CN117051019B - Novel coronavirus vaccine, preparation method and application thereof - Google Patents
Novel coronavirus vaccine, preparation method and application thereof Download PDFInfo
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- CN117051019B CN117051019B CN202310507350.6A CN202310507350A CN117051019B CN 117051019 B CN117051019 B CN 117051019B CN 202310507350 A CN202310507350 A CN 202310507350A CN 117051019 B CN117051019 B CN 117051019B
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Abstract
The invention provides a novel coronavirus vaccine, a preparation method and application thereof, and the novel coronavirus vaccine comprises components derived from immunodominant strains and epidemic dominant strains, and can show obviously improved immune effects on different strains.
Description
Technical Field
The invention belongs to the technical field of biomedical engineering, and relates to a novel coronavirus mRNA vaccine, a preparation method and application thereof.
Background
Infection with the novel coronavirus (SARS-CoV-2) can lead to coronavirus diseases (COVID-19), common signs of fever, cough, sore throat, etc., and in more severe cases, infection can lead to dyspnea, hypoxia, acute respiratory distress syndrome, and even death. The novel coronavirus can be transmitted from person to person via the respiratory tract and droplet pathways, and there is also the possibility of transmission through the air and digestive tract.
Novel coronaviruses continue to evolve during transmission, and a number of representative mutants have been detected. Vaccines currently developed or under development are only directed against one strain and cannot produce neutralizing antibodies against a different strain.
Currently, mRNA vaccines are becoming a research hotspot in new coronavirus vaccines. mRNA vaccine is prepared through introducing mRNA containing coded antigen protein into human body, and direct translation to form corresponding antigen protein, so as to induce organism to produce specific immune response and prevent immunity. The mRNA vaccine is a third-generation vaccine after the inactivated vaccine, the attenuated live vaccine, the subunit vaccine and the viral vector vaccine, breaks through the immune activation mode of the traditional inactivated vaccine, and creatively utilizes the cells of the human body to produce the antigen so as to activate specific immunity. The mRNA vaccine has the characteristics of high response speed to pathogen variation, simple production process, easy scale expansion and the like, and is particularly suitable for preventing diseases caused by continuously evolving novel coronaviruses.
Disclosure of Invention
The present invention aims to provide an mRNA encoding a novel coronavirus antigen comprising receptor binding regions of different strain S proteins, which mRNA has a higher immunogenicity, and is capable of eliciting the production of neutralizing antibodies against the different strains, thus being capable of significantly enhancing the immune effect.
To achieve the object of the present invention, in one aspect, the present invention provides an mRNA encoding a novel coronavirus antigen comprising a first antigen derived from an immunodominant strain and a second antigen derived from an epidemic dominant strain, each antigen comprising a receptor binding region of the S protein or a portion of the receptor binding region, respectively.
In some embodiments, the immunodominant strain is a novel coronavirus WH01 strain or Beta (Beta) strain.
In some embodiments, the epidemic dominant strain is a novel coronavirus Delta (Delta) strain or an Omicron (Omicron) strain including subtype variants ba.1, ba.1.1, ba.2, ba.2.12.1, ba.3, ba.4, ba.5, bq.1.1, bf.7, xbb.1.5, and xbb.1.16.
In some embodiments, the novel coronavirus antigen further comprises one or more antigens derived from strains other than the immunodominant strain and the epidemic dominant strain.
In some embodiments, the novel coronavirus antigen further comprises a third antigen derived from a strain other than the immunodominant strain and the epidemic dominant strain.
In some embodiments, the novel coronavirus antigen further comprises a fourth antigen derived from the immunodominant strain and a strain other than the epidemic dominant strain.
In some embodiments, the antigens are mixed to form a composition, or the antigens are linked directly or through an amino acid linker to form a fusion protein.
In some embodiments, the immunodominant strain and the strain other than the epidemic dominant strain are selected from the following strains: alpha (Alpha) strain, gamma (Gamma) strain, ai Puxi dragon (Epsilon), truncated tower (Zeta) strain, eta (Eta) strain, theta (Theta) strain, idota (iotata) strain, kappa (Kappa) strain, lambda (Lambda) strain, mu (Mu) strain, and the like.
In some embodiments, the mRNA comprises an mRNA sequence encoding a Receptor Binding Domain (RBD) or a portion of a receptor binding domain of an S protein derived from 2-4 novel coronavirus strains.
In some embodiments, the mRNA comprises a plurality of mRNA sequences, each of which encodes a Receptor Binding Domain (RBD) of the S protein of at least one novel coronavirus strain, respectively.
In some embodiments, the amino acid sequence of each antigen comprises the sequence set forth in any one of SEQ ID NOs 1-8, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs 1-8, respectively.
In some embodiments, the novel coronavirus antigen further comprises an N-terminal domain (NTD) of a novel coronavirus S protein.
In some embodiments, the amino acid sequence of each antigen comprises the sequence set forth in any one of SEQ ID NOs 12 and 15-22, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of SEQ ID NOs 12 and 15-22, respectively.
In some embodiments, the mRNA comprises at least one of the following sequences:
mRNA sequence shown in 123-2750 th position in SEQ ID NO. 53,
MRNA sequence shown at positions 123-1436 of SEQ ID NO. 54,
MRNA sequence shown at positions 123-1436 of SEQ ID NO. 55,
MRNA sequence shown at positions 123-1436 of SEQ ID NO. 56,
MRNA sequence shown at positions 123-2093 in SEQ ID NO. 57,
MRNA sequence shown at positions 123-2093 in SEQ ID NO. 58,
MRNA sequence shown at positions 123-2093 in SEQ ID NO. 59,
MRNA sequence shown at 123-2093 in SEQ ID NO. 60,
MRNA sequence shown in positions 123-2351 of SEQ ID NO. 64,
MRNA sequence shown at positions 123-3008 of SEQ ID NO. 67,
MRNA sequence shown in 123-2999 of SEQ ID NO. 68,
MRNA sequence shown in positions 123-3002 of SEQ ID NO. 69,
MRNA sequence shown in 123-2999 of SEQ ID NO. 70,
MRNA sequence shown in 123-2999 of SEQ ID NO. 71,
MRNA sequence shown in positions 123-2351 of SEQ ID NO. 72,
MRNA sequence shown in positions 123-2342 of SEQ ID NO. 73,
MRNA sequence shown at positions 123-2342 of SEQ ID NO. 74.
