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CN119060197A - Novel coronavirus recombinant chimeric antigen, preparation method and application thereof - Google Patents

Novel coronavirus recombinant chimeric antigen, preparation method and application thereof Download PDF

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Publication number
CN119060197A
CN119060197A CN202410579155.9A CN202410579155A CN119060197A CN 119060197 A CN119060197 A CN 119060197A CN 202410579155 A CN202410579155 A CN 202410579155A CN 119060197 A CN119060197 A CN 119060197A
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chimeric antigen
rbd
recombinant
active fragment
ntd
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Inventor
罗士强
邓家荔
吴双
张元杰
郗永义
姚文荣
胡颖嵩
洪坤学
刘勇
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Abzymo Biosciences Co ltd
Jiangsu Ruike Biotechnology Co ltd
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Abzymo Biosciences Co ltd
Jiangsu Ruike Biotechnology Co ltd
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Publication of CN119060197A publication Critical patent/CN119060197A/en
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Abstract

本发明涉及一种新型冠状病毒重组嵌合抗原、其制备方法和应用。本发明所述重组嵌合抗原包含新型冠状病毒毒株的表面刺突蛋白的受体结合区RBD或其功能活性片段以及N端结构域NTD或其功能活性片段，其中所述受体结合区RBD或其功能活性片段与所述N端结构域NTD或其功能活性片段源自不同的新型冠状病毒毒株，且至少一个源自Omicron变异株。本发明的重组嵌合抗原能够更高效地激活广谱保护性抗体，对D614G变异株以及多种Omicron亚型变异株都能起到很好的预防或治疗效果。

The present invention relates to a recombinant chimeric antigen of a novel coronavirus, a preparation method and an application thereof. The recombinant chimeric antigen of the present invention comprises a receptor binding region RBD or a functionally active fragment thereof and an N-terminal domain NTD or a functionally active fragment thereof of a surface spike protein of a novel coronavirus strain, wherein the receptor binding region RBD or a functionally active fragment thereof and the N-terminal domain NTD or a functionally active fragment thereof are derived from different novel coronavirus strains, and at least one is derived from an Omicron variant. The recombinant chimeric antigen of the present invention can more efficiently activate broad-spectrum protective antibodies, and can have a good preventive or therapeutic effect on the D614G variant and a variety of Omicron subtype variants.

Description

Novel coronavirus recombinant chimeric antigen, preparation method and application thereof
Technical Field
The present application belongs to the field of biomedical engineering, in particular to recombinant chimeric antigens comprising a Receptor Binding Domain (RBD) or functionally active fragment thereof and an N-terminal domain (NTD) or functionally active fragment thereof derived from S proteins of different novel coronavirus strains, a method for their preparation and their use in the field of immunotherapy and prophylaxis.
Background
Infection with the novel coronavirus (SARS-CoV-2) can lead to acute respiratory infections. The novel coronavirus belongs to the genus beta-coronavirus of the family coronaviridae, has a envelope, and is a positive strand RNA virus.
The Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein (S protein) is considered to be the most predominant antigen target region for inducing the production of neutralizing antibodies by the body. The RBD can be used as a vaccine to focus the neutralizing antibodies generated by the stimulation of the organism on the receptor binding aiming at the virus, so that the immunogenicity and the immune efficiency of the vaccine can be improved. The N-terminal domain (NTD) of SARS-CoV-2 spike protein (S protein) is a sequence N-terminal to the viral S protein that binds to a protein or glycoprotein of a host cell, mediating viral invasion of the host cell, and thus may comprise an epitope that induces the production of neutralizing antibodies.
The novel coronavirus has a plurality of variant strains, wherein the number of the mutation sites of the omacron variant strain is as high as 37 on the virus S protein, and the mutant strains have serious immune escape to neutralizing antibody drugs and vaccine activated humoral immune response developed based on the novel coronavirus prototype strain. However, it has been reported that the vaccine developed with omacron sequence activates immune response, though strong against omacron variant strain, but weak against cross reaction of prototype strain and other strain, and does not adapt to the situation that the current prototype strain and various variant strains coexist and epidemic variant strain is still changing fast, so that it is necessary to develop a vaccine with strong protection effect against current global epidemic strain and capable of inducing broad-spectrum immune response, which can play a vital role in prevention and control of novel coronal infection.
Disclosure of Invention
The invention aims to provide a novel coronavirus recombinant chimeric antigen, an immunogenic composition containing the same, a preparation method and application. The recombinant chimeric antigen according to the invention is formed by the direct concatenation of a receptor binding domain RBD, or a functionally active fragment thereof, derived from the surface spike protein of at least one novel coronavirus strain, and an N-terminal domain NTD, or a functionally active fragment thereof, derived from the surface spike protein of another novel coronavirus strain, through a suitable linking sequence. The antibody can activate the broad-spectrum protective antibody more efficiently, and particularly has good prevention or treatment effects on prototype strains, D614G variant strains and various current omacron subtype variant strains.
The technical scheme of the invention is as follows:
The first aspect of the present invention provides a novel coronavirus recombinant chimeric antigen comprising a receptor binding domain RBD of a surface spike protein of a novel coronavirus strain or a functionally active fragment thereof and an N-terminal domain NTD or a functionally active fragment thereof, wherein said receptor binding domain RBD or a functionally active fragment thereof and said N-terminal domain NTD or a functionally active fragment thereof are derived from a different novel coronavirus strain and at least one from omacron variants.
In some embodiments, the omacron variant comprises at least one of subtype variants ba.1, ba.1.1, ba.2, ba.2.12.1, ba.3, ba.4, ba.5, bq.1.1, bf.7, xbb.1.5, xbb.1.16, eg.5, and jn.1, and the other strain is selected from at least one of a novel coronavirus prototype, alpha variant, beta variant, gamma variant, delta variant, lambda variant, mu variant.
In some embodiments, the RBD or functionally active fragment thereof is derived from at least one of a prototype strain, a Beta variant, a Delta variant, and an Omicron variant, and the NTD or functionally active fragment thereof is derived from an Omicron variant.
