WO2021259206A1 - 针对sars-cov-2病毒的dna疫苗及其用途 - Google Patents

针对sars-cov-2病毒的dna疫苗及其用途 Download PDF

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WO2021259206A1
WO2021259206A1 PCT/CN2021/101269 CN2021101269W WO2021259206A1 WO 2021259206 A1 WO2021259206 A1 WO 2021259206A1 CN 2021101269 W CN2021101269 W CN 2021101269W WO 2021259206 A1 WO2021259206 A1 WO 2021259206A1
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cov
sars
seq
amino acid
acid sequence
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PCT/CN2021/101269
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French (fr)
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吴炯
黄勇
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艾立克(北京)生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6043—Heat shock proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the field of vaccines, in particular to a DNA vaccine specific to SARS-COV-2 virus.
  • SARS-COV in 2003 and MERS-COV in 2012 have infected many people worldwide; the SARS-COV-2 group infection that broke out at the end of 2019 also has a great impact, and there is an urgent need to develop an effective vaccine against SARS-COV-2 , Such as DNA vaccines.
  • people seek various ways to enhance the effectiveness of antigens the results of efforts in this area are still limited and unpredictable.
  • the method of enhancing the effectiveness of the antigen tends to show a disadvantage that it is difficult to apply in other cases.
  • Hsp70 which has been attempted to enhance antigen efficacy, induces immune tolerance in autoimmune disease models (Prakken B J, Wendling U, van der Zee R, et al.
  • the present invention provides a DNA vaccine that continuously and efficiently induces the body to produce both a humoral immune response and a cellular immune response against SARS-COV-2 and has improved safety, which meets the current urgent need for a SARS-COV-2 virus vaccine need.
  • the present invention provides a fusion protein comprising the amino acid sequence at positions 14-1294 in SEQ ID NO: 10 or the mature polypeptide encoded by SEQ ID NO: 9.
  • the present invention provides a fusion protein comprising the amino acid sequence of positions 14-1094 in SEQ ID NO: 12 or the mature polypeptide encoded by SEQ ID NO: 11.
  • the present invention provides a polynucleotide encoding the fusion protein described herein or a nucleic acid molecule comprising the polynucleotide sequence.
  • the present invention provides a vector comprising the polynucleotide sequence described herein.
  • the present invention provides a composition comprising:
  • the present invention provides the fusion protein described herein, or the polynucleotide described herein, or the vector described herein, or the composition described herein is prepared for use in a subject in need In the treatment or prevention of SARS-COV-2 virus infection and/or related diseases.
  • the present invention provides a method for treating or preventing SARS-COV-2 virus infection and/or related diseases in a subject in need, which comprises combining the fusion protein described herein, or the fusion protein described herein, or The polynucleotide, or the vector described herein, or the composition described herein is administered to the subject.
  • the present invention provides a kit for treating or preventing SARS-COV-2 virus infection and/or related diseases in subjects in need, which includes:
  • a cytokine selected from one or more of human GM-CSF, IL-2, IFN- ⁇ and/or IL-17, or a polynucleotide encoding the cytokine, or comprising the polynucleus Carrier of glycidyl acid.
  • the present invention provides a method for treating or preventing SARS-COV-2 virus infection and/or related diseases in a subject in need, which includes:
  • a cytokine selected from one or more of human GM-CSF, IL-2, IFN- ⁇ and/or IL-17, or a polynucleotide encoding the cytokine, or comprising the The polynucleotide vector is administered to the subject; then
  • Figure 1 Construction of SARS-COV-2 S1-Hsp70 fusion antigen protein and its expression vector.
  • Figure 1A Schematic diagram of the structural composition of the fusion antigen protein;
  • Figure 1B Schematic diagram of the construction of the molecular cloning expression vector of the fusion antigen protein
  • FIG. 1 Detection of SARS-COV-2 S1-Hsp70 fusion antigen protein expression in 293 cells (WB).
  • Figure 3 Cytokine expression of 293 cells 48 hours after transfection of different cytokine DNA expression vectors.
  • the left column represents the control cell supernatant
  • the right column represents the transfected cell supernatant.
  • Figure 4 Fluorescence imaging image of the expression of the GFP vector after transfection.
  • FIG. 5 Immune mice with SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) DNA expression vectors, and induce the mice to produce SARS-COV-2 S1 protein-specific antibodies.
  • SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70 ( ⁇ 200) fusion antigen proteins induce the production of Hsp70 protein-specific antibodies.
  • Figure 7 The construction of SARS-COV-2 S1-Hsp70 fusion antigen protein promotes the effective elimination of its DNA expression vector in the body.
  • Lane 1 pVax1-SARS-COV-2 S1+pVax1-Hsp70
  • Lane 2 pVax1-SARS-COV-2S1-Hsp70
  • Lane 3 pVax1-SARS-COV-2 S1
  • Lane 4 pVax1-Hsp70
  • Lane 5 PVax1-SARS-COV-2 S1-Hsp70( ⁇ 200).
  • Figure 8 SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) DNA expression vectors induced SARS-COV-2 S1 specific antibody to SARS-COV-2 S1 protein and Inhibition of alveolar epithelial cell binding.
  • FIG. 9 Immunization of mice with SARS-COV-2-S1-Hsp70 DNA and SARS-COV-2 S1-Hsp70( ⁇ 200) DNA expression vectors stimulates SARS-COV-2 S1 specific T cells to produce IL-2 and IFN -gamma factor.
  • Figure 10 The killing effect of T cells induced by mice immunized with SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) DNA expression vectors on 3T3 cells expressing SARS-COV-2 S1 protein.
  • SARS-COV-2 virus is similar to SARS (SARS) virus and Middle East Respiratory Syndrome (MERS), and belongs to the beta coronavirus genus.
  • SARS SARS
  • MERS Middle East Respiratory Syndrome
  • the current research results confirm that the coronavirus has four structural proteins (Zhou J Y, Wu J X, Cheng L Q, et al. Expression of immunogenic S1 glycoprotein of infectious bronchitis virus in transgenic potatoes[J]. Journal of virology, 2003, 77(16): 9090-9093).
  • the 21563-25384 segment of the SARS-COV-2 virus genetic coding sequence is the coding sequence of the S protein.
  • the S protein contains two functional subunits: S1 and S2.
  • the inventors have unexpectedly discovered that the DNA sequence encoding the fusion antigen protein contained in the S protein that mediates the entry of the new coronavirus into the host cell and is responsible for the binding of the host cell receptor to the functional subunit S1 fused with the antigen helper sequence It exerts excellent DNA vaccine immunity, including humoral immunity (see Examples 5 and 8) and cellular immunity (see Examples 9 and 10).
  • the present inventors have also unexpectedly discovered that the use of the specific fusion antigen framework of the present invention realizes the timely elimination of the DNA vaccine encoding the specific fusion antigen framework in the host and improves the safety of the DNA vaccine (see Example 7).
  • the present inventors have also unexpectedly discovered that the use of the specific truncated fusion antigen framework of the present invention greatly reduces the undesired immunogenicity caused by the antigen auxiliary part of the fusion antigen protein, so that the body’s immunity is further focused on the fusion
  • the S1 part of the antigen protein improves the utilization of the body's immune system resources and also improves the safety of the fusion DNA vaccine (see Example 6).
  • the present invention provides a fusion protein, which comprises the amino acid sequence at positions 14-1294 in SEQ ID NO: 10 or the mature polypeptide encoded by SEQ ID NO: 9 or more than 80%, more than 85% of the mature polypeptide. , 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more of 99% sequence identity.
  • the fusion protein may be composed of the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of positions 14-1294 of SEQ ID NO: 10 or the mature polypeptide encoded by SEQ ID NO: 9 or more than 80% of the mature polypeptide. , 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more of 99% sequence identity Sequence composition.
  • amino acid sequence at positions 14-667 in SEQ ID NO: 10 in the fusion protein can be interchanged with the amino acid sequence at positions 671-1294.
  • the present invention provides a fusion protein comprising the amino acid sequence at positions 14-1094 in SEQ ID NO: 12 or the mature polypeptide encoded by SEQ ID NO: 11 or more than 80%, more than 85%, An amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity.
  • the fusion protein may be composed of the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of positions 14-1094 of SEQ ID NO: 12, or the mature polypeptide encoded by SEQ ID NO: 11, or more than 80% of the mature polypeptide. , 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more of 99% sequence identity Sequence composition.
  • amino acid sequence at positions 14-667 in SEQ ID NO: 12 in the fusion protein can be interchanged with the amino acid sequence at positions 671-1094.
  • the present invention provides a polynucleotide encoding the fusion protein described herein or a nucleic acid molecule comprising the polynucleotide sequence.
  • the polynucleotide may consist of SEQ ID NO: 9 or SEQ ID NO: 11.
  • the present invention provides a vector comprising the polynucleotide sequence described herein.
  • the present invention provides a composition comprising:
  • the present invention provides the fusion protein described herein, or the polynucleotide described herein, or the vector described herein, or the composition described herein is prepared for use in a subject in need In the treatment or prevention of SARS-COV-2 virus infection and/or related diseases.
  • the treatment or prevention may further include administering to the subject selected from the group consisting of human GM-CSF, IL-2, IFN- ⁇ , and/or before administering the drug to the subject.
  • administering to the subject selected from the group consisting of human GM-CSF, IL-2, IFN- ⁇ , and/or before administering the drug to the subject.
  • the present invention provides a method for treating or preventing SARS-COV-2 virus infection and/or related diseases in a subject in need, which comprises combining the fusion protein described herein, or the fusion protein described herein, or The polynucleotide, or the vector described herein, or the composition described herein is administered to the subject.
  • the method may further include administering to the subject selected from human GM-CSF, IL-2, IFN- ⁇ and/or IL before administering the drug to the subject -17 one or more cytokines, or polynucleotide encoding the cytokine, or a vector containing the polynucleotide.
  • the present invention provides a kit for treating or preventing SARS-COV-2 virus infection and/or related diseases in subjects in need, which includes:
  • a cytokine selected from one or more of human GM-CSF, IL-2, IFN- ⁇ and/or IL-17, or a polynucleotide encoding the cytokine, or comprising the polynucleus Carrier of glycidyl acid.
  • the present invention provides a method for treating or preventing SARS-COV-2 virus infection and/or related diseases in a subject in need, which includes:
  • a cytokine selected from one or more of human GM-CSF, IL-2, IFN- ⁇ and/or IL-17, or a polynucleotide encoding the cytokine, or comprising the The polynucleotide vector is administered to the subject; then
  • the composition may be an immunological composition.
  • the related disease may be COVID-19.
  • the related disease may be caused by the SARS-COV-2 virus and/or its mutants.
  • the fusion protein described herein may contain one or more conservative amino acid substitutions.
  • the vector described herein may be a vector suitable for eukaryotic cells.
  • the vector described herein may be a viral vector.
  • the vectors described herein may include regulatory elements, such as promoters and enhancers, operably linked to the polynucleotide.
  • polynucleotides or nucleic acid molecules or vectors described herein can be codon optimized.
  • the polynucleotide or nucleic acid molecule or vector described herein may be a degenerate version thereof.
  • the cytokine selected from one or more of human GM-CSF, IL-2, IFN- ⁇ and/or IL-17 may be selected from human GM-CSF, IL-2, IFN -One, two, three or four cytokines in ⁇ and/or IL-17.
  • suitable routes such as enteral, parenteral, transdermal, intramuscular, transmucosal, nasal, and inhalation.
  • the cytokine described herein, or the polynucleotide encoding the cytokine, or the vector containing the polynucleotide can be used in the administration of the fusion protein described herein, or the polynucleotide described herein
  • the acid, or the carrier as described herein, or the composition as described herein is administered to a subject in need one day, two days, three days, four days, or five days before, preferably two days.
