Patent Application
20230143228
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 20121PCT_ST25.txt. The text file is 240 KB, was created on April 20, 2021, and is being submitted electronically via EFS-Web.
About 10% of common colds are due to certain coronavirus (CoV) strains associated with mild symptoms. More dangerous human strains such as severe acute respiratory syndrome associated coronavirus (SARS-CoV-1) and SARS-CoV-2 (also referred to as COVID-19) are believed to result from coronavirus strains jumping to humans by secondary zoonotic transfers, e.g., from bats to cats and cats to humans. In humans, SARS-CoV-2 can be transferred from individuals who have mild symptoms or are asymptomatic and has caused numerous deaths worldwide. Thus, there is a need to find an effective vaccine.
The SARS-CoV-2 genome has about 30 kb that can be directly read by ribosomes with host cells. The RNA forms a ribonucleoprotein complex within virus particles having a viral lipid envelope membrane made up of membrane (M) glycoproteins, trimeric spike (S) glycoproteins and envelope (E) proteins. The trimeric units of the spike protein contain a receptor binding domain and a fusion domain that anchors it into lipid membrane.
Walls et al. report that the SARS-CoV-2 spike protein is involved in viral cell entry by recognizing human ACE2. Cell, 2020, 180, 1-12.
Andersen et al. report six receptor binding domain amino acids L455, F486, Q493, S494, N501 and Y505 are involved in binding to ACE2 receptors in SARS-CoV-2. Nat Med, 2020.
Altenburg et al. report modified vaccinia virus Ankara (MVA) as a production platform for vaccines against influenza and other viral respiratory diseases. Viruses, 2014, 6(7):2735-61.
Graham et al. report prefusion coronavirus spike proteins and uses. See WO 2018/081318.
References cited herein are not an admission of prior art.
This disclosure relates to methods of promoting immune responses against coronavirus, such as SARS-CoV-2, and compositions related thereto. In certain embodiments, this disclosure relates to methods of vaccinating for coronavirus comprising administering to the subject a composition disclosed herein. In certain embodiments, the composition comprises a recombinant virus such as recombinant MVA that encodes a coronavirus spike protein. In certain embodiments, the coronavirus spike protein comprises a proline mutation at position 986. In certain embodiments, the coronavirus spike protein comprises a proline mutation at position 987.
In certain embodiments, this disclosure relates to methods of vaccinating or immunizing a human subject comprising administering an effective amount of coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein under conditions such that a spike protein and/or virus-like particles with a spike protein are formed in the subject.
In certain embodiments, this disclosure relates to methods of vaccinating or immunizing a human subject comprising administering an effective amount of coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein in combination with a nucleic acid encoding a T cell stimulating chimeric protein under conditions such that a spike protein and/or virus-like particles with a spike protein are formed in the subject.
In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising a coronavirus spike protein or segment thereof as disclosed herein and variants thereof. In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising virus-like particles having a coronavirus spike protein or segment thereof as disclosed herein and variants thereof. In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein and variants thereof.
In certain embodiments, this disclosure contemplates nucleic acids, recombinant vectors, viral vectors, and bacterial plasmids encoding a coronavirus spike protein or segment thereof as disclosed herein which forms trimeric protein complexes and uses in vaccination methods disclosed herein.
In certain embodiments, this disclosure relates to cells and other expression vectors and expression systems for use in producing a coronavirus spike protein or segment thereof as disclosed herein and trimeric coronavirus spike proteins or segment thereof as disclosed herein, or variants thereof.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly, the terms “comprising”, “including” and “having” can be used interchangeably. It is further noted that the claims may be drafted to exclude any optional element.
“Subject” refers to any animal, preferably a human patient, livestock, rodent, monkey or domestic pet. The term is used herein to encompasses apparently healthy, non-infected individuals or a patient who is known to be infected with, diagnosed with, a pathogen.
As used herein, the term “combination with” when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.
The term “comprising” in reference to a peptide having an amino acid sequence refers a peptide that may contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to include the amino acid sequence within a larger peptide. The term “consisting of in reference to a peptide having an amino acid sequence refers a peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide may consist of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids specified in the claim however the C-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide may consist of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids specified in the claim however the N-terminus may be connected to additional amino acids, e.g., as part of a larger peptide.
The terms “protein” and “peptide” refer to polymers comprising amino acids joined via peptide bonds and are used interchangeably. Amino acids may be naturally or non-naturally occurring. A “chimeric protein” or “fusion protein” is a molecule in which different portions of the protein are derived from different origins such that the entire molecule is not naturally occurring. A chimeric protein may contain amino acid sequences from the same species of different species as long as they are not arranged together in the same way that they exist in a natural state. Examples of a chimeric protein include sequences disclosed herein that are contain one, two or more amino acids attached to the C-terminal or N-terminal end that are not identical to any naturally occurring protein, such as in the case of adding an amino acid containing an amine side chain group, e.g., lysine, an amino acid containing a carboxylic acid side chain group such as aspartic acid or glutamic acid, a polyhistidine tag, e.g. typically four or more histidine amino acids. Contemplated chimeric proteins include those with self-cleaving peptides such as P2A-GSG. See Wang. Scientific Reports 5, Article number: 16273 (2015).
In certain embodiments, the disclosure relates to recombinant polypeptides comprising sequences disclosed herein or variants or fusions thereof wherein the amino terminal end or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.
A “label” refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as 35S or 131I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
In certain embodiments, this disclosure contemplates that chimeric proteins disclosed herein may be variants. Variants may include 1 or 2 amino acid substitutions or conserved substitutions. Variants may include 3 or 4 amino acid substitutions or conserved substitutions. Variants may include 5 or 6 or more amino acid substitutions or conserved substitutions. Variant include those with not more than 1% or 2% of the amino acids are substituted. Variant include those with not more than 3% or 4% of the amino acids are substituted. Variants include proteins with greater than 80%, 89%, 90%, 95%, 98%, or 99% identity or similarity.
Variant peptides can be produced by mutating a vector to produce appropriate codon alternatives for polypeptide translation. Active variants and fragments can be identified with a high probability using computer modeling. Shihab et al. report an online genome tolerance browser. BMC Bioinformatics, 2017, 18(1):20. Ng et al. report methods of predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet, 2006, 7:61-80. Teng et al. Approaches and resources for prediction of the effects of non-synonymous single nucleotide polymorphism on protein function and interactions. Curr Pharm Biotechnol, 2008, 9(2): 123-33.
Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, RaptorX, ESyPred3D, HHpred, Homology Modeling Professional for HyperChem, DNAStar, SPARKS-X, EVfold, Phyre, and Phyre2 software. See also Saldano et al. Evolutionary Conserved Positions Define Protein Conformational Diversity, PLoS Comput Biol. 2016, 12(3):e1004775; Marks et al. Protein structure from sequence variation, Nat Biotechnol. 2012, 30(11):1072-80; Mackenzie et al. Curr Opin Struct Biol, 2017, 44:161-167 Mackenzie et al. Proc Natl Acad Sci U S A. 113(47):E7438-E7447 (2016); Joseph et al. J R Soc Interface, 2014, 11(95):20131147, Wei et al. Int. J. Mol. Sci. 2016, 17(12), 2118. Variants can be tested in functional assays. Certain variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).
Sequence “identity” refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. For example, the polypeptides GGGGGG (SEQ ID NO: 32) and GGGGT (SEQ ID NO: 33) have a sequence identity of 4 out of 5 or 80%. For example, the polypeptides GGGPPP (SEQ ID NO: 34) and GGGAPPP (SEQ ID NO: 35) have a sequence identity of 6 out of 7 or 85%. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic—F Y W; hydrophobic-A V I L; Charged positive: R K H; Charged negative—D E; Polar—S T N Q. The amino acid groups are also considered conserved substitutions.
Percent identity can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J Mol Biol 1970 48:443), as revised by Smith and Waterman (Adv Appl Math 1981 2:482). Briefly, the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess (Nucl Acids Res 1986 14:6745), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 1979, pp. 353-358); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
The term “recombinant vector” when made in reference to vectors and nucleic acids refers to a nucleic acid molecule that is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term recombinant nucleic acid is distinguished from the natural recombinants that result from crossing-over between homologous chromosomes. Recombinant nucleic acids as used herein are an unnatural union of nucleic acids from nonhomologous sources, usually from different organisms.
The terms “expression vector ” refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
In certain embodiments, a vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col E1 origin of replication, f1 origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.
Protein “expression systems” refer to in vivo and in vitro (cell free) systems. Systems for recombinant protein expression typically utilize somatic cells transfected with a DNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Also, some proteins are recovered using denaturants and protein-refolding procedures. In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids and nucleotides. In the presence of an expression vector, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labelling of the protein with modified amino acids. Some cell free systems incorporated encoded components for translation into the expression vector. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol., 19, 751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): e141, both hereby incorporated by reference in their entirety.
A “selectable marker” is a nucleic acid introduced into a vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-5′ phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5-fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptII), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (at1D), UDP-glucose:galactose-1-phosphate uridyltransferasel (galT), feedback-insensitive α subunit of anthranilate synthase (OASA1D), 2-deoxyglucose (2-DOGR), benzyladenine-N-3-glucuronide, E. coli threonine deaminase, glutamate 1-semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPS1), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSB1), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase (dsdA).
In certain embodiments, this disclosure relates to methods of vaccinating or immunizing a human subject comprising administering an effective amount of a coronavirus spike protein, a trimeric spike protein complex, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein under conditions such that spike protein, trimeric complex, and/or virus-like particles with spike protein are formed in the subject.
In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising a coronavirus spike protein, trimeric complex, or segment thereof as disclosed herein and variants thereof. In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising virus-like particles having a coronavirus spike protein or segment thereof as disclosed herein and variants thereof. In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein and variants thereof.
In certain embodiments, this disclosure contemplates nucleic acids, recombinant vectors, viral vectors, and bacterial plasmids encoding a coronavirus spike protein or segment thereof as disclosed herein which form trimeric protein complexes and uses in vaccination methods disclosed herein.
In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, further comprises a proline mutation at position 987. In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 1)
MF VFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKT SVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNF SQILPDP SKP SKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof. In certain embodiments, the amino acid position of a coronavirus protein is in relation to SEQ ID NO: 1.
In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation in the furin cleavage site at position 682, 683, 684 or 685, In certain embodiments, the mutation in the furin cleavage site is at position 682. In certain embodiments, the coronavirus spike protein comprises a serine mutation at position 682. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, further comprises a proline mutation at position 987. In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 23)
MFVFLVLLPLVS SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS SVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVL SFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPSRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof.
In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a lysine mutation at position 484, an asparagine mutation at position 417, a tyrosine mutation at position 501, or combinations thereof and a mutation in the furin cleavage site at position 682, 683, 684 or 685, a serine mutation at position 682, a proline mutation at position 986, a proline mutation at position 987, or combinations thereof.
In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation at position 484. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a lysine mutation at position 484 optionally in combination with other mutations below. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation in the furin cleavage site at position 682, 683, 684 or 685. In certain embodiments, the mutation in the furin cleavage site is at position 682. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a serine mutation at position 682. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein further comprises a proline mutation at position 987.
In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 28) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS SVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF SALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVL SFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPSRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof.
In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation at position 417. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising an asparagine mutation at position 417 optionally in combination with other mutations below. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation in the furin cleavage site at position 682, 683, 684 or 685, In certain embodiments, the mutation in the furin cleavage site is at position 682. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a serine mutation at position 682. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein further comprises a proline mutation at position 987.
In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 29)
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS SVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GNIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPSRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNF SQILPDP SKP SKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof.
In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation at position 501. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a tyrosine mutation at position 501 optionally in combination with other mutations below. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation in the furin cleavage site at position 682, 683, 684 or 685, In certain embodiments, the mutation in the furin cleavage site is at position 682. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a serine mutation at position 682. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein further comprises a proline mutation at position 987.
In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 30)
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPSRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof.
In certain embodiments, the coronavirus spike protein further comprises a heterologous N-terminal signal sequence.
In certain embodiments, the coronavirus spike protein further comprises a C-terminal trimerization sequence.
In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 2)
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIR AAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQE KNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGI VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKN LNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCC SCLKGCCSCGS CCKFDEDDSEPVLKGVKLHYT (S-Tri) or variants thereof.
In certain embodiments, the coronavirus spike protein comprises a coronavirus M protein sequence downstream from the C-terminal end of the coronavirus spike protein sequence, and wherein the M protein sequence and the coronavirus spike sequence are separated by a self-cleaving sequence. In certain embodiments, the coronavirus spike protein comprises a coronavirus E protein sequence downstream from the C-terminal end of the M protein sequence, and wherein the E protein sequence and the coronavirus M protein sequence are separated by a self-cleaving sequence.
In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 3)
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVL SFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIR AAEIRASANLAATKMSECVLGQ SKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQE KNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGI VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKN LNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCC SCLKGCCSCGS CCKFDEDDSEPVLKGVKLHYTGSGATNFSLLKQAGDVEENPGPMADSNGTITVEELKKL LEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWI TGGIAIAMACLVGLMWLSYFIASFRLFARTRSMW SFNPETNILLNVPLHGTILTRPLLESE LVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYS RYRIGNYKLNTDHSSSSDNIALLVQGSGATNFSLLKQAGDVEENPGPMYSFVSEETGTLI VNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKP SFYVYSRVKNLNS SRVPDL LV (S-VLP) or variants thereof.
With specific regard to coronavirus proteins disclosed herein, any amino acid substitution is permissible so long as the activity of the protein is not significantly affected. In this regard, it is appreciated in the art that amino acids can be classified into groups based on their physical properties. Examples of such groups include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Preferred variants that contain substitutions are those in which an amino acid is substituted with an amino acid from the same group. Such substitutions are referred to as conservative substitutions.
In certain embodiments, this disclosure relates to virus-like particles comprising a coronavirus spike protein disclosed herein.
In certain embodiments, this disclosure relates nucleic acids comprising a sequence encoding a coronavirus spike protein disclosed herein in operable combination with a heterologous promotor.
