Patent Application
20240417697
Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and an immune system that shows unusual tolerance for viruses that cause life-threatening diseases in humans (e.g., SARS-CoVs, MERS-CoV, Ebola). Recent comparative genomic studies uncovered bat-specific changes to key immunity genes and exposed numerous integrated viral sequences, suggesting a particularly intimate and deep-rooted accord between bats and viruses. Still, what makes bats most distinctive is that they are home to the richest virosphere among mammals with some of the bat-related viruses causing significant outbreaks, including SARS, Ebola, and COVID-19. Remarkably, bats can be infected with viruses that are lethal to other mammals without causing any symptoms. Even more, the bat genome seems to act as a sponge for viral sequences. While endowed with a small genome, bats house a spacious number of ancient and contemporary viral insertions of retroviral and non-retroviral origin. Because some of the viral sequences are full length and even of non-bat origin, bats might supply an essential template for zoonotic viruses and act as super-spreaders. Nonetheless, how bats deal with viruses so well is poorly understood. It is clear that, although bats are a critically needed new model organism, limited access to animal and cell models has hindered their study. Bat breeding colonies are notoriously challenging to establish, and bat primary cell lines typically have a limited lifespan in vitro. Therefore, induced pluripotent stem cells would offer a research tool for bat research.
In one aspect, the disclosure provides a composition for an induced pluripotent bat stem cell (bat IPSC), wherein the cell is in a pluripotent state. In some embodiments the bat IPS cell is in a pluripotent state characterized by the expression of one or more factors for example of Klf4, Klf17, Essrb, Tfcp2l1, Tfe3, Dppa, Oct4, Sox2, Nanog, and Dusp6. In some embodiments, the IPSC cell is in a naïve pluripotent state. In some embodiments, the cell is characterized by the expression of one or more factors for example Otx2 or Zic2. In some embodiments the cell is a bat fibroblast or a bat embryonic fibroblast. In some embodiments the bat is a Rhinolophus bat or a Rhinolophus ferrumequinum bat, alternatively the bat is a Myotis bat or a Myotis myotis bat. In some embodiments, the IPS cell is capable of differentiating into embryonic bodies. In some embodiments, the embryonic bodies are capable of differentiating into three-dimensional structures comprising three germ layer markers.
In another aspect, the disclosure provides a method of producing induced pluripotent bat stem cells (bat IPSCs), the method comprising: (i) reprogramming isolated bat cells with Oct4, Sox2, cMyc, and Klf4 factors, (ii) culturing the reprogrammed cells on feeder cells in a medium comprising FGF, Leukemia inhibitory factor (Lif), SCF, and Forskolin until colonies appear; and (iii) splitting cells using a low concentration EDTA buffer; thereby producing IPSCs from bats. In some embodiments, the isolated bat cell is a fibroblast or an embryonic fibroblast. In some embodiments the cell is derived from a bat is a Rhinolophus bat or a Rhinolophus ferrumequinum bat, alternatively the bat is a Myotis bat or a Myotis myotis bet. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 U/ml. In some embodiments, the FGF is at a concentration of 100 ng/ml. In some embodiments, the SCF is at a concentration of 100 ng/ml. In some embodiments, the Forskolin is at a concentration of 20 nM. In some embodiments, the feeder cell is a mouse CFT mouse embryonic fibroblasts (MEF). In some embodiments, the method further comprises passaging the bat IPSCs every 5 days onto feeder cells. In some embodiments, the bat IPSC is further differentiated into embryonic bodies. In some embodiments, the embryonic bodies are further differentiated into three-dimensional structures comprising three germ layer markers.
In another aspect the disclosure provides a method of producing induced pluripotent bat stem cells (bat IPSCs), the method comprising: (i) reprogramming isolated bat cells with Oct4, Sox2, cMyc, and Klf4 factors; (ii) culturing the reprogrammed cells in feeder free medium comprising FGF, Leukemia inhibitory factor (Lif), SCF, and Forskolin until colonies appear; and (iii) splitting cells using a low concentration EDTA buffer thereby producing IPSCs from bats.
In another aspect the disclosure provides a composition for reprogramming a bat cell to produce pluripotent stem cells comprising a medium comprising FGF, Leukemia inhibitory factor (Lif), SCF, and Forskolin. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 U/ml. In some embodiments, the FGF is at a concentration of 100 ng/ml. In some embodiments the SCF is at a concentration of 100 ng/ml. In some embodiments, the Forskolin is at a concentration of 20 nM.
In another aspect the disclosure provides a method of obtaining viral sequences from bat IPSCs, the method comprising obtaining bat IPSCs; identifying viral sequences residing in the bat iPSC genome or intracellular virus genome; and assembling the viral sequences; thereby obtaining viral sequences from the bat iPSCs. In some embodiments, the identifying comprises sequencing the bat genome or the genome of viral particles residing in the bat IPSCs, or of viral particles shed by the bat IPSCs. In some embodiments, identifying comprises sequencing the RNA of the bat genome or the genome of viral particles residing in the bat IPSCs, or of viral particles shed by the bat IPSCs. In some embodiments, the identifying the proteins and peptides produced by the viral genome by proteomics e.g., LC-MS. In some embodiments, the method comprises translating the sequence into a protein sequence and determining whether the translated sequence has a significant homology to a known protein sequence in a viral protein database. In some embodiments, the sequence is selected from SEQ ID NO: 1-349. In some embodiments, the virus is selected from the group of a SARS-CoV-2 virus, endogenous retrovirus (RfRV), and sindbis virus. In some embodiments, the virus is a coronavirus. In some embodiments, the sequence encodes a gag protein, a pol protein, or an env protein.
In another aspect the disclosure provides a method of obtaining viral sequences from virus particles shed by bat IPSCs or cells derived from bat IPSCs, the method comprising obtaining bat IPSCs or cells derived from bat IPSCs; culturing the bat IPSCs or cells derived from bat IPSCs under conditions that allows shedding of virus particles into the culture media; collecting the culture media; identifying viral sequences residing in the culture media; and assembling the viral sequences, thereby obtaining viral sequences from virus particles shed by bat iPSCs or cells derived from bat IPSCs.
In another aspect the disclosure provides for the use of any one of the viral sequences described above for the development of a vaccine.
In another aspect the disclosure provides for a recombinant nucleic acid molecule, comprising a promoter, and a nucleic acid selected from SEQ ID NO: 1-349 encoding for a viral protein or fragment thereof. In some embodiments, a recombinant, replication deficient adenovirus, comprising nucleic acid described above is provided. In some embodiments, mRNA comprising the nucleic acid described above is provided.
In another aspect the disclosure provides for an expression vector comprising a promoter and a nucleic acid set forth in SEQ ID NO: 1-349 encoding for a viral protein or fragment thereof.
In another aspect the disclosure provides for an isolated protein or peptide comprising an amino acid sequence encoded in a nucleic acid set forth in SEQ ID NO: 1-349, wherein the peptide is no more than 100 amino acids in length, and an optional pharmaceutically acceptable carrier. In some embodiments, the protein or peptide is no more than 30 amino acids in length or 20 amino acids in length. In some embodiments, the protein or peptide is synthetic.
In another aspect the disclosure provides for a pharmaceutical composition comprising the adenovirus of described above, the mRNA described above, or the protein or peptide of any described above and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) proteins or peptides described above and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a nucleic acid encoding the mRNA described above or the protein or peptide described above and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises one or more nucleic acids encoding a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mRNAs of described above or proteins or peptides of described above, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises a liposome, wherein the protein or peptide or the nucleic acid encoding the protein or peptide is disposed within the liposome. In some embodiments, the pharmaceutical composition further comprises a lipid nanoparticle, wherein the protein or peptide or the nucleic acid encoding the protein or peptide is disposed within the lipid nanoparticle. In some embodiments, the pharmaceutical composition comprises an immunogenicity enhancing adjuvant.
In another aspect the disclosure provides for a vaccine that stimulates a T cell mediated immune response when administered to a subject, the vaccine comprising the pharmaceutical composition described above. In some embodiments, the vaccine is a priming vaccine and/or a booster vaccine.
In another aspect the disclosure provides for a recombinant cell comprising a nucleic acid or a portion of a nucleic acid set forth in SEQ ID NO: 1-349. In some embodiments, the recombinant cell comprises a protein or a portion of a protein encoded by a nucleic acid set forth in SEQ ID NO: 1-349.
In another aspect the disclosure provides for a composition comprising an inhibitor of a protein encoded by a nucleic acid selected from SEQ ID NO: 1-349.
For a fuller understanding of the nature and advantages of the present disclosure, reference should be had to the ensuing detailed description taken in conjunction with the accompanying figures. The present disclosure is capable of modification in various respects without departing from the present disclosure. Accordingly, the figures and description of these embodiments are not restrictive.
These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings, where:
Various features and aspects of the disclosure are discussed in more detail below.
The disclosure is based, in part, upon the discovery that induced pluripotent bat stem cells can be produced and are stable in culture, readily differentiate into all three germ layers, and form complex embryoid bodies, including organoids. Bat iPSCs (BiPS) and their differentiated progeny can be used for example as an accessible and versatile tool required to advance bats as a new model system. Further, BiPS can provide the platform to further understand the role bats play as virus reservoirs and enable new insights into emerging viruses, such as SARS-CoV-2, and better prepare for future pandemics. BiPS can enable studies that directly impact every aspect of bats' particular biology, including this mammal's unique adaptations of flight, echolocation, extreme longevity, and unique immunity. Further, BiPS are also useful for example in understanding of bats' asymptomatic response to viral pathogens.
Accordingly, the disclosure provides BiPS, methods of producing and using BiPS, and compositions for reprogramming bat cells.
In another aspect, the disclosure is based in part on the discovery of viruses and viral nucleic acids and proteins in BiPS. The viruses, viral nucleic acids, viral proteins, viral nucleic acid sequences, and protein sequences are useful in the development of therapeutics and prophylactics for viral diseases, such as vaccines, antibodies, and small molecule antivirals.
Accordingly, the disclosure provides viral nucleic acid and protein sequences, expression constructs, vectors comprising the expression constructs, methods of making and using therapeutics and prophylactics against viral diseases such as vaccines, antibodies, and small molecule antivirals.
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 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, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).
In general, terms used in the claims and the specification are intended to be construed as having the plain meaning understood by a person of ordinary skill in the art. Certain terms are defined below to provide additional clarity. In case of conflict between the plain meaning and the provided definitions, the provided definitions are to be used.
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.
It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.
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” and “an” 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.
Where aspects or embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members in an embodiment of the disclosure.
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 disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R. I. Freshney, ed. (2010)) (which is entirely incorporated by reference herein).
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.
As used herein, “residue” refers to a position in a protein and its associated amino acid identity.
As used herein the term “antigen” is a substance that induces an immune response. An antigen can be a neoantigen.
As used herein the term “antigen-based vaccine” is a vaccine composition based on one or more antigens, e.g., a plurality of antigens. The vaccines can be nucleotide-based (e.g., virally based, RNA based, or DNA based), protein-based (e.g., peptide based), or a combination thereof.
As used herein the term “coding region” is the portion(s) of a gene that encode protein.
As used herein the term “coding mutation” is a mutation occurring in a coding region.
As used herein the term “ORF” means open reading frame.
As used herein the term “epitope” is the specific portion of an antigen typically bound by an antibody or T cell receptor.
As used herein the term “immunogenic” is the ability to elicit an immune response, e.g., via T cells, B cells, or both.
As used herein the term “HLA binding affinity” “MHC binding affinity” means affinity of binding between a specific antigen and a specific MHC allele.
As used herein the term “ELISPOT” means Enzyme-linked immunosorbent spot assay—which is a common method for monitoring immune responses in humans and animals.
The term “lipid” includes hydrophobic and/or amphiphilic molecules. Lipids can be cationic, anionic, or neutral. Lipids can be synthetic or naturally derived, and in some instances biodegradable. Lipids can include cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethylenegly col (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, fats, and fat-soluble vitamins. Lipids can also include dilinoleylmethyl-4-dimethylaminobutyrate (MC3) and MC3-like molecules.
The term “lipid nanoparticle” or “LNP” includes vesicle like structures formed using a lipid containing membrane surrounding an aqueous interior, also referred to as liposomes. Lipid nanoparticles includes lipid-based compositions with a solid lipid core stabilized by a surfactant. The core lipids can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants. Biological membrane lipids such as phospholipids, sphingomyelins, bile salts (sodium taurocholate), and sterols (cholesterol) can be utilized as stabilizers. Lipid nanoparticles can be formed using defined ratios of different lipid molecules, including, but not limited to, defined ratios of one or more cationic, anionic, or neutral lipids. Lipid nanoparticles can encapsulate molecules within an outer-membrane shell and subsequently can be contacted with target cells to deliver the encapsulated molecules to the host cell cytosol. Lipid nanoparticles can be modified or functionalized with non-lipid molecules, including on their surface. Lipid nanoparticles can be single-layered (unilamellar) or multi-layered (multilamellar). Lipid nanoparticles can be complexed with nucleic acid. Unilamellar lipid nanoparticles can be complexed with nucleic acid, wherein the nucleic acid is in the aqueous interior. Multilamellar lipid nanoparticles can be complexed with nucleic acid, wherein the nucleic acid is in the aqueous interior or and/or can be sandwiched between the layers.
Unless specifically stated or otherwise apparent from context, as used herein the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
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., methylphosphonates, 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)NRi (“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 (—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.
The term “expression”, as used herein, generally refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
As used herein, “operably linked”, “operable linkage”, “operatively linked”, or grammatical equivalents thereof generally refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a regulatory element, which may comprise promoter and/or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.
A “vector” as used herein, generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. The vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.
As used herein, “an expression cassette” and “a nucleic acid cassette” are used interchangeably generally to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression. In some cases, an expression cassette refers to the combination of regulatory elements and a gene or genes to which they are operably linked for expression.
As used herein, the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
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.
