CN113637695B - Novel coronavirus mRNA vaccine for targeted stimulation of humoral immunity and cellular immunity - Google Patents

Novel coronavirus mRNA vaccine for targeted stimulation of humoral immunity and cellular immunity Download PDF

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CN113637695B
CN113637695B CN202111195423.XA CN202111195423A CN113637695B CN 113637695 B CN113637695 B CN 113637695B CN 202111195423 A CN202111195423 A CN 202111195423A CN 113637695 B CN113637695 B CN 113637695B
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程功
太万博
冯胜勇
童良琴
庞慕加
蔡珠明
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Shenzhen Bay Laboratory
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Abstract

The invention discloses a novel coronavirus mRNA vaccine for targeted stimulation of humoral immunity and cellular immunity. A first aspect of the present application provides an isolated combination of DNA molecules comprising a first DNA molecule and at least one of a second DNA molecule and a third DNA molecule. Through the combination of the first DNA molecule and the second DNA molecule and/or the third DNA molecule, mRNA finally synthesized by the first DNA molecule is used for inducing high-titer cross-neutralizing antibodies, and mRNA finally synthesized by the second DNA molecule and/or the third DNA molecule is used for inducing cytotoxic T lymphocytes specific to the new coronavirus, so that the relatively independent humoral immune response and cellular immune response are efficiently and simultaneously activated, and the breakthrough infection caused by a mutant strain generated in the process of epidemic propagation of the new coronavirus is coped with.

Description

Novel coronavirus mRNA vaccine for targeted stimulation of humoral immunity and cellular immunity
Technical Field
The application relates to the technical field of immunization, in particular to a novel coronavirus mRNA vaccine for targeted stimulation of humoral immunity and cellular immunity.
Background
Compared with other traditional vaccines, the mRNA vaccine has the advantages of short development period and relatively low development cost, and meanwhile, the physicochemical properties such as solubility and the like of the mRNA vaccine are suitable for drug development and high in safety; and the side effect is low, and the risk of integration, gene mutation induction and exogenous virus infection does not exist, so the vaccine is a hot spot for the development of the current vaccines. On the aspect of target antigen gene design, different antigen sequences are selected for construction according to the type of targeted induced protective immune response, and ideal vaccine design can effectively induce pathogen-specific humoral immunity and cellular immune response.
However, the current mRNA vaccines mainly have the following two problems: on one hand, two new coronavirus mRNA vaccines which are successfully marketed at present tend to induce neutralizing antibodies to exert protective immune responses, and specific design for targeting induction of T cell immune responses is not explored yet. On the other hand, some dominant variants of the new coronavirus, including Alpha variant (B.1.1.7), Beta variant (B.1.351), Gamma variant (P.1), Delta variant (B.1.617.2), etc., which are generated by continuous mutation during circulation, may have severe immune escape against the antibodies induced by the current mRNA vaccine. Therefore, it is necessary to provide an mRNA vaccine capable of simultaneously stimulating humoral immunity and cellular immunity in a targeted manner and effectively inducing the production of cross-neutralizing antibodies.
Disclosure of Invention
The present application is directed to solving at least one of the problems in the prior art. Therefore, the application provides an isolated DNA molecule combination, and an mRNA vaccine which can stimulate humoral immunity and cellular immunity in a targeted mode and can effectively induce cross-neutralizing antibodies can be prepared by using the DNA molecule combination.
In a first aspect of the present application, there is provided an isolated combination of DNA molecules comprising:
a. a first DNA molecule, and
b. at least one of a second DNA molecule and a third DNA molecule;
wherein the first DNA molecule comprises a first coding region, the first coding region comprises a sequence shown as SEQ ID No.1 for coding a signal peptide and a sequence shown as SEQ ID No.3 for coding an RBD-SA fragment;
the second DNA molecule comprises a second coding region, the second coding region comprises a ubiquitination sequence as shown in SEQ ID No.2 and a sequence as shown in SEQ ID No.6 which codes rMN fragment;
the third DNA molecule comprises a third coding region, wherein the third coding region comprises a ubiquitination sequence shown as SEQ ID No.2 and a sequence shown as SEQ ID No.8 for coding NSPs fragments.
The combination of the isolated DNA molecules according to the embodiments of the present application has at least the following beneficial effects:
(1) the first DNA molecule has a sequence for coding tPA signal peptide and a sequence for coding RBD-SA segment, and mRNA synthesized by using the DNA molecule as a template mediates the expression and secretion of neutralizing antigen of the RBD-SA segment by using the signal peptide after being delivered into a body. The RBD-SA fragment comprises amino acids 331-524 of the Spike protein of the new coronavirus, and also fuses some key amino acid mutation sites of the mutant strain, so that high-titer neutralizing antibodies can be induced, and the RBD-SA fragment has good cross-neutralization effect, so that the level of the neutralizing antibodies is not remarkably reduced when aiming at different mutant virus strains.
(2) The second DNA molecule has ubiquitination sequence and rMN segment encoding sequence, and after the mRNA synthesized with the DNA molecule as template is delivered into body, the expressed rMN segment is degraded and forms T cell epitope owing to ubiquitination of antigen. The rMN fragment comprises fragments of epitope enrichment regions of M protein and N protein of the new coronavirus, and is rearranged, so that CTLs epitopes in the structural protein M, N are retained on the premise of preventing the structural protein from playing normal biological functions to promote virus replication, the degradation and efficient presentation of antigens can be promoted, and an Antibody Dependence Enhancement (ADE) effect is effectively eliminated.
(3) The third DNA molecule has ubiquitination sequence and sequence for encoding NSPs segment, and after mRNA synthesized by using the DNA molecule as a template is delivered into a body, the expressed NSPs segment is degraded due to ubiquitination of antigen and forms T cell epitope. The NSPs fragments comprise fragments of epitope enrichment regions of NSP proteins such as neocoronavirus NSP3, NSP4 and NSP6, are rearranged, retain CTLs epitopes in the NSPs on the premise of preventing NSP from playing normal biological functions and promoting virus replication, can promote degradation and efficient presentation of antigens, and effectively eliminate Antibody Dependence Enhancement (ADE) effect.
