WO2022253193A1 - 一种新型冠状病毒疫苗多肽及其纳米乳制剂在预防新冠野毒株和突变株感染中的应用 - Google Patents

一种新型冠状病毒疫苗多肽及其纳米乳制剂在预防新冠野毒株和突变株感染中的应用 Download PDF

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WO2022253193A1
WO2022253193A1 PCT/CN2022/096047 CN2022096047W WO2022253193A1 WO 2022253193 A1 WO2022253193 A1 WO 2022253193A1 CN 2022096047 W CN2022096047 W CN 2022096047W WO 2022253193 A1 WO2022253193 A1 WO 2022253193A1
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vaccine
polypeptide
seq
cov
peptide
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PCT/CN2022/096047
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English (en)
French (fr)
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宫丽崑
任进
黄蔚
甘勇
郑永唐
孙建华
张馨欣
龙益如
秦秋平
刘婷婷
唐峰
余盼
缪云秋
蔡泳龙
秦勉
宋天章
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中国科学院上海药物研究所
中科中山药物创新研究院
中国科学院昆明动物研究所
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Publication of WO2022253193A1 publication Critical patent/WO2022253193A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the fields of polypeptide drugs and polypeptide vaccines, and in particular, relates to the application of novel coronavirus polypeptide vaccines, polypeptide compositions and nanoemulsion preparations in preventing the infection of new coronavirus wild strains and mutant strains.
  • SARS-CoV-2 The new coronavirus (SARS-CoV-2) is spreading on a large scale around the world, and its uncontrolled epidemic seriously threatens global public health security and economic development. To develop and apply a safe, economical and efficient new crown vaccine to achieve herd immunity, It is the fundamental way to contain the epidemic.
  • the existing COVID-19 vaccines mainly adopt the following technical routes: live attenuated vaccines, inactivated vaccines, recombinant virus vector vaccines, recombinant protein vaccines and nucleic acid vaccines. These vaccine technical routes are currently mainly designed for wild strains of SARS-CoV-2.
  • the purpose of the present invention is to provide a novel coronavirus polypeptide vaccine and its vaccine nanoemulsion preparation, which can produce blocking anti-SARS-CoV-2 antibodies in the vaccinated person, and can deal with SARS-CoV-2 wild strains Infection with mutant strains.
  • a novel coronavirus pneumonia vaccine polypeptide is provided, and the vaccine polypeptide has an amino acid sequence derived from the S protein of SARS-CoV-2 wild strain and mutant strain;
  • Described vaccine polypeptide is selected from following group:
  • the "substantially the same function” means that the derived polypeptide has substantially the same immunogenicity for stimulating cellular and humoral immune responses, and can induce primates to produce wild-type RBD specificity binding antibodies and neutralizing antibodies that block wild-type and/or mutant RBD binding to ACE2.
  • mutant strains of SARS-CoV-2 include but are not limited to D614G mutant strain, mutant strain B.1.1.7, mutant strain B.1.351, mutant strain P.1, mutant strain B.1.617 and the B.1.1.529 mutant strain.
  • the vaccine polypeptide can induce cellular immunity and humoral immunity in primates.
  • the vaccine polypeptide can induce primates to produce wild-type RBD-specific binding antibodies and neutralizing antibodies that block the binding of wild-type RBD to ACE2.
  • the wild type RBD is the receptor binding domain of the S protein of the SARS-CoV-2 wild strain.
  • the vaccine polypeptide can stimulate primates to produce mutated RBD-specific binding antibodies and neutralizing antibodies that block the mutated RBD from binding to ACE2.
  • the mutated RBD is the receptor binding domain of the S protein of the SARS-CoV-2 mutant strain.
  • the mutant RBD is a mutant RBD protein formed by adding one or more amino acids, replacing one or more amino acids or deleting one or more amino acids based on the wild-type RBD.
  • the mutations in the mutated RBD include but are not limited to one or more of the following: K417N, K417T, N439K, L452R, Y453F, S447N, E484Q, E484K and N501Y.
  • the antiserum after the vaccine polypeptide immunizes primates can block the invasion of cells by the wild strain of SARS-CoV-2.
  • the antiserum after the vaccine polypeptide is immunized to primates can block the invasion of cells by the mutant strain of SARS-CoV-2.
  • the primates include humans and non-human primates (such as monkeys).
  • the length of the vaccine polypeptide is 8-100 amino acids, preferably 10-80 amino acids.
  • the vaccine polypeptide is an artificially synthesized or recombinant vaccine polypeptide.
  • X is a core fragment, wherein the sequence of the core fragment is selected from one or more of SEQ ID NO: 1-15 (see Table A);
  • X1 and X2 are each 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;
  • the vaccine polypeptide is selected from Table A:
  • C(C) in SEQ ID No:3 and 5 means that the cysteine on the peptide chain forms a disulfide bond with the cysteine on another non-peptide chain.
  • the sulfhydryl group of cysteine on the main chain of PSS forms a disulfide bond structure with a sulfhydryl group of cysteine, and its structure and schematic diagram are shown in FIG. 1 .
  • the sulfhydryl groups of the three cysteines on the main chain of LPSS respectively form disulfide bond structures with the sulfhydryl groups of cysteine.
  • the structure and schematic diagram are shown in FIG. 2 .
  • the three vaccine polypeptides shown in SEQ ID Nos: 6, 7 and 8 are used in combination, wherein any one, two or three of SEQ ID Nos: 6-8 can also be based on According to the variation of SARS-CoV-2, the corresponding mutation sites (such as those shown in Figure 19 falling in the sequence of SEQ ID No: 6-8) are introduced in time, so as to form a timely response to SARS-CoV- 2 variant strain (or mutant strain), thereby improving the protection of the vaccine of the present invention to the mutant strain.
  • a representative vaccine polypeptide set (or combination) that is more suitable for dealing with mutant strains includes: vaccine polypeptides shown in SEQ ID Nos: 9, 10 and 11 (mutated amino acids of mutant strains are introduced respectively).
  • the present invention corresponds to one or more vaccine polypeptides shown in SEQ ID Nos: 6, 7 and 8 corresponding to wild-type virus strains, and can also be combined with one or more vaccine polypeptides corresponding to mutant strains (such as SEQ ID Nos. Vaccine polypeptides shown in ID No:9, 10 and 11) are combined to form, for example, vaccine polypeptide sets shown in SEQ ID No:9, 7 and 11; or vaccine polypeptides shown in SEQ ID No:9, 10 and 8 set.
  • X1 and X2 are each independently none, K, C, G, L, and A.
  • X1 is none, K, or C.
  • X2 is none, K, or C.
  • the vaccine polypeptide has at least one T cell epitope and/or at least one B cell epitope of the SARS-CoV-2 S protein.
  • the vaccine polypeptide has at least one T cell epitope and/or at least one B cell epitope of the RBD region of the SARS-CoV-2 S protein.
  • the vaccine polypeptide has at least one T cell epitope and/or at least one B cell epitope of the RBM region of the SARS-CoV-2 S protein.
  • the vaccine polypeptide has at least one T cell epitope and/or at least one B cell epitope of the NTD region of the SARS-CoV-2 S protein.
  • the vaccine polypeptide has at least one T cell epitope, preferably 1, 2, 3 or 4 T cell epitopes, more preferably 1 or 2 T cell epitopes.
  • the vaccine polypeptide has at least one B cell epitope, preferably 1, 2, 3 or 4 B cell epitopes, more preferably 1 or 2 B cell epitopes.
  • the vaccine polypeptide has 1-2 T cell epitopes and 0-2 B cell epitopes, preferably 1-2 T cell epitopes and 0-1 B cell epitopes bit.
  • an isolated peptide collection is provided, and the peptide collection includes at least two novel coronavirus pneumonia vaccine polypeptides described in the first aspect of the present invention.
  • the peptide set contains at least 2-15 kinds (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 kinds) The vaccine polypeptide.
  • the peptide set includes vaccine polypeptides selected from the group consisting of:
  • (Z3) One or more (such as 1, 2, 3, 4 or 5) vaccine polypeptides selected from SEQ ID No: 1-12, and 1, 2 selected from SEQ ID No: 13-15 or 3 vaccine peptides.
  • the peptide set includes (or consists of) vaccine polypeptides selected from the following group:
  • a pharmaceutical composition which contains the novel coronavirus pneumonia vaccine polypeptide described in the first aspect of the present invention or the peptide set described in the second aspect of the present invention and pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a vaccine composition.
  • the vaccine composition is monovalent or multivalent.
  • the pharmaceutical composition further contains an adjuvant, and the selected adjuvant includes but not limited to aluminum salt, Titermax, emulsion, liposome, and virus vector.
  • the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.
  • the dosage form of the pharmaceutical composition is liquid, solid, or gel.
  • the pharmaceutical composition is administered in a manner selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oronasal spray and aerosol inhalation.
  • a vaccine formulation which comprises the vaccine polypeptide of the novel coronavirus pneumonia described in the first aspect of the present invention or the peptide set described in the second aspect or the peptide set described in the third aspect
  • the pharmaceutical composition, the vaccine preparation is preferably a nanoemulsion preparation.
  • the vaccine formulation comprises nanoemulsion based on squalene and emulsifier.
  • the squalene is derived from shark liver
  • the emulsifier is phospholipid, polysorbate 80, or a combination of polysorbate 80 and phospholipid, and sucrose esters, citric acid fatty acid glycerides , one or more of fatty acid glycerides, fatty acid sorbitan, cyclodextrin, polyoxyethylene fatty acid esters, etc.
  • the vaccine preparation contains 0-20% of other acceptable raw and auxiliary materials in injection dosage forms.