In certain embodiments, the nucleic acid the sequence encoding a coronavirus spike protein comprises (SEQ ID NO: 4)
ATGTGGTTACAAGGACTACTATTACTAGGTACTGTTGCCTGTTCAATTTCACA ATGTGTAAATCTAACTACAAGAACTCAATTACCGCCTGCCTATACTAATTCTTTTACA AGAGGAGTATATTATCCTGATAAAGTTTTTAGATCTTCTGTATTACATTCTACACAAG ATTTGTTTTTACCATTTTTCTCTAATGTTACTTGGTTTCATGCAATACATGTATCTGGA ACTAATGGAACAAAAAGATTTGATAATCCAGTATTACCTTTTAATGATGGAGTTTAT TTTGCTTCTACTGAAAAATCTAATATAATTAGAGGATGGATATTTGGAACTACATTA GATTCTAAAACACAATCTCTACTAATTGTTAATAATGCAACTAATGTAGTTATAAAA GTATGTGAATTTCAATTTTGTAATGATC CATTTTTGGGAGTTTATTATCATAAAAATA ATAAGTCTTGGATGGAATCTGAATTCAGAGTATATTCTTCTGCTAATAATTGTACATT TGAATATGTATCTCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAACTTTAA AAATTTGAGAGAATTTGTTTTTAAAAATATTGATGGATACTTTAAAATCTATTCTAA ACATACTCCAATTAATCTAGTAAGAGATTTGCCTCAAGGATTTTCTGCTTTAGAACC ACTAGTAGATTTGC CTATAGGAATTAATATTACTAGATTTCAAACATTATTAGC TTTA CATAGATCTTATTTGACACCTGGAGATTCTTCTTCTGGATGGACTGCAGGAGCTGCA GCTTATTATGTTGGATATTTGCAACCAAGAACATTTTTGTTAAAATATAATGAAAAT GGAACTATAACAGATGCAGTTGATTGTGCTTTAGATCCTCTATCTGAAACTAAATGT ACTTTAAAATCTTTTACTGTAGAAAAAGGAATCTATCAAACATCTAACTTTAGAGTA CAACCAACTGAATCTATTGTTAGATTTCCAAATATAACAAATCTATGTCCTTTTGGA GAAGTTTTTAATGCAACTAGATTTGCTTCTGTATATGCATGGAATAGAAAAAGAATA TCTAATTGCGTAGCTGATTATTCTGTATTATATAATTCTGCATCTTTTTCTACTTTTAA ATGTTATGGAGTATCTCCAACAAAATTGAATGATCTATGTTTTACTAATGTTTATGCA GATTCTTTTGTAATAAGAGGAGATGAAGTTAGACAAATAGCTCCTGGACAAACAGG AAAAATAGCAGATTATAATTATAAATTACCAGATGATTTCACTGGATGCGTAATTGC TTGGAATTCTAATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTTGTATAG ATTGTTTAGAAAATCTAATTTGAAACCTTTTGAAAGAGATATTTCTACAGAAATCTA TCAAGCAGGATCTACTCCATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACTA CAATCTTATGGATTTCAACCTACAAATGGAGTAGGATATCAACCATATAGAGTAGTT GTATTATC TTTTGAATTATTACATGCACCAGCTACAGTATGTGGACC TAAAAAATCT ACTAATTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGATTAACTGGAACA GGAGTTTTAACTGAATCTAATAAGAAATTTTTGCCTTTTCAACAATTTGGAAGAGAT ATTGCTGATACTACAGATGCAGTAAGAGATCCTCAAACTTTAGAAATATTGGATATT ACACCATGTTCTTTTGGAGGAGTTTCTGTAATAACACCAGGAACTAATACATCTAAT CAAGTTGCTGTATTATATCAAGATGTTAATTGTACTGAAGTTCCTGTAGCAATTCATG CTGATCAATTAACTCCAACATGGAGAGTATATTCTACTGGATCTAATGTTTTTCAAA CAAGAGCTGGATGTCTAATTGGAGCAGAACATGTAAATAATTCTTATGAATGTGATA TTCCTATAGGAGCTGGAATATGTGCATCTTATCAAACTCAAACAAATTCTCCAAGAA GAGCTAGATCTGTTGCATCTCAATCTATAATTGCTTATACAATGTCTTTAGGAGCTGA AAATTCTGTAGCATATTCTAATAATTCTATTGCAATTCCTACTAACTTTACTATTTCT GTAACTACAGAAATATTGCCAGTTTCTATGACTAAAACATCTGTAGATTGTACAATG TATATATGTGGAGATTCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTTTTT GTACTCAATTGAATAGAGCTTTAACAGGAATAGCAGTAGAACAAGATAAAAATACA CAAGAAGTTTTTGCTCAAGTAAAACAAATCTATAAAACTCCACCTATAAAAGATTTT GGAGGTTTTAATTTTTCTCAAATATTGCCAGATCCTTCTAAACCTTCTAAAAGATCTT TTATTGAAGATTTGTTGTTTAATAAGGTTACATTAGCAGATGCTGGTTTTATAAAACA ATATGGAGATTGTTTAGGAGATATTGCAGCTAGAGATTTGATTTGTGC TCAAAAGTT TAATGGATTAACTGTATTACCACCTCTACTAACAGATGAAATGATAGCACAATATAC ATCTGCATTATTAGCTGGAACTATTACATCTGGATGGACTTTTGGAGCTGGAGCAGC TTTACAAATACCATTTGCTATGCAAATGGCATATAGATTCAATGGAATTGGAGTTAC TCAAAATGTATTATATGAAAATCAAAAACTAATTGCTAATCAATTCAATTCTGCAAT TGGAAAAATTCAAGATTCTCTATCTTCTACAGCATCTGCTTTAGGAAAACTACAAGA TGTTGTAAATCAAAATGCACAAGCTTTAAATACTCTAGTTAAACAACTATCTTCTAA TTTTGGAGCTATTTCTTCTGTTTTAAATGATATATTGTCTAGACTAGATCCACCT (encoding spike sequence amino acids 1 to 987) or variants with great than 85% identity thereto. In certain embodiments, variants are synonymous or non-synonymous codons.
In certain embodiments, the disclosure relates to recombinant viral vectors, recombinant vectors, and recombinant plasmids comprising nucleic acids encoding coronavirus spike proteins disclosed herein. In certain embodiments, this disclosure relates to expression systems comprising nucleic acids and vectors disclosed herein.
Nucleic acids, vectors, and expression constructs can be introduced in vivo via lipofection (DNA transfection via liposomes prepared from synthetic cationic lipids). Synthetic cationic lipids can be used to prepare liposomes to encapsulate a nucleic acid, vector, or expression construct of the disclosure. A nucleic acid, vector, or expression construct can also be introduced as naked DNA or RNA using methods known in the art, such as transfection, microinjection, electroporation, calcium phosphate precipitation, and by biolistic methods.
If a recombinant virus vector of this disclosure is constructed starting with a vaccinia virus, the majority of the nucleic acid molecules and proteins in the recombinant virus vector will come from vaccinia virus and thus the final recombinant virus vector can be referred to, for example, as a recombinant vaccinia virus vector or a vaccinia-based recombinant virus vector. In certain embodiments, the recombinant virus vector is selected from the group consisting of a recombinant poxvirus vector, a recombinant vaccinia virus vector, a recombinant chordopoxvirus vector, a recombinant iridovirus vector, a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant SV40 virus vector, a recombinant Epstein-Barr virus vector, a recombinant herpes virus vector, and a recombinant JC virus vector.
In certain embodiments, this disclosure contemplates that methods disclosed herein are used with recombinant virus, preferably recombinant modified vaccinia virus Ankara (MVA). MVA is an attenuated strain of vaccinia virus originally developed as a vaccine for smallpox. The ability of MVA to infect mammalian, including human host cells, is restricted due to known deletions in the virus genome. In addition to the safe use in human vaccinations, Wyatt et al. report mice with severe combined immunodeficiency disease remained healthy when inoculated with MVA. Proc Natl Acad Sci USA. 2004, 101(13):4590-5.
MVA can be engineered in deleted regions to express heterologous genes to induce protective immunity to other viruses. Combined DNA and recombinant modified vaccinia Ankara (MVA62B) vaccines can produce virus-like particles that display membrane-bound trimeric forms of envelope proteins. As a result of extensive passage in cell culture, the MVA virus genome contains six major deletions, referred to as Del I, II, III, IV, V and VI. Historically, the region around Del II and Del III has been used for insertion of heterologous nucleic acid sequences.
As used herein, the term heterologous is a comparative term, and refers to a molecule that is from an organism different from that to which it is being referenced or that is made synthetically. The molecule can be a protein or a nucleic acid sequence (i.e., RNA or DNA). For example, a heterologous nucleic acid sequence in a recombinant virus vector refers to the fact that the heterologous nucleic acid sequence is or may be from an organism other than the base virus used to construct the recombinant virus vector. As a further example, a heterologous nucleic acid sequence in a recombinant vaccinia virus vector refers to the fact that the heterologous nucleic acid sequence is from an organism other than vaccinia virus or that was made synthetically.
A heterologous nucleic acid sequence can be inserted at any location in a recombinant virus vector genome, as long as such insertion does not unintentionally alter the functioning of the resulting recombinant virus vector. For example, a nucleic acid sequence can be inserted into a non-essential region. Such non-essential regions include, but are not limited to, naturally occurring deletions within the viral genome (e.g., Del I, II, III, etc. of modified vaccinia virus Ankara (MVA)), intergenic regions or non-essential genes. A non-essential region is a genomic region, the alteration of which has no, or almost no, discernible effect on viral replication and the production of progeny virus. One example of a non-essential region is a non-essential gene such as, for example, the vaccinia virus hemagglutinin gene.
Alternatively, a nucleic acid sequences can be inserted into an essential region of the genome (e.g., an essential gene). It will be appreciated that interruption of an essential region will result in a recombinant virus vector unable to complete the virus life cycle and produce progeny virus. However, such recombinant virus vectors can produce progeny virus when grown in cells that provide the missing function. Such a cell can be referred to as a complementing cell because it provides the function usually provided by the essential gene. That is, it “complements” the recombinant virus vector. Conversely, a cell that is unable to provide the missing viral function can be referred to as a non-commenting cell. Such culture systems are contemplated herein. At least one heterologous nucleic acid sequence may be inserted into the gene required for expression of post-replicative viral genes.
This disclosure relates to methods of promoting immune responses against coronavirus, such as SARS-CoV-2, and compositions related thereto. In certain embodiments, this disclosure relates to methods of vaccinating for coronavirus, such as SARS-CoV-2, comprising administering to the subject a composition disclosed herein. In certain embodiments, the composition comprises a coronavirus spike protein, VLP containing the same, or a recombinant virus such as recombinant MVA that encodes a coronavirus, such as SARS-CoV-2 spike protein. In certain embodiments, the coronavirus spike protein comprises a proline mutation at position 986. In certain embodiments, the coronavirus spike protein comprises a proline mutation at position 987.
In certain embodiments, this disclosure relates to methods of vaccinating or immunizing comprising administering to a human subject an effective amount of coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein under conditions such that spike protein and/or virus-like particles with spike protein are formed in the subject.
In certain embodiments, the methods are conducted in combination with an adjuvant. In certain embodiments, methods include using a coronavirus spike protein, trimeric complex or virus-like particle or nucleic acid encoding the same in combination with an adjuvant.
In certain embodiments, administering is to the skin, muscle, or buccal cavity. In certain embodiments, administration is by syringe, microneedle, topically, or using pressurized devices, e.g., device comprising a nozzle to push a solution into tissue by means of pressure, e.g., spring-powered without the use of a needle (needle-free devices).
DNA-based vaccines typically use bacterial plasmids to express protein immunogens in vaccinated hosts. Recombinant DNA technology is used to clone cDNAs encoding immunogens of interest into eukaryotic expression plasmids. Vaccine plasmids are then amplified in bacteria, purified, and directly inoculated into the hosts being vaccinated. DNA typically is inoculated by a needle injection of DNA in saline, or by a gene gun device that delivers DNA-coated gold beads into skin. The plasmid DNA is taken up by host cells, the vaccine protein is expressed, processed and presented in the context of self-major histocompatibility (MHC) class I and class II molecules, and an immune response against the DNA-encoded immunogen is generated.
In certain embodiments the present disclosure is a method to generate an immune response against coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein. Such a response can be a CD8+T cell immune response or an antibody response. More particularly, the present disclosure relates to “prime and boost” immunization regimes in which the immune response induced by administration of a priming composition is boosted by administration of a boosting composition. The present disclosure is based on experimental demonstration that effective priming can be achieved using modified vaccinia Ankara (MVA) vectors, following boosting with coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein.
A major protective component of the immune response against a number of pathogens is mediated by T lymphocytes of the CD8+type, also known as cytotoxic T lymphocytes (CTL). An important function of CD8+cells is secretion of gamma interferon (IFNγ), and this provides a measure of CD8+T cell immune response. A second component of the immune response is antibody directed to the proteins of the pathogen.
It is contemplated that a vaccination regime using needle-free, intradermal, intramuscular, or mucosal immunization for both prime and boost can be employed, constituting a general immunization regime suitable for inducing CD8+T cells and also eliciting an antibody response, e.g., in humans. An immune response to coronavirus spike protein, trimeric complex or virus-like particle thereof may be primed by immunization with plasmid DNA, recombinant virus, or by infection with an infectious agent.
A further aspect of this disclosure provides a method of inducing a CD8+T cell immune response to a coronavirus spike protein, trimeric complex or virus-like particle thereof in an individual, and also eliciting an antibody response.
A further aspect provides for use of coronavirus spike protein, trimeric complex or virus-like particle thereof as disclosed herein, in the manufacture of a medicament for administration to a mammal to boost a CD8+T cell immune response and also eliciting an antibody response. Such a medicament is generally for administration following prior administration of a priming composition comprising nucleic acid and/or recombinant virus encoding the antigen.
The priming composition may comprise DNA encoding a coronavirus spike protein, trimeric complex or virus-like particle thereof, such DNA being in the form of a circular plasmid that is not capable of replicating in mammalian cells. Any selectable marker should preferably not be resistance to an antibiotic used clinically, so for example Kanamycin resistance is preferred to Ampicillin resistance. Antigen expression should be driven by a promoter which is active in mammalian cells, for instance the cytomegalovirus immediate early (CMV IE) promoter.
In particular embodiments of the various aspects of the present disclosure, administration of a priming composition is followed by boosting with a boosting composition, or first and second boosting compositions, the first and second boosting compositions being the same or different from one another.
In certain embodiments, the subject is a human subject. In certain embodiments, the human subject is of advanced age or elderly e.g., more than 45, 55, or 65 years old.
