Patent Application
20210260182
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 26, 2021, is named 104545-0047-101-SL.txt and is 766,062 bytes in size.
On Dec. 31, 2019 the Wuhan Health Commission reported a cluster of atypical pneumonia cases in the city of Wuhan, China. The first patients began experiencing symptoms of illness in mid-December 2019. Clinical isolates were found to contain a novel coronavirus. As of Jan. 28, 2020, there are in excess of 4,500 laboratory-confirmed cases, with >100 known deaths. The novel coronavirus is currently referred to as SARS-CoV-2 or 2019-nCoV and is related to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), although with only approximately 80% similarity at the nucleotide level. Ralph et al. J Infect Dev Ctries. 2020 Jan. 31; 14(1):3-17.
Coronaviruses are enveloped single stranded RNA viruses with positive-sense RNA genomes ranging from 25.5 to ˜32 kb in length. The spherical virus particles range from 70-120 nm in diameter with four structural proteins.
Despite the fact that a much effort is currently being invested into methods of providing vaccines and delivery vectors for SARS-CoV-2, there is still a need to provide additional and improved approaches against this coronavirus.
An aspect of the present disclosure provides a recombinant poxvirus comprising a nucleic acid encoding a SARS-CoV-2 virus protein, methods for producing such viruses and the use of such viruses, for example, as immunogens, in immunogenic formulations against SARS-CoV-2 virus. Another aspect of the present disclosure provides a recombinant synthetic poxvirus comprising a nucleic acid encoding a SARS-CoV-2 virus protein, methods for producing such viruses and the use of such viruses, for example, as immunogens, in immunogenic formulations against SARS-CoV-2 virus. In some embodiments, the synthetic poxviruses are assembled and replicated from chemically synthesized DNA which are safe, reproducible and free of contaminants. Because chemical genome synthesis is not dependent on a natural template, a plethora of structural and functional modifications of the viral genome are possible. Chemical genome synthesis is particularly useful when a natural template is not available for genetic replication or modification by conventional molecular biology methods.
In one aspect, the disclosure relates to recombinant poxviruses comprising a nucleic acid encoding a SARS-CoV-2 virus protein, wherein the SARS-CoV-2 protein is selected from the group consisting of the spike protein (S), the membrane protein (M) and the nucleocapsid protein (N), or combinations of two or more of said proteins.
In another aspect, the disclosure relates to pharmaceutical compositions comprising the recombinant poxviruses of the disclosure.
In another aspect, the disclosure relates to cells infected with the recombinant poxviruses of the disclosure.
In another aspect, the disclosure relates to methods for selecting a cell that expresses a SARS-CoV-2 virus protein, comprising infecting said cell with the recombinant poxvirus of the disclosure and selecting the infected cell expressing said SARS-CoV-2 virus protein.
In another aspect, the disclosure relates to methods of inducing an immune response against a SARS-CoV-2 virus in a subject in need or at risk therefor, comprising administering to said subject an immunologically effective amount of a recombinant poxvirus of the disclosure.
In another aspect, the disclosure relates to methods of generating the recombinant poxviruses of the disclosure, the methods comprising: (a) infecting a host cell with a poxvirus; (b) transfecting the infected cell of step (a) with a nucleic acid encoding a SARS-CoV-2 virus protein to generate a recombinant poxvirus; and (c) selecting a recombinant poxvirus, wherein the nucleic acid encoding a SARS-CoV-2 virus protein is located, upon transfection, in a region of the poxvirus that is not essential for the replication of the poxvirus.
For the purpose of illustrating the disclosure that are shown in the drawings and various embodiment(s) of this disclosure. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities shown in the drawings.
Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art. In case of conflict, the present specification, including definitions, will control.
The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, virology and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, N Y (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, N Y (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).
Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, and chemical analyses.
Throughout this specification and embodiments, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
The term “including” is used to mean “including but not limited to.” “Including” and “including but not limited to” are used interchangeably.
Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.
Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
The articles “a”, “an” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.
Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application. The materials, methods, and examples are illustrative only and not intended to be limiting.
The following terms, unless otherwise indicated, shall be understood to have the following meanings:
The terms “chimeric” or “engineered” or “modified” (e.g., chimeric poxvirus, engineered polypeptide, modified polypeptide, engineered nucleic acid, modified nucleic acid) or grammatical variations thereof are used interchangeably herein to refer to a non-native sequence that has been manipulated to have one or more changes relative a native sequence.
As used herein, the term “essential gene for replication” or “essential region for replication” refers to those gene(s) or region(s) indispensable for the replication of an organism, and therefore are considered a foundation of life. In the context of a virus, a gene or region is considered essential (i.e. has a role in cell culture) if its deletion results in a decrease in virus titer of greater than 10-fold in either a single or multiple step growth curve. Most of the essential genes are thought to encode proteins that maintain a central metabolism, replicate DNA, translate genes into proteins, maintain a basic cellular structure, and mediate transport processes into and out of the cell. Genes involved in virion production, actin tail formation, and extracellular virion release are typically also considered as essential. Two main strategies have been employed to identify essential genes on a genome-wide basis: directed deletion of genes and random mutagenesis using transposons. In the first case, individual genes (or ORFs) are completely deleted from the genome in a systematic way. In random mutagenesis, transposons are randomly inserted in as many positions in a genome as possible, aiming to inactivate the targeted genes. Insertion mutants that are still able to survive or grow are not in essential genes. (Zhang, R., 2009 & Gerdes, S., 2006).
The term “expression cassette” or “transcription unit”, as used herein, defines a nucleic acid sequence region that contains one or more genes to be transcribed. The nucleotide sequences encoding the to be transcribed gene(s), as well as the polynucleotide sequences containing the regulatory elements contained within an expression cassette, are operably linked to each other. The genes are transcribed from a promoter and transcription is terminated by at least one polyadenylation signal. In some embodiments, each of the one or more genes are transcribed from one promoter. In some embodiments, the one or more genes are transcribed from one single promoter. In that case, the different genes are at least transcriptionally linked. More than one protein or product can be transcribed and expressed from each transcription unit (multicistronic transcription unit). Each transcription unit will comprise the regulatory elements necessary for the transcription and translation of any of the selected sequences that are contained within the unit. Each transcription unit may contain the same or different regulatory elements.
“Homologous,” in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,” including proteins from superfamilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. “Homologous” may also refer to a nucleic acid which is native to the virus.
In common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
“Heterologous,” in all its grammatical forms and spelling variations, may refer to a nucleic acid which is non-native to the virus. It means derived from a different species or a different strain than the nucleic acid of the organism to which the nucleic acid is described as being heterologous relative to. In a non-limiting example, the viral genome of the synVACV comprises heterologous terminal hairpin loops. Those heterologous terminal hairpin loops can be derived from a different viral species or from a different VACV strain.
As used herein, a “host cell” includes an individual cell or cell culture that can be or has been a recipient for the virus of the disclosure. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected and/or transformed in vivo with a poxvirus of this disclosure.
An “immunologically effective amount” refers to the amount to be administered of a composition of matter that comprises at least one antigen, or immunogenic portion thereof, which is able to elicit an immunological response in the host cell or an antibody-mediated immune response to the composition. An immunologically effective amount of a recombinant poxvirus, as disclosed herein, refers to the amount of poxviral particles necessary to deliver a SARS-CoV-2 virus protein and elicit an immune response against said SARS-CoV-2 virus protein. In some embodiments, an immunologically effective amount of the recombinant poxvirus of the present disclosure is an amount within the range of 102-109 PFU. In some embodiments, an immunologically effective amount of the recombinant poxvirus of the present disclosure is from about 103-105 PFU. In some embodiments, an immunologically effective amount of the recombinant poxvirus of the present disclosure is about 105 PFU.
The terms “operative linkage” and “operatively linked” (or “operably linked”) or variations thereof, as used herein, are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, the nucleic acid encoding a SARS-CoV-2 virus protein may be operatively linked to a promoter. The nucleic acid sequence encoding a SARS-CoV-2 virus protein may be operatively linked in cis with a poxvirus specific promoter nucleic acid sequence, but does not need to be directly adjacent to it. For example, a linker sequence can be located between both sequences.
As used herein, the phrase “multiplicity of infection” or “MOI” is the average number of viruses per infected cell. The MOI is determined by dividing the number of virus added (ml added×plaque forming units (PFU)) by the number of cells added (ml added×cells/ml).
The terms “patient”, “subject”, or “individual” are used interchangeably herein and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, camels, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).
As known in the art, “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog; internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.); those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.); those containing alkylators; those with modified linkages (e.g., alpha anomeric nucleic acids, etc.); as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether and (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to chains of amino acids of any length. The chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as single chains or associated chains.
“Percent (%) sequence identity” or “sequence % identical to” with respect to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical with the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
As outlined elsewhere herein, certain positions of the viral genome can be altered. By “position” as used herein is meant a location in the genome sequence. Corresponding positions are generally determined through alignment with other parent sequences.
As used herein, “purify,” and grammatical variations thereof, refers to the removal, whether completely or partially, of at least one impurity from a mixture containing the polypeptide and one or more impurities, which thereby improves the level of purity of the polypeptide in the composition (i.e., by decreasing the amount (ppm) of impurity(ies) in the composition). As used herein “purified” in the context of viruses refers to a virus which is substantially free of cellular material and culture media from the cell or tissue source from which the virus is derived. The language “substantially free of cellular material” includes preparations of virus in which the virus is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a virus that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of cellular protein (also referred to herein as a “contaminating protein”). The virus may also be substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the virus preparation. A virus can be “purified” using routine methods known to one of skill in the art including, but not limited to, chromatography and centrifugation.
As used herein, the term “recombinant poxvirus” refers to a poxvirus comprising an exogenous or heterologous sequence in its genome generated by artificial manipulation of the viral genome, i.e. generation by recombinant DNA technology. The recombinant poxvirus contains an exogenous polynucleotide sequence encoding a polypeptide of interest. In some embodiments, the recombinant poxvirus comprises a nucleic acid encoding a SARS-CoV-2 virus protein.
As used herein, the term “rescue poxvirus” or “rescue virus” or “rescue system” refers to a virus or system which relies on a helper virus to provide the machinery necessary to produce recombinant viruses, by assembling the fragmented genome, while simultaneously integrating the targeted gene or expression cassette. Rice et al. Viruses. 2011 March; 3(3): 217-232.
As used herein, the term “residue” in the context of a polypeptide refers to an amino-acid unit in the linear polypeptide chain. It is what remains of each amino acid, i.e. —NH—CHR—C—, after water is removed in the formation of the polypeptide from α-amino-acids, i.e. NH2-CHR—COOH.