  • the vaccine adjuvant composition contains ⁇ -tocopherol, and the amount of the ⁇ -tocopherol is not more than 15% by weight;
  • the vaccine adjuvant composition contains a block copolymer, and the block copolymer can be methoxy Polyethylene glycol poly(lactic-co-glycolic acid) copolymer, methoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid), poloxamer or one or more, the content is not more than 5%; it also contains isotonicity regulator, The isotonic regulator is 0.1-8% (w/w); the vaccine adjuvant composition also contains a pH regulator, and the pH regulator does not exceed 1% (w/w).
  • novel coronavirus pneumonia vaccine polypeptide described in the first aspect of the present invention or the peptide collection described in the second aspect or the pharmaceutical composition described in the third aspect or the pharmaceutical composition described in the fourth invention The purposes of the preparation, be used for preparing the medicine that prevents coronavirus SARS-CoV-2 wild strain and mutant strain infection or its related diseases.
  • 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).
  • a cell preparation comprising (a) immunization with the vaccine polypeptide described in the first aspect of the present invention or the peptide collection described in the second aspect of the present invention to activate the immune system cells; and (b) a pharmaceutically acceptable carrier.
  • the immune cells are selected from the group consisting of dendritic cells, natural killer cells (NK), lymphocytes, monocytes/macrophages, granulocytes, or combinations thereof.
  • NK natural killer cells
  • the activation is in vitro activation.
  • the in vitro activation includes: culturing the immune cells for a period of time (such as 6-48 hours) in the presence of the vaccine polypeptide, so as to obtain immune-activated immune cells.
  • the cell preparation is a liquid preparation containing living cells.
  • the cell preparation is reinfused through intravenous administration.
  • a method of generating an immune response against coronavirus SARS-CoV-2 wild strains and mutant strains comprising the steps of: administering the vaccine described in the first aspect of the present invention to a subject in need
  • the polypeptide, the peptide collection of the second aspect, the pharmaceutical composition of the third aspect, or the preparation of the fourth aspect comprising the steps of: administering the vaccine described in the first aspect of the present invention to a subject in need.
  • the subject includes a human or a non-human mammal.
  • the non-human mammals include non-human primates (such as monkeys).
  • the method induces in the subject neutralizing antibodies against wild strains and mutant strains of coronavirus SARS-CoV-2.
  • the neutralizing antibody blocks the binding of coronavirus SARS-CoV-2 wild strains and mutant strains to human ACE2 protein.
  • a fusion protein which includes a carrier protein and the vaccine polypeptide according to the first aspect of the present invention fused with the fusion protein.
  • the fusion protein has the structure of formula IIIa or IIIb:
  • P1 is the vaccine polypeptide described in the first aspect of the present invention
  • P2 is a carrier protein
  • the P1 may be a single vaccine polypeptide, or multiple identical or different vaccine polypeptides (or antigenic polypeptides) connected in series.
  • a pharmaceutical composition which includes (a) immune cells activated by or by the fusion protein described in the seventh aspect of the present invention; and (b) a pharmaceutically acceptable carrier.
  • the fusion protein described in the seventh aspect of the present invention or the pharmaceutical composition described in the eighth aspect which are used to prepare and prevent coronavirus SARS-CoV-2 infection or its related Medicines for diseases.
  • Figure 1 shows the specific structure of the polypeptide PSS.
  • Figure 2 shows the specific structure of the polypeptide LPSS.
  • Figure 3 shows the results of MHC class II molecular epitope analysis of the RBD region of SARS-CoV-2.
  • Figure 4 shows the results of MHC class I molecular epitope analysis of the RBD region of SARS-CoV-2.
  • Figure 5 shows the localization of vaccine polypeptides LY54, LY54mut, P67, PSS, LPSS and LP67 derived from wild-type RBD in RBD proteins.
  • Figure 6 shows the localization of vaccine polypeptides MP1, MP2, MP3, MP477, MP484 and MP501 derived from mutant RBDs in RBD proteins.
  • Figure 7 shows that cynomolgus monkeys immunized with vaccine polypeptides produced high levels of RBD-specific binding antibodies on day 34.
  • Fig. 8 shows that after the cynomolgus monkey was immunized with the vaccine polypeptide, a high level of neutralizing antibody that could block the interaction between RBD and ACE2 was produced after 34 days.
  • Figure 9 shows that after immunization of cynomolgus monkeys with vaccine polypeptides, high levels of RBD-specific binding antibodies remained after 105 days.
  • Figure 10 shows that after the cynomolgus monkey was immunized with the vaccine polypeptide, a high level of neutralizing antibody that can block the interaction between RBD and ACE2 was still retained after 105 days.
  • Figure 11 shows that cynomolgus monkeys immunized with polypeptide nanoemulsion formulations produced high levels of bound antibodies on day 42.
  • Figure 12 shows that the polypeptide nanoemulsion preparation produced a high level of neutralizing antibodies that could block the interaction between RBD and ACE2 after 42 days after the cynomolgus monkey was immunized.
  • Figure 13 shows that the antiserum after the polypeptide nanoemulsion preparation immunizes cynomolgus monkeys has high neutralizing activity of blocking wild strain pseudovirus from invading cells.
  • Figure 14 shows that the antiserum after vaccine polypeptide immunization in cynomolgus monkeys has a high neutralization titer of blocking live virus of wild strains from invading cells.
  • Figure 15 shows that cynomolgus monkeys were immunized with polypeptide nanoemulsion preparations to produce high levels of mutant RBD-specific binding antibodies.
  • Figure 16 shows that the antiserum after the polypeptide nanoemulsion preparation immunized cynomolgus monkeys has high neutralizing activity of blocking mutant B.1.1.7 pseudovirus from invading cells.
  • Figure 17 shows that mixed vaccine polypeptides MP477, MP484, MP501 and P67 derived from mutant RBD immunized cynomolgus monkeys to produce high levels of specific binding antibodies against the key RBD mutation sites of B.1.351 and P.1 mutants .
  • Figure 18 shows that mixed immunization of cynomolgus monkeys with vaccine polypeptides MP477, MP484, MP501 and P67 derived from mutant RBD produced a high level of specific binding antibodies against the key RBD mutation site of the B.1.617 mutant strain.
  • Figure 19 shows the mutated amino acid sites and mutation types of the S protein region of several new coronavirus mutants compared with wild strains.
  • Figure 20 shows that the antiserum after the polypeptide nanoemulsion preparation immunized cynomolgus monkeys has high neutralizing activity of blocking mutant B.1.1.529 pseudovirus from invading cells.
  • the inventors analyzed the S protein of wild strains and mutant strains.
  • the immunogenicity of the virus, and the key region of the interaction between the wild strain and the mutant strain S protein and the host ACE2 protein was analyzed.
  • the present invention screens and determines the vaccine polypeptide that can effectively induce the primate body to produce an immune response against the coronavirus SARS-CoV-2.
  • the vaccine polypeptide of the present invention can cause cynomolgus monkeys to produce a high-level and long-lasting humoral immune response against SARS-CoV-2.
  • the antiserum after the vaccine polypeptide and polypeptide nanoemulsion vaccine preparation of the present invention immunizes cynomolgus monkeys has high-titer RBD-binding antibodies and neutralizing antibodies that block the combination of RBD and ACE2, and can be tested in SARS-CoV-2 pseudovirus models and Both showed high levels of blocking effects on live virus models.
  • the binding activity of cynomolgus monkey antiserum against various RBD mutant proteins such as S477N, E484K and N501Y was not affected, and showed a high level of blocking in the B.1.1.7 mutant strain pseudovirus model
  • the polypeptide vaccine of the present invention can be used to prevent the infection of novel coronavirus wild strains and various mutant strains such as B.1.1.7, B.1.351 and B.1.1.529. The present invention has been accomplished on this basis.
  • Coronavirus (Coronavirus, CoV) belongs to the family Coronaviridae of Nidovirales.
  • HCoV-229E and HCoV-NL63 belong to the ⁇ -coronaviruses
  • HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all ⁇ -coronaviruses.
  • the new coronavirus (SARS-CoV-2) that broke out at the end of 2019 has about 80% similarity with SARS-CoV and 40% similarity with MERS-CoV, and also belongs to the ⁇ -coronavirus.
  • the genome of this type of virus is a single-stranded positive-strand RNA, which is one of the largest RNA viruses in the genome, encoding replicase, spike protein, envelope protein, envelope protein, and nucleocapsid protein.
  • the genome is translated into two peptide chains of several thousand amino acids, the precursor polyprotein (Polyprotein), and then the precursor protein is cleaved by proteases to generate non-structural proteins (such as RNA polymerase and unwinding enzymes) and structural proteins (such as spike proteins) and auxiliary proteins.
  • the S protein is a major structural protein of the coronavirus SARS-CoV-2, in which the RBD is responsible for the structure of the human ACE2 receptor, and the RBM region contains a motif (motif) that binds to the human ACE2.
  • the amino acid sequence of a typical S protein is shown in SEQ ID No:16.
  • the RBD region of the coronavirus SARS-CoV-2 is located at positions 333-527 of the S protein, and a representative amino acid sequence is shown at positions 333-527 in SEQ ID No:16.
  • the RBM region of the coronavirus SARS-CoV-2 is located at positions 438-506 of the S protein, and a representative amino acid sequence is shown at positions 438-506 in SEQ ID No:17.
  • S protein, RBD region and RBM region all include wild type and mutant type.
  • mutant strains of SARS-CoV-2 which are different from the wild strain Wuhan-Hu-1, have emerged and are widely prevalent.
  • a mutant strain called B.1.1.7 or 501Y.V1 which has been detected in many countries around the world, may be more contagious and have an increased risk of death.
