CN116549627A - Broad-spectrum new crown vaccine based on adenovirus vector and application thereof - Google Patents

Broad-spectrum new crown vaccine based on adenovirus vector and application thereof Download PDF

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CN116549627A
CN116549627A CN202210106932.9A CN202210106932A CN116549627A CN 116549627 A CN116549627 A CN 116549627A CN 202210106932 A CN202210106932 A CN 202210106932A CN 116549627 A CN116549627 A CN 116549627A
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周东明
邢嫚
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Suzhou Xiangyi Biotechnology Co ltd
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Abstract

The invention provides a broad-spectrum novel crown vaccine based on an adenovirus vector and application thereof. The recombinant adenovirus expresses the Spike protein, T cell epitope string and Receptor Binding Motif (RBM) of Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). The recombinant adenovirus of the invention can activate organism to generate strong immune response against SARS-COV-2 as vaccine, and the generated antibody has strong neutralization effect on various new coronavirus mutant strains and broad spectrum; and can improve the virus removing capacity of the organism.

Description

Broad-spectrum new crown vaccine based on adenovirus vector and application thereof
Technical Field
The invention belongs to the fields of biomedicine and virology, and in particular relates to a broad-spectrum novel coronal vaccine based on an adenovirus vector and application thereof.
Background
In recent years, the new coronaviruses have resulted in a large number of cases of viral pneumonia. The sequence of the novel coronavirus was first published in month 1 2020 and was subsequently named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the pneumonia caused by this virus infection was named "COVID-19" (Corona Virus Disease 2019). People of all ages of the virus are susceptible, and no effective therapeutic medicine exists. At present, the control of the virus is mainly dependent on physical isolation and vaccination.
In the art, currently, the development of vaccines is mainly divided into 5 technical routes, respectively: inactivated vaccines, nucleic acid vaccines, recombinant protein vaccines, adenovirus vector vaccines, and attenuated influenza virus vector vaccines. Adenovirus has high transduction efficiency, can carry exogenous genes, is easy to amplify and purify, can excite effective T cell immune response aiming at exogenous genes, is regarded as an excellent vaccine carrier, and has been widely applied to research of vaccines for various infectious diseases, including HIV, influenza, ebola, malaria, rabies and the like. A current generation of new coronal vaccines based on adenovirus vectors, which have been approved for urgent use, are: adY25 adenovirus vector vaccine of AdNetherlands, adHu26 adenovirus vector vaccine of Duchesnea, adHu5 adenovirus vector vaccine of China Kang Xinuo, adHu26 and AdHu5 combined adenovirus vector vaccine of Russia.
These already developed adenovirus vaccines demonstrate the safety and effectiveness of new coronavirus vaccines based on adenovirus vectors. However, as viruses continue to spread, more and more viral mutants, such as those listed as relevant by the World Health Organization (WHO), appear: b.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617 (Delta), listed as variants of interest (VOI): c.37 (Lambda) and the like. Some mutants produced a different degree of immune evasion than the original strain. For example, beta, gamma, delta, lambda mutant can significantly escape the neutralizing antibody response induced by individuals who had previously naturally infected the original strain, while Alpha mutant has less effect. More current vaccines are also developed based on the original strain design of the virus, and part of mutant strains have obvious immune escape, so that the vaccine effect is influenced, and the public worry is caused. Therefore, there is an urgent need to develop a broad spectrum of novel crown vaccines.
Disclosure of Invention
The invention aims to provide a broad-spectrum novel coronal vaccine based on an adenovirus vector and application thereof.
In a first aspect of the invention, there is provided a method of preparing a vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising:
(1) Introducing the following elements into an adenovirus vector to obtain a recombinant adenovirus vector:
(1) a coding gene of mutant Spike protein or a coding gene of mutant Spike protein-T cell epitope string; wherein the T cell epitope string is derived from the ORF1, ORF3 and M proteins of the virus;
(2) a gene encoding a Receptor Binding Motif (RBM) or a mutant receptor binding motif;
(2) And (3) packaging the virus based on the recombinant adenovirus vector of (1) to obtain the recombinant adenovirus vaccine.
In one or more embodiments, the mutant (protein) comprises: point mutation (protein), truncate (protein), and protein with added amino acid at N or C terminal.
In one or more embodiments, the point mutation is a mutation at one or more sites.
In one or more embodiments, the mutant Spike protein comprises: two heterologous RBD dimers (4 RBD) or two heterologous RBD truncate dimers (4 tRBD); preferably, the two heterologous RBD dimers (4 RBD) comprise: one heterodimer RBD delta and another heterodimer RBD beta gamma, wherein one RBD monomer in the RBD delta generates an L452R mutation at position 452 and a T478K mutation at position 478 of the Spike protein sequence, the other RBD monomer does not undergo amino acid change, and two different RBD monomers are connected in series to form a heterodimer; the RBDβγ is mutated at positions 417, 484, 501 corresponding to the Spike protein sequence, wherein one RBD monomer is K417T, E484K, N501Y and the other RBD monomer is K417N, E484K, N501Y (preferred combination sequence and amino acid mutation of the heterodimer RBD include but are not limited to those described above, such as RBD monomers comprising the RBD mutation site of the Omicron mutant), and two different RBD monomers are connected in series to form a heterodimer; preferably, the RBD truncations only have amino acid sequences corresponding to positions 319-537 of the Spike protein sequence; the mutant Spike or heterodimeric RBD protein is preferably directed for expression with a signal peptide (more preferably the signal peptide includes but is not limited to the signal peptide of Spike protein).
In one or more embodiments, the mutant Spike protein comprises: spike proteins from severe acute respiratory syndrome coronavirus 2 original or variant strains; preferably, the Spike protein from the variant includes (but is not limited to) mutations of: K417N, N440K, G446S, L452R, S N, T478K, E484K, E484A, T K, Q493R, G496S, Q498R, N501Y, Y505H, A570L, T572I, D614G, P681H, R682S, R G, F855Y, N856I, K986P, V987P, or a combination thereof; preferably, the Spike protein is the protein (vS) of the amino acid sequence shown in SEQ ID NO:3, or the Spike protein (vSC) mutated with N501Y, A570L, T572I, D614G, P681H, R682S, R685G, F855Y, N I, K986P and V987P, or the Spike protein (vS delta) mutated with L452R, T487K, N501Y, D614G, P681H, R682S, R685G, K986P, V987P, or the Spike protein (vSO) mutated with K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, D614G, P681H, R682S, R685G, K986P, V987P.
In one or more embodiments, the T cell epitope string is concatenated with an epitope derived from ORF1 at positions 2-48, an epitope derived from ORF3 at positions 49-153 and an epitope derived from M protein at positions 154-208 of SEQ ID NO. 2.
In one or more embodiments, the receptor binding motif comprises: receptor binding motifs from severe acute respiratory syndrome coronavirus 2 original or variant strains; the mutant receptor binding motif comprises a truncated receptor binding motif, preferably selected from the group consisting of: the protein with the amino acid sequence shown in any one of SEQ ID NO 4-7.
In one or more embodiments, the adenovirus vector further comprises genes encoding the following elements: t cell epitopes derived from influenza M1, M2 and NP proteins; preferably, it is located in the vicinity of the T cell epitope string of severe acute respiratory syndrome coronavirus 2 (near or in operative connection with the T cell epitope string of SARS-CoV-2, such as at the N-or C-terminus of the T cell epitope string of SARS-CoV-2); preferably, the amino acid sequence is shown as 209-354 in SEQ ID NO. 2.
In one or more embodiments, the adenovirus vector further comprises genes encoding the following elements: HA protein derived from H1N1 influenza virus; preferably, it is located near the T cell epitope string (at the N-or C-terminus of the T cell epitope string, linked by a 2A sequence in the middle); preferably, the amino acid sequence is as in GenBank accession No.: ACP41953.1.
In one or more embodiments, the adenovirus vector comprises: adenovirus vectors AdC68XY, adC6, adC7, adC 5, adC63, adC 26; preferably, the AdC68XY comprises a chimpanzee-type adenovirus AdC68 genome based sequence in which the E1 and E3 sequences are deleted and the E4 sequence is engineered; the engineering comprises replacing the E4 sequence or fragment thereof in the AdC68 genome with the corresponding E4 sequence or fragment thereof in the human serum type 5 adenovirus AdHu5 genome.
In one or more embodiments, the adcs 68XY include (but are not limited to) the following sets of engineered vectors:
(a) Replacing the ad c68 genomic E4 sequence with an ad hu5 genomic E4 sequence; more preferably, the deletion region is the 33518-36105 sequence of the AdC68 genome, and the 32914-35641 sequence of the AdHu5 genome is inserted in the deletion region;
(b) Replacing ORF6 and ORF6/7 region sequences in the E4 sequence of the AdC68 genome with ORF6 and ORF6/7 region sequences in the E4 sequence of the AdHu5 genome; more preferably, the deletion region is the 33518-34671 locus of the AdC68 genome, and the 32914-34077 locus of the AdHu5 genome is inserted into the deletion region; or (b)
(c) Replacing ORF 1-ORF 6/7 sequences in the E4 sequence of the AdC68 genome with the ORF 3-ORF 6 sequences in the E4 sequence of the AdHu5 genome; more preferably, the deletion region is the 33518-36105 sequence of the AdC68 genome, and the 33193-34703 sequence of the AdHu5 genome is inserted in the deletion region.
