WO2022179318A1 - 基于s蛋白r815位点的冠状病毒干预的方法和产品 - Google Patents

基于s蛋白r815位点的冠状病毒干预的方法和产品 Download PDF

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WO2022179318A1
WO2022179318A1 PCT/CN2022/070763 CN2022070763W WO2022179318A1 WO 2022179318 A1 WO2022179318 A1 WO 2022179318A1 CN 2022070763 W CN2022070763 W CN 2022070763W WO 2022179318 A1 WO2022179318 A1 WO 2022179318A1
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protein
vaccine
present
antigenic peptide
cells
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孟广勋
喻实
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中国科学院上海巴斯德研究所
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Definitions

  • the invention belongs to the field of biomedicine, and in particular, the invention relates to methods and products for coronavirus intervention based on S protein R815 site.
  • Novel coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) is a member of the genus coronavirus beta and is the pathogen that causes COVID19. Since December 2019, the COVID19 epidemic has spread to more than 80 countries and regions on all continents, more than 100 million people have been diagnosed, and more than 2 million people have died worldwide; in addition, it has also paralyzed the operation of the international community, and unprecedented Economic property damage. Infected people not only obstructed lung function, but also caused a variety of complications in high-risk groups due to systemic inflammation.
  • the purpose of the present invention is to provide an anti-novel coronavirus vaccine that can effectively inhibit the cell fusion effect induced by the new coronavirus, thereby preventing viral infection.
  • Another purpose of the present invention is to discover the most important amino acid functional sites of the new crown S protein; single point mutation maximizes the retention of the original S protein structure; the present invention includes functional sites effective for mutant strains; effectively avoid and reduce wild type Cell fusion and side effects induced by SARS-CoV-2 S protein; a protocol for intervention targeting wild-type S protein.
  • the first aspect of the present invention provides an antigenic peptide, the antigenic peptide is derived from the new coronavirus S protein, and the antigenic peptide is corresponding to the new coronavirus S protein amino acid sequence of SEQ ID NO.: 1 No. 1 Arginine at position 815 is mutated.
  • the mutation includes amino acid insertion, deletion or substitution.
  • the arginine at position 815 is mutated to any amino acid.
  • the arginine at position 815 is mutated to one or more amino acids selected from the group consisting of alanine (A), asparagine (N), lysine (K) , aspartic acid (D) and glutamic acid (E).
  • A alanine
  • N asparagine
  • K lysine
  • D aspartic acid
  • E glutamic acid
  • the arginine at position 815 is mutated to one or more amino acids selected from the group consisting of alanine (A) and asparagine (N).
  • the mutation is selected from the group consisting of R815A, R815N, or a combination thereof.
  • the new coronavirus S protein includes a new coronavirus mutant strain (such as British B.1.1.7 (Alpha), South Africa B.1.351 (Beta), India B.1.617.2 (Delta) or South Africa B.1.1.529 (Omicron)) of the S protein.
  • a new coronavirus mutant strain such as British B.1.1.7 (Alpha), South Africa B.1.351 (Beta), India B.1.617.2 (Delta) or South Africa B.1.1.529 (Omicron) of the S protein.
  • amino acid sequence of the antigenic peptide is shown in any of SEQ ID NO.3-5.
  • the antigenic peptide is a polypeptide having an amino acid sequence shown in any of SEQ ID NO.3-5, an active fragment thereof, or a conservative variant polypeptide thereof.
  • amino acid sequence of the antigenic peptide is identical or substantially identical to the sequence shown in SEQ ID NO.: 1 except for the mutation (such as position 815).
  • the remaining amino acid sequences of the antigenic peptide include C-terminal 19 amino acid truncated ( ⁇ CT19), C-terminal TM truncated ( ⁇ TM), and Truncated S protein after arginine at position 815.
  • the substantially identical is that at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids are different, wherein, The difference includes amino acid substitution, deletion or addition, and the antigenic peptide has the activity of inhibiting the cell fusion and infection effects of the novel coronavirus S protein.
  • the homology between the antigenic peptide and the sequence shown in SEQ ID NO.: 1 is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, most preferably A good location is at least 98% or 99%.
  • the antigenic peptide competes with the S protein of the novel coronavirus to bind to the human ACE2 receptor (including known or unknown co-receptors necessary for its function).
  • the "competitive binding” refers to the binding of the antigenic peptide and the S protein of the novel coronavirus to the same or substantially the same binding domain (or amino acid segment) of the human ACE2 protein.
  • the antigenic peptide and the S protein of the novel coronavirus bind to the same binding segment on the human ACE2 protein.
  • the competitive binding includes blocking or non-blocking competitive binding.
  • the antigenic peptide is a synthetic or recombinant antigenic peptide.
  • the antigenic peptide is a recombinant protein expressed by yeast cells.
  • the antigenic peptide is a recombinant protein expressed by insect cells.
  • insect cells are selected from the group consisting of Sf9, Sf21, Tni, Hi5-Sf cells, or a combination thereof.
  • the yeast includes Pichia pastoris.
  • the antigenic peptide is selected from the following group:
  • the "substantially the same function” means that the derivative polypeptide has the activity of inhibiting the cell fusion and infection effects of the novel coronavirus S protein.
  • the second aspect of the present invention provides a vaccine polypeptide, the vaccine polypeptide includes the antigenic peptide described in the first aspect of the present invention.
  • the vaccine polypeptide can stimulate primates and rodents to produce neutralizing antibodies that block the binding of RBD to ACE2 and inhibit the cell fusion and infection effects of the novel coronavirus S protein.
  • the vaccine polypeptide can induce cellular immunity and humoral immunity in primates.
  • the primates include humans and non-human primates.
  • the third aspect of the present invention provides an mRNA vaccine, the vaccine contains mRNA encoding the antigenic peptide described in the first aspect of the present invention, and a DNA expression vector.
  • the packaging carrier of the mRNA vaccine is protamine containing a Toll-like receptor binding agent, a nanoparticle artificial membrane, a chemically synthesized polymer, and a liposome.
  • the fourth aspect of the present invention provides an isolated polynucleotide encoding the antigenic peptide of the first aspect of the present invention or the vaccine polypeptide of the second aspect of the present invention.
  • the fifth aspect of the present invention provides an expression vector, the expression vector contains the polynucleotide according to the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides a host cell, the host cell contains the expression vector of the fifth aspect of the present invention, or integrates the polynucleotide of the fourth aspect of the present invention into the genome.
  • the host cells include prokaryotic cells and eukaryotic cells.
  • the host cells include yeast, Drosophila S2 cells, insect Hi5-Sf cells, Escherichia coli, monkey-derived Vero E6 cells, hamster CHO cells, DC cells, or a combination thereof.
  • the seventh aspect of the present invention provides an antigenic virus strain that loses the cell fusion effect of the new coronavirus S protein, wherein the genome of the virus strain corresponds to the 815th SEQ ID NO.: 1 of the amino acid sequence of the new coronavirus S protein.
  • the arginine at position is mutated.
  • the mutation includes amino acid insertion, deletion or substitution.
  • nucleotide corresponding to the 815th arginine of SEQ ID NO.:1 in the genome of the virus strain is mutated based on the encoding of SEQ ID NO.:2.
  • the nucleotide mutation refers to: the nucleotide is mutated to a nucleotide encoding any amino acid other than arginine.
  • the nucleotide mutation refers to: the nucleotide is mutated to a nucleotide encoding one or more amino acids selected from the group consisting of alanine (A), asparagine (N), lysine (K), aspartic acid (D) and glutamic acid (E).
  • A alanine
  • N asparagine
  • K lysine
  • D aspartic acid
  • E glutamic acid
  • the nucleotide mutation refers to: the nucleotide is mutated to a nucleotide encoding one or more amino acids selected from the group consisting of alanine (A), asparagine (N).
  • the eighth aspect of the present invention provides a pharmaceutical composition
  • the composition contains the antigenic peptide described in the first aspect of the present invention, the vaccine polypeptide described in the second aspect of the present invention, or the mRNA described in the third aspect of the present invention
  • the pharmaceutical composition is a vaccine composition.
  • the vaccine composition is monovalent or multivalent.
  • the pharmaceutical composition further contains adjuvants, preferably various aluminum adjuvants.
  • the drug is an inhibitor for arginine at position 815 of SEQ ID NO.: 1 of the S protein amino acid sequence of the novel coronavirus.
  • the molar ratio or weight ratio of antigenic peptide, immune polypeptide, mRNA vaccine or virus strain, and adjuvant (such as aluminum) in the pharmaceutical composition is between 1:100, preferably 1:40 between 1:60.
  • the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.
  • the dosage form of the pharmaceutical composition is liquid, solid, or gel state.
  • the pharmaceutical composition is administered in a manner selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal injection and aerosol inhalation.
  • the ninth aspect of the present invention provides a vaccine composition, the composition contains the antigenic peptide described in the first aspect of the present invention, the vaccine polypeptide described in the second aspect of the present invention, or the mRNA described in the third aspect of the present invention
  • the vaccine composition further contains an adjuvant.
  • the adjuvant includes: particulate adjuvant and non-particulate adjuvant.
  • the particulate adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or a combination thereof.
  • the non-particulate adjuvant is selected from the group consisting of muramyl dipeptide and its derivatives, saponins, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and their products such as branches Bacillus (Mycobacterium tuberculosis, BCG), Bacillus pumilus, Bacillus pertussis, Propolis, or a combination thereof.
  • the adjuvant includes alumina, saponin, quil A, muramyl dipeptide, mineral oil or vegetable oil, vesicle-based adjuvant, nonionic block copolymer or DEAE dextran , cytokines (including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12, and CpG).
  • cytokines including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12, and CpG.
  • the vaccine composition includes an injection dosage form.
  • the tenth aspect of the present invention provides the antigenic peptide according to the first aspect of the present invention or the vaccine polypeptide according to the second aspect of the present invention or the mRNA vaccine according to the third aspect of the present invention or the virus according to the seventh aspect of the present invention
  • Use of the strain or the pharmaceutical composition according to the eighth aspect of the present invention or the vaccine composition according to the ninth aspect of the present invention (a) for the preparation of antibodies against the novel coronavirus; and/or (b) for the preparation of Medicines to prevent and/or treat coronavirus infection or its related diseases.
  • the antibody comprises a blocked antibody against the mutation at the 815th arginine site of the novel coronavirus S protein.
  • the coronavirus infection or its related disease is selected from the following group: respiratory tract infection, pneumonia and its complications, Middle East respiratory syndrome, human respiratory disease new coronary pneumonia, swine gastrointestinal disease and its complications , or a combination thereof.
  • the coronavirus-related disease includes novel coronavirus pneumonia (COVID-19).
  • the coronavirus is selected from the following group: SARS-CoV-2, SARS-CoV, MERS-CoV (Middle East Respiratory Syndrome), SADS-CoV (Swine Acute Diarrhea Syndrome), or a combination thereof .
  • the treatment includes treatment by the method of gene therapy.
  • the eleventh aspect of the present invention provides a method for preparing the antigenic peptide described in the first aspect of the present invention, comprising the steps of:
  • step (i) of the method the transformed yeast single colonies are respectively inoculated into BMGY medium, and the supernatant is removed by centrifugation after culturing, and the cells are resuspended in BMMY medium at 28-30°C (preferably, the temperature is 29.5°C), and the induction culture is carried out for 36-48 hours (preferably 48 hours).
  • the twelfth aspect of the present invention provides a method for generating an immune response against the coronavirus SARS-CoV-2, characterized in that it comprises the steps of: administering the antigenic peptide described in the first aspect of the present invention, the present invention
  • the subject includes a human or a non-human mammal.
  • the non-human mammals include non-human primates (such as monkeys).
  • the method induces the production of neutralizing antibodies against the coronavirus SARS-CoV-2 in the subject.
  • the neutralizing antibody can inhibit the cell fusion and infection effects of the novel coronavirus S protein.
  • the thirteenth aspect of the present invention provides a method for inhibiting the cell fusion effect of the novel coronavirus S protein, comprising the steps of: the antigenic peptide described in the first aspect of the present invention, the vaccine polypeptide described in the second aspect of the present invention, the In the presence of the mRNA vaccine described in the third aspect of the present invention or the virus strain described in the seventh aspect of the present invention, or the pharmaceutical composition described in the eighth aspect of the present invention or the vaccine composition described in the ninth aspect of the present invention, culture and express ACE2 protein, thereby inhibiting the cell fusion and infection effects of the new coronavirus S protein.