In some embodiments, the recombinant chimeric antigen comprises a first amino acid sequence A-B and a second amino acid sequence C-D of a receptor binding domain RBD, or functionally active fragment thereof, derived from surface spike proteins of different novel coronavirus strains, wherein C-D is chimeric directly or indirectly in A-B through substitution of amino acid residues in A-B through a linker sequence to form A- (C-D) n -B, n represents the number of amino acid sequences C-D, n is an integer greater than or equal to 1, preferably n is an integer selected from 1-10, more preferably an integer selected from 1-5.
In some embodiments, the recombinant chimeric antigen further comprises a third amino acid sequence E-F derived from the receptor binding domain RBD of the surface spike protein of a different novel coronavirus strain or a functionally active fragment thereof, wherein E-F is chimeric directly or indirectly via a linker sequence in A-B by substituting the amino acid residues in A-B to form A- (C-D) n-(E-F)m -B, m represents the number of amino acid sequences E-F, m is an integer of 1 or more, preferably m is an integer selected from 1 to 10, further preferably an integer selected from 1 to 5.
In some embodiments, the number of amino acid residues of C-D or E-F is 10N60, e.g., 10 amino acid residues, 15 amino acid residues, 20 amino acid residues, 25 amino acid residues, 30 amino acid residues, 35 amino acid residues, 40 amino acid residues, 45 amino acid residues, 50 amino acid residues, 55 amino acid residues, or 60 amino acid residues.
In some embodiments, the N-terminus of the RBD or functionally active fragment thereof is directly linked to the C-terminus of the NTD or functionally active fragment thereof or linked by a linker sequence.
In some embodiments, the omacron variant is selected from at least one of bq.1.1, bf.7, xbb.1.5, xbb.1.16, eg.5, and jn.1.
In some preferred embodiments, the recombinant chimeric antigen further comprises a foldon domain or functionally active fragment thereof.
In some embodiments, the N-terminus of the foldon domain or functionally active fragment thereof is directly linked to the C-terminus of the RBD or functionally active fragment thereof or is linked by a linker sequence, and the N-terminus of the RBD or functionally active fragment thereof is directly linked to the C-terminus of the NTD or functionally active fragment thereof or is linked by a linker sequence. In some preferred embodiments, the linker sequence is gsgsgsg.
In some embodiments, the RBD or functionally active fragment thereof of the recombinant chimeric antigen is derived from a bq.1.1 subtype variant of an Omicron variant, the NTD or functionally active fragment thereof is derived from a xbb.1.5 subtype variant of an Omicron variant, and the recombinant chimeric antigen is NTD (xbb.1.5) -RBD (bq.1.1). In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD (xbb.1.5) -RBD (bq.1.1) -foldon.
In some embodiments, the RBD or functionally active fragment thereof of the recombinant chimeric antigen is derived from an xbb.1.5 subtype variant of an Omicron variant, the NTD or functionally active fragment thereof is derived from a bq.1.1 subtype variant of an Omicron variant, and the recombinant chimeric antigen is NTD (bq.1.1) -RBD (xbb.1.5). In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD (bq.1.1) -RBD (xbb.1.5) -foldon.
In some embodiments, the RBD or functionally active fragment thereof of the recombinant chimeric antigen is derived from an xbb.1.16 subtype variant of an Omicron variant and the NTD or functionally active fragment thereof is derived from an xbb.1.5 subtype variant of an Omicron variant, and the recombinant chimeric antigen is NTD (xbb.1.5) -RBD (xbb.1.16). In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD (xbb.1.5) -RBD (xbb.1.16) -foldon.
In some embodiments, the RBD or functionally active fragment thereof of the recombinant chimeric antigen is derived from an xbb.1.5 subtype variant of an Omicron variant and the NTD or functionally active fragment thereof is derived from an xbb.1.16 subtype variant of an Omicron variant, and the recombinant chimeric antigen is NTD (xbb.1.16) -RBD (xbb.1.5). In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD (xbb.1.16) -RBD (xbb.1.5) -foldon.
In some embodiments, the RBD or functionally active fragment thereof of the recombinant chimeric antigen is derived from a prototype strain and an Omicron variant strain of subtype bq.1.1, and the NTD or functionally active fragment thereof is derived from an Omicron variant strain of subtype xbb.1.5, and the recombinant chimeric antigen is NTD (xbb.1.5) -RBD (prototype strain-bq.1.1). In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD (xbb.1.5) -RBD (prototype strain-bq.1.1) -foldon.
In some embodiments, the RBD or functionally active fragment thereof of the recombinant chimeric antigen is derived from a prototype strain and an xbb.1.5 subtype variant of an Omicron variant, and the NTD or functionally active fragment thereof is derived from a bq.1.1 subtype variant of an Omicron variant, and the recombinant chimeric antigen is NTD (bq.1.1) -RBD (prototype strain-xbb.1.5). In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD (bq.1.1) -RBD (prototype strain-xbb.1.5) -foldon.
In some embodiments, the RBD or functionally active fragment thereof of the recombinant chimeric antigen is derived from a prototype strain and a bq.1.1h and bf.7 subtype variant strain of an Omicron variant strain, and the NTD or functionally active fragment thereof is derived from an xbb.1.5 subtype variant strain of an Omicron variant strain, and the recombinant chimeric antigen is NTD (xbb.1.5) -RBD (prototype strain-bq.1.1-bf.7). In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD (xbb.1.5) -RBD (prototype strain-bq.1.1-bf.7) -foldon.
In some embodiments, the RBD or functionally active fragment thereof of the recombinant chimeric antigen is derived from a prototype strain and an xbb.1.5 and bf.7 subtype variant strain of an Omicron variant strain, and the NTD or functionally active fragment thereof is derived from a bq.1.1 subtype variant strain of an Omicron variant strain, and the recombinant chimeric antigen is NTD (bq.1.1) -RBD (prototype strain-xbb.1.5-bf.7). In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD (bq.1.1) -RBD (prototype strain-xbb.1.5-bf.7) -foldon.
