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WO2022171182A1 - 一种治疗或者预防冠状病毒病突变株的疫苗试剂 - Google Patents

一种治疗或者预防冠状病毒病突变株的疫苗试剂 Download PDF

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WO2022171182A1
WO2022171182A1 PCT/CN2022/075895 CN2022075895W WO2022171182A1 WO 2022171182 A1 WO2022171182 A1 WO 2022171182A1 CN 2022075895 W CN2022075895 W CN 2022075895W WO 2022171182 A1 WO2022171182 A1 WO 2022171182A1
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Prior art keywords
amino acid
seq
polypeptide
polynucleotide
rna
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PCT/CN2022/075895
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English (en)
French (fr)
Inventor
张军
李航文
张育坚
姚卫国
林昂
赵凡凡
马晓颦
黄雷
张静
张玉强
章小铃
沈明云
陈春秀
沈海法
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斯微(上海)生物科技股份有限公司
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Priority to CN202280003537.6A priority Critical patent/CN115427432A/zh
Publication of WO2022171182A1 publication Critical patent/WO2022171182A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • 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
    • 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

Definitions

  • the present invention relates to the field of biomedicine, in particular to a vaccine for preventing or treating coronavirus infection.
  • the present invention provides polypeptides, polynucleotides (especially mRNAs) encoding the same, and compositions for preventing or treating novel coronavirus infection.
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic. SARS-CoV-2 has the characteristics of high transmissibility and high lethality, which can cause severe viral pneumonia and respiratory disease in infected people, called “coronavirus disease 2019 (COVID-19)".
  • SARS-CoV-2 A variety of vaccines against SARS-CoV-2 have been developed, including inactivated virus vaccines, viral vector-based vaccines, recombinant protein vaccines, DNA vaccines, and mRNA vaccines.
  • SARS-CoV-2 has high variability, and multiple mutant strains have been developed, and some of them have shown high immune escape characteristics, posing new challenges to existing vaccines. There is an urgent need for drugs and methods for preventing and/or treating coronavirus infection.
  • CN111218458A discloses an mRNA encoding SARS-CoV-2 virus antigen, a vaccine and a preparation method of the vaccine, the mRNA encoding SARS-CoV-2 virus antigen at least contains S protein and N protein encoding SARS-CoV-2 virus.
  • the present invention provides a polypeptide comprising the S1 subunit and the S2 subunit of the SARS-CoV-2 S protein from the N-terminus to the C-terminus, wherein the S1 subunit comprises an inactive furin cleavage site, the The inactive furin cleavage site is located at the C-terminus of the S1 subunit and has the amino acid sequence of QSAQ.
  • the amino acid of the polypeptide at the positions corresponding to amino acids 986 and 987 of SEQ ID NO: 1 is proline. In one embodiment, the amino acids of the polypeptide at positions corresponding to amino acids 383 and 985 of SEQ ID NO: 1 are cysteine. In one embodiment, the amino acid of the polypeptide at the positions corresponding to amino acids 817, 892, 899 and 942 of SEQ ID NO: 1 is proline.
  • the amino acid of the polypeptide at a position corresponding to amino acid 614 of SEQ ID NO: 1 is glycine.
  • the amino acid of the polypeptide at the position corresponding to amino acid 614 of SEQ ID NO: 1 is glycine
  • the amino acid at the position corresponding to amino acid 417 of SEQ ID NO: 1 is asparagine
  • the amino acid at the position corresponding to amino acid 484 of SEQ ID NO:1 is lysine
  • the amino acid at the position corresponding to amino acid 501 of SEQ ID NO:1 is tyrosine
  • the amino acid at the position corresponding to amino acid 501 of SEQ ID NO:1 is tyrosine.
  • amino acid at the position of amino acid 80 is alanine
  • amino acid at the position corresponding to amino acid 215 of SEQ ID NO:1 is glycine
  • amino acid at the position corresponding to amino acid 701 of SEQ ID NO:1 is valine amino acid.
  • polypeptide further comprises one or more of the following amino acid modifications:
  • the polypeptide comprises at least 80%, 85%, 90%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical. In one embodiment, the polypeptide comprises the amino acid sequence of amino acids 14-1273, 15-1273, 16-1273, or 17-1273 of any one of SEQ ID NOs: 2-7. In one embodiment, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-7.
  • the present invention provides a polynucleotide encoding the polypeptide of the present invention.
  • the polynucleotide is RNA, and optionally the RNA is modified by comprising one or more modified nucleosides. In one embodiment, the RNA is modified by replacing one or more uracils with 1-methylpseudouracil, pseudouracil, 5-methyl-uracil, or a combination thereof. In one embodiment, the RNA comprises the nucleotide sequence of any one of SEQ ID NOs: 8-13.
  • the RNA further comprises one or more of a 5' cap, 5' UTR, 3' UTR and a poly(A) sequence.
  • the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 14-19.
  • the present invention provides a composition comprising a polynucleotide of the present invention and a lipid that encapsulates the polynucleotide.
  • the composition comprises lipid nanoparticles or lipid multimeric complexes.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptide, polynucleotide, composition or vaccine formulation of the present invention; and a pharmaceutically acceptable carrier.
  • the present invention provides polypeptides, polynucleotides, compositions, vaccine formulations or pharmaceutical compositions of the present invention for preventing and/or treating SARS-CoV-2 infection.
  • the present invention also provides the use of the polypeptide, polynucleotide, composition, vaccine preparation or pharmaceutical composition of the present invention in the preparation of a medicament for preventing and/or treating SARS-CoV-2 infection.
  • the present invention also provides a method for preventing and/or treating SARS-CoV-2 infection in a subject, the method comprising administering a therapeutically effective amount of a polypeptide, polynucleotide (especially RNA) of the present invention , composition or pharmaceutical composition.
  • Figure 1 shows a schematic diagram of the structure of the DNA template sequence in the constructed plasmid. From the 5' end to the 3' end, it includes: T7 promoter, 5' UTR, ORF, 3' UTR and poly(A) tail.
  • Figure 2 shows the expression results of candidate mRNAs in DC2.4 cells analyzed by flow cytometry.
  • the expressions “comprising”, “comprising”, “containing” and “having” are open ended and mean that recited elements, steps or components are included but not excluded from other unrecited elements, steps or components.
  • the expression “consisting of” excludes any element, step or component not specified.
  • the expression “consisting essentially of” means that the scope is limited to the specified elements, steps or components, plus optional elements, steps or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions “consisting essentially of” and “consisting of” are encompassed within the meaning of the expression “comprising”.
  • polypeptide refers to a polymer comprising two or more amino acids covalently linked by peptide bonds.
  • a “protein” may comprise one or more polypeptides, wherein the polypeptides interact covalently or non-covalently. Unless otherwise indicated, “polypeptide” and “protein” are used interchangeably.
  • wild-type (WT) in reference to a polypeptide means that the polypeptide is naturally occurring and has not been artificially modified, including naturally occurring mutants.
  • a "variant" of a reference polypeptide refers to a polypeptide that differs from the reference polypeptide by virtue of at least one amino acid modification.
  • the reference polypeptide can be naturally occurring or a modified form of the wild-type polypeptide.
  • polypeptide variant and “mutant polypeptide” have the same meaning.
  • Polypeptide variants can be, for example, mutants, post-translationally modified variants, isoforms, species variants, species homologues, and the like.
  • Polypeptide variants can be prepared by recombinant DNA techniques, for example by modifying known amino acid sequences by altering the coding sequence.
  • Polypeptide variants can also be prepared by chemical synthesis or enzymatic methods.
  • the S protein variant may have an ability to induce an immune response comparable to or higher than that of the wild-type S protein, ie, exhibit an equivalent or enhanced immunogenicity to that of the wild-type S protein.
  • modifications to amino acid sequences can include, for example, amino acid substitutions, additions and/or deletions.
  • amino acid addition refers to the addition of one or more amino acids to an amino acid sequence. Amino acid additions can occur anywhere in the amino acid sequence, including but not limited to the middle, amino-terminal and/or carboxy-terminal ends of the amino acid sequence. Amino acid additions that occur in the middle of an amino acid sequence may also be referred to as "amino acid insertions.”
  • Amino acid deletion refers to the removal of one or more amino acids from an amino acid sequence. Amino acid deletions can occur anywhere in the amino acid sequence.
  • amino acid deletions that occur at the N- and/or C-terminus can also be referred to as truncations. Truncated variants may also be referred to as "fragments.”
  • amino acid substitution refers to the replacement of an amino acid residue at a particular amino acid position with another amino acid residue.
  • amino acid modification may also be referred to as "mutation”. Preference is given to conservative substitutions at non-conservative amino acid positions between homologous polypeptides. Preferably, amino acid substitutions in polypeptide variants are conservative amino acid substitutions.
  • amino acid X aa at amino acid position N of a given amino acid sequence (ie, the N-th amino acid is X aa , where N is an integer greater than or equal to 1) may be represented as "NX aa ".
  • Substituting amino acid Xbb for amino acid NXaa in a given amino acid sequence can be represented as " XaaNXbb " .
  • the amino acid sequence of a reference polypeptide or a portion of an amino acid sequence can be determined by optimally aligning the amino acid sequences of the two with the amino acid sequence of another polypeptide (eg, as described herein) domains) or between specified amino acid positions in both.
  • a polypeptide variant comprising an amino acid substitution at the amino acid N corresponding to the reference polypeptide or "a polypeptide variant comprising an amino acid substitution compared to the reference polypeptide” means that the polypeptide variant is identical to the reference polypeptide at the point corresponding to the reference polypeptide.
  • the amino acid position N of the polypeptide contains a different amino acid, but the amino acid at other positions of the polypeptide variant is not limited, that is, the amino acid at other positions may be the same as or different from the amino acid at the corresponding position in the reference polypeptide).
  • the polypeptide variant has an amino acid at the position corresponding to amino acid N of the reference polypeptide is X aa ", only means that the amino acid of the polypeptide variant at the amino acid position N corresponding to the reference polypeptide is X aa , but for Amino acids at other positions of the polypeptide variant are not limiting.
  • % identity or “percent identity” in reference to sequences refer to the percentage of nucleotides or amino acids that are identical in an optimal alignment between the sequences being compared. Differences between two sequences can be distributed over local regions (segments) or the entire length of the sequences being compared. The percent identity between two sequences is usually determined after optimal alignment of segments or "comparison windows". Optimal alignment can be performed manually, or with the aid of algorithms known in the art, including but not limited to those described in Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443 Homology algorithm, similarity search method described in Pearson and Lipman, 1988, Proc.
  • Percent identity is obtained by determining the number of identical positions corresponding to the sequences being compared, dividing this number by the number of positions being compared (eg, in the reference sequence), and multiplying this result by 100.
  • the degree of identity is given to a region of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. In some embodiments, the degree of identity is given over the entire length of the reference sequence.
  • Alignment to determine sequence identity can be performed using tools known in the art, preferably using optimal sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • nucleotides include deoxyribonucleotides and ribonucleotides and derivatives thereof.
  • ribonucleotide refers to a nucleotide having a hydroxyl group at the 2' position of a ⁇ -D-ribofuranosyl group.
  • Nucleotides are generally referred to by the single letter representing the bases in them: “A(a)” refers to deoxyadenosine or adenylate, “C(c)” refers to deoxycytidine or cytidine, “G(c)” refers to deoxyguanylic acid or guanylic acid, “U(u)” refers to uridylic acid, and “T(t)” refers to deoxythymidylic acid.
  • polynucleotide and “nucleic acid” are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA) ).
  • Polynucleotide sequence and “nucleotide sequence” are used interchangeably to refer to the ordering of nucleotides in a polynucleotide.
  • DNA coding strand (sense strand) and the RNA it encodes can be regarded as having the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridylic acid in the RNA sequence it encodes .
  • vector refers to a vehicle used to introduce nucleic acid into a host cell.
  • Vectors can include expression vectors and cloning vectors.
  • an expression vector contains the desired coding sequence and the appropriate DNA sequences necessary to express the operably linked coding sequence in a particular host organism (eg, bacteria, yeast, plant, insect, or mammal) or in an in vitro expression system .
  • Cloning vectors are typically used to engineer (perform recombinant DNA manipulation) and amplify desired DNA fragments, and may lack functional sequences required to express the desired DNA sequence.
  • vectors include, but are not limited to, plasmids, cosmids, bacteriophage (eg, lambda phage) vectors, viral vectors (eg, retrovirus, adenovirus, or baculovirus vectors), or artificial chromosomes (eg, bacterial artificial chromosomes (BAC), yeast artificial chromosome (YAC) or P1 artificial chromosome (PAC) vector.
  • plasmids eg, cosmids, bacteriophage (eg, lambda phage) vectors
  • viral vectors eg, retrovirus, adenovirus, or baculovirus vectors
  • artificial chromosomes eg, bacterial artificial chromosomes (BAC), yeast artificial chromosome (YAC) or P1 artificial chromosome (PAC) vector.
  • BAC bacterial artificial chromosomes
  • YAC yeast artificial chromosome
  • PAC P1 artificial chromosome
  • the term "expression” includes transcription and/or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and/or polypeptides.
  • transcription refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcript).
  • in vitro transcription refers to the in vitro synthesis of RNA, especially mRNA, in a cell-free system (eg, in a suitable cell extract) (see, eg, Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). ).In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation.
  • transcripts also known as “transcription vectors”
  • transcription vectors contain the regulatory sequences required for transcription.
  • transcription encompasses "in vitro transcription”.
  • the term "host cell” refers to a cell used to receive, maintain, replicate, express a polynucleotide or vector.
  • the host cell can be a cell in which a polypeptide of the invention is expressed.
  • isolated refers to a substance (eg, a polynucleotide or polypeptide) that is separated from the source or environment in which it exists.
  • An isolated polynucleotide or polypeptide can exist in substantially pure form (eg, in a composition), or can exist in a non-natural environment, eg, a host cell.
  • the polypeptides and polynucleotides of the invention are isolated.
  • naturally occurring refers to the fact that an object can be found in nature. For example, polypeptides or polynucleotides that are present in organisms (including viruses) and that can be isolated from natural sources and have not been intentionally modified by humans in the laboratory are naturally occurring.
  • recombinant means "produced by genetic engineering”.
  • recombinant molecules eg, recombinant proteins and recombinant nucleic acids
  • polypeptides and polynucleotides of the present invention may be recombinant molecules.
  • the term "expressed on the cell surface” means that a molecule, such as an antigen, is associated with and located on the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space and accessible from outside the cell, for example, by being located at Antibodies outside the cell.
  • the polypeptides of the invention can be expressed on the surface of a suitable host cell.
  • the polypeptides of the invention are expressed at higher levels on the surface of the host cell compared to the S protein comprising an active furin cleavage site (eg, a furin cleavage site having the amino acid sequence RRAR).
  • the polypeptides of the invention are expressed on the surface of a suitable host cell and are capable of binding the human ACE2 protein (hACE2).
  • the term "vaccine” refers to a composition comprising an active ingredient (eg, a polypeptide antigen of the invention or a polynucleotide encoding the same) that induces an immune response when vaccinated into a subject, the The immune response is sufficient to prevent and/or alleviate at least one symptom associated with infection with a pathogen or disease.
  • an active ingredient eg, a polypeptide antigen of the invention or a polynucleotide encoding the same
  • the polypeptides, polynucleotides, compositions or pharmaceutical compositions described herein can be used as vaccines to provide prophylactic and/or therapeutic immunity against SARS-CoV-2 in a subject in need thereof .
  • neutralizing antibody refers to an antibody or fragment thereof capable of neutralizing, ie preventing, inhibiting, reducing or interfering with the ability of a pathogen to initiate and/or maintain infection in a host (eg, a host organism or host cell).
  • a host eg, a host organism or host cell.
  • neutralizing antibodies against SARS-CoV-2 S protein or SARS-CoV-2 can be produced in a subject vaccinated with the vaccine of the present invention, eg in the subject's immune serum.
  • Neutralizing antibody titer levels in immune serum can be measured using methods known in the art.
  • neutralizing antibody levels in immune sera are measured by pseudovirus neutralization assay (pVNT), and neutralizing antibody titer levels measured by pVNT are expressed as 50 % inhibitory dilution (ID50), which represents pseudovirus Immune serum dilution corresponding to 50% inhibition of pseudovirus after neutralization with immune serum.
  • ID50 50 % inhibitory dilution
  • the specific value is mainly expressed as the expression level of the reporter gene carried by the pseudovirus itself (for example: the luminescence intensity generated by the luciferase-catalyzed substrate; or the fluorescence intensity of the GFP protein), for example, as described in Example 4 of the present application.
  • antigen refers to a substance that contains an epitope against which an immune response can be raised.
  • the antigen may bind to a T cell epitope or T or B cell receptor, or to an immunoglobulin such as an antibody.
  • polypeptide antigen refers to a polypeptide as an antigen, including but not limited to the polypeptide antigen itself or a processed product thereof (eg, an antigen that is processed and presented in vivo).
  • the polypeptide of the present invention or a processed product thereof may be a polypeptide antigen and induce an immune response as an immunogenic active ingredient in a vaccine.
  • transfection refers to the introduction of a polynucleotide into a host cell.
  • Host cells for transfection of the polynucleotides described herein can exist in vitro or in vivo.
  • the host cells can be cells of a subject, particularly a patient, eg, a patient infected with the novel coronavirus.
  • Transfection can be transient or stable. In general, transient transfection does not involve integration into the host cell genome. Stable transfection can be achieved by transfection using viral or transposon-based systems.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • novel coronavirus and “SARS-CoV-2” are used interchangeably.
  • SARS-CoV-2 is known to be the causative agent of "Coronavirus Disease 2019 (COVID-19)”.
  • SARS-CoV-2 is a positive-sense single-stranded RNA ((+)ssRNA) enveloped virus belonging to the ⁇ genus of the family Coronaviridae.
  • SARS-CoV-2 encodes 4 structural proteins: spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N).
  • S protein mediates the specific binding of the virus to the host cell and the fusion of the viral envelope and the host cell membrane, so it is a key molecule for the virus to infect the host cell.
  • the present invention provides a polypeptide comprising a spike protein variant of SARS-CoV-2 (also referred to herein as a "S protein variant") comprising an inactive S protein variant the furin cleavage site, and wherein the inactive furin cleavage site has the amino acid sequence of QSAQ.
  • S protein variant spike protein variant of SARS-CoV-2
  • the inactive furin cleavage site has the amino acid sequence of QSAQ.
  • Polypeptides of the invention may comprise amino acid modifications, such as modifications, such as additions, substitutions and/or deletions of amino acids, relative to the wild-type S protein (an exemplary amino acid sequence is shown in SEQ ID NO: 1).
  • the polypeptides of the present invention can be used as polypeptide antigens to induce a protective immune response against SARS-CoV-2 infection in a subject.
  • the polypeptide comprises a polypeptide antigen described herein.
  • the polypeptide consists of a polypeptide antigen described herein.
  • the polypeptide comprises a polypeptide antigen and a signal peptide as described herein.
  • SARS-CoV-2 spike protein As used herein, "SARS-CoV-2 spike protein”, “SARS-CoV-2 S protein” or “S protein” refers to the spike protein of SARS-CoV-2.
  • the SARS-CoV-2 S protein is synthesized as a glycoprotein of approximately 1273-1300 amino acids (an exemplary amino acid sequence is shown in SEQ ID NO:1) that includes an N-terminal signal peptide (approximately corresponding to amino acid 1 of SEQ ID NO:1) -13, 1-14, 1-15, or 1-16), the S1 subunit (corresponding approximately to amino acids 14-685 of SEQ ID NO: 1, eg, amino acids 15-685, 16-685, or 17-685), and the S2 subunit base (approximately corresponding to amino acids 686-1273 of SEQ ID NO: 1, eg, amino acids 686-1213).
  • the S1 subunit contains an N-terminal domain (approximately corresponding to amino acids 14-305 of SEQ ID NO: 1, eg, amino acids 15-305, 16-305 or 17-305), a receptor binding domain (RBD) (approximately corresponding to SEQ ID NO: 1) Amino acids 319-527 of ID NO: 1, eg, amino acids 328-527 or 331-524) and subdomains 1 and 2 (SD1/2) (approximately corresponding to amino acids 528-685 of SEQ ID NO: 1).
  • the S2 subunit comprises a fusion peptide (FP) (approximately corresponding to amino acids 788-806 of SEQ ID NO: 1), a heptad repeat HR1 (approximately corresponding to amino acids 912-984 of SEQ ID NO: 1), HR2 (approximately corresponding to amino acids 912-984 of SEQ ID NO: 1), HR2 (approximately corresponding to amino acids 788-806 of SEQ ID NO: 1) NO: 1 amino acids 1163-1213), transmembrane domain (corresponding approximately to amino acids 1213-1237 of SEQ ID NO: 1) and cytoplasmic domain (corresponding approximately to amino acids 1237-1273 of SEQ ID NO: 1).
  • FP fusion peptide
  • HR1 approximately corresponding to amino acids 912-984 of SEQ ID NO: 1
  • HR2 approximately corresponding to amino acids 912-984 of SEQ ID NO: 1
  • HR2 approximately corresponding to amino acids 788-806 of SEQ ID NO: 1
  • RBD of the S1 subunit recognizes target host cells by interacting with the specific receptor angiotensin-converting enzyme 2 (ACE2), while the S2 subunit is responsible for membrane fusion.
  • ACE2 angiotensin-converting enzyme 2
  • the S protein exists on the virus surface in a metastable prefusion trimer conformation.
  • host proteases such as Furin
  • cleaves the S1/S2 cleavage site of the S protein destabilizing the prefusion trimer, resulting in shedding of the S1 subunit and The S2 subunit transitions to the stable conformation after fusion.
  • the Furin cleavage site is an exposed loop structure containing multiple arginine residues, which contains the amino acid motif Arg-X aa -X bb -Arg (wherein X aa is any amino acid; X bb is any amino acid, preferably is Arg or Lys.
  • the amino acid sequence of the Furin cleavage site is Arg-Arg-Ala-Arg ("RRAR"), corresponding to amino acids 682-685 in SEQ ID NO:1.
  • Polypeptide antigens of the present invention may contain an inactive Furin cleavage site.
  • the polypeptide antigens of the present invention comprise an inactivated Furin cleavage site Gln-Ser-Ala-Gln (QSAQ), thereby having higher expression levels in host cells and/or inducing stronger immune response.
  • an "inactive Furin cleavage site” refers to an amino acid sequence that cannot be recognized and cleaved by Furin.
  • an “active furin cleavage site” or “furin cleavage site” refers to an amino acid sequence capable of being recognized and cleaved by furin.
  • the polypeptide of the invention comprises the S1 subunit and the S2 subunit of the SARS-CoV-2 S protein from the N-terminus to the C-terminus, wherein the S1 subunit comprises an inactive furin cleavage site,
  • the inactive furin cleavage site is located at the C-terminus of the S1 subunit and has the amino acid sequence of QSAQ.
  • mutant SARS-CoV-2 S proteins have been identified.
  • such mutant SARS-CoV-2 S protein may comprise mutations, such as amino acid deletions and/or substitutions, as compared to wild-type SARS-CoV-2 S protein (eg, SEQ ID NO: 1).
  • the polypeptides of the invention may have one or more amino acids deleted, for example, amino acids 69, 70, 144, 145, 242-244, 689-715, 715-724, One or more of the amino acids at positions 788-806 and 819-828.
  • one or more amino acids in the polypeptides of the invention may be substituted with other amino acids, such as at amino acids 18, 20, 26, 80, 138, 152, 190, 215, One or more of the amino acids at positions 242, 246, 417, 439, 452, 453, 484, 501, 570, 614, 655, 681, 701, 716, 982, 1027 and 1118 may be substituted with other amino acids.