  • the cells are cells cultured in vitro.
  • the method is an in vitro method.
  • the cells are selected from the group consisting of lung epithelial cells, intestinal epithelial cells, liver and kidney epithelial cells, or a combination thereof.
  • the cells are selected from the group consisting of HEK293T-ACE2, Vero E6-ACE2, Caco2, Calu3 cells, or a combination thereof.
  • the fourteenth aspect of the present invention provides a treatment method, which comprises administering the antigenic peptide of the first aspect of the present invention, the vaccine polypeptide of the second aspect of the present invention, the mRNA vaccine of the third aspect of the present invention to a subject in need,
  • the polynucleotide of the fourth aspect of the present invention or the expression vector of the fifth aspect of the present invention or the host cell of the sixth aspect of the present invention or the virus strain of the seventh aspect of the present invention or the eighth aspect of the present invention The pharmaceutical composition or the vaccine composition of the ninth aspect of the present invention.
  • the treatment method includes a gene therapy method.
  • the indicated treatment method comprises in vitro transplantation of human DC cells transfected by electroporation, and injection of lymphatic mRNA vaccine.
  • a fifteenth aspect of the present invention provides a specific binding agent that specifically recognizes or binds to the antigenic peptide of the first aspect of the present invention.
  • the specific binding agent specifically recognizes a mutation site comprising the 815th arginine mutation site (R815) in the antigenic peptide.
  • the specific binding agent specifically recognizes the arginine mutation site (R815) at position 815 in the antigenic peptide.
  • the specific binding agent recognizes a sequence epitope.
  • the specific binding agent recognizes a conformational epitope.
  • the specific binding agent is selected from the group consisting of polypeptides, antibodies, small molecule compounds, or a combination thereof.
  • the antibody includes a monoclonal antibody or a polyclonal antibody.
  • the antibody includes antibody fragments, such as camelid VHH nanobodies.
  • the sixteenth aspect of the present invention provides a method for screening candidate compounds for preventing and/or treating coronavirus SARS-CoV-2 infection or related diseases, including:
  • the compound in the test compound library binds to the antigen peptide described in the first aspect of the present invention, it indicates that the compound that binds to the antigen peptide described in the first aspect of the present invention is the candidate compound.
  • the binding comprises the binding of the compound in the test compound library to the arginine mutation site (R815) at position 815 in the antigenic peptide.
  • the method includes step (b): applying the candidate compound determined in step (a) to cells expressing ACE2 in vitro, and measuring its effect on cell fusion and new coronavirus infection.
  • the method includes step (b): administering the candidate compound determined in step (a) to a mammalian model to determine its effect on the mammal.
  • the mammal is a mammal suffering from a coronavirus SARS-CoV-2 infection or a related disease thereof.
  • the method is non-diagnostic and non-therapeutic.
  • Figure 1 shows the cell fusion effect of SARS-CoV-2 S protein and host ACE2 cells.
  • HEK293T single cells expressing SARS-CoV-2 S protein (A); or cell complexes (B) after fusion with ACE2 cells were photographed by light microscopy.
  • S2' protein band (C) was generated after cell fusion induced by S protein.
  • D The sequence alignment of the amino acid sites of the S protein of 4 known coronaviruses and the structural diagram of the S2' position and cleavage site of the SARS-CoV-2 S protein. Scale distance represents 20 ⁇ m.
  • FIG. 2 shows the loss of fusion effect of the S protein mutated at the R815 site.
  • Western blot showing wild-type (WT) S, R815N, and R815A mutant S protein sizes (A); when ACE2 cells were added to the last three rows, wild-type S protein produced an S2' band, while R815N and R815A mutants Both inhibited the production of S2' (A).
  • Figure 3 shows that the R815 site effectively prevents the fusion effect of the S protein of other mutants.
  • the fusion effect of natural D614G S protein mutant strains captured by light microscope photos can be completely inhibited by R815N and R815A mutations (A); the artificially made C-terminal 19 amino acid (S-CT ⁇ 19) truncated function-enhancing mutation can also be completely inhibited by R815N and the R815A mutation inhibited (B).
  • C The Alpha, Beta and Delta mutant strains of the global pandemic SARS-CoV-2 can all fuse with ACE2-expressing cells (C, top), however, the single point mutation of R815A can make the spike protein function of different SARS-CoV-2 mutants lose their function (C, bottom). Scale distance represents 20 ⁇ m.
  • FIG. 4 shows that the R815 site mutation effectively prevents the pseudovirus from infecting ACE2 cells.
  • Scale distance represents 20 ⁇ m.
  • Figure 5 shows that the R815 site mutation reduces the cell fusion function of the wild-type S protein. Because the S protein forms trimers during expression, the monomers of the mutant S protein can assemble into the trimer of the SARS-CoV-2 S protein and interfere with its membrane fusion function (A). After co-immunoprecipitation with HA-protein tag, Western blotting showed that both wild-type and R815A mutant could bind to wild-type S protein containing Myc tag (B); Western blotting showed that the co-expressed R815A mutant S protein could Effectively reduces the cell fusion phenomenon of wild-type S protein with ACE2 cells and the production of S2' (C).
  • A membrane fusion function
  • the inventors have unexpectedly found that the mutation of arginine at position 815 of SEQ ID NO.: 1 of the amino acid sequence of the S protein of the novel coronavirus can effectively prevent the S protein from producing cell fusion and infection effects , and the site mutation of R815 can also effectively prevent the fusion effect of various S protein mutant strains, thereby effectively preventing the infection of the new coronavirus.
  • Coronavirus SARS-CoV-2 infection or its related diseases On this basis, the present inventors have completed the present invention.
  • AxxB means that amino acid A at position xx is changed to amino acid B, eg, "L87I” means that amino acid L at position 87 is mutated to I, and so on.
  • Coronavirus belongs to the family Coronaviridae of the order Nidovirales. It is an enveloped positive-strand RNA virus.
  • HCoV-229E and HCoV-NL63 belong to alpha coronaviruses
  • HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all beta coronaviruses Virus.
  • the novel coronavirus (SARS-CoV-2), which broke out at the end of 2019, is about 80% similar to SARS-CoV and 40% similar to MERS-CoV, and also belongs to the beta coronavirus.
  • the genome of this type of virus is a single-stranded positive-stranded RNA, which is one of the largest RNA viruses with the encoding including replicase, spike protein, envelope protein, envelope protein and nucleocapsid protein.
  • the genome is translated into two peptide chains of several thousand amino acids, the precursor polyprotein (Polyprotein), and then the precursor protein is cleaved by proteases to generate non-structural proteins (such as RNA polymerase and unwinding). enzymes) and structural proteins (such as spike proteins) and accessory proteins.
  • the S protein is a major structural protein of the coronavirus SARS-CoV-2, in which RBD is responsible for binding to the human ACE2 receptor.
  • S protein YP_009724390.1
  • the S protein includes wild type and mutant type.
  • the main goal of the present invention is to develop a vaccine that can induce the body to produce neutralizing antibodies targeting the coronavirus SARS-CoV-2, which is used to prevent the fusion effect of the new mutant strain of the new coronavirus, thereby preventing the infection of the new coronavirus, and It can effectively block infection caused by new coronavirus mutant strains.
  • Another objective of the present invention is to develop a drug containing S protein mutants that can effectively treat coronavirus SARS-CoV-2 infection or its related diseases.
  • the present invention provides an antigenic peptide derived from novel coronavirus S protein, and the antigenic peptide is mutated at the arginine at position 815 of SEQ ID NO.: 1 corresponding to the amino acid sequence of novel coronavirus S protein.
  • the arginine at position 815 is mutated to one or more amino acids selected from the group consisting of alanine (A), asparagine (N), lysine (K) , aspartic acid (D) and glutamic acid (E).
  • A alanine
  • N asparagine
  • K lysine
  • D aspartic acid
  • E glutamic acid
  • amino acid numbering in the antigenic peptide of the present invention is based on SEQ ID NO.: 1, and when the homology of a specific antigen peptide and the sequence shown in SEQ ID NO.: 1 reaches 80% or more, the antigenic peptide’s homology reaches 80% or more.
  • Amino acid numbering may be misplaced relative to the amino acid numbering of SEQ ID NO.: 1), such as 1-5 positions to the N-terminus or C-terminus of the amino acid, and those skilled in the art can use conventional sequence alignment techniques in the art.
  • the antigenic peptides of the present invention are synthetic or recombinant proteins, ie, may be the product of chemical synthesis, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants) using recombinant techniques. Depending on the host used in the recombinant production protocol, the muteins of the present invention may be glycosylated or may be non-glycosylated. The muteins of the invention may or may not also include an initial methionine residue.
  • the present invention also includes fragments, derivatives and analogs of the antigenic peptides.
  • fragment refers to proteins that retain substantially the same biological function or activity of the antigenic peptide.
  • the antigenic peptide fragments, derivatives or analogs of the present invention may be (i) muteins having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acids
  • the residue may or may not be encoded by the genetic code, or (ii) an antigenic peptide with a substitution group in one or more amino acid residues, or (iii) a mature mutein combined with another compound (such as an elongation mutein).
  • Half-life compounds such as polyethylene glycol
  • an additional amino acid sequence is fused to this antigenic peptide sequence to form an antigenic peptide (such as a leader sequence or a secretory sequence or used to purify the antigenic peptide).
  • sequence or proprotein sequence, or a fusion protein formed with an antigenic IgG fragment are well known to those skilled in the art in light of the teachings herein.
  • conservatively substituted amino acids are preferably produced by amino acid substitutions according to Table 1.
  • the active antigenic peptide of the present invention has basically the same immunogenicity for stimulating an immune response, has the activity of competitively binding human ACE2 protein with the S protein of the novel coronavirus, and has the cell fusion and infection effects of completely inhibiting the S protein of the novel coronavirus activity.
  • the antigenic peptide is shown in any of SEQ ID NO.3-5.
  • R815A mutant full-length S protein sequence (SEQ ID NO. 3):
  • the mutein of the present invention generally has higher homology (identity) compared with the sequence shown in any one of SEQ ID NO.: 3-5, preferably, the mutein and SEQ ID NO.
  • the homology of the sequences shown in any of NO.:3-5 is at least 80%, preferably at least 85%-90%, more preferably at least 95%, most preferably at least 98%, most preferably ground, ⁇ 99%.
  • the antigenic peptide of the present invention can also be modified.
  • Modified (usually without altering the primary structure) forms include chemically derivatized forms such as acetylation or carboxylation of the antigenic peptide in vivo or in vitro.
  • Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the antigenic peptide or in further processing steps. This modification can be accomplished by exposing the antigenic peptide to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases.
  • Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine).
  • antigenic peptides that have been modified to increase their resistance to proteolysis or to optimize their solubility properties.
  • polynucleotide encoding an antigenic peptide may include a polynucleotide encoding an antigenic peptide of the present invention, or a polynucleotide that also includes additional coding and/or non-coding sequences; nucleotides include ribonucleic acid (RNA, Ribonucleic Acid), and deoxyribonucleic acid (DNA, Deoxyribonucleic Acid).
  • RNA Ribonucleic Acid
  • DNA Deoxyribonucleic Acid
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or antigenic peptides having the same amino acid sequence as the present invention.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but which does not substantially alter the mutated protein it encodes. Function.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that are hybridizable under stringent conditions (or stringent conditions) to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%.
  • antigenic peptides and polynucleotides of the present invention are preferably provided in isolated form, more preferably, purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercial cDNA libraries or cDNAs prepared by conventional methods known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the polynucleotides of the present invention.
  • the RACE method RACE-cDNA Rapid Amplification of cDNA Ends
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods.
  • Amplified DNA/RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • epitopope peptides of the present invention refer to vaccine polypeptides conforming to the second aspect of the present invention.
  • vaccine polypeptides also include other forms, such as pharmaceutically acceptable salts, conjugates, or fusion proteins.
  • the vaccine polypeptide comprises one or more (such as 1-5, preferably 1-3) amino acid additions, one or more (such as 1-5, preferably 1-3) amino acid substitutions and/or 1-3 amino acid deletions to form a derivative polypeptide, the derivative polypeptide has substantially the same function as the original polypeptide before derivatization.
  • the vaccine polypeptide comprises 1-3 amino acid additions (preferably at the N-terminus or C-terminus), and/or 1-2 amino acid substitutions ( Conservative amino acid substitutions are preferred) and still have substantially the same function as the original polypeptide before derivatization.