Similarly, the recombinant chimeric antigen may also be NTD(XBB.1.5)-RBD(Delta-BQ.1.1)、NTD(BQ.1.1)-RBD(Delta-XBB.1.5)、NTD(XBB.1.5)-RBD(Delta-BQ.1.1-BF.7)、NTD(BQ.1.1)-RBD(Delta-XBB.1.5-BF.7)、NTD(BQ.1.1)-RBD(Delta-EG.5)、NTD(BQ.1.1)-RBD(Delta-JN.1) or the like. In the case where the recombinant chimeric antigen comprises a foldon domain or functionally active fragment thereof, the recombinant chimeric antigen is NTD(XBB.1.5)-RBD(Delta-BQ.1.1)-foldon、NTD(BQ.1.1)-RBD(Delta-XBB.1.5)-foldon、NTD(XBB.1.5)-RBD(Delta-BQ.1.1-BF.7)-foldon、NTD(BQ.1.1)-RBD(Delta-XBB.1.5-BF.7)-foldon、NTD(BQ.1.1)-RBD(Delta-EG.5)-foldon、NTD(BQ.1.1)-RBD(Delta-JN.1)-foldon or the like.
In some embodiments, the recombinant chimeric antigen has the amino acid sequence set forth in any one of SEQ ID NOs 1-3.
In a second aspect the invention provides a recombinant nucleic acid comprising a polynucleotide sequence encoding said recombinant chimeric antigen, said polynucleotide being a codon optimised nucleotide sequence, which may be DNA, mRNA or circRNA. Preferably, the DNA sequence of the recombinant nucleic acid is shown in any one of SEQ ID NOs 6 to 8.
In a third aspect the present invention provides a nucleic acid construct comprising a recombinant nucleic acid as described in the second aspect above, and optionally at least one expression control element operably linked to the recombinant nucleic acid.
In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid construct as described in the third aspect above.
In a fifth aspect the invention provides a host cell comprising a nucleic acid encoding a recombinant chimeric antigen as described in the second aspect above, a nucleic acid construct as described in the third aspect above or an expression vector as described in the fourth aspect above, said host cell comprising a mammalian cell, an insect cell, a yeast cell or a bacterial cell, optionally said mammalian cell comprising a HEK293T cell, a HEK293F cell, an Expi293F cell or a CHO cell, optionally said bacterial cell comprising an e.
In a sixth aspect the present invention provides a method of preparing a recombinant chimeric antigen according to the first aspect comprising the steps of:
(1) Constructing a recombinant expression plasmid by utilizing a nucleotide sequence for encoding the novel coronavirus recombinant chimeric antigen;
(2) Transforming the constructed recombinant expression plasmid into host bacteria, and screening the correct recombinant expression plasmid;
(3) Transfecting a mammalian host cell with the recombinant expression plasmid obtained by screening for expression;
(4) Collecting cell culture supernatant and purifying to obtain the recombinant chimeric antigen.
In a seventh aspect the present invention provides an immunogenic composition comprising a recombinant chimeric antigen as described in the first aspect, a recombinant nucleic acid as described in the second aspect, a nucleic acid construct as described in the third aspect, an expression vector as described in the fourth aspect or a host cell as described in the fifth aspect.
In some embodiments, the immunogenic composition comprises a first component comprising an NTD or functionally active fragment thereof derived from the XBB.1.5 variant and an RBD or functionally active fragment thereof derived from the prototype strain, i.e., NTD (XBB.1.5) -RBD (prototype strain), and a second component comprising an NTD or functionally active fragment thereof derived from the BQ.1.1 variant and an RBD or functionally active fragment thereof derived from the Delta variant, i.e., NTD (BQ.1.1) -RBD (Delta). In a preferred embodiment, each component further comprises a foldon domain or functionally active fragment thereof, i.e. the immunogenic composition comprises two components, NTD (xbb.1.5) -RBD (prototype strain) -foldon and NTD (bq.1.1) -RBD (Delta) -foldon.
In some embodiments, the immunogenic composition comprises a first component comprising an NTD or functionally active fragment thereof derived from an XBB.1.5 variant and an RBD or functionally active fragment thereof derived from a prototype strain, i.e., NTD (XBB.1.5) -RBD (prototype strain), and a second component comprising an NTD or functionally active fragment thereof derived from an XBB.1.16 variant and an RBD or functionally active fragment thereof derived from a Delta variant, i.e., NTD (XBB.1.16) -RBD (Delta). In a preferred embodiment, each component further comprises a foldon domain or functionally active fragment thereof, i.e. the immunogenic composition comprises two components, NTD (xbb.1.5) -RBD (prototype strain) -foldon and NTD (xbb.1.16) -RBD (Delta) -foldon.
Similarly, the immunogenic composition may comprise at least one component selected from the group consisting of NTD (xbb.1.5) -RBD (prototype strain -BF.7)、NTD(XBB.1.5)-RBD(Delta-BF.7)、NTD(BQ.1.1)-RBD(Delta-XBB.1.5)、NTD(XBB.1.16)-RBD(Delta-BF.7)、NTD(XBB.1.5)-RBD( prototype strain-xbb.1.16), NTD (xbb.1.5) -RBD (Delta-xbb.1.16), and the like. In a preferred embodiment, each component further comprises a foldon domain or functionally active fragment thereof, i.e., each component may be NTD (xbb.1.5) -RBD (prototype strain -BF.7)-foldon、NTD(XBB.1.5)-RBD(Delta-BF.7)-foldon、NTD(BQ.1.1)-RBD(Delta-XBB.1.5)-foldon、NTD(XBB.1.16)-RBD(Delta-BF.7)-foldon、NTD(XBB.1.5)-RBD( prototype strain-xbb.1.16) -foldon, NTD (xbb.1.5) -RBD (Delta-xbb.1.16) -foldon, or the like. In some embodiments, the immunogenic composition may further comprise NTD or functionally active fragment thereof and RBD or functionally active fragment thereof derived from the same strain, e.g., NTD (prototype strain) -RBD (prototype strain), NTD (Delta) -RBD (Delta), NTD (prototype strain) -RBD (prototype strain) -foldon, NTD (Delta) -RBD (Delta) -foldon, etc.
According to an eighth aspect of the present invention there is provided a novel coronavirus recombinant protein vaccine comprising the recombinant chimeric antigen as described in the first aspect above, or the immunogenic composition as described in the seventh aspect above, and an adjuvant selected from one or any combination of aluminium adjuvants, lipopolysaccharides, saponins, polyI: C, cpG and emulsion adjuvants.