  • the S1 subunit comprises an N-terminal domain, a receptor binding domain, and subdomains 1 and 2.
  • the N-terminal domain, receptor binding domain and subdomains 1 and 2 respectively, have at least the corresponding portion of the SARS-CoV-2 S protein having the amino acid sequence of SEQ ID NO: 1. 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical.
  • the S2 subunit comprises a fusion peptide (FP), a heptapeptide repeat HR1, HR2, a transmembrane domain, and a cytoplasmic domain.
  • the fusion peptide (FP), heptapeptide repeat HR1, HR2, transmembrane domain and cytoplasmic domain are respectively associated with the SARS-CoV-2 S protein having the amino acid sequence of SEQ ID NO: 1 Corresponding portions in are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.
  • the S1 subunit comprises at least 80%, 85%, 90%, 95% of the amino acid sequence of amino acids 14-685, 15-685, 16-685 or 17-685 of SEQ ID NO: 1 %, 96%, 97%, 98% or 99% identical amino acid sequences.
  • the S2 subunit comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of amino acids 686-1273 of SEQ ID NO: 1 % identical amino acid sequences.
  • the polypeptide comprises at least 80%, 85%, 90%, 95%, 80%, 85%, 90%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical.
  • the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1.
  • the amino acid in the polypeptide at positions corresponding to amino acids 986 and 987 of SEQ ID NO: 1 is proline (P).
  • the amino acid in the polypeptide at positions corresponding to amino acids 383 and 985 of SEQ ID NO: 1 is cysteine (C).
  • the amino acid at positions corresponding to amino acids 817, 892, 899, and 942 of SEQ ID NO: 1 in the polypeptide is proline (P).
  • the amino acids in the polypeptide at positions corresponding to amino acids 986 and 987 of SEQ ID NO: 1 are proline (P)
  • the amino acids at positions corresponding to amino acids 383 and 985 of SEQ ID NO: 1 are proline (P).
  • the amino acid at the position is cysteine (C).
  • the amino acid at positions corresponding to amino acids 986, 987, 817, 892, 899, and 942 of SEQ ID NO: 1 in the polypeptide is proline (P).
  • the amino acid in the polypeptide at positions corresponding to amino acids 986, 987, 817, 892, 899, and 942 of SEQ ID NO: 1 is proline (P), and the amino acid at positions corresponding to amino acids 986, 987, 817, 892, 899, and 942 of SEQ ID NO:1
  • the amino acids at positions 383 and 985 of amino acids 383 and 985 of :1 are cysteine (C).
  • polypeptide further comprises one or more of the following amino acid modifications:
  • the amino acid in the polypeptide at a position corresponding to amino acid 614 of SEQ ID NO: 1 is glycine (G).
  • the amino acid in the polypeptide at a position corresponding to amino acid 614 of SEQ ID NO: 1 is glycine (G) and the amino acid at a position corresponding to amino acid 417 of SEQ ID NO: 1 is Glycine Paraparagine (N)
  • the amino acid at the position corresponding to amino acid 484 of SEQ ID NO: 1 is lysine (K)
  • the amino acid at the position corresponding to amino acid 501 of SEQ ID NO: 1 is tyrosine (Y)
  • the amino acid at the position corresponding to amino acid 80 of SEQ ID NO:1 is alanine (A)
  • the amino acid at the position corresponding to amino acid 215 of SEQ ID NO:1 is glycine (G)
  • the amino acid at the position corresponding to amino acid 701 of SEQ ID NO: 1 is valine (V).
  • polypeptides of the invention comprise amino acid modifications, such as additions, substitutions and/or deletions of amino acids, relative to the wild-type S protein (an exemplary amino acid sequence is shown in SEQ ID NO: 1).
  • polypeptide antigens of the invention further comprise one or more amino acid substitutions compared to the wild-type S protein.
  • a polypeptide antigen can comprise amino acid substitutions at one or more of amino acid positions 986, 987, 383, 985, 817, 892, 899, and 942 corresponding to SEQ ID NO:1.
  • polypeptide antigen may further comprise one or more of the following amino acid substitutions compared to SEQ ID NO: 1: K986P, V987P, S383C, D985C, F817P, A892P, A899P and A942P.
  • the polypeptide antigen comprises an amino acid sequence having the amino acid sequence QSAQ at amino acid positions 682-685 corresponding to SEQ ID NO:1.
  • the polypeptide antigen further comprises amino acid substitutions of K986P and V987P compared to SEQ ID NO:1.
  • the polypeptide antigen further comprises amino acid substitutions of S383C and D985C compared to SEQ ID NO:1.
  • the polypeptide antigen further comprises the following amino acid substitutions compared to SEQ ID NO: 1: F817P, A892P, A899P and A942P.
  • the polypeptide antigen further comprises amino acid substitutions at one or more of amino acid positions 614, 417, 484, 501, 80, 215, and 701 corresponding to SEQ ID NO:1.
  • the amino acid substitution is one or more of the following amino acid substitutions compared to SEQ ID NO: 1: D614G, K417N, E484K, N501Y, D80A, D215G, and A701V.
  • the amino acid substitution includes D614G compared to SEQ ID NO:1.
  • the amino acid substitutions include D614G, K417N, E484K and N501Y compared to SEQ ID NO:1.
  • the amino acid substitutions include any combination of the following compared to SEQ ID NO: 1:
  • the polypeptide antigen comprises at least 80%, 85%, 90% of the amino acid sequence of amino acids 14-1273, 15-1273, 16-1273 or 17-1273 of any one of SEQ ID NOs: 2-7 , 95%, 96%, 97%, 98% or 99% identical amino acid sequences. In one embodiment, the polypeptide antigen comprises at least 80%, 85%, 90% of the amino acid sequence of amino acids 14-1273, 15-1273, 16-1273 or 17-1273 of any one of SEQ ID NOs: 2-7 , immunogenic fragments of amino acid sequences that are 95%, 96%, 97%, 98%, or 99% identical.
  • the polypeptide antigen comprises the amino acid sequence of amino acids 14-1273, 15-1273, 16-1273, or 17-1273 of any one of SEQ ID NOs: 2-7. In one embodiment, the polypeptide antigen comprises an immunogenic fragment of the amino acid sequence of amino acids 14-1273, 15-1273, 16-1273, or 17-1273 of any one of SEQ ID NOs: 2-7. In a preferred embodiment, the polypeptide antigen comprises an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of any one of SEQ ID NOs: 2-7.
  • the polypeptides of the present invention may also comprise a signal peptide.
  • signal peptides facilitate secretion and/or transport of polypeptides, eg, to the cell surface, endoplasmic reticulum (ER) or endosomal-lysosomal compartment.
  • Exemplary signal peptides can be about 15-30 amino acids in length.
  • the signal peptide may comprise the signal peptide sequence of the SARS-CoV-2 S protein or a variant thereof, in particular comprising amino acids 1-13, 1-14, 1-15 or 1- of SEQ ID NO: 1 16 amino acid sequence.
  • signal peptides may include, but are not limited to, the signal peptide sequences of immunoglobulins, such as the signal peptide sequences of the heavy chain variable regions of immunoglobulins, preferably human immunoglobulins.
  • the signal peptide directs a nascent polypeptide (eg, a polypeptide antigen described herein) into the endoplasmic reticulum for glycosylation modification.
  • a polypeptide of the invention following expression of a polypeptide of the invention in a host cell, the N-terminal signal peptide is cleaved, resulting in a mature S protein variant (eg, a polypeptide antigen described herein) that is secreted into the extracellular space.
  • the signal peptide can be fused directly or through a linker to the polypeptide antigen as described herein.
  • the signal peptide is N-terminal to the polypeptide antigen.
  • the signal peptide is fused directly to the N-terminus of the polypeptide antigen.
  • a polynucleotide of the present invention may comprise a nucleotide sequence encoding a polypeptide antigen and a signal peptide, wherein the signal peptide is fused to the N-terminus of the polypeptide antigen.
  • the signal peptide comprises the amino acid sequence of amino acids 1-13, 1-14, 1-15, or 1-16 of SEQ ID NO:1. In a preferred embodiment, the signal peptide comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO:1.
  • the polypeptide of the present invention comprises a polypeptide antigen as described above and a signal peptide as described above, the signal peptide being fused directly to the N-terminus of the polypeptide antigen.
  • the polypeptide of the invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7 sexual amino acid sequence. In one embodiment, the polypeptide of the invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7
  • the immunogenic fragment of the amino acid sequence comprises the amino acid sequence of any one of SEQ ID NOs: 2-7. In one embodiment, the polypeptide of the invention comprises an immunogenic fragment of the amino acid sequence of any one of SEQ ID NOs: 2-7.
  • the present invention provides polynucleotides encoding the polypeptides described herein.
  • Polynucleotides can be single-stranded or double-stranded.
  • Polynucleotides include, but are not limited to, DNA, cDNA, RNA (eg, mRNA), recombinantly produced, and chemically synthesized polynucleotides.
  • the polynucleotide can be contained in a vector.
  • the polynucleotides of the present invention can include naturally occurring, synthetic and modified nucleotides.
  • the polynucleotides of the present invention are used to express the polypeptides of the present invention in host cells to provide polypeptide antigens.
  • the polypeptide antigen can induce an immune response against the SARS-CoV-2 S protein, preferably against SARS-CoV-2, in a suitable subject.
  • a polynucleotide may comprise one or more nucleotide sequences (eg, 1, 2, 3, 4, 5, 6, 7, 8 sequences).
  • a polynucleotide can comprise a coding sequence for a polypeptide of interest (eg, polypeptides and polypeptide antigens described herein).
  • a polynucleotide may comprise a coding sequence for a polypeptide of interest as well as regulatory sequences (including, but not limited to, transcriptional and translational regulatory sequences).
  • the regulatory sequences comprise one or more of the following: a promoter sequence, a 5' untranslated region (5' UTR) sequence, a 3' untranslated region (3' UTR) sequence, and a poly(A) sequence.
  • coding sequence refers to a nucleotide sequence in a polynucleotide that can serve as a template for the synthesis of a defined nucleotide sequence (eg, tRNA and mRNA) or a defined amino acid sequence in a biological process. Coding sequences can be DNA sequences or RNA sequences. A DNA sequence or mRNA sequence can be considered to encode a polypeptide if the mRNA corresponding to the DNA sequence (including the same coding strand as the mRNA sequence and the template strand complementary thereto) is translated into the polypeptide in a biological process.
  • cognid refers to a sequence of three consecutive nucleotides in a polynucleotide (also known as a triplet codon) that encodes a specific amino acid. Synonymous codons (codons encoding the same amino acid) are used with different frequencies in different species, known as “codon bias”. It is generally believed that, for a given species, coding sequences that use its preferred codons can have higher translation efficiency and accuracy in expression systems for that species. Thus, polynucleotides can be "codon-optimized,” ie, changing the codons in the polynucleotide to reflect the codons preferred by the host cell, preferably without changing the amino acid sequence it encodes.
  • the polynucleotides of the present invention may comprise coding sequences that differ (eg, about 50% from the coding sequences described herein) , 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical) but encode the same amino acid sequence.
  • the RNAs of the invention comprise codons optimized for the cells of the host (eg, a subject, particularly a human), such that the polypeptides of the invention are optimally expressed in the host (eg, a subject, particularly a human) .
  • the polynucleotides of the invention comprise a coding sequence for a polypeptide antigen as described herein. In one embodiment, the polynucleotides of the present invention comprise nucleotide sequences complementary to the coding sequences described herein. In some embodiments, the polynucleotides of the invention comprise a coding sequence for a polypeptide antigen as described herein. In some embodiments, the polynucleotides of the present invention comprise a coding sequence for a polypeptide as described herein. In one embodiment, the coding sequence contains a start codon at its 5' end and a stop codon at its 3' end. In one embodiment, the coding sequence comprises an open reading frame (ORF) as described herein.
  • ORF open reading frame
  • the coding sequence of the polypeptide antigen comprises a nucleotide sequence encoding: (1) amino acids 14-1273, 15-1273, 16 comprising any one of SEQ ID NOs: 2-7 - the amino acid sequence of 1273 or 17-1273; (2) having at least 80%, 85% of the amino acid sequence of amino acids 14-1273, 15-1273, 16-1273 or 17-1273 of any one of SEQ ID NOs: 2-7 %, 90%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences; (3) amino acids 14-1273, 15-1273, 16- of any one of SEQ ID NOs: 2-7 an immunogenic fragment of the amino acid sequence of 1273 or 17-1273; or (4) having Immunogenic fragments of amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.
  • the coding sequence of the polypeptide antigen comprises a nucleotide sequence encoding: (1) an amino acid sequence comprising amino acids 17-1273 of any one of SEQ ID NOs: 2-7; (2) ) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of amino acids 17-1273 of any one of SEQ ID NOs: 2-7 (3) an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of any one of SEQ ID NOs: 2-7; or (4) an amino acid of any one of amino acids 17-1273 of SEQ ID NO: 2-7 An immunogenic fragment of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the coding sequence of the polypeptide of the present invention comprises a nucleotide sequence encoding a polypeptide antigen as described above and a nucleotide sequence encoding a signal peptide as described above, the signal peptide being directly fused to the polypeptide antigen. N-terminal.
  • the coding sequence for the signal peptide comprises the nucleotide sequence of nucleotides 1-39, 1-42, 1-45, or 1-48 of SEQ ID NO:8.
  • the coding sequence of the polypeptide of the present invention comprises a nucleotide sequence encoding: (1) the amino acid sequence comprising any one of SEQ ID NOs: 2-7; (2) the same as SEQ ID NO: 2-7 An amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity in any of the amino acid sequences of ID NOs: 2-7; (3) SEQ ID NO: 2 - an immunogenic fragment of any one of the amino acid sequences in 7; or (4) having at least 80%, 85%, 90%, 95%, 96%, 97%, Immunogenic fragments of amino acid sequences that are 98% or 99% identical.
  • the polynucleotides of the present invention are RNA.
  • RNA encompasses single-stranded, double-stranded, linear and circular RNAs.
  • the RNAs of the present invention may be chemically synthesized, recombinantly produced, and in vitro transcribed RNAs.
  • the RNA of the invention is used to express the polypeptide of the invention in a host cell.
  • the RNA of the invention is single-stranded RNA.
  • the RNA of the invention is in vitro transcribed RNA (IVT-RNA).
  • IVT-RNA can be obtained by in vitro transcription using a DNA template by RNA polymerase (eg, as described herein).
  • the RNA of the invention is messenger RNA (mRNA).
  • mRNA may comprise a 5'UTR sequence, a coding sequence for a polypeptide, a 3'UTR sequence, and optionally a poly(A) sequence.
  • mRNA can be produced, for example, by in vitro transcription or chemical synthesis.
  • the mRNA of the invention is obtained by in vitro transcription using a DNA template by an RNA polymerase (eg, T7 RNA polymerase).
  • the mRNA of the invention comprises (1) an optional 5' cap, (2) a 5' UTR, (3) a coding sequence, (4) a 3' UTR, and (5) an optional poly (A) Sequence.
  • the 5' cap, 5' UTR, coding sequence, 3' UTR and poly(A) sequence are as described herein.
  • the mRNA of the invention is a nucleoside-modified mRNA.
  • the RNA of the invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, the RNA of the invention comprises a coding sequence for a polypeptide as described herein.
  • the RNAs of the invention further comprise structural elements that contribute to the stability and/or translational efficiency of the RNA, including but not limited to 5' caps, 5' UTRs, 3' UTRs and poly(A) sequences .
  • the term "untranslated region (UTR)” generally refers to a region in RNA (eg, mRNA) that is not translated into an amino acid sequence (noncoding region), or a corresponding region in DNA.
  • RNA eg, mRNA
  • a UTR located 5' (upstream) of the open reading frame (start codon) may be referred to as a 5' untranslated region 5' UTR;
  • a UTR located 3' (downstream) of the open reading frame (stop codon) may be called 3'UTR.
  • the 5' UTR is located downstream of the 5' cap, eg, directly adjacent to the 5' cap.
  • an optimized "Kozak sequence” may be included in the 5'UTR, eg, adjacent to the initiation codon, to improve translation efficiency.
  • the "3'UTR” does not contain a poly(A) sequence.
  • the 3'UTR is located upstream of the poly(A) sequence, eg, directly adjacent to the poly(A) sequence.
  • the RNA of the invention comprises a 5'UTR.
  • the 5'UTR comprises the nucleotide sequence of any one of SEQ ID NOs: 33-44.
  • the 5'UTR comprises the nucleotide sequence of SEQ ID NO:42.
  • the RNA of the invention comprises a 3'UTR.
  • the 3' UTR comprises the nucleotide sequence of any one of SEQ ID NOs: 45-55.
  • the 3'UTR comprises the nucleotide sequence of SEQ ID NO:55.
  • the RNA of the invention comprises a 5'UTR and a 3'UTR.
  • the 5'UTR comprises the nucleotide sequence of SEQ ID NO:42 and the 3'UTR comprises the nucleotide sequence of any one of SEQ ID NOs:45-55.
  • the 5'UTR comprises the nucleotide sequence of SEQ ID NO:42 and the 3'UTR comprises the nucleotide sequence of SEQ ID NO:55.
  • poly(A) sequence or “poly(A) tail” refers to a nucleotide sequence comprising contiguous or discontinuous adenine nucleotides.
  • the poly(A) sequence is usually located at the 3' end of the RNA, such as the 3' end (downstream) of the 3' UTR.
  • the poly(A) sequence contains no nucleotides other than adenylate at its 3' end.
  • Poly(A) sequences can be generated by DNA-dependent RNA polymerase transcription from the coding sequence of the DNA template during the preparation of IVT-RNA, or ligated to the IVT by a DNA-independent RNA polymerase (poly(A) polymerase) - the free 3' end of the RNA, eg the 3' end of the 3' UTR.
  • poly(A) polymerase DNA-independent RNA polymerase
  • the RNAs of the invention comprise poly(A) sequences.
  • the poly(A) sequence comprises contiguous adenosine nucleotides.
  • the poly(A) sequence may comprise at least 20, 30, 40, 50, 60, 70, 80 or 100 and as much as 120, 150, 180, 200, 300 adenosine.
  • the contiguous adenylate sequence in the poly(A) sequence is interrupted by a sequence comprising U, C or G nucleotides.
  • the poly(A) sequence may comprise at least 20, 30, 40, 50, 60, 70, 80 or 100 and up to 120, 150, 180, 200, 300 nucleotides. In one embodiment, the poly(A) sequence comprises at least 50 nucleotides. In one embodiment, the poly(A) sequence comprises at least 80 nucleotides. In one embodiment, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 70, 80, 90, 100, 120 or 150 nucleotides. In a specific embodiment, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:56.
  • the term “5'cap” generally refers to an N7-methylguanosine structure (also referred to as “ m7G cap", “ m7Gppp " linked to the 5' end of the mRNA by a 5' to 5' triphosphate bond -").
  • the 5' cap can be co-transcribed to the RNA during in vitro transcription (eg, using an anti-reverse cap analog "ARCA"), or can be attached to the RNA post-transcriptionally using a capping enzyme.
  • cap analogs are used to generate 5' cap-modified RNAs.
  • a description of "cap analogs” can be found, for example, in Contreas, R. et al. (1982). Nucl. Acids Res.. 10, 6353-6363 and US7074596B2.
  • Examples of cap analogs include, but are not limited to, N7-methylguanosine-5'-triphosphate-5'guanosine (m 7 G(5')ppp(5')G), N7-methylguanosine-5 '-5'-adenosine triphosphate (m 7 G(5')ppp(5')A) and 3'-O-Me-m 7 G(5')ppp(5')G(ARCA).
  • RNA eg, IVT-RNA
  • a capping enzyme eg, vaccinia virus capping enzyme
  • additional methylation occurs at the ribose 2'O position of the nucleotide immediately adjacent to the m7G cap in the CapO RNA, resulting in "Capl RNA”.
  • the RNA of the invention comprises a 5' cap. In some embodiments, the RNA of the invention is a CapO RNA. In some embodiments, the RNA of the invention is Capl RNA.
  • RNAs of the invention are transcribed into CapO RNAs having the following structure:
  • Base represents the base of the starting nucleotide of the RNA.
  • the RNA of the invention comprises a coding sequence for a polypeptide antigen as described herein and a coding sequence for a signal peptide as described herein fused directly to the N-terminus of the polypeptide antigen.
  • the coding sequence for the signal peptide comprises the nucleotide sequence of nucleotides 1-39, 1-42, 1-45 or 1-48 of SEQ ID NO:8. In one embodiment, the coding sequence for the signal peptide comprises the nucleotide sequence of nucleotides 1-48 of SEQ ID NO:8.
  • the RNA of the invention comprises the nucleotide sequence of any one of SEQ ID NOs: 8-13. In one embodiment, the RNA of the invention comprises the nucleotide sequence of any one of SEQ ID NOs: 14-19.
  • the RNA (a) of the invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or a nucleotide sequence that is 99% identical; and (b) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:2.
  • the RNA (a) of the invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or a nucleotide sequence that is 99% identical; and (b) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:3.
  • the RNA(a) of the invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or a nucleotide sequence that is 99% identical; and (b) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:4.
  • the RNA (a) of the invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or a nucleotide sequence that is 99% identical; and (b) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:5.
  • the RNA (a) of the invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or a nucleotide sequence that is 99% identical; and (b) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:6.
  • the RNA (a) of the invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or a nucleotide sequence that is 99% identical; and (b) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:7.
  • the nucleotides in the RNA (eg, mRNA) of the invention can be naturally occurring nucleotides (eg, naturally occurring ribonucleotides) and modified nucleotides.
  • Modified nucleotides can be, for example, nucleotides not found in naturally occurring RNA, such as non-standard nucleotides or deoxynucleotides. Modification of nucleotides can occur on nucleosides, eg, on ribose moieties and/or nucleobase moieties. Modified nucleotides can be incorporated during transcription (eg, in vitro transcription) or added during chemical RNA synthesis.
  • the RNA is modified by comprising one or more modified nucleosides. In one embodiment, the RNA is modified by replacing one or more uracils with modified uridines. In one embodiment, the modified uridine includes 1-methylpseudouracil, pseudouracil, 5-methyl-uracil, or a combination thereof.
  • modified uridines may include, but are not limited to: 1-methyluridine, 1-methyl-pseudouridine, 3-methyl-uridine, 3-methyl-pseudouridine, 2-methoxy - uridine, 5-methoxy-uridine, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine , uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxy Hydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2
  • the RNA (eg, mRNA) of the invention is modified by comprising one or more modified nucleobases.
  • the modified nucleobase includes modified cytosine, modified uracil, or a combination thereof.
  • the modified uracil is independently selected from pseudouracil, 1-methyl-pseudouracil, 5-methyl-uracil, or a combination thereof.
  • the modified cytosines are independently selected from 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof.
  • the ratio of modified nucleobases in the RNA of the present invention is 10%-100%, that is, the RNA of the present invention can be modified by replacing 10%-100% of the nucleobases with modified nucleobases. retouch.
  • the RNA (eg, mRNA) of the invention is modified by replacing one or more uracils with modified uracils.
  • the modified uracil includes 1-methylpseudouracil, pseudouracil, 5-methyl-uracil, or a combination thereof.
  • the modified uracil includes pseudouracil.
  • the modified uracil includes 5-methyl-uracil.
  • the modified uracil includes 1-methyl-pseudouracil.
  • the RNA is modified by replacing at least one uracil with a modified uracil. In one embodiment, the RNA is modified by replacing all uracils with modified uracils. In one embodiment, the ratio of modified uracil in the RNA is 10%-100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In one embodiment, the proportion of modified uracil in the RNA is 20%-100%. In one embodiment, 20%-100% of the uracils in the RNA are replaced by 1-methylpseudouracil. In a preferred embodiment, 100% of the uracil in the RNA is replaced by 1-methylpseudouracil.