  • Another mutant strain, called B.1.351 or 501Y.V2 began to circulate in countries other than South Africa and showed a strong immune escape ability of vaccines and neutralizing antibodies.
  • Mutant strain P.1 the immune escape ability of this mutant strain seems to be stronger than that of mutant strain B.1.351.
  • the Omicron (B.1.1.529) mutant showed more significant immune escape ability and transmission ability.
  • epitopope peptide of the present invention in the present invention, "epitope peptide of the present invention”, “vaccine polypeptide of the present invention”, “antigenic polypeptide of the present invention”, and “polypeptide of the present invention” can be used interchangeably, referring to the vaccine polypeptide described in the first aspect of the present invention . It should be understood that the term includes not only one vaccine polypeptide of the present invention, but also a peptide set (or peptide combination) formed by multiple vaccine polypeptides of the present invention.
  • vaccine polypeptides also include other forms, such as pharmaceutically acceptable salts, conjugates, or fusion proteins.
  • the vaccine polypeptide includes one or more (such as 1-5, preferably 1-3) amino acid additions, one or more (such as 1- A derivative polypeptide formed by substitution of 5, preferably 1-3) amino acids and/or deletion of 1-3 amino acids, the derivative polypeptide has substantially the same function as the original polypeptide before derivation.
  • the vaccine polypeptide includes 1-3 amino acid additions (preferably added at the N-terminal or C-terminal), and/or 1-2 amino acid substitutions (preferably conservative) to any sequence in SEQ ID No: 1-15 amino acid substitution) and still have substantially the same function as the original polypeptide before derivation.
  • the conservative amino acid substitution is performed according to Table C.
  • the three vaccine polypeptides shown in SEQ ID Nos: 6, 7 and 8 are used in combination, wherein any one, two or three of SEQ ID Nos: 6-8 can also be based on According to the variation of SARS-CoV-2, the corresponding mutation sites (such as those shown in Figure 19 falling in the sequence of SEQ ID No: 6-8) are introduced in time, so as to form a timely response to SARS-CoV- 2 variant strain (or mutant strain), thereby improving the protection of the vaccine of the present invention to the mutant strain.
  • a representative vaccine polypeptide set (or combination) that is more suitable for dealing with mutant strains includes: vaccine polypeptides shown in SEQ ID Nos: 9, 10 and 11 (mutated amino acids of mutant strains are introduced respectively).
  • the present invention corresponds to one or more vaccine polypeptides shown in EQ ID Nos: 6, 7 and 8 of the wild-type virus strain, and can also be combined with one or more vaccine polypeptides corresponding to mutant strains (such as SEQ ID NO: ID No: 9, 10 and 11 vaccine polypeptides) combined to form, for example, the vaccine polypeptide set shown in SEQ ID No: 9, 7 and 11; or the vaccine polypeptide shown in SEQ ID No: 9, 10 and 8 set.
  • mutant strains such as SEQ ID NO: ID No: 9, 10 and 11 vaccine polypeptides
  • peptide collection refers to a peptide collection consisting of at least two vaccine polypeptides of the present invention or derivative polypeptides thereof.
  • the peptide set of the present invention contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 peptides selected from the first aspect of the present invention.
  • Vaccine polypeptides or derived polypeptides thereof (including conjugated peptides); more preferably, said peptide set contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 vaccine polypeptides or derivative polypeptides selected from SEQ ID No: 1-15.
  • the peptide collection also includes antigenic peptides or proteins of other coronaviruses SARS-CoV-2 other than SEQ ID No: 1-15.
  • the peptide set includes vaccine polypeptides selected from the group consisting of:
  • (Z3) One or more (such as 1, 2, 3, 4 or 5) vaccine polypeptides selected from SEQ ID No: 1-12, and 1, 2 selected from SEQ ID No: 13-15 or 3 vaccine peptides.
  • the peptide set includes (or consists of) vaccine polypeptides selected from the following group:
  • isolated means that the material is separated from its original environment (if the material is native, the original environment is the natural environment).
  • the polypeptide in the natural state in living cells is not isolated and purified, but the same polypeptide is isolated and purified if it is separated from other substances that exist together in the natural state.
  • isolated peptide refers to a polypeptide of the present invention that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify polypeptides of the invention using standard protein purification techniques. A substantially purified polypeptide (fusion protein) yields a single major band on a non-reducing polyacrylamide gel.
  • polypeptide of the present invention may be a recombinant polypeptide or a synthetic polypeptide, preferably a synthetic polypeptide.
  • sequence of the vaccine polypeptide is relatively short (such as ⁇ 70aa, more preferably ⁇ 60aa)
  • related peptide sequences can be directly synthesized by chemical methods.
  • the sequence of the vaccine polypeptide is relatively long or the vaccine polypeptide is provided in the form of a fusion protein
  • recombinant methods can also be used to obtain related peptide sequences in large quantities.
  • the coding sequence encoding the antigenic polypeptide or its fusion protein is cloned into a vector, then transformed into cells, and then the related antigenic polypeptide or fusion protein is isolated from the proliferated host cells by conventional methods.
  • the invention also provides a pharmaceutical composition.
  • the pharmaceutical compositions of the invention may be therapeutic or prophylactic (eg vaccines).
  • the pharmaceutical composition of the present invention includes an effective amount of the vaccine polypeptide or peptide collection of the present invention, or immune cells activated with the vaccine polypeptide (such as dendritic cells sensitized with the vaccine polypeptide of the present invention or dendritic cells induced with the vaccine polypeptide). T cells), and at least one pharmaceutically acceptable carrier, diluent or excipient.
  • these (vaccine) compositions comprise immunogenic antigens (including vaccine polypeptides of the invention, peptide collections or derivatives thereof), and are usually combined with "pharmaceutically acceptable carriers", which include Any carrier of antibodies harmful to the individual receiving the composition.
  • suitable carriers include, but are not limited to, proteins, lipid aggregates (such as oil droplets or liposomes), and the like. These vectors are well known to those of ordinary skill in the art.
  • these carriers can act as immunostimulants ("adjuvants").
  • compositions according to the invention may also contain additional adjuvants.
  • vaccine adjuvants include (but are not limited to) the following categories: inorganic adjuvants, such as aluminum hydroxide, alum, etc.; synthetic adjuvants, such as artificially synthesized double-stranded polynucleotides (double-stranded polyadenosine acid, uridine acid), levamisole, isoprinosine, etc.; oil agents, such as Freund's adjuvant, peanut oil emulsification adjuvant, mineral oil, vegetable oil, etc.
  • vaccine or immunogenic compositions are prepared as injectables, such as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the formulation can also be emulsified or encapsulated in liposomes for enhanced adjuvant effect.
  • compositions can be presented in unit or multiple dosage form.
  • Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients.
  • the formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, oral, or topical administration.
  • a safe and effective amount of a vaccine polypeptide or peptide collection of the invention is administered to a human, wherein the safe and effective amount is generally 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.
  • the vaccine polypeptides and polypeptide nanoemulsion vaccine preparations used in the present invention can produce high-level and persistent wild-type and multiple mutant RBD-specific binding antibodies in the body of primates, and block wild-type and mutant RBD binding neutralizing antibody to ACE2.
  • the antiserum of the polypeptide nanoemulsion vaccine preparation adopted in the present invention after immunizing primates has a high level of neutralizing activity to block wild strain pseudoviruses and live viruses from invading cells.
  • Example 1 Determination of antigenic polypeptides based on SARS-CoV-2 wild strain and mutant strain S protein
  • the inventors took the RBD region interacting with human ACE2 in the S protein of the wild strain and mutant SARS-CoV-2 as the analysis object, and predicted its immunodominant antigenic site.
  • the present inventors used software to predict and comprehensively analyze the immunogenicity of RBD sequences, mainly including MHC class II binding epitopes ( FIG. 3 ) and MHC class I binding epitopes ( FIG. 4 ).
  • the inventor finally screened 15 polypeptides, the amino acid sequence of which is as SEQ ID NO: 1-15 As shown, the screened vaccine polypeptides mainly correspond to the RBD region and adjacent regions, and the representative locations on the RBD of the wild strain and mutant SARS-CoV-2 are shown in Figure 5 and Figure 6, respectively.
  • polypeptide shown in SEQ ID NO: 1-15 was prepared using a polypeptide solid-phase synthesizer.
  • the synthetic peptides are identified and purified by HPLC to ensure correct properties and a purity of over 95%.
  • 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 polypeptide vaccine of the present invention induces a high level, SARS-CoV-2 specific, blocking activity and persistent humoral immune response in cynomolgus monkeys.
  • the droplet size of the prepared nanoemulsion is less than 220nm, to meet the Filter sterilization required.
  • the optimal prescription is:
  • Table 1 The prescription of nanoemulsion vaccines with different doses and types of polypeptides as antigens
  • the preferred prescription can adapt to different types and different dosages of polypeptides, and the particle size of the prepared nanoemulsion droplets is less than 220nm, which meets the requirements of filtration sterilization.
  • Embodiment 6 In vivo immune effect evaluation of polypeptide vaccine nanoemulsion preparation
  • LY54 and P67 1 mg each of LY54 and P67 was prepared into a nanoemulsion (LY54+P67 (1:1, F2)) according to the formula (F2) in Example 5. Cynomolgus monkeys were grouped into intramuscular injections and immunized on days 0, 14, and 28.
  • Example 7 The pseudovirus neutralization experiment of the antiserum of the polypeptide vaccine nanoemulsion immune cynomolgus monkey
  • a pseudovirus test system was used to evaluate the pseudovirus blocking neutralization activity of cynomolgus monkey antiserum.
