WO2022100729A1 - 含有利塞膦酸锌铝的佐剂及其应用 - Google Patents

含有利塞膦酸锌铝的佐剂及其应用 Download PDF

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WO2022100729A1
WO2022100729A1 PCT/CN2021/130588 CN2021130588W WO2022100729A1 WO 2022100729 A1 WO2022100729 A1 WO 2022100729A1 CN 2021130588 W CN2021130588 W CN 2021130588W WO 2022100729 A1 WO2022100729 A1 WO 2022100729A1
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adjuvant
immunogen
protein
coronavirus
zinc
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PCT/CN2021/130588
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English (en)
French (fr)
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赵勤俭
黄小芬
聂梅凤
张志刚
袁权
张天英
李少伟
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Priority to JP2023528748A priority Critical patent/JP2023550353A/ja
Priority to US18/253,160 priority patent/US20240000813A1/en
Priority to EP21891249.1A priority patent/EP4245316A1/en
Publication of WO2022100729A1 publication Critical patent/WO2022100729A1/zh

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Definitions

  • the present application relates to the field of biopharmaceutical technology.
  • the present application relates to an adjuvant comprising zinc aluminum risedronate, an immunogenic composition comprising the adjuvant and an immunogen, and the use of the adjuvant and the immunogenic composition.
  • Adjuvants are a class of substances or mixtures that can specifically or non-specifically bind to immunogens and stimulate and induce long-term and effective specific immune responses in the body.
  • the immunobiological effects of adjuvants include reducing the amount of immunogen used, enhancing the immunogenicity of antigen, and changing the type of immune response.
  • Adjuvants currently approved for human vaccines include aluminum adjuvant, MF59, AS04, AS01B, CpG1018, etc., and several new adjuvants are in clinical trials, including: AS03, RC-529, Advax, Matrix-M Wait.
  • Aluminum adjuvant is the first adjuvant approved for human vaccines. It has been used for nearly 90 years and is recognized as the most widely used, safe and effective adjuvant.
  • aluminum adjuvant stimulates the induction of Th2-type immunodominant responses, and has a limited effect on inducing Th1-type immune responses, which also limits the application of aluminum adjuvants in therapeutic vaccines, such as chickenpox Herpes zoster virus vaccine, hepatitis B therapeutic vaccine and tumor vaccine, etc.
  • therapeutic vaccines such as chickenpox Herpes zoster virus vaccine, hepatitis B therapeutic vaccine and tumor vaccine, etc.
  • aluminum adjuvants have weaker activity and are not ideal for immuno-enhancing effects on most other genetically engineered antigens other than virus-like particle antigens, making their application in non-polymerized protein antigen vaccines. restricted.
  • Aluminum-containing adjuvant system refers to the adjuvant system based on aluminum adjuvant and formed with other adjuvant components or delivery systems (such as MPL, CPG, QS21 and liposomes, etc.), which can induce relatively balanced Th1 and Th2 immune response.
  • adjuvant components or delivery systems such as MPL, CPG, QS21 and liposomes, etc.
  • NPJ Vaccines. 2018, 10; 3: 51. have been approved for marketing or entered clinical trials.
  • TLR-4 Toll like receptor 4, TLR4
  • MPL 3-O-deacyl -4"-Monophosphoryl lipid A
  • AS04 has been successfully used in hepatitis B vaccine (Fendrix, 2005 approved marketed) and HPV16/18 bivalent cervical cancer vaccine (Cervarix, approved for marketing in 2007). Compared with aluminum adjuvant alone, it can significantly increase the neutralizing antibody of HPV16/18 bivalent cervical cancer vaccine (Cervarix). titer and prolong the duration of immune protection, and one or two immunizations with this vaccine can produce protection similar to that provided by three doses of immunization.
  • CpG a receptor agonist of TLR-9, combined with aluminum adjuvant for malaria Vaccine adjuvants and hookworm vaccine adjuvants have entered clinical trials.
  • the adsorption of aluminum adjuvant to antigen is one of the reasons for its immune-enhancing mechanism.
  • Ligand exchange is the strongest interaction force between adjuvant and antigen, which is produced by the ligand exchange between the phosphate group in the antigen and the hydroxyl group in aluminum hydroxide or aluminum phosphate.
  • the concept of "phosphorophilicity" of the white surface of aluminum adjuvant was proposed in 2001 (Analytical Biochemistry, 2001, 295(1):76-81).
  • the aforementioned AS04, MPL forms a new type of composite adjuvant through the adsorption of phosphate group and aluminum adjuvant, which has been well used in clinical practice.
  • Bisphosphonates (BPs) drugs are a class of synthetic pyrophosphoric acid analogs with P-C-P as the central skeleton. They are organic bisphosphonic acids. Because of their phosphoric acid groups, they are highly compatible with calcium, aluminum, zinc, magnesium and other ions. It is used for the treatment of bone diseases and calcium metabolism diseases, such as osteoporosis, osteitis deformans, hypercalcemia and bone pain caused by bone metastasis of malignant tumors.
  • bone diseases and calcium metabolism diseases such as osteoporosis, osteitis deformans, hypercalcemia and bone pain caused by bone metastasis of malignant tumors.
  • osteoporosis osteoporosis
  • osteitis deformans hypercalcemia
  • bone pain caused by bone metastasis of malignant tumors.
  • clinical studies have shown that the use of bisphosphonates in the adjuvant treatment of multiple myeloma, breast cancer, kidney cancer, prostate cancer, etc. can reduce the incidence of bone-related
  • bisphosphonic acid exhibits adjuvant activity due to its positive immune regulation effect, and it has also been reported as an immune enhancer for the patented invention of vaccine preparations (Chinese Patent: CN103768595B; US Patent: US20170281759A1; China Patent: CN 108289902A).
  • Bisphosphonates are usually administered orally or intravenously in clinical practice, which has a strong mucosal stimulatory effect, while in the use of vaccines, immunization is usually performed by intramuscular injection.
  • bisphosphonates have potential side effects that still require further improvement.
  • Coronavirus is an enveloped, non-segmented, single-stranded positive-stranded RNA virus belonging to the Coronaviridae family of the order Nidovirales, the subfamily Orthocoronavirinae. Divided into alpha, beta, gamma and delta four genera.
  • coronaviruses have been found to infect humans, among which the seasonally predominant coronaviruses include: 229E and NL63 of alpha genus, OC43 and HKU1 of beta genus, and pandemic coronavirus: severe acute respiratory syndrome Symptom-related coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2).
  • SARS-CoV severe acute respiratory syndrome Symptom-related coronavirus
  • MERS-CoV Middle East respiratory syndrome-related coronavirus
  • SARS-CoV-2 novel coronavirus
  • coronaviruses can infect vertebrates such as humans, mice, pigs, cats, dogs, minks, guinea pigs, hamsters, rhesus monkeys, and birds.
  • the coronavirus spike protein is a type I transmembrane protein composed of two parts, the S1 and S2 protein subunits, which mediate binding to host cell surface receptors and fusion of cell membranes to enter target cells.
  • S protein The coronavirus spike protein
  • a number of evidences have shown that antibodies against the spike protein may play a key role in the immune prevention and treatment of 2019-nCoV pneumonia, and are currently the key target for the development and design of 2019-nCoV recombinant protein vaccines.
  • Some studies have found that acute new coronavirus infection will weaken its long-lasting neutralizing antibody response, but it can still achieve immune memory through virus-specific memory T cells (Cell, 2020, 183(1): 13-15), indicating that The importance of T cell responses in the prevention of novel coronavirus infection.
  • the subunit protein vaccine expressed by recombinant DNA technology has good safety and can achieve multiple booster immunizations, but its immunogenicity is weak.
  • the World Health Organization (WHO) formulated the target product characteristics of the 2019 coronavirus disease (COVID-19) vaccine in April 2020.
  • the vaccine In the event of an outbreak, the vaccine must take effect quickly, provide protection within 2 weeks, and at the same time 1 injection achieves basic immunity, and the dosing regimen does not exceed 2 doses, and requires at least 6 months of protection in terms of durability of protection.
  • a novel adjuvant comprising zinc and 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.
  • the adjuvant of the present application when used in combination with an immunogen, such as the novel coronavirus spike protein (S protein), in Balb/c mice, minks, guinea pigs, Syrian golden hamsters and cynomolgus monkeys It can stimulate or induce high levels of functional antibodies and balanced cellular and humoral immune responses in various animals such as the present application. Therefore, the adjuvant of the present application can improve the druggability of vaccines (such as the novel coronavirus S protein vaccine).
  • an immunogen such as the novel coronavirus spike protein (S protein)
  • the adjuvant of the present application can effectively prevent the infection of the novel coronavirus (SARS-CoV-2) when used in combination with the novel coronavirus spike protein (S protein).
  • SARS-CoV-2 novel coronavirus spike protein
  • a vaccine containing the adjuvant of the present application and the new coronavirus S protein can effectively prevent or reduce the replication of SARS-CoV-2 in the upper and lower respiratory tract of animals (such as minks), and prevent the new coronavirus from affecting animals (such as minks) lung infection and damage.
  • the application provides an adjuvant comprising risedronate zinc aluminum (also referred to herein as risedronate zinc aluminum adjuvant), wherein the molar concentration ratio of zinc:risedronate is 1:1: in the range of 1 to 16:1 (eg, in the range of 2:1 to 16:1); and the zinc:aluminum molar concentration ratio is in the range of 1:1 to 50:1 (eg, in the range of 5:1 to 50:1) 1 range).
  • risedronate zinc aluminum also referred to herein as risedronate zinc aluminum adjuvant
  • the zinc aluminum risedronate is present in particulate form. In certain embodiments, the zinc aluminum risedronate is present in nanoscale or microscale particles. In certain embodiments, the particle size is 0.01-100 ⁇ m, eg, 0.01-60 ⁇ m, 0.01-50 ⁇ m, 0.1-60 ⁇ m, 0.1-30 ⁇ m, 0.4-30 ⁇ m, 0.4-20 ⁇ m.
  • the adjuvant zinc:risedronic acid molar ratio is 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 zinc:risedronic acid 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.
  • the adjuvant zinc:risedronic acid molar ratio is no more than 16:1, no more than 14:1, no more than 12:1, no more than 10:1, no more than 8:1 , no more than 7:1, no more than 6:1, no more than 5:1, no more than 4:1, no more than 3:1, or no more than 2:1.
  • the adjuvant 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 adjuvant zinc:risedronic acid molar ratio is 4:1 or 4.5:1.
  • the adjuvant zinc:aluminum molar ratio is 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.
  • the adjuvant zinc:aluminum 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 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.
  • the adjuvant zinc:aluminum molar ratio is no more than 50:1, no more than 40:1, no more than 30:1, no more than 20:1, no more than 15:1, no more than 12:1, no more than 10:1, no more than 8:1, no more than 6:1, no more than 5:1, no more than 4:1, no more than 3:1, or no more than 2:1.
  • the adjuvant zinc:aluminum molar ratio is 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 zinc:aluminum molar ratio is 10:1.
  • the adjuvant has a zinc:risedronic acid molar ratio in the range of 2:1 to 8:1, eg, 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 (eg, in the range of 5:1 to 20:1), such as 2:1 to 3:1 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.
  • the adjuvant has a zinc:risedronic acid molar ratio of 4:1 and a zinc:aluminum molar ratio of 10:1.
  • the adjuvant has a zinc:risedronic acid molar ratio of 4.5:1 and a zinc:aluminum molar ratio of 10:1.
  • the pH of the adjuvant is 5.0-8.0, eg, 5.0-7.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, or 7.5-8.0.
  • the adjuvant pH is between 7.0 and 7.5.
  • the adjuvant pH is 5.5 to 6.5.
  • the adjuvant pH is 5.8 to 6.3.
  • the adjuvant has a point of zero charge between 3.0 and 8.0, eg, 4.0-8.0, 3.0-4.0, 4.0-5.0, 5.0-6.0, 6.0-7.0, or 7.0-8.0. In certain embodiments, the adjuvant has a point of zero charge between 4.0 and 6.0.
  • the adjuvants of the present application have good adsorption rates for immunogens (eg, proteins).
  • the adjuvant of the invention 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 an immunogen (eg, a protein). %.
  • the adjuvants of the present invention can be used in combination with various immunogens to enhance the immunogenicity of the immunogen.
  • immunogens include, but are not limited to, proteins, nucleic acids, polysaccharides, or immunogenic portions thereof, and the like.
  • the application provides an immunogenic composition comprising an immunogen and an adjuvant as described above.
  • the immunogens include, but are not limited to, proteins, nucleic acids, polysaccharides, or immunogenic portions thereof, and the like.
  • the immunogen is derived from a viral, bacterial, fungal, etc. pathogen.
  • the immunogen is a protein or an immunogenic fragment thereof, eg, a protein or immunogenic fragment thereof derived from a viral, bacterial, fungal, etc. pathogen.
  • the viruses include, but are not limited to, respiratory viruses (eg, influenza virus, parainfluenza virus, rhinovirus, coronavirus, respiratory syncytial virus), enteroviruses (eg, EV71 virus, rotavirus) ), varicella-zoster virus (VZV).
  • the immunogen is a protein of a coronavirus (eg, the spike protein) or an immunogenic fragment thereof.
  • the coronavirus is selected from the group consisting of Orthocoronaviridae alpha viruses (e.g. 229E and NL63), Orthocoronaviridae beta viruses (e.g. OC43 and HKU1), severe acute respiratory syndrome-associated coronavirus virus (SARS-CoV), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2).
  • the coronavirus is selected from the group consisting of severe acute respiratory syndrome-associated coronavirus (SARS-CoV), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2) .
  • the coronavirus is a novel coronavirus (SARS-CoV-2).
  • the immunogen is a structural protein of the novel coronavirus or an immunogenic fragment thereof, such as the spike protein (S protein) or an immunogenic fragment thereof.
  • the immunogen is a protein of varicella zoster virus or an immunogenic fragment thereof, eg, varicella zoster virus gE protein or an immunogenic fragment thereof.
  • the immunogen is an influenza virus protein or an immunogenic fragment thereof, eg, an influenza virus HA protein or an immunogenic fragment thereof.
  • the immunogen is a rotavirus protein or an immunogenic fragment thereof, eg, a rotavirus VP4 protein or an immunogenic fragment thereof.
  • the immunogenic composition further comprises pharmaceutically acceptable adjuvants such as excipients, preservatives, antibacterial agents, buffers and/or additional immunological adjuvants.
  • additional immunological adjuvant is selected from the group consisting of aluminum adjuvants (eg, aluminum hydroxide), Freund's adjuvants (eg, complete and incomplete Freund's adjuvants), Corynebacterium pumilus, Lipopolysaccharide, cytokine, or any combination thereof.
  • the immunogenic composition further comprises a second immunogen.
  • the second immunogen includes, but is not limited to, proteins, nucleic acids, polysaccharides, or immunogenic portions thereof, and the like.
  • the immunogenic composition is a vaccine.
  • the application provides a method for preparing an adjuvant as described above, comprising the steps of:
  • step (2) Mix the soluble salt solution in step (1) with the risedronate base solution to obtain the adjuvant.
  • the method further includes the step of sterilizing the adjuvant obtained in step (2).
  • the adjuvant is sterilized by filter sterilization or autoclaving.
  • the adjuvant is sterilized by sterilization at 121°C for at least 15 minutes (eg, at least 30 minutes, eg, 30-60 minutes).
  • the molar ratio of zinc:aluminum in the soluble salt solution is in the range of 1:1 to 50:1 (eg, in the range of 5:1 to 50:1).
  • the zinc:aluminum molar ratio is from 1:1 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 4:1 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 1, 20:1 to 30:1, 30:1 to 40:1, or 40:1 to 50:1.
  • the zinc:aluminum 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 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.
  • the zinc:aluminum molar concentration ratio is no more than 50:1, no more than 40:1, no more than 30:1, no more than 20:1, no more than 15:1 1. Not more than 12:1, not more than 10:1, not more than 8:1, not more than 6:1, not more than 5:1, not more than 4:1, not more than 3:1, or not more than 2:1 .
  • the zinc:aluminum molar ratio is 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 zinc:aluminum molar ratio is 10:1 in the soluble salt solution.
  • step (2) at a zinc:risedronic acid molar concentration ratio in the range of 1:1 to 16:1 (eg, in the range of 2:1 to 16:1),
  • the soluble salt solution is mixed with the risedronate base solution.
  • the zinc:risedronic acid molar ratio is 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.
  • the zinc:risedronic acid 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 7:1, at least 8:1, at least 10:1, at least 12:1, at least 14:1, or at least 15:1.
  • the zinc:risedronic acid molar concentration ratio is no more than 16:1, no more than 14:1, no more than 12:1, no more than 10:1, no more than 8:1, no more than 12:1 More than 7:1, not more than 6:1, not more than 5:1, not more than 4:1, not more than 3:1, or not more than 2:1.
  • 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.
  • step (2) the soluble salt solution is mixed with the risedronate base solution in such a way that zinc ions, aluminum ions, and risedronate are co-precipitated.
  • the risedronic acid base solution is added dropwise to the soluble salt solution to co-precipitate zinc ions, aluminum ions, and risedronic acid.
  • step (2) during the process of mixing the soluble salt solution with the risedronate base solution, zinc ions, aluminum ions and risedronate are co-precipitated to produce risedronate Zinc aluminum phosphonate particles.
  • the resulting risedronate zinc aluminum particles have a particle size size of 0.01-100 ⁇ m, such as 0.01-60 ⁇ m, 0.01-50 ⁇ m, 0.1-60 ⁇ m, 0.1-30 ⁇ m, 0.4-30 ⁇ m, 0.4-20 ⁇ m .
  • the risedronate base solution is selected from the group consisting of risedronate sodium hydroxide, risedronate phosphate solution (eg, risedronate disodium phosphate solution, risedronate hydrogen phosphate solution) sodium solution), or any combination thereof.
  • the risedronate base solution is risedronate sodium hydroxide solution or risedronate disodium hydrogen phosphate solution.
  • the soluble salt solution of 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.
  • the application also provides the use of the adjuvant for the preparation of an immunogenic composition or as a carrier for the delivery of an immunogen or as an immunoenhancer for an immunogen. In one aspect, the application also provides the use of the adjuvant for enhancing the immunogenicity of an immunogen. In one aspect, the application also provides the use of the adjuvant for enhancing the level of immune response to an immunogen in a subject. In one aspect, the application also provides the use of the adjuvant in the manufacture of a formulation for enhancing the level of an immune response to an immunogen in a subject. It will be readily understood that the various descriptions above for such adjuvants and immunogens, etc. apply equally to these aspects.
  • the immune response is a cellular immune response and/or a humoral immune response.
  • the cellular immune response is a T cell immune response.
  • the T cell immune response is an immune response Th1 and/or a Th2 immune response.
  • the present application also provides a method of preparing an immunogenic composition comprising the step of admixing the adjuvant and the immunogen.
  • the application provides a method of increasing the immunogenicity of an immunogen comprising the step of admixing the immunogen with the adjuvant.
  • the adjuvants of the present invention can be used in combination with various possible immunogens to enhance the immunogenicity of the immunogen. Accordingly, the various descriptions above for such adjuvants and immunogens, etc. apply equally to these aspects.
  • the immunogens include, but are not limited to, proteins, nucleic acids, polysaccharides, or immunogenic portions thereof, and the like.
  • the immunogen is derived from a viral, bacterial, fungal, etc. pathogen.
  • the immunogen is a protein or an immunogenic fragment thereof, eg, a protein or immunogenic fragment thereof derived from a viral, bacterial, fungal, etc. pathogen.
  • the viruses include, but are not limited to, respiratory viruses (eg, influenza virus, parainfluenza virus, rhinovirus, coronavirus, respiratory syncytial virus), enteroviruses (eg, EV71 virus, rotavirus) ). Varicella zoster virus (VZV).
  • the immunogen is a protein of a coronavirus (eg, the spike protein) or an immunogenic fragment thereof.
  • the coronavirus is selected from the group consisting of Orthocoronaviridae alpha viruses (e.g. 229E and NL63), Orthocoronaviridae beta viruses (e.g. OC43 and HKU1), severe acute respiratory syndrome-associated coronavirus virus (SARS-CoV), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2).
  • the coronavirus is selected from the group consisting of severe acute respiratory syndrome-associated coronavirus (SARS-CoV), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2) .
  • the coronavirus is a novel coronavirus (SARS-CoV-2).
  • the immunogen is a structural protein of the novel coronavirus or an immunogenic fragment thereof, such as the spike protein (S protein) or an immunogenic fragment thereof.
  • the immunogen is a protein of varicella zoster virus or an immunogenic fragment thereof, eg, varicella zoster virus gE protein or an immunogenic fragment thereof.
  • the immunogen is an influenza virus protein or an immunogenic fragment thereof, eg, an influenza virus HA protein or an immunogenic fragment thereof.
  • the immunogen is a rotavirus protein or an immunogenic fragment thereof, eg, a rotavirus VP4 protein or an immunogenic fragment thereof.
  • the method further includes the step of adding a pharmaceutically acceptable excipient.
  • the adjuvant is selected from, for example, excipients, preservatives, antibacterial agents, buffers and/or additional immunological adjuvants.
  • the additional immunological adjuvant is selected from the group consisting of aluminum adjuvants (eg, aluminum hydroxide), Freund's adjuvants (eg, complete and incomplete Freund's adjuvants), Corynebacterium pumilus, Lipopolysaccharide, cytokine, or any combination thereof.
  • the method further comprises the step of adding a second immunogen.
  • the second immunogen includes, but is not limited to, proteins, nucleic acids, polysaccharides, or immunogenic portions thereof, and the like.
  • the immunogenic composition is a vaccine.
  • the application provides the use of the immunogenic composition in the manufacture of a medicament for the prevention and/or treatment of a disease in a subject, the disease being an immune response that can be induced by the immunogen Disease to be prevented or treated.
  • the various descriptions above for such adjuvants, immunogens, immunogenic compositions, etc. are equally applicable in this regard.
  • various immunogens can be included in the immunogenic composition; accordingly, the prepared medicament can be used to prevent or treat various corresponding diseases.
  • the immunogenic composition of the present application can be used in the preparation for the prevention and/or treatment of pathogen infection in a subject (such as viral infections) or diseases associated with pathogenic infections (eg viral infections).
  • the immunogen is derived from a coronavirus (eg, novel coronavirus), and the disease is or is associated with a coronavirus (eg, novel coronavirus) infection disease.
  • the immunogen is a protein of a coronavirus (eg, novel coronavirus) (eg, a spike protein) or an immunogenic fragment thereof, and the disease is a coronavirus (eg, novel coronavirus) infection or illnesses associated with infection with coronaviruses such as the novel coronavirus.
  • the immunogen is a structural protein of the novel coronavirus (eg, the spike protein (S protein)) or an immunogenic fragment thereof; and, the disease is infection with the novel coronavirus or is associated with the novel coronavirus Infection-related illness (eg, novel coronavirus pneumonia (COVID-19)).
  • S protein spike protein
  • COVID-19 novel coronavirus pneumonia
  • the immunogen is derived from varicella zoster virus
  • the disease is varicella zoster virus infection or a disease associated with varicella zoster virus infection.
  • the immunogen is a varicella-zoster virus protein (eg, gE protein) or an immunogenic fragment thereof, and the disease is varicella-zoster virus infection or a combination with varicella-zoster virus Infection-related diseases.
