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WO2023138334A1 - Vaccin à nouvelle protéine de coronavirus recombinante, son procédé de préparation et son application - Google Patents

Vaccin à nouvelle protéine de coronavirus recombinante, son procédé de préparation et son application Download PDF

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WO2023138334A1
WO2023138334A1 PCT/CN2022/142937 CN2022142937W WO2023138334A1 WO 2023138334 A1 WO2023138334 A1 WO 2023138334A1 CN 2022142937 W CN2022142937 W CN 2022142937W WO 2023138334 A1 WO2023138334 A1 WO 2023138334A1
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protein
novel coronavirus
recombinant
vaccine
nucleic acid
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Inventor
李启明
张靖
梁宇
苏计国
杜丽芳
唐芳
邵帅
张学峰
雷泽华
刘兆明
韩子泊
刘宁
靳玉琴
张�浩
侯俊伟
侯亚楠
马智静
陈实
郑凡
沈福杰
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National Vaccine And Serum Institute Nvsi
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention relates to the field of biomedicine, in particular to a recombinant novel coronavirus protein vaccine, its preparation method and application.
  • coronavirus belongs to Nidovirales, Coronaviridae, Orthocoronavirus subfamily, Betacoronavirus genus, Sarbecovirus subgenus, SARS-like virus species, single-stranded positive-sense RNA virus, has an envelope, and the full length of the genome is about 29.9kb. in,), M protein (membrane protein), E protein (envelope protein) and N protein (nucleo protein).
  • M protein membrane protein
  • E protein envelope protein
  • N protein nucleo protein
  • accessory proteins 3a, 3b, p6, 7a, 7b, 8b, 9b and orf14, which are all involved in virus assembly.
  • the S, M and E proteins constitute the viral envelope and are the main surface antigens on which the virus induces an immune response.
  • the S protein is a transmembrane glycoprotein with a molecular weight of about 150kDa, which forms a prominent homotrimer on the surface of the virus.
  • S consists of two functional subunits that are cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage point), which remain non-covalently associated in the prefusion conformation.
  • the S2 subunit is also composed of multiple structural domains, and its function is mainly to mediate the fusion of the virus and the host cell.
  • the distal S1 subunit is structurally divided into four different structural domains: N-terminal domain (NTD), receptor-binding domain (RBD), C-terminal domain 1 (CTD1) and C-terminal domain 2 (CTD2).
  • NTD N-terminal domain
  • RBD receptor-binding domain
  • CCD1 C-terminal domain 1
  • CCD2 C-terminal domain 2
  • ACE2 receptor angiotensin converting enzyme 2
  • the new coronavirus is an RNA virus and is more prone to mutations. More than seven million new coronavirus mutants have been found in the world.
  • the main mutants include: Alpha (B.1.1.7) mutant, Beta (B.1.351) mutant, Gamma (P1) mutant, Epsilon (B.1.429) mutant, Delta (B.1.617.2) mutant, Kappa (B.1.617.1) mutant and Omic ron (B.1.1.529) mutant strain, the emergence of mutant strains has affected the protective effect of existing vaccines to varying degrees.
  • VOCs variants of concern
  • the Omicron mutant was first detected in South Africa on November 9, 2021. On November 26, 2021, the World Health Organization defined it as the fifth "variant strain of concern", named it the Greek letter Omicron (Omicron) mutant strain.
  • the mutant contains a total of 36 amino acid mutation sites, including K417N/T, E484K/Q/A, N501Y, and D6 that frequently appear in the dominant epidemic strains Alpha (B.1.1.7) mutant, Beta (B.1.351) mutant, Gamma (P1) mutant, Delta (B.1.617.2) mutant and Kappa (B.1.617.1) mutant 14G, P681H/R mutation sites. Omicron strains have gradually become the dominant and prevalent strains in most parts of the world.
  • One aspect of the present invention is aimed at the lack of broad-spectrum novel coronavirus (SARS-CoV-2) vaccines in the prior art, especially the lack of vaccines that have good neutralizing activity against five VOCs variant strains, especially Omicron strains, and provides a single-component broad-spectrum recombinant protein vaccine that can produce good cross-neutralizing activity against multiple novel coronavirus epidemic strains, especially Omicron, Beta, Delta, Alpha, Gamma and other variant strains.