In some embodiments, the aluminum adjuvant is selected from aluminum hydroxide and aluminum phosphate.
In some embodiments, the saponin is selected from the group consisting of QS-7, QS-17, QS-18, QS21, TQL-1055, and the like. In a preferred embodiment, the saponin is selected from QS21 or TQL-1055.
In some embodiments, the adjuvant is a liposome adjuvant or emulsion adjuvant comprising a saponin.
In some embodiments, the adjuvant in a human dose comprises QS21 in an amount of 5-50 μg, for example 6μg、7μg、8μg、9μg、10μg、11μg、12μg、13μg、14μg、15μg、16μg、17μg、18μg、19μg、20μg、21μg、22μg、23μg、24μg、25μg、26μg、27μg、28μg、29μg、30μg、31μg、32μg、33μg、34μg、35βg、36μg、37μg、38μg、39μg、40μg、41μg、42μg、43μg、44μg、44μg、45μg、46μg、47μg、48μg or 49 μg.
In some embodiments, the lipopolysaccharide comprises gram negative bacterial lipopolysaccharide LPS, glucopyranosyl Lipid A (GLA), MPL (3D-MPL).
In some embodiments, the adjuvant for human dosage comprises MPL in an amount of 1-50 μg, e.g. 2μg、3μg、4μg、5μg、6μg、7μg、8μg、9μg、10μg、11μg、12μg、13μg、14μg、15μg、16μg、17μg、18μg、19μg、20μg、21μg、22μg、23μg、24μg、25μg、26μg、27μg、28μg、29μg、30μg、31μg、32μg、33μg、34μg、35μg、36μg、37μg、38μg、39μg、40μg、41μg、42μg、43μg、44μg、44μg、45μg、46μg、47μg、48μg or 49 μg.
In some embodiments, the liposome adjuvant comprising a saponin specifically comprises QS21 and MPL.
In some embodiments, the human dose of the immunogenic composition comprises 25 μg MPL and 25 μg gQS a 21. In other embodiments, the human dose of the immunogenic composition comprises 10 μg MPL and 10 μg QS21.
In some embodiments, the liposome contains a neutral lipid, such as phosphatidylcholine, which may be selected from, for example, egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), or dilauroyl phosphatidylcholine.
In some embodiments, the liposomes contain sterols, suitable sterols include p-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. In a preferred embodiment, the adjuvant composition comprises cholesterol as sterol.
In some embodiments, the liposome adjuvant comprising a saponin comprises TQL-1055 and MPL.
In a preferred embodiment 1 human dose of AS01 adjuvant is used with a PBS buffer comprising 50 μg MPL, 50 μg QS21, 1mg DOPC and 0.25mg cholesterol per 0.5 ml.
In some embodiments, the emulsion comprises an oil phase and an aqueous phase.
In some embodiments, the oil phase comprises a metabolizable oil, preferably, the metabolizable oil is squalene.
In some embodiments, wherein the oil phase further comprises alpha-tocopherol.
In some embodiments, wherein the weight ratio of squalene to alpha-tocopherol is 0.8-1, e.g. 0.85-0.95, preferably 0.9, 1 human dose AS03 is used having an emulsion composition of 4.86mg polysorbate 80 (tween 80), 10.69mg squalene and 11.86mg alpha-tocopherol per 0.5 ml.
In some embodiments, wherein the oil phase further comprises span 85, preferably the oil-in-water emulsion is MF59, comprising 1% squalene, 0.5% tween 80, and 0.5% span 85.
In some embodiments, wherein the weight ratio of squalene to span 85 is 8-10, preferably 8.4.
In some embodiments, the emulsion adjuvant comprising a saponin comprises QS21 and MPL.
In some embodiments, the emulsion adjuvant comprises squalene, tocopherol, and tween 80.
In some embodiments, the emulsion adjuvant is an oil-in-water emulsion comprising squalene, span 85, and tween 80.
In some embodiments, the adjuvant comprises an aluminum adjuvant and 3D-MPL.
In some embodiments, the adjuvant comprises an emulsion and nano-aluminum particles uniformly dispersed in the emulsion.
In some embodiments, the emulsion comprises squalene, tocopherol, tween 80, and span 85.
In some embodiments, the adjuvant comprises PolyI:C and 3D-MPL.
In a ninth aspect the present invention provides a novel coronavirus recombinant nucleic acid vaccine comprising a recombinant nucleic acid as described in the second aspect.
In some embodiments, the recombinant nucleic acid vaccine is a DNA vaccine comprising a eukaryotic expression vector and a DNA sequence encoding the recombinant chimeric antigen as described in the first aspect above constructed into the eukaryotic expression vector, optionally the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS, and pCDNA series vectors.
In some embodiments, the recombinant nucleic acid vaccine is an mRNA vaccine comprising an mRNA sequence encoding the recombinant chimeric antigen described in the first aspect above and a Lipid Nanoparticle (LNP), the mRNA being encapsulated in the Lipid Nanoparticle (LNP).
In some embodiments, the recombinant nucleic acid vaccine is a circRNA vaccine comprising a circular RNA sequence encoding the recombinant chimeric antigen as described in the first aspect above.
In a tenth aspect the present invention provides the use of a recombinant chimeric antigen as described in the first aspect, a recombinant nucleic acid as described in the second aspect, a nucleic acid construct as described in the third aspect, an expression vector as described in the fourth aspect, a host cell as described in the fifth aspect, an immunogenic composition as described in the seventh aspect, a novel coronavirus recombinant protein vaccine as described in the eighth aspect, a novel coronavirus recombinant nucleic acid vaccine as described in the ninth aspect for the manufacture of a medicament for the prevention of novel coronavirus infection.
Compared with the prior art, the invention has the following beneficial effects:
the invention screens out NTD sequences and RBD sequences with immune advantage, has strong immunogenicity and broad spectrum of corresponding recombinant chimeric antigen, can provide balanced and efficient protection effect for different strains of novel coronavirus, especially various Omicron variant strains, even Alpha, beta and Delta variant strains which commonly contain a key mutation site D614G.