  • a varicella-zoster virus protein eg, gE protein
  • the disease is varicella-zoster virus infection or a combination with varicella-zoster virus Infection-related diseases.
  • the immunogen is derived from an influenza virus
  • the disease is an influenza virus infection or a disease associated with an influenza virus infection.
  • the immunogen is an influenza virus protein (eg, HA protein) or an immunogenic fragment thereof, and the disease is an influenza virus infection or a disease associated with an influenza virus infection.
  • influenza virus protein eg, HA protein
  • the disease is an influenza virus infection or a disease associated with an influenza virus infection.
  • the immunogen is derived from a rotavirus
  • the disease is a rotavirus infection or a disease associated with a rotavirus infection.
  • the immunogen is a rotavirus protein (eg, VP4 protein) or an immunogenic fragment thereof, and the disease is a rotavirus infection or a disease associated with a rotavirus infection.
  • a rotavirus protein eg, VP4 protein
  • the disease is a rotavirus infection or a disease associated with a rotavirus infection.
  • the subject is an animal, such as an avian or mammal.
  • the subject is a rodent, porcine, feline, canine, equine, primate, or avian.
  • the subject is a non-human subject.
  • the subject is a mouse, mink, guinea pig, Syrian golden hamster, or cynomolgus monkey.
  • the subject is a human.
  • the present application provides a method of stimulating or enhancing an immune response to an immunogen in a subject comprising administering to the subject an effective amount of an immunogenic composition comprising the immunogen and an adjuvant of the present invention A step of. It will be readily understood that the various descriptions above for such adjuvants, immunogens, immunogenic compositions, subjects, etc. apply equally to this aspect.
  • the immune response is a cellular immune response and/or a humoral immune response.
  • the cellular immune response is a T cell immune response.
  • the T cell immune response is an immune response Th1 and/or a Th2 immune response.
  • the immunogenic composition is administered by a route selected from the group consisting of intramuscular injection, subcutaneous injection, intradermal, intranasal, oral, transdermal, or intravenous injection. In certain embodiments, the immunogenic composition is administered by intramuscular injection.
  • the present application provides a method of preventing or treating a disease comprising the step of administering to a subject an effective amount of an immunogenic composition, wherein the disease is an immune response capable of being elicited by the immunogen Disease to be prevented or treated.
  • the various descriptions above for such adjuvants, immunogens, immunogenic compositions, subjects, etc. apply equally to this aspect.
  • various immunogens can be included in the immunogenic composition; accordingly, the immunogenic composition can be used to prevent or treat various corresponding diseases.
  • the immunogenic compositions of the present application can be used to prevent and/or treat a pathogenic infection (eg, viral infection) in a subject when the immunogenic composition contains an immunogen derived from a pathogen (eg, a virus) ) or diseases associated with pathogenic infections such as viral infections.
  • the disease is a coronavirus (eg, novel coronavirus) infection or a disease associated with a coronavirus (eg, novel coronavirus) infection.
  • the immunogen is derived from a coronavirus (eg, a novel coronavirus).
  • the immunogen is a protein (eg, the spike protein) of a coronavirus (eg, novel coronavirus) or an immunogenic fragment thereof.
  • the disease is a novel coronavirus infection or a disease associated with a novel coronavirus infection, such as novel coronavirus pneumonia (COVID-19).
  • the immunogen is derived from a novel coronavirus.
  • the immunogen is a structural protein of the novel coronavirus (eg, the spike protein (S protein)) or an immunogenic fragment thereof.
  • the disease is varicella-zoster virus infection or a disease associated with varicella-zoster virus infection.
  • the immunogen is derived from the varicella zoster virus.
  • the immunogen is a protein of the varicella-zoster virus (eg, the gE protein) or an immunogenic fragment thereof.
  • the disease is an influenza virus infection or a disease associated with an influenza virus infection.
  • the immunogen is derived from an influenza virus.
  • the immunogen is an influenza virus protein (eg, HA protein) or an immunogenic fragment thereof.
  • the disease is a rotavirus infection or a disease associated with a rotavirus infection.
  • the immunogen is derived from a rotavirus.
  • the immunogen is a rotavirus protein (eg, VP4 protein) or an immunogenic fragment thereof.
  • the subject is an animal, such as an avian or mammal.
  • the subject is a rodent, porcine, feline, canine, equine, primate, or avian.
  • the subject is a non-human subject.
  • the subject is a mouse, mink, guinea pig, Syrian golden hamster, or cynomolgus monkey.
  • the subject is a human.
  • S protein refers to the novel coronavirus spike protein, which belongs to the type I transmembrane protein and consists of two parts, S1 and S2 protein subunits, which mediate the binding to host cell surface receptors and the binding of cell membranes. fusion to enter target cells.
  • the amino acid sequence of wild-type S protein is known to those skilled in the art, and its typical amino acid sequence can be found in Genbank: QHD43416.1. In certain embodiments, the amino acid sequence of the wild-type S protein is set forth in SEQ ID NO:1.
  • the amino acid sequence of the engineered S protein is set forth in SEQ ID NO:4.
  • S protein should be construed to cover wild-type S protein as well as engineered S protein.
  • gE protein is an envelope glycoprotein of varicella-zoster virus (VZV).
  • VZV varicella-zoster virus
  • the amino acid sequence of wild-type gE protein is known to those skilled in the art, and its typical amino acid sequence can be found in Genbank: DQ008355.1.
  • the amino acid sequence of the gE protein is set forth in SEQ ID NO:2.
  • HA protein refers to influenza virus hemagglutinin (HA), which is a key protein that mediates virus invasion into host cells and is also the main target of anti-influenza virus neutralizing antibodies.
  • HA protein hemagglutinin
  • the amino acid sequence of the HA protein of each subtype of influenza virus is well known to those skilled in the art, for example, a typical amino acid sequence of a type B Victoria influenza virus can be found in Genbank: AIU46088.
  • the amino acid sequence of the HA protein is set forth in SEQ ID NO:3.
  • VP4 protein is the outer capsid protein of rotavirus.
  • VP4 protein is an important neutralizing antigen that can stimulate the body to produce neutralizing antibodies.
  • the amino acid sequence of wild-type VP4 protein is known to those skilled in the art, and its typical amino acid sequence can be found in Genbank: KP752474 or GenBank: MG729832.
  • gE proteins, HA proteins and VP4 proteins described herein are to be construed to cover their corresponding wild-type proteins as well as their corresponding engineered proteins.
  • the term "adjuvant” refers to a non-specific immune enhancer which, when delivered to the body together with or in advance of an antigen, can enhance or alter the type of immune response of the body to an antigen.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as 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 used more in clinical experiments.
  • the term "pharmaceutically acceptable adjuvant" may be, for example, inert diluents such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzyl benzoate, propylene glycol, 1,3-butanediol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol Alcohols, fatty acid esters of sorbitan, and mixtures thereof; besides inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring agents, flavoring agents and preservatives; suspension formulations may also contain suspending agents such as e
  • isotonic agents such as sugars, sodium chloride, and the like in the composition.
  • prolonged absorption of the injectable pharmaceutical form can be brought about by the incorporation of substances which delay absorption such as aluminum monostearate and gelatin.
  • the term “immunogenicity” refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It not only means that the antigen can stimulate specific immune cells, activate, proliferate, and differentiate the immune cells, and finally produce immune effector substances such as antibodies and sensitized lymphocytes, but also means that after the antigen stimulates the body, the body's immune system can form antibodies or Specific immune responses of sensitized T lymphocytes. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the reactivity of the host and the way of immunity.
  • immunogenic fragment refers to a polypeptide fragment which at least partially retains the immunogenicity of the protein from which it is derived.
  • an immunogenic fragment of the coronavirus spike protein refers to a fragment of the coronavirus spike protein that at least partially retains immunogenicity.
  • the term "additional immune adjuvant” includes, but is not limited to, stabilizers; emulsifiers; pH adjusting agents, such as sodium hydroxide, hydrochloric acid, etc.; liposomes; iscom adjuvants; synthetic glycopeptides, such as cell walls Acyl dipeptides; bulking agents, such as dextran; carbopol; bacterial cell walls, such as mycobacterial cell wall extracts; their derivatives, such as Corynebacterium parvum; Propionibacterium acnes ; Mycobacterium bovis (eg, Bovine Calmette Guerin, BCG); vaccinia or animal pox virus proteins; subviral particle adjuvants such as circovirus; cholera toxin; N,N-dioctadecane yl-N',N'-bis(2-hydroxyethyl)-propanediamine (pyridine); monophosphoryl lipid A; dimethyldioctadecy
  • suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ-amine or NZ-amine AS.
  • emulsifiers include, but are not limited to, mineral oil, vegetable oil, peanut oil, and other standard, metabolizable, non-toxic oils useful in injectable or intranasal vaccine compositions.
  • the term "subject" can be any animal, such as an avian or mammal.
  • the subject is a rodent, porcine, feline, canine, equine, primate, or avian.
  • the subject is a non-human subject.
  • the subject is a mouse, mink, guinea pig, Syrian golden hamster, or cynomolgus monkey.
  • the subject is a human.
  • COVID-19 and COVID-19 refer to pneumonia caused by SARS-CoV-2 infection, and both have the same meaning.
  • the term "effective amount” refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • a prophylactically effective amount refers to an amount sufficient to prevent, prevent, or delay the onset of a disease (eg, COVID-19);
  • a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent an existing The disease and the amount of its complications in the patient with the disease. Determining such effective amounts is well within the ability of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently and many more.
  • the present invention provides an adjuvant comprising zinc and 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 the antigen, and its effect is better than The effect of Al adjuvant.
  • the adjuvant of the present application when used in combination with an immunogen (such as the novel coronavirus spike protein (S protein)), in a variety of animals such as Balb/c mice, minks, guinea pigs, Syrian golden hamsters and cynomolgus monkeys , capable of stimulating or inducing high levels of functional antibodies with balanced cellular and humoral immune responses. Therefore, the adjuvant of the present application can improve the druggability of vaccines (eg, novel coronavirus S protein vaccine).
  • an immunogen such as the novel coronavirus spike protein (S protein)
  • Fig. 1 shows the appearance of the suspension of aluminum adjuvant (Al001) (Fig. 1A) and risedronate zinc-aluminum adjuvant (FH002C) (Fig. 1B) and the morphology of particles under optical microscope and electron microscope.
  • Figures 4A to 4F are the appearance of the suspension of FH002C adjuvant with different ratios of zinc, aluminum and risedronic acid and the morphology of the particles under the electron microscope.
  • Figure 5 shows the antigenicity detection results of the new coronavirus S recombinant protein without adjuvant and the dissociated vaccine preparation (FH002C combined with S recombinant protein), the figure shows the specific binding based on the ACE2 receptor at different protein concentrations ELISA test results.
  • the abscissa represents the molar concentration ratio of zinc: aluminum: risedronic acid.
  • Figure 17 shows the results of ELISA assay for T cell responses after immunizing Balb/c mice with FH002C or Al001 combined with novel coronavirus S recombinant protein
  • Figure A is the captured image
  • Figure B is the counting result of the captured image
  • *p ⁇ 0.05; ***p ⁇ 0.01; ***p ⁇ 0.001; ***p ⁇ 0.0001; n 8, mean ⁇ SD.
  • Victoria/WT means Victoria wild-type HA protein without deglycosylase (Pngase) treatment
  • Victoria/Pngase means Victoria wild-type HA protein treated with Pngase.
  • the reagents used in the preparation example are as follows:
  • Risedronate sodium (C 7 H 10 NNaO 7 P 2 ), purchased from Hunan Huateng Pharmaceutical Co., Ltd.;
  • Anhydrous zinc chloride (ZnCl 2 ), purchased from Xilong Chemical Industry;
  • Aluminum chloride hexahydrate (AlCl 3 ⁇ 6H 2 O), purchased from Xilong Chemical Industry;
  • Disodium hydrogen phosphate dodecahydrate Na 2 HPO 4 ⁇ 12H 2 O, purchased from Xilong Chemical Industry;
  • the risedronate zinc-aluminum adjuvant by co-precipitating the solution A and B in a volume ratio of 1:1, that is, the prepared solution B is dropwise in a volume ratio of 1:1 until all A is added.
  • the solution forms a suspension.
  • the suspension obtained after mixing was sterilized once at 121°C for 60 minutes, and the physicochemical properties such as pH, particle size and particle shape after sterilization were measured.
  • phosphate/Al molar concentration ratio 0.15:1, prepare 0.5L 124mM aluminum chloride solution, which is defined as A solution; prepare 0.5L 18.6mM disodium hydrogen phosphate solution, which is defined as B solution, in B solution Contains 140mM sodium hydroxide, 0.22 ⁇ m filter membrane for use.
  • the preparation process of aluminum adjuvant Al001 suspension can refer to the preparation of FH002C suspension.
  • Example 1 Determination of physicochemical properties of zinc aluminum risedronate (FH002C) and aluminum adjuvant (Al001)
  • the FH002C suspension (preparation example 1) and Al001 suspension (preparation example 3) obtained after mixing were sterilized once at 121°C for 60 minutes, and the pH, particle size, particle shape, zero charge point, etc. after sterilization were measured. physicochemical properties.
  • the aluminum adjuvant Al001 and the risedronate zinc aluminum adjuvant FH002C were diluted 50 and 100 times with deionized water, respectively, and then observed using a JEM-2100 transmission electron microscope (Transmission Electron Microscopy, TEM) from Japan Electronics.
  • the specific steps are as follows: drop the adjuvant sample on the copper mesh, let it adsorb for 10 min, wipe off the residual liquid with filter paper, send it into the sample chamber of the transmission electron microscope, observe the shape of the sample, and take a picture.
  • test sample place it at room temperature to equilibrate for at least 30 minutes, and measure it with a Sartorius acidity meter.
  • Err error message
  • the blank background and the size of the test standard sample should be corrected before each start-up measurement.
  • the sample cell is placed on the sample slot and the door is closed.
  • the Al001 adjuvant was pH adjusted to 6.5, 7.0, 7.5, 8.0, 8.5 and the FH002C adjuvant was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0 using 36%-38% hydrochloric acid and 10M NaOH.
  • Passivate the electrode add 3-4 mL of adjuvant to the sample tube, after the electrode is inserted, set the cycle in the SOP to: 50, and run the instrument to passivate the electrode.
  • Running the instrument After fully shaking the adjuvant of different pH, draw 2 mL into the plastic sample cell in turn, insert the electrode, put it into the sample tank, connect the instrument with the data of the sample tank, click "start” to start the measurement, and then Proceed to the next sample test.
  • the content of zinc was determined by the flame method of atomic absorption spectrometer:
  • Preparation of the test solution Dilute the sample with 0.2% nitric acid solution until the concentration is within the range of the standard curve (Abs reading value is between 0.2 and 0.8), and shake and mix with a vortex mixer before each step of dilution.
  • Select element select the "Select element” option on the “Element selection” page, select “Zn” on the “Load parameter” page, select the [Flame Continuous] measurement method, the "Use ASC” option does not need to be checked, select [Normal lamp] , click ⁇ OK>. No special settings are required on the "Calibration Curve Setting” and “Edit Parameters” pages, click ⁇ OK>. On the "Unconnected Instrument/Send Parameters” page, click ⁇ Connect/Send Parameters>.
  • Burner origin position adjustment select [Instrument] ⁇ [Maintenance] ⁇ [Burner Origin Position Adjustment] in turn, adjust the front and rear position knobs and the wrench on the burner to control the position of the burner, and observe the launch with the AA-6300C standard card
  • For the position of the light use the scale on the card to make the light passing through the entire burner slit on the same horizontal line.
  • After adjustment wait for the Abs value to stabilize; adjust the vertical height of the burner through the computer, ⁇ move up> or ⁇ down Move >, first coarse adjustment and then fine adjustment, make the Abs value half of the maximum indication (to ensure that 1/2 of the maximum light intensity passes through the burner), select ⁇ Origin Memory> and close the current page, cover the burner for safety cover.
  • the content of aluminum was determined by the graphite method of atomic absorption spectrometer:
  • Select element Select “Select element” option in “Element selection” page, select “Al” in “Load parameter” page, check “Use ASC” option, select [Normal light], click ⁇ Next>. No special settings are required on the “Calibration Curve Setting” and “Edit Parameters” pages, click ⁇ OK>. Turn on the power switch wrench (nEAT) of the GFA-EX7i, and click ⁇ Connect/Send Parameters> on the "Connect Host/Send Parameters” page. The "Instrument not connected, would you like to connect?" dialog box appears, click ⁇ Yes>.
  • Graphite furnace atomizer origin position adjustment select [Instrument], [Maintenance], and [Graphite Furnace Origin Position Adjustment] in turn.
  • the manual front and rear position knobs to move the graphite furnace atomizer until When the Abs indication reaches the maximum, use the WizAArd software to adjust the upper and lower positions of the atomizer, firstly adjust the [Fast] and then use the [Slow] to adjust until the indication reaches the maximum, and click ⁇ Origin Memory>.
  • Start-up preheating Turn on the main power switch on the right rear side of the instrument, and generally preheat for 10-20 minutes.
  • Option measurement as required Select Fixed Wavelength in the optional column on the top left corner of the screen to perform measurement.
  • Fixed Wavelength Click the Edit Method icon to display the wavelength setting interface, select the 262nm wavelength in the Number of wavelengths column, and click OK after setting. Put the sample in the blank tube into a clean cuvette, put it into the colorimetric rack, click the BLK icon to deduct the blank, then add the samples separately, and click Read.
  • the novel coronavirus S recombinant protein was designed and prepared as follows. Briefly, furin cleavage was removed by fusing the trimerization domain of the T4 phage fibritin protein to the C-terminus of the full-length extracellular segment of the wild-type S protein of SARS-CoV-2 (SEQ ID NO: 1). site, and fused 6*His tag at the C-terminus to design the recombinant novel coronavirus S protein (S recombinant protein, SEQ ID NO: 4).
  • the S recombinant protein was expressed using the Chinese hamster ovarian cancer cells (CHO) expression system (purchased from Thermo Scientific, A29133) and purified by nickel-sepharose column (Cytiva, 17-5318-03). , so as to obtain S recombinant protein with a purity greater than 95%. Purified S recombinant protein was quantified by the BCA method (Pierce TM BCA Protein Assay Kit, Thermo Scientific, 23227).
  • (1) Plate coating use 1x CB9.6 coating buffer to coat the 36H6 monoclonal antibody (made in the laboratory, the 36H6 monoclonal antibody is an antibody that recognizes the RBD epitope on the new crown S protein, prepared by hybridoma technology, the method is referred to Li , et al. Emerging Microbes Infection. 2020) diluted to 1 ⁇ g/mL, 100 ⁇ L/well was added to a polystyrene 96-well plate, and coated overnight at 4°C.
  • 1x CB9.6 coating buffer to coat the 36H6 monoclonal antibody (made in the laboratory, the 36H6 monoclonal antibody is an antibody that recognizes the RBD epitope on the new crown S protein, prepared by hybridoma technology, the method is referred to Li , et al. Emerging Microbes Infection. 2020) diluted to 1 ⁇ g/mL, 100 ⁇ L/well was added to a polystyrene 96-well plate, and coated
  • Blocking Discard the coating solution in the well, wash the plate once with PBST washing solution, spin dry, add blocking solution-1, 200 ⁇ L/well, and block at 25°C for 4 h.
  • enzyme-labeled antibody (85F7-HRP) (self-made in the laboratory): discard the liquid in the well, wash the plate 5 times with PBST, spin dry, add enzyme-labeled antibody (85F7-HRP, press 1:5000 (V:V) ) dilution, ED-11 as enzyme dilution) 100 ⁇ L/well, incubate the reaction at 25°C for 1h.
  • Color development Discard the enzyme-labeled antibody in the well, wash the plate 5 times with PBST, spin dry, add 100 ⁇ L/well of equal volume of color developing solution A and B mixed, and react at 25°C for 10 min.
  • Termination 50 ⁇ L/well, 2M sulfuric acid stop solution, to terminate the reaction.
  • Plate reading Set the detection dual wavelengths to 450 nm and 630 nm on the microplate reader, and measure the OD value of each reaction well.
  • the adjuvant adsorption rate of the vaccine preparation of FH002C compatible with S recombinant protein can reach more than 95% when it is adsorbed for 30 hours.
  • the adsorption rates were all above 95% (96.5%-99.9%).
  • Example 2 Determination of physicochemical properties of risedronate zinc-aluminum adjuvant (FH002C) with different ratios of zinc, aluminum and risedronate
  • the FH002C suspension (preparation example 2) and the Al001 suspension (preparation example 3) obtained after mixing were sterilized at 121°C for 60 min once, and the pH, adsorption rate, metal precipitation rate, appearance and particles after sterilization were measured. physical and chemical properties.
  • the pH measurement method is the same as the pH measurement process in Example 1.
  • BSA preparation 100mM NaAc is used as dilution buffer, a certain amount of BSA sample is weighed and diluted into EP tube, the final concentration is 1mg/mL.
  • adsorption rate The absorbance value of the supernatant at 280 nm was directly measured by an ultraviolet spectrophotometer, and the reading value was between 0.2 and 0.8.
  • Calculation of adsorption rate The OD reading of the diluted sample well is brought into the standard curve, and the corresponding protein concentration of the sample well is obtained, and then multiplied by the corresponding dilution factor, which is the protein concentration in the supernatant of the original sample.
  • Adjuvant adsorption rate (total protein concentration in original sample-protein concentration in supernatant)/total protein concentration in original sample*100%.
  • Preparation of mixed metal element standard solution Take a 12mL centrifuge tube and dilute the four metal single element standard solutions with 5% nitric acid to the highest concentration point STD7 of the standard curve. Then it was diluted with 1% nitric acid step by step (6 mL of hydrochloric acid plus 6 mL of the previous standard working solution) to obtain STD6 ⁇ STD1 solutions, and the final volume of each mixed standard solution was 6 mL to ensure complete injection.
  • test solution (at least 4mL): take the test sample and equilibrate to room temperature, mix thoroughly with a vortex mixer, accurately measure the test sample and dilute it with 5% nitric acid solution to an appropriate multiple, and let it stand for 1 hour after mixing. That is, the solution to be tested (200 times of dilution is recommended for the determination of calcium, magnesium and zinc content, and 50 times of dilution for the determination of aluminum content).
  • the appearance of the adjuvant can be observed in the form of particles by referring to the detection process in Example 1.
  • Example 3 Determination of antigenic activity of novel coronavirus S recombinant protein preparations containing FH002C (based on ACE2 receptor binding)
  • the FH002C (as prepared in Preparation Example 1) and the novel coronavirus S recombinant protein were mixed in a volume ratio of 1:1 to form a vaccine preparation, and placed at 4°C for 1 week (complete adsorption) to detect its antigenicity.
  • the unadsorbed new coronavirus S recombinant protein stock solution was used as a control, the antigenicity of the adjuvant after the antigen was adsorbed and dissociated was compared.
  • Antibody coating solution 1X CB 9.6 buffer (15 mM Na 2 CO 3 ; 35 mM NaHCO 3 ).
  • Blocking solution blocking solution-1, ELISA kit from INNOVAX company.
  • Color developing solution A ELISA kit from INNOVAX company.
  • Color developing solution B ELISA kit from INNOVAX company.
  • Blocking discard the coating solution in the well, wash the plate once with PBST washing solution, spin dry, add 200 ⁇ L/well of blocking solution, and block at room temperature for 4 hours.