  • pseudovirus neutralization test 100 ⁇ L of serum samples of different dilutions were mixed with 50 ⁇ L of the supernatant containing the pseudovirus of the wild strain of SARS-CoV-2. 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 hours. 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.
  • Example 8 The live virus neutralization experiment of the antiserum of the polypeptide vaccine nanoemulsion immune cynomolgus monkey
  • the cytopathic method of live virus was used to evaluate the activity of cynomolgus monkey antiserum. Virus blocking neutralizing activity.
  • SARS-CoV-2 field strain virus propagates in VERO E6 cells.
  • Serum samples (antiserum produced after immunization of cynomolgus monkeys with peptide vaccine preparation LY54 (Titermax)) were heat-inactivated at 56°C for 30 minutes; 50% tissue culture virus solution was mixed. 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
  • Example 9 Humoral immune response induced by polypeptide vaccine nanoemulsion against SARS-CoV-2 mutant strain
  • the peptide vaccine nanoemulsion can induce a high-level, protective and durable humoral immune response against wild-type SARS-CoV-2.
  • the present invention uses a variety of mutated RBD proteins and the B.1.1.7 mutant strain pseudovirus system to test the effect of the polypeptide vaccine.
  • the wild-type RBD was replaced by various mutant RBD proteins with N439K mutation, Y453F mutation, S477N mutation, and N501Y mutation for detection.
  • the present invention tested the blocking activity of the antiserum of the polypeptide vaccine nanoemulsion immunized cynomolgus monkeys against the mutant strain B.1.1.7 which is widely prevalent at present.
  • the present invention adopts the pseudovirus system of B.1.1.7, and the basic method is as described in Example 5.
  • the test found that the antiserum of cynomolgus monkeys has a high level of blocking activity against the mutant strain B.1.1.7, and the titer is as high as 1 : 512 (Fig. 16).
  • the polypeptides MP477, MP484 and MP501 determined for the screening of the mutant strain of SARS-CoV-2 in Example 1 were mixed with P67 to form a combined epitope polypeptide, and prepared Cynomolgus monkeys were immunized into nanoemulsions. Cynomolgus monkeys were immunized by intramuscular injection on days 0 and 14, and blood samples were collected on day 21 (7 days after the second immunization) to detect mutant RBD-specific binding antibodies in serum.
  • the high level of RBD-binding antibody induced by immunization of cynomolgus monkeys against the vaccine polypeptide of the mutant strain also had a high level of binding activity to the E484K mutant RBD with the immune escape effect of the new crown vaccine and neutralizing antibody drugs (Figure 17), It is suggested that the nanoemulsion of polypeptide vaccine can protect the body from the infection of mutant strain B.1.351 and mutant strain P.1 with E484K mutation.
  • the vaccine antiserum (7 days after the third immunization) was also identified to have the ability to neutralize the Omicron mutant strain, and the neutralization titer was 1:218 (Fig. 20), suggesting that the polypeptide vaccine nanoemulsion can protect the body against the infection of the Omicron mutant strain.
  • Peptide vaccine is a type of vaccine that can be rapidly designed and mass-produced against pathogenic proteins, and it is safe in nature, specific in action, and low in cost. Therefore, peptide vaccines are suitable for dealing with sudden outbreaks and mutations of pathogens.
  • the new crown vaccines that have been tested in clinical trials are all designed based on the wild strain of SARS-CoV-2. Studies have shown that the B.1.1.7 mutant strains, B.1.351 mutant strains, P.1 mutant strains, B.1.617 mutant strains and B.1.1.529 mutant strains that are currently popular on a large scale have the property of immune escape. This may make the existing new crown vaccines and neutralizing antibody drugs invalid and weaken the protective effect. Therefore, there is an urgent need for vaccine products that can deal with the mutant strain of the new crown.
  • the technical route of the polypeptide vaccine combination of the present invention is different from that of the existing COVID-19 vaccine, and it is equipped with an optimized nanoemulsion adjuvant preparation.
  • the immune effect of the polypeptide nanoemulsion vaccine of the present invention is still highly effective against the new crown mutant strain and can block The effect has been verified on the pseudovirus and live virus system of wild strains and mutant strains, and has a development prospect to deal with the prevalence of mutant strains.

