Patent Grant
10953085
The present disclosure relates to genetically engineered Foot and Mouth Disease Virus (FMDV), and related proteins, polynucleotides, compositions, methods and systems.
FMDV is the etiological agent of Foot-and-mouth disease (FMD) a rapidly spreading viral disease of cloven hoofed animals and other mammals.
Various forms of FMD vaccines have been developed. The success of such vaccinations however is directly linked to the level of coverage that the vaccine produces against the virus circulating in a particular geographical location. Additional challenges on FMD control relates to the lack of cross-protection offered by strains of different serotypes and even within the same serotype among different subtypes to another. Often times within a given serotype, protection offered by a given vaccine could be insufficient if there is no antigenic match.
Accordingly, despite the efforts provided in recent years, prevention and treatment of FMD remains challenging.
Provided herein are genetically engineered FMD viruses and related components, modified by the insertion of protein tags into selected regions of the capsid of the FMD virus which in several embodiments provide tagged FMD viruses suitable to be used in various diagnostic and therapeutic applications as well as in applications wherein production of the FMD virus and/or its components are desired.
According to a first aspect, a genetically modified FMD virus is described. The genetically modified FMD virus has a viral capsid comprising a genetically modified VP1 protein. The genetically modified VP1 protein is a VP1 protein of the FMD virus engineered to fuse at a C-terminus of the VP1 protein, a 2A protein of the FMDV virus, with an N-terminus of the 2A protein fused to the C-terminus of the VP1 protein. The genetically modified VP1 protein, is further engineered to include a protein tag formed by up to 100 amino acids at an insertion position selected from any one of position −1 to position +6 relative to the N-terminus of the 2A protein, or from any one of position −1 to position −7 relative to the C-terminus of the FMD VP1 protein. In the genetically modified FMD virus, the protein tag of the modified VP1 protein is presented on an external surface of the viral capsid.
According to a second aspect, a genetically modified FMD virus is described. The genetically modified FMD virus has a viral capsid comprising a modified VP1 protein. The modified VP1 protein is a VP1 protein of the FMD virus engineered to insert a protein tag formed by up to 100 amino acids in the VP1 protein at an insertion position selected from any one of position −1 to position −7 relative to the C-terminus of the VP1 protein. In the genetically modified FMD virus, the protein tag of the modified VP1 protein is presented on an external surface of the viral capsid. According to a third aspect a genetically modified VP1 protein of a FMD virus is described. The genetically modified VP1 protein is a VP1 protein of the FMD virus genetically engineered to fuse at a C-terminus of the VP1 protein, a 2A protein of the FMDV virus, with an N-terminus of the 2A protein fused to the C-terminus of the VP1 protein. The genetically modified VP1 protein is further engineered to include a protein tag formed by up to 100 amino acids within the VP1 protein or the 2A protein, at an insertion position selected from any one of position −1 to position +6 relative to the N-terminus of the 2A protein, or selected from any one of position −1 to position −7 relative to the C-terminus of the FMD VP1 protein.
According to a fourth aspect, a genetically engineered VP1 protein of an FMD virus is described. The genetically engineered VP1 protein is a VP1 protein of the FMD virus engineered to include a protein tag formed by up to 100 amino acids inserted at an insertion position selected from any one of position −1 to position −7 relative to the C-terminus of the VP1 protein.
According to a fifth aspect, a genetically engineered polynucleotide is described. The genetically engineered polynucleotide is configured to encode one or more of the modified FMD viruses and/or genetically modified VP1 proteins herein described.
According to a sixth aspect, an FMDV functionalized nanolipoprotein particle is described. The FMDV functionalized nanolipoprotein particle comprises a scaffold protein and a functionalized membrane forming lipid presenting a tag substrate. In the nanolipoprotein particle a tag substrate on the functionalized membrane forming lipid is configured to be attached to a corresponding protein tag presented on one or more genetically engineered FMD virus herein described. In some embodiments, the FMDV functionalized nanolipoprotein particle herein described further comprises a one or more genetically engineered FMD virus herein described attached to the FMDV functionalized nanolipoprotein particle through attachment of the protein tag with corresponding functionalized membrane forming lipid of the FMDV functionalized nanolipoprotein particle.
According to a seventh aspect, a composition is described comprising one or more genetically modified FMD virus, one or more genetically modified VP1 and/or one or more FMD functionalized nanolipoprotein particles together with a suitable vehicle. In some embodiments, the composition is a pharmaceutical composition comprising one or more genetically modified FMD virus, and/or one or more FMD functionalized nanolipoprotein particles together with a pharmaceutically acceptable vehicle.
According to an eighth aspect, a vaccine is described, the vaccine comprising one or more genetically modified FMD viruses, one or more nanolipoprotein particle comprising a scaffold protein and a functionalized membrane forming lipid presenting a tag substrate and/or one or more functionalized FMD nanolipoprotein particles herein described together with a pharmaceutically acceptable vehicle.
According to a ninth aspect, a method to treat or prevent FMD in a cloven-hoofed animal, the method comprising administering to the cloven-hoofed animal an effective amount of a pharmaceutical composition vaccine herein described.
According to a tenth aspect, a method and system to provide a tagged FMD virus are described. The method comprises genetically engineering a FMD virus comprising a VP1 protein genetically engineered to fuse an N-terminus of a 2A protein of the FMD virus with a C-terminus of the VP1 protein and further genetically engineering the FMD virus to insert a protein tag formed by up to 100 amino acids located at an insertion position selected from any one of position −1 to position +6 relative to the N-terminus of the 2A protein, or selected from any one of position −1 to position −7 relative to the C-terminus of the FMD VP1 protein, or genetically engineering a FMD virus comprising a VP1 protein to insert a protein tag formed by up to 100 amino acids at an insertion position selected from any one of position −1 to position −7 relative to the C-terminus of the VP1 protein.
The system comprises at least one polynucleotide encoding for at least one protein tag formed by up to 100 amino acids and reagents for the introduction of the at least one polynucleotide in the FMDV virus.
According to an eleventh aspect, an array to is described, the array comprising one or more genetically modified FMD viruses herein described attached to a substrate. In some embodiments, the array can be used for detection of antibody against one or more strains of FMD virus for investigation, research and/or diagnostic purpose.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide rationally designed FMDV vaccines displaying tags that interact with tag substrates, nanolipoprotein particles (NLP) presenting tag substrates and NLPs containing adjuvant molecules.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide a mutant FMDV vaccine that can be used in a chemically-inactivated form is capable of protecting an animal from clinical FMD when challenged with virulent FMDV wherein said vaccine comprises FMDV modified to display tags on surface-exposed regions of the FMD viral capsid, wherein the latter are capable of interacting with adjuvanted NLP nanoconstructs.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide a genetically modified FMDV encoded by the isolated polynucleotide molecule recited above where the modification enables affinity purification of virus via binding on the tag to tag substrates on columns, beads, and plates.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide a genetically modified FMDV encoded by the isolated polynucleotide molecule recited above where the modification enables recognition of tags by antibodies that specifically recognize the tag sequence.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide a FMDV cDNA clone that is further modified to include unique restriction endonuclease sites that facilitate the exchange of DNA cassettes representing relevant capsid coding regions of other FMDV strains/serotypes/and subtypes of FMDV field isolates.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide a marker vaccine which allows a serological distinction between vaccinated animals and animals infected with FMDV.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide a method for protecting an animal against FMD by administering an effective amount of rationally designed and chemically-inactivated marker FMDV vaccine.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide a method for delaying onset or severity of FMD in an animal by administering an effective amount of rationally designed and chemically-inactivated marker FMDV vaccine formulation.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described allow in several embodiments to provide effective vaccine formulations as well as opportunities for purification through binding to divalent cations and anti-tag antibody recognition protocols.
Genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems herein described can be used in connection with various applications wherein detectability, purification of FMD virus and/or treatment and/or diagnosis of FMD is desired. For example, genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems can be used for delivery of a genetically modified FMD virus to a specific target destination, as a platform for immunostimulating agents, vaccine development and use, and/or to contain cell-targeting moieties. Additional exemplary applications include uses of genetically modified FMD virus, genetically engineered proteins and polynucleotides, FMDV functionalized nanolipoprotein particle, compositions, vaccines, methods and systems in several fields including basic biology research, applied biology, bio-engineering, bio-energy, medical research, medical diagnostics, therapeutics, bio-fuels, and in additional fields identifiable by a skilled person upon reading of the present disclosure.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and example sections, serve to explain the principles and implementations of the disclosure. Exemplary embodiments of the present disclosure will become more fully understood from the detailed description and the accompanying drawings, wherein:
Provided herein are genetically engineered FMD viruses that are modified by the insertion of protein tags into selected regions of the capsid of the FMD virus.