In certain embodiments, an “effective amount” in the context of administration of a therapy to a subject refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a viral infection, disease or symptom associated therewith; (ii) reduce the duration of a viral infection, disease or symptom associated therewith; (iii) prevent the progression of a viral infection, disease or symptom associated therewith; (iv) cause regression of a viral infection, disease or symptom associated therewith; (v) prevent the development or onset of a viral infection, disease or symptom associated therewith; (vi) prevent the recurrence of a viral infection, disease or symptom associated therewith; (vii) reduce or prevent the spread of a viral from one cell to another cell, one tissue to another tissue, or one organ to another organ; (viii) prevent or reduce the spread of a viral from one subject to another subject; (ix) reduce organ failure associated with a viral infection; (x) reduce hospitalization of a subject; (xi) reduce hospitalization length; (xii) increase the survival of a subject with a viral infection or disease associated therewith; (xiii) eliminate a viral infection or disease associated therewith; (xiv) inhibit or reduce viral replication; (xv) inhibit or reduce the entry of an virus into a host cell(s); (xvi) inhibit or reduce replication of the virus genome; (xvii) inhibit or reduce synthesis of virus proteins; (xviii) inhibit or reduce assembly of virus particles; (xix) inhibit or reduce release of virus particles from a host cell(s); (xx) reduce virus titer; and/or (xxi) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
In certain embodiments, the effective amount does not result in complete protection from a coronavirus infection but results in a lower titer or reduced number of viruses compared to an untreated subject with a viral infection. In certain embodiments, the effective amount results in a 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200 fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greater reduction in titer of virus relative to an untreated subject with a viral infection. Benefits of a reduction in the titer, number or total burden of virus include, but are not limited to, less severe symptoms of the infection, fewer symptoms of the infection and a reduction in the length of the disease associated with the infection.
Compositions described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, transdermal, intravenous, conjunctival and subcutaneous routes. In some embodiments, a composition is formulated for topical administration, for example, for application to the skin. In specific embodiments, the route of administration is nasal, e.g., as part of a nasal spray. In certain embodiments, a composition is formulated for intramuscular administration. In some embodiments, a composition is formulated for subcutaneous administration. In certain embodiments, a composition is not formulated for administration by injection.
In certain embodiments, immunogenic compositions disclosed herein are administered intradermally. In certain embodiments, this disclosure contemplates administration using a transdermal patch for diffusion of the drug across the skin or by microneedle injection. In certain embodiments, it may be desirable to introduce the pharmaceutical compositions into the lungs by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray.
In certain embodiments, this disclosure contemplates a combination vaccine that is designed to induce a strong neutralizing antibody response and broad cytotoxic CD8 T cell response against the SARS-CoV-2 providing long-lasting protection against SARS-CoV-2 and other SARS corona viruses. To achieve one combine DNA and modified vaccinia Ankara (MVA) vaccines such that both neutralizing antibodies and CD8 T cells are induce. The DNA and MVA immunogens express nucleocapsid, membrane and envelope proteins and a string of conserved epitopes from other proteins of SARS-CoV-2. In certain embodiments, priming with a DNA or MVA construct disclosed herein plus chimeric construction disclosed herein and boosting with DNA or MVA constructs disclosed herein. The DNA or MVA constructs for the priming and boosting may be the same or different. T cell epitopes in DNA and MVA vaccines promotes T cells against SARS corona viruses that could potentially provide protection even when the virus escapes from antibody responses providing induction of high levels of neutralizing antibodies and CD8 T cells with fewer immunizations.
In certain embodiments, this disclosure relates to vaccination methods using nucleic acids encoding T cell stimulating chimeric proteins. In certain embodiments, this disclosure relates to methods of vaccinating or immunizing a human subject comprising administering an effective amount of coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein in combination with a nucleic acid encoding a T cell stimulating chimeric protein under conditions such that a spike protein and/or virus-like particles with a spike protein are formed in the subject.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M (SEQ ID NO: 24), N-M, SdRBD-N-M_dTM (SEQ ID NO: 25), N-M_dTM, NSP3-4-6 (SEQ ID NO: 26) NSP3-4-6_dTM (SEQ ID NO: 27) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M (SEQ ID NO: 24), N-M, SdRBD-N-M_dTM (SEQ ID NO: 25), N-M_dTM, NSP3-4-6 (SEQ ID NO: 26), NSP3-4-6_dTM (SEQ ID NO: 27) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses; and the boost is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein. In certain embodiments the prime is a DNA coronavirus construct, and the boost is an MVA coronavirus construct.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M (SEQ ID NO: 24) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses, and the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M_dTM (SEQ ID NO: 25) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses, and the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding NSP3-4-6 (SEQ ID NO: 26) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses, and the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding NSP3-4-6_dTM (SEQ ID NO: 27) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses; and the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a DNA or MVA or combination of a DNA and MVA coronavirus construct disclosed herein.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a DNA or MVA or combination of a DNA and MVA coronavirus construct disclosed herein and the boost is a DNA or MVA or combination of a DNA and MVA coronavirus construct disclosed herein.
In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is any coronavirus vaccine, e.g., mRNA vaccines viral vector coronavirus vaccines conventionally known by their supplier/brand name such as the Pfizer-BioNTech COVID-19 vaccine, Moderna COVID-19 vaccine, Johnson & Johnson's Janssen COVID-19 vaccine, AstraZeneca COVID-19 vaccine, and Novavax COVID-19 vaccine and the boost is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA or combination of a DNA and MVA coronavirus construct disclosed herein.
In certain embodiments, the boost is in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M (SEQ ID NO: 24), N-M, SdRBD-N-M_dTM (SEQ ID NO: 25), N-M_dTM, NSP3-4-6 (SEQ ID NO: 26), NSP3-4-6_dTM (SEQ ID NO: 27) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses.
In certain embodiments, the boost is vaccine comprises mutation E484K, K417N, N501Y, or combinations thereof. In certain embodiments, the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof.
In certain embodiments, the boost is administered more than one or two weeks after the prime. In certain embodiments, the boost is administered more than one or two months after the prime. In certain embodiments, the boost is administered more than six months after the prime. In certain embodiments, the boost is administered more than one year after the prime.
Four forms (
Construction and Characterization of rMVAs:
Full-length consensus spike protein sequences of SARS-CoV-2 was modified recombinant methods. DNA sequences encoding the proteins were codon-optimized for vaccinia virus codon usage, synthesized, and subcloned in between Xma1 and BamH1 restriction sites of the plasmid transfer vector pLW-73 (see Patent EP2402451). Inserts are transfer in between two essential genes I8R and G1L of MVA, under the control of an independent early/late vaccinia virus promoter (modified H5 [mH5]) to generate stable MVAs. Recombinant MVAs are characterized for protein expression using Western blotting and flow cytometry, grown in large-scale in chicken embryo fibroblasts, purified, quality tested, and titrated. Expression data for two of the MVA recombinants MVA/S-Tri and MVA/S-VLP are shown in
Plasmid Sequence (SEQ ID NO: 5) and Sequence encoding spike protein fusion (bold, SEQ ID NO: 6)
TCTAGTTTTGCTACCGTTGGTTTCAAGTCAATGTGTAAATCTAACTACAAGAAC