  • the conservative amino acid substitutions are amino acid substitutions according to Table II.
  • isolated refers to the separation of a substance from its original environment (in the case of a natural substance, the original environment is the natural environment).
  • the polypeptide in the natural state in living cells is not isolated and purified, but the same polypeptide is isolated and purified if it is separated from other substances existing in the natural state.
  • an "isolated peptide” refers to a polypeptide of the invention that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated.
  • One skilled in the art can purify the polypeptides of the invention using standard protein purification techniques. Substantially purified polypeptides (fusion proteins) produce a single major band on a non-reducing polyacrylamide gel.
  • polypeptides of the present invention may be recombinant polypeptides, or synthetic polypeptides, preferably synthetic polypeptides.
  • the sequence of the vaccine polypeptide is relatively short (eg ⁇ 70aa, more preferably ⁇ 60aa)
  • the related peptide sequence can be directly synthesized by chemical methods.
  • the sequence of the vaccine polypeptide is longer or the vaccine polypeptide is provided in the form of a fusion protein
  • recombinant methods can also be used to obtain the relevant peptide sequences in large quantities.
  • the coding sequence encoding the antigenic polypeptide or its fusion protein is cloned into a vector, and then transferred into cells, and then the relevant antigenic peptide or fusion protein is isolated from the propagated host cells by conventional methods.
  • the present invention also provides an mRNA vaccine for preventing novel coronavirus.
  • the method of the present invention includes: first obtaining the expressed gene (or coding sequence) of the antigenic protein by PCR, and then obtaining the corresponding mRNA by in vitro transcription technology, thereby obtaining an efficient mRNA vaccine.
  • mRNA vaccine is a kind of mRNA with expression activity prepared in vitro, and its main structure includes 5' and 3' UTR and an open reading frame containing the expressed antigen. In contrast to DNA vaccines, it does not require any nuclear localization signal and has no risk of integration into the genome.
  • the mRNA vaccine of the present invention is the mRNA encoding the antigenic peptide of the first aspect of the present invention.
  • the present invention also provides a vector comprising the coding sequence of the antigenic peptide of the present invention, and a host cell containing the vector.
  • the vector has an expression cassette for expressing the antigenic peptide gene, and the expression cassette has the following elements in order from 5' to 3': a promoter, an antigenic peptide gene, and a terminator.
  • a preferred synthesis method is the asymmetric PCR method.
  • Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods.
  • Amplified DNA/RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the polynucleotide sequence of the present invention can express or produce the target protein (antigenic peptide) by conventional recombinant DNA technology, including steps:
  • expression vectors preferably commercially available vectors such as pPink ⁇ HC or pMT/BiP/V5-HisA, containing DNA sequences encoding the proteins of the invention and suitable transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
  • a vector comprising the above DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to express the protein of interest.
  • the host cells capable of expressing the antigenic peptides of the present invention can be prokaryotic cells, such as E. coli; or lower eukaryotic cells, such as yeast cells (Pichia pastoris, Saccharomyces cerevisiae); or higher eukaryotic cells, such as insect cells; preferably for yeast cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Engineered cells can be of a fast methanol-utilizing form (Mut + ) or a slow methanol-using form (Mut s ).
  • the engineered cells can be cultured under suitable conditions to express the proteins encoded by the gene sequences of the present invention.
  • the medium used in the culture can be selected from various conventional medium, and the culture is carried out under conditions suitable for the growth of the host cell.
  • the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • DO dissolved oxygen
  • the types of feeding should preferably include carbon sources such as glycerol, methanol, and glucose, which can be fed individually or in combination.
  • the engineered cells expressing the target protein can be purified by chromatographic techniques.
  • Chromatography techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography and other techniques. Commonly used chromatography methods include:
  • Anion exchange chromatography media include (but are not limited to): Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermentation sample is high, which affects the binding to the ion exchange medium, the salt concentration needs to be reduced before ion exchange chromatography.
  • the sample can be exchanged for the equilibration buffer by means of dilution, ultrafiltration, dialysis, gel filtration chromatography, etc., until it is similar to the corresponding ion exchange column equilibration solution system, and then the sample is loaded for gradient elution of salt concentration or pH.
  • Hydrophobic chromatography media include, but are not limited to: Phenyl-Sepharose, Butyl-Sepharose, Octyle-Sepharose.
  • the sample is increased in salt concentration by adding NaCl, (NH 4 ) 2 SO 4 , etc., and then loaded, and eluted by reducing the salt concentration. Impurity proteins with large differences in hydrophobicity were removed by hydrophobic chromatography.
  • Hydrophobic chromatography media include, but are not limited to: Sephacryl, Superdex, Sephadex classes.
  • the buffer system is exchanged by gel filtration chromatography, or further purified.
  • Affinity chromatography media include, but are not limited to: HiTrap TM Heparin HP Columns.
  • the antigenic peptide (polypeptide) of the present invention may be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptides of the present invention may be chemically synthesized, or recombinant.
  • the polypeptides of the present invention can be artificially synthesized by conventional methods or produced by recombinant methods.
  • a preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques such as Boc solid phase method, Fmoc solid phase method or a combination of both methods.
  • Solid-phase synthesis can quickly obtain samples, and an appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide.
  • the preferred solid phase carrier in the Fmoc system is Wang resin linked to the C-terminal amino acid in the peptide, the Wang resin structure is polystyrene, and the arm between the amino acid is 4-alkoxybenzyl alcohol; with 25% hexahydropyridine /Dimethylformamide was treated at room temperature for 20 minutes to remove the Fmoc protecting group and extended from the C-terminus to the N-terminus one by one according to the given amino acid sequence. After the synthesis, the synthesized proinsulin-related peptide was cleaved from the resin with trifluoroacetic acid containing 4% p-cresol and the protective group was removed.
  • the desired peptide was purified by gel filtration and reversed-phase high pressure liquid chromatography.
  • the preferred resin is PAM resin linked with the C-terminal amino acid in the peptide, the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethyl phenylacetamide; synthesized in Boc
  • Boc Boc
  • the protecting group Boc was removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane. The condensation of the peptide chain was completed.
  • the peptide chain was cleaved from the resin with hydrogen fluoride (HF) containing p-cresol (5-10%) for 1 hour at 0 °C, and the protective group was removed at the same time.
  • HF hydrogen fluoride
  • a small amount of mercaptoethanol) to extract the peptide the solution is lyophilized and further separated and purified by molecular sieve Sephadex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide.
  • Various couplings known in the field of peptide chemistry can be used Reagents and coupling methods to couple the amino acid residues, for example dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea hexafluorophosphate can be used (HBTU) for direct coupling.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • HBTU 1,1,3,3-tetraurea hexafluorophosphate
  • the antigenic peptide of the present invention is prepared by solid-phase synthesis according to its sequence, and purified by high performance liquid chromatography to obtain a high-purity lyophilized powder of the target peptide, which is stored at -20°C.
  • Another approach is to use recombinant techniques to produce the polypeptides of the invention.
  • the antigenic peptides of the present invention can be expressed or produced using the polynucleotides of the present invention by conventional recombinant DNA techniques. Generally there are the following steps:
  • polynucleotide (or variant) of the antigenic peptide of the present invention or use the recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the recombinant polypeptide can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the polypeptide of the present invention is short, it can be considered to connect multiple polypeptides in series, and after recombinant expression, the expression product in the form of a multimer can be obtained, and then the desired small peptide can be formed by methods such as enzymatic cleavage.
  • the present invention also provides a method for preparing the vaccine composition, specifically, comprising the steps:
  • the antigenic peptide prepared by the present invention is mixed with a pharmaceutically acceptable vaccine adjuvant to form a vaccine composition.
  • the adjuvant is aluminum adjuvant, GLA adjuvant, preferably GLA adjuvant.
  • the present invention also provides a composition
  • a composition comprising: (i) the recombinant antigenic peptide or vaccine polypeptide prepared by the method of the present invention, and (ii) a pharmaceutically or immunologically acceptable excipient or adjuvant agent.
  • the term “comprising” means that various components can be applied or present together in the composition of the present invention.
  • the terms “consisting essentially of” and “consisting of” are encompassed by the term “comprising”.
  • compositions of the present invention include pharmaceutical compositions and vaccine compositions.
  • the compositions of the present invention may be monovalent or polyvalent.
  • the pharmaceutical composition or vaccine composition of the present invention can be prepared into various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.
  • the pharmaceutical composition of the present invention includes an effective amount of the antigenic peptide or vaccine polypeptide prepared by the method of the present invention, and the antigenic peptide or vaccine polypeptide may be monovalent or multivalent.
  • the term "effective amount" refers to an amount of a therapeutic agent that treats, ameliorates, or prevents a target disease or condition, or an amount that exhibits a measurable therapeutic or prophylactic effect. This effect can be detected, for example, by antigen levels. The therapeutic effect also includes a reduction in physical symptoms.
  • the precise effective amount for a subject depends on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is useless to prespecify the exact effective amount. However, for a given situation, routine experimentation can be used to determine this effective amount.
  • an effective dose is about 0.2 micrograms/kg to 2 micrograms/kg administered to an individual.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent (eg, an antigenic peptide or other therapeutic agent).
  • a therapeutic agent eg, an antigenic peptide or other therapeutic agent.
  • the term refers to pharmaceutical carriers that do not themselves induce the production of antibodies detrimental to the individual receiving the composition, and are not undue toxicity upon administration.
  • Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, and the like. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • compositions can include liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the compositions can be prepared as injectables, such as liquid solutions or suspensions; solid forms suitable for solution or suspension, liquid vehicles prior to injection can also be prepared. Liposomes are also included in the definition of pharmaceutically acceptable carrier.
  • the vaccine compositions of the present invention may be prophylactic (ie, preventing infection) or therapeutic.
  • the vaccine compositions described comprise immunizing antigens (including the proteins of the invention or self-assembling virus-like particles), and are usually combined with "pharmaceutically acceptable carriers" that do not by themselves induce the production of Any carrier of antibodies that are detrimental to the individual.
  • Suitable carriers are usually large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (eg, oil droplets or liposomes), and the like. These vectors are well known to those of ordinary skill in the art. Additionally, these carriers can act as immunostimulatory agents ("adjuvants").
  • the antigen may be conjugated to bacterial toxoids (eg, toxoids of pathogens such as diphtheria, tetanus, cholera, Helicobacter pylori, etc.).
  • Preferred adjuvants for enhancing the effect of the immune composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO90/14837), (b) SAF, and (c) Ribi TM Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT), (3) Saponin Adjuvant; (4) Freund Complete Adjuvant ( CFA) and Freund's incomplete adjuvant (IFA); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.
  • alum aluminum salts
  • oil-in-water emulsion formulations for example, (a) MF59 (see WO90/14837), (b) SAF, and (c) Ribi TM Adjuvant System (RA
  • interferon such as gamma interferon
  • macrophage colony stimulating factor M-CFS
  • tumor necrosis factor TNF
  • bacterial ADP-ribosylation toxins such as cholera toxin CT, pertussis toxin PT) or E. coli heat labile toxin LT
  • WO93/13302 and WO92/19265 other substances that act as immunostimulants to enhance the effect of the composition.
  • Vaccine compositions including immunogenic compositions (for example, which may include antigens, pharmaceutically acceptable carriers, and adjuvants), typically contain diluents such as water, saline, glycerol, ethanol, and the like. Additionally, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like can be present in such vehicles.
  • vaccines comprising an immunologically effective amount of an immunogenic polypeptide, as well as other desired components as described above.
  • An "immunologically effective amount” refers to an amount administered to a subject in a single dose or as part of a continuous dose that is effective for treatment or prophylaxis. The amount may depend on the health and physiology of the individual being treated, the type of individual being treated (eg, human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating physician's assessment of the medical condition, and other relevant factors. This amount is expected to be in a relatively wide range and can be determined by routine experimentation.
  • vaccine compositions or immunogenic compositions can be prepared as injectables, such as liquid solutions or suspensions; solid forms suitable for solution or suspension, liquid vehicles prior to injection can also be prepared.
  • the formulation can also be emulsified or encapsulated in liposomes to enhance adjuvant effects.
  • the composition can be administered directly to a subject.
  • the subject can be a human or a non-human mammal, preferably a human.
  • the virus-like particles of the present invention can be administered directly to an individual by known methods. These vaccines are typically administered using the same route of administration as conventional vaccines and/or mimicking pathogen infection routes.