  • Sample preparation S recombinant protein antigen stock solution (before adsorption), S recombinant protein vaccine preparation (the S recombinant protein antigen concentration in the preparation is 100 ⁇ g/mL, and the adjuvant is FH002C) after dissociation of the antigen.
  • the antigen concentration in the first well was 4 ⁇ g/mL. Discard the blocking solution in the well, wash the plate once with PBST, spin dry, add 200 ⁇ L/well of the serum to be tested in the first well, add 100 ⁇ L of sample diluent to each subsequent well, 2-fold gradient dilution, and incubate the reaction at 25°C for 1 h.
  • Color development Discard the enzyme-labeled antibody in the well, wash the plate 5 times with PBST, spin dry, add 100 ⁇ L/well of equal volume of color developing solution A and B mixed, and react at 25°C for 10 min.
  • Termination 50 ⁇ L/well of 2M sulfuric acid stop solution was added to terminate the reaction.
  • Plate reading Set the detection dual wavelengths to 450 nm and 630 nm on the microplate reader, and measure the OD value of each reaction well.
  • Example 4 Study on dose-response relationship of novel coronavirus S recombinant protein containing risedronate zinc-aluminum adjuvant
  • the FH002C adjuvant prepared according to Preparation Example 1 and the Al001 adjuvant prepared according to Preparation Example 3 were mixed with the new coronavirus recombinant S recombinant protein as adjuvants by volume ratio of 1: 1 to form a vaccine, and then intramuscularly injected into mice , to determine the specific antibody binding titer and neutralizing titer in serum.
  • the specific method is as follows:
  • mice purchased from Shanghai Slack Laboratory Animal Co., Ltd., item number: 2017000502480
  • 6-8 weeks 5 mice/group, female.
  • Immunization scheme S recombinant protein 1 ⁇ g/mouse or 10 ⁇ g/mouse, adjuvant and S recombinant protein were mixed at a volume ratio of 1:1 to form a vaccine, and then intramuscularly injected into mice, 100 ⁇ L per mouse, and 50 ⁇ L in each hind leg of the mouse.
  • immunization that is, 3 weeks after the first immunization of the animals according to the immunization group, blood was collected from the orbit, and the specific antibody titer in the serum was determined.
  • Booster immunization was performed in the third week, and orbital blood was collected every week thereafter, and the specific antibody binding titer in serum was determined by ELISA.
  • Antibody coating solution 1X CB 9.6 buffer (15 mM Na 2 CO 3 ; 35 mM NaHCO 3 ).
  • Blocking solution blocking solution-1, ELISA kit from INNOVAX company.
  • Color developing solution A ELISA kit from INNOVAX company.
  • Color developing solution B ELISA kit from INNOVAX company.
  • Plate coating Dilute the antigen S recombinant protein to a certain concentration with CB9.6 coating buffer. 100 ⁇ L/well was added to a polystyrene 96-well plate and coated overnight at 4°C.
  • Blocking discard the coating solution in the well, wash the plate once with PBST washing solution, spin dry, add 200 ⁇ L/well of blocking solution, and block at room temperature for 4 hours.
  • Color development Discard the enzyme-labeled antibody in the well, wash the plate 5 times with PBST, spin dry, add 100 ⁇ L/well of equal volume of color developing solution A and B mixed, and react at 25°C for 10 min.
  • Termination 50 ⁇ L/well of 2M sulfuric acid stop solution was added to terminate the reaction.
  • Plate reading Set the detection dual wavelengths to 450 nm and 630 nm on the microplate reader, and measure the OD value of each reaction well.
  • the antibody titer of the mice in the FH002C adjuvant group was higher than that in the aqueous solution group and the Al001 adjuvant group, and was 1-1.5 orders of magnitude higher than that in the aluminum adjuvant group.
  • the antibody titer of the FH002C adjuvant group was 1.5 orders of magnitude higher than that of the aluminum adjuvant group, and 3 orders of magnitude higher than that of the aqueous solution group.
  • the antibody levels of the FH002C adjuvant group and the Al001 group were comparable, and both were 2 orders of magnitude higher than the aqueous solution group.
  • the antibody titer of the FH002C adjuvant group was still significantly better than that of the Al001 group (0.5-1 order of magnitude) and the aqueous solution group (2.5-3 orders of magnitude).
  • mice in the FH002C adjuvant group After one injection of immunization, only the mice in the FH002C adjuvant group produced neutralizing antibodies in the third week, while the mice in the aqueous solution group and the Al001 adjuvant group did not.
  • the neutralizing titer of the mice in the FH002C adjuvant group was higher than that in the Al001 adjuvant group (0.5-1.5 higher than that in the high-dose group and the low-dose group). orders of magnitude) and aqueous solutions (1.5-2.5 orders of magnitude higher).
  • Example 5 Specific antibody binding titers produced by FH002C adjuvant with different ratios of zinc, aluminum and risedronic acid combined with novel coronavirus S recombinant protein
  • the prepared FH002C (see preparation example 2 for the preparation method) was mixed with the new coronavirus S recombinant protein as an adjuvant to form a vaccine by volume ratio of 1:1, and then intramuscularly injected into Balb/C mice to measure the specific antibodies produced. titer.
  • the specific method is as follows:
  • mice Balb/C mice, 6-7 weeks, female, 5 mice per group
  • Immunization scheme 1 ⁇ g of antigen per animal, 100 ⁇ L per animal (half for left and right legs). Two injections of immunization were given at 0 and 3 weeks, and the antibody titer in the serum was detected after weekly orbital blood collection from 0 to 6 weeks.
  • Antibody binding titers were detected by enzyme-linked immunosorbent assay (ELISA), as follows:
  • Serum dilution add 100 ⁇ L of 300-fold diluted serum to the first well, 3-fold dilution for 10 gradients, and incubate at 37° C. for 1 h.
  • Example 6 Specific antibody binding titers produced by risedronate zinc-aluminum adjuvant combined with novel coronavirus S recombinant protein
  • the prepared Al001 (preparation example 3) and FH002C (preparation example 1) were mixed with the new coronavirus S recombinant protein as adjuvants respectively by volume ratio of 1: 1 to form a vaccine, and then intramuscularly injected Syrian golden hamsters (also referred to herein as "Syrian hamster") and cynomolgus monkeys, and the specific antibody titers produced were determined.
  • the specific method is as follows:
  • Cynomolgus monkey immunization scheme antigen 20 ⁇ g/mouse, adjuvant and novel coronavirus S recombinant protein were mixed by volume ratio of 1:1 to form a vaccine, and then intramuscular injection of cynomolgus monkeys, 500 ⁇ L each. 0 and 2 weeks of immunization with 2 injections, and weekly blood collection for antibody binding titer detection from 0 to 6 weeks.
  • Antibody binding titers were detected by enzyme-linked immunosorbent assay (ELISA), as follows:
  • Serum dilution 100 ⁇ L of 50- or 100-fold diluted serum was added to each well, and incubated at 37°C for 1 h.
  • the antibody titer of the FH002C group was higher than that of the Al001 group 2 weeks after the immunization of Syrian golden hamsters, which was 15 times that of the aluminum adjuvant group, which had the characteristics of fast onset. After two injections of immunization, its humoral immunity enhancement was still obvious. At the 4th week, the antibody titer of the FH002C group was 8 times that of the aluminum adjuvant group. After three injections of immunization, the antibody titer of the FH002C group was 11 times higher than that of the control group at the 7th week.
  • the antibody titer of the FH002C group was higher than that of the Al001 group, which was 6 times that of the aluminum adjuvant group, which had the characteristics of fast onset. After two doses of immunization, the antibody titer of the FH002C group was comparable to that of the aluminum adjuvant group at the 4th week (Fig. 9).
  • Example 7 Neutralizing antibody titers produced by risedronate zinc-aluminum adjuvant combined with novel coronavirus S recombinant protein
  • Example 6 Using the experimental procedure described in Example 6, Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were combined with the new coronavirus S recombinant protein to immunize Syrian golden hamsters and cynomolgus monkeys by intramuscular injection. and antibody titers. The immunization procedure of Syrian golden hamster and cynomolgus monkey is the same as that of Example 6.
  • Antibody neutralization titers were detected by Pseudovirus neutralization (H.L.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, 1-38 (2020)), as follows:
  • the vesicular stomatitis virus (vesicular stomatitis virus) carrying the SARS-CoV-2 S protein (Wuhan-Hu-1 strain, gene bank: QHD43416.1) was recombined with the recombinant plasmids pCAG-nCoVSdel18 and VSVdG-EGFP-G virus (Addgene, 31842).
  • VSV VSV pseudovirus
  • hACE2-expressing BHK21 (BHK21-hACE2) cells were plated into 96-well plates, followed by high-glucose DMEM medium (Sigma) containing 10% FBS (GIBCO, 10099141) and penicillin-streptomycin (GIBCO, 15140122).
  • high-glucose DMEM medium (Sigma) containing 10% FBS (GIBCO, 10099141) and penicillin-streptomycin (GIBCO, 15140122).
  • -Aldrich, D6429 Serum was diluted 50-fold followed by a 3-fold serial dilution.
  • the mixture was transferred to a plate plated with BHK21-hACE2 cells, followed by incubation in an incubator containing 5% CO 2 at 37° C. for 12 h.
  • Fluorescence image data was captured by the Opera Phenix or Operetta CLS High Content Analysis System (PerkinElmer) and the counts of GFP positive cells per well were analyzed using the Columbus system (PerkinElmer).
  • Each plate contained 8 serum-free wells as virus controls.
  • Serum neutralization titer was defined as the serum dilution (ID50) corresponding to a 50 % reduction in the number of GFP-positive cells compared to positive wells.
  • the antibody titer of the FH002C group was higher than that of the Al001 group, 1.8 times that of the aluminum adjuvant group, 2 weeks after the Syrian golden hamster was immunized with one injection, which has the characteristics of fast onset. After two injections of immunization, its humoral immunity enhancement was still obvious. At the 4th week, the antibody titer of the FH002C group was 2.6 times that of the aluminum adjuvant group. After three injections of immunization, the antibody titer of the FH002C group was 6.5 times that of the control group at the 7th week.
  • the antibody titer of the FH002C group was higher than that of the Al001 group, which was 2.6 times that of the aluminum adjuvant group, which had the characteristics of fast onset. After two doses of immunization, the antibody titer of the FH002C group was 3.5 times that of the aluminum adjuvant group at the 4th week (Fig. 11).
  • Example 8 Cell-based Spike blocking experiment with risedronate zinc-aluminum adjuvant combined with novel coronavirus S recombinant protein immune serum
  • hACE2-mRuby3 293T (293T-ACE2iRb3) were plated on 96-well plates pretreated with poly-D-lysine at 2 ⁇ 10 4 cells/well 24 h in advance.
  • Serum of Syrian golden hamster and cynomolgus monkey was diluted with 2-fold serial dilution in DMEM medium containing 10% FBS.
  • 11 ⁇ L of Bacillus fused to SARS-CoV-2 S (STG) probe was mixed with 44 ⁇ L of diluted serum to give a final probe concentration of 2.5 nM.
  • Example 9 Effect of risedronate zinc-aluminum adjuvant combined with novel coronavirus S recombinant protein on antibody affinity
  • Example 6 Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were combined with the new coronavirus S recombinant protein to immunize mice and Syrian golden hamsters by intramuscular injection, and serum antibodies were detected. affinity.
  • the immunization procedure of Syrian golden hamsters was the same as that in Example 6, but there were 16 FH002C and Al001 in each group, half male and half female.
  • the immunization program of the mice is as follows:
  • mice purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • 6-8 weeks 4 or 5 mice/group, female
  • mice immunization protocol antigen 1 ⁇ g/mouse, adjuvant and novel coronavirus S recombinant protein were mixed at a volume ratio of 1:1 to form a vaccine, and then Balb/C mice were intramuscularly injected, 150 ⁇ L per mouse. Three injections of immunization were performed at 0, 2, and 4 weeks, and the antibody avidity in serum was detected after weekly orbital blood collection from 0 to 6 weeks.
  • Antibody affinity was detected by enzyme-linked immunosorbent assay (ELISA), as follows:
  • Serum antibody affinity detection add 100 ⁇ L of serum to each well, repeat each sample in two wells, and incubate at 37°C for 1 h. After the plate was washed once with PBST buffer, 100 ⁇ L of PBS was added to one well of the double well, and 100 ⁇ L of 4M urea (prepared with PBS) was added to the other well, and the plate was washed with PBST buffer for 20 min at 37°C. Second-rate.
  • the prepared Al001 (preparation example 3) and FH002C (preparation example 1) were mixed with the novel coronavirus S recombinant protein as adjuvants to form a vaccine by volume ratio of 1: 1, and then intramuscularly injected into mice, and the immunization program was as in the example 9.
  • Enzyme-linked immunosorbent assay to detect antibody subtype levels as follows:
  • Plate coating and blocking The purified S recombinant protein was diluted to 2 ⁇ g/mL with carbonate buffer and then coated, added to the ELISA plate according to 100 ⁇ L/well, placed at 37°C for 2 hours of incubation, and then coated with PBST buffer ( That is, the plate was washed once with 0.05% Tween-20 in PBS, and 200 ⁇ L of PBS containing 20% NBS was added to each well, and then incubated at 37° C. for 2 h for blocking. Remove excess liquid and shake dry.
  • Detection of serum antibody subtype levels Add 100 ⁇ L of serum to each well, incubate at 37°C for 1 h, and incubate at 37°C for 1 h. After washing the plate for 5 times with PBST buffer, 100 ⁇ L of HRP-conjugated goat anti-mouse IgG1 (AbD Serotec, STAR132P) or goat anti-mouse IgG2a (AbD Serotec, STAR133P) or goat anti-mouse IgG2b (AbD Serotec, STAR133P) or goat anti-mouse IgG2b (AbD Serotec, STAR133P) was added. , STAR134P), incubated at 37°C for 0.5h.
  • the FH002C adjuvant group can stimulate higher levels of IgG2a and IgG2b subtype antibodies, and the ratio of IgG1 to IgG2a and IgG2b is lower than that of the aluminum adjuvant group , indicating that it has a certain stimulating effect on the Th1 immune pathway.
  • Example 11 Influence of risedronate zinc-aluminum adjuvant with novel coronavirus S recombinant protein on T cell response
  • the prepared Al001 (preparation example 3) and FH002C (preparation example 1) were mixed with the novel coronavirus S recombinant protein as adjuvants to form a vaccine by volume ratio of 1:1, and then intramuscularly injected into mice to measure the T cells produced. answer.
  • the specific method is as follows:
  • mice C57BL/6, 6-8 weeks, 8 animals/group, female.
  • Immunization scheme of C57BL/6 mice antigen 10 ⁇ g/mouse, adjuvant and novel coronavirus S recombinant protein were mixed at a volume ratio of 1:1 to form a vaccine, and then intramuscularly injected, 150 ⁇ L per mouse. Two injections of immunization were given at 0 and 3 weeks, and the spleen and lymph nodes were separated for T cell immune response experiment after sacrifice at the 4th week.
  • Enzyme-linked immunospot assay (ELISPOT) to detect T cell responses
  • mice spleen and lymph nodes Take the mouse spleen and lymph nodes, prepare a single cell suspension, and plate them on mouse IFN- ⁇ -coated ELISPOT plates ( DAKEWEI , 2210005). Then, the cells were stimulated with PBS or a 15-unit SARS-CoV-2S peptide library with 11 amino acid overlaps (Genscript, RP30020) for 20 h. Subsequently, the detection was performed according to the kit instructions. use (Cellular Technology Limited) for image capture and spot counting. The number of IFN- ⁇ secreting cells was calculated by subtracting PBS-stimulated wells from spike peptide pool-stimulated wells.
  • Example 12 The specific antibody binding titer produced by the combination of aluminum adjuvant (Al001) and FH002C adjuvant with varicella zoster virus gE protein (VZV gE)
  • the prepared Al001 (Preparation Example 3) and FH002C (Preparation Example 1) were respectively mixed with the varicella zoster virus gE protein (VZV gE, SEQ ID NO: 2) as adjuvants to form a vaccine in a volume ratio of 1:1, Balb/C mice were then injected intramuscularly, and the specific antibody titers produced were determined.
  • the gE protein is obtained by using E. coli expression system (purchased from Shanghai Weidi Biotechnology Co., Ltd., EC1060).
  • mice Balb/C mice, 6-7 weeks, 5 in each group, female.
  • Balb/C mouse immunization scheme antigen 5 ⁇ g/mouse, adjuvant and varicella zoster virus gE protein (VZV gE) were mixed in a volume ratio of 1:1 to form a vaccine, and then intramuscularly injected into Balb/C mice, 100 ⁇ L per mouse . Two injections of immunization were given at 0, 2, and 4 weeks, and the antibody titer in serum was detected after weekly orbital blood collection from 0 to 4 weeks.
  • VZV gE varicella zoster virus gE protein
  • Antibody binding titers were detected by enzyme-linked immunosorbent assay (ELISA), as follows:
  • Serum dilution 100 ⁇ L of 50- or 100-fold diluted serum was added to each well, and incubated at 25° C. for 1 h.
  • Example 13 The specific antibody binding titer produced by the combination of Freund's adjuvant and FH002C adjuvant with influenza virus HA protein
  • influenza virus HA treated with or without deglycosylase was mixed in a volume ratio of 1:1 to form a vaccine, and then injected subcutaneously into Balb/C mice, and the specific antibody titers produced were determined.
  • influenza virus HA protein was expressed by using a baculovirus expression system (purchased from Invitrogen, USA, 10359016).
  • the deglycosylase was purchased from NEB, item number P0705S; the deglycosylase treatment process was carried out with reference to the description of the deglycosylase.
  • mice purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • mice/group in FH002C group 10 mice/group in Freund's adjuvant group.
  • Immunization scheme (1) Antigen 30 ⁇ g/mouse, Freund’s adjuvant and influenza virus HA protein were mixed by volume ratio of 1:1 to form a vaccine, and then subcutaneously injected into Balb/C mice, 300 ⁇ L per mouse; (2) Antigen 30 ⁇ g/ Only, FH002C and influenza virus HA protein were mixed in a volume ratio of 1:1 to form a vaccine, and then Balb/C mice were intramuscularly injected with 200 ⁇ L each. Antibody titers in serum were measured 14 days after immunization by orbital blood collection.
  • Antibody binding titers were detected by enzyme-linked immunosorbent assay (ELISA), as follows:
  • Serum dilution 100 ⁇ L of serum diluted in different times was added to each well, and incubated at 37° C. for 1 h.
  • Example 14 Binding titers of neutralizing antibodies produced by aluminum adjuvant (Al001) and FH002C adjuvant combined with rotavirus VP4 protein
  • the prepared Al001 (preparation example 3) and FH002C (preparation example 1) were mixed with the rotavirus VP4 protein as adjuvants respectively to form a vaccine by volume ratio of 1:1, and then intramuscularly injected into Balb/C mice and guinea pigs, and determined. Neutralizing antibody titers produced.
  • the VP4 protein was expressed by E. coli expression system (purchased from Shanghai Weidi Biotechnology Co., Ltd., EC1060).
  • mice purchased from Shanghai Slack Laboratory Animal Co., Ltd., 6-8 weeks old, guinea pigs, 450-500 g, purchased from Shanghai Songlian Experimental Animal Farm.
  • mice group 5 mice/group in FH002C group and 5 mice/group in Al001 group, all female.
  • Guinea pig group FH002C group 5/group, Al001 group 5/group, all female.
  • Immunization scheme 10 ⁇ g/mouse immunization antigen, 10 ⁇ g/guinea pig immunizing antigen, adjuvant and rotavirus VP4 protein were mixed in a volume ratio of 1:1 to form a vaccine, and then intramuscularly injected into mice, guinea pigs, and mice and guinea pigs. 200 ⁇ L per animal, blood was collected before the first immunization and 2 weeks after the third immunization, and the sera were separated to detect the neutralizing antibody titer.
  • ELISPOT enzyme-linked immunospot assay
  • MA104 cells are plated in a 96-well cell plate: after the cells are digested with trypsin, the cells are blown up in a cell culture medium containing 10% FBS, and then counted in a cell counting plate, and the medium is added to dilute to 25,000 cells/mL, and then evenly added to 96-well plates, 100 ⁇ L/well, incubated at 37°C for 20 h, and the concentration of CO 2 was 5%.
  • Virus digestion treatment add 4 ⁇ L of 2.5 ⁇ g/ ⁇ L trypsin to 1 mL of rotavirus solution, mix well and place it in, and dilute it with serum-free DMEM medium containing 1 ⁇ g/mL trypsin after treatment virus to a certain titer.
  • Serum complement inactivation treatment 10 ⁇ L of serum samples were taken in a 1.5 mL EP tube, and then heat-treated at 56° C. for 30 min. After the treatment, the serum samples were diluted with DMEM containing 1 ⁇ g/ml trypsin, and the double-well detection was repeated.
  • Serum and virus reaction 100 ⁇ L of virus in (2) and serum in (3) were mixed, and neutralized at 37° C. for 1 h.
  • Cell fixation spin dry the cell supernatant in (6), be careful not to damage the cells, then fix the cells with 0.1% glutaraldehyde-containing PBS solution, 100 ⁇ L/well, fix at room temperature for 1 h, pay attention to Fix in dark conditions to avoid photodecomposition of glutaraldehyde and affect the fixation effect.
  • Enzyme-labeled antibody reaction Drain the rinse solution, and then dilute the HRP-labeled VP6 antibody (made in the laboratory, the monoclonal antibody is The antibody recognizing VP6 was prepared by hybridoma technology, the method was referred to Li, et al. Emerging Microbes Infection. 2020, 1:5000 dilution) was added to the cells, and reacted at 37°C for 1 h.
  • mice and guinea pigs could produce neutralizing antibodies after three injections of immunization, and the neutralizing antibody titer of the FH002C group was higher than that of the Al001 group.
  • the serum neutralization titer of the FH002C adjuvant group was about 16 times that of the Al001 adjuvant group ( Figure 20); while in guinea pigs, the serum neutralization titer of the FH002C adjuvant group was about 4 times that of the Al001 group. times ( Figure 21).