“Percent (%) sequence identity” or “percent (%) 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. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alternatively, sequence similarity or dissimilarity can be established by the combined presence or absence of particular nucleotides, or, for translated sequences, amino acids at selected sequence positions (e.g., sequence motifs).
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
“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.
However, 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.
The term “transgene” refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and/or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA.
As used herein, “isolated molecule” (where the molecule is, for example, a polypeptide, a polynucleotide, or fragment thereof) is a molecule that by virtue of its origin or source of derivation (1) is not associated with one or more naturally associated components that accompany it in its native state, (2) is substantially free of one or more other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature.
The term “subject” encompasses a cell, tissue, or organism, human or non-human, whether in vivo, ex vivo, or in vitro, male or female. The term subject is inclusive of mammals including humans.
The term “mammal” encompasses both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, pteropines, and porcines.
As used herein, a “vector,” refers to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo. A “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e. a nucleic acid sequence not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid is flanked by at least one inverted terminal repeat sequence (ITR). In some embodiments, the recombinant nucleic acid is flanked by two ITRs.
The phrase “pharmaceutical composition” refers to a mixture containing a specified amount of a therapeutic, e.g., a therapeutically effective amount, of a therapeutic compound in a pharmaceutically acceptable carrier to be administered to a mammal, e.g., a human, in order to treat a disease.
The phrase “pharmaceutically acceptable carrier” means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
Each embodiment described herein may be used individually or in combination with any other embodiment described herein.
The disclosure is based, in part, upon the discovery that bat induced pluripotent stem cells (iPSC) (BiPS) can be produced and are stable in culture, proliferate, readily differentiate into all three germ layers, and form complex embryoid bodies, including organoids.
Accordingly, compositions and methods of making and using the BiPS are provided herein.
In some embodiments, BiPS are provided. In some embodiments the pluripotent state of the BiPS is characterized by the expression of one or more factors selected from the group of Klf4, Klf17, Essrb, Tfcp2l1, Tfe3, Dppa, Oct4, Sox2, Nanog, and Dusp6. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10 factors are expressed in the BiPS. Pluripotent stem cells can be classified into at least naïve and primed stem cell states based on the growth characteristics in vitro and their potential rise to all somatic lineages and the germ line in chimeras. In some embodiments, the BiPS are in a naïve pluripotent state. In some embodiments, the BiPS are further characterized by the expression pf one or more factors for example Otx2 or Zic2.
Bats are divided in two groups: fruit-eating megabats, and the echolocating microbats. Megabats are further divided into Yinpterochiroptera that include the Pteropodidae, or megabat family, as well as the family of Rhinolophoidea, and Yangochiroptera. Rhinolophoidea can be further divided into Hipposideridae, Craseonycteridae, Megadermatidae, Rhinopomatidae and Rhinolophidae. In some embodiments, the BiPS can be derived from isolated source bat cells from embryonic, young, or adult bats. In some embodiments, the bat is a Rhinolophus bat. In some embodiments the bat is a wild horseshoe bat (Rhinolophus ferrumequinum). In some embodiments, the bat is a Myotis bat or a Myotis myotis bat. In some embodiments, embryonic fibroblasts (BEF) cells can be isolated from the bat. In some embodiments, adult fibroblasts cells can be isolated from the bat.
A BiPS of the disclosure may be isolated, substantially isolated, purified or substantially purified. The iPSC is isolated or purified if it is completely free of any other components, such as culture medium, other cells of the disclosure or other cell types. The iPSC is substantially isolated if it is mixed with carriers or diluents, such as culture medium, which will not interfere with its intended use. Alternatively, the iPSC of the disclosure may be present in a growth matrix or immobilized on a surface as discussed below.
In some embodiments, the BiPS are further differentiated into embryonic bodies. In some embodiments, the BiPS can be further differentiated into endoderm (Afp+), ectoderm (Tbxt+), and mesoderm (Pax6+). The embryonic bodies derived from the BiPS can be further differentiated into three-dimensional structures comprising the three germ layer markers.
Techniques for producing and culturing iPSCs are well known to a person skilled in the art. Suitable conditions are discussed below.
The one aspect, the disclosure also provides a method of producing a population of BiPS, comprising culturing source bat cells under conditions which reprogram the source bat cells to produce the BiPS. Any of the source bat cells discussed above may be used.
Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell that can be generated (reprogrammed) from a non-pluripotent cell of a multicellular organism, such as a somatic cell. iPSCs are characterized in that they propagate indefinitely and can differentiate into the three germ layers endoderm, mesoderm and ectoderm, form embryonic bodies, develop into teratomas in vivo, and can form fully differentiated tissues including but not limited to neurons, cardiomyocytes, hepatocytes, and immune cells. Typically, iPSCs express a group of markers for stem cells on the surface of the cell such as SSEA-4, TRA-1-60, and CD30, though expressed markers and timing of expression for the markers can vary (for example as described in Pomeroy et al., Stem Cells Transl Med. (2016) 5(7): 870-882). Recently, two protocols to produce bat reprogrammed stem cells were published (Mo et al., Theriogenology (2014)15; 82(2):283-93, Aurine et al., BioRxiv (2019)). However, neither of the protocols provides for BiPS that are able to differentiate into the three germ layers or form embryonic bodies or teratomas in vivo. Thus, lack of access to robust cell models has hindered further understanding of bat asymptomatic response to viral pathogens.
To establish bats as new model study species, initially the Yamanaka reprogramming protocol based on four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) (Takahashi K. et al., Cell (2006) 25; 126(4):663-76, and. Hochedlinger K. et al., Cold Spring Harb Perspect Biol. (2015) 7(12): a019448), that is highly effective in mice, humans, and other mammalian species (e.g., dog, pig, marmoset) was tried to produce induced pluripotent stem cells (iPSCs) from a wild horseshoe bat (Rhinolophus ferrumequinum). However, the protocol failed to produce BiPS that were stable in culture, and that proliferated. Though the protocols failed, the Yamanaka factors triggered the formation of rudimentary stem cell-like colonies even though they ceased to expand.
Here, methods of making BiPS are provided that overcome these problems.
The method preferably comprises culturing the source bat cells with a Sendai virus system, a retroviral system, a lentiviral system, microRNA or other reprogramming factors which is/are capable of reprogramming the source bat cells to produce the BiPS. In some embodiments, the method of making bat iPSCs comprises (i) reprogramming isolated bat cells with Oct4, Sox2, cMyc, and Klf4 factors; (ii) culturing the reprogrammed cells in a medium comprising FGF, Leukemia inhibitory factor (Lif), SCF, and Forskolin until colonies appear; and (iii) splitting cells using a low concentration EDTA buffer.
In some embodiments, the reprogramming factors can be delivered to the bat cells with viruses such as a Sendai virus, retrovirus, AAV, nonviral vector systems, physical delivery, mechanical and chemical methods, or with mRNA delivery. In some embodiments, the reprogramming factors comprise Oct4, Sox2, cMyc, and Klf4 factors. In some embodiments, the reprogramming factors comprise additional factors.
In some embodiments, the method comprises culturing the cells in a feeder free medium. In some embodiments, the cells can be cultured on feeder cells, such as CFT mouse embryonic fibroblasts.
In some embodiments, the feeder cell free or the feeder cell culture medium comprises FGF, Leukemia inhibitory factor (Lif), SCF, and Forskolin. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 U/ml. In some embodiments, the FGF is at a concentration of 100 ng/ml. In some embodiments, the SCF is at a concentration of 100 ng/ml. In some embodiments, the Forskolin is at a concentration of 20 nM. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 U/ml, the FGF is at a concentration of 100 ng/ml, the SCF is at a concentration of 100 ng/ml and the Forskolin is at a concentration of 20 nM. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 to 10≡U/ml. In some embodiments, the FGF is at a concentration of 100 ng/ml. In some embodiments, the SCF is at a concentration of 10-100 ng/ml. In some embodiments, the Forskolin is at a concentration of 5-20 nM. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 to 10≡U/ml, the FGF is at a concentration of 4-100 ng/ml, the SCF is at a concentration of 10-100 ng/ml and the Forskolin is at a concentration of 5-20 nM. In some embodiments, the concentration of Lif is 40%, 30%, 20%, 10%, or 5% more or less than 10{circumflex over ( )}4 U/ml. In some embodiments, the concentration of FGF is 40%, 30%, 20%, 10%, or 5% more or less than 100 ng/ml. In some embodiments, the concentration of SCF is 40%, 30%, 20%, 10%, or 5% more or less than 100 ng/ml. In some embodiments, the concentration of Forskolin is 40%, 30%, 20%, 10%, or 5% more or less than 20 nM. In some embodiments, the concentration of Lif is about 10{circumflex over ( )}4 U/ml. In some embodiments, the concentration of FGF is about 100 ng/ml. In some embodiments, the concentration of SCF is about 100 ng/ml. In some embodiments, the concentration of Forskolin is about 20 nM.
In some embodiments, the BiPS are passaged, i.e. moved into fresh media. In some embodiments the BiPS are passaged every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the BiPS are passaged every 5 days. In some embodiments, the BiPS are passaged when they are 50%, 60%, 70%, 80%, 90%, or 100% confluent. In some embodiments, the BiPS are passaged before they are confluent. In some embodiments, the feeder cells are freshly changed every passage. In some embodiments, the feeder cells are irradiated. In some embodiments, the BiPS are passaged using a low concentration EDTA buffer. In some embodiments, the BiPS are passaged using a low concentration EDTA buffer with a EDTA concentration less than 0.48 mM EDTA. In some embodiments the BiPS can be passaged indefinitely. In some embodiments the BiPS can be passaged at least to passage 78.
In some embodiments, the BiPS are further differentiated into embryonic bodies. In some embodiments, the BiPS can be further differentiated into endoderm (Afp+), ectoderm (Tbxt+), and mesoderm (Pax6+). The embryonic bodies can be further differentiated into three-dimensional structures comprising the three germ layer markers.
In some embodiments, a medium is provided that is conducive to producing and maintaining BiPS comprising FGF, Leukemia inhibitory factor (Lif), SCF, and Forskolin. In some embodiments, the medium comprises FGF at a concentration of 20 nM, Leukemia inhibitory factor (Lif) at a concentration of 10{circumflex over ( )}4 U/ml, SCF at a concentration of 100 ng/ml, and Forskolin at a concentration of 100 ng/ml. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 U/ml, the FGF is at a concentration of 100 ng/ml, the SCF is at a concentration of 100 ng/ml and the Forskolin is at a concentration of 20 nM. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 to 10≡U/ml. In some embodiments, the FGF is at a concentration of 100 ng/ml. In some embodiments, the SCF is at a concentration of 10-100 ng/ml. In some embodiments, the Forskolin is at a concentration of 5-20 nM. In some embodiments, the Lif is at a concentration of 10{circumflex over ( )}4 to 10≡U/ml, the FGF is at a concentration of 4-100 ng/ml, the SCF is at a concentration of 10-100 ng/ml and the Forskolin is at a concentration of 5-20 nM. In some embodiments, the medium comprises FGF at a concentration of 40%, 30%, 20%, 10%, or 5% more or less than 20 nM, Leukemia inhibitory factor (Lif) at a concentration of 40%, 30%, 20%, 10%, or 5% more or less than 10{circumflex over ( )}4 U/ml, SCF at a concentration of 40%, 30%, 20%, 10%, or 5% more or less than 100 ng/ml, and Forskolin at a concentration of 40%, 30%, 20%, 10%, or 5% more or less than 100 ng/ml.
An important method for reprogramming is the use of messenger RNA specific for the reprogramming factors since this does not involve any genetic modification of the cells and the risk of tumorigenesis. Another method is to produce from the reprogramming genes, recombinant proteins modified to permit their penetration of the plasma and nuclear membranes. Other reprogramming factors include, but are not limited to, small compounds synthesized through medicinal chemistry.
The method preferably further comprises isolating clonal lines of BiPS of the disclosure. For instance, the method preferably further comprises isolating clonal lines of BiPS of the disclosure by limiting dilution or the manual ‘picking’ of individual colonies.
Standard methods known in the art may be used to determine the detectable expression and level of expression of the various markers discussed above. Suitable methods include, but are not limited to, immunocytochemistry, flow cytometry, western blotting and quantitative PCR.
Provided herein are also methods and compositions for using the viruses and viral sequences identified herein from the bat pluripotent stem cells. In particular, viruses, viral families, and viral sequences are disclosed herein.
In some embodiments, the method of obtaining viral sequences from bat IPSCs, comprises obtaining bat IPSCs; identifying viral sequences residing in the bat iPSC genome or intracellular virus genome; and assembling the viral sequences. In some embodiments, the bat IPSCs (BiPS) are produced by the methods described above. In some embodiments, the nucleic acid sequences are obtained by sequencing RNA transcripts such as RNA seq, long read sequencing such ss Iso-seq (PacBio), or sequencing the genomic DNA such as by DNA sequencing of samples derived from the BiPS. In some embodiments, amino acid sequences can be obtained by LC-MS or amino acid sequencing of samples derived from the BiPS. In some embodiments the samples can be derived directly from the BiPS or the medium BiPS were grown in. In some embodiments, the samples can be derived from differentiated cells derived from the BiPS.
In some embodiments, the obtained nucleic acid sequences are assembled into longer nucleic acid sequences. Short and long assembled sequences can be classified as potentially viral origin or non-viral origin for example as described in Example 10. The sequences can be further classified into virus clades by comparing with known sequences from virus nucleic acids in databases such as the NCBI Assembly database (www.ncbi.nlm.nih.gov/assembly) or Virus Pathogen Resource (www.viprbrc.org/brc/home.spg?decorator=vipr). Nucleic acid sequences can be also classified using metagenomic classifiers, such as Kraken2.
TABLE 1 Exemplary virus families and viruses found in a taxonomic distribution of virome reads from BiPS as determined by the metagenomic classifier Kraken2.
More exemplary viral families, viruses and sequences identified from the BiPS are shown in TABLE A.