As used herein, the term “Foot-and-mouth disease” or “FMD” indicates an infectious and sometimes fatal viral disease that affects mammals and in particular, cloven-hoofed animals, including domestic and wild bovides and swines. “Mammals” include any warm-blooded vertebrates of the Mammalia class, including humans. The terms “porcine” and “swine” are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig. Exemplary susceptible animals include cattle, water buffalo, sheep, goats, pigs, antelope, deer, and bison, hedgehogs and elephants; llamas and alpacas. In laboratory experiments, mice, rats, and chickens have been successfully infected by artificial means, but they are not believed to contract the disease under natural conditions. Humans are very rarely infected. Symptoms of FMD comprise high fever for approximately two to six days, followed by blisters inside the mouth and on the feet that may rupture and cause lameness. FMD has severe implications for animal farming, since it is highly infectious and can be spread by infected animals through aerosols, through contact with contaminated farming equipment, vehicles, clothing, or feed, and by domestic and wild predators. In particular symptoms in cattle and swine caused by a FMDV infection. Containment of the FMD demands considerable efforts in vaccination, strict monitoring, trade restrictions, and quarantines, and occasionally the killing of animals [5-7].
FMD etiological agent is the foot-and-mouth disease virus (FMDV), a picornavirus which is the prototypical member of the Aphthovirus genus.
The term “virus” indicates an infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. Viruses display a wide diversity of shapes and sizes, called morphologies. In general, viruses are much smaller than bacteria. Most viruses that have been studied have a diameter between 20 and 300 nm. Most viruses cannot be seen with an optical microscope so scanning and transmission electron microscopes are used to visualize virions.
The term “virion” indicates the complete, infective form of a virus outside a host cell. While not inside an infected cell or in the process of infecting a cell, viruses exist in the form of independent particles. These virions, also known as viral particles, consist of two or three parts: (i) the genetic material made from either DNA or RNA; (ii) a protein coat, called the capsid, which surrounds and protects the genetic material; and in some cases (iii) an envelope of lipids that surrounds the protein coat when they are outside a cell.
A “functional virion” is a virus particle that is able to enter a cell capable of hosting the virus, and express genes of its particular genome (either an unmodified genome or a genetically modified genome as described herein) within the cell. Cells capable of hosting a FMDV include baby hamster kidney, strain 21, cells (BHK-21) and BHK-αVβ6 cells expressing bovine αVβ6 integrin. Other mammalian cells, especially other bovine and porcine cells, can also serve as suitable host cells for FMDV virions.
A picornavirus is a virus belonging to the family Picornaviridae, a family of viruses in the order Picornavirales. Vertebrates, including humans, serve as natural hosts. Picornaviruses are nonenveloped viruses that represent a large family of small, cytoplasmic, plus-strand RNA (˜7.5 kb) viruses with a 30 nm icosahedral capsid.
The term “capsid” refers to the protein shell of a virus. It consists of several oligomeric structural subunits made of protein. The observable 3-dimensional morphological subunits, which may or may not correspond to individual proteins, are called capsomeres. The capsid encloses the genetic material of the virus. Capsids are broadly classified according to their structure. The majority of viruses have capsids with either helical or icosahedral structure. The icosahedral shape, which has 20 equilateral triangular faces, approximates a sphere, while the helical shape resembles the shape of a spring, taking the space of a cylinder but not being a cylinder itself. The capsid faces can consist of one or more proteins. For example, the foot-and-mouth disease virus capsid has faces consisting of three proteins named VP1-3. Some viruses are enveloped, meaning that the capsid is coated with a lipid membrane known as the viral envelope. The envelope is acquired by the capsid from an intracellular membrane in the virus' host; examples include the inner nuclear membrane, the golgi membrane, and the cell's outer membrane. Once the virus has infected a cell and begins replicating itself, new capsid subunits are synthesized according to the genetic material of the virus, using the protein biosynthesis mechanism of the cell. During the assembly process, a portal subunit is assembled at one vertex of the capsid. Through this portal, viral DNA or RNA is transported into the capsid. Structural analyses of major capsid protein (MCP) architectures can been used to categorize viruses into families. For example, the bacteriophage PRD1, Paramecium bursaria Chlorella algal virus, and mammalian adenovirus have been placed in the same family. Although the icosahedral structure is extremely common among viruses, size differences and slight variations exist between virions. Given an asymmetric subunit on a triangular face of a regular icosahedron, with three subunits per face 60 such subunits can be placed in an equivalent manner. Most virions, because of their size, have more than 60 subunits. These variations have been classified on the basis of the quasi-equivalence principle proposed by Donald Caspar and Aaron Klug [8].
In particular, FMD viruses possess an icosahedral capsid shell formed by various copies (typically 60) of each viral protein VP1, VP2, VP3 and VP4 which are cleavage products of the P1 capsid polypeptide precursor. The structural proteins, VP1, VP2, and VP3, fold into an eight-stranded wedge-shaped β-barrel to form the outer capsid structure. The VP4 protein is buried within the capsid and is myristoylated at its N-terminus. By electron microscopy, the FMD virion appears to be a round particle with a smooth surface and a diameter of about 25-30 nm. The integrity of the FMDV capsid structure is very relevant in eliciting protective immune responses and in determining serotype and subtype specificities. Previous studies have revealed surface-exposed regions on the FMDV capsid as sites suitable for the insertion or substitution of foreign epitopes [9-11]. Given the flexibility of the hypervariable G-H loop [12], replacing the FMDV G-H loop of VP1 by those of other serotypes is well tolerated by the virus capsid [2, 3, 9, 13].
The synthesis of FMDV capsid VP proteins and other viral proteins typically include 2A ‘cleavage’ during translation.
The term “cleavage” and “cleaved” as used herein refers to the breaking of a peptide bond between two amino acids within a di-peptide, polypeptide, protein, or polyprotein. In particular, cleavage can refer to the separation of two fused protein segments via breaking a peptide bond. Cleavage of peptide bonds typically occurs via hydrolysis reaction mediated by an enzyme such as a protease. For example, cleavage of the FMD virus polyprotein to separate FMD virus proteins VP1 and 2A occurs via enzymatic cleavage at the N-terminus of the 2A protein, catalyzed by the enzyme 3Cpro in a cleavage during translation process.
This process involves proteases that inhibit the synthesis of normal cell proteins, and other proteins that interact with components of the host cell. The FMDV genome is an ssRNA molecule of mRNA sense of approximately 8500 nucleotides (
In embodiments herein described, genetically engineered FMDV are modified by the strategic insertion of a protein tag into viral capsid proteins in targeted regions predicted to be exposed on the surface of the viral capsid.
The terms “genetically engineered” or “genetically modified”, as used herein and unless otherwise indicated, indicates genetically mutated proteins or polynucleotides (e.g. by having one or more nucleotides or amino acids replaced, deleted and/or added). Polynucleotide and protein molecules can be genetically mutated using recombinant techniques known to those of ordinary skill in the art, including by site-directed mutagenesis, or by random mutagenesis such as by exposure to chemical mutagens or to radiation, as known in the art.
As used herein, in relation to proteins, the term “insertion” of a first protein in a second protein refers to the introduction of the second protein in between two adjacent amino acids of the second protein. As a result, an inserted first protein is located in between a first segment of the second protein having one of the adjacent amino acids at its C-terminus and a second segment of the second protein having the other one of the adjacent amino acids at its N-terminus
In particular, an insertion of a first protein in a second protein is performed by forming a first covalent bond between the N-terminal amino acid of the first protein with a first amino acid of the two adjacent amino acids the second protein, and a second covalent bond between the C-terminal amino acid of the first protein with a second amino acid of the two adjacent amino acids of the second protein. As would be understood by a skilled person, a covalent bond between two amino acids in a protein is typically a peptide bond, which is a covalent bond between a carboxyl group and an amino group of two molecules or portions thereof, which results in releasing a molecule of water.
Accordingly, an insertion of a second protein in a first protein when performed at a protein level typically results in breaking the peptide bond between the two adjacent amino acids of the first protein and forming two new peptide bonds: one between one of the two adjacent amino acids of the first protein and the N-terminal amino acid of the second protein and the other peptide bond formed between the other one of the two adjacent amino acid of the first protein and the C-terminal amino acid of the second protein.
In embodiments herein described, genetically modified FMD viruses are described comprising a genetically modified VP1 protein comprising a VP1 protein of the FMD virus engineered to insert a protein tag up to 100 amino acids at insertion positions in the VP1 protein of the FMD virus. Having the ability to mark (tag) the FMDV capsid without affecting virus viability or altering its ability to enter susceptible cells, provides opportunities for the mutant viruses to bind to tag-specific antibodies, or other complex molecules, depending on the tag selection.
