WO2022253191A1 - 一种基于tlr7激动剂偶联肽的新型冠状病毒纳米乳疫苗及其制备 - Google Patents

一种基于tlr7激动剂偶联肽的新型冠状病毒纳米乳疫苗及其制备 Download PDF

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WO2022253191A1
WO2022253191A1 PCT/CN2022/096041 CN2022096041W WO2022253191A1 WO 2022253191 A1 WO2022253191 A1 WO 2022253191A1 CN 2022096041 W CN2022096041 W CN 2022096041W WO 2022253191 A1 WO2022253191 A1 WO 2022253191A1
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vaccine
nanoemulsion
preparation
adjuvant
polypeptide
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French (fr)
Inventor
张馨欣
宫丽崑
任进
靳广毅
黄蔚
甘勇
孙建华
刘婷婷
龙益如
缪云秋
秦秋平
唐峰
余盼
郭琳苗
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Shanghai Institute of Materia Medica of CAS
Shenzhen University
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Shanghai Institute of Materia Medica of CAS
Shenzhen University
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Priority to US18/565,013 priority Critical patent/US20240261391A1/en
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Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to the preparation of a coronavirus SARS-CoV-2 nanoemulsion vaccine based on a TLR7 agonist-coupled peptide and its role in preventing coronavirus SARS-CoV-2 wild strains and mutations Application in strain infection.
  • Severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), is a positive-sense single-stranded RNA virus with an envelope, belonging to the family Coronaviridae Betacoronavirus. Syndrome-associated coronavirus species.
  • the host of the virus includes mammals and birds, and it is the pathogen that causes coronavirus disease 2019 (COVID-19).
  • COVID-19 coronavirus disease 2019
  • 192 countries and regions around the world have reported more than 132 million confirmed cases, of which more than 2.873 million have died and 75.221 million have been cured. The number is still rising rapidly. As the virus spreads rapidly, it is also mutating. In the urgent situation of fighting against COVID-19, peptide vaccines are expected to save time and cost for the development and development of vaccines.
  • viral epitope peptide vaccines are more suitable for coping with virus mutations; they can meet the requirements of rapid and efficient production and reduce the cost of vaccine production; there is no complete virus structure in the vaccine, which is safe higher.
  • modern vaccines composed of genetically engineered recombinant antigens or chemically synthesized polypeptides often have problems such as weak immunogenicity, so immune adjuvants are an important part of synthetic polypeptide vaccine preparations.
  • Adjuvants in vaccines can effectively enhance the body's immune response to antigens or change the type of immune response.
  • vaccine adjuvants include aluminum adjuvants, emulsions, liposomes, virosomes, etc.
  • Aluminum adjuvant is the most widely used immune adjuvant, which can be used as an "antigen library" to slowly release antigens, prolong the immune stimulation effect, and at the same time promote the response of macrophages at the injection site.
  • emulsion-type adjuvants have a long history of research and development, and are currently mainly used as reserve adjuvants for epidemics such as influenza, Leishmaniasis, and malaria, and have been used in more than 30 countries so far.
  • emulsion-type immune adjuvants mainly deliver antigens indirectly, which can enhance the phagocytosis and pinocytosis of antigen-presenting cells (APCs), stimulate monocytes, macrophages, Granulocytes secrete factors such as CCL2, CXCL8, CCL3, and CCL4, and promote the differentiation of monocytes into DCs.
  • APCs antigen-presenting cells
  • Emulsion adjuvant does not directly target DC to enhance antigen uptake, but mediates the upstream DC precursor cell recruitment and subsequent differentiation to play an adjuvant role. Recent studies have found that emulsion adjuvants can also drain antigens from the injection site to lymph nodes, stimulate reactions with immune cells, thereby greatly improving immune responses and producing higher levels of protective antibodies.
  • the oil-in-water nanoemulsion based on squalene can increase the stability of the antigen, which is beneficial to reduce the vaccine dose or antigen concentration, and can also enhance the cellular immune response.
  • Patent document CN201010247976.0 discloses a sub-microemulsion adjuvant containing squalene, polyether, and polyoxyethylene castor oil, but polyoxyethylene castor oil has greater toxicity in injection administration, and is likely to cause allergic reactions, Toxic renal injury, neurotoxicity, cardiovascular toxicity, etc.
  • Patent document CN200910193930.2 discloses an oil-in-water compound vaccine adjuvant, but the composition of propolis in it is complex and contains many unknown ingredients, and 70% ethanol is needed in the preparation process, which is easy to cause solvent residue, and is only suitable for animal Use the vaccine.
  • the object of the present invention is to provide a kind of coronavirus SARS-CoV-2 nanoemulsion vaccine based on TLR7 agonist coupling peptide and preparation method thereof.
  • the first aspect of the present invention provides a coronavirus SARS-CoV-2 nanoemulsion vaccine formulation, said vaccine formulation comprising:
  • a coronavirus SARS-CoV-2 vaccine polypeptide comprising an antigenic polypeptide and optionally a TLR7 agonist coupled to the antigenic polypeptide;
  • the adjuvant comprises the following components: squalene 1-15% (w/w), ⁇ -tocopherol 0-15% (w/w), emulsifier 0.1-10.0% ( w/w), and block copolymer 0.005-10% (w/w), based on the total weight of the formulation.
  • the adjuvant further includes an aqueous medium.
  • the adjuvant includes an oil phase part and a water phase part.
  • the oil phase part of the adjuvant contains: squalene 1-15% (w/w), ⁇ -tocopherol 0-15% (w/w), and emulsifier 0.1-10.0% % (w/w);
  • the water phase part of the adjuvant comprises: block copolymer 0.005-10% (w/w) and water phase medium, calculated according to the total weight of the preparation.
  • the adjuvant contains 1-5% (w/w) squalene, preferably 2-2.5% (w/w).
  • the adjuvant contains ⁇ -tocopherol 0-5% (w/w), preferably 2.5-4% (w/w).
  • the adjuvant contains emulsifier 1-5% (w/w), preferably 1-2% (w/w).
  • the adjuvant comprises 0.01-5% (w/w) of block copolymer, preferably 0.01-0.5% (w/w).
  • the squalene is derived from shark liver (especially thorn shark, beaked shark, southern black shark, etc.), olive oil, palm oil, wheat germ oil and/or yeast, etc. .
  • the emulsifier is selected from the group consisting of phospholipids, polysorbates, sucrose esters, citric acid fatty acid glycerides, fatty acid glycerides, fatty acid sorbitans, cyclodextrins, polyoxyethylene Fatty acid esters, polyoxyethylene fatty alcohol ethers, polyethylene glycol, chitin, chitosan, cholic acid and its salts, or combinations thereof.
  • the emulsifier is selected from the group consisting of phospholipids, polysorbates, or combinations thereof.
  • the emulsifier includes polysorbate 80.
  • the emulsifier includes a combination of polysorbate 80 and phospholipids.
  • the block copolymer is a medical block copolymer.
  • the number average molecular weight or weight average molecular weight of the block copolymer is 300-200,000, preferably 500-100,000.
  • the block copolymer is selected from the group consisting of methoxypolyethylene glycol-polycaprolactone, methoxypolyethylene glycol polylactic acid-glycolic acid, polylactic acid glycolic acid-poly Ethylene imine, polylactic acid-polyethylene glycol, polyphosphate diblock copolymer, polyoxyethylene polyoxypropylene ether block copolymer, polyethylene oxide-polypropylene oxide-polyethylene oxide three block copolymers, or combinations thereof.
  • the block copolymer is selected from the group consisting of: methoxy polyethylene glycol-polycaprolactone (mPEG-PCL, PEG: 350, 550, 750, 1000, 3400, 5000, 10000 , 20000; PCL: 2000, 5000), methoxypolyethylene glycol polylactic acid-glycolic acid (mPEG-PLGA, PEG: 1000, 2000, 3400, 5000, 10000, 20000; PLGA: 1000, 2000, 5000, 10000 , 15000, 20000, 40000), polylactic glycolic acid-polyethyleneimine (PLGA-PEI, PLGA: 1000, 2000, 5000, 10000, 15000, 20000, 40000; PEI: 600, 1800, 10000, 70000), poly Lactic acid-polyethylene glycol (PLA-PEG, PLA: 2000, 5000; PEG: 1000, 2000, 3400, 5000, 10000, 20000), polyphosphate diblock copolymer (PCL-b
  • the block copolymer includes methoxy polyethylene glycol-polycaprolactone copolymer.
  • the aqueous medium is selected from the group consisting of physiological saline, sterilized water, buffered saline, glucose solution, cyclodextrin solution, or combinations thereof.
  • the adjuvant further contains an isotonic regulator, and the content of the isotonic regulator is 0.1-8% (w/w).
  • the adjuvant also contains a preservative, and the content of the preservative does not exceed 0.1% (w/w).
  • the adjuvant has one or more of the following characteristics:
  • the adjuvant co-localizes with the vaccine polypeptide at the injection site, induces transient cytokine and chemokine responses, and increases the recruitment of innate immune cells from the bloodstream to the injection site;
  • the adjuvant enhances the recruitment of innate immune cells at the local draining lymph nodes.
  • the pharmaceutically acceptable carrier, excipient or diluent is a physiologically acceptable buffer such as phosphate or citrate.
  • the vaccine polypeptide has the structure of formula I or an oligomer comprising the structure of formula I:
  • Z is an antigenic polypeptide, and the antigenic polypeptide has at least one T cell epitope and/or at least one B cell epitope of the new coronavirus S protein; and, the antigenic polypeptide has an amino acid sequence derived from the RBM region of the S protein ;
  • U are each independently a TLR7 agonist
  • n 0 or a positive integer
  • J is a chemical bond or linker
  • the vaccine polypeptide is selected from the group consisting of LY54-101, P67-101, or a combination thereof.
  • the nanoemulsion vaccine preparation is prepared as an injection.
  • the pH of the nanoemulsion vaccine formulation is 6.0-8.0.
  • the droplet size in the nanoemulsion vaccine formulation is less than 220nm, preferably 80-180nm, more preferably 100-150nm.
  • the content of the vaccine polypeptide in the nanoemulsion vaccine preparation is 0.1-4 mg/mL.
  • the nanoemulsion vaccine formulation has one or more characteristics selected from the following group:
  • the stability is good, and the particle size does not change by more than 1% when placed at 4°C and 40°C for 1-2 months.
  • the particle diameters are all less than 0.22 ⁇ m, meeting the requirements for filtration sterilization.
  • a second aspect of the present invention provides a method for preparing the vaccine formulation described in the first aspect of the present invention, the method comprising the following steps:
  • the method includes the following steps:
  • the vaccine polypeptide is mixed with adjuvant oil.