In some embodiments the nucleic acid sequences are derived from sequencing transcripts derived from the BiPS by Iso-seq. Exemplary Iso-Seq derived sequences are set forth in SEQ ID NO: 1-7. The sequences can be classified using Kraken 2. Exemplary Kraken 2 classification of Iso-Seq derived sequences and bat genome sequences are presented in TABLE 2. Exemplary full-length retrovirus sequence identified are RFe-V-MD1, RFe-V-MD2 RFe-V-MD3 RFe-V-MD4, and RFe-V-MD5, set forth in SEQ ID NO: 1-7. A detailed analysis of the sequence of RFe-V-MD1 is shown in
In some embodiments, exemplary nucleic acid sequences and an alignment with known viruses such as Scotophilus bat coronavirus 512 are shown in TABLE 3 and RaTG13 bat coronavirus are shown in TABLE 4.
Other viral sequences such as presented in TABLE 3 and TABLE 4, or SEQ ID NO: 1-349 can be identified. Translated into amino acid sequences, and aligned with known viral sequences as described herein.
Methods for identifying antigens (e.g., antigens derived from an infectious disease organism) include identifying antigens that are likely to be presented on a cell surface (e.g., presented by MHC on an infected cell or an immune cell, including professional antigen presenting cells such as dendritic cells), and/or are likely to be immunogenic. As an example, one such method may comprise the steps of: obtaining at least one of exome, transcriptome or whole genome nucleotide sequencing and/or expression data from an infected cell or an infectious disease organism (e.g., RFe-V-MD1, RFe-V-MD2 RFe-V-MD3 RFe-V-MD4, and RFe-V-MD5, Columbid/Falconid herpesvirus, and Sindbis virus), wherein the nucleotide sequencing data and/or expression data is used to obtain data representing peptide sequences of each of a set of antigens (e.g., antigens derived from the infectious disease organism); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical likelihoods that each of the antigens is presented by one or more MHC alleles on a cell surface, such as an infected cell of the subject, the set of numerical likelihoods having been identified at least based on received mass spectrometry data; and selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens. Antigens can include nucleotides or polypeptides. For example, an antigen can be an RNA sequence that encodes for a polypeptide sequence. Antigens useful in vaccines can therefore include nucleotide sequences or polypeptide sequences. Antigens can be selected that are predicted to be presented on the cell surface of a cell, such as an infected cell or an immune cell, including professional antigen presenting cells such as dendritic cells. Antigens can be selected that are predicted to be immunogenic. Exemplary antigens predicted using the methods described herein to be presented on the cell surface by an MHC include predicted MHC class I epitopes and predicted MHC class II epitopes. Exemplary nucleic acid sequences or polypeptide sequences for antigen prediction are presented in SEQ ID NO: 1-349,
Protein sequences for the desired antigen are analyzed for potential HLA specific antigens by using for example the SYFPEITHI algorithm (Rammensee et al. (1999) Immunogenetics 50:213-219), and the artificial neural network (ANN) and stabilized matrix method (SMM) algorithms from IEDB (Peters et al. (2005) PLoS Biol. 3:e91). Peptides are selected based on a predicted binding value of either >21 for SYFPEITHY, <6000 for ANN, or <600 for SMM. Selected peptides are synthesized.
Binding assays can be performed using a fluorescence polarization (FP) assay as previously described (e.g., Buchi et al. (2004) Biochemistry 43:14852-14863; Sette et al. (1994) Mol. Immunol. 31:813-822). To determine binding capacity of the peptides, percentage inhibition relative to controls can be determined in an FP competition assay with the placeholder peptide.
In some embodiments, the peptides bound to the pMHC multimers are from an unbiased library of peptides derived from the antigen. In some embodiments, the peptides are 9-mers. In some embodiments, the peptides bound to the pMHCI multimers are 9-mers which include an HLA-A2 binding motif with key amino acids at positions 2 and 9 which can include isoleucine (I), valine (V) or leucine (L).
In some embodiments, the library comprises all k-mer peptides produced by transcription and translation of any polynucleotide sequence of interest, for example, in silico production of the transcription and translation products of both the forward and reverse strands of a genome or metagenome in all six reading frames.
In some embodiments, a library of the disclosure comprises all k-mer peptides that can be derived from in silico translation of an exome of interest. In some embodiments, a library of the disclosure comprises all k-mer peptides that can be derived from in silico translation of a transcriptome of interest. In some embodiments, a library of the disclosure comprises all k-mer peptides that can be derived from a proteome of interest. In some embodiments, a library of the disclosure comprises all k-mer peptides that can be derived from in silico translation of an ORFeome of interest. In some embodiments, an algorithm can be used to select peptides in a peptide library. For example, an algorithm can be used to predict peptides most likely to fold or dock in an MHC/HLA binding pocket, and peptides above a certain threshold value can be selected for inclusion in the library.
In some embodiments, a library of the disclosure comprises all peptides that can be derived from in silico transcription and translation or translation of a group of genomes, proteomes, transcriptomes, ORFeomes, or any combination thereof. In some embodiments, the peptides are derived from in silico transcription and translation or translation of polynucleotide sequences from a group of samples, for example, clinical samples from a patient population, or a group of pathogen genomes.
One or more polypeptides encoded by an antigen nucleotide sequence can comprise at least one of: a binding affinity with MHC with an IC50 value of less than 1000 nM, for MHC Class I peptides a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, presence of sequence motifs within or near the peptide promoting proteasome cleavage, and presence or sequence motifs promoting TAP transport. For MHC Class II peptides a length 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, presence of sequence motifs within or near the peptide promoting cleavage by extracellular or lysosomal proteases (e.g., cathepsins) or HLA-DM catalyzed HLA binding.
One or more antigens can be presented on the surface of an infected cell (e.g., a., RFe-V-MD1, RFe-V-MD2 RFe-V-MD3 RFe-V-MD4, and RFe-V-MD5, Columbid/Falconid herpesvirus, or Sindbis virus infected cell).
One or more antigens can be immunogenic in a subject having or suspected to have an infection (e.g., a RFe-V-MD1, RFe-V-MD2 RFe-V-MD3 RFe-V-MD4, and RFe-V-MD5, Columbid/Falconid herpesvirus, or Sindbis virus infection), e.g., capable of eliciting a T cell response or a B cell response in the subject. One or more antigens can be immunogenic in a subject at risk of an infection (e.g., a RFe-V-MD1, RFe-V-MD2 RFe-V-MD3 RFe-V-MD4, and RFe-V-MD5, Columbid/Falconid herpesvirus, or Sindbis virus infection), e.g., capable of eliciting a T cell response or a B cell response in the subject that provides immunological protection (i.e., immunity) against the infection, e.g., such as stimulating the production of memory T cells, memory B cells, or antibodies specific to the infection.
One or more antigens can be capable of eliciting a B cell response, such as the production of antibodies that recognize the one or more antigens (e.g., antibodies that recognize a RFe-V-MD1, RFe-V-MD2 RFe-V-MD3 RFe-V-MD4, and RFe-V-MD5, Columbid/Falconid herpesvirus, and Sindbis virus antigen and/or virus). Antibodies can recognize linear polypeptide sequences or recognize secondary and tertiary structures. Accordingly, B cell antigens can include linear polypeptide sequences or polypeptides having secondary and tertiary structures, including, but not limited to, full-length proteins, protein subunits, protein domains, or any polypeptide sequence known or predicted to have secondary and tertiary structures. In general, antigens capable of eliciting a B cell response to an infection are antigens found on the surface of an infectious disease organism (e.g., RFe-V-MD1, RFe-V-MD2 RFe-V-MD3 RFe-V-MD4, and RFe-V-MD5, Columbid/Falconid herpesvirus, and Sindbis virus). Exemplary antigens capable of eliciting a B cell response include, but are not limited to, ORF1ab, spike (S), envelope (E), membrane (M), and nucleocapsid (N).
One or more antigens that induce an autoimmune response in a subject can be excluded from consideration in the context of vaccine generation for a subject.
The size of at least one antigenic peptide molecule (e.g., an epitope sequence) can comprise, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or greater amino molecule residues, and any range derivable therein. In specific embodiments the antigenic peptide molecules are equal to or less than 50 amino acids.
Antigenic peptides and polypeptides can be: for MHC Class I 15 residues or less in length and usually consist of between about 8 and about 11 residues, particularly 9 or 10 residues; for MHC Class II, 6-30 residues, inclusive.
In some embodiments, a recombinant cell is provided comprising a nucleic acid or polypeptide set forth in SEQ ID NO: 1-349. The recombinant cells can be used in therapeutic development, such as vaccines, small molecules and biologics. In some embodiments, a recombinant cell is provided comprising a nucleic acid or protein or part thereof set forth in
The present disclosure also features pharmaceutical compositions that contain a therapeutically effective amount of one or more T cell epitopes, nucleic acids coding for T cells epitopes or peptides. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation.
In various embodiments, the pharmaceutical compound includes an acceptable pharmaceutically acceptable carrier. The carrier(s) should be “acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the subject. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. In one embodiment the pharmaceutical composition is administered orally and includes an enteric coating suitable for regulating the site of absorption of the encapsulated substances within the digestive system or gut.
Pharmaceutical compositions containing a therapeutic, such as those disclosed herein, can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).
Pharmaceutical formulations, in some embodiments, are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
Disclosed herein is an immunogenic composition, e.g., a vaccine composition, capable of raising a specific immune response, e.g., a tumor-specific immune response. Vaccine compositions typically comprise a plurality of viral antigens, e.g., selected using a method described herein. Vaccine compositions can also be referred to as vaccines.
The viral nucleic acids, proteins, antigens, and T cell epitopes can be used to design prophylactic or therapeutic vaccines comprising such composition (e.g., pharmaceutical compositions) for immunizing subjects at risk of contracting, or subjects having already contacted, a virus set forth in TABLE 1 or TABLE A. In certain embodiments, the vaccine is a subunit vaccine. In certain embodiments, the vaccine elicits a protective immune reaction against a plurality of viruses (e.g., RFe-V-MD1, RFe-V-MD2 RFe-V-MD3 RFe-V-MD4, or RFe-V-MD5). In certain embodiments, the vaccine elicits a protective immune reaction against a virus set forth in TABLE 1 or TABLE A.
In some embodiments, the vaccine comprises a recombinant nucleic acid molecule comprising one or more promoter and a nucleic acid encoding for a T cell epitope. In some embodiments the nucleic acid is set forth in SEQ ID NO: 1-349, TABLE 3, TABLE 4, or a functional portion thereof.
A vaccine composition of the disclosure can comprise a peptide composition(s) comprising the T cell epitope(s). Alternatively, a vaccine composition of the disclosure can comprise a nucleic acid composition, e.g., an RNA composition or DNA composition, encoding the T cell epitope(s). For such nucleic acid vaccines, suitable regulatory sequences are included such that the peptide epitope is expressed from the nucleic acid (RNA or DNA) in cells of the subject being immunized. In some embodiments, the nucleic acids or the peptides are synthetic.
A vaccine can contain between 1 and 30 peptides, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different peptides, 6, 7, 8, 9, 10 11, 12, 13, or 14 different peptides, or 12, 13 or 14 different peptides. Peptides can include post-translational modifications. A vaccine can contain between 1 and 100 or more nucleotide sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different nucleotide sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different nucleotide sequences, or 12, 13 or 14 different nucleotide sequences. A vaccine can contain between 1 and 30 viral antigen sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different viral antigen sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different viral antigen sequences, or 12, 13 or 14 different viral antigen sequences.
In some embodiments, the pharmaceutical composition comprises a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) proteins or peptides and a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition comprising a nucleic acid encoding the mRNA of claim 44 or the protein or peptide of any one of claims 46-48 and a pharmaceutically acceptable carrier or excipient.
In some embodiments, the pharmaceutical composition comprises one or more nucleic acids encoding a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mRNAs and a pharmaceutically acceptable carrier or excipient.
In one embodiment, antigens or T cell epitopes are for example ORF1ab, spike (S), envelope (E), membrane (M) and nucleocapsid (N), RNA polymerases, kinases, and viral proteases. Exemplary antigens are shown in
In certain embodiments, the two or more of the T cell peptides collectively recognize MHC molecules in at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the human population. In certain embodiments, the vaccine contains individualized components according to the personal need (e.g., MHC variants) of the particular patient.
In one embodiment, different peptides and/or polypeptides or nucleotide sequences encoding them are selected so that the peptides and/or polypeptides capable of associating with different MHC molecules, such as different MHC class I molecule. In some aspects, one vaccine composition comprises coding sequence for peptides and/or polypeptides capable of associating with the most frequently occurring MHC class I molecules. Hence, vaccine compositions can comprise different fragments capable of associating with at least 2 preferred, at least 3 preferred, or at least 4 preferred MHC class I molecules.
The vaccine composition can be capable of raising a specific cytotoxic T-cell response and/or a specific helper T-cell response.
A vaccine composition of the disclosure can comprise one or more short (e.g., 8-35 amino acids) peptides as the immunostimulatory agent. In certain embodiments, a cell surface antigen sequence is incorporated into a larger carrier polypeptide or protein, to create a chimeric carrier polypeptide or protein that comprises the T cell epitope(s). This chimeric carrier polypeptide or protein can then be incorporated into the vaccine composition.
Recombinant cells can be engineered to express proteins and peptides of the disclosure. Vectors can be designed for the expression of cell surface antigens (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, cell surface antigens can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. The cell surface antigens can be purified from the recombinant cells and used in antibody development or further formulated into pharmaceutical compositions. Additionally or alternatively, the recombinant cells expressing the cell surface antigens can be used for producing antibodies or T cells specific to the cell surface antigens.