  • Routes of administration of the pharmaceutical or vaccine compositions of the present invention include, but are not limited to, intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, intravaginal, oral, or other parenteral routes of administration. Routes of administration can be combined, if desired, or adjusted according to disease conditions.
  • Vaccine compositions may be administered in single or multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.
  • VLP vaccines should be administered in an "effective amount", that is, the amount of VLPs in the selected route of administration is sufficient to elicit an immune response and can effectively promote the protection of the host against novel coronavirus infection.
  • each dose of vaccine is sufficient to contain about 1 ⁇ g to 1000 ⁇ g, preferably 1 ⁇ g to 100 ⁇ g, more preferably 10 ⁇ g to 50 ⁇ g of protein or VLP after infection of host cells.
  • the optimal dosage for a particular vaccine can be determined using standard research methods involving observation of antibody titers and other responses in subjects.
  • the need for a booster dose can be determined by monitoring the level of immunity provided by the vaccine. After assessment of antibody titers in serum, optional booster immunization may be required.
  • the administration of adjuvants and/or immunostimulants can increase the immune response to the proteins of the invention.
  • the preferred method is to administer the immunogenic composition by injection from the parenteral (subcutaneous or intramuscular) route.
  • the present invention finds for the first time that after the arginine mutation at position 815 of SEQ ID NO.: 1 of the amino acid sequence of the new coronavirus S protein, the S protein can be effectively prevented from producing a cell fusion effect, and the site mutation of R815 also It can effectively prevent various S protein mutant strains from exerting the fusion effect, thereby effectively preventing the infection of the new coronavirus.
  • the present invention finds for the first time that the protein containing the site mutation of R815 can also effectively treat the coronavirus SARS-CoV-2 infection or its related diseases.
  • the present invention finds for the first time that the amino acid site mutation of the Spike protein can effectively inhibit the cell fusion effect induced by the novel coronavirus, thereby preventing virus infection.
  • the present invention finds for the first time that the genetically modified Spike protein has low toxicity to cells, thereby contributing to the development of safe vaccines.
  • the present invention finds for the first time that the spike protein site (that is, the R815 site) of the present invention can also become a broad-spectrum small molecule drug target, so as to screen out the special drug for coronavirus in the later stage. Therefore, the discovered specific drugs can provide prospective knowledge reserves and clinical application value for the prevention and control of similar coronavirus infections in the future.
  • the present invention finds for the first time that the replacement of the amino acid at the single R815 site can completely lead to the loss of the virus fusion function, thereby effectively blocking future infections caused by the new coronavirus mutant.
  • the present invention finds for the first time that the S protein produced by mutation can maximize the retention of the structure and shape of the original virus, activate the host immune response, and at the same time avoid tissue side effects or body damage caused by the fusion effect of the S protein.
  • the present invention finds for the first time that the mutant S protein (ie, the antigenic peptide) of the present invention can prepare a safer viral vector and mRNA vaccine without side effects.
  • Human HEK293T cells were transfected with SARS-CoV-2 to express wild-type or mutant S protein for 24 hours, and then mixed with HEK293T cells stably expressing or not expressing ACE2 receptors in cell culture dishes for 16 hours. The state of confluent cells was captured by a 20X light microscope. After fusion, the cells were lysed and boiled with 1x Laemmli protein loading solution, and subjected to SDS-PAGE running, and mouse monoclonal antibodies against SARS-CoV-2 S2 rabbit polyclonal antibody, HA- and Myc-tagged proteins were used. Anti-Western blotting. co-immunoprecipitation
  • Human HEK293T cells were simultaneously transfected with Lipofectamine 2000 to express HA-wild-type or mutant S protein and Myc-tagged wild-type S protein for 24 hours, and the cell lysis buffer was collected with 0.5% NP-40 cell lysis buffer. sample.
  • the cell solution was prepared by adding HA-tagged mouse mAb overnight and using magnetic beads to precipitate the S protein. Co-immunoprecipitated samples were washed three times with lysate before Western blotting.
  • Example 1 Cell fusion effect of new crown S protein and host ACE2 cells
  • the Spike glycoprotein (S) on the surface of SARS-CoV-2 is the only exposed protein on the outer membrane of the virus that can recognize host receptors and play a binding role.
  • the S protein has the function of recognizing the host receptor until it undergoes structural changes to fuse the virus into the host cell. After binding to the receptor, S triggers the instability of the trimer, and then the S1 and S2 subunits are generated after enzymatic cleavage at the 685 amino acid site in the middle of S, resulting in the shedding of the S1 subunit, and at the same time assisting the S2 subunit to form post-fusion stability structure, and promote the membrane fusion reaction between the new coronavirus and the host after binding to the cell.
  • the S gene of SARS-CoV-2 is 93.1% similar to the S gene of bat RaTG13 coronavirus, and is less than 80% identical to that of SARS-CoV in previous years; therefore, how to pass the S gene locus? It is of great significance to explore and develop broad-spectrum small molecule inhibitors to explore the mechanism of cell fusion of coronaviruses.
  • the present invention firstly studies the fusion function of the Spike spike protein through a cell-cell fusion experiment.
  • HEK293T cells expressing S protein alone no changes in cell morphology can be clearly observed under the microscope (Fig. 1A); after the addition of HEK293T cells expressing human ACE2 receptor, the two cells present a multicellular aggregation and fusion state (Fig. 1B). .
  • S2' Fig. 1C
  • the S2' fragment is 816-1273aa of the S protein (Fig. 1D); and positioned this band to be cleaved by the host cell membrane protease, which is an amino acid P0