(4) Through the combination of the first DNA molecule and the second DNA molecule and/or the third DNA molecule, mRNA finally synthesized by the first DNA molecule is used for inducing high-titer cross-neutralizing antibodies, and mRNA finally synthesized by the second DNA molecule and/or the third DNA molecule is used for inducing cytotoxic T lymphocytes specific to the new coronavirus, so that the relatively independent humoral immune response and cellular immune response are efficiently and simultaneously activated, and the breakthrough infection caused by a mutant strain generated in the process of epidemic propagation of the new coronavirus is coped with.
Wherein, the nucleotide sequence of the coding signal peptide is as follows:
ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC (SEQ ID No. 1). The signal peptide is tPA signal peptide, and is one of the most widely used signal peptides at present. After the first DNA molecule is transcribed to form mRNA, the tPA signal peptide can improve the translatability of the neutralizing antigen, thereby effectively improving the expression and secretion level of the neutralizing antigen and enhancing the immune response.
The ubiquitination sequence is as follows:
ATGCAGATTTTCGTGAAAACCCTGACCGGGAAAACCATCACCCTGGAAGTCGAGCCCAGCGACACCATCGAGAACGTCAAGGCCAAAATCCAGGACAAGGAGGGCATCCCCCCCGACCAGCAGAGGCTGATCTTCGCCGGCAAGCAGCTGGAAGACGGCAGAACACTGAGCGACTACAACATCCAGAAGGAAAGCACCCTGCACCTGGTGCTGAGACTGAGAGGCGCC (SEQ ID No. 2). The highly expressed T cell antigen can promote self degradation and T cell epitope formation through ubiquitination.
The nucleotide sequence encoding the RBD-SA fragment is as follows:
AACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGTGTGTACGCCTGGAACAGAAAGAGGATCTCCAACTGCGTGGCCGATTACTCTGTGCTGTATAATAGCGCCTCCTTCTCTACCTTCAAATGCTATGGCGTGTCCCCCACAAAGCTGAACGATCTGTGTTTTACCAACGTGTACGCCGACTCCTTCGTGATCAGAGGCGACGAGGTGAGGCAGATCGCCCCCGGACAGACCGGAAATATCGCCGATTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAACCTGGACAGTAAGGTGGGGGGAAACTACAACTACCTGTACCGGCTGTTCCGCAAGAGCAACCTGAAGCCCTTTGAGAGAGACATCAGCACAGAGATTTATCAGGCCGGCAGCACCCCCTGCAACGGAGTGAAAGGATTCAACTGCTACTTCCCACTGCAAAGCTACGGCTTCCAGCCCACCTATGGCGTGGGATACCAGCCCTACAGAGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCCCCCGCCACCGTG(SEQ ID No.3)。
the amino acid sequence of the RBD-SA fragment is shown below:
NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATV (SEQ ID No. 4). The RBD-SA fragment utilizes the 331-524 amino acid segment of the S protein of the novel coronavirus and introduces key amino acid mutation sites K417N, E484K and N501Y of the novel coronavirus.
The large fragment rearrangement antigen rMN fragment comprises a cytotoxic T lymphocyte epitope, the amino acid sequence of which is shown below:
GHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQAAYLIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLAAYMADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVAAYQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQAAAYIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSAAYMSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDAAYNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYAAYGKPIPNPLLGLDST (SEQ ID No. 5). rMN the antigen-enriched regions of M and N proteins of new coronavirus are rearranged in segments, and the segments are connected by AAY.
The nucleotide sequence encoding the rMN fragment is shown below:
GGCCACCTGCGCATCGCCGGCCATCACCTGGGCAGATGCGACATCAAGGATCTGCCTAAAGAAATCACCGTGGCCACGAGCCGGACCCTGTCTTACTACAAACTGGGCGCCTCCCAGAGAGTGGCCGGCGATAGCGGATTTGCCGCCTATTCTAGATACCGGATCGGCAACTACAAGCTGAACACCGATCACAGCAGCAGCAGCGACAACATCGCCCTGCTGGTCCAGGCCGCCTACCTGATCTTCCTGTGGCTGCTGTGGCCTGTGACCCTGGCCTGTTTTGTGCTTGCTGCTGTCTACAGAATCAACTGGATCACCGGCGGCATCGCCATCGCCATGGCCTGCCTGGTGGGCCTGATGTGGCTGAGCTACTTCATCGCATCATTTAGACTGTTCGCCAGAACCCGGTCCATGTGGAGCTTCAACCCCGAGACAAACATCCTGCTCAATGTGCCGCTGCACGGAACCATCCTGACACGGCCTCTGCTTGAGAGCGAGCTGGTTATCGGAGCTGTGATCCTGAGAGGCCACCTGAGAATCGCCGGACACCACCTGGCTGCCTATATGGCTGATAGCAACGGCACCATCACAGTGGAGGAATTAAAGAAACTGCTGGAACAGTGGAACCTGGTGATCGGATTTCTGTTCTTGACATGGATCTGCCTGCTGCAGTTCGCCTATGCCAATAGAAATAGATTCCTGTACATCATCAAACTGATCTTTCTGTGGCTGCTCTGGCCCGTGGCTGCCTATCAGGAGCTGATTCGCCAGGGCACCGACTACAAGCACTGGCCTCAAATAGCCCAGTTTGCCCCTAGCGCCAGCGCCTTCTTCGGAATGAGCAGAATCGGCATGGAAGTGACTCCTAGCGGCACATGGCTCACATACACAGGCGCCATCAAGCTCGACGATAAGGACCCCAACTTCAAGGATCAGGTGATCCTGCTGAACAAGCACATCGACGCCTACAAGACATTCCCCCCCACCGAGCCCAAGAAGGACAAGAAGAAGAAGGCCGACGAGACACAGGCCCTGCCCCAGCGGCAAAAGAAGCAGCAAACAGTGACCCTGCTTCCTGCCGCCGACCTGGACGACTTCAGCAAGCAGCTGCAGCAAAGCATGAGCTCCGCCGACAGCACCCAAGCCGCTGCCTACATCCGGGGCGGAGACGGCAAGATGAAGGACCTGAGCCCCAGATGGTACTTCTACTACCTGGGCACCGGCCCAGAGGCCGGCCTGCCATACGGAGCCAACAAGGATGGCATCATCTGGGTGGCCACAGAAGGCGCCCTGAATACCCCTAAGGACCACATCGGCACCCGGAACCCTGCTAATAACGCCGCAATCGTGCTGCAACTGCCACAAGGCACGACCCTGCCTAAAGGCTTCTACGCCGAGGGATCCCGGGGAGGCAGCCAGGCCTCTAGCAGAAGCTCTTCTCGGAGCCGTAACAGCTCTCGCAACAGCACACCCGGCTCCGCCGCCTACATGAGCGATAACGGCCCTCAGAACCAGAGAAATGCTCCTCGGATCACCTTCGGCGGCCCTTCTGACAGCACCGGTAGCAACCAGAACGGCGAGAGAAGTGGAGCTAGAAGCAAGCAAAGAAGGCCTCAGGGCCTGCCTAACAACACAGCCTCTTGGTTCACCGCCCTGACACAGCACGGGAAAGAGGACCTGAAGTTCCCTAGAGGCCAGGGGGTGCCTATTAACACCAACAGCTCTCCAGACGACCAGATCGGCTACTACAGACGCGCCACCAGACGAATCAGAGGCGGAGACGGCAAGATGAAAGATGCCGCTTACAATTCTAGCCGGAATAGCACACCCGGCAGCTCTAGAGGAACAAGCCCTGCTAGAATGGCCGGAAACGGCGGCGACGCCGCTCTGGCCCTGCTGTTGCTGGATAGACTGAACCAGCTGGAATCCAAGATGAGCGGCAAGGGCCAGCAGCAGCAGGGCCAGACCGTGACAAAGAAATCTGCTGCCGAGGCCTCTAAGAAACCTAGACAGAAAAGAACAGCCACCAAGGCCTACAACGTGACCCAGGCTTTCGGCAGACGGGGCCCCGAGCAGACCCAGGGCAACTTCGGGGACCAGGAGCTGATCAGACAGGGCACCGACTACGCCGCTTACGGCAAGCCCATCCCCAATCCTCTGCTGGGCCTGGACAGCACCTGA(SEQ ID No.6)。
the large-fragment rearranged antigen NSPs comprise cytotoxic T lymphocyte epitopes, and the amino acid sequences of the large-fragment rearranged antigen NSPs are shown as follows:
SYCTGYREGYLNSTNVTIATYCTGSIPCSVCLSGLDSLDTYPSLETIQITISSFKWDLTAFGLVAEWFLAYILFTRFFYVLGLAAIMQLFFSYFAVHFISNSWLMWLIINLVQMAPISAMVRMYIFFASFYYVWKSYVHVVDGCNSSTCMMCYKRNRATRVECAAYGVCVSTSGRWVLNNDYYRSLPGVFCGVDAVNLLTNMFTPLIQPIGALDISASIVAGGIVAIVVTCLAYYFMRFRRAFGEYSHVVAFNTLLFLMSFTVLCLTPVYSFLPGVYSVIYLYLTFYLTNDVSFLAHIQWMVMFTPLVPFWITIAYIICISTKHFYWFFSNYLKRRVVFNGVSFSTFEEAALCTFLLNKEMYLKLRSDVLLPLTQYNRYAAYSAVKRTIKGTHHWLLLTILTSLLVLVQSTQWSLFFFLYENAFLPFAMGIIAMSAFAMMFVKHKHAFLCLFLLPSLATVAYFNMVYMPASWVMRIMTWLDMVDTSLSGFKLKDCVMYASAVVLLILMTARTVYDDGARRVWTLMNVLTLVYKVYYGNALDQAISMWALIISVTSNYSGVVTTVMFLARGIVFMCVEYCPIFF (SEQ ID No. 7). Wherein the NSPs fragments carry out sectional rearrangement on antigen enrichment regions of nonstructural proteins such as NSP3, NSP4, NSP6 and the like, and the fragments are connected by AAY.
The nucleotide sequence encoding the NSPs fragment is shown below:
AGCTACTGCACCGGATACAGAGAGGGATATCTGAACAGCACAAACGTGACCATCGCCACCTACTGCACAGGCAGCATCCCCTGCAGCGTGTGCCTGAGCGGCCTGGATTCACTGGATACCTACCCCTCACTGGAGACCATCCAGATCACCATCAGCAGCTTCAAGTGGGACCTGACCGCCTTTGGCCTGGTGGCCGAGTGGTTCCTGGCCTATATCCTGTTCACCCGCTTCTTTTACGTCCTGGGCCTGGCCGCCATCATGCAGCTGTTCTTCTCCTACTTCGCCGTGCACTTCATCAGCAATTCCTGGCTGATGTGGCTGATCATTAACCTGGTGCAGATGGCCCCTATCAGCGCCATGGTGAGAATGTACATCTTCTTCGCCTCCTTCTACTACGTGTGGAAGTCCTACGTCCACGTGGTGGACGGCTGCAACTCCTCTACCTGCATGATGTGCTACAAGAGAAACAGAGCCACCAGAGTGGAGTGCGCCGCCTATGGAGTGTGTGTGAGCACCAGCGGGCGCTGGGTGCTGAATAATGACTATTACAGGTCCCTGCCCGGAGTGTTCTGCGGCGTGGACGCAGTGAATCTGCTGACCAACATGTTCACCCCCCTGATTCAGCCTATCGGAGCCCTGGACATTTCTGCCAGCATCGTGGCCGGCGGAATCGTGGCTATTGTGGTGACCTGTCTGGCTTACTATTTCATGAGATTCAGAAGGGCCTTCGGCGAGTACAGCCACGTGGTGGCCTTTAACACCCTGCTGTTCCTGATGTCCTTTACCGTGCTGTGTCTGACTCCCGTGTATTCTTTCCTGCCCGGCGTGTACAGCGTGATCTATCTGTATCTGACCTTCTACCTGACCAATGACGTGAGCTTCCTGGCCCACATCCAGTGGATGGTGATGTTTACCCCCCTGGTCCCCTTCTGGATCACCATCGCCTACATCATCTGCATCTCCACCAAACACTTCTACTGGTTCTTCTCCAACTACCTGAAAAGACGCGTGGTCTTCAACGGCGTGTCCTTCTCCACCTTCGAGGAGGCCGCCCTGTGCACCTTCCTGCTGAACAAGGAGATGTACCTGAAGCTGCGGTCCGACGTCCTGCTGCCCCTGACACAGTATAATAGATACGCTGCTTACTCCGCCGTGAAGAGAACAATCAAGGGCACCCACCACTGGCTGCTGCTGACCATCCTGACCTCCCTGCTGGTGCTGGTGCAGAGCACCCAGTGGAGCCTGTTCTTCTTTCTGTATGAGAACGCCTTTCTGCCCTTCGCCATGGGCATCATCGCCATGTCCGCCTTCGCCATGATGTTCGTGAAGCATAAGCACGCCTTCCTGTGCCTGTTTCTGCTGCCCTCCCTGGCTACCGTGGCCTACTTCAACATGGTGTACATGCCCGCCAGCTGGGTGATGAGAATCATGACCTGGCTGGATATGGTGGACACCTCCCTGTCCGGCTTCAAGCTGAAAGACTGCGTGATGTATGCTTCCGCCGTGGTGCTGCTGATCCTGATGACTGCCCGGACCGTGTACGACGACGGCGCTAGAAGAGTGTGGACCCTGATGAACGTCCTGACCCTGGTGTACAAAGTGTATTACGGCAATGCCCTGGATCAGGCCATTTCCATGTGGGCCCTGATTATCTCCGTGACCTCCAATTACAGCGGAGTGGTGACCACCGTGATGTTCCTGGCCCGGGGCATCGTGTTCATGTGCGTGGAGTATTGCCCCATCTTTTTC(SEQ ID No.8)。
in some embodiments of the present application, the first, second and third coding regions further have 5 'untranslated regions (5' -UTRs) and 3 'untranslated regions (3' -UTRs), respectively, upstream and downstream of the first, second and third coding regions.