In some embodiments, the mRNA comprises a 5' -UTR; preferably, the 5'-UTR is selected from the group consisting of the 5' -UTR of albumin genes, alpha-globin genes or beta-globin genes.
In some embodiments, the 5' -UTR comprises an mRNA sequence corresponding to the DNA sequence shown in SEQ ID NO. 25, or an mRNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said mRNA sequence.
In some embodiments, the mRNA comprises a 3' -UTR; preferably, the 3'-UTR is selected from the group consisting of the 3' -UTR of albumin genes, alpha-globin genes or beta-globin genes.
In some embodiments, the 3' -UTR comprises an mRNA sequence corresponding to the DNA sequence shown in SEQ ID NO. 29, or an mRNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said mRNA sequence.
In some embodiments, the mRNA comprises at least one of the sequences of SEQ ID NOS 53-60, 64 and 67-74 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of the sequences of SEQ ID NOS 53-60, 64 and 67-74.
In some embodiments, the mRNA has 3' -polyA; preferably, the 3' -polyA comprises 50 to 300 adenylates, preferably 60 to 200 adenylates, more preferably 80 to 150 adenylates.
In some embodiments, the mRNA has a 5' cap structure; preferably, the 5' Cap structure is selected from one of a modified or unmodified Cap0 type Cap structure, cap1 type Cap structure, cap2 type Cap structure, anti-reverse Cap analogue (ARCA).
In some embodiments, the mRNA has a nucleoside modification comprising at least one of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methoxy-uridine, and 2' -O-methyl uridine.
In another aspect, the present invention provides a method for preparing the mRNA, comprising:
(1) Constructing an expression vector encoding the novel coronavirus antigen, wherein the expression vector comprises a DNA sequence encoding a polyadenylic acid (polyA);
(2) Transforming host cells by using the expression vector, and preparing linearization plasmids after amplification culture;
(3) In vitro transcription is performed using the linearized plasmid to obtain mRNA.
In yet another aspect, the invention provides the use of the mRNA in the preparation of a composition for preventing novel coronavirus infection.
In yet another aspect, the invention provides a composition comprising the mRNA and a delivery vehicle.
In some embodiments, the delivery vehicle comprises a lipid nanoparticle.
In some embodiments, the lipid nanoparticle comprises a cationic lipid and a helper lipid.
In some embodiments, the cationic lipid is selected from at least one of 1, 2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyl Dioctadecyl Ammonium (DDAB), 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP), 1, 2-dioleoyl-3-dimethylammonium propane (DOTAP), 1, 2-dioleoxy-3-dimethylammonium propane, 1, 2-dialkoxy-3-dimethylammonium propane, dioctadecyl dimethylammonium chloride (dotac), 1, 2-dimyristoxypropyl-1, 3-dimethylhydroxyethyl ammonium (dmrii), and 2, 3-dioleoyloxy-N- [2 (spermidine) ethyl ] -N, N-dimethyl-1-trifluoropropyl ammonium acetate (DOSPA).
In some embodiments, the helper lipid is selected from at least one of 1, 2-di- (9Z-octadecenoyl) -sn-glycerol-3-phosphate ethanolamine (DOPE), 1, 2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, cephalin, sterols, and cerebrosides; preferably, the helper lipid comprises DOPC and cholesterol.
In some embodiments, the composition further comprises an adjuvant.
In some embodiments, the adjuvant is selected from one or more of aluminum adjuvants, MF59, MPL, QS-21, GLA, cpG, poly I C, AS01, AS02, AS03, AS04 adjuvants.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the novel coronavirus mRNA can encode at least two antigens from immunodominant strains and epidemic dominant strains, can induce the generation of neutralizing antibodies aiming at different strains, and experiments show that the vaccine prepared by the novel coronavirus mRNA can show obviously improved immune effect on different strains.
Drawings
FIG. 1 is an electrophoretogram of linearized plasmids prepared in examples of the present invention;
FIG. 2 is an electrophoretogram of mRNA prepared in the example of the present invention.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
An "immunodominant strain" in the present invention refers to a variant that has higher immunogenicity of an antigen and is capable of providing better cross-protection to other variants than a non-immunodominant variant.
The term "dominant strain" in the present invention refers to the major variant currently in use, and is generally understood to be the "variant of interest" (Variants of concern, VOC) currently in use. The definition of VOC can be referred to the working definition of the World Health Organization (WHO), namely: consistent with the definition of "variants to be attended to" (Variants of interest (VOI)), and by comparative evaluation, SARS-CoV-2 variants have been demonstrated to be associated with changes that have some global public health significance to one or more of the following:
increased transmissibility or a detrimental change in COVID-19 epidemiology; or alternatively
Increased toxicity or changes in clinical disease manifestations; or alternatively
The effectiveness of public health and social measures or available diagnostic methods, vaccines and therapeutic methods is reduced.