  • fusion antigen protein refers to a linear single-chain protein that includes a polypeptide component based on one or more parent proteins, polypeptides, or fragments thereof (for example, antigenic peptides), and which is not naturally Exist in host cells.
  • the fusion antigen protein may contain two or more naturally-occurring amino acid sequences that are linked together in a way that would not naturally occur.
  • the fusion antigen protein may have two or more parts derived from the same protein or fragments thereof (eg, antigenic fragments) that are connected in a manner not common in cells or proteins, or the fusion antigen protein may have Portions from 2, 3, 4, 5, or more different proteins (e.g., antigenic portions) that are linked in ways that are not common in cells. And, the fusion antigen protein may have two or more copies of the same part of the protein or fragments thereof (e.g., antigenic fragments).
  • the fusion antigen protein can be encoded by a nucleic acid molecule, wherein the polynucleotide sequence encoding one protein or part thereof (for example, an antigen) and the nucleic acid molecule encoding one or more proteins or parts thereof (for example, the same or different antigen) are in Attached in frame, the two or more proteins or parts thereof are optionally separated by nucleotides encoding linkers, spacers, cleavage sites, linking amino acids, or combinations thereof.
  • linking amino acid refers to one or more (for example, about 2-10) amino acid residues which are in two adjacent motifs, regions or structures of a polypeptide Between domains, such as between antigenic peptides or between antigenic peptides and adjacent peptides encoded by multiple translation leader sequences, or between antigenic peptides and spacers or cleavage sites.
  • the linking amino acid may be derived from the design of the construct of the fusion antigen protein (for example, the amino acid residues caused by the use of restriction enzyme sites in the process of constructing the nucleic acid molecule encoding the fusion antigen protein).
  • conservative amino acid substitution is recognized in the art as replacing one amino acid with another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, for example, WO 97/09433, page 10, published on March 13, 1997; Lehninger, Biochemistry, 2nd edition; Worth Publishers, Inc. NY: NY (1975) ), pages 71-77).
  • nucleic acid molecule vaccine or “DNA vaccine” as used herein refers to nucleic acid molecule immunity as defined herein Nucleic acid molecules encoding one or more antigens or antigenic epitopes used in vaccination.
  • the term "immune composition” as used herein refers to a composition that can stimulate or cause an immune response.
  • the immune response is a cellular and humoral immune response, such as an adaptive immune response mediated by T-cells (eg, CD8 + T cells or CD4 + T cells).
  • the immune composition is a pharmaceutical preparation.
  • the immune composition is an antigenic peptide immune composition, a nucleic acid immune composition, a cellular immune composition, or a combination thereof.
  • antigen immune composition or "peptide immune composition” as used herein refers to an immune composition comprising one or more antigens capable of promoting or stimulating cellular and humoral immune responses.
  • nucleic acid immune composition refers to an immune composition that includes nucleic acid molecules that encode one or more antigens or antigenic epitopes and which can be contained in a vector (e.g., plasmid, virus) middle.
  • the nucleic acid immune composition can be introduced into host cells in vitro or in vivo for expression of one or more antigenic peptides in a subject.
  • nucleic acid or “nucleic acid molecule” as used herein means, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR) or by in vitro translation Any one of them, and a fragment produced by any one or more of ligation, cleavage, endonuclease action, or exonuclease action.
  • the nucleic acid of the present invention is produced by PCR.
  • Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., naturally occurring nucleotides). The ⁇ -enantiomeric form of) or a combination thereof.
  • the modified nucleotide may have a modification in or in place of the sugar moiety, or the pyrimidine or purine base moiety, or the pyrimidine or purine base moiety.
  • Nucleic acid monomers can be connected via phosphodiester bonds or analogs of such bonds.
  • nucleic acid molecule also includes "peptide nucleic acids” (PNAs), which comprise naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acid molecules can be single-stranded or double-stranded.
  • construct refers to any polynucleotide containing recombinant nucleic acid.
  • the construct can be present in a vector (e.g., bacterial vector, viral vector), or can be integrated into the genome.
  • a "vector” is a nucleic acid molecule capable of transporting another nucleic acid.
  • the vector can be, for example, a plasmid, cosmid, virus, RNA vector or linear or circular DNA or RNA molecule, which can include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid.
  • Exemplary vectors are those capable of autonomously replicating (episomal vectors) and/or expressing the nucleic acids to which they are linked (expression vectors).
  • Vectors such as viral vectors used herein include retroviruses, adenoviruses, parvoviruses (for example, adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (for example, influenza virus), rhabdoviruses (for example, Rabies and vesicular stomatitis virus), paramyxoviruses (for example, measles and Sendai), positive-stranded RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses, including adenovirus and herpes virus (E.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus) and pox viruses (e.g., vaccinia, fowlpox, and canarypox).
  • retroviruses for example, adeno-associated virus
  • coronaviruses negative-strand RNA viruses
  • viruses include, for example, Norwalk virus, toga virus, flavivirus, reovirus, papilloma virus, hepatotropic DNA virus, and hepatitis virus.
  • retroviruses include avian leukocyte tissue hyperplasia-sarcoma, mammalian C-type, B-type virus, D-type virus, HTLV-BLV collection, lentivirus, foam virus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, third edition, BNFields, et al., ed., Lippincott-Raven Publishers, Philadelphia, 1996).
  • lentiviral vector refers to HIV-based lentiviral vectors because of their relatively large packaging capacity, reduced immunogenicity and their ability to stably transduce a wide range of different cell types with high efficiency. Ability to be used for gene delivery. Lentiviral vectors are often produced after transient transfection of three or more plasmids (e.g., packaging, envelope, and transfer) into producer cells. Like HIV, lentiviral vectors enter target cells through the interaction of virus surface glycoproteins with receptors on the cell surface. After entry, the viral RNA undergoes reverse transcription, which is mediated by the reverse transcriptase complex of the virus. The product of reverse transcription is double-stranded linear viral DNA, which is a substrate for viral integration in the DNA of the infected cell.
  • plasmids e.g., packaging, envelope, and transfer
  • signal peptide and leader sequence as used herein are used interchangeably herein and refer to an amino acid sequence that can be linked to the amino terminus of the protein set forth herein.
  • the signal peptide/leader sequence usually directs the localization of the protein.
  • the signal peptide/leader sequence used herein preferably promotes the secretion of the protein from the cell where it is produced.
  • the signal peptide/leader sequence is often cleaved from the rest of the protein (usually called the mature protein) after being secreted from the cell.
  • the signal peptide/leader sequence is attached to the N-terminus of the protein and is about 9 to 200 nucleotides (3 to 60 nucleic acids) in length.
  • the signal peptide used in the present invention can be the signal peptide sequence of the SARS-COV-2 virus S protein or the signal peptide sequence from other eukaryotic/viral proteins.
  • promoter may mean a molecule of synthetic or natural origin capable of conferring, activating or enhancing nucleic acid expression in a cell.
  • the promoter may contain one or more specific transcription control sequences to further enhance its expression and/or change its spatial expression and/or temporal expression. Promoters can also contain distal enhancer or repressor elements, which can be located up to several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals.
  • Promoters can be constitutively or differentially relative to the cells, tissues or organs in which expression occurs, relative to the developmental stage at which expression occurs or in response to external stimuli such as physiological stress, pathogens, metal ions or inducers Regulate the expression of gene components.
  • promoters include phage T7 promoter, phage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE Promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.
  • operably linked means that the expression of a gene is under the control of a promoter spatially linked to it.
  • the promoter can be located 5'(upstream) or 3'(downstream) of the gene under its control.
  • the distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, changes in this distance can be adjusted without losing promoter function.
  • expression vector refers to a DNA construct containing a nucleic acid molecule operably linked to a suitable control sequence, which can realize the expression of the nucleic acid molecule in a suitable host.
  • control sequences include a promoter for achieving transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls the termination of transcription and translation.
  • the vector can be a plasmid, phage particle, virus, or simply a potential genomic insert.
  • Viral vectors can be based on DNA (e.g., adenovirus or vaccinia virus) or RNA, including oncolytic virus vectors (e.g., VSV), which can or cannot be replicated. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, can be integrated into the genome itself.
  • DNA e.g., adenovirus or vaccinia virus
  • RNA including oncolytic virus vectors (e.g., VSV)
  • VSV oncolytic virus vectors
  • expression refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene.
  • the process includes transcription and translation.
  • Translation can start with an unconventional start codon, such as the CUG codon, or translation can start with several start codons (standard AUG and unconventional) to produce more protein than the mRNA produced (based on per mole quantity).
  • the term "introduction” as used herein refers to “transfection” or “transformation” or “transduction”, and includes the integration of a nucleic acid sequence into a eukaryotic or prokaryotic cell
  • introduction refers to "transfection” or “transformation” or “transduction”
  • the nucleic acid sequence can be integrated into the genome of the cell (for example, chromosome, plasmid, plastid or mitochondrial DNA), transformed into an autonomous replicon, or expressed temporarily (for example, transfected mRNA).
  • the term "antigen-specific T-cell response" as used herein refers to an immune response mediated by T-cells against cells expressing a specific antigen.
  • the T-cell response is a CD8 + T-cell response, a CD4 + T-cell response, or a combination thereof.
  • nucleic acid molecule immunization refers to the introduction of nucleic acid molecules encoding one or more antigens into a host or host cell to express the one or more antigens in vivo.
  • the nucleic acid molecule immunization can be by direct administration into the host, such as by standard injection (e.g., intramuscular, intradermal), transdermal particle delivery, inhalation, topically, orally, intranasally, or mucosally.
  • the nucleic acid molecule can be introduced into a host cell ex vivo (for example, a host cell or a cell derived from a donor HLA that matches the host), and the transfected host cell can be administered into the host, so that the target can be caused by The immune response to one or more antigens encoded by the nucleic acid molecule.
  • a host cell ex vivo for example, a host cell or a cell derived from a donor HLA that matches the host
  • the transfected host cell can be administered into the host, so that the target can be caused by The immune response to one or more antigens encoded by the nucleic acid molecule.
  • adjuvant refers to a natural or synthetic substance that promotes the body's T cell or B cell response by enhancing the activity of macrophages, and participates in the immune response of a hapten or antigen. Cytokines, as new molecular adjuvants, can enhance the specific immune response of vaccines.
  • IFN interferon
  • TNF- ⁇ TNF- ⁇
  • chemokines such as CCL21, eosinophil chemokine, HMGB1, SA100- 8 ⁇
  • GCSF eosinophil chemokine
  • treatment refers to the medical management of a disease, disorder, or condition of a subject (eg, patient), which can be therapeutic, preventive/preventive, or their combined treatment .
  • Treatment can improve or reduce the severity of at least one symptom of the disease, delay the deterioration or progression of the disease, or delay or prevent the onset of another related disease.
  • Reducing the risk of developing a disease means preventing or delaying the onset of a disease (eg, cancer) or the recurrence of one or more symptoms of the disease.
  • the term "therapeutically effective amount (or dose)" or “effective amount (or dose)” of a compound or composition means that it is sufficient to cause one or more symptoms of the disease being treated in a statistically significant manner The amount of improved compound. The precise amount depends on many factors, for example, the activity of the composition, the delivery method employed, the immunostimulatory ability of the composition, the anticipated patient and patient considerations, etc., and can be easily determined by a person of ordinary skill in the art.
  • the therapeutic effect may directly or indirectly include the alleviation of one or more symptoms of the disease, and the therapeutic effect may also directly or indirectly include the stimulation of the cellular immune response.
  • subject may be any organism capable of undergoing a cellular immune response, such as humans, pets, domestic animals, display animals, zoo samples, or other animals.
  • the subject may be a human, non-human primate, dog, cat, immune, rat, mouse, guinea pig, horse, cow, sheep, goat, pig, etc.
  • Subjects who need to administer the therapeutic agent as described herein include those who have been infected with SARS-COV-2 virus or even have viral infection-related diseases, or are at risk of SARS-COV-2 virus infection.