The term “sequence similarity,” in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
As used herein, “synthetic virus” refers to a virus initially derived from synthetic DNA (e.g., chemically synthesized DNA, PCR amplified DNA, engineered DNA, polynucleotides comprising nucleoside analogs, etc., or combinations thereof) and includes its progeny, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent synthetic virus due to natural, accidental, or deliberate mutation. In some embodiments, the synthetic virus refers to a virus where substantially all of the viral genome is initially derived from synthetic DNA (e.g., chemically synthesized DNA, PCR amplified DNA, engineered DNA, polynucleotides comprising nucleoside analogs, etc., or combinations thereof). In a preferred embodiment, the synthetic virus is derived from chemically synthesized DNA.
As used herein, “substantially pure” refers to material which is at least 50% pure (i.e., free from contaminants), more preferably, at least 90% pure, more preferably, at least 95% pure, yet more preferably, at least 98% pure, and most preferably, at least 99% pure.
The term “vaccine”, as used herein, refers to a composition comprising at least one immunologically active component that induces an immunological response in an animal and possibly, but not necessarily, one or more additional components that enhance the immunological activity of the active component. A vaccine may additionally comprise further components typical to pharmaceutical compositions. The immunologically active component of a vaccine may comprise complete virus particles in either their original form or as attenuated particles (modified live vaccine), or particles inactivated by appropriate methods (killed or inactivated vaccine). In other embodiments, the immunologically active component of a vaccine may comprise appropriate elements of the organisms (subunit vaccines) that best stimulate the immune system. The immunologically active component may be a protein of the viral envelope. The immunologically active component may be a protein forming part of the nucleocapsid. In some embodiments, the immunologically active component of a vaccine against SARS-CoV-2 is an envelope protein. Non-limiting examples of such proteins are the Spike protein (S), the Membrane protein (M) and the Hemagglutinin-Esterase protein (HE). In some embodiments, the immunologically active component of a vaccine against SARS-CoV-2 is the nucleocapsid protein (N).
The term “viral vector”, as used herein, describes a genetically modified virus which was manipulated by a recombinant DNA technique in a way so that its entry into a host cell is capable of resulting in a specific biological activity, e.g. the expression of a foreign target gene carried by the vector. A viral vector may or may not be replication competent in the target cell, tissue, or organism. A viral vector can incorporate sequences from the genome of any known organism. The sequences can be incorporated in their native form or can be modified in any way to obtain a desired activity. For example, the sequences can comprise insertions, deletions or substitutions. A viral vector can also incorporate an insertion site for an exogenous polynucleotide sequence. In some embodiments, the viral vector is a poxvirus. In some embodiments, the viral vector is a horsepox viral vector. In some embodiments, the viral vector is a synthetic horsepox viral vector.
As used herein, the terms “wild type virus”, “wild type genome”, “wild type protein,” or “wild type nucleic acid” refer to a sequence of amino or nucleic acids that occurs naturally within a certain population (e.g., a particular viral species, etc.).
Each embodiment described herein may be used individually or in combination with any other embodiment described herein.
Poxviruses are large (˜200 kbp) DNA viruses that replicate in the cytoplasm of infected cells. The Orthopoxvirus (OPV) genus comprises a number of poxviruses that vary greatly in their ability to infect different hosts. Vaccinia virus (VACV), for example, can infect a broad group of hosts, whereas variola virus (VARV), the causative agent of smallpox, only infects humans. A feature common to many, if not all poxviruses, is their ability to non-genetically “reactivate” within a host. Non-genetic reactivation refers to a process wherein cells infected by one poxvirus can promote the recovery of a second “dead” virus (for example one inactivated by heat) that would be non-infectious on its own.
Purified poxvirus DNA is not infectious because the virus life cycle requires transcription of early genes via the virus-encoded RNA polymerases that are packaged in virions. However, this deficiency can be overcome if virus DNA is transfected into cells previously or subsequently infected with a helper poxvirus, providing the necessary factors needed to transcribe, replicate, and package the transfected genome in trans (Sam C K, Dumbell K R. Expression of poxvirus DNA in coinfected cells and marker rescue of thermosensitive mutants by subgenomic fragments of DNA. Ann Virol (Inst Past). 1981; 132:135-50). Although this produces mixed viral progeny, a desired virus can be obtained by performing a reactivation reaction in a cell line that supports the propagation of both viruses, and then eliminating the helper virus by plating the mixture of viruses on cells that do not support the helper virus' growth (Scheiflinger F, Dorner F, Falkner F G. Construction of chimeric vaccinia viruses by molecular cloning and packaging. Proceedings of the National Academy of Sciences of the United States of America. 1992; 89(21):9977-81).
Any of the synthetic poxviruses disclosed in US 2018/0251736 and WO 2019/213452, the entire disclosure of each is incorporated by reference herein, may be used in the present disclosure.
In one aspect, the present disclosure provides recombinant poxviruses comprising a nucleic acid encoding a SARS-CoV-2 virus protein, wherein the SARS-CoV-2 protein is selected from the group consisting of the spike protein (S), the membrane protein (M) and the nucleocapsid protein (N), or combinations of two or more of said proteins.
In some embodiments, the poxvirus belongs to the Chordopoxvirinae subfamily. In some embodiments, the poxvirus belongs to a genus of Chordopoxvirinae subfamily selected from Avipoxvirus, Capripoxvirus, Cervidpoxvirus, Crocodylipoxvirus, Leporipoxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, or Yatapoxvirus. In some embodiments, the recombinant poxvirus is an Orthopoxvirus. In some embodiments, the Orthopoxvirus is selected from the group consisting of camelpox virus (CMLV), cowpox virus (CPXV), ectromelia virus (ECTV, “mousepox agent”), horsepox virus (HPXV), monkeypox virus (MPXV), rabbitpox virus (RPXV), raccoonpox virus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus, vaccinia virus (VACV), variola virus (VARV) and volepox virus (VPV). In some embodiments, the poxvirus is a Parapoxvirus. In some embodiments, the Parapoxvirus is selected from orf virus (ORFV), pseudocowpox virus (PCPV), bovine popular stomatitis virus (BPSV), squirrel parapoxvirus (SPPV), red deer parapoxvirus, Ausdyk virus, Chamois contagious ecythema virus, reindeer parapoxvirus, or sealpox virus. In some embodiments, the poxvirus is a Molluscipoxvirus. In some embodiments, the Molluscipoxvirus is molluscum contagiousum virus (MCV). In some embodiments, the poxvirus is a Yatapoxvirus. In some embodiments, the Yatapoxvirus is selected from Tanapox virus or Yaba monkey tumor virus (YMTV). In some embodiments, the poxvirus is a Capripoxvirus. In some embodiments, the Capripoxvirus is selected from sheepox, goatpox, or lumpy skin disease virus. In some embodiments, the poxvirus is a Suipoxvirus. In some embodiments, the Suipoxvirus is swinepox virus. In some embodiments, the poxvirus is a Leporipoxvirus. In some embodiments, the Leporipoxvirus is selected from myxoma virus, Shope fibroma virus (SFV), squirrel fibroma virus, or hare fibroma virus. In some embodiments, the poxvirus is an HPXV. In some embodiments, the horsepox virus is strain MNR-76. In other embodiments, the poxvirus is a VACV. In some embodiments, the VACV is selected from the group of strains consisting of: Western Reserve, Western Reserve Clone 3, Tian Tian, Tian Tian clone TP5, Tian Tian clone TP3, NYCBH, NYCBH clone Acambis 2000, Wyeth, Copenhagen, Lister, Lister 107, Lister-LO, Lister GL-ONC1, Lister GL-ONC2, Lister GL-ONC3, Lister GL-ONC4, Lister CTC1, Lister IMG2 (Turbo FP635), IHD-W, LC16m18, Lederle, Tashkent clone TKT3, Tashkent clone TKT4, USSR, Evans, Praha, L-IVP, V-VET1 or LIVP 6.1.1, Ikeda, EM-63, Malbran, Duke, 3737, CV-1, Connaught Laboratories, Serro 2, CM-01, NYCBH Dryvax clone DPP13, NYCBH Dryvax clone DPP15, NYCBH Dryvax clone DPP20, NYCBH Dryvax clone DPP17, NYCBH Dryvax clone DPP21, VACV-IOC, Chorioallantoid Vaccinia virus Ankara (CVA), Modified vaccinia Ankara (MVA), and MVA-BN. New poxviruses (e.g. Orthopoxviruses) are still being constantly discovered. It is understood that a poxvirus of the disclosure may be based on such a newly discovered poxvirus.
Chemical viral genome synthesis opens up the possibility of introducing a large number of useful modifications to the resulting genome or to specific parts of it. The modifications may improve ease of cloning to generate the virus, provide sites for introduction of recombinant gene products, improve ease of identifying reactivated viral clones and/or confer a plethora of other useful features (e.g. introducing a desired antigen, producing an oncolytic virus, etc.). In some embodiments, the modifications may include the attenuation or deletion of one or more virulence factors. In some embodiments, the modifications may include the addition or insertion of one or more virulence regulatory genes or gene-encoding regulatory factors.
Traditionally, the terminal hairpins of poxviruses have been difficult to clone and to sequence. As a result, some of the published genome sequences (e.g., VACV, ACAM 2000 and HPXV MNR-76) are incomplete. The published sequence of the HPXV genome is likewise incomplete, probably missing ˜60 bp from the terminal ends. In an exemplary embodiment, 129 nt ssDNA fragments were chemically synthesized using the published sequence of the VACV terminal hairpins as a guide and ligated onto dsDNA fragments comprising left and right ends of the HPXV genome. In some embodiments, the terminal hairpins of the poxvirus of the disclosure are derived from VACV. In some embodiments, the terminal hairpins are derived from CMLV, CPXV, ECTV, HPXV, MPXV, RPXV, raccoonpox virus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus or VPV. In some embodiments, the terminal hairpins are based on the terminal hairpins of any poxvirus whose genome has been completely sequenced or a natural isolate of which is available for genome sequencing. In some embodiments, the poxviruses are synthetic versions of HPXV comprising the terminal hairpins of VACV (GenBank accession number KY349117; see US 2018/0251736, incorporated by reference herein).