  • 1-Methyl-pseudouracil has the following structure:
  • the mRNA of the invention comprises the nucleotide sequence of any one of SEQ ID NOs: 14-19, and wherein 100% of the uracils are replaced by 1-methylpseudouracil.
  • the polynucleotides of the present invention are DNA.
  • DNA may be, for example, a DNA template for in vitro transcription of the RNA of the invention or a DNA vaccine for expression of a polypeptide antigen in a host cell.
  • DNA can be double-stranded, single-stranded, linear and circular DNA.
  • the DNA template can be provided in a suitable transcription vector.
  • a DNA template can be a double-stranded complex comprising a nucleotide sequence identical to the coding sequence described herein (the coding strand) and a nucleotide sequence complementary to the coding sequence described herein (the template strand).
  • the DNA template may comprise a promoter, 5'UTR, coding sequence, 3'UTR and optionally a poly(A) sequence.
  • the promoter may be a suitable RNA polymerase (particularly DNA-dependent RNA polymerase) available promoter known to those skilled in the art, including but not limited to promoters of SP6, T3 and T7 RNA polymerases.
  • the 5'UTR, coding sequence, 3'UTR and poly(A) sequences in the DNA template are or are complementary to the corresponding sequences contained in the RNAs described herein.
  • Polynucleotides as DNA vaccines can be provided in plasmid vectors (eg, circular plasmid vectors).
  • the DNA of the invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, the DNA of the present invention comprises a coding sequence for a polypeptide as described herein. In some embodiments, the DNA of the invention comprises (1) a T7 promoter, (2) a 5' UTR, (3) a coding sequence, (4) a 3' UTR as described herein from the 5' end to the 3' end and (5) an optional poly(A) sequence. In some embodiments, the DNA of the present invention comprises the nucleotide sequences of SEQ ID NOs: 57-59.
  • the present invention also provides a composition comprising a polypeptide or polynucleotide (particularly RNA) of the present invention.
  • the compositions of the present invention are used to provide prophylactic and/or therapeutic immunity against SARS-CoV-2 in a subject.
  • the compositions of the present invention comprise a polypeptide or polypeptide antigen as described herein.
  • the compositions of the present invention comprise polynucleotides of the present invention.
  • the compositions of the present invention comprise the DNA of the present invention.
  • the compositions of the present invention comprise RNAs of the present invention.
  • the RNA is in vitro transcribed RNA.
  • the RNA is mRNA.
  • the compositions of the present invention are formulated as pharmaceutical compositions.
  • compositions of the present invention comprise a polynucleotide (particularly RNA, eg, mRNA) as described herein and a lipid that encapsulates the polynucleotide.
  • lipid refers to an organic compound comprising a hydrophobic moiety and optionally also a hydrophilic moiety. Lipids are generally poorly soluble in water but soluble in many organic solvents. Generally, amphiphilic lipids comprising hydrophobic and hydrophilic moieties can be organized into lipid bilayers in an aqueous environment, eg, in the form of vesicles. Lipids can include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, and steroids and cholesterol esters, among others.
  • compositions of the present invention can be, for example, lipid nanoparticles (LNPs) and lipid polyplexes (LPPs) as described herein. Methods of preparing such compositions can be found in, for example, Kaczmarek, J.C. et al., 2017, Genome Medicine 9, 60 or as described herein.
  • the compositions of the present invention comprise lipid nanoparticles (LNPs) or lipid polyplexes (LPPs).
  • the compositions of the present invention are lipid nanoparticles (LNPs) or lipid polyplexes (LPPs) comprising RNAs of the present invention.
  • the lipids encapsulating the polynucleotides comprise cationic lipids and non-cationic lipids.
  • the cationic lipid is an ionizable cationic lipid.
  • the cationic lipid comprises DOTMA, DOTAP, DDAB, DOSPA, DODAC, DODAP, DC-Chol, DMRIE, DMOBA, DLinDMA, DLenDMA, CLinDMA, DMORIE, DLDMA, DMDMA, DOGS), N4-cholesteryl- Spermine, DLin-KC2-DMA, DLin-MC3-DMA, or a combination thereof.
  • the cationic lipid comprises M5, which has the following structure: In one embodiment, the cationic lipid comprises DOTMA. In one embodiment, the cationic lipid comprises DOTAP. In one embodiment, the cationic lipid comprises DOTMA and DOTAP.
  • the non-cationic lipid comprises a phospholipid as described herein. In one embodiment, the non-cationic lipid comprises a steroid as described herein. In one embodiment, the non-cationic lipid comprises a phospholipid and a steroid as described herein. In one embodiment, the phospholipid comprises DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, and DSPE, or a combination thereof. In one embodiment, the steroid is cholesterol. In one embodiment, the non-cationic lipid comprises DOPE. In one embodiment, the non-cationic lipid comprises DSPC. In one embodiment, the non-cationic lipid comprises cholesterol. In one embodiment, the non-cationic lipid comprises DOPE and cholesterol. In one embodiment, the non-cationic lipid comprises DSPC and cholesterol.
  • the cationic lipid comprises M5 and the non-cationic lipid comprises DOPE and cholesterol. In one embodiment, the cationic lipid comprises M5 and the non-cationic lipid comprises DSPC and cholesterol.
  • the lipid encapsulating the polynucleotide further comprises a polyethylene glycol-modified lipid.
  • the polyethylene glycol-modified lipid comprises DMG-PEG (eg, DMG-PEG 2000), DOGPEG, and DSPE-PEG, or a combination thereof.
  • the polyethylene glycol-modified lipid comprises DSPE-PEG.
  • the polyethylene glycol-modified lipid comprises DMG-PEG (eg, DMG-PEG 2000).
  • compositions of the present invention further comprise a cationic polymer associated with the polynucleotide in a complex, co-encapsulated in the lipid.
  • the cationic polymer comprises poly-L-lysine, protamine, polyethyleneimine (PEI), or a combination thereof. In one embodiment, the cationic polymer is protamine. In one embodiment, the cationic polymer is polyethyleneimine.
  • the amount of lipid in the composition is calculated in mole percent (mol %) determined based on the total moles of lipid in the composition.
  • the amount of cationic lipid in the composition is from about 10 to about 70 mole percent. In some embodiments, the amount of cationic lipid in the composition is about 20 to about 60 mole %, about 30 to about 50 mole %, about 35 to about 45 mole %, about 38 to about 45 mole %, about 40- About 45 mole %, about 40 to about 50 mole %, or about 45 to about 50 mole %.
  • the amount of phospholipid in the composition is from about 10 to about 70 mole percent. In one embodiment, the amount of phospholipid in the composition is about 20 to about 60 mol %, about 30 to about 50 mol %, about 10 to about 30 mol %, about 10 to about 20 mol %, or about 10 to about 15 mol % mol%.
  • the amount of cholesterol in the composition is from about 10 to about 70 mole percent. In one embodiment, the amount of cholesterol in the composition is about 20 to about 60 mole %, about 30 to about 50 mole %, about 35 to about 40 mole %, about 35 to about 45 mole %, about 40 to about 45 mole % mole % or from about 45 to about 50 mole %.
  • the amount of polyethylene glycol-modified lipid in the composition is from about 0.05 to about 20 mole percent. In one embodiment, the amount of polyethylene glycol-modified lipid in the composition is about 0.5 to about 15 mole %, about 1 to about 10 mole %, about 5 to about 15 mole %, about 1 to about 5 mole % %, about 1.5 to about 3 mole %, or about 2 to 5 mole %.
  • RNAs (particularly mRNAs) of the invention are formulated as lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • lipid nanoparticles or “LNPs” refer to particles formed from lipids in which nucleic acid (eg, mRNA) is encapsulated.
  • the LNP comprises the RNA of the invention and an RNA-encapsulating lipid, wherein the RNA-encapsulating lipid comprises a cationic lipid, a phospholipid, cholesterol, and a polyethylene glycol-modified lipid.
  • the cationic lipid is M5.
  • the phospholipid is DSPC.
  • the polyethylene glycol-modified lipid is DMG-PEG 2000.
  • the cationic lipid is M5, the phospholipid is DSPC, and the polyethylene glycol-modified lipid is DMG-PEG 2000.
  • the RNA-encapsulating lipid comprises 50 mol % M5, 10 mol % DSPC, 38.5 mol % cholesterol, and 1.5 mol % DMG-PEG 2000.
  • the RNA (particularly mRNA) of the invention is formulated as a lipopolyplex (LPP).
  • lipopolyplex or “LPP” refers to a core-shell structure comprising a nucleic acid core encapsulated by a lipid outer shell, the nucleic acid core comprising nucleic acid (e.g., mRNA) associated with a polymer.
  • the LPP comprises an RNA of the invention, which is associated with a cationic polymer as a complex; and a lipid that encapsulates the complex, wherein the lipid that encapsulates the complex comprises a cationic lipid, Non-cationic lipids and polyethylene glycol-modified lipids.
  • the non-cationic lipid comprises a phospholipid and a steroid.
  • the non-cationic lipid comprises a lipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), or a combination thereof. Phospholipids and cholesterol.
  • the cationic polymer comprises protamine.
  • the polyethylene glycol modified lipid comprises DMG-PEG 2000.
  • the cationic lipid comprises M5, which has the structure:
  • the non-cationic lipid comprises a phospholipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), or a combination thereof, and cholesterol;
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DSPC distearoylphosphatidylcholine
  • cholesterol cholesterol
  • the polyethylene glycol-modified lipid comprises 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000);
  • the cationic polymer comprises protamine.
  • the cationic polymer is protamine
  • the cationic lipid is M5
  • the phospholipid is DOPE
  • the polyethylene glycol-modified lipid is DMG-PEG 2000.
  • the lipid of the encapsulating complex comprises 40 mol % M5, 15 mol % DOPE, 43.5 mol % cholesterol and 1.5 mol % DMG-PEG 2000.
  • the composition of the present invention is a vaccine formulation (also referred to as a "vaccine agent”) comprising a nucleic acid sequence, and 10-70 mol % M5, 10-70 mol % DOPE, 10-70 mol % % cholesterol and 0.05-20 mol% DMG-PEG 2000,
  • nucleic acid sequence encodes a polypeptide of the present invention.
  • the nucleic acid sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 8-13.
  • the vaccine formulation comprises a polynucleotide encoding a polypeptide of the invention and a lipid encapsulating the polynucleotide, the lipid comprising 10-70 mol % M5, 10-70 mol % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 10-70 mol% cholesterol and 0.05-20 mol% 1,2-dimyristoyl-rac-glycerol-3- Methoxy polyethylene glycol (DMG-PEG) 2000,
  • the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 8-13.
  • the vaccine formulation further comprises a cationic polymer, wherein the cationic polymer is associated with the polynucleotide as a complex, co-encapsulated in a lipid to form a lipid multimeric complex.
  • Cationic lipids are lipids with a net positive charge at a specified pH. Lipids with a net positive charge can associate with nucleic acids through electrostatic interactions.
  • cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium-propane (1,2-di-O-octadecenyl-3-trimethylammonium-propane, DOTMA), 1 , 2-dioleoyl-3-trimethylammonium-propane (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), Didecyldimethylammonium bromide (DDAB), 2, 3-Dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propylaminium trifluoroacetate (2,3-dioleoyloxy-N-[2 (spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate, DOSPA), dioctadecyld
  • the cationic lipid is preferably an ionizable cationic lipid.
  • Ionizable cationic lipids have a net positive charge at, eg, acidic pH, and are neutral at higher pH (eg, physiological pH).
  • ionizable cationic lipids include, but are not limited to: dioctadecylamidoglycyl spermine (DOGS), N4-cholesteryl-spermine (N4-cholesteryl-spermine), 2,2- Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane , DLin-KC2-DMA), triheptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (heptatriaconta-6,9,28,31 -tetraen-19-yl-4-(dimethylamino)butanoate, DLin-MC3-DMA), heptadecan-9-yl-8-((2-hydroxyethyl)
  • the cationic lipid comprises M5, which has the following structure:
  • non-cationic lipids refer to lipids that do not carry a net positive charge at a given pH, such as anionic lipids and neutral lipids.
  • neutral lipid refers to a lipid that exists in an uncharged, neutral or zwitterionic form at physiological pH. Neutral lipids can include, but are not limited to, phospholipids and steroids.
  • phospholipids include, but are not limited to: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1-palmitoyl-2-oil Acylphosphatidylethanolamine (1-palmitoyl-2-oleoylphosphatidylethanolamine, POPE), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidylethanolamine (DSPE), dioleoyl phospholipid Dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC) , Dibehenoylphosphatidylcholine (DBPC), Ditricosanoylphosphatidylcholine (DTPC),
  • steroids examples include, but are limited to, for example, cholesterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and the like. derivative.
  • polyethylene glycol modified lipid refers to a molecule comprising a polyethylene glycol moiety and a lipid moiety.
  • polyethylene glycol-modified lipids include, but are not limited to: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol, DMG-PEG), 1,2-dioleoyl-rac-glycerol, methoxy-polyethylene glycol (1,2-Dioleoyl-rac-glycerol, methoxypolyethylene Glycol, DOGPEG)) and 1,2- Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol), DSPE-PEG).
  • the polyethylene glycol-modified lipid is DMG-PEG, such as DMG-PEG 2000.
  • DMG-PEG 2000 has the following structure:
  • n has an average value of 44.
  • cationic polymer refers to any ionic polymer capable of carrying a net positive charge to electrostatically bind nucleic acids at a specified pH.
  • examples of cationic polymers include, but are not limited to, poly-L-lysine, protamine, and polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • the polyethyleneimine can be linear or branched polyethyleneimine.
  • protamine refers to an arginine-rich, low molecular weight basic protein that is present in the sperm cells of various animals, especially fish, and binds DNA instead of histones.
  • the cationic polymer is protamine (eg, protamine sulfate).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptide, polynucleotide (especially RNA, eg mRNA) of the present invention, the composition and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises LNP or LPP as described herein.
  • pharmaceutically acceptable refers to the non-toxicity of a substance that does not interact with the action of the active components of the pharmaceutical composition.
  • “Pharmaceutically acceptable carrier” includes, but is not limited to, excipients, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, isotonic agents , flavoring agents and coloring agents.
  • Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and biocompatible polymers (eg lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers). Additional descriptions of pharmaceutically acceptable carriers can also be found, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R Gennaro edit. 1985).
  • the pharmaceutical composition of the present invention is used to induce an immune response against SARS-CoV-2 in a subject. In one embodiment, the pharmaceutical compositions of the present invention prevent and/or treat SARS-CoV-2 infection in a subject in need thereof.
  • the pharmaceutical composition of the present invention is an immunogenic composition, such as a vaccine.
  • the pharmaceutical compositions of the present invention may also contain adjuvants.
  • adjuvant refers to a substance capable of promoting, prolonging and/or enhancing an immune response.
  • adjuvants include, but are not limited to, oil emulsions (eg, Freund's adjuvant), aluminum hydroxide, mineral oil, bacterial products (eg, pertussis toxin).
  • compositions of the present invention are preferably administered parenterally.
  • parenteral administration refers to administration by any means other than through the gastrointestinal tract.
  • the pharmaceutical compositions of the present invention are administered intravenously, subcutaneously, intradermally, intramuscularly.
  • the pharmaceutical composition of the present invention is administered by subcutaneous, intradermal or intramuscular injection.
  • the present invention provides polypeptides, polynucleotides (especially RNA), compositions or pharmaceutical compositions of the present invention for preventing and/or treating SARS-CoV-2 infection.
  • the present invention provides the use of the polypeptide, polynucleotide (especially RNA), composition or pharmaceutical composition of the present invention in the preparation of a medicament for preventing and/or treating SARS-CoV-2 infection.
  • the present invention provides a method for preventing and/or treating SARS-CoV-2 infection in a subject, the method comprising administering a therapeutically effective amount of a polypeptide, polynucleotide (especially RNA), composition or pharmaceutical composition.
  • the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an mRNA of the invention, particularly a pharmaceutical composition comprising LNP or LPP as described herein.
  • therapeutically effective amount refers to an amount sufficient to prevent or inhibit the onset of a disease or symptom and/or slow, alleviate, delay the development or severity of the disease or symptom.
  • a therapeutically effective amount is affected by factors including, but not limited to, the speed and severity of the disease or condition, the age, sex, weight and physical condition of the subject, the duration of treatment, and the particular route of administration.
  • a therapeutically effective amount can be administered in one or more doses.
  • a therapeutically effective amount can be achieved by continuous or intermittent administration.
  • a therapeutically effective amount is provided in one or more administrations. In some embodiments, the therapeutically effective amount is provided in two administrations. In some embodiments, the therapeutically effective amount is provided in three administrations.
  • the polypeptides of the invention can be provided by expressing in a host cell (eg, a cell of a subject) a polynucleotide encoding the same (as described herein).
  • the polynucleotides of the invention are transiently expressed in cells of a subject.
  • the polypeptides of the invention are expressed on the cell surface of a subject.
  • the polypeptides of the invention are expressed as mature S proteins that lack a signal peptide.
  • the polypeptides of the invention are processed in antigen-presenting cells (eg, macrophages and dendritic cells) of a subject and presented as polypeptide antigens on the cell surface.
  • the polypeptides or polynucleotides (particularly mRNA) of the invention induce immune sera against SARS-CoV-2 or SARS-CoV-2 S protein in a subject.
  • the polypeptides or polynucleotides (particularly mRNA) of the invention induce an immune response against SARS-CoV-2 or SARS-CoV-2 S protein in a subject.
  • the polypeptides or polynucleotides (particularly mRNA) of the invention induce neutralizing antibodies against the SARS-CoV-2 S protein in a subject.
  • the mRNA is formulated as LNP and LPP as described herein.
  • the SARS-CoV-2 is wild-type SARS-CoV-2. In one embodiment, the SARS-CoV-2 is a SARS-CoV-2 B.1.351 variant (also known as "South African variant", “beta variant” or "501.V2 variant”).
  • the SARS-CoV-2 has the wild-type SARS-CoV-2 S protein.
  • the wild-type SARS-CoV-2 S protein comprises the amino acid sequence of SEQ ID NO:1.
  • the SARS-CoV-2 has a mutant SARS-CoV-2 S protein.
  • the mutant SARS-CoV-2 S protein may contain one or more amino acid modifications, such as amino acid additions, substitutions and/or deletions.
  • the mutant SARS-CoV-2 S protein comprises aspartic acid (D) at amino acid position 614 corresponding to SEQ ID NO:1.
  • the mutant SARS-CoV-2 S protein comprises a lysine (K) at amino acid position 417 corresponding to SEQ ID NO:1.
  • the mutant SARS-CoV-2 S protein comprises glutamic acid (E) at amino acid position 484 corresponding to SEQ ID NO:1.
  • the mutant SARS-CoV-2 S protein comprises glutamine (N) at amino acid position 501 corresponding to SEQ ID NO:1. In one embodiment, the mutant SARS-CoV-2 S protein comprises the following amino acids at amino acid positions 614, 417, 484 and 501 corresponding to SEQ ID NO: 1: D614, K417, E484 and N501.
  • the mutant SARS-CoV-2 S protein comprises a glycine (G) at amino acid position 614 corresponding to SEQ ID NO:1. In one embodiment, the mutant SARS-CoV-2 S protein comprises glutamine (N) at amino acid position 417 corresponding to SEQ ID NO:1. In one embodiment, the mutant SARS-CoV-2 S protein comprises a lysine (K) at amino acid position 484 corresponding to SEQ ID NO:1. In one embodiment, the mutant SARS-CoV-2 S protein comprises a tyrosine (Y) at amino acid position 501 corresponding to SEQ ID NO:1.
  • the mutant SARS-CoV-2 S protein comprises one or more of the following amino acid substitutions compared to SEQ ID NO: 1: D614G, K417N, E484K and N501Y. In one embodiment, the mutant SARS-CoV-2 S protein comprises the following amino acid substitutions compared to SEQ ID NO: 1: N501Y and D614G. In one embodiment, the mutant SARS-CoV-2 S protein comprises the following amino acid substitutions compared to SEQ ID NO: 1: K417N, N501Y and D614G. In one embodiment, the mutant SARS-CoV-2 S protein comprises the following amino acid substitutions compared to SEQ ID NO: 1: E484K, N501Y and D614G.
  • the mutant SARS-CoV-2 S protein comprises the following amino acid substitutions compared to SEQ ID NO: 1: D80A, D215G, K417N, E484K, N501Y, D614G and A701V. In one embodiment, the mutant SARS-CoV-2 S protein comprises the following amino acid substitutions compared to SEQ ID NO: 1: L18F, K417N, E484K, N501Y, D614G, D80A, D215G and A701V; and optionally Deletion of amino acids 242-244.
  • a polypeptide comprising the S1 subunit and the S2 subunit of the SARS-CoV-2 S protein from the N-terminal to the C-terminal, wherein the S1 subunit comprises an inactive furin cleavage site, and the inactive furin cleavage site is The active furin cleavage site is located at the C-terminus of the S1 subunit and has the amino acid sequence of QSAQ.
  • polypeptide of item 1 wherein the amino acids at positions corresponding to amino acids 986 and 987 of SEQ ID NO: 1 are proline.
  • polypeptide of item 1 or 2 wherein the amino acids at positions corresponding to amino acids 383 and 985 of SEQ ID NO: 1 are cysteine.
  • polypeptide of any one of items 1-4 wherein the amino acid at the position corresponding to amino acid 614 of SEQ ID NO:1 is glycine, and the amino acid at the position corresponding to amino acid 417 of SEQ ID NO:1 is asparagine, the amino acid at the position corresponding to amino acid 484 of SEQ ID NO: 1 is lysine, the amino acid at the position corresponding to amino acid 501 of SEQ ID NO: 1 is tyrosine, and the amino acid at the position corresponding to amino acid 501 of SEQ ID NO: 1 is tyrosine.
  • the amino acid at the position corresponding to amino acid 80 of SEQ ID NO:1 is alanine
  • the amino acid at the position corresponding to amino acid 215 of SEQ ID NO:1 is glycine
  • the amino acid at the position corresponding to amino acid 701 of SEQ ID NO:1 is glycine.
  • the amino acid at the position is valine.
  • the S1 subunit comprises an N-terminal domain, a receptor binding domain, and subdomains 1 and 2; preferably, the N-terminal domain, receptor
  • the body-binding domain and subdomains 1 and 2 have at least 80%, 85%, 90%, 95%, 96%, respectively, with the corresponding portion in the SARS-CoV-2 S protein having the amino acid sequence of SEQ ID NO: 1 , 97%, 98% or 99% identical.
  • polypeptide of any one of items 1-7 wherein the S1 subunit comprises at least 80 amino acid sequences with amino acids 14-685, 15-685, 16-685 or 17-685 of SEQ ID NO: 1 %, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said S2 subunit comprising the amino acid sequence of amino acids 686-1273 of SEQ ID NO: 1 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 80%, 85%, 90%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical.
  • polypeptide of any one of items 1-9 comprising the amino acid sequence of amino acids 14-1273, 15-1273, 16-1273, or 17-1273 of any one of SEQ ID NOs: 2-7.
  • polypeptide of any one of items 1-9 comprising the amino acid sequence of any one of SEQ ID NOs: 2-7.
  • the polynucleotide of item 12, which is RNA, and optionally the RNA is modified by a nucleoside comprising one or more modifications.
  • RNA is modified by replacing one or more uracils with 1-methylpseudouracil, pseudouracil, 5-methyl-uracil, or a combination thereof.
  • RNA comprises the nucleotide sequence of any one of SEQ ID NOs: 8-13.
  • RNA further comprises a 5' cap.