In some embodiments of the present application, the first coding region, the second coding region, and the third coding region further have a poly A (polyA) downstream.
In order to solve the problems, structural elements with modification effects, such as 5 'untranslated region (5' -UTR), 3 'untranslated region (3' -UTR), poly (A) (polyA) and the like, are added to a DNA template, so that the finally synthesized mRNA keeps an integral structure, the stability of the mRNA is facilitated, the half-life period of the mRNA is prolonged, and the expression capacity of the mRNA is improved.
In some embodiments of the present application, the number of polyadenylic acid adenylates is more than 100.
In some embodiments of the present application, the DNA molecule further has a promoter upstream of the coding region, the promoter being selected from any one of the T7 promoter, SP6 promoter.
In some embodiments of the present application, the coding region further comprises a reporter gene. In order to clarify the expression of different antigens or for other purposes, a reporter gene may be inserted into the antigen coding region to facilitate detection.
In some embodiments of the present application, the reporter gene is selected from at least one of a fluorescent protein, a luciferase, a protein tag.
In some embodiments of the present application, the protein tag is selected from at least one of an HA tag, a Flag tag, a His tag, or a V5 tag.
In a second aspect of the present application, there is provided an antigen expression vector combination comprising:
a. a first antigen expression vector, and
b. at least one of a second antigen expression vector and a third antigen expression vector;
wherein the first antigen expression vector comprises a first DNA molecule of the aforementioned combination of DNA molecules; the second antigen expression vector comprises a second DNA molecule of the aforementioned combination of DNA molecules; the third antigen expression vector comprises a third DNA molecule of the aforementioned combination of DNA molecules.
In a third aspect of the present application, there is provided an isolated combination of mRNA molecules prepared from the aforementioned combination of antigen expression vectors.
After the mRNA molecule combination formed by in vitro transcription of the antigen expression vector enters the body, on one hand, the signal peptide can be used for mediating the expression and secretion of the neutralizing domain antigen, so that a neutralizing antibody with high titer is induced, and the level of the neutralizing antibody is not remarkably reduced when aiming at different mutant virus strains; on the other hand, ubiquitinated fusion T cell epitope enrichment antigen expression promotes degradation and formation of T cell epitopes; thereby inducing the production of relatively independent neutralizing antibodies and T cell immune responses simultaneously.
In a fourth aspect of the present application, there is provided a novel coronavirus mRNA vaccine comprising a combination of mRNA molecules as described above.
In some embodiments of the present application, a pharmaceutically acceptable carrier is also included.
In some embodiments of the present application, the carrier is a lipid carrier. Non-limiting examples of lipid carriers include liposomes, such as Lipid Nanoparticles (LNPs), prepared by methods including, but not limited to, thin film hydration, reverse evaporation, extrusion, homogenization, etc., and incubated with mRNA vaccines to obtain the vaccines. It is understood that other molecules such as cationic lipid complexes (LPX), lipid polyplexes (LPP), Polymeric Nanoparticles (PNP), Inorganic Nanoparticles (INP), Cationic Nanoemulsions (CNE), etc. can be delivered as carriers of mRNA as well.
In some embodiments of the present application, the lipid carrier is a cationic lipid nanoparticle.
In a fifth aspect of the application, there is provided a formulation comprising the novel coronavirus mRNA vaccine as described above.
In conclusion, the scheme discloses the separated DNA molecule combination, the antigen expression vector combination, the separated mRNA molecule combination, the vaccine and the preparation, and the materials can be applied to the preparation of products for preventing and/or treating the new coronavirus, so that the independent humoral immune response and cellular immune response can be efficiently activated simultaneously, and the breakthrough infection caused by the mutant strains generated in the process of spread of the new coronavirus can be responded.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.
Drawings
FIG. 1 is a map of pCA-RBD-SA antigen expression vector in example 1 of the present application.
FIG. 2 is a map of the pCA-Ub-rMN antigen expression vector in example 1 of the present application.
FIG. 3 is a map of the pCA-Ub-NSPs antigen expression vector in example 1 of the present application.
FIG. 4 shows the Western Blot results of antigen expression levels in protein expression verification experiments of the present application.
FIG. 5 shows the results of flow cytometry for antigen expression levels in protein expression validation experiments of the present application.
FIG. 6 shows the test results of the mRNA vaccine immunization evaluation safety test of the present application. Where A is the fluorescence result of the in vivo imaging and B and C are the counting results of different tissue distributions.
FIG. 7 shows the results of ELISA detection of neutralizing antibodies of different mutants of the mRNA vaccine immunoassay test of the present application.
