CN116925195A - mRNA vaccine based on novel coronavirus - Google Patents
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- CN116925195A CN116925195A CN202310435527.6A CN202310435527A CN116925195A CN 116925195 A CN116925195 A CN 116925195A CN 202310435527 A CN202310435527 A CN 202310435527A CN 116925195 A CN116925195 A CN 116925195A
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Abstract
The present disclosure relates to a novel coronavirus-based mRNA vaccine, in particular for preventing or treating coronavirus infection, and a method for synthesizing the same and an RNA composition. In particular, the present disclosure relates to mRNA vaccines for preventing coronavirus infection by inducing an effective coronavirus antigen-specific immune response. The disclosure also describes methods for preparing the vaccine and immunological evaluation of the vaccine.
Description
Technical Field
The present disclosure relates to the field of prevention or treatment of coronavirus infection, in particular to a novel coronavirus SARS-CoV-2mRNA vaccine, a method for preparing the same and applications thereof. In particular, the present disclosure relates to methods and agents for vaccinating against coronavirus infection and inducing an effective coronavirus antigen-specific immune response such as an antibody and/or T cell response. These methods and agents are particularly useful for preventing or treating coronavirus infections. The disclosure also describes methods for preparing the vaccine and immunological evaluation of the vaccine.
Background
2019 novel coronavirus (SARS-CoV-2) is a novel strain of coronavirus that has never been found in humans before, which is the seventh coronavirus (CoV) that can infect humans. The incubation period of human SARS-CoV-2 infection is generally 1-14 days, and the common signs after infection are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. The latest data of the Shiwei organization website shows that 259502031 definite cases and 5183003 dead cases are reported in a global accumulated way by 2021, 11 and 26 days.
The novel coronavirus is a plus-sense single-stranded RNA ((+) ssRNA) enveloped virus encoding 4 structural proteins: spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N). The S protein is divided into two subdomains S1 and S2, the S1 domain being responsible for recognizing virus-specific receptors and binding to host cells, S2 having a transmembrane domain responsible for membrane fusion. The SARS-CoV-2Delta variant strain was found in India earlier than 10 months in 2020, and at least 185 countries and regions have been transmitted later, becoming the virus variant strain that is mainly prevalent worldwide, while the neutralizing ability of the novel coronavaccine developed based on the early-stage epidemic strain against the SARS-CoV-2Delta variant strain is significantly reduced.
The novel coronavirus vaccines on the market and under research at present mainly comprise mRNA vaccines, inactivated vaccines, adenovirus vector vaccines, DNA vaccines, recombinant protein vaccines and the like. mRNA vaccines are directed to the delivery of in vitro transcribed mRNA to cells, which are translated to produce protein, which in turn elicits a specific immune response in the body. Unlike other nucleic acid vaccines, mRNA vaccines do not require nuclear entry to complete expression only in the cytoplasm, and thus do not risk causing insertion mutations in the host genome. In addition, the quick and simple preparation method and low cost of mRNA are also one of the advantages of the vaccine, so that the reaction time for treating the sudden infectious diseases is greatly shortened, and the prevention and control cost is reduced. The invention aims to prepare a novel coronavirus mRNA vaccine.
Disclosure of Invention
In view of the importance of the S protein in host cell recognition and entry and in the induction of virus neutralizing antibodies by the host immune system, we used the S protein of SARS-CoV-2 for vaccine development. More specifically, the present invention provides an mRNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding an antigen of a SARS-CoV-2Delta mutant strain. SARS-CoV-2 antigen includes spike protein (S protein) and variants thereof. The pre-fusion conformation of the S protein is critical for establishing an effective immune system, so that in order to develop a more targeted mRNA vaccine, we selected the coding sequence of the S protein gene of the Delta virus strain and the substitution variants of specific proline sites performed on the basis of the coding sequence, so as to obtain the antigen sequences of Delta S, delta S-2P (K984P, V985P) and Delta S-6P (F815P, A890P, A897P, A940P, K984P and V985P) respectively.
In one aspect, the invention provides an RNA comprising an open reading frame encoding an antigenic polypeptide of SARS-CoV-2, or an immunogenic fragment or variant thereof, wherein said antigenic polypeptide is selected from the group consisting of the receptor binding domain, S protein, variant or immunogenic fragment thereof of SARS-CoV-2, preferably said SARS-CoV-2 is a SARS-CoV-2Delta variant virus strain.
In some embodiments, for the RNA, wherein the antigenic polypeptide or immunogenic fragment or variant thereof comprises one or more immunogenic epitopes of a SARS-CoV-2 polypeptide or variant thereof; for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more immunogenic epitopes; preferably, the antigenic polypeptide or immunogenic fragment or variant thereof is selected from the group consisting of the (full length) S protein of a variant strain of SARS-CoV-2Delta, preferably the (full length) S protein variant of a variant strain of SARS-CoV-2Delta, more preferably the S protein variant is selected from the group consisting of Delta S-2P comprising the mutations K984P and V985P, and Delta S-6P comprising the mutations F815P, A890P, A897P, A940P, K984P and V985P,
preferably, the antigenic polypeptide or immunogenic fragment or variant thereof comprises the amino acid sequence of SEQ ID NO: 1. 2 or 3, amino acid sequence of amino acids 17-1271, which corresponds to SEQ ID NO: 1. 2 or 3, the amino acid sequence of amino acids 17-1271 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical; and/or
The RNA encoding the antigenic polypeptide or an immunogenic fragment or variant thereof comprises SEQ ID NO: 4. 5 or 6, and nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4. 5 or 6, the nucleotide sequence of nucleotides 49-3813 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
In some embodiments, the open reading frame encoding the antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof further comprises a secretion signal peptide, preferably fused to the antigenic polypeptide or immunogenic fragment or variant thereof via the N-terminus, which is preferably the secretion signal peptide of the S protein. Preferably, the secretion signal peptide comprises SEQ ID NO: 1. 2 or 3, amino acid sequence of amino acids 1-16, which corresponds to SEQ ID NO: 1. 2 or 3, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1. 2 or 3 or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1. 2 or 3, the amino acid sequence of amino acids 1-16 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or
The RNA encoding the secretion signal peptide comprises SEQ ID NO: 4. 5 or 6, and nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6 or a fragment of the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6, the nucleotide sequence of nucleotides 1-48 having a fragment of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
In some preferred embodiments of this aspect, the RNAs are mRNA, circular RNA (cRNA), and self-replicating RNA (saRNA), preferably the RNAs are suitable for intracellular expression of the polypeptide.
In some embodiments, the RNA is a modified RNA that is modified by substitution of some or all of the uridine residues with modified uridine residues, preferably the modified uridine is N1-methyl-pseudouridine.
In a preferred embodiment, the RNA further comprises one or more structural elements optimized for maximum efficacy of the RNA in terms of stability and translation efficiency, preferably the structural elements comprise: 5' cap, 5' UTR, 3' UTR and polyA tail sequences.
Preferably, the 5' cap is or comprises a cap1 structure; more preferably, the 5 'cap is m7G (5') ppp (5 ') (2' -OMeA) pG.
Preferably, the 5'-UTR is the 5' -UTR sequence of human β -globin mRNA, optionally with an optimized Kozak sequence; more preferably, the 5' utr comprises SEQ ID NO:7, or a nucleotide sequence that hybridizes to SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity,
preferably, wherein the 3'-UTR is two repeated 3' -UTRs of human β -globin mRNA; more preferably, the 3' utr comprises SEQ ID NO:8, or a nucleotide sequence that hybridizes to SEQ ID NO:8, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
Preferably, the polyA tail sequence comprises at least 50, at least 60 or at least 100 a nucleotides; more preferably, the polyA tail sequence comprises SEQ ID NO:9, or a nucleotide sequence consisting of SEQ ID NO: 9.
In another aspect, the invention provides a composition comprising an RNA as described herein.
In some embodiments, the composition is formulated or to be formulated as a liquid, solid, or combination thereof, preferably the composition is formulated or to be formulated for injection or other modes of administration, preferably the composition is formulated or to be formulated for intramuscular injection.
In some embodiments of the invention, the RNA is complexed with a protein and/or lipid to produce an RNA-particle for administration.
In a composition of a further embodiment of the invention, the RNA is formulated in a lipid nanoparticle comprising a cationic/ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG) -lipid; preferably the lipid nanoparticle comprises ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-caprate) bis (4-hydroxybutyl) bis (2-hexydecanoa te), 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide (2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide), 1, 2-Distearoyl-sn-glycero-3-phosphorylcholine (1, 2-Distearoyl-sn-glycero-3-phosphorylcholine) and cholesterol; more preferably the lipid nanoparticle comprises SM-102, distearoyl phosphatidylcholine, cholesterol and DMG-PEG2000.
In the lipid nanoparticle, the molar ratio of cations/ionizable lipid, phospholipid, cholesterol, and polyethylene glycol (PEG) -lipid is (40-55): (10-15): (35-45): (0.5-2.5),
preferably the molar ratio of cation/ionizable lipid, phospholipid, cholesterol and polyethylene glycol (PEG) -lipid is 50:10:38.5:1.5.
In the compositions of some embodiments of the invention, the RNA is formulated or to be formulated as a colloid; preferably, the RNA is formulated as particles, 50% or more, 75% or more, or 85% or more RNA being present in the colloidal dispersed phase formed; more preferably the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase, wherein preferably the organic phase comprises ethanol; also preferably, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase, wherein preferably the lipids dispersed in the aqueous phase form liposomes.
In some embodiments of the invention, the RNA is present in the composition in an amount ranging from 1 μg to 100 μg per dose.
In yet another aspect, the invention provides the use of an RNA or composition described herein in the manufacture of a medicament, the medicament being a vaccine, the medicament further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.
In some embodiments, the medicament of the invention is for inducing an immune response against coronavirus, preferably a specific immune response against coronavirus antigen, in a subject.