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Abstract

公开了一种冠状病毒SARS-CoV-2疫苗多肽、多肽组合物及其纳米乳制剂在预防冠状病毒SARS-CoV-2野毒株和突变株感染中的应用。具体地，提供了具有衍生自SARS-CoV-2野毒株和突变株的S蛋白的氨基酸序列的冠状病毒SARS-CoV-2疫苗多肽，疫苗多肽可使机体产生针对SARS-CoV-2的高水平且持久的体液免疫反应，产生高滴度的RBD结合抗体和阻断RBD与ACE2结合的中和抗体。疫苗多肽可用于预防SARS-CoV-2野毒株和B.1.1.7、B.1.351、B.1.617、B.1.1.529等多种突变株的感染。

Description

一种新型冠状病毒疫苗多肽及其纳米乳制剂在预防新冠野毒株和突变株感染中的应用 技术领域
本发明涉及多肽药物和多肽疫苗领域，具体地，涉及新型冠状病毒多肽疫苗、多肽组合物及其纳米乳制剂在预防新冠野毒株和突变株感染中的应用。
背景技术
新型冠状病毒(SARS-CoV-2)在世界范围大规模传播，其不受控的流行严重威胁着全球公共卫生安全和经济发展，开发并应用安全、经济和高效的新冠疫苗以达成群体免疫，是阻遏疫情的根本途径。
现有的新冠疫苗主要采取以下技术路线：减毒活疫苗、灭活疫苗、重组病毒载体疫苗、重组蛋白疫苗和核酸疫苗。这些疫苗技术路线目前主要为针对SARS-CoV-2野毒株设计。
然而新冠肺炎疫情的持续失控，病毒在快速传播的同时也在快速进化，目前已有多种病毒突变株出现并流行，包括D614G突变、N501.V1(B.1.1.7)突变株、N501.V2(B.1.351)突变株、P.1突变株和Omicron突变株(B.1.1.529)等。一些突变株已表现出对现有疫苗和中和抗体药物的免疫逃逸，例如研究显示，科兴的灭活疫苗CoronaVac针对B.1.1.7突变株的抗体有显著下降，而辉瑞和阿斯利康的新冠疫苗对B.1.351突变株的中和活性下降8-9倍。新冠病毒的快速突变可能导致已开发的疫苗产品接种后难以预防新冠病毒突变株的感染，从而难以实现群体免疫控制新冠疫情。
因此，目前急需开发安全、高效、经济且可应对新冠病毒突变的第二代新冠疫苗，以最低经济代价实现大规模生产供给世界范围人民的疫苗接种需求，应对突变株的流行，实现世界范围群体免疫，有效控制新冠疫情。
发明内容
本发明目的就是提供一种新型冠状病毒多肽疫苗及其疫苗纳米乳制剂，该疫苗可在接种者中产生阻断型的抗SARS-CoV-2抗体，并可应对SARS-CoV-2野毒株和突变株感染。
在本发明的第一方面，提供了一种新型冠状病毒肺炎的疫苗多肽，所述的疫苗多肽具有衍生自SARS-CoV-2野毒株和突变株的S蛋白的氨基酸序列；
所述的疫苗多肽选自下组：
(a)具有SEQ ID No:1-15中任一所示的氨基酸序列的多肽；
(b)对(a)中多肽的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基 酸的取代或1-3个氨基酸缺失所形成的衍生多肽，所述衍生多肽与衍生前的原始多肽具有基本相同的功能。
在另一优选例中，所述的“基本相同的功能”指所述的衍生多肽具有基本相同的激发细胞免疫和体液免疫反应的免疫原性，以及可诱导灵长动物产生野生型RBD特异性的结合抗体和阻断野生型和/或突变型RBD与ACE2结合的中和抗体。
在另一优选例中，所述的SARS-CoV-2突变株包括但不限于D614G突变株、突变株B.1.1.7、突变株B.1.351、突变株P.1、突变株B.1.617和B.1.1.529突变株。
在另一优选例中，所述的疫苗多肽可诱导灵长动物产生细胞免疫和体液免疫。
在另一优选例中，所述的疫苗多肽可诱导灵长动物产生野生型RBD特异性的结合抗体和阻断野生型RBD与ACE2结合的中和抗体。
在另一优选例中，所述的野生型RBD为SARS-CoV-2野毒株的S蛋白的受体结合结构域。
在另一优选例中，所述的疫苗多肽可激发灵长动物产生突变的RBD特异性的结合抗体和阻断突变的RBD与ACE2结合的中和抗体。
在另一优选例中，所述的突变的RBD为SARS-CoV-2突变株的S蛋白的受体结合结构域。
在另一优选例中，所述的突变的RBD为在野生型RBD基础上发生了一个或多个氨基酸添加、一个或多个氨基酸的取代或一个或多个氨基酸缺失所形成的突变RBD蛋白。
在另一优选例中，所述的突变的RBD发生的突变包括但不限于以下的一种或多种：K417N、K417T、N439K、L452R、Y453F、S447N、E484Q、E484K和N501Y。
在另一优选例中，所述的疫苗多肽免疫灵长动物后的抗血清可阻断SARS-CoV-2野毒株侵入细胞。
在另一优选例中，所述的疫苗多肽免疫灵长动物后的抗血清可阻断SARS-CoV-2突变株侵入细胞。
在另一优选例中，所述的灵长动物包括人、非人灵长类动物(如猴)。
在另一优选例中，所述疫苗多肽的长度为8-100个氨基酸，较佳地10-80个氨基酸。
在另一优选例中，所述的疫苗多肽为人工合成的或重组的疫苗多肽。
在另一优选例中，所述的疫苗多肽的结构如式Ⅰ所示：
X1-X-X2          (Ⅰ)，
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:1-15(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；
(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)。
在另一优选例中，所述疫苗多肽选自表A：
表A疫苗多肽
Figure PCTCN2022096047-appb-000001
其中，SEQ ID No:3和5中的“ C(C)”表示肽链上的半胱氨酸与另一个非肽链上的半胱氨酸形成二硫键。
具体地，PSS的主链上的半胱氨酸的巯基与一个半胱氨酸的巯基形成二硫键结构，其结构和示意图见图1。
LPSS(也称为LP67-1)中主链上的3个半胱氨酸的巯基分别与半胱氨酸的巯基形成二硫键结构，其结构和示意图见图2。
在另一优选例中，SEQ ID No:6、7和8所示的三种疫苗多肽被组合使用，其中，SEQ ID No:6-8中的任何一种、二种或三种还可基于SARS-CoV-2的变异情况，及时引入相应的突变位点(例如图19中所示的落于SEQ ID No:6-8序列中的突变位点)，从而形成能够及时应对SARS-CoV-2的变异株(或突变株)，从而提高本发明疫苗对突变株的保护力。
一种代表性的更适合应对突变株的疫苗多肽集合(或组合)包括：SEQ ID No:9、10和11所示的疫苗多肽(分别引入了突变株的突变氨基酸)。
此外，本发明对应于野生型病毒株的如SEQ ID No:6、7和8所示的一种或多种疫苗多肽，也可与对应于突变株的一种或多种疫苗多肽(如SEQ ID No:9、10和11所示的疫苗多肽)进行组合，形成例如SEQ ID No:9、7和11所示的疫苗多肽集；或SEQ ID No:9、10和8所示的疫苗多肽集。
在另一优选例中，X1、X2各自独立地为无、K、C、G、L、A。
在另一优选例中，X1为无、K、或C。
在另一优选例中，X2为无、K、或C。
在另一优选例中，所述疫苗多肽具有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。
在另一优选例中，所述疫苗多肽具有SARS-CoV-2S蛋白RBD区的至少一个T细胞表位和/或至少一个B细胞表位。
在另一优选例中，所述疫苗多肽具有SARS-CoV-2S蛋白RBM区的至少一个T细胞表位和/或至少一个B细胞表位。
在另一优选例中，所述疫苗多肽具有SARS-CoV-2S蛋白NTD区的至少一个T细胞表位和/或至少一个B细胞表位。
在另一优选例中，所述的疫苗多肽具有至少一个T细胞表位，较佳地1、2、3或4个T细胞表位，更佳地1或2个T细胞表位。
在另一优选例中，所述的疫苗多肽具有至少一个B细胞表位，较佳地1、2、3或4个B细胞表位，更佳地1或2个B细胞表位。
在另一优选例中，所述的疫苗多肽具有1-2个T细胞表位和0-2个B细胞表位，较佳地1-2个T细胞表位和0-1个B细胞表位。
在本发明的第二方面，提供了一种分离的肽集合，所述的肽集合包括至少两种的本发明第一方面所述的新型冠状病毒肺炎的疫苗多肽。
在另一优选例中，所述的肽集合中至少含有2-15种(如2、3、4、5、6、7、8、9、10、11、12、13、14或15种)所述的疫苗多肽。
在另一优选例中，所述的肽集合包括选自下组的疫苗多肽：
(Z1)选自SEQ ID No:1和12中的1或2种疫苗多肽，和选自SEQ ID No:2-5中的1、2、3或4种疫苗多肽；
(Z2)选自SEQ ID No:6和9中的1或2种疫苗多肽，选自SEQ ID No:7和10中的1或2种疫苗多肽，和选自SEQ ID No:8和11中的1或2种疫苗多肽；
(Z3)选自SEQ ID No:1-12中的一种或多种(如1、2、3、4或5种)疫苗多肽，和选自SEQ ID No:13-15中的1、2或3种疫苗多肽。
在另一优选例中，所述的肽集合包括选自下组的疫苗多肽(或由自下组的疫苗多肽构成)：
(s1)SEQ ID No:1所示的疫苗多肽，和选自SEQ ID No:2所示的疫苗多肽；
(s2)选自SEQ ID No:1和12中的1或2种疫苗多肽，和选自SEQ ID No:2-5中的1、2、3或4种疫苗多肽，并且所述肽集合至少包括选自SEQ ID No:3、4、5和12的一种或多种疫苗多肽；
(s3)SEQ ID No:6、7和8所示的疫苗多肽；
(s4)SEQ ID No:9、10和11所示的疫苗多肽；
(s5)SEQ ID No:6、10和11所示的疫苗多肽；
(s6)SEQ ID No:9、7和11所示的疫苗多肽；
(s7)SEQ ID No:9、10和8所示的疫苗多肽；
(s8)SEQ ID No:2、9、10和11所示的疫苗多肽；
(s9)SEQ ID No:2、6、7和8所示的疫苗多肽。
在本发明的第三方面，提供了一种药物组合物，所述的药物组合物含有本发明第一方面所述的新型冠状病毒肺炎的疫苗多肽或本发明第二方面所述的肽集合和药学上可接受的载体。
在另一优选例中，所述的药物组合物为疫苗组合物。
在另一优选例中，所述疫苗组合物为单价或多价。
在另一优选例中，所述的药物组合物还含有佐剂，所选用的佐剂包括但不限于铝盐、Titermax、乳剂、脂质体、病毒载体。
在另一优选例中，所述的药物组合物包括单方药物、复方药物、或协同药物。
在另一优选例中，所述的药物组合物的剂型为液态、固体、或凝胶态。