  • subject in need refers to a subject who is at high risk of or suffers from a disease, disorder, or condition that is suitable for use with the compounds provided herein or The composition treats or improves.
  • the subject in need is a human.
  • the desired result is a safe product capable of inducing durable protective immunity with minimal side effects, and compared with other strategies (for example, whole live or attenuated pathogens) , Inexpensively produced, minimize or eliminate contraindications that have been otherwise (usually) associated with the application of intact or attenuated viral immune compositions, and have an extended shelf life (because it is based on nucleic acid and/or synthetic Peptide-based).
  • Other strategies for example, whole live or attenuated pathogens
  • contraindications that have been otherwise (usually) associated with the application of intact or attenuated viral immune compositions, and have an extended shelf life (because it is based on nucleic acid and/or synthetic Peptide-based).
  • the ability to respond quickly to infectious disease emergencies is a benefit of the effective application of the embodiments disclosed herein, whether in the context of biological defense or immunotherapy or technology.
  • DNA vaccines can contain nucleic acids in the form of plasmids (Li et al., J. Biotechnol. 162:171, 2012) or incorporated into the nucleic acids of viral vectors used for delivery.
  • the plasmid DNA includes a promoter that drives the expression of one or more transcription units described herein.
  • Nucleic acid-based vaccines can be administered by, for example, intramuscular injection, subcutaneously, intranasally, transmucosal presentation, intravenously, or by intradermal or subcutaneous administration.
  • Example 1 Connecting the coding sequence of SARS-COV-2 S1 protein and antigen auxiliary sequence to construct a DNA vector expressing SARS-COV-2 S1-Hsp70 fusion antigen protein
  • SEQ ID NO: 10 The N-terminal sequence of SARS-COV-2 S1-Hsp70 fusion antigen protein sequence SEQ ID NO: 10 is derived from the amino acid sequence 1-667 of the SARS-COV-2 S1 protein, and the amino acid sequence 1-13 is the S protein The signal peptide sequence. Then, the S1 protein was linked to the amino acid sequence 2-625 of the antigen auxiliary sequence Hsp70 through a linker composed of three glycines (see Figure 1). The total length of the precursor of the fusion antigen protein is 1294 amino acid residues, and the signal peptide sequence composed of amino acid residues 1-13 is excised in the mature fusion antigen protein.
  • a DNA sequence encoding a full length of 1294 amino acid residues was designed with human cells preferentially expressing codons, and then a 3882bp long cDNA fragment was artificially synthesized, and KpnI and BamHI restriction sites were added to the 5'and 3'ends, respectively. Perform molecular cloning operations in accordance with "Molecular Cloning: A laboratory manual” (ed by J. Sambrook & D. W. Russel, published by CSHLP, 4th edition). The synthetic DNA fragment (2 ⁇ g) and pVax1 vector (2 ⁇ g) were digested at 37°C for 2 hours with KpnI and BamHI endonuclease (New England Biolabs).
  • the digested DNA fragments are separated by agarose gel electrophoresis, and the separated DNA fragments are purified. Further, the purified SARS-COV-2 S1-Hsp70 DNA fragment and the pVax1 plasmid fragment were subjected to a DNA ligation reaction.
  • the total DNA in the standard ligation reaction is about 100ng, and the ratio of vector plasmid to insert is about 1:3.
  • transform competent E. coli 1-2 ⁇ l of ligation reaction solution is added to 100 ⁇ l of competent colon cells DH5alpha TOP10). Heat shock at 42°C for 60 seconds and put it in ice water for 3 minutes.
  • Example 2 Link SARS-COV-2 S1 protein with the coding sequence of the truncated antigen auxiliary sequence Hsp70 ( ⁇ 200) to construct a DNA expression vector expressing SARS-COV-2 S1-Hsp70 ( ⁇ 200) fusion antigen protein
  • the fusion of SARS-COV-2 S1 and Hsp70 ( ⁇ 200) was prepared and tested using the same method as in Example 1.
  • the antigen protein SARS-COV-2 S1-Hsp70( ⁇ 200) (see SEQ ID NO: 12), and the result obtained is also consistent with the design (not shown in the figure).
  • Example 3 Construction of human IL-2, GM-CSF, IL-17 and IFN- ⁇ eukaryotic expression vectors
  • the cDNA sequences encoding human IL-2, GM-CSF, IL-17 and IFN- ⁇ can be obtained from GenBank ( https://www.ncbi.nlm) .nih.gov/nuccore ), for example, human IL-2 cDNA GenBank number is NM_000586, human GM-CSF cDNA GenBank number is M11220, human IFN- ⁇ cDNA GenBank number is NM_000619, human IL-17 cDNA GenBank number is U32659) clone Into p C DNA-3.1 eukaryotic expression vector, p C DNA-3.1-IL-2, p C DNA-3.1-GM-CSF, p C DNA-3.1-IL-17 and p C DNA-3.1-IFN are obtained -gamma expression vector plasmid.
  • the plasmid mass preparation and purification kit produced by QIAGEN was used to prepare 10 mg of various cytokine expression vectors for use.
  • the above cytokine expression plasmids were transfected into 293 cell lines, and the respective cell culture supernatants were collected after 48 hours, and tested with human IL-2, GM-CSF, IL-17 and IFN- ⁇ cytokine ELISA from R&D Systems
  • the kit detects the expression of cytokines in the cell culture supernatant. The results are shown in Figure 3, these cytokine expression vectors can respectively mediate the synthesis of corresponding specific cytokine proteins in human 293 cells.
  • a large-scale preparation of highly purified and high-quality DNA expression vectors was made with the kit produced by QIAGEN.
  • the DNA was dissolved in the optimized DNA cell introduction buffer (see Table 1) before immunization, and the DNA concentration was adjusted to 1 ⁇ g/ ⁇ l.
  • Two days before immunization with pVax1-SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) expression plasmids Two days before immunization with pVax1-SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) expression plasmids, Balb/c mice were injected with IL-2, GM-CSF, IL-17 and IFN - ⁇ DNA expression vector. The immune DNA solution was injected into the mouse muscle or subcutaneously in a volume of 30 ⁇ l.
  • SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70 ( ⁇ 200) fusion antigen expression plasmids were injected into the same muscle or subcutaneously for immunization.
  • the mice were immunized once every two weeks. After a total of three immunizations, the peripheral blood of the mice was drawn, lymphocytes were separated and serum was prepared to determine the cellular and humoral immune response of the immunized mice to the SARS-COV-2 S1 protein.
  • the optimized DNA immunization method of the present invention is used to mediate the green fluorescent protein (GFP) DNA expression vector into mouse muscle cells for expression very effectively.
  • GFP green fluorescent protein
  • Example 5 SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70 ( ⁇ 200) fusion antigen expression vector induces SARS-COV-2 S1 specific antibody production in mice
  • Example 4 the SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70 ( ⁇ 200) expression vectors were used for DNA immunization, and the mice 5 weeks after the first immunization were collected from the tail vein to prepare serum.
  • the SARS-COV-2 S1 protein expression vector was used to immunize mice according to the same DNA immunization program as a control, and their sera were also prepared before and 5 weeks after the first DNA immunization.
  • the S1 specific antibody titer in the mouse serum was determined according to the standard enzyme-linked immunoassay (ELISA) method of "Antibody: A Laboratory Manual, Second edition, CSHL Press".
  • ELISA enzyme-linked immunoassay
  • the SARS-COV-2 S1 protein (Sino Biological) 1 ⁇ g/ml was prepared in a carbonate buffer of pH 9.6 and coated on a 96-well microtiter plate (100 ⁇ l/well), and placed at 4°C overnight. The coating solution was removed the next day, and washed 3 times with washing buffer (PBS containing 0.05% Tween-20) for 5 minutes each time. Block with PBS buffer containing 10% BSA and 0.05% Tween-20 at 37°C for 1 hour. Wash once with washing buffer (PBS containing 0.05% Tween-20).
  • the mouse serum was diluted in PBS buffer in different proportions and added to an enzyme-labeled plate (100 ⁇ l/well) to react with S1 protein, and reacted at 37°C for 1 hour. Then wash with washing buffer (PBS containing 0.05% Tween-20) 3 times, 5 minutes each time. Then, an anti-mouse IgG-HRP-labeled secondary antibody (Cell Signaling Technology, Inc) (100 ⁇ l/well) diluted with dilution buffer was added to detect mouse IgG in the microtiter plate, and reacted at 37°C for 1 hour. Then wash with washing buffer (containing 0.05% Tween-20) 3 times, 5 minutes each time.
  • SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) DNA expression vector immunization induced the production of S1 protein-specific antibodies significantly higher than SARS-COV-2
  • the amount of antibodies induced by immunization with the S1 protein DNA expression vector is almost twice the titer of the non-fusion antigen protein at each dilution.
  • SARS-COV-2 S1-Hsp70 SARS-COV-2 S1-Hsp70 ( ⁇ 200), which is truncated up to 200 amino acid residues, is produced by the immune induction of its DNA expression vector
  • the amount of S1 protein specific antibody is equivalent to that of SARS-COV-2 S1-Hsp70 DNA expression vector.
  • Table 2 The amount of S1 protein specific antibodies induced by the fusion antigen expression vector
  • Example 6 Removal of the 200 amino acid residues at the N-terminus of the antigen helper sequence Hsp70 protein reduces the production of Hsp70-specific antibodies induced by the fusion antigen protein.
  • mice After immunizing mice with pVax1-SARS-COV-2 S1-Hsp70 and pVax1-SARS-COV-2 S1-Hsp70( ⁇ 200) expression vectors for 5 weeks, blood was collected from the tail vein to prepare serum. Serum before immunization was also prepared as a control. Then, the Hsp70-specific antibody titer in the mouse serum was determined according to the standard enzyme-linked immunoassay (ELISA) method of "Antibody: A Laboratory Manual, Second edition, CSHL Press".
  • ELISA enzyme-linked immunoassay
  • the Hsp70 protein (Creative Biomart) 1 ⁇ g/ml was prepared in a carbonate buffer of pH 9.6 and coated on a 96-well microtiter plate (100 ⁇ l/well), and placed at 4°C overnight. The coating solution was removed the next day, and washed 3 times with washing buffer (PBS containing 0.05% Tween-20) for 5 minutes each time. Block with PBS buffer containing 10% BSA and 0.05% Tween-20 at 37°C for 1 hour. Wash once with washing buffer (PBS containing 0.05% Tween-20).
  • the mouse serum was diluted in PBS buffer in different proportions and added to an enzyme-labeled plate (100 ⁇ l/well) to react with S1 protein, and reacted at 37°C for 1 hour. Then wash with washing buffer (PBS containing 0.05% Tween-20) 3 times, 5 minutes each time. Then, an anti-mouse IgG-HRP-labeled secondary antibody (Cell Signaling Technology, Inc) (100 ⁇ l/well) diluted with a dilution buffer was added to detect mouse IgG in the microtiter plate, and reacted at 37°C for 1 hour. Then wash with washing buffer (containing 0.05% Tween-20) 3 times, 5 minutes each time.
  • the amount of induced antibody confirmed that the use of the specific truncated fusion antigen framework of the present invention greatly reduces the undesired immunogenicity caused by the antigen auxiliary part of the fusion antigen protein, and makes the body’s immune ability further focus on the fusion antigen protein.
  • the S1 part improves the utilization rate of the body's immune system resources. In this way, SARS-COV-2 S1-HSP70 ( ⁇ 200) basically maintains its ability to induce neutralizing antibodies, while also greatly reducing undesirable immunogenicity, showing surprising advantages in terms of safety.
  • Table 3 The amount of Hsp70 protein-specific antibodies induced by the fusion antigen expression vector
  • Example 7 The construction of SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70 ( ⁇ 200) fusion antigen protein promotes the elimination of its pVax1 DNA expression vector in the body.