In some embodiments, the medicament of the invention is used in the treatment or prophylactic treatment of coronavirus infection.
In a preferred embodiment, the coronavirus is a β coronavirus, preferably the coronavirus is sand Bei Bingdu (sarbecovirus), more preferably the coronavirus is SARS-CoV-2, further preferably the coronavirus comprises: new coronavirus original strain (GD 108), SARS-CoV-2Alpha variant virus strain, SARS-CoV-2Beta variant virus strain, SARS-CoV-2Delta variant virus strain and SARS-CoV-2Omicron variant virus strain.
In said aspect of the invention, detectable expression of said antigenic polypeptide or immunogenic fragment or variant thereof is effected when said RNA, composition or medicament is administered to a human cell, and preferably such expression is for a period of at least 24 hours or more.
In this aspect of the invention, administration of the RNA, composition or medicament produces an immune effect in a subject, the immune effect comprising production of SARS-CoV-2 neutralizing antibodies and/or T cell responses, in particular a robust TH1 type T cell response, preferably a cd4+ and/or cd8+ T cell response.
In this aspect of the invention, administration of the RNA, composition or medicament produces an immune response in the subject, preferably the immune response comprises production of a bound antibody titer against the S1 subunit of SARS-CoV-2 spike protein, more preferably the immune response comprises production of a neutralizing antibody titer against SARS-CoV-2 virus.
In this aspect of the invention, the serum of the subject (e.g., a mouse) shows the production of antibodies to the polypeptide encoded by the open reading frame 7 days after administration of the RNA, composition or medicament to the subject.
In this aspect of the invention, the serum of a subject (e.g., a mouse) exhibits virus neutralization activity 14 days after administration of the RNA, composition or drug to the subject.
In the aspect of the invention, the subject is a mammal, preferably the subject is a mouse, and further preferably the subject is a human.
The invention also provides a method of preparing a vaccine comprising formulating an RNA as described herein in a lipid nanoparticle comprising a cationically ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG) -lipid; preferably the lipid nanoparticle comprises ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (hexyl 2-decanoate), 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine and cholesterol; more preferably the lipid nanoparticle comprises SM-102, distearyl phosphatidylcholine (DSPC), cholesterol and DMG-PEG2000.
The invention also provides methods of inducing an immune response in a subject against a coronavirus, preferably a specific immune response against a coronavirus antigen. In another aspect, methods for the therapeutic or prophylactic treatment of coronavirus infections are also provided. Preferably, the method comprises administering to a subject an RNA, composition or medicament described herein. Preferably, the subject is a mammal, preferably the subject is a mouse, further preferably the subject is a human.
In some embodiments of the methods of the invention, the coronavirus is a β coronavirus, preferably the coronavirus is a saber virus, more preferably the coronavirus is SARS-CoV-2, further preferably the coronavirus comprises: new coronavirus original strain (GD 108), SARS-CoV-2Alpha variant virus strain, SARS-CoV-2Beta variant virus strain, SARS-CoV-2Delta variant virus strain and SARS-CoV-2Omicron variant virus strain.
Drawings
FIG. 1 expression of LNP-mRNA in 293T cells.
Fig. 2: immunological evaluation of novel coronavirus mRNA vaccines on mouse models.
Fig. 3: immunological evaluation of novel coronavirus mRNA vaccines on rhesus model.
Fig. 4: physiological evaluation of novel coronavirus mRNA vaccine after immunization of rhesus monkeys.
Fig. 5: rhesus monkey lung pathology was scored.
Detailed Description
The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and synthetic biology, etc., which are within the skill of the art. Such techniques are well explained in the literature: "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al, 1989); "Oligonucleotide Synthesis" (M.J.Gait, 1984); "Animal Cell Culture" (r.i. freshney, 1987); "Methods in Enzymology" (Academic Press, inc.); "Current Protocols in Molecular Biology" (F.M. Ausubel et al, 1987, and periodic updates); "PCR: the Polymerase Chain Reaction," (Mullis et al, 1994); singleton et al, second edition Dictionary of Microbiology and Molecular Biology, J.Wiley & Sons (New York, N.Y. 1994) and March's Advanced Organic Chemistry Reactions, fourth edition Mechanisms and Structure, john Wiley & Sons (New York, N.Y. 1992), provide one of ordinary skill in the art with a general guidance for many of the terms used in the present application.
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. For the purposes of the present invention, the following terms are defined below.
The articles "a" and "an" and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The use of alternatives (e.g., "or") should be understood to mean either, both, or any combination thereof. The term "and/or" should be understood to mean either or both of the alternatives.
As used herein, the term "about" or "approximately" means an amount, level, value, quantity, frequency, percentage, dimension, size, quantity, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference amount, level, value, quantity, frequency, percentage, dimension, size, quantity, weight, or length.
Throughout this specification, unless the context requires otherwise, the terms "comprise," "comprising," "includes," "including," and "having" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms "comprising," "including," "containing," and "having" are used synonymously.
"consisting of … …" is meant to include, but is not limited to, any following the phrase "consisting of … …". Thus, the phrase "consisting of … …" is an indication that the listed elements are required or mandatory, and that no other element may be present.
"consisting essentially of … …" is intended to include any element listed after the phrase "consisting essentially of … …" and is limited to other elements not interfering with or contributing to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of … …" is an indication that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.
Reference throughout this specification to "one embodiment," "some embodiments," "a particular embodiment," and the like means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
As used herein, "mutant" and "variant" refer to molecules that retain the same or substantially the same biological activity as the original sequence. The mutants or variants may be from the same or different species, or may be synthetic sequences based on natural or existing molecules. In some embodiments, the terms "mutant" and "variant" refer to polypeptides having an amino acid sequence that differs from the corresponding wild-type polypeptide by at least one amino acid. For example, mutants and variants may comprise conservative amino acid substitutions: i.e. the original corresponding amino acid is replaced by an amino acid having similar properties. Conservative substitutions may be polar versus polar amino acids (glycine (G, gly), serine (S, ser), threonine (T, thr), tyrosine (Y, tyr), cysteine (C, cys), asparagine (N, asn), and glutamine (Q, gin)); nonpolar to nonpolar amino acids (alanine (a, ala), valine (V, val), tryptophan (W, trp), leucine (L, leu), proline (P, pro), methionine (m, met), phenylalanine (F, phe)); acidic para-acidic amino acids (aspartic acid (D, asp), glutamic acid (E, glu)); basic para-basic amino acids (arginine (R, arg), histidine (H, his), lysine (K, lys)); charged amino acids versus charged amino acids (aspartic acid (D, asp), glutamic acid (E, glu), histidine (H, his), lysine (K, lys), and arginine (R, arg)); and hydrophobic para-hydrophobic amino acids (alanine (a, ala), leucine (ule), isoleucine (I, ile), valine (V, val), proline (P, pro), phenylalanine (F, phe), tryptophan (W, trp), and methionine (M, met)). In some other embodiments, the mutant or variant may also comprise a non-conservative substitution.
In some embodiments, the mutant or variant polypeptide may have substitutions, additions, insertions, or deletions of amino acids of a range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more, or any two of the foregoing values. The mutant or variant may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or a range of any two of the foregoing values, as compared to the unaltered polypeptide.
A polynucleotide or polypeptide has a certain "sequence identity" or "percent identity" to another polynucleotide or polypeptide, meaning that the percent bases or amino acids are identical and in the same relative position when the two sequences are aligned. Determining the percent identity of two amino acid sequences or two nucleotide sequences may include aligning and comparing amino acid residues or nucleotides at corresponding positions in the two sequences. A sequence is considered 100% identical if all positions in both sequences are occupied by the same amino acid residue or nucleotide. Sequence identity can be determined in a number of different ways, for example, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.).
The invention generally includes immunotherapy of a subject comprising administering RNA, i.e., vaccine RNA, that encodes an amino acid, i.e., vaccine antigen, comprising SARS-CoV-2S protein, or an immunogenic fragment or variant thereof. Thus, the vaccine antigen comprises an epitope of the SARS-CoV-2S protein for inducing an immune response against the coronavirus S protein, in particular the SARS-CoV-2S protein, in a subject. RNA encoding the vaccine antigen is administered to provide (after expression of the polynucleotide by a suitable target cell) the antigen for inducing, i.e. stimulating, eliciting and/or expanding an immune response, e.g. antibodies and/or immune effector cells, which target the target antigen (coronavirus S protein, in particular SARS-CoV-2S protein) or processed products thereof. The immune response induced according to the present disclosure is a B cell mediated immune response, i.e. an antibody mediated immune response, in particular an anti-SARS-CoV-2 immune response.
The vaccines described herein comprise as an active ingredient single stranded RNA which can be translated into the corresponding protein upon entry into the recipient cell. In addition to wild-type, mutant or codon-optimized sequences encoding antigen sequences, the RNA may also comprise one or more structural elements that are optimized for the maximum potency of the RNA in terms of stability and translation efficiency (5 ' cap, 5' utr, 3' utr, polyA tail). The m7G (5 ') ppp (5') (2 '-OMeA) pG formed by CleanCap can be used as a specific capping structure for the 5' -end of RNA drug substance. As the 5'-UTR sequence, a 5' -UTR sequence of human β -globin mRNA may be used, optionally with an optimized "Kozak sequence" to increase translation efficiency. The 3'-UTR may be two repeated 3' -UTRs of human beta-globin mRNA. In addition, polyA tail sequences of 50-120 nucleotides in length may be used.
In addition, the secretion signal peptide (sec) may be fused to the antigen encoding region, preferably in such a manner that sec is translated into an N-terminal tag. In one embodiment, sec corresponds to the secretion signal peptide of the S protein.
The vaccine RNAs described herein may be complexed with proteins and/or lipids (preferably lipids) to produce RNA-particles for administration. If a combination of different RNAs is used, the RNAs may be complexed together or separately with proteins and/or lipids to produce RNA-particles for administration.