In some embodiments, the modifications introduced in a poxvirus genome may include the deletion of one or more restriction sites. In some embodiments, the modifications may include the introduction of one or more restriction sites. In some embodiments, the restriction sites to be deleted from the genome or added to the genome may be selected from one or more of restriction sites such as but not limited to AanI, AarI, AasI, AatI, AatII, AbaSI, AbsI, Acc65I, AccI, AccII, AccIII, AciI, AcII, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BcoDI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpu10I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaXI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFαI, BsrGI, BsrI, BssHII, BssSαI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ171, Bsu36I, BtgI, BtgZI, BtsαI, BtsCI, BtsIMutI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DpnII, DraI, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO1091, EcoP15I, EcoRI, EcoRV, FatI, FauI, Fnu4HI, FokI, FseI, FspEI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, I-CeuI, I-SceI, KasI, KpnI, LpnPI, MboI, MboII, MfeI, MluCI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspA1I, MspI, MspJI, MwoI, NaeI, NarI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, PacI, PaeR7I, PciI, PflFI, PflMI, PleI, PluTI, PmeI, PmII, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsnII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmII, SnaBI, SpeI, SphI, SrfI, SspI, StuI, StyD4I, StyI, SwaI, TaqαI, TfiI, TseI, Tsp45I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI, or ZraI. It is understood that any desired restriction site(s) or combination of restriction sites may be inserted into the genome or mutated and/or eliminated from the genome. In some embodiments, one or more AarI sites are deleted from the viral genome. In some embodiments, one or more BsaI sites are deleted from the viral genome. In some embodiments, one or more restriction sites are completely eliminated from the genome (e.g. all the AarI sites in the viral genome may be eliminated). In some embodiments, one or more AvaI restriction sites are introduced into the viral genome. In some embodiments, one or more StuI sites are introduced into the viral genome. In some embodiments, the one or more modifications may include the incorporation of recombineering targets including but not limited to loxP or FRT sites.
In some embodiments, the poxvirus modifications may include the introduction of fluorescence markers such as but not limited to green fluorescent protein (GFP), enhanced GFP, yellow fluorescent protein (YFP), cyan/blue fluorescent protein (BFP), red fluorescent protein (RFP), or variants thereof, etc.; selectable markers such as but not limited to drug resistance markers (e.g. E. coli xanthine-guanine phosphoribosyl transferase gene (gpt), Streptomyces alboniger puromycin acetyltransferase gene (pac), neomycin phosphotransferase I gene (nptI), neomycin phosphotransferase gene II (nptII), hygromycin phosphotransferase (hpt), sh ble gene, etc.; protein or peptide tags such as but not limited to MBP (maltose-binding protein), CBD (cellulose-binding domain), GST (glutathione-S-transferase), poly(His), FLAG, V5, c-Myc, HA (hemagglutinin), NE-tag, CAT (chloramphenicol acetyl transferase), DHFR (dihydrofolate reductase), HSV (Herpes simplex virus), VSV-G (Vesicular stomatitis virus glycoprotein), luciferase, protein A, protein G, streptavidin, T7, thioredoxin, Yeast 2-hybrid tags such as B42, GAL4, LexA, or VP16; localization tags such as an NLS-tag, SNAP-tag, Myr-tag, etc. It is understood that other selectable markers and/or tags known in the art may be used. In some embodiments, the modifications include one or more selectable markers to aid in the selection of reactivated clones (e.g. a fluorescence marker such as YFP, a drug selection marker such as gpt, etc.) to aid in the selection of reactivated viral clones. In some embodiments, the one or more selectable markers are deleted from the reactivated clones after the selection step.
In some embodiments, the poxviruses are synthetic horsepox viruses (scHPXV). In some embodiments, the synthetic horsepox viruses have been produced by recombination of overlapping DNA fragments of the viral genome and reactivation of the functional poxvirus is carried out in cells previously infected with a helper virus. Briefly, overlapping DNA fragments that encompass all or substantially all of the viral genome of the horsepox are chemically synthesized and transfected into helper virus-infected cells. The transfected cells are cultured to produce mixed viral progeny comprising the helper virus and reactivated horsepox virus. Next, the mixed viral progeny is plated on host cells that do not support the growth of the helper virus but allow the synthetic poxvirus to grow, in order to eliminate the helper virus and recover the synthetic poxviruses.
In some embodiments, substantially all of the synthetic poxviral genome is derived from chemically synthesized DNA. In some embodiments, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, over 99%, or 100% of the synthetic poxviral genome is derived from chemically synthesized DNA. In some embodiments, the poxviral genome is derived from a combination of chemically synthesized DNA and naturally occurring DNA.
The number of overlapping DNA fragments used to generate the synthetic poxvirus will depend on the size of the poxviral genome. Practical considerations such as reduction in recombination efficiency as the number of fragments increases on the one hand and difficulties in synthesizing very large DNA fragments as the number of fragments decreases on the other hand will also inform the number of overlapping fragments used. In some embodiments, the synthetic poxviral genome may be synthesized as a single fragment. In some embodiments, the synthetic poxviral genome is assembled from 2-14 overlapping DNA fragments. In some embodiments, the synthetic poxviral genome is assembled from 4-12 overlapping DNA fragments. In some embodiments, the synthetic poxviral genome is assembled from 6-10 overlapping DNA fragments. In some embodiments, the synthetic poxviral genome is assembled from 8-12 overlapping DNA fragments. In some embodiments, the synthetic poxviral genome is assembled from 10 overlapping DNA fragments. In an exemplary embodiment of the disclosure, a synthetic horsepox virus (scHPXV) is reactivated from 10 chemically synthesized overlapping double-stranded DNA fragments. In some embodiments, all of the fragments encompassing the poxviral genome are chemically synthesized. In some embodiments, one or more of the fragments are chemically synthesized and one or more of the fragments are derived from naturally occurring DNA (e.g. by PCR amplification or by well-established recombinant DNA techniques).
In some embodiments, the terminal hairpin loops are synthesized separately and ligated onto the fragments comprising the left and right ends of the poxviral genome. In some embodiments, terminal hairpin loops may be derived from a naturally occurring template. In some embodiments, the terminal hairpins of the synthetic poxvirus are derived from VACV. In some embodiments, the terminal hairpins of the recombinant synthetic poxvirus are derived from CMLV, CPXV, ECTV, HPXV, MPXV, RPXV, raccoonpox virus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus or VPV. In some embodiments, the terminal hairpins of the recombinant scHPXV are derived from VACV. In some embodiments, the terminal hairpins of the recombinant scHPXV are derived from CMLV, CPXV, ECTV, HPXV, MPXV, RPXV, raccoonpox virus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus or VPV. In some embodiments, the terminal hairpins of the poxvirus are based on the terminal hairpins of any poxvirus whose genome has been completely sequenced or a natural isolate of which is available for genome sequencing.
The size of the overlapping fragments used to generate the poxvirus of the disclosure will depend on the size of the poxviral genome. It is understood that there can be wide variations in fragment sizes and various practical considerations such as the ability to chemically synthesize very large DNA fragments, will inform the choice of fragment sizes. In some embodiments, the fragments range in size is from about 2000 bp to about 50000 bp. In some embodiments, the fragments range in size is from about 3000 bp to about 45000 bp. In some embodiments, the fragments range in size is from about 4000 bp to 40000 bp. In some embodiments, the fragments range in size is from about 5000 bp to 35000 bp. In some embodiments, the largest fragments are about 20000 bp, 21000 bp, 22000 bp, 23000 bp, 24 000 bp, 25000 bp, 26000 bp, 27000 bp, 28000 bp, 29000 bp, 30000 bp, 31000 bp, 32000 bp, 33000 bp, 34000 bp, 35000 bp, 36000 bp, 37000 bp, 38000 bp, 39000 bp, 40000 bp, 41000 bp, 42000 bp, 43000 bp, 44000 bp, 45000 bp, 46000 bp, 47000 bp, 48000 bp, 49000 bp, or 50000 bp. In some embodiments, a scHPXV is reactivated from 10 chemically synthesized overlapping double-stranded DNA fragments ranging in size from about 8500 bp to about 32000 bp (Table 2).
The poxviruses of the present disclosure can be propagated in any substrate that allows the virus to grow to titers that permit the uses of the recombinant poxvirus described herein. The poxvirus of the present disclosure may be grown in cells (e.g. avian cells, bat cells, bovine cells, camel cells, canary cells, cat cells, deer cells, equine cells, fowl cells, gerbil cells, goat cells, human cells, monkey cells, pig cells, rabbit cells, raccoon cells, seal cells, sheep cells, skunk cells, vole cells, etc.) that are susceptible to infection by the poxviruses. In some embodiments, the poxvirus is grown in adherent cells. In some embodiments, the poxvirus is grown in suspension cells. In some embodiments, the poxvirus is grown in mammalian cells. Such methods are well-known to those skilled in the art. Representative mammalian cells include, but are not limited to, BHK, MRC, BGMK, BRL3A, BSC-40, CEF, CEK, CHO, COS, CVI, HaCaT, HEL, HeLa cells, HEK293, human bone osteosarcoma cell line 143B, MDCK, NIH/3T3, Vero cells, etc. For virus isolation, the recombinant poxvirus is removed from cell culture and separated from cellular components, typically by well-known clarification procedures, e.g., such as gradient centrifugation and column chromatography, and may be further purified as desired using procedures well known to those skilled in the art, e.g., plaque assays. In some embodiments, the poxvirus is grown in Vero cells. In some embodiments, the poxvirus is grown in ACE2 Knockout Vero cells. In some embodiments, the poxvirus is grown in Vero adherent cells. In other embodiments, the poxvirus is grown in Vero suspension cells. In some embodiments, the poxvirus is grown in BSC-40 cells. In some embodiments, the poxvirus is grown in BHK-21 cells. In some embodiments, the poxvirus is grown in MRC-5 cells. In some embodiments, the poxvirus is grown in MRC-5 cells in the presence of for example, 5% serum, including but not limited to fetal calf serum. In some embodiments, the poxvirus is grown in avian cells. Such methods are well-known to those skilled in the art. Representative avian cells include, but are not limited to, chicken embryo fibroblasts, DF-1 cells (see, e.g., Himly et al., Virology, (1998) 248:295-304), duck embryo-derived cells, EB66® cells (see, e.g., Leon et al. Vaccine, (2016) 34: 5878-5885), AGE1. CR cells, including but not limited to AGE1.CRpIX® cells, DF-1 cells (see, e.g., Lohr et al., Vaccine, (2009) 36:4975-4982), etc. In some embodiments, the poxvirus is grown in chicken embryo fibroblasts. In some embodiments, the poxvirus is grown in duck embryo-derived cells. In some embodiments, the poxvirus is grown in EB66® cells. In some embodiments, the poxvirus is grown in AGE1.CRpIX® cells. In some embodiments, the poxvirus is grown in DF-1 cells.
In some embodiments, the method of producing a synthetic poxvirus comprises a step of chemically synthesizing overlapping DNA fragments that correspond to substantially all of the viral genome of the poxvirus and, optionally, chemically synthesizing the terminal hairpin loops from another virus or from another strain of virus; (ii) transfecting the overlapping DNA fragments into helper virus-infected cells; (iii) culturing said cells to produce a mixture of helper virus and synthetic poxvirus particles in said cells; and (iv) plating the mixture on host cells specific to the poxvirus to recover the synthetic poxvirus.