  • SARS-CoV-2 broad-spectrum novel coronavirus
  • a recombinant novel coronavirus protein is in the form of a trimer, including three subunits composed of the 319th to 537th amino acid fragment in the RBD region of the novel coronavirus S protein,
  • the recombinant novel coronavirus protein includes:
  • the subunit of the recombinant new coronavirus protein constructed in the present invention contains at least two artificially constructed non-natural RBD fragments, and the recombinant vaccine as the target antigen has broad-spectrum protection ability across epidemic strains.
  • the receptor-binding domain (RBD) of the novel coronavirus spike (S) protein is directly involved in the binding of host cell receptors and plays a key role in the process of virus invasion of host cells.
  • RBD contains major neutralizing epitopes. Therefore, RBD is one of the main target antigens for the development of new crown vaccines.
  • RBD monomers are not highly immunogenic due to their small molecular size.
  • the natural S protein is a trimeric structure, and RBD also exists in a trimerized form. The trimerized RBD is constructed to simulate its natural structure to the greatest extent.
  • the molecular size of the antigen can be increased through trimerization to realize the repetitive and regular arrangement of the antigen and enhance the cross-linking of B cell receptors, which can significantly improve the immunogenicity of the RBD.
  • trimerization of antigens a common method is to introduce exogenous tethers or trimerization motifs.
  • exogenous sequences may bring about unexpected immune responses and has certain safety risks.
  • trimerization of the RBD without introducing a trimerization motif.
  • RBD has the following structural features: (1) The N-terminal and C-terminal of RBD have a long flexible loop structure.
  • RBD interception scheme that is, to intercept the 319-537 amino acid fragment.
  • This interception scheme retains the loop structure at both ends of the RBD as much as possible, and at the same time, ensures that the distance between the N-terminal and C-terminal interfaces is relatively close.
  • the three truncated RBD regions were connected end to end in series to form a new RBD trimerization fusion protein.
  • a mosaic-type RBD trimerization antigen was constructed.
  • the inventors analyzed the mutation sites and screened and integrated the key mutation sites. Among the 20 mutation sites with the highest frequency, residue mutations with strong immune escape ability were selected. At the same time, considering the distribution of these mutations in the spatial structure and avoiding mutual influence in the spatial structure, two unnatural RBDs were artificially designed.
  • One of the artificial RBDs contained K417N, L452R, T A total of 5 residue mutations of 478K, F490S and N501Y (that is, the first subunit, can be recorded as artificial RBD-1), and another artificially constructed RBD contains a total of 3 residue mutations of K417T, S477N and E484K (that is, the second subunit, can be recorded as artificial RBD-2).
  • the Omicron variant contains 15 mutated RBD regions including G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H.
  • RBD regions including G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H.
  • the present invention uses computational biology methods to design a brand-new fusion protein, which contains three RBD domains, and can form a trimer form with a stable antigen conformation without introducing any exogenous linker or other irrelevant components, and realize the trimerization of the RBD protein.
  • the RBD trimeric protein is expressed and purified by genetic engineering technology, it is mixed with an adjuvant to prepare a vaccine.
  • protective neutralizing antibodies against a variety of novel coronavirus epidemic strains can be produced for the treatment and/or prevention of SARS-CoV-2 infection and/or novel coronavirus disease (COVID-19).
  • the RBD region Due to the clear function and structure of the RBD region, it is responsible for recognizing the ACE2 receptor of the host cell. At the same time, the antibody produced against the RBD has a clear function and target specificity, and avoids inducing the body to produce antibody-dependent enhancement (Antibody Dependent Enhancement, ADE) to the greatest extent.
  • ADE Antibody Dependent Enhancement
  • the three subunits of the recombinant novel coronavirus protein may also contain RBD fragments of other novel coronavirus variant strains in addition to the above two artificially constructed non-natural RBD fragments.
  • the recombinant novel coronavirus protein further includes a subunit composed of the 319th to 537th amino acid fragment of the S protein RBD region of the novel coronavirus Omicron mutant strain.
  • the above subunits contain five or more mutation sites selected from G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y or Y505H mutation sites .
  • Some combinations of the above mutation sites are shown in Table 1, for example.
  • the primary structure of the three subunits of the above-mentioned recombinant novel coronavirus protein is that the three subunits are first connected in series from the N-terminus to the C-terminus.
  • first subunit/RBD-1 first subunit/RBD-1
  • second subunit/RBD-2 second subunit/RBD-2
  • third subunit third subunit
  • the three subunits of the recombinant novel coronavirus protein are connected in series in the order of the first subunit (RBD-1), the second subunit (RBD-2), and the third subunit.