It is understood that a peptide can be expressed from a nucleic acid (e.g., an mRNA) in a cell of the subject. Exemplary methods of producing peptides by translation in vitro or in vivo are described in U.S. Patent Application Publication No. 2012/0157513 and He et al., J. Ind. Microbiol. Biotechnol. (2015) 42(4):647-53. The present disclosure provides a composition (e.g., pharmaceutical composition) comprising one or more nucleic acids (e.g., mRNAs) encoding one or more cell surface antigens or derived peptides. It is understood that a peptide can be expressed from a nucleic acid (e.g., an mRNA) in a cell of the subject. Exemplary methods of producing peptides by translation in vitro or in vivo are described in U.S. Patent Application Publication No. 2012/0157513 and He et al., J. Ind. Microbiol. Biotechnol. (2015) 42(4):647-53. The present disclosure provides a composition (e.g., pharmaceutical composition) comprising one or more nucleic acids (e.g., mRNAs) encoding one or more peptides disclosed herein, optionally further comprising a pharmaceutically acceptable carrier or excipient. In certain embodiments, the composition comprises nucleic acid sequences encoding two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 11 or more, 12 or more, 13 or more, 14, or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more) of the peptides disclosed herein. In certain embodiments, the two or more peptides are derived from the same cell surface antigen. In certain embodiments, the two or more peptides are derived from at least two different cell surface antigens. In certain embodiments, the two or more peptides collectively are recognized by MHC molecules in at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the human population. In certain embodiments, the vaccine contains individualized components according to the personal need (e.g., MHC variants) of the particular patient. In certain embodiments, each of the nucleic acids further comprises one or more expression control sequences (e.g., promoter, enhancer, translation initiation site, internal ribosomal entry site, and/or ribosomal skipping element) operably linked to one or more of the peptide coding sequences.
A vaccine composition can further comprise an adjuvant and/or a carrier. Examples of useful adjuvants and carriers are given herein below. A composition can be associated with a carrier such as e.g. a protein or an antigen-presenting cell such as e.g. a dendritic cell (DC) capable of presenting the peptide to a T-cell.
Adjuvants are any substance whose admixture into a vaccine composition increases or otherwise modifies the immune response to a viral antigen. Carriers can be scaffold structures, for example a polypeptide or a polysaccharide, to which a viral antigen, is capable of being associated. Optionally, adjuvants are conjugated covalently or non-covalently.
The ability of an adjuvant to increase an immune response to an antigen is typically manifested by a significant or substantial increase in an immune-mediated reaction, or reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion. An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th response into a primarily cellular, or Th response.
Suitable adjuvants include, but are not limited to 1018 ISS, alum, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Dupuis M, et al., Cell Immunol. 1998; 186(1):18-27; Allison A C; Dev Biol Stand. 1998; 92:3-11). Also cytokines can be used. Several cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418).
CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting. Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.
Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g. CpR, Idera), Poly(I:C)(e.g. polyi:CI2U), non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and/or as an adjuvant. The amounts and concentrations of adjuvants and additives can readily be determined by the skilled artisan without undue experimentation. Additional adjuvants include colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).
A vaccine composition of the disclosure can comprise one or more short (e.g., 8-35 amino acids) peptides as the immunostimulatory agent. In certain embodiments, a T cell epitope sequence is incorporated into a larger carrier polypeptide or protein, to create a chimeric carrier polypeptide or protein that comprises the T cell epitope(s). This chimeric carrier polypeptide or protein can then be incorporated into the vaccine composition.
A vaccine composition can comprise more than one different adjuvant. Furthermore, a therapeutic composition can comprise any adjuvant substance including any of the above or combinations thereof. It is also contemplated that a vaccine and an adjuvant can be administered together or separately in any appropriate sequence.
A carrier (or excipient) can be present independently of an adjuvant. The function of a carrier can for example be to increase the molecular weight of in particular mutant to increase activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life. Furthermore, a carrier can aid presenting peptides to T-cells. A carrier can be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell. A carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. For immunization of humans, the carrier is generally a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers. Alternatively, the carrier can be dextrans for example sepharose.
Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptide bound to an MHC molecule rather than the intact foreign antigen itself. The MHC molecule itself is located at the cell surface of an antigen presenting cell. Thus, an activation of CTLs is possible if a trimeric complex of peptide antigen, MHC molecule, and APC (antigen presenting cell) is present. Correspondingly, it may enhance the immune response if not only the peptide is used for activation of CTLs, but if additionally APCs with the respective MHC molecule are added. Therefore, in some embodiments a vaccine composition additionally contains at least one antigen presenting cell.
Viral antigens can also be included in viral vector-based vaccine platforms, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentivirus, including but not limited to second, third or hybrid second/third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880). Dependent on the packaging capacity of the above mentioned viral vector-based vaccine platforms, this approach can deliver one or more nucleotide sequences that encode one or more viral antigen peptides. The sequences may be flanked by non-mutated sequences, may be separated by linkers or may be preceded with one or more sequences targeting a subcellular compartment (See, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22 (4):433-8, Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352 (6291):1337-41, Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13):3401-10). Upon introduction into a host, infected cells express the viral antigens, and thereby elicit a host immune (e.g., CTL) response against the peptide(s). Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration or immunization of viral antigens, e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein. In some embodiments, the viral vector is a adenovirus vector.
The compositions (e.g., pharmaceutical compositions) disclosed herein may be formulated for delivery into cells (e.g., APCs, such as dendritic cells, monocytes, macrophages, or artificial APCs). In certain embodiments, the composition comprises an agent that facilitate transfection in vitro or in vivo, such as a liposome or a nanoparticle (e.g., lipid nanoparticle). In certain embodiments, the liposome or nanoparticle further comprises a binding moiety (e.g., an antibody or an antigen-binding fragment thereof) for delivering the liposome or nanoparticle to a target T cell (e.g., a professional APC). Another delivery method employs virus particles (e.g., adenovirus, adeno-associated virus, vaccinia virus, fowlpox virus, self-replicating alphavirus, marabavirus, or lentivirus). In certain embodiments, the composition comprises a pharmaceutically acceptable carrier or excipient, such as a diluent, an isotonic solution, water, etc. Excipients also can be selected for enhancement of delivery of the composition.
Suitable routes of administration and dosages for vaccines are known in the art and can be determined by a person of medical skill. In certain embodiments, the vaccine is administered parenterally, e.g., by intramuscular, intradermal, subcutaneous, intravenous, topical, nasal, or local administration. In certain embodiments, the vaccine comprising peptide(s) is administered via skin scarification. In certain embodiments, the vaccine comprising peptide(s) is administered at a dosage of 0.1-10 mg, e.g., 0.1-0.5 mg, 0.5-1 mg, 1-3 mg, 1-5 mg, or 5-10 mg of total amount per human patient. In certain embodiments, the vaccine comprises a plurality of different peptides, wherein each peptide is provided at a dosage of 0.01-0.05 mg, 0.05-0.1, or 0.1-0.5 mg per human patient. Stimulation of an anti-virus T cell immune response in a subject by the vaccine can be monitored by methods established in the art, e.g., by isolating T cells from the subject and measuring reactivity of the T cells to the viral T cell epitope(s) contained within the vaccine (see, e.g., Immunohistochemistry, ELISPOT, binding assays such as Biacore and ELISA, and LC-MC techniques).
Small molecule drug therapeutics generally refer to therapeutics of low molecular weight (e.g., below 1 kDa) that modulate cellular behavior to treat a disease. Such small molecule drugs bind one or more biological targets of a target cell, thereby causing a change in the activity or function of the biological target of the target cell. Given their size, small molecule drug therapeutics are able to penetrate cellular membranes, thereby enabling them to bind or affect biological targets located within cells.
In various embodiments, small molecule drug therapeutics are inhibitors that serve to inhibit a biologic target that is involved in a disease. For example, small molecule drug therapeutics may be kinase inhibitors, proteasome inhibitors, proteinase inhibitors, or protein inhibitors. Additionally, small molecule drug therapeutics can be chemotherapeutics that prevent cell replication such as alkylating agents, anti-microtubule agents, topoisomerase inhibitors, DNA intercalators, and the like.
More comprehensive lists of small molecule drug therapeutics are found in publicly available databases such as DrugBank, ChemSpider, ChEMBL, KEGG, and PubChem. In some embodiments, the small molecule is an inhibitor of a protein or portion thereof encoded by the nucleic acid sequence set forth in SEQ ID NO: 1-349. In some embodiments, the small molecule is an inhibitor of a protein or portion thereof set forth in
Biologics generally refer to therapeutics that are manufactured from biologic sources (e.g., produced in cells). Biologics are larger than small molecule drugs and often times more complex in structure and molecular makeup. In various embodiments, biologics are synthesized through manufacturing methods that include 1) inserting a DNA sequence encoding for the biologic or a portion of the biologic into a living cell, 2) having the cell produce transcribe/translate the DNA sequence into a protein, 3) isolating the protein from the cells, where the protein serves as the biologic or a component of the biologic. Example of biologics include antibodies (e.g., monoclonal or polyclonal antibodies), cytokines, growth factors, enzymes, immunomodulators, recombinant proteins, vaccines, allergenics, blood components, hormones, therapeutic cells (e.g., stem cells), tissues, carbohydrates, and nucleic acids.
In some embodiments, any of the BiPS or viral sequences disclosed herein is assembled into a pharmaceutical or diagnostic or research kit to facilitate their use in therapeutic, diagnostic or research applications. A kit may include one or more containers housing any of the vectors, nucleic acids, proteins, peptides, or viruses disclosed herein and instructions for use.
The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration.
Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.
In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.
Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and/or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and disclosure. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the disclosure described and depicted herein.
It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.
The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context
Where the use of the term “about” is before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.
It should be understood that the order of steps or order for performing certain actions is immaterial so long as the embodiments remain operable. Moreover, two or more steps or actions may be conducted simultaneously.
The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the embodiments and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.
The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.
This example describes the isolation of embryonic fibroblasts from bats. An embryo (approximately developmental stage 20) acquired from a Spanish Rhinolophus ferrumequinum bat (wild horseshoe bat) was cut into several pieces while removing the head and as much as the inner organ tissue as possible. The pieces were then flushed with PBS and processed separately. The tissue was covered with 0.05% trypsin, minced with a scalpel, and incubated in a cell culture incubator at 37° C. and 5% CO2 for 45 minutes. The trypsin was deactivated with fibroblast medium consisting of DMEM (Life Technologies, CA), 10% fetal bovine serum (Sigma, MO), 0.1 mM MEM Non-essential amino acids (Life Technologies, CA), 2 mM GlutaMax supplement (Life Technologies, CA), and Penicillin-Streptomycin (10 U/ml and 10 μg/ml, respectively; Life Technologies, CA). The cells were broken up by pipetting up and down 20 times, collected by centrifugation, transferred to a gelatin-coated (Sigma-Aldrich, MO) T75 cell culture treated flasks (Corning, AZ) in 15 ml of fibroblast medium, and cultured at 37° C. and 5% CO2. After 3 days, when reaching ˜80% confluency, the attached cells were washed with DPBS (Life Technologies, CA), treated with 0.05% trypsin-EDTA, (Life Technologies, CA) to obtain a single cell solution and either split at a ratio of 1:4 or used directly in a reprogramming experiment.
This example describes the isolation of fibroblasts from tail biopsies from adult bats.
M. myotis bats were sampled in Morbihan, Brittany in North-West France in accordance with the permits and ethical guidelines issued by ‘Arrêté’ by the Préfet du Morbihan and the University College Dublin ethics committee. This population has been transponded and followed since 2010 as part of on-going mark-recapture studies by Bretagne Vivante and the Teeling laboratory (Huang et al., 2019). Once captured, all bats were placed in individual cloth bags before processing. A single 3 mm biopsy was taken from the outstretched uropatagium of each bat using a sterile biopsy punch and immediately submerged in a Cryotube with 2 ml of DMEM cell culture medium supplemented with 20% FBS, 1% NEA, and 1% Antibiotic-Antimycotic containing Streptomycin, Amphotericin B and Penicillin, maintaining as sterile conditions as possible. All bats were offered food and water and rapidly released after processing. Biopsies were then stored at 4° C. and transported to the laboratory for processing within 6 days. Samples were further processed through a cell extraction methodology similar to a previously established protocol (Kacprzyk et al., 2021) with a few modifications. The samples were rinsed with DPBS and cut finely within a minimal amount of cell culture medium using sterile blades to result in six 0.5 mm pieces. These pieces were then transferred aseptically to a cryotube containing cell culture medium and incubated for 18 hours with collagenase type II at 37° C. with 5% CO2 to allow for digestion. The pieces were collected by centrifugation for 5 minutes at 300 rcf, resuspended in 2 ml of fresh cell culture medium and transferred to a 35 mm cell culture treated plate for initial P1 expansion. Cells were then fed every 2-3 days with cell culture medium as above but a reduced 0.2% concentration of antibiotic-antimycotic. For the first feeding a % media change was performed to avoid sudden changes in antibiotic-antimycotic concentration from 1% to 0.2%. When the cells reached 70% confluency, they were transferred to a T25 flask in cell culture medium after treatment with 0.05% Trypsin and were fed every 2-3 days as necessary. At 85% confluency, the cells were trypsinized as before and 1×10{circumflex over ( )}6 cells were frozen in 1 ml cell culture medium containing 10% DMSO.