Drawings
The following drawings are only for purposes of illustration and explanation of the present invention and are not intended to limit the scope of the invention. Wherein:
FIG. 1 is an absorbance curve of NTD (BQ.1.1) -RBD (XBB.1.5) -foldon recombinant chimeric protein purified by HPLC-SEC as described in example 2 of the present invention, and SDS-PAGE and WB identification results of the collected elution peaks.
FIG. 2 is an absorbance curve of NTD (XBB.1.5) -RBD (prototype strain-BQ.1.1-BF.7) -foldon recombinant chimeric protein purified by HPLC-SEC, and SDS-PAGE and WB identification results of collected elution peaks, described in example 2 of the present invention.
FIG. 3 is an absorbance curve of NTD (BQ.1.1) -RBD (prototype strain-XBB.1.5-BF.7) -foldon recombinant chimeric protein purified by HPLC-SEC, and SDS-PAGE and WB identification results of collected elution peaks, described in example 2 of the present invention.
FIG. 4 is a graph showing the absorbance curves of NTD (BQ.1.1) -RBD (BQ.1.1) -foldon recombinant chimeric proteins purified by HPLC-SEC and the SDS-PAGE and WB identification results of the collected elution peaks, as described in example 2 of the present invention.
FIG. 5 is an absorbance curve of NTD (XBB.1.5) -RBD (XBB.1.5) -foldon recombinant chimeric protein purified by HPLC-SEC as described in example 2 of the present invention, and SDS-PAGE and WB identification results of the collected elution peaks.
FIG. 6 shows the results of neutralizing antibody titers of pseudoviruses of various novel coronavirus variants in serum collected 14 days after the second immunization of mice according to example 5 of the present invention.
FIG. 7 shows the results of neutralizing antibody titers of pseudoviruses of various novel coronavirus variants in serum collected 14 days after the first immunization of a bivalent vaccine in mice according to example 6 of the present invention.
Detailed Description
The invention will be further illustrated by the following non-limiting examples, which are well known to those skilled in the art, that many modifications can be made to the invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as embodiments are necessarily varied. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, the scope of the present invention being defined in the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Preferred methods and materials of the invention are described below, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. The following experimental methods are all methods described in conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.
Definition of terms
Reference throughout this specification to "one embodiment" means that a particular parameter, step, or the like described in that embodiment is included in at least one embodiment according to the present application. Thus, references to "one embodiment according to the present application," "in an embodiment," and the like are not intended to be interpreted as referring to the same embodiment, and references to "in another embodiment," "in a different embodiment according to the present application," "in another embodiment," and the like are not intended to mean that the recited feature is included in only a specific different embodiment. It will be appreciated by those of skill in the art that the specific parameters, steps, etc. disclosed in one or more of the embodiments of the application can be combined in any suitable manner.
In the present application, the term "foldon domain" generally refers to a residue at the C-terminal end of bacteriophage T4 fibrin. In the present application, the foldon domain may be 27 residues or mutants of the C-terminal end of bacteriophage T4 fibrin. In the present application, the foldon domain may be a truncated or increased N-terminal or C-terminal 1, 2, 3, 4, 5, or 6 or 10 amino acid truncated or increased 27 residues from the C-terminal end of bacteriophage T4 fibrin. In the present application, "mutant" generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids.
In the present application, the term "adjuvant" refers to a substance having an immune response enhancing function that is clinically applicable to the human body or has a prospect of application to the human body, and includes various adjuvants that are currently approved and may be approved in the future, such as, but not limited to, aluminum adjuvants and various forms of adjuvant compositions.
In the present application, the term "comprising" generally means containing, summarizing, containing or comprising. In some cases, the meaning of "as", "consisting of.
EXAMPLE 1 construction of recombinant expression plasmid
The constructs shown in Table 1 were designed by directly concatenating NTD sequences and RBD sequences/RBD chimeric sequences derived from different strains, ligating a signal peptide (shown as SEQ ID NO: 11) at the N-terminus thereof, ligating a foldon domain (shown as SEQ ID NO: 12) at the C-terminus thereof via the ligating sequence, and finally adding 6 histidine tags (HHHHHH) at the C-terminus of the foldon domain to give the constructs shown in Table 1.
Table 1 construction scheme for each construct
Amino acid sequence Recombinant expression plasmid DNA sequence
SEQ ID NO:1 NTD(BQ.1.1)-RBD(XBB.1.5)-foldon-6*His SEQ ID NO:6
SEQ ID NO:2 NTD (xbb.1.5) -RBD (prototype strain-bq.1.1-bf.7) -foldon-6 his SEQ ID NO:7
SEQ ID NO:3 NTD (BQ.1.1) -RBD (prototype strain-XBB.1.5-BF.7) -foldon-6 x his SEQ ID NO:8
SEQ ID NO:4 NTD(BQ.1.1)-RBD(BQ.1.1)-foldon-6*His SEQ ID NO:9
SEQ ID NO:5 NTD(XBB.1.5)-RBD(XBB.1.5)-foldon-6*His SEQ ID NO:10
The amino acid sequence of the above construct was codon optimized according to the host CHO cell.
Cloning fragments, namely amplifying primers of each construct by a PCR method to obtain fragment PCR products, and recombining the fragment PCR products into a target vector pcDNA3.1 (+) (BamHI-XhoI digestion vector) by a multistage recombination method to obtain each recombinant expression plasmid. The reaction system is shown in Table 2, and the connection solution was connected for 30 minutes at a constant temperature of 52 ℃.
TABLE 2 ligation reaction system of treated target fragment and vector
Name of the name Volume of
Carrier after enzyme cutting 5μl
Purified PCR products 5μl
Seamless assembled MIX 10μl
Total volume of 20μl
Transformation 1-3. Mu.l of each recombinant expression plasmid described in example 1 was added to 100. Mu.l of competent cells at a concentration of 100 ng/. Mu.l, gently swirled and rotated to mix, placed on ice for 3 minutes, placed in a 42℃water bath for 90s without shaking, placed in an ice bath for about 3 minutes, and added with 500-800. Mu.l of LB medium pre-warmed at 37℃per tube, and gently shaken at 200rpm for 40 minutes in a 37℃shaker.