In one aspect, the invention relates to a composition or pharmaceutical product (medical preparation) comprising RNA encoding an amino acid sequence comprising a SARS-CoV-2S protein or an immunogenic fragment or variant thereof.
In one embodiment, the amino acid sequence comprising the SARS-CoV-2S protein or immunogenic fragment or variant thereof is capable of forming a multimeric complex, particularly a trimeric complex. For this purpose, the amino acid sequence comprising the SARS-CoV-2S protein or immunogenic fragment or variant thereof may comprise a domain that allows the formation of a multimeric complex, in particular a trimeric complex comprising the amino acid sequence of the SARS-CoV-2S protein or immunogenic fragment or variant thereof. In one embodiment, the domain that allows for the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain described herein.
In one embodiment, the amino acid sequence comprising the SARS-CoV-2S protein or immunogenic fragment or variant thereof is encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to the wild-type coding sequence, wherein the codon optimization and/or G/C content increase preferably does not alter the sequence of the encoded amino acid sequence.
In one embodiment, (i) the RNA encoding SARS-CoV-2S protein or an immunogenic fragment or variant thereof comprises the sequence of SEQ ID NO: 4. 5 or 6, and nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4. 5 or 6, the nucleotide sequence of nucleotides 49-3813 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or
(ii) the SARS-CoV-2S protein or immunogenic fragment or variant thereof comprises the amino acid sequence of SEQ ID NO: 1. 2 or 3, amino acid sequence of amino acids 17-1271, which corresponds to SEQ ID NO: 1. 2 or 3, and the amino acid sequence of amino acids 17-1271 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
In one embodiment, the amino acid sequence comprising a SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment or variant of said SARS-CoV-2S protein or an immunogenic fragment thereof comprises a secretion signal peptide.
In one embodiment, the secretion signal peptide is fused, preferably by N-terminal fusion, to the SARS-CoV-2S protein or immunogenic fragment or variant thereof.
In one embodiment, (i) the RNA encoding the secretion signal peptide comprises SEQ ID NO: 4. 5 or 6, and nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6 or a fragment of the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6, the nucleotide sequence of nucleotides 1-48 having a fragment of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical; and/or
(ii) the secretion signal peptide comprises SEQ ID NO: 1. 2 or 3, amino acid sequence of amino acids 1-16, which corresponds to SEQ ID NO: 1. 2 or 3, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1. 2 or 3 or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1. 2 or 3, and the amino acid sequence of amino acids 1-16 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity.
In one embodiment, the RNA is a modified RNA, in particular a stable mRNA. In one embodiment, the RNA comprises a modified nucleoside in place of uridine. In one embodiment, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).
In one embodiment, the RNA encoding an amino acid sequence comprising a SARS-CoV-2S protein or an immunogenic fragment or variant thereof comprises a 5' utr comprising the amino acid sequence of SEQ ID NO:7, or a nucleotide sequence that hybridizes to SEQ ID NO:7 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
In one embodiment, the RNA encoding an amino acid sequence comprising a SARS-CoV-2S protein or an immunogenic fragment or variant thereof comprises a 3' utr comprising the amino acid sequence of SEQ ID NO:8, or a nucleotide sequence that hybridizes to SEQ ID NO:8, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
In one embodiment, the RNA encoding an amino acid sequence comprising a SARS-CoV-2S protein or an immunogenic fragment or variant thereof comprises a polyA tail sequence. In one embodiment, the polyA tail sequence comprises at least 100 nucleotides. In one embodiment, the polyA tail sequence comprises SEQ ID NO:9, or a nucleotide sequence consisting of SEQ ID NO: 9.
In one embodiment, the RNA or composition is formulated or to be formulated as a liquid, a solid, or a combination thereof. In one embodiment, the RNA or composition is formulated or to be formulated for injection or other modes of administration. In one embodiment, the RNA or composition is formulated or to be formulated for intramuscular injection.
In one embodiment, the RNA is formulated or to be formulated as particles. In one embodiment, the particle is a Lipid Nanoparticle (LNP).
In one embodiment, the LNP particles comprise (4-hydroxybutyl) azetidinyl) bis (hexane-6, 1-diyl) bis (2-caprate) bis (hexane-6, 1-diyl) bis (2-hexydecanoa), 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide (2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide), 1, 2-Distearoyl-sn-glycero-3-phosphorylcholine (1, 2-Distearoyl-sn-glycero-3-phosphorylcholine), and cholesterol.
In one embodiment, the RNA is formulated or to be formulated as a colloid. In one embodiment, the RNA is formulated or to be formulated as particles, forming a colloidal dispersed phase. In one embodiment, 50% or more, 75% or more, or 85% or more RNA is present in the dispersed phase. In one embodiment, the RNA is formulated or to be formulated as particles comprising RNA and lipid. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase. In one embodiment, the organic phase comprises ethanol. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase. In one embodiment, the lipid dispersed in the aqueous phase forms liposomes.
In one embodiment, the RNA is mRNA, circular RNA (cRNA), and self-replicating RNA (saRNA).
In one embodiment, the composition or pharmaceutical product is a pharmaceutical composition. In one embodiment, the composition or pharmaceutical product is a vaccine. In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
In one aspect, the present invention relates to a composition or pharmaceutical product as described herein for use in pharmaceutical applications. In one embodiment, the pharmaceutical use comprises inducing an immune response against a coronavirus in a subject. In one embodiment, the pharmaceutical use includes the treatment or prophylactic treatment of a coronavirus infection. In one embodiment, the coronavirus is a beta coronavirus. In one embodiment, the coronavirus is sand Bei Bingdu (sarbecovirus). In one embodiment, the coronavirus is SARS-CoV-2. In a further preferred embodiment, the coronavirus comprises: new coronavirus original strain (GD 108), SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Delta variant and SARS-CoV-2Omicron variant
In one aspect, the invention relates to a method of inducing an immune response against a coronavirus in a subject, the method comprising administering to the subject a composition comprising RNA encoding an amino acid sequence comprising a SARS-CoV-2S protein or an immunogenic fragment or variant thereof.
In one embodiment, the method is a method of vaccinating against coronavirus. In one embodiment, the method is a method for the therapeutic or prophylactic treatment of a coronavirus infection. In one embodiment, the subject is a mouse. In one embodiment, the coronavirus is a beta coronavirus. In one embodiment, the coronavirus is sand Bei Bingdu (sarbecovirus). In one embodiment, the coronavirus is SARS-CoV-2. In a further preferred embodiment, the coronavirus comprises: new coronavirus original strain (GD 108), SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Delta variant and SARS-CoV-2Omicron variant
In one aspect, the invention relates to a composition or pharmaceutical product described herein for use in a method described herein.
Wherein the present disclosure demonstrates that a composition comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., is or comprises an epitope) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein) can achieve a detectable antibody titer against the epitope in serum within 7 days after administration to a mouse according to the regimen (including administration of at least one dose of the vaccine composition).
The present disclosure records compositions provided in which nucleotides within the mRNA are modified (e.g., compositions comprising lipid nanoparticle-coated mRNA encoding at least a portion (e.g., is or comprises an epitope) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein)) and/or methods involving the provision of such compositions are characterized by the absence of intrinsic adjuvant effects or reduced intrinsic adjuvant effects as compared to other comparable compositions (or methods) having unmodified results. Alternatively or additionally, in some embodiments, such compositions (or methods) are characterized in that they induce an antibody response and/or a cd4+ T cell response. In some embodiments involving modified nucleotides, such modified nucleotides may be present in, for example, a 3'utr sequence, an antigen coding sequence, and/or a 5' utr sequence. In some embodiments, the modified nucleotide is or includes one or more modified uracil residues.
Wherein the disclosure records compositions (e.g., compositions comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., is or comprises an epitope) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein)) and/or methods characterized by sustained expression of the encoded polypeptide (e.g., SARS-CoV-2 encoded protein (e.g., S protein)), which may be or comprise an epitope thereof in some embodiments. For example, in some embodiments, such compositions and/or methods are characterized in that they achieve detectable polypeptide expression when administered to a human cell, and in some embodiments, such expression is continued for a period of at least 24 hours or more.
Those of skill in the art will further appreciate from reading this disclosure that it describes various mRNA constructs comprising the nucleic acid sequence of a full-length SARS-CoV-2 spike protein (e.g., including embodiments in which such encoded SARS-CoV-2 spike protein may comprise at least one or more amino acid substitutions, e.g., proline substitutions as described herein, and/or embodiments in which the mRNA sequence is codon optimized for a subject (e.g., mammal, e.g., human). Still further, one of ordinary skill in the art will appreciate upon reading this disclosure that it describes certain features and/or advantages of certain mRNA constructs comprising a nucleic acid sequence encoding a full-length SARS-CoV-2 spike protein. Without wishing to be bound by any particular theory. In some embodiments, one of ordinary skill in the art will appreciate that the provided mRNA constructs encoding full-length SARS-CoV-2S proteins can be particularly useful and/or effective for use as or in immunogenic compositions (e.g., vaccines) to achieve an immune effect as described herein (e.g., to produce SARS-CoV-2 neutralizing antibodies, and/or T cell responses (e.g., cd4+ and/or cd8+ T cell responses)).
In some embodiments, the disclosure provides RNAs (e.g., mrnas) comprising an open reading frame encoding a full-length SARS-CoV-2S protein (e.g., a full-length SARS-CoV-2S protein having one or more amino acid substitutions) that are suitable for intracellular expression of a polypeptide. In some embodiments, such RNAs can be formulated in lipid nanoparticles (e.g., lipid nanoparticles described herein).
