchased from SIGMA-ALDRICH, Cat. No.: F5881) and FH002C adjuvant (Preparation Example 1) were mixed with influenza virus HA protein (SEQ ID NO: 3) treated with or without deglycosylation enzyme at a volume ratio of 1:1 to form a vaccine, which was then subcutaneously injected into Balb / C mice to measure the titer of the specific antibody produced. Here, the influenza virus HA protein was obtained by expression using a baculovirus expression system (purchased from Invitrogen, USA, 10359016). The deglycosylation enzyme was purchased from NEB (Cat. No.: P0705S), and the deglycosylation enzyme treatment process was carried out according to the instructions for the deglycosylation enzyme.

[0280] The specific method was as follows: Experimental animals: Balb / C mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.), 6 to 7 weeks old, 10 mice / group in the FH002C group, 10 mice / group in the Freund's adjuvant group.

[0281] Experimental grouping: (1) HA + Freund's adjuvant; (2) HA + FH002C. Immunization protocol: (1) 30 μg of antigen per animal was mixed with Freund's adjuvant at a volume ratio of 1:1 to form a vaccine, which was then injected subcutaneously into Balb / C mice at 300 μL per animal; (2) 30 μg of antigen per animal was mixed with FH002C at a volume ratio of 1:1 to form a vaccine, which was then injected intramuscularly into Balb / C mice at 200 μL per animal. 14 days after immunization, retro-orbital blood was collected for detection of serum antibody titers.

[0282] Enzyme-linked immunosorbent assay (ELISA) was used to detect antibody binding titers, which is detailed as follows: The reagents used in the ELISA were those described in Example 4.

[0283] (1) Coating and blocking: Recombinant HA protein or ultracentrifuged influenza virus was diluted to 2 μg / mL in CB9.6 or added at 100 μL / well to a 96-well microtiter plate, placed, and incubated at 37° C. for 2 hours. After that, the plate was washed once with PBST buffer (i.e., PBS containing 0.05% Tween-20), and 200 μL of a commercially available blocking solution was added to each well, followed by incubation for 2 hours at 37° C. for blocking. After removing excess liquid, it was centrifuged and dried for later use.

[0284] (2) Serum detection: 100 μL of serum diluted at various dilutions was added to each well and incubated at 37° C. for 1 hour.

[0285] (3) Addition of enzyme-labeled antibody: After washing the plate five times with PBST buffer, 100 μL of enzyme-labeled goat anti-mouse antibody GAM-HRP (self-prepared in the laboratory) diluted 5000 times was added and incubated at 37°C for 0.5 hours.

[0286] (4) Develop, stop, and read. Experimental results: As shown in Figure 19, two weeks after Balb / C mice were immunized with a single shot, the antibody titers in the FH002C adjuvant groups against influenza virus HA proteins treated with or without deglycosylation enzymes were all higher than those in the Freund's adjuvant group, indicating that the FH002C adjuvant had the property of rapidly exerting its effect.

[0287] Example 14: Neutralizing antibody titers induced by aluminum adjuvant (Al001) and FH002C adjuvant in combination with rotavirus VP4 protein The prepared Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were used as adjuvants and mixed with rotavirus VP4 protein at a volume ratio of 1:1 to form vaccines, which were then intramuscularly injected into Balb / C mice and guinea pigs to determine the titer of neutralizing antibodies produced, in which the VP4 protein was expressed in an Escherichia coli expression system (purchased from Shanghai Weidi Biotechnology Co., Ltd., EC1060).

[0288] The specific method was as follows: Experimental animals: Balb / C mice, purchased from Shanghai Slack Experimental Animal Co., Ltd., 6-8 weeks old; guinea pigs, 450g-500g, purchased from Shanghai Songlian Experimental Animal Farm).

[0289] Mouse groups: 5 mice / group in FH002C group, 5 mice / group in Al001 group, all animals were female.

[0290] Guinea pig groups: 5 guinea pigs / group in group FH002C, 5 guinea pigs / group in group Al001, all animals were female.