Preparing agar plate with relative resistance, spreading 100 mu l bacterial liquid on the surface of agar plate, using aseptic glass spreader to lightly spread bacteria on the surface of plate, placing the plate at 37 deg.C for 15 min, inverting the plate for 12-16 h, culturing at 37 deg.C for 12-16 h to form colony, selecting bacteria, shaking at 37 deg.C for 14 h at 250r/min, PCR identification with bacterial liquid, and sequencing the positive clone bacterial liquid. The correct plasmid was sequenced and aligned, and double digestion was performed with BamHI-XhoI to obtain the desired fragment.
Recombinant expression plasmid extraction 1% of E.coli cells (Stbl 3) containing each recombinant expression plasmid were inoculated in 2ml of LB medium and cultured overnight at 37℃with shaking. After the cells were treated, 100. Mu.g of plasmid was extracted using a plasmid extraction kit. The correct plasmid was sequenced and aligned, and double digestion was performed with BamHI-XhoI to obtain the desired fragment.
EXAMPLE 2 cell transfection and protein purification
Cell transfection the resulting plasmid was introduced into CHO cells by transient transfection, and the protein was collected and purified after culture. CHO cells (EXPICHO from Thermo) were subcultured and expanded with ExpiCHO Expression Medium, gently swirled to mix host cells during all cell manipulations, avoiding vigorous mixing/pipetting. CHO cells were subcultured and expanded until the cell density reached 4 x 10 6～6×106/mL. On day-1, cells were expanded, cultured to 3X 10 6N4×106 cells/mL, and allowed to grow overnight. On day 0, cells were transfected, viable cell density and viability were determined, cell density should be 7X 10 6～10×106/mL, viability was 95-99% transfected, fresh cell expression medium, pre-warmed to 37℃and cells diluted to a final density of 6X 10 6/mL, 50mm amplitude incubator at 90rpm, 37℃, the culture was performed under the condition of 8% CO 2. Preparation of transfection reagent and recombinant expression plasmid complexes (4 ℃) using OPti-PRO SFM Medium 1ml of cells were prepared 40. Mu. l OPti-PRO SFM added with 0.8. Mu.g of the corresponding recombinant expression plasmid, mixed well and placed for 5min, prepared 40. Mu. l OPti-PRO SFM added with 3. Mu. l Expi Fectamine CHO reagent, mixed well and placed for 5min, at room temperature for 1-5 min, then the solution was slowly transferred to shake flask and shaken, during addition, the shake flask was gently shaken. Cells were incubated in a 50mm amplitude incubator at 90rpm, 37℃at 8% CO 2. 18-22 hours after transfection, add the enhancers and ExpiCHO Feed, perform standard protocols, e.g., 1ml cells with 6. Mu. L ENHANCER and 0.24. 0.24ml ExpiCHO Feed, place the cells in a 50mm amplitude incubator at 90rpm for 37℃culture, 8% CO 2. Collecting after 8 days of transfection, purifying by HPLC-SEC to obtain trimeric protein with purity more than or equal to 95%, namely the required recombinant chimeric purified protein.
SDS-PAGE and Western Blot verification are carried out on the obtained recombinant chimeric purified protein, and the results are shown in figures 1-5.
EXAMPLE 3 preparation of recombinant protein vaccine
Each recombinant chimeric purified protein obtained in example 2 was diluted to 80. Mu.g/ml with a diluent (1 XPBS buffer) and thoroughly mixed with an equal volume of AS03 adjuvant, wherein the adjuvant components were 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg polysorbate 80, 3.53mg sodium chloride, 0.09mg potassium chloride, 0.51mg disodium hydrogen phosphate, 0.09mg monopotassium phosphate and water for injection per 0.5 ml.
EXAMPLE 4 construction of pseudoviruses
The test and detection limited company of the national Country (Beijing) entrusts to construct the pseudoviruses of D614G, omicron-BQ.1.1 and Omicron-XBB.1.5, and carries out corresponding detection for later in-vitro neutralization test.
EXAMPLE 5 immunogenicity evaluation of vaccine
BALB/c mice (females, 6-8 weeks old, purchased from the institute of laboratory animals at the national academy of medicine) were used for group immunization, randomly divided into 5 groups of 5 mice each, and first received basic immunization (2-needle inactivated vaccine, 21 days apart). The 1 st needle booster immunization was performed 10 months after the basal immunization, and blood was collected and tested 14 days later. The 2 nd needle booster immunization was performed 21 days after the 1 st needle booster immunization, and blood was collected and tested 14 days after the 1 st needle booster immunization. At boost, 100 μl of immune sample (containing 4 μg antigen and 1/10 of the human being immunized with dose AS03 adjuvant) was injected per muscle. Collected blood samples were left at 37℃for 1 hour and at 4℃for 1 hour, centrifuged at 8000r/min for 10 minutes, and serum was collected and stored at-20 ℃. The grouping and immunizing dose of the mice are shown in Table 3.
Table 3 novel coronavirus recombinant chimeric protein vaccine immunized mice group and dose
Using the novel coronavirus pseudoviruses constructed in example 4, the collected immunized mouse serum was each tested for 50% pseudovirus neutralization titers of pseudoviruses for each variant of the novel coronavirus, including the D614G, omicron-BQ.1.1 and Omicron-XBB.1.5 pair variants.
The method for detecting neutralizing antibody titer of novel coronavirus pseudovirus (hereinafter referred to as pseudovirus) comprises diluting immune mouse serum with 2-fold gradient ratio in 96-well plate, mixing with pseudovirus, and incubating at 37deg.C for 1 hr. The immune serum-pseudovirus mixture was transferred to 96-well plates that were confluent with Vero cells. After 15 hours, positive cell numbers were calculated by a CQ1 confocal cell imager (Yokogawa), then fitted curves were drawn in GRAPHPAD PRISM software, and the reciprocal of the corresponding serum dilution at 50% neutralization was calculated as 50% pseudovirus neutralization titer. The results of the pseudovirus neutralizing antibody titer measurements are shown in FIG. 6 (groups 1-5 in sequence from left to right).