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Abstract

一种含有利塞膦酸锌铝的佐剂，一种包含所述佐剂和免疫原的免疫原性组合物，以及所述佐剂和免疫原性组合物的用途。

Description

含有利塞膦酸锌铝的佐剂及其应用 技术领域
本申请涉及生物制药技术领域。具体地，本申请涉及一种含有利塞膦酸锌铝的佐剂，一种包含所述佐剂和免疫原的免疫原性组合物，以及所述佐剂和免疫原性组合物的用途。
技术背景
佐剂是一类能与免疫原特异或者非特异性结合，并刺激诱导机体产生长期有效的特异性免疫应答的物质或混合物。佐剂的免疫生物学作用包括降低免疫原的使用量、增强抗原的免疫原性、改变免疫应答类型等。目前被批准用于人用疫苗的佐剂有铝佐剂、MF59、AS04、AS01B、CpG1018等，并有数种新型佐剂处于临床试验阶段，其包括：AS03、RC-529、Advax、Matrix-M等。铝佐剂是第一个被批准用于人用疫苗的佐剂，至今已有近90年的使用历史，是公认的应用最为广泛、安全、有效的佐剂。然而，随着疫苗研究品种的增加，发现铝佐剂刺激诱导Th2类免疫优势应答，在诱导产生Th1类免疫应答上作用有限，这也限制了铝佐剂在治疗性疫苗中的应用，比如水痘带状疱疹病毒疫苗、乙肝治疗性疫苗以及肿瘤疫苗等。并且，与许多新型疫苗佐剂相比，铝佐剂活性较弱，对类病毒颗粒抗原以外的其它大多数基因工程抗原免疫增强作用不理想，使其在非多聚化蛋白抗原疫苗中的应用受到限制。
含铝佐剂系统是指以铝佐剂为基础，与其他佐剂成分或递呈系统(如MPL、CPG、QS21和脂质体等)形成的佐剂系统，可诱导相对平衡的Th1和Th2免疫应答。目前已有数种含铝佐剂系统批准上市或进入临床试验(NPJ Vaccines.2018，10；3：51.)。例如，由葛兰素史克公司(GlaxoSmithKline，GSK)研发的AS04，该佐剂系统是在铝佐剂的基础上添加TLR-4(Toll like receptor 4，TLR4)激动剂：3-O-去酰基-4”-单磷酰脂质A(Monophosphoryl lipid A，MPL)，因其氨基葡萄糖磷酸化，与Al 3+有很高的亲和力，被铝佐剂吸附形成复合型佐剂，其可提高抗原特异性CD4 +T细胞γ-干扰素(Interferon-γ，IFN-γ)的表达(IFN-γ为T细胞应答的重要指标之一)。目前AS04已成功应用于乙肝疫苗(Fendrix，2005获批上市)和HPV16/18双价宫颈癌疫苗(Cervarix，2007获批上市)中。相较于单独使用铝佐剂而言，其可明显提高HPV16/18 双价宫颈癌疫苗(Cervarix)中和抗体滴度并延长免疫保护有效期，并且该疫苗免疫一或两次可产生类似于免疫三针次所提供的保护效果。CpG，为TLR-9的受体激动剂，其与铝佐剂联合作为疟疾疫苗佐剂、钩虫疫苗佐剂已进入临床试验中。
铝佐剂与抗原的吸附作用是其免疫增强机制的原因之一。配体交换是佐剂和抗原间最强的一种相互作用力，由抗原中的磷酸基团与氢氧化铝或者磷酸铝中的羟基基团发生配体交换而产生，此为发明人Zhao在2001年提出的铝佐剂白表面“亲磷性”的概念(Analytical Biochemistry，2001，295(1)：76-81)。前述的AS04，MPL通过磷酸基团与铝佐剂吸附而形成新型的复合佐剂在临床中已得到了很好的应用。
双膦酸盐(Bisphosphonates，BPs)药物是一类以P-C-P为中心骨架的人工合成焦磷酸类似物，为有机双膦酸，因其含有磷酸基团使得与钙、铝、锌、镁等离子具有高度亲和力，被用于骨疾病、钙代谢性疾病的治疗，例如骨质疏松症，变形性骨炎，恶性肿瘤骨转移引起的高钙血症和骨痛症等。同时，临床研究显示将双膦酸药物用于多发性骨髓瘤、乳腺癌、肾癌、前列腺癌等的辅助治疗可降低患者骨相关疾病的发生率，癌症复发率以及提高患者的存活期并改善临床结局。除此之外，双膦酸因具有免疫正向调节作用而表现出佐剂活性，其作为免疫增强剂被用于疫苗制剂的专利发明亦有报道(中国专利：CN103768595B；美国专利：US20170281759A1；中国专利：CN 108289902A)。双膦酸药物在临床上通常采用口服或静脉注射给药，其具有较强的粘膜刺激作用，而在疫苗的使用中，通常以肌肉注射的方式进行免疫。然而，双膦酸具有潜在的副作用，仍需要进一步改进。
冠状病毒(Coronavirus，CoV)是一种具有包膜的、非节段的单股正链RNA病毒，属于巢病毒目(Nidovirales)冠状病毒科(Coronaviridae)，正冠状病毒亚科(Orthocoronavirinae)，被分为α、β、γ和δ四个属。截止目前为止，发现有7种冠状病毒可感染人类，其中以季节性流行为主的冠状病毒包括：α属的229E和NL63，β属的OC43和HKU1，以及大流行冠状病毒：严重急性呼吸综合征相关冠状病毒(SARS-CoV)、中东呼吸综合征相关冠状病毒(MERS-CoV)和新型冠状病毒(SARS-CoV-2)。目前已知冠状病毒可感染人、鼠、猪、猫、犬、水貂、豚鼠、地鼠、恒河猴以及禽类等脊椎动物，同时荷兰病毒学家证实新型冠状病毒可由人类传播给水貂再传回给人类，证实新型冠状病毒动物传人传播链。SARS-CoV、MERS-CoV以及SARS-CoV-2具有较强的传播能力和致病性。目前新型冠状病毒的全球大流行，给人 们的生命健康安全和社会活动造成重大威胁并引起巨大的经济损失，而疫苗是控制和预防该病毒感染的最有效手段。
冠状病毒刺突蛋白(S蛋白)是一种I型跨膜蛋白，由S1和S2蛋白亚基两部分组成，介导与宿主细胞表面受体的结合和细胞膜的融合以进入靶细胞。多项证据表明，针对刺突蛋白的抗体可能在新型冠状病毒性肺炎的免疫预防和治疗中发挥关键作用，是目前新型冠状病毒重组蛋白疫苗研制和设计的关键靶点。有研究发现，急性新型冠状病毒感染会使其长效的中和抗体反应减弱，但仍可通过病毒特异性记忆T细胞实现免疫记忆(Cell，2020，183(1)：13-15)，说明T细胞应答在预防新型冠状病毒感染的重要性。重组DNA技术表达制备的亚单位蛋白疫苗具有较好的安全性，并可实现多针次加强免疫，但其免疫原性较弱。同时，世界卫生组织(WHO)与2020年4月份制定了2019冠状病毒病(COVID-19)疫苗的目标产品特性，在疫情暴发时，疫苗须快速起效，在2周内可提供保护，同时1针实现基础免疫，并不超过2剂的给药方案，同时在保护持久性上要求至少提供6个月的保护。
因此，本领域仍然需要开发新的佐剂，以提高疫苗(例如COVID-19疫苗)的功效。
发明内容
本申请的发明人经过深入的研究，开发获得了一种包含利塞膦酸锌铝的新型佐剂，其可作为疫苗佐剂或药物递送载体等，可用于有效提高抗原的免疫原性。
特别地，已惊讶地发现，当本申请的佐剂与免疫原(例如新型冠状病毒刺突蛋白(S蛋白)联用时，在Balb/c小鼠、水貂、豚鼠、叙利亚金黄地鼠和食蟹猴等多种动物上，能够刺激或诱导高水平的功能性抗体与平衡的细胞和体液免疫应答。因此，本申请的佐剂可提高疫苗(例如新型冠状病毒S蛋白疫苗)的成药性。
此外，还已发现，当本申请的佐剂与新型冠状病毒刺突蛋白(S蛋白)联用时，能有效预防新型冠状病毒(SARS-CoV-2)的感染。例如，含有本申请的佐剂和新型冠状病毒S蛋白的疫苗能有效阻止或降低SARS-CoV-2在动物(例如水貂)的上下呼吸道中的复制，并防止新型冠状病毒对动物(例如水貂)的肺部的感染和损害。
佐剂
在一个方面，本申请提供了一种包含利塞膦酸锌铝的佐剂(在本文中也简称利塞 膦酸锌铝佐剂)，其中，锌∶利塞膦酸摩尔浓度比在1∶1至16∶1的范围内(例如，2∶1至16∶1的范围内)；并且，锌∶铝摩尔浓度比在1∶1至50∶1的范围内(例如5∶1至50∶1的范围内)。
在某些实施方案中，利塞膦酸锌铝以颗粒形式存在。在某些实施方案中，利塞膦酸锌铝以纳米级颗粒或微米级颗粒形式存在。在某些实施方案中，所述颗粒的粒径大小为0.01-100μm，例如0.01-60μm，0.01-50μm，0.1-60μm，0.1-30μm，0.4～30μm，0.4-20μm。
在某些实施方案中，所述佐剂锌∶利塞膦酸摩尔浓度比在1∶1至2∶1，2∶1至4∶1，4∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至14∶1或者14∶1至16∶1的范围内。在某些实施方案中，所述佐剂锌∶利塞膦酸摩尔浓度比为至少1∶1，至少2∶1，至少3∶1，至少4∶1，至少5∶1，至少6∶1，至少7∶1，至少8∶1，至少10∶1，至少12∶1，至少14∶1，或者至少15∶1。在某些实施方案中，所述佐剂锌∶利塞膦酸摩尔浓度比为不超过16∶1，不超过14∶1，不超过12∶1，不超过10∶1，不超过8∶1，不超过7∶1，不超过6∶1，不超过5∶1，不超过4∶1，不超过3∶1，或者不超过2∶1。
在某些实施方案中，所述佐剂锌∶利塞膦酸摩尔浓度比为1∶1、2∶1、4∶1、4.5∶1、6∶1、8∶1、10∶1、12∶1、14∶1或16∶1。在某些实施方案中，所述佐剂锌∶利塞膦酸摩尔浓度比为4∶1或4.5∶1。
在某些实施方案中，所述佐剂锌∶铝摩尔浓度比在1∶1至2∶1，2∶1至3∶1，3∶1至4∶1，4∶1至5∶1，5∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至15∶1，15∶1至20∶1，20∶1至30∶1，30∶1至40∶1，或者40∶1至50∶1的范围内。在某些实施方案中，所述佐剂锌∶铝摩尔浓度比为至少1∶1，至少2∶1，至少3∶1，至少4∶1，至少5∶1，至少6∶1，至少8∶1，至少10∶1，至少12∶1，至少15∶1，至少20∶1，至少30∶1，或者至少40∶1。在某些实施方案中，所述佐剂锌∶铝摩尔浓度比为不超过50∶1，不超过40∶1，不超过30∶1，不超过20∶1，不超过15∶1，不超过12∶1，不超过10∶1，不超过8∶1，不超过6∶1，不超过5∶1，不超过4∶1，不超过3∶1，或者不超过2∶1。
在某些实施方案中，所述佐剂锌∶铝摩尔浓度比为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、或1∶1。在某些实施方案中，所述佐剂锌∶铝摩尔浓度比为10∶1。
在某些实施方案中，所述佐剂中，锌∶利塞膦酸摩尔浓度比在2∶1至8∶1的范围内， 例如2∶1至4∶1，4∶1至6∶1，或6∶1至8∶1，并且，锌∶铝摩尔浓度比在2∶1至50∶1的范围内(例如5∶1至20∶1的范围内)，例如2∶1至3∶1，3∶1至4∶1，4∶1至5∶1，5∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至15∶1，或15∶1至20∶1。
在某些实施方案中，所述佐剂中，锌∶利塞膦酸摩尔浓度比为4∶1，并且，锌∶铝摩尔浓度比为10∶1。
在某些实施方案中，所述佐剂中，锌∶利塞膦酸摩尔浓度比为4.5∶1，并且，锌∶铝摩尔浓度比为10∶1。
在某些实施方案中，所述佐剂的pH为5.0-8.0，例如5.0-7.0，5.0-5.5，5.5-6.0，6.0-6.5，6.5-7.0，7.0-7.5，或7.5-8.0。在某些实施方案中，所述佐剂pH为7.0～7.5。在某些实施方案中，所述佐剂pH为5.5～6.5。在某些实施方案中，所述佐剂pH为5.8～6.3。
在某些实施方案中，所述佐剂零电荷点为3.0～8.0，例如4.0-8.0，3.0-4.0，4.0-5.0，5.0-6.0，6.0-7.0，或7.0-8.0。在某些实施方案中，所述佐剂零电荷点为4.0～6.0。
本申请的佐剂对免疫原(例如蛋白)具有良好的吸附率。在某些实施方案中，本发明的佐剂对免疫原(例如蛋白)的吸附率为至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，或至少95％。
免疫原性组合物
不受理论限制，本发明的佐剂可以与各种免疫原联用，用于增强免疫原的免疫原性。此类免疫原包括但不限于蛋白，核酸，多糖，或其免疫原性部分等。
因此，在一个方面，本申请提供了一种免疫原性组合物，其包含免疫原和如上所述的佐剂。
在某些实施方案中，所述免疫原包括但不限于蛋白，核酸，多糖，或其免疫原性部分等。在某些实施方案中，所述免疫原来源于病毒、细菌、真菌等病原体。
在某些实施方案中，所述免疫原为蛋白或其免疫原性片段，例如来源于病毒、细菌、真菌等病原体的蛋白或其免疫原性片段。在某些实施方案中，所述病毒包括但不限于，呼吸道病毒(例如，流感病毒、副流感病毒、鼻病毒、冠状病毒、呼吸道合胞病毒)，肠道病毒(例如EV71病毒，轮状病毒)，水痘带状疱疹病毒(VZV)。
在某些实施方案中，所述免疫原为冠状病毒的蛋白(例如刺突蛋白)或其免疫原性片段。在某些实施方案中，所述冠状病毒选自正冠状病毒亚科α属病毒(例如229E和NL63)，正冠状病毒亚科β属病毒(例如OC43和HKU1)、严重急性呼吸综合征 相关冠状病毒(SARS-CoV)、中东呼吸综合征相关冠状病毒(MERS-CoV)，和新型冠状病毒(SARS-CoV-2)。在某些实施方案中，所述冠状病毒选自严重急性呼吸综合征相关冠状病毒(SARS-CoV)、中东呼吸综合征相关冠状病毒(MERS-CoV)和新型冠状病毒(SARS-CoV-2)。在某些实施方案中，所述冠状病毒为新型冠状病毒(SARS-CoV-2)。在某些实施方案中，所述免疫原为新型冠状病毒的结构蛋白或其免疫原性片段，例如刺突蛋白(S蛋白)或其免疫原性片段。
在某些实施方案中，所述免疫原是水痘带状疱疹病毒的蛋白或其免疫原性片段，例如水痘带状疱疹病毒gE蛋白或其免疫原性片段。
在某些实施方案中，所述免疫原是流感病毒的蛋白或其免疫原性片段，例如流感病毒HA蛋白或其免疫原性片段。
在某些实施方案中，所述免疫原是轮状病毒的蛋白或其免疫原性片段，例如轮状病毒VP4蛋白或其免疫原性片段。
在某些实施方案中，所述免疫原性组合物还包含药学上可接受的辅料，例如赋形剂、防腐剂、抗菌剂、缓冲剂和/或额外的免疫佐剂。在某些实施方案中，所述额外的免疫佐剂选自铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子，或其任何组合。
在某些实施方案中，所述免疫原性组合物还包含第二免疫原。在某些实施方案中，所述第二免疫原包括但不限于蛋白，核酸，多糖，或其免疫原性部分等。
在某些实施方案中，所述免疫原性组合物为疫苗。
佐剂制备方法
在一个方面，本申请提供了制备如上所述的佐剂的方法，其包括如下步骤：
1)提供含有锌离子、铝离子的可溶性盐溶液；
2)将步骤(1)中的可溶性盐溶液与利塞膦酸碱溶液混合，从而获得所述佐剂。
在某些实施方案中，所述方法还包括，将步骤(2)获得的佐剂进行灭菌的步骤。在某些实施方案中，通过过滤灭菌或高温高压灭菌来对所述佐剂进行灭菌。在某些实施方案中，通过在121℃下灭菌至少15分钟(例如至少30分钟，例如30-60分钟)，对所述佐剂进行灭菌。
在某些实施方案中，在所述可溶性盐溶液中，锌∶铝的摩尔浓度比在1∶1至50∶1的范围内(例如5∶1至50∶1的范围内)。在某些实施方案中，在所述可溶性盐溶液中， 锌∶铝摩尔浓度比在1∶1至2∶1，2∶1至3∶1，3∶1至4∶1，4∶1至5∶1，5∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至15∶1，15∶1至20∶1，20∶1至30∶1，30∶1至40∶1，或者40∶1至50∶1的范围内。在某些实施方案中，在所述可溶性盐溶液中，锌∶铝摩尔浓度比为至少1∶1，至少2∶1，至少3∶1，至少4∶1，至少5∶1，至少6∶1，至少8∶1，至少10∶1，至少12∶1，至少15∶1，至少20∶1，至少30∶1，或者至少40∶1。在某些实施方案中，在所述可溶性盐溶液中，锌∶铝摩尔浓度比为不超过50∶1，不超过40∶1，不超过30∶1，不超过20∶1，不超过15∶1，不超过12∶1，不超过10∶1，不超过8∶1，不超过6∶1，不超过5∶1，不超过4∶1，不超过3∶1，或者不超过2∶1。
在某些实施方案中，在所述可溶性盐溶液中，锌∶铝摩尔浓度比为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、或1∶1。在某些实施方案中，在所述可溶性盐溶液中，锌∶铝摩尔浓度比为10∶1。
在某些实施方案中，在步骤(2)中，以在1∶1至16∶1的范围内(例如2∶1至16∶1的范围内)的锌∶利塞膦酸摩尔浓度比，将所述可溶性盐溶液与利塞膦酸碱溶液混合。在某些实施方案中，所述锌∶利塞膦酸摩尔浓度比在1∶1至2∶1，2∶1至4∶1，4∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至14∶1或者14∶1至16∶1的范围内。在某些实施方案中，所述锌∶利塞膦酸摩尔浓度比为至少1∶1，至少2∶1，至少3∶1，至少4∶1，至少5∶1，至少6∶1，至少7∶1，至少8∶1，至少10∶1，至少12∶1，至少14∶1，或者至少15∶1。在某些实施方案中，所述锌∶利塞膦酸摩尔浓度比为不超过16∶1，不超过14∶1，不超过12∶1，不超过10∶1，不超过8∶1，不超过7∶1，不超过6∶1，不超过5∶1，不超过4∶1，不超过3∶1，或者不超过2∶1。
在某些实施方案中，所述锌∶利塞膦酸摩尔浓度比为1∶1、2∶1、4∶1、4.5∶1、6∶1、8∶1、10∶1、12∶1、14∶1或16∶1。在某些实施方案中，所述锌∶利塞膦酸摩尔浓度比为4∶1或4.5∶1。
在某些实施方案中，在步骤(2)中，以使锌离子、铝离子和利塞膦酸共同沉淀的方式，将所述可溶性盐溶液与利塞膦酸碱溶液混合。在某些实施方案中，在步骤(2)中，将利塞膦酸碱溶液逐滴加入所述可溶性盐溶液，使锌离子、铝离子和利塞膦酸共同沉淀。在某些实施方案中，在步骤(2)中，在将所述可溶性盐溶液与利塞膦酸碱溶液混合的过程中，锌离子、铝离子和利塞膦酸发生共同沉淀，产生利塞膦酸锌铝颗粒。在某些实施方案中，所产生的利塞膦酸锌铝颗粒的粒径大小为0.01-100μm，例如 0.01-60μm，0.01-50μm，0.1-60μm，0.1-30μm，0.4～30μm，0.4-20μm。
在某些实施方案中，所述利塞膦酸碱溶液选自利塞膦酸氢氧化钠，利塞膦酸磷酸盐溶液(例如利塞膦酸磷酸氢二钠溶液，利塞膦酸磷酸氢钠溶液)，或其任何组合。在某些实施方案中，所述利塞膦酸碱溶液为利塞膦酸氢氧化钠溶液或利塞膦酸磷酸氢二钠溶液。
在某些实施方案中，步骤(1)所述可溶性盐溶液选自例如硫酸盐溶液、氯酸盐溶液、醋酸盐溶液、或其任何组合。在某些实施方案中，所述可溶性盐溶液为氯酸盐溶液或醋酸盐溶液。
佐剂用途
在一个方面，本申请还提供了所述佐剂用于制备免疫原性组合物或用做递送免疫原的载体或用做免疫原的免疫增强剂的用途。在一个方面，本申请还提供了所述佐剂用于增强免疫原的免疫原性的用途。在一个方面，本申请还提供了所述佐剂用于增强受试者对免疫原的免疫应答水平的用途。在一个方面，本申请还提供了所述佐剂在制备用于增强受试者对免疫原的免疫应答水平的制剂中的用途。易于理解，上文对于所述佐剂和免疫原等的各种描述也同样适用于这些方面。
在某些实施方案中，所述免疫应答为细胞免疫应答和/或体液免疫应答。在某些实施方案中，所述细胞免疫应答为T细胞免疫应答。在某些实施方案中，所述T细胞免疫应答为免疫应答Th1和/或Th2免疫应答。
免疫原性组合物制备方法/提高免疫原的免疫原性的方法
在一个方面，本申请还提供了制备免疫原性组合物的方法，其包括：将所述佐剂与免疫原进行混合的步骤。在一个方面，本申请提供了一种提高免疫原的免疫原性的方法，其包括将免疫原与所述佐剂混合的步骤。易于理解，可以将本发明的佐剂与各种可能的免疫原联用，以增强免疫原的免疫原性。因此，上文对于所述佐剂和免疫原等的各种描述也同样适用于这些方面。
在某些实施方案中，所述免疫原包括但不限于蛋白，核酸，多糖，或其免疫原性部分等。在某些实施方案中，所述免疫原来源于病毒、细菌、真菌等病原体。
在某些实施方案中，所述免疫原为蛋白或其免疫原性片段，例如来源于病毒、细菌、真菌等病原体的蛋白或其免疫原性片段。在某些实施方案中，所述病毒包括但不限于，呼吸道病毒(例如，流感病毒、副流感病毒、鼻病毒、冠状病毒、呼吸道合胞 病毒)，肠道病毒(例如EV71病毒，轮状病毒)。水痘带状疱疹病毒(VZV)。
在某些实施方案中，所述免疫原为冠状病毒的蛋白(例如刺突蛋白)或其免疫原性片段。在某些实施方案中，所述冠状病毒选自正冠状病毒亚科α属病毒(例如229E和NL63)，正冠状病毒亚科β属病毒(例如OC43和HKU1)、严重急性呼吸综合征相关冠状病毒(SARS-CoV)、中东呼吸综合征相关冠状病毒(MERS-CoV)，和新型冠状病毒(SARS-CoV-2)。在某些实施方案中，所述冠状病毒选自严重急性呼吸综合征相关冠状病毒(SARS-CoV)、中东呼吸综合征相关冠状病毒(MERS-CoV)和新型冠状病毒(SARS-CoV-2)。在某些实施方案中，所述冠状病毒为新型冠状病毒(SARS-CoV-2)。在某些实施方案中，所述免疫原为新型冠状病毒的结构蛋白或其免疫原性片段，例如刺突蛋白(S蛋白)或其免疫原性片段。
在某些实施方案中，所述免疫原是水痘带状疱疹病毒的蛋白或其免疫原性片段，例如水痘带状疱疹病毒gE蛋白或其免疫原性片段。
在某些实施方案中，所述免疫原是流感病毒的蛋白或其免疫原性片段，例如流感病毒HA蛋白或其免疫原性片段。
在某些实施方案中，所述免疫原是轮状病毒的蛋白或其免疫原性片段，例如轮状病毒VP4蛋白或其免疫原性片段。
在某些实施方案中，所述方法还包括，添加药学上可接受的辅料的步骤。在某些实施方案中，所述辅料选自例如赋形剂、防腐剂、抗菌剂、缓冲剂和/或额外的免疫佐剂。在某些实施方案中，所述额外的免疫佐剂选自铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子，或其任何组合。
在某些实施方案中，所述方法还包括，添加第二免疫原的步骤。在某些实施方案中，所述第二免疫原包括但不限于蛋白，核酸，多糖，或其免疫原性部分等。
在某些实施方案中，所述免疫原性组合物为疫苗。
免疫原性组合物用途
在一个方面，本申请提供了所述的免疫原性组合物在制备用于预防和/或治疗受试者的疾病的药物中的用途，所述疾病是能够被所述免疫原诱发的免疫应答所预防或治疗的疾病。
易于理解，上文对于所述佐剂、免疫原、免疫原性组合物等的各种描述也同样适 用于本方面。还易于理解的是，免疫原性组合物中可以包含各种免疫原；相应地，所制备的药物可用于预防或治疗各种对应的疾病。例如，当所述免疫原性组合物中含有来源于病原体(例如病毒)的免疫原时，本申请的免疫原性组合物可用于制备用于预防和/或治疗受试者中的病原体感染(例如病毒感染)或与病原体感染(例如病毒感染)相关的疾病的药物。