  • RNA further comprises a 5'UTR; preferably, the 5'UTR comprises the nucleotide sequence of any one of SEQ ID NOs: 33-44 ; More preferably, the 5'UTR comprises the nucleotide sequence of SEQ ID NO:42.
  • RNA further comprises a 3'UTR; preferably, the 3'UTR comprises the nucleotide sequence of any one of SEQ ID NOs:45-55 ; More preferably, the 3'UTR comprises the nucleotide sequence of SEQ ID NO:55.
  • RNA further comprises a poly(A) sequence; preferably, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:56.
  • composition comprising the polynucleotide of any of items 12-22 and a lipid that encapsulates the polynucleotide.
  • composition of item 23 comprising lipid nanoparticles or lipid multimeric complexes.
  • composition of item 23 or 24, wherein the lipid encapsulating the polynucleotide comprises a cationic lipid, a non-cationic lipid, and a polyethylene glycol-modified lipid; optionally, the composition also further A cationic polymer is included, wherein the cationic polymer is associated with the polynucleotide as a complex, co-encapsulated in a lipid to form a lipid polyplex.
  • a vaccine formulation comprising a polynucleotide encoding the polypeptide of any one of items 1-11 and a lipid encapsulating the polynucleotide, wherein the lipid comprises 10-70 mole % M5 , 10-70 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 10-70 mol% of cholesterol and 0.05-20 mol% of 1,2-dimyristoyl- rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG) 2000,
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DMG-PEG 1,2-dimyristoyl- rac-glycerol-3-methoxypolyethylene glycol
  • the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 8-13;
  • the vaccine formulation further comprises a cationic polymer, wherein the cationic polymer is associated with the polynucleotide as a complex, co-encapsulated in a lipid to form a lipid multimeric complex.
  • a pharmaceutical composition comprising the polypeptide of any one of items 1-11, the polynucleotide of any one of items 12-22, the composition of any one of items 23-25, or the The vaccine formulation of item 26; and a pharmaceutically acceptable carrier.
  • polypeptide of any one of items 1-11, the polynucleotide of any one of items 12-22, the composition of any one of items 23-25, the vaccine formulation of item 26, or the item 27 Use of the pharmaceutical composition of the item in the preparation of a medicament for preventing and/or treating SARS-CoV-2 infection.
  • polypeptides, polynucleotides, compositions, pharmaceutical compositions and methods of the present invention have at least one of the following beneficial effects:
  • S protein variants with design numbers 213, 215 and 217, respectively, and the amino acid sequences are shown in SEQ ID NOs: 2-4. wherein S protein variants 213, 215 and 217 all contain an inactive Furin cleavage site "QSAQ" and amino acid substitutions K986P, V987P and D614G (213, 215 and 217) compared to the wild-type S protein of SEQ ID NO: 1 ), S protein variants 215 and 217 further comprise the following amino acid substitutions, respectively: S383C and D985C (215); or F817A, A892P, A899P and A942P (217) (Table 1A).
  • S protein variants numbered 223, 225 and 227 were designed and the amino acid sequences are shown in SEQ ID NOs: 5-7. These S protein variants compared to S protein variants 213, 215 and 217 further contained the major mutations present in the beta variant strains: K417N, E484K, N501Y, D80A, D215G and A701V (Table 1B).
  • the control S protein variants 212, 214, 216, 222, 224 and 226 contain the active Furin cleavage site "RRAR", and the other mutations correspond to the S protein variants 213, 215, 217, 223, 225 and 227, respectively (Table 1). 1A and 1B).
  • DNA open reading frame (ORF) sequences encoding the S protein variants described in Example 1.1 were designed, codon-optimized for optimal expression in human cells.
  • the DNA ORF sequences encoding S protein variants 213, 215 and 217 are shown in SEQ ID NOs: 57-59.
  • the RNA ORF sequences corresponding to the DNA ORF sequences encoding S protein variants 213, 215, 217, 223, 225 and 227 are shown in SEQ ID NOs: 8-13.
  • the RNA ORF sequences corresponding to the DNA ORF sequences encoding the control S protein variants 212, 214, 216, 222, 224 and 226 are shown in SEQ ID NOs: 26-31 (Table 2).
  • T7 promoter sequence (SEQ ID NO:32), 5'UTR sequence (SEQ ID NO:33-44), 3'UTR sequence (SEQ ID NO:45-55) and poly(A) sequence (SEQ ID NO:45-55) were also designed NO:56).
  • the plasmid DNA template was finally linearized using restriction enzymes using a pair of primers (upstream primer: 5'TTGGACCCTCGTACAGAAGCTAATACG 3'; and downstream poly(T) long primer: 5'TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • CN113186203A The method of preparing in vitro transcribed mRNA using DNA template is described in CN113186203A. Briefly, using the DNA template prepared as in Example 1.2 as a template, a co-transcription capping reaction was performed using T7 RNA polymerase to perform in vitro transcription of RNA to generate Cap1 mRNA. 1-methyl-pseudouracil nucleotide (1-methyl-pseudouridine) was added to the reaction system instead of uridine triphosphate (UTP), therefore, the 1-methyl-pseudouracil in the in vitro transcribed Cap1 mRNA The modification ratio is 100%. After transcription, the DNA template was digested with DNaseI (Thermo Fisher Scientific Co., Ltd.) to reduce the risk of residual DNA template.
  • DNaseI Thermo Fisher Scientific Co., Ltd.
  • Cap1 mRNA was purified using DynabeadsMyone (Thermo Fisher Scientific). Purified Cap1 mRNA was dissolved in sodium citrate solution.
  • the nucleotide sequences of mRNAs numbered 213, 215, 217, 223, 225 and 227 are shown in SEQ ID NOs: 14-19 (Table 2).
  • the full-length sequences of mRNAs numbered 212, 214, 216, 222, 224 and 226 are not shown.
  • the difference between the unshown mRNA sequences and the mRNA sequences of SEQ ID NOs: 14-19 is that the RNA ORF sequences in the unshown mRNA sequences are RNA ORF sequences selected from the group consisting of SEQ ID NOs: 26-31.
  • Example 1.3 Expression of candidate mRNAs prepared as in Example 1.3 was verified in DC2.4 cells (mouse bone marrow-derived dendritic cell line; ATCC). Briefly, 2 ⁇ g of mRNA was transfected into DC2.4 cells using the transfection reagent Lipofectamine MessengerMax (Invitrogen). Place the transfected cells in a cell incubator and continue to culture at 37°C 5% CO for 18-24h. Cells were then collected and counted after washing with PBS. Take 1x10 6 cells into a flow tube and centrifuge to discard the supernatant.
  • Cationic lipid M5 was synthesized by microorganisms; auxiliary phospholipids (DOPE and DSPC) were purchased from CordenPharma; cholesterol was purchased from Sigma-Aldrich; mPEG2000-DMG (ie DMG-PEG 2000) was purchased from Avanti Polar Lipids, Inc.; PBS was purchased from Invitrogen ; Protamine sulfate was purchased from Beijing Silian Pharmaceutical Co., Ltd.
  • DOPE and DSPC auxiliary phospholipids
  • cholesterol was purchased from Sigma-Aldrich
  • mPEG2000-DMG ie DMG-PEG 2000
  • PBS was purchased from Invitrogen
  • Protamine sulfate was purchased from Beijing Silian Pharmaceutical Co., Ltd.
  • mRNA aqueous solution Each mRNA prepared as in Example 1.3 was diluted to 0.35 mg/mL mRNA aqueous solution with 50 mM citric acid-sodium citrate buffer (pH 3-4).
  • lipid solution cationic lipid (M5): DSPC: cholesterol: DMG-PEG 2000 was dissolved in ethanol solution at a molar ratio of 50:10:38.5:1.5 to prepare a 10 mg/mL lipid solution.
  • Centrifugal ultrafiltration The LNP-mRNA solution was added to the ultrafiltration tube for centrifugal ultrafiltration concentration (centrifugal force 3400g, centrifugation time 60min, temperature 4°C), and the serial numbers were 213, 215, 217, 223, 225, 227, 212, LNP-mRNA preparations of 214, 216, 222, 224 and 226.
  • mRNA aqueous solution Each mRNA prepared as in Example 1.3 was diluted to 0.35 mg/mL mRNA aqueous solution with 50 mM citric acid-sodium citrate buffer (pH 3-4).
  • lipid solution cationic lipid (M5): DOPE: cholesterol: DMG-PEG 2000 was dissolved in absolute ethanol at a molar ratio of 40:15:43.5:1.5 to prepare a 10 mg/mL lipid solution.
  • M5 cationic lipid
  • DOPE cholesterol: DMG-PEG 2000 was dissolved in absolute ethanol at a molar ratio of 40:15:43.5:1.5 to prepare a 10 mg/mL lipid solution.
  • protamine sulfate solution Protamine sulfate was dissolved in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.2 mg/mL.
  • Centrifugal ultrafiltration The LPP-mRNA solution was centrifuged to remove ethanol by ultrafiltration (centrifugation force 3400g, centrifugation time 60min, temperature 4°C), and the numbers were 213, 215, 217, 223, 225, 227, 212, 214, 216, 222 , 224 and 226 LPP-mRNA preparations.
  • the LPP-mRNA preparation prepared as in Example 3.2 was used to immunize C57BL/6 mice (Shanghai Lingchang Biotechnology Co., Ltd.) and divided into 12 groups (respectively LPP-mRNA 213, 215, 217, 223, 225, 227, 212 , 214, 216, 222, 224 and 226 groups), 8 mice in each group. Mice were immunized on day 0 (primary immunization) and day 14 (secondary immunization) by intramuscular injection, with a single immunization dose of 10 ⁇ g mRNA per mouse.
  • the mouse immune serum was collected on the 14th day (ie, the 28th day) after the second immunization, and a commercial wild-type or B.1.351 mutant pseudovirus kit (Beijing Tiantan Pharmaceutical Biotechnology Development Co., Ltd.; wild-type pseudovirus product number) was used. : 80033; B.1.351 variant strain pseudovirus Cat. No. 80044), to evaluate the titer level of neutralizing antibodies in immune serum.
  • the pseudovirus uses a plasmid expressing wild-type or B.1.351 SARS-CoV-2 S protein instead of a plasmid expressing VSV-G protein, and carries a luciferase reporter gene.
  • a pseudovirus is used to infect cells that express ACE2 on their surface, the S protein binds to ACE2 to mediate the entry of the pseudovirus into the cell, resulting in the expression of luciferase.
  • the ability of immune serum to inhibit pseudovirus infection of ACE2-expressing cells can be characterized by the rate of inhibition, which can be measured by the luminescence intensity of the luciferase-catalyzed substrate luciferin from a sample of immune serum compared to a positive control (eg, a serum-free control). The percentage of decline is calculated.
  • the S protein used for the wild-type pseudovirus has the amino acid sequence of SEQ ID NO:1.
  • the S protein for the B.1.351 variant pseudovirus contains the following mutations relative to SEQ ID NO: 1: amino acid substitutions L18F, D80A, D215G, K417N, E484K, N501Y, D614G, and A701V; and deletions of amino acids 242-244.
  • the immune sera from each group were diluted 20, 60, 180, 540, 1620 and 4860-fold; pseudoviruses were added to the diluted immune sera or equal volume of cell culture medium (as a serum-free control) and incubated for 1 hours; then a certain amount of Huh7 cells (a human hepatoma cell line expressing endogenous hACE2; ATCC) was added to the serum-pseudovirus mixture; after 24 hours, the supernatant was discarded, the cells were lysed and luciferin was added; using the enzyme
  • the immune sera induced by vaccines 215 and 217 further introduced other mutations have the neutralization ability of both wild-type and B.1.351 S protein pseudoviruses. Further improve.
  • Vaccines 213, 215 and 217 showed significant differences compared to 223, 225 and 227 in specificity for wild-type and B.1.351 S protein pseudoviruses: vaccines 213, 215 and 217 induced immune sera to wild-type S The neutralization capacity of the protein pseudovirus was higher, and the neutralization capacity of the B.1.351 S protein pseudovirus was higher by the immune sera induced by vaccines 223, 225 and 227. This indicates that vaccines 223, 225 and 227 induce stronger B.1.351 variant-specific immune responses.

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Abstract