TCAATTACCGCCTGCCTATACTAATTCTTTTACAAGAGGAGTATATTATCCTGA
TAAAGTTTTTAGATCTTCTGTATTACATTCTACACAAGATTTGTTTTTACCATTT
TTCTCTAATGTTACTTGGTTTCATGCAATACATGTATCTGGAACTAATGGAACA
AAAAGATTTGATAATCCAGTATTACCTTTTAATGATGGAGTTTATTTTGCTTCTA
CTGAAAAATCTAATATAATTAGAGGATGGATATTTGGAACTACATTAGATTCTA
AAACACAATCTCTACTAATTGTTAATAATGCAACTAATGTAGTTATAAAAGTAT
GTGAATTTCAATTTTGTAATGATCCATTTTTGGGAGTTTATTATCATAAAAATAA
TAAGTCTTGGATGGAATCTGAATTCAGAGTATATTCTTCTGCTAATAATTGTAC
ATTTGAATATGTATCTCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAA
CTTTAAAAATTTGAGAGAATTTGTTTTTAAAAATATTGATGGATACTTTAAAATC
TATTCTAAACATACTCCAATTAATCTAGTAAGAGATTTGCCTCAAGGATTTTCT
GCTTTAGAACCACTAGTAGATTTGCCTATAGGAATTAATATTACTAGATTTCAA
ACATTATTAGCTTTACATAGATCTTATTTGACACCTGGAGATTCTTCTTCTGGAT
GGACTGCAGGAGCTGCAGCTTATTATGTTGGATATTTGCAACCAAGAACATTTT
TGTTAAAATATAATGAAAATGGAACTATAACAGATGCAGTTGATTGTGCTTTAG
ATCCTCTATCTGAAACTAAATGTACTTTAAAATCTTTTACTGTAGAAAAAGGAA
TCTATCAAACATCTAACTTTAGAGTACAACCAACTGAATCTATTGTTAGATTTCC
AAATATAACAAATCTATGTCCTTTTGGAGAAGTTTTTAATGCAACTAGATTTGC
TTCTGTATATGCATGGAATAGAAAAAGAATATCTAATTGCGTAGCTGATTATTC
TGTATTATATAATTCTGCATCTTTTTCTACTTTTAAATGTTATGGAGTATCTCCA
ACAAAATTGAATGATCTATGTTTTACTAATGTTTATGCAGATTCTTTTGTAATAA
GAGGAGATGAAGTTAGACAAATAGCTCCTGGACAAACAGGAAAAATAGCAGAT
TATAATTATAAATTACCAGATGATTTCACTGGATGCGTAATTGCTTGGAATTCT
AATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTTGTATAGATTGTTT
AGAAAATCTAATTTGAAACCTTTTGAAAGAGATATTTCTACAGAAATCTATCAA
GCAGGATCTACTCCATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACTA
CAATCTTATGGATTTCAACCTACAAATGGAGTAGGATATCAACCATATAGAGTA
GTTGTATTATCTTTTGAATTATTACATGCACCAGCTACAGTATGTGGACCTAAA
AAATCTACTAATTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGATTA
ACTGGAACAGGAGTTTTAACTGAATCTAATAAGAAATTTTTGCCTTTTCAACAA
TTTGGAAGAGATATTGCTGATACTACAGATGCAGTAAGAGATCCTCAAACTTTA
GAAATATTGGATATTACACCATGTTCTTTTGGAGGAGTTTCTGTAATAACACCA
GGAACTAATACATCTAATCAAGTTGCTGTATTATATCAAGATGTTAATTGTACT
GAAGTTCCTGTAGCAATTCATGCTGATCAATTAACTCCAACATGGAGAGTATAT
TCTACTGGATCTAATGTTTTTCAAACAAGAGCTGGATGTCTAATTGGAGCAGAA
CATGTAAATAATTCTTATGAATGTGATATTCCTATAGGAGCTGGAATATGTGCA
TCTTATCAAACTCAAACAAATTCTCCAAGAAGAGCTAGATCTGTTGCATCTCAA
TCTATAATTGCTTATACAATGTCTTTAGGAGCTGAAAATTCTGTAGCATATTCTA
ATAATTCTATTGCAATTCCTACTAACTTTACTATTTCTGTAACTACAGAAATATT
GCCAGTTTCTATGACTAAAACATCTGTAGATTGTACAATGTATATATGTGGAGA
TTCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTTTTTGTACTCAATTG
AATAGAGCTTTAACAGGAATAGCAGTAGAACAAGATAAAAATACACAAGAAGT
TTTTGCTCAAGTAAAACAAATCTATAAAACTCCACCTATAAAAGATTTTGGAGG
TTTTAATTTTTCTCAAATATTGCCAGATCCTTCTAAACCTTCTAAAAGATCTTTT
ATTGAAGATTTGTTGTTTAATAAGGTTACATTAGCAGATGCTGGTTTTATAAAA
CAATATGGAGATTGTTTAGGAGATATTGCAGCTAGAGATTTGATTTGTGCTCAA
AAGTTTAATGGATTAACTGTATTACCACCTCTACTAACAGATGAAATGATAGCA
CAATATACATCTGCATTATTAGCTGGAACTATTACATCTGGATGGACTTTTGGA
GCTGGAGCAGCTTTACAAATACCATTTGCTATGCAAATGGCATATAGATTCAAT
GGAATTGGAGTTACTCAAAATGTATTATATGAAAATCAAAAACTAATTGCTAAT
CAATTCAATTCTGCAATTGGAAAAATTCAAGATTCTCTATCTTCTACAGCATCT
GCTTTAGGAAAACTACAAGATGTTGTAAATCAAAATGCACAAGCTTTAAATACT
CTAGTTAAACAACTATCTTCTAATTTTGGAGCTATTTCTTCTGTTTTAAATGATA
TATTGTCTAGACTAGATCCACCTGAAGCAGAAGTACAAATTGATAGACTAATTA
CAGGAAGATTACAATCTCTACAAACTTATGTAACACAACAACTAATTAGAGCAG
CTGAAATAAGAGCATCTGCTAATTTGGCAGCTACTAAAATGTCTGAATGCGTAT
TAGGACAATCTAAAAGAGTAGATTTTTGTGGAAAAGGATATCATTTGATGTCTT
TTCCACAATCTGCTCCTCATGGAGTAGTATTTTTGCATGTTACATATGTACCTG
CACAAGAAAAGAACTTTACTACAGCACCAGCTATATGTCATGATGGAAAAGCTC
ATTTTCCTAGAGAAGGAGTTTTTGTATCTAATGGAACTCATTGGTTTGTTACAC
AAAGAAACTTTTATGAACCACAAATTATAACTACAGATAATACATTTGTATCTG
GAAATTGTGATGTTGTAATTGGAATTGTTAATAATACTGTATATGATCCACTAC
AACCTGAACTAGATTCTTTTAAAGAAGAACTAGATAAATACTTTAAAAATCATA
CTTCTCCTGATGTTGATTTGGGAGATATATCTGGAATTAATGCTTCTGTTGTAA
ATATTCAAAAAGAAATAGATAGATTGAATGAAGTAGCAAAAAATTTGAATGAAT
CTCTAATTGATTTGCAAGAATTAGGAAAATATGAACAATATATCAAATGGCCAT
TTATGTTATGTTGTATGACATCTTGTTGTTCTTGTCTAAAAGGATGTTGTTCTTG
TGGATCTTGTTGTAAATTTGATGAAGATGATTCTGAACCTGTTTTGAAAGGTGT
TAAACTACATTATACTGGATCTGGAGCAACTAATTTTTCTTTGTTAAAACAAGC
TGGAGATGTAGAAGAAAATCCAGGACCTATGGCTGATTCTAATGGAACTATAA
CAGTTGAAGAATTGAAAAAACTATTAGAACAATGGAATTTGGTAATAGGATTTT
TGTTTTTAACATGGATTTGTTTATTACAATTTGCATATGCTAATAGAAATAGATT
TTTGTATATCATAAAACTAATATTTTTGTGGTTATTATGGCCAGTTACTTTAGCA
TGTTTTGTTTTAGCAGCTGTATATAGAATTAATTGGATTACAGGAGGAATTGCA
ATAGCTATGGCATGTCTAGTAGGATTAATGTGGCTATCTTACTTTATAGCATCT
TTTAGACTATTTGCTAGAACTAGATCTATGTGGTCTTTTAATCCTGAAACAAAT
ATATTGTTAAATGTACCATTACATGGAACTATATTGACAAGACCTCTACTAGAA
TCTGAATTAGTTATTGGAGCAGTAATATTAAGAGGACATTTGAGAATTGCTGGA
CATCATTTGGGAAGATGTGATATCAAAGATTTGCCTAAAGAAATTACTGTTGCT
ACATCTAGAACTTTATCTTATTATAAACTAGGAGCATCTCAAAGAGTAGCTGGA
GATTCTGGATTTGCAGCTTATTCTAGATATAGAATTGGAAATTATAAATTGAAT
ACTGATCATTCTTCTTCTTCTGATAATATTGCATTATTAGTACAAGGATCTGGA
GCTACAAATTTTTCTTTGTTAAAACAGGCAGGAGATGTTGAAGAAAATCCAGGA
CCAATGTATTCTTTTGTATCTGAAGAAACTGGAACATTAATTGTTAATTCTGTAT
TATTGTTTTTAGCTTTTGTAGTATTTTTGCTAGTTACATTAGCAATATTGACTGC
TTTAAGATTATGTGCATATTGTTGTAATATTGTTAATGTATCTTTAGTAAAACCA
TCTTTTTATGTATATTCAAGAGTTAAAAATCTAAATTCATCAAGAGTTCCTGATC
TATTGGTATAATAATTTTTATGGATCCTCTAGAGTCGACCTGCAGTCAAACTCTAAT
Plasmid Sequence (SEQ ID NO: 7) and Sequence encoding spike protein (bold, SEQ ID NO: 8)
TCTAGTTTTGCTACCGTTGGTTTCAAGTCAATGTGTAAATCTAACTACAAGAAC
TCAATTACCGCCTGCCTATACTAATTCTTTTACAAGAGGAGTATATTATCCTGA
TAAAGTTTTTAGATCTTCTGTATTACATTCTACACAAGATTTGTTTTTACCATTT
TTCTCTAATGTTACTTGGTTTCATGCAATACATGTATCTGGAACTAATGGAACA
AAAAGATTTGATAATCCAGTATTACCTTTTAATGATGGAGTTTATTTTGCTTCTA
CTGAAAAATCTAATATAATTAGAGGATGGATATTTGGAACTACATTAGATTCTA
AAACACAATCTCTACTAATTGTTAATAATGCAACTAATGTAGTTATAAAAGTAT
GTGAATTTCAATTTTGTAATGATCCATTTTTGGGAGTTTATTATCATAAAAATAA
TAAGTCTTGGATGGAATCTGAATTCAGAGTATATTCTTCTGCTAATAATTGTAC
ATTTGAATATGTATCTCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAA
CTTTAAAAATTTGAGAGAATTTGTTTTTAAAAATATTGATGGATACTTTAAAATC
TATTCTAAACATACTCCAATTAATCTAGTAAGAGATTTGCCTCAAGGATTTTCT
GCTTTAGAACCACTAGTAGATTTGCCTATAGGAATTAATATTACTAGATTTCAA
ACATTATTAGCTTTACATAGATCTTATTTGACACCTGGAGATTCTTCTTCTGGAT
GGACTGCAGGAGCTGCAGCTTATTATGTTGGATATTTGCAACCAAGAACATTTT
TGTTAAAATATAATGAAAATGGAACTATAACAGATGCAGTTGATTGTGCTTTAG
ATCCTCTATCTGAAACTAAATGTACTTTAAAATCTTTTACTGTAGAAAAAGGAA
TCTATCAAACATCTAACTTTAGAGTACAACCAACTGAATCTATTGTTAGATTTCC
AAATATAACAAATCTATGTCCTTTTGGAGAAGTTTTTAATGCAACTAGATTTGC
TTCTGTATATGCATGGAATAGAAAAAGAATATCTAATTGCGTAGCTGATTATTC
TGTATTATATAATTCTGCATCTTTTTCTACTTTTAAATGTTATGGAGTATCTCCA
ACAAAATTGAATGATCTATGTTTTACTAATGTTTATGCAGATTCTTTTGTAATAA
GAGGAGATGAAGTTAGACAAATAGCTCCTGGACAAACAGGAAAAATAGCAGAT
TATAATTATAAATTACCAGATGATTTCACTGGATGCGTAATTGCTTGGAATTCT
AATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTTGTATAGATTGTTT
AGAAAATCTAATTTGAAACCTTTTGAAAGAGATATTTCTACAGAAATCTATCAA
GCAGGATCTACTCCATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACTA
CAATCTTATGGATTTCAACCTACAAATGGAGTAGGATATCAACCATATAGAGTA
GTTGTATTATCTTTTGAATTATTACATGCACCAGCTACAGTATGTGGACCTAAA
AAATCTACTAATTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGATTA
ACTGGAACAGGAGTTTTAACTGAATCTAATAAGAAATTTTTGCCTTTTCAACAA
TTTGGAAGAGATATTGCTGATACTACAGATGCAGTAAGAGATCCTCAAACTTTA
GAAATATTGGATATTACACCATGTTCTTTTGGAGGAGTTTCTGTAATAACACCA
GGAACTAATACATCTAATCAAGTTGCTGTATTATATCAAGATGTTAATTGTACT
GAAGTTCCTGTAGCAATTCATGCTGATCAATTAACTCCAACATGGAGAGTATAT
TCTACTGGATCTAATGTTTTTCAAACAAGAGCTGGATGTCTAATTGGAGCAGAA
CATGTAAATAATTCTTATGAATGTGATATTCCTATAGGAGCTGGAATATGTGCA
TCTTATCAAACTCAAACAAATTCTCCAAGAAGAGCTAGATCTGTTGCATCTCAA
TCTATAATTGCTTATACAATGTCTTTAGGAGCTGAAAATTCTGTAGCATATTCTA
ATAATTCTATTGCAATTCCTACTAACTTTACTATTTCTGTAACTACAGAAATATT
GCCAGTTTCTATGACTAAAACATCTGTAGATTGTACAATGTATATATGTGGAGA
TTCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTTTTTGTACTCAATTG
AATAGAGCTTTAACAGGAATAGCAGTAGAACAAGATAAAAATACACAAGAAGT
TTTTGCTCAAGTAAAACAAATCTATAAAACTCCACCTATAAAAGATTTTGGAGG
TTTTAATTTTTCTCAAATATTGCCAGATCCTTCTAAACCTTCTAAAAGATCTTTT
ATTGAAGATTTGTTGTTTAATAAGGTTACATTAGCAGATGCTGGTTTTATAAAA
CAATATGGAGATTGTTTAGGAGATATTGCAGCTAGAGATTTGATTTGTGCTCAA
AAGTTTAATGGATTAACTGTATTACCACCTCTACTAACAGATGAAATGATAGCA
CAATATACATCTGCATTATTAGCTGGAACTATTACATCTGGATGGACTTTTGGA
GCTGGAGCAGCTTTACAAATACCATTTGCTATGCAAATGGCATATAGATTCAAT
GGAATTGGAGTTACTCAAAATGTATTATATGAAAATCAAAAACTAATTGCTAAT
CAATTCAATTCTGCAATTGGAAAAATTCAAGATTCTCTATCTTCTACAGCATCT
GCTTTAGGAAAACTACAAGATGTTGTAAATCAAAATGCACAAGCTTTAAATACT
CTAGTTAAACAACTATCTTCTAATTTTGGAGCTATTTCTTCTGTTTTAAATGATA
TATTGTCTAGACTAGATCCACCTGAAGCAGAAGTACAAATTGATAGACTAATTA
CAGGAAGATTACAATCTCTACAAACTTATGTAACACAACAACTAATTAGAGCAG
CTGAAATAAGAGCATCTGCTAATTTGGCAGCTACTAAAATGTCTGAATGCGTAT
TAGGACAATCTAAAAGAGTAGATTTTTGTGGAAAAGGATATCATTTGATGTCTT
TTCCACAATCTGCTCCTCATGGAGTAGTATTTTTGCATGTTACATATGTACCTG
CACAAGAAAAGAACTTTACTACAGCACCAGCTATATGTCATGATGGAAAAGCTC
ATTTTCCTAGAGAAGGAGTTTTTGTATCTAATGGAACTCATTGGTTTGTTACAC
AAAGAAACTTTTATGAACCACAAATTATAACTACAGATAATACATTTGTATCTG
GAAATTGTGATGTTGTAATTGGAATTGTTAATAATACTGTATATGATCCACTAC
AACCTGAACTAGATTCTTTTAAAGAAGAACTAGATAAATACTTTAAAAATCATA
CTTCTCCTGATGTTGATTTGGGAGATATATCTGGAATTAATGCTTCTGTTGTAA
ATATTCAAAAAGAAATAGATAGATTGAATGAAGTAGCAAAAAATTTGAATGAAT
CTCTAATTGATTTGCAAGAATTAGGAAAATATGAACAATATATCAAATGGCCAT
GGTATATTTGGCTAGGTTTTATAGCTGGATTAATAGCAATTGTTATGGTAACTA