在某些实施方案中，所述免疫原来源于冠状病毒(例如新型冠状病毒)，并且，所述疾病为冠状病毒(例如新型冠状病毒)感染或与冠状病毒(例如新型冠状病毒)感染相关的疾病。在某些实施方案中，所述免疫原为冠状病毒(例如新型冠状病毒)的蛋白(例如刺突蛋白)或其免疫原性片段，并且，所述疾病为冠状病毒(例如新型冠状病毒)感染或与冠状病毒(例如新型冠状病毒)感染相关的疾病。
在某些实施方案中，所述免疫原为新型冠状病毒的结构蛋白(例如刺突蛋白(S蛋白))或其免疫原性片段；并且，所述疾病为新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如，新型冠状病毒性肺炎(COVID-19))。
在某些实施方案中，所述免疫原来源于水痘带状疱疹病毒，并且，所述疾病为水痘带状疱疹病毒感染或与水痘带状疱疹病毒感染相关的疾病。
在某些实施方案中，所述免疫原为水痘带状疱疹病毒的蛋白(例如gE蛋白)或其免疫原性片段，并且，所述疾病为水痘带状疱疹病毒感染或与水痘带状疱疹病毒感染相关的疾病。
在某些实施方案中，所述免疫原来源于流感病毒，并且，所述疾病为流感病毒感染或与流感病毒感染相关的疾病。
在某些实施方案中，所述免疫原为流感病毒的蛋白(例如HA蛋白)或其免疫原性片段，并且，所述疾病为流感病毒感染或与流感病毒感染相关的疾病。
在某些实施方案中，所述免疫原来源于轮状病毒，并且，所述疾病为轮状病毒感染或与轮状病毒感染相关的疾病。
在某些实施方案中，所述免疫原为轮状病毒的蛋白(例如VP4蛋白)或其免疫原性片段，并且，所述疾病为轮状病毒感染或与轮状病毒感染相关的疾病。
在某些实施方案中，所述受试者是动物，例如禽类动物或哺乳动物。在某些实施方案中，所述受试者是啮齿类动物、猪科动物、猫科动物、犬科动物、马科动物、灵长类动物或禽类动物。在某些实施方案中，所述受试者是非人受试者。在某些实施方 案中，所述受试者是小鼠、水貂、豚鼠、叙利亚金黄地鼠或食蟹猴。在某些实施方案中，所述受试者是人。
刺激/增强免疫应答的方法
在一个方面，本申请提供了一种刺激或增强受试者对免疫原的免疫应答的方法，其包括给予受试者有效量的含有所述免疫原和本发明佐剂的免疫原性组合物的步骤。易于理解，上文对于所述佐剂、免疫原、免疫原性组合物、受试者等的各种描述也同样适用于本方面。
在某些实施方案中，所述免疫应答为细胞免疫应答和/或体液免疫应答。在某些实施方案中，所述细胞免疫应答为T细胞免疫应答。在某些实施方案中，所述T细胞免疫应答为免疫应答Th1和/或Th2免疫应答。
在某些实施方案中，所述免疫原性组合物是通过选自下列的途径进行给药的：肌内注射、皮下注射、皮内、鼻内、口服、透皮或静脉注射。在某些实施方案中，所述免疫原性组合物通过肌内注射给药。
疾病治疗/预防方法
在一个方面，本申请提供了一种预防或治疗疾病的方法，其包括给予受试者有效量的免疫原性组合物的步骤，其中，所述疾病是能够被所述免疫原诱发的免疫应答所预防或治疗的疾病。
易于理解，上文对于所述佐剂、免疫原、免疫原性组合物、受试者等的各种描述也同样适用于本方面。还易于理解的是，免疫原性组合物中可以包含各种免疫原；相应地，免疫原性组合物可用于预防或治疗各种对应的疾病。例如，当所述免疫原性组合物中含有来源于病原体(例如病毒)的免疫原时，本申请的免疫原性组合物可用于预防和/或治疗受试者中的病原体感染(例如病毒感染)或与病原体感染(例如病毒感染)相关的疾病。
在某些实施方案中，所述疾病为冠状病毒(例如新型冠状病毒)感染或与冠状病毒(例如新型冠状病毒)感染相关的疾病。相应地，在此类实施方案中，所述免疫原来源于冠状病毒(例如新型冠状病毒)。在某些实施方案中，所述免疫原为冠状病毒(例如新型冠状病毒)的蛋白(例如刺突蛋白)或其免疫原性片段。
在某些实施方案中，所述疾病为新型冠状病毒感染或与新型冠状病毒感染相关的疾病，例如新型冠状病毒性肺炎(COVID-19)。相应地，在此类实施方案中，所述免 疫原来源于新型冠状病毒。在某些实施方案中，所述免疫原为新型冠状病毒的结构蛋白(例如刺突蛋白(S蛋白))或其免疫原性片段。
在某些实施方案中，所述疾病为水痘带状疱疹病毒感染或与水痘带状疱疹病毒感染相关的疾病。相应地，在此类实施方案中，所述免疫原来源于水痘带状疱疹病毒。在某些实施方案中，所述免疫原为水痘带状疱疹病毒的蛋白(例如gE蛋白)或其免疫原性片段。
在某些实施方案中，所述疾病为流感病毒感染或与流感病毒感染相关的疾病。相应地，在此类实施方案中，所述免疫原来源于流感病毒。在某些实施方案中，所述免疫原为流感病毒的蛋白(例如HA蛋白)或其免疫原性片段。
在某些实施方案中，所述疾病为轮状病毒感染或与轮状病毒感染相关的疾病。相应地，在此类实施方案中，所述免疫原来源于轮状病毒。在某些实施方案中，所述免疫原为轮状病毒的蛋白(例如VP4蛋白)或其免疫原性片段。
在某些实施方案中，所述受试者是动物，例如禽类动物或哺乳动物。在某些实施方案中，所述受试者是啮齿类动物、猪科动物、猫科动物、犬科动物、马科动物、灵长类动物或禽类动物。在某些实施方案中，所述受试者是非人受试者。在某些实施方案中，所述受试者是小鼠、水貂、豚鼠、叙利亚金黄地鼠或食蟹猴。在某些实施方案中，所述受试者是人。
本申请中相关术语的说明及解释
在本申请中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。
根据本发明，术语“S蛋白”是指新型冠状病毒刺突蛋白，其属于I型跨膜蛋白，由S1和S2蛋白亚基两部分组成，介导与宿主细胞表面受体的结合和细胞膜的融合以进入靶细胞。野生型S蛋白的氨基酸序列是本领域技术人员所公知的，其典型氨基酸序列可参见Genbank：QHD43416.1。在某些实施方案中，所述野生型S蛋白的氨基酸序列如SEQ ID NO：1所示。
易于理解，本领域技术人员可对野生型S蛋白进行改造，例如，去除弗林酶切位点，增加蛋白多聚化结构域(例如增加T4噬菌体fibritin蛋白质的三聚化结构域)，和/或，增加标签以获得期望的性能。在某些实施方案中，所述经改造的S蛋白的氨基 酸序列如SEQ ID NO：4所示。
除非本文另外指明或根据上下文明显矛盾，否则，术语“S蛋白”应被解释成覆盖野生型S蛋白以及经改造的S蛋白。
根据本发明，“gE蛋白”为水痘带状疱疹病毒(varicella-zoster virus，VZV)的一种囊膜糖蛋白。野生型gE蛋白的氨基酸序列是本领域技术人员所公知的，其典型氨基酸序列可参见Genbank：DQ008355.1。在某些实施方案中，所述gE蛋白的氨基酸序列如SEQ ID NO：2所示。
根据本发明，术语“HA蛋白”是指流感病毒血凝素(hemagglutinin，HA)，其为介导病毒入侵宿主细胞的关键蛋白，也是抗流感病毒中和抗体的主要靶点。各亚型流感病毒HA蛋白的氨基酸序列是本领域技术人员所公知的，例如，乙型Victoria系流感病毒的一个典型氨基酸序列可参见Genbank：AIU46088。在某些实施方案中，所述HA蛋白的氨基酸序列如SEQ ID NO：3所示。
根据本发明，“VP4蛋白”为轮状病毒的外层衣壳蛋白。VP4蛋白是一种重要的中和抗原，能够刺激机体产生中和抗体。野生型VP4蛋白的氨基酸序列是本领域技术人员所公知的，其典型氨基酸序列可参见Genbank：KP752474或GenBank：MG729832。
除非本文另外指明或根据上下文明显矛盾，否则，本文所述gE蛋白、HA蛋白和VP4蛋白应被解释成覆盖其相应的野生型蛋白以及其相应的经改造的蛋白。
根据本发明，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
根据本发明，术语“药学上可接受的辅料”可以是例如惰性稀释剂，例如水或其他溶剂、增溶剂和乳化剂，如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苄醇、苯甲酸苄酯、丙二醇、1，3-丁二醇、油(具体而言为棉籽油、花生油、玉米油、胚芽油、橄榄油、蓖麻油和芝麻油)、甘油、四氢糠醇、聚乙二醇、脱水山梨糖醇的脂肪酸酯，以及它们的混合物；除了惰性稀释剂之外，口服组合物还可包含佐剂如湿润剂、乳化剂和悬浮剂、甜味剂、矫味剂、着色剂、香味剂和防腐剂；混悬剂制剂还可含有悬浮剂，例如乙氧基化异十八醇、聚氧乙烯山梨醇和脱水山梨糖醇酯、微晶纤维素、偏氢氧化铝、膨润 土、琼脂和黄蓍胶以及它们的混合物；这些组合物还可含有辅剂如湿润剂、乳化剂和分散剂。还可能需要在组合物中包含等渗剂，如糖类、氯化钠等。另外，可通过掺入能延迟吸收的物质如单硬脂酸铝和明胶，实现可注射药物形式的延长吸收。
如本文中使用的，术语“免疫原性”是指，能够刺激机体形成特异抗体或致敏淋巴细胞的能力。其既指，抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质如抗体和致敏淋巴细胞的特性，也指抗原刺激机体后，机体免疫系统能形成抗体或致敏T淋巴细胞的特异性免疫应答。免疫原性是抗原最重要的性质，一种抗原能否成功地诱导宿主产生免疫应答取决于三方面的因素：抗原的性质、宿主的反应性和免疫方式。
根据本发明，术语“免疫原性片段”是指这样的多肽片段，其至少部分地保留了所源自的蛋白的免疫原性。例如，冠状病毒刺突蛋白(S蛋白)的免疫原性片段是指，冠状病毒刺突蛋白的至少部分地保留了免疫原性的片段。
根据本发明，术语“额外的免疫佐剂”包括但不限于稳定剂；乳化剂；pH调节剂，如氢氧化钠、盐酸等；脂质体；iscom佐剂；合成的糖肽，如胞壁酰二肽；增量剂，如右旋糖苷；聚羧乙烯；细菌细胞壁，如分枝杆菌细胞壁提取物；它们的衍生物，如短小棒状杆菌(Corynebacterium parvum)；痤疮丙酸杆菌(Propionibacterium acne)；牛分支杆菌(Mycobacterium bovis)，例如卡介菌(Bovine Calmette Guerin，BCG)；牛痘或动物痘病毒蛋白；亚病毒颗粒佐剂，如环状病毒；霍乱毒素；N，N-双十八烷基-N′，N′-双(2-羟乙基)-丙二胺(吡啶)；单磷酰脂质A；二甲基双十八烷基溴化铵；它们的合成物和混合物。合适的稳定剂的例子包括但不限于蔗糖、明胶、蛋白胨、消化的蛋白质提取物如NZ-胺或NZ-胺AS。乳化剂的例子包括但不限于矿物油、植物油、花生油和其他可用于注射剂或鼻内疫苗组合物的标准、可代谢、无毒的油。
如本文中所使用的，术语“受试者”可以是任何动物，例如禽类动物或哺乳动物。在某些实施方案中，所述受试者是啮齿类动物、猪科动物、猫科动物、犬科动物、马科动物、灵长类动物或禽类动物。在某些实施方案中，所述受试者是非人受试者。在某些实施方案中，所述受试者是小鼠、水貂、豚鼠、叙利亚金黄地鼠或食蟹猴。在某些实施方案中，所述受试者是人。
如本文中所使用的，术语“新型冠状病毒肺炎”和“COVID-19”是指，因 SARS-CoV-2感染而导致的肺炎，二者具有相同的含义。
根据本发明，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如COVID-19)有效量是指，足以预防，阻止，或延迟疾病(例如COVID-19)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
与现有技术相比，本发明提供了一种包含利塞膦酸锌铝的佐剂，其可作为疫苗佐剂或药物递送载体等，可用于有效提高抗原的免疫原性，其效果优于Al佐剂的效果。
特别地，当本申请的佐剂与免疫原(例如新型冠状病毒刺突蛋白(S蛋白))联用时，在Balb/c小鼠、水貂、豚鼠、叙利亚金黄地鼠和食蟹猴等多种动物上，能够刺激或诱导高水平的功能性抗体与平衡的细胞和体液免疫应答。因此，本申请的佐剂可提高疫苗(例如新型冠状病毒S蛋白疫苗)的成药性。
附图说明
图1为铝佐剂(Al001)(图1A)与利塞膦酸锌铝佐剂(FH002C)(图1B)悬浊液外观以及在光学显微镜和电镜下的颗粒形态图。
图2为Al001(图2A)与FH002C(图2B)的颗粒粒径测量结果。n＝3，mean±SD
图3为Al001与FH002C在不同pH值下Zeta电位的测量结果。n＝3，mean±SEM
图4A至图4F为不同锌、铝、利塞膦酸比例的FH002C佐剂的悬浊液外观以及在电镜下的颗粒形态图。
图5为未吸附佐剂的新型冠状病毒S重组蛋白与经解离的疫苗制剂(FH002C联合S重组蛋白)的抗原性检测结果，图中显示的是不同蛋白浓度下基于ACE2受体特异性结合的ELISA检测结果。
图6为用不同免疫原组分(S重组蛋白1μg，S重组蛋白10μg，S重组蛋白+Al001  1μg，S重组蛋白+Al001 10μg，S重组蛋白+FH002C 1μg，和S重组蛋白+FH002C 10μg)分别免疫小鼠后，小鼠血清中抗体结合滴度及中和滴度的检测结果。＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；＊＊＊＊p<0.0001；n＝5，mean±SEM。
图7为不同锌、铝、利塞膦酸比例的FH002C佐剂联合新型冠状病毒S重组蛋白免疫Balb/C小鼠后的特异性抗体结合滴度，＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；n＝5，mean±SEM。注：横坐标表示锌∶铝∶利塞膦酸的摩尔浓度比例。
图8为FH002C或Al001联合新型冠状病毒S重组蛋白免疫叙利亚金黄地鼠后的特异性抗体结合滴度，＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；＊＊＊＊p<0.0001；n＝22或16，Geometric mean±Geometric mean SD。
图9为FH002C或Al001联合新型冠状病毒S重组蛋白免疫食蟹猴后的特异性抗体结合滴度，n＝2，Geometric mean±Geometric mean SD。
图10为FH002C或Al001联合新型冠状病毒S重组蛋白免疫叙利亚金黄地鼠后血清的中和抗体滴度，＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；＊＊＊＊p<0.0001；n＝22或16，Geometric mean±Geometric mean SD。
图11为FH002C或Al001联合新型冠状病毒S重组蛋白免疫食蟹猴的中和抗体滴度，n＝2，Geometric mean±Geometric mean SD。
图12为FH002C或Al001联合新型冠状病毒S重组蛋白免疫叙利亚金黄地鼠后的血清阻断滴度，＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；＊＊＊＊p<0.0001；n＝22或16，Geometric mean±Geometric mean SD。
图13为FH002C或Al001联合新型冠状病毒S重组蛋白免疫食蟹猴的血清阻断滴度，n＝2，Geometric mean±Geometric mean SD。
图14为FH002C或Al001联合新型冠状病毒S重组蛋白免疫Balb/c小鼠后的血清抗体亲合力，n＝4或5，mean±SEM。
图15为FH002C或Al001联合新型冠状病毒S重组蛋白免疫叙利亚金黄地鼠后的血清抗体亲合力，n＝16，mean±SEM。
图16为FH002C或Al001联合新型冠状病毒S重组蛋白免疫Balb/c小鼠后的血清抗体亚型，＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；＊＊＊＊p<0.0001；n＝4或5，mean±SEM。
图17为酶联免疫斑点法测定FH002C或Al001联合新型冠状病毒S重组蛋白免疫Balb/c小鼠后的T细胞应答的结果，图A为捕获的图像，图B为对捕获图像的计数结 果，＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；＊＊＊＊p<0.0001；n＝8，mean±SD。
图18为FH002C或Al001联合水痘带状疱疹病毒gE蛋白免疫Balb/C小鼠后的特异性抗体结合滴度，＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；＊＊＊＊p<0.0001；n＝5，mean±SEM。
图19为FH002C或弗氏佐剂联合流感病毒HA蛋白免疫Balb/C小鼠后的特异性抗体结合滴度，＊p<0.05；＊＊p<0.01；＊＊＊p<0.001；＊＊＊＊p<0.0001；n＝10，mean±SEM。注：“Victoria/WT”表示未经去糖基化酶(Pngase)处理的Victoria野生型HA蛋白，“Victoria/Pngase”表示经Pngase处理的Victoria野生型HA蛋白。
图20为FH002C或Al001联合轮状病毒VP4蛋白免疫Balb/C小鼠的中和抗体滴度，n＝5，mean±SEM。
图21为FH002C或Al001联合轮状病毒VP4蛋白免疫豚鼠的中和抗体滴度，n＝5，mean±SEM。
序列信息
本申请涉及的序列的描述提供于下表中。
表1序列信息
Figure PCTCN2021130588-appb-000001
Figure PCTCN2021130588-appb-000002
Figure PCTCN2021130588-appb-000003
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
制备例所用试剂如下：
利塞膦酸钠(C 7H 10NNaO 7P 2)，购自湖南华腾制药有限公司；
无水氯化锌(ZnCl 2)，购自西陇化工；
六水合氯化铝(AlCl 3·6H 2O)，购自西陇化工；
十二水合磷酸氢二钠(Na 2HPO 4·12H 2O)，购自西陇化工；
氢氧化钠(NaOH)，购自西陇化工
制备例1：利塞膦酸锌铝佐剂(FH002C)的制备
(1)溶液配制：
按照1∶0.1∶0.22的Zn/Al/利塞膦酸摩尔浓度比，配制0.5L(47mM氯化锌+4.7mM氯化铝)溶液，定义为A溶液；配制0.5L(10.3mM利塞膦酸+55mM氢氧化钠+63mM磷酸氢二钠)溶液，定义为B液，A溶液和B溶液用0.22μm滤膜过滤备用。
(2)利塞膦酸锌铝佐剂FH002C悬浊液的制备：
将A和B溶液按照1∶1体积配比，以共同沉淀的方式来制备利塞膦酸锌铝佐剂，即将配制好的B溶液，按照体积比1∶1的方式逐滴直至全部加入A溶液形成的悬浊液。对混合后获得悬浊液进行一次121℃，60min灭菌，测定灭菌之后的pH、粒径及颗粒形态等理化性质。
制备例2：不同锌、铝、利塞膦酸比例的利塞膦酸锌铝佐剂(FH002C)的制备
(1)溶液配制：
以Zn的摩尔浓度为1，设计了Zn/Al/利塞膦酸摩尔浓度比为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以及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∶0.05∶1以及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以及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以及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以及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，分别配制0.5L(47mM氯化锌+0.94、2.35、4.7、7.05、11.75、23.5mM氯化铝)溶液，定义为A溶液；配制0.5L(4.7、7.05、10.3、15.51、23.5、47mM利塞膦酸+35～250mM氢氧化钠+63mM磷酸氢二钠)溶液，定义为B液，A溶液和B溶液用0.22μm滤膜过滤备用。
2)悬浊液的制备：
将A和B溶液按照1∶1体积配比，以共同沉淀的方式来制备利塞膦酸锌铝佐剂，即将配制好的B溶液，按照体积比1∶1的方式逐滴直至全部加入A溶液形成的悬浊液。对混合后获得悬浊液进行一次121℃，60min灭菌。
制备例3：铝佐剂Al001的制备
按照0.15∶1的磷酸根/Al摩尔浓度配比，配制0.5L 124mM的氯化铝溶液，定义为A溶液；配制0.5L 18.6mM浓度的磷酸氢二钠溶液，定义为B液，B溶液中含有140mM氢氧化钠，0.22μm滤膜过滤备用。
铝佐剂Al001悬浊液的制备过程可参考FH002C悬浊液的制备。
实施例1：利塞膦酸锌铝(FH002C)以及铝佐剂(Al001)的理化性质测定
对混合后获得的FH002C悬浊液(制备例1)以及Al001悬浊液(制备例3)进行一次121℃，60min灭菌，测定灭菌之后的pH、粒径及颗粒形态、零电荷点等理化性质。
(1)佐剂外观以颗粒形态观察
将利塞膦酸锌铝佐剂FH002C用100mM NaAc缓冲溶液稀释1倍以及铝佐剂Al001用生理盐水稀释1倍后，用中国华为mate305G手机摄像机进行拍摄，记录其 佐剂的外观；同时将利用中国Motic的AE31E倒置生物显微镜进行颗粒形态的观察与拍摄，目镜和物镜的放大倍数为10x。
将铝佐剂Al001和利塞膦酸锌铝佐剂FH002C用去离子水稀释分别稀释50和100倍后，利用日本Electronics的JEM-2100透射电子显微镜(Transmission Electron Microscopy，TEM)进行观察。具体的步骤如下：将佐剂样品滴于铜网上，令其吸附10min，用滤纸擦去残液，送入透射电镜的样品室里，观察样品形态，拍照。
实验结果：如图1所示，利塞膦酸锌铝佐剂FH002C以及铝佐剂Al001均为乳白色悬浊物；光学显微镜下两者均为无定型的团簇状形态；在电镜下FH002C为无规则的片状结构，而Al001佐剂可以清晰看到丝状结构。
(2)pH测定
取供试品，放置室温平衡至少30分钟，利用Sartorius酸度计进行测量。
选择标准缓冲液(pH 7.00)、标准缓冲液(pH 4.01)、标准缓冲液(pH 10.01)按照使用说明书要求校正仪器。
按“Mode”(转换)键可以在pH和mV模式之间进行切换。通常测定溶液pH值将模式至于pH状态。
按“SETUP”键，显示屏显示Clear buffer，按“ENTER”键确认，清除以前的校准数据。
按“SETUP”键直至显示屏显示缓冲溶液组“4.01，7.00，10.01”，按“ENTER”键确以。