  • pVAX1-SARS-COV-2 S1-Hsp70 and pVAX1-SARS-COV-2 S1-Hsp70( ⁇ 200) DNA expression vectors to immunize Balb/c mice once according to the method of Example 4. After 5 weeks, the immune parts of the mice will be taken Cell lysates were prepared from the muscle tissues of the patients, and the pVax1 expression vector present in them was determined by the PCR method. The PCR experiment operation is as described in the literature (Green, M.R: Molecular cloning, A Laboratory Manual (4th), Cold Spring Harbor Press, 2012).
  • oligonucleotide primers (1) 5’GGAGGATTGGGAAG 3’; (2) 5’CCACCGCTGGTAG3’.
  • Primer 1 corresponds to the 1001-1014 nucleotide sequence of pVax1 plasmid
  • primer 2 corresponds to the 2401-2413 nucleotide sequence of pVax1 plasmid.
  • the reactant was placed at 94°C for 5 minutes before the reaction.
  • the PCR reaction environment is 94°C for 1 minute; 55°C for 1 minute; 72°C for 1 minute.
  • the reaction is 35 cycles. Set at 72°C for 10 minutes in the last cycle.
  • 1 ⁇ l of the PCR reaction product was subjected to electrophoresis analysis in a 1% agarose electrophoresis gel. As shown in Figure 7, a DNA band with a size of 1.4 kb was seen, which was consistent with the sequence size of the amplified product corresponding to the aforementioned primer pair.
  • PCR detection revealed that five weeks after immunization, the DNA expression vectors of pVAX1-SARS-COV-2 S1-Hsp70 (lane 2) and pVAX1-SARS-COV-2 S1-Hsp70 ( ⁇ 200) (lane 5) were all completely removed from the mouse immunization site. Disappeared from the tissue, which means that the cells containing the DNA expression vector are completely eliminated. In contrast, pVAX1-SARS-COV-2 S1 (lane 3), pVAX1-Hsp70 (lane 4) or a mixture of the two (lane 1) did not induce the corresponding pVAX1 expression vector to be cleared from body cells ( Figure 7) .
  • SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) fusion antigen protein vaccine design stimulates the body to produce an effective protective immune response, while also avoiding foreign DNA carriers in the body.
  • the long-term residence of the vaccine eliminates the adverse reactions that may be caused by the long-term residence, such as toxic side effects, and enhances the safety of the vaccine.
  • Example 8 SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) fusion antigen protein expression vector induced S1 specific neutralizing antibody to block the binding of S1 protein to human lung epithelial cells effect
  • HPAEpiC Human primary alveolar epithelial cells
  • ACCEGEN Biotechnology, USA Human primary alveolar epithelial cells
  • the medium was HPAEPiC complete medium provided by the company. After the cells were cultured in a 37°C 5% CO 2 incubator for 24 hours, the medium was removed and washed twice with PBS. Then fix with 4% Formaldehyde (100 ⁇ l/well) for 20 minutes at room temperature. Wash three times with PBS buffer containing 0.05% Tween-20. Then, 1% H 2 O 2 containing PBS buffer (100/well) was added and left at room temperature for 25 minutes. Wash three times with PBS buffer containing 0.05% Tween-20.
  • Example 5 the mouse antiserum in Example 5 was diluted in PBS according to different ratios, and then the diluted mouse antiserum was diluted 1:1 with the biotinylated SARS-COV-2 S1 protein solution (10 ⁇ g/ml in PBS) Mix and incubate at 37°C for 30 minutes. Then add it to the cell culture plate (100 ⁇ l/well) and incubate at 37°C for 1 hour.
  • SARS-COV-2 S1-Hsp70 DNA expression vector In mice immunized with SARS-COV-2 S1-Hsp70 DNA expression vector, SARS-COV-2 S1-Hsp70( ⁇ 200) DNA expression vector and SARS-COV-2 S1 DNA expression vector, respectively Both S1 and antibody serum significantly inhibit the binding of S1 protein to human alveolar epithelial cells, thus confirming that the S1 protein-specific antibodies induced by each are effective neutralizing antibodies against human cell lines.
  • SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70 ( ⁇ 200) DNA vaccines was significantly higher than that of the non-fusion SARS-COV-2 S1 DNA vaccine.
  • antibody serum did not significantly decrease the ability of preventing S1 protein from binding to human alveolar epithelial cells.
  • Unimmunized mouse serum does not have this neutralizing effect (see Figure 8 and Table 4).
  • Example 9 SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) DNA vaccine immunization induced mouse T cell immune response.
  • mice immunized with SARS-COV-2 S1-Hsp70, SARS-COV-2 S1-Hsp70 ( ⁇ 200) and SARS-COV-2 S1 DNA expression vectors in Example 5 were taken 5 weeks after the first immunization
  • the spleen was prepared as a cell suspension in RPMI 1640 medium with 10% serum.
  • nylon wool column method to separate T lymphocytes.
  • the mouse spleen cell suspension 2ml (containing 5x10 8 splenocytes) was added to the column, incubated for 30 min counter 37 °C. Then, the T lymphocytes were eluted with RPMI 1640 medium containing 10% fetal bovine serum. Wash the T cells with this medium once, then adjust the cell density to 5X10 6 /ml in the RPMI 1640 medium containing 10% fetal bovine serum, and add the T cells to the 96-well cell culture plate at 200 ⁇ l cell suspension/well nourish. The pre-culture plate containing 1x10 4 / hole mouse spleen adherent cells.
  • Mouse spleen adherent cells were isolated and prepared according to conventional methods (Rosenwasser, LJ & Rosenthal, AS: J. Immunol. 120: 1991, 1978). Then 1ng/well SARS-COV-2 S1 protein was added to stimulate T cells, and they were cultured in a 37°C CO 2 incubator. After 24 hours, the culture supernatant was taken, and the production of IL-2 and IFN- ⁇ was measured with the mouse IL-2 and IFN- ⁇ ELISA kit of R&D Systems. The results are shown in Figure 9 and Table 5.
  • the mouse T cells immunized with SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) DNA vector are better than SARS-COV-2 S1 DNA vector.
  • the levels of IL-2 and IFN- ⁇ produced by the immunized mouse T cells were significantly increased by about 100% and the two were comparable, while the unimmunized mouse T cells produced very low levels of IL-2 and IFN- ⁇ .
  • Table 5 The amount of cytokines produced by mouse T cells immunized with fusion DNA vaccine