In some embodiments, an immunogenic composition provided herein can comprise a plurality (e.g., at least 2 or more, including, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.) immunogenic epitopes of a SARS-CoV-2 polypeptide or variant thereof. In some such embodiments, such multiple immunogenic epitopes can be encoded by a single RNA (e.g., mRNA). Without wishing to be bound by any particular theory, in some embodiments, when considering the genetic diversity of SARS-CoV-2 variants, the provided multi-epitope immunogenic compositions (including, for example, those encoding full-length SARS-CoV-2 spike proteins) may be particularly useful in providing protection against a variety of viral variants and/or may provide greater opportunities to develop diverse and/or robust neutralizing antibodies and/or T cell responses, particularly robust TH1 type T cell (e.g., cd4+ and/or cd8+ T cell) responses.
In some embodiments, the present disclosure records that the provided compositions and/or methods are characterized in that they achieve one or more specific therapeutic results (e.g., an effective immune response and/or detectable expression of the encoded SARS-CoV-2S protein as described herein) with a single administration.
In some embodiments, the immune response may include generating a binding antibody titer against the S1 subunit of SARS-CoV-2 spike protein. In some embodiments, the immune response may include generating neutralizing antibody titers against SARS-CoV-2 virus.
In some embodiments, the neutralizing antibody titer is (e.g., established as) a titer sufficient to reduce or block binding of virus to serum of vaccinated mice as observed relative to an appropriate control (e.g., unvaccinated control mice).
Examples
Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.
The experimental methods used in the examples below are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 preparation of novel coronavirus mRNA vaccine
S protein Gene Synthesis and vector construction
1.1S protein Gene Synthesis and optimization
The target antigen of the novel coronavirus mRNA vaccine is the full length of the S protein of a Delta variant virus strain, and the coding sequence of the gene is shown in GenBank: QWU18818.1, and on the basis of this, combined mutation of different sites is carried out to obtain Delta S-2P (K984P, V985P) and Delta S-6P (F815P, A890P, A897P, A940P, K984P, V985P), respectively. According to the amino acid sequence, the coded nucleotide is reversely deduced and synthesized by a gene synthesis company after human codon optimization and related cleavage site exclusion.
1.2 vector construction
And (3) performing BamHI and AscI double digestion on the synthesized S sequence, performing 1% agarose gel electrophoresis gel cutting recovery on the digested product, performing a ligation reaction, and replacing the luc coding sequence in the pUC57-luc vector with the 5'UTR, the target gene, the 3' UTR and the polyA tail sequence in sequence with the S gene sequence to form the pUC57-S recombinant plasmid. After monoclonal plate preliminary screening with kana antibiotics, the miniplasmids were submitted and positive clone verified by BamHI and AscI restriction enzyme digestion and DNA sequencing.
Preparation of mRNA
2.1 plasmid linearization
The plasmid contains the T7 promoter, 5'UTR, ORF, 3' UTR and the polyA tail sequence, followed by a SapI cleavage site after the last A of the polyA tail sequence. The plasmid containing the target gene was linearized with restriction enzyme SapI and the reaction system is shown in Table 1 and digested at 37℃for 3h.
TABLE 1 plasmid linearization cleavage System
| 10xCutsmart buffer | 5μL |
| SapI enzyme (10000U/mL) | 1μL |
| Plasmid(s) | 10μg |
| ddH 2 O | Supplement to 50 mu L |
mu.L of the digested product was subjected to 1% agarose gel electrophoresis to determine whether the plasmid was completely linearized. Linearized plasmids were purified using PCR product recovery kit (convalaver century).
2.2 in vitro transcription
In vitro transcription was performed using the linearized recombinant plasmid as a template, and the components (for example, 20. Mu.L of the reaction system) were added according to the following system (Table 2), and reacted at 37℃for 2 hours after mixing.
TABLE 2 in vitro transcription System
| 10x reaction buffer | 2μL |
| ATP(100mM) | 2μL |
| ΨTP(100mM) | 2μL |
| CTP(100mM) | 2μL |
| GTP(100mM) | 2μL |
| Enzyme mixture | 2μL |
| DNA template | 500ng-1μg |
| CleanCap AG(100mM) | 1μL |
| Nuclease-free H 2 O | Supplement to 20 mu L |
After completion of the transcription reaction, 1. Mu.L of DNase I was added, and the reaction was carried out at 37℃for 15 minutes, followed by addition of 15. Mu.L of a reaction terminator.
2.3RNA purification
To the in vitro transcription reaction system was added 1/3 volume of 7.5M LiCl (to give a final concentration of 2.5M), -left at 20℃for 30min.12000g was centrifuged for 15min, RNA was precipitated at the bottom, and the supernatant was discarded. RNA was washed by adding 1mL of 70% ethanol, centrifuged at 12000g for 5min, and the supernatant was discarded. After air-drying, 50. Mu.L of RNase-free water was added to dissolve the precipitate, and mRNA was quantified using an ultraviolet spectrophotometer to obtain capped in vitro transcribed mRNA.
LNP entrapment
mRNA stock solution was dispersed in 20mM acetic acid solution (pH 6.0) to give a final concentration of 200. Mu.g/mL. The mRNA and lipid mixtures (formulation see table 3) were mixed T-mix at a 3:1 volume ratio by controlling the flow rates of the aqueous and oil phase side syringe pumps to obtain LNP-entrapped mRNA. Then the solution was replaced and concentrated by ultrafiltration. The concentration of the liposome mRNA is measured by using a Ribogreen method, free RNA is directly sampled and measured, total RNA is measured after 5% OTG is cracked, and the encapsulation rate is calculated to be more than 90%. The particle size of the liposome mRNA was measured by a Markov Zetasizer particle size analyzer and was about 60 nm.
Table 3 lipid mixture formulation
Expression identification of S target antigen
293T cells were seeded in six well plates and 24h later 2. Mu.g of LNP-coated mRNA was added to the cell culture medium for intracellular expression. After 24h, cells were collected, and 100. Mu.L of 1 Xprotein loading buffer was added to each well of cell pellet and lysed in a metal bath at 100℃for 30min. Protein samples were stored at-20℃or-80 ℃. Expression of S protein was detected by immunoblotting: mu.L of protein lysate was subjected to 10% SDS-PAGE to separate protein samples. After 75V constant pressure wet transfer for 1.2 hours using PVDF membrane, 5% skim milk was blocked for 1 hour at room temperature. Incubation with primary antibody (anti-SARS-CoV-2S protein rabbit antibody, 1:1000) followed by secondary antibody (goat anti-rabbit, 1:5000) and final ECL development. The results are shown in FIG. 1, where three mRNA vaccines, delta S-2P and Delta S-6P, detect the expression of S protein on a cellular level.
EXAMPLE 2 immunological evaluation of different novel coronavirus mRNA vaccines on mouse model 1 immunization and serum separation of mice
SPF-class female BALB/c mice (6-8 weeks old) were immunized by injection with the mRNA vaccine of the present invention, and the immunization protocol is shown in FIG. 2A. The immune group was LNP control group and different mRNA vaccine group. The serum used in this experiment was the serum 14 days after the secondary immunization. 2. Neutralization activity detection of vaccine immune serum on SARS-CoV-2 pseudovirus
The neutralizing activity of serum against SARS-CoV-2Delta, omicron strain pseudotype virus was tested in a Hela-ACE2 cell line (described in "Liu X, wei L, xu F, zhao F, huang Y, fan Z, mei S, hu Y, zhai L, guo J, zheng A, cen S, liang C, guo F.SARS-CoV-2spike protein-induced cell fusion activates the cGAS-STING pathway and the interferon response.Sci Signal.2022Apr 12;15 (729): eabg8744.Doi: 10.1126/scissign.abg8744.Epub 2022Apr 12.PMID:35412852.") which stably overexpresses the SARS-CoV-2 primary receptor ACE 2. Pseudotype virus is a single round of infection pseudotype virus packaged by S (Spike) protein and using slow virus as a core and firefly luciferase reporter gene. Serum to be tested was serially diluted 2-fold with DMEM medium for a total of 8 gradients, from 1:128 to 1:16384. Pseudoviruses were diluted to 1.5X10 in P2 laboratory with DMEM medium 4 TCID 50 /mL. Mixing serum with diluted virus solution, and mixing with 5% CO at 37deg.C 2 Incubate in incubator for 1 hour. The incubated virus serum mixture was added to a pre-inoculated Hela-ACE2 cell plate at 100. Mu.l per well and placed at 37℃with 5% CO 2 Culturing in an incubator. After 48 hours, firefly luciferase activity was measured and pseudoviral infectivity was calculated with the reciprocal of the highest serum dilution inhibiting 50% luciferase activity as the endpoint titer. Statistical analysis of the two-tailed student t-test (.xp) for significant differences between the Delta-S (6P) group and the other groups using GraphPad Prism 8 software<0.05vs.Delta-S(6P))。
Referring to the results in FIG. 2B, it was shown that the serum of the mice immunized with Delta S-6P had a higher neutralizing activity against the Delta pseudovirus than that of Delta S and Delta S-2P. The neutralizing activity against Omicron strain pseudovirus (PNT 50) induced by the serum of mice after two immunizations with Delta S-6P reached 992, which is 2-4 times that of the Delta S and Delta S-2P vaccines.