在另一优选例中，所述的药物组合物用选自下组的方式施用：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、口服、或口鼻腔喷入和雾化吸入。
在本发明的第四方面，提供了一种疫苗制剂，所述疫苗制剂包含本发明第一方面所述的新型冠状病毒肺炎的疫苗多肽或第二方面所述的肽集合或第三方面所述的药物组合物，所述的疫苗制剂优选纳米乳制剂。
在另一优选例中，所述的疫苗制剂包含以角鲨烯、乳化剂为基础的纳米乳剂。
在另一优选例中，所述角鲨烯来源于鲨鱼肝脏，所述乳化剂是磷脂，聚山梨酯80，或者是聚山梨酯80和磷脂的组合，及蔗糖酯、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等的一种或多种。
在另一优选例中，所述的疫苗制剂包含0-20％的其他注射剂型中可接受的原辅料。所述的疫苗佐剂组合物包含α-生育酚，所述α-生育酚用量不超过15wt％；所述的疫苗佐剂组合物包含嵌段共聚物，所述嵌段共聚物可为甲氧基聚乙二醇聚乳酸羟基乙酸共聚物、甲氧基聚乙二醇聚乳酸-羟基乙酸、泊洛沙姆中的一种或多 种，含量不超过5％；还含有等渗调节剂，所述的等渗调节剂为0.1-8％(w/w)；所述的疫苗佐剂组合物还含有pH调节剂，所述的pH调节剂不超过1％(w/w)。
在本发明的第五方面，提供了本发明第一方面所述的新型冠状病毒肺炎的疫苗多肽或第二方面所述的肽集合或第三方面所述的药物组合物或第四发明所述的制剂的用途，用于制备预防冠状病毒SARS-CoV-2野毒株和突变株感染或其相关疾病的药物。
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)。
在本发明的第六方面，提供了一种细胞制剂，所述的细胞制剂包括(a)用本发明第一方面所述的疫苗多肽或本发明第二方面所述的肽集合免疫激活的免疫细胞；和(b)药学上可接受的载体。
在另一优选例中，所述的免疫细胞选自下组：树突状细胞、自然杀伤细胞NK、淋巴细胞、单核/巨噬细胞、粒细胞、或其组合。
在另一优选例中，所述的激活为体外激活。
在另一优选例中，所述的体外激活包括：在所述的疫苗多肽存在下，培养所述的免疫细胞一段时间(如6-48小时)，从而获得经免疫激活的免疫细胞。
在另一优选例中，所述的细胞制剂为含活细胞的液态制剂。
在另一优选例中，所述的细胞制剂通过静脉给药方式回输。
在本发明的第七方面，提供了一种产生针对冠状病毒SARS-CoV-2野毒株和突变株的免疫反应的方法，包括步骤：给需要的对象施用本发明第一方面所述的疫苗多肽、第二方面所述的肽集合、第三方面所述的药物组合物、或第四方面所述的制剂。
在另一优选例中，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述的非人哺乳动物包括非人灵长动物(如猴)。
在另一优选例中，所述方法在所述对象中诱导产生针对冠状病毒SARS-CoV-2野毒株和突变株的中和抗体。
在另一优选例中，所述中和抗体阻断冠状病毒SARS-CoV-2野毒株和突变株与人ACE2蛋白的结合。
在本发明的第八方面，提供了一种融合蛋白，所述的融合蛋白包括载体蛋白以及与所述融合蛋白融合的本发明第一方面所述的疫苗多肽。
在另一优选例中，所述的融合蛋白具有式Ⅲa或Ⅲb结构：
P1-P2         (Ⅲa)
P2-P1        (Ⅲb)
式中，P1为本发明第一方面所述的疫苗多肽，P2为载体蛋白。
在另一优选例中，所述的P1可以是单个疫苗多肽，也可以是串联的多个相同或不同的疫苗多肽(或抗原多肽)。
在本发明的第九方面，提供了一种药物组合物，它包括(a)用本发明第七方面所述的融合蛋白或被其免疫激活的免疫细胞；和(b)药学上可接受的载体。
在本发明的第十方面，提供了本发明第七方面所述的融合蛋白或第八方面所述的药物组合物的用途，它们被用于制备预防冠状病毒SARS-CoV-2感染或其相关疾病的药物。
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显示了多肽PSS的具体结构。
图2显示了多肽LPSS的具体结构。
图3显示了SARS-CoV-2的RBD区域的MHC二类分子表位分析结果。
图4显示了SARS-CoV-2的RBD区域的MHC一类分子表位分析结果。
图5显示了衍生自野生型RBD的疫苗多肽LY54、LY54mut、P67、PSS、LPSS和LP67在RBD蛋白中的定位。
图6显示了衍生自突变RBD的疫苗多肽MP1、MP2、MP3、MP477、MP484和MP501在RBD蛋白中的定位。
图7显示了疫苗多肽免疫食蟹猴后在第34天后产生了高水平的RBD特异性结合抗体。
图8显示了疫苗多肽免疫食蟹猴后在第34天后产生了高水平的可阻断RBD与ACE2作用的中和抗体。
图9显示了疫苗多肽免疫食蟹猴后在第105天后仍旧保留高水平的RBD特异性结合抗体。
图10显示了疫苗多肽免疫食蟹猴后在第105天后仍旧保留高水平的可阻断RBD与ACE2作用的中和抗体。
图11显示了多肽纳米乳制剂免疫食蟹猴后在第42天产生高水平的结合抗体。
图12显示了多肽纳米乳制剂免疫食蟹猴后在第42天后产生高水平的可阻断RBD与ACE2作用的中和抗体。
图13显示了多肽纳米乳制剂免疫食蟹猴后的抗血清具有阻断野毒株假病毒侵入细胞的高中和活性。
图14显示了疫苗多肽免疫食蟹猴后的抗血清具有阻断野毒株活病毒侵入细胞的高中和滴度。
图15显示了多肽纳米乳制剂免疫食蟹猴后产生了高水平的突变RBD特异性结合抗体。
图16显示了多肽纳米乳制剂免疫食蟹猴后的抗血清具有阻断突变株B.1.1.7假病毒侵入细胞的高中和活性。
图17显示了衍生自突变RBD的疫苗多肽MP477、MP484、MP501和P67混合免疫食蟹猴后产生了高水平的针对B.1.351和P.1突变株关键的RBD突变位点的特异性结合抗体。
图18显示了衍生自突变RBD的疫苗多肽MP477、MP484、MP501和P67混合免疫食蟹猴后产生了高水平的针对B.1.617突变株关键的RBD突变位点的特异性结合抗体。
图19显示了几种新冠突变株的S蛋白区域相较于野毒株的突变氨基酸位点和突变类型。
图20显示了多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。
具体实施方式
本发明人通过广泛而深入的研究，基于对SARS-CoV-2野毒株和多种突变株的S蛋白序列分析和S蛋白与宿主ACE2作用结构分析，分析了野毒株和突变株S蛋白的免疫原性，且分析了野毒株和突变株S蛋白与宿主ACE2蛋白作用的关键区域。本发明筛选并确定了可有效诱导灵长动物机体产生针对冠状病毒SARS-CoV-2的免疫反应的疫苗多肽。实验表明，本发明的疫苗多肽可使食蟹猴产生针对SARS-CoV-2的高水平且持久的体液免疫反应。本发明的疫苗多肽及多肽纳米乳疫苗制剂免疫食蟹猴后的抗血清具有高滴度的RBD结合抗体和阻断RBD与ACE2结合的中和抗体，并在SARS-CoV-2假病毒模型和活病毒模型上均显示出高水平的阻断作用。同时，与野生型RBD相比，食蟹猴抗血清针对S477N、E484K和N501Y等多种RBD突变蛋白的结合活性不受影响，且在B.1.1.7突变株假病毒模型显示高水平阻断作用，说明本发明的多肽疫苗可用于预防新型冠状病毒野毒株和B.1.1.7、B.1.351和B.1.1.529等多种突变株的感染。在此基础上完成了本发明。
术语
冠状病毒SARS-CoV-2
冠状病毒(Coronavirus，CoV)属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)，是一种有包膜的正链RNA病毒，其亚科包含α、β、δ及γ四属。目前已知的感染人的冠状病毒中，HCoV-229E和HCoV-NL63属于α属冠状病毒，HCoV-OC43、SARS-CoV、HCoV-HKU1、MERS-CoV和SARS-CoV-2均为β属冠状病毒。
2019年年底爆发的新型冠状病毒(SARS-CoV-2)与SARS-CoV有约80％相似性、与MERS-CoV有40％的相似性，也属于β属冠状病毒。
该类病毒的基因组是一条单股正链RNA，是基因组最大的RNA病毒之一，编码包括复制酶、刺突蛋白、囊膜蛋白、包膜蛋白和核壳蛋白等。在病毒复制的初始阶段，基因组被翻译成两条长达几千个氨基酸的肽链即前体多聚蛋白(Polyprotein)，随后前体蛋白被蛋白酶切割生成非结构蛋白(如RNA聚合酶和解旋酶)和结构蛋白(如刺突蛋白)及辅助蛋白。
S蛋白是冠状病毒SARS-CoV-2的一种主要的结构蛋白，其中，RBD负责与人ACE2受体结构，而RBM区域含有与人ACE2结合的基序(motif)。一种典型的S蛋白的氨基酸序列如SEQ ID No:16所示。
>S蛋白
Figure PCTCN2022096047-appb-000002
Figure PCTCN2022096047-appb-000003
冠状病毒SARS-CoV-2的RBD区域位于S蛋白的第333-527位，一种代表性的氨基酸序列如SEQ ID No:16中第333-527位所示。
>RBD(333-527)
Figure PCTCN2022096047-appb-000004
冠状病毒SARS-CoV-2的RBM区域位于S蛋白的第438-506位，一种代表性的氨基酸序列如SEQ ID No:17中第438-506位所示。
>RBM(438-506)
Figure PCTCN2022096047-appb-000005
应理解，在本发明中，S蛋白、RBD区域和RBM区域均包括野生型和突变型。
目前，SARS-CoV-2已出现区别于野毒株Wuhan-Hu-1的多种突变株并广泛流行。例如一种突变株称为B.1.1.7或501Y.V1，该突变株已在世界许多国家中检测到，可能具有更强的传染性和增加的死亡风险。另一种突变株称为B.1.351或501Y.V2，该突变株开始在南非以外国家流行，并表现出强力的疫苗和中和抗体免疫逃逸能力。突变株P.1，该突变株的免疫逃逸能力似乎还要强于突变株B.1.351。Omicron(B.1.1.529)突变株则表现出更为显著的免疫逃逸能力和传播能力。
部分突变株与野毒株的S蛋白区域对比的突变位置和突变类型如图19所示。
疫苗多肽
在本发明中，“本发明表位肽”、“本发明疫苗多肽”、“本发明抗原多肽”、“本发明多肽”可互换使用，指符合本发明第一方面中所述的疫苗多肽。应理解，所述术语不仅包括一种本发明疫苗多肽，还包括多种本发明的疫苗多肽所形成的肽集合(或肽组合)。
在本发明中，疫苗多肽还包括其他形式，例如药学上可接受的盐、偶联物、或融合蛋白。
在本发明中，疫苗多肽包括对SEQ ID No:1-15中任一序列进行一个或多个 (如1-5个，优选地1-3个)氨基酸添加、一个或多个(如1-5个，优选地1-3个)氨基酸的取代和/或1-3个氨基酸缺失所形成的衍生多肽，所述衍生多肽与衍生前的原始多肽具有基本相同的功能。