  • Example 10 The killing effect of mouse T cells immunized with SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70 ( ⁇ 200) DNA vaccines on mouse 3T3 cells expressing SARS-COV-2 S1 protein.
  • the mouse 3T3 cells were transfected with pcDNA3.1 plasmid expressing the full-length SARS-COV-2 S1 protein.
  • the transfected cells were labeled with 51 Cr according to a conventional method (Nelson, DL et al: Current Protocols in Immunology, 1993. https://doi.org/10.1002/0471142735.im0727s08).
  • the labeled cells were added to a 96-well cell culture plate (5 ⁇ 10 4 cells/well) in DMEM medium containing 10% fetal bovine serum, and placed in a 37°C 5% CO 2 incubator for 4 hours, then added to the examples T lymphocytes isolated in 9 (the same DMEM medium containing 10% fetal bovine serum, 1x10 6 cells/well).
  • T cell killing activity (%) [(experimental group CPM-spontaneous release CPM)/(maximum release CPM-spontaneous release CPM)] ⁇ 100%.
  • Mouse T cells immunized with SARS-COV-2 S1-Hsp70 and SARS-COV-2 S1-Hsp70( ⁇ 200) DNA vaccines have a positive effect on the expression of SARS-COV-2 S1 protein.
  • the killing effect of 3T3 cells was comparable, and it was significantly enhanced by about 100% compared with mouse T cells immunized with SARS-COV-2 S1 DNA expression vector.
  • Mouse T cells that have not been immunized have no killing effect on the 3T3 cells transfected with the SARS-COV-2 S1 expression vector.

Abstract

本发明属于疫苗领域，提供了特异性针对SARS-COV-2病毒的DNA疫苗。公开了一种融合蛋白，其包含SEQ ID NO：10中14-1294位的氨基酸序列或SEQ ID NO：9编码的成熟多肽；一种融合蛋白，其包含SEQ ID NO：12中14-1094位的氨基酸序列或SEQ ID NO：11编码的成熟多肽；以及编码该融合蛋白的多核苷酸或包含该多核苷酸序列的核酸分子。还公开了相关的组合物、方法、用途和试剂盒。

Description

针对SARS-COV-2病毒的DNA疫苗及其用途 技术领域
本发明涉及疫苗领域，尤其涉及特异性针对SARS-COV-2病毒的DNA疫苗。
背景技术
2003年的SARS-COV和2012年的MERS-COV在世界范围内感染了众多人群；2019年底爆发的SARS-COV-2群体感染也影响甚大，目前迫切需要开发针对SARS-COV-2的有效疫苗，如DNA疫苗。尽管人们寻求各种增强抗原效力的方式，但在这方面的努力成果仍然是有限且难以预料的。在一些个案中表现出抗原效力增强的方式往往叉在另一些个案中表现出难以应用的劣势。例如，曾被试图用于增强抗原效力的Hsp70在自身免疫性疾病模型中引发免疫耐受(Prakken B J，Wendling U，van der Zee R，et al.Induction of IL-10 and inhibition of experimental arthritis are specific features of microbial heat shock proteins that are absent for other evolutionarily conserved immunodominant proteins[J].The Journal of Immunology，2001，167(8)：4147-4153.)，甚至有些情况下Hsp70与肿瘤抗原Her2融合不但降低Her2 DNA疫苗的有效性，而且还促进肿瘤进展(Pakravan N，Soudi S，Hassan Z M.N-terminally fusion of Her2/neu to HSP70 decreases efficiency of Her2/neu DNA vaccine[J].Cell Stress and Chaperones，2010，15(5)：631-638.)。而且，当试图把SARS COV-2的功能性亚基S1作为靶抗原用于制备疫苗时，由于该亚基S1的性质与常见抗原不同，例如其大小远超常见抗原，使得通常的抗原增效技术的可用性存疑，例如产生的应答(如有)是否是免疫有用的，产生的抗体(如有)是否是中和性/阻断性的，是否具有保护作用和/或细胞杀伤作用，都是未知的。试图使用辅助序列使抗原增效的努力其结果不但难以预料，而且甚至有可能导致负面效果。可见，增强抗原效力的尝试需要大量的努力和实验验证。另外，从安全性的角度，还需要克服DNA疫苗普遍存在的体内长期存续带来的潜在安全性问题，以及机体针对增效组分产生的不期望的抗体应答(即不期望的免疫原性)的安全性问题。
发明内容
本发明提供了持续、高效地诱导机体产生针对SARS-COV-2的体液免疫应答和细胞免疫应答两者且具有改善的安全性的DNA疫苗，满足了目前对SARS-COV-2病毒疫苗的迫切需要。
因此，在一个方面，本发明提供了一种融合蛋白，其包含SEQ ID NO：10中14-1294位的氨基酸序列或SEQ ID NO：9编码的成熟多肽。
在另一个方面，本发明提供了一种融合蛋白，其包含SEQ ID NO：12中14-1094位的氨基酸序列或SEQ ID NO：11编码的成熟多肽。
在叉一个方面，本发明提供了一种编码本文所述的融合蛋白的多核苷酸或包含所述多核苷酸序列的核酸分子。
在叉一个方面，本发明提供了一种载体，其包含本文所述的多核苷酸序列。
在叉一个方面，本发明提供了一种组合物，其包含：
(1)本文所述的融合蛋白，或本文所述的多核苷酸，或本文所述的载体；和
(2)药学上可接受的载体和/或赋形剂。
在叉一个方面，本发明提供了本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物在制备用于在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的药物中的用途。
在叉一个方面，本发明提供了一种在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的方法，其包括将本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物施用于所述受试者。
在叉一个方面，本发明提供了一种用于在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的试剂盒，其包括：
(1)本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物；和
(2)选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体。
在叉一个方面，本发明提供了一种在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的方法，其包括：
(1)将选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体施用于所述受试者；然后
(2)将本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物施用于所述受试者。
当参考下面的详细描述和附图时，本发明的这些和其他实施方式将变得显而易见。此外，本申请中提到的专利和非专利文献均完整引入本文作为参考。
附图说明
图1：SARS-COV-2 S1-Hsp70融合抗原蛋白及其表达载体的构建。图1A.融合抗原蛋白结构组成示意图；图1B.融合抗原蛋白的分子克隆表达载体构建示意图
图2：SARS-COV-2 S1-Hsp70融合抗原蛋白在293细胞中的表达的检测(WB)。
图3：不同细胞因子DNA表达载体转染293细胞48小时后的细胞因子表达。每组柱中，左侧柱表示对照细胞上清，右侧柱表示转染细胞上清。
图4：转染后的GFP载体的表达的荧光成像图。
图5：用SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA表达载体免疫小鼠，诱导小鼠产生SARS-COV-2 S1蛋白特异性抗体。
图6：SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)融合抗原蛋白诱导产生Hsp70蛋白特异性抗体。
图7：SARS-COV-2 S1-Hsp70融合抗原蛋白的构建促进其DNA表达载体在机体内的有效清除。泳道1：pVax1-SARS-COV-2 S1+pVax1-Hsp70；泳道2：pVax1-SARS-COV-2S1-Hsp70；泳道3：pVax1-SARS-COV-2 S1；泳道4：pVax1-Hsp70；泳道5：pVax1-SARS-COV-2 S1-Hsp70(Δ200)。
图8：SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA表达载体免疫小鼠诱导产生的SARS-COV-2 S1特异性抗体对SARS-COV-2 S1蛋白与肺泡上皮细胞结合的抑制作用。
图9：用SARS-COV-2-S1-Hsp70 DNA和SARS-COV-2 S1-Hsp70(Δ200)DNA表达载体免疫小鼠，刺激SARS-COV-2 S1特异性T细胞产生IL-2和IFN-γ因子。
图10：SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA表达载体免疫小鼠诱导的T细胞对表达SARS-COV-2 S1蛋白的3T3细胞的杀伤作用。
具体实施方式
SARS-COV-2病毒与萨斯(SARS)病毒和中东呼吸综合征(MERS)类似，同属于beta冠状病毒属。目前的研究结果证实，冠状病毒具有表面的刺突蛋白(Spike蛋白或S蛋白)、外膜蛋白(E蛋白)、膜蛋白(M蛋白)及核鞘蛋白(N蛋白)4种结构蛋白(Zhou J Y，Wu J X，Cheng L Q，et al.Expression of immunogenic S1 glycoprotein of infectious bronchitis virus in transgenic potatoes[J].Journal of virology，2003，77(16)：9090-9093)。SARS-COV-2病毒遗传编码序列的21563-25384区段为S蛋白的编码序列。S蛋白含有两个功能性亚基：S1和S2。
本发明人已经出人意料地发现，介导新冠病毒进入宿主细胞的S蛋白所包含的、负责与宿主细胞受体结合的功能性亚基S1与抗原辅助序列融合而成的融合抗原蛋白的编码DNA序列发挥出优异的DNA疫苗免疫作用，包括体液免疫(参见实施例5和8)和细胞免疫(参见实施例9和10)两方面。本发明人还已经出人意料地发现，采用本发明的特定融合抗原架构，实现了编码该特定融合抗原架构的DNA疫苗在宿主体内的及时清除，改善了DNA疫苗的安全性(参见实施例7)。本发明人还已经出人意料地发现，采用本发明的特定截短融合抗原架构，极大地减小了融合抗原蛋白中的抗原辅助部分导致的不期望的免疫原性，使得机体免疫能力进一步聚焦于融合抗原蛋白的S1部分，提高了机体免疫系统资源的利用率，也改善了融合DNA疫苗的安全性(参见实施例6)。
因此，在一个方面，本发明提供了一种融合蛋白，其包含SEQ ID NO：10中14-1294位的氨基酸序列或SEQ ID NO：9编码的成熟多肽或与其具有80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上或99％以上的序列一致性的氨基酸序列。
在一些实施方式中，所述融合蛋白可以由SEQ ID NO：10的氨基酸序列或SEQ ID NO：10的14-1294位的氨基酸序列或SEQ ID NO：9编码的成熟多肽或与其具有80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上或99％以上的序列一致性的氨基酸序列组成。
在一些实施方式中，所述融合蛋白中SEQ ID NO：10中14-667位的氨基酸序列可以与671-1294位的氨基酸序列的位置互换。
在另一个方面，本发明提供了一种融合蛋白，其包含SEQ ID NO：12中14-1094位的氨基酸序列或SEQ ID NO：11编码的成熟多肽或与其具有80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上或99％以上的序列一致性的氨基酸序列。
在一些实施方式中，所述融合蛋白可以由SEQ ID NO：12的氨基酸序列或SEQ ID NO：12的14-1094位的氨基酸序列或SEQ ID NO：11编码的成熟多肽或与其具有80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上或99％以上的序列一致性的氨基酸序列组成。
在一些实施方式中，所述融合蛋白中SEQ ID NO：12中14-667位的氨基酸序列可以与671-1094位的氨基酸序列的位置互换。