  • the cleavage site is conserved at the same S2' position as other coronaviruses.
  • the expression of full-length S protein after mutation was not different from that of wild-type S protein (Fig.
  • Example 3 The R815 site effectively prevents the fusion effect of the S protein of other mutant strains
  • D614G is a new variant of the new coronavirus S protein mutation (S-D614G), which occurs in the S1 domain and causes the S protein to enhance the ability to infect.
  • S-D614G new coronavirus S protein mutation
  • the present invention also tested the function-enhancing mutation of the C-terminal 19 amino acid (S-CT ⁇ 19) truncation of the S protein, and also tested R815N and R815A.
  • S-CT ⁇ 19 cells had an extremely strong cell fusion effect with ACE2 cells, but consistent with the above figure, both R815N and R815A site mutations prevented S-CT ⁇ 19 cells from fusion (Fig. 3B).
  • the existing popular mutants contain multi-site complex mutations in the S protein.
  • the present invention produced retroviral vector particles (Pseudotype particles, PPs) containing wild type and single amino acid site mutations of R815N and R815A.
  • Viral vector particles that do not package the S protein cannot infect HEK293T cells stably expressing ACE2; while the viral vector particles that assemble the wild-type S protein efficiently infect cells and express the green fluorescent GFP protein tag that can be photographed by fluorescence microscopy ( Figure 4A). , Figure 4B). Consistent with the cell fusion results, the viral vector particles assembled with the R815N and R815A mutant S proteins failed to successfully infect ACE2 cells (Fig. 4A, Fig. 4B). These data suggest that the R815 site mutation is particularly important for the fusion infection effect of the SARS-CoV-2 S protein.
  • Example 5 The fusion effect of the mutant S protein at the R815 site directly interferes with the wild-type S protein
  • the new crown wild-type S protein needs to form a homotrimer to exert its infection and fusion effect on the virus or cell surface. If the mutant S protein expressed in cells can co-assemble with the wild-type S protein, it can interfere with the membrane fusion function of the S protein (Fig. 5A).
  • HEK293T cells we expressed HA-tagged wild-type or R815A mutant S protein and wild-type Myc-tagged S protein in HEK293T cells, respectively. Within 24 hours, the lysate was collected and checked to see if the mutant S protein in the cells could assemble with the wild type S protein.
  • the R815 site of the S protein of SARS-CoV-2 is currently the amino acid site found in the present invention, which has the most significant inhibition of cell fusion and infection. Since it has no cell fusion effect, the mutant S protein can prepare a safer viral vector and mRNA vaccine without side effects.

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Abstract

本发明提供了基于S蛋白R815位点的新型冠状病毒干预方法和产品，具体地，本发明提供了抗原肽和新型冠状病毒的疫苗多肽，本发明还提供含有所述抗原肽或疫苗多肽的疫苗组合物及其应用。实验表明，本发明的抗原肽或疫苗多肽可有效防止S蛋白产生细胞融合和感染效应，从而有效防止新型冠状病毒的感染。

Description

基于S蛋白R815位点的冠状病毒干预的方法和产品 技术领域
本发明属于生物医药领域，具体地说，本发明涉及基于S蛋白R815位点的冠状病毒干预的方法和产品。
背景技术
新型冠状病毒(severe acute respiratory syndrome coronavirus-2,SARS-CoV-2)属于冠状病毒β属的一成员，是造成COVID19的病原。自2019年12月起，COVID19疫情已蔓延至各个大洲超过80个国家和地区，超过1亿人口确诊，并造成全球超过200万人死亡；除此之外还导致国际社会运行瘫痪，以及前所未有的经济财产损失。受感染人群不光肺部功能受阻，同时产生的系统性炎症也造成多种并发症在高危人群中发病。
然而目前市面上没有任何一款直接针对病毒或宿主病症反应的特效药，因此新的药物开发、运用与普及都迫在眉睫。
因此，本领域迫切需要开发一种能有效抑制新型冠状病毒所诱导的细胞融合效果，从而防止病毒感染的抗新型冠状病毒疫苗。
发明内容
本发明的目的在于提供一种能有效抑制新型冠状病毒所诱导的细胞融合效果，从而防止病毒感染的抗新型冠状病毒疫苗。
本发明的另一目的在于，发现新冠S蛋白的最重要氨基酸功能位点；单点突变最大化保留原有S蛋白结构；本发明包含对突变株有效的功能位点；有效避免、减少野生型新冠S蛋白促使的细胞融合和副作用；一种可针对野生型S蛋白进行干预的方案。
本发明的第一方面，提供了一种抗原肽，所述抗原肽衍生自新型冠状病毒S蛋白，并且所述抗原肽在对应于新型冠状病毒S蛋白氨基酸序列的SEQ ID NO.:1的第815位的精氨酸发生突变。
在另一优选例中，所述突变包括氨基酸的插入、缺失或替换。
在另一优选例中，所述第815位的精氨酸突变为任何氨基酸。
在另一优选例中，所述第815位的精氨酸突变为选自下组的一种或多种氨基酸：丙氨酸(A)、天冬酰胺(N)、赖氨酸(K)、天冬氨酸(D)和谷氨酸(E)。
在另一优选例中，所述第815位的精氨酸突变为选自下组的一种或多种氨基酸：丙氨酸(A)、天冬酰胺(N)。
在另一优选例中，所述的突变选自下组：R815A、R815N、或其组合。
在另一优选例中，所述新型冠状病毒S蛋白包括新冠病毒突变株(比如英国B.1.1.7(Alpha)、南非B.1.351(Beta)、印度B.1.617.2(Delta)或南非B.1.1.529(Omicron))的S蛋白。
在另一优选例中，所述抗原肽的氨基酸序列如SEQ ID NO.3－5中任一所示。
在另一优选例中，所述的抗原肽为具有SEQ ID NO.3－5中任一所示氨基酸序列的多肽、其活性片段、或其保守性变异多肽。
在另一优选例中，所述的抗原肽除所述突变(如815位)外，其余的氨基酸序列与SEQ ID NO.:1所示的序列相同或基本相同。
在另一优选例中，所述抗原肽除所述突变(如815位)外，其余的氨基酸序列包括C端19氨基酸截短(ΔCT19)型、C端TM截短(ΔTM)型、以及第815位的精氨酸后的截短型S蛋白。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-20个，更佳地为1-10个、更佳地1-5个)氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加，且所述的抗原肽具有抑制新型冠状病毒S蛋白的细胞融合和感染效应的活性。
在另一优选例中，所述抗原肽与SEQ ID NO.:1所示序列的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，最佳地至少为98％或99％。
在另一优选例中，所述的抗原肽与新型冠状病毒的S蛋白竞争性结合人ACE2受体(包括其发挥作用所必须的已知或未知的辅助受体)。
在另一优选例中，所述的“竞争性结合”指所述的抗原肽与新型冠状病毒的S蛋白结合于相同或基本相同的人ACE2蛋白的结合域(或氨基酸区段)。
在另一优选例中，所述的抗原肽与新型冠状病毒的S蛋白结合于人ACE2蛋白上的相同的结合区段。
在另一优选例中，所述的竞争性结合包括阻断性或非阻断性的竞争性结合。
在另一优选例中，所述的抗原肽为人工合成的或重组的抗原肽。
在另一优选例中，所述抗原肽为酵母细胞表达的重组蛋白。
在另一优选例中，所述抗原肽为昆虫细胞表达的重组蛋白。
在另一优选例中，所述昆虫细胞选自下组：Sf9、Sf21、Tni、Hi5-Sf细胞、或其组合。
在另一优选例中，所述酵母包括毕赤酵母。
在另一优选例中，所述的抗原肽选自下组：
(a)具有SEQ ID NO.:3－5中任一所示的多肽；
(b)对(a)中多肽的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生多肽，所述衍生多肽与衍生前的原始多肽具有基本相同的功能。
在另一优选例中，所述的“基本相同的功能”指所述的衍生多肽具有抑制新型冠状病毒S蛋白的细胞融合和感染效应的活性。
本发明第二方面提供了一种疫苗多肽，所述疫苗多肽包括本发明第一方面所述的抗原肽。
在另一优选例中，所述的疫苗多肽可激发灵长动物和啮齿动物产生阻断RBD与ACE2结合，且抑制新型冠状病毒S蛋白的细胞融合和感染效应的中和抗体。
在另一优选例中，所述的疫苗多肽可激发灵长动物产生细胞免疫和体液免疫。
在另一优选例中，所述的灵长动物包括人、非人灵长类动物。
本发明第三方面提供了一种mRNA疫苗，所述的疫苗含有用于表达本发明第一方面所述抗原肽的编码mRNA、以及DNA表达载体。
在另一优选例中，所述的mRNA疫苗的包装载体为含有Toll样受体结合剂的鱼精蛋白、纳米颗粒人工膜、化学合成多聚体、以及脂质体。
本发明第四方面提供了一种分离的多核苷酸，所述的多核苷酸编码本发明第一方面所述的抗原肽或本发明第二方面所述的疫苗多肽。
本发明第五方面提供了一种表达载体，所述表达载体含有本发明第四方面所述的多核苷酸。
本发明第六方面提供了一种宿主细胞，所述的宿主细胞含有本发明第五方面所述的表达载体，或者在基因组中整合有本发明第四方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞和真核细胞。
在另一优选例中，所述的宿主细胞包括酵母、果蝇S2细胞、昆虫Hi5-Sf 细胞、大肠杆菌、猴来源Vero E6细胞、仓鼠CHO细胞、DC细胞、或其组合。
本发明第七方面提供了一种丧失新型冠状病毒S蛋白细胞融合效应的抗原病毒株，所述病毒株的基因组中的对应于新型冠状病毒S蛋白氨基酸序列的SEQ ID NO.:1的第815位的精氨酸发生突变。
在另一优选例中，所述突变包括氨基酸的插入、缺失或替换。
在另一优选例中，所述病毒株的基因组中的基于SEQ ID NO.:2的编码对应于SEQ ID NO.:1的第815位精氨酸的核苷酸发生突变。
在另一优选例中，所述核苷酸发生突变指：核苷酸突变为编码精氨酸以外的任何氨基酸的核苷酸。
在另一优选例中，所述核苷酸发生突变指：所述核苷酸突变为编码选自下组的一种或多种氨基酸的核苷酸：丙氨酸(A)、天冬酰胺(N)、赖氨酸(K)、天冬氨酸(D)和谷氨酸(E)。
在另一优选例中，所述核苷酸发生突变指：所述核苷酸突变为编码选自下组的一种或多种氨基酸的核苷酸：丙氨酸(A)、天冬酰胺(N)。
本发明第八方面提供了一种药物组合物，所述的组合物含有本发明第一方面所述的抗原肽、本发明第二方面所述的疫苗多肽或本发明第三方面所述的mRNA疫苗或本发明第四方面所述的多核苷酸或者本发明第五方面所述的表达载体或者本发明第六方面所述的宿主细胞或本发明第七方面所述的病毒株，以及药学上可接受的载体和/或辅料。
在另一优选例中，所述的药物组合物为疫苗组合物。
在另一优选例中，所述疫苗组合物为单价或多价。
在另一优选例中，所述的药物组合物还含有佐剂，首选各种铝佐剂。
在另一优选例中，所述的药物为针对新型冠状病毒S蛋白氨基酸序列的SEQ ID NO.:1的第815位精氨酸的抑制剂。
在另一优选例中，所述药物组合物中的抗原肽、免疫多肽、mRNA疫苗或病毒株、和佐剂(如铝)的摩尔数或重量比在1:100之间，优选为1:40到1:60之间。
在另一优选例中，所述的药物组合物包括单方药物、复方药物、或协同药物。
在另一优选例中，所述的药物组合物的剂型为液态、固体、或凝胶态。
在另一优选例中，所述的药物组合物用选自下组的方式施用：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、口服、或口鼻腔喷入和雾化吸入。