  • the adjuvant oil phase is prepared by the following method: under the protection of inert gas, squalene 1-15% (w/w), ⁇ -tocopherol 0-15% (w/w ), emulsifier 0.1-10.0% (w/w), stirring and mixing until a uniform oil phase is formed, thereby obtaining an adjuvant oil phase, the percentage is based on the total weight of the preparation; preferably, the inert gas for nitrogen.
  • the aqueous adjuvant phase is prepared by the following method: adding 0.01-5% of a block copolymer to the aqueous phase medium, stirring and dissolving until a uniform aqueous phase is formed, thereby obtaining the aqueous adjuvant phase.
  • the method further includes step (iv), filtering, sterilizing and packaging the vaccine preparation obtained in step (iii).
  • the third aspect of the present invention provides a use of the coronavirus SARS-CoV-2 nanoemulsion vaccine preparation for the preparation of drugs for preventing coronavirus SARS-CoV-2 infection or related diseases.
  • the coronavirus SARS-CoV-2 includes wild strains and/or mutant strains.
  • the mutant strain is selected from the following group: B.1.1.7, B.1.617, B.1.351, P.1 and B.1.1.529.
  • the coronavirus SARS-CoV-2 related disease is selected from the group consisting of respiratory tract infection, pneumonia and its complications, or a combination thereof.
  • the coronavirus SARS-CoV-2 related disease is novel coronavirus pneumonia (COVID-19).
  • Figure 1 shows the chemical structure information of LY54-101.
  • Figure 2 shows the chemical structure information of P67-101.
  • Figure 3 shows the thermal stability analysis comparison of F2 nanoemulsion and AS03 nanoemulsion of LY54-101.
  • Figure 4 shows the results of fluorescence quantification of isolated tissue heart, liver, spleen, lung, and kidney after intramuscular injection of free conjugated peptide and conjugated peptide nanoemulsion preparations
  • Figure 5 shows the quantitative results of fluorescence imaging of inguinal lymph nodes after intramuscular injection of free conjugated peptides and conjugated peptide nanoemulsion preparations.
  • Figure 6 shows serum RBD-binding antibody levels after 35 days of immunization with conjugated peptide nanoemulsion formulations in cynomolgus monkeys.
  • Figure 7 shows serum RBD-binding antibody levels after 70 days of immunization with conjugated peptide nanoemulsion formulations in cynomolgus monkeys.
  • Figure 8 shows the level of serum neutralizing antibody in cynomolgus monkeys 35 days after the conjugated peptide nanoemulsion preparation was immunized.
  • Figure 9 shows the level of serum neutralizing antibody after 70 days of immunization with conjugated peptide nanoemulsion formulations in cynomolgus monkeys.
  • Figure 10 shows that the antiserum after the conjugated peptide nanoemulsion preparation immunized cynomolgus monkeys blocked the invasion of host cells by the pseudovirus of the wild strain of SARS-CoV-2.
  • Figure 11 shows that the antiserum after the conjugated peptide nanoemulsion preparation immunized cynomolgus monkeys blocked the pseudovirus of the British strain of the new coronavirus from invading host cells.
  • Figure 12 shows that antisera from cynomolgus monkeys immunized with conjugated peptide nanoemulsion formulations are resistant to various amino acid mutations on the RBD.
  • Figure 13 shows that the antiserum after the conjugated peptide nanoemulsion preparation immunized cynomolgus monkeys has the high neutralizing activity of blocking mutant B.1.1.529 pseudovirus from invading cells.
  • Figure 14 shows that the antiserum after the conjugated peptide nanoemulsion preparation immunized cynomolgus monkeys blocked the invasion of the host cells by the true virus of the new coronavirus.
  • Figure 15 shows viral load monitoring of nasal and throat swabs during challenge.
  • Figure 16 shows the viral load in lung tissue of cynomolgus monkeys after challenge.
  • a vaccine formulation suitable for TLR7 agonist-conjugated peptides After extensive and in-depth research and a large number of screenings, the inventors first developed a vaccine formulation suitable for TLR7 agonist-conjugated peptides, and used the formulation to prepare a novel TLR7-based agonist-conjugated peptide Nanoemulsion vaccine against coronavirus SARS-CoV-2.
  • a preferred vaccine formulation is determined by mixing TLR7 agonist-conjugated peptides with different formulations of adjuvants to prepare conjugated peptide-adjuvant compositions, and testing the physicochemical properties of each prepared composition.
  • nanoemulsion vaccine prepared by using the preferred formula of the present invention can induce a higher level of humoral immune response after entering the body, stimulate the body to produce a higher titer of neutralizing antibodies, and effectively block virus invasion Host cells have a nearly complete protective effect on the upper and lower respiratory tracts of the body.
  • the antiserum obtained by using the nanoemulsion vaccine of the present invention to immunize the body not only has a high level of neutralizing activity to the wild strain of coronavirus SARS-CoV-2, but also has a high level of neutralizing activity to mutant strains (such as B.1.1.7, B1 .1.529) also has a considerable neutralizing effect. Therefore, the nanoemulsion vaccine of the present invention can not only be used to prevent the infection of the wild strain of coronavirus SARS-CoV-2, but also has a good preventive effect on the infection of mutant strains.
  • the present invention provides a vaccine formulation containing a coronavirus SARS-CoV-2 vaccine polypeptide, the vaccine polypeptide comprising an antigenic polypeptide and a TLR7 agonist optionally coupled to the antigenic polypeptide.
  • the vaccine polypeptide has the structure of formula I or an oligomer comprising the structure of formula I:
  • Z is an antigen polypeptide, and the antigen polypeptide has at least one T cell epitope and/or at least one B cell epitope of the coronavirus SARS-CoV-2 S protein; and, the antigen polypeptide has an RBM region derived from the S protein amino acid sequence;
  • U are each independently a TLR7 agonist
  • n 0 or a positive integer
  • J is a chemical bond or linker
  • the vaccine polypeptide can stimulate primates and rodents to produce neutralizing antibodies that block the combination of RBD and ACE2.
  • the vaccine polypeptide can stimulate primates to produce cellular immunity and humoral immunity.
  • the primates include humans and non-human primates.
  • the length of the antigen polypeptide is 8-100 amino acids, preferably 10-80 amino acids.
  • the antigen polypeptide has an amino acid sequence derived from the RBD region of the coronavirus SARS-CoV-2 S protein.
  • the antigen polypeptide has an amino acid sequence derived from the RBM region of the RBD region.
  • the RBM region refers to amino acids 438-506 of the coronavirus SARS-CoV-2 RBD protein.
  • the antigenic polypeptide "has an amino acid sequence derived from the RBM region of the RBD protein” means that the amino acid sequence of the antigenic polypeptide has homology (or identity) with the RBM region, and the Homology ⁇ 80%, preferably ⁇ 85%, more preferably ⁇ 90%, most preferably ⁇ 95%.
  • the antigenic polypeptide is a synthetic or recombinant antigenic polypeptide.
  • X is a core fragment, wherein the sequence of the core fragment is selected from one or more of SEQ ID NO: 1-12 (see Table A);
  • X1 and X2 are independently none, 1, 2 or 3 amino acids, and the sum of the amino acid numbers of X1 and X2 is ⁇ 4, preferably 3, 2, 1, more preferably 0 or 1; In another preferred example, X1 and X2 are each independently none, K, C, G, L, and A.
  • the number n of molecules of the TLR agonist is 1, 2, 3, 4, 5 or 6; preferably 1, 2, 3 or 4.
  • the TLR7 agonist is a small molecule agonist.
  • the TLR7 agonists include: SZU-101:
  • the TLR7 agonist (such as SZU-101) is linked to the terminal amino group or side chain amino group of the antigenic polypeptide.
  • the TLR7 agonist (such as SZU-101) is linked to the sulfhydryl group of the antigenic polypeptide.
  • SZU-101 is connected to the amino group of the antigenic polypeptide to form the structure shown in S1:
  • the SZU-101 is connected to the sulfhydryl group of the antigenic polypeptide and forms the structure shown in S2:
  • the vaccine polypeptide is selected from the following group of conjugated peptides:
  • SZU-101 is connected with the N-terminal amino group of L and the side chain amino group of K in the amino acid sequence through the S1 structure;
  • SZU-101 is connected to the sulfhydryl group on C in the polypeptide through the S2 structure;
  • the squalene described in the present invention is a fully metabolizable lipid synthesized by the human body through the cholesterol synthesis pathway.
  • Squalene is mainly derived from the liver of sharks, especially sharks with high squalene content such as spiny sharks, beaked sharks, and southern black sharks. It can also be obtained from olive oil, palm oil, wheat germ oil and yeast extract.
  • the nanoemulsion adjuvant with squalene as the main oil phase can enhance the humoral immune response and cellular immune response, stimulate the maturation of plasma cells, and allow the body to produce sufficient antibodies to fight viruses. In human phase I to III clinical trials, it has been proved that there is no obvious toxic and side effect, and it is safe and reliable.
  • the ⁇ -tocopherol of the present invention acts as an immunostimulant in the emulsion.
  • ⁇ -tocopherol is one of the eight isoforms of vitamin E, the most widely distributed in nature.
  • the addition of ⁇ -tocopherol to the emulsion can increase the uptake of antigen by monocytes, enhance the production of cytokines, and generate a higher antibody response.
  • the ⁇ -tocopherol used in the present invention is mainly obtained through synthetic routes.
  • the emulsifier described in the present invention can be selected from polysorbate 80 and other polysorbates, phospholipids, sucrose esters, citric acid fatty acid glycerides, fatty acid glycerides, fatty acid sorbitan, cyclodextrin, polyoxygen One or more of ethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, polyethylene glycol, chitin, chitosan, cholic acid and its salts, etc.
  • the block copolymer of the present invention is a biodegradable polymer, which can spontaneously form spherical nanoparticles with a core-shell structure with the hydrophilic group facing outward and the hydrophobic group facing inward in water, which can improve Drug entrapment efficiency and biocompatibility, and promote lymph node drainage.
  • the block copolymer is a medical block copolymer.
  • the number average molecular weight or weight average molecular weight of the block copolymer is 300-200,000, preferably 500-100,000.
  • the block copolymer of the present invention comprises methoxy polyethylene glycol-polycaprolactone (mPEG-PCL, PEG:350,550,750,1000,3400,5000,10000,20000; PLA:2000,5000 ), methoxypolyethylene glycol polylactic-glycolic acid (mPEG-PLGA, PEG: 1000, 2000, 3400, 5000, 10000, 20000; PLGA: 1000, 2000, 5000, 10000, 15000, 20000, 40000), Polylactic acid glycolic acid-polyethyleneimine (PLGA-PEI, PLGA: 1000, 2000, 5000, 10000, 15000, 20000, 40000; PEI: 600, 1800, 10000, 70000), polylactic acid-polyethylene glycol (PLA -PEG, PLA: 2000, 5000; PEG: 1000, 2000, 3400, 5000, 10000, 20000), polyphosphate diblock copolymer (PCL-b-PHEP, PEG: 1000, 2000, 3400,
  • Nanoemulsion vaccine of the present invention is provided.