The definition of "variants to be noted" (Variants of interest (VOI)) in the present invention can be defined with reference to the World Health Organization (WHO) work, namely: a variant SARS-CoV-2 having the following characteristics:
genetic changes that are predicted or known to affect viral characteristics, such as transmissibility, disease severity, immune escape, diagnostic or therapeutic escape; and
Confirming that significant community transmission or multiple COVID-19 aggregate cases occur in multiple countries with increasing relative prevalence, increasing numbers of cases, or other apparent epidemiological effects that indicate that global public health is facing new risks.
"Nucleic acid" in the present invention includes deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The nucleic acid may be single-stranded or double-stranded.
"DNA" in the present invention refers to a deoxyribonucleic acid molecule, i.e., a polymer composed of deoxynucleotides. "deoxynucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the beta-D-ribofuranosyl group.
"RNA" in the present invention refers to ribonucleic acid molecules, i.e.polymers consisting of nucleotides. "ribonucleotides" relate to nucleotides having a hydroxyl group at the 2' -position of the beta-D-ribofuranosyl group.
"RNA" in the present invention includes, and preferably relates to, "mRNA," which means "messenger RNA," mRNA (messenger RNA) generally provides a nucleotide sequence that can be translated into an amino acid sequence of a particular peptide or protein.
The RNA can be suitably modified by the addition of a5 '-cap structure or 5' -cap analogue to stabilize the RNA and/or enhance RNA translation. "5 '-cap structure" refers to a modified nucleotide, particularly a guanine nucleotide, located at the 5' end of an RNA molecule, such as an mRNA molecule. The 5' -cap structure is linked to the RNA by a 5' -5' -triphosphate linkage. Suitable 5' -Cap structures include Cap0 (first nucleobase methylation, e.g., m7 GpppN), cap1 (ribose additional methylation of nucleotides adjacent to m7 GpppN), cap2 (ribose additional methylation of second nucleotide downstream of m7 GpppN), cap3 (ribose additional methylation of third nucleotide downstream of m7 GpppN), cap4 (ribose additional methylation of fourth nucleotide downstream of m7 GpppN), ARCA (anti-reverse Cap analogue), modified ARCA (e.g., phosphorothioate modified ARCA). 5' -cap analogs include, but are not limited to, those selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., gpppG); chemical structure of a dimethyl cap analogue (e.g., m2,7 GpppG), a trimethyl cap analogue (e.g., m2,7 GpppG), a dimethyl symmetrical cap analogue (e.g., m7Gpppm G), or an anti-reverse cap analogue (e.g., ARCA; m7,2'ome GpppG, m7,2' dgppg, m7,3'ome GpppG, m7,3' dgppg, and tetraphosphate derivatives thereof).
According to an embodiment of the invention, the RNA further comprises a 5 '-or 3' -untranslated region (UTR) and a terminal polyadenylation sequence (poly (a) tail). The term "5' -untranslated region" or "5' -UTR element" in the present invention refers to a portion of a nucleic acid molecule that is located 5' (i.e., upstream) of a coding sequence and that is not translated into a protein. The 5'-UTR may be a portion of the RNA located 5' of the coding sequence. Typically, the 5' -UTR begins at the transcription start site and ends before the start codon of the coding sequence.
The term "3 '-untranslated region", "3' -UTR" or "3'-UTR element" in the present invention refers to a portion of a nucleic acid molecule that is located 3' (i.e., downstream) of a coding sequence and is not translated into a protein. The 3' -UTR may be a portion of RNA that is located between the coding sequence and the terminal polyadenylation sequence.
In some embodiments, the 5'-UTR or 3' -UTR of the present invention is a UTR that is not related to the coding region of RNA, e.g., a 5'-UTR or 3' -UTR derived from an albumin gene, an α -globin gene, a β -globin gene, preferably a 5'-UTR or 3' -UTR of a β -globin gene, more preferably a 5'-UTR or 3' -UTR of a human β -globin gene.
In the present invention, "poly (A)", "poly (A) tail" or "polyA sequence" refers to a sequence of an adenine-based (A) residue located at the 3' -end of an RNA molecule. poly (a) tails or polyA sequences can increase the stability and/or expression of RNA. According to some embodiments of the invention, the polyA sequence is 10 to 500, preferably 30 to 300, more preferably 50 to 200 adenosine residues in length. In some preferred embodiments, the polyA sequence is 100 to 150 adenosine residues in length. In a particularly preferred embodiment, the polyA sequence is about 120 adenosine residues in length.
In the present invention, the RNA may be a modified RNA, wherein modification refers to chemical modification, which includes backbone modification as well as sugar modification or base modification. Wherein the backbone modification is a modification in which the phosphate of the nucleotide backbone in the RNA is chemically modified; sugar modification is the chemical modification of the sugar of a nucleotide in RNA; base modification is the chemical modification of the base portion of a nucleotide in an RNA. In general, the immunogenicity of RNA can be reduced or inhibited by chemical modification.
In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methoxy-uridine, and 2' -O-methyl uridine.
In some embodiments, the chemically modified nucleoside replaces one or more uridine, e.g., at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% uridine.
According to an embodiment of the invention, the prepared mRNA is mixed with a delivery vehicle to produce an mRNA vaccine. The delivery vehicle is preferably a Lipid Nanoparticle (LNP) comprising a cationic lipid.
Cationic lipids generally have a lipophilic moiety, such as a sterol, acyl, or diacyl chain, and carry a net positive charge. Examples of cationic lipids include, but are not limited to, 1, 2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyl Dioctadecyl Ammonium (DDAB), 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP), 1, 2-dioleoyl-3-dimethylammonium propane (DODAP), 1, 2-diacyloxy-3-dimethylammonium propane, 1, 2-dialkoxy-3-dimethylammonium propane, dioctadecyl dimethylammonium chloride (DODAC), 1, 2-dimyristoxypropyl-1, 3-dimethylhydroxyethyl ammonium (DMRIE), and 2, 3-dioleoyloxy-N- [2 (spermidine carboxamide) ethyl ] -N, N-dimethyl-1-trifluoroacetic acid propylamine (DOSPA); DOTMA, DOTAP, DODAC and DOSPA are preferred, with DOTMA being most preferred.