  • the amino acid sequence of the above-mentioned recombinant novel coronavirus protein is as shown in SEQ ID No. 1-11 or a sequence having more than 95% homology with the amino acid sequence except the mutation site. More preferably, the amino acid sequence of the above-mentioned recombinant novel coronavirus protein is the amino acid sequence shown in SEQ ID No. 1 or a sequence having more than 95% homology with the amino acid sequence except the mutation site.
  • the amino acid sequence in SEQ ID No.1-11 except the mutation site can be replaced, deleted, or inserted with one or more amino acids to obtain a new amino acid sequence.
  • the new protein composed of the amino acid sequence has the same or substantially the same immunological activity as the protein composed of the amino acid sequence in SEQ ID No.1-11.
  • the new amino acid sequence is also considered to be included in the protection scope of the present invention.
  • sequence having more than 95% homology with it refers to an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the recombinant novel coronavirus protein or the fusion protein except for the mutation site.
  • Those skilled in the art can carry out random or engineered point mutations in an appropriate manner on the amino acid sequence of the fusion protein described in this specification. The purpose can be, for example, to obtain better affinity and/or dissociation properties, improve expression performance, etc.
  • sequences can have the same or substantially the same immunological activity as the recombinant novel coronavirus RBD trimer protein or the fusion protein, and these mutated amino acid sequences are all included within the protection scope of the present invention.
  • trimerization motifs can also be introduced into the trimer form of the above-mentioned recombinant novel coronavirus protein, for example, T4 bacteriophage fibritin, which is also considered to be within the protection scope of the present invention.
  • T4 bacteriophage fibritin which is also considered to be within the protection scope of the present invention.
  • its safety is weaker than that of the recombinant novel coronavirus protein in the present invention, and it may also lead to unexpected immune reactions.
  • Another aspect of the present invention is to provide a fusion protein, which comprises the above-mentioned recombinant novel coronavirus protein.
  • the fusion protein further comprises one or more selected from signal peptides, tags, or immune-enhancing peptides.
  • the function of the above-mentioned signal peptide can be more conducive to the expression of the protein; the above-mentioned label can be, for example, Flag tag, enhanced green fluorescent protein (eGFP), glutathione sulfhydryl transferase (GST), etc., and its function can be used for detection, purification, separation and the like.
  • the above functional sequences can be used in any combination.
  • Another aspect of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned recombinant novel coronavirus protein, or encoding the above-mentioned fusion protein.
  • the inventors optimized the codons of the trimeric protein, and the resulting nucleotide sequence was shown in SEQ ID No. 12-22 or a sequence having more than 95% homology therewith.
  • sequence having more than 95% homology with it refers to a nucleotide sequence having 95%, 96%, 97%, 98% or 99% identity with the said nucleotide sequence.
  • the method for preparing the above-mentioned nucleic acid molecule can be prepared by known techniques such as chemical synthesis or PCR amplification based on the above-mentioned nucleotide sequence.
  • the codons encoding the amino acids of the above-mentioned domains can be optimized to optimize their expression in host cells.
  • Information on the above base sequence can be obtained by searching known literature or databases such as NCBI (https://www.ncbi.nlm.nih.gov/).
  • Another aspect of the present invention is to provide a vector, the above-mentioned vector comprises the above-mentioned nucleic acid molecule.
  • the above-mentioned carrier may be a linear carrier or a circular carrier. It may be a non-viral vector such as a plasmid, or a viral vector (such as an adenovirus vector, a measles virus vector, a mumps virus vector, a rubella virus vector, a varicella virus vector, a poliovirus vector, a yellow fever virus vector), or a vector using a transposon.
  • the vector may contain regulatory sequences such as promoters and terminators, and marker sequences such as drug resistance genes and reporter genes.
  • the above-mentioned vector is an expression vector of the nucleic acid molecule described in the present invention, and is used to express the recombinant novel coronavirus protein of the present invention.
  • Another aspect of the present invention provides a host cell comprising the above nucleic acid molecule or the above vector.
  • the above-mentioned host cells are Escherichia coli, yeast cells, insect cells or mammalian cells;
  • the above-mentioned host cells are CHO cells.
  • Another aspect of the present invention provides a method for preparing the above-mentioned recombinant novel coronavirus protein or the above-mentioned fusion protein, comprising the following steps:
  • Step A) preparing the nucleic acid molecule, constructing the expression vector, transforming or transfecting the expression vector into the host cell;
  • Step B performing protein expression using the product of step A);
  • Step C) purifying the expression product obtained in step B) to obtain the above-mentioned recombinant novel coronavirus protein or fusion protein.