  • the invention provides a coronavirus SARS-CoV-2 nanoemulsion vaccine preparation, said vaccine preparation comprising:
  • a coronavirus SARS-CoV-2 vaccine polypeptide comprising an antigenic polypeptide and optionally a TLR7 agonist coupled to the antigenic polypeptide;
  • the adjuvant comprises the following components: squalene 1-15% (w/w), ⁇ -tocopherol 0-15% (w/w), emulsifier 0.1-10.0% (w/w) , and block copolymer 0.005-10% (w/w), based on the total weight of the formulation.
  • the adjuvant also includes an aqueous medium.
  • the adjuvant used in the vaccine formulation of the present invention comprises an oil phase part and an aqueous phase part.
  • the oil phase part of the adjuvant comprises: squalene 1-15% (w/w), ⁇ -tocopherol 0-15% (w/w), and emulsifier 0.1-10.0% (w/w );
  • the water phase part of the adjuvant comprises: block copolymer 0.005-10% (w/w) and water phase medium, calculated according to the total weight of the preparation.
  • the novel coronavirus-conjugated peptide vaccine of the present invention uses nanoemulsion as an adjuvant, which co-localizes with the antigen at the injection site, induces transient cytokine and chemokine responses, and increases innate immune cells from the bloodstream to Recruitment at the injection site.
  • monocytes are activated to become antigen-presenting cells, loaded with antigens, and migrate to draining lymph nodes.
  • the adjuvant enhances the recruitment of innate immune cells at the local draining lymph nodes.
  • Antigen-presenting cells activate naive CD4+ T cells, and activated CD4+ T cells interact with antigen-specific B cells to induce a large number of memory B cells and antibody-secreting plasma cells.
  • the present invention also provides a method for preparing the vaccine preparation described in the first aspect of the present invention, characterized in that the method comprises the following steps:
  • the method includes the following steps:
  • the vaccine polypeptide is mixed with adjuvant oil.
  • the adjuvant oil phase is prepared by the following method: under the protection of inert gas, squalene 1-15% (w/w), ⁇ -tocopherol 0-15% (w/w ), emulsifier 0.1-10.0% (w/w), stirring and mixing until a uniform oil phase is formed, thereby obtaining an adjuvant oil phase, the percentage is based on the total weight of the preparation; preferably, the inert gas for nitrogen.
  • the aqueous adjuvant phase is prepared by the following method: adding 0.01-5% block copolymer to the aqueous medium, stirring and dissolving until a uniform aqueous phase is formed, thereby obtaining the aqueous adjuvant phase.
  • the method further includes step (iv), filtering, sterilizing and packaging the vaccine preparation obtained in step (iii).
  • coronavirus SARS-CoV-2 nanoemulsion vaccine preparation of the present invention can be used to prepare medicines for preventing coronavirus SARS-CoV-2 infection or related diseases.
  • the coronavirus SARS-CoV-2 infection includes infections caused by wild strains and/or mutant strains.
  • the coronavirus SARS-CoV-2 related diseases include but are not limited to respiratory tract infection, pneumonia and its complications, such as novel coronavirus pneumonia (COVID-19).
  • the vaccine formulations of the present invention can be administered as injectables, such as liquid solutions or suspensions.
  • the vaccine formulations of the present invention can be made into unitary or multiple dosage forms.
  • Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients.
  • the formulated vaccine formulations can be administered by conventional routes including, but not limited to, intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, oral, or topical administration.
  • a safe and effective amount of a vaccine polypeptide or peptide collection according to the invention is administered to a human being, wherein the safe and effective amount is usually at least about 1 microgram peptide/kg body weight, and in most cases no more than About 8 mg peptide/kg body weight, preferably the dose is about 1 microgram to 1 mg peptide/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • novel coronavirus-conjugated peptide vaccine provided by the present invention uses nanoemulsion as an adjuvant, which can enhance the immune response and antibodies produced by polypeptide antigens by enhancing the recruitment of immune cells in local draining lymph nodes.
  • the raw materials used in the nanoemulsion adjuvant of the present invention are highly safe, and will not cause potential safety hazards while exerting the auxiliary immune function.
  • nanoemulsion adjuvant of the present invention and the corresponding nanoemulsion vaccine preparation have good stability, which can greatly simplify the storage and transportation conditions of the vaccine preparation.
  • the vaccine formula provided has good compatibility with the conjugated peptide, and also has good compatibility in manufacturing, and can be applied to the combination of one or more different conjugated peptides.
  • Example 1 Preparation of nanoemulsion vaccine with TLR7 agonist-coupled peptide LY54-101 as antigen
  • LY54-101 As shown in Table 1 below, a nanoemulsion vaccine using TLR7 agonist-coupled peptide LY54-101 (2 mg/mL) as antigen was prepared.
  • the structure of LY54-101 is shown in Figure 1. The preparation method is as follows:
  • nanoemulsion After the nanoemulsion is filtered through a filter membrane of 0.22 ⁇ m, it is filled, filled with nitrogen, and sealed.
  • Example 2 Preparation of nanoemulsion vaccines with different doses and types of conjugated peptides as antigens
  • nanoemulsion After the nanoemulsion is filtered through a filter membrane of 0.22 ⁇ m, it is filled, filled with nitrogen, and sealed.
  • the optimal formulation can adapt to different types and different dosages of coupled peptides, and the particle size of the prepared nanoemulsion droplets is less than 220nm, which meets the requirements of filtration sterilization.
  • the structure of the coupled peptide P67-101 is shown in FIG. 2 .
  • Embodiment 3 Stability determination of nanoemulsion vaccine
  • Single conjugated peptide preparation group AS03 nanoemulsion and F2 nanovaccine emulsion containing LY54-101 2mg/mL;
  • the formulations were stored in stability boxes at 4°C and 40°C, respectively, and samples were taken on days 0, 7, 14, 30, and 60, and the particle size was measured with a Malvern Nano-ZS90 dynamic light scattering particle size potentiometer.
  • the nanoemulsion vaccine preparation of the present invention is placed at 4°C and 40°C for 60 days, there is no discoloration and stratification, and the particle size is basically unchanged, indicating that the nanoemulsion vaccine preparation of the present invention is stable in properties.
  • the active ingredients of the nanoemulsion vaccine are identified by high performance liquid chromatography (HPLC). Both the F2 nanoemulsion vaccine and the AS03 nanoemulsion vaccine of the present invention can keep the entrapped polypeptide components stable at 4°C. In addition, even if the F2 nanoemulsion vaccine of the present invention is placed at 40°C for one month, its active ingredient LY54-101 remains stable without degradation, while the active ingredient LY54-101 in the nanoemulsion vaccine using AS03 as a control Degradation has occurred (Figure 3). This shows that the nanoemulsion of the present invention is more suitable for coupling peptides with TLR7 agonists, and can protect the coupled peptides to maintain stable properties under high temperature conditions.
  • Embodiment 4 the lymph node drainage effect of nanoemulsion vaccine
  • the preparations all use LY54-101 linked to Cy5 as the antigen, and the preparation method is the same as that described in Example 1.
  • Titermax group adopts water-in-oil type adjuvant.
  • the rats were anesthetized with isoflurane, and the left leg of the rat was depilated, and placed in an in vivo imager for imaging before administration (0h).
  • 100 ⁇ L of PBS, free conjugated peptide, AS03, F1 and F2 preparations were injected intramuscularly into the left inner thigh.
  • the rats were dissected, and the heart, liver, spleen, lung, kidney and left inguinal lymph node were taken out for tissue fluorescence imaging in vitro, using an excitation wavelength of 640nm and an emission wavelength of 680nm.
  • Fig. 4 The results showed (Fig. 4): the free conjugated peptide group and the Titermax group were mainly distributed in the liver and kidney, indicating that they were mainly metabolized by the liver and kidney.
  • the target emulsion groups AS03, F1, and F2 had higher fluorescence distribution in the kidney, indicating that the vaccine-conjugated peptide emulsion was mainly metabolized by the kidney.
  • Cynomolgus monkeys were grouped into intramuscular injections and immunized on days 0, 14, and 28.
  • the scheme is as follows:
  • Group 1 blank control group, simply given the same volume of normal saline.
  • RBD-binding antibodies were determined using a standard bridging ELISA method.
  • the specific detection method is as follows: 1 ⁇ g/mL RBD-His was coated on a 96-well ELISA plate, and left overnight at 4°C. After washing three times with PBST buffer (0.05% Tween-20 in PBS), the ELISA plate was blocked with 200 ⁇ L of 1% BSA solution at 37° C. for 1 hour. After washing, 100 ⁇ L of serum serial dilutions were added to the 96-well plate and incubated at 37°C for 1 hour.
  • the neutralizing antibody is detected by a competitive ELISA method, and the specific determination method is as follows: samples and controls are pre-incubated with HRP-RBD, so that the neutralizing antibody to be tested is combined with HRP-RBD. The mixture was then added to capture plates pre-coated with hACE2 protein. Unbound HRP-RBD as well as HRP-RBD bound to non-neutralizing antibodies will be captured on the plate, while neutralizing antibody/HRP-RBD complexes in the sample remain in the supernatant and are removed during washing. remove. After the washing step, TMB solution was added to change the color to blue. The reaction was quenched and the color changed to yellow by adding stop solution. This final solution can be read at 450 nm in a microplate reader. The absorbance of a sample is inversely proportional to the titer of anti-SARS-CoV-2 neutralizing antibody.
  • the LY54-101+P67-101 (2:1, 2mg+1mg, F2) group showed the highest RBD-binding antibody titer, up to 1:72900, indicating that the best conjugated peptide combination of the present invention is LY54 A 2:1 combination of -101 and P67-101.
  • the nanoemulsion developed by the present invention has an immune effect better than that of AS03 nanoemulsion when applied to the preparation development of conjugated peptide vaccines.
  • the conjugated peptide nanoemulsion vaccine of the present invention induces a high level of humoral immune response in cynomolgus monkeys, and the serum neutralizing antibody level is extremely high and persistent after immunization of cynomolgus monkeys, suggesting that it has the effect of blocking virus invasion.
  • Embodiment 6 Nanoemulsion vaccine blocks virus invasion
  • pseudovirus and true virus test systems were used to evaluate the pseudovirus blocking neutralization activity of cynomolgus monkey antiserum.