This example describes the reprogramming of bat embryonic fibroblasts for the generation of bat iPSCs. First, the original Yamanaka reprogramming protocol (Takahashi et al., Cell (2006) 126, 663-676) based on four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) was tried, because it provides the most direct way to generate pluripotent stem cells in most species. Strikingly, the standard protocol that is highly effective in mice, humans and other mammalian species (domestic dog, (Canis familiaris), domestic pig, (Sus scrofa), common marmoset (Callithrix jacchus)) failed in bats. Even though the standard reprogramming protocol failed, it provided the crucial insight that the Yamanaka factors triggered the formation of rudimentary stem cell-like colonies even though the reprogrammed cells ceased to expand. Thus, the core pluripotency network might be conserved in bats. However, the signaling cascades that usually shield this network from differentiation cues are different. An exemplary bat pluripotent stem cell derivation strategy is illustrated in
Briefly, 150,000 embryonic Rhinolophus ferrumequinum fibroblasts at passage 2, adult Myotis myotis at passage 3, or CF1 mouse embryonic fibroblasts at passage 3 were resuspended in 1 ml of fibroblast medium and mixed with Sendai-virus particles containing the reprogramming factors Oct4, Sox2, cMyc, and Klf4 (CytoTune iPS 2.0, Life Technologies, CA) with a final multiplicity of infection (MOI) of 10, 10, 10, and 15, respectively. The cells were plated on one gelatin-coated well of a 6-well plate and cultured at 37° C. with 5% CO2. The medium was replaced every 24 hours. 6 days after transduction, the cells of each well were collected by treatment with 0.05% trypsin-EDTA, seeded at a density of 50,000 cells per 60 cm2 on irradiated CF1 mouse embryonic fibroblasts (MEFs; ThermoFisher, MA) in fibroblast medium. After 24 hours, the medium was switched to 50% fibroblast medium and 50% pluripotent stem cell (PSC) medium consisting of DMEM/F-12 (Life Technologies, CA), 20% knockout serum replacement, 0.1 mM MEM Non-essential amino acids, 2 mM GlutaMax supplement, Penicillin-Streptomycin (10 U/ml and 10 μg/ml, respectively), 100 μM 2-mercaptoethanol, and 40 ng/ml FGF2. From then on, the medium was replaced every day with PSC medium until day 14 when the FGF concentration was increased to 100 ng/ml and the medium was supplemented with 10{circumflex over ( )}4 U/ml Leukemia inhibitory factor (Lif), 100 ng/ml SCF (R&D Systems, MN) and 20 nM Forskolin Forskolin. Colonies appeared 14 to 16 days after transduction, were picked on day 20 and expanded on irradiated MEFs with Gentle Cell dissociation Reagent (StemCell Technologies, MA). After that, cells were passaged approximately every 5 days, or when they were confluent, at a ratio of 1:6 to 1:12 onto irradiated MEFs. Cell and colony morphology were recorded with an EVOS digital inverted microscope (Invitrogen, MA).
Thus, specific ratios of reprogramming factors, and the addition of Lif, Scf, the Pka activator forskolin and Fgf2 to the culture medium allowed for the uninterrupted growth of bat pluripotent stem cells. Under these conditions, bat stem cell colonies typically appeared after 14-16 days of culture. These initial stem cell colonies were, however, not readily pickable and expandable using conventional EDTA- (Versene), collagenase- or trypsin-based methods that are normally used to passage pluripotent stem cells from other species. To split cells for further passaging and growth cells were lightly flushed off the feeder cell layer after gentle treatment with low concentrations of EDTA. Exemplary cell morphology of the reprogrammed bat iPSCs is shown in
This example illustrates the characterization of the reprogrammed cells. After reprogramming, cells were analyzed for karyotype, chromatin organization, and gene and RNA expression.
This example illustrates the karyotyping of reprogrammed cells. Briefly, cells were treated with 100 ng/ml KaryMax Colcemid Solution in HBSS (Life Technologies, CA) for 16 hours, then treated with 0.05% trypsin-EDTA for 15 minutes and filtered through a 40 μm cell strainer to remove clumps. Cells were collected by centrifugation, resuspended in 1 ml 0.075 M potassium chloride (Sigma-Aldrich, MO) and incubated for 20 minutes at room temperature. 0.5 ml fixative (1 part glacial acetic (Fisher Scientific, MA) mixed with 3 parts methanol (Sigma-Aldrich, MO) were added, cells were collected as before, resuspended in 4 ml fixative, and incubated for 20 minutes at room temperature. The fixation step was repeated, the cells collected as before and all but about 200 μl of the fixative was removed. The cells were resuspended in the remaining fixative and dropped onto slides that were precooled at −20° C. The slides were airdried and the cells stained for 10 minutes with Giemsa Staining solution consisting of 1 part KaryoMax Giemsa solution (Life Technologies, CA) and 3 parts Gurr buffer (Invitrogen, MA). The slides were washed with water, dried, and mounted in Cytoseal 60 (Thermo Scientific, MA). High-resolution pictures of chromosome spreads were acquired with an AxioObserver microscope (Zeiss) using the 100× oil objective. Even after prolonged culture (over 50 passages), the cells retained a normal karyotype, with most cells containing 56 chromosomes (
mRNA was extracted with the RNeasy Mini Kit (Qiagen). 500 ng of each sample were used to generate cDNA by reverse transcription using the SuperScript™ IV VILO™ Master Mix (Invitrogen). 2 μl of the cDNA were used to detect the presence of Sendai virus transcripts using GoTaq Green Polymerase (Promega), and the oligos as recommended in the CytoTune iPS 2.0 kit (Invitrogen). Gapdh was amplified as loading control using oligos with the following sequence: Z25-132:GAPDH_F1_GHB: TGGTGAAGGTCGGAGTGAAC (SEQ ID NO: 350) and Z25-133:GAPDH_R1_GHB: GAAGGGGTCATTGATGGCGA (SEQ ID NO: 351)). The PCR products were analyzed on a 2% agarose gel containing ethidium bromide.
For immunofluorescence staining, cells were plated on pt-slides (Ibidi, Germany). After 4 days, cells were washed once with DPBS and fixed with Cytofix/Cytoperm solution (Becton Dickinson, NJ) for 20 minutes at 4° C. Cells were rinsed with Perm/Wash buffer (Becton Dickinson, NJ) and then incubated overnight at 4° C. in Perm/Wash buffer containing primary anti-Afp (R&D Systems, MN) anti-Pax6 (BioLegend, CA), J2 anti-dsRNA (Scicons, Hungary), anti-(gag/pol) HERVK (Austrial Biological) or FIPV3-70 anti-Pan Corona (Life Technologies, CA) or directly conjugated anti-Oct3/4-AF488 (Santa Cruz, CA) or anti-Brachyury (R&D Systems, MN) anti-Otx2 (R&D Systems), anti-Zic2 (Abcam), anti-Tfe3 (Sigma Aldrich) or anti-Tfcp2l1 (R&D Systems) in a 1:50 (anti-Oct3/4) or 1:100 dilution (all others). Cells were rinsed and washed 3 times for 2 minutes with Perm/Wash solution at room temperature followed by a 1-hour incubation with a 1:200 dilution of the corresponding secondary antibodies (Donkey anti-chicken-Cy3, Millipore, AP194C; Goat anti-chicken-AF488; Donkey anti-rabbit-AF647; Goat anti-rabbit-AF488, Goat anti-mouse-AF488) in Perm/Wash buffer. Cells were rinsed, washed twice for 2 minutes with Perm/Wash Buffer and then incubated for 5 minutes with Perm/Wash buffer containing 2 drops per ml NucBlue Dapi stain (Invitrogen, MA). The buffer was removed, and the cells were cover-slipped in Prolong Dimond antifade mounting medium (Invitrogen, MA). Images were acquired with an AxioObserver fluorescence microscope with Apotome (Zeiss). For the simulated emission depletion (STED) microscopy (super-resolution), the cells were plated on coverslips that were placed in wells of 6-well plates. The staining was performed as described above but with a 1:200 dilution of the Abberior Star 635P secondary antibody in Perm/Wash buffer. Cells were rinsed, washed twice for 2 minutes with Perm/Wash Buffer and then incubated for 5 minutes with Perm/Wash buffer containing 2 drops per ml DyeCycle Violet stain. The coverslips were mounted face down on glass slides with Prolong Dimond antifade mounting medium (Invitrogen). Images were acquired with a TCS SP8 confocal microscope with STED 3× and White Light Laser (Leica) with a 100× oil objective. 405 nm and 594 nm lasers were used for excitation and 775 nm laser for depletion. Image resolution obtained was 19.8 μm by 19.8 μm using a zoom factor of 6×. Exemplary immunofluorescent detection of Oct4/Pou5f2 in BiPS cells shows that the cells were positive for the pluripotency factor Oct4 (
For RNA-seq, RNA was extracted from BiPS cells at passage 22 and BEFs at passage 3. RNA was extracted with the RNeasy RNA isolation kit (Qiagen, Germany) following the manufacturer's recommendations including the DNase digest (Qiagen, Germany) and eluted in 50 μl RNase/DNase free H2O. The libraries were prepared with the SMART-Seq v4 Ultra Low Input kit (Takara Bio, undifferentiated cells) or the Stranded Total RNA with Ribo-Zero Plus kit (Illumina, differentiated cells) and 100 bp paired-end sequencing reads were (PE100) were generated by Illumina sequencing (NovaSeq 6000 S1) to a depth of 50 million reads (100 million total reads).
The quality of the reads from the RNA sequencing was analysed with FastQC v0.11.9 (Andrews, 2010), and visualized using MultiQC (Ewels et al., 2016. With the mean phred score of around Q35 across each base position no filter or processing was performed. To carry out the differential expression analysis, the genome of Rhinolophus ferrumequinum was used as reference genome, RefSeq assembly accession GCF_004115265.1, assembled and annotated by the Vertebrate Genomes Project (www.vertebrategenomesproject.org). The reads were mapped with HISAT2 v2.2.1 (Kim et al., 2019), the .sam files resulting from each mapping were converted into .bam files and indexed using samtools v1.10 (Li et al., 2009). The reads were mapped against each gene using featureCounts v2.0.1 (Liao et al., 2014) and the differential expression analysis was performed with DESeq2 v1.10.1 (Love et al., 2014). To visualize the RNA-seq data in the UCSC genome browser, bigwig files were generated using the bamCoverage command from deepTools (www.deeptools.readthedocs.io/en/develop/content/tools/bamCoverage.html; Ramirez et al., 2016).
The MA plots were generated based on the DESeq2 (see above) results with the ggmaplot function (www.rpkgs.datanovia.com/ggpubr/reference/ggmaplot.html) from the R package ggpubr (www.rpkgs.datanovia.com/ggpubr/). Genes are indicated by dots, plotted by their log 2 fold change between bat fibroblast and pluripotent stem cells and the log 2 mean of normalized counts (ratio of means). Blue dots indicate genes with an adjusted p value of (or FDR) of <0.05 and a fold change of 2 (log 2 fold change of 1), red dots indicate genes with an adjusted p value (or FDR) of <0.05 and fold change of −2 (log 2 fold change of −1). Dotted lines are drawn at fold change of 2/−2 (log 2 fold change of 1/−1).
RNA-seq analyses revealed the induced expression of canonical pluripotency-associated genes (
However, closer data inspection revealed that the expression profile did not necessarily match any known pluripotency state. Instead, factors indicative of the so-called naive pluripotent state (Klf4, Klf17, Essrb, Tfcp2l1, Tfe3, Dppa, and Dusp6) were expressed alongside genes typically found in the more advanced primed pluripotent cells (e.g., Otx2, Zic2). Double immunostainings detecting four of the most commonly used primed/naïve factors, Otx2/Tfe3 and Tfcp2l1/Zic2, respectively, showed co-expression of naïve and primed markers in most cells (
To analyze the effects of the reprogramming approach on the bat chromatin and epigenetic structures a global epigenetic landscape survey using ATAC-seq was performed. ATAC-seq and bioinformatics analysis to detect open chromatin in bat fibroblasts and bat pluripotent stem cells was performed by Active Motif, CA from 100,000 cryopreserved cells (ATAC-seq service). In brief, nuclei were isolated and libraries of open chromatin were prepared with the Nextera Library Prep Kit (Illumina) by Tn5 tagmentation. The tagmented DNA was purified using the MinElute PCR purification kit (Qiagen, Germany), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter, CA). 42 bp paired-end sequencing reads (PE42) were generated by Illumina sequencing (using NextSeq 500) to a depth of at least 83 million total reads and mapped to the GCA_004115265.2 genome (Ensembl, annotation version 102) using the BWA algorithm with default settings (“bwa mem”). Alignment information for each read was stored as BAM file. Only reads that passed the Illumina's purity filter, aligned with no more than 2 mismatches, and mapped uniquely to the genome were used in the subsequent analysis. Duplicate reads (“PCR duplicates”) were removed. Genomic regions with high levels of transposition/tagging events were then determined using the MACS2 peak calling algorithm (Zhang et al., Genome Biology (2008) 9:R137). To identify the density of transposition events along the genome, the genome was divided into 32 bp bins and the number of fragments in each bin was determined. The data were then normalized by reducing the tag number of all samples by random sampling to the number of tags present in the smallest sample. Peak metrics between samples were compared by grouping overlapping Intervals into “Merged Regions,” which are defined by the start coordinate of the most upstream Interval and the end coordinate of the most downstream Interval (=union of overlapping Intervals; “merged peaks”). In locations where only one sample has an Interval, this Interval defines the Merged Region. Intervals and Merged Regions, their genomic locations along with their proximities to gene annotations and other genomic features were determined and average and peak (i.e. at “summit”) fragment densities were compiled. The sequencing tracks (number of fragments in each 32 bp bin stored as .bigwig file) were visualized with the UCSC genome browser.