  • the nucleic acid molecule in step A) comprises a nucleotide sequence encoding the above-mentioned recombinant novel coronavirus protein, or encoding the above-mentioned fusion protein.
  • the above-mentioned nucleotide sequence is as shown in SEQ ID No. 12-24 or a sequence having more than 95% homology therewith.
  • the nucleic acid molecules may be prepared from the nucleotide sequences described in this specification using any suitable molecular biology method.
  • the construction of the expression vector in step A) can use any suitable method to construct the above nucleotide sequence in the corresponding expression vector of the host cell.
  • the expression vector is then transformed or transfected into the host cell.
  • the inventors after constructing the CHO cell expression vector, the inventors transfected it into HEK293FT cells or CHO cells to construct recombinant cell lines.
  • the protein expression in step B) can express the recombinant protein according to different expression systems used.
  • the inventors obtained cell lines capable of stably secreting and expressing recombinant novel coronavirus proteins or fusion proteins through screening by limiting dilution method.
  • the purification in step C) can be any suitable method.
  • suitable method for example, salting out, precipitation, dialysis or ultrafiltration, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and the like.
  • ion exchange and hydrophobic chromatography are used to purify the recombinant novel coronavirus protein or fusion protein.
  • the collection process of the target protein should also be included before the purification step, eg.
  • the collection of the supernatant of the cell culture medium rich in the target protein for example, any suitable disrupting method such as ultrasonic disruption, repeated freeze-thaw disruption, and chemical treatment can be used.
  • any suitable disrupting method such as ultrasonic disruption, repeated freeze-thaw disruption, and chemical treatment can be used.
  • the above-mentioned collection process of host cells should also be understood as included within the scope of the purification.
  • Another aspect of the present invention is to provide the above-mentioned recombinant new coronavirus protein, the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned vector or the use of the host cell in the preparation of drugs for treating and/or preventing new coronavirus infection and/or diseases caused by new coronavirus.
  • the disease caused by the novel coronavirus is preferably novel coronavirus pneumonia (COVID-19).
  • Another aspect of the present invention is to provide a vaccine, the above-mentioned vaccine comprising the above-mentioned recombinant novel coronavirus protein or the above-mentioned fusion protein, and an adjuvant.
  • the above-mentioned vaccine is a recombinant protein vaccine (or called a genetically engineered subunit vaccine).
  • the above-mentioned vaccine can also be a genetically engineered vector vaccine, or can be a nucleic acid vaccine, and the above-mentioned vaccine comprises the nucleotide sequence described in this specification or encodes the amino acid sequence described in this specification.
  • any suitable adjuvant may be included.
  • the above-mentioned adjuvant is aluminum hydroxide, aluminum phosphate, MF59 or CpG. More preferably, the above-mentioned adjuvant is aluminum hydroxide.
  • Another aspect of the present invention provides a method for preparing the above-mentioned vaccine, wherein the purified above-mentioned recombinant novel coronavirus protein or the above-mentioned fusion protein is mixed with the adjuvant.
  • Another aspect of the present invention provides the use of the above-mentioned vaccine in the treatment and/or prevention of novel coronavirus infection and/or diseases caused by novel coronavirus.
  • Another aspect of the present invention provides the above-mentioned recombinant new coronavirus protein, the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned carrier or the use of the host cell in the preparation of a drug for boosting immunization of people who have been vaccinated with the new coronavirus vaccine;
  • the new coronavirus vaccine is preferably an inactivated new coronavirus vaccine.
  • the disease caused by the novel coronavirus is preferably novel coronavirus pneumonia (COVID-19).
  • Another aspect of the present invention provides a pharmaceutical composition, which comprises the vaccine and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be any pharmaceutically acceptable additive, for example, physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and their compositions, stabilizers, surfactants, preservatives, isotonic agents, etc.
  • the pharmaceutical composition of the present invention can also be used in combination with other drugs for treating and/or preventing novel coronavirus infection and/or diseases caused by novel coronavirus at effective and safe doses.
  • Another aspect of the present invention provides a method for eliciting an immune response against a novel coronavirus in a subject or treating a subject for a novel coronavirus infection, by administering an effective dose of the vaccine or the pharmaceutical composition to the subject.