In some embodiments, the method of producing a synthetic horsepox virus comprises a step of (i) chemically synthesizing overlapping DNA fragments that correspond to substantially all of the viral genome of the horsepox virus and chemically synthesizing the terminal hairpin loops from another poxvirus (such as VACV, strain WB or NYCBH clone ACAM 2000); (ii) transfecting the overlapping DNA fragments into helper virus-infected cells; (iii) culturing said cells to produce a mixture of helper virus and synthetic horsepox virus particles in said cells; and (iv) plating the mixture on host cells specific to the horsepox virus to recover the synthetic horsepox virus.
In some embodiments, the poxvirus is a synthetic horsepox virus. In some embodiments, the synthetic horsepox virus genome is based on the published genome sequence described for horsepox virus (GenBank accession DQ792504) and the terminal hairpins are based on the published genome sequence similar to VACV strain NYCBH clone ACAM2000 (GenBank accession MN974380). In some embodiments, the synthetic horsepox virus comprises the sequence deposited in GenBank as accession number KY349117; see US 2018/0251736, incorporated by reference herein. In some embodiments, the synthetic horsepox virus is characterized by a nucleic acid encoding a SARS-CoV-2 virus S protein comprises the sequence set forth in SEQ ID NO: 43.
In some embodiments, the poxvirus is a synthetic recombinant vaccinia virus (synVACV). In some embodiments, the synthetic vaccinia genome is based on the published genome sequence described for VACV strain NYCBH clone ACAM2000 (GenBank accession AY313847; Osborne J D et al. Vaccine. 2007; 25(52):8807-32). In some embodiments, the synthetic vaccinia genome is based on the published genome sequence similar to VACV strain NYCBH clone ACAM2000 (GenBank accession MN974380; see WO 2019/213452, incorporated by reference herein). In some embodiments, the synthetic vaccinia virus comprises the sequence deposited in GenBank as accession number MN974381 (see WO 2019/213452, incorporated by reference herein). In some embodiments, the synthetic vaccinia virus is characterized by a nucleic acid encoding a SARS-CoV-2 virus S protein comprises the sequence set forth in SEQ ID NO: 44.
Any of the synthetic poxviruses disclosed in US 2018/0251736 and WO 2019/213452, may be used to generate a recombinant poxvirus comprising a SARS-CoV-2 protein, as disclosed herein.
In one aspect, the present disclosure relates to a recombinant poxvirus comprising a nucleic acid encoding a SARS-CoV-2 virus protein, wherein the SARS-CoV-2 protein is selected from the group consisting of the spike protein (S), the membrane protein (M) and the nucleocapsid protein (N), or combinations of two or more of said proteins. In some embodiments, the nucleotide sequence of the SARS-CoV-2 virus is any one of the published genome sequences, including, but not limited, to the genome sequences of the Wuhan strain, the UK strain B.1.1.7 strain, the South African B. 1.351 strain, the Brazilian B.1.1.28 strain, other emerging variants and any of their variants. In some embodiments, the nucleotide sequence of the SARS-CoV-2 virus is selected from the group consisting of GenBank accession numbers NC045512.2, LC521925.1, MN988668.1, MN985325.1, MN975262.1, MN938384.1, LR757998.1, LR757996.1, LR757995.1 and MN908947.3. In some embodiments, the nucleotide sequence of the SARS-CoV-2 virus is characterized by the sequence set forth in GenBank Accession Number MN988668.1; SEQ ID NO: 46. In some embodiments, the nucleotide sequence of the SARS-CoV-2 virus is further selected from the group consisting of GenBank accession numbers QQX99439 (e.g., B.1.1.7 United Kingdom variant), TEGALLY (e.g., B.1.351 South Africa variant), YP_009724390 (e.g., a Wuhan variant), and FARIA (e.g., B.1.1.28 Brazil variant).
The viral envelope of the SARS-CoV-2 virus is covered by characteristic spike-shaped glycoproteins (S) as well as the envelope (E) and membrane (M) proteins. The S protein mediates host cell attachment and entry. The helical nucleocapsid, comprised of the viral genome encapsidated by the nucleocapsid protein (N), resides within the viral envelope. In some embodiments, the poxvirus or synthetic poxvirus comprises a nucleic acid encoding a SARS-CoV-2 envelope protein. Non-limiting examples of such proteins are the Spike protein (S), the Membrane protein (M) and the Hemagglutinin-Esterase protein (HE). In some embodiments, the poxviruses or synthetic poxviruses comprise a nucleic acid encoding the S protein (SEQ ID NO: 9). In some embodiments, the poxviruses or synthetic poxviruses comprise a nucleic acid encoding the S protein (SEQ ID NO: 47). In some embodiments, the poxviruses or synthetic poxviruses comprise a nucleic acid encoding the M protein (SEQ ID NO: 10). In some embodiments, the poxviruses or synthetic poxviruses comprise a nucleic acid encoding the M protein (SEQ ID NO: 48). In some embodiments, the poxviruses or synthetic poxviruses comprise a nucleic acid encoding the N protein (SEQ ID NO: 11). In some embodiments, the poxviruses or synthetic poxviruses comprise a nucleic acid encoding the N protein (SEQ ID NO: 49). In some embodiments, the poxviruses or synthetic poxviruses comprise a nucleic acid encoding the HE protein (protein E or HE of Wuhan-HU-1, Accession LC521925.1; SEQ ID NO: 12). In some embodiments, the poxviruses or synthetic poxviruses comprise a combination of S protein and M protein. In some embodiments, the poxviruses or synthetic poxviruses comprise a combination of S protein and N protein. In some embodiments, the poxviruses or synthetic poxviruses comprises a combination of M protein and N protein.
In some embodiments, the SARS-CoV-2 virus is a Wuhan seafood market pneumonia virus 2019-nCoV isolate. GenBank accession number LC521925.1; SEQ ID NO: 13. In some embodiments, the SARS-CoV-2 virus is a Wuhan seafood market pneumonia virus 2019-nCoV isolate. GenBank accession number MN988668.1; SEQ ID NO: 46.
In some embodiments, the amino acid sequence of the SARS-CoV-2 virus protein is modified with reference to a wild type protein.
In some embodiments, the nucleotide sequence encoding the S protein is modified with reference to a wild type nucleotide sequence. In some embodiments, the amino acid sequence of the S protein is modified with reference to the wild type protein (protein S of Wuhan-HU-1, Accession LC521925.1; SEQ ID NO: 9). In some embodiments, the amino acid sequence of the S protein is modified with reference to the wild type protein (protein S of Wuhan-HU-1, Accession MN988668.1; SEQ ID NO: 47). In some embodiments, the amino acid sequence of the S protein is modified with reference to the wild type protein (protein S of Wuhan-Hu-1, Accession NC_045512.2; SEQ ID NO: 53) In some embodiments, the amino acid sequence of the SARS-CoV-2 virus protein is modified with reference to a wild type protein, so that the modified protein is adapted to infect mice. See Roberts et al. PLoS Pathog 3(1): e5. doi:10.1371; incorporated herein by reference in its entirety. In some embodiments, Tyrosine at position 459 is substituted by Histidine (Y459H) in the S protein with reference to the wild type protein (SEQ ID NO: 47). In some embodiments, the S protein comprises one or more mutations that enable antibody-dependent enhancement. In some embodiments, Aspartic acid at position 614 is substituted by Glycine (D614G) in the S protein with reference to the wild type protein (SEQ ID NO: 47). See Korber et al. bioRxiv 2020.04.29.069054; incorporated herein by reference in its entirety. In some embodiments, the S protein comprises one or more mutations in the fusion core of the HR1 region. In some embodiments, Serine at position 943 is substituted by Proline (S943P) in the S protein with reference to the wild type protein (SEQ ID NO: 47). In some embodiments, the S protein comprises one or more mutations that stabilize the S protein in an antigenically optimal prefusion conformation, which results in increased expression, conformational homogeneity and elicitation of potent antibody responses. In some embodiments, the mutations that stabilize the S protein in the prefusion conformation are located at the beginning of the central helix. See Pallesen et al. Proc Natl Acad Sci USA. 2017; 114(35); incorporated herein by reference in its entirety. In some embodiments, Lysine at position 986 is substituted by Proline (K986P) in the S protein with reference to the wild type protein (SEQ ID NO: 47). In some embodiments, Valine at position 987 is substituted by Proline (V987P) in the S protein with reference to the wild type protein (SEQ ID NO: 47). In some embodiments, the S protein comprises any one of substitutions Y459H, D614G, S943P, K986P and V987P, or a combination thereof, with reference to the wild type protein (SEQ ID NO: 47).
In some embodiments, the amino acid sequence of the M protein is modified with reference to the wild type protein (protein M of Wuhan-HU-1, Accession LC521925.1; SEQ ID NO: 10). In some embodiments, the amino acid sequence of the M protein is modified with reference to the wild type protein (protein M of Wuhan-HU-1, Accession MN988668.1; SEQ ID NO: 48). In some embodiments, Glutamic acid at position 11 is substituted by a Lysine in the M protein with reference to the wild type protein. In some embodiments, Glutamic acid at position 11 is substituted by a Lysine in the M protein with reference to the wild type protein (SEQ ID NO: 10). In some embodiments, Glutamic acid at position 11 is substituted by a Lysine in the M protein with reference to the wild type protein (SEQ ID NO: 48).
In some embodiments, the amino acid sequence of the N protein is modified with reference to the wild type protein (protein N of Wuhan-HU-1, Accession LC521925.1; SEQ ID NO: 11). In some embodiments, the amino acid sequence of the N protein is modified with reference to the wild type protein (protein N of Wuhan-HU-1, Accession MN988668.1; SEQ ID NO: 49).
In some embodiments, the nucleic acid sequence encoding the SARS-CoV-2 virus protein is modified with reference to the wild type protein. In some embodiments, the nucleic acid sequence encoding the SARS-CoV-2 virus protein is modified with reference to the wild type protein (SEQ ID NO: 9). In some embodiments, the nucleic acid sequence encoding the SARS-CoV-2 virus protein is modified with reference to the wild type protein (SEQ ID NO: 47). In some embodiments, the nucleic acid sequence encoding the SARS-CoV-2 virus protein is modified with reference to the wild type protein for efficient expression of transgenes in poxviruses. In some embodiments, the heterologous gene coding sequences containing the vaccinia Early Transcription Terminator Signal (ETTS) (TTTTTNT; also called T5NT (SEQ ID NO: 14)) are removed. See Earl et al. Journal of Virology, 1990; 2448-2451; incorporated herein by reference in its entirety. In some embodiments, the poxvirus genome retains two overlapping endogenous ETTS. In some embodiments, the heterologous gene coding sequences containing the vaccinia Early Transcription Terminator Signal (ETTS) (TTTTTNT; also called T5NT (SEQ ID NO: 14)) are removed with reference to the nucleic sequence encoding the S protein of the SARS-CoV-2 virus (protein S of Wuhan-HU-1, Accession MN988668.1; SEQ ID NO: 47).