[0291] Experimental grouping: (1) VP4+Al001; (2) VP4+FH002C. Immunization protocol: 10 μg of antigen per animal for mouse immunization, and 10 μg of antigen per animal for guinea pig immunization, i.e., adjuvant was individually mixed with rotavirus VP4 protein at a volume ratio of 1:1 to form a vaccine, and then 200 μL per animal for both mice and guinea pigs was intramuscularly injected into mice and guinea pigs. Before the first shot of immunization and 2 weeks after the third shot of immunization, blood was collected and serum was separated for detection of neutralizing antibody titers.

[0292] Enzyme-linked immunospot assay (ELISPOT) was used to detect neutralizing antibody titers, which is detailed as follows: (1) Seeding of MA104 cells on 96-well cell plates: After digesting the cells with trypsin solution, the cells were suspended by pipetting with cell culture medium containing 10% FBS, and then counted using a cell counting plate. Culture medium was added to dilute to 25,000 cells / mL, which was then evenly added to the 96-well plate at 100 μL / well and incubated at 37°C with 5% CO2 for 20 hours.

[0293] (2) Virus digestion: 4 μL of 2.5 μg / μL trypsin was added to 1 mL of rotavirus solution, mixed thoroughly, and placed for digestion; after digestion, the virus was diluted to a certain titer with serum-free DMEM culture medium containing 1 μg / mL trypsin.

[0294] (3) Serum complement inactivation: 10 μL of serum sample was collected and placed in a 1.5 mL EP tube, followed by heat treatment at 56° C. for 30 minutes. After treatment, the serum sample was diluted with DMEM containing 1 μg / mL trypsin, and duplicate wells were used for detection.

[0295] (4) Serum-virus reaction: 100 μL of the virus from (2) was mixed with 100 μL of the serum from (3), and the neutralization reaction was carried out at 37° C. for 1 hour.

[0296] (5) Replacing the cell culture medium: The cell supernatant in (1) was removed, and the MA104 cells in (1) were rinsed with serum-free DMEM medium containing 1 μg / mL trypsin for 5 minutes, and the washing was repeated three times.

[0297] (6) Infection: After the final rinse, the rinsing medium was removed, and the neutralization reaction mixture from (4) was added to the cells, followed by incubation at 37°C and 5% CO 2 for 14 hours.

[0298] (7) Cell fixation: The cell supernatant of (6) is gently centrifuged to dryness to avoid cell damage, and then the cells are fixed with 100 μL / well of PBS solution containing 0.1% glutaraldehyde. Fixation is carried out at room temperature for 1 hour, and should be carried out under dark conditions to avoid light, which can prevent photodecomposition of glutaraldehyde from affecting the fixation effect.

[0299] (8) Cell permeabilization: The glutaraldehyde fixation solution from (7) was removed, and 100 μL of PBS solution containing 0.3% Triton X-100 was added to perform permeabilization treatment at room temperature for 30 minutes.

[0300] (9) Oxidation: After removing the Triton X-100 permeabilization solution from (8), 100 μL of 3% H2O2 in PBS solution was added and the treatment was carried out at room temperature for 15 minutes.

[0301] (10) Washing: The oxidizing solution was removed, and the cells were rinsed with PBST washing solution five times, for 5 minutes each.

[0302] (11) Enzyme-labeled antibody reaction: After removing the rinse solution by centrifugal drying, HRP-labeled VP6 antibody (self-produced in the laboratory; the monoclonal antibody is an antibody that recognizes VP6, prepared by hybridoma technology; the method was based on Li et al., Emerging Microbes Infection. 2020) was diluted at a dilution ratio of 1:5000 with a pre-prepared enzyme-labeled antibody reaction solution (enzyme diluent, ED-13), added to the cells, and reacted at 37°C for 1 hour.

[0303] (12) Washing: The enzyme-labeled antibody reaction solution was removed, and the cells were rinsed with PBST washing solution five times, for 5 minutes each time.

[0304] (13) Color development: After the rinsing solution was dried by spin-drying, 100 μL / well of TMB color development solution prepared in situ was added and reacted at room temperature for 15 minutes in the dark to avoid the reaction of the color development solution with light, which may affect the experimental results.

[0305] (14) Plate reading: The color developing solution was spun dry, and the above 96-well plate was read and counted using an ELISPOT plate reader.