将电极小心从电极储存液中取出，用去离子水充分冲洗电极，冲洗干净后用滤纸吸干表面水(注意不要擦拭电极)。
将电极浸入第一种缓冲溶液(pH 7.00)，等到数值稳定并出现“S”时，按“STANDARDIZE”键，等待仪器自动校准。校准成功后，将显示“7.00”和电极斜率。
将电极从第一种缓冲溶液中取出，用去离子水充分淋洗电极，将电极依次浸入第二种缓冲溶液(pH 4.01)，等到数值稳定并出现“S”时，按“STANDARDIZE”键，等待仪器自动校准。校准成功后，将显示“4.01 7.00”和信息“Slope”。Slope显示测量的电极斜率值，该测量值在90-105％范围内可以接受。
如果与理论值有很大偏差，将显示错误信息(Err)，电极应清洗，并重复上述步骤校准。
重复以上操作完成第三点(pH 10.01)校准。
校准完成后，用去离子水充分淋洗电极，然后滤纸轻轻吸干。摇匀供试品溶液，将玻璃电极浸入供试品溶液中，至pH值的读数在1分钟内改变不超过±0.05时进行读数。
摇匀供试品溶液，重新测量，两次pH值的度数相差应不超过0.1。取两次读数的平均值作为供试品pH值。
实验结果：Al001佐剂灭菌前pH为8.00～8.40，灭菌后6.50～6.90；FH002C佐剂灭菌前pH为7.00～7.50，灭菌后5.80～6.30。
(3)粒径大小的测定
打开Beckman LS 13320激光粒径测试仪，预热15min。
打开仪器控制软件和样品池封闭舱，将样品池从样品槽中取出并加入12mL纯化水。
将样品池置于样品槽上，关闭舱门。
打开LS13320软件后，点击弹窗中“OK”进行仪器自检。点击菜单栏“run”，选择“Use Optical Module”，打开检测模块。点击菜单栏“control”，选择“stirrer on”启动样品池内置的自动搅拌器。点击“start cycle”，依次选择“Measure Offsets”、“Align”、“Measure Background”，最后点击“start”，在弹出的对话框中点击“OK”开始校正空白背景。
取出样品池，加入一定质量的标准样品(仪器自带)，点击“start cycle”，依次选择“Measure Loading”、“Enter Sample Info”、“Enter run setting”、“start runs”，最后点击“start”，在弹出的对话框中输入标准样品名称，待软件中“Obscuration”参数为8％-12％时点击“OK”，进行标准样品的测试。
为保证实验数据的准确性和可靠性，每次开机测量之前均需校正空白背景和测试标准样品的大小。
打开样品池封闭舱，取出样品池。
将样品池内含有标准样品的水溶液倒弃，倒入去离子水清洗样品池3遍。
清洗完毕后加入12mL去离子水，将样品池置于样品槽上，关闭舱门。
点击“start cycle”，依次选择“Measure Offsets”、“Align”、“Measure Background”，最后点击“start”，在弹出的对话框中点击“OK”开始校正空白背景。
取出样品池，加入一定体积的检测样品，打开样品测试舱门，将
样品池置于样品槽上，关闭舱门。
点击“start cycle”，选择“Enter Sample Info”、“Enter run setting”、“start runs”，最后点击“start”，在弹出的对话框中输入样品名称，待软件中“Obscuration”参数为8％-12％时点击“OK”，记录样品测得的粒径大小；对铝佐剂Al001和利塞膦酸锌铝FH002C分别进行三次重复测量。
实验结果：如图2所示，Al001的颗粒分布为单峰，大小在0.4-60μm之间，主要分布在15-16μm(图2A)；利塞膦酸锌铝佐剂FH002C颗粒分布为双峰，大小在0.4-25μm之间，主峰的颗粒大小为5.0μm左右(图2B)。
(4)PZC(零电荷点)检测
仪器检测：Nanobrook Omni(Brookhaven)
实验操作：打开电源，将多角度粒度及高灵敏Zeta电位仪预热30min。
使用36％-38％盐酸和10M NaOH将Al001佐剂调节pH至6.5、7.0、7.5、8.0、8.5以及FH002C佐剂调节至3.0、4.0、5.0、6.0、7.0。
钝化电极：向样品管中加入3-4mL佐剂，电极插入后，SOP中将cycle设置为：50，运行仪器，使得电极钝化。
样品检测：电极取出后，去离子水冲洗下端，加入相应样品，SOP中点击“instrument parameters”，在“cell type”中选择“square polystyrene cell”，在“electrode assembly”中选择“BI-SREL(1250μL)”后，点击“advanced settings”，设置“equilibration time”为120seconds，“inter-cycle delay”为1seconds，在“time dependent”中选择“measurements”为3，“time interval”为0，点击“liquid”，pH设置为各样品对应的pH，点击“Data Analysis”中的“model”，选择“smoluchowski”，最后点击“save”，点击“OK”，完成参数设置。
运行仪器：将不同pH的佐剂充分摇匀后，依次吸取2mL于塑料样品池中，插入电极，放入样品槽中，将仪器与样品槽的数据连接起来，点击“start”开始测量，再进行下一次样品检测。
数据处理：得到不同pH值下对应的Zeta电位，运行仪器自带软件，得到PZC值，再采用GraphPad Prism 8.0.2(厂商：GraphPad Software，LLC)进行绘图。
实验结果：如图3所示，铝佐剂Al001的PZC为7.52；利塞锌铝佐剂FH002C的 PZC为4.75。
(5)FH002C中Zn/Al/利塞膦酸的沉淀率
实验方法：游离上清中锌、铝含量采用原子吸收光谱仪(岛津，AA6300C(P/N206-52430))进行检测，利塞膦酸采用紫外分光光度仪(贝克曼，DU800)进行检测，具体如下：
采用原子吸收光谱仪的火焰法进行锌元素含量测定：
标准曲线的配制：将锌单元素标准溶液(北京有色金属研究总院，GSB04-1761-2004)用0.2％硝酸稀释成0，500，1000，1500，2000ng/mL的锌标准溶液。
供试品溶液的准备：用0.2％硝酸溶液将样品进行梯度稀释直至浓度在标准曲线范围内(Abs读值在0.2～0.8之间)，每步稀释之前需用涡旋混和仪震动混匀。
登录WizAArd：双击WizzAArd图表打开软件，在“操作”一栏中点击“测定”图标，在“Login ID”一栏中输入“Admin”，无需密码点击<OK>即可。
Wizard选择：选择“元素选择”，点击<OK>。
选择元素：在“元素选择”页面中选择“选择元素”选项，在“装载参数”页面中选择“Zn”，选择[火焰连续]测定方法，“使用ASC”选项无需打钩，选择[普通灯]，点击<确定>。“校准曲线设置”和“编辑参数”页面无需特殊设定，点击<确定>。“未连接仪器/发送参数”页面，点击<连接/发送参数>。
在“仪器初始化”页面中，有关石墨炉法的ASC检查、GFA检查选项无需检查；出现“检查燃气”对话框，选择<否>；出现“现在可以检查安全装置，希望检查助燃器压力监控器？”对话框，选择<是>；出现“请供应空气，在供应空气后点击确定键，空气排放”对话框，选择<确定>；出现“检查NO 2”对话框，选择<否>；出现“不能使用NO 2-C 2H 2”对话框，选择<确定>；出现“把废液探头提到液面之上”，按照提示操作并向废液罐中加入足够多的超纯水，直至水从废液管流出，防止回火；出现“移动废液探头到液面之下”，按照提示操作并旋紧废液罐瓶口，所有项目检查完成后，点击<确定>。
在“火焰分析的仪器检查目录”中逐项检查并打钩，点击<OK>。
在“光学参数”页面中设定波长[213.86nm]，狭缝宽[0.7]，点灯方式[发射]，[灯位设置]保证Zn空心阴极灯的实际位置与设定位置相同，选择[点灯]，待屏幕下方显示“就绪”，点击<确定>。
“谱线搜索”页面：当“谱线搜索”及“光束平衡”均显示[OK]时，点击[关闭]，回到“光 学参数”页面，点击<下一步>。在“原子化器/气体流量设置”页面中，无需更改，点击<完成>。
燃烧器原点位置调节：依次选择[仪器]→[维护保养]→[燃烧器原点位置调节]，调节前后位置旋钮及燃烧器上的扳手，控制燃烧器的位置，用AA-6300C标准卡片观察发射光的位置，利用卡片上的刻度，使通过整个燃烧器缝上的光处于同一水平线上，调好后，等待Abs值稳定；通过计算机调节燃烧器的垂直高度，<向上移动>或<向下移动>，先粗调后细调，使Abs值为最大示数的一半(保证有1/2最大光强通过燃烧器)，选择<原点记忆>并关闭当前页面，在燃烧器上方罩上安全罩。
在菜单栏中选择[参数]→[编辑参数]→将点灯方式改为<BGC-D2>。重新进行“谱线搜索”当“谱线搜索”及“光束平衡”均显示[OK]时，点击<关闭>。
点火：确保C 2H 2打开且压力达到要求后，同时按下主机上的PURGE和IGNITE键，直至点火。
自动调零：正式检测样品前，选择[自动调零]，除去燃烧器中残余的杂质。
在MRT工作单上设置空白组(BLK)、标准品(STD)、待测样品(UNK)，输入标准品的理论浓度以及样品名称，通过雾化器延伸出来的上样管进行手动上样，每次上样量最少1mL，选择[开始]进行检测。
数据处理：根据标准品的Abs和其浓度在Excel中制作散点图，添加趋势线，显示公式和相关系数R 2，R 2≥99％证明相关性良好，可用于计算样品浓度。
采用原子吸收光谱仪的石墨法进行铝元素含量测定：
标准曲线的配制：将铝单元素标准溶液(国家有色金属及电子材料分析测试中心，GNM-SAl-002-2013)，用2％硝酸稀释成0，20，50，100，150，200ng/mL的铝标准溶液。
供试品溶液的准备：将样品用0.2％进行稀释直至浓度在标准曲线范围内(Abs读值在0.2～0.8之间)。
登录WizAArd：双击WizzAArd图表打开软件，在“操作”一栏中点击“测定”图标，在”Login ID”一栏中输入“Admin”，无需密码点击<OK>即可。Wizard选择：选择“元素选择”，点击<OK>。
选择元素：在“元素选择”页面中选择“选择元素”选项，在“装载参数”页面中选择“Al”，“使用ASC”选项打钩，选择[普通灯]，点击<下一步>。“校准曲线设置”和“编 辑参数”页面无需特殊设定，点击<确定>。打开GFA-EX7i的电源开关扳手(nEAT)，在“连接主机/发送参数”页面，点击<连接/发送参数>。出现“未连接仪器，希望连接？”对话框，点击<是>。
在“仪器初始化”页面中，有关火焰法的C2H2阀、助燃器、燃气、火焰监控器、燃烧器、废液探头选项无需检查，点击<NO>，所有项目检查完成后，点击<确定>。绕过火焰分析：出现“将要进行火焰分析？”对话框，点击<否>。
“光学参数”标签页中，设定波长[396.2nm]，狭缝宽[0.7nm]，点灯方式[发射]，[灯位设置]保证铝灯处于设定位置，在[点灯]选项处打钩待屏幕右下角显示[就绪]，点击<确定>。
“谱线搜索”页面：当“谱线搜索”及“光束平衡”均显示[OK]时，点击<关闭>，回到“光学参数”页面，点击<下一步>。
在“石墨炉程序”页面中，“最大阶段号”选择[7]，“采样阶段号”选择[6]，“石墨管类型”选择[未知]，点击<完成>。
石墨炉原子化器原点位置调节：依次选择[仪器]，[维护保养]，[石墨炉原点位置调节]，待原子化器位置示数稳定后，使用手动前后位置旋钮移动石墨炉原子化器直至Abs示数达到最大，利用WizAArd软件调节原子化器上下位置，先[快速]调节后改用[慢速]调节直至示数达到最大，点击<原点记忆>。
检查ASC管口位置：从菜单栏中依次选择[仪器]→[石墨炉管口位置]，根据计算机提示，使吸样管口先移向ASC转盘，而后移向石墨炉原子化器，根据WizAArd软件提示，先放松臂引导螺丝，点击<向下移动>使吸样管口下移，接近石墨炉注入孔时，调节ASC工作台的注入位置调节旋钮，前后左右水平调节吸样管口的位置，直至位于注入孔的正中央。利用<向上移动>和<向下移动>调节上样位置，先粗调后细调，用ASC配备的观察镜观察吸样管口在注入孔内的位置，吸样管口处距离注入孔底端1/3时为最佳位置。点击<确定>保存当前位置并关闭当前页面。
重复检查ASC管口位置：旋紧旋钮，从菜单栏中依次选择[仪器]→[石墨炉管口位置]，当吸样管口移向原子化器后，选择[移向管]，若吸样管口位于注入孔正中央则无需调节，若吸样管口位置偏移则重新调至注入孔正中央，并选择<取消>，直至吸样管口位于注射孔正中央。
在菜单栏中选择[参数]→[编辑参数]→将点灯方式改为<BGC-D2>。重新进行“谱线 搜索”当“谱线搜索”及“光束平衡”均显示[OK]时，点击<关闭>。清洁：正式检测样品前，选择<清洁>，除去石墨管中残余的杂质。
在MRT工作单上依次设置空白组(BLK)、标准品(STD)、待测样品(UNK)的位置及上样量(10μL)，输入标准品的理论浓度以及待测样品的样品名称，选择<开始>进行检测。
结果处理：根据标准品的Abs值和理论浓度在excel中制作散点图，添加趋势线，显示公式和相关系数R2，R2≥99％证明相关性良好，可用于计算样品浓度。
紫外分光光度仪对FH002C上清的利塞膦酸含量进行测定：
开机预热：打开仪器右后侧的总电源开关，一般预热10-20分钟。
根据要求进行选项测定：在屏幕左上角第一行可选栏中选择Fixed Wavelength(固定波长测定)进行测定。
Fixed Wavelength(固定波长测定)：点击Edit Method图标出现波长设定界面，Number of wavelengths栏中选择262nm波长，设定完毕点击OK。将空白管的样本装上干净的比色皿中，放入比色架，点击BLK图标，扣除空白，再分别加入样品，点击Read即可。
实验结果：根据得出的标准曲线，分别计算FH002C上清中的锌离子、铝离子以及利塞膦酸，根据公式：(各成分总含量-上清中各成分的含量)/各成分总浓度＊100％，即为各成分的沉淀率，通过计算得出利塞膦酸锌铝佐剂FH002C中的锌的沉淀率大于99.0％、铝和利塞膦酸的沉淀率大于99.9％(表2)。
表2 FH002C中锌离子、铝离子以及利塞膦酸的沉淀率
Figure PCTCN2021130588-appb-000004
(6)吸附率测定
如下设计和制备新型冠状病毒S重组蛋白。简言之，通过在SARS-CoV-2的野生型S蛋白(SEQ ID NO：1)全长胞外段的C末端融合T4噬菌体fibritin蛋白质的三聚化结构域，去除其中的弗林酶切位点，并在C端融合6＊His标签设计重组新型冠状病毒S蛋白(S重组蛋白，SEQ ID NO：4)。利用中国仓鼠卵巢癌细胞(Chinese hamster ovarian cancer cells，CHO)表达系统(购自Thermo Scientific，A29133)表达S重 组蛋白，并通过镍-琼脂糖凝胶柱(Cytiva，17-5318-03)进行纯化，从而获得纯度大于95％的S重组蛋白。通过BCA方法(Pierce TM BCA Protein Assay Kit，Thermo Scientific，23227)对经纯化的S重组蛋白进行定量。将S重组蛋白稀释至400μg/mL，与FH002C佐剂混合，S重组蛋白∶佐剂＝1∶1(体积比)，将混合后的样品制剂摇匀后4℃静置吸附，在各个时间点分别进行取样。取样前充分摇匀，500μL/次；取出的样品按13000rpm/min离心5min后取上清，再根据预实验结果将样品上清稀释相应倍数，待用。
绘制标准曲线：将S重组蛋白原液作为标准品进行梯度稀释，样稀液SD-1为稀释缓冲液，将标准品稀释至EP管中规定的浓度梯度：50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.12ng/mL、1.56ng/mL，待用。
实验过程：
(1)包板：用1x CB9.6包被缓冲液将36H6单克隆抗体(实验室自制，36H6单克隆抗体为识别新冠S蛋白上RBD表位的抗体，采用杂交瘤技术制备，方法参考Li，et al.Emerging Microbes Infection.2020)稀释至1μg/mL，100μL/孔加至聚苯乙烯96孔板中，4℃包被过夜。
(2)封闭：弃去孔内包被液，PBST洗液洗板1次，甩干，加封闭液-1，200μL/孔，25℃封闭4h。
(3)弃去孔内封闭液，PBST洗板1次，甩干，将稀释好的标准品和样品上清加入相应的96孔板内，25℃孵育反应1h。
(4)加酶标抗体(85F7-HRP)(实验室自制)：弃去孔内液体，PBST洗板5次，甩干，加酶标抗体(85F7-HRP，按1∶5000(V∶V)稀释，ED-11作为酶稀液)100μL/孔，25℃孵育反应1h。
(5)显色：弃去孔内酶标抗体，PBST洗板5次，甩干，以100μL/孔加入A、B等体积混匀的显色液，25℃反应10min。
(6)终止：50μL/孔，2M硫酸终止液，终止反应。
(7)读板：在酶标仪上设置检测双波长为450nm和630nm，测各反应孔的OD值。
FH002C佐剂吸附率计算：将稀释后的样品孔OD读值带入标准曲线内，求得该样品孔相应的蛋白浓度，再乘以相应的稀释倍数，即为原样品上清中蛋白浓度。佐剂 吸附率＝(原样品中蛋白总浓度-上清中蛋白浓度)/原样品中蛋白总浓度＊100％。
实验结果如表3所示：根据吸附率计算公式计算，FH002C配伍S重组蛋白的疫苗制剂在吸附30h时，佐剂吸附率可到达95％以上，4℃放置70天，该疫苗制剂中的蛋白吸附率均在95％以上(96.5％-99.9％)。
表3.FH002C佐剂对新冠S重组蛋白的吸附率
Figure PCTCN2021130588-appb-000005
实施例2：不同锌、铝、利塞膦酸比例的利塞膦酸锌铝佐剂(FH002C)的理化性质测定
对混合后获得的FH002C悬浊液(制备例2)以及Al001悬浊液(制备例3)进行一次121℃，60min灭菌，测定灭菌之后的pH、吸附率、金属沉淀率、外观与颗粒形态等理化性质。
(1)pH测定
pH测定方法同实施例1中的pH测定过程。
实验结果：不同锌、铝、利塞膦酸比例的FH002C佐剂灭菌前pH为7.20～7.90，灭菌后6.00～6.60。
(2)吸附率测定
BSA标准品绘制标准曲线：100mM NaAc为稀释缓冲液，将BSA标准品(2mg/mL)进行系列稀释，用紫外分光光度计检测其在280nm处的吸光值，绘制标曲。
BSA配制：100mM NaAc为稀释缓冲液，称取一定量BSA样品，稀释至EP管中，最终浓度为1mg/mL。
BSA与佐剂混合：将佐剂摇匀后，按照1mg/mL BSA∶佐剂＝1∶1(体积比)进行混合，室温吸附1h，期间摇晃5次，离心(13000rpm/min，3min)，取上清，待用。
吸附率测定：采用紫外分光光度计直接测定上清在280nm处的吸光值，使读值在0.2-0.8之间。吸附率计算：将稀释后的样品孔OD读值带入标准曲线内，求得该样品孔相应的蛋白浓度，再乘以相应的稀释倍数，即为原样品上清中蛋白浓度。佐剂吸附 率＝(原样品中蛋白总浓度-上清中蛋白浓度)/原样品中蛋白总浓度＊100％。
实验结果如表4所示：根据吸附率计算公式计算，不同锌、铝、利塞膦酸比例的FH002C佐剂吸附率在39％～96％之间。以75％为标准，选取吸附率大于75％以上的佐剂进行金属沉降率观察。
表4.不同锌、铝、利塞膦酸比例的FH002C佐剂对0.5mg/mL BSA的吸附率结果
Figure PCTCN2021130588-appb-000006
注：以Zn的摩尔浓度为1对铝以及利塞膦酸摩尔浓度比例的换算
(3)金属沉降率检测
混合金属元素标准溶液制备：取12mL离心管，用5％硝酸稀释四种金属单元素标准溶液至标曲最高浓度点STD7。再将其用1％硝酸逐级对倍稀释(6mL盐酸加6mL上一级标准工作液)，依次得到STD6～STD1溶液，最终每份混合标曲溶液体积为6mL，以确保进样完全。
表5.混合金属标准曲线
Figure PCTCN2021130588-appb-000007
注：表中单位“ppm”等同于“μg/mL”。
供试品溶液制备(至少4mL)：取供试品平衡至室温，用旋涡混合仪充分混匀， 精密量取供试品用5％硝酸溶液稀释至合适倍数，混匀后静置1小时，即为供试品溶液(测定钙、镁和锌的含量建议稀释倍数200倍，测定铝含量建议稀释50倍)。
空白对照溶液制备：取5％硝酸溶液作为空白对照
测定：打开氩气瓶(调节气压为0.6MPa左右)；吹气平衡：打开ICP-OES仪器设备及软件，在软件界面中的“Dashboard”选择“Purge Gas Flow”，点击选择下拉选项中“Normal”模式。在该状态下至少平衡仪器1个小时。平衡时观察软件界面“Torch compartment”、“Plasma Gas Pressure”、“Purge Gas Pressure”、“Drain Flow”、“Exhaust Flow”和“Optics Temperature”应变为绿色状态。打卡水箱，等待约2分钟，待仪器状态栏“Detector Water Flow”和“Detector Temperature”状态变绿。连接仪器管路并清洗：将导管进样口放在超纯水中，正确连接导管至蠕动泵上，选择界面中“Pump Speed”改为50，点击“Apply”，观察导管出口是否能正常流出液体。待1-2分钟清洗后，可进行点火。点火：选择软件界面中的“Dashboard”，单击操作界面上方中间蓝色图标“Get Ready”，弹出窗口，点击“OK”，大约等2-5分钟，点火成功。若第一次点火未识别为正常情况，待仪器自清洗完全后，再次进行点火操作。在日志查看Carbon line中x值与y值。两者的绝对值分别均应小于10。建立LabBook：点击右侧菜单栏“LabBooks”，在“Name”栏输入待做实验名称(实验日期+实验内容+操作人)，选择“Create a new LabBook from an exsiting LabBook”，打开“Risedronate Sodium-Zinc-Aluminum adjuvant Method Template”，点击界面下方“Create LabBook”，即成功在利塞膦酸锌铝佐剂模板中新建当次实验LabBook；参数选择：进入新创建的模板界面，在右侧“Analytes”栏检查待测元素及其检测波长Zn(213.856nm)、Al(396.152nm)、Ca(396.847nm)、Mg(279.553nm)。在“Measure Modes”栏检查观测方向是否为Radial。建立样品进样序列：在右侧栏选择“Sample list”将样品信息及进样位置输入序列中并保存，点击左上角绿色三角形图标“Start”，再点击左下角绿色三角形图标即可开始运行序列。每个样品测样时将导管放入离心管中，每一针测完后系统提示放入wash中清洗前，需用无尘纸(或滤纸)擦去导管上残留液滴。将导管从wash中放回下一个样品中时同样需擦去导管上液滴。结果计算：以混合标准溶液的浓度为横坐标，以相应的峰面积为纵坐标绘制4条标准曲线，拟定直线回归方程。将稀释后的供试品溶液对应的各单元素峰面积代入回归方程，乘以稀释倍数，求出供试品溶液的金属元素浓度(ppm)。