TTATGTTATGTTGTATGACATCTTGTTGTTCTTGTCTAAAAGGATGTTGTTCTTG
TGGATCTTGTTGTAAATTTGATGAAGATGATTCTGAACCTGTTTTGAAAGGTGT
TAAACTACATTATACTTAATAATTTTTATGGATCCTCTAGAGTCGACCTGCAGTCAA
Plasmid Sequence (SEQ ID NO: 9) and Sequence encoding spike protein (bold, SEQ ID NO: 10)
TACAAGGACTACTATTACTAGGTACTGTTGCCTGTTCAAT
TTCACAATGTGTAAATCTAACTACAAGAACTCAATTACCG
CCTGCCTATACTAATTCTTTTACAAGAGGAGTATATTATC
CTGATAAAGTTTTTAGATCTTCTGTATTACATTCTACACA
AGATTTGTTTTTACCATTTTTCTCTAATGTTACTTGGTTT
CATGCAATACATGTATCTGGAACTAATGGAACAAAAAGAT
TTGATAATCCAGTATTACCTTTTAATGATGGAGTTTATTT
TGCTTCTACTGAAAAATCTAATATAATTAGAGGATGGATA
TTTGGAACTACATTAGATTCTAAAACACAATCTCTACTAA
TTGTTAATAATGCAACTAATGTAGTTATAAAAGTATGTGA
ATTTCAATTTTGTAATGATCCATTTTTGGGAGTTTATTAT
CATAAAAATAATAAGTCTTGGATGGAATCTGAATTCAGAG
TATATTCTTCTGCTAATAATTGTACATTTGAATATGTATC
TCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAAC
TTTAAAAATTTGAGAGAATTTGTTTTTAAAAATATTGATG
GATACTTTAAAATCTATTCTAAACATACTCCAATTAATCT
AGTAAGAGATTTGCCTCAAGGATTTTCTGCTTTAGAACCA
CTAGTAGATTTGCCTATAGGAATTAATATTACTAGATTTC
AAACATTATTAGCTTTACATAGATCTTATTTGACACCTGG
AGATTCTTCTTCTGGATGGACTGCAGGAGCTGCAGCTTAT
TATGTTGGATATTTGCAACCAAGAACATTTTTGTTAAAAT
ATAATGAAAATGGAACTATAACAGATGCAGTTGATTGTGC
TTTAGATCCTCTATCTGAAACTAAATGTACTTTAAAATCT
TTTACTGTAGAAAAAGGAATCTATCAAACATCTAACTTTA
GAGTACAACCAACTGAATCTATTGTTAGATTTCCAAATAT
AACAAATCTATGTCCTTTTGGAGAAGTTTTTAATGCAACT
AGATTTGCTTCTGTATATGCATGGAATAGAAAAAGAATAT
CTAATTGCGTAGCTGATTATTCTGTATTATATAATTCTGC
ATCTTTTTCTACTTTTAAATGTTATGGAGTATCTCCAACA
AAATTGAATGATCTATGTTTTACTAATGTTTATGCAGATT
CTTTTGTAATAAGAGGAGATGAAGTTAGACAAATAGCTCC
TGGACAAACAGGAAAAATAGCAGATTATAATTATAAATTA
CCAGATGATTTCACTGGATGCGTAATTGCTTGGAATTCTA
ATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTT
GTATAGATTGTTTAGAAAATCTAATTTGAAACCTTTTGAA
AGAGATATTTCTACAGAAATCTATCAAGCAGGATCTACTC
CATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACT
ACAATCTTATGGATTTCAACCTACAAATGGAGTAGGATAT
CAACCATATAGAGTAGTTGTATTATCTTTTGAATTATTAC
ATGCACCAGCTACAGTATGTGGACCTAAAAAATCTACTAA
TTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGA
TTAACTGGAACAGGAGTTTTAACTGAATCTAATAAGAAAT
TTTTGCCTTTTCAACAATTTGGAAGAGATATTGCTGATAC
TACAGATGCAGTAAGAGATCCTCAAACTTTAGAAATATTG
GATATTACACCATGTTCTTTTGGAGGAGTTTCTGTAATAA
CACCAGGAACTAATACATCTAATCAAGTTGCTGTATTATA
TCAAGATGTTAATTGTACTGAAGTTCCTGTAGCAATTCAT
GCTGATCAATTAACTCCAACATGGAGAGTATATTCTACTG
GATCTAATGTTTTTCAAACAAGAGCTGGATGTCTAATTGG
AGCAGAACATGTAAATAATTCTTATGAATGTGATATTCCT
ATAGGAGCTGGAATATGTGCATCTTATCAAACTCAAACAA
ATTCTCCAAGAAGAGCTAGATCTGTTGCATCTCAATCTAT
AATTGCTTATACAATGTCTTTAGGAGCTGAAAATTCTGTA
GCATATTCTAATAATTCTATTGCAATTCCTACTAACTTTA
CTATTTCTGTAACTACAGAAATATTGCCAGTTTCTATGAC
TAAAACATCTGTAGATTGTACAATGTATATATGTGGAGAT
TCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTT
TTTGTACTCAATTGAATAGAGCTTTAACAGGAATAGCAGT
AGAACAAGATAAAAATACACAAGAAGTTTTTGCTCAAGTA
AAACAAATCTATAAAACTCCACCTATAAAAGATTTTGGAG
GTTTTAATTTTTCTCAAATATTGCCAGATCCTTCTAAACC
TTCTAAAAGATCTTTTATTGAAGATTTGTTGTTTAATAAG
GTTACATTAGCAGATGCTGGTTTTATAAAACAATATGGAG
ATTGTTTAGGAGATATTGCAGCTAGAGATTTGATTTGTGC
TCAAAAGTTTAATGGATTAACTGTATTACCACCTCTACTA
ACAGATGAAATGATAGCACAATATACATCTGCATTATTAG
CTGGAACTATTACATCTGGATGGACTTTTGGAGCTGGAGC
AGCTTTACAAATACCATTTGCTATGCAAATGGCATATAGA
TTCAATGGAATTGGAGTTACTCAAAATGTATTATATGAAA
ATCAAAAACTAATTGCTAATCAATTCAATTCTGCAATTGG
AAAAATTCAAGATTCTCTATCTTCTACAGCATCTGCTTTA
GGAAAACTACAAGATGTTGTAAATCAAAATGCACAAGCTT
TAAATACTCTAGTTAAACAACTATCTTCTAATTTTGGAGC
TATTTCTTCTGTTTTAAATGATATATTGTCTAGACTAGAT
CCACCTGAAGCAGAAGTACAAATTGATAGACTAATTACAG
GAAGATTACAATCTCTACAAACTTATGTAACACAACAACT
AATTAGAGCAGCTGAAATAAGAGCATCTGCTAATTTGGCA
GCTACTAAAATGTCTGAATGCGTATTAGGACAATCTAAAA
GAGTAGATTTTTGTGGAAAAGGATATCATTTGATGTCTTT
TCCACAATCTGCTCCTCATGGAGTAGTATTTTTGCATGTT
ACATATGTACCTGCACAAGAAAAGAACTTTACTACAGCAC
CAGCTATATGTCATGATGGAAAAGCTCATTTTCCTAGAGA
AGGAGTTTTTGTATCTAATGGAACTCATTGGTTTGTTACA
CAAAGAAACTTTTATGAACCACAAATTATAACTACAGATA
ATACATTTGTATCTGGAAATTGTGATGTTGTAATTGGAAT
TGTTAATAATACTGTATATGATCCACTACAACCTGAACTA
GATTCTTTTAAAGAAGAACTAGATAAATACTTTAAAAATC
ATACTTCTCCTGATGTTGATTTGGGAGATATATCTGGAAT
TAATGCTTCTGTTGTAAATATTCAAAAAGAAATAGATAGA
TTGAATGAAGTAGCAAAAAATTTGAATGAATCTCTAATTG
ATTTGCAAGAATTAGGAAAATATGAACAAGGATCTGCTGG
ATATATTCCAGAAGCACCTAGAGATGGACAAGCGTATGTT
AGAAAAGATGGTGAATGGGTATTATTGAGTACATTTTTGT
AATAATTTTTATGGATCCTCTAGAGTCGACCTGCAGTCAA
Plasmid Sequence (SEQ ID NO: 11) and Sequence encoding spike protein (bold, SEQ ID NO: 12)
TACAAGGACTACTATTACTAGGTACTGTTGCCTGTTCAAT
TTCACAATGTGTAAATCTAACTACAAGAACTCAATTACCG
CCTGCCTATACTAATTCTTTTACAAGAGGAGTATATTATC
CTGATAAAGTTTTTAGATCTTCTGTATTACATTCTACACA
AGATTTGTTTTTACCATTTTTCTCTAATGTTACTTGGTTT
CATGCAATACATGTATCTGGAACTAATGGAACAAAAAGAT
TTGATAATCCAGTATTACCTTTTAATGATGGAGTTTATTT
TGCTTCTACTGAAAAATCTAATATAATTAGAGGATGGATA
TTTGGAACTACATTAGATTCTAAAACACAATCTCTACTAA
TTGTTAATAATGCAACTAATGTAGTTATAAAAGTATGTGA
ATTTCAATTTTGTAATGATCCATTTTTGGGAGTTTATTAT
CATAAAAATAATAAGTCTTGGATGGAATCTGAATTCAGAG
TATATTCTTCTGCTAATAATTGTACATTTGAATATGTATC
TCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAAC
TTTAAAAATTTGAGAGAATTTGTTTTTAAAAATATTGATG
GATACTTTAAAATCTATTCTAAACATACTCCAATTAATCT
AGTAAGAGATTTGCCTCAAGGATTTTCTGCTTTAGAACCA
CTAGTAGATTTGCCTATAGGAATTAATATTACTAGATTTC
AAACATTATTAGCTTTACATAGATCTTATTTGACACCTGG
AGATTCTTCTTCTGGATGGACTGCAGGAGCTGCAGCTTAT
TATGTTGGATATTTGCAACCAAGAACATTTTTGTTAAAAT
ATAATGAAAATGGAACTATAACAGATGCAGTTGATTGTGC
TTTAGATCCTCTATCTGAAACTAAATGTACTTTAAAATCT
TTTACTGTAGAAAAAGGAATCTATCAAACATCTAACTTTA
GAGTACAACCAACTGAATCTATTGTTAGATTTCCAAATAT
AACAAATCTATGTCCTTTTGGAGAAGTTTTTAATGCAACT
AGATTTGCTTCTGTATATGCATGGAATAGAAAAAGAATAT
CTAATTGCGTAGCTGATTATTCTGTATTATATAATTCTGC
ATCTTTTTCTACTTTTAAATGTTATGGAGTATCTCCAACA
AAATTGAATGATCTATGTTTTACTAATGTTTATGCAGATT
CTTTTGTAATAAGAGGAGATGAAGTTAGACAAATAGCTCC
TGGACAAACAGGAAAAATAGCAGATTATAATTATAAATTA
CCAGATGATTTCACTGGATGCGTAATTGCTTGGAATTCTA
ATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTT
GTATAGATTGTTTAGAAAATCTAATTTGAAACCTTTTGAA
AGAGATATTTCTACAGAAATCTATCAAGCAGGATCTACTC
CATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACT
ACAATCTTATGGATTTCAACCTACAAATGGAGTAGGATAT
CAACCATATAGAGTAGTTGTATTATCTTTTGAATTATTAC
ATGCACCAGCTACAGTATGTGGACCTAAAAAATCTACTAA
TTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGA
TTAACTGGAACAGGAGTTTTAACTGAATCTAATAAGAAAT
TTTTGCCTTTTCAACAATTTGGAAGAGATATTGCTGATAC
TACAGATGCAGTAAGAGATCCTCAAACTTTAGAAATATTG
GATATTACACCATGTTCTTTTGGAGGAGTTTCTGTAATAA
CACCAGGAACTAATACATCTAATCAAGTTGCTGTATTATA
TCAAGATGTTAATTGTACTGAAGTTCCTGTAGCAATTCAT
GCTGATCAATTAACTCCAACATGGAGAGTATATTCTACTG
GATCTAATGTTTTTCAAACAAGAGCTGGATGTCTAATTGG
AGCAGAACATGTAAATAATTCTTATGAATGTGATATTCCT
ATAGGAGCTGGAATATGTGCATCTTATCAAACTCAAACAA
ATTCTCCAAGAAGAGCTAGATCTGTTGCATCTCAATCTAT
AATTGCTTATACAATGTCTTTAGGAGCTGAAAATTCTGTA
GCATATTCTAATAATTCTATTGCAATTCCTACTAACTTTA
CTATTTCTGTAACTACAGAAATATTGCCAGTTTCTATGAC
TAAAACATCTGTAGATTGTACAATGTATATATGTGGAGAT
TCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTT
TTTGTACTCAATTGAATAGAGCTTTAACAGGAATAGCAGT
AGAACAAGATAAAAATACACAAGAATAATAATTTTTATGG
Four DNA vaccines are made using the coronavirus spike protein as shown in
pGA8-nCoV S-VLP Plasmid sequences (SEQ ID NO: 13) and Sequence encoding spike protein (bold, SEQ ID NO: 14)
GTTTGTCTTCCTGGTCCTGCTGCCTCTGGTCTCCTCACAGTGCGTCAATCTGAC
TACCCGAACTCAGCTGCCCCCCGCCTACACCAACTCCTTCACCCGGGGCGTGT
ACTATCCAGACAAGGTGTTTAGAAGCTCCGTGCTGCACTCCACCCAGGATCTGT
TTCTGCCCTTCTTTTCTAATGTGACATGGTTCCACGCCATCCACGTGAGCGGCA
CCAACGGCACAAAGAGGTTCGACAACCCTGTGCTGCCATTCAATGATGGCGTG
TACTTTGCCTCCACCGAGAAGTCTAACATCATCCGCGGCTGGATCTTTGGCACC
ACACTGGACTCCAAGACCCAGTCCCTGCTGATCGTGAACAATGCCACAAACGT
GGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAACGATCCTTTCCTGGGCGTGTA
CTATCACAAGAACAATAAGTCTTGGATGGAGAGCGAGTTTAGGGTGTATTCTA
GCGCCAACAATTGCACCTTCGAGTACGTGTCCCAGCCATTTCTGATGGACCTG
GAGGGCAAGCAGGGCAATTTCAAGAACCTGCGGGAGTTCGTGTTTAAGAACAT
CGACGGCTACTTCAAGATCTACTCCAAGCACACCCCCATCAACCTGGTGCGGG
ACCTGCCACAGGGCTTCTCTGCCCTGGAGCCTCTGGTGGATCTGCCAATCGGC
ATCAACATCACACGGTTTCAGACCCTGCTGGCCCTGCACAGAAGCTACCTGAC
CCCTGGCGACTCCTCTAGCGGATGGACAGCAGGAGCAGCAGCATACTATGTGG
GCTATCTGCAGCCACGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATC
ACAGACGCCGTGGATTGCGCCCTGGATCCACTGTCTGAGACAAAGTGTACACT
GAAGAGCTTTACAGTGGAGAAGGGCATCTATCAGACCAGCAACTTCAGGGTGC
AGCCCACAGAGTCCATCGTGCGCTTTCCAAATATCACCAACCTGTGCCCCTTCG
GCGAGGTGTTTAATGCCACAAGATTCGCCAGCGTGTACGCCTGGAACAGGAAG
CGCATCTCCAATTGCGTGGCCGACTATTCTGTGCTGTACAACTCTGCCAGCTTC
TCCACCTTTAAGTGCTATGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTC
ACAAACGTGTACGCCGATTCCTTTGTGATCAGGGGCGACGAGGTGCGCCAGAT
CGCACCAGGACAGACCGGCAAGATCGCAGACTACAACTATAAGCTGCCCGACG
ATTTCACAGGCTGCGTGATCGCCTGGAATTCCAACAATCTGGATTCTAAAGTGG
GCGGCAACTACAATTATCTGTACAGGCTGTTCCGCAAGTCTAACCTGAAGCCTT
TTGAGCGGGACATCTCCACCGAGATCTACCAGGCCGGCTCTACACCATGCAAC
GGCGTGGAGGGCTTCAATTGTTATTTTCCCCTGCAGAGCTACGGCTTCCAGCCT
ACCAATGGCGTGGGCTATCAGCCATACAGAGTGGTGGTGCTGTCTTTTGAGCT
GCTGCACGCACCAGCAACCGTGTGCGGACCTAAGAAGAGCACAAATCTGGTGA
AGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGAACAGGCGTGCTG
ACCGAGTCCAACAAGAAGTTCCTGCCCTTTCAGCAGTTCGGCAGGGACATCGC
AGATACCACAGACGCCGTGCGCGACCCCCAGACACTGGAGATCCTGGATATCA
AACCAGGTGGCCGTGCTGTATCAGGACGTGAACTGTACAGAGGTGCCAGTGGC
CATCCACGCCGATCAGCTGACCCCCACATGGCGGGTGTACTCCACAGGCTCTA