  • the aforementioned subjects may be humans or other animals.
  • the above-mentioned administration may be intramuscular injection, intraperitoneal injection or subcutaneous injection.
  • the recombinant new coronavirus protein of the present invention contains at least two artificially constructed non-natural RBD fragments, and the recombinant vaccine used as the target antigen has broad-spectrum protection ability across epidemic strains.
  • the multivalent broad-spectrum protection effect is achieved on one antigen molecule, which has obvious advantages in terms of time cost, economic cost and vaccine production capacity of vaccine preparation.
  • Figure 1 is a structural simulation diagram of the C05G12 protein in Example 1 of the present invention.
  • Lane 1 is C05G12 protein
  • M is a protein marker (molecular weight standards: kDa: 250, 130, 100, 70, 55, 35, 25, 15, 10);
  • Example 3 is a Western-blot identification result diagram of the protein purified in Example 2 of the present invention, wherein, lanes 4-6 are C05G12 proteins, and M is a protein marker (molecular weight standards: kDa: 250, 130, 100, 70, 55, 35, 25, 15, 10);
  • Fig. 4 is the binding curve of the recombinantly expressed protein and MM43 neutralizing monoclonal antibody in Example 3 of the present invention
  • Figure 5 is a graph showing the binding curve between the recombinantly expressed protein and MM57 neutralizing monoclonal antibody in Example 3 of the present invention.
  • Figure 6 is a graph showing the binding curve between the recombinantly expressed protein and MM117 neutralizing monoclonal antibody in Example 3 of the present invention.
  • Fig. 7 is the binding curve of the recombinantly expressed protein and R001 neutralizing monoclonal antibody in Example 3 of the present invention.
  • Figure 8 is a graph showing the binding curve between the recombinantly expressed protein and R117 neutralizing monoclonal antibody in Example 3 of the present invention.
  • Fig. 9 is a graph showing the binding curve between the recombinantly expressed protein and R118 neutralizing monoclonal antibody in Example 3 of the present invention.
  • Fig. 10 is the result figure of the neutralizing antibody titer of the mouse immune serum detected by wild virus microneutralization test in Example 5 of the present invention.
  • Fig. 11 is the result figure of the neutralizing antibody titer that utilizes pseudovirus microneutralization test to detect mouse immune serum in the embodiment of the present invention 5;
  • Fig. 12 is a graph showing the results of neutralizing antibody titer of rat immune serum detected by wild virus microneutralization test in Example 6 of the present invention.
  • SEQ ID No.1-11 is the amino acid sequence of the recombinant novel coronavirus protein in the embodiment of the present invention respectively;
  • SEQ ID No.12 is the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No.1, that is, the nucleotide sequence encoding the C05G12 protein;
  • SEQ ID No.13-22 are the nucleotide sequences encoding the amino acid sequence shown in SEQ ID No.2-11 respectively;
  • SEQ ID No.23 is the amino acid sequence of the C05 protein in Example 3 of the present invention.
  • SEQ ID No.24 is the amino acid sequence of the C05C protein in Example 3 of the present invention.
  • the invention discloses a recombinant novel coronavirus protein vaccine, its preparation method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to realize it. It should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously make changes or appropriate changes and combinations to the content described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.
  • RBD represents the RBD domain of the spike protein of the novel coronavirus, which can be understood as being interchangeable with “RBD” or "the novel coronavirus RBD region”.
  • new coronavirus that is, SARS-CoV-2, belongs to the order Nidovirales, Coronaviridae, Orthocoronavirus subfamily, Betacoronavirus genus, Sarbecovirus subgenus, SARS-like virus species, single-stranded positive-sense RNA virus, has an envelope, and the full length of the genome is about 29.9kb. protein), M protein (membrane protein), E protein (envelope protein) and N protein (nucleo protein), in addition to several accessory proteins: 3a, 3b, p6, 7a, 7b, 8b, 9b and orf14, these proteins are all involved in virus assembly.
  • the S, M and E proteins constitute the viral envelope and are the main surface antigens on which the virus induces an immune response.
  • the S protein is a transmembrane glycoprotein with a molecular weight of about 150kDa, which forms a prominent homotrimer on the surface of the virus.
  • S consists of two functional subunits that are cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage point), which remain non-covalently associated in the prefusion conformation.
  • the S2 subunit is also composed of multiple structural domains, and its function is mainly to mediate the fusion of the virus and the host cell.