In one or more embodiments, the Spike protein encoding gene, HA protein encoding gene, T cell epitope string encoding gene, and receptor binding motif encoding gene are operably linked in an adenovirus vector; preferably, the sequence of each protein-encoding gene is variable.
In one or more embodiments, the coding gene of the mutant Spike protein or the coding gene of the mutant Spike protein-T cell epitope string-influenza HA protein is introduced into the E1 deletion region, the E3 deletion region, the E4 region, and/or the like of the adenovirus.
In one or more embodiments, the receptor binding motif is inserted into the adenovirus vector at the structural protein Fiber encoding gene or structural protein Hexon/Penton, preferably at the Fiber HI loop.
In one or more embodiments, the mutant Spike protein further comprises a junction sequence between the gene encoding the mutant Spike protein and the gene encoding the T cell epitope string; preferably the linker sequence is a 2A sequence; more preferably the linker sequence is E2A, P2A, T a or F2A.
In one or more embodiments, the two heterologous RBD dimers (4 RBD) are linked by a 2A sequence; preferably, the T cell epitope string is located between two heterologous RBD dimers, and the protein coding gene sequences are linked by a 2A sequence.
In one or more embodiments, the two heterologous RBD truncating dimers (4 tRBD) are linked by a 2A sequence; preferably, the T cell epitope string is located between two heterologous RBD truncations, and the protein coding gene sequences are linked by a 2A sequence.
In one or more embodiments, in (2), the method further comprises: purifying the culture solution containing the virus.
In one or more embodiments, in (2), an immunoadjuvant, such as, but not limited to, an aluminum hydroxide adjuvant, is also added to the recombinant adenovirus vaccine.
In one or more embodiments, in (2), the recombinant adenovirus vaccine further comprises a pharmaceutically acceptable carrier.
In one or more embodiments, the coding gene is a codon optimized DNA sequence or a native DNA sequence.
In another aspect of the invention, there is provided a recombinant adenovirus vector into which are introduced the following sets of elements: (1) a coding gene of mutant Spike protein, or a coding gene of mutant Spike protein-T cell epitope string-influenza HA protein; wherein the T cell epitope string is derived from ORF1, ORF3 and M proteins of severe acute respiratory syndrome coronavirus 2 or ORF1, ORF3 and M proteins of a novel coronavirus and M1, M2 and NP proteins of an influenza virus; (2) severe acute respiratory syndrome coronavirus 2 receptor binding motif or mutant severe acute respiratory syndrome coronavirus 2 receptor binding motif.
In another aspect of the invention, there is provided a recombinant adenovirus obtained from the recombinant adenovirus vector package described previously.
In one or more embodiments, the recombinant adenovirus vector is transfected into a virus-producing cell, thereby packaging the recombinant adenovirus.
In one or more embodiments, the virus-producing cell is a cell that can achieve viral packaging; preferably a cell which integrates the adenovirus E1 gene; preferred include (but are not limited to): HEK293 cells.
In another aspect of the invention, there is provided the use of said recombinant adenovirus for the preparation of a pharmaceutical composition or kit for inhibiting severe acute respiratory syndrome coronavirus 2 infection or influenza virus infection; preferably, the severe acute respiratory syndrome coronavirus 2 infection or the disease caused by influenza virus infection comprises: viral pneumonia, severe acute respiratory infection, intestinal disease, heart failure, renal failure, or severe acute respiratory syndrome; preferably, the pharmaceutical composition is a vaccine.
In another aspect of the present invention, there is provided a pharmaceutical composition or kit for inhibiting severe acute respiratory syndrome coronavirus 2 and/or influenza virus infection, said pharmaceutical composition or kit comprising: an effective amount of said recombinant adenovirus, optionally together with an immunoadjuvant; and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is a vaccine.
In one or more embodiments, the vaccine is a liquid vaccine or a spray vaccine.
In one or more embodiments, the vaccine includes, but is not limited to: intranasal administration, intramuscular administration, intravenous administration, intradermal administration, or subcutaneous administration.
In another aspect of the invention, there is provided a kit for preparing a vaccine against severe acute respiratory syndrome coronavirus 2 and/or influenza virus infection, comprising: the recombinant adenovirus vector described above; virus-producing cells.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1, C57BL/6 mice were raised against binding antibodies to Spike (A) and Nucleoprotein (B).
FIG. 2, C57BL/6 mice were immunized with neutralizing antibody titres (A-B) 2 weeks, 4 weeks and 8 weeks after AdC68XY3-S, adC XY3-vSN, and neutralizing antibody titres (C) 2 weeks after immunization with AdC68XY 3-vSmNL-vtrBM.
FIG. 3, adC68XY3-vSN and AdC68XY3-vSmNL-vtrBM induced T cell responses of mice against Spike: a is nasal immunization (i.n.) results and B is muscle immunization (i.m.) results.
FIG. 4, adC68XY3-vSN and AdC68XY3-vSmNL-vtrBM induced T cell responses of mice against Nucleoprotein: a is nasal cavity immune result, B is muscle immunity.
Figures 5A-C, binding antibody levels of the candidate vaccine after optimization.
FIG. 6, neutralizing antibody levels of the optimized candidate vaccine AdC68XY 3-vS-vtrBM.
FIG. 7A-B, levels of neutralizing antibodies induced by optimized candidate vaccines AdC68XY3-4RBDT-C.37, adC68XY3-4 tRBBDT-C.37, adC68XY3-4RBDTHA-C.37 and AdC68XY3-4 tBDTHA-C.37 and AdC68XY 3-vST-vtrBM.
FIG. 8, optimized candidate vaccine AdC68XY3-vST-vtrBM induced T cell immune response. A is a Spike-specific T cell immune response; b is a T cell immune response specific for ORF1, ORF3 and M.
FIG. 9, T cell immune responses induced by the optimized candidate vaccines AdC68XY3-4RBDT-C.37 and AdC68XY3-4 tRBDT-C.37. A is an RBD-specific T cell immune response; b is a T cell immune response specific for the inserted novel crown T cell epitopes (ORF 1, ORF3 and M).
Detailed Description
The inventor of the present invention has conducted intensive studies to reveal a broad-spectrum recombinant adenovirus vaccine against a novel coronavirus based on adenovirus. The recombinant adenovirus expresses Spike protein, T cell epitope string and Receptor Binding Motif (RBM) of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). The recombinant adenovirus of the invention can activate the organism to generate strong response to SARS-COV-2 as vaccine, has ideal control effect on the replication of various novel coronavirus mutant strains and has broad spectrum; and can improve the virus removing capacity of the organism.
Terminology
As used herein, the terms "novel coronavirus", "2019-nCov" and "SARS-CoV-2" are used interchangeably and are abbreviated as "severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2)".
As used herein, the terms "surface Spike protein", "Spike protein" and "S protein" have the same meaning and are used interchangeably to refer to a membrane protein of SARS-CoV-2.
As used herein, the terms "novel coronavirus pneumonia" and "COVID-19" refer to pneumonia caused by SARS-CoV-2 infection, which have the same meaning and are used interchangeably.
As used herein, the terms "original," "wild," or "natural" are used interchangeably. When these terms are used to describe a nucleic acid molecule, polypeptide (protein) or virus, it means that the nucleic acid molecule, polypeptide (protein) or virus is present in nature, found in nature, and has not been subjected to any artificial modification or processing. As used herein, the Spike protein of the original SARS-CoV-2 (original strain) refers to a naturally occurring, biologically active Spike protein. The amino acid sequences of Spike proteins can be conveniently obtained by those skilled in the art from various public databases, such as the GenBank database.
As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or artificial chromosomes of P1 origin (PACs); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, adenoviruses, adeno-associated viruses, retroviruses (including lentiviruses), herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma vacuolated viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.
As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and includes, but is not limited to, a prokaryotic cell such as e.g. e.coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK293 (a) cells or human cells.
As used herein, the term "element" refers to a series of functional nucleic acid sequences useful for the expression of proteins, where the term "element" is systematically constructed to form an expression construct (adenovirus vector). The sequences of the "elements" may be those provided in the present invention, and include variants thereof, as long as the variants substantially retain the function of the "elements" obtained by inserting or deleting some bases (e.g., 1 to 50bp; preferably 1 to 30bp, more preferably 1 to 20bp, still more preferably 1 to 10 bp), or performing random or site-directed mutation, etc.
As used herein, the term "operably linked" or "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.
As used herein, the term "construct" refers to a single-or double-stranded DNA molecule that has been made to contain DNA fragments that are assembled and arranged according to sequences that are not found in nature by human intervention. The "construct" includes an expression vector; alternatively, the "construct" is contained in the expression vector as part of the expression vector.