In some embodiments, the lipid nanoparticle of the present invention further comprises a neutral lipid as a helper lipid to increase delivery efficiency. Examples of neutral lipids include, but are not limited to, 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, cephalin, sterols, and cerebrosides. Among them, DOPE and/or DOPC are preferable, and DOPE is most preferable. In a preferred embodiment, the lipid nanoparticle of the invention further comprises cholesterol. In the case where the lipid nanoparticle comprises a cationic lipid and a neutral lipid, the molar ratio of cationic lipid to neutral lipid may be appropriately adjusted to improve the stability of the liposome.
In some embodiments, the lipid nanoparticle is a lipid complex comprising DOTMA and DOPE, the molar ratio of DOTMA to DOPE is 9:1 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5.
In some embodiments, the lipid nanoparticle is a lipid complex comprising DOTMA and cholesterol in a molar ratio of DOTMA to cholesterol of 9:1 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5.
In some embodiments, the lipid nanoparticle is a lipid complex comprising DOTAP and DOPE in a molar ratio of DOTAP to DOPE of 9:1 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5.
The nucleic acid may fill the void space of the lipid nanoparticle such that the lipid nanoparticle captures the nucleic acid, or the nucleic acid may be bound by covalent, ionic or hydrogen bonding, or by association with the lipid nanoparticle via a non-specific bond, thereby forming a nucleic acid-lipid nanoparticle complex.
According to an embodiment of the invention, the mRNA is present in the mRNA vaccine in an amount of about 100ng to about 500. Mu.g, about 1. Mu.g to about 200. Mu.g, about 1. Mu.g to about 100. Mu.g, about 5. Mu.g to about 100. Mu.g, preferably about 10. Mu.g to about 50. Mu.g, in particular about 5. Mu.g, about 10. Mu.g, about 15. Mu.g, about 20. Mu.g, about 25. Mu.g, about 30. Mu.g, about 35. Mu.g, about 40. Mu.g, about 45. Mu.g, about 50. Mu.g, about 55. Mu.g, about 60. Mu.g, about 65. Mu.g, about 70. Mu.g, about 75. Mu.g, about 80. Mu.g, about 85. Mu.g, about 90. Mu.g, about 95. Mu.g or about 100. Mu.g.
According to an embodiment of the invention, the mRNA vaccine further comprises an adjuvant. In some embodiments, the adjuvant is selected from one or more of an aluminum adjuvant, an MF59 adjuvant, an MPL adjuvant, a QS-21, GLA, cpG, AS, an AS02, an AS03, an AS04 adjuvant.
Example 1 novel coronavirus antigen design
1. Novel coronavirus antigen design
In the study of novel coronavirus antigens, the inventors of the present invention first conducted a preliminary study from both immunodominant strain RBD screening and addition of N-terminal domain (NTD).
(1) Immunodominant strain RBD screening
The invention first examines the immunogenicity of different candidate antigens against different strains.
TABLE 1
As shown in Table 1, the results of the study showed that the dimer candidate antigen (RBD (WH 01) -RBD (WH 01)) formed by the RBD combination of the WH01 prototype strain had a higher geometric mean titer against the WH01 strain and the D614G strain pseudoviruses, but the geometric mean titer against the Delta strain and the Omicron-BA.1 strain pseudoviruses was significantly decreased, whereas the dimer candidate antigen (RBD (Delta) -RBD (Delta)) formed by the RBD combination of the Delta strain had a higher geometric mean titer against the Delta strain and the Omicron-BA.1 strain pseudoviruses, and the geometric mean titer against the WH01 strain and the D614G strain pseudoviruses was significantly decreased. This suggests that when the RBD is from the same strain, the candidate antigen has poor cross-protection against different strains.
When RBD is derived from different strains, the geometric mean titer of the RBD candidate antigen (RBD (WH 01) -RBD (Delta)) formed by the RBD combination of the WH01 strain and the Delta strain is improved relative to that of the RBD candidate antigen (RBD (WH 01) -RBD (Delta)) of the Delta strain and the Omicron-BA.1 strain pseudovirus, but the geometric mean titer of the RBD candidate antigen relative to the WH01 strain and the D614G strain is obviously reduced, and the change trend is the same as that of the RBD (Delta) -RBD (Delta), but the geometric mean titer relative to the different strains is still lower than that of the RBD (Delta) -RBD (Delta).
While the geometric mean titers of pseudoviruses for WH01 strain, D614G strain, delta strain and Omicron-BA.1 strain all remained high when the RBDs of Beta strain and Delta strain were combined to form a dimer candidate antigen (RBD (Beta) -RBD (Delta)), the geometric mean titers of pseudoviruses for especially Omicron-BA.1 strain reached the highest value at the above-mentioned different combinations, which suggests that RBDs comprising immunodominant strains can significantly improve the cross-protective capacity of candidate antigens against different strains when forming dimer candidate antigens comprising RBDs of different strains, and that optimal cross-protective capacity can be achieved when RBDs of immunodominant strains and RBDs of epidemic strains form dimer candidate antigens.
(2) Influence of NTD addition
The invention further examined the effect of adding NTD to candidate antigens.
TABLE 2
As shown in Table 2, when RBD is derived from the same strain, the geometric mean titers of the RBD candidate antigen formed by the combination of NTD and RBD of strain WH01 (NTD-RBD (WH 01) -RBD (WH 01)) and the dimer candidate antigen formed by the combination of NTD and RBD of strain Delta (NTD-RBD (Delta) -RBD (Delta)) against different pseudoviruses are all increased to different extents as compared to RBD (WH 01) -RBD (WH 01) and RBD (Delta) -RBD (Delta).