本发明第九方面提供了一种疫苗组合物，所述的组合物含有本发明第一方面所述的抗原肽、本发明第二方面所述的疫苗多肽或本发明第三方面所述的mRNA疫苗或本发明第四方面所述的多核苷酸或者本发明第五方面所述的表达载体或者本发明第六方面所述的宿主细胞或本发明第七方面所述的病毒株，以及免疫学上可接受的载体和/或辅料。
在另一优选例中，所述的疫苗组合物还含有佐剂。
在另一优选例中，所述佐剂包括：颗粒型和非颗粒型佐剂。
在另一优选例中，所述颗粒型佐剂选自下组：铝盐、油包水乳剂、水包油乳剂、纳米颗粒、微小颗粒、脂质体、免疫刺激复合物，或其组合。
另一优选例中，所述非颗粒型佐剂选自下组：胞壁酰二肽及其衍生物、皂苷、脂质A、细胞因子、衍生多糖、细菌毒素，微生物及其产物如分枝杆菌(结核杆菌、卡介苗)、短小杆菌、百日咳杆菌、蜂胶、或其组合。
在另一优选例中，所述的佐剂包括氧化铝、皂苷、quil A、胞壁酰二肽、矿物油或植物油、基于囊泡的佐剂、非离子嵌段共聚物或DEAE葡聚糖、细胞因子(包括IL-1、IL-2、IFN-r、GM-CSF、IL-6、IL-12、和CpG)。
在另一优选例中，所述的疫苗组合物包括注射剂型。
本发明第十方面提供了如本发明第一方面所述的抗原肽或本发明第二方面所述的疫苗多肽或本发明第三方面所述的mRNA疫苗或本发明第七方面所述的病毒株或本发明第八方面所述的药物组合物或本发明第九方面所述的疫苗组合物的用途，(a)用于制备针对新型冠状病毒的抗体；和/或(b)用于制备预防和/或治疗冠状病毒感染或其相关疾病的药物。
在另一优选例中，所述抗体包括针对新型冠状病毒S蛋白第815位精氨酸位点突变的封闭型抗体。
在另一优选例中，所述的冠状病毒感染或其相关疾病选自下组：呼吸道感染、肺炎及其并发症、中东呼吸综合症、人类呼吸系统疾病新冠肺炎、猪肠胃疾病及其并发症、或其组合。
在另一优选例中，所述的冠状病毒相关疾病包括新型冠状病毒肺炎(COVID-19)。
在另一优选例中，所述冠状病毒选自下组：SARS-CoV-2、SARS-CoV、MERS-CoV(中东呼吸综合征)、SADS-CoV(猪急性腹泻综合征)、或其组合。
在另一优选例中，所述治疗包括用基因治疗的方法进行的治疗。
本发明第十一方面提供了一种制备本发明第一方面所述的抗原肽的方法，包括步骤：
(i)在适宜条件下培养本发明第六方面所述的宿主细胞，从而表达本发明第一方面所述的抗原肽；
(ii)纯化所述抗原肽。
在另一优选例中，所述方法步骤(i)中将转化的酵母单菌落分别接种到BMGY培养基中，培养后离心去除上清，用BMMY培养基重悬菌体，28－30℃(优选为29.5℃)，诱导培养36－48小时(优选为48小时)。
本发明第十二方面提供了一种产生针对冠状病毒SARS-CoV-2的免疫反应的方法，其特征在于，包括步骤：给需要的对象施用本发明第一方面所述的抗原肽、本发明第二方面所述的疫苗多肽、本发明第三方面所述的mRNA疫苗或本发明第七方面所述的病毒株或本发明第八方面所述的药物组合物或本发明第九方面所述的疫苗组合物。
在另一优选例中，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述的非人哺乳动物包括非人灵长动物(如猴)。
在另一优选例中，所述方法在所述对象中诱导产生针对冠状病毒SARS-CoV-2的中和抗体。
在另一优选例中，所述中和抗体能抑制新型冠状病毒S蛋白的细胞融合和感染效应。
本发明第十三方面提供了一种抑制新型冠状病毒S蛋白的细胞融合效应的方法，包括步骤：在本发明第一方面所述的抗原肽、本发明第二方面所述的疫苗多肽、本发明第三方面所述的mRNA疫苗或本发明第七方面所述的病毒株或本发明第八方面所述的药物组合物或本发明第九方面所述的疫苗组合物存在下，培养表达ACE2蛋白的细胞，从而抑制新型冠状病毒S蛋白的细胞融合和感染效应。
在另一优选例中，所述细胞为体外培养的细胞。
在另一优选例中，所述方法为体外的方法。
在另一优选例中，所述细胞选自下组：肺部上皮细胞、肠上皮细胞、肝与肾上皮细胞、或其组合。
在另一优选例中，所述细胞选自下组：HEK293T-ACE2、Vero E6-ACE2、Caco2、Calu3细胞、或其组合。
本发明第十四方面提供了一种治疗方法，给需要的对象施用本发明第一方 面所述的抗原肽、本发明第二方面所述的疫苗多肽、本发明第三方面所述mRNA疫苗、本发明第四方面所述的多核苷酸或者本发明第五方面所述的表达载体或者本发明第六方面所述的宿主细胞或本发明第七方面所述的病毒株或本发明第八方面所述的药物组合物或本发明第九方面所述的疫苗组合物。
在另一优选例中，所述治疗方法包括基因治疗方法。
在另一优选例中，所示治疗方法包括体外使用电穿孔技术转染的人类DC细胞移植，淋巴mRNA疫苗注射。
本发明第十五方面提供了一种特异性结合剂，所述特异性结合剂特异性识别或结合本发明第一方面所述的抗原肽。
在另一优选例中，所述特异性结合剂特异性识别所述抗原肽中包含第815位的精氨酸突变位点(R815)的突变位点。
在另一优选例中，所述特异性结合剂特异性识别所述抗原肽中第815位的精氨酸突变位点(R815)。
在另一优选例中，所述特异性结合剂识别序列性表位。
在另一优选例中，所述特异性结合剂识别构象性表位。
在另一优选例中，所述特异性结合剂选自下组：多肽、抗体、小分子化合物、或其组合。
在另一优选例中，所述抗体包括单克隆抗体或多克隆抗体。
在另一优选例中，所述抗体包括抗体片段，例如骆驼类VHH纳米抗体。
本发明第十六方面提供了一种筛选预防和/或治疗冠状病毒SARS-CoV-2感染或其相关疾病的候选化合物的方法，包括：
(a)将权利要求1所述的抗原肽与化合物库混合，用模拟及筛选方法测定化合物库中的化合物与本发明第一方面所述的抗原肽的结合情况；其中所述模拟及筛选方法包括化学结构信息学、系统细胞生物学、分子蛋白组学；
其中，如果所述测试化合物库中的化合物与本发明第一方面所述的抗原肽有结合，则表明所述与本发明第一方面所述的抗原肽结合的化合物为所述的候选化合物。
在另一优选例中，所述结合包括所述测试化合物库中的化合物与所述的抗原肽中第815位的精氨酸突变位点(R815)的结合。
在另一优选例中，所述方法包括步骤(b)：将步骤(a)中所确定的候选化合物 施用于体外表达ACE2的细胞，测定其对细胞融合和新冠病毒感染的效果。
在另一优选例中，所述方法包括步骤(b)：将步骤(a)中所确定的候选化合物施用于哺乳动物模型，测定其对哺乳动物的影响。
在另一优选例中，所述哺乳动物为患有冠状病毒SARS-CoV-2感染或其相关疾病的哺乳动物。
在另一优选例中，所述的方法是非诊断和非治疗性的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了新冠S蛋白与宿主ACE2细胞的细胞融合效应。通过光学显微镜拍摄到表达SARS-CoV-2 S蛋白的HEK293T单细胞(A)；或与ACE2细胞融合后的细胞复合体(B)。通过蛋白质印迹法，我们发现S蛋白诱导的细胞融合后产生S2’蛋白条带(C)。(D)4种已知冠状病毒S蛋白氨基酸位点的序列比对及SARS-CoV-2 S蛋白S2’位置及切割位点的结构图。标尺距离代表20μm。
图2显示了R815位点突变S蛋白的融合效应缺失。蛋白质印迹法展示了野生型(WT)S、R815N及R815A突变型S蛋白大小(A)；后三排加入ACE2细胞的情况下，野生型S蛋白产生了S2’条带，而R815N和R815A突变均抑制了S2’的产生(A)。光学显微镜照片拍摄到的野生型(WT)S与ACE2细胞融合效应，并且能够完全被R815N和R815A突变抑制(B)。标尺距离代表20μm。
图3显示了R815位点有效防止其它突变株S蛋白的融合效应。光学显微镜照片拍摄到的天然D614G S蛋白突变株融合效应，能够完全受R815N及R815A突变抑制(A)；人工制作的C端19氨基酸(S-CTΔ19)截短的功能增强突变也能够完全被R815N和R815A突变抑制(B)。(C)全球大流行的新冠病毒Alpha，Beta以及Delta突变株均能与表达ACE2的细胞发生融合(C，上)，然而R815A的单点突变能够让不同的新冠病毒突变株刺突蛋白功能丧失(C，下)。标尺距离代表20μm。
图4显示了R815位点突变有效防止假病毒感染ACE2细胞。荧光显微镜图 片(A)、及蛋白质印迹法展示的绿色荧光体(B)，只在受野生型(WT)S蛋白逆转录病毒感染的ACE2-HEK293T细胞中表达；R815N和R815A突变型逆转录病毒颗粒均不具有感染力。标尺距离代表20μm。
图5显示了R815位点突变减少野生型S蛋白发挥细胞融合功能。因为S蛋白在表达过程中形成三聚体，突变型S蛋白的单体能够组装入新冠S蛋白的三聚体，并干扰其膜融合功能(A)。HA-蛋白标签免疫共沉淀后，蛋白质印迹法显示野生型与R815A突变型，均能与含有Myc标签的野生型S蛋白结合(B)；蛋白质印迹法显示共同表达的R815A突变型S蛋白，能有效减少野生型S蛋白与ACE2细胞的细胞融合现象和S2’的产生(C)。
具体实施方式
本发明人通过广泛而深入的研究，意外地发现，新型冠状病毒S蛋白氨基酸序列的SEQ ID NO.:1的第815位的精氨酸突变后，可有效防止S蛋白产生细胞融合和感染效应，并且R815的位点突变还可有效阻止各种S蛋白突变株发挥融合效应，从而有效防止新型冠状病毒的感染，此外本发明人还首次发现，含有R815的位点突变的蛋白还可有效治疗冠状病毒SARS-CoV-2感染或其相关疾病。在此基础上，本发明人完成了本发明。
术语
如本文所用，术语“AxxB”表示第xx位的氨基酸A变为氨基酸B，例如“L87I”表示第87位的氨基酸L突变为I，以此类推。
冠状病毒SARS-CoV-2
冠状病毒(Coronavirus，CoV)属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)，是一种有包膜的正链RNA病毒，其亚科包含α、β、δ及γ四属。
目前已知的感染人的冠状病毒中，HCoV-229E和HCoV-NL63属于α属冠状病毒，HCoV-OC43、SARS-CoV、HCoV-HKU1、MERS-CoV和SARS-CoV-2均为β属冠状病毒。
2019年年底爆发的新型冠状病毒(SARS-CoV-2)与SARS-CoV有约80％相似性、与MERS-CoV有40％的相似性，也属于β属冠状病毒。
该类病毒的基因组是一条单股正链RNA，是基因组最大的RNA病毒之一，编码 包括复制酶、刺突蛋白、囊膜蛋白、包膜蛋白和核壳蛋白等。在病毒复制的初始阶段，基因组被翻译成两条长达几千个氨基酸的肽链即前体多聚蛋白(Polyprotein)，随后前体蛋白被蛋白酶切割生成非结构蛋白(如RNA聚合酶和解旋酶)和结构蛋白(如刺突蛋白)及辅助蛋白。
S蛋白是冠状病毒SARS-CoV-2的一种主要的结构蛋白，其中，RBD负责与人ACE2受体结合。
>全长Spike蛋白的氨基酸序列(S蛋白，YP_009724390.1)
Figure PCTCN2022070763-appb-000001
编码全长Spike蛋白(S蛋白，YP_009724390.1)的核苷酸序列如SEQ ID NO.:2所示：
Figure PCTCN2022070763-appb-000002
Figure PCTCN2022070763-appb-000003
Figure PCTCN2022070763-appb-000004
应理解，在本发明中，S蛋白包括野生型和突变型。
本发明主要目标是研发出一种能诱导机体产生靶向冠状病毒SARS-CoV-2的中和抗体的疫苗，用于防止新型突变株新冠病毒的融合效应，从而预防新型冠状病毒的感染，并可有效阻断因新冠病毒突变株导致的感染。
此外，本发明的另一目标是研发出能够有效治疗冠状病毒SARS-CoV-2感染或其相关疾病的含有S蛋白突变体的药物。
抗原肽
本发明提供了一种衍生自新型冠状病毒S蛋白的抗原肽，并且所述抗原肽在对应于新型冠状病毒S蛋白氨基酸序列的SEQ ID NO.:1的第815位的精氨 酸发生突变。
在一优选实施方式中，所述第815位的精氨酸突变为选自下组的一种或多种氨基酸：丙氨酸(A)、天冬酰胺(N)、赖氨酸(K)、天冬氨酸(D)和谷氨酸(E)。
应理解，本发明抗原肽中的氨基酸编号基于SEQ ID NO.:1作出，当某一具体抗原肽与SEQ ID NO.:1所示序列的同源性达到80％或以上时，抗原肽的氨基酸编号可能会有相对于SEQ ID NO.:1)的氨基酸编号的错位，如向氨基酸的N末端或C末端错位1-5位，而采用本领域常规的序列比对技术，本领域技术人员通常可以理解这样的错位是在合理范围内的，且不应当由于氨基酸编号的错位而使同源性达80％(如90％、95％、98％)的、具有相同或相似的具有抑制新型冠状病毒S蛋白的细胞融合和感染效应的活性的抗原肽不在本发明抗原肽的范围内。
本发明抗原肽是合成蛋白或重组蛋白，即可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物)中产生。根据重组生产方案所用的宿主，本发明的突变蛋白可以是糖基化的，或可以是非糖基化的。本发明的突变蛋白还可包括或不包括起始的甲硫氨酸残基。
本发明还包括所述抗原肽的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持所述抗原肽相同的生物学功能或活性的蛋白。
本发明的抗原肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的突变蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的抗原肽，或(iii)成熟突变蛋白与另一个化合物(比如延长突变蛋白半衰期的化合物，例如聚乙二醇)融合所形成的抗原肽，或(iv)附加的氨基酸序列融合到此抗原肽序列而形成的抗原肽(如前导序列或分泌序列或用来纯化此抗原肽的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。本发明中，保守性替换的氨基酸最好根据表I进行氨基酸替换而产生。
表I
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明的活性抗原肽具有基本相同的激发免疫反应的免疫原性，与新型冠状病毒的S蛋白具有竞争性结合人ACE2蛋白的活性，且具有完全抑制新型冠状病毒S蛋白的细胞融合和感染效应的活性。
优选地，所述抗原肽如SEQ ID NO.3－5中任一所示。
R815A突变型全长S蛋白序列(SEQ ID NO.3)：
Figure PCTCN2022070763-appb-000005
Figure PCTCN2022070763-appb-000006
R815N突变型全长S蛋白序列(SEQ ID NO.:4)
Figure PCTCN2022070763-appb-000007
R815K突变型全长S蛋白序列(SEQ ID NO.5)
Figure PCTCN2022070763-appb-000008
应理解，本发明突变蛋白与SEQ ID NO.:3－5中任一所示的序列相比，通常具有较高的同源性(相同性)，优选地，所述的突变蛋白与SEQ ID NO.:3－5中任一所示序列的同源性至少为80％，较佳地至少为85％-90％，更佳地至少为95％，最佳地至少为98％，最佳地，≥99％。
此外，还可以对本发明的抗原肽进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的抗原肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在抗原肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的抗原肽。这种修饰可以通过将抗原肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的抗原肽。