涉及生物医药领域，具体涉及预防或治疗冠状病毒感染的疫苗。特别地，提供用于预防或治疗新型冠状病毒感染的多肽、编码其的多核苷酸(特别是mRNA)以及组合物。

Description

一种治疗或者预防冠状病毒病突变株的疫苗试剂
本申请要求2021年2月10日提交的，题为“一种治疗或者预防冠状病毒病突变株的疫苗试剂”的第202110184680.7号和题为“一种治疗或者预防冠状病毒病的疫苗试剂”的第202110184684.5号中国专利申请的优先权，该申请的内容整体援引加入本文。
技术领域
本发明涉及生物医药领域，具体涉及预防或治疗冠状病毒感染的疫苗。特别地，本发明提供用于预防或治疗新型冠状病毒感染的多肽、编码其的多核苷酸(特别是mRNA)以及组合物。
背景技术
严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引发全球大流行。SARS-CoV-2具有传播能力强、致死率高的特性，可在感染者中导致严重的病毒性肺炎和呼吸系统疾病，称为“冠状病毒疾病2019(COVID-19)”。
目前已开发多种针对SARS-CoV-2的疫苗，包括灭活病毒疫苗、基于病毒载体的疫苗、重组蛋白疫苗、DNA疫苗和mRNA疫苗。SARS-CoV-2具有高变异性，已发展出多个变异毒株，并且其中部分已显示出较高的免疫逃逸特性，对现有疫苗提出了新的挑战。目前亟需用于预防和/或治疗冠状病毒感染的药物和方法。
CN111218458A公开了一种编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法，该编码SARS-CoV-2病毒抗原的mRNA至少含有编码SARS-CoV-2病毒的S蛋白和N蛋白中的至少一个蛋白，和/或至少一个蛋白的片段的编码区。
发明内容
本发明提供一种多肽，其从N端至C端包含SARS-CoV-2 S蛋白的S1亚基和S2亚基，其中所述S1亚基包含失活的弗林蛋白酶切割位点，所述失活的弗林蛋白酶切割位点位于所述S1亚基的C端并且具有QSAQ的氨基酸序列。
在一实施方案中，所述多肽在对应于SEQ ID NO:1的氨基酸986和987的位置处的氨基酸为脯氨酸。在一实施方案中，所述多肽在对应于SEQ ID NO:1的氨基酸383和985的位置处的氨基酸为半胱氨酸。在一实施方案中，所述多肽在对应于SEQ ID NO:1的氨基酸817、892、899和942的位置处的氨基酸为脯氨酸。
在一实施方案中，所述多肽在对应于SEQ ID NO:1的氨基酸614的位置处的氨基酸为甘氨酸。
在一实施方案中，所述多肽在对应于SEQ ID NO:1的氨基酸614的位置处的氨基酸为甘氨酸，在对应于SEQ ID NO:1的氨基酸417的位置处的氨基酸为天冬酰胺，在对应于SEQ ID NO:1的氨基酸484的位置处的氨基酸为赖氨酸，在对应于SEQ ID NO:1的氨基酸501的位置处的氨基酸为酪氨酸，在对应于SEQ ID NO:1的氨基酸80的位置处的氨基酸为丙氨酸、在对应于SEQ ID NO:1的氨基酸215的位置处的氨基酸为甘氨酸，并且在对应于SEQ ID NO:1的氨基酸701的位置处的氨基 酸为缬氨酸。
在一实施方案中，所述多肽进一步包含以下氨基酸修饰中的一个或多个：
(a)在对应于SEQ ID NO:1的氨基酸69、70、144、145、242-244、689-715、715-724、788-806和819-828的位置处的氨基酸中的一个或多个的缺失；
(b)在对应于SEQ ID NO:1的氨基酸18、20、26、80、138、152、190、215、242、246、417、439、452、453、484、501、570、614、655、681、701、716、982、1027和1118的位置处的氨基酸中的一个或多个的取代。
在一实施方案中，所述多肽包含与SEQ ID NO:1的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一实施方案中，所述多肽包含SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列。在一实施方案中，所述多肽包含SEQ ID NO:2-7中任一个的氨基酸序列。
在另一方面，本发明提供一种多核苷酸，其编码本发明的多肽。
在一实施方案中，所述多核苷酸为RNA，并且任选地所述RNA通过包含一个或多个修饰的核苷进行修饰。在一实施方案中，所述RNA通过用1-甲基假尿嘧啶、假尿嘧啶、5-甲基-尿嘧啶或其组合代替一个或多个尿嘧啶进行修饰。在一实施方案中，所述RNA包含SEQ ID NO:8-13中任一个的核苷酸序列。
在一实施方案中，所述RNA进一步包含5’帽、5’UTR、3’UTR和poly(A)序列中的一个或多个。
在一实施方案中，所述多核苷酸包含SEQ ID NO:14-19中任一个的核苷酸序列。
在又一方面，本发明提供一种组合物，其包含本发明的多核苷酸以及包封所述多核苷酸的脂质。在一实施方案中，所述组合物包含脂质纳米颗粒或脂质多聚复合物。
本发明还提供一种药物组合物，其包含本发明的多肽、多核苷酸、组合物或疫苗制剂；以及药学上可接受的载剂。
本发明提供本发明的多肽、多核苷酸、组合物、疫苗制剂或药物组合物，用于预防和/或治疗SARS-CoV-2感染。
本发明还提供本发明的多肽、多核苷酸、组合物、疫苗制剂或药物组合物在制备用于预防和/或治疗SARS-CoV-2感染的药物中的用途。
本发明还提供一种用于在受试者中预防和/或治疗SARS-CoV-2感染的方法，所述方法包括给药治疗有效量的本发明的多肽、多核苷酸(特别是RNA)、组合物或药物组合物。
附图说明
图1显示构建的质粒中DNA模板序列的结构示意图，从5’端至3’端依次包含：T7启动子、5’UTR、ORF、3’UTR和poly(A)尾。
图2显示通过流式细胞术分析的候选mRNA在DC2.4细胞中的表达结果。
图3显示通过假病毒中和试验分析的候选mRNA疫苗制剂诱导的免疫血清中针对野生型假病毒的中和抗体的滴度水平(ID 50)，显示的是平均值±SEM，N＝8。
图4显示通过假病毒中和试验分析的候选mRNA疫苗制剂诱导的免疫血清中针对B.1.351变异株假病毒的中和抗体的滴度水平(ID 50)，显示的是平均值±SEM，N＝8。
具体实施方式
一般术语和定义
本文引用的所有专利、专利申请、科学出版物、制造商的说明书和指南等，无论上文或下文，均整体援引加入本文。本文中的任何内容均不应理解为承认本公开无权先于这样的公开。
除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白和核酸化学、分子生物学、细胞和组织培养、微生物学相关术语均为相应领域内广泛使用的术语(参见，例如，Molecular Cloning:A Laboratory Manual,2 nd Edition,J.Sambrook et al.eds.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989)。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文所用，表述“包括”、“包含”、“含有”和“具有”是开放式的，表示包括所列举的元素、步骤或组分但不排除其他未列举的元素、步骤或组分。表述“由……组成”不包括未指定的任何元素、步骤或组分。表述“基本上由……组成”是指范围限于指定的元素、步骤或组分，加上不显著影响要求保护的主题的基本和新颖性质的任选存在的元素、步骤或组分。应当理解，表述“基本上由……组成”和“由……组成”涵盖在表述“包含”的含义之内。
如本文所用，除非上下文另外指明，单数形式的表述“一个”、“一种”或“这个”包括复数指代。术语“一个或多个”或者“至少一个”涵盖1、2、3、4、5、6、7、8、9个或更多个。
本文中值的范围的列举仅为了用作单独提到落在所述范围内的每个不同值的速记方法。除非本文另有说明，否则每个单独的值如其在本文中单独列举地加入本说明书。除非明确指出相反，在本文示出的数值或范围均由“约”修饰，表示所列举或声称的数值或范围±20％、±10％、±5％或±3％。
除非另有说明，否则本文描述的所有方法可以以任何合适的顺序进行。
如本文所用，术语“多肽”指包含通过肽键共价连接的两个以上氨基酸的聚合物。“蛋白”可以包含一条或多条多肽，其中多肽之间通过共价或非共价方式相互作用。除非另有说明，“多肽”和“蛋白”可以互换使用。
如本所用，关于多肽的术语“野生型(WT)”表示该多肽是天然存在的并且未经人为修饰的，包括天然存在的突变体。
如本文所用，参考多肽的“变体”表示由于至少一个氨基酸修饰而与参考多肽不同的多肽。参考多肽可以是天然存在的，也可以是野生型多肽的修饰形式。在本文中，“多肽变体”和“突变型多肽”具有相同含义。多肽变体可以是例如突变体、翻译后修饰变体、同种型、物种变体和物种同源物等。多肽变体可以通过重组DNA技术制备，例如通过改变编码序列对已知氨基酸序列进行修饰。多肽变体还可以通过化学合成或酶促方法制备。根据本发明，S蛋白变体可以具有与野生型S蛋白相当或更高的诱导免疫应答的能力，即表现出与野生型S蛋白相当或增强的免疫原性。
如本文所用，对于氨基酸序列的修饰可以包括例如氨基酸取代、添加和/或缺失。“氨基酸添加”是指将一个或多个氨基酸添加到氨基酸序列。氨基酸添加可以发生在氨基酸序列中的任何位置，包括但不限于氨基酸序列的中间、氨基末端和/或羧基末端。发生在氨基酸序列中间的氨基酸添加也可以称为“氨基酸插入”。“氨基酸缺失”是指从氨基酸序列去除一个或多个氨基酸。氨基酸缺失可以发生在氨基酸序列中的任何位置。发生在N和/或C末端的氨基酸缺失也可以称为截短。截短变体也可以称为“片段”。“氨基酸取代”是指将特定氨基酸位置处的氨基酸残基替换为另一个氨基酸残基。在本文中，“氨基酸修饰”也可以称为“突变”。优先考虑在同源多肽之间非保守氨基酸位置进行保守取 代。优选地，多肽变体中的氨基酸取代是保守的氨基酸取代。
在本文中，给定氨基酸序列的氨基酸位置N处的氨基酸X aa(即第N位氨基酸为X aa，其中N为大于等于1的整数)可以表示为“NX aa”。用氨基酸X bb取代给定氨基酸序列中的氨基酸NX aa可以表示为“X aaNX bb”。
如本文所用，可以通过将参考多肽与另一多肽的氨基酸序列进行最佳比对(例如如本文所述)后确定二者氨基酸序列或氨基酸序列的部分(例如亚基、结构域或亚结构域)之间的对应性或者二者中指定氨基酸位置之间的对应性。在本文中，“多肽变体在对应于参考多肽的氨基酸N处包含氨基酸取代”或者“多肽变体包含与参考多肽相比的氨基酸取代”，表示所述多肽变体与参考多肽在对应于参考多肽的氨基酸位置N处包含不同的氨基酸，但对多肽变体的其他位置的氨基酸不构成限制，即其他位置的氨基酸可以与参考多肽的对应位置上的氨基酸相同或不同)。类似地，“多肽变体在对应于参考多肽的氨基酸N的位置处的氨基酸为X aa”，仅表示所述多肽变体在对应于参考多肽的氨基酸位置N处的氨基酸为X aa，但对多肽变体的其他位置的氨基酸不构成限制。
如本文所用，关于序列的术语“％相同性”或“百分比相同性”是指在待比较的序列之间的最佳比对中相同的核苷酸或氨基酸的百分比。两个序列之间的差异可以分布在待比较序列的局部区域(区段)或整个长度上。通常在对区段或“比较窗口”最佳比对之后，确定两个序列之间的百分比相同性。最佳比对可以手动进行，或者借助于本领域已知算法，包括但不限于Smith and Waterman,1981,Ads App.Math.2,482和Neddleman and Wunsch,1970,J.Mol.Biol.48,443描述的局部同源性算法，Pearson and Lipman,1988,Proc.Natl Acad.Sci.USA 88,2444描述的相似性搜索方法，或使用计算机程序，例如Wisconsin Genetics Software Package,Genetics Computer Group,575 Science Drive,Madison,Wis.中的GAP、BESTFIT、FASTA、BLAST P、BLAST N和TFASTA进行。例如，可以利用美国国家生物技术信息中心(NCBI)网站公共可用的BLASTN或BLASTP算法确定两个序列的百分比相同性。
通过确定待比较的序列对应的相同位置的数目，用这个数目除以比较的位置数目(例如，参考序列中的位置数目)，并将这个结果乘以100，获得百分比相同性。在一些实施方案中，对参考序列整个长度的至少约50％、至少约60％、至少约70％、至少约80％、至少约90％或约100％的区域给出相同性程度。在一些实施方案中，对参考序列的整个长度给出相同性程度。可以用本领域已知的工具进行确定序列相同性的比对，优选利用最佳序列比对，例如，利用Align，利用标准设置，优选EMBOSS::needle、Matrix:Blosum62、Gap Open 10.0、Gap Extend 0.5。
在本文中，“核苷酸”包括脱氧核糖核苷酸和核糖核苷酸及其衍生物。如本文所用，“核糖核苷酸”是指在β-D-呋喃核糖(β-D-ribofuranosyl)基团的2’位置具有羟基的核苷酸。“核苷酸”通常由代表其中碱基的单字母来指代：“A(a)”指脱氧腺苷酸或腺苷酸，“C(c)”指脱氧胞苷酸或胞苷酸，“G(c)”指脱氧鸟苷酸或鸟苷酸，“U(u)”指尿苷酸，“T(t)”指脱氧胸苷酸。
如本文所用，术语“多核苷酸”和“核酸”可以互换使用，用于指脱氧核糖核苷酸的聚合物(脱氧核糖核酸，DNA)或核糖核苷酸的聚合物(核糖核酸，RNA)。“多核苷酸序列”、“核酸序列”和“核苷酸序列”可以互换使用，用来表示多核苷酸中核苷酸的排序。本领域人员应当理解，DNA编码链(有义链)与其编码的RNA可以看作具有相同的核苷酸序列，DNA编码链序列中的脱氧胸苷酸对应其编码的RNA序列中的尿苷酸。
如本文所用，术语“载体”是指用于将核酸导入宿主细胞的媒介物。载体可以包括表达载体和克隆载体。通常而言，表达载体包含期望的编码序列以及在特定宿主生物体(例如，细菌、酵母、植物、昆虫或哺乳动物)或体外表达系统中表达可操作地连接的编码序列所必需的适当DNA序列。克隆载 体一般用来工程化(进行重组DNA操作)和扩增期望的DNA片段，并且可以缺少表达期望的DNA序列所需要的功能序列。载体的实例包括但不限于质粒、粘粒、噬菌体(如λ噬菌体)载体、病毒载体(如逆转录病毒、腺病毒或杆状病毒载体)或者人工染色体(如细菌人工染色体(BAC)、酵母人工染色体(YAC)或P1人工染色体(PAC))载体。
如本文所用，术语“表达”包括核苷酸序列的转录和/或翻译。因此，表达可以涉及转录物和/或多肽的产生。术语“转录”涉及将DNA序列中的遗传密码转录为RNA(转录物)的过程。术语“体外转录”指在不含细胞的系统中(例如在适当的细胞提取物中)体外合成RNA，特别是mRNA(参见例如，Pardi N.,Muramatsu H.,Weissman D.,KarikóK.(2013).In:Rabinovich P.(eds)Synthetic Messenger RNA and Cell Metabolism Modulation.Methods in Molecular Biology(Methods and Protocols),vol 969.Humana Press,Totowa,NJ.)。可以用于产生转录物的载体又称为“转录载体”，其中包含转录所需的调控序列。术语“转录”涵盖“体外转录”。
如本文所用，术语“连接”和“融合”可互换使用，用于表示两个或更多个元件、区段或者结构域连接在一起。
如本文所用，术语“宿主细胞”指用于接受、保持、复制、表达多核苷酸或载体的细胞。在一些实施方案中，宿主细胞可以是在其中表达本发明的多肽的细胞。
如本文所用，“分离的”是指物质(例如多核苷酸或多肽)与其存在的来源或环境是分离的。分离的多核苷酸或多肽可以以基本上纯的形式存在(例如在组合物中)，或者可以存在于非天然环境中，例如，宿主细胞。在一些实施方案中，本发明的多肽和多核苷酸是分离的。术语“天然存在”是指物体可以在自然中发现这一事实。例如，存在于生物体(包括病毒)中且可以分离自自然来源并且尚未被人在实验室中有意修饰的多肽或多核苷酸是天然存在的。
如本文所用，术语“重组”表示通过“遗传工程制备”。一般而言，重组分子(例如重组蛋白和重组核酸)是非天然存在的。本发明的多肽和多核苷酸可以是重组分子。
术语“在细胞表面上表达”表示分子如抗原与细胞的质膜相互关联并位于细胞的质膜上，其中至少一部分分子面向胞外空间，并且可从所述细胞的外部接近，例如，通过位于细胞外部的抗体。在一些实施方案中，本发明的多肽可以在合适的宿主细胞表面上表达。在一些实施方案中，与包含活性弗林蛋白酶切割位点(例如具有氨基酸序列RRAR的弗林蛋白酶切割位点)的S蛋白相比，本发明的多肽在宿主细胞表面上表达的水平更高。在一些实施方案中，本发明的多肽在合适的宿主细胞表面上表达，并且能够结合人ACE2蛋白(hACE2)。
如本文所用，术语“疫苗”是指包含活性成分(例如本发明的多肽抗原或者编码其的多核苷酸)的组合物，所述组合物在接种至受试者中时诱导免疫应答，所述免疫应答足以预防和/或减轻与病原体或疾病感染相关的至少一种症状。根据本发明，本文所述的多肽、多核苷酸、组合物或药物组合物可以作为疫苗，用于在有需要的受试者中提供针对SARS-CoV-2的预防性和/或治疗性免疫。
如本文所用，术语“中和抗体”是指能够中和，即防止、抑制、降低或干扰病原体在宿主(例如宿主生物体或宿主细胞)中引发和/或保持感染的能力的抗体或其片段。根据本发明，用本发明的疫苗进行接种的受试者中可以产生针对SARS-CoV-2 S蛋白或SARS-CoV-2的中和抗体，例如在受试者的免疫血清中。可以使用本领域已知的方法测量免疫血清中的中和抗体滴度水平。在一实施方案中，通过假病毒中和试验(pVNT)测量免疫血清中的中和抗体水平，pVNT测量的中和抗体滴度水平表示为50％抑制稀释度(ID 50)，其表示假病毒在与免疫血清发生中和后，50％假病毒受抑制时对应的免疫血清稀释度。具体的数值主要表现为假病毒自身携带的报告基因的表达水平(例如：荧光素酶催化底 物产生的发光强度；或GFP蛋白的荧光强度)，例如如本申请实施例4所述。
术语“抗原”是指其中包含表位的物质，针对所述表位可以产生免疫应答。在特定实施方案中，抗原可以与T细胞表位或者T或B细胞受体结合，或者与免疫球蛋白例如抗体结合。
在本文中，“多肽抗原”是指作为抗原的多肽，包括但不限于多肽抗原本身或者其加工产物(例如在体内经过加工和呈递的抗原)。根据本发明，本发明的多肽或其加工产物可以是多肽抗原，并作为疫苗中的免疫原性活性成分诱导免疫应答。
术语“转染”涉及将多核苷酸导入宿主细胞。用于转染本文所述多核苷酸的宿主细胞可以存在于体外或体内。在一些实施方案中，宿主细胞可以是受试者(特别是患者，例如感染有新型冠状病毒的患者)的细胞。转染可以是瞬时或稳定的。一般而言，瞬时转染不涉及整合入宿主细胞基因组。稳定转染可以通过使用基于病毒或转座子的系统进行转染来实现。
冠状病毒
如本文所用，“严重急性呼吸综合征冠状病毒2”、“新型冠状病毒”和“SARS-CoV-2”可以互换使用。已知SARS-CoV-2是导致“冠状病毒病2019(COVID-19)”的病原体。
SARS-CoV-2是一种正义单链RNA((+)ssRNA)包膜病毒，属于冠状病毒科的β属。SARS-CoV-2编码4种结构蛋白：刺突蛋白(S)、包膜蛋白(E)、膜蛋白(M)和核衣壳蛋白(N)。其中，刺突蛋白(S蛋白)介导病毒对宿主细胞的特异性结合以及病毒囊膜与宿主细胞膜的融合，因此是病毒感染宿主细胞的关键分子。
多肽
在一总的方面，本发明提供一种多肽，其包含SARS-CoV-2的刺突蛋白变体(在本文中又称为“S蛋白变体”)，所述S蛋白变体包含失活的弗林蛋白酶切割位点，并且其中所述失活的弗林蛋白酶切割位点具有QSAQ的氨基酸序列。
本发明的多肽可以包含氨基酸修饰，例如相对于野生型S蛋白(示例性氨基酸序列示于SEQ ID NO:1)包含修饰，例如氨基酸的添加、取代和/或缺失。本发明的多肽可以作为多肽抗原用于在受试者中诱导针对SARS-CoV-2感染的保护性免疫应答。在一些实施方案中，所述多肽包含本文所述的多肽抗原。在一些实施方案中，所述多肽由本文所述的多肽抗原组成。在一些实施方案中，所述多肽包含本文所述的多肽抗原和信号肽。
如本文所用，“SARS-CoV-2刺突蛋白”、“SARS-CoV-2 S蛋白”或“S蛋白”是指SARS-CoV-2的刺突蛋白。SARS-CoV-2 S蛋白合成为具有约1273-1300个氨基酸的糖蛋白(示例性氨基酸序列示于SEQ ID NO:1)，其包含N端信号肽(大约对应SEQ ID NO:1的氨基酸1-13、1-14、1-15或1-16)、S1亚基(大约对应SEQ ID NO:1的氨基酸14-685，例如氨基酸15-685、16-685或17-685)和S2亚基(大约对应SEQ ID NO:1的氨基酸686-1273，例如氨基酸686-1213)。S1亚基中包含N端结构域(大约对应SEQ ID NO:1的氨基酸14-305，例如氨基酸15-305、16-305或17-305)、受体结合结构域(RBD)(大约对应SEQ ID NO:1的氨基酸319-527，例如氨基酸328-527或331-524)和亚结构域1和2(SD1/2)(大约对应SEQ ID NO:1的氨基酸528-685)。S2亚基包含融合肽(FP)(大约对应SEQ ID NO:1的氨基酸788-806)，七肽重复序列HR1(大约对应SEQ ID NO:1的氨基酸912-984)、HR2(大约对应SEQ ID NO:1的氨基酸1163-1213)，跨膜结构域(大约对应SEQ ID NO:1的氨基酸1213-1237)和胞质结构域(大约对应SEQ ID NO:1的氨基酸1237-1273)。有关SARS-CoV-2 S蛋白的描述还可以参见例如Huang Y et al.,Acta Pharmacol Sin.2020；41(9):1141-1149。
研究表明，S1亚基的RBD通过与特异性受体血管紧张素转化酶2(ACE2)识别靶宿主细胞，而S2亚基负责膜融合。在自然状态下，S蛋白以亚稳定的融合前三聚体构象存在于病毒表面。在感染期间，RBD与宿主细胞受体结合，宿主蛋白酶(如弗林蛋白酶(Furin))切割S蛋白的S1/S2切割位点，破坏融合前三聚体的稳定性，导致S1亚基脱落和S2亚基转变为融合后的稳定构象。Furin切割位点是一个含有多个精氨酸残基的暴露的环型结构，其包含氨基酸基序Arg-X aa-X bb-Arg(其中X aa为任意氨基酸；X bb为任意氨基酸，优选为Arg或Lys。在一实施方案中，Furin切割位点的氨基酸序列为Arg-Arg-Ala-Arg(“RRAR”)，对应于SEQ ID NO:1中氨基酸682-685。
本发明的多肽抗原可以包含失活的Furin切割位点。具体地，本发明的多肽抗原包含失活的Furin切割位点Gln-Ser-Ala-Gln(QSAQ)，从而在宿主细胞中具有更高的表达水平和/或在受试者中诱导更强的免疫应答。如本文所用，“失活的弗林蛋白酶(Furin)切割位点”是指不能被弗林蛋白酶识别和切割的氨基酸序列。如本文所用，“活性弗林蛋白酶切割位点”或“弗林蛋白酶切割位点”是指能够被弗林蛋白酶识别并切割的氨基酸序列。
在一些实施方案中，本发明的多肽从N端至C端包含SARS-CoV-2 S蛋白的S1亚基和S2亚基，其中所述S1亚基包含失活的弗林蛋白酶切割位点，所述失活的弗林蛋白酶切割位点位于所述S1亚基的C端并且具有QSAQ的氨基酸序列。
已经发现多种突变型SARS-CoV-2 S蛋白。例如，这类突变型SARS-CoV-2 S蛋白可以包含与野生型SARS-CoV-2 S蛋白(例如SEQ ID NO:1)相比的突变，例如氨基酸缺失和/或取代。
在一些实施方案中，本发明的多肽可以缺失一个或多个氨基酸，例如可以缺失对应于SEQ ID NO:1的氨基酸69、70、144、145、242-244、689-715、715-724、788-806和819-828的位置处的氨基酸中的一个或多个。在一些实施方案中，本发明的多肽中的一个或多个氨基酸可以被其他氨基酸取代，例如在对应于SEQ ID NO:1的氨基酸18、20、26、80、138、152、190、215、242、246、417、439、452、453、484、501、570、614、655、681、701、716、982、1027和1118的位置处的氨基酸中的一个或多个可以被其他氨基酸取代。
在一些实施方案中，所述S1亚基包含N端结构域、受体结合结构域和亚结构域1和2。在一实施方案中，所述N端结构域、受体结合结构域和亚结构域1和2分别与具有SEQ ID NO:1的氨基酸序列的SARS-CoV-2 S蛋白中相应的部分具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性。在一些实施方案中，所述S2亚基包含融合肽(FP)、七肽重复序列HR1、HR2、跨膜结构域和胞质结构域。在一实施方案中，所述融合肽(FP)、七肽重复序列HR1、HR2、跨膜结构域和胞质结构域分别与具有SEQ ID NO:1的氨基酸序列的SARS-CoV-2 S蛋白中相应的部分具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性。
在一些实施方案中，所述S1亚基包含与SEQ ID NO:1的氨基酸14-685、15-685、16-685或17-685的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一些实施方案中，所述S2亚基包含与SEQ ID NO:1的氨基酸686-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一些实施方案中，所述多肽包含与SEQ ID NO:1的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一些实施方案中，所述多肽包含与SEQ ID NO:1的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。
在一些实施方案中，所述多肽中在对应于SEQ ID NO:1的氨基酸986和987的位置处的氨基酸为脯氨酸(P)。在一些实施方案中，所述多肽中在对应于SEQ ID NO:1的氨基酸383和985的位置处的氨基酸为半胱氨酸(C)。在一些实施方案中，所述多肽中在对应于SEQ ID NO:1的氨基酸817、892、899和942的位置处的氨基酸为脯氨酸(P)。在一些实施方案中，所述多肽中在对应于SEQ ID NO:1的氨基酸986和987的位置处的氨基酸为脯氨酸(P)，在对应于SEQ ID NO:1的氨基酸383和985的位置处的氨基酸为半胱氨酸(C)。在一些实施方案中，所述多肽中在对应于SEQ ID NO:1的氨基酸986、987、817、892、899和942的位置处的氨基酸为脯氨酸(P)。在一些实施方案中，所述多肽中在对应于SEQ ID NO:1的氨基酸986、987、817、892、899和942的位置处的氨基酸为脯氨酸(P)，在对应于SEQ ID NO:1的氨基酸383和985的位置处的氨基酸为半胱氨酸(C)。
在一些实施方案中，所述多肽进一步包含以下氨基酸修饰中的一个或多个：
(a)在对应于SEQ ID NO:1的氨基酸69、70、144、145、242-244、689-715、715-724、788-806和819-828的位置处的氨基酸中的一个或多个的缺失；
(b)在对应于SEQ ID NO:1的氨基酸18、20、26、80、138、152、190、215、242、246、417、439、452、453、484、501、570、614、655、681、701、716、982、1027和1118的位置处的氨基酸中的一个或多个的取代。
在一些实施方案中，所述多肽中在对应于SEQ ID NO:1的氨基酸614的位置处的氨基酸为甘氨酸(G)。在一些实施方案中，所述多肽中在对应于SEQ ID NO:1的氨基酸614的位置处的氨基酸为甘氨酸(G)，在对应于SEQ ID NO:1的氨基酸417的位置处的氨基酸为天冬酰胺(N)，在对应于SEQ ID NO:1的氨基酸484的位置处的氨基酸为赖氨酸(K)，在对应于SEQ ID NO:1的氨基酸501的位置处的氨基酸为酪氨酸(Y)，在对应于SEQ ID NO:1的氨基酸80的位置处的氨基酸为丙氨酸(A)、在对应于SEQ ID NO:1的氨基酸215的位置处的氨基酸为甘氨酸(G)，并且在对应于SEQ ID NO:1的氨基酸701的位置处的氨基酸为缬氨酸(V)。