When RBD comes from different strains, compared with RBD (WH 01) -RBD (Delta), the geometric average titer of a dimer candidate antigen (NTD-RBD (WH 01) -RBD (Delta)) formed by the combination of the RBD of the WH01 strain and the RBD of the RBD strain and the RBD of the Delta strain against different pseudoviruses is obviously improved; the dimeric candidate antigen (NTD-RBD (Beta) -RBD (Delta)) formed by the combination of the NTD and RBD of the Beta strain and the RBD of the Delta strain has reduced, but still maintained at a higher level, geometric mean titers against the pseudoviruses of the D614G strain and the Delta strain, while the geometric mean titers against the pseudoviruses of the WH01 strain and the Omicron-BA.1 strain are increased, especially against the pseudoviruses of the Omicron-BA.1 strain, to the maximum value of the combinations. Thus, overall, adding NTD on an RBD basis may improve cross-protection capability.
Based on the above studies, the present inventors designed various novel coronavirus antigens (amino acid sequences SEQ ID NOs: 1-22) using the receptor binding region and N-terminal domain (NTD) of the S proteins of novel coronavirus WH01 prototype strain, beta (Beta) strain, delta (Delta) strain and Omicron (Omicron) BA.1 strain, as shown in Table 3.
TABLE 3 Table 3
Adding a tag DYKDDDDKHHHHHHHH (SEQ ID NO: 23) at the C end of the antigen, optimizing the DNA sequence encoding the antigen according to a host cell, and sequentially adding a T7 RNA polymerase binding sequence (SEQ ID NO: 24), a 5'UTR (5'UTR,SEQ ID NO:25 of Homo sapiens Beta-globin), a kozak sequence (gccacc, SEQ ID NO: 26) and a signal peptide (novel crown self signal peptide SP: SEQ ID NO:27; DNA sequence optimized according to the homosapiens system: SEQ ID NO: 28) at the 5 'end, and sequentially adding a 3' UTR (3'UTR,SEQ ID NO:29 of two copies of Homo sapiens Beta-globin), ployA (120) and a cleavage site (SEQ ID NO: 30) at the 3' end to obtain DNA sequences SEQ ID NO:31-52.
Example 2 preparation of transcription templates
The DNA sequences SEQ ID NOS.31-52 were synthesized and cloned into the expression vector pUC57-kan, and the target gene was verified by sequencing.
The expression vector was transformed into E.coli competent cells. Transformants were picked and liquid-cultured and streaked on LB agar plates (containing 100. Mu.g/ml ampicillin). One single clone was picked from the plate and inoculated into 300ml of LB medium for expansion culture.
After culturing the working strain, performing alkaline lysis, centrifuging, collecting supernatant, performing ultrafiltration concentration, performing ammonium sulfate treatment, adding ammonium sulfate according to the amount of 265g ammonium sulfate/kg of the first ultrafiltration concentrated solution, uniformly mixing, centrifuging at 12000g for 15min, collecting supernatant, and performing filtration and impurity removal by using a filter element with the diameter of 0.45 μm.
The ammonium sulfate treated solution was subjected to size exclusion chromatography using an NW Rose TM FF column (su sodium micro technologies). The chromatographic column is balanced by 1 time of the volume of a bed by using a solution A (2.0M (NH 4)2SO4 +10mM EDTA+100mM Tris,pH7.5), loading is started after a base line is stable, the loading amount is 20 percent of the volume of the bed, the loading flow rate is 60cm/h, the volume of the bed is 2 times of the volume of the solution A after loading is completed, the flow rate is 60cm/h, the ultraviolet absorption value of 254nm (260 nm) is monitored, the eluting peak with the UV of more than or equal to 100mAu is collected as size exclusion chromatographic sample liquid, and the chromatographic column is continuously flushed by using water for injection until the UV is parallel to the base line.
The size exclusion chromatography lower sample was subjected to hydrophobic/affinity composite chromatography, which was NW Rose TM Plasmid chromatography (Souzhou sodium micro technologies Co., ltd.). The chromatographic column is balanced by 2 times of column bed volume by the solution A, the flow rate is 200cm/h, the sample is loaded after the baseline is stable, the loading flow rate is 100cm/h, and the loading plasmid amount is 1mg/ml filler volume; after loading was completed, 2 bed volumes were rinsed with solution A at a flow rate of 100cm/h until UV254nm (260 nm) was substantially parallel to baseline; eluting with solution B (1.7M (NH 4)2SO4 +0.3M NaCl+10mM EDTA+100mM Tris,pH7.5) for 3 times of column bed volume, wherein the eluting flow rate is 100cm/h, monitoring ultraviolet absorption value at 254nm (260 nm), and collecting eluting sample when UV > 20mAu is less than 100mAu until UV < 100mAu is reached, wherein the collected eluting peak is the hydrophobic/affinity composite chromatography lower sample liquid.
An enzyme digestion system (restriction enzyme BspQ I, northenan Biotechnology Co., ltd.) was prepared at a plasmid concentration of 200. Mu.g/ml, the enzyme amount was 1U/. Mu.g of the plasmid, the enzyme digestion reaction was carried out at 50℃in a water bath for 10 hours, and the reaction was stopped at 80℃for 30 minutes.