The term “tag” as used herein means protein tags comprising peptide sequences introduced onto a recombinant protein. Tags can be removable by chemical agents or by enzymatic means, such as proteolysis or splicing. Tags can be attached to proteins for various purposes: Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique. These include chitin binding protein (CBP), and the poly(His) tag. The poly(His) tag is a widely-used protein tag; it binds to metal matrices. Chromatography tags can be used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique. Often, these consist of polyanionic amino acids, such as FLAG-tag. Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include V5-tag, Myc-tag, HA-tag and NE-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification. Protein tags can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging). Tags can be combined, in order to connect proteins to multiple other components. However, with the addition of each tag comes the risk that the native function of the protein may be abolished or compromised by interactions with the tag. Therefore, after purification, tags are sometimes removed by specific proteolysis (e.g. by TEV protease, Thrombin, Factor Xa or Enteropeptidase).
Exemplary tags comprise the following, among others known to persons skilled in the art: Peptide tags, such as: AviTag, a peptide allowing biotinylation by the enzyme BirA and so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE (SEQ ID NO:18)); Calmodulin-tag, a peptide that can be bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO:19)); polyglutamate tag, a peptide binding efficiently to anion-exchange resin such as Mono-Q (EEEEEE (SEQ ID NO:20)); E-tag, a peptide recognized by an antibody (GAPVPYPDPLEPR (SEQ ID NO:21)); FLAG-tag, a peptide recognized by an antibody (DYKDDDDK (SEQ ID NO:22)); HA-tag, a peptide from hemagglutinin recognized by an antibody (YPYDVPDYA (SEQ ID NO:23)); His-tag, typically 5-10 histidines that can be bound by a nickel or cobalt chelate (HHHHHH (SEQ ID NO:24)); Myc-tag, a peptide derived from c-myc recognized by an antibody (EQKLISEEDL (SEQ ID NO:25)); NE-tag, a novel 18-amino-acid synthetic peptide (TKENPRSNQEESYDDNES (SEQ ID NO:26)) recognized by a monoclonal IgG1 antibody, which is useful in a wide spectrum of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; S-tag, a peptide derived from Ribonuclease A (KETAAAKFERQHMDS (SEQ ID NO:27)); SBP-tag, a peptide which binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO:28)); Softag 1, for mammalian expression (SLAELLNAGLGGS (SEQ ID NO:29)); Softag 3, for prokaryotic expression (TQDPSRVG (SEQ ID NO:30)); Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK (SEQ ID NO:31)); TC tag, a tetracysteine tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC (SEQ ID NO:32)); V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST (SEQ ID NO:33)); VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK (SEQ ID NO:34)); Xpress tag (DLYDDDDK (SEQ ID NO:35)); Covalent peptide tags such as: Isopeptag, a peptide which binds covalently to pilin-C protein (TDKDMTITFTNKKDAE (SEQ ID NO:36)); SpyTag, a peptide which binds covalently to SpyCatcher protein (AHIVMVDAYKPTK (SEQ ID NO:37)); SnoopTag, a peptide which binds covalently to SnoopCatcher protein (KLGDIEFIKVNK (SEQ ID NO:38)). In embodiments described herein, any of the tags of SEQ ID NO:18-38, and other tags known to those skilled in the art, can comprise one or more amino acid substitutions, insertions, or deletions that do not alter the function of the tag, and can further comprise one or more additional amino acids, up to a maximum tag length of 100 amino acids. In preferred embodiments, the tag comprises up to a maximum of 20 amino acids in length.
In some embodiments, the protein tag can be a polyhistidine tag. A polyhistidine-tag is an amino acid motif in proteins that typically consists of six histidine (His) residues typically, often at the N- or C-terminus of the protein. It is also known as hexahistidine-tag, 6×His-tag, His6 tag and by the trademarked name His-tag (registered by EMD Biosciences). The total number of histidine residues can vary in the tag. N- or C-terminal his-tags can also be followed or preceded, respectively, by a suitable amino acid sequence that facilitates a removal of the polyhistidine-tag using endopeptidases. This extra sequence is not necessary if exopeptidases are used to remove N-terminal His-tags (e.g., Qiagen TAGZyme). Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a pull-down assay. Fluorescent hexahistidine CyDye tags are also available. These use Nickel covalent coordination to EDTA groups attached to fluorophores in order to create dyes that attach to the polyhistidine tag. This technique has been shown to be effective for following protein migration and trafficking. This technique may also be effective in order to measure distance via Fluorescent Resonance Energy Transfer.
In embodiments herein described, insertion of a protein tag into a FMD virus VP1 capsid protein results in the formation of a peptide bond between the N-terminal amino acid of the protein tag and a first amino acid of a protein of the FMD virus VP1 capsid protein or 2A protein fused thereto, as well as a peptide bond between the C-terminal amino acid of the tag and a second amino acid of the FMD virus VP1 capsid protein or 2A protein fused thereto, the second amino acid of the FMDV VP1 capsid protein or 2A protein fused thereto adjacent to the first amino acid of the FMDV VP1 capsid protein or 2A protein fused thereto.
As would be understood by those skilled in the art, an insertion as described above can be obtained through different approaches identifiable by a skilled person.
For example, in some embodiments, an insertion of a protein tag into a VP1 protein or 2A protein fused thereto can be performed at a protein level by first providing the VP1 protein or 2A protein fused thereto and the protein tag and then performing the insertion by breaking a peptide bond between two adjacent amino acids of the VP1 protein or 2A protein fused thereto and then forming new peptide bonds between the VP1 protein or 2A protein fused thereto and the protein tag, as described above. For example, the VP1 protein or 2A protein fused thereto can be digested with a protease to break a peptide bond between two adjacent amino acids in the VP1 protein or 2A protein fused thereto, followed by insertion of the protein tag between the previously adjacent amino acids of the VP1 protein or 2A protein fused thereto, for example using native chemical ligation methods known to those skilled in the art [15].
In some embodiments, a protein comprising the VP1 protein or 2A protein fused thereto with the protein tag inserted can be synthesized as single protein by design. Proteins can be synthesized using biosynthetic methods, such as cell-based methods or cell-free methods known to those skilled in the art. Protein biosynthesis can be performed by translation of DNA or RNA polynucleotides encoding the protein. Thus, protein biosynthesis can be performed by providing cell-based or cell-free protein translation systems with DNA or RNA polynucleotides encoding the protein. For example, protein biosynthesis can be performed in cells transfected with in vitro transcribed RNA encoding the protein (Example 2). Proteins can also be produced by liquid-phase or solid-phase chemical protein synthetic methods known to those skilled in the art [16].
In some embodiments, insertion of a protein tag into a VP1 protein can also be performed by fusion of the tag with two VP1 protein segments, for example where one protein, comprising a VP1 protein segment already having a protein tag at the N-terminal or C-terminal end, is “fused” to a second VP1 protein segment, with the result that the protein tag is covalently joined between the first and second VP1 protein segments.
In some embodiments, insertion of a protein tag in a VP1 protein or 2A protein fused thereto to obtain a genetically engineered VP1 protein can be performed at a polynucleotide level through to an in-frame insertion of a protein tag-coding polynucleotide in between two codons of a VP1 protein-coding polynucleotide, or in between two codons of a 2A protein fused thereto. An in-frame insertion can be performed in several steps, by first providing the VP1 coding and the protein tag-coding polynucleotides and performing the insertion by breaking a bond (typically a phosphodiester bond) between two adjacent nucleotide bases of the first polynucleotide and then forming new bonds between the VP1-coding polynucleotide and the protein tag-coding polynucleotide. For example, the VP1 coding polynucleotide can be digested with one or more restriction endonucleases and then the protein tag-coding polynucleotide inserted by ligation (e.g., using T7 DNA ligase) into compatible site(s) allowing formation of phosphodiester bonds between the first and second polynucleotide bases. Compatible DNA ligation sites can be “sticky” ends, digested with restriction endonuclease producing an overhang (e.g. EcoRI), or can be “blunt ends” with no overhang, as would be understood by those skilled in the art.
In some embodiments, the VP1-coding and the protein tag-coding polynucleotides can be provided within a single polynucleotide by design. For example, a tag can be added by inserting the polynucleotide encoding a protein of interest in a vector that has the tag ready to fuse at the N-terminus or C-terminus. The tag can be added using PCR primers encoding the tag; using PCR the tag can be fused to the N-terminus or C-terminus of the protein-coding polynucleotide, or can be inserted at an internal location, using internal epitope tagging [17], among other methods known to those skilled in the art. Other methods such as overlap extension PCR and infusion HD cloning can be used to insert a tag at a site between the N-terminus and C-terminus of a protein-coding polynucleotide (see Example 2). Optionally, a polynucleotide encoding a ‘linker’ (such as a sequence encoding a short polypeptide or protein sequence, e.g., gly-gly-gly or gly-ser-gly can be placed between the protein of interest and the tag; this can be useful to prevent the tag from affecting the activity of the protein being tagged.