The global epigenetic landscape survey using ATAC-seq revealed significant chromatin configuration changes when bat fibroblasts transitioned into the pluripotent state (
Reduced Representation Bisulfite Sequencing (RRBS) of Bat iPSCs
Reduced representation bisulfite sequencing of bat fibroblasts and pluripotent stem cells was performed by Active Motif, CA(RRBS Service, Active Motif, CA). Briefly, 500,000 cells were provided as a frozen pellet. Genomic DNA was isolated, and 100 ng were digested with TaqaI (NEB, MA) at 65° C. for 2 hours followed by MspI (NEB, MA) at 37° C. overnight. Following enzymatic digestion, samples were used for library generation with the Ovation RRBS Methyl-Seq System (Tecan, Switzerland) following the manufacturer's instructions. In brief, digested DNA was randomly ligated, and, following fragment end repair, bisulfite converted using the EpiTect Fast DNA Bisulfite Kit (Qiagen, Germany) following the Qiagen protocol. After conversion and clean-up, samples were amplified resuming the Ovation RRBS Methyl-Seq System protocol for library amplification and purification. 75 bp single-end sequencing reads (SE75) were generated by Illumina sequencing (using NextSeq 500) to a depth of at least 27 million reads (total of 54 million reads), with at least 2.9 million covered CpGs. The reads were mapped to the GCA_004115265.2 genome (Ensembl, annotation version 102) and the percentage of methylation at CpG sites across the genome was calculated. To visualize the methylation ratios aligned to the gnome with the UCSC genome browser, the methylation ratio files containing the methylation ratio for each chromosomal position were first converted to bed files, that were then used to generate bigwig files with the bedGraphToBigWig v4 tool (www.encodeproject.org/software/bedgraphtobigwig/). Correlation scatter plots were generated to show the level of methylation at common CpG sites. To visualize the global differences between bat fibroblast and pluripotent stem cells, the RRBS methylation data were combined for all samples based on chromosome position, the ratios of the duplicates were averaged and the methylation ratio for each chromosomal position was plotted using the ggplot2 function “stat_density_2d_filled” with fill based on density. Only chromosomal positions that were present in all replicates were included in the analysis.
Similarly, mapping the DNA methylome by RRBS exposed significant CpG methylation changes across the genome (
5 million cells were fixed cells in 1% formaldehyde by adding 1/10 volume of freshly prepared Formaldehyde Solution (11% formaldehyde, 0.1 M NaCl, 1 mM EDTA, pH 8.0, 50 mM HEPES, pH 7.9) to the existing medium. Cells were agitated for 15 minutes at room temperature and the fixation was stopped by addition of 1/20 volume of 2.5 M glycine solution (final concentration of 0.125 M) to the existing medium and incubation at room temperature for 5 minutes. The cells were scraped off the wells, collected by centrifugation at 800 g and washed with 10 ml chilled 0.5% Igepal in PBS per tube by pipetting up and down. Cells were pelleted by centrifugation as before and resuspended in 10 ml chilled PBS-Igepal containing 1 mM PMSF. Cells were collected as before, and the cell pellet was snap-frozen in liquid nitrogen. Further processing, chromatin immunoprecipitation and bioinformatics analysis to detect H3K4me3 and H3K27me3 was performed by Active Motif, CA(HistoPath ChIP-seq service). In brief, chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp with Active Motif's EpiShear probe sonicator. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by SPRI beads clean up (Beckman Coulter, CA) and quantitation with Clariostar (BMG Labtech). An aliquot of chromatin (20 μg) was precleared with protein A agarose beads (Life Technologies, CA). Genomic DNA regions of interest were isolated using 4 μg of antibody against H3K4me3 (Active Motif, CA) or H3K27me3 (Active Motif, CA). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65° C., and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were generated from the ChIP and Input DNAs with the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, 75-nt single-end (SE75) sequence reads were generated by Illumina sequencing (using NextSeq 500) to a depth of at least 36 million reads per sample and mapped to the GCA_004115265.2 genome (Ensembl, annotation version 102) using the BWA algorithm with default settings. Duplicate reads were removed, and only uniquely mapped reads (mapping quality >=25) were used for further analysis. Alignments were extended in silico at their 3′-ends to a length of 200 bp, which is the average genomic fragment length in the size-selected library and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. To find peaks, the generic term “Interval” was used to describe genomic regions with local enrichments in tag numbers. Intervals were defined by the chromosome number and a start and end coordinate. Peak locations were determined using the MACS algorithm (v2.1.0) with a cutoff of p-value=1e-7 (Zhang et al., 2008). Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations. No normalization was performed on the H3K27me3 data, while standard normalization was applied to the H3K4me3 data. The tag number of all samples (within a comparison group) was reduced by random sampling to the number of tags present in the smallest sample. To compare peak metrics between 2 or more samples, overlapping Intervals were grouped into “Merged Regions,” which are defined by the start coordinate of the most upstream Interval and the end coordinate of the most downstream Interval (=union of overlapping Intervals; “merged peaks”). In locations where only one sample has an Interval, this Interval defines the Merged Region. The sequencing tracks (number of fragments in each 32 bp bin stored as bigwig file) were visualized with the UCSC genome browser.
ChIP-seq analysis showed that histone marks associated with active (H3K4me3) and developmentally repressed genes (H3K27me3) showed many changes (
Collectively, the results establish that the bat pluripotent stem cells are reprogrammed both transcriptionally and epigenetically.
This example illustrates the further functional characterization of the reprogrammed bat IPS cells. After reprogramming, cells were analyzed in pluripotency assays for pluripotency potential.
The differentiation of bat pluripotent stem cells was carried out with the STEMdiff Trilineage differentiation kit (StemCell Technologies, MA) following the manufacturer's protocol. Cells were plated at the desired densities in mTeSR medium (StemCell Technologies, MA), and plated on Vitronectin-coated (StemCell Technologies, MA) cell culture plates. After 5 days (endoderm or mesoderm) or 7 days (ectoderm) in culture as directed by the manufacturer. For the ectoderm differentiation, the floating three-dimensional structures were then replated and grown for 4 additional days in fibroblast medium. The cells were stained with antibodies detecting the appropriate lineage markers as described above or cells were collected (surface area of 10 cm2 per replicate) for RNA isolation and RNAseq after addition of 600 μl lysis buffer RTL (part of the RNeasy kit; Qiagen, Germany).
Results show that the bat iPSCs differentiate into ectodermal, mesodermal, and endodermal fates (
To analyze the bat stem cells' developmental plasticity, the cells were subjected to embryoid body (EB) differentiation. Briefly, bat pluripotent stem cells grown on irradiated mouse embryonic fibroblasts from a total area of 60 cm2 were washed with PBS, treated for 10 minutes with Gentle Cell Dissociation Reagent (StemCell Technologies, MA), collected by centrifugation and resuspended in 12 ml differentiation medium consisting of DMEM/F-12 (Life Technologies, CA), 10% fetal bovine serum (Sigma, MO), 0.1 mM MEM Non-essential amino acids (Life Technologies, CA), 2 mM GlutaMax supplement (Life Technologies, CA), Penicillin-Streptomycin (10 U/ml and 10 μg/ml, respectively; Life Technologies, 15140122) and 100 μM 2-mercaptoethanol (Fluka, NC). The cells were then transferred to one uncoated 60 cm2 petri dish (Corning, 351029). After 3 days in culture, as much as possible of the medium (about ⅔) was carefully exchanged without disturbing and removing the floating EBs that had formed. The floating EBs were collected after 3 more days (total of 6 days) in culture, fixed in Cytofix/Cytoperm fixation buffer (Becton Dickinson, NJ) overnight, and then stained with antibodies against as described above to detect differentiation markers of all three germ-layers by immunofluorescence. For RNA isolation and RNA-seq, EBs were formed as described, collected, resuspended in 6 ml differentiation medium, and distributed into three wells of cell-culture treated 6-well plates (10 cm2 each). After 2 more days in culture, the cells were washed with PBS, lysed with 600 μl buffer RTL (part of the RNeasy kit; Qiagen, 74104) and RNA was isolated as described above.
In the assay, cells differentiated and formed the for EBs' typical spherical arrangements. They subsequently matured into elaborate three-dimensional structures that were positive for all three germ layer markers (
To assay the potential of the bat iPSCs to form teratomas in vivo, cells were injected into immunocompromised mice and then analyzed. Briefly, two 6-well plates (12 wells) of bat pluripotent stem cells grown on irradiated mouse embryonic fibroblasts were scraped off in 2 ml DMEM/F-12 medium (Life Technologies, CA), collected by centrifugation and resuspended in 500 μl DMEM/F-12 medium. 100 μl of the cell suspension were injected into the hindleg muscle of 8-week-old male Fox Chase SCID Beige Mice (Charles River, MA). Tumor tissue that had formed after 16 weeks was harvested, fixed in 10% Formalin (Fisher Scientific, MA) overnight and then transferred to 70% ethanol. The tissue was embedded in paraffin and hematoxylin and stained with eosin of 5 μm sections. Images were acquired with an AxioObserver microscope (Zeiss) and analyzed.
The analysis showed, that the bat iPSCs formed a particular tumor (teratoma) at the injection site after four to five months albeit infrequently (33%) and very small (2-4 mm). The tumors were comprised of immature tissue with epithelial, neural and stromal characteristics (
To analyze the potential of the iPSCs to form embryoid structures, the cells were subjected to a modified blastoid protocol. Cells were harvested and plated as described for the embryonic body formation above. After 3 days in culture, 100 ng/ml BMP4 (R&D Systems, 314-BP-010) were added to the medium. 24 later the supernatant was diluted with ⅔ of fresh medium and transferred to two fresh uncoated petri dishes. The medium was exchanged after 3 more days in culture and floating blastoids were harvested 4 days later (total of 12 days of differentiation). The blastoids were fixed in Cytofix/Cytoperm fixation buffer (Becton Dickinson, BDB554714) overnight, and stained as described above to detect the expression of Oct4 by immunofluorescence microscopy.
Further analysis showed, that bat blastoids recapitulate critical aspects of preimplantation embryos, including an Oct4-positive inner cell mass, the cystic cavity and a bilayered epithelium consisting of trophoblastic and yolk sac cells (
Embryonic stem cell lines were derived from these outgrowths, confirming these embryoids' blastocyst nature.
The differentiation studies exemplify the unique potential of the described pluripotent bat cells to recapitulate important developmental events and serve as a powerful model to study the unique physiological adaptations of bats.
To assay distinct characteristics of pluripotent bat stem cells, gene expression patterns in bat stem cells were analyzed such as the ground state transcriptome and then compared to other species. Transcriptome profiles of pluripotent stem cells from an assorted set of species (Bats, mouse, pig, dog, marmoset, human) and different cell types (EF, iPSCs, MEF, ESC) were assembled and principal component analysis was performed to obtain a high-level overview of the number of commonalities and differences between bats and other mammals (
The DESeq2 output files of the RNA-seq analyses described above were subjected to a Variance Stabilizing Transformation (VST) using within-group-variability (Anders and Huber, 2010) to compare the bat pluripotent stem cell transcriptional profile with that of other species. The first two principal components of this result were plotted using the ggscatter function (https://rpkgs.datanovia.com/ggpubr/reference/ggscatter.html) from the R package ggpubr (www.cran.r-project.org/web/packages/ggpubr/index.html). The datasets used in the PCA were: GSM4616525, GSM4616526 and GSM4616527 (dog iPS), GSM4617887, GSM4617889, GSM4617890, GSM4617891, GSM4617895, GSM4617900 and GSM4617901 (marmoset iPS), GSM4616532 (human iPS), GSM4616535 and GSM4616536 (pigIPS) from study GSE152493 (Yoshimatsu et al., 2021), and GSM1287734, GSM1287745 and GSM1287746 (mouse ESC) and GSM1287736, GSM1287747 and GSM1287748 (mouse iPS) from GSE53212 (Carter et al., 2014), as well as GSM2718393 and GSM2718399 (mouse iPS) from GSE101905 (Knaupp et al., 2017).
PCA showed that bats were unique to all mammals, even the more distant ones like dogs, clustered together in the PCA plot, while bats formed a separate distinctive group (
When analyzing the enrichment of any KEGG pathway, by far the most significantly enriched category was “Corona virus disease” (
Further, data were analyzed for the enrichment of transcription factor footprints in the mapping of open chromatin regions to these genes in the ATAC-seq data. Surprisingly, only two transcription factor motifs were significantly enriched, Klf5 and Ctcf Notably, however, these factors accompanied the majority of the genes in this set. Klf5 is a canonical pluripotency factor, which is essential for early embryogenesis and self-renewal of pluripotent stem cells. The recruitment of Klf5 binding sites to a new set of genes makes it likely that bat stem cells acquired novel features under the influence of this transcription factor. Ctcf, on the other hand, contributes to the establishment of higher-order genome structures (topologically associating domains), which are evolutionarily stable.
The leading-edge genes showed that they were under a purifying and positive selection. Of the 655 orthologous genes analyzed, a significant intensifying, purifying selection was observed in only five (Rsph1, Nes, Col3a1, Rgs5, and Lamb).
First, the ATAC-seq regions were identified that showed a shrunkelog2 fold change of 5 between bat fibroblast and pluripotent stem cells and an adjusted p value of less than 0.1 that were within 10 kb (i.e., any interval within 10 kb upstream or downstream) of any gene that is part of the top 5% of genes contributing to the differences in PC1 in the PCA analysis described above. The DNA sequences corresponding to these ATAC-seq regions were extracted from the GCF_004115265.1 reference genome und used in a MEME-ChIP motif search to identify sequence motifs (6-15 bp in width) for protein binding sites that are enriched in this set of genes (Machanick and Bailey, 2011; www.meme-suite.org/meme/tools/meme-chip). The sequence motifs with a p-value below 0.05 were then used in a FIMO analysis to identify the genomic positions and gene association of these motifs within the gene set. The number of genes associated with each motif within the gene set was then plotted against the factor known to bind to the and labeled with the protein know to bind to the motif
To explore evidence of positive selection in R. ferrumequinum for the 674 genes identified as part of the “leading” edge in the PCA analysis described above, all gene alignments were extracted that were available for these transcripts (n=491) and had previously been annotated (Jebb et al., 2020), in addition to annotating 169 alignments that had been made available as part of BATIK but were currently unannotated. These alignments contained a maximum of 48 species from all eutherian mammalian superorders, with the species tree published by Jebb et al. (2020) used for all selection analyses. A total of 660 of these alignments contained representative genes for R. ferrumequinum and were analysed for positive selection using the branch-site models in the codeml package of the PAML suite of software (Yang, 2007). Positive selection was inferred using likelihood-derived dN/dS (o) values under both a null (foreground and background ω constrained to be less than 1) and alternative (foreground ω can vary) model. The R. ferrumequinum lineage was designated as foreground branch to detect unique instances of taxon-specific positive selection. A likelihood ratio test (LRT, 2*lnLalt-lnLnull) was used to compare the fit of both models, with a p-value calculated assuming chi-squared distributed LRTs. P-values were corrected for multiple testing using the Benjamin-Hochberg False Discovery Rate (FDR) method via ‘padjust’ implemented in R. Any significant gene showing a p-value greater than 0.05 with ω>1 was explored further. Significant sites showing positive selection were identified using Bayes Empirical Bayes (BEB) scores with a probability >0.95. All significant genes were subject to a visual inspection of the alignment, to rule out potential false positive results having occurred due to misaligned sequences. In addition to R. ferrumequinum, the Myotis myotis (n=637 representative genes), Homo sapiens (n=652), Mus musculus (n=628), Canis lupus (n=593) and Felis catus (n=603) lineages were also independently designated as foreground branches for all genes containing a representative sequence shared with R. ferrumequinum. This served as a means of determining whether positive selection identified in R. ferrumequinum was truly unique to the species lineage or a consequence of bat-specific, Laurasiatherian-specific, or eutherian mammal-specific instances of sequence evolution.