实验结果：如表6、7、8所示，Zn基本存在于沉淀中，Mg和Ca有部分存在沉淀，部分存在于上清中。根据Mg和Ca的沉淀率结果选择标灰的部分进行佐剂外观和颗粒形态观察。
表6.不同锌、铝、利塞膦酸比例的FH002C佐剂中锌的沉淀率
Figure PCTCN2021130588-appb-000008
注：以Zn的摩尔浓度为1，换算铝和利塞膦酸的摩尔浓度比例。“/”表示未测量
表7.不同锌、铝、利塞膦酸比例的FH002C佐剂中镁的沉淀率
Figure PCTCN2021130588-appb-000009
注：以Zn的摩尔浓度为1，换算铝和利塞膦酸的摩尔浓度摩尔浓度比例。“/”表示未测量
表8.不同锌、铝、利塞膦酸比例的FH002C佐剂中钙的沉淀率
Figure PCTCN2021130588-appb-000010
注：以Zn的摩尔浓度为1，换算铝和利塞膦酸的摩尔浓度比例。“/”表示未测量
(4)佐剂外观以颗粒形态观察
佐剂外观以颗粒形态观察可参考实施例1中的检测过程。
实验结果：如图4A-图4F所示，不同锌、铝、利塞膦酸比例的利塞膦酸锌铝佐剂均为乳白色悬浊物，混浊度不一；在电镜下大部分为无规则的片状结构，个别为丝状结构。
实施例3：含FH002C的新型冠状病毒S重组蛋白制剂抗原活性的测定(基于ACE2受体结合)
将FH002C(如制备例1所制备的)与新型冠状病毒S重组蛋白通过体积比为1∶1混合形成疫苗制剂，4℃放置1周后(吸附完全)，以检测其抗原性。以未吸附的新型冠状病毒S重组蛋白原液作对照，对佐剂吸附抗原并解离后的抗原性进行比较。
基于ACE2受体结合的体外相对效力实验检测蛋白的抗原性：
(1)抗体包被液：1X CB 9.6缓冲液(15mM Na 2CO 3；35mM NaHCO 3)。
(2)洗涤液：PBST，INNOVAX公司ELISA试剂盒
(3)封闭液：封闭液-1，INNOVAX公司ELISA试剂盒。
(4)显色液A：INNOVAX公司ELISA试剂盒。
(5)显色液B：INNOVAX公司ELISA试剂盒。
(6)终止液：INNOVAX公司ELISA试剂盒。
实验过程：
(1)包板：用1X CB9.6包被缓冲液将新型冠状病毒S蛋白抗体45C3(实验室自制)稀释至1μg/mL。100μL/孔加至聚苯乙烯96孔板中，4℃包被过夜。
(2)封闭：弃去孔内包被液，PBST洗液洗板1次，甩干，加封闭液200μL/孔，室温封闭4h。
(3)样品准备：S重组蛋白抗原原液(吸附前)，S重组蛋白疫苗制剂(制剂中S重组蛋白抗原浓度为100μg/mL，佐剂为FH002C)经解离后的抗原。首孔抗原浓度均为4μg/mL。弃去孔内封闭液，PBST洗板1次，甩干，首孔加待检血清200μL/孔，后面的每孔加入100μL样品稀释液，2倍梯度稀释，25℃孵育反应1h。
(4)加ACE2-hFc(购自SinoBiological，货号：10108-H02H)：弃去孔内液体， PBST洗板5次，甩干，加ACE2-hFc，1μg/mL，100μL/孔，25℃孵育反应1h。
(5)加酶标抗体(MAH-HRP)(购自SouthernBiotech，货号：9042-05)：弃去孔内液体，PBST洗板5次，甩干，加酶标抗体(MAH-HRP，V∶V＝1∶5000)100μL/孔，25℃孵育反应1h。
(5)显色：弃去孔内酶标抗体，PBST洗板5次，甩干，以100μL/孔加入A、B等体积混匀的显色液，25℃反应10min。
(6)终止：50μL/孔加2M硫酸终止液，终止反应。
(7)读板：在酶标仪上设置检测双波长为450nm和630nm，测各反应孔的OD值。
实验结果如图5所示：利用基于ACE2受体结合的抗原性检测结果表明，FH002C联合新型冠状病毒S重组蛋白疫苗制剂经解离液解离后，其抗原性与S重组蛋白原液抗原性相当(rEC 50＝EC 50，原液/EC 50，解离后＝0.9)，说明含FH002C的新型冠状病毒S重组蛋白疫苗制剂具有良好的稳定性。
实施例4：含利塞膦酸锌铝佐剂的新型冠状病毒S重组蛋白量效关系研究
将按制备例1制备好的FH002C佐剂和按制备例3制备好的Al001佐剂作为佐剂分别与新型冠状病毒重组S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射小鼠，测定血清中特异性抗体结合滴度和中和滴度。具体方法如下：
实验动物：Balb/C小鼠(购自上海斯莱克实验动物有限责任公司，货号：2017000502480)，6-8周，5只/组，雌性。
实验分组：(1)S重组蛋白水溶液低剂量组(1μg)；(2)S重组蛋白水溶液高剂量组(10μg)；(3)S重组蛋白+Al001低剂量组(1μg)；(4)S重组蛋白+Al001高剂量组(10μg)；(5)S重组蛋白+FH002C低剂量组(1μg)；(6)S重组蛋白+FH002C高剂量组(10μg)。
免疫方案：S重组蛋白1μg/只或10μg/只，佐剂与S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射小鼠，每只100μL，小鼠每条后腿各50μL。0，3周免疫，即动物按照免疫分组首次免疫后3周，眼眶采血，测定血清中特异性抗体滴度。第3周加强免疫，其后每周进行眼眶取血，采用ELISA法测定血清中特异性抗体结合滴度。
酶联免疫吸附法(ELISA)检测抗体结合滴度：
(1)抗体包被液：1X CB 9.6缓冲液(15mM Na 2CO 3；35mM NaHCO 3)。
(2)洗涤液：PBST，INNOVAX公司ELISA试剂盒
(3)封闭液：封闭液-1，INNOVAX公司ELISA试剂盒。
(4)显色液A：INNOVAX公司ELISA试剂盒。
(5)显色液B：INNOVAX公司ELISA试剂盒。
(6)终止液：INNOVAX公司ELISA试剂盒。
实验过程：
(1)包板：用CB9.6包被缓冲液将抗原S重组蛋白稀释至一定浓度。100μL/孔加至聚苯乙烯96孔板中，4℃包被过夜。
(2)封闭：弃去孔内包被液，PBST洗液洗板1次，甩干，加封闭液200μL/孔，室温封闭4h。
(3)加一定稀释度的血清：弃去孔内封闭液，PBST洗板1次，甩干，首孔加待检血清200μL/孔，后面的每孔加入100μL样品稀释液，2倍梯度稀释，25℃孵育反应1h。
(4)加酶标抗体(GAM-HRP)(购自Bio-Rad Laboratories Inc.，货号：1706516)：弃去孔内血清稀释液，PBST洗板5次，甩干，加酶标抗体(GAM-HRP，V∶V＝1∶5000)100μL/孔，25℃孵育反应1h。
(5)显色：弃去孔内酶标抗体，PBST洗板5次，甩干，以100μL/孔加入A、B等体积混匀的显色液，25℃反应10min。
(6)终止：50μL/孔加2M硫酸终止液，终止反应。
(7)读板：在酶标仪上设置检测双波长为450nm和630nm，测各反应孔的OD值。
血清中特异性抗体中和滴度检测方法参见H.L.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，1-38(2020).
实验结果如图6所示：
小鼠免疫一针后3周，相同免疫剂量下，FH002C佐剂组小鼠抗体滴度高于水溶 液组和Al001佐剂组，高于铝佐剂组1-1.5个数量级，具有起效快的特点。免疫两针后，其体液免疫增强优势依然明显。4周时，低剂量组中，FH002C佐剂组抗体滴度高于铝佐剂组1.5个数量级，高于水溶液组3个数量级。高剂量组中，FH002C佐剂组与Al001组抗体水平相当，均高于水溶液组2个数量级。第5、6周时，在高低剂量组中，FH002C佐剂组抗体滴度仍然明显优于Al001组(0.5-1个数量级)和水溶液组(2.5-3个数量级)。
免疫一针后，第三周仅有FH002C佐剂组小鼠产生中和抗体，水溶液组及Al001佐剂组小鼠均未产生。第二针加免后，第4，5，6周中，无论在高剂量组还是低剂量组，FH002C佐剂组小鼠中和滴度均高于Al001佐剂组(高于0.5-1.5个数量级)和水溶液组(高于1.5-2.5个数量级)。
实施例5：不同锌、铝、利塞膦酸比例的FH002C佐剂联合新型冠状病毒S重组蛋白产生的特异性抗体结合滴度
将制备好的FH002C(制备方法参见制备例2)作为佐剂分别与新型冠状病毒S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射Balb/C小鼠，测定产生的特异性抗体滴度。具体方法如下：
实验动物：Balb/C小鼠，6-7周，雌性，每组5只
免疫方案：抗原1μg/只，每只100μL(左右腿各半)。0，3周免疫二针，0-6周每周经眼眶采血后进行血清中的抗体滴度检测。
酶联免疫吸附法(ELISA)检测抗体结合滴度，具体如下：
ELISA所用试剂可参考实施例4。
实验过程：
(1)包板与封闭：将S重组蛋白使用CB9.6稀释至1μg/mL按照100μL/孔加入到96孔酶标板中，放置于37℃下孵育2h后用PBST缓冲液(即PBS中含0.05％Tween-20)洗板1次，向每孔中添加200μL含20％NBS的PBS，再放置于37℃下孵育2h进行封闭。除去多余的液体并甩干备用。
(2)血清稀释：向首孔中添加100μL稀释300倍的后血清，3倍稀释10个梯度，在37℃下孵育1h。
(3)加酶标抗体：用PBST缓冲液将板洗涤5次后，添加100μL稀释一定倍数 的羊抗鼠-辣根过氧化物酶酶标抗体GAM-HRP(实验室自制)，在37℃下孵育0.5h。
(4)显色、终止、读值。
实验结果：如图7所示，不同锌、铝、利塞膦酸比例的FH002C佐剂联合新型冠状病毒S重组蛋白均能产生特异性结合抗体。
实施例6：利塞膦酸锌铝佐剂联合新型冠状病毒S重组蛋白产生的特异性抗体结合滴度
将制备好的Al001(制备例3)和FH002C(制备例1)作为佐剂分别与新型冠状病毒S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射叙利亚金黄地鼠(本文也称“叙利亚仓鼠”)以及食蟹猴，测定产生的特异性抗体滴度。具体方法如下：
实验动物：叙利亚金黄地鼠(购自上海斯莱克实验动物有限责任公司)，6-7周，FH002C组22只/组，Al001组16只/组，雌雄各半。成年食蟹猴(购自广西雄森灵长类实验动物养殖开发有限公司)，2只/组，雌雄各半。
实验分组：(1)S重组蛋白+Al001；(2)S重组蛋白+FH002C
叙利亚金黄地鼠免疫方案：抗原10μg/只，佐剂与新型冠状病毒S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射叙利亚金黄地鼠，每只200μL。0，2，6周免疫三针，0-7周每周经眼眶采血后进行血清中的抗体滴度检测。
食蟹猴免疫方案：抗原20μg/只，佐剂与新型冠状病毒S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射食蟹猴，每只500μL。0，2周免疫2针，0-6周每周进行采血进行抗体结合滴度检测。
酶联免疫吸附法(ELISA)检测抗体结合滴度，具体如下：
ELISA所用试剂可参考实施例4。
实验过程：
(1)包板与封闭：将S重组蛋白使用CB9.6稀释至2μg/mL按照100μL/孔加入到96孔酶标板中，放置于37℃下孵育2h后用PBST缓冲液(即PBS中含0.05％Tween-20)洗板1次，向每孔中添加200μL含20％NBS的PBS，再放置于37℃下孵育2h进行封闭。除去多余的液体并甩干备用。
(2)血清稀释：向各孔中添加100μL稀释50或100倍的后血清，在37℃下孵 育1h。
(3)加酶标抗体：用PBST缓冲液将板洗涤5次后，添加100μL稀释一定倍数的HRP偶联的山羊抗人IgG(H+L)(BEYOTIME，A0201)或山羊叙利亚金黄地鼠IgG H&L(Abcam，ab6892)，在37℃下孵育0.5h。
(4)显色、终止、读值。
实验结果：如图8所示，叙利亚金黄地鼠免疫一针后2周，FH002C组的抗体滴度高于Al001组，即为铝佐剂组15倍，具有起效快的特点。免疫两针后，其体液免疫增强优势依然明显，第4周时，FH002C组的抗体滴度是铝佐剂组的8倍。免疫三针后，第7周时，FH002C组的抗体滴度是对照组的11倍。
食蟹猴免疫一针后2周，FH002C组的抗体滴度高于Al001组，即为铝佐剂组6倍，具有起效快的特点。免疫两针后，第4周时，FH002C组的抗体滴度与铝佐剂组相当(图9)。
实施例7：利塞膦酸锌铝佐剂联合新型冠状病毒S重组蛋白后产生的中和抗体滴度
采用实施例6所述的实验流程，将Al001(制备例3)与FH002C(制备例1)佐剂分别配伍新型冠状病毒S重组蛋白通过肌肉注射来免疫叙利亚金黄地鼠和食蟹猴，检测血清中和抗体滴度。叙利亚金黄地鼠以及食蟹猴免疫程序同实施例6。
通过假病毒中和法(Pseudovirus neutralization)检测抗体中和滴度(H.L.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，1-38(2020))，具体如下：
通过重组质粒pCAG-nCoVSdel18和VSVdG-EGFP-G病毒(Addgene，31842)将承载了SARS-CoV-2 S蛋白(Wuhan-Hu-1株，基因库：QHD43416.1)的水疱性口炎病毒(VSV)假病毒(rVSV-SARS-CoV-2)进行了包装。提前24h将hACE2表达的BHK21(BHK21-hACE2)细胞铺到96孔板中，接着用含10％FBS(GIBCO，10099141)和青霉素-链霉素(GIBCO，15140122)的高糖DMEM培养基(Sigma-Aldrich，D6429)将血清稀释50倍，然后进行3倍梯度稀释。将稀释了的rVSV-SARS-CoV-2病毒(MOI＝0.05)与稀释后的血清混合，并在37℃下孵育1h。紧接着，将混合物转入铺了 BHK21-hACE2细胞的板中，随后在含5％CO 2，37℃的培养箱孵育12h。通过Opera Phenix或Operetta CLS高含量分析系统(PerkinElmer)捕获荧光图像数据，再用Columbus系统(PerkinElmer)分析每个孔的GFP阳性细胞的计数。每个板包含8个无血清的孔作为病毒对照。血清中和滴度定义为与阳性孔相比，GFP阳性细胞数量减少了50％时对应的血清稀释倍数(ID 50)。
实验结果：如图10所示，叙利亚金黄地鼠免疫一针后2周，FH002C组的抗体滴度高于Al001组，即为铝佐剂组1.8倍，具有起效快的特点。免疫两针后，其体液免疫增强优势依然明显，第4周时，FH002C组的抗体滴度是铝佐剂组的2.6倍。免疫三针后，第7周时，FH002C组的抗体滴度是对照组的6.5倍。
食蟹猴免疫一针后2周，FH002C组的抗体滴度高于Al001组，即为铝佐剂组2.6倍，具有起效快的特点。免疫两针后，第4周时，FH002C组的抗体滴度为铝佐剂组的3.5倍(图11)。
实施例8：利塞膦酸锌铝佐剂联合新型冠状病毒S重组蛋白免疫血清基于细胞的Spike阻断实验
采用实施例6所述免疫程序，将Al001(制备例3)与FH002C(制备例1)佐剂分别配伍新型冠状病毒S重组蛋白通过肌肉注射来免疫叙利亚金黄地鼠和食蟹猴，用基于细胞的Spike血清阻断实验(Y.Zhang et al.，Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors.bioRxiv，(2020))，具体如下：
将表达hACE2-mRuby3 293T(293T-ACE2iRb3)的细胞以2×10 4个/孔提前24h铺在用聚-D-赖氨酸预处理的96孔板上。用含10％FBS的DMEM培养基以2倍梯度稀释的方式稀释叙利亚金黄地鼠及食蟹猴血清。将11μL芽孢杆菌与SARS-CoV-2 S(STG)融合的探针与44μL稀释的血清混合，使得探针终浓度为2.5nM。将铺有293T-ACE2iRb3细胞孔除去一半的培养基后，吸取50μL混合物到细胞中，并在37℃，5％CO 2的条件下孵育1h。随后，通过Opera Phenix高内涵分析系统以共聚焦模式捕获细胞图像。
数据分析：采用Columbus System进行图像数据定量分析。首先，根据293T-ACE2iRb3细胞都表达定位在细胞核中的融合H2B的iRFP670的原理，通过近 红外通道(Ex：640/Em：670)找到所有已经接种的细胞。又根据融合了mRuby3的hACE2会位于膜上的原理，通过红色通道(Ex：561/Em：590)定义细胞边界。接着通过计算细胞质区域(cMFI，外边界：20％，内边界：45％)中STG探针通道(Ex：488/Em：525)的MFI，可得到其抑制比：[(cMFIpc-cMFItst)/(cMFIpc-cMFIblk)]×100％。cMFItst是指测试孔的cMFI值，cMFIpc和cMFIblk分别是仅探针(阳性对照)孔和仅细胞孔的cFMI值。血清阻断水平用ID 50进行表示。
实验结果：如图12所示，在叙利亚金黄地鼠FH002C组的血清阻断率均高于Al001组；如图13所示，在食蟹猴中，两个佐剂组的血清阻断率相当。
实施例9：利塞膦酸锌铝佐剂联合新型冠状病毒S重组蛋白对抗体亲合力的影响
采用实施例6所述的实验流程，将Al001(制备例3)与FH002C(制备例1)佐剂分别配伍新型冠状病毒S重组蛋白通过肌肉注射来免疫小鼠与叙利亚金黄地鼠，检测血清抗体亲合力。叙利亚金黄地鼠免疫程序同实施例6，但FH002C和Al001每组各16只，雌雄各半。小鼠的免疫程序具体如下：
实验动物：Balb/C小鼠(购自上海斯莱克实验动物有限公司)，6-8周，4或5只/组，雌性
实验分组：(1)S重组蛋白+Al001；(2)S重组蛋白+FH002C
小鼠免疫方案：抗原1μg/只，佐剂与新型冠状病毒S重组蛋白过体积比为1：1混合形成疫苗，而后肌肉注射Balb/C小鼠，每只150μL。0，2，4周免疫三针，0-6周每周经眼眶采血后进行血清中的抗体亲合力检测。
酶联免疫吸附法(ELISA)检测抗体亲合力，具体如下：
ELISA所用试剂可参考实施例4。
实验过程：
包板与封闭：将纯化的S重组蛋白使用CB9.6稀释至2μg/mL后进行包被，按照100μL/孔加入到ELISA板中，放置于37℃下孵育2h后用PBST缓冲液(即PBS中含0.05％Twenn-20)洗板1次，向每孔中添加200μL含20％NBS的PBS，再放置于37℃下孵育2h进行封闭。除去多余的液体并甩干。
血清抗体亲合力检测：向各孔中添加100μL血清，每份样品进行双孔重复，在 37℃下孵育1h。用PBST缓冲液将板洗涤1次后，向双孔重复的其中一孔添加100μL PBS，另一孔添加100μL 4M尿素(用PBS配制)，放于37℃20min后用PBST缓冲液将板洗涤4次。接着加入稀释一定倍数的HRP偶联的山羊抗小鼠IgG(H+L)(Proteintech，SA00001-1)或者山羊抗叙利亚仓鼠IgG H&L(Abcam，ab6892)，在37℃下孵育0.5h。用洗涤缓冲液洗涤板5次。向每孔添加100μL显色底物混合液，并使用PHOMO Microplate读取器读取在450nm处的吸光度。抗体亲合力可用抗体比例表示，即尿素处理组的EC 50与未处理组的EC 50的比值。
实验结果：如图14和15所示，不管在小鼠模型还是叙利亚金黄地鼠模型上，FH002C组比Al001组能产生具有更高比例的高亲合力抗体。
实施例10：利塞膦酸锌铝佐剂联合新型冠状病毒S重组蛋白免疫小鼠产生特异性抗体亚型
将制备好的Al001(制备例3)与FH002C(制备例1)作为佐剂分别与新型冠状病毒S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射小鼠，免疫程序如实施例9。
酶联免疫吸附法(ELISA)检测抗体亚型水平，具体如下：
ELISA所用试剂可参考实施例4。
实验过程：
包板与封闭：将纯化的S重组蛋白使用碳酸盐缓冲液稀释至2μg/mL后进行包被，按照100μL/孔加入到ELISA板中，放置于37℃下孵育2h后用PBST缓冲液(即PBS中含0.05％Tween-20)洗板1次，向每孔中添加200μL含20％NBS的PBS，再放置于37℃下孵育2h进行封闭。除去多余的液体并甩干。
血清抗体亚型水平检测：向各孔中添加100μL血清，在37℃下孵育1h，在37℃下孵育1h。用PBST缓冲液将板洗涤5次后，添加100μL稀释一定倍数的HRP偶联的山羊抗鼠IgG1(AbD Serotec，STAR132P)或者山羊抗鼠IgG2a(AbD Serotec，STAR133P)或者山羊抗鼠IgG2b(AbD Serotec，STAR134P)，在37℃下孵育0.5h。用洗涤缓冲液洗涤板5次。向每孔添加100μL稀释3倍后的显色底物混合液，并使用PHOMO Microplate读取器读取在450nm处的吸光度。每块板中包含5孔阴性血清作为阴性对照。
实验结果：如图16所示，与铝佐剂组相比，FH002C佐剂组能激起更高水平的IgG2a和IgG2b亚型抗体，且IgG1与IgG2a和IgG2b的比例较铝佐剂组更低，表明起在Th1免疫途径具有一定的刺激作用。
实施例11：利塞膦酸锌铝佐剂配伍新型冠状病毒S重组蛋白对T细胞应答的影响
将制备好的Al001(制备例3)与FH002C(制备例1)作为佐剂分别与新型冠状病毒S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射小鼠，测定产生的T细胞应答。具体方法如下：
实验动物：C57BL/6，6-8周，8只/组，雌性。
实验分组：(1)空白组；(2)S重组蛋白+Al001；(3)S重组蛋白+FH002C