术语“编码抗原肽的多核苷酸”可以是包括编码本发明抗原肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸；核苷酸包括核糖核酸(RNA，Ribonucleic Acid)，和脱氧核糖核酸(DNA，Deoxyribonucleic Acid)。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或抗原肽的片段、类似物和衍生物。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的突变蛋白的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的抗原肽和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可 用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
疫苗多肽
在本发明中，“本发明表位肽”、“本发明疫苗多肽”、“本发明多肽”可互换使用，指符合本发明第二方面中所述的疫苗多肽。
在本发明中，疫苗多肽还包括其他形式，例如药学上可接受的盐、偶联物、或融合蛋白。
在本发明中，疫苗多肽包括对SEQ ID No:3－5中任一所示的序列进行一个或多个(如1-5个，优选地1-3个)氨基酸添加、一个或多个(如1-5个，优选地1-3个)氨基酸的取代和/或1-3个氨基酸缺失所形成的衍生多肽，所述衍生多肽与衍生前的原始多肽具有基本相同的功能。
优选地，疫苗多肽包括对SEQ ID No:3－5中任一所示的序列经过1-3个氨基酸添加(优选添加在N端或C端)、和/或1-2个氨基酸的取代(优选保守性氨基酸替换)并仍具有与衍生前的原始多肽具有基本相同的功能。
优选地，所述的保守性氨基酸替换根据表II进行氨基酸替换。
表II
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多肽是没有分离纯化的，但同样的多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的肽”是指本发明多肽基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化本发明多肽。基本上纯化的多肽(融合蛋白)在非还原聚丙烯酰胺凝胶上能产生单一的主带。
本发明的多肽可以是重组多肽、或合成多肽，优选合成多肽。
在本发明中，当疫苗多肽的序列较短(如≤70aa，更佳地≤60aa时)，可用化学方法直接合成相关肽序列。
当疫苗多肽的序列较长或以融合蛋白形式提供疫苗多肽时，也可以用重组法来大批量地获得相关肽序列。这通常是将编码所述抗原多肽或其融合蛋白的编码序列克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到相关的抗原肽或融合蛋白。
mRNA疫苗
本发明还提供了用于预防新型冠状病毒的mRNA疫苗。
典型地，本发明方法包括：首先通过PCR方法获得抗原蛋白的表达基因(或编码序列)，之后利用体外转录技术获得相应的mRNA，从而获得高效的mRNA疫苗。
mRNA疫苗是一种体外制备的具有表达活性的mRNA，其主要结构包括5'和3'UTR以及含有表达抗原的开放读码框。相比于DNA疫苗，它不需要任何核定位信号，并且无整合到基因组上的风险。
在本发明的mRNA疫苗中，是编码本发明第一方面所述抗原肽的mRNA。
载体和宿主细胞
本发明还提供了一种包含本发明的抗原肽编码序列的载体，以及含所述载体的宿主细胞。
在本发明的一个优选例中，所述载体具有表达所述抗原肽基因的表达盒，所述表达盒从5’-3’依次具有下述元件：启动子，抗原肽基因，和终止子。
本领域的普通技术人员可以使用的常规方法获得所述抗原肽的上述优化基因序列，例如全人工合成或PCR法合成。一种优选的合成法为不对称PCR法。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明的多核苷酸序列可以通过常规的重组DNA技术，表达或生产目的蛋白(抗原肽)，包括步骤：
(1)用编码本发明蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞，较佳地为酵母或果蝇S2细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体，优选市售的载体如pPinkαHC或pMT/BiP/V5-HisA。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。此外，表达载体优选包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状。
包含上述DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，表达目的蛋白。能够表达本发明抗原肽的宿主细胞可以是原核细胞，如大肠杆菌；或是低等真核细胞，如酵母细胞(毕赤酵母、酿酒酵母)；或是高等真核细胞，如昆虫细胞；优选为酵母细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。工程细胞可以是快速利用甲醇型(Mut +)或慢速利用甲醇型(Mut s)。
工程细胞的培养和目的蛋白发酵生产
在获得工程细胞后，便可在适合的条件下培养工程细胞，表达本发明的基因序列所编码的蛋白。根据宿主细胞的不同，培养中所用的培养基可选自各种常规培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将 细胞再培养一段时间。
在本发明中，可采用常规的发酵条件。代表性的条件包括(但并不限于)：
(a)就温度而言，本发明的抗原肽的发酵及诱导温度保持在28-30℃；
(b)就诱导期的pH值而言，诱导期pH控制在3-9；
(c)就溶氧(DO)而言，DO控制在20-90％，溶氧的维持可以用氧气/空气混合气体的通入来解决；
(d)就补料而言，补料种类宜包括甘油、甲醇、葡萄糖等碳源，可单独补料或混合补料。
工程细胞表达目的蛋白可以采用层析技术进行纯化。层析技术包括阳离子交换层析、阴离子交换层析、凝胶过滤层析、疏水层析、亲和层析等技术。常用的层析方法包括：
1.阴离子交换层析
阴离子交换层析介质包括(但不限于)：Q-Sepharose、DEAE-Sepharose。如果发酵样品的盐浓度较高，影响与离子交换介质的结合，则在进行离子交换层析前需降低盐浓度。样品可以用稀释、超滤、透析、凝胶过滤层析等手段进行平衡缓冲液的更换，直至与对应的离子交换柱平衡液系统相似，然后上样，进行盐浓度或pH的梯度洗脱。
2.疏水层析
疏水层析介质包括(但不限于)：Phenyl-Sepharose、Butyl-Sepharose、Octyle-Sepharose。样品通过添加NaCl、(NH 4) 2SO 4等方式提高盐浓度，然后上样，通过降低盐浓度方法洗脱。通过疏水层析除去疏水性有较大差异的杂蛋白。
3.凝胶过滤层析
疏水层析介质包括(但不限于)：Sephacryl、Superdex、Sephadex类。通过凝胶过滤层析更换缓冲体系，或进一步精纯。
4.亲和层析
亲和层析介质包括(但不限于)：HiTrap TM Heparin HP Columns。
制备方法
本发明的抗原肽(多肽)可以是重组多肽或合成多肽。本发明的多肽可以是化学合成的，或重组的。相应地，本发明多肽可用常规方法人工合成，也可用重组方法生产。
一种优选的方法是使用液相合成技术或固相合成技术，如Boc固相法、Fmoc固相法或是两种方法联合使用。固相合成可快速获得样品，可根据目的肽的序列特征选用适当的树脂载体及合成系统。例如，Fmoc系统中优选的固相载体如连接有肽中C端氨基酸的Wang树脂，Wang树脂结构为聚苯乙烯，与氨基酸间的手臂是4-烷氧基苄醇；用25％六氢吡啶/二甲基甲酰胺室温处理20分钟，以除去Fmoc保护基团，并按照给定的氨基酸序列由C端逐个向N端延伸。合成完成后，用含4％对甲基苯酚的三氟乙酸将合成的胰岛素原相关肽从树脂上切割下来并除去保护基，可过滤除树脂后乙醚沉淀分离得到粗肽。将所得产物的溶液冻干后，用凝胶过滤和反相高压液相层析法纯化所需的肽。当使用Boc系统进行固相合成时，优选树脂为连接有肽中C端氨基酸的PAM树脂，PAM树脂结构为聚苯乙烯，与氨基酸间的手臂是4-羟甲基苯乙酰胺；在Boc合成系统中，在去保护、中和、偶联的循环中，用TFA/二氯甲烷(DCM)除去保护基团Boc并用二异丙基乙胺(DIEA/二氯甲烷中和。肽链缩合完成后，用含对甲苯酚(5-10％)的氟化氢(HF),在0℃下处理1小时，将肽链从树脂上切下，同时除去保护基团。以50-80％乙酸(含少量巯基乙醇)抽提肽，溶液冻干后进一步用分子筛Sephadex G10或Tsk-40f分离纯化，然后再经高压液相纯化得到所需的肽。可以使用肽化学领域内已知的各种偶联剂和偶联方法偶联各氨基酸残基，例如可使用二环己基碳二亚胺(DCC)，羟基苯骈三氮唑(HOBt)或1,1,3,3-四脲六氟磷酸酯(HBTU)进行直接偶联。对于合成得到的短肽，其纯度与结构可用反相高效液相和质谱分析进行确证。
在一实施方式中，本发明的抗原肽，按其序列，采用固相合成的方法制备，行高效液相色谱纯化，获得高纯度目的肽冻干粉，-20℃贮存。
另一种方法是用重组技术产生本发明多肽。通过常规的重组DNA技术，可利用本发明的多核苷酸来表达或生产本发明的抗原肽。一般来说有以下步骤：
(1).用本发明的抗原肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于： 常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
由于本发明多肽较短，因此可以考虑将多个多肽串联在一起，重组表达后获得多聚体形式的表达产物，然后通过酶切等方法形成所需的小肽。
制备疫苗组合物
本发明还提供了一种制备疫苗组合物的方法，具体地，包括步骤：
将本发明制备的抗原肽与药学上可接受的疫苗佐剂混合，从而形成疫苗组合物。
在另一优选例中，所述的佐剂为铝佐剂、GLA佐剂，较佳的GLA佐剂。
组合物和施用方法
本发明还提供了一种组合物，所述组合物含有：(i)用本发明方法制备的重组抗原肽或疫苗多肽，以及(ii)药学上或免疫学上可接受的赋形剂或佐剂。本发明中，术语“含有”表示各种成分可一起应用于或存在于本发明的组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
本发明的组合物包括药物组合物和疫苗组合物。本发明的组合物可以是单价的，也可以是多价的。
本发明的药物组合物或疫苗组合物可制备成各种常规剂型，其中包括(但并不限于)：注射剂、粒剂、片剂、丸剂、栓剂、胶囊、悬浮液、喷雾剂等。
(i)药物组合物
本发明的药物组合物包括有效量的用本发明方法制备的抗原肽或疫苗多肽，所述抗原肽或疫苗多肽可以是单价的，也可以是多价的。
本文所用的术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。该效果可通过例如抗原水平来检测。治疗效果也包括生理性症状的减少。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的状况而言，可以用常规实验来确定该有效量。
为了本发明的目的，有效的剂量为给予个体约0.2微克/千克至2微克/千 克。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂(例如抗原肽或其它治疗剂)给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。合适的载体可以是大的、代谢缓慢的大分子，如蛋白质、多糖、聚乳酸(polylactic acid)、聚乙醇酸等。这些载体是本领域普通技术人员所熟知的。在Remington’s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的载体或赋形剂的充分讨论。
组合物中药学上可接受的载体可包括液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。通常，可将组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液、液体赋形剂的的固体形式。脂质体也包括在药学上可接受的载体的定义中。
(ii)疫苗组合物
本发明的疫苗组合物可以是预防性的(即预防感染)，也可以是治疗性的。所述的疫苗组合物包含免疫性抗原(包括本发明蛋白或自组装的病毒样颗粒)，并且通常与“药学上可接受的载体”组合，这些载体包括本身不诱导产生对接受该组合物的个体有害的抗体的任何载体。合适的载体通常是大的、代谢缓慢的大分子，如蛋白质、多糖、聚乳酸、聚乙醇酸、氨基酸聚合物、氨基酸共聚物、脂质凝集物(如油滴或脂质体)等。这些载体是本领域普通技术人员所熟知的。另外，这些载体可起免疫刺激剂(“佐剂”)作用。另外，抗原也可以和细菌类毒素(如白喉、破伤风、霍乱、幽门螺杆菌等病原体的类毒素)偶联。
增强免疫组合物效果的优选佐剂包括但不限于：(1)铝盐(alum)，如氢氧化铝、磷酸铝、硫酸铝等；(2)水包油型乳剂配方，例如，(a)MF59(参见WO90/14837)，(b)SAF，和(c)Ribi TM佐剂系统(RAS)(Ribi Immunochem，Hamilton，MT)，(3)皂素佐剂；(4)Freund完全佐剂(CFA)和Freund不完全佐剂(IFA)；(5)细胞因子，如白介素(如IL-1、IL-2、IL-4、IL-5、IL-6、IL-7、IL-12等)、干扰素(如γ干扰素)、巨噬细胞集落刺激因子(M-CFS)、肿瘤坏死因子(TNF)等；(6)细菌ADP-核糖基化毒素(如霍乱毒素CT，百日咳毒素PT或大肠杆菌热不稳定毒素LT)的脱毒变异体，参见例如WO93/13302和WO92/19265；以及(7)作为免疫刺激剂来增强组合物效果的其它物质。
包括免疫原性组合物在内的疫苗组合物(例如，可包括抗原、药学上可接受的载体以及佐剂)，通常含有稀释剂，如水，盐水，甘油，乙醇等。另外，辅助性物质，如润湿剂或乳化剂、pH缓冲物质等可存在于这类运载体中。
更具体地，包括免疫原性组合物在内的疫苗，包含免疫学有效量的免疫原性多肽，以及上述其它所需的组分。“免疫学有效量”指以单剂或连续剂一部分给予个体的量对治疗或预防是有效的。该用量可根据所治疗个体的健康状况和生理状况、所治疗个体的类别(如人)、个体免疫系统合成抗体的能力、所需的保护程度、疫苗的配制、治疗医师对医疗状况的评估、及其它的相关因素而定。预计该用量将在相对较宽的范围内，可通过常规实验来确定。
通常，可将疫苗组合物或免疫原性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液、液体赋形剂的固体形式。该制剂还可乳化或包封在脂质体中，以增强佐剂效果。
(iii)给药途径和剂量
所述组合物可以直接给予对象。对象可以是人或非人哺乳动物，较佳地为人。当用作疫苗时，可用已知的方法将本发明的病毒样颗粒直接施用于个体。通常采用与常规疫苗相同的施用途径和/或模拟病原体感染路径施用这些疫苗。