  • pseudovirus neutralization assay 100 ⁇ L of serum samples at different dilutions were mixed with 50 ⁇ L of the supernatant containing the SARS-CoV-2 pseudovirus. The mixture was incubated at 37°C for 1 hour. Then 100 ⁇ L of Huh-7/ACE2 cells were added to the mixture of pseudovirus and serum samples and incubated at 37 °C for another 24 h. Then, remove the supernatant and add 100 ⁇ L of luciferase assay solution to each well. After 2 min of incubation, luciferase activity was measured using a microplate luminometer.
  • the wild strain of SARS-CoV-2 virus was propagated in VERO E6 cells. Serum samples were heat inactivated at 56°C for 30 min; serial 2-fold dilutions starting at 1:4 were then mixed with an equal volume of virus solution containing 50% tissue culture. The serum-virus mixture was incubated for 1 hour at 37°C in a humidified environment with 5% CO2. After incubation, 100 ⁇ L of each dilution of the mixture was added in duplicate to cell plates containing hemi-confluent VERO E6 monolayers. The plates were incubated at 37°C for 4 days. After 4 days of culture, the cytopathic effect (CPE) of each well was recorded under a microscope. The highest serum dilution that can protect more than 50% of the cells from CPE was taken as the neutralization titer.
  • CPE cytopathic effect
  • the nanoemulsion vaccine of the present invention can effectively protect cells from infection of the mutant strain B.1.1.7, with a titer as high as 1:512 (FIG. 11, mutant strain B.1.1.7).
  • a titer as high as 1:512 (FIG. 11, mutant strain B.1.1.7).
  • the present invention adopts the RBD-binding antibody detection method based on Example 5, and replaces wild-type RBD with various single amino acid mutant RBD proteins of K417N, N439K, L452R, Y453F, S477N, E484K, E484Q and N501Y for detection.
  • the antiserum of cynomolgus monkeys has a three-fold reduction in the binding ability to the RBD of the E484K mutation (Fig. E484K and N501Y) and P.1 (comprising E484K and N501Y) provided good immune protection.
  • the cynomolgus monkey antiserum (62 days after the third immunization) still had significant neutralizing activity against the Omicron mutant strain, with a neutralizing titer of 1:346 (Fig. 13), suggesting that the conjugated peptide nanoemulsion vaccine can protect the body against the infection of the Omicron mutant strain.
  • the conjugated peptide nanoemulsion vaccine of the present invention can produce a high level of protective neutralizing antibodies after immunization of cynomolgus monkeys, and can prevent the infection of the new coronavirus wild strain and mutant strains.
  • Example 7 The challenge test evaluates the protective effect of vaccine preparations in vivo
  • the present invention uses the highest viral load (1 ⁇ 10 7 TCID 50 ) at home and abroad to attack the virus (commonly used 1 ⁇ 10 6 TCID 50 ), followed by nasal swabs on the first day, the third day, the fifth day and the seventh day And throat swabs were used to monitor the viral load of the upper respiratory tract. On the seventh day, the cynomolgus monkeys were euthanized, and lung tissues were taken to detect the viral load of each lung lobe. Detection of viral load was performed by qRT-PCR.
  • the viral load monitoring results of nasal swabs and throat swabs showed that the upper respiratory tract of cynomolgus monkeys in the saline group showed high viral load, while the conjugated peptide nanoemulsion vaccine of the present invention immunized Cynomolgus monkeys only detected low levels of viral load in nasal swabs on the first day post-challenge, after which no viral RNA was detected in either the nose or pharynx (Figure 15).
  • the detection results of the viral load in the lung tissue showed that viral RNA was detected in both the left and right lungs of the cynomolgus monkeys immunized with the conjugated peptide nanoemulsion vaccine of the present invention, while the cynomolgus monkeys in the normal saline group had viral RNA detected in the left and right lungs. There were high levels of viral load in each lobe of the lung (Fig. 16).
  • the conjugated peptide nanoemulsion vaccine of the present invention has an excellent protective effect, and at a very high challenge dose, it can still prevent cynomolgus monkeys from infecting the wild strain of SARS-CoV-2. Has a nearly complete protective effect.
  • the present invention develops a safe and effective new adjuvant for human use, which can assist the coupling peptide to achieve the best immune effect in vivo.
  • the new nanoemulsion developed by the present invention shows better properties than the clinically used AS03 nanoemulsion. For example, the thermal stability of the prepared coupled peptide nanoemulsion preparation is better, and it can withstand high temperatures of 40°C; another example is the development of the present invention.