As used herein, the term "exogenous" or "heterologous" refers to a relationship between two or more nucleic acid or protein sequences from different sources, or a relationship between a nucleic acid from different sources and a host cell. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, the nucleic acid is heterologous to the host cell. The particular sequence is "exogenous" to the cell or organism into which it is inserted.
As used herein, the terms "comprising," "having," or "including" include, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are under the notion of "comprising", "having" or "including".
As used herein, an "effective amount" or "effective dose" refers to that amount which is functional or active in and acceptable to a human and/or animal as used herein.
As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
Optimized novel adenovirus vaccine
The invention establishes an optimized adenovirus vector for preparing SARS-CoV-2 virus vaccine. The adenovirus vector comprises the following operably linked elements: a coding gene of mutant Spike protein or a coding gene of mutant Spike protein-T cell epitope string; and, a gene encoding a Receptor Binding Motif (RBM) or a mutant receptor binding motif.
Spike mutations
SARS-CoV-2 virus belongs to the genus beta coronavirus and is a single-stranded positive sense RNA virus containing an envelope. The genomic sequence of SARS-CoV-2 virus is known to those skilled in the art and can be found, for example, in GenBank accession number: nc_045512.2.SARS-CoV-2 virus contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2 virus is specifically combined with angiotensin transferase 2 (ACE 2) on host cell through receptor binding domain (Receptor binding domain, RBD) on S protein, and the S protein plays a critical role in the process of virus infection of cell. Spike proteins are abbreviated as S proteins, and can be used as antigens, and can also be called "S antigens".
In the invention, spike proteins derived from original strains are included, and Spike proteins derived from various variant strains or artificially mutated Spike are also included; in an optimized version of the invention, mutant spikes are used, for example, the mutant spikes referred to as vS, vSC, vS delta, vSO in the examples of the invention.
In optimizing the candidate vaccine, the inventors have applied the RBD region of Spike (amino acids 319-541 of Spike), preferably the region corresponding to amino acids 319-541 of Spike. It is contemplated that cysteine may affect the stability of RBD proteins.
In another preferred embodiment, the inventors use a truncate of the RBD (tRBD), which in the examples exhibits good stability. In the present invention, RBD monomers derived from original strains are included, and RBD monomers derived from each mutant strain or artificially mutated RBD monomers are also included, and the inventors connect RBD monomers containing two different mutations in series to form a dimer, i.e., a heterologous RBD dimer. The two heterologous RBD dimers or tRBD dimers are operatively linked to form a 4RBD or 4tRBD. It will be appreciated that in addition to the mutant RBDs of the specific sequences provided by the present invention, further other mutant RBDs may be included, such as RBDs containing Omacron mutant mutation sites. Although two heterologous RBD dimers or tRBD dimers are preferred in this patent, three or four heterologous RBD dimers may be employed, and the combination and arrangement of the different mutated RBDs may be varied.
T cell epitope string
SARS-COV-2 belongs to RNA virus, and is easy to mutate in the transmission process, and mutant strain is produced. In the process of virus prevention and control, besides lgG-mediated humoral immunity, cellular immunity plays an important role in virus elimination. Mutations in SARS-COV-2 are concentrated in Spike and other genes are relatively conserved. In optimizing the candidate vaccine, the present inventors isolated a plurality of T cell epitopes on ORF1 and ORF3 of SARS-COV-2 and M gene, and constituted tandem epitope sequences (fusion sequences), and inserted them into adenovirus for expression. The inventors have surprisingly found that this can very effectively activate the body to produce a T cell response against the conserved regions of the new coronavirus. It will be appreciated that in addition to the epitope string structure of the specific sequences provided by the present invention, further T cell epitopes may be included. Although the COV2T (SEQ ID NO:2, positions 2-208) sequence is preferred in the present invention, the order of the epitope sequences derived from the ORF1, ORF3 and M genes, respectively, may vary.
In addition to the use of T cell epitope strings from SARS-COV-2, epitopes from other viruses can be further introduced to form a combination vaccine. The other virus is preferably a virus that causes an epidemic disease. For example, influenza virus, other coronaviruses, herpes zoster virus, herpes simplex virus, respiratory syncytial virus. In a preferred embodiment of the invention, T cell epitopes from influenza M1, M2 and NP proteins and HA proteins are used. Experimental results show that the invention successfully prepares and obtains the combined vaccine, and can generate neutralizing antibodies against influenza virus on the basis of generating neutralizing antibodies against SARS-COV-2.
Receptor binding motifs
The adenovirus expression vector also comprises a Receptor Binding Motif (RBM) or a truncated form (tRBM) thereof, and the RBM is preferably displayed on a structural protein Fiber of adenovirus, so that the organism can be further induced to generate neutralizing antibodies against the novel coronavirus; in addition, it can be displayed on other structural proteins of adenovirus, such as Hexon, penton, etc.
Although truncated RBMs (corresponding to bits 451-501 of the Spike sequence) are preferred in the present invention, RBMs of full length sequences are also useful (corresponding to bits 451-525 of the Spike sequence). The truncated protein reduces the length of the protein compared with the full-length protein, and under the condition that the exogenous gene loading capacity on the adenovirus structural protein is limited, the truncated RBM and the variant thereof can display the binding site of the novel coronavirus and the receptor hACE2 as much as possible, so that the corresponding neutralizing antibody is caused.
In the present invention, tandem proteins (fusion proteins), mutant proteins and/or truncated proteins are provided. Through a series of modifications, the invention successfully obtains the optimized novel adenovirus vaccine.
The tandem proteins (fusion proteins), mutant proteins or truncated proteins of the invention may be synthetic or recombinant proteins, i.e. may be chemically synthesized products, or may be produced from a host using recombinant techniques.
The invention also includes fragments, derivatives and analogs of the tandem, mutant or truncated proteins that retain substantially the same biological function or activity of the mutant protein. The fragment, derivative or analogue may be (i) a mutein having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a mutein having a substituent group in one or more amino acid residues. In addition, the mutant proteins of the present invention may be modified.
The present invention also includes proteins having 85% or more (preferably 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more) sequence identity (homology) to the sequence, based on providing a specific sequence. However, it is to be understood that for the particular mutation sites specified in the present invention, the amino acids of these sites should exist conservatively as specified in the present invention.
The invention also includes genes encoding these proteins. The sequence of the "coding gene" can be obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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. The synthesis of the relevant sequences, optionally introducing mutations into the sequences, by synthetic methods is also a common technique in the art.
The invention provides vectors comprising the coding sequences of the tandem, mutant or truncated proteins, which are adenoviral vectors.
The genome of adenovirus is relatively large and in previous studies, the inventors have tried various adenovirus engineering protocols, providing a preferred chimpanzee-based adenovirus engineering strategy. In a preferred mode of the invention, the replication-defective recombinant adenovirus vector deleted in E1/E3 is prepared by using chimpanzee adenovirus AdC68 as a template and using the genome sequence thereof. The adenovirus genome contains the E1-E4 early genes, and E1A activates the promoters of E1B, E, 2A, E, 2B, E, and E4. The adenovirus E4 region comprises 7 open reading frames, the products of which are ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6 and ORF6/7, respectively. In a more preferred embodiment, the present inventors replaced all or part of the E4 sequence of AdC68 with the E4 sequence of AdHu5, and designed a plurality of recombinant AdC68 vectors each carrying a different E4 sequence, designated AdC68XY1, adC68XY2, adC68XY3, adC68XY4, respectively.
In a preferred form of the invention, there is provided a recombinant adenovirus vector (plasmid) comprising: an engineered chimpanzee adenovirus AdC68 genomic sequence; wherein the E1 complete sequence and the E3 partial sequence are deleted and the E4 sequence is modified; the engineering comprises replacing the E4 sequence or fragment thereof in the AdC68 genome with the corresponding E4 sequence or fragment thereof in the human serum type 5 adenovirus AdHu5 genome. As a more preferred mode of the invention, the modification is selected from: (a) an AdHu5 genomic E4 sequence replaces the AdC68 genomic E4 sequence; (b) Replacing ORF6 and ORF6/7 region sequences in the E4 sequence of the AdC68 genome with ORF6 and ORF6/7 region sequences in the E4 sequence of the AdHu5 genome; or (c) replacing the ORF 1-ORF 6/7 sequences in the E4 sequence of the AdC68 genome with the ORF 3-ORF 6 sequences in the E4 sequence of the AdHu5 genome.
Optionally, the adenovirus vector may further comprise a reporter gene, an inverted terminal repeat, and/or a cleavage site. It will be appreciated that the design of the reporter gene, cleavage site, etc. elements is sometimes intended to provide a convenient way of observation or detection when studied, but is not necessary when the recombinant adenovirus vector or recombinant adenovirus of the invention is applied in the field of clinical pharmacy; alternatively, a gene capable of expressing a protein of interest having pharmaceutical activity may be substituted at the position of the reporter gene.