在一些实施方案中，本发明的多肽相对于野生型S蛋白(示例性氨基酸序列示于SEQ ID NO:1)包含氨基酸修饰，例如氨基酸的添加、取代和/或缺失。在进一步的实施方案中，与野生型S蛋白相比，本发明的多肽抗原进一步包含一个或多个氨基酸取代。例如，多肽抗原可以在对应于SEQ ID NO:1的氨基酸位置986、987、383、985、817、892、899和942中的一个或多个处包含氨基酸取代。例如，多肽抗原可以进一步包含与SEQ ID NO:1相比的以下一个或多个氨基酸取代：K986P、V987P、S383C、D985C、F817P、A892P、A899P和A942P。
在一实施方案中，多肽抗原包含在对应于SEQ ID NO:1的氨基酸位置682-685处具有氨基酸序列QSAQ的氨基酸序列。在进一步的实施方案中，多肽抗原进一步包含与SEQ ID NO:1相比的K986P和V987P的氨基酸取代。在更进一步的实施方案中，多肽抗原进一步包含与SEQ ID NO:1相比的S383C和D985C的氨基酸取代。在更进一步其他的实施方案中，多肽抗原进一步包含与SEQ ID NO:1相比的以下氨基酸取代：F817P、A892P、A899P和A942P。
在进一步的实施方案中，多肽抗原还包含在对应于SEQ ID NO:1的氨基酸位置614、417、484、501、80、215和701中的一个或多个处的氨基酸取代。在一些实施方案中，氨基酸取代为与SEQ ID NO:1相比的以下氨基酸取代中的一个或多个：D614G、K417N、E484K、N501Y、D80A、D215G和A701V。在一实施方案中，氨基酸取代包括与SEQ ID NO:1相比的D614G。在一实施方案中，氨基酸取代包括与SEQ ID NO:1相比的D614G、K417N、E484K和N501Y。在具体实施方案中， 所述氨基酸取代包括与SEQ ID NO:1相比的以下任一组合：
(1)D614G；
(2)D614G、K417N、E484K和N501Y；
(3)D614G、K417N、E484K、N501Y、D80A、D215G和A701V。
在一实施方案中，多肽抗原包含与SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一实施方案中，多肽抗原包含与SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列的免疫原性片段。在一实施方案中，多肽抗原包含SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列。在一实施方案中，多肽抗原包含SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列的免疫原性片段。在一优选实施方案中，多肽抗原包含SEQ ID NO:2-7中任一个的氨基酸17-1273的氨基酸序列的免疫原性片段。
在更进一步的实施方案中，本发明的多肽还可以包含信号肽。一般而言，信号肽促进多肽的分泌和/或转运，例如转运至细胞表面、内质网(ER)或内体-溶酶体区室。示例性信号肽可以长约15-30个氨基酸。在优选实施方案中，信号肽可以包含SARS-CoV-2 S蛋白或其变体的信号肽序列，特别是包含SEQ ID NO:1的氨基酸1-13、1-14、1-15或1-16的氨基酸序列。其他示例性信号肽可以包括但不限于免疫球蛋白的信号肽序列，例如免疫球蛋白(优选人免疫球蛋白)重链可变区的信号肽序列。在一些实施方案中，信号肽引导新生多肽(例如本文所述多肽抗原)进入内质网进行糖基化修饰。在一些实施方案中，本发明的多肽在宿主细胞内表达后，N端信号肽被切除，从而产生分泌到胞外空间的成熟S蛋白变体(例如本文所述多肽抗原)。
信号肽可以直接或通过接头融合至如本文所述的多肽抗原。在优选的实施方案中，信号肽位于多肽抗原的N端。在一实施方案中，信号肽直接融合至多肽抗原的N端。在一实施方案中，本发明的多核苷酸可以包含编码多肽抗原和信号肽的核苷酸序列，其中所述信号肽融合至所述多肽抗原的N端。
在一实施方案中，信号肽包含SEQ ID NO:1的氨基酸1-13、1-14、1-15或1-16的氨基酸序列。在优选实施方案中，信号肽包含SEQ ID NO:1的氨基酸1-16的氨基酸序列。
在一实施方案中，本发明的多肽包含如上所述多肽抗原以及如上所述信号肽，所述信号肽直接融合至所述多肽抗原的N端。
在一实施方案中，本发明的多肽包含与SEQ ID NO:2-7中任一个氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一实施方案中，本发明的多肽包含与SEQ ID NO:2-7中任一个氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列的免疫原性片段。在一实施方案中，本发明的多肽包含SEQ ID NO:2-7中任一个的氨基酸序列。在一实施方案中，本发明的多肽包含SEQ ID NO:2-7中任一个氨基酸序列的免疫原性片段。
多核苷酸
在另一方面，本发明提供编码本文所述多肽的多核苷酸。多核苷酸可以是单链的或双链的。多核苷酸包括但不限于DNA、cDNA、RNA(例如mRNA)、重组产生和化学合成的多核苷酸。多核苷酸可以包含在载体中。本发明的多核苷酸可以包括天然存在的、合成的和修饰的核苷酸。在一些实 施方案中，本发明的多核苷酸用于在宿主细胞中表达本发明的多肽以提供多肽抗原。在一些实施方案中，所述多肽抗原可以在合适的受试者中诱导针对SARS-CoV-2 S蛋白、优选针对SARS-CoV-2的免疫应答。
多核苷酸可以包含一个或多个核苷酸序列(例如1、2、3、4、5、6、7、8个序列)。多核苷酸可以包含感兴趣多肽(例如本文所述多肽和多肽抗原)的编码序列。在特定实施方案中，多核苷酸可以包含感兴趣多肽的编码序列以及调控序列(包括但不限于转录和翻译调控序列)。在一实施方案中，调控序列包含以下的一个或多个：启动子序列、5’非翻译区(5’UTR)序列、3’非翻译区(3’UTR)序列和poly(A)序列。
编码序列
如本文所用，“编码序列”是指多核苷酸中可以作为模板用于在生物过程中合成具有确定的核苷酸序列(例如tRNA和mRNA)或确定的氨基酸序列的核苷酸序列。编码序列可以是DNA序列或RNA序列。如果对应于DNA序列(包括与mRNA序列相同的编码链和与之互补链的模板链)的mRNA在生物过程中翻译成多肽，可以认为所述DNA序列或mRNA序列编码所述多肽。
如本文所用，“密码子”指多核苷酸中三个连续的核苷酸序列(又称三联体密码)，其编码特定的氨基酸。同义密码子(编码相同氨基酸的密码子)在不同物种中使用的频率不同，称为“密码子偏好性”。通常认为，对于给定物种，使用其偏好的密码子的编码序列可以在该物种表达系统中具有较高的翻译效率和准确率。因此，可以对多核苷酸进行“密码子优化”，即改变多核苷酸中的密码子以反映宿主细胞偏好的密码子，而优选不改变其编码的氨基酸序列。如本领域技术人员会理解的，由于密码子的简并性，本发明的多核苷酸可以包含这样的编码序列，其与本文所述编码序列不同(例如与本文所述编码序列具有约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％相同性)但编码相同的氨基酸序列。在特定实施方案中，本发明的RNA包含针对宿主(例如受试者，特别是人)细胞优化的密码子，使得本发明的多肽在宿主(例如受试者，特别是人)中最佳表达。
在一实施方案中，本发明的多核苷酸包含如本文所述多肽抗原的编码序列。在一实施方案中，本发明的多核苷酸包含与本文所述编码序列互补的核苷酸序列。在一些实施方案中，本发明的多核苷酸包含如本文所述多肽抗原的编码序列。在一些实施方案中，本发明的多核苷酸包含如本文所述多肽的编码序列。在一实施方案中，编码序列在其5’端包含起始密码子，并且在其3’端包含终止密码子。在一实施方案中，编码序列包含本文所述的开放阅读框(ORF)。
在一实施方案中，多肽抗原的编码序列包含核苷酸序列，所述核苷酸序列编码：(1)包含SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列；(2)与SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列；(3)SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列的免疫原性片段；或者(4)与SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列的免疫原性片段。
在一实施方案中，多肽抗原的编码序列包含核苷酸序列，所述核苷酸序列编码：(1)包含SEQ ID NO:2-7中任一个的氨基酸17-1273的氨基酸序列；(2)与SEQ ID NO:2-7中任一个的氨基酸17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列；(3)SEQ ID NO:2-7中任一个的氨基酸17-1273的氨基酸序列的免疫原性片段；或者(4)与SEQ ID NO:2-7中任一个的氨基酸17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或 99％相同性的氨基酸序列的免疫原性片段。
在一实施方案中，本发明的多肽的编码序列包含编码如上所述多肽抗原的核苷酸序列以及编码如上所述信号肽的核苷酸序列，所述信号肽直接融合至所述多肽抗原的N端。在一实施方案中，信号肽的编码序列包含SEQ ID NO:8的核苷酸1-39、1-42、1-45或1-48的核苷酸序列。
在一实施方案中，本发明的多肽的编码序列包含核苷酸序列，所述核苷酸序列编码：(1)包含SEQ ID NO:2-7中任一个的氨基酸序列；(2)与SEQ ID NO:2-7中任一个氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列；(3)SEQ ID NO:2-7中任一个氨基酸序列的免疫原性片段；或者(4)与SEQ ID NO:2-7中任一个氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列的免疫原性片段。
RNA
在一些实施方案中，本发明的多核苷酸是RNA。如本文所用，“RNA”的定义涵盖单链、双链、线性和环状RNA。本发明的RNA可以是通过化学合成的、重组产生的和体外转录的RNA。在一实施方案中，本发明的RNA用于在宿主细胞中表达本发明的多肽。
在一实施方案中，本发明的RNA是单链RNA。在一实施方案中，本发明的RNA是体外转录的RNA(IVT-RNA)。IVT-RNA可以通过RNA聚合酶利用DNA模板进行体外转录获得(例如如本文所述)。
在一些实施方案中，本发明的RNA是信使RNA(mRNA)。一般而言，mRNA可以包含5’UTR序列、多肽的编码序列、3’UTR序列和任选存在的poly(A)序列。mRNA可以例如通过体外转录或化学合成产生。在一实施方案中，本发明的mRNA通过RNA聚合酶(例如T7RNA聚合酶)利用DNA模板进行体外转录获得。在一实施方案中，本发明的mRNA包含(1)任选存在的5’帽、(2)5’UTR、(3)编码序列、(4)3’UTR和(5)任选存在的poly(A)序列。所述5’帽、5’UTR、编码序列、3’UTR和poly(A)序列如本文所述。在一实施方案中，本发明的mRNA是核苷修饰的mRNA。
在一些实施方案中，本发明的RNA包含如本文所述多肽抗原的编码序列。在一些实施方案中，本发明的RNA包含如本文所述多肽的编码序列。
在一些实施方案中，本发明的RNA还包含有助于提高RNA的稳定性和/或翻译效率的结构元件，包括但不限于5’帽、5’UTR、3’UTR和poly(A)序列。
如本文所用，术语“非翻译区(UTR)”一般指RNA中(如mRNA)中不翻译为氨基酸序列的区域(非编码区)，或者DNA中的相应区域。通常，位于开放阅读框(起始密码子)的5’(上游)的UTR可以称为5’非翻译区5’UTR；位于开放阅读框(终止密码子)的3’(下游)的UTR可以称为3’UTR。在5’帽存在的情况下，5’UTR位于5’帽的下游，例如，与5’帽直接相邻。在特定实施方案中，可以在5’UTR中，例如在临近起始密码子的位置，包含优化的“Kozak序列”以提高翻译效率。优选地，“3’UTR”不包含poly(A)序列。在poly(A)序列存在的情况下，3’UTR位于poly(A)序列的上游，例如与poly(A)序列直接相邻。
在一些实施方案中，本发明的RNA包含5’UTR。在一实施方案中，5’UTR包含SEQ ID NO:33-44中任一个的核苷酸序列。在一优选实施方案中，5’UTR包含SEQ ID NO:42的核苷酸序列。在一些实施方案中，本发明的RNA包含3’UTR。在一实施方案中，3’UTR包含SEQ ID NO:45-55中任一个的核苷酸序列。在一优选实施方案中，3’UTR包含SEQ ID NO:55的核苷酸序列。在一些实施方案中，本发明的RNA包含5’UTR和3’UTR。在一实施方案中，5’UTR包含SEQ ID NO:42的核苷酸序列，3’UTR包含SEQ ID NO:45-55中任一个的核苷酸序列。在一具体实施方案中，5’UTR包 含SEQ ID NO:42的核苷酸序列，3’UTR包含SEQ ID NO:55的核苷酸序列。
如本文所用，术语“poly(A)序列”或“poly(A)尾”是指包含连续或不连续腺苷酸的核苷酸序列。poly(A)序列通常位于RNA的3’端，例如3’UTR的3’端(下游)。在一些实施方案中，poly(A)序列在其3’端不包含腺苷酸以外的核苷酸。Poly(A)序列可以在制备IVT-RNA期间，由DNA依赖性RNA聚合酶根据DNA模板的编码序列转录产生，或者通过不依赖于DNA的RNA聚合酶(poly(A)聚合酶)连接至IVT-RNA的游离3’端，例如3’UTR的3’端。
在一些实施方案中，本发明的RNA包含poly(A)序列。在一实施方案中，poly(A)序列包含连续的腺苷酸。在一实施方案中，poly(A)序列可以包含至少20、30、40、50、60、70、80或100以及多达120、150、180、200、300个腺苷酸。在一实施方案中，poly(A)序列中的连续腺苷酸序列被包含U、C或G核苷酸的序列中断。
poly(A)序列可以包含至少20、30、40、50、60、70、80或100以及多达120、150、180、200、300个核苷酸。在一实施方案中，poly(A)序列包含至少50个核苷酸。在一实施方案中，poly(A)序列包含至少80个核苷酸。在一实施方案中，poly(A)序列包含至少100个核苷酸。在一些实施方案中，poly(A)序列包含约70、80、90、100、120或150个核苷酸。在一具体实施方案中，poly(A)序列包含SEQ ID NO:56的核苷酸序列。
如本文所用，术语“5’帽”一般涉及通过5’至5’三磷酸键连接至mRNA的5’端的N7-甲基鸟苷结构(又称为“m 7G帽”、“m 7Gppp-”)。5’帽可以在体外转录中共转录加至RNA中(例如使用抗反向帽类似物“ARCA”)，或者可以利用加帽酶在转录后连接至RNA。
在一些实施方案中，使用帽类似物来产生5’帽修饰的RNA。关于“帽类似物”的描述可以参见例如Contreas,R.et al.(1982).Nucl.Acids Res..10,6353-6363和US7074596B2。帽类似物的实例包括但不限于N7-甲基鸟苷-5’-三磷酸-5’鸟苷(m 7G(5’)ppp(5’)G)、N7-甲基鸟苷-5’-三磷酸-5’-腺苷(m 7G(5’)ppp(5’)A)和3’-O-Me-m 7G(5’)ppp(5’)G(ARCA)。
在一些实施方案中，使用加帽酶(例如牛痘病毒加帽酶)将RNA(例如IVT-RNA)修饰为“Cap0RNA”。在一些实施方案中，Cap0RNA中紧邻m 7G帽的核苷酸的核糖2’O位置上发生额外的甲基化(例如通过2’O-甲基转移酶)，产生“Cap1RNA”。
在一些实施方案中，本发明的RNA包含5’帽。在一些实施方案中，本发明的RNA是Cap0RNA。在一些实施方案中，本发明的RNA是Cap1RNA。
在一实施方案中，本发明的RNA转录为具有如下结构的Cap0RNA：
Figure PCTCN2022075895-appb-000001
其中Base表示RNA的起始核苷酸的碱基。
在一些实施方案中，本发明的RNA包含如本文所述多肽抗原的编码序列以及如本文所述信号肽的编码序列，所述信号肽直接融合至所述多肽抗原的N端。
在一实施方案中，信号肽的编码序列包含SEQ ID NO:8的核苷酸1-39、1-42、1-45或1-48的 核苷酸序列。在一实施方案中，信号肽的编码序列包含SEQ ID NO:8的核苷酸1-48的核苷酸序列。
在一实施方案中，本发明的RNA包含SEQ ID NO:8-13中任一个的核苷酸序列。在一实施方案中，本发明的RNA包含SEQ ID NO:14-19中任一个的核苷酸序列。
在一实施方案中，本发明的RNA(a)包含与SEQ ID NO:8或14的核苷酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的核苷酸序列；并且(b)编码氨基酸序列，所述氨基酸序列包含SEQ ID NO:2的氨基酸序列。
在一实施方案中，本发明的RNA(a)包含与SEQ ID NO:9或15的核苷酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的核苷酸序列；并且(b)编码氨基酸序列，所述氨基酸序列包含SEQ ID NO:3的氨基酸序列。
在一实施方案中，本发明的RNA(a)包含与SEQ ID NO:10或16的核苷酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的核苷酸序列；并且(b)编码氨基酸序列，所述氨基酸序列包含SEQ ID NO:4的氨基酸序列。
在一实施方案中，本发明的RNA(a)包含与SEQ ID NO:11或17的核苷酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的核苷酸序列；并且(b)编码氨基酸序列，所述氨基酸序列包含SEQ ID NO:5的氨基酸序列。
在一实施方案中，本发明的RNA(a)包含与SEQ ID NO:12或18的核苷酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的核苷酸序列；并且(b)编码氨基酸序列，所述氨基酸序列包含SEQ ID NO:6的氨基酸序列。
在一实施方案中，本发明的RNA(a)包含与SEQ ID NO:13或19的核苷酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的核苷酸序列；并且(b)编码氨基酸序列，所述氨基酸序列包含SEQ ID NO:7的氨基酸序列。
修饰的核苷酸
在一些实施方案中，本发明的RNA(例如mRNA)中的核苷酸可以是天然存在的核苷酸(例如天然存在的核糖核苷酸)和修饰的核苷酸。修饰的核苷酸可以是例如不存在于天然存在的RNA中的核苷酸，例如非标准核苷酸或脱氧核苷酸。核苷酸的修饰可以发生在核苷上，例如在核糖部分和/或核碱基部分上。修饰的核苷酸可以在转录(例如体外转录)过程中掺入，也可以在RNA化学合成的过程中添加。
在一实施方案中，所述RNA通过包含一个或多个修饰的核苷进行修饰。在一实施方案中，所述RNA通过用修饰的尿苷代替一个或多个尿嘧啶进行修饰。在一实施方案中，修饰的尿苷包括1-甲基假尿嘧啶、假尿嘧啶、5-甲基-尿嘧啶或其组合。
修饰的尿苷的实例可以包括但不限于：1-甲基尿苷、1-甲基-假尿苷、3-甲基-尿苷、3-甲基-假尿苷、2-甲氧基-尿苷、5-甲氧基-尿苷、5-氮杂-尿苷、6-氮杂-尿苷、2-硫代-5-氮杂-尿苷、2-硫代-尿苷、4-硫代-尿苷、4-硫代-假尿苷、2-硫代-假尿苷、5-羟基-尿苷、5-氨基烯丙基-尿苷、5-卤代-尿苷、尿苷5-氧乙酸、尿苷5-氧乙酸甲基酯、5-羧基甲基-尿苷、1-羧基甲基-假尿苷、5-羧基羟基甲基-尿苷、5-羧基羟基甲基-尿苷甲基酯、5-甲氧基羰基甲基-尿苷、5-甲氧基羰基甲基-2-硫代-尿苷、5-氨基甲基-2-硫代-尿苷、5-甲基氨基甲基-尿苷、1-乙基-假尿苷、5-甲基氨基甲基-2-硫代-尿苷、5-氨甲酰基甲基-尿苷、5-羧基甲基氨基甲基-尿苷、5-羧基甲基氨基甲基-2-硫代-尿苷、5-丙炔基-尿苷、1-丙炔基-假尿苷、5-牛磺酸甲基-尿苷、1-牛磺酸甲基-假尿苷、5-牛磺酸甲基-2-硫代-尿苷、1-牛磺酸甲基-4-硫代-假尿苷、5-甲基-2-硫代-尿苷、1-甲基-4-硫代-假尿苷、4-硫代-1-甲基-假尿苷、2-硫代-1-甲基- 假尿苷、1-甲基-1-脱氮-假尿苷、2-硫代-1-甲基-1-脱氮-假尿苷、二氢尿苷(D)、二氢假尿苷、5,6-二氢尿苷、5-甲基-二氢尿苷、2-硫代-二氢尿苷、2-硫代-二氢假尿苷、2-甲氧基-4-硫代-尿苷、4-甲氧基-假尿苷、4-甲氧基-2-硫代-假尿苷、3-(3-氨基-3-羧基丙基)尿苷、5-(异戊烯基氨基甲基)尿苷、5-(异戊烯基氨基甲基)-2-硫代-尿苷、α-硫代-尿苷、2’-O-甲基-尿苷、5,2’-O-二甲基-尿苷、2’-O-甲基-假尿苷、2-硫代-2’-O-甲基-尿苷、5-甲氧基羰基甲基-2’-O-甲基-尿苷、5-氨甲酰基甲基-2’-O-甲基-尿苷、5-羧基甲基氨基甲基-2’-O-甲基-尿苷、3,2’-O-二甲基-尿苷、5-(异戊烯基氨基甲基)-2’-O-甲基-尿苷、1-硫代-尿苷、5-(2-甲氧羰基乙烯基)尿苷和5-[3-(1-E-丙烯基氨基)尿苷。
在一实施方案中，本发明的RNA(例如mRNA)通过包含一个或多个修饰的核碱基进行修饰。在一实施方案中，修饰的核碱基包括修饰的胞嘧啶、修饰的尿嘧啶或其组合。在一实施方案中，修饰的尿嘧啶独立地选自假尿嘧啶、1-甲基-假尿嘧啶、5-甲基-尿嘧啶或其组合。在一实施方案中，修饰的胞嘧啶独立地选自5-甲基胞嘧啶、5-羟甲基胞嘧啶或其组合。在一实施方案中，本发明的RNA中修饰的核碱基的比例为10％-100％，即本发明的RNA可以通过用修饰的核碱基代替其中10％-100％的核碱基来修饰。
在一些实施方案中，本发明的RNA(例如mRNA)通过用修饰的尿嘧啶代替一个或多个尿嘧啶进行修饰。在一实施方案中，修饰的尿嘧啶包括1-甲基假尿嘧啶、假尿嘧啶、5-甲基-尿嘧啶或其组合。在一实施方案中，修饰的尿嘧啶包括假尿嘧啶。在一实施方案中，修饰的尿嘧啶包括5-甲基-尿嘧啶。在一实施方案中，修饰的尿嘧啶包括1-甲基-假尿嘧啶。
在一实施方案中，所述RNA通过用修饰的尿嘧啶代替至少一个尿嘧啶进行修饰。在一实施方案中，所述RNA通过用修饰的尿嘧啶代替所有尿嘧啶进行修饰。在一实施方案中，所述RNA中修饰的尿嘧啶的比例为10％-100％，例如10％、20％、30％、40％、50％、60％、70％、80％、90％或100％。在一实施方案中，所述RNA中修饰的尿嘧啶的比例为20％-100％。在一实施方案中，所述RNA中20％-100％的尿嘧啶被1-甲基假尿嘧啶代替。在优选的实施方案中，所述RNA中100％的尿嘧啶被1-甲基假尿嘧啶代替。1-甲基-假尿嘧啶具有如下结构：
Figure PCTCN2022075895-appb-000002
在一具体实施方案中，本发明的mRNA包含SEQ ID NO:14-19中任一个的核苷酸序列，并且其中100％的尿嘧啶被1-甲基假尿嘧啶代替。
DNA
在一些实施方案中，本发明的多核苷酸是DNA。这样的DNA可以是例如用于在体外转录本发明的RNA的DNA模板或者用于在宿主细胞中表达多肽抗原的DNA疫苗。DNA可以是双链、单链、线性和环状DNA。
DNA模板可以在合适的转录载体中提供。一般而言，DNA模板可以是双链复合物，其包含与本文所述编码序列相同的核苷酸序列(编码链)和与本文所述编码序列互补的核苷酸序列(模板链)。如本领域技术人员已知的，DNA模板可以包含启动子、5’UTR、编码序列、3’UTR和任选存在的poly(A)序列。启动子可以是本领域技术人员已知的合适RNA聚合酶(特别是DNA依赖性RNA聚合酶)可用的启动子，包括但不限于SP6、T3和T7RNA聚合酶的启动子。在一些实施方案中，DNA模板中的 5’UTR、编码序列、3’UTR和poly(A)序列为本文所述RNA中包含的相应序列或者与之互补。作为DNA疫苗的多核苷酸可以在质粒载体(例如环状质粒载体)中提供。
在一些实施方案中，本发明的DNA包含如本文所述多肽抗原的编码序列。在一些实施方案中，本发明的DNA包含如本文所述多肽的编码序列。在一些实施方案中，本发明的DNA从5’端至3’端包含如本文所述的(1)T7启动子、(2)5’UTR、(3)编码序列、(4)3’UTR和(5)任选存在的poly(A)序列。在一些实施方案中，本发明的DNA包含SEQ ID NO:57-59的核苷酸序列。
组合物
本发明还提供一种组合物，其包含本发明的多肽或多核苷酸(特别是RNA)。在一实施方案中，本发明的组合物用于在受试者中提供针对SARS-CoV-2的预防性和/或治疗性免疫。在一些实施方案中，本发明的组合物包含如本文所述多肽或多肽抗原。在一些实施方案中，本发明的组合物包含本发明的多核苷酸。在一些实施方案中，本发明的组合物包含本发明的DNA。在一些实施方案中，本发明的组合物包含本发明的RNA。在一实施方案中，所述RNA为体外转录的RNA。在一实施方案中，所述RNA为mRNA。在一些实施方案中，本发明的组合物配制为药物组合物。
在一些实施方案中，本发明的组合物包含如本文所述的多核苷酸(特别是RNA，例如mRNA)以及包封所述多核苷酸的脂质。
如本文所用，术语“脂质”是指包含疏水部分并且任选地还包含亲水部分的有机化合物。脂质通常难溶于水但可溶于许多有机溶剂。通常，包含疏水部分和亲水部分的两亲性脂质可以在水环境中组织为脂质双层结构，例如以囊泡形式存在。脂质可以包括但不限于：脂肪酸、甘油酯、磷脂、鞘脂、糖脂和类固醇和胆固醇酯等。
特别优选的核酸组合物可以是例如本文所述的脂质纳米颗粒(LNP)和脂质多聚复合物(LPP)。制备这类组合物的方法可以参见例如Kaczmarek,J.C.et al.,2017,Genome Medicine 9,60或者如本文所述。在一些实施方案中，本发明的组合物包含脂质纳米颗粒(LNP)或脂质多聚复合物(LPP)。在一些实施方案中，本发明的组合物为包含本发明的RNA的脂质纳米颗粒(LNP)或脂质多聚复合物(LPP)。
在一些实施方案中，包封多核苷酸的脂质包含阳离子脂质和非阳离子脂质。在一优选实施方案中，所述阳离子脂质为可电离阳离子脂质。
在一实施方案中，阳离子脂质包含DOTMA、DOTAP、DDAB、DOSPA、DODAC、DODAP、DC-Chol、DMRIE、DMOBA、DLinDMA、DLenDMA、CLinDMA、DMORIE、DLDMA、DMDMA、DOGS)、N4-胆固醇基-精胺、DLin-KC2-DMA、DLin-MC3-DMA或其组合。
在一实施方案中，阳离子脂质包含M5，其具有如下结构：
Figure PCTCN2022075895-appb-000003
在一实施方案中，阳离子脂质包含DOTMA。在一实施方案中，阳离子脂质包含DOTAP。在一实施方案中，阳离子脂质包含DOTMA和DOTAP。
在一实施方案中，非阳离子脂质包含如本文所述的磷脂。在一实施方案中，非阳离子脂质包含如本文所述的类固醇。在一实施方案中，非阳离子脂质包含如本文所述的磷脂和类固醇。在一实施方案中，所述磷脂包含DSPC、DPPC、DMPC、DOPC、POPC、DOPE、DOPG、DPPG、POPE、 DPPE、DMPE和DSPE或其组合。在一实施方案中，所述类固醇为胆固醇。在一实施方案中，非阳离子脂质包含DOPE。在一实施方案中，非阳离子脂质包含DSPC。在一实施方案中，非阳离子脂质包含胆固醇。在一实施方案中，非阳离子脂质包含DOPE和胆固醇。在一实施方案中，非阳离子脂质包含DSPC和胆固醇。