在叉一个方面，本发明提供了一种编码本文所述的融合蛋白的多核苷酸或包含所述多核苷酸序列的核酸分子。
在一些实施方式中，所述多核苷酸可以由SEQ ID NO：9或SEQ ID NO：11组成。
在叉一个方面，本发明提供了一种载体，其包含本文所述的多核苷酸序列。
在叉一个方面，本发明提供了一种组合物，其包含：
(1)本文所述的融合蛋白，或本文所述的多核苷酸，或本文所述的载体；和
(2)药学上可接受的载体和/或赋形剂。
在叉一个方面，本发明提供了本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物在制备用于在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的药物中的用途。
在一些实施方式中，所述治疗或预防还可以包括在向所述受试者施用所述药物之前，向所述受试者施用选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体。
在叉一个方面，本发明提供了一种在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的方法，其包括将本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物施用于所述受试者。
在一些实施方式中，所述方法可以还包括在向所述受试者施用所述药物之前，向所述受试者施用选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体。
在叉一个方面，本发明提供了一种用于在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的试剂盒，其包括：
(1)本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物；和
(2)选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体。
在叉一个方面，本发明提供了一种在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的方法，其包括：
(1)将选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体施用于所述受试者；然后
(2)将本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物施用于所述受试者。
在一些实施方式中，所述组合物可以是免疫组合物。
在一些实施方式中，所述相关疾病可以是COVID-19。
在一些实施方式中，所述相关疾病可以是由SARS-COV-2病毒和/或其突变体引起。
在一些实施方式中，本文所述的融合蛋白可以包含一个或多个保守氨基酸置换。
在一些实施方式中，本文所述的载体可以是适用于真核细胞的载体。
在一些实施方式中，本文所述的载体可以是病毒载体。
在一些实施方式中，本文所述的载体可以包含与所述多核苷酸可操作地连接的调节元件，如启动子、增强子。
在一些实施方式中，本文所述的多核苷酸或核酸分子或载体可以被密码子优化。
在一些实施方式中，本文所述的多核苷酸或核酸分子或载体可以是其简并版本。
在一些实施方式中，选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子可以是选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种、两种、三种或四种的细胞因子。
在一些实施方式中，本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物、或本文所述的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体可以经肠、肠胃外、经皮、肌肉内、经粘膜、经鼻和吸入等合适的途径施用于需要的受试者。
在一些实施方式中，本文所述的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体可以在施用本文所述的融合蛋白、或本文所述的多核苷酸、或本文所述的载体、或本文所述的组合物之前1天、2天、3天、4天或5天施用于有需要的受试者，优选2天。
本文中使用的术语“融合抗原蛋白”表示这样的线性单链蛋白：其包括基于一种或多种亲本蛋白、多肽或其片段(例如，抗原性肽)的多肽组分，且其不天然地存在于宿主细胞中。融合抗原蛋白可以含有两种或更多种天然存在的氨基酸序列，所述氨基酸序列以不会天然存在的方式连接在一起。例如，融合抗原蛋白可以具有来自相同蛋白或其片段(例如，抗原性片段)的两个或更多个部分，所述部分以在细胞或蛋白中不常见的方式连接，或融合抗原蛋白可以具有来自2种、3种、4种、5或更多种不同蛋白的部分(例如，抗原性部分)，所述部分以在细胞中不常见的方式连接。并且，融合抗原蛋白可以具有蛋白或其片段(例如，抗原性片段)的相同部分的两个或更多个拷贝。融合抗原蛋白可以由核酸分子编码，其中编码一个蛋白或其部分(例如，抗原)的多核苷酸序列与编码一个或多个蛋白或其部分(例如，相同的或不同的抗原)的核酸分子在框架内附接，所述两个或更多个蛋白或其部分任选地被编码接头、间隔区、切割位点、连接氨基酸或它们的组合的核苷酸隔开。
本文中使用的术语“连接氨基酸”或“连接氨基酸残基”表示这样的一个或多个(例如，约2-10个)氨基酸残基：其在多肽的两个邻近的基序、区域或结构域之间，诸如在抗原性肽之间或在抗原性肽和由多重翻译前导序列编码的邻近肽之间，或在抗原性肽和间隔区或切割位点之间。连接氨基酸可以源自融合抗原蛋白的构建体设计(例如，在构建编码融合抗原蛋白的核酸分子的过程中由限制性酶位点的使用引起的氨基酸残基)。
本文中使用的术语“保守氨基酸置换”在本领域中被公认为将一个氨基酸置换为另一个具有类似性能的氨基酸。示例性的保守置换是本领域众所周知的(参见，例如，WO 97/09433，第10页，1997年3月13日公开；Lehninger，Biochemistry，第2版；Worth Publishers，Inc.NY：NY(1975)，第71-77页)。
本文中使用的术语“抗原、“抗原性肽”或其变体表示可以刺激细胞产生免疫应答的多肽。本文中使用的术语“核酸分子疫苗”或“DNA疫苗”表示在本文定义的核酸分子免疫接种中使用的、编码一种或多种抗原或抗原性表位的核酸分子。
本文中使用的术语“免疫组合物”表示可以刺激或引起免疫应答的组合物。优选地，所述免疫应答是细胞和体液免疫应答，诸如由T-细胞(例如，CD8 +T细胞或CD4 +T细胞)介导的适应性免疫应答。在某些实施方式中，免疫组合物是药物制剂。在其它实施方式中，免疫组合物是抗原性肽免疫组合物、核酸免疫组合物、细胞免疫组合物或它们的组合。
本文中使用的术语“抗原免疫组合物”或“肽免疫组合物”表示包括一种或多种抗原的免疫组合物，所述抗原能够促进或刺激细胞和体液免疫应答。
本文中使用的术语“核酸免疫组合物”表示包括核酸分子的免疫组合物，所述核酸分子编码一种或多种抗原或抗原性表位且其可以被包含在载体(例如，质粒、病毒)中。可以将核酸免疫组合物引入离体或体内宿主细胞中用于在受试者中表达一种或多种抗原性肽。
本文中使用的术语“核酸”或“核酸分子”表示，例如通过聚合酶链式反应(PCR)或通过体外翻译产生的脱氧核糖核酸(DNA)、核糖核酸(RNA)、寡核苷酸、片段中的任一种，和通过连接、切割、内切核酸酶作用或外切核酸酶作用中的任意一种或多种产生的片段。在某些实施方式中，本发明内容的核酸通过PCR产生。核酸可以由单体组成，所述单体是天然存在的核苷酸(诸如脱氧核糖核苷酸和核糖核苷酸)、天然存在的核苷酸的类似物(例如，天然存在的核苷酸的α-对映异构形式)或它们的组合。修饰的核苷酸可以具有在糖部分、或嘧啶或嘌呤碱基部分中或替代糖部分、或嘧啶或嘌呤碱基部分的修饰。核酸单体可以通过磷酸二酯键或这样的键的类似物而连接。磷酸二酯键的类似物包括硫代磷酸酯、二硫代磷酸酯、硒代磷酸酯、二硒代磷酸酯、苯胺基硫代磷酸酯(phosphoroanilothioate)、phosphoranilidate、氨基磷酸酯、吗啉代等。术语“核酸分子”也包括“肽核酸”(PNAs)，其包含与聚酰胺主链连接的天然存在的或经修饰的核酸碱基。核酸分子可以是单链的或双链的。
本文中使用的术语“构建体”表示含有重组核酸的任何多核苷酸。构建体可以存在于载体(例如，细菌载体、病毒载体)中，或可以整合进基因组中。“载体”是能够运输另一种核酸的核酸分子。载体可以是，例如，质粒、粘粒、病毒、RNA载体或线性或圆形DNA或RNA分子，其可以包括染色体、非染色体、半合成的或合成的核酸。示例性的载体是能够自主复制(附加型载体)和/或表达它们所连接的核酸(表达载体)的那些载体。
本文中使用的载体如病毒载体包括逆转录病毒、腺病毒、细小病毒(例如，腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疹和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒，和双链DNA病毒，所述双链DNA病毒包括腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、爱泼斯坦-巴尔病毒、巨细胞病毒)和痘病毒(例如，牛痘、鸡痘和金丝雀痘)。其它病毒包括例如诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒和肝炎病毒。逆转录病毒的例子包括禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒、泡沫病毒(Coffin，J.M.，Retroviridae：The viruses and their replication，In FundamentalVirology，第三版，B.N.Fields，等人，编，Lippincott-Raven Publishers，Philadelphia，1996)。
本文中使用的术语“慢病毒载体”是指基于HIV的慢病毒载体，其因为它们的相对大的包装能力、降低的免疫原性和它们以高效率稳定地转导大范围的不同细胞类型的能力而可用于基因递送。慢病毒载体经常在三种或更多种质粒(例如，包装、包膜和转移)向生产细胞中的瞬时转染以后产生。像HIV一样，慢病毒载体通过病毒表面糖蛋白与细胞表面上的受体的相互作用而进入靶细胞中。在进入后，病毒RNA经历反转录，其由病毒的逆转录酶复合物介导。反转录的产物是双链线性病毒DNA，其为受感染的细胞的DNA中的病毒整合的底物。
本文中使用的术语“信号肽”和“前导序列”在本文可互换使用并且是指可以连接在本文阐述的蛋白质的氨基末端的氨基酸序列。信号肽/前导序列通常指导蛋白质的定位。本文所用的信号肽/前导序列优选地促进蛋白质从产生其的细胞中分泌。信号肽/前导序列常常在从细胞分泌后从蛋白质的其余部分(通常称为成熟蛋白质)切割下来。信号肽/前导序列连接在所述蛋白质的N端，长度为约9至200个核苷酸(3至60个核酸)。本发明使用的信号肽可以是SARS-COV-2病毒S蛋白的信号肽序列或来自其它真核/病毒蛋白的信号肽序列。
本文中使用的术语“启动子”可以意指能够在细胞中赋予、激活或增强核酸表达的合成或天然来源的分子。启动子可包含一个或多个特异性转录调控序列以进一步增强其表达和/或改变其空间表达和/或时间表达。启动子还可以包含远端增强子或阻遏元件，其可以位于距转录起始位点多达几千个碱基对处。启动子可以源自包括病毒、细菌、真菌、植物、昆虫和动物的来源。启动子相对于其中发生表达的细胞、组织或器官，相对于发生表达时 所处的发育阶段或响应于外部刺激诸如生理应激、病原体、金属离子或诱导剂，可以组成性地或差异性地调控基因构件的表达。启动子的代表性实例包括噬菌体T7启动子、噬菌体T3启动子、SP6启动子、lac操纵子-启动子、tac启动子、SV40晚期启动子、SV40早期启动子、RSV-LTR启动子、CMV IE启动子、SV40早期启动子或SV40晚期启动子以及CMV IE启动子。
本文中使用的术语“可操作地连接”意指基因的表达处于在空间上与之连接的启动子的控制之下。启动子可以定位在处于其控制之下的基因的5′(上游)或者3′(下游)。所述启动子和基因之间的距离可与在该启动子所源自的基因中该启动子与其控制的基因之间的距离大致相同。如本领域所已知，该距离的变化可以在不失去启动子功能的情况下进行调节。
本文中使用的术语“表达载体”表示含有与合适的控制序列可操作地连接的核酸分子的DNA构建体，所述控制序列能够实现所述核酸分子在合适的宿主中的表达。这样的控制序列包括用于实现转录的启动子、任选的用于控制这样的转录的操纵基因序列、编码合适的mRNA核糖体结合位点的序列、和控制转录和翻译的终止的序列。所述载体可以是质粒、噬菌体颗粒、病毒，或简单地是潜在基因组插入物。病毒载体可以是基于DNA(例如，腺病毒或痘苗病毒)或RNA的，包括溶瘤病毒载体(例如，VSV)，能复制的或不能复制的。一旦转化进合适的宿主中，所述载体可以独立于宿主基因组而复制和起作用，或在某些情况下，可以整合进基因组本身中。在本说明书中，“质粒”、“表达质粒”、“病毒”和“载体”经常互换使用。
本文中使用的术语“表达”表示基于基因的核酸序列而生产多肽的过程。所述过程包括转录和翻译。翻译可以开始于非常规起始密码子，诸如CUG密码子，或翻译可以开始于几种起始密码子(标准的AUG和非常规的)以产生比产生的mRNA更多的蛋白(基于每摩尔量)。
在将核酸序列插入细胞中的背景下，本文中使用的术语“引入”是指“转染”或“转化”或“转导”，且包括对核酸序列向真核或原核细胞中的整合的提及，其中所述核酸序列可以整合进细胞的基因组(例如，染色体、质粒、质体或线粒体的DNA)中，转化成自主复制子，或短暂地表达(例如，转染的mRNA)。
用于在细胞中表达外源或异源核酸的重组方法是本领域众所周知的。这样的方法可以参见，例如，Sambrook等人，Molecular Cloning：A Laboratory Manual，第三版， ColdSpring Harbor Laboratory，New York(2001)；和Ausubel等人，Current Protocols inMolecular Biology，John Wiley and Sons，Baltimore，MD(1999)。对编码融合抗原蛋白的核酸分子的遗传修饰可以给从它的天然存在状态改变的重组或非天然细胞赋予生化或代谢能力。
本文中使用的术语“抗原特异性的T-细胞应答”表示由针对表达特定抗原的细胞的T-细胞介导的免疫应答。在某些实施方式中，所述T-细胞应答是CD8 +T-细胞应答、CD4 +T-细胞应答或它们的组合。