The linearized plasmid samples were subjected to anion exchange chromatography, which was performed using a NanoQ-30L column (Sony micro technologies Co., ltd.). The column was first washed with 0.5M sodium hydroxide solution at a flow rate of 60cm/h for 4 bed volumes. The column was then rinsed with water for injection at a flow rate of 100cm/h for 2 bed volumes until the post column conductance was parallel to baseline. Balancing 2 times of bed volume with solution C (0.4M NaCl+10mM EDTA+100mM Tris,pH7.5) at a flow rate of 100cm/h, and starting loading after the baseline is stable, wherein the loading flow rate is 100cm/h, and the loading plasmid amount is 0.5mg/ml of filler volume; after loading, washing 2 times of the volume of the bed by using the solution C, wherein the washing flow rate is 1100cm/h; eluting with solution D (1.0M NaCl+10mM EDTA+100mM Tris,pH7.5) at a flow rate of 80cm/h by 3 times of column bed volume, monitoring ultraviolet absorption value at 254nm (260 nm), and collecting eluting sample when UV > 20mAu is less than 100mAu, wherein the collected eluting peak is the anion exchange chromatography lower sample liquid.
The prepared mRNA sequence is SEQ ID NO. 53-74, wherein,
The 123-2750 th bit of SEQ ID NO. 53 is an antigen coding region,
The 123 th to 1436 th positions of SEQ ID NO. 54 are antigen coding regions,
The 123 th to 1436 th positions of SEQ ID NO. 55 are antigen coding regions,
The 123-1436 position of SEQ ID NO. 56 is an antigen coding region,
The 123-2093 position of SEQ ID NO. 57 is an antigen encoding region,
Positions 123-2093 of SEQ ID NO. 58 are the antigen encoding region,
The 123-2093 of SEQ ID NO. 59 is an antigen encoding region,
The 123-2093 of SEQ ID NO. 60 is an antigen encoding region,
The 123-2750 bit of SEQ ID NO. 61 is an antigen coding region,
The 123-2093 of SEQ ID NO. 62 is an antigen encoding region,
The 123-1436 position of SEQ ID NO. 63 is an antigen coding region,
The 123 th to 2351 th positions of SEQ ID NO. 64 are antigen coding regions,
The 123 th to 2999 th positions of SEQ ID NO. 65 are antigen coding regions,
The 123 th to 2342 th positions of SEQ ID NO. 66 are antigen coding regions,
The 123-3008 th bit of SEQ ID NO. 67 is an antigen coding region,
The 123 th to 2999 th positions of SEQ ID NO. 68 are antigen coding regions,
The 123-3002 position of SEQ ID NO. 69 is an antigen encoding region,
The 123 th to 2999 th positions of SEQ ID NO. 70 are antigen coding regions,
The 123 th to 2999 th positions of SEQ ID NO. 71 are antigen coding regions,
The 123 th to 2351 th positions of SEQ ID NO. 72 are antigen coding regions,
The 123 th to 2342 th positions of SEQ ID NO. 73 are antigen coding regions,
The 123-2342 th bit of SEQ ID NO. 74 is an antigen encoding region.
FIG. 1 is an electrophoretogram of the linearized plasmid corresponding to SEQ ID NO. 41.
Example 3 preparation of mRNA
1. In vitro transcription
In vitro transcription was performed using T7 HIGH YIELD RNA Transcription Kit (Novoprotein, CAT: E131-01A) according to the instructions. 1. Mu.g of linearized plasmid was used in a reaction volume of 20. Mu.l, as follows:
(1) Mixing the components except T7 RNA Polymerase Mix, centrifuging for a short time, collecting at the bottom of the tube, and storing on ice for use;
(2) The components shown in the following Table 4 were added:
TABLE 4 Table 4
(3) Gently mixing the components by a pipette, and briefly centrifugally collecting, and incubating at 37 ℃ for 3 hours;
(4) Adding 3 μl of DNase I into the reaction system, incubating at 37deg.C for 15min, and digesting the transcribed DNA template;
(5) Purification of mRNA by lithium chloride precipitation purification:
mu.l of mRNA was added to 95. Mu.l Lithium Chloride Precipitation Solution (7.5M Lithium Chloride,50mM EDTA) and 95. Mu. L RNASE FREE WATER, respectively;
b. mixing, and standing at-20deg.C for 30min;
c.4deg.C 12000rpm for 15min, removing supernatant, and collecting precipitate;
d. the mixture was washed once with precooled 70% ethanol. (centrifugation at 12000rpm at 4 ℃ C., discarding supernatant);
e, detecting the concentration after the re-dissolution of 50 mu L RNASE FREE WATER.
MRNA capping
MRNA capping was performed using Cap1 CAPPING SYSTEM (Novoprotein, CAT: M082) according to the instructions. 100 μl of the reaction system was suitable for capping 50 μg mRNA, and the following steps were performed:
(1) Diluting an appropriate amount of RNA to 536. Mu.l by RNase-FREE WATER;
(2) Heating RNA at 65deg.C for 5min, and standing on ice for 5min;
(3) The components shown in the following Table 5 were added in order:
TABLE 5
(4) The reaction was carried out at 37℃for 1h.
MRNA purification
The volume of 800 μl after capping reaction was uniformly divided into four parts, and purification was performed by lithium chloride precipitation purification, specifically as follows:
a.200. Mu.l volumes were added 300. Mu.l Lithium Chloride Precipitation Solution (7.5M Lithium Chloride,50mM EDTA) and 300. Mu. L RNASE FREE WATER (Lithium Chloride final concentration to be maintained at 2.5-2.8M), respectively;
b. mixing, and standing at-20deg.C for 30min;
c.4deg.C 12000rpm for 15min, removing supernatant, and collecting precipitate;
d. the mixture was washed once with precooled 70% ethanol. (centrifugation at 12000rpm at 4 ℃ C., discarding supernatant);
e, detecting the concentration after the re-dissolution of 50 mu L RNASE FREE WATER;
FIG. 2 is an electrophoretogram of mRNA corresponding to SEQ ID NO. 63.