Accordingly, in several embodiments, a protein can be tagged using standard molecular biology techniques suitable to be use for an insertion in the sense of the disclosure as would be understood by those skilled in the art. For example, a His-tag or other protein tag can be inserted in a protein of interest by cloning a polynucleotide encoding a protein of interest in a vector that has the tag ready to fuse at the N-terminus or C-terminus. The His-tag or other protein tags can be added using primers containing the tag; using PCR the tag can be fused to the N-terminus or C-terminus of the gene. Alternatively, methods such as overlap extension PCR and infusion HD cloning can be used to insert a tag at a site between the N-terminus and C-terminus of a protein-coding polynucleotide (see Example 2). Optionally, a linker (such as gly-gly-gly or gly-ser-gly) can be placed between the protein of interest and the 6 His tag. This can be used to prevent the polyhistidine tag from affecting the activity of the protein being tagged.
The choice of the location where a tag is added to a protein sequence depends mainly on the structural and functional features of a protein and the intended downstream methods employing the use of the tag.
In embodiments herein described, the insertion location of a protein tag in a genetically engineered VP1 protein of an FMDV virus is performed at insertion position selected to have the tag presented on the external surface-exposed position of the FMDV without compromising the integrity of the FDMV capsid.
The term “insertion point”, “insertion site” or “insertion position” as used herein refers to the position into which a second protein is inserted into a first protein, and can be described relative to a reference amino acid in the first protein's amino acid sequence. In particular, an insertion point in engineered VP1 can be described as upstream (towards the N-terminus) or downstream (towards the C-terminus) with respect to the reference amino acid.
For example, in a protein sequence, a position adjacently upstream of the reference amino acid (removed 1 residue from the reference amino acid towards the N terminus) can be referred to as “−1”, whereas a position adjacently downstream of the reference amino acid (removed 1 residue from the reference amino acid towards the C-terminus) can be referred to as “+1”.
Examples of reference amino acids in a protein sequence are the N-terminus of a protein sequence or the C-terminus of a protein sequence. The term “N-terminus” of a protein sequence as used herein refers to an amino acid that is not covalently linked via its amino group to another amino acid of the same protein or of a same segment thereof. The term “C-terminus” of a protein sequence as used herein refers to an amino acid that is not covalently linked via its carboxyl group to another amino acid of the same protein or of a same segment thereof.
In embodiments described herein, the protein tag formed by up to 100 amino acids can be inserted at an insertion position selected from any one of position −1 to position +6 relative to the N-terminus of the 2A protein segment of the FMD virus, or from any one of position −1 to position −7 relative to the C-terminus of the VP1 protein segment of the FMD virus. The inserted protein tag residues will be presented on the viral capsid surface. Tag insertion site is a disordered (flexible) loop structure formed by the C-terminus of VP1 or 2A protein fused thereto, therefore by a protein tag insertion the length of an existing loop is extended and the conformation of tag-loop allows its surface exposure.
Diagrams of insertion points of some embodiments herein described are shown in
In some embodiments, the genetically modified VP1 protein is engineered to include a protein tag formed by up to 100 amino acids at an insertion position selected from any one of position −1 to position −7 relative to the C-terminus of the FMD VP1 protein.
In some embodiments, the genetically modified VP1 protein can be engineered to fuse the N-terminus of the 2A protein to the C-terminus of the VP1 protein and to include a protein tag formed by up to 100 amino acids at an insertion position selected from any one of position −1 to position +6 relative to the N-terminus of the 2A protein, or at an insertion position selected from any one of position −1 to position −7 relative to the C-terminus of the VP1 protein.
The term “fused” as used herein in relation to two proteins or related segments refers to a state of being covalently linked via a peptide bond, or two polynucleotides being covalently linked, typically via a phosphodiester bond. In particular, as used herein in some embodiments, FMD virus protein VP1 is said to be fused to FMD virus protein 2A, wherein the C-terminus of protein VP1 is covalently linked to the N-terminus of protein 2A via a peptide bond.
In some embodiments, the tag is comprised in a genetically modified VP1 protein of the FMD virus engineered so that the N-terminus of the 2A protein is fused to the C-terminus of the VP1 protein, wherein a 3Cpro cleavage site is absent, and wherein the tag is inserted at an insertion position selected from any one of position −1 to position +6 relative to the N-terminus of the 2A protein segment of the FMD virus, or from any one of position −1 to position −7 relative to the C-terminus of the VP1 protein segment of the FMD virus. In these embodiments, the sequence of the inserted protein tag eliminates the 3Cpro cleavage site, abolishing 3Cpro-mediated cleavage of the 2A protein from the VP1 protein, resulting in a non-cleaved VP1-2A fusion protein. An exemplary tagged VP1-2A fusion protein described herein comprises the genetically modified VP1 protein comprised in the exemplary genetically engineered FMDV, A24 FMD 2A6H, where insertion of the sequence HHHHHHKQ (SEQ ID NO:16) at position −1 relative to the N-terminus of FMDV protein 2A abolished the 3Cpro cleavage site. In embodiments wherein the tag is inserted into the 2A protein sequence, a 3Cpro cleavage site is absent to prevent cleavage of the 2A protein segment comprising the tag from the VP1 protein
In embodiments described herein, a 3Cpro cleavage site means a cleavage site between the C-terminal amino acid of FMDV protein VP1 and the N-terminal amino acid of FMDV protein 2A. An exemplary 3Cpro cleavage site in exemplary FMDV A24 Cruzeiro is PAKQLLNFDL (SEQ ID NO:40), wherein 3Cpro-mediated cleavage occurs at the site “↓” shown in
In some embodiments, the tag is comprised in a genetically modified VP1 protein of the FMD virus engineered with an insertion of a protein tag located at an insertion position selected from any one of position −1 to position −7 relative to the C-terminus of the VP1 protein. In these embodiments, the sequence of the inserted protein tag preserves the 3Cpro cleavage site, enabling 3Cpro-mediated cleavage of the 2A protein from the VP1 protein, resulting in a VP1 protein that lacks protein 2A. An exemplary tagged VP1 protein described herein comprises the genetically modified VP1 protein comprised in the exemplary genetically engineered FMDV, A24 FMDV P16H, wherein the insertion of the sequence HHHHHHIIAPAK (SEQ ID NO:17) at position −1 relative to the C-terminus of VP1 preserves the 3Cpro cleavage site. In this example, the sequence IIAPAKQ (SEQ ID NO:39) follows the 6×His residues to preserve 3Cpro cleavage at the VP1-2A junction. However, as shown in the exemplary genetically engineered FMDV, A24 FMD 2A6H, a 3Cpro cleavage site is not necessary as 3Cpro cleavage is not required for FMD virus viability.
Tags such as 6 consecutive histidine residues (6×-His) and other protein tags can be used for affinity purification and concentration of recombinant proteins using ion affinity chromatography supports, e.g. Ni2+ or Co2+ beads or affinity columns, and also can be used for binding to other substrates such as nanolipoproteins particles presenting chelated Ni2+ ions (NiNLP) enabling new strategies for enhancing immunogenicity, among other applications known to those skilled in the art and disclosed herein. Primary protein sequence information and structural modeling can be used to predict the three-dimensional location of an inserted tag. For example, protein crystal structure can be visualized using software such as Pymol Viewer, among others known to those skilled in the art.
In some embodiments, a genetically modified FMD virus is provided comprising a genetically engineered VP1 protein according to an embodiment herein described. In some embodiments, the genetically modified FMDV comprises a 6×-His tag-bearing mutant FMD virus.
In particular, in some embodiments, the genetically modified FMD virus has a viral capsid comprising a genetically modified VP1 protein, wherein the VP1 protein of the FMD virus is engineered to fuse at a C-terminus of the VP1 protein, a His tag formed by a HIS segment of sequence HHHHHHKQ (SEQ ID NO: 16) or another protein tag, and a 2A protein of the FMDV virus. In the HIS-tag of the genetically modified VP1 protein, the 2A protein is fused to the C-terminus of the HIS segment. In the genetically modified FMD virus, the HIS-tag or the another protein tag, of the modified VP1 protein is presented on an external surface of the viral capsid. An exemplary embodiment of this modified FMDV is referred to herein as A24 FMD 2A6H. In other embodiments, the genetically modified FMD virus has a viral capsid comprising a modified VP1 protein, wherein the VP1 protein of the FMD virus is engineered to insert a HIS tag having sequence HHHHHHIIAPAK (SEQ ID No: 17) at position 210 of the VP1 protein. In the genetically modified FMD virus, the HIS-tag of the modified VP1 protein is presented on an external surface of the viral capsid. An exemplary embodiment of this modified FMDV is referred to herein as A24 FMDV P16H. The two mutant 6×His FMDV are designed to express 60 copies of 6×His tags decorating the viral capsid, but the location of the His tag differs in the two mutants. To design the mutant constructs, the structural model of the FMDV A24 capsid was generated by homology modeling using the crystal structure of FMDV O1 BFS serotype (PDB, 1FOD) [18]. The structure was visualized on Pymol Molecular Viewer and analyzed for surface-exposed residues/stretches. The structural analysis revealed that the C-terminus of VP1 is largely surface-exposed and composed of variable residues indicating that insertions placed in this region are unlikely to negatively impact the capsid structure. An insertion was preferred in the design of these viruses, rather than substitutions in the VP1 C-term residues, in order to minimize alteration of the actual viral sequences in this region.