3. Neutralizing antibody detection of vaccine immune serum to SARS-CoV-2 real virus
Detection of immune mouse serum on Vero-E6 cells against 5 strains of SARS-CoV-2 (epidemic strain (i.e. new coronariesNeutralizing antibody titers of the initial strain (GD 108)), alpha, beta, delta and omacron strains). The serum to be tested is inactivated at 56℃for 30 minutes. Serial dilutions were performed 2-fold with DMEM medium, starting with 1:8 dilution. Viruses were diluted to working concentration in DMEM medium in P3 laboratory based on their original titer. Mixing diluted virus solution with serum of each dilution gradient, and concentrating at 37deg.C with 5% CO 2 Incubate in incubator for 1 hour. The incubated virus serum mixture was added to a suspension Vero-E6 cell plate at 100. Mu.l per well and placed at 37℃in 5% CO 2 Serum neutralizing antibody titers (EC) were calculated by determining cellular CPE after 72 hours of incubation in incubators 50 ). The cross-protective effect of serum neutralizing antibodies of immunized rhesus monkeys was also assessed. The experiment was performed in the P3 laboratory of the institute of medical biology, national academy of medical science. Statistical analysis of two-tailed student t-test for significance differences between Delta S-6P group and other groups using GraphPad Prism 8 software (P)<0.01vs.Delta S-6P；*p<0.05vs.Delta S-6P)。
The results in fig. 2C show that the three vaccines produced neutralizing antibodies with cross-protection against multiple strains. The serum of the mice immunized by Delta S-6P shows strong neutralization activity on the infection of the five strains, and particularly the neutralization activity of the mice immunized by Delta S-6P is obviously better than that of Delta S and Delta S-2P.
Example 3 immunological evaluation of different novel coronavirus mRNA vaccines on rhesus model
1. Immunization of rhesus monkeys
The rhesus monkeys were immunized by intramuscular injection with Delta S-6P mRNA vaccine at the time points D0 (primary) and D21 (secondary). The immune group was LNP control group, low dose group (30. Mu.g) mRNA vaccine, high dose group (100. Mu.g) mRNA vaccine. Toxin challenge was performed 28 days after the second immunization. The IND strain (Delta) was used for all 3 groups of experimental animals (virulent strain 21V05P0345/21V05P0346/21V05P0347, titres 1X 10 were used) 6 TCID 50 Per mL), the toxicity is removed by nasal drip and tracheal injection inoculation of 500 mu L each, and the toxicity removing amount of each monkey is 1 multiplied by 10 6 TCID 50 . Immunization protocols are shown in fig. 3A.
2. Antigen-specific IgG antibody detection in serum
Serum from all 12 animals from the control, low and high dose groups was collected before immunization (D0) and after immunization (D7, D14, D21 (secondary), D27, D35) and assayed for antigen-specific binding antibody IgG levels by ELISA (enzyme-linked immunosorbent assay). The 96-well ELISA plate is coated with NTD protein with relatively high conservation of S protein of the original strain, wherein the coating concentration is 100 ng/well, the volume is 100 mu L, and the coating is carried out at 2-8 ℃ overnight. The next day, after 3 washes with wash solution (PBST), blocking was performed for 2 hours at 25℃using blocking solution (PBST solution containing 2% BSA). Further, the serum samples after the gradient dilution were added to a 96-well plate at 100. Mu.L/well and incubated at 25℃for 2 hours. Serum dilution was started at 1:100, 2-fold dilution; after incubation was completed, the cells were washed 3 times with PBST, HRP-conjugated anti-monkey IgG was added, and incubated at 25 ℃ for 1 hour. Finally, the mixture was washed 5 times with PBST, TMB was added at 100. Mu.L/well, developed in the dark, and 100. Mu.L/Kong Zhongzhi solution was added. And reading data on an enzyme label instrument, taking 610nm as a reference wavelength, and reading an OD value at 450nm to further calculate the serum IgG titer.
The results in fig. 3B show that: all animals in the low dose and high dose groups began producing high levels of binding antibodies at D14 after immunization with mRNA vaccine. All 4 animals of the control group had no production of new coronavirus-specific antibodies.
3. Neutralization potency detection of vaccine
Experimental monkey serum was collected and neutralization titer detection was performed using the new coronavirus euvirus according to the same experimental method as in example 1.
The results in FIGS. 3C-G show that: the serum of experimental monkeys after the high dose group and the low dose group were subjected to the secondary immunization, and the neutralizing antibodies were detected to a certain degree, wherein the neutralizing titer of the immune group against the original strain and the Delta strain was higher. No neutralizing antibodies were detected in the control group.
The serum used for further experiments was serum 14 days after the secondary immunization. Serum was tested for neutralizing activity against SARS-CoV-2 original strain, delta, omicron BA.1, omicron BA.2.12.1, omicron BA.4/5 strain pseudotyped virus according to the same experimental method as in example 1. The junction shown in FIG. 3HThe results show that the vaccine immune group has a strong neutralizing effect against pseudoviruses of the original strain and the Delta strain. High and low dose group NT against Omicron strain 50 The values are as follows: for BA.1 plants, respectively, are-826 and-1172; about 570 and about 905 for the BA.2.12.1 strain; for the BA4/5 strains-364 and-463 respectively, these all indicate that the vaccine has the potential to induce broad-spectrum neutralizing antibodies in rhesus monkeys.
Samples of PBMCs from day 14 after the high dose and low dose groups were analyzed using a commercial Assay kit from Mabtech (cat# PBMC IFN-. Gamma.ELISPOT Assay:3421M-4APW-2; PBMC IL-2ELISpot Assay:3421M-4 AST-2) using ELISPOT (enzyme-linked immune absorbent spot) to detect the ability of antigen-specific T cells to secrete IFN-. Gamma.and IL-2 following Spike protein stimulation of the original, delta and Omicron strains. The results in fig. 3I show that: the vaccine group can significantly activate the cellular immune response after the second immunization. There was no significant dose-group difference in the level of cellular immune response generated by the omacron strain and the original strain against the new coronavirus Delta strain.
Example 4 physiological evaluation of novel coronavirus mRNA vaccine after immunization of rhesus monkeys 1. Animal viral load detection
Rhesus monkeys were anesthetized before and after challenge on days 1,3,5,7, and nasal swabs, pharyngeal swabs, and anal swabs were collected. Swab sample processing: nasal, pharyngeal, anal swabs were lysed with 800 μl Trizol, 200 μl of which was used to extract RNA template using an automatic nucleic acid extractor, and SARS-CoV-2 genomic RNA levels were determined using qRT-PCR (one-step method).
The results in FIG. 4A show that the level of SARS-CoV-2 genomic RNA was significantly reduced in Delta S-6P immunized rhesus nasal swabs, pharyngeal swabs, anal swabs compared to the control group.
2. Animal pulmonary viral load detection
On day 7 after the toxin is attacked, the general pathological changes of the lung are observed through dissection, and each monkey takes left lung (upper, middle and lower lobes) and right lung (upper, middle and lower lobes) tissues respectively. Each lung was randomly sampled at multiple points (6 points), the sampling points were run through the whole lung lobe, the total weight was weighed to approximately 100mg, tissue homogenization was performed with 800 μl Trizol, 400 μl of the extracted RNA template was taken, and qRT-PCR (one-step method) was used to detect viral load.
The results in FIG. 4B show a significant reduction in lung lobe tissue viral load in experimental monkeys vaccinated with Delta S-6P compared to the control group, indicating a strong inhibition of SARS-CoV-2 viral replication. Furthermore, the 100 μg Delta S-6P immunized group as a whole showed better viral clearance than the 30 μg dose.
3. Animal lung pathology analysis
Each rhesus monkey was stained with hematoxylin-eosin (H & E) and scanned in whole images per lobe lung tissue (upper left, middle left, lower left, upper right, middle right, lower right, 6 lobes total). The rhesus monkey is classified for pulmonary inflammation, pulmonary structural changes, hemorrhage, etc., and the scoring criteria of each index are shown in table 4 below.
TABLE 4 rhesus monkey pulmonary pathology change scoring Table
Scoring the pathology maps of each lung of each rhesus lung tissue pathology according to a scoring table, wherein the scoring is mainly aimed at the main pathological characteristics of the new coronavirus infection: and (3) evaluating characteristic indexes such as the thickening or actual transformation degree of the lung interval, the bleeding degree of the lung interval, the inflammatory cell infiltration degree, the vascular thrombosis, the dust cell distribution area and the like, wherein the aggregate of the scores of all indexes is the pathological score. At least 5 fields of view were selected for scoring, and the average of pathology scores for all lung lobes was the combined pathology score for the entire lung of the monkey. The results in fig. 5 show that the low and high dose groups have significantly lower scores for lung pathological lesions than the control group (p < 0.01).