In some embodiments, the nucleic acid encoding a SARS-CoV-2 virus protein is operatively linked to a promoter. In some embodiments, the promoter is a poxvirus-specific promoter. In some embodiments, the promoter is located between the left flanking arm and the ATG of the transgene expression cassette. In some embodiments, the poxvirus promoter is a vaccinia virus early promoter. In some embodiments, the poxvirus promoter is an optimized vaccinia virus early promoter (AAAATTGAAANNNTANNNNNNNNNNNNNNNNNN; SEQ ID NO: 3). In some embodiments, the poxvirus promoter is a synthetic vaccinia virus late promoter (TTTTTTTTTTTTTTTTTTTNNNNNNTAAATG; SEQ ID NO: 4). In some embodiments, the poxvirus promoter is an overlapping synthetic early/late promoter (AAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATA; SEQ ID NO: 5). See
In some embodiments, the vaccinia virus late promoter nucleotide sequence comprises the sequence set forth in SEQ ID NO: 6 (TTTTATTTTTTTTTTTTGGAATATAAATA). In some embodiments, the vaccinia virus late promoter is the sequence set forth in SEQ ID NO: 6. In some embodiments, the vaccinia virus late promoter nucleotide sequence comprises the sequence set forth in SEQ ID NO: 7 (AAAATTGAAAAAATA). In some embodiments, the poxvirus promoter is an overlapping synthetic early/late promoter comprising the sequence set forth in SEQ ID NO: 8 (TTTTATTTTTTTTTTTTGGAATATAAATATCCGGT AAAATTGAAAAAATA). In some embodiments, the poxvirus promoter is an overlapping synthetic early/late promoter comprising a nucleic acid spacer sequence of 38-160 nucleotides 3′ of the early promoter and between the RNA start site and the ATG. In some embodiments, the spacer is 160 nucleotides long, resulting in enhanced levels of expression. See
In some embodiments, the protein of the SARS-CoV-2 is inserted into a non-essential gene for replication. In some embodiments, the SARS-CoV-2 protein is inserted into the Thymidine Kinase (TK) locus (Gene ID HPXV095; positions 992077-92610; SEQ ID NO: 1) of the horsepox virus or the synthetic horsepox virus. In some embodiments, the SARS-CoV-2 protein is inserted into the Thymidine Kinase (TK) locus (Gene ID synVACV_105; positions 83823-84344; SEQ ID NO: 2) of the vaccinia virus or the synthetic vaccinia virus. The TK locus provides a stable insertion site for foreign genes of interest. The TK locus also provides a selection marker to identify those clones where the nucleic acid encoding a SARS-CoV-2 protein has been inserted. The clones where the nucleic acid encoding a SARS-CoV-2 protein is inserted are not capable of growing in the presence of 5-bromo-2-deoxyuridine (BrdU), which is an analogue of the pyrimidine deoxynucleoside thymidine, due to not having the TK gene.
An exemplary method to generate a recombinant poxvirus of the disclosure comprising the S protein of SARS-CoV-2 virus comprises:
Any of the synthetic poxviruses disclosed in US 2018/0251736 and WO 2019/213452, may be used in any of the methods disclosed herein.
Any of the recombinant poxviruses comprising a nucleic acid encoding a SARS-CoV-2 virus protein described in the present disclosure may be used in any of the methods disclosed herein.
In one aspect, the disclosure relates to a method for selecting a cell that expresses a SARS-CoV-2 virus protein, comprising infecting said cell with the recombinant poxvirus of the disclosure and selecting the infected cell expressing said SARS-CoV-2 virus protein.
In another aspect, the disclosure relates to a method of inducing an immune response against a SARS-CoV-2 virus in a subject, comprising administering to said subject an immunologically effective amount of the recombinant poxvirus of the disclosure.
In another aspect, the disclosure relates to a method of generating a recombinant poxvirus of the disclosure, the method comprising:
(a) Infecting a host cell with a poxvirus;
(b) Transfecting the infected cell of step (a) with a nucleic acid encoding a SARS-CoV-2 virus protein to generate a recombinant poxvirus; and
(c) Selecting a recombinant poxvirus, wherein the nucleic acid encoding a SARS-CoV-2 virus protein is located, upon transfection, in a region of the poxvirus that is not essential for the replication of the poxvirus.
In some embodiments, the recombinant poxvirus of the disclosure is used as a vaccine to express a SARS-CoV-2 virus protein. Methods to assess the safety, immunogenicity and protective capacity of the recombinant poxvirus are known in the art. See Kremer M et al. 2012. p 59-92. In Isaacs S N (ed), Vaccinia virus and poxvirology, vol 890. Humana Press, Totowa, N.J. In some embodiments, the immunization is via a subcutaneous route. In some embodiments, the immunization is via an intramuscular route. In some embodiments, the immunization is via an intranasal route. In some embodiments, the immunization is via scarification. In some embodiments, a range between about 104 and about 108 PFU of the recombinant poxvirus is used. In some embodiments, about 104, about 105, about 106, about about 107 or about 108 PFU of recombinant poxvirus is used for the immunization. In some embodiments, about 105 PFU of the recombinant poxvirus is used for the immunization. A physician will be able to determine the adequate PFU dosage for each subject. In some embodiments, one dose is administered to the subject. In some embodiments, more than one dose is administered to the subject.
In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against a SARS-CoV-2 virus in a subject, comprising administering to said subject an immunologically effective amount of a recombinant poxvirus or a pharmaceutical composition. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against the SARS-CoV-2 virus in a subject, wherein the immunologically effective amount of the recombinant poxvirus is administered by scarification. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against a SARS-CoV-2 virus in a subject, wherein the immune response comprises antibodies that are capable of neutralizing the SARS-CoV-2 virus. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against a SARS-CoV-2 virus in a subject, wherein the immunologically effective amount of a recombinant poxvirus is capable of protecting the subject from SARS-CoV-2 virus. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against a SARS-CoV-2 virus in a subject, wherein the immunologically effective amount of a recombinant poxvirus reduces or prevents the progression of the virus after SARS-CoV-2 infection in the subject. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against a SARS-CoV-2 virus in a subject, wherein the immune response is a T-cell immune response.
In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against a SARS-CoV-2 virus and a poxvirus comprising administering to said subject an immunologically effective amount of a recombinant poxvirus or pharmaceutical composition. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against the SARS-CoV-2 virus and the poxvirus, wherein said immunologically effective amount of the recombinant poxvirus is administered by scarification. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against the SARS-CoV-2 virus and the poxvirus, wherein said immune response comprises antibodies that are capable of neutralizing the SARS-CoV-2 virus and the poxvirus. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against the SARS-CoV-2 virus and the poxvirus, wherein the immunologically effective amount of a recombinant poxvirus is capable of protecting the subject from the SARS-CoV-2 virus and the variola virus. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against the SARS-CoV-2 virus and the poxvirus, wherein the immunologically effective amount of a recombinant poxvirus reduces or prevents the progression of the SARS-CoV-2 virus infection and/or poxvirus infection in the subject. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against the SARS-CoV-2 virus and the poxvirus, wherein the immune response is a T-cell immune response. In some embodiments, the recombinant poxvirus is useful towards the method of inducing an immune response against the SARS-CoV-2 virus and the poxvirus, wherein the poxvirus is vaccinia virus, variola, horsepox virus or monkeypox virus.
In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against a SARS-CoV-2 virus comprising administering to said subject an immunologically effective amount of a recombinant poxvirus or pharmaceutical composition. In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against the SARS-CoV-2 virus, wherein said immunologically effective amount of the recombinant poxvirus is administered by scarification. In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against the SARS-CoV-2 virus, wherein the immunologically effective amount of a recombinant poxvirus is capable of protecting the subject from SARS-CoV-2 virus. In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against the SARS-CoV-2 virus, wherein the immunologically effective amount of a recombinant poxvirus reduces or prevents the progression of the virus after SARS-CoV-2 infection in the subject.
In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against a SARS-CoV-2 virus and a poxvirus comprising administering to a subject an immunologically effective amount of the recombinant poxvirus reduces or pharmaceutical composition. In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against the SARS-CoV-2 virus and the poxvirus, wherein said immunologically effective amount of the recombinant poxvirus is administered by scarification. In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against the SARS-CoV-2 virus and the poxvirus, wherein the immunologically effective amount of a recombinant poxvirus is capable of protecting the subject from the SARS-CoV-2 virus and the poxvirus. In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against the SARS-CoV-2 virus and the poxvirus, wherein the immunologically effective amount of a recombinant poxvirus reduces or prevents the progression of the virus after SARS-CoV-2 infection and/or variola virus infection in the subject. In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against the SARS-CoV-2 virus and the poxvirus, wherein the poxvirus is vaccinia virus, variola, horsepox virus or monkeypox virus.
In some embodiments, the recombinant poxvirus is useful towards the method of reducing or preventing the progression of a SARS-CoV-2 virus infection in a subject in need or at risk thereof comprising administering to said subject an immunologically effective amount of the recombinant poxvirus or pharmaceutical composition.
In some embodiments, the recombinant poxvirus is useful towards the method of reducing or preventing the progression of a SARS-CoV-2 virus and a poxvirus infection in a subject in risk thereof comprising administering to said subject an immunologically effective amount of the recombinant poxvirus or pharmaceutical composition. In some embodiments, the recombinant poxvirus is useful towards the method of reducing or preventing the progression of the SARS-CoV-2 virus and the poxvirus infection, wherein the poxvirus is vaccinia virus, variola, horsepox virus or monkeypox virus.
In some embodiments, the recombinant poxvirus is useful for a vaccine against a SARS-CoV-2 virus comprising a recombinant virus or a pharmaceutical composition.
In some embodiments, the recombinant poxvirus is useful for a bivalent vaccine against a SARS-CoV-2 virus and a poxvirus comprising a recombinant virus or a pharmaceutical composition. In some embodiments, the recombinant poxvirus is useful for a bivalent vaccine against a SARS-CoV-2 virus, wherein the poxvirus is a vaccinia virus, variola, horsepox virus or monkeypox.
The synthetic horsepox virus (scHPXV) is generated following the methods disclosed in US 2018/0251736, incorporated herein by reference in its entirety.
The design of the synthetic HPXV genome is based on the previously described genome sequence for HPXV (strain MNR-76; GenBank accession DQ792504) (Tulman E R, Delhon G, Afonso C L, Lu Z, Zsak L, Sandybaev N T, et al. Genome of horsepox virus. Journal of virology. 2006; 80(18):9244-58). The 212,633 bp genome is divided into 10 overlapping fragments. These fragments are designed so that they shared at least 1.0 kbp of overlapping sequence (i.e. homology) with each adjacent fragment, to provide sites where homologous recombination will drive the assembly of full-length genomes. The fragments generated are shown in Table 2. These overlapping sequences will provide sufficient homology to accurately carry out recombination between the co-transfected fragments
8505-24,761
The resulting synthetic HPXV has been deposited in GenBank as accession number KY349117.