本文中使用的术语“核酸分子免疫接种”或“DNA免疫接种”表示将编码一种或多种抗原的核酸分子引入宿主或宿主细胞中以便在体内表达所述一种或多种抗原。核酸分子免疫接种可以是通过直接施用进宿主中，诸如通过标准注射(例如，肌肉内、真皮内)、透皮颗粒递送、吸入、局部地、口服地、鼻内地或粘膜地。可替换地，可以将核酸分子离体引入宿主细胞(例如，宿主细胞或来自与宿主匹配的供体HLA的细胞)中，并可以将转染的宿主细胞施用进宿主中，使得可以引起针对由所述核酸分子编码的一种或多种抗原的免疫应答。
本文中使用的术语“佐剂”指通过增强巨噬细胞活性促进机体T细胞或B细胞的反应，参与半抗原或抗原免疫应答的天然的或合成的物质。细胞因子作为新型分子佐剂能增强疫苗的特异性免疫反应。可与本发明的免疫组合物共同施用的分子佐剂包括但不限于干扰素(IFN)、TNF-α、TNF-β、趋化因子如CCL21、嗜酸性粒细胞趋化因子、HMGB1、SA100-8α、GCSF、GMCSF、颗粒溶素、乳铁蛋白、卵白蛋白、CD-40L、CD28激动剂、PD-1、可溶性PD1、L1或L2、或白介素，如IL-1、IL-2、IL-4、IL-6、IL-7、IL-10、IL-12、IL-13、IL-21、IL-23、IL-15、IL-17和IL-18。
本文中使用的术语“治疗”或“改善”表示受试者(例如，患者)的疾病、障碍或病症的医学管理，其可以是治疗性的、预防性的/防止性的或它们的联合治疗。治疗可以改善或减轻疾病的至少一种征状的严重程度，延迟疾病的恶化或进展，或延迟或阻止另外相关疾病的发作。“减小发生疾病的风险”表示预防或延迟疾病(例如，癌症)的发作或疾病的一种或多种征状的复发。
本文中使用的术语化合物或组合物的“治疗有效量(或剂量)”或“有效量(或剂量)”表示，足以以统计上显著的方式导致正在治疗的疾病的一种或多种征状的改善的化合物的量。精确量取决于众多因素，例如，组合物的活性、采用的递送的方法、组合物的免疫刺激能力、 预期的患者和患者考虑因素等，且可以由本领域普通技术人员容易地确定。治疗效果可以直接地或间接地包括疾病的一种或多种征状的减轻，治疗效果还可以直接地或间接地包括细胞免疫应答的刺激。
本文中使用的术语“受试者”可以是能够发生细胞免疫应答的任何生物体，诸如人类、宠物、家畜、展示动物、动物园样本或其它动物。例如，受试者可以是人、非人灵长类动物、狗、猫、免、大鼠、小鼠、豚鼠、马、牛、绵羊、山羊、猪等。需要施用如本文中所述的治疗剂的受试者包括已被SARS-COV-2病毒感染甚至已经出现病毒感染相关疾病，或者处于SARS-COV-2病毒感染风险中的受试者。
本文中使用的术语“有需要的受试者”表示处于疾病、障碍或病症的高危中或遭受疾病、障碍或病症的受试者，所述疾病、障碍或病症适合用本文所提供的化合物或其组合物治疗或改善。在某些实施方式中，有需要的受试者是人。
对于包含如本文中所述的核酸分子的免疫组合物，期望的结果是能够以最小副作用诱导持久保护性免疫的安全产品，并且与其它策略(例如，完整活的或减毒的病原体)相比，廉价地生产，使已经在其它方面(通常)与完整的或减毒的病毒免疫组合物的应用相关联的禁忌最小化或消除，具有延长的贮存期限(因为它是以核酸和/或合成肽为基础的)。对传染性疾病紧急事件(天然爆发、大范围流行或生物恐怖主义)快速响应的能力是本文公开的实施方式的有效应用的一个益处，无论在生物防御还是免疫疗法或技术的背景下。
与常规的基于多肽的疫苗不同，DNA疫苗可以包含质粒形式的核酸(Li等人，J.Biotechnol.162：171，2012)或掺入用于递送的病毒载体的核酸中。如本领域普通技术人员会理解的，所述质粒DNA包括驱动本文所述的一个或多个转录单元的表达的启动子。通过例如肌肉内注射、皮下地、鼻内地、经粘膜呈递、静脉内地或通过真皮内或皮下施用，可以施用基于核酸的疫苗。
以下将对有关SARS-COV-2病毒的DNA疫苗发明通过具体实施的方式，对其进行举例说明。但应当理解这些实施例不以任何形式限制本发明的范围。
实施例
实施例1：将SARS-COV-2 S1蛋白与抗原辅助序列的编码序列连接构建表达SARS-COV-2 S1-Hsp70融合抗原蛋白表达DNA载体
SARS-COV-2 S1-Hsp70融合抗原蛋白序列SEQ ID NO：10的N端序列来源于SARS-COV-2 S1蛋白质的1-667位氨基酸序列，其中第1-13位氨基酸序列为该S蛋白质的信号肽序列。然后将S1蛋白通过三个甘氨酸组成的连接物(linker)与抗原辅助序列Hsp70的第2-625位氨基酸序列连接在一起(参见图1)。融合抗原蛋白的前体总长度为1294氨基酸残基，其中1-13位氨基酸残基组成的信号肽序列在成熟的融合抗原蛋白中被切除。
采用人体细胞优先表达密码子设计一编码全长1294氨基酸残基的DNA序列，然后人工合成3882bp长的cDNA片段，并在其5’和3’端分别加上KpnI和BamHI酶切位点。按照《Molecular Cloning：A laboratory manual》(ed by J.Sambrook&D.W.Russel，CSHLP出版，4th edition)进行分子克隆操作。用KpnI和BamHI内切酶(New England Biolabs)分别对合成DNA片段(2μg)和pVax1载体(2μg)置37℃消化2小时。然后在琼脂糖凝胶电泳分离消化的DNA片段，将分离获得的DNA片段纯化。进一步将纯化的SARS-COV-2 S1-Hsp70 DNA片段与pVax1质粒片段进行DNA连接反应。标准连接反应中的总DNA约为100ng，载体质粒与插入片段比例约为1∶3。连接后进行转化感受态大肠杆菌(1-2μl的连接反应液加入到100μl感受态大肠细胞DH5alpha TOP10中)。42℃热休克60秒，放入冰水中3分钟。吸取10μl在kanamycin琼脂糖平板上涂板，37℃过夜，选择菌斑培养，用QIAGEN公司生产的质粒纯化试剂药盒提取质粒DNA并进行酶切验证。这样得到3882bp长的SARS-COV-2 S1-Hsp70融合抗原蛋白编码cDNA片段克隆到真核表达载体pVax1中得目的表达载体。进一步将pVax1-SARS-COV-2 S1-Hsp70表达载体转染人293细胞系。将细胞裂解产物进行Western Blot分析，结果如图2所示。用抗SARS-COV-2 S蛋白抗体可以检测到分子量为180KDa左右的SARS-COV-2 S1-Hsp70融合抗原蛋白，这与设计结果相一致。
实施例2：将SARS-COV-2 S1蛋白与截短的抗原辅助序列Hsp70(Δ200)的编码序列连接构建表达SARS-COV-2 S1-Hsp70(Δ200)融合抗原蛋白的DNA表达载体
使用与实施例1相同的方法制备并检测了SARS-COV-2 S1与Hsp70(Δ200)(与前述融合抗原蛋白中包含的抗原辅助序列Hsp70相比，缺少N端200个氨基酸残基)的融合抗原蛋白SARS-COV-2 S1-Hsp70(Δ200)(参见SEQ ID NO：12)，所得结果同样与设计一致(图中未示出)。
实施例3：构建人IL-2、GM-CSF、IL-17和IFN-γ真核表达载体
按照实施例1中的常规分子克隆方法将编码人IL-2、GM-CSF、IL-17和IFN-γ的cDNA序列(所有这些cDNA序列均可自GenBank( https：//www.ncbi.nlm.nih.gov/nuccore)获得，例如人IL-2 cDNA GenBank编号为NM_000586，人GM-CSF cDNA GenBank编号为M11220，人IFN-γcDNA GenBank编号为NM_000619，人IL-17 cDNA GenBank编号为U32659)克隆到p CDNA-3.1真核表达载体中，得到p CDNA-3.1-IL-2、p CDNA-3.1-GM-CSF、p CDNA-3.1-IL-17和p CDNA-3.1-IFN-γ表达载体质粒。用QIAGEN公司生产的质粒大量制备纯化试剂盒分别制备各种细胞因子表达载体10mg备用。将上述细胞因子表达质粒分别转染293细胞系，48小时后收集各自的细胞培养上清液，用R&D Systems公司的人IL-2、GM-CSF、IL-17和IFN-γ细胞因子ELISA检测试剂盒检测细胞培养上清的细胞因子表达情况。结果如图3所示，这些细胞因子表达载体都可以在人源293细胞中分别介导合成相应的特异性细胞因子蛋白。
实施例4：DNA免疫
用QIAGEN公司生产的试剂盒大规模制备高度纯化的高质量的DNA表达载体，在免疫前将DNA溶于经优化的DNA细胞导入缓冲液(见表1)中，调节DNA浓度为1μg/μl。在免疫pVax1-SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)表达质粒前两天先给Balb/c小鼠注射IL-2、GM-CSF、IL-17和IFN-γDNA表达载体。在小鼠肌肉或皮下注射免疫DNA溶液，体积为30μl。然后将30μg/30μl SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)融合抗原表达质粒进行同一肌肉或皮下注射免疫。每2周免疫小鼠一次，共免疫三次后，抽取小鼠外周血，分离淋巴细胞和制备血清，以测定免疫小鼠对SARS-COV-2 S1蛋白质的细胞和体液免疫应答。
作为验证，如图4所示，使用本发明优化的DNA免疫方法非常有效地将绿色荧光蛋白(GFP)DNA表达载体介导进入小鼠肌肉细胞中表达。
表1.化学分子DNA细胞导入缓冲液(pH6.7)
Figure PCTCN2021101269-appb-000001
Figure PCTCN2021101269-appb-000002
*购自MedKoo Biosciences公司(CAT#555350)，先用酒精溶解DLin-KC2-DMA制备成储存液50mg/ml，用前加入到缓冲液中。
实施例5：SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)融合抗原表达载体在小鼠中诱导SARS-COV-2 S1特异性抗体产生
将实施例4中经SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)表达载体进行DNA免疫以及首次免疫后5周后的小鼠经尾静脉取血制备血清。同时用SARS-COV-2 S1蛋白表达载体按相同DNA免疫程序免疫小鼠作为对照，也分别制备其免疫前和首次DNA免疫5周后的血清。然后根据《Antibody：A Laboratory Manual，Second edition，CSHL Press》标准酶联免疫检测(ELISA)方法测定小鼠血清中的S1特异性抗体滴度。将SARS-COV-2 S1蛋白(Sino Biological)1μg/ml配置于pH9.6的碳酸盐缓冲液中包被于96孔酶标板上(100μl/孔)，置4℃过夜。次日移去包被液，用洗涤缓冲液(PBS含0.05％吐温-20)洗3次，每次5分钟。再用含10％BSA和0.05％吐温-20的PBS缓冲液封闭37℃ 1小时。用洗涤缓冲液(PBS含0.05％吐温-20)洗1次。接着将小鼠血清于PBS缓冲液中按不同比例稀释加入酶标板中(100μl/孔)与S1蛋白进行反应，37℃作用1小时。然后用洗涤缓冲液(PBS含0.05％吐温-20)洗3次，每次5分钟。再加入用稀释缓冲液稀释的抗小鼠IgG-HRP标记的二抗(Cell Signaling Technology，Inc)(100μl/孔)检测酶标板中的小鼠IgG，37℃作用1小时。再用洗涤缓冲液(含0.05％吐温-20)洗3次，每次5分钟。加入底物溶液(OPD)(100μl/孔)，室温作用30分钟。每孔加2M H 2SO 450μl。用酶标比色计测定(492nm)OD值。结果如图5和表2所示，SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA表达载体免疫诱导产生S1蛋白特异性抗体量显著高于SARS-COV-2 S1蛋白DNA表达载体免疫所诱导的抗体量，在各稀释 滴度都几乎达到非融合抗原蛋白的两倍。通过与抗原辅助序列Hsp70蛋白融合而针对S1抗原显著增强的体液免疫效应令人惊讶。同时，预料不到的是，相比SARS-COV-2 S1-Hsp70，截短了长达200个氨基酸残基的SARS-COV-2 S1-Hsp70(Δ200)，其DNA表达载体免疫诱导产生的S1蛋白特异性抗体的量与SARS-COV-2 S1-Hsp70的DNA表达载体相当。
表2：融合抗原表达载体诱导产生的S1蛋白特异性抗体量
Figure PCTCN2021101269-appb-000003
实施例6：除去抗原辅助序列Hsp70蛋白的N端的200个氨基酸残基减少了融合抗原蛋白诱导的Hsp70特异性抗体生成。
用pVax1-SARS-COV-2 S1-Hsp70和pVax1-SARS-COV-2 S1-Hsp70(Δ200)表达载体免疫小鼠5周后自尾静脉取血制备血清。作为对照也制备其免疫前的血清。然后根据《Antibody：A Laboratory Manual，Second edition，CSHL Press》标准酶联免疫检测(ELISA)方法测定小鼠血清中的Hsp70特异性抗体滴度。将Hsp70蛋白(Creative Biomart)1μg/ml配置于pH9.6的碳酸盐缓冲液中包被于96孔酶标板上(100μl/孔)，置4℃过夜。次日移去包被液，用洗涤缓冲液(PBS含0.05％吐温-20)洗3次，每次5分钟。再用含10％BSA和0.05％吐温-20的PBS缓冲液封闭37℃ 1小时。用洗涤缓冲液(PBS含0.05％吐温-20)洗1次。接着将小鼠血清于PBS缓冲液中按不同比例稀释加入酶标板中(100μl/孔)与S1蛋白进行反应，37℃作用1小时。然后用洗涤缓冲液(PBS含0.05％吐温-20)洗3次，每次5分钟。再加入用稀释缓冲液稀释的抗小鼠IgG-HRP标记的二抗(Cell Signaling Technology，Inc)(100μl/孔)检测酶标板中的小鼠IgG，37℃作用1小时。再用洗涤缓冲液(含0.05％吐温-20)洗3次，每次5分钟。加入底物溶液(OPD)(100μl/孔)，室温作用30分钟。每孔加2M H 2SO 450μl。用酶标比色计测定(492nm)OD值。结果如图6和表3所示，SARS-COV-2 S1-HSP70(Δ200)DNA表达载体免疫诱导产生的 Hsp70蛋白特异性抗体量明显低于SARS-COV-2 S1-Hsp70 DNA表达载体免疫所诱导的抗体量，证实采用本发明的特定截短融合抗原架构，极大地减小了融合抗原蛋白中的抗原辅助部分导致的不期望的免疫原性，使得机体免疫能力进一步聚焦于融合抗原蛋白的S1部分，提高了机体免疫系统资源的利用率。如此，SARS-COV-2 S1-HSP70(Δ200)在基本保持其诱导产生中和抗体能力的同时，还大幅降低了不期望的免疫原性，在安全性方面显示了令人惊讶的优势。