Example 4 preparation of mRNA vaccine
Mixing each mRNA obtained in example 3 with lipid nanoparticles to obtain a nucleic acid-lipid nanoparticle complex, and thoroughly mixing with an equal volume of adjuvant to obtain an immune sample, wherein the lipid nanoparticles comprise DOTMA and DOPE in a molar ratio of 1:1; the mRNA content in the complex is 100 mug/ml, and the mass ratio of the lipid nanoparticle to the mRNA is 10:1; the adjuvant ingredients were 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg polysorbate 80, 3.53mg sodium chloride, 0.09mg potassium chloride, 0.51mg disodium hydrogen phosphate, 0.09mg potassium dihydrogen phosphate and water for injection per 0.5 ml.
Example 5 preparation of mRNA vaccine
Mixing each mRNA obtained in example 3 with a lipid nanoparticle to obtain a nucleic acid-lipid nanoparticle complex, and thoroughly mixing with an equal volume of adjuvant to obtain an immune sample, wherein the lipid nanoparticle comprises DOTAP and DOPE in a molar ratio of 2:1; the mRNA content in the complex is 200 mug/ml, and the mass ratio of the lipid nanoparticle to the mRNA is 12:1; the adjuvant ingredients were 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg polysorbate 80, 3.53mg sodium chloride, 0.09mg potassium chloride, 0.51mg disodium hydrogen phosphate, 0.09mg potassium dihydrogen phosphate and water for injection per 0.5 ml.
Example 6 preparation of mRNA vaccine
Mixing each mRNA obtained in example 3 with lipid nanoparticles to obtain a nucleic acid-lipid nanoparticle complex, and thoroughly mixing with an equal volume of adjuvant to obtain an immune sample, wherein the lipid nanoparticles comprise DOTMA and cholesterol in a molar ratio of 1:1; the mRNA content in the complex is 100 mug/ml, and the mass ratio of the lipid nanoparticle to the mRNA is 10:1; the adjuvant composition comprises 19.5mg squalene, 2.35mg Tween 80,2.35mg Span85 and water for injection per 0.5 ml.
Example 7 preparation of mRNA vaccine
Mixing each mRNA obtained in example 3 with lipid nanoparticles to obtain a nucleic acid-lipid nanoparticle complex, and thoroughly mixing with an equal volume of adjuvant to obtain an immune sample, wherein the lipid nanoparticles comprise DOTMA and DOPE in a molar ratio of 2:1; the mRNA content in the complex is 200 mug/ml, and the mass ratio of the lipid nanoparticle to the mRNA is 8:1; the adjuvant components include DOPC 1mg, cholesterol 0.25mg, MPL 50 μg and QS-21 μg per 0.5 ml.
Example 8, mouse immunization experiment
BALB/c mice (purchased from the company beversham fukang biotechnology limited, beijing) of 8 to 10 weeks old were group immunized with each mRNA vaccine obtained in example 4, 5 mice per group. Mice were immunized on days 0 and 21, respectively, with 50 μl of each immunization sample (containing 5 μg mRNA) intramuscular injection, and blood was collected on days 0, 21, and 14 days after the second immunization. Collected blood samples were placed at 37℃for 1 hour, 4℃for 1 hour, centrifuged at 8000r/min for 10 minutes, and serum was collected and stored at-20℃for specific antibody detection and pseudovirus neutralization detection.
Example 9 pseudovirus neutralization experiments
The novel coronavirus S protein containing the mutation site of each strain is adopted to construct pseudoviruses, and a novel coronavirus neutralizing antibody detection method (chemiluminescence method) based on a VSV system is used for detecting serum obtained by separation after immunization. Taking 30-fold initial gradient dilution as an example, the specific steps are as follows:
(1) 30-fold initial dilution: a96-well plate was prepared, 150. Mu.l/well of DMEM complete medium (1% diabody, 10% FBS, 1% nonessential amino acids, 1% HEPES) was added to column 2 (cell control CC), 100. Mu.l/well of DMEM complete medium was added to column 3 (virus control VC), 100. Mu.l/well of DMEM complete medium was added to C4-G11 wells, and 142.5. Mu.l/well of DMEM complete medium was added to B4-B11 wells. Sample 1 was added to wells B4 and B5: 7.5 μl/well. 7.5 μl/well.
(2) Sample dilution: the multichannel pipettor is adjusted to 50 mu l, the liquid in the holes B4-B11 is gently and repeatedly mixed uniformly, then 50 mu l of the liquid is transferred to the corresponding holes C4-C11, the liquid is gently and repeatedly blown and sucked for 6-8 times and then transferred to the holes D4-D11, so that the liquid is pushed, and finally 50 mu l of the liquid is sucked from the holes G4-G11.
(3) The pseudoviruses were diluted to 1.3X10 4(1×104～2×104) TCID50/ml in DMEM complete medium and 50. Mu.l per well was added to columns 3-11 to give the pseudoviruses an amount of 650TCID 50/well.
(4) The 96-well plate was placed in a cell incubator (37 ℃ C., 5% CO 2) and incubated for 1h.
(5) When the incubation time reaches half an hour, the prepared Vero cells (the confluence rate reaches 80% -90%) in the incubator are taken out, the cells are counted after pancreatin digestion, and the cells are diluted to 2X 10 5 cells/ml by using a DMEM complete medium.
(6) The 96-well plate is gently rocked back and forth and left and right to disperse cells uniformly in the well, and the 96-well plate is put into a cell incubator to be cultured for 24 hours at 37 ℃ with 5% CO 2.
(7) The 96-well plate was removed from the cell incubator, 150. Mu.l of the supernatant was pipetted from each well with a multi-channel pipette, and 100. Mu.l of luciferase assay reagent was added thereto, and the reaction was performed at room temperature in a dark place for 2min.