  • the distal S1 subunit is structurally divided into four different structural domains: N-terminal domain (NTD), receptor-binding domain (RBD), C-terminal domain 1 (CTD1) and C-terminal domain 2 (CTD2).
  • NTD N-terminal domain
  • RBD receptor-binding domain
  • CCD1 C-terminal domain 1
  • CCD2 C-terminal domain 2
  • ACE2 receptor angiotensin converting enzyme 2
  • trimeric form is a type of higher order structure of proteins. Containing three protein subunits is the trimeric form.
  • At least one can be understood as two of the three amino acid sequences being the same or the three amino acid sequences being different.
  • primary structure is the linear sequence of amino acids in a peptide or protein.
  • primary structure of a protein refers to the amino-terminal (N) terminal to the carboxy-terminal (C) terminal.
  • fusion protein refers to the expression product of one, two or more gene recombination obtained by DNA recombination technology. Fusion protein technology is a purposeful gene fusion and protein expression method to obtain a large number of standard fusion proteins. Using fusion protein technology, new target proteins with multiple functions can be constructed and expressed.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector can 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.
  • the vector may also contain an origin of replication.
  • host cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection of viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • treating refers to reducing the possibility of disease pathology, reducing the occurrence of disease symptoms, for example, to the extent that the subject has a longer survival period or less discomfort.
  • Treatment can refer to the ability of a therapy to reduce symptoms, signs or causes of a disease when administered to a subject. Treating also refers to alleviating or reducing at least one clinical symptom and/or inhibiting or delaying the progression of a condition and/or preventing or delaying the onset of a disease or disorder.
  • subject refers to any human or other animal, especially other mammals, receiving prophylaxis, treatment, diagnosis.
  • Other mammals can include, for example, dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, and the like.
  • Example 1 Novel coronavirus RBD trimeric protein designed based on protein structure and computational biology
  • the new coronavirus continues to mutate, and the emergence of multiple mutant strains has led to multiple rounds of outbreaks.
  • a large number of studies have confirmed that many mutant strains have strong immune escape capabilities.
  • the recent outbreak of the Omicron mutant strain preliminary research evidence shows that the mutant strain has both a very strong ability to spread and a strong immune escape ability.
  • the emergence of strains with strong immune escape ability, especially the Omicron variant has caused great concern about the effectiveness of the existing new crown vaccine.
  • the development of a new generation of vaccines with cross-protection ability is an effective means to deal with these mutant strains. Integrating multiple mutant strains into the same immunogen to construct a mosaic-type antigen molecule is a feasible strategy for realizing a single-component cross-protective vaccine.
  • the receptor-binding domain (RBD) of the novel coronavirus spike (S) protein is directly involved in the binding of host cell receptors and plays a key role in the process of virus invasion of host cells.
  • RBD contains major neutralizing epitopes. Therefore, RBD is one of the main target antigens for the development of new crown vaccines.
  • RBD monomers are not highly immunogenic due to their small molecular size.
  • the natural S protein is a trimeric structure, and RBD also exists in a trimerized form. The trimerized RBD is constructed to simulate its natural structure to the greatest extent.
  • the molecular size of the antigen can be increased through trimerization to realize the repetitive and regular arrangement of the antigen and enhance the cross-linking of B cell receptors, which can significantly improve the immunogenicity of the RBD.
  • trimerization of antigens a common method is to introduce exogenous tethers or trimerization motifs.
  • exogenous sequences may bring about unexpected immune responses and has certain safety risks.
  • trimerization of the RBD without introducing a trimerization motif.
  • RBD has the following structural features: (1) The N-terminal and C-terminal of RBD have a long flexible loop structure.
  • RBD interception scheme that is, to intercept the 319-537 amino acid fragment.
  • This interception scheme retains the loop structure at both ends of the RBD as much as possible, and at the same time, ensures that the distance between the N-terminal and C-terminal interfaces is relatively close.
  • the three truncated RBD regions were connected end to end in series to form a new RBD trimerization fusion protein.
  • a mosaic-type RBD trimerization antigen was constructed, in which three RBDs integrated key mutation sites of various variants.
  • One of the RBDs is from the Omicron mutant strain, which contains 15 mutations including G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H.
  • the other two RBDs are artificially constructed non-natural RBDs that integrate key mutation sites that frequently appear in different mutant strains and have strong immune escape capabilities.