The results show that for the D614G strain, the neutralizing antibody titers GMT for the NTD (bq.1.1) -RBD (xbb.1.5) -foldon (group 1), NTD (xbb.1.5) -RBD (prototype strain-bq.1.1-bf.7) -foldon (group 2) and NTD (bq.1.1) -RBD (prototype strain-xbb.1.5-bf.7) -foldon (group 3) were 12265, 44723 and 18627, respectively, significantly higher than for the non-chimeric antigens NTD (bq.1.1) -RBD (bq.1.1) -foldon (group 4) and NTD (xbb.1.5) -RBD (xbb.1.5) -foldon (group 5) 7791 and 2233, respectively, indicating that the chimeric antigens of the invention can significantly increase neutralizing antibody levels against the D614G strain.
For BQ.1.1 strain, the neutralizing antibody titer GMT for NTD (BQ.1.1) -RBD (BQ.1.1) -foldon (group 4) was highest (up to 39074), and it was expected that the neutralizing antibody titers GMT for NTD (BQ.1.1) -RBD (XBB.1.5) -foldon (group 1) and NTD (BQ.1.1) -RBD (prototype-XBB.1.5-BF.7) -foldon (group 3) were also higher, 11355 and 7826, respectively, than for non-chimeric antigen NTD (XBB.1.5) -RBD (XBB.1.5) -foldon (group 5) and chimeric antigen NTD (XBQ.1.5) -RBD (prototype-BQ.1.1-BF.7) -foldon (group 2).
For the XBB.1.5 strain, unexpectedly, the neutralizing antibody titers GMT for NTD (BQ.1.1) -RBD (XBB.1.5) -foldon (group 1) were highest (up to 15728), even higher than 7737 for NTD (XBB.1.5) -RBD (XBB.1.5) -foldon (group 5), the neutralizing antibody titers GMT for NTD (XBB.1.5) -RBD (prototype-BQ.1.1-BF.7) -foldon (group 2) and NTD (BQ.1.1) -RBD (BQ.1.1) -foldon (group 4) were 2906 and 1973, respectively, and the neutralizing antibody titers GMT for NTD (BQ.1.1) -RBD (prototype-XBB.1.5) -foldon (group 3) were lowest, only 610.
Taken together, the chimeric antigens NTD (bq.1.1) -RBD (xbb.1.5) -foldon (group 1) of the invention have a greater neutralizing antibody titer GMT for both D614G and xbb.1.5 strains than the non-chimeric antigens NTD (bq.1.1) -RBD (bq.1.1) -foldon (group 4), and the three strains D614G, bq.1.1 and xbb.1.5 have a greater neutralizing antibody titer GMT for both non-chimeric antigens NTD (xbb.1.5) -RBD (xbb.1.5) -foldon (group 5); the neutralizing antibody titer GMT of the chimeric antigen NTD (XBB.1.5) -RBD (prototype strain-BQ.1.1-BF.7) -foldon (group 2) of the invention against both strains D614G and XBB.1.5 is greater than that of the non-chimeric antigen NTD (BQ.1.1) -RBD (BQ.1.1) -foldon (group 4), the neutralizing antibody titer GMT of the chimeric antigen NTD (BQ.1.1) -RBD (XBB.1.5) -foldon (group 5) of the invention against one strain of D614G is greater than that of the non-chimeric antigen NTD (BQ.1.5) -RBD (XBB.1.5) -foldon (group 5), the neutralizing antibody titer GMT of the chimeric antigen NTD (BQ.1.1.1) -RBD (prototype strain-XBB.1.5-BF.7) -foldon (group 3) of the invention against both strains of the non-chimeric antigen NTD (BQ.1.1.1) -RBD (group 1.5) is greater than that of the non-chimeric antigen NTD (BQ.1.1) -RBD (group 1.5) of the invention against both strains of the chimeric antigen (BQ.1.1-BQ.1) -RBD (group 5) of the chimeric antigen (BQ.1.1.1-BB.5) of the chimeric antigen of the invention against both strains of the chimeric antigen of the strain of the invention, this suggests that the chimeric antigens of the invention can provide a more balanced and efficient protection against different strains of novel coronaviruses, especially various omacron variants (e.g., bq.1.1, xbb.1.5, xbb.1.16, etc.), even Alpha, beta, and Delta variants that together contain the critical mutation site D614G.
EXAMPLE 6 evaluation of immunogenicity of bivalent vaccine
BALB/c mice (females, 6-8 weeks old, purchased from the institute of laboratory animals at the national academy of medicine) were used for group immunization, randomly divided into 5 groups of 5 mice each, and first received basic immunization (2-needle inactivated vaccine, 21 days apart). The 1 st needle booster immunization was performed 10 months after the basal immunization, and blood was collected and tested 14 days later. At boost, 100. Mu.l of immune sample (containing 4. Mu.g antigen and 1/10 of the human being immunized with dose AS03 adjuvant) was injected per muscle. Collected blood samples were left at 37℃for 1 hour and at 4℃for 1 hour, centrifuged at 8000r/min for 10 minutes, and serum was collected and stored at-20 ℃. The grouping and immunizing dose of the mice are shown in Table 4.
Table 4 novel coronavirus recombinant chimeric protein vaccine immunized mice group and dose
The collected immune mouse serum was assayed for 50% pseudovirus neutralization titer of pseudoviruses of the novel coronavirus omacron-xbb.1.5 variant, respectively, using the novel coronavirus pseudoviruses constructed in example 4.
The results of the pseudovirus neutralizing antibody titer measurements are shown in FIG. 7 (groups 1-6 in sequence from left to right). The results show that NTD (bq.1.1) -RBD (xbb.1.5) -foldon (group 3) and NTD (xbb.1.5) -RBD (bq.1.1) -foldon (group 4) have higher neutralizing antibody titers GMT against the Omicron-xbb.1.5 variant, 820 and 541 respectively, which we speculate to be due to the higher immunogenicity of the RBD component, which results in when RBD is derived from the xbb.1.5 variant (i.e., NTD (bq.1.1) -RBD (xbb.1.5) -foldon) its corresponding neutralizing antibody titers GMT are greater than the immunogens from the bq.1.1 variant (i.e., NTD (xbb.1.5) -RBD (bq.1.1) -foldon).