A yfp/gpt cassette under the control of a poxvirus early late promoter is introduced into the HPXV095/J2R locus within GA_Fragment_3, so that reactivation of HPXV (scHPXV YFP-gpt::095) will be easy to visualize under a fluorescence microscope. SFV-catalyzed recombination and reactivation of poxvirus DNA to assemble recombinant poxviruses has previously been described (Yao X D et al. Journal of virology. 2003; 77(13):7281-90; and Yao X D et al. Methods Mol Biol. 2004; 269:51-64; the entire disclosures of each are incorporated by reference herein). Several biological features make this an attractive model system. First, SFV has a narrow host range, productively infecting rabbit cells and certain monkey cell lines, like BGMK. It can infect, but grows very poorly on cells like BSC-40. Second, it grows more slowly compared to Orthopoxviruses, taking approximately 4-5 days to form transformed “foci” in monolayers of cells, a characteristic that is very different from Orthopoxviruses, which produce plaques within 1-2 days in culture. This difference in growth between Leporipoxviruses and Orthopoxviruses allows differentiation of these viruses by performing the reactivation assays in BGMK cells and plating the progeny on BSC-40 cells. In some embodiments, other helper viruses (such as, but not limited to, fowlpox virus) may be used. In some embodiments, different cell combinations may be used.
BGMK cells are infected with SFV at a MOI of 0.5 and then transfected with 5 μg of digested GA_HPXV fragments 2 h later. Five days post transfection, all of the infectious particles are recovered by cell lysis and re-plated on BSC-40 cells, which only efficiently support growth of HPXV. The resulting reactivated scHPXV YFP-gpt::095 plaques are visualized under a fluorescence microscope. The visualization is enabled by the yfp/gpt selectable marker in the HPXV095/J2R locus within Frag_3. Virus plaques are detected in BSC-40 monolayers within 48 h of transfection. The efficiency of recovering scHPXV YFP-gpt::095 is dependent on a number of factors, including DNA transfection efficiency, but ranges up to a few PFU/μg of DNA transfected.
A yfp/gpt cassette under the control of a poxvirus early late promoter is also introduced into the HPXV200 locus within GA_Fragment_7, so that reactivation of HPXV (scHPXV YFP-gpt::200) will be easy to visualize under a fluorescence microscope. SFV-catalyzed recombination and reactivation of poxvirus DNA to assemble recombinant poxviruses has previously been described (Yao X D et al. Journal of virology. 2003; 77(13):7281-90; and Yao X D et al. Methods Mol Biol. 2004; 269:51-64; the entire disclosures of each are incorporated by reference herein). Several biological features make this an attractive model system. First, SFV has a narrow host range, productively infecting rabbit cells and certain monkey cell lines, like BGMK. It can infect, but grows very poorly on cells like BSC-40. Second, it grows more slowly compared to Orthopoxviruses, taking approximately 4-5 days to form transformed “foci” in monolayers of cells, a characteristic that is very different from Orthopoxviruses, which produce plaques within 1-2 days in culture. This difference in growth between Leporipoxviruses and Orthopoxviruses allows differentiation of these viruses by performing the reactivation assays in BGMK cells and plating the progeny on BSC-40 cells. In some embodiments, other helper viruses (such as, but not limited to, fowlpox virus) may be used. In some embodiments, different cell combinations may be used.
BGMK cells are infected with SFV at a MOI of 0.5 and then transfected with 5 μg of digested GA_HPXV fragments 2 hours later. Five days post transfection, all of the infectious particles are recovered by cell lysis and re-plated on BSC-40 cells, which only efficiently support growth of HPXV. The resulting reactivated scHPXV YFP-gpt::200 plaques are visualized under a fluorescence microscope. The visualization is enabled by the yfp/gpt selectable marker in the HPXV200 locus within Frag_7. Virus plaques are detected in BSC-40 monolayers within 48 hours of transfection. The efficiency of recovering scHPXV YFP-gpt::200 is dependent on a number of factors, including DNA transfection efficiency, but ranges up to a few PFU/μg of DNA transfected.
The synthetic vaccinia virus ACAM2000 was generated using the methods disclosed in WO 2019/213452, incorporated herein by reference in its entirety.
The design of the synthetic VACV (synVACV) genome was based on the previously described genome sequence for VACV ACAM2000 (GenBank accession AY313847) (Osborne J D et al. Vaccine. 2007; 25(52):8807-32). The genome was divided into 9 overlapping fragments (
The resulting synthetic VACV, ACAM 2000 has been deposited in GenBank as accession number MN974381.
The nucleotide sequence alignment of the synthetic HPXV (Accession number KY349117) and the synthetic VACV (Accession number MN974381) indicates a nucleotide sequence identity of 99% throughout the 4 Kb TK gene locus and a co-linearity (Start and Stop) of the TK gene sequences, which were used for the construction of the ΔTK insertion locus or knockout TK locus. See
The TK gene is non-essential for viral replication in tissue culture. It also provides a stable insertion site for foreign gene(s) of interest and a selection marker (TK−) in the presence of the nucleotide analog 5-Bromodeoxyuridine (5-BrdU).
Because of the high level of sequence identity between the synthetic HPXV and the synthetic VACV, the PCR sequence manipulations used for the generation of the expression cassette containing the promoter/gene sequences allow for the use of the same expression cassette with the two different rescue viruses. For the rescue of the transfected PCR fragment comprising the engineered SARS-CoV-2 S protein, virus specific sequences (recombination left and right flanking arms, corresponding to HPXV094 and HPXV096, respectively) allows the recombination of the expression cassette into the viral TK locus. See
A nucleotide sequence alignment of the Spike (S) gene of different SARS-CoV-2 isolates is performed. The viral isolates aligned are the ones published under the following accession numbers NC045512.2, LC521925.1, MN988668.1, MN985325.1, MN975262.1, MN938384.1, LR757998.1, LR757996.1, LR757995.1 and MN908947.3. The alignment of the S genes indicates 100% homology at the nucleotide level between the S gene of the different viral isolates. All viral isolates sequences are isolates with complete genome sequence entries from China, Japan and the US. Early indications from isolate sequence analysis seems to indicate little viral drift. However, if drift is ultimately observed, the same techniques can be used with the modified virus and its proteins and nucleic acid sequences.
The nucleotide sequence encoding the S protein of the SARS-CoV-2 comprises the nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 47. The SARS-CoV-2 is not well adapted for infection in mice. Therefore, genomic adaptative mutations are introduced to adapt the virus for infection in mice. In particular, a mutation in the nucleotide sequence is introduced, the mutation resulting in a S protein comprising a Y459H substitution. Table 4 shows genomic adaptative mutations in SARS-CoV virus, that can be adapted and introduced into other regions of the SARS-CoV-2 virus. See Roberts A et al. PLoS Pathog. 2007 January; 3(1): e5. doi: 10.1371.
The six mutations found in a SARS-CoV virus resulting from fifteen passages (and the resulting virus called MA15) and that are lethal for mice following intranasal inoculation are listed in Table 4. The labels in Table 4 are as follows: ORFa: open reading frame; CDSb coding sequence, sequence of nucleotides that corresponds with the sequence of amino acids in a protein (location includes start and stop codon); nspc; non-structural protein, cleavage product of ORF lab; Mainpro: main 3C-like protease; Hel: helicase; RBMd: receptor binding motif (amino acids 424-494).
For efficient expression of transgenes from poxvirus vectors, heterologous gene coding sequences containing the vaccinia Early Transcription Terminator Signal (ETTS) should be removed, in one embodiment of this disclosure, through coding silent mutagenesis to generate full length transcripts during the early phase of the infection. These sequences have the following sequence: TTTTTNT (T5NT); SEQ ID NO: 14. Removing the ETTS in the S protein coding sequence can positively impact the generation of robust immune responses. See Earl P L et al. J Virol. 1990 May; 64(5):2448-51.
Examples of other mutations introduced in the S protein (SEQ ID NO: 47) in other embodiments of this disclosure are the following: D614G, S943P, K986P and V987P. One or more of these mutations can be introduced in the S protein in those embodiments.
Poxvirus replication occurs in the cytoplasm of the infected cell. The viruses do not enter the nucleus of the infected cell during the replication cycle, and therefore do not utilize the host cell transcriptional apparatus. Because of the cytoplasmic location of replication, poxviruses encode their own transcriptional machinery including the viral RNA polymerase and their own regulatory promoter recognition signals. Therefore, for efficient high-level expression from eukaryotic transgene expression has to be driven from poxvirus promoters. Poxvirus gene expression is controlled by early, intermediate and late promoters and can be defined as early (8 Hours before infection) and late (8 hours post-infection). DNA synthesis occurs 8 hours post infection and is referred to as the temporal boundary for the initiation of late gene expression. Highest levels of transgene antigenic load have usually been achieved through the use of a combination of Early and Late Promoter signals. The promoter used to control transcription of the S protein is an overlapping synthetic early/late promoter comprising the sequence (TTTTATTTTTTTTTTTTGGAATATAAATATCCGGTAAAATTGAAAAAATA SEQ ID NO: 8) including a 160 nucleotides long spacer 3′ of the early promoter and between the RNA start site and the ATG (SEQ ID NO: 42). See
The expression cassette generated comprises the engineered SARS-CoV-2 S protein adapted for mouse infection and where the ETTS sequences have been removed and controlled under the transcription of the overlapping tandem early/late promoter.
An exemplary method to generate a recombinant horsepox comprising the S protein of SARS-CoV-2 virus is shown in
Once a recombinant poxvirus has been identified, a variety of methods can be used to assay the expression of the polypeptide encoded by the inserted gene. These methods include, but are not limited to, black plaque assay (an in situ enzyme immunoassay performed on viral plaques), Western blot analysis, radioimmunoprecipitation (RIPA), and enzyme immunoassay (EIA). Antibodies that recognize the SARS-CoV-2 S may be used.
The sequence of one embodiment of a synthetic horsepox virus comprising a nucleic acid encoding a SARS-CoV-2 virus S protein is SEQ ID NO: 43. The sequence of one embodiment of a synthetic vaccinia virus comprising a nucleic acid encoding a SARS-CoV-2 virus S protein is SEQ ID NO: 44.
Primary chicken embryo fibroblasts (CEF) cells prepared from 10-day-old embryos are grown in minimum essential medium supplemented with 10% FBS and used to propagate and titer the recombinant poxvirus.