[0306] Experimental Results: As shown in Figures 20 and 21, both mice and guinea pigs were able to produce neutralizing antibodies after three immunization shots, and the neutralizing antibody titers in the FH002C group were higher than those in the Al001 group. Among them, in mice, the serum neutralizing titers in the FH002C adjuvant group were about 16-fold higher than those in the Al001 adjuvant group (Figure 20), while in guinea pigs, the serum neutralizing titers in the FH002C adjuvant group were about 4-fold higher than those in the Al001 group (Figure 21).

[0307] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that, in accordance with all the teachings disclosed, various modifications and changes can be made to the details, and all of these modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof. < / ok> < / ok> < / no> < / ok> < / no> < / yes> < / ok> < / ok> < / ok> < / ok> < / ok> < / ok> < / ok> < / no> < / ok> < / yes> < / no> < / ok> < / ok> < / ok> < / ok>

Claims

1. 1. An adjuvant comprising zinc aluminum risedronate, said adjuvant having a zinc:risedronate molar concentration ratio in the range of 1:1 to 16:1 and a zinc:aluminum molar concentration ratio in the range of 1:1 to 50:

1.

2. 2. The adjuvant of claim 1, wherein the zinc:risedronic acid molar concentration ratio is in the range of 2:1 to 16:1, and / or the zinc:aluminum molar concentration ratio is in the range of 5:1 to 50:

1.

3. One or more of the following: (1) The adjuvant is present in the form of particles; (2) The adjuvant is present in the form of particles having a particle size of 0.01 μm to 100 μm, 0.01 μm to 60 μm, 0.01 μm to 50 μm, 0.1 μm to 60 μm, 0.1 μm to 30 μm, 0.4 μm to 30 μm, or 0.4 μm to 20 μm. (3) The adjuvant has a zinc:risedronic acid molar concentration ratio ranging from 1:1 to 2:1, 2:1 to 4:1, 4:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 14:1, or 14:1 to 16:1, or the adjuvant has a zinc:risedronic acid molar concentration ratio of 4:1 or 4.5:

1. (4) The adjuvant has a zinc:aluminum molar concentration ratio in the range of 1:1 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 15:1, 15:1 to 20:1, 20:1 to 30:1, 30:1 to 40:1, or 40:1 to 50:1, or the adjuvant has a zinc:aluminum molar concentration ratio of 10:

1. (5) The adjuvant has a zinc:risedronic acid molar concentration ratio ranging from 2:1 to 8:1, 2:1 to 4:1, 4:1 to 6:1, or 6:1 to 8:1, and a zinc:aluminum molar concentration ratio ranging from 2:1 to 50:1, 5:1 to 20:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 15:1, or 15:1 to 20:

1. Or, the adjuvant has a zinc:risedronic acid molar ratio of 4:1 and a zinc:aluminum molar ratio of 10:

1. Or, the adjuvant has a zinc:risedronic acid molar ratio of 4.5:1 and a zinc:aluminum molar ratio of 10:

1. (6) The adjuvant has a pH of 5.0 to 8.0, 5.0 to 7.0, 5.0 to 5.5, 5.5 to 6.0, 6.0 to 6.5, 6.5 to 7.0, 7.0 to 7.5, or 7.5 to 8.

0. (7) The adjuvant has a point of zero charge of 3.0 to 8.0, 4.0 to 8.0, 3.0 to 4.0, 4.0 to 5.0, 5.0 to 6.0, 6.0 to 7.0, or 7.0 to 8.

0. (8) The adjuvant has an adsorption rate of at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% for the immunogen; and (9) The adjuvant has an adsorption rate of at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% for the immunogen, and the immunogen is a protein.

3. The adjuvant according to claim 1 or 2, characterized by:

4. A method for preparing the adjuvant according to any one of claims 1 to 3, comprising the steps of: 1) providing a soluble salt solution containing zinc ions and aluminum ions; 2) mixing the soluble salt solution of step (1) with an alkaline risedronate solution to obtain an adjuvant; A method comprising:

5. One or more of the following: (i) the method further comprises a step of sterilizing the adjuvant obtained in step (2); (ii) the method further comprises a step of sterilizing the adjuvant obtained in step (2), wherein the adjuvant obtained in step (2) is sterilized by filter sterilization or high-temperature and high-pressure sterilization; (iii) the soluble salt solution has a zinc:aluminum molar concentration ratio in the range of 1:1 to 50:1, or the soluble salt solution has a zinc:aluminum molar concentration ratio in the range of 5:1 to 50:1, or the soluble salt solution has a zinc:aluminum molar concentration ratio in the range of 1:1 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 15:1, 15:1 to 20:1, 20:1 to 30:1, 30:1 to 40:1, or 40:1 to 50:1; (iv) In step (2), the soluble salt solution is mixed with the alkaline risedronate solution having a zinc:risedronate molar concentration ratio in the range of 1:1 to 16:1, or the soluble salt solution is mixed with the alkaline risedronate solution having a zinc:risedronate molar concentration ratio in the range of 2:1 to 16:1, or the soluble salt solution is mixed with the alkaline risedronate solution having a zinc:risedronate molar concentration ratio in the range of 1:1 to 2:1, 2:1 to 4:1, 4:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, 10:1 to 12:1, 12:1 to 14:1, or 14:1 to 16:

1. (v) In step (2), the soluble salt solution is mixed with the alkaline risedronic acid solution in such a manner that zinc ions, aluminum ions and risedronic acid are co-precipitated. (vi) In the method, zinc ions, aluminum ions and risedronate are precipitated in a co-precipitation manner to obtain risedronate zinc aluminum particles. (vii) In step (2), the alkaline risedronic acid solution is added dropwise to the soluble salt solution so that zinc ions, aluminum ions and risedronic acid are coprecipitated. (viii) the alkaline risedronic acid solution is selected from the group consisting of a solution of risedronic acid and sodium hydroxide, a solution of risedronic acid and phosphate, and any combination thereof; (ix) the alkaline risedronic acid solution is selected from the group consisting of a solution of risedronic acid and sodium hydroxide, a solution of risedronic acid and a phosphate, and any combination thereof, wherein the solution of risedronic acid and a phosphate is a solution of risedronic acid and disodium hydrogen phosphate or a solution of risedronic acid and sodium dihydrogen phosphate; and (x) the soluble salt solution in step (1) is selected from a sulfate solution, a chlorate solution, an acetate solution, and any combination thereof, or the soluble salt solution in step (1) is a chlorate solution or an acetate solution; 5. The method of claim 4, characterized by:

6. An immunogenic composition comprising an immunogen and the adjuvant of any one of claims 1 to 3.

7. The immunogen of claim 1, wherein the immunogen is one or more of the following: (1) The immunogen is selected from the group consisting of proteins, nucleic acids, polysaccharides, and immunogenic portions thereof; (2) The immunogen is derived from a pathogen, or the immunogen is derived from a virus, a bacterium, or a fungus; (3) The immunogen is a protein derived from a pathogen or an immunogenic fragment thereof. (4) The immunogen is a protein or an immunogenic fragment thereof derived from a virus, a bacterium, or a fungus; (5) The immunogen is a protein or an immunogenic fragment thereof derived from a virus, a bacterium, or a fungus, and the virus is selected from the group consisting of a respiratory virus, an enterovirus, and a varicella-zoster virus (VZV). (6) The immunogen is a protein or immunogenic fragment thereof derived from a virus, bacterium, or fungus, and the virus is selected from the group consisting of a respiratory virus, an enterovirus, and a varicella-zoster virus (VZV), wherein (i) the respiratory virus is influenza, parainfluenza, rhinovirus, coronavirus, or respiratory syncytial virus, and / or (ii) the enterovirus is EV71 virus or rotavirus. (7) The immunogen is a coronavirus spike protein or an immunogenic fragment thereof. (8) The immunogen is a coronavirus spike protein or an immunogenic fragment thereof, wherein the coronavirus is selected from the group consisting of an Orthocoronavirus alpha virus, an Orthocoronavirus beta virus, a severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and a SARS-CoV-2. (9) The immunogen is a coronavirus spike protein or an immunogenic fragment thereof. (10) The immunogen is a coronavirus spike protein or an immunogenic fragment thereof, wherein the coronavirus is selected from the group consisting of an Orthocoronavirus alpha virus, an Orthocoronavirus beta virus, a severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and a SARS-CoV-2. (11) The immunogen is SARS-CoV-2 S protein or an immunogenic fragment thereof, varicella-zoster virus gE protein or an immunogenic fragment thereof, influenza virus HA protein or an immunogenic fragment thereof, or rotavirus VP4 protein or an immunogenic fragment thereof. The immunogenic composition of claim 6, characterized by:

8. One or more of the following: (1) The immunogenic composition further comprises a pharmaceutically acceptable adjuvant material; (2) The immunogenic composition further comprises a pharmaceutically acceptable auxiliary material, wherein the pharmaceutically acceptable auxiliary material is selected from an excipient, a preservative, an antibacterial agent, a buffer, an additional immunoadjuvant, and any combination thereof. (3) The immunogenic composition further comprises a pharmaceutically acceptable auxiliary material, wherein the pharmaceutically acceptable auxiliary material is selected from the group consisting of an excipient, a preservative, an antibacterial agent, a buffer, an additional immunoadjuvant, and any combination thereof, wherein the additional immunoadjuvant is selected from the group consisting of aluminum adjuvant, Freund's adjuvant, Corynebacterium pumilus, lipopolysaccharide, cytokine, and any combination thereof. (4) The immunogenic composition further comprises a pharmaceutically acceptable auxiliary material, the pharmaceutically acceptable auxiliary material being selected from an excipient, a preservative, an antibacterial agent, a buffer, an additional immunoadjuvant, and any combination thereof, wherein the additional immunoadjuvant is selected from the group consisting of an aluminum adjuvant, a Freund's adjuvant, Corynebacterium pumilus, a lipopolysaccharide, a cytokine, and any combination thereof, wherein (i) the aluminum adjuvant is aluminum hydroxide, and / or (ii) the Freund's adjuvant is a complete Freund's adjuvant or an incomplete Freund's adjuvant. (5) The immunogenic composition further comprises a second immunogen. (6) The immunogenic composition further comprises a second immunogen, wherein the second immunogen is a protein, a nucleic acid, a polysaccharide, or an immunogenic portion thereof; and (7) The immunogenic composition is a vaccine. The immunogenic composition according to claim 6 or 7, characterized by:

9. 4. Use of an adjuvant according to any one of claims 1 to 3 for preparing an immunogenic composition, or as a carrier for delivering an immunogen, or as an immunostimulant for an immunogen, or for preparing a formulation for enhancing the immunogenicity of an immunogen, or for enhancing the immune response to an immunogen in a subject.

10. The use described in claim 9, wherein the immunogen is as defined in claim 7.

11. A method for preparing an immunogenic composition, comprising the step of mixing the adjuvant according to any one of claims 1 to 3 with an immunogen.

12. One or more of the following: (1) The immunogen is as defined in claim 7. (2) The method further comprises the step of adding a pharmaceutically acceptable auxiliary material. (3) The method further comprises adding a pharmaceutically acceptable auxiliary material, wherein the auxiliary material is selected from the group consisting of an excipient, a preservative, an antimicrobial agent, a buffer, an additional immune adjuvant, and any combination thereof. (4) The method further comprises the step of adding a pharmaceutically acceptable auxiliary material, wherein the auxiliary material is selected from the group consisting of an excipient, a preservative, an antibacterial agent, a buffer, an additional immunoadjuvant, and any combination thereof, and the additional immunoadjuvant is selected from the group consisting of an aluminum adjuvant, a Freund's adjuvant Corynebacterium pumilus, a lipopolysaccharide, a cytokine, and any combination thereof. (5) The method further comprises the step of adding a pharmaceutically acceptable auxiliary material, wherein the auxiliary material is selected from the group consisting of an excipient, a preservative, an antibacterial agent, a buffer, an additional immunoadjuvant, and any combination thereof, and the additional immunoadjuvant is selected from the group consisting of an aluminum adjuvant, a Freund's adjuvant Corynebacterium pumilus, a lipopolysaccharide, a cytokine, and any combination thereof, wherein (i) the aluminum adjuvant is aluminum hydroxide, and / or (ii) the Freund's adjuvant is a complete Freund's adjuvant or an incomplete Freund's adjuvant. (6) The method further comprises the step of adding a second immunogen. (7) The method further comprises the step of adding a second immunogen, wherein the second immunogen is a protein, a nucleic acid, a polysaccharide, or an immunogenic portion thereof; and (8) The immunogenic composition is a vaccine.

12. The method of claim 11, characterized by:

13. A pharmaceutical composition comprising the immunogenic composition of any one of claims 6 to 8 for the prevention and / or treatment of a disease in a subject, wherein the disease is a disease that can be prevented or treated by an immune response induced by the immunogen.