  • the mutation sites of various mutant strains of the new coronavirus were analyzed. Among the 20 mutation sites with the highest frequency, residue mutations with strong immune escape ability were selected. At the same time, the distribution of these mutations in the spatial structure was considered to avoid mutual influence in the spatial structure.
  • the two artificially constructed RBDs integrated a total of 8 key site mutations, one of which contained 5 residue mutations of K417N, L452R, T478K, F490S, and N501Y, and the other artificially constructed RBD contained 3 residue mutations of K417T, S477N, and E484K.
  • the specific series method was: the C-terminus of the RBD containing the "K417N, L452R, T478K, F490S and N501Y” mutations was connected to the N-terminus of the RBD containing the "K417T, S477N and E484K” mutations, and the RBD containing the "K417T, S477N and E484K” mutations The C-terminus of the Omicron mutant is connected to the N-terminus of the RBD.
  • SEQ ID No.1 is the C05G12 protein.
  • the possible spatial structure of the mosaic trimeric RBD fusion protein was constructed, and the results are shown in Figure 1.
  • the figure shows that the fusion protein contains three independent RBD domains, which can form a trimeric form with stable antigen conformation.
  • the protein contains a total of 23 mutation sites, and these mutation sites are shown in Figure 1 by a ball and stick model. It is theoretically speculated that the recombinant vaccine using this as the target antigen covers the key mutation sites that frequently appear in a large number of mutant strains, and has broad-spectrum protection across epidemic strains.
  • the nucleotide sequence encoding the recombinant protein C05G12 (amino acid sequence shown in SEQ ID NO.1) was codon optimized, and the optimized nucleotide sequence was shown in SEQ ID NO.12.
  • the CHO cell expression vector was constructed and then transfected into 293FT cells or CHO cells to construct a recombinant cell line.
  • the cell line capable of stably secreting and expressing the recombinant protein C05G12 was screened by the limiting dilution method.
  • the supernatant was harvested after cell culture, and the recombinant protein C05G12 with a purity of ⁇ 95% was obtained after serial chromatography purification.
  • the SDS-PAGE test results are shown in Figure 2.
  • the molecular weight of the protein is 70-100kD, and some product-related substances can be seen, such as dimer protein and monomer protein.
  • the purified C05G12 protein was electrophoresed by SDS-PAGE, it was transferred to PVDF membrane and identified by Western-blot using RBD-specific antibody (manufacturer: Beijing Yiqiao Shenzhou Technology Co., Ltd.; article number: 40591-T62; dilution: 2000 times) (the results are shown in Figure 3). It can be seen that C05G12 protein can bind to RBD-specific antibody and has good biological activity.
  • the purified C05G12 protein was analyzed by size exclusion chromatography using TSKgel G2500PW gel chromatography column, and the protein purity was greater than 90%.
  • C05G12 protein the amino acid sequence is shown in SEQ ID No. 23, obtained by recombinant expression in 293FT cells or CHO cells, and purified by chromatography
  • C05C protein the amino acid sequence is shown in SEQ ID No.
  • the prototype strain RBD protein (manufacturer: Beijing Sino Biological Science and Technology Co., Ltd.; product number: 40592-V08B), and RBD protein with the same mutation site of Beta strain virus (K417N, E484K, N501Y; manufacturer: Beijing Sino Biological Science and Technology Co., Ltd.; product number: 40592-V08H85), RBD protein with the same mutation site of Delta strain virus (L452R, T478K; manufacturer: Beijing Sino Biological Technology Co., Ltd.; product number: 40592-V02H3), and Omicron strain virus with the same mutation site RBD protein (G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H; manufacturer: Beijing
  • Figure 4 shows the binding activity to MM43 monoclonal antibody
  • Figure 5 shows the binding activity to MM57 monoclonal antibody
  • Figure 6 shows the binding activity to MM117 monoclonal antibody
  • Figure 7 shows the binding activity to R001 monoclonal antibody
  • Figure 8 shows the binding activity to R117 monoclonal antibody
  • Figure 9 shows the binding activity to R118 monoclonal antibody.
  • Embodiment 4 Preparation of recombinant novel coronavirus vaccine
  • the residual protein content of the supernatant should be less than 10% of the total protein content.
  • the prepared recombinant new coronavirus vaccine (wherein, the C05G12 protein vaccine is the vaccine prepared in Example 4 of the present invention; the C05 protein in the C05 protein vaccine is a recombinant protein in the form of a homotrimer composed of the 319th to 537th amino acid fragments of the S protein RBD region of the three original strains of the new coronavirus, and the C05 protein vaccine is prepared by the same method as in Example 4 of the present invention), and immunized by intraperitoneal injection.