  • the new nanoemulsion has a better immune effect, can induce a higher level of neutralizing antibodies against RBD, and can provide high-efficiency protection against SARS-CoV-2 wild strains and mutant strains, which may be related to its good lymphatic drainage ability .
  • the TLR7 agonist-coupled peptide nanoemulsion COVID-19 vaccine of the present invention has a prominent effect on preventing the infection of SARS-CoV-2 wild strains and mutant strains, can prevent SARS-CoV-2 infection and has a nearly complete effect on the upper and lower respiratory tracts of the body. Protective effect, with clinical value.

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Abstract

本发明涉及一种以TLR7激动剂偶联肽为抗原，乳剂为佐剂的新型冠状病毒疫苗。其中，所述偶联肽的抗原多肽是衍生自冠状病毒SARS-CoV-2的S蛋白的多肽，所述佐剂为含有角鲨烯的水包油型纳米乳剂。本发明的偶联肽纳米乳疫苗制剂具有热稳定性，在食蟹猴中可以引发高水平的保护性体液免疫反应，食蟹猴免疫后抗血清的中和抗体滴度高，可阻断野毒株和突变株新型冠状病毒入侵。在食蟹猴SARS-CoV-2攻毒试验中，本发明的疫苗对食蟹猴上下呼吸道均具有接近完全的保护作用。本发明的纳米乳疫苗制备快速简便，可在短期内实现规模生产用于应对新型冠状病毒突发疫情。

Description

一种基于TLR7激动剂偶联肽的新型冠状病毒纳米乳疫苗及其制备 技术领域
本发明属于生物医药技术领域，具体地，涉及一种基于TLR7激动剂偶联肽的冠状病毒SARS-CoV-2纳米乳疫苗的制备及其在预防冠状病毒SARS-CoV-2野毒株和突变株感染中的应用。
背景技术
严重急性呼吸系统综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)，是一种具有包膜的正链单股RNA病毒，属于冠状病毒科乙型冠状病毒属严重急性呼吸道综合征相关冠状病毒种。该病毒的宿主包括哺乳动物和禽类动物，是导致2019冠状病毒病(COVID-19)的病原体。截至2021年4月7日，全球已有192个国家和地区累计报告逾1.32亿名确诊病例，其中逾287.3万人死亡、7,522.1万人治愈，目前数字仍在迅速攀升中。病毒迅速传播的同时也在发生变异。在抗击COVID-19的紧迫形势下,多肽疫苗有望为疫苗的开发和研制节省时间和成本。
与减毒疫苗和灭活疫苗相比，病毒抗原表位多肽疫苗更适用于应对病毒的变异；能达到快速、高效生产的要求，降低疫苗制作的成本；疫苗中无完整的病毒结构，安全性较高。但是与传统的灭活或活体疫苗相比，由基因工程重组抗原或化学合成多肽组成的现代疫苗往往存在免疫原性弱等问题，因此免疫佐剂是合成多肽疫苗制剂中的重要组成部分。
疫苗中的佐剂可有效增强机体对抗原的免疫应答或改变免疫反应的类型。目前已研发和批准上市了多种疫苗佐剂包括铝佐剂、乳剂、脂质体、病毒体等。铝佐剂是应用最广泛的免疫佐剂，可作为“抗原库”，缓慢释放抗原，延长免疫刺激效果，同时促进注射部位巨噬细胞的应答。另外，乳剂型佐剂研发历史悠久，目前主要用于流感、利仕曼病、疟疾等疫情的储备佐剂，迄今已应于用30多个国家。与铝佐剂的作用机制不同乳剂型免疫佐剂主要以间接方式递送抗原，它可增强抗原递呈细胞(APC)的对抗原物质的吞噬及胞饮作用，刺激单核细胞、巨噬细胞、粒细胞分泌CCL2、CXCL8、CCL3和CCL4等因子，并促进单核细胞向DC分化。乳剂佐剂不是直接以DC为靶标增强抗原摄取，而是介导其上游的DC前体细胞募集及其后续的分化来发挥佐剂作用。近来的研究发现乳剂佐剂还能将抗原从注射位点引流到淋巴结，与免疫细胞激发反应从而极大地提高免疫应答，产生更高水平的保护性抗体。
前期研究显示铝佐剂与高纯度的小分子蛋白抗原共同使用时存在激发细胞免疫应答能力不足的问题，不适用于多肽疫苗的开发。而基于角鲨烯的水包油型纳 米乳剂可增加抗原的稳定性，有利于降低疫苗剂量或抗原浓度，还可以增强细胞免疫应答。
已批准上市的角鲨烯的水包油型纳米乳剂，如MF-59 TM、AS03，处方设计不利于COVID-19多肽抗原发挥最佳的免疫应答效应。专利文献CN201010247976.0公开了一种含角鲨烯、聚醚、聚氧乙烯蓖麻油的亚微乳佐剂，但聚氧乙烯蓖麻油在注射给药中具有较大毒性，易引起变态反应、中毒性肾损伤、神经毒性、心脏血管毒性等。专利文献CN200910193930.2公开了一种水包油型复方疫苗佐剂，但其中的蜂胶组成复杂，含多种不明成分，且制备过程需要用到70％乙醇，易造成溶剂残留，仅适用于兽用疫苗。
因此，本领域迫切需要研发更为安全有效的人用新型佐剂，配合TLR7激动剂偶联肽发挥作用，并且需要制备快速简便，可在短期内实现大规模生产用于应对突发疫情。
发明内容
本发明的目的在于提供一种基于TLR7激动剂偶联肽的冠状病毒SARS-CoV-2纳米乳疫苗及其制备方法。
本发明的第一方面，提供了一种冠状病毒SARS-CoV-2纳米乳疫苗制剂，所述疫苗制剂包含：
(a)冠状病毒SARS-CoV-2疫苗多肽，所述疫苗多肽包括抗原多肽以及任选地与所述抗原多肽偶联的TLR7激动剂；
(b)佐剂，所述佐剂是基于角鲨烯的水包油型纳米乳剂；和
(c)药学上可接受的载体、赋形剂或稀释剂。
在另一优选例中，所述佐剂包含以下组分：角鲨烯1-15％(w/w)，α-生育酚0-15％(w/w)，乳化剂0.1-10.0％(w/w)，和嵌段共聚物0.005-10％(w/w)，按所述制剂的总重量计。
在另一优选例中，所述佐剂还包含水相介质。
在另一优选例中，所述佐剂包含油相部分和水相部分。
在另一优选例中，所述佐剂的油相部分包含：角鲨烯1-15％(w/w)，α-生育酚0-15％(w/w)，和乳化剂0.1-10.0％(w/w)；所述佐剂的水相部分包含：嵌段共聚物0.005-10％(w/w)和水相介质，按所述制剂的总重量计。
在另一优选例中，所述佐剂包含角鲨烯1-5％(w/w)，较佳地2-2.5％(w/w)。
在另一优选例中，所述佐剂包含α-生育酚0-5％(w/w)，较佳地2.5-4％(w/w)。
在另一优选例中，所述佐剂包含乳化剂1-5％(w/w)，较佳地1-2％(w/w)。
在另一优选例中，所述佐剂包含嵌段共聚物0.01-5％(w/w)，较佳地 0.01-0.5％(w/w)。
在另一优选例中，所述角鲨烯来源于鲨鱼肝脏(特别是同齿刺鲨鱼、喙吻田氏鲨、南方乌鲨等)、橄榄油、棕榈油、麦胚油和/或酵母等。
在另一优选例中，所述乳化剂选自下组：磷脂、聚山梨酯类、蔗糖酯、柠檬酸脂肪酸甘油酯类、脂肪酸甘油脂类、脂肪酸山梨坦类、环糊精、聚氧乙烯脂肪酸酯类、聚氧乙烯脂肪醇醚类、聚乙二醇、甲壳质、甲壳胺、胆酸及其盐类、或其组合。
在另一优选例中，所述乳化剂选自下组：磷脂、聚山梨酯、或其组合。
在另一优选例中，所述乳化剂包括聚山梨酯80。
在另一优选例中，所述乳化剂包括聚山梨酯80和磷脂的组合。
在另一优选例中，所述嵌段共聚物为医用嵌段共聚物。
在另一优选例中，所述嵌段共聚物的数均分子量或重均分子量为300-200000，较佳地500-100000。
在另一优选例中，所述嵌段共聚物选自下组：甲氧基聚乙二醇-聚己内酯、甲氧基聚乙二醇聚乳酸-羟基乙酸、聚乳酸羟基乙酸-聚乙烯亚胺、聚乳酸-聚乙二醇、聚磷酸酯两嵌段共聚物、聚氧乙烯聚氧丙烯醚嵌段共聚物、聚环氧乙烷-聚环氧丙烷-聚环氧乙烷三嵌段共聚物、或其组合。
在另一优选例中，所述嵌段共聚物选自下组：甲氧基聚乙二醇-聚己内酯(mPEG-PCL，PEG:350、550、750、1000、3400、5000、10000、20000；PCL：2000、5000)，甲氧基聚乙二醇聚乳酸-羟基乙酸(mPEG-PLGA，PEG:1000、2000、3400、5000、10000、20000；PLGA：1000、2000、5000、10000、15000、20000、40000)，聚乳酸羟基乙酸-聚乙烯亚胺(PLGA-PEI，PLGA：1000、2000、5000、10000、15000、20000、40000；PEI:600、1800、10000、70000)，聚乳酸-聚乙二醇(PLA-PEG，PLA：2000、5000；PEG:1000、2000、3400、5000、10000、20000)，聚磷酸酯两嵌段共聚物(PCL-b-PHEP，PEG:1000、2000、3400、5000、10000、20000)，聚氧乙烯聚氧丙烯醚嵌段共聚物(泊洛沙姆188等)，聚环氧乙烷-聚环氧丙烷-聚环氧乙烷三嵌段共聚物(PEO-PPO-PEO)，或其组合。
在另一优选例中，所述嵌段共聚物包括甲氧基聚乙二醇-聚己内酯共聚物。
在另一优选例中，所述水相介质选自下组：生理盐水、灭菌水、缓冲盐水、葡萄糖溶液、环糊精溶液、或其组合。
在另一优选例中，所述佐剂中还含有等渗调节剂，所述等渗调节剂的含量为0.1-8％(w/w)。
在另一优选例中，所述佐剂中还含有防腐剂，所述防腐剂的含量不超过0.1％(w/w)。
在另一优选例中，所述佐剂具有以下的一种或多种特征：
(1)所述佐剂在注射部位与疫苗多肽共定位，诱导短暂的细胞因子和趋化因子反应，并增加先天免疫细胞从血流中向注射部位的募集；
(2)所述佐剂增强先天免疫细胞在局部引流淋巴结处的募集。
在另一优选例中，所述药学上可接受的载体、赋形剂或稀释剂为磷酸盐或柠檬酸盐等生理上可接受的缓冲剂。
在另一优选例中，所述的疫苗多肽具有式I结构或包含式I结构的寡聚体：
Z-(J-U)n         (I)
式中，
Z为抗原多肽，所述抗原多肽具有新型冠状病毒S蛋白的至少一个T细胞表位和/或至少一个B细胞表位；并且，所述的抗原多肽具有衍生自S蛋白的RBM区域的氨基酸序列；
U各自独立地为TLR7激动剂；
n为0或正整数；
J为化学键或连接子。
在另一优选例中，所述疫苗多肽选自下组：LY54-101、P67-101、或其组合。
在另一优选例中，所述纳米乳疫苗制剂被制备为注射剂。
在另一优选例中，所述纳米乳疫苗制剂的pH为6.0-8.0。