(8) After the reaction is finished, the mixture is evenly mixed by vibrating in a flat-plate oscillator, and the mixture is put into a multifunctional plate reader to read the luminous value.
(9) And (3) calculating the neutralization inhibition rate: inhibition ratio = [1- (mean of luminous intensity of sample group-mean of cell control CC)/(mean of luminous intensity of virus control group VC-mean of cell control CC) ] ×100%.
(10) The 50% neutralizing antibody titer ND 50 was calculated from the cumulative positive rate.
The neutralizing antibody titers and geometric mean titers GMT for the 5 serum samples of each group against the different pseudoviruses are shown in table 6.
TABLE 6
It can be seen that the average of the neutralizing antibody titres GMT of the vaccine prepared from the mRNA (SEQ ID NO: 63) encoding RBD (Beta) -RBD (Beta) against different pseudoviruses was 31133 and the average of the neutralizing antibody titres GMT against different pseudoviruses was 58513 for the vaccine prepared from the mRNA (SEQ ID NO: 53) encoding RBD (Beta) -RBD (BA.1) against the pseudoviruses, the average of the neutralizing antibody titres GMT against different pseudoviruses prepared from the mRNA (SEQ ID NO: 54) encoding RBD (Beta) -RBD (BA.1) against the mRNA (SEQ ID NO: 55) encoding RBD (Beta) -RBD (Beta) against the pseudoviruses, and the average of the neutralizing antibody titres 58513 for the different pseudoviruses prepared from the mRNA (Beta) -RBD (Beta) against the mRNA (Beta) against the human strain and the average of the vaccine prepared from the mRNA (Beta) -RBD (Beta) against the mRNA (Beta) against the strain (Beta) against the pseudoviruses, and the average of the neutralizing antibody titres GMT against the vaccine prepared from the strain (Beta) -RBD (Beta) against the pseudoviruses was up to 99781.
It can be seen that the mRNA vaccine prepared from the mRNA encoding the immunogenic substance of the invention has also very good immune effects against different strains. It is expected that vaccines prepared from the immunogenic material of the invention and nucleic acids encoding the immunogenic material will still have a better prophylactic effect against mutants of the new coronavirus that may occur in the future. Therefore, the invention has important guiding significance under the condition that the novel coronavirus is continuously mutated.
The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present invention and are more fully described herein with reference to certain specific embodiments thereof, it being understood that the invention is not limited to the specific embodiments shown, but is intended to cover various modifications, equivalents, alternatives, and improvements made within the spirit and principles of the invention.
Claims (18)
1. An mRNA encoding a novel coronavirus antigen, wherein said mRNA comprises SEQ ID No. 53, 56 or 60, or said mRNA comprises SEQ ID nos. 54 and 55.
2. The mRNA of claim 1, wherein the mRNA has 3' -polyA.
3. The mRNA of claim 2, wherein the 3' -polyA comprises 50-300 adenylates.
4. The mRNA of claim 3, wherein the 3' -polyA comprises 60-200 adenylates.
5. The mRNA of claim 4, wherein the 3' -polyA comprises 80-150 adenylates.
6. The mRNA of claim 1, wherein the mRNA has a 5' cap structure.
7. The mRNA of claim 6, wherein the 5' Cap structure is selected from one of a modified or unmodified Cap 0-type Cap structure, cap 1-type Cap structure, cap 2-type Cap structure, anti-reverse Cap analogue.
8. The mRNA of claim 1, wherein the mRNA has a nucleoside modification comprising at least one of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methoxy-uridine, and 2' -O-methyl-uridine.
9. A method for producing an mRNA according to any one of claims 1 to 8, comprising:
(1) Constructing an expression vector encoding the novel coronavirus antigen, wherein the expression vector comprises a DNA sequence encoding a polyadenylic acid (polyA);
(2) Transforming host cells by using the expression vector, and preparing linearization plasmids after amplification culture;
(3) In vitro transcription is performed using the linearized plasmid to obtain mRNA.
10. Use of an mRNA according to any one of claims 1 to 8 for the preparation of a composition for the prevention of a novel coronavirus infection.
11. A composition comprising the mRNA of any one of claims 1-8 and a delivery vehicle.
12. The composition of claim 11, wherein the delivery vehicle comprises lipid nanoparticles.
13. The composition of claim 12, wherein the lipid nanoparticle comprises a cationic lipid and a helper lipid.
14. The composition of claim 13, wherein the cationic lipid is selected from at least one of 1, 2-di-O-octadecenyl-3-trimethylammonium propane, dimethyl dioctadecyl ammonium, 1, 2-dioleoyl-3-trimethylammonium propane, 1, 2-dioleoyl-3-dimethylammonium propane, 1, 2-dioleyloxy-3-dimethylammonium propane, 1, 2-dialkoxy-3-dimethylammonium propane, dioctadecyl dimethylammonium chloride, 1, 2-dimyristoxypropyl-1, 3-dimethylhydroxyethyl ammonium 2, 3-dioleoyloxy-N- [2 (spermidine carboxamide) ethyl ] -N, N-dimethyl-1-trifluoroacetic acid propylammonium.
15. The composition of claim 13, wherein the helper lipid is selected from at least one of1, 2-bis- (9Z-octadecenoyl) -sn-glycerol-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycerol-3-phosphate choline, diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, cephalin, sterols, and cerebrosides.
16. The composition of claim 15, wherein the helper lipid comprises 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine and cholesterol.
17. The composition of claim 11, wherein the composition further comprises an adjuvant.
18. The composition of claim 17, wherein the adjuvant is selected from one or more of aluminum adjuvants, MF59, MPL, QS-21, GLA, cpG, polyI: C, AS01, AS02, AS03, AS04 adjuvants.
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