Tolerance for insertion of short tags in the conformationally flexible FMDV VP1 G-H loop, such as FLAG, HA and 6×His tags has previously been reported [9, 10, 19, 20], In particular, histidine tag insertions at amino acid position 9 downstream the VP1 ROD (RGD+9) and FLAG at position 8 downstream of (RGD+8) rendered viable viruses [10, 19, 20]. Although the aforementioned studies reported the preservation of virus infectivity and detection of tag expression, the appearance of two alternate forms of FLAG tag in the FLAG tagged FMDVs and a threonine to arginine (T to R) mutations at 158 of VP1 in HA tagged FMDVs have been also documented.
In some embodiments, to generate the first exemplary genetically engineered FMDV, A24 FMD 2A6H, insertion of the sequence HHHHHHKQ (SEQ ID NO:16) in 2A in this infectious cDNA clone results in a mutant FMDV genome in which the mutated 2A protein is fused at C-terminus of VP1 on the capsid exposed surface. This modification produced a mutant virus capsid wherein 2A contains the HHHHHHKQ (SEQ ID NO:16) tag. There is precedent in the literature [21] for the tolerance of FMDV to contain a modified 2A protein as part of the capsid structure and still be able to infect cell lines and give productive infections in the natural host [21]. A major difference in the construct design described herein is that the FMDV 2A is modified with the 6×-His tag insertion toward the N-terminus. The FMDV capsid is composed of 60 copies of VP1; therefore, the capsid of the mutant A24 FMD 2A6H virus is decorated with 60 6×-His sequences. A24 FMDV P16H, the second exemplary mutant virus, carries the 6×His tag insertion within the C-terminus sequence of VP1; the amino acid sequence HHHHHHIIAPAK (SEQ ID NO:17) was inserted at position 210 of VP1. This mutant was engineered to maintain the cleavage site amino acid sequence at the VP1-2A junction which is hydrolyzed by viral proteinase 3Cpro. The mutant virus carries 60 copies of 6×-His tag within the VP1 and has no 2A protein fused to the capsid; it more closely resembles the natural capsid form having no 2A attached but with the 6×His insertion in VP1 C-terminus.
As will be apparent from the present disclosure, the exemplary A24 FMD 2A6H and A24 FMDV P16H FMDV virus constructs display the following characteristics: (1) a fully viable virus presenting a 6×-His tag fused to either VP1 or 2A (A24 FMDV P16H or FMDV 2A6H, respectively); (2) both 6×-His tagged viruses capable of binding divalent cations such as Ni2+ and Co2+ thus allowing for one step purification using an ion affinity chromatography (IAC); (3) both 6×-His tagged viruses recognized by anti-His antibody and capable of binding to Ni2+-coated plates with potential for use as a diagnostic; (4) other 6×-His-tagged FMDV serotypes are easily made using conveniently located unique restriction sites that are engineered into the viral genome and that flank the capsid coding region in the A24Cru backbone (cassette design); (5) 6×-His tagged viruses capable of binding NiNLP/MPLA nanoconstructs [22] and possibly improving FMDV vaccine efficacy 6) more specifically, bromo ethyleneimine (BEI)-inactivated mutant A24 FMDV P16H-NiNLP-MPLA formulation might be effective as a vaccine formulation in FMDV animal model challenge studies compared to/with parental (wild type, WT) A24 FMDV. In a mouse model of FMDV described herein, it is shown that percentage of survival after lethal challenge with FMDV A24Cru improves in animals vaccinated with low doses (0.025 ug/animal) BEI-inactivated mutant A24 FMDV P16H formulated with NiNLP-MPLA as compared with animals vaccinated with the same dose of BEI-inactivated A24 FMDV P16H virus alone. These results indicate that exemplary formulation of FMDV P16H-Ni-NLP-MPLA has potential as an improved vaccine against FMD.
The exemplary 6×-His tagged FMDVs display growth characteristics similar to the parental WT-A24 FMDV and are able to bind to complex molecules such as nickel (Ni) substrate-containing nanolipoprotein particles (NLP) further comprising adjuvants such as MLPA (NiNLP-MPLA) that are visualized by electron microscopy (EM) and detected by His-specific antibodies. These mutant viruses can be purified in one step by using a Co2+-charged affinity supports (e.g. Co-beads or Co-affinity columns) with up to 95% recovery of infectious viral particles. Remarkably a BEI-inactivated 6×His mutant A24 FMDV P16H formulated with nanolipoproteins conjugated with an MPLA adjuvant (Ni-NLP-MPLA) was shown to be an effective vaccine using a validated FMD mouse model. In particular, animals vaccinated with the mutant virus conjugated with NiNLP-MPLA developed significant levels of neutralizing antibodies at 7 dpv and showed a boost after challenge. Similar vaccine potency study is expected with the A24 FMDV P16H formulated with either Ni-NLP-MPLA or a commercially available adjuvant (Sepic ISA-206) in cattle, a FMDV natural host.
The presence of multiple copies of 6×-His decorating the FMDV capsid surface allows the mutant viruses to attach to nickel-coated ELISA plates. As described herein, binding of 6×His A24 viruses to these plates could be disrupted in a concentration-dependent manner suggesting that addition of Ni-NLP was competitive. Similarly, 6×His mutant virus attachment to Ni and Co complex particles (NiNLP-MPLA, Co-beads and Co-columns) is also described herein.
In some embodiments, a genetically modified VP1 protein of a FMD virus is provided. In a particular embodiment, the genetically modified VP1 protein is a VP1 protein of the FMD virus genetically engineered to fuse at a C-terminus of the VP1 protein a HIS tag at formed by a HIS segment of sequence HHHHHHKQ (SEQ ID NO: 16) and a 2A protein of the FMDV virus fused at a C-terminus of the HIS segment; an exemplary genetically modified VP1 protein comprises the VP1 of FMDV 2A6H. In another particular embodiment, the genetically engineered VP1 protein is a VP1 protein of the FMD virus engineered to insert a HIS tag having sequence HHHHHHIIAPAK (SEQ ID No: 17) at position 210 of the VP1 protein; an exemplary genetically modified VP1 protein comprises the VP1 of FMDV P16H. According to these embodiments, it is expected that a genetically engineered FMDV VP1 comprising a tag can be produced using recombinant viral protein production techniques. For example, a polynucleotide encoding a genetically engineered VP1 can be used to express an isolated sequence encoding an engineered FMDV VP1 protein, for example in cell culture, or in a cell-free expression system.
The approaches, configurations and methods described with reference to the exemplary HIS tag and related insertion points can be applied to other protein tags in accordance with the guidance provided by the instant disclosure as will be understood by a skilled person.
In some embodiments, a genetically engineered polynucleotide is provided encoding one or more of the engineered FMD viruses and/or genetically modified VP1 protein herein described.
The term “polynucleotide” or “polynucleotide molecule” as used herein indicates an organic polymer composed of two or more monomers including nucleotides, or analogs thereof. The isoelectric point of a polynucleotide in the sense of the disclosure is less than 7 as will be understood by a skilled person. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or pyrimidine base and to a phosphate group and that is the basic structural unit of nucleic acids. The term “nucleotide analog” refers respectively to a nucleotide in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term “polynucleotide” includes nucleic acids of any length, and in particular DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called “nucleotidic oligomer” or “oligonucleotide.”. In particular, polynucleotides in the sense of the disclosure comprise biological molecules comprising a plurality of nucleotides. Exemplary nucleic acids include deoxyribonucleic acids, ribonucleic acids, and synthetic analogues thereof, including peptide nucleic acids. Polynucleotides can typically be provided in single-stranded form or double-stranded form and in liner or circular form as will be understood by a person of ordinary skill in the art.
In some embodiments herein described, the polynucleotide is a DNA molecule that can be in a linear or circular form, and encodes the desired polypeptide under the control of a promoter specific to an enzyme such as an RNA polymerase, that is capable of transcribing the encoded portion of the DNA.
The isolated polynucleotide molecules of the present disclosure encode FMD viruses or VP1 proteins that can be used to prepare chemically inactivated vaccines using art-recognized methods for protecting cattle and swine from infection by a FMDV, as described in further detail below. Furthermore, these isolated polynucleotide molecules are useful because they can be mutated using molecular biology techniques to encode genetically-modified FMD viruses useful, inter alia, as vaccines for protecting cattle and swine from FMD infection. Such genetically-modified FMD viruses, as well as vaccines comprising them, are described in further detail below. In addition to targeted mutagenesis by recombinant techniques random approaches such as chemical treatment or use of error prone DNA polymerases can also be used for virus attenuation, or antigenic modification.