The expression vector typically also contains an origin of replication and/or a marker gene, etc. According to the teachings of the present invention, methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter (e.g., CMV) in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
The elements of the expression vectors of the invention are operably linked to facilitate packaging of the virus or efficient expression of the protein of interest.
After the adenovirus expression vector is obtained, the adenovirus expression vector is transfected into virus-producing cells to reproduce viruses. After a period of time following transfection, the virus may be harvested. The virus-producing cells may be various cells known in the art capable of propagating adenoviruses, such as HEK293 (A) cells, and the like.
As a preferred mode of the invention, the harvested virus may be repeatedly infected with virus-producing cells and passaged continuously. Viral Titer (TCID) 50 ) The determination of (2) may be performed according to methods conventional in the art. The recombinant adenoviruses obtained are also encompassed by the invention.
Vaccine composition and kit
The invention also provides a composition (vaccine composition) comprising an effective amount of said adenovirus, and a pharmaceutically acceptable carrier. The adenovirus can express Spike protein, T cell epitope string and RBM.
Typically, the adenovirus is formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8.
The pharmaceutical composition of the present invention can be formulated into administration forms such as injection, lyophilized powder, spray, etc., for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. For example, the adenovirus vaccine may be an intranasal injection, an intramuscular injection, an intravenous injection, an intradermal injection, or a subcutaneous injection. In practical application, the preparation can be adjusted and selected according to clinical requirements such as transfection efficiency, local immunity monitoring and the like, for example, single dosage form is selected for injection immunization, or multiple mixed dosage forms are selected for injection immunization. The amount of active ingredient (recombinant adenovirus) administered is a therapeutically effective amount, for example, from about 0.1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
The pharmaceutical composition is in unit dosage form.
The invention also provides a kit/medicine box, which comprises the adenovirus expression vector or the adenovirus. The kit/kit may also include virus-producing cells (e.g., 293 cells), culture medium, and the like. In addition, the kit/kit may also include instructions for using the adenovirus after the expression method.
Aiming at the novel coronavirus, the adenovirus vaccine (composition) has broad spectrum, can play a better role in controlling the replication of various novel coronavirus mutant strains, improves the virus clearance capability, and further enhances the broad spectrum of the vaccine.
The present invention also provides the use of a recombinant adenovirus vaccine (composition) according to the foregoing, for preventing or treating SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection in a subject. The disease is, for example, novel coronavirus pneumonia. The subject is a mammal, such as a human, capable of being infected with SARS-CoV-2.
The present invention also provides a method of preventing or treating SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection in a subject comprising administering to a subject in need thereof an effective amount of a recombinant adenovirus vaccine or composition according to the foregoing.
The adenovirus vaccine of the present invention has excellent immunogenicity, and can activate body to produce strong response to SARS-COV-2 and induce high level antibody expression. The adenovirus vaccine of the invention has ideal control effect on the replication of a plurality of novel coronavirus mutant strains and has broad spectrum; and can improve the virus removing capacity of the organism.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which are not specifically noted in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, or according to the manufacturer's recommendations.
Material
Plasmid, strain, cell and animal
Adenovirus plasmid pAdC68XY3-EGFP was constructed by the inventors; based on an AdC68 genome sequence (GenBank accession number: AC_ 000011.1), deleting partial E1 and E3 sequences, wherein the deletion size of E1 is 2552bp, and the deletion size of E3 is 3721bp; the E4 region was also engineered, substituting ORF6/7 and ORF6 with the corresponding ORFs of AdHu5 origin (see China patent application 201910777937.2).
The target gene is optimized according to human codon, and then cloned into the multicloning site of pUC57 to obtain recombinant plasmid. Plasmids pUC57-Spike, pUC57-mNL, pUC57-COV2FluT and pUC57-HA carrying the target gene were synthesized by Tianjin engine biotechnology Co. Wherein the amino acid sequence of mNL is shown as SEQ ID NO. 1; the amino acid sequence of COV2FluT codes is shown as SEQ ID NO. 2. Wherein, the amino acid sequence of the Spike of the original virus strain is shown in GenBank accession number: yp_009724390.1. The amino acid sequence of the influenza HA code is shown in GenBank accession number: ACP41953.1.
The amino acid sequence of the novel coronavirus nucleocapsid protein N (N protein) is shown in GenBank accession number: yp_009724397.2.
The novel coronavirus original strain is SARS-CoV-2, also labeled Origin strain in the invention, and the 5 mutant strains are respectively: b.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617 (Delta), C.37 (Lambda).
HEK 293 cell lines were purchased from the Shanghai cell bank of the national academy of sciences.
Coli strain DH 5. Alpha. And Stbl 2 were purchased from Shanghai Weidi Biotechnology Co.
Experimental mice 6-8 week old C57 mice were purchased from si Bei Fu (beijing) biotechnology limited.
Main reagent
All restriction enzymes were purchased from New England Biolabs.
Primers were synthesized in Beijing Optimu Biotechnology Co.
Plasmid miniprep purification kit, DNA gel recovery and purification kit were purchased from the astronomical biochemistry technologies ltd.
DMEM medium, fetal bovine serum, 0.25% pancreatin, secondary antibodies were purchased from Hyclone.
Luciferase assay kit was purchased from Promega.
Flow antibodies were purchased from Biolegend.
Fixation/Permeabilization Solution Kit with BD GolgiPlug was purchased from BD Bioscience.
The RBD and S1 peptide libraries and the RBD peptide libraries were synthesized by Kirschner Biotech Inc.
ELISPot detection kit was purchased from Mabtech.
Sequence design
mNL (SEQ ID NO: 1) (modified N protein, signal peptide of LAMP-1 protein added to the N-terminus, transmembrane region of LAMP-1 protein added to the C-terminus):
MAAPGSARRPLLLLLLLLLLGLMHCASASDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGI IWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTAAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQALIPI AVGGALAGLVLIVLIAYLVGRKRSHAGYQTI
COV2FluT (SEQ ID NO: 2) (which is a tandem product of a plurality of T cell epitopes obtained by screening against novel coronavirus and influenza virus genomes, wherein positions 2 to 48 are T cell epitopes derived from novel coronavirus ORF1, positions 49 to 153 are T cell epitopes derived from novel coronavirus ORF3, positions 154 to 208 are T cell epitopes derived from novel coronavirus M protein, and positions 209 to 354 are T cell epitope strings derived from influenza virus): MTTDPSFLGRYCTDDNALAYYYLITPVHVMFYYVWKSYVNSFSGYLKLRGVYYPDKVFRSSVKGIYQTSNFRVQPTESIVRFTSNQVAVLYQDVNCTEVCRSKNPLLYDANYFLCWHTNCYDYCIPYNSVTSSIVYFLQSINFALSKGVHFVRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGILGFVFTLILSPLTKGIILGFVFTLTRMVLASTTALGFVFTLTVVTTEVAFGLILHLILWILLLTEVETPIIIGILHLILATYQRTRALRRSGAAGAAVKELRSRYWAILPFERATVMSLENFRAYVCELTDSSWICELTDSSWI
vS (SEQ ID NO: 3) (mutant of Spike protein):
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSHSRAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
vtRBM (SEQ ID NO: 4) (corresponding to tRBM with an l→r mutation at amino acid 2):
YRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN
vtRBM (SEQ ID NO: 5) (corresponding to tRBM, amino acid 2 has an L.fwdarw.R mutation, and 34 has an E.fwdarw.K mutation):
YRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTN
tRBM (SEQ ID NO: 6) (truncated receptor binding motif (receptor binding motif, RBM), t stands for "truncated"):
YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN
RBMC.37 (SEQ ID NO: 7) (corresponding to tRBM, mutation of L.fwdarw.Q in amino acid 2, mutation of F.fwdarw.S in amino acid 40)
YQYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYSPLQSYGFQPTN
Example 1 construction of recombinant adenovirus plasmid
And (3) carrying out SrfI digestion on the adenovirus plasmid pAdC68XY3-EGFP, designing a primer, taking pUC57-Spike as a template, obtaining a target gene expression frame carrying an adenovirus vector homology arm through PCR, and cloning the original strain Spike expression frame into an E1 deletion region in pAdC68XY3 by using a homologous recombination method to obtain a first-generation recombinant adenovirus plasmid pAdC68XY3-S based on the original strain Spike.
Adenovirus plasmid pAdHu5 (NCBI accession number: AC_000008.1, with this as a backbone, the E1 and E3 regions were deleted, and the deleted E1 region was added to the I-CeuI and PI-SecI cleavage sites) and digested with both I-CeuI and PI-SecI. The pAdC68XY3-S is used as a template, a primer PCR is designed to obtain a Spike expression frame (CMV-Spike-BGH ployA), and the expression frame is inserted into an E1 deletion region of the linearized pAdHu5 by a homologous recombination method to obtain pAdHu5-S.