ACGTGTTCCAGACCAGAGCAGGATGCCTGATCGGAGCAGAGCACGTGAACAAT
AGCTATGAGTGCGACATCCCCATCGGCGCCGGCATCTGTGCCTCCTACCAGAC
CCAGACAAACTCCCCTCGGAGAGCCAGGTCTGTGGCCTCTCAGAGCATCATCG
CCTATACCATGAGCCTGGGCGCCGAGAACTCCGTGGCCTACAGCAACAATTCC
ATCGCCATCCCCACCAATTTCACAATCTCCGTGACCACAGAGATCCTGCCCGTG
AGCATGACCAAGACAAGCGTGGACTGCACCATGTATATCTGTGGCGATTCCAC
AGAGTGCTCTAATCTGCTGCTGCAGTACGGCTCTTTTTGTACACAGCTGAACCG
CGCCCTGACCGGAATCGCAGTGGAGCAGGACAAGAATACCCAGGAGGTGTTCG
CCCAGGTGAAGCAGATCTACAAGACACCCCCTATCAAGGACTTTGGCGGCTTC
AACTTTAGCCAGATCCTGCCTGATCCATCTAAGCCTAGCAAGAGGTCCTTCATC
GAGGACCTGCTGTTTAATAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCA
GTATGGCGATTGCCTGGGCGACATCGCAGCACGCGACCTGATCTGTGCCCAGA
AGTTTAACGGCCTGACAGTGCTGCCACCCCTGCTGACCGATGAGATGATCGCA
CAGTACACCTCTGCCCTGCTGGCCGGAACCATCACAAGCGGATGGACATTCGG
CGCAGGAGCCGCCCTGCAGATCCCATTCGCCATGCAGATGGCCTATCGGTTTA
ATGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAATCAGAAGCTGATCGCC
AATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACTCTCTGTCCTCTACCGCC
AGCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAA
CACACTGGTGAAGCAGCTGAGCTCCAATTTCGGCGCCATCTCTAGCGTGCTGA
ACGATATCCTGAGCAGGCTGGACAAGGTGGAGGCCGAGGTGCAGATCGACCG
GCTGATCACCGGCAGACTGCAGTCCCTGCAGACCTACGTGACACAGCAGCTGA
TCAGGGCAGCAGAGATCAGGGCCTCTGCCAACCTGGCAGCAACAAAGATGAGC
GAGTGCGTGCTGGGACAGTCCAAGAGGGTGGACTTTTGTGGCAAGGGCTATCA
CCTGATGAGCTTCCCACAGTCCGCCCCACACGGAGTGGTGTTTCTGCACGTGA
CCTACGTGCCTGCCCAGGAGAAGAATTTCACCACAGCCCCAGCCATCTGCCAC
GATGGCAAGGCACACTTCCCAAGGGAGGGCGTGTTTGTGAGCAATGGCACACA
CTGGTTCGTGACCCAGAGAAACTTTTACGAGCCTCAGATCATCACCACAGACAA
CACCTTCGTGAGCGGCAATTGTGACGTGGTCATCGGCATCGTGAACAATACAG
TGTATGATCCCCTGCAGCCTGAGCTGGACTCTTTCAAGGAGGAGCTGGATAAG
TACTTTAAGAACCACACCAGCCCCGACGTGGATCTGGGCGACATCTCCGGCAT
CAACGCCTCTGTGGTGAATATCCAGAAGGAGATCGACAGACTGAATGAGGTGG
CCAAGAATCTGAACGAGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTATGAG
CAGTACATCAAGTGGCCATGGTATATCTGGCTGGGCTTCATCGCCGGCCTGAT
CGCCATCGTGATGGTGACAATCATGCTGTGCTGTATGACCTCTTGCTGTAGCTG
CCTGAAGGGCTGCTGTTCCTGTGGCTCTTGCTGTAAGTTCGATGAGGACGATT
CCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTACACAGGCTCCGGCGCCACC
AACTTTTCTCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAACCCAGGACCTAT
GGCCGATAGCAATGGCACCATCACAGTGGAGGAGCTGAAGAAGCTGCTGGAGC
AGTGGAACCTGGTCATCGGCTTCCTGTTTCTGACCTGGATCTGCCTGCTGCAGT
TCGCCTATGCCAATCGGAACAGATTTCTGTACATCATCAAGCTGATCTTCCTGT
GGCTGCTGTGGCCAGTGACCCTGGCCTGCTTCGTGCTGGCCGCCGTGTATCGG
ATCAACTGGATCACAGGCGGCATCGCCATCGCCATGGCCTGTCTGGTGGGCCT
GATGTGGCTGAGCTACTTCATCGCCTCCTTTAGACTGTTCGCCAGGACCCGCA
GCATGTGGTCCTTTAATCCCGAGACAAACATCCTGCTGAATGTGCCTCTGCACG
GCACCATCCTGACAAGGCCACTGCTGGAGTCCGAGCTGGTCATCGGAGCCGTG
ATCCTGAGGGGACACCTGAGAATCGCAGGACACCACCTGGGCCGCTGCGATAT
CAAGGACCTGCCTAAGGAGATCACCGTGGCCACATCTAGGACCCTGAGCTACT
ATAAGCTGGGAGCCAGCCAGAGGGTGGCAGGCGACAGCGGATTCGCAGCATA
TTCCCGGTACAGAATCGGCAACTACAAGCTGAATACCGATCACTCCTCTAGCTC
CGACAATATCGCCCTGCTGGTGCAGGGATCTGGAGCAACAAACTTCAGCCTGC
TGAAGCAGGCCGGCGATGTGGAAGAAAACCCAGGACCCATGTATTCTTTTGTG
AGCGAGGAGACAGGCACACTGATCGTGAATAGCGTGCTGCTGTTTCTGGCCTT
CGTGGTGTTTCTGCTGGTGACACTGGCCATCCTGACCGCCCTGAGACTGTGCG
CCTACTGCTGTAATATCGTGAACGTGTCTCTGGTGAAGCCCAGCTTTTACGTGT
ATAGTAGGGTGAAGAATCTGAACTCAAGTAGGGTGCCCGATCTGCTGGTCTAA
pGA8-nCoV-S-Tri Plasmid sequences (SEQ ID NO: 15) and Sequence encoding spike protein (bold, SEQ ID NO: 16)
GTTCGTGTTTCTGGTCTTGCTGCCCCTGGTGTCCAGCCAGTGCGTCAACCTGAC
AACCAGAACCCAACTGCCCCCAGCCTACACCAACTCCTTCACAAGAGGCGTGT
ATTACCCTGACAAGGTGTTTCGGAGCAGCGTGCTGCACTCCACCCAGGACTTG
TTTCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGC
ACCAATGGAACCAAGAGATTCGACAATCCTGTGCTCCCCTTCAACGACGGCGT
CTACTTCGCCAGCACCGAAAAGTCTAACATCATCAGGGGCTGGATCTTCGGCA
CAACACTGGACAGCAAGACCCAGTCCCTGCTGATTGTGAACAACGCCACAAAT
GTGGTGATCAAGGTGTGCGAATTCCAGTTTTGCAACGATCCCTTTTTGGGCGTG
TATTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAATTCCGGGTGTACAG
CAGCGCCAACAACTGTACCTTCGAATACGTGAGCCAGCCTTTCCTGATGGACCT
GGAAGGCAAACAGGGCAACTTCAAGAACCTGCGGGAATTCGTGTTCAAGAACA
TCGACGGGTACTTCAAGATCTACTCTAAGCACACCCCTATCAACCTGGTCAGAG
ACCTGCCTCAAGGCTTTAGCGCCCTGGAACCTCTGGTGGACCTGCCGATCGGC
ATTAACATCACCAGATTCCAGACACTGCTGGCTCTGCACAGATCCTACCTGACC
CCTGGCGATAGCTCCAGCGGCTGGACCGCCGGAGCTGCTGCTTACTACGTGGG
CTACCTGCAACCAAGAACCTTTCTGCTGAAGTACAACGAAAACGGCACCATCAC
AGACGCCGTGGACTGCGCCCTGGATCCTCTCAGCGAGACAAAGTGTACCCTCA
AGTCGTTCACCGTGGAAAAGGGCATATACCAGACCTCTAACTTCAGAGTGCAG
CCTACAGAGAGCATCGTAAGATTCCCTAACATCACCAACCTCTGTCCCTTTGGC
GAGGTTTTCAACGCCACCAGATTCGCCAGCGTATACGCCTGGAACAGAAAGAG
AATCTCCAATTGCGTGGCCGACTACAGCGTGCTGTACAATTCTGCATCTTTTAG
CACATTCAAATGCTACGGCGTGTCCCCAACCAAGCTAAACGACCTGTGCTTCAC
CAACGTCTACGCCGACTCATTTGTGATTCGGGGCGACGAAGTGCGCCAGATCG
CCCCTGGCCAGACCGGCAAAATCGCCGATTACAACTACAAGCTGCCAGATGAC
TTCACCGGCTGTGTGATCGCCTGGAACAGCAATAATCTGGACAGCAAGGTTGG
AGGAAACTACAACTACCTGTATCGGCTGTTCAGAAAGAGCAACCTGAAGCCTTT
CGAGCGGGACATCAGTACAGAGATCTACCAGGCTGGCTCCACGCCATGCAATG
GCGTGGAGGGCTTCAACTGCTACTTCCCCCTGCAGAGCTATGGCTTCCAGCCC
ACAAACGGCGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCT
GCTTCATGCCCCTGCTACAGTCTGCGGCCCTAAGAAAAGCACCAATCTGGTGA
AAAATAAATGCGTGAACTTCAACTTTAACGGCCTGACCGGAACTGGAGTCCTTA
CCGAGAGCAACAAGAAGTTCCTGCCTTTTCAGCAGTTCGGAAGAGATATCGCC
GACACCACCGATGCCGTGCGGGATCCCCAGACCCTGGAGATCCTGGATATCAC
CCCCTGCAGCTTCGGCGGCGTGTCTGTGATCACGCCCGGCACCAACACCAGCA
ACCAGGTGGCCGTTCTGTACCAGGATGTGAATTGCACCGAGGTGCCTGTGGCC
ATCCACGCCGATCAGCTGACACCCACCTGGCGGGTGTATAGCACCGGATCTAA
TGTGTTCCAGACAAGAGCCGGATGTCTGATCGGAGCCGAACACGTGAACAATA
GCTACGAGTGTGACATCCCTATCGGCGCCGGAATCTGCGCCAGCTACCAAACA
CAGACTAACAGCCCTCGGAGAGCCAGAAGCGTGGCCTCTCAGTCAATCATCGC
CTACACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAACAACAGCA
TCGCGATTCCTACCAACTTTACCATCAGCGTTACGACAGAGATCCTGCCTGTGA
GCATGACCAAAACCTCCGTGGACTGCACAATGTACATCTGCGGCGACAGCACC
GAGTGCAGCAACCTGCTGCTGCAATACGGAAGCTTCTGCACCCAGCTGAATCG
GGCCCTGACCGGCATCGCCGTTGAACAGGACAAGAACACTCAGGAGGTGTTTG
CCCAGGTCAAGCAGATATACAAGACCCCTCCTATCAAGGACTTCGGCGGATTT
AACTTTTCTCAGATCCTGCCTGACCCCAGCAAACCTTCCAAAAGAAGCTTCATC
GAAGACCTGCTGTTCAACAAGGTGACACTCGCCGACGCCGGATTTATCAAGCA
GTACGGCGATTGCCTGGGAGACATCGCCGCTAGAGATCTGATCTGCGCCCAAA
AATTCAACGGCCTGACAGTGCTGCCTCCACTGCTGACAGACGAGATGATCGCC
CAATACACCTCTGCCCTGCTGGCCGGAACCATCACAAGCGGCTGGACCTTCGG
CGCCGGCGCAGCCCTGCAAATCCCCTTCGCCATGCAGATGGCTTATAGATTCA
ATGGCATCGGCGTCACACAGAACGTGCTGTACGAGAATCAGAAGCTGATCGCC
AACCAGTTCAACTCTGCTATCGGCAAAATCCAGGATTCACTAAGCAGCACCGCC
TCAGCCCTGGGCAAACTGCAGGATGTGGTTAATCAGAATGCCCAGGCCCTGAA
CACACTGGTGAAGCAACTGTCCAGCAATTTCGGGGCTATTAGCAGTGTGCTGA
ACGATATCCTGAGTAGGCTGGATCCACCTGAGGCCGAAGTGCAGATCGACCGG
CTCATCACAGGGAGACTGCAGTCCCTGCAGACCTACGTGACCCAGCAGCTCAT
CAGAGCTGCTGAGATACGGGCCTCTGCTAATCTGGCCGCTACCAAAATGAGCG
AGTGCGTGCTGGGCCAGTCTAAGCGGGTAGATTTCTGCGGCAAGGGCTATCAC
CTGATGAGCTTCCCACAGAGCGCTCCGCACGGCGTAGTGTTCTTACATGTGAC
ATACGTCCCTGCCCAGGAGAAGAACTTCACCACAGCTCCTGCCATCTGTCACG
ATGGCAAGGCCCACTTCCCCAGAGAGGGCGTGTTCGTGTCCAACGGCACCCAC
TGGTTCGTGACGCAGCGGAACTTCTACGAGCCTCAGATTATCACAACAGACAA
CACCTTCGTGTCTGGAAATTGCGACGTTGTCATCGGCATCGTCAACAACACCGT
GTACGACCCACTGCAGCCTGAGCTGGACAGCTTCAAGGAAGAGCTGGACAAGT
ACTTCAAGAACCACACCAGCCCCGATGTGGACCTGGGCGACATCAGCGGAATC
AACGCCTCTGTGGTGAACATCCAGAAGGAAATCGACAGACTGAACGAGGTGGC
CAAGAACCTGAATGAGTCACTTATTGACCTGCAGGAACTGGGCAAATACGAAC
AGTACATCAAATGGCCCTGGTACATCTGGCTGGGATTCATCGCTGGCCTGATC
GCCATCGTGATGGTGACAATCATGCTGTGTTGCATGACATCTTGTTGTAGCTGC
CTGAAGGGCTGCTGTAGCTGTGGCTCTTGTTGCAAGTTCGACGAGGACGACAG
CGAGCCCGTGCTCAAGGGAGTGAAGCTGCACTATACCTAAACCATGATATTCGG
pGA8-nCoV S-Tri-sec Plasmid sequences (SEQ ID NO: 17) and Sequence encoding spike protein (bold, SEQ ID NO: 18)
GTGGCTGCAGGGCCTGCTGCTGCTGGGCACCGTGGCATGCAGTATCAGCCAAT
GTGTGAACCTGACCACCAGAACCCAACTGCCTCCTGCCTACACCAACTCTTTCA
CCAGAGGCGTGTACTACCCTGACAAGGTGTTCAGAAGCAGCGTGCTGCATTCT
ACCCAGGACCTGTTTCTGCCATTCTTCAGCAACGTCACCTGGTTCCACGCCATC
CACGTGTCTGGCACCAATGGCACTAAGAGATTCGACAACCCCGTGCTGCCTTT
CAACGACGGCGTGTACTTTGCCTCAACTGAGAAGAGCAACATCATCAGAGGAT
GGATCTTCGGCACCACACTTGACTCAAAGACACAGTCACTGCTGATCGTGAAC
AATGCTACCAATGTGGTGATCAAGGTGTGTGAATTCCAGTTTTGCAACGATCCT
TTCCTGGGTGTATACTACCACAAGAACAACAAGTCTTGGATGGAGAGCGAGTT
CCGGGTGTATAGTAGCGCCAACAACTGCACCTTCGAATACGTGAGCCAGCCTT
TCCTCATGGACCTGGAAGGCAAGCAAGGCAACTTCAAGAACCTGAGAGAGTTC
GTGTTTAAGAACATTGATGGCTACTTCAAGATCTACAGCAAGCACACCCCCATC
AACCTGGTGCGGGACCTCCCTCAGGGCTTCAGCGCCCTGGAACCCTTGGTTGA
TCTGCCAATTGGCATCAATATCACTCGGTTCCAAACCCTGCTGGCCCTGCACAG
AAGCTATCTGACACCTGGAGACAGCAGCAGCGGCTGGACCGCCGGAGCCGCC
GCCTACTACGTGGGCTACCTGCAGCCCCGGACCTTCCTGCTGAAGTACAACGA
GAACGGGACCATTACCGACGCCGTCGACTGCGCCCTGGATCCTCTGAGCGAAA
CCAAGTGCACACTTAAAAGCTTCACAGTGGAGAAGGGCATCTACCAAACCTCC
AATTTTCGGGTCCAGCCAACCGAGAGCATCGTTAGATTCCCCAACATCACCAAC
TTGTGCCCCTTCGGAGAAGTGTTCAACGCCACAAGATTCGCCAGCGTCTACGC
CTGGAACAGAAAGAGAATTTCCAATTGCGTCGCAGACTACTCTGTGCTGTACAA
CAGCGCCAGCTTTTCTACATTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGA
ACGACCTATGCTTCACAAACGTGTACGCCGACAGCTTTGTGATCCGGGGCGAC
GAGGTGCGCCAGATCGCGCCAGGACAGACCGGTAAGATCGCCGATTACAATTA
CAAACTGCCTGACGACTTCACCGGCTGCGTCATCGCTTGGAACAGCAACAACC
TGGACTCTAAGGTGGGCGGAAACTACAACTACCTGTACCGGCTGTTTAGAAAG
AGCAACCTGAAGCCTTTTGAACGGGACATCTCTACAGAGATCTACCAGGCCGG
ATCTACCCCTTGTAATGGCGTGGAGGGCTTTAATTGCTACTTCCCCCTGCAATC
GTACGGCTTCCAGCCGACAAACGGCGTCGGCTACCAGCCTTACAGAGTGGTGG