Accordingly, the present disclosure further provides a method for making a genetically modified FMDV, which method comprises mutating the DNA sequence encoding an infectious RNA molecule which encoding a genetically modified FMDV in accordance with the present disclosure, and expressing the genetically modified FMDV using a suitable expression system. A FMDV, either wild-type or genetically modified, can be expressed from an isolated polynucleotide molecule using suitable expression systems generally known in the art, examples of which are described in this application. For example, the isolated polynucleotide molecule can be in the form of a plasmid capable of expressing the encoded virus in a suitable host cell in vitro. Furthermore, FMD virus like particle production in plants, insect cells, yeast and virus vectors such as adenovirus are other alternatives that can provide additional flexibility of producing high quantity recombinant preparations that can be explored further.
In several embodiments, one or more tagged FMD viruses can be provided by methods and/or systems herein described. In particular, in some embodiments, a method is described that comprises genetically engineering a FMD virus comprising a VP1 protein to fuse a HIS tag formed by a HIS segment of sequence HHHHHHKQ (SEQ ID NO: 16), or another protein tag, at a C-terminus of the VP1 protein and to fuse a 2A protein of the FMDV virus at a C-terminus of the HIS segment, or genetically engineering a FMD virus comprising a VP1 protein to insert a HIS segment having sequence HHHHHHIIAPAK (SEQ ID No: 17) or another protein tag, at position 210 of the VP1 protein or at another insertion position of the present disclosure.
The system comprises at least one polynucleotide encoding for at least one of HIS tag formed by a HIS segment of sequence HHHHHHKQ (SEQ ID NO: 16) and HIS tag having sequence HHHHHHIIAPAK (SEQ ID No: 17) and/or another protein tag in accordance with the present disclosure, and reagents for the introduction of the at least one polynucleotide in the FMDV virus. Site directed mutagenesis and overlap-extension PCR can be utilized to introduce or delete sequences amenably in FMDV and in many other viruses.
Production and manipulation of the isolated polynucleotide molecules described herein are within the skill in the art and can be carried out according to recombinant techniques described, inter alia, in Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [23] and ref: Innis et al. (eds). 1995. PCR Strategies, Academic Press, Inc., San Diego, [24] which are incorporated herein by reference.
The present disclosure provides vectors comprising genetically modified nucleic acid sequences that encode genetically modified infectious RNA molecules that encode genetically modified Foot and Mouth Disease (FMD) Viruses. Viral vectors such as adenovirus or ms2-phage, VSV, NDV can be utilized to express tagged capsids, virus like particles and sub unit vaccines as suitable.
In particular, the instant disclosure provides isolated polynucleotide molecules encoding genetically modified infectious RNA molecules that encode genetically modified FMD viruses; for example, the vaccine candidate viruses A24 FMDV P16H and A24 FMDV 2A6H.
It is understood that terms herein referring to nucleic acid molecules such as “isolated polynucleotide molecule” and “nucleotide sequence” include any polynucleotides such as DNA and RNA molecules and include both single-stranded and double-stranded molecules whether it is natural or synthetic origin.
For example, SEQ ID NO:1 is a DNA sequence corresponding to the genetically modified RNA genome of a genetically modified FMDV. Thus, a DNA sequence complementary to the DNA sequence set forth in SEQ ID NO:1 is a template for, is complementary to or “encodes”, the RNA genome of the FMD virus (RNA that encodes the FMDV) and in particular, an infectious RNA molecule.
An “infectious RNA molecule”, for purposes of the present disclosure, is an RNA molecule that encodes the necessary elements for viral replication, transcription, and translation into a functional virion in a suitable host cell.
Terms such as “suitable host cell” and “appropriate host cell”, unless otherwise indicated, refer to cells into which RNA molecules (or isolated polynucleotide molecules or viral vectors comprising DNA sequences encoding such RNA molecules) of the present disclosure can be transformed or transfected. “Suitable host cells” for transfection with such RNA molecules, isolated polynucleotide molecules, or viral vectors, comprise mammalian, particularly bovine, hamster (e.g. BHK-21 cell line) and porcine cells, and are described in further detail below.
In some embodiments, the polynucleotide of the FMDV can be a homolog of SEQ ID NO: 1. For purposes of the present disclosure, two nucleotide (RNA or DNA) sequences are substantially homologous when at least 80% (preferably at least 85% and most preferably at least 90%) of the nucleotides match over the defined length of the sequence using algorithms such as CLUSTAL or PHILIP. Sequences that are substantially homologous can be identified in a Northern hybridization experiment under stringent conditions as is known in the art. See, for example, Sambrook et al. [23]. Sambrook et al. describe highly stringent conditions as a hybridization temperature 5-10° C. below the Tm of a perfectly matched target and probe; thus, sequences that are “substantially homologous” would hybridize under such conditions.
As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant disclosure such as deletion or insertion of nucleotides that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the disclosure encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. Alterations in a nucleic acid fragment that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, can be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell (eukaryotic, such as plant, yeast, fungi, or algae; prokaryotic, such as bacteria) can comprise the steps of: constructing an isolated polynucleotide of the present disclosure or an isolated chimeric gene of the present disclosure; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.
High-resolution crystal structures for several FMDV strains, such as FMDV O1 BFS serotype (PDB, 1FOD) [18] are available in databases such as Protein Databank, and others known to those skilled in the art. Thus, polynucleotides encoding homologs of FMDV described herein that are capable of properly folding and maintaining an icosahedral virion structure can be determined by visualizing the crystal structure of FMDV homologs using programs such as Pymol Molecular Viewer and others, as would be understood by persons skilled in the art. The structure of FMDV is also described in published articles such as Fry et al., 1990 [25].
Moreover, substantially similar nucleic acid fragments can also be characterized by their ability to hybridize. Estimates of such homology are provided by DNA-DNA, DNA-RNA, or RNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art [26]. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.
Thus, isolated sequences that encode a modified FMDV, for example, A24 FMDV 2A6H (SEQ ID NO:1) and A24 FMDV P16H (SEQ ID NO:3) and which hybridize under stringent conditions, as described herein, to the exemplary modified FMDVs, A24 FMDV 2A6H and A24 FMDV P16H sequences disclosed herein, (SEQ ID NO:1, SEQ ID NO:3), or to fragments thereof, are encompassed by the present disclosure.
Substantially similar nucleic acid fragments of the instant disclosure can also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller [27], the local homology algorithm of Smith et al. [28]; the homology alignment algorithm of Needleman and Wunsch [29]; the search-for-similarity-method of Pearson and Lipman [30]; the algorithm of Karlin and Altschul [31], modified as in Karlin and Altschul [32].
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or sub-genomic sequence, or the complete cDNA or gene sequence. A reference sequence can comprise, for example, a sequence identifiable a database such as GenBank and others known to those skilled in the art.
The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970) [29].
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein.
By “variants” substantially similar sequences are intended. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, can encode the exemplary A24 FMDV 2A6H and A24 FMDV P16H sequences, the amino acid sequences of the disclosure. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR), a technique used for the amplification of specific DNA segments. Generally, variants of a particular nucleotide sequence of the disclosure will have generally at least about 90%, preferably at least about 95% and more preferably at least about 98% sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein.
By “variant protein” a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein is intended. Variant proteins encompassed by the present disclosure are biologically active, that is they possess the desired biological activity, that is, a modified A24 FMDV 2A6H and/or A24 FMDV P16H activity. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of modified A24 FMDV 2A6H and A24 FMDV P16H activities of the disclosure will have at least about 90%, preferably at least about 95%, and more preferably at least about 98% sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein. A biologically active variant of a protein of the disclosure may differ from that protein by as few as 1-15 amino acid residues, or even 1 amino acid residue.
The polypeptides of the disclosure can be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Novel proteins having properties of interest can be created by combining elements and fragments of proteins of the present disclosure, as well as with other proteins. Methods for such manipulations are generally known in the art. Thus, the genes and nucleotide sequences of the disclosure include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the disclosure encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired modified A24 FMDV 2A6H and/or A24 FMDV P16H activities. The mutations that will be made in the DNA encoding the variant does not place the sequence out of reading frame and preferably will not disrupt secondary mRNA structure with potential involvement as cis-actin elements of the viral genome.
In particular, variants of FMDV described herein can include those comprising mutations in regions of the FMDV genome that do not encode highly conserved mRNA structure, thus indicating that the RNA structure of these regions is not predicted to have any role in other viral biological functions. RNA structure can be predicted from primary RNA sequence using programs such as Mfold web server v.2.3 [33] among others known to those skilled in the art.