Gene ontology and KEGG pathways that are enriched within a group of genes were identified with the Enrichr tool (Xie et al., 2021; www.maayanlab.cloud/Enrichr/). The odd ratios were then plotted with ggplot2 (Wickham, 2016; www.cran.r-project.org/web/packages/ggplot2/index.html) with the odds ratio displayed on the x-axis, the dot size reflecting the gene count (number of genes present in the top 5% of PC1 contributing genes) and the dot color reflecting the p-value.
In order to understand if the leading-edge genes that make horseshoe bats unique were enriched for any particular functional gene ontology category (
The differential expression analysis was performed between bat (this study) and mouse iPS cells (GEO accession number: GSM1287736, GSM1287747 and GSM1287748 from Study GSE53212 (Carter et al., 2014) using DESeq2 (Love et al., 2014). The Corona virus disease-related genes were then illustrated with Cytoscape (Version 3.8.2, Shannon et al., 2003) using the STRING protein query with a 0.8 confidence score cutoff. The nodes were colored based on the log 2FoldChange with a negative (blue) fold change indicating down-regulation and a positive (red) fold change indicating upregulation in bat pluripotent stem cells cells. Bold borders indicate proteins that were present in the top 5% of PC1 in the PCA analysis described above.
When analyzing the enrichment of any KEGG pathway, by far the most significantly enriched category was “Corona virus disease” (
This example describes the identification of virus like structures in bat IPSCs.
Briefly, bat IPSCs were imaged with differential interference contrast microscopy and Image-based flow cytometry. Images of the bat IPSCs highlighted prominent cytoplasmic vesicles. Bat stem cells were observed to be packed with small, luminescent vesicles that filled a significant proportion of the cytoplasm (
In order to analyze the vesicles, ultrastructural studies were performed using electron microscopy. Cells were grown in chambered Permanox slides (LabTek, MI) on irradiated mouse embryonic fibroblasts as described above for 5 days and then further processed by the Biorepository and Pathology core at the Icahn School of Medicine at Mount Sinai. Briefly, the cells were rinsed once with DPBS and fixed overnight with 2% paraformaldehyde and 2% glutaraldehyde in 0.01 M sodium cacodylate buffer at 4° C. Sections were rinsed in 0.1 M sodium cacodylate buffer, followed by a quick rinse with ddH2O. Cells were post fixed with 1% aqueous osmium tetroxide for 1 hour, followed with an En bloc stain of 2% aqueous uranyl acetate for 1 hour. Sections were washed again in ddH2O, dehydrated through graduated ethanol (25-100%), infiltrated through an ascending ethanol/epoxy resin mixture (Embed 812, EMS), and then covered with pure resin overnight. Chambers were separated from the slides, and a modified #3 BEEM embedding capsule (EMS) was placed over defined areas containing cells. Capsules were filled with pure resin and placed in vacuum oven to polymerize at 60° C. for 72 hours. Immediately after polymerization, the capsules were snapped from the substrate to dislodge the cells from the slide. Semithin sections (0.5-1 μm) were obtained using a Leica UC7 ultramicrotome (Leica, Buffalo Grove, IL), counterstained with 1% Toluidine Blue, cover slipped and viewed under a light microscope to identify successful dislodging of cells. Ultra-thin sections (85 nms) were collected on 300 hexagonal mesh copper grids (EMS) using a Coat-Quick adhesive pen (EMS). Sections were counter-stained with uranyl acetate and lead citrate and imaged with a Hitachi 7700 Electron Microscope (Hitachi High-Technologies) using an advantage CCD camera (Advanced Microscopy Techniques). Images were adjusted for brightness, contrast, and size using Adobe Photoshop CS4 11.0.1.
Data analysis showed that the vesicles were lipid or glycogen-filled vesicles and autophagosomes (
Interestingly, the virion structures did not belong to a uniform set of virus categories. While some exhibited features of (endogenous) retroviruses, other virus-like particles were packed in highly electron-dense material and resembled DNA viruses. Finally, numerous intermediate assemblies were much smaller than the more “mature viruses” but could also be defective exogenous retroviruses and many of them were embedded in double-membrane structures (
Cells were seeded onto 6-well plates and separated from irradiated MEFs via two-stage trypsinization after four days. Wells were dosed and incubated with 0.25 ml prewarmed (37° C.) trypsin which was removed and discarded at 4 minutes. An additional 0.25 ml trypsin was added and the plate was again incubated. After eight minutes cells were removed and pelleted via centrifugation. The cells were washed twice in PBS containing 0.5% BSA, fixed and permeabilized with Cytofix/Cytoperm. The Primary antibody was added at a dilution of 1:200 in wash buffer incubated overnight at 4° C. The cells were washed twice with 0.5% BSA/PBS, resuspended in wash buffer containing the secondary antibody at a 1:200 dilution Cells were then resuspended in wash buffer, the secondary goat anti-mouse AF568 antibody and incubated for 1 hour at 4° C. The cells were washed as before resuspended in 0.5% BSA/PBS containing two drops/ml DyeCycle Violet to stain the nuclei.
Imaging was conducted with the ImageStream MkII, at 60× magnification with the extended depth of field mode for probe resolution. Images were acquired using the INSPIRE 2.0 software at the lowest flow speed. Fluorophores were excited by the 405 nm and 568 nm lasers at 60 mW and 100 mW, respectively. Cells in focus were gated via histogram of brightfield gradient R. M.S. values and an aspect ratio vs. area plot was used to select the population of single cells. 5000 individual images of focused single cells were taken. Gating was refined further post-acquisition via the IDEAS 6.2 software suite by the same methods and plots, yielding n=1846 (BiPS). This software was used also for image processing, in which a set of custom masks defined by logical operators were used to denote vesicles and sensitively assess probes. For vesicles, it was observed that they may be selected from other cell component by contrast (bright and dark) and also by aspect ratio, and therefore are defined here by “Dilate(Range(Dilate(Range(System(Peak. (Threshold(M01, BF, 70), BF, Bright, 1), BF, 20), 0-5000, 0.4-1), 1), 0-5000, 0.4-1), 1) Or Range (AdaptiveErode(LevelSet(M01, BF, Dim, 5), BF, 75), 0-5000, 0.5-1).” BF and BF2 represent each brightfield image taken of a single cell from each of the two cameras, M01 and M09 represent the corresponding channel masks for each channel and the remaining terms represent mask modifiers and their associated values in the IDEAS software. For resolving immunofluorescence, “Peak(System(M05, Ch05, 3), Ch05, Bright, 1)” where Ch05 represents the staining of interest and M05 represents the corresponding channel mask. Modification was necessary to sensitively include all representative fluorescence, and to distinguish individual foci. The nuclear mask corresponding to DyeCycle Violet staining was defined “Object(M07, Ch07, Tight)” and the cytoplasm was defined through subtraction of the nuclear and vesicle masks from the cell mask through the logical operator available in the software (“Not”). Vesicle-nucleus overlap was determined in favor of vesicles by excluding them from the nuclear mask (“Not”). Probe localization was then defined according to these entities using the respective definitions and the operator “And.” Statistics for foci were generated using the Spot Count feature with a connectedness of 4. Prism 9 was used for graphs and statistics.
The results show that the bat stem cells were positive for coronavirus antigen in western blots and immunostaining (
This example describes the identification of retroviral sequences in the bat IPSC.
2 ml of tissue culture medium were collected, and retroviral particle concentrations were determined using the QuickTiter Retrovirus Quantitation Kit (Cell Biolabs) according to the manufacturer's instructions.
Reverse transcriptase enzyme levels were determined with the colorimetric reverse transcriptase kit (Roche) per the manufacturer protocol. Cells lines represented were lysed in RIPA buffer, frozen at −80° C., thawed on ice, collected and resuspended in the kit lysis buffer (10 μL pellet in 40 μL lysis buffer per colorimetric well). Incubation duration (15 h at 37° C.) was selected for maximal sensitivity to the limit of the kit (1-5 pg RT). Absorbance at 405 nm was measured by microtiter ELISA plate reader. Sample absorbance measurements were fitted to a linear regression of the measured HIV-1 RT standards (Y=2.549×) to obtain RT concentrations in units of ng/well. The results show, that some of the virus-like particles shed from the BiPS into the supernatant as substantial levels of viral particles (1.21*1010 viral particles per mL as determined in a retroviral assay and 0.3 ng/well in a direct reverse transcriptase assay) were detected in the culture medium.
Supernatants were centrifuged at 10000 rpm for 5 min to remove cellular debris, and the cleared lysates transferred to new tubes. Lysates were then diluted in 10-fold dilutions 6 times. Quantification of infectious titer was then performed by plaque assays in comparison to SARS-CoV-2 infection as positive control. Briefly, Vero-E6 cells were plated as confluent monolayers in 12 well dishes. Media was removed, and wells washed in 1 ml of PBS. 200 ul of diluted lysates was then added per well and allowed to incubate for 1 hour at 37° C. After viral adsorption, lysates were removed from the well and cells were overlaid with Minimum Essential Media supplemented with 2% FBS, 4 mM L-glutamine, 0.2% BSA, 10 mM HEPES and 0.12% NaHCO3 and 0.7% agar. 72 h post infection, agar plugs were fixed in 10% formalin for 24 h before being removed. Plaques were visualized by staining with TrueBlue substrate (KPL-Seracare) and viral titers calculated and expressed as PFU/ml. Immunostaining with an antibody detected the endogenous retrovirus protein Herv K or a Pan Corona antibody in Rhinolophus ferrumequinum embryonic fibroblasts. Immunostaining with a Pan corona antibody in Myotis myotis fibroblasts or induced pluripotent stem cells (iPS) is shown in FIG. The results show that inoculated Vero cells with cell culture supernatant of the bat iPSCs in the plaque assay did not detect any measurable cytotoxic effects in contrast to acute infectious virus particles that served as positive controls (SARS-CoV-2 particles).
50,000 mouse ES cells (R1) or BiPS cells were plated per well of a 12-well plate on irradiated CF1 mouse embryonic fibroblasts using mouse and bat culture medium respectively. After 24 hours, culture medium containing human Metapneumovirus with GFP (MPV-GFP) (ViralTree) with a final multiplicity of infection (MOI) of 3. Medium was changed daily, and samples were dissociated at 3 and 5 dpi using trypsin/EDTA and the infection rate was determined by fluorescence activated cell sorting (FACS).
In line with the pro-viral environment that was observed transcriptionally, bat stem cells infected with an exogenous Metapneumovirus (MPV) in comparison with mouse stem cells revealed a particularly permissive environment for viral persistence, further underscoring the supportive nature of bat stem cells for viruses. These results suggest that bat stem cells execute a program that in other mammalian cells is activated only after a virus infection.
This example describes the identification of viral sequences in the bat IPSC transcriptome.
Endogenization of an unusually varied group of viral genomes has occurred in bats (for example described in Banerjee et al. 2020; Katzourakis and Gifford 2010; Jebb et al. 2020). Endogenized viral sequences are reactivated and tolerated by all pluripotent stem cells (Grow et al. 2015). As a result, bat pluripotent stem cells should express and tolerate a particularly wide range of endogenized viral sequences. First, endogenous retroviruses, which are abundant and diverse in bat genomes (Jebb et al. 2020; Hayward et al. 2013; Skirmuntt and Katzourakis et al. 2019) were analyzed. As a starting point, anchor points of retroviral sequences that had been previously mapped (Jebb et al. 2020) were picked. To obtain a broader portrait of the virus-like particles and approximate their identity more specifically, RNA-seq data was re-analyzed and additional long-read RNA sequencing (iso-seq) was performed.
Cells were lyzed in 400 μl Trizol reagent (Life Technologies) and total RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen) including a DNase digest to remove any potential contamination from carryover of genomic DNA using RNase-free DNase (Qiagen,) according to the manufacturer's instructions. The extracted RNA was then purified using 1.8×RNAClean XP beads (Beckman Coulter) to remove any molecular impurities. Iso-Seq SMRTbell libraries were prepared as recommended by the manufacturer (Pacific Biosciences). Briefly, 300 nanograms of total RNA (RIN>8) from each sample was used as input for cDNA synthesis using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB,), which employs a modified oligodT primer and template switching technology to reverse-transcribe full-length polyadenylated transcripts. Following double-stranded cDNA amplification and purification, the full-length cDNA was used as input into SMRTbell library preparation, using SMRTbell Express Template Preparation Kit v2.0. Briefly, a minimum of 100 ng of cDNA from each sample were treated with a DNA Damage Repair enzyme mix to repair nicked DNA, followed by an End Repair and A-tailing reaction to repair blunt ends and polyadenylate each template. Next, overhang SMRTbell adapters were ligated onto each template and purified using 0.6×AMPure PB beads to remove small fragments and excess reagents (Pacific Biosciences). The completed SMRTbell libraries were further treated with the SMRTbell Enzyme Clean Up Kit to remove unligated templates. The final libraries were then annealed to sequencing primer v4 and bound to sequencing polymerase 3.0 before being sequenced on one SMRTcell 8M on the Sequel II system with a 24-hour movie each. After data collection, the raw sequencing subreads were imported to the SMRTLink analysis suite, version 10.1 for processing. Intramolecular error correcting was performed using the circular consensus sequencing (CCS) algorithm to produce highly accurate (>Q10) CCS reads, each requiring a minimum of 3 polymerase passes. The polished CCS reads were then passed to the lima tool to remove Iso-Seq and template-switching oligo sequences and orient the isoforms into the correct 5′ to 3′ direction. The refine tool was then used to remove polyA tails and concatemers from the full-length reads to generate final full-length, non-chimeric (FLNC) isoforms. The FLNC isoforms were then clustered together using the cluster tool to generate final, polished consensus isoforms per sample.