BALB/c mice are immunized by single-shot and prime-boost vaccination with 105, 106, 107 or 108 PFU of recombinant synthetic horsepox virus expressing SARS-CoV-2 protein via either scarification, intranasally, intramuscular or subcutaneous inoculations. Animals inoculated with non-recombinant virus (WT) or phosphate-buffered saline (Mock) are used as controls.
Four weeks after the immunization, animals are challenged intranasally with 104 tissue culture 50% infective dose (TCID50) of SARS-CoV-2 as described. (Subbarao, K et al. (2004) J. Virol. 78, 3572-3577). Two days later, the lungs and nasal turbinates of four animals in each group are removed and the SARS-CoV-2 titers are determined.
Subjects at risk for infection by SARS-CoV-2 S are vaccinated using a recombinant poxvirus engineered SARS-CoV-2 S protein of this disclosure through scarification with a bifurcated needle (standard dose, 2.5×105 to 12.5×105 plaque-forming units) typically into the upper arm. The recombinant poxvirus engineered SARS-CoV-2 S protein can also be administered as a single dose one-shot vaccine (e.g., 1×106 PFU TNX-1800), in which vials containing 100 doses per vial are manufactured. The vaccination protects them from infection. However, subsequent vaccinations may be useful to boost immunity.
Methods regarding clinical trial testing of a vaccine have been previously described (Sadoff, J. et al. (2020) Safety and immunogenicity of the Ad26.COV2.S COVID-19 vaccine candidate: interim results of a phase 1/2a, double-blinded, randomized, placebo-controlled trial, MedRxiv, Pages 1-28; incorporated herein by reference in its entirety). A multi-center phase 1/2a randomized, double-blind, placebo-controlled clinical study designed to assess the safety, reactogenicity and immunogenicity of recombinant poxvirus engineered SARS-CoV-2 S protein is conducted. The engineered SARS-CoV-2 S protein is administered at a dose level, for example, between about 5×1010 to 1×1011 viral particles (vp) per vaccination, either as a single dose or as a two-dose schedule spaced by, for example, 56 days in healthy adults (18-55 years old) and healthy elderly (≥65 years old). Vaccine elicited S specific antibody levels are measured, for example, by ELISA and neutralizing titers are measured, for example, in a microneutralization assay (see, e.g., methods in Example 11). CD4+T-helper (Th)1 and Th2, and CD8+ immune responses are assessed, for example, by intracellular cytokine staining (ICS).
The SARS-CoV-2 Spike protein (SEQ ID NO: 45) was codon-optimized (SARS-CoV-2-Spike-co; SEQ ID NO: 50) for expression during poxvirus infection and was synthesized by GenScript. The synthesized DNA also contains a poxvirus synthetic early/late promoter at nucleotide position 10-48. The synthesized DNA was subcloned into a plasmid containing homology to either the HPXV095 gene locus (SEQ ID NO: 51) or the HPXV200 gene locus (SEQ ID NO: 52). Homologous recombination was used to insert the synthesized DNA by replacing the selectable markers that were previously inserted into the synthetic VACV (synVACV) or synthetic HPXV (scHPXV). The selectable markers were inserted as a fusion between yellow fluorescent protein (YFP) and guanine phosphoriosyltransferase (GPT) into either of the HPXV095 or A2K105 genes, respectively (see methods as disclosed in US 2018/0251736, incorporated herein by reference in its entirety).
The YFP-GPT selectable marker in the synVACV (see Example 2) thymidine kinase (TK) locus (also referred to as the A2K105 gene locus) is replaced using, for example, homologous recombination with a codon-optimized SARS-CoV-2 Spike (SARS-CoV-2-co) nucleotide sequence to generate the synthetic vaccinia virus TNX-2200. One exemplary procedure is as follows.
Approximately 20 μgrams of plasmid containing the SARS-CoV-2-Spike-co nucleotide sequence flanked by approximately 400 nucleotides homologous to the A2K105 gene was linearized using the restriction enzyme SacI. Following restriction enzyme digestion, the linearized plasmid was further purified to remove residual enzyme. BSC-40 cells were infected with synVACV expressing YFP-GPT in the A2K105 gene locus (synVACVΔ A2K105yfp-gpt) at a MOI of 0.1 for 1 hour. Following infection, the virus inoculum was replaced with OptiMEM media and was incubated for an additional 30 minutes at 37° C. Approximately 5 μgrams of purified linearized plasmid was mixed with Lipofectamine 2000 (ThermoFisher Scientific) at a ratio of 1 μgram of DNA to 3 μL of Lipofectamine 2000 in a total volume of 2 mL of OptiMEM. A DNA-lipid complex formed during approximately 10 minutes of incubation. It was then added to the virus-infected BSC-40 cells.
BSC-40 cells were incubated for 48 hours to allow for homologous recombination to occur. After 48 hours, the plates were scraped to lift virus-infected cells and the mixture was transferred to a conical tube. The cells were lysed following three rounds of freezing at −80° C. and thawing. An appropriate dilution, which can range from 1×10−2 to 1×10−5, of the infection/transfection mixture was plated onto BSC-40 cells followed by an agar overlay. Infected cell plates were incubated until non-fluorescent “recombinant” plaques were visualized. These non-fluorescent plaques were marked, and agar plugs were picked and added into a 10 mM Tris pH 8.0 solution. The plaques were subsequently used to infect BSC-40 cells in a second round of infection. This plaque picking process and infection of BSC-40 cells was repeated until YFP was undetectable in the infected cells (ranges between 4-6 rounds of purification). PCR analysis using primers (sA2K J2R Flank Forward Primer 5′ to 3′: ATGCGATTCAAAAAAGAATCAGC (SEQ ID NO: 56) and sA2K J2R Flank Reverse Primer 5′ to 3′: CAATTTCCTCAAAATACATAAACGG (SEQ ID NO: 57)) that amplify the A2K105 gene locus was performed to confirm that the SARS-CoV-2 Spike gene was inserted into the A2K105 locus.
Western blot analysis was performed to test for SARS-Spike-co protein expression in the BSC-40 cells infected with synVACVΔA2K105yfp-gpt or synVACVΔA2K105SARSCoV2-SPIKE-co:nm (TNX-2200) clones 1.1.1.1.1 or 2.1.1.1.1 (
Viral genomic DNA from synVACVΔA2K105SARSCoV2-SPIKE-co::nm (TNX-2200) clones 1.1.1.1.1 and 2.1.1.1.1 was isolated and the DNA was sequenced using Next Generation Sequencing (NGS) with the Illumina MiSeq platform. The sequencing data were analyzed by de novo assembly and mapped to reference software using the CLC Genomics Workbench software (Qiagen).
The YFP-GPT selectable marker in the scHPXV (see Example 7) thymidine kinase (TK) locus (also referred to as the HPXV095 gene locus) was replaced using, for example, homologous recombination with a codon-optimized SARS-CoV-2 Spike (SARS-CoV-2-co) nucleotide sequence to generate the synthetic vaccinia virus TNX-1800a. One exemplary procedure is as follows.
Approximately 20 μgrams of plasmid containing the SARS-CoV-2-Spike-co nucleotide sequence flanked by approximately 400 nucleotides homologous to the HPXV095 gene was linearized using the restriction enzyme, SacI. Following restriction enzyme digestion, the linearized plasmid was further purified to remove residual enzyme. BSC-40 cells were infected with scHPXV expressing YFP-GPT in the HPXV095 gene locus at a MOI of 0.1 for 1 hour. Following infection, the virus inoculum was replaced with OptiMEM media and was incubated for an additional 30 minutes at 37° C. Approximately 5 μgrams of purified linearized plasmid was mixed with Lipofectamine 2000 (ThermoFisher Scientific) at a ratio of 1 μgram of DNA to 3 of Lipofectamine 2000 in a total volume of 2 mL of OptiMEM. A DNA-lipid complex formed during approximately 10 minutes of incubation. It was then added to the virus-infected BSC-40 cells.
BSC-40 cells were incubated for 48 hours to 72 hours to allow for homologous recombination to occur. Subsequently, the plates were scraped to lift virus-infected cells and the mixture was transferred to a conical tube. The cells were lysed following 3 rounds of freezing at −80° C. and thawing. An appropriate dilution, which can range from 1×10−2 to 1×10−5, of the infection/transfection mixture was plated onto BSC-40 cells followed by an agar overlay. Infected cell plates were incubated until non-fluorescent “recombinant” plaques were visualized. These non-fluorescent plaques were marked, and agar plugs were picked and added into a 10 mM Tris pH 8.0 solution. The plaques were subsequently used to infect BSC-40 cells in a second round of infection. This plaque picking process and infection of BSC-40 cells was repeated until YFP was undetectable in the infected cells (ranges between 4-6 rounds of plaque purification). One non-fluorescent plaque was isolated from the low efficiency of homologous recombination in the HPXV-infected cells.
PCR analysis using primers (sA2K/HPXV J2R Flank Forward Primer 5′-3′: TATCGCATTTTCTAACGTGATGG (SEQ ID NO: 58) and sA2K/HPXV J2R Flank Reverse Primer 5′-3′: CCTCATTTGCACTTTCTGGTTC (SEQ ID NO: 59)) that amplify the HPXV095 gene locus was performed to confirm that the SARS-Spike-co gene was inserted into the HPXV095 locus. The viral genomic DNA was subsequently isolated from a preparation of sucrose-purified virus particles and used in Next Generation Sequencing with the Illumina MiSeq platform. The sequence data was analyzed by de novo assembly and mapped to reference software using the CLC Genomics Workbench software (Qiagen).
The YFP-GPT selectable marker in the scHPXV (see Example 7) HPXV200 gene locus (also referred to as the Variola virus B22R homolog locus) was replaced using, for example, homologous recombination with a codon-optimized SARS-CoV-2 Spike (SARS-CoV-2-co) nucleotide sequence to generate the synthetic vaccinia virus TNX-1800b. One exemplary procedure is as follows.
Approximately 20 μgrams of plasmid containing SARS-CoV-2-Spike-co flanked by approximately 400 nucleotides homologous to the HPXV200 gene was linearized using the restriction enzyme, SacI. Following restriction enzyme digestion, the linearized plasmid was further purified to remove residual enzyme. BSC-40 cells were infected with scHPXV expressing YFP-GPT in the HPXV200 gene locus at a MOI of 0.1 for 1 hour. Following infection, the virus inoculum was replaced with OptiMEM media and incubated for an additional 30 minutes at 37° C. Approximately 5 μgrams of purified linearized plasmid was mixed with Lipofectamine 2000 (ThermoFisher Scientific) at a ratio of 1 μgram of DNA to 3 μL of Lipofectamine 2000 in a total volume of 2 mL of OptiMEM. A DNA-lipid complex formed during approximately 10 minutes of incubation. It was then added to the virus-infected BSC-40 cells.