The term “open reading frame”, or “ORF”, as used herein, means the minimal nucleotide sequence required to encode a particular FMDV protein without an intervening stop codon.
Accordingly, the instant disclosure relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein considering codon degeneracy. “Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.
As used herein, unless otherwise indicated, “coding regions” refer to those sequences of RNA from which FMDV proteins are expressed, and also refer to cDNA that encodes such RNA sequences. Likewise, “ORFs” refer both to RNA sequences that encode FMDV proteins and to cDNA sequences encoding such RNA sequences.
It is furthermore to be understood that the isolated polynucleotide molecules and the isolated RNA molecules of the present disclosure include both synthetic molecules and molecules obtained through recombinant techniques, such as by in vitro cloning and transcription.
In some embodiments, a polynucleotide is provided encoding one or more FMDV cDNA clones that further comprise unique restriction endonuclease sites that facilitate the exchange of DNA cassettes representing relevant capsid coding regions of other FMDV strains/serotypes and subtypes of FMDV field isolates. Other FMDV strains/serotypes and subtypes of FMDV field isolates are identifiable by those skilled in the art, such as those described in Rieder et al. (1993) and Uddowla et al. (2012) [34, 35]. For example, exemplary unique restriction sites R1 and R2 in
In some embodiments, an FMDV functionalized nanolipoprotein particle is provided. The term “nanolipoprotein particle” “nanodisc” “rHDL” or “NLP” as used herein indicates a supramolecular complex formed by a membrane forming lipid arranged in a lipid bilayer stabilized by a scaffold protein. The membrane forming lipids and scaffold protein are components of the NLP. In particular, the membrane forming lipid component is part of a total lipid component, (herein also membrane lipid component or lipid component) of the NLP together with additional lipids such as functionalized lipids and/or lysolipids, that can further be included in the NLPs as will be understood by a skilled person upon reading of the present disclosure. The scaffold protein component is part of a protein component of the NLP together with additional proteins such as membrane proteins, target proteins and other proteins that can be further included as components of the NLPs as will be understood by a skilled person upon reading of the present disclosure. Additional components can be provided as part of the NLP herein described as will be understood by a skilled person. In particular, the membrane lipid bilayer can attach membrane proteins or other amphipathic compounds through interaction of respective hydrophobic regions with the membrane lipid bilayer. The membrane lipid bilayer can also attach proteins or other molecule through anchor compounds or functionalized lipids as will be understood by a skilled person upon reading of the disclosure. In a nanolipoprotein particle, the membrane lipid bilayer can be confined in a discoidal configuration by the scaffold protein. Predominately discoidal in shape, nanolipoprotein particles typically have diameters between 5 to 25 nm, share uniform heights between 3 to 6 nm and can be produced in yields ranging between 30 to 90%.
In particular, in embodiments herein described the nanolipoprotein particle can be formed by a lipid bilayer confined in a discoidal configuration by a scaffold protein. In this configuration, the lipid bilayer confined by the scaffold protein can be 3-6 nanometers in thickness, the nanolipoprotein particle can have an overall diameter of 5-25 nanometers, and the scaffold protein on the particle can have a thickness of 1-2 nanometers. In some embodiments, an entire NLP structure can be up to 600 kilodaltons in weight.
The particular membrane forming lipid, scaffold protein, the lipid to protein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles as will be understood by a skilled person. In the nanolipoprotein particle the membrane forming lipid are typically arranged in a membrane lipid bilayer confined by the scaffold protein in a discoidal configuration as will be understood by a skilled person.
The term “membrane forming lipid” or “amphipathic lipid” as used herein indicates a lipid possessing both hydrophilic and hydrophobic moieties that in an aqueous environment assembles into a lipid bilayer structure that consists of two opposing layers of amphipathic molecules know as polar lipids. Each polar lipid has a hydrophilic moiety, such a polar group such as, a derivatized phosphate or a saccharide group, and a hydrophobic moiety, such as a long hydrocarbon chain. Exemplary polar lipids include phospholipids, sphingolipids, glycolipids, ether lipids, sterols, alkylphosphocholines and the like. Amphipathic lipids include but are not limited to membrane lipids, such as amphipathic lipids that are constituents of a biological membrane, such as phospholipids like dimyristoylphosphatidylcholine (DMPC) or dioleoylphosphoethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC), or dipalmitoylphosphatidylcholine (DPPC).
The term “scaffold protein” as used herein indicates any amphipathic protein that is capable of self-assembly with amphipathic lipids in an aqueous environment, organizing the amphipathic lipids into a bilayer disc, and comprise apolipoproteins, lipophorins, derivatives thereof (such as truncated and tandemly arrayed sequences) and fragments thereof (e.g. peptides) which maintains the amphipathic nature and capability of self-assembly, such as apolipoprotein E4 (22 Kd fragment), lipophorin III, apolipoprotein A-1 and the like. In general, scaffold proteins have an alpha helical secondary structure in which a plurality of hydrophobic amino acids form a hydrophobic face and a plurality of hydrophilic amino acids form an opposing hydrophilic face. In some embodiments, rationally designed amphipathic peptides and synthetic apolipoproteins which maintain an amphipathic structure and capability of self-assembly can serve as a scaffold protein of the NLP.
The term “apolipoprotein” as used herein indicates an amphipathic protein that binds lipids to form lipoproteins. The term “amphipathic” pertains to a molecule containing both hydrophilic and hydrophobic properties. Exemplary amphipathic molecules comprise molecules having hydrophobic and hydrophilic regions/portions in its structure. Examples of biomolecules which are amphipathic include but not limited to phospholipids, cholesterol, glycolipids, fatty acids, bile acids, saponins, and additional lipids identifiable by a skilled person. A “lipoprotein” as used herein indicates a biomolecule assembly that contains both proteins and lipids. In particular, in lipoproteins, the protein component surrounds or solubilizes the lipid molecules enabling particle formation. Exemplary lipoproteins include the plasma lipoprotein particles classified under high-density (HDL) and low-density (LDL) lipoproteins, which enable fats and cholesterol to be carried in the blood stream, the transmembrane proteins of the mitochondrion and the chloroplast, and bacterial lipoproteins. In particular, the lipid components of lipoproteins are insoluble in water, but because of their amphipathic properties, apolipoproteins such as certain Apolipoproteins A and Apolipoproteins B and other amphipathic protein molecules can organize the lipids in a bilayer orientation with exposed hydrophilic moieties, creating the lipoprotein particle that is itself water-soluble, and can thus be carried through water-based circulation (e.g. blood, lymph in vivo or in vitro). Apolipoproteins known to provide the protein components of the lipoproteins can be divided into six classes and several sub-classes, based on the different structures and functions. Exemplary apolipoprotein known to be able to form lipoproteins comprise Apolipoproteins A (apo A-I, apo A-II, apo A-IV, and apo A-V), Apolipoproteins B (apo B48 and apo B100), Apolipoproteins C (apo C-I, apo C-II, apo and apo C-IV), Apolipoproteins D, Apolipoproteins E, and Apolipoproteins H. For example, apolipoproteins B can form low-density lipoprotein particles, and have mostly beta-sheet structure and associate with lipid droplets irreversibly, while Apolipoprotein A1 comprise alpha helices and can associate with lipid droplets reversibly forming high-density lipoprotein particles.
The term “protein” as used herein indicates a polypeptide with a particular secondary and tertiary structure that can interact with another molecule and in particular, with other biomolecules including other proteins, DNA, RNA, lipids, metabolites, hormones, chemokines, and/or small molecules. The term “polypeptide” as used herein indicates an organic linear, circular, or branched polymer composed of two or more amino acid monomers and/or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full-length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer, peptide, or oligopeptide. In particular, the terms “peptide” and “oligopeptide” usually indicate a polypeptide with less than 100 amino acid monomers. In particular, in a protein, the polypeptide provides the primary structure of the protein, wherein the term “primary structure” of a protein refers to the sequence of amino acids in the polypeptide chain covalently linked to form the polypeptide polymer. A protein “sequence” indicates the order of the amino acids that form the primary structure. Covalent bonds between amino acids within the primary structure can include peptide bonds or disulfide bonds, and additional bonds identifiable by a skilled person. Polypeptides in the sense of the present disclosure are usually composed of a linear chain of alpha-amino acid residues covalently linked by peptide bond or a synthetic covalent linkage. The two ends of the linear polypeptide chain encompassing the terminal residues and the adjacent segment are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Unless otherwise indicated, counting of residues in a polypeptide is performed from the N-terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond to the C-terminal end (—COOH group) which is the end where a COOH group is not involved in a peptide bond. Proteins and polypeptides can be identified by x-ray crystallography, direct sequencing, immunoprecipitation, and a variety of other methods as understood by a person skilled in the art. Proteins can be provided in vitro or in vivo by several methods identifiable by a skilled person. In some instances where the proteins are synthetic proteins in at least a portion of the polymer two or more amino acid monomers and/or analogs thereof are joined through chemically-mediated condensation of an organic acid (—COOH) and an amine (—NH2) to form an amide bond or a “peptide” bond.