在一实施方案中，阳离子脂质包含M5，非阳离子脂质包含DOPE和胆固醇。在一实施方案中，阳离子脂质包含M5，非阳离子脂质包含DSPC和胆固醇。
在一些实施方案中，包封多核苷酸的脂质进一步包含聚乙二醇修饰的脂质。在一实施方案中，聚乙二醇修饰的脂质包含DMG-PEG(例如DMG-PEG 2000)、DOGPEG和DSPE-PEG或其组合。在一实施方案中，聚乙二醇修饰的脂质包含DSPE-PEG。在一实施方案中，聚乙二醇修饰的脂质包含DMG-PEG(例如DMG-PEG 2000)。
在一些实施方案中，本发明的组合物进一步包含阳离子聚合物，所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在所述脂质中。
在一实施方案中，阳离子聚合物包含聚-L-赖氨酸、鱼精蛋白、聚乙烯亚胺(PEI)或其组合。在一实施方案中，阳离子聚合物为鱼精蛋白。在一实施方案中，阳离子聚合物为聚乙烯亚胺。
在一实施方案中，组合物中脂质的量以摩尔百分比(摩尔％)来计算，所述摩尔百分比基于组合物中脂质的总摩尔来确定。
在一实施方案中，组合物中阳离子脂质的量为约10-约70摩尔％。在一些实施方案中，组合物中阳离子脂质的量为约20-约60摩尔％、约30-约50摩尔％、约35-约45摩尔％、约38-约45摩尔％、约40-约45摩尔％、约40-约50摩尔％或约45-约50摩尔％。
在一实施方案中，组合物中磷脂的量为约10-约70摩尔％。在一实施方案中，组合物中磷脂的量为约20-约60摩尔％、约30-约50摩尔％、约10-约30摩尔％、约10-约20摩尔％或约10-约15摩尔％。
在一实施方案中，组合物中胆固醇的量为约10-约70摩尔％。在一实施方案中，组合物中胆固醇的量为约20-约60摩尔％、约30-约50摩尔％、约35-约40摩尔％、约35-约45摩尔％、约40-约45摩尔％或约45-约50摩尔％。
在一实施方案中，组合物中聚乙二醇修饰的脂质的量为约0.05-约20摩尔％。在一实施方案中，组合物中聚乙二醇修饰的脂质的量为约0.5-约15摩尔％、约1-约10摩尔％、约5-约15摩尔％、约1-约5摩尔％、约1.5-约3摩尔％或约2-5摩尔％。
在一些实施方案中，本发明的RNA(特别是mRNA)配制为脂质纳米颗粒(LNP)。如本文所用，“脂质纳米颗粒”或“LNP”涉及由脂质形成的颗粒，核酸(例如mRNA)被包封在脂质中。
在一实施方案中，LNP包含本发明的RNA以及包封RNA的脂质，其中所述包封RNA的脂质包含阳离子脂质、磷脂、胆固醇和聚乙二醇修饰的脂质。在一实施方案中，所述阳离子脂质为M5。在一实施方案中，所述磷脂为DSPC。在一实施方案中，所述聚乙二醇修饰的脂质为DMG-PEG 2000。在一实施方案中，所述阳离子脂质为M5，所述磷脂为DSPC，所述聚乙二醇修饰的脂质为DMG-PEG 2000。
在一实施方案中，所述包封RNA的脂质包含50摩尔％的M5、10摩尔％的DSPC、38.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG 2000。
在一些实施方案中，本发明的RNA(特别是mRNA)配制为脂质多聚复合物(lipopolyplex，LPP)。如本文所用，“脂质多聚复合物”或“LPP”是指包含由脂质外壳包封的核酸内核的核壳结构，所述核酸 内核包含与聚合物缔合的核酸(例如mRNA)。
在一实施方案中，LPP包含本发明的RNA，其与阳离子聚合物缔合为复合物；以及包封所述复合物的脂质，其中所述包封复合物的脂质包含阳离子脂质、非阳离子脂质和聚乙二醇修饰的脂质。在一实施方案中，所述非阳离子脂质包含磷脂和类固醇。在一实施方案中，所述非阳离子脂质包含选自1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、二硬脂酰基磷脂酰胆碱(DSPC)或其组合的磷脂以及胆固醇。在一实施方案中，所述阳离子聚合物包含鱼精蛋白。在一实施方案中，所述聚乙二醇修饰的脂质包含DMG-PEG 2000。
在一实施方案中，所述阳离子脂质包含M5，其具有如下结构：
Figure PCTCN2022075895-appb-000004
所述非阳离子脂质包含选自1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、二硬脂酰基磷脂酰胆碱(DSPC)或其组合的磷脂以及胆固醇；
所述聚乙二醇修饰的脂质包含1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇2000(DMG-PEG2000)；
所述阳离子聚合物包含鱼精蛋白。
在一实施方案中，所述阳离子聚合物为鱼精蛋白，所述阳离子脂质为M5，所述磷脂为DOPE，所述聚乙二醇修饰的脂质为DMG-PEG 2000。
在一实施方案中，所述包封复合物的脂质包含40摩尔％的M5、15摩尔％的DOPE、43.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG 2000。
在一实施方案中，本发明的组合物为疫苗制剂(又称为“疫苗试剂”)，其包含核酸序列，以及10～70摩尔％的M5、10～70摩尔％的DOPE、10～70摩尔％的胆固醇和0.05～20摩尔％的DMG-PEG 2000，
Figure PCTCN2022075895-appb-000005
其中所述核酸序列编码本发明的多肽。
在一实施方案中，所述核酸序列包含SEQ ID NO:8-13中任一个的核苷酸序列。
在一实施方案中，所述疫苗制剂包含编码本发明的多肽的多核苷酸以及包封所述多核苷酸的脂质，所述脂质包含10～70摩尔％的M5、10～70摩尔％的1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、10～70摩尔％的胆固醇和0.05～20摩尔％的1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)2000，
Figure PCTCN2022075895-appb-000006
在一实施方案中，所述多核苷酸包含SEQ ID NO:8-13中任一个的核苷酸序列。任选地，所述疫苗制剂还包含阳离子聚合物，其中所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在脂质中形成脂质多聚复合物。
阳离子脂质
阳离子脂质是在指定pH下带有净正电荷的脂质。带有净正电荷的脂质可以通过静电相互作用与核酸缔合。
阳离子脂质的实例包括但不限于1,2-二-O-十八烯基-3-三甲基铵丙烷(1,2-di-O-octadecenyl-3-trimethylammonium-propane，DOTMA)、1,2-二油酰基-3-三甲基铵-丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、双十烷基二甲基溴化铵(Didecyldimethylammonium bromide，DDAB)、2,3-二油酰基氧基-N-[2(精胺羧酰胺)乙基]-N,N-二甲基-l-丙胺鎓三氟乙酸盐(2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate，DOSPA)、双十八烷基二甲基氯化铵(dioctadecyldimethyl ammonium chloride，DODAC)、1,2-二油酰基-3-二甲基铵-丙烷(1,2-dioleoyl-3-dimethylammonium-propane，DODAP)、3-(N—(N′,N′-二甲基氨基乙烷)-氨甲酰基)胆固醇(3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol，DC-Chol)、2,3-二(十四烷基氧基)丙基-(2-羟基乙基)-二甲基氨鎓(2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium，DMRIE)、N,N-二甲基-3,4-二油基氧基苄胺(N,N-dimethyl-3,4-dioleyloxybenzylamine，DMOBA)、1,2-二亚油基氧基-N,N-二甲基氨基丙烷(1,2-dilinoleyloxy-N,N-dimethylaminopropane，DLinDMA)、1,2-二亚油烯基氧基-N,N-二甲基氨基丙烷(1,2-dilinolenyloxy-N,N-dimethylaminopropane，DLenDMA)、3-二甲基氨基-2-(胆甾-5-烯-3-β-氧基丁烷-4-氧基)-1-(顺式,顺式-9,12-十八碳二烯基氧基)丙烷(3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane，CLinDMA)、N-(2-氨基乙基)-N,N-二甲基-2,3-双(十四烷基氧基)丙烷-1-胺鎓溴化物(N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide，DMORIE)、N,N-二甲基-2,3-双(十二烷基氧基)丙烷-1-胺(N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine，DLDMA)、N,N-二甲基-2,3-双(十四烷基氧基)丙烷-1-胺(N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine，DMDMA)、双十八烷基酰氨基甘氨酰基精胺(dioctadecylamidoglycyl spermine，DOGS)、N4-胆固醇基-精胺(N4-cholesteryl-spermine)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧戊环(2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane，DLin-KC2-DMA)、三十七烷基-6,9,28,31-四烯-19-基-4-(二甲基氨基)丁酸酯(heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate，DLin-MC3-DMA)、十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((癸氧基)己基)氨基)辛酸酯)(heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate)、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)。
在一些实施方案中，阳离子脂质优选为可电离阳离子脂质。可电离阳离子脂质在例如酸性pH下带有净正电荷，而在较高pH(例如生理pH)下是中性的。可电离阳离子脂质的实例包括但不限于：双十八烷基酰氨基甘氨酰基精胺(dioctadecylamidoglycyl spermine，DOGS)、N4-胆固醇基-精胺(N4-cholesteryl-spermine)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧戊环(2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane，DLin-KC2-DMA)、三十七烷基-6,9,28,31-四烯-19-基-4-(二甲基氨基)丁酸酯(heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate， DLin-MC3-DMA)、十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((癸氧基)己基)氨基)辛酸酯)(heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate)、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)。
在一实施方案中，阳离子脂质包含M5，其具有以下结构：
Figure PCTCN2022075895-appb-000007
非阳离子脂质
在本文中，“非阳离子脂质”是指在指定pH下不带有净正电荷的脂质，例如阴离子脂质和中性脂质。术语“中性脂质”指在生理pH下以不带电、中性或两性离子形式存在的脂质。中性脂质可以包括但不限于磷脂和类固醇。
磷脂的实例包括但不限于：1,2-二油酰-sn-甘油-3-磷酸乙醇胺(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine，DOPE)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(1-palmitoyl-2-oleoylphosphatidylethanolamine，POPE)、二硬脂酰基磷脂酰胆碱(distearoylphosphatidylcholine，DSPC)、二硬脂酰基-磷脂酰乙醇胺(distearoyl-phosphatidylethanolamine，DSPE)、二油酰基磷脂酰胆碱(dioleoylphosphatidylcholine，DOPC)、二肉豆蔻酰基磷脂酰胆碱(dimyristoylphosphatidylcholine，DMPC)、二棕榈酰磷脂酰胆碱(dipalmitoylphosphatidylcholine，DPPC)、二花生四烯酰基磷脂酰胆碱(diarachidoylphosphatidylcholine，DAPC)、二二十二酰基磷脂酰胆碱(dibehenoylphosphatidylcholine，DBPC)、二二十三酰基磷脂酰胆碱(ditricosanoylphosphatidylcholine，DTPC)、二二十四酰基磷脂酰胆碱(dilignoceroylphatidylcholine，DLPC)、棕榈酰油酰基-磷脂酰胆碱(palmitoyloleoyl-phosphatidylcholine，POPC)、二棕榈酰-磷脂酰乙醇胺(dipalmitoyl-phosphatidylethanolamine，DPPE)、二肉豆蔻酰基-磷脂酰乙醇胺(dimyristoyl-phosphatidylethanolamine，DMPE)和二月桂酰基-磷脂酰乙醇胺(dilauroyl-phosphatidylethanolamine，DLPE)。
类固醇的实例包括但限于例如胆固醇、胆甾烷醇、胆甾烷酮、胆甾烯酮、胆固醇基-2'-羟基乙基醚、胆固醇基-4'-羟基丁基醚、生育酚及其衍生物。
聚乙二醇修饰的脂质
如本文所用，术语“聚乙二醇修饰的脂质”是指包含聚乙二醇部分和脂质部分的分子。聚乙二醇修饰的脂质的实例包括但不限于：1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol，DMG-PEG)、1,2-二油酰基-rac-甘油,甲氧基-聚乙二醇(1,2-Dioleoyl-rac-glycerol,methoxypolyethylene Glycol，DOGPEG))和1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-聚(乙二醇)(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol)，DSPE-PEG)。
在一实施方案中，聚乙二醇修饰的脂质为DMG-PEG，例如DMG-PEG 2000。在一实施方案中，DMG-PEG 2000具有以下结构：
Figure PCTCN2022075895-appb-000008
其中n的平均值为44。
阳离子聚合物
如本文所用，术语“阳离子聚合物”涉及在指定pH下能够带有净正电荷从而与核酸静电结合的任何离子聚合物。阳离子聚合物的实例包括但不限于：聚-L-赖氨酸、鱼精蛋白和聚乙烯亚胺(PEI)。聚乙烯亚胺可以是线性或支化的聚乙烯亚胺。
术语“鱼精蛋白”是指富含精氨酸的低分子量碱性蛋白，其存在于各种动物(特别是鱼)的精细胞中并代替组蛋白与DNA结合。在一优选实施方案中，阳离子聚合物为鱼精蛋白(例如硫酸鱼精蛋白)。
药物组合物
本发明还提供一种药物组合物，其包含本发明的多肽、多核苷酸(特别是RNA，例如mRNA)、组合物以及药学上可接受的载剂。在一实施方案中，所述药物组合物包含本文所述的LNP或LPP。
术语“药学上可接受的”是指物质的无毒性，其不与药物组合物的活性组分的作用相互作用。
“药学上可接受的载剂”包括但不限于赋形剂、粘合剂、稀释剂、润滑剂、增稠剂、表面活性剂、防腐剂、稳定剂、乳化剂、缓冲剂、等渗剂、增香剂和着色剂。合适的载剂包括但不限于无菌水、林格氏液、乳酸林格氏液、无菌氯化钠溶液、等渗盐水、聚亚烷基二醇、氢化萘以及生物相容性聚合物(例如丙交酯聚合物、丙交酯/乙交酯共聚物或聚氧乙烯/聚氧-丙烯共聚物)。药学上可接受的载剂的其他描述还可参见例如Remington's Pharmaceutical Sciences,Mack Publishing Co.(A.R Gennaro edit.1985)。
在一实施方案中，本发明的药物组合物用于在受试者中诱导针对SARS-CoV-2的免疫应答。在一实施方案中，本发明的药物组合物预防和/或治疗有需要的受试者中的SARS-CoV-2感染。
在一实施方案中，本发明的药物组合物是免疫原性组合物，例如疫苗。本发明的药物组合物还可以包含佐剂。如本文所用，术语“佐剂”是指能够促进、延长和/或增强免疫应答的物质。佐剂的实例包括但不限于：油乳剂(例如，弗氏佐剂)、氢氧化铝、矿物油、细菌产物(如百日咳杆菌毒素)。
本发明的药物组合物优选通过肠胃外给药。如本文所用，术语“肠胃外给药”是指以通过胃肠道以外的任何方式给药。在一些实施方案中，本发明的药物组合物通过静脉内、皮下、皮内、肌肉内给药。在一优选实施方案中，本发明的药物组合物通过皮下、皮内或肌肉内注射给药。
治疗
本发明提供本发明的多肽、多核苷酸(特别是RNA)、组合物或药物组合物，用于预防和/或治疗SARS-CoV-2感染。
本发明提供本发明的多肽、多核苷酸(特别是RNA)、组合物或药物组合物在制备用于预防和/或治疗SARS-CoV-2感染的药物中的用途。
本发明提供一种用于在受试者中预防和/或治疗SARS-CoV-2感染的方法，所述方法包括给药治疗有效量的本发明的多肽、多核苷酸(特别是RNA)、组合物或药物组合物。在一实施方案中，所述方法包括给药治疗有效量的包含本发明的mRNA的药物组合物，特别是包含如本文所述的LNP或 LPP的药物组合物。
术语“治疗有效量”是指足以预防或抑制疾病或症状的发生和/或减缓、减轻、延迟疾病或症状的发展或严重程度的量。治疗有效量受到包括但不限于以下因素的影响：疾病或症状的发展速度和严重程度，受试者的年龄、性别、体重和生理状况，治疗的持续时间以及具体给药途径。治疗有效量可以在一个或多个剂量中施用。治疗有效量可以通过持续或间断给药实现。
在一些实施方案中，治疗有效量在一次或多次给药中提供。在一些实施方案中，治疗有效量在两次给药中提供。在一些实施方案中，治疗有效量在三次给药中提供。
作为多肽抗原给药
在一些实施方案中，本发明的多肽可以通过在宿主细胞(例如受试者的细胞)中表达编码其的多核苷酸(如本文所述)来提供。在一些实施方案中，本发明的多核苷酸在受试者的细胞中瞬时表达。在一些实施方案中，本发明的多肽在受试者的细胞表面表达。在一些实施方案中，本发明的多肽作为成熟S蛋白表达，所述成熟S蛋白缺少信号肽。在一些实施方案中，本发明的多肽在受试者的抗原呈递细胞(例如巨噬细胞和树突状细胞)中加工并在所述细胞表面呈递为多肽抗原。
在一些实施方案中，本发明的多肽或多核苷酸(特别是mRNA)在受试者中诱导针对SARS-CoV-2或SARS-CoV-2 S蛋白的免疫血清。
在一些实施方案中，本发明的多肽或多核苷酸(特别是mRNA)在受试者中诱导针对SARS-CoV-2或SARS-CoV-2 S蛋白的免疫应答。在一些实施方案中，本发明的多肽或多核苷酸(特别是mRNA)在受试者中诱导针对SARS-CoV-2 S蛋白的中和抗体。在一些实施方案中，所述mRNA配制为本文所述的LNP和LPP。
在一实施方案中，所述SARS-CoV-2为野生型SARS-CoV-2。在一实施方案中，所述SARS-CoV-2为SARS-CoV-2 B.1.351变异株(又称为“南非变异株”、“β变异株”或“501.V2变异株”)。
在特定实施方案中，所述SARS-CoV-2具有野生型SARS-CoV-2 S蛋白。在一实施方案中，野生型SARS-CoV-2 S蛋白包含SEQ ID NO:1的氨基酸序列。
在其他特定实施方案中，所述SARS-CoV-2具有突变型SARS-CoV-2 S蛋白。与野生型SARS-CoV-2 S蛋白相比，突变型SARS-CoV-2 S蛋白可以包含一个或多个氨基酸修饰，例如氨基酸的添加、取代和/或缺失。在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置614处包含天冬氨酸(D)。在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置417处包含赖氨酸(K)。在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置484处包含谷氨酸(E)。在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置501处包含谷氨酰胺(N)。在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置614、417、484和501处包含以下氨基酸：D614、K417、E484和N501。
在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置614处包含甘氨酸(G)。在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置417处包含谷氨酰胺(N)。在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置484处包含赖氨酸(K)。在一实施方案中，突变型SARS-CoV-2 S蛋白在对应于SEQ ID NO:1的氨基酸位置501处包含酪氨酸(Y)。
在一实施方案中，突变型SARS-CoV-2 S蛋白包含与SEQ ID NO:1相比的以下氨基酸取代中的 一个或多个：D614G、K417N、E484K和N501Y。在一实施方案中，突变型SARS-CoV-2 S蛋白包含与SEQ ID NO:1相比的以下氨基酸取代：N501Y和D614G。在一实施方案中，突变型SARS-CoV-2 S蛋白包含与SEQ ID NO:1相比的以下氨基酸取代：K417N、N501Y和D614G。在一实施方案中，突变型SARS-CoV-2 S蛋白包含与SEQ ID NO:1相比的以下氨基酸取代：E484K、N501Y和D614G。在一实施方案中，突变型SARS-CoV-2 S蛋白包含与SEQ ID NO:1相比的以下氨基酸取代：D80A、D215G、K417N、E484K、N501Y、D614G和A701V。在一实施方案中，突变型SARS-CoV-2 S蛋白包含与SEQ ID NO:1相比的以下氨基酸取代：L18F、K417N、E484K、N501Y、D614G、D80A、D215G和A701V；以及任选存在的氨基酸242-244的缺失。
本发明的实施方案可以列举如下：
1.一种多肽，其从N端至C端包含SARS-CoV-2 S蛋白的S1亚基和S2亚基，其中所述S1亚基包含失活的弗林蛋白酶切割位点，所述失活的弗林蛋白酶切割位点位于所述S1亚基的C端并且具有QSAQ的氨基酸序列。
2.第1项的多肽，其中在对应于SEQ ID NO:1的氨基酸986和987的位置处的氨基酸为脯氨酸。
3.第1或2项的多肽，其中在对应于SEQ ID NO:1的氨基酸383和985的位置处的氨基酸为半胱氨酸。
4.第1-3中任一项的多肽，其中在对应于SEQ ID NO:1的氨基酸817、892、899和942的位置处的氨基酸为脯氨酸。
5.第1-4项中任一项的多肽，其中在对应于SEQ ID NO:1的氨基酸614的位置处的氨基酸为甘氨酸。
6.第1-4项中任一项的多肽，其中在对应于SEQ ID NO:1的氨基酸614的位置处的氨基酸为甘氨酸，在对应于SEQ ID NO:1的氨基酸417的位置处的氨基酸为天冬酰胺，在对应于SEQ ID NO:1的氨基酸484的位置处的氨基酸为赖氨酸，在对应于SEQ ID NO:1的氨基酸501的位置处的氨基酸为酪氨酸，在对应于SEQ ID NO:1的氨基酸80的位置处的氨基酸为丙氨酸、在对应于SEQ ID NO:1的氨基酸215的位置处的氨基酸为甘氨酸，并且在对应于SEQ ID NO:1的氨基酸701的位置处的氨基酸为缬氨酸。
7.第1-4项中任一项的多肽，进一步包含以下氨基酸修饰中的一个或多个：
(a)在对应于SEQ ID NO:1的氨基酸69、70、144、145、242-244、689-715、715-724、788-806和819-828的位置处的氨基酸中的一个或多个的缺失；
(b)在对应于SEQ ID NO:1的氨基酸18、20、26、80、138、152、190、215、242、246、417、439、452、453、484、501、570、614、655、681、701、716、982、1027和1118的位置处的氨基酸中的一个或多个的取代。
8.第1-7项中任一项的多肽，其中所述S1亚基包含N端结构域、受体结合结构域和亚结构域1和2；优选地，所述N端结构域、受体结合结构域和亚结构域1和2分别与具有SEQ ID NO:1的氨基酸序列的SARS-CoV-2 S蛋白中相应的部分具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性。
9.第1-7项中任一项的多肽，其中所述S1亚基包含与SEQ ID NO:1的氨基酸14-685、15-685、16-685或17-685的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的 氨基酸序列，所述S2亚基包含与SEQ ID NO:1的氨基酸686-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列；
优选地，所述多肽包含与SEQ ID NO:1的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。
10.第1-9项中任一项的多肽，其包含SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列。
11.第1-9项中任一项的多肽，其包含SEQ ID NO:2-7中任一个的氨基酸序列。
12.一种多核苷酸，其编码第1-11项中任一项的多肽。
13.第12项的多核苷酸，其为DNA。
14.第12项的多核苷酸，其为RNA，并且任选地所述RNA通过包含一个或多个修饰的核苷进行修饰。
15.第14项的多核苷酸，其中所述RNA通过用1-甲基假尿嘧啶、假尿嘧啶、5-甲基-尿嘧啶或其组合代替一个或多个尿嘧啶进行修饰。
16.第14项的多核苷酸，其中所述RNA中20％-100％的尿嘧啶被1-甲基假尿嘧啶代替；优选地，所述RNA中100％的尿嘧啶被1-甲基假尿嘧啶代替。
17.第14-16项中任一项的多核苷酸，其中所述RNA包含SEQ ID NO:8-13中任一个的核苷酸序列。
18.第14-17项中任一项的多核苷酸，其中所述RNA进一步包含5’帽。
19.第14-18项中任一项的多核苷酸，其中所述RNA进一步包含5’UTR；优选地，所述5’UTR包含SEQ ID NO:33-44中任一个的核苷酸序列；更优选地，所述5’UTR包含SEQ ID NO:42的核苷酸序列。