C57BL/6小鼠免疫方案：抗原10μg/只，佐剂与新型冠状病毒S重组蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射，每只150μL。0，3周免疫两针，第4周处死后分离脾脏与淋巴结进行T细胞免疫应答实验。
酶联免疫斑点法(Enzyme-linked immunospot assay，ELISPOT)检测T细胞应答
取小鼠脾脏和淋巴结，制备成单细胞悬液，按照每孔10 6个细胞(脾)或每孔4×10 5个细胞(淋巴结)铺到小鼠IFN-γ包被的ELISPOT板(DAKEWEI，2210005)中。然后，用PBS或者具有11个氨基酸重叠的15个单元的SARS-CoV-2S肽库刺激(Genscript，RP30020)培养20h。随后，根据试剂盒说明书进行检测。使用
Figure PCTCN2021130588-appb-000011
(Cellular Technology Limited)进行图像捕获和点计数。将刺突肽库刺激的孔中减去PBS刺激的孔来计算分泌IFN-γ的细胞的点数。
实验结果：如图17所示，FH002C和Al001组分泌IFN-γ的细胞数量在脾脏中分别增加了28.9倍和5.8倍，在淋巴结中分别增加了14.0倍和2.3倍，与Al001相比，FH002C组可以诱导较高水平的T细胞应答。
实施例12：铝佐剂(Al001)、FH002C佐剂配伍水痘带状疱疹病毒gE蛋白(VZV gE)产生的特异性抗体结合滴度
将制备好的Al001(制备例3)、FH002C(制备例1)作为佐剂分别与水痘带状疱疹病毒gE蛋白(VZV gE，SEQ ID NO：2)通过体积比为1∶1混合形成疫苗，而后肌肉注射Balb/C小鼠，测定产生的特异性抗体滴度。其中，所述gE蛋白为利用大肠 杆菌表达系统(购自上海唯地生物技术有限公司，EC1060)表达所得。
具体方法如下：
实验动物：Balb/C小鼠，6-7周，每组5只，雌性。
实验分组：(1)VZV gE+Al001；(2)VZV gE+FH002C
Balb/C小鼠免疫方案：抗原5μg/只，佐剂与水痘带状疱疹病毒gE蛋白(VZV gE)通过体积比为1∶1混合形成疫苗，而后肌肉注射Balb/C小鼠，每只100μL。0，2，4周免疫二针，0-4周每周经眼眶采血后进行血清中的抗体滴度检测。
酶联免疫吸附法(ELISA)检测抗体结合滴度，具体如下：
ELISA所用试剂可参考实施例4。
实验过程：
(1)包板与封闭：将VZV gE使用PB7.4+NaCl稀释至1μg/mL按照100μL/孔加入到96孔酶标板中，放置于4℃下孵育过夜后用PBST缓冲液(即PBS中含0.05％Tween-20)洗板1次，向每孔中添加200μL含20％NBS的PBS，再放置于37℃下孵育2h进行封闭。除去多余的液体并甩干备用。
(2)血清稀释：向各孔中添加100μL稀释50或100倍的后血清，在25℃下孵育1h。
(3)加酶标抗体：用PBST缓冲液将板洗涤5次后，添加100μL稀释一定倍数的酶标抗体(GAM-HRP)(购自Bio-Rad Laboratories Inc.，货号：1706516)，25℃孵育反应1h。
(4)显色、终止、读值。
实验结果：如图18所示，免疫两针后FH002C佐剂组的结合滴度明显高于Al001组，大约10倍左右。
实施例13：弗式佐剂、FH002C佐剂配伍流感病毒HA蛋白产生的特异性抗体结合滴度
将商品化的弗氏佐剂(购自SIGMA-ALDRICH，货号：F5881)、FH002C佐剂(制备例1)分别与经去糖基化酶处理或未经去糖基化酶处理的流感病毒HA蛋白(SEQ ID NO：3)通过体积比为1∶1混合形成疫苗，而后皮下注射Balb/C小鼠，测定产生的特异性抗体滴度。其中，所述流感病毒HA蛋白为利用杆状病毒表达系统(购 自美国Invitrogen，10359016)表达所得。所述去糖基化酶购自NEB，货号P0705S；所述去糖基化酶处理过程参照所述去糖基化酶的说明书进行。
具体方法如下：
实验动物：Balb/C小鼠(购自上海斯莱克实验动物有限责任公司)，6-7周，FH002C组10只/组，弗氏佐剂组10只/组。
实验分组：(1)HA+弗氏佐剂；(2)HA+FH002C
免疫方案：(1)抗原30μg/只，弗氏佐剂与流感病毒HA蛋白通过体积比为1∶1混合形成疫苗，而后皮下注射Balb/C小鼠，每只300μL；(2)抗原30μg/只，FH002C与流感病毒HA蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射Balb/C小鼠，每只200μL。免疫后14天经眼眶采血后进行血清中的抗体滴度检测。
酶联免疫吸附法(ELISA)检测抗体结合滴度，具体如下：
ELISA所用试剂可参考实施例4。
(1)包板与封闭：将重组HA蛋白或流感超离病毒使用CB9.6稀释至2μg/mL或者按照100μL/孔加入到96孔酶标板中，放置于37℃下孵育2h后用PBST缓冲液(即PBS中含0.05％Tween-20)洗板1次，向每孔中添加200μL商品化封闭液，再放置于37℃下孵育2h进行封闭。除去多余的液体并甩干备用。
(2)血清稀释：向各孔中添加100μL稀释不同倍数的血清，在37℃下孵育1h。
(3)加酶标抗体：用PBST缓冲液将板洗涤5次后，添加100μL稀释5000倍的酶标羊抗鼠抗体GAM-HRP(实验室自制)，在37℃下孵育0.5h。
(4)显色、终止、读值。
实验结果：如图19所示，Balb/C小鼠免疫一针后2周，针对经去糖基化酶处理和未经去糖基化酶处理的流感病毒HA蛋白，FH002C佐剂组的抗体滴度均高于弗式佐剂组，具有起效快的特点。
实施例14：铝佐剂(Al001)、FH002C佐剂配伍轮状病毒VP4蛋白产生的中和抗体结合滴度
将制备好的Al001(制备例3)与FH002C(制备例1)作为佐剂分别与轮状病毒VP4蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射Balb/C小鼠和豚鼠，测定产生的中和抗体滴度。其中，所述VP4蛋白由大肠杆菌表达系统(购自上海唯地生物技 术有限公司，EC1060)表达。
具体方法如下：
实验动物：Balb/C小鼠，购自上海斯莱克实验动物有限责任公司，6-8周，豚鼠，450-500g，购自上海松联实验动物场。
小鼠组：FH002C组5只/组，Al001组5只/组，均为雌性。
豚鼠组：FH002C组5只/组，Al001组5只/组，均为雌性。
实验分组：(1)VP4+Al001；(2)VP4+FH002C
免疫方案：小鼠免疫抗原10μg/只，豚鼠免疫抗原10μg/只，佐剂与轮状病毒VP4蛋白通过体积比为1∶1混合形成疫苗，而后肌肉注射小鼠，豚鼠体内，小鼠豚鼠均每只200μL，第一针免疫前以及第三针免疫后2周采血，分离血清检测中和抗体滴度。
酶联免疫斑点实验(ELISPOT)检测中和抗体滴度，具体如下：
(1)MA104细胞铺96孔细胞板：将细胞用胰酶消化液消化后，然后用含10％FBS的细胞培养基将细胞吹悬起来，然后利用细胞计数板计数之后，添加培养基稀释至25000个/mL，然后均匀的添加到96孔板中，100μL/孔，37℃培养20h，CO 2浓度为5％。
(2)病毒酶切处理：往1mL轮状病毒溶液中加入4μL的2.5μg/μL的胰酶，混匀后置于，处理结束后用无血清的含1μg/mL胰酶的DMEM培养基稀释病毒至一定滴度。
(3)血清补体灭活处理：取10μL血清样本于1.5mL的EP管中，然后56℃热处理30min。处理结束后用含1μg/ml胰酶的DMEM稀释血清样品，双孔重复检测。
(4)血清与病毒反应：将(2)中的病毒和(3)中的血清各取100μL混合，37℃中和反应1h。
(5)更换细胞培养基：将(1)中的细胞上清移去，用无血清的含1μg/mL胰酶的DMEM培养基漂洗(1)中的MA104细胞5min，重复漂洗三次。
(6)感染：最后一次漂洗结束后，移去漂洗的培养基，并将(4)中的中和反应混合液加入细胞中，在37℃培养14h后，CO2浓度为5％。
(7)细胞固定：将(6)中的细胞上清甩干，注意轻缓，避免细胞受损，然后用含0.1％戊二醛的PBS溶液固定细胞，100μL/孔，室温固定1h，注意在避光条件下固定，避免戊二醛见光分解影响固定效果。
(8)细胞通透：移去(7)中戊二醛固定液，并加入100μL含0.3％Triton X-100的PBS溶液，室温通透处理30min。
(9)氧化：移去(8)中的TritonX-100通透液，然后加入100μL含3％的H 2O 2的PBS溶液，室温处理15min。
(10)洗涤：移去氧化液，并用PBST洗液漂洗细胞5次，每次漂洗5min。
(11)酶标抗体反应：将漂洗液甩干，然后将事先配好的酶标抗体反应液(酶稀释液，ED-13)稀释标记HRP的VP6抗体(实验室自制，该单克隆抗体为识别VP6的抗体，采用杂交瘤技术制备，方法参考Li，et al.Emerging Microbes Infection.2020，1∶5000稀释)加入到细胞上，37℃反应1h。
(12)洗涤：移去酶标抗体反应液，并用PBST洗液漂洗细胞5次，每次漂洗5min。
(13)显色：将漂洗液甩干，然后现配的TMB显色液，100μL/孔，室温反应15min，注意避光，避免显色液见光反应，影响实验结果。
(14)读板：甩干显色反应液，利用ELISPOT读板仪对上述96孔板进行读板和计数。
实验结果：如图20和21所示，小鼠和豚鼠免疫三针后均可产生中和抗体，且FH002C组的中和抗体滴度高于Al001组。其中，在小鼠中，FH002C佐剂组血清中和滴度约为Al001佐剂组的16倍(图20)；而在豚鼠中，FH002C佐剂组血清中和滴度约为Al001组的4倍(图21)。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (9)

  1. 一种包含利塞膦酸锌铝的佐剂，其中，锌∶利塞膦酸摩尔浓度比在1∶1至16∶1的范围内(例如，2∶1至16∶1的范围内)；并且，锌∶铝摩尔浓度比在1∶1至50∶1的范围内(例如5∶1至50∶1的范围内)；
    优选地，所述佐剂以颗粒形式存在；优选地，所述颗粒的粒径大小为0.01-100μm，例如0.01-60μm，0.01-50μm，0.1-60μm，0.1-30μm，0.4～30μm，0.4-20μm；
    优选地，所述佐剂锌∶利塞膦酸摩尔浓度比在1∶1至2∶1，2∶1至4∶1，4∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至14∶1，或者14∶1至16∶1的范围内；优选地，所述佐剂锌∶利塞膦酸摩尔浓度比为4∶1或4.5∶1；
    优选地，所述佐剂锌∶铝摩尔浓度比在1∶1至2∶1，2∶1至3∶1，3∶1至4∶1，4∶1至5∶1，5∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至15∶1，15∶1至20∶1，20∶1至30∶1，30∶1至40∶1，或者40∶1至50∶1的范围内；优选地，所述佐剂锌∶铝摩尔浓度比为10∶1；
    优选地，所述佐剂锌∶利塞膦酸摩尔浓度比在2∶1至8∶1的范围内，例如2∶1至4∶1，4∶1至6∶1，或6∶1至8∶1，并且，锌∶铝摩尔浓度比在2∶1至50∶1的范围内(例如5∶1至20∶1的范围内)，例如2∶1至3∶1，3∶1至4∶1，4∶1至5∶1，5∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至15∶1，或15∶1至20∶1；
    优选地，所述佐剂锌∶利塞膦酸摩尔浓度比为4∶1，并且，锌∶铝摩尔浓度比为10∶1；
    优选地，所述佐剂锌∶利塞膦酸摩尔浓度比为4.5∶1，并且，锌∶铝摩尔浓度比为10∶1；
    优选地，所述佐剂pH为5.0-8.0，例如5.0-7.0，5.0-5.5，5.5-6.0，6.0-6.5，6.5-7.0，7.0-7.5，或7.5-8.0；
    优选地，所述佐剂零电荷点为3.0～8.0，例如4.0-8.0，3.0-4.0，4.0-5.0，5.0-6.0，6.0-7.0，或7.0-8.0；
    优选地，所述佐剂对免疫原(例如蛋白)的吸附率为至少60％，至少70％，至少75％，至少80％，至少85％，至少90％，或至少95％。
  2. 制备权利要求1所述的佐剂的方法，其包括如下步骤：
    1)提供含有锌离子、铝离子的可溶性盐溶液；
    2)将步骤(1)中的可溶性盐溶液与利塞膦酸碱溶液混合，从而获得所述佐剂；
    优选地，所述方法还包括，将步骤(2)获得的佐剂进行灭菌的步骤；优选地，通过过滤灭菌或高温高压灭菌来对所述佐剂进行灭菌，例如通过在121℃下灭菌至少15分钟(例如至少30分钟，例如30-60分钟)，对所述佐剂进行灭菌；
    优选地，在所述可溶性盐溶液中，锌∶铝的摩尔浓度比在1∶1至50∶1的范围内(例如5∶1至50∶1的范围内)；优选地，在所述可溶性盐溶液中，锌∶铝摩尔浓度比在1∶1至2∶1，2∶1至3∶1，3∶1至4∶1，4∶1至5∶1，5∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至15∶1，15∶1至20∶1，20∶1至30∶1，30∶1至40∶1，或者40∶1至50∶1的范围内；
    优选地，在步骤(2)中，以在1∶1至16∶1的范围内(例如2∶1至16∶1的范围内)的锌∶利塞膦酸摩尔浓度比，将所述可溶性盐溶液与利塞膦酸碱溶液混合；优选地，所述锌∶利塞膦酸摩尔浓度比在1∶1至2∶1，2∶1至4∶1，4∶1至6∶1，6∶1至8∶1，8∶1至10∶1，10∶1至12∶1，12∶1至14∶1或者14∶1至16∶1的范围内；
    优选地，在步骤(2)中，以使锌离子、铝离子和利塞膦酸共同沉淀的方式，将所述可溶性盐溶液与利塞膦酸碱溶液混合；
    所述方法为以共同沉淀的方式使锌离子、铝离子和利塞膦酸产生沉淀，从而获得利塞膦酸锌铝颗粒；
    优选地，在步骤(2)中，将利塞膦酸碱溶液逐滴加入所述可溶性盐溶液，使锌离子、铝离子和利塞膦酸共同沉淀；
    优选地，所述利塞膦酸碱溶液选自利塞膦酸氢氧化钠，利塞膦酸磷酸盐溶液(例如利塞膦酸磷酸氢二钠溶液，利塞膦酸磷酸氢钠溶液)，或其任何组合；
    优选地，步骤(1)所述可溶性盐溶液选自例如硫酸盐溶液、氯酸盐溶液、醋酸盐溶液、或其任何组合，优选氯酸盐溶液或醋酸盐溶液。
  3. 一种免疫原性组合物，其包含免疫原和权利要求1所述的佐剂；
    优选地，所述免疫原选自蛋白，核酸，多糖，或其免疫原性部分；
    优选地，所述免疫原来源于病毒、细菌、真菌等病原体；
    优选地，所述免疫原是蛋白或其免疫原性片段；优选地，所述免疫原是来源于病毒、细菌、真菌等病原体的蛋白或其免疫原性片段；优选地，所述病毒选自，呼吸道 病毒(例如，流感病毒、副流感病毒、鼻病毒、冠状病毒、呼吸道合胞病毒)，肠道病毒(例如EV71病毒，轮状病毒)，水痘带状疱疹病毒(VZV)；优选地，所述免疫原是冠状病毒的蛋白或其免疫原性片段，例如冠状病毒刺突蛋白(S蛋白)或其免疫原性片段；
    优选地，所述冠状病毒选自正冠状病毒亚科α属病毒(例如229E和NL63)，正冠状病毒亚科β属病毒(例如OC43和HKU1)、严重急性呼吸综合征相关冠状病毒(SARS-CoV)、中东呼吸综合征相关冠状病毒(MERS-CoV)，和新型冠状病毒(SARS-CoV-2)；优选地，所述冠状病毒选自严重急性呼吸综合征相关冠状病毒(SARS-CoV)、中东呼吸综合征相关冠状病毒(MERS-CoV)和新型冠状病毒(SARS-CoV-2)，优选为新型冠状病毒(SARS-CoV-2)；
    优选地，所述免疫原为新型冠状病毒刺突蛋白(S蛋白)或其免疫原性片段，水痘带状疱疹病毒gE蛋白或其免疫原性片段，流感病毒HA蛋白或其免疫原性片段，或者，轮状病毒VP4蛋白或其免疫原性片段；
    优选地，所述免疫原性组合物还包含药学上可接受的辅料，例如赋形剂、防腐剂、抗菌剂、缓冲剂和/或额外的免疫佐剂；优选地，所述额外的免疫佐剂为选自铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子，或其任何组合；
    优选地，所述免疫原性组合物还包含第二免疫原；优选地，所述第二免疫原包括但不限于蛋白，核酸，多糖，或其免疫原性部分；
    优选地，所述免疫原性组合物为疫苗。
  4. 权利要求1所述的佐剂的用途，其用于制备免疫原性组合物，用做递送免疫原的载体，用做免疫原的免疫增强剂，用于增强免疫原的免疫原性，用于增强受试者对免疫原的免疫应答水平，或用于制备用于增强受试者对免疫原的免疫应答水平的制剂；
    优选地，所述免疫原如权利要求3所定义；
    优选地，所述免疫应答为细胞免疫应答和/或体液免疫应答；优选地，所述细胞免疫应答为T细胞免疫应答；优选地，所述T细胞免疫应答为免疫应答Th1和/或Th2免疫应答。
  5. 一种制备免疫原性组合物的方法，其包括：将权利要求1所述的佐剂与免疫原进行混合的步骤；
    优选地，所述免疫原如权利要求3所定义；
    优选地，所述方法还包括，添加药学上可接受的辅料的步骤；
    优选地，所述辅料是例如赋形剂、防腐剂、抗菌剂、缓冲剂和/或额外的免疫佐剂；优选地，所述额外的免疫佐剂为选自铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子，或其任何组合；
    优选地，所述方法还包含添加第二免疫原的步骤；优选地，所述第二免疫原包括但不限于蛋白，核酸，多糖，或其免疫原性部分等；
    优选地，所述免疫原性组合物为疫苗。
  6. 权利要求3所述的免疫原性组合物在制备用于预防和/或治疗受试者的疾病的药物中的用途，所述疾病是能够被所述免疫原诱发的免疫应答所预防或治疗的疾病；
    优选地，所述免疫原来源于冠状病毒(例如新型冠状病毒)，并且，所述疾病为冠状病毒(例如新型冠状病毒)感染或与冠状病毒(例如新型冠状病毒)感染相关的疾病；
    优选地，所述免疫原为冠状病毒(例如新型冠状病毒)的蛋白(例如刺突蛋白)或其免疫原性片段，并且，所述疾病为冠状病毒(例如新型冠状病毒)感染或与冠状病毒(例如新型冠状病毒)感染相关的疾病；
    优选地，所述免疫原为新型冠状病毒的结构蛋白(例如刺突蛋白(S蛋白))或其免疫原性片段；并且，所述疾病为新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如，新型冠状病毒性肺炎(COVID-19))；
    优选地，所述免疫原来源于水痘带状疱疹病毒，并且，所述疾病为水痘带状疱疹病毒感染或与水痘带状疱疹病毒感染相关的疾病；
    优选地，所述免疫原为水痘带状疱疹病毒的蛋白(例如gE蛋白)或其免疫原性片段，并且，所述疾病为水痘带状疱疹病毒感染或与水痘带状疱疹病毒感染相关的疾病；
    优选地，所述免疫原来源于流感病毒，并且，所述疾病为流感病毒感染或与流感病毒感染相关的疾病；
    优选地，所述免疫原为流感病毒的蛋白(例如HA蛋白)或其免疫原性片段，并 且，所述疾病为流感病毒感染或与流感病毒感染相关的疾病；
    优选地，所述免疫原来源于轮状病毒，并且，所述疾病为轮状病毒感染或与轮状病毒感染相关的疾病；
    优选地，所述免疫原为轮状病毒的蛋白(例如VP4蛋白)或其免疫原性片段，并且，所述疾病为轮状病毒感染或与轮状病毒感染相关的疾病；
    优选地，所述受试者是动物，例如禽类动物或哺乳动物；
    优选地，所述受试者是啮齿类动物、猪科动物、猫科动物、犬科动物、马科动物、灵长类动物或禽类动物；
    优选地，所述受试者是小鼠、水貂、豚鼠、叙利亚金黄地鼠或食蟹猴；
    优选地，所述受试者是人。
  7. 一种提高免疫原的免疫原性的方法，其包括将免疫原与权利要求1所述的佐剂混合的步骤；优选地，所述免疫原如权利要求3所定义。
  8. 一种刺激或增强受试者对免疫原的免疫应答的方法，其包括给予受试者有效量的含有所述免疫原和权利要求1所述的佐剂的免疫原性组合物的步骤；
    优选地，所述免疫原如权利要求3所定义；
    优选地，所述受试者是动物，例如禽类动物或哺乳动物；优选地，所述受试者是啮齿类动物、猪科动物、猫科动物、犬科动物、马科动物、灵长类动物或禽类动物；优选地，所述受试者是小鼠、水貂、豚鼠、叙利亚金黄地鼠或食蟹猴；优选地，所述受试者是人；
    优选地，所述免疫应答为细胞免疫应答和/或体液免疫应答；优选地，所述细胞免疫应答为T细胞免疫应答；优选地，所述T细胞免疫应答为免疫应答Th1和/或Th2免疫应答；
    优选地，所述免疫原性组合物是通过选自下列的途径进行给药的：肌内注射、皮下注射、皮内、鼻内、口服、透皮或静脉注射；
    优选地，所述免疫原性组合物通过肌内注射给药。
  9. 一种预防或治疗疾病的方法，其包括给予受试者有效量的权利要求3所述的免 疫原性组合物的步骤，所述疾病是能够被所述免疫原诱发的免疫应答所预防或治疗的疾病；
    优选地，所述疾病为冠状病毒(例如新型冠状病毒)感染或与冠状病毒(例如新型冠状病毒)感染相关的疾病，并且，所述免疫原来源于冠状病毒(例如新型冠状病毒)；例如，所述免疫原为冠状病毒(例如新型冠状病毒)的蛋白(例如刺突蛋白)或其免疫原性片段；
    优选地，所述疾病为新型冠状病毒感染或与新型冠状病毒感染相关的疾病，例如新型冠状病毒性肺炎(COVID-19)，并且，所述免疫原来源于新型冠状病毒；例如，所述免疫原为新型冠状病毒的结构蛋白(例如刺突蛋白(S蛋白))或其免疫原性片段；
    优选地，所述疾病为水痘带状疱疹病毒感染或与水痘带状疱疹病毒感染相关的疾病，并且，所述免疫原来源于水痘带状疱疹病毒；例如，所述免疫原为水痘带状疱疹病毒的蛋白(例如gE蛋白)或其免疫原性片段；
    优选地，所述疾病为流感病毒感染或与流感病毒感染相关的疾病，并且，所述免疫原来源于流感病毒；例如，所述免疫原为流感病毒的蛋白(例如HA蛋白)或其免疫原性片段；
    优选地，所述疾病为轮状病毒感染或与轮状病毒感染相关的疾病，并且，所述免疫原来源于轮状病毒；例如，所述免疫原为轮状病毒的蛋白(例如VP4蛋白)或其免疫原性片段；
    优选地，所述受试者是动物，例如禽类动物或哺乳动物；优选地，所述受试者是啮齿类动物、猪科动物、猫科动物、犬科动物、马科动物、灵长类动物或禽类动物；优选地，所述受试者是小鼠、水貂、豚鼠、叙利亚金黄地鼠或食蟹猴；优选地，所述受试者是人。
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