优选地，疫苗多肽包括对SEQ ID No:1-15中任一序列经过1-3个氨基酸添加(优选添加在N端或C端)、和/或1-2个氨基酸的取代(优选保守性氨基酸替换)并仍具有与衍生前的原始多肽具有基本相同的功能。
优选地，所述的保守性氨基酸替换根据表C进行氨基酸替换。
表C
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
在另一优选例中，SEQ ID No:6、7和8所示的三种疫苗多肽被组合使用，其中，SEQ ID No:6-8中的任何一种、二种或三种还可基于SARS-CoV-2的变异情况，及时引入相应的突变位点(例如图19中所示的落于SEQ ID No:6-8序列中的突变位点)，从而形成能够及时应对SARS-CoV-2的变异株(或突变株)，从而提高本发明疫苗对突变株的保护力。
一种代表性的更适合应对突变株的疫苗多肽集合(或组合)包括：SEQ ID No:9、10和11所示的疫苗多肽(分别引入了突变株的突变氨基酸)。
此外，本发明对应于野生型病毒株的如EQ ID No:6、7和8所示的一种或多种疫苗多肽，也可与对应于突变株的一种或多种疫苗多肽(如SEQ ID No:9、10 和11所示的疫苗多肽)进行组合，形成例如SEQ ID No:9、7和11所示的疫苗多肽集；或SEQ ID No:9、10和8所示的疫苗多肽集。
如本文所用，术语“肽集合”所述的肽集合由至少两种本发明疫苗多肽或其衍生多肽构成。
优选地，本发明所述的肽集合中至少含有2、3、4、5、6、7、8、9、10、11、12、13、14或15种选自本发明第一方面所述的疫苗多肽或其衍生多肽(包括偶联肽)；更优选地，所述肽集合至少含有2、3、4、5、6、7、8、9、10、11、12、13、14或15种选自SEQ ID No:1-15的疫苗多肽或其衍生多肽。此外，所述的肽集合还包括可以除SEQ ID No:1-15以外的其他冠状病毒SARS-CoV-2的抗原肽或蛋白。
在另一优选例中，所述的肽集合包括选自下组的疫苗多肽：
(Z1)选自SEQ ID No:1和12中的1或2种疫苗多肽，和选自SEQ ID No:2-5中的1、2、3或4种疫苗多肽；
(Z2)选自SEQ ID No:6和9中的1或2种疫苗多肽，选自SEQ ID No:7和10中的1或2种疫苗多肽，和选自SEQ ID No:8和11中的1或2种疫苗多肽；
(Z3)选自SEQ ID No:1-12中的一种或多种(如1、2、3、4或5种)疫苗多肽，和选自SEQ ID No:13-15中的1、2或3种疫苗多肽。
在另一优选例中，所述的肽集合包括选自下组的疫苗多肽(或由自下组的疫苗多肽构成)：
(s1)SEQ ID No:1所示的疫苗多肽，和选自SEQ ID No:2所示的疫苗多肽；
(s2)选自SEQ ID No:1和12中的1或2种疫苗多肽，和选自SEQ ID No:2-5中的1、2、3或4种疫苗多肽，并且所述肽集合至少包括选自SEQ ID No:3、4、5和12的一种或多种疫苗多肽；
(s3)SEQ ID No:6、7和8所示的疫苗多肽；
(s4)SEQ ID No:9、10和11所示的疫苗多肽；
(s5)SEQ ID No:6、10和11所示的疫苗多肽；
(s6)SEQ ID No:9、7和11所示的疫苗多肽；
(s7)SEQ ID No:9、10和8所示的疫苗多肽；
(s8)SEQ ID No:2、9、10和11所示的疫苗多肽；
(s9)SEQ ID No:2、6、7和8所示的疫苗多肽。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多肽是没有分离纯化的，但同样的多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的肽”是指本发明多肽基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化本发明多肽。基本上纯化的多肽(融合蛋白)在非还原聚丙烯酰胺凝胶上能产生 单一的主带。
本发明的多肽可以是重组多肽、或合成多肽，优选合成多肽。
在本发明中，当疫苗多肽的序列较短(如≤70aa，更佳地≤60aa时)，可用化学方法直接合成相关肽序列。
当疫苗多肽的序列较长或以融合蛋白形式提供疫苗多肽时，也可以用重组法来大批量地获得相关肽序列。这通常是将编码所述抗原多肽或其融合蛋白的编码序列克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到相关的抗原多肽或融合蛋白。
药物组合物和给药方式
本发明还提供了一种药物组合物。本发明的药物组合物可以是治疗性的或预防性的(如疫苗)。本发明的药物组合物包括有效量的本发明的疫苗多肽或肽集合、或用该疫苗多肽激活的免疫细胞(例如用本发明疫苗多肽致敏的树突状细胞或用树突状细胞诱导的T细胞)，以及至少一种药学上可接受的载体、稀释剂或赋形剂。
在本发明中，这些(疫苗)组合物包含免疫性抗原(包括本发明疫苗多肽、肽集合或其衍生物)，并且通常与“药学上可接受的载体”组合，这些载体包括本身不诱导产生对接受该组合物的个体有害的抗体的任何载体。合适的载体的例子包括(但并不限于)蛋白质、脂质凝集物(如油滴或脂质体)等。这些载体是本领域普通技术人员所熟知的。另外，这些载体可起免疫刺激剂(“佐剂”)作用。
此外，本发明的(疫苗)组合物还可含有额外的佐剂。代表性的疫苗佐剂包括(但并不限于)以下种类：无机佐剂，如氢氧化铝，明矾等；合成佐剂，如人工合成的双链多聚核苷酸(双链多聚腺苷酸、尿苷酸)、左旋咪唑、异丙肌苷等；油剂，如弗氏佐剂、花生油乳化佐剂、矿物油、植物油等。
通常，可将疫苗组合物或免疫原性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液、液体赋形剂的固体形式。该制剂还可乳化或包封在脂质体中，以增强佐剂效果。
组合物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。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：静脉内、肌内、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。
本发明的主要优点包括：
(a)本发明所采用的疫苗多肽以及多肽纳米乳疫苗制剂能够在灵长动物的机体内产生高水平且持久的野生型和多种突变的RBD特异性结合抗体，和阻断野生型和突变的RBD与ACE2结合的中和抗体。
(b)本发明所采用的多肽纳米乳疫苗制剂免疫灵长动物后的抗血清具有阻断野毒株假病毒和活病毒侵入细胞的高水平中和活性。
(c)本发明所采用的多肽纳米乳疫苗制剂免疫灵长动物后的抗血清具有阻断突变株B.1.1.7假病毒侵入细胞的高水平中和活性。
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基于SARS-CoV-2野毒株和突变株S蛋白的抗原多肽确定
在本实施例中，本发明人以野毒株和突变株SARS-CoV-2的S蛋白中与人ACE2相互作用的RBD区域作为分析对象，预测了其免疫显性的抗原部位。
本发明人利用软件预测综合分析了RBD序列的免疫原性情况，主要包括MHC二类分子结合表位(图3)和MHCⅠ类分子结合表位(图4)。
根据以上针对野毒株和突变株SARS-CoV-2的S蛋白免疫原性分析，以及肽段的空间位置等，本发明人最终筛选到15条多肽，氨基酸序列如SEQ ID NO:1-15所示，筛选得到的疫苗多肽主要对应于RBD区以及邻近区域，在野毒株和突变株SARS-CoV-2的RBD上的代表性定位分别如图5和图6所示。
实施例2多肽的制备
在本实施例中，应用多肽固相合成仪，制备SEQ ID NO:1-15示多肽。合成的多肽经由HPLC鉴定和纯化，确保性质正确，且纯度在95％以上。
实施例3多肽疫苗的食蟹猴免疫实验
在本实施例中，进一步在食蟹猴中验证多肽疫苗对SARS-CoV-2的免疫效果。
以(a)LY54+P67,(b)LY54+LP67,(c)LY54，分别以TiterMax佐剂制备免疫制剂，采用在第0天和第14天给与食蟹猴肌肉注射多肽疫苗制剂的免疫程序。疫苗多肽的免疫剂量为1mg/只。免疫后的食蟹猴血清用于RBD结合抗体和中和抗体的检测，以及SARS-CoV-2假病毒和活病毒的入侵阻断测试。
实施例4多肽疫苗诱导的体液免疫反应检测
为评估多肽疫苗在食蟹猴体内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：多肽疫苗免疫后的食蟹猴产生了高水平的RBD特异性结合抗体，在免疫后的第34天，多肽疫苗免疫后食蟹猴的血清中RBD特异性结合抗体滴度高达1:72900(图7)。而且多肽疫苗免疫诱导了高水平的阻断RBD与ACE2结合的中和抗体，在免疫后的第34天，多肽疫苗免疫后食蟹猴的血清中中和抗体滴度高达1：160(图8)。意外地，即使在免疫的三个月后(第105天)检测食蟹猴血清抗体，食蟹猴血清中仍保留高达1：24300滴度的RBD特异性结合抗体(图9)。出乎意料的是，三个月后，食蟹猴血清中的中和抗体滴度不降反升，高达1：320(图10)。
因此，本发明的多肽疫苗在食蟹猴体内诱导产生了高水平的、SARS-CoV-2特异性的、具有阻断活性的和持久的体液免疫反应。
实施例5多肽疫苗纳米乳制剂的开发
在固定量的多肽中，加入2.0-5.0％的角鲨烯，0.0-4.0％乳化剂，0-20％注射剂型中可接受的原辅料，制备纳米乳的液滴粒径小于220nm，以符合过滤除菌要求。
其中，最优处方为：
Figure PCTCN2022096047-appb-000006
并以此配方(命名为F2)进行不同剂量及种类多肽为抗原的纳米乳剂疫苗的制备(表一)。
表1以不同剂量及种类多肽为抗原的纳米乳剂疫苗的处方
Figure PCTCN2022096047-appb-000007
按照表1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多肽，制备纳米乳液滴粒径小于220nm，符合过滤除菌要求。
实施例6多肽疫苗纳米乳制剂的体内免疫效果评价
LY54和P67各1mg，并按照实施例5中的配方(F2)制备成纳米乳剂(LY54+P67(1:1,F2))。将食蟹猴分组进行肌肉注射，在第0、14、28天免疫。
食蟹猴免疫后的血清中RBD特异性结合抗体和中和抗体水平检测显示多肽疫苗纳米乳制剂免疫后42天诱导了高水平的体液免疫反应，血清中RBD结合抗体的滴度高达1:24300(图11)，中和抗体的滴度高达1:1280(图12)。这说明，本发明的多肽疫苗纳米乳制剂可在体内诱导高水平保护性的体液免疫反应，预期可保护机体免受病毒感染。
实施例7多肽疫苗纳米乳剂免疫食蟹猴的抗血清的假病毒中和实验