在另一优选例中，所述纳米乳疫苗制剂中的液滴粒径小于220nm，较佳地，80-180nm，更佳地，100-150nm。
在另一优选例中，所述纳米乳疫苗制剂中疫苗多肽的含量为0.1-4mg/mL。
在另一优选例中，所述纳米乳疫苗制剂具有选自下组的一个或多个特征：
(1)稳定性好，在4℃与40℃下放置1-2个月，粒径变化不超过1％。
(2)粒径均小于0.22μm，满足过滤除菌要求。
本发明的第二方面，提供了一种制备本发明第一方面所述的疫苗制剂的方法，所述方法包括以下步骤：
(S1)提供一疫苗多肽；
(S2)将所述疫苗多肽与佐剂和药学上可接受的载体、赋形剂或稀释剂混合，从而制得本发明所述的疫苗制剂。
具体地，所述方法包括以下步骤：
(i)提供一疫苗多肽；
(ii)将疫苗多肽与佐剂水相和/或佐剂油相充分混合；
(iii)将佐剂油相与佐剂水相混合，通过剪切搅拌或高压均质形成水包油的乳剂，从而获得所述疫苗制剂。
在另一优选例中，将疫苗多肽与佐剂油相混合。
在另一优选例中，所述佐剂油相通过以下方法制备：在惰性气体保护下，将角鲨烯1-15％(w/w)、α-生育酚0-15％(w/w)、乳化剂0.1-10.0％(w/w)，搅拌混合，直至形成均一的油相，从而获得佐剂油相，所述百分比按所述制剂的总重量计；优选地，所述惰性气体为氮气。
在另一优选例中，所述佐剂水相通过以下方法制备：在水相介质中，加入嵌段共聚物0.01-5％，搅拌溶解直至形成均一的水相，从而获得佐剂水相。
在另一优选例中，所述方法还包括步骤(iv)，将步骤(iii)所得的疫苗制剂进行过滤、灭菌和包装。
本发明的第三方面，提供了一种所述的冠状病毒SARS-CoV-2纳米乳疫苗制剂的用途，用于制备预防冠状病毒SARS-CoV-2感染或其相关疾病的药物。
在另一优选例中，所述冠状病毒SARS-CoV-2包括野毒株和/或突变株。
在另一优选例中，所述的突变株选自下组：B.1.1.7、B.1.617、B.1.351、P.1和B.1.1.529。
在另一优选例中，所述的冠状病毒SARS-CoV-2相关疾病选自下组：呼吸道感染、肺炎及其并发症、或其组合。
在另一优选例中，所述的冠状病毒SARS-CoV-2相关疾病为新型冠状病毒肺炎(COVID-19)。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了LY54-101的化学结构信息。
图2显示了P67-101的化学结构信息。
图3显示了LY54-101的F2纳米乳和AS03纳米乳的热稳定性分析对比。
图4显示了游离偶联肽和偶联肽纳米乳制剂在肌肉注射8h后，离体组织心、肝、脾、肺、肾的荧光定量结果
图5显示了游离偶联肽和偶联肽纳米乳制剂在肌肉注射8h后，腹股沟淋巴结的的荧光成像定量结果。
图6显示了偶联肽纳米乳制剂免疫食蟹猴35后的血清RBD结合抗体水平。
图7显示了偶联肽纳米乳制剂免疫食蟹猴70后的血清RBD结合抗体水平。
图8显示了偶联肽纳米乳制剂免疫食蟹猴35后的血清中和抗体水平。
图9显示了偶联肽纳米乳制剂免疫食蟹猴70后的血清中和抗体水平。
图10显示了偶联肽纳米乳制剂免疫食蟹猴后的抗血清阻断新冠病毒野毒株假病毒入侵宿主细胞。
图11显示了偶联肽纳米乳制剂免疫食蟹猴后的抗血清阻断新冠病毒英国株假病毒入侵宿主细胞。
图12显示了偶联肽纳米乳制剂免疫食蟹猴后的抗血清耐受多种RBD上氨基酸的突变。
图13显示了偶联肽纳米乳制剂免疫食蟹猴后的抗血清具有阻断突变株B.1.1.529假病毒侵入细胞的高中和活性。
图14显示了偶联肽纳米乳制剂免疫食蟹猴后的抗血清阻断新冠病毒真病毒入侵宿主细胞。
图15显示了攻毒过程中鼻拭子和咽拭子的病毒载量监测。
图16显示了攻毒后食蟹猴肺组织病毒载量。
具体实施方式
本发明人经过广泛而深入的研究和大量的筛选，首次研发出一种适合TLR7激动剂偶联肽的疫苗制剂配方，并使用所述配方制备得到了一种新型的基于TLR7激动剂偶联肽的冠状病毒SARS-CoV-2纳米乳疫苗。在本发明中，通过将TLR7激动剂偶联肽与不同配方的佐剂混合制备成偶联肽-佐剂组合物，并检测制备的各组合物的理化性质，确定了优选的疫苗制剂配方。体内外实验证明，使用本发明的优选配方制备得到的纳米乳疫苗在进入机体内后，可以诱导更高水平的体液免疫反应，刺激机体产生更高滴度的中和抗体，有效阻断病毒入侵宿主细胞，对机体的上下呼吸道均具有接近完全的保护作用。
实验证明，使用本发明的纳米乳疫苗免疫机体得到的抗血清不仅对冠状病毒SARS-CoV-2的野毒株具有高水平的中和活性，同时对突变株(如B.1.1.7、B1.1.529)也具有相当的中和效果。因此，本发明的纳米乳疫苗不仅可以用于预防冠状病毒SARS-CoV-2野毒株的感染，对突变株感染也具有良好的预防作用。
在此基础上，完成了本发明。
疫苗多肽
本发明提供了一种含有冠状病毒SARS-CoV-2疫苗多肽的疫苗制剂，所述疫苗多肽包括抗原多肽以及任选地与所述抗原多肽偶联的TLR7激动剂。
在另一优选例中，所述的疫苗多肽具有式I结构或包含式I结构的寡聚体：
Z-(J-U)n         (I)
式中，
Z为抗原多肽，所述抗原多肽具有冠状病毒SARS-CoV-2S蛋白的至少一个T 细胞表位和/或至少一个B细胞表位；并且，所述的抗原多肽具有衍生自S蛋白的RBM区域的氨基酸序列；
U各自独立地为TLR7激动剂；
n为0或正整数；
J为化学键或连接子。
在另一优选例中，所述的疫苗多肽可激发灵长动物和啮齿动物产生阻断RBD与ACE2结合的中和抗体。
在另一优选例中，所述的疫苗多肽可激发灵长动物产生细胞免疫和体液免疫。
在另一优选例中，所述的灵长动物包括人、非人灵长类动物。
在另一优选例中，所述抗原多肽的长度为8-100个氨基酸，较佳地10-80个氨基酸。
在另一优选例中，所述的抗原多肽具有衍生自冠状病毒SARS-CoV-2S蛋白的RBD区域的氨基酸序列。
在另一优选例中，所述的抗原多肽具有衍生自RBD区域的RBM区域的氨基酸序列。
在另一优选例中，所述的RBM区域指冠状病毒SARS-CoV-2 RBD蛋白的第438-506位氨基酸。
在另一优选例中，所述的抗原多肽“具有衍生自RBD蛋白的RBM区域的氨基酸序列”指所述抗原多肽的氨基酸序列与RBM区域的具有同源性(或同一性)，且所述同源性≥80％，较佳地≥85％，更佳地≥90％，最佳地≥95％。
在另一优选例中，所述的抗原多肽为人工合成的或重组的抗原多肽。
在另一优选例中，所述的抗原多肽的结构如式II所示：
X1-X-X2       (Ⅱ)，
式中，
(a)X为核心片段,其中，所述的核心片段的序列选自SEQ ID NO:1-12(见表A)中的一个或多个；
(b)X1、X2各自独立地为无、1、2或3个氨基酸，且X1和X2的氨基酸个数总和≤4，较佳地，3、2、1，更佳地为0或1；在另一优选例中，X1、X2各自独立地为无、K、C、G、L、A。
(c)“-”表示肽键、肽接头、或其他连接子(即X1与X之间和/或X与X2之间，以肽键、肽接头(如1-15个氨基酸构成的柔性接头)或其他连接子相连)。
表A抗原多肽
Figure PCTCN2022096041-appb-000001
Figure PCTCN2022096041-appb-000002
在另一优选例中，TLR激动剂的分子数n为1、2、3、4、5或6；较佳地为1，2，3或4。
在另一优选例中，所述的TLR7激动剂为小分子激动剂。
在另一优选例中，所述的TLR7激动剂包括：SZU-101:
Figure PCTCN2022096041-appb-000003
在另一优选例中，TLR7激动剂(如SZU-101)连接于抗原多肽的末端氨基或侧链氨基。
在另一优选例中，TLR7激动剂(如SZU-101)连接于抗原多肽的巯基。
在另一优选例中，所述的SZU-101连接于抗原多肽的氨基，并形成S1所示结构：
Figure PCTCN2022096041-appb-000004
或者
所述的SZU-101连接于抗原多肽的巯基，并形成S2所示结构：
Figure PCTCN2022096041-appb-000005
在另一优选例中，所述疫苗多肽选自下组的偶联肽：
(S1)-LFRK(-S1)SNLK(-S1)PFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY(LY54-101,SEQ ID No:1)
其中，SZU-101与氨基酸序列中L的N端氨基和K的侧链氨基通过S1结构连接；
GYAWNRKRISNC(-S2)VADYSVLYNSASFSTFK(P67-101，SEQ ID No:2)
其中，SZU-101与多肽中C上的巯基通过S2结构相连；
并且S1和S2结构如上定义。
角鲨烯
本发明所述的角鲨烯是一种人体经胆固醇合成途径合成的可完全代谢的脂类。角鲨烯主要来源于鲨鱼肝脏，特别是同齿刺鲨鱼、喙吻田氏鲨、南方乌鲨等角鲨烯含量较高的鲨鱼，也可从橄榄油、棕榈油、麦胚油和酵母中提取。以角鲨烯为主要油相的纳米乳佐剂可增强体液免疫应答和细胞免疫应答，刺激浆细胞的成熟，让机体产生足够的抗体，对抗病毒。在人体I期到III期临床试验中证明没有明显的毒副作用，安全可靠。
α-生育酚
本发明所述的α-生育酚作为一种免疫刺激剂在乳剂中发挥作用。α-生育酚是维生素E的8种同工型之一，自然界分布最广。添加α-生育酚的乳剂能够增加单核细胞对抗原的摄取，增强细胞因子的产生，产生更高的抗体应答。本发明中使用的α-生育酚主要通过合成途径获得。
乳化剂
本发明所述所述乳化剂可选自聚山梨酯80及其它聚山梨酯类、磷脂、蔗糖酯、柠檬酸脂肪酸甘油酯类、脂肪酸甘油脂类、脂肪酸山梨坦类、环糊精、聚氧乙烯脂肪酸酯类、聚氧乙烯脂肪醇醚类、聚乙二醇、甲壳质、甲壳胺、胆酸及其盐类等的一种或多种。
嵌段共聚物
本发明所述的嵌段共聚物是一种可生物降解的聚合物，在水中可以自发的形成亲水基团朝外、疏水基团朝内的具有核-壳结构的球形纳米粒子，能够提高药物包封率和生物相容性，并促使淋巴结引流。在另一优选例中，所述嵌段共聚物为医用嵌段共聚物。在另一优选例中，所述嵌段共聚物的数均分子量或重均分子量为300-200000，较佳地500-100000。
本发明所述的嵌段共聚物包括甲氧基聚乙二醇-聚己内酯(mPEG-PCL，PEG:350、550、750、1000、3400、5000、10000、20000；PLA：2000、5000)，甲氧基聚乙二醇聚乳酸-羟基乙酸(mPEG-PLGA，PEG:1000、2000、3400、5000、10000、20000；PLGA：1000、2000、5000、10000、15000、20000、40000)，聚乳酸羟基乙酸-聚乙烯亚胺(PLGA-PEI，PLGA：1000、2000、5000、10000、15000、20000、40000；PEI:600、1800、10000、70000)，聚乳酸-聚乙二醇(PLA-PEG，PLA：2000、5000；PEG:1000、2000、3400、5000、10000、20000)，聚磷酸酯两嵌段共聚物(PCL-b-PHEP，PEG:1000、2000、3400、5000、10000、20000)，聚氧乙烯聚氧丙烯醚嵌段共聚物(泊洛沙姆188等)，聚环氧乙烷-聚环氧丙烷-聚环氧乙烷三嵌段共聚物(PEO-PPO-PEO)中的一种或多种。