While preferred embodiments of the present invention have been shown and described herein, it should be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
Partial sequence information:
SEQ ID NO:1Delta S amino acid sequence
MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS
NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV
NNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDL
EGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTL
LALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET
KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN
CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA
DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSK
PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN
KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS
VITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH
VNNSYECDIPIGAGICASYQTQTNSRRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPT
NFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK
NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY
GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP
FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQN
AQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAA
EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKN
FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN
NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN
ESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT**
SEQ ID NO:2Delta S-2P amino acid sequence
MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS
NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV
NNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDL
EGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTL
LALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET
KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN
CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA
DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSK
PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN
KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS
VITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH
VNNSYECDIPIGAGICASYQTQTNSRRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPT
NFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK
NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY
GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP
FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQN
AQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAA
EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKN
FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN
NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN
ESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT**
SEQ ID NO:3Delta S-6P amino acid sequence
MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS
NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV
NNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDL
EGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTL
LALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET
KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN
CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA
DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSK
PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN
KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS
VITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH
VNNSYECDIPIGAGICASYQTQTNSRRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPT
NFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK
NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQY
GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGPALQIPF
PMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQNVVNQNAQ
ALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIR
ASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFT
TAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNT
VYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNES
LIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT**
SEQ ID NO:4Delta S nucleotide sequence
ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAATGCGTGAACCTGAGA
ACAAGAACACAGCTGCCCCCCGCCTACACCAACAGCTTCACAAGAGGCGTGTACTA
CCCCGACAAGGTGTTCAGAAGCTCCGTGCTGCACAGCACCCAAGACCTGTTTCTGC
CCTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAACGGC
ACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCTAG
CACCGAAAAGAGCAACATCATCAGAGGCTGGATCTTCGGCACCACCCTGGACTCCA
AGACACAGAGCCTGCTGATCGTCAACAACGCCACCAACGTGGTGATCAAGGTGTGC
GAGTTTCAGTTCTGCAACGACCCCTTCCTGGACGTGTACTACCACAAGAACAACAA
GAGCTGGATGGAGAGCGGCGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACG
TGAGCCAACCCTTCCTGATGGACCTGGAGGGCAAGCAAGGCAATTTCAAGAACCTG
AGAGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACAC
CCCCATCAACCTGGTGAGAGACCTGCCCCAAGGCTTCAGCGCCCTGGAGCCCCTGG
TGGACCTGCCCATCGGCATCAACATCACAAGATTCCAAACCCTGCTGGCCCTGCAC
CGGAGCTACCTGACCCCTGGCGACTCCTCCTCCGGCTGGACAGCTGGCGCCGCCGC
TTACTACGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACG
GCACAATTACAGACGCCGTGGACTGCGCCCTGGACCCCCTGAGCGAGACCAAGTGC
ACCCTCAAGAGCTTCACCGTGGAGAAGGGCATCTATCAGACAAGCAACTTCAGAGT
GCAGCCCACCGAGAGCATCGTGAGATTCCCCAACATCACCAACCTGTGCCCCTTCG
GCGAGGTGTTCAACGCCACAAGATTCGCTAGCGTGTACGCTTGGAATAGAAAAAGA
ATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCTAGCTTCAGCAC
CTTCAAGTGCTACGGCGTCAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACG
TGTACGCCGACAGCTTCGTGATCAGAGGCGACGAGGTGAGACAGATCGCCCCCGGG
CAGACCGGCAAGATCGCCGACTACAATTACAAGCTGCCCGACGACTTCACCGGCTG
CGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAGGTGGGCGGCAACTACAAC
TACAGATACAGACTGTTCAGAAAGAGCAACCTGAAGCCCTTCGAGAGAGACATCAG
CACCGAGATCTACCAAGCCGGCAGCAAGCCCTGCAACGGCGTGGAGGGCTTCAACT
GCTACTTCCCCCTGCAGAGCTACGGCTTTCAGCCCACCAACGGCGTGGGCTATCAGC
CCTACAGAGTGGTCGTGCTGAGCTTCGAGCTGCTGCACGCCCCTGCCACAGTGTGC
GGCCCCAAAAAGAGCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAA
CGGGCTGACCGGCACCGGCGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCTTTTC
AGCAGTTCGGCAGAGACATCGCCGACACCACAGACGCCGTGAGAGACCCTCAGAC
CCTGGAGATCCTGGACATCACACCCTGCAGCTTCGGCGGCGTGAGCGTGATCACCC
CCGGCACCAACACAAGCAACCAAGTGGCCGTGCTGTACCAAGGCGTGAACTGCACC
GAGGTGCCCGTGGCCATCCACGCCGATCAGCTGACCCCCACCTGGAGAGTGTACAG
CACCGGCAGCAACGTGTTTCAGACAAGAGCCGGCTGCCTGATCGGCGCCGAGCACG
TGAACAACAGCTACGAGTGCGACATCCCCATCGGCGCCGGCATCTGCGCTAGCTAT
CAGACACAGACCAACAGCAGACGGAGAGCTAGAAGCGTGGCTAGCCAAAGCATCA
TCGCCTACACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAACAACAGC
ATCGCCATCCCCACCAACTTCACCATCAGCGTGACCACCGAGATTCTGCCCGTGAGC
ATGACCAAGACAAGCGTGGACTGCACCATGTACATCTGCGGCGACAGCACCGAGTG
CAGCAACCTGCTCCTGCAGTACGGCAGCTTCTGCACACAGCTGAACAGAGCCCTGA
CCGGCATCGCCGTGGAGCAAGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAA
GCAGATCTACAAGACCCCCCCCATCAAGGACTTCGGCGGCTTCAACTTCAGCCAAA
TCCTGCCCGACCCTAGCAAGCCTAGCAAGAGAAGCTTCATCGAGGACCTGCTGTTC
AACAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCTCGG
CGACATCGCCGCTAGAGACCTGATCTGCGCTCAGAAGTTCAACGGCCTGACCGTGC
TGCCCCCCCTGCTGACCGACGAGATGATCGCTCAGTACACAAGCGCCCTCCTGGCC
GGCACCATTACATCCGGCTGGACATTCGGGGCCGGCGCCGCCCTGCAGATCCCCTT
CGCCATGCAGATGGCCTACAGATTCAACGGCATCGGCGTGACACAGAACGTGCTGT
ACGAGAATCAGAAGCTGATCGCCAATCAGTTCAACAGCGCCATCGGCAAGATCCAA
GACAGCCTGAGCAGCACCGCTAGCGCCCTGGGCAAGCTGCAGAACGTGGTGAATCA
GAACGCCCAAGCCCTGAACACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCA
TCTCCTCCGTGCTGAACGACATCCTGAGCAGACTGGACAAGGTGGAGGCCGAGGTG
CAGATCGACAGACTGATCACCGGCAGACTGCAGAGCCTGCAGACCTACGTGACACA
GCAGCTGATCAGAGCCGCCGAGATCAGAGCTAGCGCCAACCTGGCCGCCACCAAG
ATGAGCGAGTGCGTGCTGGGGCAGAGCAAGAGAGTGGACTTCTGCGGCAAGGGCT
ACCACCTGATGAGCTTCCCTCAGAGCGCCCCCCACGGCGTGGTGTTCCTGCACGTGA
CCTACGTGCCCGCCCAAGAGAAGAACTTCACCACCGCCCCCGCCATCTGCCACGAC
GGCAAGGCCCACTTCCCTAGAGAGGGCGTGTTCGTGAGCAACGGCACCCACTGGTT
CGTGACACAGAGAAACTTCTACGAGCCTCAGATCATCACCACCGACAACACCTTCG
TGAGCGGCAACTGCGACGTGGTGATCGGCATCGTCAACAATACAGTGTACGACCCC
CTGCAGCCCGAACTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAATCA
CACAAGCCCCGACGTGGACCTGGGCGACATTAGCGGCATCAACGCTAGCGTGGTGA
ACATTCAGAAGGAGATCGATAGACTGAACGAGGTGGCCAAGAACCTGAACGAGAG
CCTGATCGACCTGCAAGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGT
ACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGC
TGTGCTGCATGACAAGCTGTTGCAGCTGCCTGAAAGGCTGCTGTAGCTGTGGCAGCT
GCTGCAAGTTCGACGAGGACGACAGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCA
CTACACCTGATGA
SEQ ID NO:5Delta S-2P nucleotide sequence
ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAATGCGTGAACCTGAGA
ACAAGAACACAGCTGCCCCCCGCCTACACCAACAGCTTCACAAGAGGCGTGTACTA
CCCCGACAAGGTGTTCAGAAGCTCCGTGCTGCACAGCACCCAAGACCTGTTTCTGC
CCTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAACGGC
ACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCTAG
CACCGAAAAGAGCAACATCATCAGAGGCTGGATCTTCGGCACCACCCTGGACTCCA