TCCTGTCCTTCGAGCTGCTGCATGCCCCTGCTACAGTGTGCGGCCCTAAGAAA
AGCACCAACCTGGTGAAGAACAAGTGTGTGAACTTCAATTTCAATGGCCTGACT
GGCACCGGAGTGCTGACCGAATCCAACAAGAAGTTCCTGCCCTTCCAGCAGTT
CGGCAGAGACATCGCAGACACTACCGATGCTGTGCGGGATCCTCAGACACTGG
AGATCCTGGATATCACCCCCTGCAGCTTCGGAGGCGTGAGCGTGATCACACCT
GGCACAAACACATCCAACCAGGTGGCCGTGCTGTACCAGGATGTGAACTGCAC
ACTCCACAGGCAGCAACGTGTTCCAAACCAGAGCTGGCTGTCTGATCGGCGCT
GAACACGTGAACAATAGCTATGAGTGCGACATCCCAATCGGCGCCGGTATCTG
CGCCTCCTATCAGACGCAGACGAACAGCCCTAGGCGGGCTAGAAGCGTGGCCA
GCCAGAGCATCATCGCATATACAATGAGCCTGGGCGCCGAAAACTCTGTCGCC
TACAGCAACAACAGCATCGCTATCCCTACCAACTTCACCATAAGCGTAACAACC
GAGATCCTGCCTGTGTCCATGACAAAGACCAGCGTGGACTGTACAATGTACAT
CTGTGGCGACTCCACCGAGTGCAGCAACCTGCTCCTGCAATACGGCTCTTTCT
GCACCCAGCTGAATCGCGCCTTAACAGGCATTGCCGTGGAACAGGATAAGAAC
ACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTATAAGACCCCACCCATCAA
GGACTTCGGCGGATTCAATTTCAGTCAAATCCTGCCCGATCCTAGCAAGCCCA
GTAAGAGATCTTTCATCGAGGACCTGCTTTTCAACAAAGTGACCCTGGCGGAC
GCCGGATTTATCAAACAGTACGGCGACTGTCTGGGCGACATCGCCGCTAGAGA
TCTGATCTGCGCCCAGAAATTCAACGGCCTGACGGTGCTGCCTCCTCTGCTGA
CAGATGAGATGATCGCCCAGTATACCAGCGCCCTGCTGGCTGGAACCATCACC
TCTGGCTGGACATTTGGCGCCGGTGCCGCTCTCCAGATCCCCTTTGCCATGCA
GATGGCCTACAGATTCAATGGAATCGGCGTGACCCAGAACGTGCTGTACGAGA
ACCAGAAGCTGATCGCTAATCAGTTCAACTCTGCCATTGGCAAAATCCAGGACA
GCCTGTCTTCCACCGCCAGCGCCCTGGGCAAACTGCAAGACGTGGTGAATCAA
AACGCCCAGGCCCTGAACACTCTGGTGAAGCAGCTGTCCAGCAACTTCGGAGC
CATCAGCAGCGTGCTGAACGACATACTGAGCAGACTGGACCCTCCGGAGGCCG
AGGTGCAGATCGACAGGCTGATCACAGGCAGACTGCAGAGCCTGCAGACCTAC
GTCACACAGCAGCTGATCAGAGCCGCTGAGATCCGAGCTTCTGCCAATCTCGC
CGCGACAAAGATGTCTGAGTGCGTGCTCGGCCAGAGCAAAAGAGTGGATTTCT
GCGGAAAAGGCTATCACCTGATGAGCTTCCCTCAGTCTGCCCCACACGGCGTC
GTGTTCCTGCACGTGACCTACGTGCCTGCCCAGGAAAAAAACTTTACCACCGC
CCCGGCCATCTGCCACGACGGCAAGGCCCACTTCCCTAGAGAAGGCGTGTTCG
TGAGCAATGGCACCCACTGGTTCGTGACACAAAGAAACTTCTACGAGCCTCAA
ATCATCACAACAGATAACACCTTCGTGTCAGGCAACTGTGACGTGGTCATCGG
AATCGTGAATAATACCGTGTACGACCCCCTGCAGCCTGAACTGGACAGCTTTAA
GGAGGAACTGGACAAGTACTTCAAAAACCACACATCTCCTGATGTGGACCTGG
GGGATATCAGCGGCATCAACGCTTCTGTGGTGAACATCCAGAAGGAAATCGAC
AGACTGAACGAGGTGGCCAAGAATCTCAACGAAAGCCTCATTGACCTTCAGGA
GCTGGGGAAGTACGAGCAGGGCTCTGCCGGCTACATCCCTGAGGCTCCTAGGG
ACGGCCAGGCCTACGTGCGGAAGGACGGGGAGTGGGTGCTGCTGAGCACATT
CCTGTAAACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCT
pGA8-nCoV GMCSF-S1 Plasmid sequences (SEQ ID NO: 19) and Sequence encoding spike protein (bold, SEQ ID NO: 20)
AACCGTGGCTTGCTCCATTTCTCAGTGCGTCAATC
TGACTACCCGAACTCAGCTGCCCCCCGCCTACACC
AACTCCTTCACCCGGGGCGTGTACTATCCAGACAA
GGTGTTTAGAAGCTCCGTGCTGCACTCCACCCAGG
ATCTGTTTCTGCCCTTCTTTTCTAATGTGACATGG
TTCCACGCCATCCACGTGAGCGGCACCAACGGCAC
AAAGAGGTTCGACAACCCTGTGCTGCCATTCAATG
ATGGCGTGTACTTTGCCTCCACCGAGAAGTCTAAC
ATCATCCGCGGCTGGATCTTTGGCACCACACTGGA
CTCCAAGACCCAGTCCCTGCTGATCGTGAACAATG
CCACAAACGTGGTCATCAAGGTGTGCGAGTTCCAG
TTTTGTAACGATCCTTTCCTGGGCGTGTACTATCA
CAAGAACAATAAGTCTTGGATGGAGAGCGAGTTTA
GGGTGTATTCTAGCGCCAACAATTGCACCTTCGAG
TACGTGTCCCAGCCATTTCTGATGGACCTGGAGGG
CAAGCAGGGCAATTTCAAGAACCTGCGGGAGTTCG
TGTTTAAGAACATCGACGGCTACTTCAAGATCTAC
TCCAAGCACACCCCCATCAACCTGGTGCGGGACCT
GCCACAGGGCTTCTCTGCCCTGGAGCCTCTGGTGG
ATCTGCCAATCGGCATCAACATCACACGGTTTCAG
ACCCTGCTGGCCCTGCACAGAAGCTACCTGACCCC
TGGCGACTCCTCTAGCGGATGGACAGCAGGAGCAG
CAGCATACTATGTGGGCTATCTGCAGCCACGGACC
TTCCTGCTGAAGTACAACGAGAATGGCACCATCAC
AGACGCCGTGGATTGCGCCCTGGATCCACTGTCTG
AGACAAAGTGTACACTGAAGAGCTTTACAGTGGAG
AAGGGCATCTATCAGACCAGCAACTTCAGGGTGCA
GCCCACAGAGTCCATCGTGCGCTTTCCAAATATCA
CCAACCTGTGCCCCTTCGGCGAGGTGTTTAATGCC
ACAAGATTCGCCAGCGTGTACGCCTGGAACAGGAA
GCGCATCTCCAATTGCGTGGCCGACTATTCTGTGC
TGTACAACTCTGCCAGCTTCTCCACCTTTAAGTGC
TATGGCGTGAGCCCCACCAAGCTGAACGACCTGTG
CTTCACAAACGTGTACGCCGATTCCTTTGTGATCA
GGGGCGACGAGGTGCGCCAGATCGCACCAGGACAG
ACCGGCAAGATCGCAGACTACAACTATAAGCTGCC
CGACGATTTCACAGGCTGCGTGATCGCCTGGAATT
CCAACAATCTGGATTCTAAAGTGGGCGGCAACTAC
AATTATCTGTACAGGCTGTTCCGCAAGTCTAACCT
GAAGCCTTTTGAGCGGGACATCTCCACCGAGATCT
ACCAGGCCGGCTCTACACCATGCAACGGCGTGGAG
GGCTTCAATTGTTATTTTCCCCTGCAGAGCTACGG
CTTCCAGCCTACCAATGGCGTGGGCTATCAGCCAT
ACAGAGTGGTGGTGCTGTCTTTTGAGCTGCTGCAC
GCACCAGCAACCGTGTGCGGACCTAAGAAGAGCAC
AAATCTGGTGAAGAACAAGTGCGTGAACTTCAACT
TCAACGGCCTGACCGGAACAGGCGTGCTGACCGAG
TCCAACAAGAAGTTCCTGCCCTTTCAGCAGTTCGG
CAGGGACATCGCAGATACCACAGACGCCGTGCGCG
ACCCCCAGACACTGGAGATCCTGGATATCACCCCT
TGCAGCTTCGGCGGCGTGTCCGTGATCACCCCTGG
AACCAATACAAGCAACCAGGTGGCCGTGCTGTATC
AGGACGTGAACTGTACAGAGGTGCCAGTGGCCATC
CACGCCGATCAGCTGACCCCCACATGGCGGGTGTA
CTCCACAGGCTCTAACGTGTTCCAGACCAGAGCAG
GATGCCTGATCGGAGCAGAGCACGTGAACAATAGC
TATGAGTGCGACATCCCCATCGGCGCCGGCATCTG
TGCCTCCTACCAGACCCAGACAAACTCCCCTCGGA
GAGCCAGGTCTGTGGCCTCTCAGAGCATCATCGCC
TATACCATGAGCCTGGGCGCCGAGAACTCCGTGGC
CTACAGCAACAATTCCATCGCCATCCCCACCAATT
TCACAATCTCCGTGACCACAGAGATCCTGCCCGTG
AGCATGACCAAGACAAGCGTGGACTGCACCATGTA
TATCTGTGGCGATTCCACAGAGTGCTCTAATCTGC
TGCTGCAGTACGGCTCTTTTTGTACACAGCTGAAC
CGCGCCCTGACCGGAATCGCAGTGGAGCAGGACAA
GAATACCCAGGAGTAAACCATGATATTCGGCAAGC
DNA based T cell chimera antigens we have designed encoding proteins derived from 6 genes of SARS-CoV-2 i.e., Spike, N, M, NSP3, NSP4 and NSP6. These proteins have been chosen because: 1) they show strong conservation between multiple human betacoronaviruses; and 2) they account for greater than 90% of the CoV2 specific T cell response observed in SARS-CoV-2 infected individuals. Chimera 1 carry immunodominant T cell epitopes of spike glycoprotein (S), nucleocapsid (N) and membrane (M) proteins. The other construct has regions derived from non-structural regions, expressed during virus active replication and translation. This includes non-structural protein 3 (NSP3), NSP4, and NSP6. about 66% of the N terminal region of the NSP3 protein was deleted since this region contains peptide sequences and functional domains which can disrupt the process of epitope processing and presentation. These regions include nucleic acid binding domains, viral proteinase activity domains and autophagy modulating domains. However, the remaining C-terminal region contains three immunodominant CD8 T cell epitopes that are conserved in SARS-CoV and CoV2. Versions of each construct lacking transmembrane (TM) regions were developed providing the four chimera constructs (d/delta is deleted): Chimera 1 (SdRBD-N-M) and 3 (NSP3-4-6), and the chimeras lacking TM, Chimera 2 (SdRBD-N-M_dTM) and 4 (NSP3-4-6_dTM), respectively.
The RBD region (major target of neutralization) was deleted from the S protein to avoid antibody response to this region induced by improperly folded chimeric protein which could interfere with the neutralizing antibody responses induced by properly folded RBD protein immunogen. Two versions for each construct are provided, one with and the other without the transmembrane regions from S, M, Nsp3, Nsp4 and Nsp6 proteins in order to compare their ability to induce T cell responses. The chimeric proteins without transmembrane regions are expected to be localized to the cytoplasm and will be susceptible to degradation by proteasomes. This could potentially promote class I HLA epitope presentation to generate CD8 T cell response. In addition, these chimeric proteins expressed as fusion proteins and do not have secretory signals facilitating the priming of T cell response as opposed to antibody response with the idea that the expressed chimeric proteins may not retain the proper conformation to generate a neutralizing antibody response. These chimeric immunogens that are designed to induce a broad CD4 and CD8 T cell response with cross-reactivity to other coronaviruses by use in combination with the other DNA prime/MVA boost strategies reported herein providing an improved T cell response.
Modified vaccinia Ankara (MVA) based vaccines were developed one expressing the full-length spike protein (MVA/S) that is designed to be stabilized in prefusion state and anchored on the membrane of MVA infected cells, and the other expressing the S1 region of the spike (MVA/S1) that forms trimers and is secreted (
Modified vaccinia Ankara (MVA) is a highly attenuated strain of vaccinia virus. There are several advantages to MVA based vaccines. MVA can accommodate large inserts (>10 kb) that will allow expression of multiple antigens in a single vector. MVA recombinants are quite stable and can be produced at high titer that makes vaccine manufacture feasible. MVA vaccines also induce strong CD4 and CD8 T cell responses that will be important for protection against viral infections. MVA vaccination can provide protection against multiple virus infections including SARS-CoV, MERS, Zika and Ebola viruses.
MVA recombinants, one expressing the full-length spike protein (MVA/S) that is anchored on the membrane of MVA infected cells and the other expressing the S1 portion of the spike (MVA/S1) that is secreted were developed. Both constructs contained the RBD that is the prime target for neutralizing antibody response. The MVA/S also incorporated two mutations that have been shown to keep spike in a prefusion confirmation. These two recombinants were tested in mice for their ability to generate neutralizing antibody response.