给予本发明药物组合物或疫苗组合物的途径包括(但并不限于)：肌内、皮下、皮内、肺内、静脉内、经鼻、阴道内、经口服或其它肠胃外给药途径。如果需要，可以组合给药途径，或根据疾病情况进行调节。疫苗组合物可以单剂量或多剂量给予，且可以包括给予加强剂量以引发和/或维持免疫力。
应以“有效量”给予病毒样颗粒疫苗，即病毒样颗粒的量在所选用的给药路径中足以引发免疫应答，能有效促使保护宿主抵抗新型冠状病毒感染。
在各疫苗剂份中所选用的病毒样颗粒的量，是按可引发免疫保护性应答而无明显的副作用的量而定。通常，在感染宿主细胞后，各剂的疫苗足以含有约1μg-1000μg，较佳地为1μg-100μg，更佳地10μg-50μg蛋白质或VLP。可用包括观察对象中的抗体滴定度和其它反应的标准研究方法来确定具体疫苗的最佳用量。可通过监控疫苗提供的免疫力水平来确定是否需要增强剂量。在评估了血清中的抗体滴定度后，可能需要选用增强剂量免疫接种。施用佐剂和/或免疫刺激剂就可提高对本发明的蛋白质的免疫应答。优选方法是从肠胃外(皮下或肌内)途径通过注射给予免疫原性组合物。
本发明的主要优点在于：
(1)本发明首次发现，新型冠状病毒S蛋白氨基酸序列的SEQ ID NO.:1的第815位的精氨酸突变后，可有效防止S蛋白产生细胞融合效应，并且R815的位点突变还可有效阻止各种S蛋白突变株发挥融合效应，从而有效防止新型冠状病毒的感染。
(2)本发明首次发现，含有R815的位点突变的蛋白还可有效治疗冠状病毒SARS-CoV-2感染或其相关疾病。
(3)本发明首次发现，Spike蛋白氨基酸位点突变，能有效抑制新型冠状病毒所诱导的细胞融合效果，从而防止病毒感染。
(4)本发明首次发现，通过基因修改后的Spike蛋白对细胞具有低毒性，从而有助于安全疫苗的开发。
(5)本发明首次发现，本发明的该突刺蛋白位点(即，R815位点)还能成为广谱性小分子药物靶点，从而筛选出后期针对冠状病毒的特效药。因此，所发现的特效药物可为将来类似冠状病毒感染的防控提供前瞻性知识储备和临床应用价值。
(6)本发明首次发现，单R815位点氨基酸的替换可以完全导致病毒融合功能丧失，从而有效阻断今后因新冠病毒突变株导致的感染。
(7)本发明首次发现，通过突变产生的S蛋白可以最大化地保留原有病毒的结构与形态，激活宿主免疫反应；同时避免S蛋白融合效应产生的组织副作用或机体损伤。
(8)本发明首次发现，本发明的突变S蛋白(即抗原肽)能制备无副作用的，更安全的病毒载体及mRNA疫苗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆实验指南(New York:Cold Spring Harbor Laboratory Press,1989)；或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
通用方法
突变型S制备
[根据细则9.2改正15.02.2022]　
通过SARS-CoV-2(GenBank:QHD43419.1)毒株克隆出的人类优化S基因序列，并加入可通过CMV启动子在人类细胞内表达的S蛋白载体质粒。通过自定义引物、KOD plus酶高保真性聚合酶和DpnI内切酶对S蛋白位点进行定点突变。突变S基因通过Lipofectamine 2000转入HEK293T细胞表达24小时，然后使用冰磷酸缓冲盐溶液冲脱HEK293T细胞，然后进行细胞融合实验。
细胞融合
人类HEK293T细胞通过转染SARS-CoV-2表达野生型或突变型S蛋白24小时后，在细胞培养盘中与稳定表达或不表达ACE2受体的HEK293T细胞进行混合培养16小时。融合细胞状态通过20X光学显微镜捕捉，融合后细胞通过1x Laemmli蛋白上样液裂解煮样，并进行SDS-PAGE跑胶，并且使用针对新冠S2兔多抗、HA-和Myc-标签蛋白的鼠单抗进行蛋白印迹。免疫共沉淀
人类HEK293T细胞通过Lipofectamine 2000，同时转染表达HA-野生型或突变型S蛋白和Myc-标签型野生型S蛋白24小时后，用含有0.5％的NP-40细胞裂解缓冲液，收细胞裂解液样本。细胞溶液通过加入HA-标签鼠单抗过夜，并使用磁珠沉淀S蛋白。免疫共沉淀样本通过裂解液冲洗三次后，进行进行蛋白印迹法。
实施例1 新冠S蛋白与宿主ACE2细胞的细胞融合效应
SARS-CoV-2表面的突刺蛋白(Spike glycoprotein，简称S)是病毒外膜唯一暴露的、能识别宿主受体和发挥结合作用的蛋白。S蛋白具有识别宿主受体，直到经历结构变化将病毒融合进入宿主细胞的功能。结合受体后的S引发三聚体发生不稳定性，然后在S中间685氨基酸位点酶切割后产生S1和S2亚基，从而造成S1亚基脱落，同时辅助S2亚基形成融合后的稳定结构，并促进与细胞结合后的新冠病毒与宿主发生膜融合反应。值得一提的是，SARS-CoV-2的S基因与蝙蝠RaTG13冠状病毒S基因具有93.1％的相似度，与往年的SARS-CoV只有不到80％的吻合度；因此，如何通过S基因位点，探索冠状病毒产生细胞融合的机制，对探索及研发广谱小分子抑制剂具有重要意义。
因为SARS-CoV-2 S蛋白识别的是人类血管紧张素转化酶2(ACE2)受体， 首先本发明通过一种细胞细胞融合实验研究了Spike突刺蛋白的融合功能。单独表达S蛋白的HEK293T细胞，显微镜下能清楚地观察到细胞形态不发生任何变化(图1A)；加入表达人类ACE2受体的HEK293T细胞后，两种细胞呈现多细胞聚合融合状态(图1B)。通过收集这些融合后的细胞，我们使用蛋白质印迹法，发现了只有通过细胞融合产生出来的一种大约在68kDa分子量的蛋白条带，命名为S2’(图1C)。S2’的产生只在加入ACE2细胞后产生，并且与ACE2加入的量及其相关。因此我们推测S2’的产生对细胞融合是必要的。
实施例2 R815位点突变S蛋白的融合效应缺失
通过上述片段长度和多种冠状病毒S蛋白的排列对比，我们初步分析出S2’片段为S蛋白的816-1273aa(图1D)；并且定位这个条带由宿主细胞膜蛋白酶切割，为一个氨基酸的P0切割位点，且保守与其它冠状病毒相同S2’的位置。通过设计自定义引物和制作定点突变，我们将处于S2’蛋白的精氨酸815(R815)分别突变成了天冬酰胺(R815N)和丙氨酸(R815A)。突变后的全长S蛋白表达与野生型S蛋白并无差距(图2A)；更重要的是，在加入表达ACE2的HEK293T细胞后，两种R815位点的突变都未像野生型S蛋白产生S2’蛋白条带(图2A)。这说明S蛋白的定点突变，成功防止了S2’精氨酸位置受宿主蛋白酶识别，并且避免了蛋白切割反应。我们继续通过显微镜观察了R815N和R815A突变型S细胞与宿主ACE2细胞的融合效应，发现只有野生型能继续与ACE2细胞发生融合反应，而不是上述两种突变型S蛋白(图2B)。因此R815的位点突变能造成S蛋白细胞融合效应缺失。
实施例3 R815位点有效防止其它突变株S蛋白的融合效应
因R815N和R815A位点突变能造成野生型S蛋白的功能缺失，本发明继续分析了R815位点突变能否防止新型突变株新冠病毒的融合效应。D614G是一种新型变种新冠S蛋白突变(S-D614G)，发生在S1结构域上，且造成S蛋白感染能力增强。我们首先制作了D614G型S蛋白，并在其基础上分别插入了R815N和R815A。与野生型相同，R815N和R815A点突变都能有效防止S蛋白产生细胞融合效应(图3A)。除此之外，本发明也测试了S蛋白的C端19氨基酸(S-CTΔ19)截短的功能增强突变，并也测试了R815N和R815A。S-CTΔ19细胞与ACE2细胞发生了极其强烈的细胞融合效应，但与上图一致的是，R815N和R815A位 点突变都阻止了S-CTΔ19细胞发生融合(图3B)。除此之外，现有流行突变株在S蛋白上含有多位点复合型突变。我们针对英国Alpha,南非Beta和印度Delta突变株S蛋白保守的S2’切割位点进行了突变，并且发现原精氨酸R815位点对这些突变株诱导的细胞融合都至关重要(图3C)。因此，R815的位点突变能够有效阻止多种S蛋白突变株发挥融合效应。
实施了4 R815位点突变有效防止假病毒感染ACE2细胞
为了更加强调S蛋白位点突变对病毒颗粒的作用，本发明制作了含有野生型、及带有R815N和R815A单氨基酸位点突变的逆转录病毒载体颗粒(Pseudotype particles，PPs)。不包装S蛋白的病毒载体颗粒无法感染稳定表达ACE2的HEK293T细胞；而组装了野生型S蛋白的病毒载体颗粒有效感染细胞，并且表达可通过荧光显微镜拍摄到的绿色荧光体GFP蛋白标签(图4A、图4B)。与细胞融合结果一致，组装R815N与R815A突变S蛋白的病毒载体颗粒均未能成功感染ACE2细胞(图4A、图4B)。这些数据说明R815位点突变对SARS-CoV-2 S蛋白的融合感染效应尤其重要。
实施例5 R815位点突变S蛋白直接干预野生型S蛋白的融合效应
新冠野生型S蛋白需要形成同源三聚体，从而发挥其在病毒或细胞表面的感染与融合效应。如果细胞内表达的突变S蛋白能够与野生型S蛋白共同组装，既能干扰S蛋白发挥膜融合功能(图5A)。使用HEK293T细胞，我们分别将带有HA-标签的野生型或R815A突变型的S蛋白、与野生型Myc-标签的S蛋白表达在HEK293T细胞内。24小时内，收样裂解液并且查看细胞中的突变型S蛋白是否能与野生型S蛋白组装。通过使用针对HA-标签的免疫共沉淀，我们用HA鼠单克隆抗体拉出了同样多带有Myc-标签的S蛋白(图5B)。这说明野生型和突变型S蛋白均能组装在其它的野生型S蛋白上，并形成同源三聚体。我们接下来给这些共表达S蛋白的细胞加入了表达ACE2细胞的HEK293T细胞，并且共培养16小时。通过蛋白印迹我们发现，共同表达突变型与野生型S蛋白的细胞产生了明显减少的S2’(图5C)。因此这些实验数据证明，R815A突变型S蛋白能干扰野生型S蛋白发生融合功能，从而在已经表达S蛋白的细胞中具有治疗作用。如上所述，SARS-CoV-2的S蛋白R815位点是目前是本发明发现的，对细胞融合及感染抑制最显著的氨基酸位点。因不具有细胞融合效应， 突变S蛋白能制备无副作用的，更安全的病毒载体及mRNA疫苗。这些基础现象和特征均为目前临床上使用的疫苗提供了新型理论基础和参考。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种抗原肽，其特征在于，所述抗原肽衍生自新型冠状病毒S蛋白，并且所述抗原肽在对应于新型冠状病毒S蛋白氨基酸序列的SEQ ID NO.:1的第815位的精氨酸发生突变。
  2. 一种疫苗多肽，其特征在于，所述疫苗多肽包括权利要求1所述的抗原肽。
  3. 一种mRNA疫苗，其特征在于，所述的疫苗含有用于表达权利要求1所述抗原肽的编码mRNA、以及DNA表达载体。
  4. 一种分离的多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的抗原肽或权利要求2所述的疫苗多肽。
  5. 一种表达载体，其特征在于，所述表达载体含有权利要求4所述的多核苷酸。
  6. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求5所述的表达载体，或者在基因组中整合有权利要求4所述的多核苷酸。
  7. 一种丧失新型冠状病毒S蛋白细胞融合效应的抗原病毒株，其特征在于，所述病毒株的基因组中的对应于新型冠状病毒S蛋白氨基酸序列的SEQ ID NO.:1的第815位的精氨酸发生突变。
  8. 一种药物组合物，其特征在于，所述的组合物含有权利要求1所述的抗原肽、权利要求2所述的疫苗多肽或权利要求3所述的mRNA疫苗或权利要求4所述的多核苷酸或者权利要求5所述的表达载体或者权利要求6所述的宿主细胞或权利要求7所述的病毒株，以及药学上可接受的载体和/或辅料。
  9. 一种疫苗组合物，其特征在于，所述的组合物含有权利要求1所述的抗原肽、权利要求2所述的疫苗多肽或权利要求3所述的mRNA疫苗或权利要求4所述的多核苷酸或者权利要求5所述的表达载体或者权利要求6所述的宿主细胞或权利要求7所述的病毒株，以及免疫学上可接受的载体和/或辅料。
  10. 如权利要求1所述的抗原肽或权利要求2所述的疫苗多肽或权利要求3所述的mRNA疫苗或权利要求7所述的病毒株或权利要求8所述的药物组合物或权利要求9所述的疫苗组合物的用途，其特征在于，(a)用于制备针对新型冠状病毒的抗体；和/或(b)用于制备预防和/或治疗冠状病毒感染或其相关疾病的药物。
PCT/CN2022/070763 2021-02-26 2022-01-07 基于s蛋白r815位点的冠状病毒干预的方法和产品 WO2022179318A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111848753A (zh) * 2020-07-20 2020-10-30 中国科学院过程工程研究所 新型冠状病毒抗原表位及其应用
US20200407402A1 (en) * 2020-06-29 2020-12-31 The Scripps Research Institute Stabilized Coronavirus Spike (S) Protein Immunogens and Related Vaccines
WO2021022008A1 (en) * 2019-07-30 2021-02-04 Verndari, Inc. Virus-like particle vaccines
US20210338804A1 (en) * 2020-02-21 2021-11-04 International Aids Vaccine Initiative Inc. Vaccine Compositions For Preventing Coronavirus Disease

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021022008A1 (en) * 2019-07-30 2021-02-04 Verndari, Inc. Virus-like particle vaccines
US20210338804A1 (en) * 2020-02-21 2021-11-04 International Aids Vaccine Initiative Inc. Vaccine Compositions For Preventing Coronavirus Disease
US20200407402A1 (en) * 2020-06-29 2020-12-31 The Scripps Research Institute Stabilized Coronavirus Spike (S) Protein Immunogens and Related Vaccines
CN111848753A (zh) * 2020-07-20 2020-10-30 中国科学院过程工程研究所 新型冠状病毒抗原表位及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HOFFMANN MARKUS ET AL: "SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.", CELL, US, vol. 181, no. 2, 16 April 2020 (2020-04-16), US , pages 271 - 280.e8, XP002804571, ISSN: 1097-4172 *

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