The mutation of part of Spike amino acids in pUC57-Spike by means of point mutation comprises: K417N, E484K, N501Y, D614G, P681H, R682S, R685G, K986P, V987P (see SEQ ID NO: 3) gave a vector pUC57-vS containing the mutated gene of interest. The adenovirus vector pAdC68XY3-EGFP was digested with SrfI, and the vector backbone was recovered. Designing a primer, cloning vS into a linearized pAdC68XY3 vector by using pUC57-vS as a template and utilizing a homologous recombination cloning method to obtain a target plasmid pAdC68XY3-vS. Subsequently, a novel coronavirus nucleocapsid protein N (N protein) was inserted after the vS sequence and ligated with P2A in the middle to give pAdC68XY3-vSN.
A truncated vtrBM containing an amino acid mutation (corresponding to the L.fwdarw.R mutation at amino acid 2 of tRBM) is inserted into the HI loop structure of the structural protein Fiber of adenovirus based on pAdC68XY3-vS by homologous recombination cloning, and the plasmid pAdC68XY3-vS-vtrBM is obtained as shown in SEQ ID NO: 4.
Subsequently, foreign gene mNL (SEQ ID NO: 1) was inserted after the vS sequence, and the middle was ligated with P2A to obtain pAdC68XY3-vSmNL-vtrBM.
The obtained recombinant adenovirus plasmids pAdC68XY3-vS-vtRBM and pAdC68XY3-vSmNL-vtRBM are subjected to enzyme digestion identification analysis by using Apa I, mfeI and Xho I, and the analysis is the same as that of enzyme digestion map fragment size, so that the construction of the recombinant adenovirus vectors is proved to be successful.
EXAMPLE 2 rescue, amplification and purification of recombinant adenoviruses
Recombinant adenovirus plasmids pAdC68XY3-S, pAdC XY3-vSN, pAdC68XY3-vS-vtRBM, pAdC68XY3-vSmNL-vtRBM, pAdC68XY3-vST-vtRBM were linearized by Pac I, transferred into HEK 293 cells with Lipofectatime 2000, after 8-13 days of culture, plaques appeared, cells were collected after rounding and suspension, virus supernatant was taken after three times of repeated freeze thawing to infect HEK 293 cells (75 cm 2 Cell culture flasks). The above steps were repeated until an appropriate amount of virus (30 pieces of 150 cm) 2 Cell culture dish) was purified using cesium chloride density gradient centrifugation to obtain candidate vaccines, adC68XY3-S, adC XY3-vSN, adC68XY3-vS-vtRBM, adC68XY3-vSmNL-vtRBM, adC68XY3-vST-vtRBM.
Extracting genome of each candidate vaccine, carrying out enzyme digestion and electrophoresis by using Apa I, mfeI and Xho I restriction enzymes, and comparing with a map to show that the enzyme section is correct, which indicates that the recombinant adenovirus candidate vaccine is correctly packaged, and no fragment is lost or inserted in the amplification process.
Example 3 immunological detection of candidate vaccine
1. Humoral immune response detection
C57 BL/6 female mice with the age of 8 weeks are selected and divided into six groups, and the muscle and nasal cavity immunity of the mice in the negative control group is 2 multiplied by 10 10 vp AdC68XY3-empty, oneImmunization of mice of the control group with the vaccine substitution 2X 10 10 vp AdC68XY3-S, mice of experimental group immunized with the novel crown vaccine candidate 2X 10 10 vp AdC68XY3-vSN, adC68XY3-vSmNL-vtrBM. 2 weeks, 4 weeks and 8 weeks after immunization, blood was taken through the cheek, serum was isolated, antigen-specific binding antibodies were detected by ELISA, and neutralizing antibodies were detected by pseudovirus.
Detection of bound antibody: (1) a cladding plate: diluting recombinant S protein or N protein with a wrapper buffer, coating at 4deg.C overnight at 100ng/100 ul/well; (2) closing: after three washes of PBST (0.05%), the ELISA plates were blocked with 8% skimmed milk, 200 ul/well and incubated for 2h at 37 ℃; (3) incubating the primary antibody: after three washes of PBST (0.05%), 100ul of diluted serum with 1% BSA was added and incubated at 37℃for 2h (dilution method: 1:400 start, 3-fold serial dilutions, 8 dilution gradients per sample total); (4) incubating the secondary antibody: after three washes of PBST (0.05%), 100ul of HRP-conjugated goat anti-mouse secondary antibody diluted 1:100000 was added and incubated at 37℃for 1h; (5) color development: after three washes of PBST (0.05%), 100ul TMB color development solution was added, and 50ul1M H was added after 10 minutes of reaction at room temperature in the dark 2 SO 4 The stop solution stops the color reaction, the enzyme label instrument reads the absorbance values of the OD450 and the OD630 at two wavelengths, and the Endpoint value is calculated.
The results of the binding antibody titers against Spike and Nucleoprotein 2 weeks after C57BL/6 mice immunization are shown in figure 1. As shown in fig. 1A, the candidate vaccine induced the body to produce high titres of binding antibodies against Spike 2 weeks after immunization of C57BL/6 strain mice. However, only after the ad c68XY3-vSmNL-vtRBM candidate vaccine was nasally induced in mice to produce binding antibodies to Nucleoprotein, no antibodies were produced by muscle immunization. Meanwhile, when the N protein was the original sequence, no specific antibody could be induced in the unmodified (AdC 68XY 3-vSN), both in muscle and nasal immunization (FIG. 1B).
2. Neutralizing antibody detection
The neutralizing antibody detection steps were as follows:
(1) Cell plating: after HEK293T-hACE2 cells are digested by pancreatin and resuspended in DMEM complete medium, 96-well cell culture plates are spread for 50000 cells/well, and cultured at 37 ℃ with 5% CO2 for 16-24 hours;
(2) Serum was diluted and incubated with pseudoviruses: the mouse serum was diluted with DMEM complete medium (dilution method: 1:20 initial, 3-fold serial dilutions, 9 dilution gradients were set per sample total), 50ul of diluted mouse serum was incubated with 50ul of pseudovirus at 37℃in a 5% CO2 cell incubator for 1h, and positive wells (no mouse serum, only equivalent pseudovirus) and blank wells (no mouse serum and pseudovirus) were set;
(3) Infected cells: discarding the culture medium in HEK293T-hACE2 cells, adding 100ul of the incubated serum-pseudovirus mixture, and detecting the expression of the reporter gene after 48 hours of infection;
(4) And (3) reporter gene detection: the infected cells and luciferases (firefly Luciferase) chemiluminescent substrate were allowed to stand at room temperature, 100ul of chemiluminescent substrate was added per well, the reaction was carried out at room temperature for 5 minutes, the reaction system was transferred to a 96-well blackboard, and the RLU value was detected with an microplate reader. (6) NT50 calculation: neutralization activity = ((positive well RLU value-blank well RLU value) - (serum incubation well RLU value-blank well RLU value))/(positive well RLU value-blank well RLU value) ×100%; fitting a 4-parameter regression curve, and calculating the serum dilution corresponding to the neutralization activity=50%, namely NT50.
The results of neutralizing antibody titers at 2 weeks, 4 weeks and 8 weeks after immunization of C57BL/6 mice with AdC68XY3-S, adC68XY3-vSN are shown in FIGS. 2A-2B, and the results of neutralizing antibody titers at 2 weeks after immunization with AdC68XY3-vSmNL-vtrBM are shown in FIG. 2C. As shown in fig. 2A-2B, the generation vaccine AdC68XY3-S and the candidate vaccine AdC68XY3-vSN produced higher titers of neutralizing antibodies 2 weeks after immunization, followed by further increases in neutralizing antibody levels for 4 weeks and 8 weeks, but significantly decreased neutralizing antibodies against the Beta mutant. While the ad C68XY3-vSmNL-vtRBM candidate vaccine was immunized for two weeks, the antibody levels were significantly higher than for the first two weeks after immunization (fig. 2C).
More importantly, the neutralizing antibodies induced by the AdC68XY3-vSmNL-vtRBM candidate vaccine have high inhibition level on the original strain and also on Alpha and Beta mutant strains, regardless of muscle immunity and nasal cavity immunity.
3. Cellular immune response detection
The same immunization strategy as described above was used to immunize 8-week-old C57 BL/6 females, euthanized at 14 days, spleen isolated lymphocytes were taken and tested for CD4+ and CD8+ T cell responses by flow cytometry, as shown in FIGS. 3 and 4.
Wherein FIGS. 3A-B are the T cell immune responses induced by AdC68XY3-vSN and AdC68XY3-vSmNL-vtrBM against Spike proteins. As shown, the candidate vaccines all induced the body to produce a higher proportion of specific effector T cells against Spike after immunization. The T cell response by muscle immunity is stronger than nasal immunity: s1 antigen-specific IFN-gamma in spleen upon muscle immunization + CD8 + T cell proportion reaches about 1.2%. The resulting CD 8T cell response is stronger than the CD 4T cell response, which is predominantly Th 1-type.
FIGS. 4A-B are T cell immune responses against N protein induced by AdC68XY3-vSN and AdC68XY 3-vSmNL-vtrBM. As shown, whether nasal or intramuscular, there is a need for further improvement in eliciting a strong, N-protein directed T cell response (including cd4+ T cells and cd8+ T cells) in mice.