本发明的纳米乳疫苗
本发明提供了一种冠状病毒SARS-CoV-2纳米乳疫苗制剂，所述疫苗制剂包含：
(a)冠状病毒SARS-CoV-2疫苗多肽，所述疫苗多肽包括抗原多肽以及任选地与所述抗原多肽偶联的TLR7激动剂；
(b)佐剂，所述佐剂是基于角鲨烯的水包油型纳米乳剂；和
(c)药学上可接受的载体、赋形剂或稀释剂。
具体地，所述佐剂包含以下组分：角鲨烯1-15％(w/w)，α-生育酚0-15％(w/w)，乳化剂0.1-10.0％(w/w)，和嵌段共聚物0.005-10％(w/w)，按所述制剂的总重量计。所述佐剂还包含水相介质。
本发明疫苗制剂中所用佐剂包含油相部分和水相部分。其中，所述佐剂的油相部分包含：角鲨烯1-15％(w/w)，α-生育酚0-15％(w/w)，和乳化剂0.1-10.0％(w/w)；所述佐剂的水相部分包含：嵌段共聚物0.005-10％(w/w)和水相介质，按所述制剂的总重量计。
本发明的新型冠状病毒偶联肽疫苗，以纳米乳为佐剂，该佐剂在注射部位与抗原共定位，诱导短暂的细胞因子和趋化因子反应，并增加先天免疫细胞从血流中向注射部位的募集。主要是单核细胞被激活成为抗原呈递细胞，载有抗原迁移至引流淋巴结。另外，该佐剂增强先天免疫细胞在局部引流淋巴结处的募集。抗原呈递细胞激活幼稚的CD4+T细胞，激活的CD4+T细胞与抗原特异性B细胞相互作用，诱导大量记忆B细胞和分泌抗体的浆细胞。
制备方法
本发明还提供了一种制备本发明第一方面所述的疫苗制剂的方法，其特征在于，所述方法包括以下步骤：
(S1)提供一疫苗多肽；
(S2)将所述疫苗多肽与佐剂和药学上可接受的载体、赋形剂或稀释剂混合，从而制得本发明第一方面所述的疫苗制剂。
具体地，所述方法包括以下步骤：
(i)提供一疫苗多肽；
(ii)将疫苗多肽与佐剂水相和/或佐剂油相充分混合；
(iii)将佐剂油相与佐剂水相混合，通过剪切搅拌或高压均质形成水包油的乳剂，从而获得所述疫苗制剂。
在另一优选例中，将疫苗多肽与佐剂油相混合。
在另一优选例中，所述佐剂油相通过以下方法制备：在惰性气体保护下，将角鲨烯1-15％(w/w)、α-生育酚0-15％(w/w)、乳化剂0.1-10.0％(w/w)，搅拌混合，直至形成均一的油相，从而获得佐剂油相，所述百分比按所述制剂的总重量计；优选地，所述惰性气体为氮气。
在另一优选例中，所述佐剂水相通过以下方法制备：在水相介质中，加入0.01-5％嵌段共聚物，搅拌溶解直至形成均一的水相，从而获得佐剂水相。
在另一优选例中，所述方法还包括步骤(iv)，将步骤(iii)所得的疫苗制剂进行过滤、灭菌和包装。
应用
本发明的冠状病毒SARS-CoV-2纳米乳疫苗制剂可用于制备预防冠状病毒SARS-CoV-2感染或其相关疾病的药物。
所述冠状病毒SARS-CoV-2感染包括野毒株和/或突变株引发的感染。所述的冠状病毒SARS-CoV-2相关疾病包括但不限于呼吸道感染、肺炎及其并发症等，例如新型冠状病毒肺炎(COVID-19)。
通常，可将本发明的疫苗制剂制成可注射剂施用，例如液体溶液或悬液。
本发明的疫苗制剂可制成单元或多元剂型。各剂型包含为了产生所期望的治疗效应而计算出预定量的活性物质，以及合适的药剂学赋形剂。
配制好的疫苗制剂可以通过常规途径进行给药，其中包括(但并不限于)：肌内、静脉内、腹膜内、皮下、皮内、口服、或局部给药。
使用(疫苗)组合物时，是将安全有效量的本发明所述疫苗多肽或肽集合施用于人，其中该安全有效量通常至少约1微克肽/千克体重，而且在大多数情况下不超过约8毫克肽/千克体重，较佳地该剂量是约1微克-1毫克肽/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明提供的新冠疫苗纳米乳疫苗注射剂的有益效果包括：
(1)本发明提供的新型冠状病毒偶联肽疫苗，以纳米乳为佐剂，可通过增强免疫细胞在局部引流淋巴结处的募集作用，提高多肽抗原产生的免疫应答和抗体。
(2)本发明的纳米乳佐剂所使用的原料安全性高，在发挥辅助免疫作用的同时不会造成安全隐患。
(3)本发明的纳米乳佐剂及对应的纳米乳疫苗制剂的稳定性好，可极大地简便疫苗制剂的保存和运输条件。
(4)所提供的疫苗配方与偶联肽具有良好的相容性，在制造方面也具有很好的兼容性，能够适用于一种或多种不同偶联肽的组合。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：制备以TLR7激动剂偶联肽LY54-101为抗原的纳米乳剂疫苗
如下表1所示，制备以TLR7激动剂偶联肽LY54-101(2mg/mL)为抗原的纳米乳剂疫苗。LY54-101的结构如图1所示。制备方法如下：
(1)制备油相：将角鲨烯、α-生育酚、乳化剂在氮气保护下混合均匀，直至形成均一的油相，备用；
(2)制备水相：在水相中，加入嵌段共聚物0.01-5％(w/w)或不加入，形成均一的水相，备用；
(3)在氮气保护下将多肽LY54-101与油相充分混合；
(4)混合含药油相与水相，通过剪切搅拌或高压均质形成水包油的乳剂；
(5)将纳米乳经过0.22μm的滤膜过滤后，灌装，充氮，密封。
表1 LY54-101 2mg/mL的不同处方
Figure PCTCN2022096041-appb-000006
按照表1中的粒径大小可知，在固定量多肽LY54-101中，加入2.0-5.0％的角鲨烯，0.0-4.0％α-生育酚，0.05％蛋黄卵磷脂E80，制备的纳米乳的液滴粒径 小于220nm。加入0.01-1.0％共嵌段聚合物对粒径无影响，符合过滤除菌要求，为优选处方。
实施例2：制备以不同剂量及种类偶联肽为抗原的纳米乳剂疫苗
如下表2所示，以不同剂量及种类偶联肽为抗原的纳米乳剂疫苗的制备方法如下：
(1)制备油相：将角鲨烯2.5％(w/w)、α-生育酚2.5％(w/w)、聚山梨酯801.8％(w/w)在氮气保护下混合均匀，直至形成均一的油溶液，备用；
(2)制备水相：在水相中，加入甲氧基聚乙二醇-聚乳酸嵌段共聚物0.5％(w/w)，形成均一的水溶液，备用；
(3)在氮气保护下将脂溶性多肽加入油相，水溶性多肽加入水相，充分混合使其溶解；
(4)混合溶有多肽的油相与水相，通过剪切搅拌或高压均质形成水包油的乳剂；
(5)将纳米乳经过0.22μm的滤膜过滤后，灌装，充氮，密封。
表2以不同剂量及种类偶联肽为抗原的纳米乳剂疫苗的不同处方
Figure PCTCN2022096041-appb-000007
按照表2中的粒径大小可知，优选处方能够适应不同种类及不同剂量的偶联肽，制备纳米乳液滴粒径小于220nm，符合过滤除菌要求。其中偶联肽P67-101的结构如图2所示。
实施例3：纳米乳剂疫苗的稳定性测定
1、制备实验制剂组：
Figure PCTCN2022096041-appb-000008
单种偶联肽制剂组：含LY54-101 2mg/mL的AS03纳米乳剂和F2纳米疫苗乳剂；
多种偶联肽制剂组：含LY54-101 2mg/mL+P67-101 1mg/mL的F2纳米疫苗乳剂；。
2、多肽含量的测定：
将制剂分别储存在4℃及40℃稳定性箱中，在第0、7、14、30、60天取样，采用Malvern Nano-ZS90动态光散射粒径电位测定仪测定其粒径。
表3单种偶联肽F2纳米乳疫苗制剂稳定性结果
Figure PCTCN2022096041-appb-000009
表4多种偶联肽F2纳米乳疫苗制剂的稳定性结果
Figure PCTCN2022096041-appb-000010
结果表明：本发明的纳米乳疫苗制剂在4℃与40℃下放置60天，无变色分层现象，粒径大小基本不变，表明本发明的纳米乳疫苗制剂性质稳定。
通过高效液相色谱HPLC鉴定纳米乳疫苗的有效成分，本发明所述的F2纳米乳疫苗和AS03纳米乳疫苗均能够使包载的多肽成分在4℃保持稳定。另外，本发明所述的F2纳米乳疫苗即使在40℃下放置一个月，其有效成分LY54-101仍然保持稳定，未发生降解，而作为对照的采用AS03的纳米乳疫苗中有效成分LY54-101已经发生降解(图3)。这表明，本发明的纳米乳更适合于TLR7激动剂偶联肽，可保护偶联肽在高温条件下仍保持性质稳定。
实施例4：纳米乳剂疫苗的淋巴结引流效果
1、实验制剂组：
Figure PCTCN2022096041-appb-000011
制剂均使用连接Cy5的LY54-101为抗原，制备方法同实施例1所述。Titermax组采用油包水型
Figure PCTCN2022096041-appb-000012
佐剂。
2、实验方法：
采用异氟烷将大鼠麻醉，并对大鼠左腿脱毛后，置于活体成像仪中进行给药前(0h)成像。于左大腿内侧肌内注射PBS、游离偶联肽、AS03、F1和F2制剂100μL。注射8h后，将大鼠解剖，取出心、肝、脾、肺、肾及左侧腹股沟淋巴结进行离体组织荧光成像，所采用的激发波长为640nm，发射波长为680nm。
结果表明(图4)：游离偶联肽组与Titermax组主要分布在肝、肾中，表明主要通过肝、肾代谢。目标乳剂组AS03、F1和F2在肾脏中具有较高的荧光分布，表明疫苗偶联肽乳剂主要通过肾脏代谢。
Cy5-LY54-101在淋巴结中几乎没有聚集，表明游离的疫苗偶联肽的免疫激活效果较差。乳剂AS03、F1、F2及Titermax在淋巴结中有不同程度的募集，AS03与F1的荧光强度均低于阳性对照组Titermax(图5)。乳剂F2组在淋巴结处的荧光强度最高，是Titemax的3.8倍，是乳剂AS03组的32.9倍，推测F2乳剂可能具有比AS03乳剂更好的免疫激活效果(图5)。由此说明，本发明所述的疫苗制剂处方含有嵌段共聚物，可以进一步促进药物在注射部位累积，进而在免疫激活中发挥更积极和更显著的作用。
实施例5：纳米乳剂疫苗的免疫反应水平
1、动物免疫方案：
将食蟹猴分组进行肌肉注射，在第0、14、28天免疫。方案如下：
第1组：空白对照组，单纯给予同体积的生理盐水。
第2组：LY54-101(2mg,F2)；
第3组：LY54-101(2mg,AS03)；
第4组：LY54-101+P67-101(1:1,2mg+2mg,F2)；
第5组：LY54-101+P67-101(2:1,2mg+1mg,F2)。
2、抗体水平检测：
为评估偶联肽纳米乳疫苗诱导的体液免疫反应水平，需要检测接种后食蟹猴的血清的RBD结合抗体和中和抗体。
RBD结合抗体采用标准的桥联ELISA方法测定。具体检测方法如下：1μg/mL的RBD-His包被96孔ELISA板，4℃过夜。用PBST缓冲液(PBS中含0.05％Tween-20)洗涤三次后，用200μL的1％BSA溶液在37℃下封闭ELISA板1小时。洗涤后，96孔板加入100μL的血清梯度稀释液，并在37℃孵育1小时。洗涤后，96孔板加入100μL的Protein A-HRP(1:5000用)，在37℃下孵育一小时，摇动设置在650rpm。洗涤后，用100μL的四甲基联苯胺(TMB)底物溶液在37℃、650rpm摇动下孵育96孔板20分钟。加入2M的硫酸溶液终止反应，用自动微孔板阅读器SpectraMax在450nm处测定吸光度值。