  • BALB/c mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., SPF grade, female, 6-8 weeks old
  • 0.5 ⁇ g/dose/mouse specifically after immunization with inactivated vaccine for 1 injection at 0w, and then with 1 injection of recombinant new coronavirus vaccine or inactivated vaccine at 3w, blood was collected to separate serum at 4w.
  • the purpose of adopting this test program is to investigate and simulate the neutralizing ability against multiple mutant strains after booster immunization of the population that has been vaccinated with inactivated vaccines.
  • the neutralizing activity of mouse serum against the prototype strain, Beta strain, Delta strain and Omicron strain virus after immunization was detected by the wild virus microneutralization test.
  • the results are shown in Figure 10.
  • the GMT values of serum neutralizing antibodies are shown in Table 3. It can be seen that the C05G12 protein can produce a wide range of neutralizing activities against a variety of viruses.
  • the neutralization ability against the prototype strain, Beta strain, and Delta strain virus is equivalent to that of the C05 protein.
  • the neutralizing activities of strains, Delta strains, and Omicron strains were significantly higher than those of inactivated vaccines, and they are expected to produce broad-spectrum protection. They are ideal candidate vaccines for booster immunization.
  • the neutralizing activity of mouse serum against the prototype strain, Alpha strain, Beta strain, Delta strain, Gamma strain, Lambda strain, Mu strain and Omicron strain pseudovirus after immunization was detected by pseudovirus microneutralization test.
  • the results are shown in Figure 11.
  • the serum neutralizing antibody GMT values are shown in Table 4. It can be seen that the C05G12 protein can produce a wide range of neutralizing activities against a variety of pseudoviruses, against the prototype strain, Alpha strain, Beta strain, Delta strain, Gamma strain, and Lambda strain.
  • the neutralizing ability of the pseudovirus of the Mu strain and the C05 protein is equivalent to that of the C05 protein, and the neutralizing ability of the Omicron virus is significantly better than that of the C05 protein.
  • the neutralization activity of the prototype strain, Alpha strain, Beta strain, Delta strain, Gamma strain, Lambda strain, Mu strain and Omicron strain pseudovirus is significantly higher than that of the inactivated vaccine.
  • the prepared recombinant new coronavirus vaccine (wherein, the C05G12 protein vaccine is the vaccine prepared in Example 4 of the present invention; the C05 protein in the C05 protein vaccine is a recombinant protein in the form of a homotrimer composed of the 319th to 537th amino acid fragments of the S protein RBD region of three new coronavirus original strains, and the C05 protein vaccine is prepared by the same method as in Example 4 of the present invention; Recombinant protein in the form of a heterotrimer composed of 19-537 amino acid fragments, two subunits of which are from the Beta strain and the Kappa strain, and the C05C protein vaccine is prepared by the same method as in Example 4 of the present invention), and Wistar rats (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., SPF grade, female and male, 6-8 weeks old) that have been vaccinated with an inactivated vaccine (human dose) by intramuscular injection, respectively, 10 ⁇ g/dose/rat, specifically
  • the neutralizing activity of rat serum against prototype strain, Beta strain, Delta strain and Omicron strain virus was detected by wild virus microneutralization test. The results are shown in Figure 12.
  • the GMT values of serum neutralizing antibodies are shown in Table 5. It can be seen that C05G12 protein can produce a wide range of neutralizing activities against a variety of viruses.
  • the neutralization ability against prototype strain, Beta strain, and Delta strain virus is equivalent to C05 protein and C05C protein.
  • the C05C protein has significantly higher neutralizing activity against the prototype strain, Beta strain, Delta strain and Omicron strain virus than the inactivated vaccine, and is expected to produce broad-spectrum protection. It is an ideal candidate vaccine for booster immunization.

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Abstract

L'invention concerne une nouvelle protéine de coronavirus recombinante, comprenant au moins deux fragments de RBD non naturels construits artificiellement. Un vaccin recombinant l'utilisant en tant qu'antigène cible présente une capacité de protection à large spectre dans des souches épidémiques. La présente invention permet d'obtenir un effet de protection polyvalent à large spectre.
PCT/CN2022/142937 2022-01-21 2022-12-28 Vaccin à nouvelle protéine de coronavirus recombinante, son procédé de préparation et son application Ceased WO2023138334A1 (fr)

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