MVA vaccines expressing either the full length prefusion stabilized spike or secreted Si demonstrated that while both immunogens induce strong binding antibody response to spike only the former induces a strong neutralizing antibody response against the SARS-CoV-2. The failure of MVA/S1 immunogen to induce neutralizing activity was associated with its failure to induce antibody to RBD. This was surprising given the fact that RBD is part of S1. Binding to ACE-2 revealed that S1 presents RBD in the proper confirmation at cold temperature however the stability of RBD confirmation seems to change markedly at the room temperature. This instability of S1 protein seems to contribute to induction of strong binding antibody to other regions in S1 other than RBD following immunization. Systemic MVA vaccination also induced T cell and antibody responses in the lung that will be critical for protection against respiratory infections such as SARS-CoV-2. A dose of about 108 pfu (between 107 and 109) is contemplated for human vaccination. Collectively these results demonstrate that MVA/S is a promising vaccine for SARS-CoV-2.
The full-length spike protein sequences of the SARS-CoV-2 strain was obtained from GenBank (Accession number QHD43416.1) and generated various forms of antigens for the improved immunization responses in our vaccination studies. These antigens were expressed using Modified Vaccinia Ankara (MVA) vectors. SARS-CoV-2 full-length spike (S) (aa 1 to 1273) has site-specific mutations introduced at K986P, and V987P for better stabilization and whereas, S1-mono, aa 14 to 780 of spike protein were fused at N-terminus with 16 aa long granulocyte-macrophage colony-stimulating factor (GM-C SF) signal sequences for better secretions. Inserts of rMVA were subcloned in between Xmal and BamHI restriction sites of the pLW-73 transfer vectors, to transfer the inserts into deletion III site. These inserts express under the control of an independent early/late vaccinia virus promoter (modified H5 [mH5]).
For MVA/S, the 3821-nt ORF (GenBank accession #MN996527.1_30-Dec-2019 China: Wuhan) encoding the SARS-nCoV Spike gene was codon optimized for vaccinia virus expression, and cloned into pLW-73 using the Xmal and BamH1 sites under the control of the vaccinia virus modified H5 early late promoter and adjacent to the gene encoding enhanced GFP regulated by the vaccinia virus P11 late promoter. Similarly, to develop MVA/S1, spike secreted monomeric form, GMCSF signal sequence followed with Spike DNA sequence of 14-780 AA was synthesized and cloned between Xmal and BamH1 sites of pLW-73 vector as described above. These plasmid DNAs were subsequently used to generate recombinant MVAs by transfecting transfer plasmids into DF-1 cells that were infected with 0.05 plaque forming units (pfu) of MVA per cell into the essential region of MVA 1974 strain between genes I8R and G1L. Recombinant MVA (rMVA) was isolated using standard methods, but sorting was used during the first round of selection using green fluorescent protein (GFP). Each round GFP plaque picked were characterized for the expression using anti SARS-CoV-2 spike antibody to detect cell surface spike protein expression of MVA/S. For MVA/S1, anti SARS-CoV-2 RBD antibody was used to stain intracellularly. Plaques were picked after 7 rounds to obtain GFP-negative rMVA/S, rMVA/S1 and spike DNA sequences were confirmed. The recombinants were characterized for spike expression by flow cytometry and Western blotting. Viral stocks were purified from lysates of infected DF1cells using a 36% sucrose cushion and titrated using DF-1 cells by counting pfu/ml. Absence of wildtype MVA was confirmed by PCR using recombinant specific primers of flanking sequences with rMVA/S and rMVA/S1 infected cellular DNA isolated from DF-1 cells. Absence of 542 bp (essential region) band indicates there is no wild type reverted MVAs in the preps.
To develop the MVA recombinants the full-length spike gene (amino acids 1-1273) was synthesized with stabilizing mutations (K986P, V987P) or just the S1 region with a small portion of S2 region (amino acids 14 to 780). To promote active secretion of the S 1, the first 14 amino acids of the spike sequence were replaced with the signal sequence from GM-CSF (
Both MVA/S and MVA/S1 Vaccines Induce a Strong Binding Antibody Response but with Different Specificities
Balb/c mice were immunized with MVA/S or MVA/S1 on weeks 0 and 4, and measured binding antibody responses to total and different parts of spike i.e. RBD, S1, and S (S) using ELISA at 2 weeks post prime and boost. While both vaccines induced a strong binding antibody response to S, they differentially targeted binding to RBD and S1. The MVA/S sera showed strong binding to RBD whereas MVA/S1 sera showed strong binding to S1. This was interesting considering that S1 protein includes complete RBD and suggested that binding activity in MVA/S1 sera may be targeting regions other than RBD in S1. Luminex assay were performed using sera obtained from 3 weeks post boost to measure binding to different parts of S including S2, and to determine the antibody subclass and their ability to bind different soluble FcgRs. These analyses revealed that antibody responses in MVA/S group binding equally to RBD, S1 and S2 whereas in MVA/S1 group the antibody bound primarily to S1 but not to RBD and S2. While the lack of binding to S2 is expected, poor binding to RBD was not expected. Analysis of IgG subclass and FcgR binding of RBD-specific antibody showed strong IgG2a response (Th1 biased) and binding to all three FcRs tested with strongest binding to FcR2 and FcR4 in the MVA/S group. In contrast, poor binding of RBD-specific antibody was observed in general with MVA/S1 sera. However, the S1-specific antibody showed similar results in both groups. These results demonstrated differential targeting of spike specific antibody with Th1 profile induced by MVA/S and MVA/S1 vaccines.
Experiments were performed to determine if vaccination induced immune responses in the lung, a primary site of SARS-CoV-2 virus exposure. The formation of bronchus-associated lymphoid tissue (BALT) was measure using the immunohistochemistry at 3 weeks after the MVA boost by staining for B and T cells. The naïve mice showed very little or no BALT, however, the MVA vaccinated mice showed significant induction of BALT indicating the generation of local lymphoid tissue (
Neutralization against the SARS-CoV-2 virus was tested using the FRNT-GFP assay using sera from 2 weeks post boost. Impressively, a strong neutralizing activity was observed with sera from mice vaccinated with MVA/S that ranged from 20-900 with a median of 200 (
SARS-CoV-2 S1 Exhibits Lower Affinity to ACE2 than RBD, which Further Weakens upon Incubation at 25° C.
To further understand the failure of MVA/S1 vaccine to induce strong RBD binding antibody and neutralizing antibody, we purified the S1 trimer protein expressed by MVA/S1 vaccine and determined its ability to bind to human ACE-2 using biolayer interferometry (BLI). Purified RBD protein was used as a benchmark. SARS-CoV-2 S1 bound to hu-ACE2 quite strongly but at 2-fold lower affinity than RBD (KD=70.1 nM and 36 nM respectively). S1 exhibited 10-fold lower association rate than RBD (kon(1/Ms) 1.1E+04 and 1.3E+05 respectively). However, the affinity of S1-ACE2 further decreased by 5-fold when the protein was incubated at 25° C. for 60min. In contrary, RBD was stable and retained its affinity (KD=24 nM). The data indicated the receptor binding domain of S1 to be unstable, thereby loosing association with ACE2 protein upon prolonged incubation at room temperature, unlike RBD. A 10-fold reduction in the association rate for S1-ACE2 was observed, compared to RBD which was meagerly affected.
RBD-Binding IgG Antibody Titers for Assaying Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Candidate having a Mutant Furin Cleavage Site (MVA/S-Tri-dFCS) in BALB/c Mice.
A mutation of the furin cleavage site was introduced in order to stabilize the expressed proteins of the MVA vaccines, i.e., sequence encoding RRAR was altered to produce FCS mutation—SRAG. MVA/S-tri and MVA/S-tri-dFCS recombinants were expressed as membrane anchored spike protein variants (MVA/S-tri and MVA/S-tri-dFCS) on the surface which was confirmed by flow cytometry and western blot analysis bind studies of hACE2 to MVA/S-tri and MVA/S-tri-dFCS expressing infected cells.
Female BALB/c mice were intramuscularly (i.m.) immunized on wk0 and wk4 with either MVA/S-tri (107 PFU) or MVA/S-tri-dFCS (107 PFU) (
Two MVA based vaccines which express either a membrane anchored full-length spike protein (MVA/S) stabilized in a prefusion state or the soluble secreted trimeric S1 of the spike (MVA/S1). Both immunogens contained the receptor-binding domain (RBD) which is a known target of antibody-mediated neutralization in SARS-CoV-2 infected individuals. The MVA/S also incorporated two mutations that maintain the spike protein in a prefusion confirmation.
Vaccination of rhesus macaques followed by SARS-CoV-2 challenge demonstrated MVA/S vaccine induces neutralizing antibodies and CD8 T cells and protects from SARS-CoV-2 infection and replication in the lung.
The MVA recombinants expressing the full-length spike (amino acids 1-1273) carrying the prefusion-stabilized mutations (MVA/S) or only S1 portion of spike (amino acids 14-780)(MVA-S1) were generated and confirmed by standard methods. SARS-CoV-2 (MN996527.1; Wuhan strain) S ORF was codon optimized for vaccinia virus expression, synthesized, and cloned into pLW-73 between the Xmal and BamHI sites under the control of the vaccinia virus modified H5 (mH5) early late promoter and adjacent to the gene encoding enhanced GFP (green fluorescent protein). To promote active secretion of the S1, amino acids 1-14 of the spike sequence were replaced with the signal sequence from GMCSF, SEQ ID NO: 31 (WLQGLLLLGTVACSIS). Plaques were picked for 7 rounds to obtain GFP-negative recombinants and DNA sequenced to confirm lack of any mutations. Viral stocks were purified from lysates of infected DF-1 cells using a 36% sucrose cushion and titrated using DF-1 cells by counting pfu/ml. Absence of the wildtype MVA was confirmed by PCR using recombinant specific primers, flanking the inserts.
Ten adult male rhesus macaques (Macaca mulatta), 4-5 years old, were randomly allocated into two groups; one group (n=5) received MVA empty vector (MVA-wt) and the second group (n=5) received MVA-expressing prefusion stabilized (with proline mutations) SARS-CoV-2 full-length spike protein (MVA-S). Animals received 1×108 pfu in 1 ml vaccines at week 0 and week 4 by the intramuscular (IM) route.
In addition to the neutralizing activity, the vaccine induced sera showed strong antibody dependent complement deposition (ADCD) activity and low antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent neutrophil phagocytosis (ADNP) activities. The MVA/S vaccine also generated a strong spike-specific IFNγ+CD8 T cell response that was evident as early as one week post priming immunization. The frequency of CD8 T cell response was not further boosted following the 2nd MVA/S immunization. The vaccine-induced CD8 T cells were also positive for TNFα and IL-2 and negative for IL-17. The MVA/S vaccine induced very low frequencies of IFNγ+CD4 T cells. These data demonstrated that MVA/S vaccinations induced a poly-functional CD8 T cell response capable of producing IFNγ, IL-2 and TNFα in macaques.
Following vaccination, all macaques were challenged with SARS-CoV-2 at week 8 by intratracheal (IT) and intranasal (IN) route. MVA/S vaccinated animals rapidly controlled SARS-CoV-2 replication in the lung at Day 2 (p<0.05) and Day 4 (p<0.05) compared to controls with 4 of the 5 vaccinated animals being negative in BAL. However, in the throat, all vaccinated animals tested negative at Day 2 (p<0.01) but low titer of virus replication was evident in one or two vaccinated animals on Days 4 and 7. Similarly, in nasopharynx one or two animals showed virus replication on Days 2, 4 and 7 and the virus replication was not significantly different between controls and vaccinated animals at all time points. By Day 10 all control and vaccinated animals were negative in all compartments. These results demonstrated that MVA/S vaccination provides protection from SARS-CoV2 infection or replication in the lower respiratory tract. Virus replication, lung pathology, binding and neutralizing antibody titer and T cell responses were measured. Data indicates MVA/S vaccine protects from SARS-CoV-2 infection and replication and reduces lung pathology in rhesus macaques.
In order to define protection offered by MVA vaccine expressing Spike and nucleocapsid (NC) against SARS-CoV-2 South African variant (B.1.351) one can immunize rhesus macaques with a double recombinant MVA/S-tri-dFCS-NC on weeks 0 and 4, and challenge with B.1.351. One can assess the protective immune responses generated by the vaccine by measuring antibody and T cell responses in blood and mucosal secretions following vaccination. Animals can be challenged with SARS-CoV-2 virus intranasally and intratracheally to determine vaccine protection. One can collect blood, bone marrow, LN biopsies, BAL, rectal biopsies, rectal swabs, nasal and salivary/oral swabs at multiple times during vaccination and challenge.
Vaccines were injected via intramuscular (IM) route at weeks 0 and 4. Serum collected at week 6 (peak) were used to asses neutralizing antibody titers against live Washington SARS-CoV-2, and variants of concern—UK variants, 501Y.V1, VOC 202012/01 (B.1.1.7) and South African variants (B.1.351), and Fold-Change in neutralization titers between WA virus to the variants of concern are presented. Each sample was analyzed in duplicates and repeated twice and repeated twice and GMT values for each vaccination groups were presented in table.
MVA/S study, n=5 rhesus macaques were immunized with 10{circumflex over ( )}8 pfu/macaques MVA/S-tri vaccine. MVA/S-tri-dFCS study, n=5 BALB/c mice were immunized with 10{circumflex over ( )}7 pfu/mice MVA/S-tri-dFCS vaccine. NT, not tested
BALB/c mice were primed with DNA (50 ug/mice) and boosted with 10{circumflex over ( )}7 pfu/mice with spike expressing vaccine. All the vaccines were injected via intramuscular (IM) route at weeks 0 and 4. Blood collected at week 5 (peak) was used to assess % spike-specific tetramer positive CD8 T cells analyzed using flow cytometry.
This application claims the benefit of U.S. Provisional Application No. 63/012,920 filed Apr. 21, 2020, U.S. Provisional Application No. 63/044,711 filed Jun. 26, 2020, U.S. Provisional Application No. 63/084,065 filed Sep. 28, 2020, and U.S. Provisional Application No. 63/143,191 filed Jan. 29, 2021. The entirety of each of these applications is hereby incorporated by reference for all purposes.
This invention was made with government support under AI148378 awarded by the National Institutes of Health. The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/028444 | 4/21/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63012920 | Apr 2020 | US | |
| 63044711 | Jun 2020 | US | |
| 63084065 | Sep 2020 | US | |
| 63143191 | Jan 2021 | US |