Example 4 further optimization of candidate vaccine and immunological detection thereof
1. Candidate vaccine optimization
Since the N protein does not induce a specific T cell immune response, the present inventors have further optimized the novel corona vaccine based on AdC68XY 3-vSmNL-vtrBM. Through repeated research, the optimization strategy is determined as follows:
(1) Based on AdC68XY3-vSmNL-vtrBM, the coding gene of mNL protein is removed to obtain candidate vaccine AdC68XY3-vS-vtrBM.
(2) A plurality of T cell epitopes (obtained by screening a novel coronavirus gene, especially at ORF1, ORF3 and M genes thereof) are obtained by screening a novel coronavirus genome, are connected in series, and are synthesized into a sequence named Cov2T (SEQ ID NO:2, positions 2-208). Based on AdC68XY3-vSmNL-vtrBM, mNL was replaced with Cov2T, resulting in a candidate vaccine, adC68XY3-vST-vtrBM.
(3) Varying the mutation site of Spike resulted in different mutant S, including: vSC (comprising: N501Y, A570L, T572I, D614G, P681H, R682S, R685G, F855Y, N856I, K986P, V987P), vS delta (comprising: L452R, T487K, N501Y, D614G, P681H, R682S, R685G, K986P, V987P) and vSO (comprising: K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, D614G, P681H, R682S, R685G, K986P, V987P). These mutant S were used in place of vS in pAdC68XY 3-vS. AdC68XY3-vsCT, adC68XY3-vS delta T, adC XY3-vSOT were obtained. Then, a truncated tRBM (without mutation, see SEQ ID NO: 6) or a truncated amino acid mutation-containing vtRBM (corresponding to the L.fwdarw.R mutation at amino acid position 2, E.fwdarw.K mutation at position 34, SEQ ID NO: 5) may be inserted at the HI loop structure of the adenovirus structural protein Fiber. Thus, optimized candidate vaccines AdC68XY3-vSCT-tRBM, adC68XY3-vS delta T-vtRBM, adC68XY3-vSOT-vtRBM and the like are obtained.
(4) Instead of using full-length Spike, the RBD gene (amino acids 319-541 of Spike) was used as antigen and vS was replaced with two heterologous RBD dimers (dimers) by: the pUC57-Spike is used as a template, a primer PCR is designed to obtain a signal peptide sequence sp (1-12 bits of Spike) and an RBD sequence (319-541 bits of Spike) of SARS-COV-2, and the signal peptide sequence sp and the RBD sequence are cloned into a multiple cloning site of a pShuttle-CMV vector by a homologous recombination method to obtain pShuttle-spRBD. The residues 417, 484, 501 of the RBD region of pShuttle-spRBD, which correspond to the Spike proteins, were mutated to give pShuttle-spRBD beta (K417N, E484K, N501Y). In the same manner, RBD sequences (positions 319-541 of Spike) were cloned into the multicloning site region of pUC57 vector to obtain pUC57-RBD, and based thereon, the RBD was subjected to partial amino acid mutations (corresponding to positions 452, 417, 484, 501 of Spike protein) to obtain pUC57-RBD delta (L452R) and pUC57-RBD gamma (K417T, E484K, N501Y). Designing a primer, and cloning RBD delta to pShuttle-spRBD by taking pUC57-RBD delta as a template to obtain pShuttle-2spRBD delta; in the same way, after RBD gamma is cloned into pShuttle-spRBD beta by using pUC57-RBD gamma as a template, pShuttle-2spRBD beta gamma is obtained. And cloning 2spRBDβγ in pShuttle-2spRBDβγ into pShuttle-2spRBDδ, and connecting the two with P2A to obtain pShuttle-4spRBD. The adenovirus vector pAdC68XY3-EGFP was digested with SrfI, and the vector backbone was recovered. Primers were designed and 4spRBD was cloned into SrfI-linearized pAdC68XY3-EGFP using pShuttle-4spRBD as template (4 spRBD was inserted into the ΔE1 region) to yield pAdC68XY3-4RBD.
The T cell epitope string is kept unchanged, a foreign gene COV2T is inserted behind the 4RBD sequence, and the middle is connected by E2A, so that pAdC68XY3-4RBDT is obtained. Subsequently, a truncated tRBC.37 (corresponding to tRBM, an L.fwdarw.Q mutation at amino acid 2, and an F.fwdarw.S mutation at amino acid 40) was inserted into adenovirus Fiber, see SEQ ID NO:7, to give pAdC68XY3-4RBDT-C.37.
Thus, the candidate vaccine has 4RBD genes containing different amino acid mutations, namely RBD of an original strain, RBD of a Beta mutant strain with a key site mutation, RBD of a Gamma mutant strain with a key site mutation, RBD of a Delta mutant strain with a key site mutation, and truncated RBM of a Lambda mutant strain with a key site mutation.
(5) Cysteine may affect the stability of RBD protein, so 4 RBDs are truncated on the basis of AdC68XY3-4RBDT-C.37 to obtain tRBD (amino acids 319-537 of Spike, truncated before C548), the other are kept unchanged, and the construction method is the same to obtain candidate vaccine AdC68XY3-4tRBDT-C.37.
(6) On the basis of AdC68XY3-4tRBDT-C.37 and AdC68XY3-4RBDT-C.37, an influenza T cell epitope string (the amino acid sequence is shown in SEQ ID NO:2, positions 209-354) is added behind a new crown T cell epitope string COV2T, then an HA gene of the influenza virus (the amino acid sequence is shown in GenBank accession number: ACP 41953.1) is cloned behind the T cell epitope string, and the mixture is connected by T2A in the middle, so that an influenza and new crown combined candidate vaccine AdC68XY3-4 tBDTHA-C.37 and AdC68XY3-4RBDTHA-C.37 are obtained.
2. Humoral immune response detection
Optimization (1) for above: 8-week-old C57 BL/6 females were selected and divided into four groups: muscle and nasal immunization of control mice 2X 10 10 vp AdC68XY3-empty, new crown candidate vaccine 2X 10 for muscle and nasal cavity immunization of mice in experimental group 10 vp AdC68XY3-vS-vtrBM. Blood was collected through the cheek and serum was isolated 2 and 4 weeks after immunization, and Spike antigen-specific binding antibodies and neutralizing antibodies against the novel coronavirus and mutants thereof were detected by the same method as described above.
Optimization (4) and (5) above are aimed at: 8-week-old BALB/C females were selected and divided into six groups: muscle and nasal immunization of control mice 2X 10 10 vp AdC68XY3-empty, new crown candidate vaccine 2X 10 for muscle and nasal cavity immunization of mice in experimental group 10 vp AdC68XY3-4RBDT-C.37, adC68XY3-4tRBDT-C.37. The RBD antigen-specific binding antibodies and the neutralizing antibodies to the novel coronavirus and mutants thereof were detected by the same method as described above, except that blood was taken through the cheek, serum was isolated 2 weeks, 4 weeks and 8 weeks after immunization.
For the above optimization (6): c57 BL/6C female mice at 8 weeks of age were selected and divided into three groups: control group mouse muscle 2×10 10 vp AdC68XY3-empty, candidate vaccine 2X 10 for muscle immune of mice in experimental group 10 vp AdC68XY3-4RBDTHA-C.37, adC68XY3-4tRBDTHA-C.37. The RBD antigen-specific binding antibodies and neutralizing antibodies against influenza pandemic H1N1 (pH 1N 1) were detected by the same method as described above, by taking blood through the cheek, separating serum, 2 weeks, 6 weeks and 8 weeks after immunization.
Optimization (1) for above: figure 5 is the levels of bound antibodies induced by the candidate vaccine after optimization. As shown in FIG. 5A, the AdC68XY3-vS-vtRBM muscle immunized mice were induced to produce higher titers of binding antibodies to Spike 2 weeks later with a significant increase in antibody levels. At 2 weeks, antibodies rapidly reached higher levels (than contemporaneous muscle immunity) at 4 weeks with a slight increase.
Optimization (4) and (5) above are aimed at: as shown in FIGS. 5B and 5C, binding antibodies to RBD were rapidly raised 2 weeks after mice were muscle immunized with AdC68XY3-4RBDT-C.37, adC68XY3-4 tRBBDT-C.37, adC68XY3-4RBDTHA-C.37, and AdC68XY3-4 tBDTHA-C.37, after which binding antibody levels were significantly raised at week 8/week 6.
Optimization (1) for above: the detection result of the neutralizing antibody induced by the AdC68XY3-vS-vtRBM is shown in figure 6, the neutralizing antibody with high titer is generated on the 14 th day after the candidate vaccine is immunized, the neutralizing activity against the original strain and 5 mutant strains is high, the titer of the neutralizing antibody on the 28 th day is further obviously increased, and the neutralizing antibody has better neutralizing antibody broad spectrum. Muscle immunity and nasal immunity are all suitable.