为了进一步评价多肽疫苗纳米乳剂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野毒株假病毒的上清液混合。该混合物在37℃下孵育1小时。然后 将100微升的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。
结果表明：多肽疫苗纳米乳剂免疫后的食蟹猴血清具有高水平的中和活性，可以阻断野毒株假病毒入侵宿主细胞，滴度高达1：256(图13)。
实施例8多肽疫苗纳米乳剂免疫食蟹猴的抗血清的活病毒中和实验
在本实施例中，为了进一步真实模拟多肽疫苗纳米乳剂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，采用活病毒的细胞病变法评估食蟹猴抗血清的活病毒阻断中和活性。
SARS-CoV-2野毒株病毒在VERO E6细胞中增殖。将血清样品(经多肽疫苗制剂LY54(Titermax)免疫食蟹猴后产生的抗血清)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。
结果表明：即使在免疫后的三个月，多肽疫苗纳米乳剂免疫后的食蟹猴血清具有高水平的中和活性，可以阻断活病毒入侵宿主细胞，滴度高达1：37(图14)。
实施例9多肽疫苗纳米乳剂诱导的体液免疫反应应对SARS-CoV-2突变株
以上结果表明多肽疫苗纳米乳剂可以诱导针对野生型SARS-CoV-2的高水平、保护性的且持久的体液免疫反应。为了评估多肽疫苗诱导的体液免疫反应能否应对SARS-CoV-2突变株，本发明采用了多种突变的RBD蛋白和B.1.1.7突变株假病毒体系测试了多肽疫苗的作用。
首先，采用基于实施例4的RBD结合抗体检测方法，将野生型RBD替换为N439K突变、Y453F突变、S477N突变、N501Y突变的多种突变型RBD蛋白进行检测。
结果表明，多肽疫苗纳米乳剂LY54+P67(1:1,F2)免疫食蟹猴后(第29天)，食蟹猴的抗血清与野生型RBD的结合能力对比与N439K突变、Y453F突变、S477N突变、N501Y突变的突变型RBD蛋白的结合能力均无差距(图15)，这说明多肽疫苗免疫食蟹猴诱导的高水平体液免疫反应不受以上RBD蛋白突变的影响，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。
进一步地，本发明测试了多肽疫苗纳米乳剂免疫食蟹猴后的抗血清对目前广泛流行的突变株B.1.1.7的阻断活性。本发明采用B.1.1.7的假病毒体系，基本方法如实施例5中描述，测试发现食蟹猴的抗血清针对突变株B.1.1.7具有高水平的 阻断活性，滴度高达1：512(图16)。对比食蟹猴抗血清对野毒株假病毒的中和作用，说明，突变株B.1.1.7对本发明的多肽疫苗不会产生免疫逃逸，本发明多肽疫苗可保护机体免受该突变株感染。
为了进一步提高本发明多肽疫苗纳米乳剂对于新冠病毒突变株的保护作用，采用实施例1中针对SARS-CoV-2突变株筛选确定的多肽MP477、MP484和MP501与P67混合形成组合表位多肽，制备成纳米乳剂后免疫食蟹猴。在第0天和第14天免疫肌肉注射免疫食蟹猴，在第21天(第二次免疫后7天)取血样，检测血清中的突变型RBD特异性的结合抗体。
意外地，针对突变株的疫苗多肽免疫食蟹猴诱导的高水平的RBD结合抗体对具有新冠疫苗和中和抗体药物免疫逃逸作用的E484K突变的RBD也具有高水平的结合活性(图17)，提示多肽疫苗纳米乳剂可保护机体免受具有E484K突变的突变株B.1.351和突变株P.1感染。
另外，针对突变株的疫苗多肽免疫食蟹猴后诱导的高水平的RBD结合抗体对具有新冠疫苗和中和抗体药物免疫逃逸作用的B.1.617突变株关键突变位点L452R和E484Q突变的RBD也具有高水平的结合活性(图18)，提示多肽疫苗纳米乳剂可保护机体免受具有L452R和E484Q突变的B.1.617突变株感染。
同时，通过Omicron(B.1.1.529)假病毒模型，本疫苗抗血清(第三次免疫后7天)也被鉴定具备中和Omicron突变株的能力，中和滴度为1：218(图20)，提示本多肽疫苗纳米乳剂可保护机体应对Omicron突变株的感染。
讨论
多肽疫苗是一种可针对病原体蛋白快速设计和大规模工业生产的疫苗类型，且其性质安全，作用特异，成本低廉。因此，多肽疫苗适合于应对突发疫情和病原体突变。
目前已经通过临床试验测试的新冠疫苗，均是基于SARS-CoV-2野毒株设计的。已有研究表明，目前大规模流行的B.1.1.7突变株、B.1.351突变株、P.1突变株、B.1.617突变株和B.1.1.529突变株等具有免疫逃逸的性质，这可能使得已有的新冠疫苗和中和抗体药物失效和保护作用减弱。因此，目前急需可以应对新冠突变株的疫苗产品。本发明的多肽疫苗组合的技术路线不同于现有新冠疫苗的技术路线，并且配合有优化的纳米乳佐剂制剂，本发明的多肽纳米乳疫苗的免疫效果针对新冠突变株仍然高效，可阻断效果已在野毒株和突变株的假病毒和活病毒体系上得到验证，具备应对突变株流行的开发前景。
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。
疫苗多肽的序列
Figure PCTCN2022096047-appb-000008
(注：SEQ ID No:13-15来自于S蛋白的NTD区域)

Claims (15)

  1. 一种冠状病毒SARS-CoV-2的疫苗多肽，其特征在于，所述的疫苗多肽具有衍生自SARS-CoV-2野毒株和突变株的S蛋白的氨基酸序列；
    所述的疫苗多肽选自下组：
    (a)具有SEQ ID No:1-15中任一所示的氨基酸序列的多肽；
    (b)对(a)中多肽的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生多肽，所述衍生多肽与衍生前的原始多肽具有基本相同的功能。
  2. 如权利要求1所述的疫苗多肽，其特征在于，所述的疫苗多肽可诱导灵长动物产生野生型和突变的RBD特异性的结合抗体和阻断野生型和突变的RBD与ACE2结合的中和抗体。
  3. 如权利要求1所述的疫苗多肽，其特征在于，所述的SARS-CoV-2突变株选自下组：D614G突变株、B.1.1.7突变株、B.1.351突变株、P.1突变株、B.1.617突变株和B.1.1.529突变株或其组合。
  4. 如权利要求1所述的疫苗多肽，其特征在于，所述的疫苗多肽的结构如式Ⅰ所示：
    X1-X-X2    (Ⅰ)，
    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:1-15(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；
    (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)。
  5. 如权利要求1-4任一项所述的疫苗多肽，其特征在于，所述疫苗多肽选自表A：
    表A疫苗多肽
    Figure PCTCN2022096047-appb-100001
    Figure PCTCN2022096047-appb-100002
    其中，SEQ ID No:3和5中的“ C(C)”表示肽链上的半胱氨酸与另一个非肽链上的半胱氨酸形成二硫键。
  6. 如权利要求4所述的疫苗多肽，其特征在于，X1、X2各自独立地为无、K、C、G、L、A。
  7. 如权利要求1-4任一项所述的疫苗多肽，其特征在于，所述疫苗多肽具有SARS-CoV-2 S蛋白的至少一个T细胞表位和/或至少一个B细胞表位。
  8. 一种分离的肽集合，其特征在于，所述的肽集合包括至少两种权利要求1所述的冠状病毒SARS-CoV-2的疫苗多肽。
  9. 如权利要求8所述的肽集合，其特征在于，所述的肽集合包括选自下组的疫苗多肽：
    (Z1)选自SEQ ID No:1和12中的1或2种疫苗多肽，和选自SEQ ID No:2-5中的1、2、3或4种疫苗多肽；
    (Z2)选自SEQ ID No:6和9中的1或2种疫苗多肽，选自SEQ ID No:7和10中的1或2种疫苗多肽，和选自SEQ ID No:8和11中的1或2种疫苗多肽；
    (Z3)选自SEQ ID No:1-12中的一种或多种疫苗多肽，和选自SEQ ID No:13-15中的1、2或3种疫苗多肽。
  10. 如权利要求8所述的肽集合，其特征在于，所述的肽集合包括选自下组的 疫苗多肽：
    (s1)SEQ ID No:1所示的疫苗多肽，和选自SEQ ID No:2所示的疫苗多肽；
    (s2)选自SEQ ID No:1和12中的1或2种疫苗多肽，和选自SEQ ID No:2-5中的1、2、3或4种疫苗多肽，并且所述肽集合至少包括选自SEQ ID No:3、4、5和12的一种或多种疫苗多肽；
    (s3)SEQ ID No:6、7和8所示的疫苗多肽；
    (s4)SEQ ID No:9、10和11所示的疫苗多肽；
    (s5)SEQ ID No:6、10和11所示的疫苗多肽；
    (s6)SEQ ID No:9、7和11所示的疫苗多肽；
    (s7)SEQ ID No:9、10和8所示的疫苗多肽；
    (s8)SEQ ID No:2、9、10和11所示的疫苗多肽；
    (s9)SEQ ID No:2、6、7和8所示的疫苗多肽。
  11. 一种药物组合物，其特征在于，所述药物组合物包含：
    权利要求1所述的冠状病毒SARS-CoV-2的疫苗多肽或权利要求8所述的肽集合，和药学上可接受的载体。
  12. 一种冠状病毒SARS-CoV-2疫苗多肽纳米乳制剂，其特征在于，所述制剂包含：
    (a)权利要求1-7任一项所述的冠状病毒SARS-CoV-2疫苗多肽；
    (b)佐剂，较佳地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。
  13. 一种权利要求1-7任一项所述的疫苗多肽或权利要求8所述的肽集合，权利要求11所述的药物组合物或权利要求12所述的制剂的用途，其特征在于，用于制备预防冠状病毒SARS-CoV-2野毒株和突变株感染或其相关疾病的药物。
  14. 一种细胞制剂，所述的细胞制剂包括(a)用如权利要求1-7任一项所述的疫苗多肽或如权利要求8所述的肽集合免疫激活的免疫细胞；和(b)药学上可接受的载体。
  15. 一种产生针对冠状病毒SARS-CoV-2野毒株和突变株的免疫反应的方法，包括步骤：给需要的对象施用如权利要求1-7任一项所述的疫苗多肽、权利要求8所述的肽集合、权利要求11所述的药物组合物或权利要求12所述的制剂。
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