表3：融合抗原表达载体诱导产生的Hsp70蛋白特异性抗体量
Figure PCTCN2021101269-appb-000004
实施例7：SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)融合抗原蛋白的构建促进其pVax1 DNA表达载体在机体内的清除。
按实施例4的方法用pVAX1-SARS-COV-2 S1-Hsp70和pVAX1-SARS-COV-2 S1-Hsp70(Δ200)DNA表达载体免疫Balb/c小鼠一次，5周后取小鼠免疫部位的肌肉组织制备其细胞裂解物，用PCR方法测定其中存在的pVax1表达载体。PCR实验操作如文献所述(Green，M.R：Molecular cloning，A Laboratory Manual(4th)，Cold Spring Harbour Press，2012)。合成一对寡聚核苷酸引物：(1)5’GGAGGATTGGGAAG 3’；(2)5’CCACCGCTGGTAG3’。引物1对应的是pVax1质粒1001-1014位核苷酸序列，引物2对应的是pVax1质粒2401-2413位核苷酸序列。以上述细胞裂解物为检测样本，进行如下PCR反应(反应混合物如下)：
Figure PCTCN2021101269-appb-000005
Figure PCTCN2021101269-appb-000006
反应前将反应物置94℃、5分钟。PCR反应环境为94℃、1分钟；55℃、1分钟；72℃、1分钟。反应为35个循环。最后一个循环置72℃、10分钟。然后取1μl PCR反应产物于1％琼脂糖电泳胶中进行电泳分析，结果如图7所示，可见一1.4kb大小的DNA条带，这与对应于前述引物对的扩增产物序列大小相符。
PCR检测发现，免疫后五周，pVAX1-SARS-COV-2 S1-Hsp70(泳道2)和pVAX1-SARS-COV-2 S1-Hsp70(Δ200)(泳道5)DNA表达载体均完全从小鼠免疫部位的组织中消失，这意味着含有该DNA表达载体的细胞都完全被清除。相比之下，pVAX1-SARS-COV-2 S1(泳道3)、pVAX1-Hsp70(泳道4)或二者的混合(泳道1)均未诱导其相应pVAX1表达载体从机体细胞清除(图7)。这证明SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)融合抗原蛋白疫苗的设计在刺激产生有效的机体保护性免疫应答的同时，还避免了外源DNA载体在体内的长期驻留，由此消除了长期驻留可能引发的不良反应如毒副作用，增强了疫苗安全性。
实施例8：SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)融合抗原蛋白表达载体诱导产生的S1特异性中和抗体对S1蛋白质与人肺上皮细胞结合的阻断作用
将人原代肺泡上皮细胞(HPAEpiC)(美国ACCEGEN Biotechnology)以100μl(含20000细胞)/孔接种于96孔培养板中，培养基为该公司提供的HPAEPiC完全培养基。将细胞置37℃ 5％CO 2培养箱中培养24小时后，除去培养基，用PBS洗两次。再用4％Formaldehyde(100μl/孔)室温固定20分钟。用含0.05％吐温-20的PBS缓冲液洗三次。然后加入含1％H 2O 2的PBS缓冲液(100/孔)置室温25分钟。用含0.05％吐温-20的PBS缓冲液洗三次。接着用含10％FBS和0.05％吐温-20的PBS缓冲液封闭37℃ 1小时。然后去除封闭缓冲液。用洗涤缓冲液(含0.05％吐温-20的PBS)洗1次。同时将实施例5中的小鼠抗血清按照不同比例于PBS中稀释，然后将稀释的小鼠抗血清1∶1与生物素化的SARS-COV-2 S1蛋白溶液(10μg/ml in PBS)混合，置37℃孵育30分钟。然后加入到细胞培养板中去(100μl/孔)，置37℃孵育1小时。然后用洗涤缓冲液(含0.05％吐温-20 的PBS)洗3次，每次5分钟。再加入用稀释缓冲液稀释的HRP标记的Avidin(Cell Signaling Technology，Inc)(100μl/孔)到细胞培养板中，置37℃作用1小时。再用洗涤缓冲液(PBS含0.05％吐温-20)洗3次，每次5分钟。加入底物溶液(OPD)(100μl/孔)，室温作用30分钟。每孔加2M H 2SO 450μl。用酶标比色计测定(492nm)OD值。结果如图8所示，分别用SARS-COV-2 S1-Hsp70 DNA表达载体、SARS-COV-2 S1-Hsp70(Δ200)DNA表达载体和SARS-COV-2 S1 DNA表达载体免疫的小鼠中和抗体血清均显著抑制S1蛋白与人肺泡上皮细胞的结合，由此确认各自诱导产生的S1蛋白特异性抗体是针对人细胞系有效的中和性抗体。然而出乎意料的是，发现SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA疫苗的抑制效应明显高于非融合的SARS-COV-2 S1 DNA疫苗。而且，相比SARS-COV-2 S1-Hsp70 DNA表达载体，用其中抗原辅助序列Hsp70被截短多达200个氨基酸的SARS-COV-2 S1-Hsp70(Δ200)DNA表达载体免疫的小鼠中和抗体血清在阻止S1蛋白与人肺泡上皮细胞的结合能力方面并未明显下降。未免疫的小鼠血清无此中和作用(参见图8和表4)。
表4：融合抗原表达载体诱导产生的抗体血清阻止S1蛋白与人肺泡上皮细胞的结合
Figure PCTCN2021101269-appb-000007
实施例9：SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA疫苗免疫诱导小鼠T细胞免疫应答。
将实施例5中经SARS-COV-2 S1-Hsp70、SARS-COV-2 S1-Hsp70(Δ200)和SARS-COV-2 S1 DNA表达载体分别免疫的小鼠，在首次免疫5周后取其脾脏在10％血清的RPMI 1640培养基中制成细胞悬液。然后采用尼龙毛柱法分离T淋巴细胞。将日本WAKO公司生产的尼龙毛1克填装于10ml的一次性注射器针筒中，灭菌消毒后，用含10％胎牛血清RPMI 1640培养基平衡尼龙毛柱。将小鼠脾脏细胞悬液2ml(含5x10 8个脾细胞)加入柱中，置37℃孵育30分钟。然后用含10％胎牛血清RPMI 1640培养基洗脱下T淋巴细胞。将T细胞用该培养基洗涤一次，然后在含10％胎牛血清RPMI 1640培养 基中调节细胞密度为5X10 6/ml，以200μl细胞悬液/孔将T细胞加入到96孔细胞培养板中培养。该培养板已预先含有1x10 4/孔小鼠脾脏粘附细胞。小鼠脾脏粘附细胞按常规方法分离制得(Rosenwasser，L.J.&Rosenthal，A.S.：J.Immunol.120：1991，1978)。然后加入1ng/孔SARS-COV-2 S1蛋白刺激T细胞，置37℃ CO 2培养箱中培养。24小时后取培养上清，用R&D Systems公司的小鼠IL-2和IFN-γELISA试剂盒测定其中IL-2和IFN-γ的生成情况。结果如图9和表5所示，分别用SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA载体免疫的小鼠T细胞比用SARS-COV-2 S1 DNA载体免疫的小鼠T细胞产生的IL-2和IFN-γ水平显著增高大约100％且两者相当，而未经免疫的小鼠T细胞产生极低水平的IL-2和IFN-γ。
表5：融合DNA疫苗免疫的小鼠T细胞产生的细胞因子的量
Figure PCTCN2021101269-appb-000008
实施例10：SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA疫苗免疫的小鼠T细胞对表达SARS-COV-2 S1蛋白的小鼠3T3细胞的杀伤作用。
首先用表达全长SARS-COV-2 S1蛋白的pcDNA3.1质粒转染小鼠3T3细胞。将转染的细胞按常规方法进行 51Cr标记(Nelson，D.L.et al：Current Protocols in Immunology，1993.https：//doi.org/10.1002/0471142735.im0727s08)。将标记后的细胞于含10％胎牛血清DMEM培养基中加入到96孔细胞培养板中(5x10 4细胞/孔)，置37℃ 5％CO 2培养箱中培养4小时后，加入实施例9中分离的T淋巴细胞(同一含10％胎牛血清DMEM培养基，1x10 6细胞/孔)。在CO 2培养箱中连续培养6小时后，1000g离心培养板10分钟。每孔吸取100μl上清液在液闪计数仪上测定CPM值。细胞杀伤活性按照以下公式计算：T细胞杀伤活性(％)＝[(实验组CPM-自发释放CPM)/(最大释放CPM-自发释放CPM)]x100％。结果如图10和表6所示：分别用SARS-COV-2 S1-Hsp70和SARS-COV-2 S1-Hsp70(Δ200)DNA疫苗免疫的小鼠T细胞对表达SARS-COV-2 S1蛋白的3T3细胞的杀伤作用相当，且与用SARS-COV-2 S1 DNA表达载体免疫的小鼠T细胞相比显著增强大 约100％。没有免疫的小鼠T细胞对该SARS-COV-2 S1表达载体转染的3T3细胞无杀伤作用。
表6：融合DNA疫苗免疫的小鼠T细胞对3T3细胞的杀伤活性
Figure PCTCN2021101269-appb-000009
在不脱离本发明的范围和精神的情况下，本发明所描述的产品、方法和用途的各种修改和变化对于本领域技术人员而言是显而易见的。尽管已经结合具体实施方式描述了本发明，但是应当理解，要求保护的本发明能够作进一步修改，并且不应当不适当地受限于这样的特定实施方式。实际上，对于本领域技术人员显而易见的用于实施本发明的所描述的模式的各种修改是旨在落入本发明的范围内。本申请旨在涵盖总体遵循本发明的原理且包括从本公开到本发明所属领域内的已知惯用实践内并且可以应用于本文前文说明的必要特征的这样的偏离的本发明的任何变化、用途或改编。

Claims (13)

  1. 一种融合蛋白，其包含SEQ ID NO：10中14-1294位的氨基酸序列或SEQ ID NO：9编码的成熟多肽，或与其具有80％以上序列一致性的氨基酸序列。
  2. 根据权利要求1所述的融合蛋白，其由SEQ ID NO：10的氨基酸序列或SEQ ID NO：10的14-1294位的氨基酸序列或SEQ ID NO：9编码的成熟多肽组成，或与其具有80％以上序列一致性的氨基酸序列。
  3. 权利要求1或2所述的融合蛋白，其中SEQ ID NO：10中14-667位的氨基酸序列与671-1294位的氨基酸序列的位置互换。
  4. 一种融合蛋白，其包含SEQ ID NO：12中14-1094位的氨基酸序列或SEQ ID NO：11编码的成熟多肽，或与其具有80％以上序列一致性的氨基酸序列。
  5. 根据权利要求4所述的融合蛋白，其由SEQ ID NO：12的氨基酸序列或SEQ ID NO：12的14-1094位的氨基酸序列或SEQ ID NO：11编码的成熟多肽组成，或与其具有80％以上序列一致性的氨基酸序列。
  6. 权利要求4或5所述的融合蛋白，其中SEQ ID NO：12中14-667位的氨基酸序列与671-1094位的氨基酸序列的位置互换。
  7. 一种编码权利要求1-6中任一项所述的融合蛋白的多核苷酸或包含所述多核苷酸序列的核酸分子。
  8. 根据权利要求7所述的多核苷酸，其由SEQ ID NO：9或SEQ ID NO：11组成。
  9. 一种载体，其包含权利要求8所述的多核苷酸序列。
  10. 一种组合物，其包含：
    (1)权利要求1-6中任一项所述的融合蛋白，或权利要求7或8所述的多核苷酸，或权利要求9所述的载体；和
    (2)药学上可接受的载体和/或赋形剂。
  11. 权利要求1-6中任一项所述的融合蛋白、或权利要求7或8所述的多核苷酸、或权利要求9所述的载体、或权利要求10所述的组合物在制备用于在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的药物中的用途。
  12. 根据权利要求11所述的用途，所述治疗或预防还包括在向所述受试者施用所述药物之前，向所述受试者施用选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体。
  13. 一种用于在有需要的受试者中治疗或预防SARS-COV-2病毒感染和/或相关疾病的试剂盒，其包括：
    (1)权利要求1-6中任一项所述的融合蛋白、或权利要求7或8所述的多核苷酸、或权利要求9所述的载体、或权利要求10所述的组合物；和
    (2)选自人GM-CSF、IL-2、IFN-γ和/或IL-17中的一种或多种的细胞因子、或编码所述细胞因子的多核苷酸、或包含所述多核苷酸的载体。
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EP4291212A1 (en) 2021-02-15 2023-12-20 LivingMed Biotech S.R.L. Genetically clostridium modifiedstrains expressing recombinant antigens and uses thereof
WO2022216895A1 (en) * 2021-04-09 2022-10-13 University Of Florida Research Foundation, Incorporated Methods, kits, and approaches for viral vaccines
CN114591401A (zh) * 2021-05-18 2022-06-07 深圳市因诺转化医学研究院 SARS-CoV-2编码蛋白来源的T细胞表位多肽HLVDFQVTI及其应用
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CN114150004B (zh) * 2022-02-09 2022-04-22 广州恩宝生物医药科技有限公司 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用

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