Optimization (4) and (5) above are aimed at: the results of detection of neutralizing antibodies 2 weeks, 6 weeks and 8 weeks after immunization of AdC68XY3-4RBDT-C.37, adC68XY3-4tRBDT-C.37 are shown in FIG. 7A, and neutralizing antibodies against the original strain and 5 mutants have been generated 2 weeks after immunization of the candidate vaccine. Antibody levels were significantly elevated 8 weeks after immunization. In addition, the neutralizing antibody induced by AdC68XY3-4RBDT-C.37 was higher than AdC68XY3-4tRBDT-C.37.
For the above optimization (6): the induced organisms generate broad-spectrum neutralizing antibodies against the new crown original strain and 4 mutant strains 2 weeks, 6 weeks and 8 weeks after the immunization of AdC68XY3-4RBDTHA-C.37 and AdC68XY3-4 tRBBDTHA-C.37, and the result is shown in 7A. Meanwhile, a strong neutralizing antibody against the live virus at pH1N1 was detected 6 weeks after immunization, and the result is shown in FIG. 7B.
Optimization (3) for above: in addition, the levels of neutralizing antibodies induced by the candidate vaccines AdC68XY3-vSCT-tRBM, adC68XY3-vS delta T-vvtRBM, and AdC68XY3-vSOT-vtRBM were also examined, all significantly induced the production of neutralizing antibodies against the novel coronavirus, with some differences in neutralizing antibody levels for different mutants, such as: adC68XY3-vSCT-tRBM induced high levels of antibodies against the original strain, the Alpha, gamma, delta, lambda mutant strain; the antibody level of the AdC68XY 3-vSdelta T-vtRBM induced against the original strain and the Alpha, delta, lambda mutant strain is higher; adC68XY3-vSOT-vtrBM induced higher levels of antibodies against Omicron and Delta mutants.
3. Cellular immune response detection
8-week-old C57 BL/6 females were selected and divided into four groups: muscle and nasal immunization of control mice 2X 10 10 vp AdC68XY3-empty, new crown candidate vaccine 2X 10 for muscle and nasal cavity immunization of mice in experimental group 10 vp AdC68XY3-vST-vtrBM. At day 10, mice were euthanized, spleen isolated lymphocytes were taken and the CD4 and CD 8T cell responses were examined by flow cytometry, the results of which are shown in figure 8. Wherein, fig. 8A is a T cell immune response against Spike protein, and fig. 8B is a T cell immune response against an inserted new crown T cell epitope.
Optimization (4) and (5) above are aimed at: 8-week-old C57 BL/6 females were selected and divided into four groups: muscle and nasal immunization of control mice 2X 10 10 vp AdC68XY3-empty, muscle and nasal cavity immunization of mice in experimental groupVaccine selection 2X 10 10 vp AdC68XY3-4RBDT-C.37 or AdC68XY3-4 tRBDT-C.37. At day 13, mice were euthanized, spleen isolated lymphocytes were taken and CD4 and CD 8T cell responses were detected by flow cytometry. FIG. 9 shows T cell immune responses induced by candidate vaccines AdC68XY3-4RBDT-C.37 and AdC68XY3-4tRBDT-C.37 against RBD proteins and novel crown T cell epitope strings. The candidate vaccine induced strong cd4+ and cd8+ T cell responses against RBD (fig. 9A), while cd8+ T cell responses against ORF1, ORF3 and M were induced (fig. 9B).
Optimization (2) for the above: as shown in FIG. 8A, the candidate vaccine AdC68XY3-vST-vtrBM induced the body to produce a higher proportion of Spike antigen-specific effector T cells after immunization, and the proportion of S1 antigen-specific IFN-gamma+CD8+ T cells in spleen reached 3.5% during muscle immunization + CD4 + T cells, IL-2 + CD4 + T cells and TNF-alpha + CD4 + T cell ratios were also achieved at 0.18%, 0.18% and 0.12%, respectively. As shown in fig. 8B, the optimized candidate vaccine induced the body to produce effector T cells, predominantly cd8+ T cells, against the new crown ORF1, ORF3, M. Wherein the IFN-. Gamma. + CD8+ T cell ratios for ORF1, ORF3, M were 0.95%, 0.09% and 0.01%, respectively.
For the above optimization (6): the cellular immune response induced after immunization of candidate vaccines AdC68XY3-4RBDTHA-C.37 and AdC68XY3-4tRBDTHA-C.37 was also examined, as well as a stronger T cell response against novel crown RBD, ORF1, ORF3 and M.
Conclusion(s)
The invention discloses a broad-spectrum vaccine for a novel coronavirus and a combined vaccine of the novel coronavirus and influenza virus. Experiments show that the novel crown vaccine can induce mice to generate neutralizing antibodies with higher target Alpha, beta, gamma, delta, lambda mutant strains and original strains, and has broad-spectrum neutralization. Moreover, the vaccine of the invention can generate strong cellular immune response, not only aiming at Spike easy to mutate, but also aiming at more conservative ORF1, ORF3 and M, thereby increasing the broad spectrum of candidate vaccines. The invention also provides a combined vaccine which can induce neutralizing antibodies against various novel coronaviruses and can generate T cell immunity against easy mutant RBD and more conservative ORF1, ORF3 and M; on the other hand, the neutralizing antibody aiming at the influenza pH1N1 can be induced to generate, and the applicability of the vaccine is expanded.
Therefore, the series of novel crown vaccines provided by the invention has ideal broad spectrum and has positive significance for preventing and controlling novel crown epidemic.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims. All documents referred to in this application are incorporated by reference herein as if each was individually incorporated by reference.
Sequence listing
<110> Suzhou gaming biotechnology Co., ltd
<120> broad-spectrum novel coronal vaccine based on adenovirus vector and use thereof
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Tyr Val Trp Lys Ser Tyr Val Asn Ser Phe Ser Gly Tyr Leu Lys Leu
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Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val
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Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
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Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
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His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
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Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
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Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
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Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
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Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
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Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
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Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
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Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
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Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
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Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
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Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser His Ser Arg Ala Gly Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
1010 1015 1020
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val
1025 1030 1035 1040
Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala
1045 1050 1055
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu
1060 1065 1070
Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His
1075 1080 1085
Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val
1090 1095 1100
Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr
1105 1110 1115 1120
Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr
1125 1130 1135
Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
1140 1145 1150
Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp
1155 1160 1165
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1170 1175 1180
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1185 1190 1195 1200
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile
1205 1210 1215
Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile
1220 1225 1230
Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val
1250 1255 1260
Leu Lys Gly Val Lys Leu His Tyr Thr
1265 1270
<210> 4
<211> 51
<212> PRT
<213> Artificial Sequence
<400> 4
Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
1 5 10 15
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
20 25 30
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
35 40 45
Pro Thr Asn
50
<210> 5
<211> 51
<212> PRT
<213> Artificial Sequence
<400> 5
Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
1 5 10 15
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
20 25 30
Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
35 40 45
Pro Thr Asn
50
<210> 6
<211> 51
<212> PRT
<213> Artificial Sequence
<400> 6
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
1 5 10 15
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
20 25 30
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
35 40 45
Pro Thr Asn
50
<210> 7
<211> 51
<212> PRT
<213> Artificial Sequence
<400> 7
Tyr Gln Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
1 5 10 15
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
20 25 30
Val Glu Gly Phe Asn Cys Tyr Ser Pro Leu Gln Ser Tyr Gly Phe Gln
35 40 45
Pro Thr Asn
50

Claims (10)

1. A method of preparing a vaccine against severe acute respiratory syndrome coronavirus 2 comprising:
(1) Introducing the following elements into an adenovirus vector to obtain a recombinant adenovirus vector:
(1) a coding gene of mutant Spike protein or a coding gene of mutant Spike protein-T cell epitope string; wherein the T cell epitope string is derived from the ORF1, ORF3 and M proteins of the virus;
(2) A gene encoding a receptor binding motif or a mutant receptor binding motif;
(2) And (3) packaging the virus based on the recombinant adenovirus vector of (1) to obtain the recombinant adenovirus vaccine.