The neutralizing antibody titers GMT of NTD (ba.2) -RBD (prototype strain) -foldon (group 5) and NTD (prototype strain) -RBD (prototype strain) -foldon (group 6) against the Omicron-xbb.1.5 variant were lower, just 139 and 190, respectively, due to the complete absence of components derived from the Omicron-xbb.1.5 variant in the immunogen, upon which the bivalent vaccines NTD (bq.1.1) -RBD (xbb.1.5) -foldon (prototype strain) -RBD (xbb.1.5) -foldon+ntd (prototype strain) -RBD (group 1) and NTD (bq.1.1) -RBD (xbb.5) -foldon+ntd (ba.2) -RBD (prototype strain) were raised against the Omicron-xbb.1.5, respectively, to the neutralizing antibody titers GMT of 364 and 270.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements, etc. made within the spirit and principles of the invention are to be included within the scope of the invention.

Claims (16)

1. A novel coronavirus recombinant chimeric antigen comprising a receptor binding domain RBD of a surface spike protein of a novel coronavirus strain or a functionally active fragment thereof and an N-terminal domain NTD or a functionally active fragment thereof, wherein said receptor binding domain RBD or a functionally active fragment thereof and said N-terminal domain NTD or a functionally active fragment thereof are derived from a different novel coronavirus strain and at least one is derived from an Omicron variant.
2. The recombinant chimeric antigen according to claim 1, wherein said omacron variant comprises at least one of subtype variants ba.1, ba.1.1, ba.2, ba.2.12.1, ba.3, ba.4, ba.5, bq.1.1, bf.7, xbb.1.5, xbb.1.16, eg.5 and jn.1, the remaining strains being selected from at least one of novel coronavirus prototype, alpha, beta, gamma, delta, lambda, mu variants.
3. The recombinant chimeric antigen according to claim 1, wherein said RBD or functionally active fragment thereof is derived from at least one of a prototype strain, a Beta variant, a Delta variant and an Omicron variant, and said NTD or functionally active fragment thereof is derived from an Omicron variant.
4. The recombinant chimeric antigen according to claim 3, wherein said recombinant chimeric antigen comprises a first amino acid sequence A-B and a second amino acid sequence C-D of a receptor binding domain RBD of surface spike proteins derived from different novel coronavirus strains or a functionally active fragment thereof, wherein C-D forms A- (C-D) n -B by substitution of amino acid residues in A-B directly or indirectly via a linker sequence, the number of amino acid residues of C-D is 10 to 60, n represents the number of amino acid sequences C-D, n is an integer not less than 1, preferably n is an integer selected from 1 to 10, more preferably an integer selected from 1 to 5.
5. The recombinant chimeric antigen according to claim 4, wherein the recombinant chimeric antigen further comprises a third amino acid sequence E-F derived from the receptor binding domain RBD of surface spike proteins of different novel coronavirus strains or a functionally active fragment thereof, wherein E-F is directly or indirectly chimeric in A-B through substitution of amino acid residues in A-B through a linker sequence to form A- (C-D) n-(E-F)m -B, the number of amino acid residues of E-F is 10 to 60, m represents the number of amino acid sequences E-F, m is an integer not less than 1, preferably m is an integer selected from 1 to 10, further preferably an integer selected from 1 to 5.
6. The recombinant chimeric antigen according to claim 4 or 5, wherein said Omicron variant is selected from at least one of bq.1.1, bf.7, xbb.1.5, xbb.1.16, eg.5 and jn.1.
7. The recombinant chimeric antigen according to any one of claims 1 to 6, further comprising a foldon domain or functionally active fragment thereof.
8. The recombinant chimeric antigen according to claim 7, wherein the N-terminus of the foldon domain or functionally active fragment thereof is directly linked to the C-terminus of the RBD or functionally active fragment thereof or linked by a linker sequence and the N-terminus of the RBD or functionally active fragment thereof is directly linked to the C-terminus of the NTD or functionally active fragment thereof or linked by a linker sequence.
9. The recombinant chimeric antigen according to claim 8, wherein said recombinant chimeric antigen has an amino acid sequence as set forth in any one of SEQ ID NOs 1 to 3.
10. A recombinant nucleic acid comprising a polynucleotide sequence encoding the recombinant chimeric antigen of any one of claims 1-9, said polynucleotide being DNA, mRNA or circRNA.
11. A host cell carrying the recombinant nucleic acid of claim 10, said host cell comprising a mammalian cell, an insect cell, a yeast cell, or a bacterial cell, optionally said mammalian cell comprises a HEK293T cell, a HEK293F cell, an Expi293F cell, or a CHO cell, optionally said bacterial cell comprises an e.
12. A method of preparing the recombinant chimeric antigen of any one of claims 1-9, comprising the steps of:
(1) Constructing a recombinant expression plasmid by utilizing a nucleotide sequence for encoding the novel coronavirus recombinant chimeric antigen;
(2) Transforming the constructed recombinant expression plasmid into host bacteria, and screening the correct recombinant expression plasmid;
(3) Transfecting a mammalian host cell with the recombinant expression plasmid obtained by screening for expression;
(4) Collecting cell culture supernatant and purifying to obtain the recombinant chimeric antigen.
13. An immunogenic composition comprising a first component and a second component, the first component and the second component being different, and at least one component being selected from the recombinant chimeric antigen of any one of claims 1-9.
14. A novel coronavirus recombinant protein vaccine comprising the recombinant chimeric antigen of any one of claims 1-9, or the immunogenic composition of claim 13, and an adjuvant selected from one or any combination of aluminum adjuvants, lipopolysaccharides, saponins, polyI: C, cpG, and emulsion adjuvants.
15. A novel coronavirus recombinant nucleic acid vaccine comprising the recombinant nucleic acid of claim 10.
16. Use of the recombinant chimeric antigen of any one of claims 1-9, the recombinant nucleic acid of claim 10, the host cell of claim 11, the immunogenic composition of claim 13, the novel coronavirus recombinant protein vaccine of claim 14, the novel coronavirus recombinant nucleic acid vaccine of claim 15 in the manufacture of a medicament for preventing novel coronavirus infection.
CN202410579155.9A 2023-05-11 2024-05-11 Novel coronavirus recombinant chimeric antigen, preparation method and application thereof Pending CN119060197A (en)

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