BSC-40 cells were incubated for 48 hours to 72 hours to allow for homologous recombination to occur. Subsequently, the plates were scraped to lift virus-infected cells and the mixture was transferred to a conical tube. The cells were lysed following three rounds of freezing at −80° C. and thawing. An appropriate dilution, which can range from 1×10−2 to 1×10−5, of the infection/transfection mixture was plated onto BSC-40 cells followed by an agar overlay. Infected cell plates were incubated until non-fluorescent “recombinant” plaques were visualized. These non-fluorescent plaques were marked, and agar plugs were picked and added into a 10 mM Tris pH 8.0 solution. These plaques were subsequently used to infect BSC-40 cells in a second round of infection. One non-fluorescent plaque was isolated due to low efficiency of homologous recombination in HPXV-infected cells compared to VACV-infected cells. The plaque picking process was repeated by infecting BSC-40 cells until YFP was undetectable (about 4-6 rounds of plaque purification).
PCR analysis using primers (sHPXV 200 Flank Forward Primer 5′-3′: ATAGCCACAATTATTGACGGGC (SEQ ID NO: 60) and sHPXV 200 Flank Reverse Primer 5′-3′: ggatgatatggtaatgtaactaccgatac (SEQ ID NO: 61)) that amplify the HPXV200 gene locus was performed to confirm that the SARS-Spike-co gene was inserted into the HPXV200 locus. The viral genomic DNA was subsequently isolated from a preparation of sucrose-purified virus particles and used for Next Generation Sequencing with the Illumina MiSeq platform. The sequence was analyzed by de novo assembly and mapped to reference software using the CLC Genomics Workbench software (Qiagen).
Western blot analysis was performed to assess SARS-Spike-co protein expression in the BSC-40 cells infected with TNX-801, TNX-1800a (clone TNX-1800a-1) and TNX-1800b (clone TNX-1800b-2) (
Methods of immunization and testing candidate vaccines in African Green Monkeys has been previously described (Hartman, A. et al. (2020) SARS-CoV-2 infection of African green monkeys result in mild respiratory disease discernible by PET/CT imaging and shedding of infectious virus from both respiratory and gastrointestinal tracts. PLOS Pathogens 16(9): e1008903; incorporated herein by reference in its entirety). African Green Monkeys (AGMs) were randomly separated into 6 groups (n=4) and vaccinated with different strains of a synthetic horsepox virus (HPXV). See Table 5 for strain and dose. At day 0, AGMs were vaccinated percutaneously via scarification using a bifurcated needle.
The inoculation site of the AGMs was monitored and after 7 days presented with a cutaneous reaction, also known as a “take”, when vaccinated with TNX-801, TNX-1800b-2 or TNX-1800a-1 regardless of the dose eliciting an immune response, including a T cell immune response (
A Microneutralization Assay was performed 14 days after the AGMs were vaccinated with the indicated HPXV strains to assess the anti-SARS-CoV-2 neutralizing titers in the serum. The assay was initially performed in duplicate and a third replicate was performed if the first two replicates were not within a 2-fold dilution of each other. Serum samples were initially heat inactivated at 56° C. for 30-60 minutes after being aliquoted onto a master plate. The master plates can be stored at 4-8° C. for seven days or at −20° C. for three months.
Vero E6 cells (ATCC) at a concentration 2×104 cells per well were seeded into 96-well plates 18-24 hours before addition of the serum test samples. On the day of the assay, master plates were thawed and nine serum test samples were 2-fold serial diluted from 1:5 to 1:640 on a separate 96-well plate/dilution block (columns 1-9). Additionally, each 96-well plate/dilution block contained a positive control serum (column 10), virus controls (column 11) and cell controls (column 12). After dilution, an equal volume of virus stock (1,000 TCID50/mL) is added to columns 1-11. In addition, assay quality control (QC) plates were set up at the same time consisting of positive control serum (columns 1-2), a negative control (columns 3-4), viral input back titer (columns 5-6), virus control (VC; columns 7-9) and cell controls (CC; columns 10-12). At least two QC plate were used per assay. Test and QC plates were incubated at 37° C. for 2-2.5 hours in a 5% CO2 incubator. After incubation, aliquots of mixtures (sera and virus) for both test and QC plates (including controls) were transferred onto the 96-well plates pre-seeded with Vero E6 cell and incubated for 72±4 hours. Following incubation, plates were removed from the incubator and allowed to rest at room temperature for 20-40 minutes. 100 uL of Cell Titer-Glo (Promega) was added to all wells in the plates, gently mixed and incubated at room temperature for 10-30 minutes. Luminescence was read using an appropriate photometer. Plate cut-off values were calculated using the following formula:
(Average of VC wells+Average of CC wells)/2
Samples with luminescence above or below the plate cut-off are positive and negative for neutralizing antibody, respectively. The individual replicate is assigned a titer that is the reciprocal of the dilution of the last positive dilution (i.e., 1:80=is reported as a titer of 80). Titers are reported as median and geometric mean titers of the accepted replicate titers.
Table 6 shows the level of anti-SARS-CoV-2 neutralizing titers measured in vaccinated AGMs after 14 days of a single vaccination. The AGMs vaccinated with TNX-1800b-2 and TNX1800a-1 generated neutralizing titers (≥1:40 titer) of antibodies against SARS-CoV-2. The TNX-801 (an scHPXV not carrying the S protein expression cassette) vaccinated control animals and the placebo group did not generate anti-SARS-CoV-2 neutralizing titers (≤1:10 titer). Both the 2.9×106 PFU and 1.06×106 PFU doses of TNX-801 and TNX-1800 were well-tolerated.
BSC-40, HeLa and HEK 293 cells were seeded into a 6-well plate and subsequently infected with TNX-801, TNX-1800, TNX-1200, or TNX-2200 at a MOI of 0.01. After 48 hours of infection, cells were fixed and stained with 5% formaldehyde containing crystal violet. BSC-40 cells infected with TNX-801 and TNX-1800 had a significant cytopathic effect, while HeLa and HEK 293 cells showed minor and no cytopathic effect, respectively (
BSC-40 cells were infected with HPXV clones (e.g., _TNX-801, scHPXVΔ095yfp-gpt, TNX-1800a-1, scHPXVΔ200yfp-gpt, or TNX-1800b-2; (
The cytopathic effect seen in Vero cells and BSC-40 cells infected with the various HPXV and VACV clones shows that these cell lines can be used to manufacture the viruses (e.g., TNX-1800 and TNX-801).
As illustrated in
The location of an insertion site for the heterologous transgene SARS-CoV-2 Spike (S) within the DNA nucleotide sequence of a synthetic chimeric (sc) Horsepox genome is selected (for example the TK gene locus HPXV095; positions 992077-92610; SEQ ID NO:1). The DNA nucleotide sequences proximal to the left and right of the selected HPXV insertion site, which define the Left and Right Flanking arms, are identified (see
One exemplary DNA nucleotide sequence of approximately 6 kb for a SARS-CoV-2 Spike (S) synthetic expression cassette, comprising the DNA nucleotide sequences of a Left Flanking Arm, a vaccinia virus Early/Late Promotor operably linked to the modified CoVID-SARS-2 Spike (S) nucleic acid sequence, and a Right Flanking Arm is then synthesized (e.g., by a commercial vendor (e.g., Genewiz)). See
The SARS-CoV-2 Spike (S) Synthetic expression cassette DNA is then transfected into cells (e.g., BSC-40 cells) infected with an scHPXV. Recombinant horsepox viral progeny containing the SARS-CoV-2 Spike (S) synthetic expression cassette are selected using media containing BrdU so as to prevent viral amplification of the parental virus retaining the original insertion site viral genomic DNA sequences. The recombinant virus is purified using successive rounds of plaque purification. The nucleotide sequence from the purified virus across the entire SARS-CoV-2 Spike (S) heterologous transgene cassette is confirmed by sequence analysis (e.g., PCR sequence analysis). See SEQ ID NO: 63.
Similar constructs and steps can be carried out using a horsepox virus to generate a recombinant scHPXV containing a mouse adapted spike protein expression cassette (see SEQ ID NO: 64) and a vaccinia virus, using, for example, the vaccinia TK gene locus synVACV105; positions 83823-84344 (see SEQ ID NO: 2) to generate a recombinant vaccinia virus containing a mouse adapted spike protein expression cassette (see SEQ ID NO: 65).
At day 0, African Green Monkeys (AGMs) were vaccinated percutaneously via scarification using a bifurcated needle as described in Example 12. Table 7 shows the level of anti-SARS-CoV-2 neutralizing titers measured in vaccinated AGMs after 0, 7, 15, 21, 29, 41 and 47 days of a single vaccination. The AGMs vaccinated with TNX-1800b-2 and TNX1800a-1 generated neutralizing titers (≥1:40 titer) of antibodies against SARS-CoV-2. The TNX-801 (an scHPXV not carrying the S protein expression cassette) vaccinated control animals and the placebo group did not generate anti-SARS-CoV-2 neutralizing titers (≤1:10 titer). Both the 2.9×106 PFU and 1.06×106 PFU doses of TNX-801 and TNX-1800 were well-tolerated.
At day 41, the vaccinated AGMs were anesthetized and challenged (also referred to as inoculated) with approximately 2×106 TCID50/animal wild-type SARS-CoV-2 via the 1. intranasal and 2. intratracheal route. The volume of virus was split evenly between each of the two routes (1 mL per route with a 1×106 TCID50/mL virus stock). For the intranasal route, AGMs were anesthetized and inoculated by slowly pipetting 500 μL into each are followed by inhalation. For the intratracheal route, AGMs were anesthetized, and a tube was inserted into the trachea. After the end of the tube was situated approximately at the mid-point of the trachea, a syringe containing the inoculate with the virus was attached to the tube and the inoculate was slowly instilled into the trachea followed by an equal volume of PBS to flush the tube. After the AGMs were inoculated, the animal was returned to its home cage and monitored for recovery from the anesthesia.
An oropharyngeal swab specimen and a tracheal lavage specimen were collected on Day 41 and Day 47 from the inoculated AGMs. The specimens were processed by RT-qPCR methods to measure SARS-CoV-2 copy number. Table 8 shows the SARS-CoV-2 copy number from oropharyngeal swab specimens. Table 9 shows the SARS-CoV-2 copy number from tracheal lavage specimens. At Day 47, AGMs vaccinated with TNX-1800b-2 and TNX-1800a-1 developed protective immunity against SARS-CoV-2.
This application claims priority and benefit from U.S. Provisional Application No. 62/981,997, filed Feb. 26, 2020 and U.S. Provisional Application No. 63/114,514, filed Nov. 16, 2020, the contents of which are hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63114514 | Nov 2020 | US | |
| 62981997 | Feb 2020 | US |