AGACACAGAGCCTGCTGATCGTCAACAACGCCACCAACGTGGTGATCAAGGTGTGC
GAGTTTCAGTTCTGCAACGACCCCTTCCTGGACGTGTACTACCACAAGAACAACAA
GAGCTGGATGGAGAGCGGCGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACG
TGAGCCAACCCTTCCTGATGGACCTGGAGGGCAAGCAAGGCAATTTCAAGAACCTG
AGAGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACAC
CCCCATCAACCTGGTGAGAGACCTGCCCCAAGGCTTCAGCGCCCTGGAGCCCCTGG
TGGACCTGCCCATCGGCATCAACATCACAAGATTCCAAACCCTGCTGGCCCTGCAC
CGGAGCTACCTGACCCCTGGCGACTCCTCCTCCGGCTGGACAGCTGGCGCCGCCGC
TTACTACGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACG
GCACAATTACAGACGCCGTGGACTGCGCCCTGGACCCCCTGAGCGAGACCAAGTGC
ACCCTCAAGAGCTTCACCGTGGAGAAGGGCATCTATCAGACAAGCAACTTCAGAGT
GCAGCCCACCGAGAGCATCGTGAGATTCCCCAACATCACCAACCTGTGCCCCTTCG
GCGAGGTGTTCAACGCCACAAGATTCGCTAGCGTGTACGCTTGGAATAGAAAAAGA
ATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCTAGCTTCAGCAC
CTTCAAGTGCTACGGCGTCAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACG
TGTACGCCGACAGCTTCGTGATCAGAGGCGACGAGGTGAGACAGATCGCCCCCGGG
CAGACCGGCAAGATCGCCGACTACAATTACAAGCTGCCCGACGACTTCACCGGCTG
CGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAGGTGGGCGGCAACTACAAC
TACAGATACAGACTGTTCAGAAAGAGCAACCTGAAGCCCTTCGAGAGAGACATCAG
CACCGAGATCTACCAAGCCGGCAGCAAGCCCTGCAACGGCGTGGAGGGCTTCAACT
GCTACTTCCCCCTGCAGAGCTACGGCTTTCAGCCCACCAACGGCGTGGGCTATCAGC
CCTACAGAGTGGTCGTGCTGAGCTTCGAGCTGCTGCACGCCCCTGCCACAGTGTGC
GGCCCCAAAAAGAGCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAA
CGGGCTGACCGGCACCGGCGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCTTTTC
AGCAGTTCGGCAGAGACATCGCCGACACCACAGACGCCGTGAGAGACCCTCAGAC
CCTGGAGATCCTGGACATCACACCCTGCAGCTTCGGCGGCGTGAGCGTGATCACCC
CCGGCACCAACACAAGCAACCAAGTGGCCGTGCTGTACCAAGGCGTGAACTGCACC
GAGGTGCCCGTGGCCATCCACGCCGATCAGCTGACCCCCACCTGGAGAGTGTACAG
CACCGGCAGCAACGTGTTTCAGACAAGAGCCGGCTGCCTGATCGGCGCCGAGCACG
TGAACAACAGCTACGAGTGCGACATCCCCATCGGCGCCGGCATCTGCGCTAGCTAT
CAGACACAGACCAACAGCAGACGGAGAGCTAGAAGCGTGGCTAGCCAAAGCATCA
TCGCCTACACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAACAACAGC
ATCGCCATCCCCACCAACTTCACCATCAGCGTGACCACCGAGATTCTGCCCGTGAGC
ATGACCAAGACAAGCGTGGACTGCACCATGTACATCTGCGGCGACAGCACCGAGTG
CAGCAACCTGCTCCTGCAGTACGGCAGCTTCTGCACACAGCTGAACAGAGCCCTGA
CCGGCATCGCCGTGGAGCAAGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAA
GCAGATCTACAAGACCCCCCCCATCAAGGACTTCGGCGGCTTCAACTTCAGCCAAA
TCCTGCCCGACCCTAGCAAGCCTAGCAAGAGAAGCTTCATCGAGGACCTGCTGTTC
AACAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCTCGG
CGACATCGCCGCTAGAGACCTGATCTGCGCTCAGAAGTTCAACGGCCTGACCGTGC
TGCCCCCCCTGCTGACCGACGAGATGATCGCTCAGTACACAAGCGCCCTCCTGGCC
GGCACCATTACATCCGGCTGGACATTCGGGGCCGGCGCCGCCCTGCAGATCCCCTT
CGCCATGCAGATGGCCTACAGATTCAACGGCATCGGCGTGACACAGAACGTGCTGT
ACGAGAATCAGAAGCTGATCGCCAATCAGTTCAACAGCGCCATCGGCAAGATCCAA
GACAGCCTGAGCAGCACCGCTAGCGCCCTGGGCAAGCTGCAGAACGTGGTGAATCA
GAACGCCCAAGCCCTGAACACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCA
TCTCCTCCGTGCTGAACGACATCCTGAGCAGACTGGACCCCCCCGAGGCCGAGGTG
CAGATCGACAGACTGATCACCGGCAGACTGCAGAGCCTGCAGACCTACGTGACACA
GCAGCTGATCAGAGCCGCCGAGATCAGAGCTAGCGCCAACCTGGCCGCCACCAAG
ATGAGCGAGTGCGTGCTGGGGCAGAGCAAGAGAGTGGACTTCTGCGGCAAGGGCT
ACCACCTGATGAGCTTCCCTCAGAGCGCCCCCCACGGCGTGGTGTTCCTGCACGTGA
CCTACGTGCCCGCCCAAGAGAAGAACTTCACCACCGCCCCCGCCATCTGCCACGAC
GGCAAGGCCCACTTCCCTAGAGAGGGCGTGTTCGTGAGCAACGGCACCCACTGGTT
CGTGACACAGAGAAACTTCTACGAGCCTCAGATCATCACCACCGACAACACCTTCG
TGAGCGGCAACTGCGACGTGGTGATCGGCATCGTCAACAATACAGTGTACGACCCC
CTGCAGCCCGAACTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAATCA
CACAAGCCCCGACGTGGACCTGGGCGACATTAGCGGCATCAACGCTAGCGTGGTGA
ACATTCAGAAGGAGATCGATAGACTGAACGAGGTGGCCAAGAACCTGAACGAGAG
CCTGATCGACCTGCAAGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGT
ACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGC
TGTGCTGCATGACAAGCTGTTGCAGCTGCCTGAAAGGCTGCTGTAGCTGTGGCAGCT
GCTGCAAGTTCGACGAGGACGACAGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCA
CTACACCTGATGA
SEQ ID NO:6Delta S-6P nucleotide sequence
ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAATGCGTGAACCTGAGA
ACAAGAACACAGCTGCCCCCCGCCTACACCAACAGCTTCACAAGAGGCGTGTACTA
CCCCGACAAGGTGTTCAGAAGCTCCGTGCTGCACAGCACCCAAGACCTGTTCCTGC
CCTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAACGGC
ACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCTAG
CACCGAAAAGAGCAACATCATCAGAGGCTGGATCTTCGGCACCACCCTGGACTCCA
AGACACAGAGCCTGCTGATCGTCAACAACGCCACCAACGTGGTGATCAAGGTGTGC
GAGTTTCAGTTCTGCAACGACCCCTTCCTGGACGTGTACTACCACAAGAACAACAA
GAGCTGGATGGAGAGCGGCGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACG
TGAGCCAACCCTTCCTGATGGACCTGGAGGGCAAGCAAGGCAATTTCAAGAACCTG
AGAGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACAC
CCCCATCAACCTGGTGAGAGACCTGCCCCAAGGCTTCAGCGCCCTGGAGCCCCTGG
TGGACCTGCCCATCGGCATCAACATCACAAGATTTCAGACACTCCTCGCCCTGCATA
GAAGCTACCTCACACCCGGCGATAGCAGCAGCGGCTGGACCGCTGGCGCTGCCGCC
TACTACGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGG
GACAATCACCGATGCCGTGGACTGCGCCCTGGACCCCCTGAGCGAGACCAAGTGCA
CCCTCAAGAGCTTCACCGTGGAGAAGGGCATCTATCAGACAAGCAACTTCAGAGTG
CAGCCCACCGAGAGCATCGTGAGATTCCCCAACATCACCAACCTGTGCCCCTTCGG
CGAGGTGTTCAACGCCACAAGATTCGCTAGCGTGTACGCCTGGAACCGGAAGAGAA
TCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCTAGCTTCAGCACC
TTCAAGTGTTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGT
GTACGCCGACAGCTTCGTGATCAGAGGCGACGAGGTGAGACAGATCGCCCCCGGGC
AGACCGGCAAGATCGCCGACTACAATTACAAGCTGCCCGACGACTTCACCGGCTGC
GTGATCGCTTGGAACAGCAATAACCTGGACAGCAAGGTGGGCGGCAACTACAACTA
TAGATATAGACTGTTCAGAAAGAGCAACCTGAAGCCCTTCGAGAGAGACATCAGCA
CCGAGATCTACCAAGCCGGCAGCAAGCCCTGCAACGGCGTGGAGGGCTTCAACTGC
TACTTCCCCCTGCAGAGCTACGGCTTTCAGCCCACCAACGGCGTGGGCTATCAGCCC
TACAGAGTGGTCGTGCTGAGCTTCGAGCTGCTGCACGCCCCTGCCACCGTGTGTGGC
CCCAAAAAGAGCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGG
GCTGACCGGCACCGGCGTGCTGACCGAGAGCAACAAGAAGTTTCTGCCCTTTCAGC
AATTCGGCAGAGACATCGCCGACACCACCGACGCCGTGAGAGACCCTCAGACCCTG
GAGATCCTGGACATCACCCCCTGTAGCTTCGGCGGCGTGAGCGTGATCACCCCCGG
CACCAACACAAGCAACCAAGTGGCCGTGCTGTACCAAGGCGTGAACTGCACCGAG
GTGCCCGTGGCCATCCACGCCGATCAGCTGACCCCCACCTGGAGAGTGTACAGCAC
CGGCAGCAACGTGTTTCAGACAAGAGCCGGCTGCCTGATCGGCGCCGAGCACGTGA
ACAACAGCTACGAGTGCGACATCCCCATCGGCGCCGGCATCTGCGCTAGCTATCAG
ACACAGACCAACAGCAGACGGAGAGCTAGAAGCGTGGCTAGCCAAAGCATCATCG
CCTACACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAACAACAGCATC
GCCATCCCCACCAACTTCACCATCAGCGTGACCACCGAGATCCTGCCTGTGAGCAT
GACCAAGACAAGCGTGGACTGCACCATGTACATCTGCGGCGACAGCACCGAGTGCA
GCAACCTGCTCCTGCAGTACGGCAGCTTCTGCACACAGCTGAACAGAGCCCTGACC
GGCATCGCCGTGGAGCAAGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGC
AGATCTACAAGACCCCCCCCATCAAGGACTTCGGCGGCTTCAACTTCAGCCAAATC
CTGCCCGACCCTAGCAAGCCTAGCAAGAGAAGCCCCATCGAGGACCTGCTGTTCAA
CAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCTGGGCG
ACATCGCCGCTAGAGACCTGATCTGCGCTCAGAAGTTCAACGGCCTGACCGTGCTC
CCCCCCCTGCTGACCGACGAGATGATCGCTCAGTACACAAGCGCCCTGCTCGCCGG
GACCATCACAAGCGGGTGGACATTCGGGGCCGGCCCTGCCCTGCAGATCCCCTTCC
CCATGCAGATGGCCTACAGATTCAACGGCATCGGCGTGACACAGAACGTGCTGTAC
GAGAATCAGAAGCTGATCGCCAATCAGTTCAACAGCGCCATCGGCAAGATCCAAGA
CAGCCTGAGCAGCACCCCTAGCGCCCTGGGCAAGCTGCAGAACGTGGTGAATCAGA
ACGCCCAAGCCCTGAACACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATC
AGCAGCGTGCTGAACGACATCCTGAGCAGACTGGACCCCCCCGAGGCCGAGGTGCA
AATCGACCGGCTGATTACCGGCAGACTGCAGAGCCTGCAGACCTACGTGACACAGC
AGCTGATCAGAGCCGCCGAGATCAGAGCTAGCGCCAACCTGGCCGCCACCAAGATG
AGCGAGTGCGTGCTGGGGCAGAGCAAGAGAGTGGACTTCTGCGGCAAGGGCTACC
ACCTGATGAGCTTCCCTCAGAGCGCCCCCCACGGCGTGGTGTTCCTGCACGTGACCT
ACGTGCCCGCCCAAGAGAAGAACTTCACCACCGCTCCCGCCATCTGCCACGACGGC
AAGGCCCACTTCCCTAGAGAGGGCGTGTTCGTGAGCAACGGCACCCACTGGTTCGT
GACACAGAGAAACTTCTACGAGCCTCAGATCATCACCACCGACAACACCTTCGTGA
GCGGCAACTGCGACGTGGTGATCGGCATCGTGAACAACACCGTGTACGACCCCCTG
CAGCCCGAGCTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCATAC
AAGCCCCGACGTGGACCTGGGCGATATCAGCGGCATCAACGCTAGCGTGGTGAACA
TTCAGAAGGAGATCGACAGACTGAACGAGGTGGCCAAGAACCTGAACGAGAGCCT
GATCGACCTGCAAGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGTACA
TCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGT
GCTGCATGACAAGCTGCTGCAGCTGCCTGAAAGGCTGCTGTAGCTGCGGCAGCTGC
TGCAAGTTCGACGAGGACGACAGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTA
CACCTGATGA
SEQ ID NO：7 5’UTR
acatttgcttctgacacaactgtgttcactagcaacctcaaacagacacc
SEQ ID NO：8 3’UTR
gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctgg
attctgcctgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttg
agcatctggattctgcct
SEQ ID NO：9 polyA
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa。
Claims (28)
- Rna comprising an open reading frame encoding an antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof, wherein said antigenic polypeptide is selected from the group consisting of a receptor binding domain of SARS-CoV-2, an S protein, a variant thereof or an immunogenic fragment thereof, preferably said SARS-CoV-2 is a SARS-CoV-2Delta variant virus strain.