14. One or more of the following: (1) The immunogen is derived from a coronavirus, and the disease is a coronavirus infection or a disease associated with a coronavirus infection. (2) The immunogen is derived from SARS-CoV-2, and the disease is SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection. (3) The immunogen is a coronavirus protein or an immunogenic fragment thereof, and the disease is a coronavirus infection or a disease associated with a coronavirus infection. (4) The immunogen is a coronavirus protein or an immunogenic fragment thereof, and the disease is a coronavirus infection or a disease associated with a coronavirus infection, wherein the protein is a spike protein, and / or the coronavirus is SARS-CoV-2. (5) The immunogen is a structural protein of SARS-CoV-2 or an immunogenic fragment thereof, and the disease is SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection. (6) The immunogen is a structural protein of SARS-CoV-2 or an immunogenic fragment thereof, and the disease is SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection, wherein (i) the immunogen is the S protein of SARS-CoV-2 or an immunogenic fragment thereof, and / or the disease associated with SARS-CoV-2 infection is pneumonia caused by SARS-CoV-2 (COVID-19); (7) The immunogen is derived from varicella-zoster virus, and the disease is varicella-zoster virus infection or a disease associated with varicella-zoster virus infection. (8) The immunogen is a varicella-zoster virus protein or an immunogenic fragment thereof, and the disease is varicella-zoster virus infection or a disease associated with varicella-zoster virus infection. (9) The immunogen is a gE protein of varicella-zoster virus or an immunogenic fragment thereof, and the disease is varicella-zoster virus infection or a disease associated with varicella-zoster virus infection. (10) The immunogen is derived from an influenza virus, and the disease is influenza virus infection or a disease associated with influenza virus infection. (11) The immunogen is an influenza virus protein or an immunogenic fragment thereof, and the disease is influenza virus infection or a disease associated with influenza virus infection. (12) The immunogen is an influenza virus HA protein or an immunogenic fragment thereof, and the disease is influenza virus infection or a disease associated with influenza virus infection. (13) The immunogen is derived from rotavirus, and the disease is rotavirus infection or a rotavirus infection-related disease. (14) The immunogen is a rotavirus protein or an immunogenic fragment thereof, and the disease is a rotavirus infection or a rotavirus infection-related disease. (15) The immunogen is a rotavirus VP4 protein or an immunogenic fragment thereof, and the disease is a rotavirus infection or a rotavirus infection-related disease; and (16) The subject is an animal, or the subject is a bird or a mammal. Or, the subject is a rodent, pig, cat, dog, horse, primate, or bird. Or, the subject is a mouse, mink, guinea pig, Syrian golden hamster, or cynomolgus monkey. Or, the subject is a human.

14. The pharmaceutical composition according to claim 13, characterized by:

15. A method for improving the immunogenicity of an immunogen in vitro, comprising the step of mixing the immunogen with an adjuvant described in any one of claims 1 to 3.

16. The method of claim 15, wherein the immunogen is as defined in claim 7.

17. A pharmaceutical composition comprising an immunogen and an adjuvant according to any one of claims 1 to 3 for stimulating or enhancing an immune response to the immunogen in a subject.

18. One or more of the following: (1) The immunogen is as defined in claim 7. (2) The subject is an animal, or the subject is a bird or a mammal, or the subject is a rodent, pig, cat, dog, horse, primate or bird, or the subject is a mouse, mink, guinea pig, Syrian golden hamster or cynomolgus monkey, or the subject is a human. (3) The immune response is a cellular immune response and / or a humoral immune response. (4) The immune response is a cellular immune response and / or a humoral immune response, wherein the cellular immune response is a T cell immune response. (5) The immune response is a cellular immune response and / or a humoral immune response, wherein the cellular immune response is a T cell immune response, wherein the T cell immune response is a Th1 immune response and / or a Th2 immune response; and (6) The pharmaceutical composition is administered by a route selected from intramuscular injection, subcutaneous injection, intradermal administration, intranasal administration, oral administration, transdermal administration, or intravenous injection; Alternatively, the pharmaceutical composition is administered by intramuscular injection.

18. The pharmaceutical composition according to claim 17, characterized by:

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