FIG. 8 shows the results of virus neutralization assays of different mutants of the mRNA vaccine immunoassay of the present application.
FIG. 9 shows the results of IFN- γ secretion assay of different mutants of the mRNA vaccine immunoassay test of the present application.
Fig. 10 shows the results of protective evaluation in the mRNA vaccine immunoassay test of the present application.
Detailed Description
The conception and the resulting technical effects of the present application will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present application belong to the protection scope of the present application.
The following detailed description of embodiments of the present application is provided for the purpose of illustration only and is not intended to be construed as a limitation of the application.
In the description of the present application, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present number, and the above, below, within, etc. are understood as including the present number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present application, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example" or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Example 1
This example provides a combination of antigen expression vectors for a novel coronavirus mRNA vaccine. The combination of the antigen expression vector comprises three antigen expression vectors, namely pCA-RBD-SA, pCA-Ub-rMN and pCA-Ub-NSPs.
Referring to fig. 1 to 3, the pCA-RBD-SA plasmid has an antigen coding region, the antigen coding region includes a sequence encoding tPA signal peptide and a sequence encoding RBD-SA fragment, which are sequentially connected, and a sequence encoding Flag tag and a stop codon (not shown in the figures) are further inserted into the 3' end of the sequence encoding RBD-SA fragment. The upstream and downstream of the antigen-encoding region are also linked to a T7 promoter, a 5 '-untranslated region (5' -UTR), a 3 '-untranslated region (3' -UTR), and poly (A), respectively.
The pCA-Ub-rMN plasmid has an antigen coding region including a ubiquitination sequence and a sequence encoding a rMN fragment, which are linked in sequence, and a sequence encoding a V5 tag and a stop codon (the V5 sequence and the stop codon are not shown in the figure) at the 3' end of the sequence encoding the rMN fragment. The T7 promoter, 5 '-untranslated region (5' -UTR), 3 '-untranslated region (3' -UTR), and poly (A) are also linked to the upstream and downstream of the antigen coding region.
The pCA-Ub-NSPs plasmid has an antigen coding region, the antigen coding region comprises a ubiquitination sequence and a sequence for coding NSPs fragment which are connected in sequence, and the 3' end of the sequence for coding NSPs fragment also comprises a sequence for coding a V5 label and a stop codon (the V5 sequence and the stop codon are not shown in the figure). The upstream and downstream of the antigen-encoding region are also linked to a T7 promoter, a 5 '-untranslated region (5' -UTR), a 3 '-untranslated region (3' -UTR), and poly (A), respectively.
The construction method of the antigen expression vector plasmid comprises the following steps:
(1) the pCAGGS vector was digested with restriction enzymes Xho I and Kpn I, and the large fragment (vector) was recovered.
(2) The following three gene sequences were synthesized by Biometrics:
sequence one: includes tPA signal peptide sequence (SEQ ID No. 1) and receptor binding domain fragment (RBD, SEQ ID No. 3) of Spike protein of SARS-CoV-2, and has Flag tag and stop codon inserted into 3' end.
And (2) sequence II: comprises an ubiquitination sequence (SEQ ID No. 2) and an rMN sequence (SEQ ID No. 6), and is also inserted with a V5 label and a stop codon at the 3' end.
And (3) sequence III: comprises an ubiquitination sequence (SEQ ID No. 2) and an NSPs sequence (SEQ ID No. 8), and is also inserted with a V5 label and a stop codon at the 3' end.
According to the above sequence, restriction sites Xho1 and Kpn1 were provided at both ends during synthesis, specific fragments were recovered by electrophoresis and gel, and each fragment was ligated to a vector by the T4 DNA ligase method.
(3) And E.coli transformation is carried out according to a conventional method, clone bacteria are selected the next day, the quality-improved grains are small, and sequencing is carried out by a sequencing company.
(4) After the sequence alignment is correct, the plasmid is greatly extracted by using a large-plasmid-upgrading kit, the concentration and purity of the plasmid are measured by using NanoDrop for later use, and the three plasmids are named pCA-RBD-SA, pCA-Ub-rMN and pCA-Ub-NSPs.
mRNA transcription verification experiment
(1) The antigen expression vector constructed in the embodiment 1 is subjected to linearization and electrophoretic identification by Bsa I restriction enzyme, and the electrophoretic displacement is relatively small. Treating the recovered protein kinase K, extracting with phenol and chloroform, dissolving in water without RNA enzyme, and detecting the concentration with a spectrophotometer.
(2) mRNA containing 50% pseudoUTP was synthesized by using an in vitro transcription kit and a capping kit purchased from Bio Inc according to the instruction, precipitated and purified by lithium chloride, washed once with 75% ethanol, air-dried, dissolved in RNase-free water, and the concentration was measured by a spectrophotometer.
(3) The denatured RNA gel was configured and RNA samples were mixed with 2 × RNA loading buffer 1: 1, mixing, metal bath thermal denaturation at 70 ℃ for 10 minutes, ice-cooling for 5 minutes, and electrophoresis (120V, 20 min). The results were in agreement with expectations.
Protein expression verification experiment
Plasmids containing neutralizing or cellular antigens were constructed with reference to FIGS. 1-3 and the above experimental methods, and the plasmids designated RBD-SA, rMN and NSPs were linearized and transcribed in vitro, respectively. And respectively transfecting HEK 293T cells with mRNA transcribed from RBD-SA, rMN and NSPs plasmids by using an RNA transfection reagent, and identifying the functions of related gene elements. Wherein the control group is cells not transfected with the corresponding mRNA, RBD+rMN+The group is cells which are simultaneously transfected with two mRNAs transcribed by corresponding RBD-SA and rMN plasmids, and the RBD+NSPs+The group is cells which are simultaneously transfected with two mRNAs transcribed by two plasmids of corresponding RBD-SA and NSPs, and the RBD+Group rMN+Group and NSPs+The group is cells transfected with a single mRNA transcribed from a corresponding one of the plasmids.