Briefly, the existence of viruses in the Rhinolophus ferrumequinum transcriptome was explored by analyzing the RNA-seq and Iso-seq data based on a metagenomic approach using Kraken2 v2.1.2 (Wood et al, 2019). First, the adaptors in the RNA-seq data were removed with Trimgalore v0.6.7 (Krueger et al., 2021) and all replicates for corresponding datasets were joined in one file. The reference library “RefSeq complete viral genomes/proteins” was downloaded and a custom database was built to identify matches within the processed RNA-seq or Iso-seq. To eliminate false positive hits that could be due to matches with any cellular transcript such as oncogenes that are carried by some viruses, a second analysis was performed after eliminating all reads from the RNA-seq and Iso-seq datasets that matched any annotated Rhinolophus ferrumequinum transcript. To do this, the Iso-Seq FLNC isoforms or RNA-seq trimmed fastq sequences were first mapped to the “Rhinolophus ferrumequinum genomic ma exons RefSeq” file “GCF_004115265.1_mRhiFer1_v1.p_rna_from_genomic.fna” using gmap/gsnap (doi.org/10.1093/bioinformatics/bti310). The sequences with no mappings were then used to identify viral sequences using Kraken2 as before.
To trim adapters and generate quality metrics of the fastq files, Trimmgalore v.0.6.6 (www.github.com/FelixKrueger/TrimGalore), a wrapper for Cutadapt (www.github.com/marcelm/cutadapt) and FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/) were used. Then, reads were mapped to the genome of R. ferrumequinum (Bat1K assembly HLrhiFer5) using HISAT2 v.2.2.1 (PMID: 31375807) suppressing unpaired alignments for paired reads (--no-mixed), suppressing discordant alignments for paired reads (--no-discordant), and setting a function for the maximum number of ambiguous characters per read (--n-ceil L,0,0.05). Output files were then filtered to remove any unmapped reads (-F 4), sorted and indexed using samtools (PMC2723002). Aligned reads were then assembled into transcripts using stringTie v2.2.1 (PMC4643835) in stranded mode (-rf). To generate a Ballgown readable expression output with normalized expression units of fragments per kilobase of transcript per million mapped fragments (FPKMs), the Bat1K annotation of known endogenous retrovirus (ERVs) for R. ferrumequinum (PMID: 32699395) (www.genome.senckenberg.de/) were also used as input in strigTie. Output counts were post-process and plotted with a custom R script.
Iso-Seq transcripts were mapped to the genome of R. ferrumequinum (Bat1K assembly HLrhiFer5) using minimap2 (PMC6137996) in mode for long-read/Pacbio-CCS spliced alignment (-ax splice:hq), giving priority to known splice sites from an input annotation (BatIK), to find canonical splicing sites GT-AG in the transcript strand (--junc-bed -uf), with a cost of 5 for a non-canonical GT-AG splicing (-C5), and excluding from the output any secondary alignments (--secondary=no). Output files were then filtered to remove any unmapped reads or those not aligned to the primary alignment (-F 260), sorted and indexed using samtools (PMC2723002). Aligned transcripts to the genome were intersected with known ERVs.
The trimmed reads that were identified by Kraken2 v2.1.2 to map to viral sequences with a confidence score of 0 as described above were classified as either mammalian or non-mammalian using the VIRION database (Carlson et al., 2022) based on their viral taxonomic ID assigned by Kraken2. The data were converted to FASTA format using the Seqtk v1.3 program and the reads were assembled using the Trinity v2.12 software. To check and gather successful assemblies that had produced at least one contig, a custom BASH script was applied for both groups of mammalian and non-mammalian viruses.
To determine if the assembled transcripts represented an expressed viral sequence, all transcripts were mapped to a database of viral genomes using BLAST. The viral database consisted of genomes whose host species contained either ‘human’ or ‘vertebrate’ as specified in the NCBI database. Initially this list contained over 17,000 genomes. However, this was reduced to 3,922 genomes by taking only unique virus/strain names. An additional non-mammalian virus database was generated by combining all genomic sequences of viruses identified by Kraken2 and classified as non-mammalian via VIRION.
Transcripts were also mapped to a combined database of bat, human and mouse genomes to both confirm their presence in the bat and to exclude the possibility of false positives through contamination. For each of these transcripts, expected values for both bat and viral genome BLAST results were combined into a single metric via the following formula: Log (bat-expected value+1×virus-expected value+1). A threshold of less than 0.3, representing a combined e-value of less than 1e−50 for both viral and bat hits, was used to rule out potential false positives. In addition, SQUID (www.eddylab.org/software.html) was used to shuffle the 63 (bottom-up) and 82 (top-down) sequences while preserving the dinucleotide distribution (parameter -d) to obtain a conservative threshold to distinguish bona fide viral homology from matches by random chance. Shuffled sequences were mapped to both the bat genome and viral genome databases, with the same BLAST threshold applied. All transcripts passing this threshold were extended by 5000 bp flanks within the bat genome and these regions were subsequently mapped to the viral database to confirm their presence in a viral genome.
The resulting sequencing reads were mapped against a virus database, using a metagenomic classification tool (Kraken). Mapping of the RNA-seq data revealed the expression of a widely diverse set of retroviral families in bat pluripotent stem cells, which was undetectable in BEFs. The results revealed a taxonomically highly diverse “zoo” of assigned viruses belonging to several significant viral families (
The potential for confounding effects that might impact the metagenomic assessment could be three potential sources for distortions: (i) statistical stringency, (ii) cellular genes containing viral-like sequences (e.g., oncogenes), and (iii) potential xeno sequence pollution originating from the feeder cells. To address the first point, progressively higher statistical stringency was used, yielding an expected decrease in matches. However, even under the most binding conditions, it still resulted in a sizable number of hits. To exclude potential cellular genes misinterpreted by the classification algorithm as viruses, the RNA-seq and iso-seq were depleted from all sequences that match exons, which only marginally affected the number of hits. Finally, some of the classified sequences were checked for murine origin as was the case for several retroviruses. Somatic tissue-derived cells, such as mouse fibroblasts, do not express endogenous viruses in measurable quantities. Hence, the ability to readily detect such sequences may suggest the intriguing possibility that the BiPS cells triggered their activation and expansion or even the infection of the BiPS cells. While confounding effects could affect the metagenomic classification process, it is highly likely that a significant body of proviral sequences inhabits BiPS cells.
This example describes the assembly of novel full-length viruses, shorter viral insertions, and novel, more distant viruses based on the sequencing data from BiPS cells.
As a starting point, anchor points of retroviral sequences that had been previously mapped were picked. Curation of the RNA sequences predicted to match those genomic sequences allowed the identification of not only previously described full-length bat retroviruses (RFeRV,
To investigate the nature of the viruses identified by Kraken2 systematically in detail, pipelines that integrate these sequencing reads to identify viral-like sequences with high confidence were developed (
When the pipelines were applied to the bat stem cell transcriptome data, 311 and 82 transcripts estimated to be mammalian viruses and 351 and 58 non-mammalian viruses (bottom-up and top-down, respectively) were obtained. Direct genome mapping yielded 56 hits (out of 63 transcripts, bottom-up; 25 unique) and 82 (all transcripts from top-down approach; 19 unique) mammalian virus hits against the R. ferrumequinum genome. After applying the BLAST threshold, 31 transcripts, with 13 transcripts shared between both methods, mapped to both a viral sequence and a locus in the bat genome. The BLAST step on extended sequences from both methods yielded a total of 16 sequences within the R. ferrumequinum genome that aligned with known viruses at high confidence. Validating this stringent approach, using the shuffled sequence data, no hits were found for the bottom-up sequences and only two top-down BLAST hits passed the threshold, indicating that the vast majority of the viral hits are not chance matches but reflect bona fide homology. Indeed, this was confirmed by manual inspection of the alignment hits, which showed numerous longer, well-aligning regions substantially exceeding the length and quality of the matches of randomized sequences. The results indicated a taxonomically diverse collection of attributed viruses from a number of major viral families. Included among them are Flaviviridae, Herpesviridae, Poxviridae and Retroviridae. Overall, this exhaustive analysis shows that bat stem cells contain a surprising diversity of sequences that resemble viral genomes. To implement an orthogonal metagenomic strategy, a direct alignment method using the Microsoft Research Premonition pipeline was employed. Using bat stem cell RNA-seq reads as input, this classifier positively recognized 419 different putative viral-like sequences. Again, the taxonomy included a number of important viral families, such as Paramyxoviridae, Flaviviridae, Retroviridae, Coronaviridae and Poxviridae. Manual examination of the expressed virus-sequence revealed a wide range of lengths ranging from (near) full-length viral sequences to specific viral protein encoding domains to short fragments of viral regulatory sequences. As before, the Premonition pipeline predicted sequences were mapped to the bat genome, extended 5000 bp flanks, and performed BLAST searches against the VirusDB and shoed that a total of 13 extended bat genome sequences mapped to know virus genomes, 9 of which overlapped with the bottom-up/top-down approaches, indicating a high degree of consistency. Viruses linked to Hardy-Zuckermann 4 feline sarcoma virus, Friend murine leukemia virus, Porcine endogenous retrovirus E, and PreXMRV-1 provirus were examples. Consequently, both metagenomics pipelines methods reveal a significant number of endogenized sequences that resemble viral genomes with a final count of 20 high-confidence viral hits across all methods. Exemplary sequences of possible viral origin discovered with this method are listed in SEQ ID NOs: 1-349.
This example describes the identification of viral nucleic acid sequences and viral proteins present in the bat genome and in bat cells for the use in vaccine development.
Briefly, viral DNA and RNA sequences can be identified as described in Example 8 Example 9, and Example 10. The viral DNA or RNA sequences can be assembled into long contigs such as SEQ ID NO: 1-349. The contigs can be translated into amino acid sequences. The identified amino acid sequences can be compared to known nucleic acid sequences and proteins using methods like BLAST (www.web.expasy.org/blast) and the sequences can be aligned and translated into amino acid sequences of peptides and proteins. Vital viral enzymes such as the essential genes are replicase ORF1ab, spike (S), envelope (E), membrane (M) and nucleocapsid (N), RNA polymerases, kinases, and viral proteases can be identified using homology models and sequence alignment as described in Example 10.
In order to develop a vaccine, immunogenic CD8+ T cell epitopes in the identified vital virus proteins can be predicted using for example a machine learning platform such as described in Bulik-Sullivan et al. (2018) Deep learning using tumor HLA peptide mass spectrometry datasets improves neoantigen identification. Nature Biotechnology 2018, 37(1). Predictions for these epitopes can be run for each HLA class I allele. Candidate CD8+ epitopes can be maximized for coverage of the prevalent HLA-types in a given population. The method described for generating candidate CD8/MHC class I epitopes can be used to generate peptides with sizes between 9 and 20 amino acids. Further, potential HLA-DRB, HLA-DQ, and HLA-DP MHC class II epitopes can be predicted. The predicted epitopes can then be displayed by MHCs and recognized by human T cells can be tested with methods such as mass spectrometry based HLA I and HLA II epitope binding prediction tools (e.g., Immune Epitope Database and Analysis Resource, www.iedb.org). Epitopes such as for HLA-I or HLA-II can be scored and identified for peptide sequences derived from the identified vital viral enzyme. Top-ranking peptides can be prioritized based on expected population coverage (allele frequencies). Predicted peptides can be tested for T cell responses using PBMCs from human donors and MHC multimers loaded with peptides and ranked. Further assays of T cell reactivity (e.g., interferon-gamma ELISpots, tetramers), which are stricter measures for T cell immunogenicity to epitopes, can be performed to further identify top immunogenic peptides.
The nucleotide sequences for the identified epitopes and peptides can be cloned into vectors with expression cassettes in order to express viral proteins for use in vaccines in recombinant cell. Recombinant cells for example HEK cells or CHO cells can be transfected with these vectors to produce vaccines, such as adenovirus based vaccines. mRNA based vaccines can be synthesized chemically or enzymatically and packaged into lipid particles, nanoparticles or liposomes for further delivery to a subject.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2115676.5 | Nov 2021 | GB | national |
This application claims the benefit of and priority to Great Britain Patent Application No. GB 2115676.5, filed on Nov. 1, 2021; U.S. Provisional Patent Application No. 63/360,472, filed on Oct. 4, 2020; U.S. Provisional Patent Application No. 63/248,835, filed on Sep. 27, 2021, the disclosure of each of which is hereby incorporated by reference in its entirety for all purposes.
This invention was made with U.S. government support, Grant No. HR0011-19-2-0020, awarded by DARPA and Grant No. W81XWH-20-1-0270, awarded by Department of Defense (DoD), NIAID grant U19AI135972, and CRIPT (Center for Research on Influenza Pathogenesis and Response), a NIAID supported Center of Excellence for Influenza Research and Response grant CEIRR, contract #75N93019R00028. The U.S. government has certain rights to the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/077012 | 9/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63360472 | Oct 2021 | US | |
| 63248835 | Sep 2021 | US |