中和抗体采用竞争ELISA方法检测，具体测定方法如下：样品和对照物与HRP-RBD预孵育，使待测中和抗体与HRP-RBD结合。然后将混合物加入到预包被有hACE2蛋白的捕获板上。未结合的HRP-RBD以及与非中和抗体结合的HRP-RBD将被捕获在板上，而样品中的中和抗体/HRP-RBD复合物留在上清液中，并在洗涤过程中被去除。洗涤步骤结束后，加入TMB溶液，使颜色变成蓝色。通过加入终止液，反应被淬灭，颜色变成黄色。这个最终的溶液可以在微孔板读数器中在450纳米处读取。样品的吸光度与抗SARS-CoV-2中和抗体的滴度成反比。
结果表明(图6和图7)：从食蟹猴免疫后的35天和70天的血清中的RBD特异性结合抗体水平来看，F2纳米乳的疫苗制剂诱导了显著高于AS03纳米乳的RBD结合抗体滴度，提示对于本发明的TLR7激动剂偶联肽来说，F2纳米乳为更为合适的疫苗制剂形式。同时，LY54-101+P67-101(2:1,2mg+1mg,F2)组表现出了最高的RBD结合抗体滴度，最高达1：72900，说明本发明的偶联肽组合最佳为LY54-101和P67-101的2:1的组合。
从食蟹猴免疫后的35天和70天的血清中的阻断RBD和ACE2相互作用的中和抗体水平来看(图8和图9)，意外的是，从35天到70天，血清中的中和抗体水平在持续增加，中和抗体滴度最高可达1:5120。类似地，F2纳米乳的疫苗制剂诱导了显著高于AS03纳米乳的中和抗体滴度，且LY54-101+P67-101(2:1,2mg+1mg,F2)组表现出了最高的中和抗体滴度。
以上结果说明，本发明开发的纳米乳应用到偶联肽疫苗的制剂开发中表现出了优于AS03纳米乳的免疫效果。而且，本发明的偶联肽纳米乳疫苗在食蟹猴体 内诱导了高水平的体液免疫反应，食蟹猴免疫后血清中和抗体水平极高且持久，提示具有阻断病毒入侵作用。
实施例6：纳米乳剂疫苗阻断病毒入侵
为了进一步评价疫苗免疫食蟹猴后诱导的高水平体液免疫反应是否能够阻断病毒入侵宿主细胞，采用了假病毒和真病毒测试体系评估食蟹猴抗血清的假病毒阻断中和活性。
在假病毒中和试验中，将100μL不同稀释度的血清样品与50μL含有SARS-CoV-2假病毒的上清液混合。该混合物在37℃下孵育1小时。然后将100μL的Huh-7/ACE2细胞加入到假病毒和血清样品的混合物中，在37℃下再孵育24小时。然后，取出上清液，将100μL荧光素酶检测液加入到每个孔中。孵育2分钟后，使用微孔板光度计测量荧光素酶活性。
在真病毒测试体系中，SARS-CoV-2病毒野毒株在VERO E6细胞中增殖。将血清样品在56℃下热灭活30分钟；然后将从1∶4开始的2倍连续稀释液与等体积的含有50％组织培养物的病毒溶液混合。将血清-病毒混合物在37℃、5％CO2加湿的环境中孵育1小时。孵育后，将每个稀释度的100μL混合物一式两份加入到含有半融合的VERO E6单层的细胞板上。该板在37℃下孵育4天。培养4天后，在显微镜下记录各孔的细胞病变效果(CPE)。取能保护50％以上细胞不受CPE影响的最高血清稀释度作为中和滴度。
假病毒中和实验结果表明(图10和图11)，偶联肽纳米乳疫苗免疫后的食蟹猴血清具有高水平的中和活性，可以阻断假病毒入侵宿主细胞，滴度高达1:256(图10，野毒株)。
同时，本发明的纳米乳疫苗可有效保护细胞免受突变株B.1.1.7感染，滴度高达1:512(图11，突变株B.1.1.7)。该结果表明，本发明的纳米乳疫苗对新型冠状病毒突变株的中和效果与野毒株相当，因此也可以用于预防新型冠状病毒突变株的感染。另外，本发明采用基于实施例5的RBD结合抗体检测方法，将野生型RBD替换为K417N、N439K、L452R、Y453F、S477N、E484K、E484Q和N501Y的多种单氨基酸突变型RBD蛋白进行检测。结果表明，偶联肽纳米乳疫苗免疫食蟹猴后(第35天)，食蟹猴的抗血清与野生型RBD的结合能力对比与K417N、N439K、L452R、Y453F、S477N、E484Q和N501Y的突变型RBD蛋白的结合能力均无差距(图12)，这提示RBD蛋白的多种突变不会针对本发明的多肽疫苗产生免疫逃逸，而且本发明疫苗不会被突变株B.1.1.7(包含N501Y突变)和突变株B.1.617(包含L452R和E484Q突变)产生免疫逃逸。食蟹猴的抗血清对于E484K突变的RBD的结合能力下降了三倍(图12)，但是仍保留了较高的抗体滴度，提示本发明疫苗仍能对突变株B.1.351(包含K417N、E484K和N501Y)和P.1(包含 E484K和N501Y)提供良好免疫保护。此外，通过B.1.1.529(Omicron)假病毒模型，食蟹猴抗血清(第三次免疫后62天)仍对Omicron突变株具有显著中和活性，中和滴度为1：346(图13)，提示本偶联肽纳米乳疫苗可保护机体应对Omicron突变株的感染。
真病毒中和实验结果表明(图14)，偶联肽纳米乳疫苗免疫后的食蟹猴血清具有高水平的中和活性，可以阻断真病毒(野毒株)入侵宿主细胞，滴度高达1:39。
以上结果表明，本发明的偶联肽纳米乳疫苗免疫食蟹猴后可以产生高水平的保护性中和抗体，可以预防新冠野毒株和突变株的感染。
实施例7：攻毒试验评价疫苗制剂体内保护效果
为评价本发明的偶联肽纳米乳疫苗的体内预防病毒感染的保护效果，在食蟹猴加强免疫14天后进行攻毒试验。
本发明采用国内外最高病毒载量(1×10 7TCID 50)攻毒(常用1×10 6TCID 50)，随后的第1天，第3天，第5天和第7天通过鼻拭子和咽拭子监测上呼吸道病毒载量，在第七天对食蟹猴实施安乐死，取肺组织检测各肺叶病毒载量。病毒载量的检测通过qRT-PCR进行。
在攻毒过程中，鼻拭子和咽拭子的病毒载量监测结果显示生理盐水组的食蟹猴的上呼吸道表现出高的病毒载量，而本发明的偶联肽纳米乳疫苗免疫的食蟹猴仅在攻毒后的第一天在鼻拭子中监测到低水平的病毒载量，之后不论在鼻还是咽中均未检出病毒RNA(图15)。
攻毒结束后，肺组织的病毒载量检测结果显示，本发明的偶联肽纳米乳疫苗免疫的食蟹猴的左右肺均为检出病毒RNA，而生理盐水组的食蟹猴在左右肺的各个肺叶都有高水平的病毒载量(图16)。
这些结果显示了本发明的偶联肽纳米乳疫苗具有极佳的保护效果，在极高的攻毒剂量下，仍可以预防食蟹猴感染SARS-CoV-2野毒株，对其上下呼吸道均具有接近完全的保护作用。
讨论
本发明在TLR7激动剂偶联肽的基础上，开发安全有效的人用新型佐剂，可以辅助偶联肽以在体内达到最佳的免疫效果。本发明开发的新型纳米乳表现出比临床使用的AS03纳米乳更佳的性质，如制备得到的偶联肽纳米乳制剂的热稳定性更佳，可耐受40℃高温；再如本发明开发的新型纳米乳免疫效果更佳，可以诱发更高水平的针对RBD的中和抗体，并可对SARS-CoV-2野毒株和突变株提供高效保护，这可能与它良好的淋巴引流能力相关。
本发明的TLR7激动剂偶联肽纳米乳新冠疫苗具有突出的预防SARS-CoV-2 野毒株和突变株感染的作用，可预防SARS-CoV-2感染并对机体上下呼吸道均具有接近完全的保护作用，具备临床价值。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种冠状病毒SARS-CoV-2纳米乳疫苗制剂，其特征在于，所述疫苗制剂包含：
    (a)冠状病毒SARS-CoV-2疫苗多肽，所述疫苗多肽包括抗原多肽以及任选地与所述抗原多肽偶联的TLR7激动剂；
    (b)佐剂，所述佐剂是基于角鲨烯的水包油型纳米乳剂；和
    (c)药学上可接受的载体、赋形剂或稀释剂。
  2. 如权利要求1所述的制剂，其特征在于，所述佐剂包含以下组分：角鲨烯1-15％(w/w)，α-生育酚0-15％(w/w)，乳化剂0.1-10.0％(w/w)，和嵌段共聚物0.005-10％(w/w)，按所述制剂的总重量计。
  3. 如权利要求1所述的制剂，其特征在于，所述佐剂包含油相部分和水相部分，所述佐剂的油相部分包含：角鲨烯1-15％(w/w)，α-生育酚0-15％(w/w)，和乳化剂0.1-10.0％(w/w)；所述佐剂的水相部分包含：嵌段共聚物0.005-10％(w/w)和水相介质，按所述制剂的总重量计。
  4. 如权利要求1至3任一项所述的制剂，其特征在于，所述角鲨烯来源于鲨鱼肝脏、橄榄油、棕榈油、麦胚油和/或酵母。
  5. 如权利要求2或3所述的制剂，其特征在于，所述乳化剂选自下组：磷脂、聚山梨酯类、蔗糖酯、柠檬酸脂肪酸甘油酯类、脂肪酸甘油脂类、脂肪酸山梨坦类、环糊精、聚氧乙烯脂肪酸酯类、聚氧乙烯聚氧丙烯共聚物类、聚氧乙烯脂肪醇醚类、聚乙二醇、泊洛沙姆、甲壳质、甲壳胺、胆酸及其盐类、或其组合。
  6. 如权利要求2或3所述的制剂，其特征在于，所述嵌段共聚物的数均分子量或重均分子量为300-200000，较佳地500-100000。
  7. 如权利要求2或3所述的制剂，其特征在于，所述嵌段共聚物选自下组：甲氧基聚乙二醇-聚己内酯、甲氧基聚乙二醇聚乳酸-羟基乙酸、聚乳酸羟基乙酸-聚乙烯亚胺、聚乳酸-聚乙二醇、聚磷酸酯两嵌段共聚物、聚氧乙烯聚氧丙烯醚嵌段共聚物、聚环氧乙烷-聚环氧丙烷-聚环氧乙烷三嵌段共聚物、或其组合。
  8. 如权利要求3所述的制剂，其特征在于，所述水相介质选自下组：生理盐水、灭菌水、缓冲盐水、葡萄糖溶液、环糊精溶液、或其组合。
  9. 如权利要求2所述的制剂，其特征在于，所述佐剂中还含有等渗调节剂，所述等渗调节剂的含量为0.1-8％(w/w)。
  10. 如权利要求1所述的制剂，其特征在于，所述的疫苗多肽具有式I结构或包含式I结构的寡聚体：
    Z-(J-U)n    (I)
    式中，
    Z为抗原多肽，所述抗原多肽具有新型冠状病毒S蛋白的至少一个T细胞表位和/或至少一个B细胞表位；并且，所述的抗原多肽具有衍生自S蛋白的RBM区域的氨基酸序列；
    U各自独立地为TLR7激动剂；
    n为0或正整数；
    J为化学键或连接子。
  11. 如权利要求1所述的制剂，其特征在于，所述纳米乳疫苗制剂中疫苗多肽的含量为0.1-4mg/mL。
  12. 如权利要求1所述的制剂，其特征在于，所述纳米乳疫苗制剂具有选自下组的一个或多个特征：
    (1)稳定性好，在4℃与40℃下放置1-2个月，制剂中纳米乳粒径变化不超过1％；
    (2)纳米乳粒径均小于0.22μm，满足过滤除菌要求。
  13. 一种制备权利要求1所述的疫苗制剂的方法，其特征在于，所述方法包括以下步骤：
    (S1)提供一疫苗多肽；
    (S2)将所述疫苗多肽与佐剂和药学上可接受的载体、赋形剂或稀释剂混合，从而制得权利要求1所述的疫苗制剂。
  14. 如权利要求1-12任一项所述的冠状病毒SARS-CoV-2纳米乳疫苗制剂的用途，其特征在于，用于制备预防冠状病毒SARS-CoV-2感染或其相关疾病的药物。
  15. 如权利要求14所述的用途，其特征在于，所述冠状病毒SARS-CoV-2包括野毒株和/或突变株，所述的突变株选自下组：B.1.1.7、B.1.617、B.1.351、P.1和B.1.1.529。
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