2. The method of claim 1, wherein the mutant Spike protein comprises: two heterologous RBD dimers or two heterologous RBD truncated dimers; preferably, the two heterologous RBD dimers comprise: one heterodimer RBD delta and another heterodimer RBD beta gamma, wherein one RBD monomer in the RBD delta generates an L452R mutation at position 452 and a T478K mutation at position 478 of the Spike protein sequence, the other RBD monomer does not undergo amino acid change, and two different RBD monomers are connected in series to form a heterodimer; the RBD beta gamma is mutated at the 417, 484 and 501 positions corresponding to the Spike protein sequence, wherein one RBD monomer is K417T, E484K, N501Y, the other RBD monomer is K417N, E484K, N501Y, and two different RBD monomers are connected in series to form a heterodimer; preferably, the different RBD monomers can be connected in series to form a heterodimer; the RBD monomer mutation sites that preferably constitute the heterodimer include, but are not limited to, the above four, and can be adjusted according to different new crown mutants, such as: a mutation site comprising Omicron mutant RBD: RBD monomers of G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y H, RBD monomers comprising Alpha mutant RBD mutation site N501Y, and the like; preferably, the RBD truncations only have amino acid sequences corresponding to positions 319-537 of the Spike protein sequence; preferably, the expression of the mutant Spike or heterodimeric RBD protein is directed with a signal peptide; and/or
The mutant Spike protein comprises: spike proteins from severe acute respiratory syndrome coronavirus 2 original or variant strains; preferably, the Spike protein from the variant comprises the following mutations: K417N, N440K, G446S, L452R, S N, T478K, E484K, E484A, T K, Q493R, G496S, Q498R, N501Y, Y505H, A570L, T572I, D614G, P681H, R682S, R G, F855Y, N856I, K986P, V987P, or a combination thereof; preferably, the Spike protein is the protein with the amino acid sequence shown in SEQ ID NO:3, or the Spike protein mutated with N501Y, A570L, T572I, D614G, P681H, R682S, R685G, F855Y, N856I, K986P and V987P, or the Spike protein mutated with L452R, T487K, N501Y, D614G, P681H, R682S, R685G, K986P, V987P, or the Spike protein mutated with K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, D614G, P681H, R682S, R685G, K986P, V987P; and/or
The T cell epitope string is connected with an epitope derived from ORF1 at positions 2-48, an epitope derived from ORF3 at positions 49-153 and an epitope derived from M protein at positions 154-208 in series in SEQ ID NO. 2; and/or
The receptor binding motif comprises: receptor binding motifs from severe acute respiratory syndrome coronavirus 2 original or variant strains; the mutant receptor binding motif comprises a truncated receptor binding motif, preferably selected from the group consisting of: protein with the amino acid sequence shown in any one of SEQ ID NO 4-7; and/or
The adenovirus vector also incorporates genes encoding the following elements: t cell epitopes derived from influenza M1, M2 and NP proteins; preferably, it is located in the vicinity of the T cell epitope string of severe acute respiratory syndrome coronavirus 2; preferably, the amino acid sequence is shown as 209 th to 354 th positions in SEQ ID NO. 2; and/or
The adenovirus vector also incorporates genes encoding the following elements: HA protein derived from H1N1 influenza virus; preferably, it is located near the T cell epitope string; preferably, the amino acid sequence is as in GenBank accession No.: ACP41953.1.
3. The method of claim 1, wherein the adenovirus vector comprises: adenovirus vectors AdC68XY, adC6, adC7, adC 5, adC63, adC 26; preferably, the AdC68XY comprises a chimpanzee-type adenovirus AdC68 genome based sequence in which the E1 and E3 sequences are deleted and the E4 sequence is engineered; the engineering comprises replacing the E4 sequence or fragment thereof in the AdC68 genome with the corresponding E4 sequence or fragment thereof in the human serum type 5 adenovirus AdHu5 genome.
4. The method according to claim 1 or 3, wherein the coding gene of the mutant Spike protein or the coding gene of the mutant Spike protein-T cell epitope string-influenza HA protein is introduced into the E1 deleted region, E3 deleted region and/or E4 region of adenovirus, etc.;
the receptor binding motif is inserted into the adenovirus vector on the structural protein Fiber encoding gene or on the structural protein Hexon/Penton, preferably at the Fiber's HIloop position.
5. A recombinant adenovirus vector into which the following elements are introduced:
(1) a coding gene of mutant Spike protein, or a coding gene of mutant Spike protein-T cell epitope string-influenza HA protein; wherein the T cell epitope string is derived from ORF1, ORF3 and M proteins of severe acute respiratory syndrome coronavirus 2 or ORF1, ORF3 and M proteins of a novel coronavirus and M1, M2 and NP proteins of an influenza virus;
(2) severe acute respiratory syndrome coronavirus 2 receptor binding motif or mutant severe acute respiratory syndrome coronavirus 2 receptor binding motif.
6. The recombinant adenovirus vector of claim 5, wherein the mutant Spike protein comprises: two heterologous RBD dimers or two heterologous RBD truncated dimers; preferably, the two heterologous RBD dimers comprise: one RBD delta and one RBD beta gamma, wherein one RBD monomer in the RBD delta generates L452R mutation corresponding to 452 th position of the Spike protein sequence, the 478 th position generates T478K mutation, the other RBD monomer does not change amino acid, and two different RBD monomers are connected in series to form a heterodimer; the RBD beta gamma is mutated at the 417, 484 and 501 positions corresponding to the Spike protein sequence, wherein one RBD monomer is K417T, E484K, N501Y, the other RBD monomer is K417N, E484K, N501Y, and two different RBD monomers are connected in series to form a heterodimer; preferably, the different RBD monomers can be connected in series to form a heterodimer; the RBD monomer mutation sites that preferably constitute the heterodimer include, but are not limited to, the above four, and can be adjusted according to different new crown mutants, such as: a mutation site comprising Omicron mutant RBD: RBD monomers of G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y H, RBD monomers comprising Alpha mutant RBD mutation site N501Y, and the like; preferably, the RBD truncations only have amino acid sequences corresponding to positions 319-537 of the Spike protein sequence; preferably, the expression of the mutant Spike protein is directed with a signal peptide; and/or
The mutant Spike protein comprises: spike proteins from severe acute respiratory syndrome coronavirus 2 original or variant strains; preferably, the Spike protein from the variant comprises the following mutations: K417N, N440K, G446S, L452R, S N, T478K, E484K, E484A, T K, Q493R, G496S, Q498R, N501Y, Y505H, A570L, T572I, D614G, P681H, R682S, R G, F855Y, N856I, K986P, V987P, or a combination thereof; preferably, the Spike protein is the protein with the amino acid sequence shown in SEQ ID NO:3, or the Spike protein mutated with N501Y, A570L, T572I, D614G, P681H, R682S, R685G, F855Y, N856I, K986P and V987P, or the Spike protein mutated with L452R, T487K, N501Y, D614G, P681H, R682S, R685G, K986P, V987P, or the Spike protein mutated with K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, D614G, P681H, R682S, R685G, K986P, V987P; and/or
The T cell epitope string is connected with the epitope of ORF1 derived from severe acute respiratory syndrome coronavirus 2 at the 2 nd-48 th position, the epitope derived from severe acute respiratory syndrome coronavirus ORF3 at the 49 th-153 th position and the epitope derived from severe acute respiratory syndrome coronavirus M protein at the 154 th-208 th position in series in SEQ ID NO. 2; and/or
The receptor binding motif comprises: receptor binding motifs from severe acute respiratory syndrome coronavirus 2 original or variant strains; the mutant receptor binding motif comprises a truncated receptor binding motif, preferably selected from the group consisting of: protein with the amino acid sequence shown in any one of SEQ ID NO 4-7; and/or
The adenovirus vector also incorporates genes encoding the following elements: t cell epitopes derived from influenza M1, M2 and NP proteins; preferably, it is located in the vicinity of the T cell epitope string of severe acute respiratory syndrome coronavirus 2; preferably, the amino acid sequence is shown as 209 th to 354 th positions in SEQ ID NO. 2; and/or
The adenovirus vector may also incorporate genes encoding the following elements: HA protein derived from H1N1 influenza virus; preferably, it is located near the T cell epitope string; preferably, the amino acid sequence is as in GenBank accession No.: ACP41953.1.
7. A recombinant adenovirus obtained from the recombinant adenovirus vector package of claim 5 or 6.
8. The use of the recombinant adenovirus of claim 7 for the preparation of a pharmaceutical composition or kit for inhibiting severe acute respiratory syndrome coronavirus 2 infection or influenza virus infection; preferably, the severe acute respiratory syndrome coronavirus 2 infection or the disease caused by influenza virus infection comprises: viral pneumonia, severe acute respiratory infection, intestinal disease, heart failure, renal failure, or severe acute respiratory syndrome; preferably, the pharmaceutical composition is a vaccine.
9. A pharmaceutical composition or kit for inhibiting severe acute respiratory syndrome coronavirus 2 and/or influenza virus infection, said pharmaceutical composition or kit comprising:
an effective amount of the recombinant adenovirus of claim 7, optionally further comprising an immunoadjuvant; and
a pharmaceutically acceptable carrier;
preferably, the pharmaceutical composition is a vaccine.
10. A kit for preparing a vaccine against severe acute respiratory syndrome coronavirus 2 and/or influenza virus infection, comprising:
the recombinant adenovirus vector of claim 5 or 6;
virus-producing cells.
CN202210106932.9A 2022-01-28 2022-01-28 Broad-spectrum new crown vaccine based on adenovirus vector and application thereof Pending CN116549627A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116726160A (en) * 2023-08-09 2023-09-12 中国医学科学院医学生物学研究所 Preparation and application of novel coronavirus mutant universal vaccine cRBD

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116726160A (en) * 2023-08-09 2023-09-12 中国医学科学院医学生物学研究所 Preparation and application of novel coronavirus mutant universal vaccine cRBD
CN116726160B (en) * 2023-08-09 2023-10-27 中国医学科学院医学生物学研究所 Preparation and application of novel coronavirus mutant universal vaccine cRBD

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