At 40 hours post transfection, the cell culture medium was supplemented with the proteasome inhibitor MG132 (50 μ M) and controls were set up, and 8 hours later, the cells were treated:
(1) western Blot: after cell lysis, proteins were collected and subjected to SDS-PAGE, and after blocking of the membrane, expression of B cell antigen (RBD) was identified using anti-Flag-tagged antibodies, respectively, and the results are shown in FIG. 4. As can be seen from the figure, a distinct band of RBD-SA appeared in the RBD-SA plasmid group compared to the control group.
(2) Flow cytometry: because T cell antigens are seriously ubiquitinated and WB detection is insensitive, expression pancreatin of the T cell antigens is identified to be digested into single cells by a flow method, after membrane rupture and fixation, FITC labeled anti-V5 labeled antibody and Alexa Fluor-555 (AF 555) labeled anti-Flag labeled antibody are respectively used for staining, and the expression of RBD, rMN and NSP346 antigens is identified by a flow cytometer. The results are shown in fig. 5, and it can be seen from the figure that three mrnas can respectively and effectively realize the expression of B cell antigen and T cell antigen, and two of them are co-transfected to realize the co-expression of two antigen components, which suggests that the two components can be used as vaccine for evaluating the effectiveness of the vaccine, and are in line with the design expectation.
Immunological evaluation experiment
1. Safety feature
Referring to example 1, a plasmid containing a luciferase reporter gene sequence was constructed in the same manner, linearized, transcribed in vitro, capped to obtain the corresponding Luc-mRNA molecule, and delivered to the bio-company to be loaded into mRNA liposome nanoparticles to prepare a preparation for forming an mRNA vaccine. Balb/c mice are immunized, 2 mice in each group are injected into each mouse by a muscle immunization way, the tissue distribution characteristics of mRNA are analyzed, the safety of mRNA vaccine can be reflected, 150 micrograms of luciferase substrate is injected into the abdominal cavity of each mouse 4 hours after immunization, and the tissue distribution is identified by living body imaging. The results are shown in FIG. 6, wherein A is the fluorescence result of in vivo imaging, the left side is the fluorescence of the Luc-mRNA in vivo (up) and the fluorescence of each organ after sacrifice, and the right side is the fluorescence of the control group of empty vector liposome nanoparticles in vivo (up) and the fluorescence of each organ after sacrifice (down); b and C are counts of different tissue distributions. As can be seen from the figure, fluorescence signals can be detected only in the muscle, the nearby lymph nodes and the spleen at the injection site, and this result indicates the safety of the mRNA vaccine provided in the examples of the present application.
2. Immunological evaluation
Three different mRNAs are sent to a biological company to be loaded into the mRNA liposome nanoparticles according to the grouping to prepare different mRNA vaccine preparations, Balb/c mice are immunized respectively, and the grouping condition is as follows: group 1: RBD-SA, group 2: RBD-SA + rMN, group 3: RBD-SA + NSPs, group 4: PBS control. Each group had 6 mice, and the immunization dose was 10. mu.g, and the immunization was performed twice at an interval of 3 weeks by the intramuscular route. Blood was collected 2 weeks after the second immunization to analyze the level of specific antibodies. 3 weeks after the second immunization, splenocytes were isolated, and the IFN-. gamma.level in the culture supernatant of splenocytes was detected by polypeptide stimulation, and the cellular immune response specific to the CTL epitope of cellular immune antigen was analyzed.
(1) ELISA: coating an ELISA plate with RBD recombinant proteins (1 mu g/ml) of 5 VOC new crown viruses (Wuhan strain, English strain, south Africa strain, Brazil strain and India strain-Delta), separating immune serum from blood collected 2 weeks after secondary immunization, diluting in multiple proportions, adding into the corresponding ELISA plate, sequentially adding HRP-anti-mouse IgG secondary antibody and TMB, detecting OD value by A450 after color development termination, and calculating antibody titer. The results are shown in fig. 7, and it can be seen from the figure that each experimental group of the mRNA vaccine provided in example 1 of the present application can induce the generation of RBD-specific IgG antibodies, and has a higher neutralizing antibody titer level for each variant, indicating that the mRNA vaccine can effectively generate cross-reactive specific antibodies and can effectively induce the body to generate humoral immune response.
(2) Pseudovirus neutralization: 5 kinds of VOC pseudoviruses of new corona virus (Wuhan strain, UK strain, south Africa strain, Brazil strain, India strain) were prepared using VSV pseudovirus system, and diluted serum was incubated with the same amount of pseudovirus at 37 ℃ for 1 hour, added to hACE2-293T cells, lysed cells after 48 hours to detect fluorescence intensity, and neutralized NT50 was calculated. As shown in fig. 8, the p value of each vaccine group relative to the PBS control group in different pseudovirus strains is shown as p value in the figure, and it can be seen from the figure that the mRNA vaccine provided in example 1 of the present application can effectively generate cross-reactive neutralizing active antibodies, and can effectively induce the body to generate humoral immune response.
(3) IFN-gamma secretion assay: killing the immunized mouse, taking the spleen, separating spleen cells, stimulating by adopting different polypeptides (RBD polypeptide, MN polypeptide, NSPs polypeptide and DMSO control), culturing for 3 days, and detecting the concentration of IFN-gamma in the supernatant by using a detection kit to reflect the activity of cytotoxic T lymphocytes. The results are shown in fig. 9, and 4 of the samples are set from left to right, and are respectively the detection results of the DMSO control group, the RBD polypeptide stimulation group, the MN polypeptide stimulation group and the NSPs polypeptide stimulation group. As can be seen from the figure, in the RBD polypeptide experimental group, different mRNA vaccines can generate immune response to RBD polypeptide stimulators, and IFN-gamma is secreted but is relatively low. The RBD-SA + rMN vaccine and the RBD-SA + NSPs vaccine can respectively generate immune response reaction to polypeptide stimulators of MN, NSP3, NSP4 and NSP6, secrete high-level IFN-gamma, and prove that the ubiquitinated T cell antigen can effectively induce and generate stronger cellular immune response reaction in vivo, thereby meeting design expectation.