20.第14-19项中任一项的多核苷酸，其中所述RNA进一步包含3’UTR；优选地，所述3’UTR包含SEQ ID NO:45-55中任一个的核苷酸序列；更优选地，所述3’UTR包含SEQ ID NO:55的核苷酸序列。
21.第14-20项中任一项的多核苷酸，其中所述RNA进一步包含poly(A)序列；优选地，所述poly(A)序列包含SEQ ID NO:56的核苷酸序列。
22.第14-21项中任一项的多核苷酸，其包含SEQ ID NO:14-19中任一个的核苷酸序列。
23.一种组合物，其包含第12-22中任一项的多核苷酸以及包封所述多核苷酸的脂质。
24.第23项的组合物，其包含脂质纳米颗粒或脂质多聚复合物。
25.第23或24项的组合物，其中包封所述多核苷酸的脂质包含阳离子脂质、非阳离子脂质和聚乙二醇修饰的脂质；任选地，所述组合物还包含阳离子聚合物，其中所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在脂质中形成脂质多聚复合物。
26.一种疫苗制剂，其包含编码第1-11项中任一项的多肽的多核苷酸以及包封所述多核苷酸的脂质，其中所述脂质包含10～70摩尔％的M5、10～70摩尔％的1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、10～70摩尔％的胆固醇和0.05～20摩尔％的1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)2000，
Figure PCTCN2022075895-appb-000009
优选地，所述多核苷酸包含SEQ ID NO:8-13中任一个的核苷酸序列；
任选地，所述疫苗制剂还包含阳离子聚合物，其中所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在脂质中形成脂质多聚复合物。
27.一种药物组合物，其包含第1-11项中任一项的多肽、第12-22项中任一项的多核苷酸、第23-25项中任一项的组合物或第26项的疫苗制剂；以及药学上可接受的载剂。
28.第1-11项中任一项的多肽、第12-22项中任一项的多核苷酸、第23-25项中任一项的组合物、第26项的疫苗制剂或第27项的药物组合物在制备用于预防和/或治疗SARS-CoV-2感染的药物中的用途。
有益效果
本发明的多肽、多核苷酸、组合物、药物组合物和方法具有以下有益效果中的至少一个：
(1)具有高的细胞表面表达水平；
(2)具有高的ACE2蛋白结合亲合力；
(3)在受试者中诱导高的针对野生型SARS-CoV-2/SARS-CoV-2 S蛋白的中和抗体；
(4)在受试者中诱导高的针对突变型SARS-CoV-2/SARS-CoV-2 S蛋白的中和抗体，特别是针对B.1.351变异株/B.1.351 S蛋白的中和抗体。
实施例
通过参考以下实施例进一步描述本发明。应当理解，这些实施例仅作为示例，而不对本发明构成限制。以下材料和仪器均是可商购的或根据本领域公知的方法制备。以下实验均按照制造商的说明书或根据本领域公知的方法和步骤进行。
实施例1 mRNA的制备
1.1 S蛋白变体的设计
设计编号分别为213、215和217的S蛋白变体，氨基酸序列示于SEQ ID NO:2-4。其中与SEQ ID NO:1的野生型S蛋白相比，S蛋白变体213、215和217均包含失活的Furin切割位点“QSAQ”和氨基酸取代K986P、V987P以及D614G(213、215和217)，S蛋白变体215和217分别进一步包含以下氨基酸取代：S383C和D985C(215)；或F817A、A892P、A899P和A942P(217)(表1A)。
设计编号为223、225和227的S蛋白变体，氨基酸序列示于SEQ ID NO:5-7。这些S蛋白变体与S蛋白变体213、215和217相比，进一步包含存在于β变异株中的主要突变：K417N、E484K、N501Y、D80A、D215G和A701V(表1B)。
还设计编号分别为212、214、216、222、224和226的对照S蛋白变体，氨基酸序列示于SEQ ID NO:20-25。对照S蛋白变体212、214、216、222、224和226包含活性Furin切割位点“RRAR”，其他突变分别与S蛋白变体213、215、217、223、225和227一一相应(表1A和1B)。
1.2 DNA模板的设计和合成
DNA模板的设计和合成方法参见CN113186203A的描述。
简言之，设计编码实施例1.1所述S蛋白变体的DNA开放阅读框(ORF)序列，其密码子优化为在人细胞中最佳表达。编码S蛋白变体213、215和217的DNA ORF序列示于SEQ ID NO:57-59。编码S蛋白变体213、215、217、223、225和227的DNA ORF序列对应的RNA ORF序列示于SEQ ID NO:8-13。编码对照S蛋白变体212、214、216、222、224和226的DNA ORF序列对应的RNA ORF序列示于SEQ ID NO:26-31(表2)。
还设计T7启动子序列(SEQ ID NO:32)、5’UTR序列(SEQ ID NO:33-44)、3’UTR序列(SEQ ID NO:45-55)和poly(A)序列(SEQ ID NO:56)。
然后分别将如上所述DNA ORF序列与T7启动子序列(SEQ ID NO:32)，5’UTR序列(SEQ ID NO:42)，3’UTR序列(SEQ ID NO:55)和poly(A)尾(SEQ ID NO:56)连接(参见图1)，并在T7启动子序列和5’UTR序列之间插入核苷酸序列“AGGAAA”以满足实施例1.3所述共转录加帽反应对起始序列5’AG 3’的要求，在5’UTR序列和DNA ORF的起始密码子“ATG”之间插入Kozak序列“GCCACC”，以Puc57为载体进行全基因合成(南京金斯瑞生物科技有限公司)，获得质粒DNA模板。
最后使用限制性内切酶将质粒DNA模板线性化，利用一对引物(上游引物：5’TTGGACCCTCGTACAGAAGCTAATACG 3’；和下游poly(T)长引物：5’TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGTTCTAGACCCTCACTTCCTACTCAGG 3’)和基于高保真DNA聚合酶的PCR扩增试剂盒(宝日医生物技术(北京)有限公司)进行PCR扩增(德国艾本德股份公司)获得DNA模板。
1.3 从DNA模板体外转录mRNA
使用DNA模板制备体外转录的mRNA的方法参见CN113186203A的描述。简而言之，以如实施例1.2制备的DNA模板为模板，利用T7RNA聚合酶进行共转录加帽反应，进行RNA的体外转录，从而产生Cap1mRNA。反应体系中加入1-甲基-假尿嘧啶核苷酸(1-甲基-假尿苷)代替三磷酸尿苷(UTP)，因此，体外转录的Cap1mRNA中1-甲基-假尿嘧啶的修饰比例为100％。转录结束后，使用DNaseI(赛默飞世尔科技有限公司)消化DNA模板，以降低残余DNA模板带来的风险。
使用DynabeadsMyone(赛默飞世尔科技有限公司)对Cap1mRNA进行纯化。纯化的Cap1mRNA溶解于柠檬酸钠溶液中。编号为213、215、217、223、225和227的mRNA的核苷酸序列示于SEQ ID NO:14-19(表2)。编号为212、214、216、222、224和226的mRNA的全长序列未示出。未示出的mRNA序列与SEQ ID NO:14-19的mRNA序列的区别在于，未示出的mRNA序列中的RNA ORF序列为选自SEQ ID NO:26-31的RNA ORF序列。
表1A
Figure PCTCN2022075895-appb-000010
注：“-”表示S蛋白变体在该氨基酸位置不包含突变；“+”表示S蛋白变体在该氨基酸位置包含标明 的突变。
表1B
Figure PCTCN2022075895-appb-000011
注：“-”表示S蛋白变体该位氨基酸位置不包含突变；“+”表示S蛋白变体在该位氨基酸位置包含标明的突变。
表2
Figure PCTCN2022075895-appb-000012
注：“-”表示未示出。
实施例2 候选mRNA的细胞表达验证
在DC2.4细胞(小鼠骨髓来源树突状细胞系；ATCC)中验证了如实施例1.3制备的候选mRNA的表达。简而言之，使用转染试剂Lipofectamine MessengerMax(Invitrogen)将2μg mRNA转染至DC2.4细胞中。将转染后的细胞置于细胞培养箱中，在37℃5％CO 2继续培养18-24h。然后收集细胞并用PBS洗涤后计数。取1x10 6个细胞到流式管中，离心弃上清。使用牛血清蛋白(北京索莱宝科技有限公司)、FcR封闭液(Miltenyi Biotec)和live/dead dye染料(BD Biosciences)对细胞进行孵育，PBS洗涤；然后再使用重组蛋白hACE2-Fc(金斯瑞生物科技公司)进行孵育，PBS洗涤；然后使用PE标记的抗Fc抗体(PE-anti-Fc)(BioLegend)进行孵育，PBS洗涤；加PBS重悬细胞，使用流式细胞仪(BD Biosciences)检测结合到DC2.4细胞表面的hACE2的量(表示为PE的平均荧光强度(MFI)值)。
结果显示(图1)，候选mRNA 213、215、217、223、225和227转染的DC2.4细胞表面均可以检测到较强的PE荧光信号，表明这些mRNA在细胞内正确地翻译为能够结合hACE2的功能性刺突蛋 白。此外，在mRNA 213、215、217、223、225和227转染的DC2.4细胞表面检测到的PE MFI值分别比mRNA 212、214、216、222、224和226转染的DC2.4细胞表面检测到的PE MFI值更高，表明将Furin切割位点突变为QSAQ提高S蛋白变体的表达水平和/或其对hACE2的结合亲和力。
实施例3 mRNA疫苗制剂的制备
实验材料
阳离子脂质M5为斯微生物合成；辅助磷脂(DOPE和DSPC)采购自CordenPharma；胆固醇采购于Sigma-Aldrich；mPEG2000-DMG(即DMG-PEG 2000)采购于Avanti Polar Lipids,Inc.；PBS采购于Invitrogen；硫酸鱼精蛋白采购自北京斯利安药业有限公司。
3.1 脂质纳米颗粒(LNP-mRNA)制剂的制备：
mRNA水溶液的配制：用50mM柠檬酸-柠檬酸钠缓冲液(pH 3～4)将如实施例1.3制备的每种mRNA稀释为0.35mg/mL mRNA水溶液。
脂质溶液的配制：将阳离子脂质(M5)：DSPC：胆固醇：DMG-PEG 2000以50:10:38.5:1.5的摩尔比溶解于乙醇溶液，配制成10mg/mL脂质溶液。
LNP的制备：使用微流控技术(迈安纳(上海)科技股份有限公司，型号：Inano D)，在以下条件下将脂质溶液和mRNA水溶液混合：体积(Volume)＝4.0mL；流速比(Flow rate ratio)＝3(脂质溶液):1(mRNA水溶液)，总流速(Total flow rate)＝12mL/min，获得LNP-mRNA溶液。
离心超滤：将LNP-mRNA溶液加入到超滤管中进行离心超滤浓缩(离心力3400g，离心时间60min，温度4℃)，获得编号为213、215、217、223、225、227、212、214、216、222、224和226的LNP-mRNA制剂。
3.2 脂质多聚复合物(LPP-mRNA)制剂的制备：
mRNA水溶液的配制：用50mM柠檬酸-柠檬酸钠缓冲液(pH 3～4)将如实施例1.3制备的每种mRNA稀释为0.35mg/mL mRNA水溶液。
脂质溶液的配制：将阳离子脂质(M5)：DOPE：胆固醇：DMG-PEG 2000以40:15:43.5:1.5的摩尔比溶解于无水乙醇，配制成10mg/mL脂质溶液。
硫酸鱼精蛋白溶液的配制：将硫酸鱼精蛋白溶解于无核酸酶水中配制成工作浓度为0.2mg/mL的硫酸鱼精蛋白溶液。
核纳米粒(core nanoparticle)溶液的制备：使用微流控技术，在以下条件将硫酸鱼精蛋白溶液与mRNA溶液混合获得由鱼精蛋白和mRNA形成的核纳米粒溶液：Volume＝4.0mL；Flow rate ratio＝3(mRNA):1(鱼精蛋白溶液)，Total flow rate＝12mL/min，前废(start waste)＝0.35mL，后废(end waste)＝0.1mL，室温。
LPP的制备：在以下条件下将核纳米粒溶液与脂质溶液进行二次混合：Volume＝4.0mL，Flow rate ratio＝3(脂质溶液):1(核纳米粒溶液)，Total flow rate＝12mL/min，前废＝0.35mL，后废＝0.1mL，室温，获得LPP-mRNA溶液。
离心超滤：将LPP-mRNA溶液通过超滤离心去除乙醇(离心力3400g，离心时间60min，温度4℃)，获得编号为213、215、217、223、225、227、212、214、216、222、224和226的LPP-mRNA制剂。
实施例4 候选mRNA疫苗制剂在小鼠体内诱导中和抗体的能力的评估
使用如实施例3.2制备的LPP-mRNA制剂免疫C57BL/6小鼠(上海灵畅生物科技有限公司)，分为12组(分别为LPP-mRNA 213、215、217、223、225、227、212、214、216、222、224和226组)，每组8只小鼠。采用肌肉注射的方式于第0天(初免)和第14天(二免)免疫小鼠，每只小鼠单次免疫剂量为10μg mRNA。在二免后的第14天(即第28天)收集小鼠免疫血清，使用商业化的野生型或B.1.351变异株假病毒试剂盒(北京天坛药物生物技术开发公司；野生型假病毒货号：80033；B.1.351变异株假病毒货号：80044)，评估免疫血清中的中和抗体的滴度水平。
假病毒采用表达野生型或B.1.351 SARS-CoV-2 S蛋白的质粒，来代替表达VSV-G蛋白的质粒，并且携带有荧光素酶报告基因。当使用假病毒感染在其表面表达ACE2的细胞时，S蛋白与ACE2结合从而介导假病毒进入细胞，导致荧光素酶的表达。免疫血清抑制假病毒感染表达ACE2的细胞的能力可以用表征为抑制率，其可以通过与阳性对照(例如无血清对照)相比，来自免疫血清样品的荧光素酶催化底物荧光素的发光强度下降的比例来计算。用于野生型假病毒的S蛋白具有SEQ ID NO:1的氨基酸序列。用于B.1.351变异株假病毒的S蛋白相对于SEQ ID NO:1包含以下突变：氨基酸取代L18F、D80A、D215G、K417N、E484K、N501Y、D614G和A701V；以及氨基酸242-244的缺失。
简而言之，将各组免疫血清稀释20、60、180、540、1620和4860倍；向稀释的免疫血清或者等体积细胞培养基中(作为无血清对照)中加入假病毒后共孵育1小时；随后将血清-假病毒混合物中加入一定量的Huh7细胞(表达内源性hACE2的人肝癌细胞系；ATCC)；24小时后，弃去上清，将细胞裂解并加入荧光素；使用酶标仪(Bio-Rad Laboratories)检测发光强度(表示为相对光单位(RLU))，从样品RLU中减去仅细胞对照的背景RLU并计算抑制率，抑制率＝[(RLU 无血清对照–RLU 仅细胞对照)–(RLU 免疫血清–RLU 仅细胞对照)]/(RLU 无血清对照–RLU 仅细胞对照)×100％；绘制各组免疫血清的稀释度-抑制率曲线，最终计算50％抑制率时对应的血清稀释度(ID 50)，示出的是平均值。
免疫血清针对野生型和B.1.351 S蛋白假病毒的中和测定结果分别示于图2和3。对于野生型和B.1.351 S蛋白的两种假病毒，疫苗213、215、217、223、225和227诱导的免疫血清均分别表现出优于其对应的疫苗212、214、216、222、224和226诱导的免疫血清的中和能力，表明将Furin切割位点突变为QSAQ显著提高疫苗诱导的针对野生型SARS-CoV-2毒株和B.1.351变异株的免疫应答。
此外，与疫苗213相比，在将Furin切割位点突变为QSAQ的基础上，进一步引入其他突变的疫苗215和217诱导的免疫血清对野生型和B.1.351 S蛋白假病毒的中和能力均进一步提高。
在对于野生型和B.1.351 S蛋白假病毒的特异性方面，疫苗213、215和217与223、225和227相比表现出显著差异：疫苗213、215和217诱导的免疫血清对野生型S蛋白假病毒的中和能力更高，而疫苗223、225和227诱导的免疫血清对B.1.351 S蛋白假病毒的中和能力更高。这表明疫苗223、225和227诱导更强的B.1.351变异株特异性的免疫应答。
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明精神和范围内，都可以做各种的改动与修饰，因此，本发明的保护范围应该以权利要求书所界定的为准。
序列表
Figure PCTCN2022075895-appb-000013
Figure PCTCN2022075895-appb-000014
Figure PCTCN2022075895-appb-000015
Figure PCTCN2022075895-appb-000016
Figure PCTCN2022075895-appb-000017
Figure PCTCN2022075895-appb-000018
Figure PCTCN2022075895-appb-000019
Figure PCTCN2022075895-appb-000020
Figure PCTCN2022075895-appb-000021
Figure PCTCN2022075895-appb-000022
Figure PCTCN2022075895-appb-000023
Figure PCTCN2022075895-appb-000024
Figure PCTCN2022075895-appb-000025
Figure PCTCN2022075895-appb-000026
Figure PCTCN2022075895-appb-000027
Figure PCTCN2022075895-appb-000028
Figure PCTCN2022075895-appb-000029
Figure PCTCN2022075895-appb-000030
Figure PCTCN2022075895-appb-000031
Figure PCTCN2022075895-appb-000032
Figure PCTCN2022075895-appb-000033
Figure PCTCN2022075895-appb-000034
Figure PCTCN2022075895-appb-000035
Figure PCTCN2022075895-appb-000036
Figure PCTCN2022075895-appb-000037
Figure PCTCN2022075895-appb-000038
Figure PCTCN2022075895-appb-000039
Figure PCTCN2022075895-appb-000040
Figure PCTCN2022075895-appb-000041
Figure PCTCN2022075895-appb-000042
Figure PCTCN2022075895-appb-000043
Figure PCTCN2022075895-appb-000044
Figure PCTCN2022075895-appb-000045
Figure PCTCN2022075895-appb-000046
Figure PCTCN2022075895-appb-000047
Figure PCTCN2022075895-appb-000048
Figure PCTCN2022075895-appb-000049
Figure PCTCN2022075895-appb-000050
Figure PCTCN2022075895-appb-000051
Figure PCTCN2022075895-appb-000052
Figure PCTCN2022075895-appb-000053
Figure PCTCN2022075895-appb-000054
Figure PCTCN2022075895-appb-000055
Figure PCTCN2022075895-appb-000056
Figure PCTCN2022075895-appb-000057
Figure PCTCN2022075895-appb-000058
Figure PCTCN2022075895-appb-000059

Claims (28)

  1. 一种多肽，其从N端至C端包含SARS-CoV-2 S蛋白的S1亚基和S2亚基，其中所述S1亚基包含失活的弗林蛋白酶切割位点，所述失活的弗林蛋白酶切割位点位于所述S1亚基的C端并且具有QSAQ的氨基酸序列。
  2. 权利要求1的多肽，其中在对应于SEQ ID NO:1的氨基酸986和987的位置处的氨基酸为脯氨酸。
  3. 权利要求1或2的多肽，其中在对应于SEQ ID NO:1的氨基酸383和985的位置处的氨基酸为半胱氨酸。
  4. 权利要求1-3中任一项的多肽，其中在对应于SEQ ID NO:1的氨基酸817、892、899和942的位置处的氨基酸为脯氨酸。
  5. 权利要求1-4中任一项的多肽，其中在对应于SEQ ID NO:1的氨基酸614的位置处的氨基酸为甘氨酸。
  6. 权利要求1-4中任一项的多肽，其中在对应于SEQ ID NO:1的氨基酸614的位置处的氨基酸为甘氨酸，在对应于SEQ ID NO:1的氨基酸417的位置处的氨基酸为天冬酰胺，在对应于SEQ ID NO:1的氨基酸484的位置处的氨基酸为赖氨酸，在对应于SEQ ID NO:1的氨基酸501的位置处的氨基酸为酪氨酸，在对应于SEQ ID NO:1的氨基酸80的位置处的氨基酸为丙氨酸、在对应于SEQ ID NO:1的氨基酸215的位置处的氨基酸为甘氨酸，并且在对应于SEQ ID NO:1的氨基酸701的位置处的氨基酸为缬氨酸。
  7. 权利要求1-4中任一项的多肽，进一步包含以下氨基酸修饰中的一个或多个：
    (a)在对应于SEQ ID NO:1的氨基酸69、70、144、145、242-244、689-715、715-724、788-806和819-828的位置处的氨基酸中的一个或多个的缺失；
    (b)在对应于SEQ ID NO:1的氨基酸18、20、26、80、138、152、190、215、242、246、417、439、452、453、484、501、570、614、655、681、701、716、982、1027和1118的位置处的氨基酸中的一个或多个的取代。
  8. 权利要求1-7中任一项的多肽，其中所述S1亚基包含N端结构域、受体结合结构域和亚结构域1和2；优选地，所述N端结构域、受体结合结构域和亚结构域1和2分别与具有SEQ ID NO:1的氨基酸序列的SARS-CoV-2 S蛋白中相应的部分具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性。
  9. 权利要求1-7中任一项的多肽，其中所述S1亚基包含与SEQ ID NO:1的氨基酸14-685、15-685、16-685或17-685的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列，所述S2亚基包含与SEQ ID NO:1的氨基酸686-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列；
    优选地，所述多肽包含与SEQ ID NO:1的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％相同性的氨基酸序列。
  10. 权利要求1-9中任一项的多肽，其包含SEQ ID NO:2-7中任一个的氨基酸14-1273、15-1273、16-1273或17-1273的氨基酸序列。
  11. 权利要求1-9中任一项的多肽，其包含SEQ ID NO:2-7中任一个的氨基酸序列。
  12. 一种多核苷酸，其编码权利要求1-11中任一项的多肽。
  13. 权利要求12的多核苷酸，其为DNA。
  14. 权利要求12的多核苷酸，其为RNA，并且任选地所述RNA通过包含一个或多个修饰的核苷进行修饰。
  15. 权利要求14的多核苷酸，其中所述RNA通过用1-甲基假尿嘧啶、假尿嘧啶、5-甲基-尿嘧啶或其组合代替一个或多个尿嘧啶进行修饰。
  16. 权利要求14的多核苷酸，其中所述RNA中20％-100％的尿嘧啶被1-甲基假尿嘧啶代替；优选地，所述RNA中100％的尿嘧啶被1-甲基假尿嘧啶代替。
  17. 权利要求14-16中任一项的多核苷酸，其中所述RNA包含SEQ ID NO:8-13中任一个的核苷酸序列。
  18. 权利要求14-17中任一项的多核苷酸，其中所述RNA进一步包含5’帽。
  19. 权利要求14-18中任一项的多核苷酸，其中所述RNA进一步包含5’UTR；优选地，所述5’UTR包含SEQ ID NO:33-44中任一个的核苷酸序列；更优选地，所述5’UTR包含SEQ ID NO:42的核苷酸序列。
  20. 权利要求14-19中任一项的多核苷酸，其中所述RNA进一步包含3’UTR；优选地，所述3’UTR包含SEQ ID NO:45-55中任一个的核苷酸序列；更优选地，所述3’UTR包含SEQ ID NO:55的核苷酸序列。
  21. 权利要求14-20中任一项的多核苷酸，其中所述RNA进一步包含poly(A)序列；优选地，所述poly(A)序列包含SEQ ID NO:56的核苷酸序列。
  22. 权利要求14-21中任一项的多核苷酸，其包含SEQ ID NO:14-19中任一个的核苷酸序列。
  23. 一种组合物，其包含权利要求12-22中任一项的多核苷酸以及包封所述多核苷酸的脂质。
  24. 权利要求23的组合物，其包含脂质纳米颗粒或脂质多聚复合物。
  25. 权利要求23或24的组合物，其中包封所述多核苷酸的脂质包含阳离子脂质、非阳离子脂质和聚乙二醇修饰的脂质；任选地，所述组合物还包含阳离子聚合物，其中所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在脂质中形成脂质多聚复合物。
  26. 一种疫苗制剂，其包含编码权利要求1-11中任一项的多肽的多核苷酸以及包封所述多核苷酸的脂质，其中所述脂质包含10～70摩尔％的M5、10～70摩尔％的1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、10～70摩尔％的胆固醇和0.05～20摩尔％的1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)2000，
    Figure PCTCN2022075895-appb-100001
    优选地，所述多核苷酸包含SEQ ID NO:8-13中任一个的核苷酸序列；
    任选地，所述疫苗制剂还包含阳离子聚合物，其中所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在脂质中形成脂质多聚复合物。
  27. 一种药物组合物，其包含权利要求1-11中任一项的多肽、权利要求12-22中任一项的多核苷 酸、权利要求23-25中任一项的组合物或权利要求26的疫苗制剂；以及药学上可接受的载剂。
  28. 权利要求1-11中任一项的多肽、权利要求12-22中任一项的多核苷酸、权利要求23-25中任一项的组合物、权利要求26的疫苗制剂或权利要求27的药物组合物在制备用于预防和/或治疗SARS-CoV-2感染的药物中的用途。
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