- 2. The RNA of claim 1, wherein the antigenic polypeptide or immunogenic fragment or variant thereof comprises one or more immunogenic epitopes of a SARS-CoV-2 polypeptide or variant thereof;Preferably, the antigenic polypeptide or immunogenic fragment or variant thereof is selected from the group consisting of the S protein of a variant strain of SARS-CoV-2Delta, preferably the S protein variant of a variant strain of SARS-CoV-2Delta, more preferably the S protein variant is selected from the group consisting of Delta S-2P, the mutations of which are K984P and V985P; and Delta S-6P, the mutations of which are F815P, A890P, A897P, A940P, K984P and V985P,preferably, the antigenic polypeptide or immunogenic fragment or variant thereof comprises the amino acid sequence of SEQ ID NO: 1. 2 or 3, amino acid sequence of amino acids 17-1271, which corresponds to SEQ ID NO: 1. 2 or 3, the amino acid sequence of amino acids 17-1271 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical; and/orThe RNA encoding the antigenic polypeptide or an immunogenic fragment or variant thereof comprises SEQ ID NO: 4. 5 or 6, and nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4. 5 or 6, the nucleotide sequence of nucleotides 49-3813 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
- 3. The RNA of claim 1 or 2, wherein the open reading frame encoding an antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof further comprises a secretion signal peptide, preferably fused N-terminally to said antigenic polypeptide or immunogenic fragment or variant thereof, said secretion signal peptide preferably being a secretion signal peptide of the S protein,Preferably, the secretion signal peptide comprises SEQ ID NO: 1. 2 or 3, amino acid sequence of amino acids 1-16, which corresponds to SEQ ID NO: 1. 2 or 3, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1. 2 or 3 or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1. 2 or 3, the amino acid sequence of amino acids 1-16 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/orThe RNA encoding the secretion signal peptide comprises SEQ ID NO: 4. 5 or 6, and nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6 or a fragment of the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4. 5 or 6, the nucleotide sequence of nucleotides 1-48 having a fragment of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
- 4. The RNA of claim 1 or 2, wherein the RNA is mRNA, circular RNA, and self-replicating RNA, preferably the RNA is suitable for intracellular expression of a polypeptide.
- 5. The RNA of claim 1 or 2, wherein the RNA is a modified RNA modified by substitution of some or all uridine residues with modified uridine residues, preferably the modified uridine is N1-methyl-pseudouridine.
- 6. The RNA of claim 1 or 2, wherein the RNA further comprises one or more structural elements optimized for maximum efficacy of the RNA in terms of stability and translation efficiency, preferably the structural elements comprise: 5' cap, 5' UTR, 3' UTR and polyA tail sequences.
- 7. The RNA of claim 6, wherein the 5' cap is or comprises a cap1 structure; more preferably, the 5 'cap is m7G (5') ppp (5 ') (2' -OMeA) pG.
- 8. The RNA of claim 6, wherein the 5'-UTR is a 5' -UTR sequence of human β -globin mRNA, optionally with an optimized Kozak sequence; more preferably, the 5' utr comprises SEQ ID NO:7, or a nucleotide sequence that hybridizes to SEQ ID NO:7 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
- 9. The RNA of claim 6, wherein the 3'-UTR is a double repeat 3' -UTR of human β -globin mRNA; more preferably, the 3' utr comprises SEQ ID NO:8, or a nucleotide sequence that hybridizes to SEQ ID NO:8, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
- 10. The RNA of claim 6, wherein the polyA tail sequence comprises at least 50, at least 60, or at least 100 a nucleotides; more preferably, the polyA tail sequence comprises SEQ ID NO:9, or a nucleotide sequence consisting of SEQ ID NO: 9.
- 11. A composition comprising the RNA of any one of claims 1-10.
- 12. The composition of claim 11, wherein the composition is formulated or to be formulated as a liquid, solid, or combination thereof, preferably the composition is formulated or to be formulated for injection or other modes of administration, preferably the composition is formulated or to be formulated for intramuscular injection.
- 13. The composition of claim 11 or 12, wherein the RNA is complexed with a protein and/or lipid to produce an RNA-particle for administration.
- 14. The composition of claim 13, wherein the RNA is formulated in a lipid nanoparticle comprising a cationically ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG) -lipid;preferably the lipid nanoparticle comprises ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (hexyl 2-decanoate), 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine and cholesterol.
- 15. The composition of claim 11, wherein the RNA is formulated or to be formulated as a colloid;preferably, the RNA is formulated as particles, 50% or more, 75% or more, or 85% or more RNA being present in the colloidal dispersed phase formed;more preferably the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase, wherein preferably the organic phase comprises ethanol;also preferably, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase, wherein preferably the lipids dispersed in the aqueous phase form liposomes.
- 16. The composition of claim 11 or 12, wherein the RNA is present in the composition in an amount ranging from 1 μg to 100 μg per dose.
- 17. Use of the RNA of any one of claims 1-10 or the composition of any one of claims 11-16 in the manufacture of a medicament, the medicament being a vaccine, the medicament further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.
- 18. The use of claim 17, wherein the medicament is for inducing an immune response against coronavirus, preferably a specific immune response against coronavirus antigen, in a subject.
- 19. The use of claim 17, wherein the medicament is for the treatment or prophylactic treatment of a coronavirus infection.
- 20. The use of any one of claims 17-19, wherein the coronavirus is a beta coronavirus, preferably the coronavirus is a saber virus, more preferably the coronavirus is SARS-CoV-2, further preferably the coronavirus comprises: new coronavirus original strain (GD 108), SARS-CoV-2Alpha variant virus strain, SARS-CoV-2Beta variant virus strain, SARS-CoV-2Delta variant virus strain and SARS-CoV-2Omicron variant virus strain.
- 21. The use of any one of claims 17-19, wherein the subject is a mammal, preferably the subject is a mouse or monkey, further preferably the subject is a human.
- 22. The RNA of any one of claims 1-10, the composition of any one of claims 11-16, or the use of any one of claims 17-21, wherein detectable expression of the antigenic polypeptide or immunogenic fragment or variant thereof is achieved when the RNA, composition, or drug is administered to a human cell, and preferably such expression is for a period of at least 24 hours or more.
- 23. The RNA of any one of claims 1-10, the composition of any one of claims 11-16, or the use of any one of claims 17-21, wherein administration of the RNA, composition, or medicament produces an immune effect in a subject, the immune effect comprising production of SARS-CoV-2 neutralizing antibodies and/or T cell responses, in particular a robust TH1 type T cell response, preferably a cd4+ and/or cd8+ T cell response.
- 24. The RNA of any one of claims 1-10, the composition of any one of claims 11-16 or the use of any one of claims 17-21, wherein administration of the RNA, composition or medicament generates an immune response in a subject,preferably, the immune response comprises generating a binding antibody titer against the S1 subunit of SARS-CoV-2 spike protein, more preferably the immune response comprises generating a neutralizing antibody titer against SARS-CoV-2 virus.
- 25. The RNA of any one of claims 1-10, the composition of any one of claims 11-16, or the use of any one of claims 17-21, wherein the serum of the subject shows production of antibodies to the polypeptide encoded by the open reading frame 7 days after administration of the RNA, composition, or drug to the subject.
- 26. The RNA of any one of claims 1-10, the composition of any one of claims 11-16, or the use of any one of claims 17-21, wherein the serum of the subject exhibits virus neutralization activity 14 days after administration of the RNA, composition, or drug to the subject.
- 27. The RNA, composition or use of any one of claims 23-26, wherein the subject is a mammal, preferably the subject is a mouse, further preferably the subject is a human.
- 28. A method of preparing a vaccine comprising formulating the RNA of any one of claims 1-10 in a lipid nanoparticle comprising a cationically ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG) -lipid;preferably the lipid nanoparticle comprises ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (hexyl 2-decanoate), 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine and cholesterol.
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| CN119950694A (en) * | 2025-01-25 | 2025-05-09 | 中国疾病预防控制中心病毒病预防控制所 | A combined vaccine of oral Ad5 vector COVID-19 vaccine and injectable COVID-19 vaccine and its application |
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| WO2023202711A1 (en) | 2023-10-26 |
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