3. Evaluation of protective Properties
Live coronavirus challenge test of new coronavirus, and new coronavirus south Africa mutant strain is used. Each group of vaccines (RBD-SA, RBD-SA + rMN, RBD-SA + NSPs and PBS) was immunized according to the procedure in the previous experiment, and three weeks after the second immunization, the nasal infection was detoxified (10)5 PFU/mouse), 3 days after infection, mice were sacrificed, lung tissue and trachea were isolated, and viral load in the tissue was detected using Q-PCR method. The results are shown in fig. 10, where a is the viral load results of different groups of lung tissues and B is the viral load results of different groups of airway tissues. As can be seen from the figure, the viral load of each vaccine group is significantly reduced compared with the PBS control (compared with the RBD-SA group, the p value of the vaccine group of lung tissue is less than 0.001, and the p value of the vaccine group of trachea tissue is less than 0.01), and meanwhile, the viral load cannot be detected by the two vaccine groups (RBD-SA + rMN and RBD-SA + NSPs) combining cellular immunity and humoral immunity, so that the cellular immune response can promote the overall preservation of the vaccineAnd (4) protecting the skin.
As can be seen from the above examples, the mRNA vaccine provided in the embodiments of the present application, on the one hand, can use the signal peptide to mediate the expression and secretion of the neutralizing domain antigen, thereby inducing high titer neutralizing antibodies, and the level of the neutralizing antibodies does not significantly decrease when being directed against different mutant virus strains; on the other hand, the ubiquitination fusion T cell epitope enrichment antigen expression promotes the degradation and the formation of T cell epitopes, thereby simultaneously inducing and generating relatively independent humoral immune response and T cell immune response.
The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments described above, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
SEQUENCE LISTING
<110> Shenzhen bay laboratory
TSINGHUA University
<120> novel coronavirus mRNA vaccine for targeted stimulation of humoral and cellular immunity
<130> 1
<160> 8
<170> PatentIn version 3.5
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gagctgatca gacagggcac cgactacgcc gcttacggca agcccatccc caatcctctg 2160
ctgggcctgg acagcacctg a 2181
<210> 7
<211> 583
<212> PRT
<213> Artificial sequence
<400> 7
Ser Tyr Cys Thr Gly Tyr Arg Glu Gly Tyr Leu Asn Ser Thr Asn Val
1 5 10 15
Thr Ile Ala Thr Tyr Cys Thr Gly Ser Ile Pro Cys Ser Val Cys Leu
20 25 30
Ser Gly Leu Asp Ser Leu Asp Thr Tyr Pro Ser Leu Glu Thr Ile Gln
35 40 45
Ile Thr Ile Ser Ser Phe Lys Trp Asp Leu Thr Ala Phe Gly Leu Val
50 55 60
Ala Glu Trp Phe Leu Ala Tyr Ile Leu Phe Thr Arg Phe Phe Tyr Val
65 70 75 80
Leu Gly Leu Ala Ala Ile Met Gln Leu Phe Phe Ser Tyr Phe Ala Val
85 90 95
His Phe Ile Ser Asn Ser Trp Leu Met Trp Leu Ile Ile Asn Leu Val
100 105 110
Gln Met Ala Pro Ile Ser Ala Met Val Arg Met Tyr Ile Phe Phe Ala
115 120 125
Ser Phe Tyr Tyr Val Trp Lys Ser Tyr Val His Val Val Asp Gly Cys
130 135 140
Asn Ser Ser Thr Cys Met Met Cys Tyr Lys Arg Asn Arg Ala Thr Arg
145 150 155 160
Val Glu Cys Ala Ala Tyr Gly Val Cys Val Ser Thr Ser Gly Arg Trp
165 170 175
Val Leu Asn Asn Asp Tyr Tyr Arg Ser Leu Pro Gly Val Phe Cys Gly
180 185 190
Val Asp Ala Val Asn Leu Leu Thr Asn Met Phe Thr Pro Leu Ile Gln
195 200 205
Pro Ile Gly Ala Leu Asp Ile Ser Ala Ser Ile Val Ala Gly Gly Ile
210 215 220
Val Ala Ile Val Val Thr Cys Leu Ala Tyr Tyr Phe Met Arg Phe Arg
225 230 235 240
Arg Ala Phe Gly Glu Tyr Ser His Val Val Ala Phe Asn Thr Leu Leu
245 250 255
Phe Leu Met Ser Phe Thr Val Leu Cys Leu Thr Pro Val Tyr Ser Phe
260 265 270
Leu Pro Gly Val Tyr Ser Val Ile Tyr Leu Tyr Leu Thr Phe Tyr Leu
275 280 285
Thr Asn Asp Val Ser Phe Leu Ala His Ile Gln Trp Met Val Met Phe
290 295 300
Thr Pro Leu Val Pro Phe Trp Ile Thr Ile Ala Tyr Ile Ile Cys Ile
305 310 315 320
Ser Thr Lys His Phe Tyr Trp Phe Phe Ser Asn Tyr Leu Lys Arg Arg
325 330 335
Val Val Phe Asn Gly Val Ser Phe Ser Thr Phe Glu Glu Ala Ala Leu
340 345 350
Cys Thr Phe Leu Leu Asn Lys Glu Met Tyr Leu Lys Leu Arg Ser Asp
355 360 365
Val Leu Leu Pro Leu Thr Gln Tyr Asn Arg Tyr Ala Ala Tyr Ser Ala
370 375 380
Val Lys Arg Thr Ile Lys Gly Thr His His Trp Leu Leu Leu Thr Ile
385 390 395 400
Leu Thr Ser Leu Leu Val Leu Val Gln Ser Thr Gln Trp Ser Leu Phe
405 410 415
Phe Phe Leu Tyr Glu Asn Ala Phe Leu Pro Phe Ala Met Gly Ile Ile
420 425 430
Ala Met Ser Ala Phe Ala Met Met Phe Val Lys His Lys His Ala Phe
435 440 445
Leu Cys Leu Phe Leu Leu Pro Ser Leu Ala Thr Val Ala Tyr Phe Asn
450 455 460
Met Val Tyr Met Pro Ala Ser Trp Val Met Arg Ile Met Thr Trp Leu
465 470 475 480
Asp Met Val Asp Thr Ser Leu Ser Gly Phe Lys Leu Lys Asp Cys Val
485 490 495
Met Tyr Ala Ser Ala Val Val Leu Leu Ile Leu Met Thr Ala Arg Thr
500 505 510
Val Tyr Asp Asp Gly Ala Arg Arg Val Trp Thr Leu Met Asn Val Leu
515 520 525
Thr Leu Val Tyr Lys Val Tyr Tyr Gly Asn Ala Leu Asp Gln Ala Ile
530 535 540
Ser Met Trp Ala Leu Ile Ile Ser Val Thr Ser Asn Tyr Ser Gly Val
545 550 555 560
Val Thr Thr Val Met Phe Leu Ala Arg Gly Ile Val Phe Met Cys Val
565 570 575
Glu Tyr Cys Pro Ile Phe Phe
580
<210> 8
<211> 1749
<212> DNA
<213> Artificial sequence
<400> 8
agctactgca ccggatacag agagggatat ctgaacagca caaacgtgac catcgccacc 60
tactgcacag gcagcatccc ctgcagcgtg tgcctgagcg gcctggattc actggatacc 120
tacccctcac tggagaccat ccagatcacc atcagcagct tcaagtggga cctgaccgcc 180
tttggcctgg tggccgagtg gttcctggcc tatatcctgt tcacccgctt cttttacgtc 240
ctgggcctgg ccgccatcat gcagctgttc ttctcctact tcgccgtgca cttcatcagc 300
aattcctggc tgatgtggct gatcattaac ctggtgcaga tggcccctat cagcgccatg 360
gtgagaatgt acatcttctt cgcctccttc tactacgtgt ggaagtccta cgtccacgtg 420
gtggacggct gcaactcctc tacctgcatg atgtgctaca agagaaacag agccaccaga 480
gtggagtgcg ccgcctatgg agtgtgtgtg agcaccagcg ggcgctgggt gctgaataat 540
gactattaca ggtccctgcc cggagtgttc tgcggcgtgg acgcagtgaa tctgctgacc 600
aacatgttca cccccctgat tcagcctatc ggagccctgg acatttctgc cagcatcgtg 660
gccggcggaa tcgtggctat tgtggtgacc tgtctggctt actatttcat gagattcaga 720
agggccttcg gcgagtacag ccacgtggtg gcctttaaca ccctgctgtt cctgatgtcc 780
tttaccgtgc tgtgtctgac tcccgtgtat tctttcctgc ccggcgtgta cagcgtgatc 840
tatctgtatc tgaccttcta cctgaccaat gacgtgagct tcctggccca catccagtgg 900
atggtgatgt ttacccccct ggtccccttc tggatcacca tcgcctacat catctgcatc 960
tccaccaaac acttctactg gttcttctcc aactacctga aaagacgcgt ggtcttcaac 1020
ggcgtgtcct tctccacctt cgaggaggcc gccctgtgca ccttcctgct gaacaaggag 1080
atgtacctga agctgcggtc cgacgtcctg ctgcccctga cacagtataa tagatacgct 1140
gcttactccg ccgtgaagag aacaatcaag ggcacccacc actggctgct gctgaccatc 1200
ctgacctccc tgctggtgct ggtgcagagc acccagtgga gcctgttctt ctttctgtat 1260
gagaacgcct ttctgccctt cgccatgggc atcatcgcca tgtccgcctt cgccatgatg 1320
ttcgtgaagc ataagcacgc cttcctgtgc ctgtttctgc tgccctccct ggctaccgtg 1380
gcctacttca acatggtgta catgcccgcc agctgggtga tgagaatcat gacctggctg 1440
gatatggtgg acacctccct gtccggcttc aagctgaaag actgcgtgat gtatgcttcc 1500
gccgtggtgc tgctgatcct gatgactgcc cggaccgtgt acgacgacgg cgctagaaga 1560
gtgtggaccc tgatgaacgt cctgaccctg gtgtacaaag tgtattacgg caatgccctg 1620
gatcaggcca tttccatgtg ggccctgatt atctccgtga cctccaatta cagcggagtg 1680
gtgaccaccg tgatgttcct ggcccggggc atcgtgttca tgtgcgtgga gtattgcccc 1740
atctttttc 1749

Claims (10)

1. An isolated combination of DNA molecules comprising:
a. a first DNA molecule, and
b. at least one of a second DNA molecule and a third DNA molecule;
wherein the first DNA molecule comprises a first coding region comprising the sequence shown in SEQ ID No.1 encoding a signal peptide and the sequence shown in SEQ ID No.3 encoding a RBD-SA fragment;
the second DNA molecule comprises a second coding region, and the second coding region comprises a ubiquitination sequence shown as SEQ ID No.2 and a sequence shown as SEQ ID No.6 for coding rMN fragments;
the third DNA molecule comprises a third coding region, and the third coding region comprises a ubiquitination sequence shown as SEQ ID No.2 and a sequence shown as SEQ ID No.8 for coding NSPs fragments.
2. The combination of DNA molecules of claim 1, wherein said first coding region, said second coding region and said third coding region further comprise, upstream and downstream of said first coding region, a 5 'untranslated region and a 3' untranslated region, respectively.
3. The combination of DNA molecules of claim 1 or 2, wherein said first coding region, said second coding region and said third coding region further comprise a poly a downstream.
4. An antigen expression vector combination comprising:
a. a first antigen expression vector, and
b. at least one of a second antigen expression vector and a third antigen expression vector;
wherein the first antigen expression vector comprises a first DNA molecule of the combination of DNA molecules of any one of claims 1 to 3; the second antigen expression vector comprising a second DNA molecule of the combination of DNA molecules of any one of claims 1 to 3; the third antigen expression vector comprising a third DNA molecule of the combination of DNA molecules of any one of claims 1 to 3.
5. An isolated combination of mRNA molecules produced from the combination of antigen expression vectors of claim 4.
6. A novel coronavirus mRNA vaccine comprising the mRNA molecule combination of claim 5.
7. The novel coronavirus mRNA vaccine of claim 6, further comprising a pharmaceutically acceptable carrier.
8. The novel coronavirus mRNA vaccine of claim 7, wherein the vector is a lipid vector.
9. The novel coronavirus mRNA vaccine of claim 8, wherein the lipid carrier is a cationic lipid nanoparticle.
10. A formulation comprising the novel coronavirus mRNA vaccine of any one of claims 6 to 9.
CN202111195423.XA 2021-10-14 2021-10-14 Novel coronavirus mRNA vaccine for targeted stimulation of humoral immunity and cellular immunity Active CN113637695B (en)

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