As used herein the term “amino acid”, “amino acid monomer”, or “amino acid residue” refers to organic compounds composed of amine and carboxylic acid functional groups, along with a side-chain specific to each amino acid. In particular, alpha- or α-amino acid refers to organic compounds composed of amine (—NH2) and carboxylic acid (—COOH), and a side-chain specific to each amino acid connected to an alpha carbon. Different amino acids have different side chains and have distinctive characteristics, such as charge, polarity, aromaticity, reduction potential, hydrophobicity, and pKa. Amino acids can be covalently linked to form a polymer through peptide bonds by reactions between the amine group of a first amino acid and the carboxylic acid group of a second amino acid. Amino acid in the sense of the disclosure refers to any of the twenty naturally occurring amino acids, non-natural amino acids, and includes both D an L optical isomers.
In embodiments herein described, NLPs comprise different types of scaffold protein and a lipid component membrane forming lipids and possibly other lipids, in ratios and proportions that would be identifiable by a skilled person upon reading of the present disclosure.
In some embodiments, the membrane forming lipids component of the lipid component lipids such as phospholipids, preferably including at least one phospholipid, typically soy phosphatidylcholine, egg phosphatidylcholine, soy phosphatidylglycerol, egg phosphatidylglycerol, palmitoyl-oleoyl-phosphatidylcholine distearoylphosphatidylcholine, or distearoylphosphatidylglycerol. Other useful phospholipids include, e.g., phosphatidylcholine, phosphatidylglycerol, sphingomyelin, phosphatidylserine, phosphatidic acid, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, stearoyl-palmitoyl-phosphatidylcholine, di-palmitoyl-phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, di-myrstoyl-phosphatidylserine, and dioleyl-phosphatidylcholine.
Additionally exemplary membrane forming lipids that can be comprised in various combinations together with one or more lysolipids comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-didecanoyl-sn-glycero-3-phosphocholine, 1,2-di erucoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg phosphatidylcholine extracts, soy phosphatidylcholine extracts, heart phosphatidylcholine extracts, brain phosphatidylcholine extracts, liver phosphatidylcholine extracts, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, 1,2-dilauroyl-sn-glycero-3-phosphate, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, Egg phosphatidylethanolamine extract, soy phosphatidylethanolamine extract, heart phosphatidylethanolamine extract, brain phosphatidylethanolamine extract, 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), egg phosphatidylglycerol extract, soy phosphatidylglycerol extract, 1,2-distearoyl-sn-glycero-3-phospho-L-serine, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, 1,2-dilauroyl-sn-glycero-3-phospho-L-serine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, soy phosphatidylserine extract, brain phosphatidylserine extract, 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate, cholesterol, ergosterol, sphingolipids, ceramides, sphingomyelin, gangliosides, glycosphingolipids, 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-di-O-octadecenyl-3-trimethylammonium propane.
Additional membrane forming lipids suitable for use in providing NLPs are well known to persons of ordinary skill in the art and are cited in a variety of well-known sources, e.g., McCutcheon's Detergents and Emulsifiers and McCutcheon's Functional Materials, Allured Publishing Co., Ridgewood, N.J., both of which are incorporated herein by reference.
In some embodiments, FMDV-NLPs herein described can comprise scaffold proteins such as human derived apoE4, truncated versions of human derived apoE4 (e.g. apoE4, 22 k), human derived apoE3, truncated versions of human derived apoE3 (e.g. apoE3, 22 k), human derived apoE2, truncated versions of human derived apoE2 (e.g. apoE2, 22 k), human derived apoA1, truncated versions of human derived apoA1 (e.g. Δ49ApoA1, MSP1, MSP1T2, MSP1E3D1), mouse derived apoE4, truncated versions of mouse derived apoE4 (e.g. apoE4, 22 k), mouse derived apoE3, truncated versions of mouse derived apoE3 (e.g. apoE3, 22 k), mouse derived apoE2, truncated versions of mouse derived apoE2 (e.g. apoE2, 22 k), mouse derived apoA1, truncated versions of mouse derived apoA1 (e.g. Δ49ApoA1, MSP1, MSP1T2, MSP1E3D1), rat derived apoE4, truncated versions of rat derived apoE4 (e.g. apoE4, 22 k), rat derived apoE3, truncated versions of rat derived apoE3 (e.g. apoE3, 22 k), rat derived apoE2, truncated versions of rat derived apoE2 (e.g. apoE2, 22 k), rat derived apoA1, truncated versions of rat derived apoA1 (e.g. Δ49ApoA1, MSP1, MSP1T2, MSP1E3D1), lipophorins (e.g. B. mori, M. sexta), synthetic cyclic peptides that mimic the function of apolipoproteins. Other apolipoproteins, as will be understood for a skilled person, can be used to form NLP, including but not limited to apoB and apoC.
In some embodiments, the scaffold proteins can contain amino acid additions, deletions, or substitutions. In other embodiments, the scaffold proteins can be derived from various species and more particularly derived from human, mouse, rat, guinea pig, rabbit, cow, horse, pig, dog, and non-human primates.
As would be understood by those skilled in the art, in general, NLPs form with a molar ratio of scaffold protein to lipid in the 1:150-1:300 range. NLP assembly can be assessed experimentally using methods described herein and other methods known to those skilled in the art to determine optimal NLP assembly conditions for NLPs described herein to form complexes with engineered FMDV described herein.
In several embodiments herein described, genetically engineered FMDV-NLPs show different size, compositions, and homogeneity. Composition of a NLP can be detected by various techniques known in the art, such as high performance liquid chromatography (HPLC), reverse phase high performance liquid chromatography (RP-HPLC), mass spectrometry, thin layer chromatography, NMR spectroscopy and additional techniques identifiable by a skilled person.
Size and compositions of the genetically engineered FMDV-NLPs herein described can be characterized by SEC (size exclusion chromatography) traces which are used to separate out molecules in solution by their size and in some cases their molecular weights as will be understood by a skilled person.
Any measuring technique available in the art can be used to determine properties of the NLPs herein described. For example, techniques such as dynamic light scattering, x-ray photoelectron microscopy, powder x-ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) can be used to determine average size and dispersity of the NLPs.
In embodiments, genetically herein described engineered FMDV-NLPs herein described further include functionalized amphipathic compounds that can be added during the assembly of the NLP herein described.
The term “functionalized amphipathic compounds” in the sense of the disclosure indicates compounds having a hydrophobic portion and a hydrophilic portion in a configuration where the hydrophobic portion anchor is able to anchor the compound to the lipid bilayer of the NLP and the hydrophilic portion is presented on the NLP bilayer face following NLP assembly. In the functionalized amphipathic compounds in the sense of the disclosure the hydrophilic portion of typically essentially consists or comprises a hydrophilic functional group.
The term “functional group” as used herein indicates specific groups of atoms within a molecular structure that are responsible for a characteristic chemical reaction of that structure. Exemplary functional groups include hydrocarbons, groups containing double or triple bonds, groups containing halogen, groups containing oxygen, groups containing nitrogen and groups containing phosphorus and sulfur all identifiable by a skilled person.
The term “present” as used herein with reference to a compound or functional group indicates attachment performed to maintain the chemical reactivity of the compound or functional group as attached. Accordingly, a functional group presented on an amphipathic compound, is able to perform under the appropriate conditions the one or more chemical reactions that chemically characterize the functional group.
In NLPs, the use of functionalized amphipathic compounds enables attachment of various peptides or other biologics to the surfaces of the lipid of the NLP that allows some desired target features to be obtained, such as stability, affinity for a target molecule, and the like as will be understood by a skilled person (see U.S. Pat. Nos. 8,889,623 and 8,883,729 each incorporated herein by reference in its entirety).
The term “attach” or “attached” as used herein, refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment where, for example, a first molecule is directly bound to a second molecule or material, or one or more intermediate molecules are disposed between the first molecule and the second molecule or material.
In the FMDV functionalized nanolipoprotein particle herein described, the NLP comprises a scaffold protein and a functionalized membrane forming lipid presenting a functional group that can attach one or more protein tags presented in the genetically engineered VP1 protein and in the genetically engineered FMD virus herein described (herein also protein tag substrate).
Non-limiting examples of functional groups presented on functionalized lipids include: chelated Ni atoms, azide, anhydride, alkynes, thiols, halogens, carboxy, amino, hydroxyl, and phosphate groups, and additional groups identifiable by a skilled person upon reading of the present disclosure.
In some embodiments, the functional group on the functionalized amphipathic compound can be a reactive chemical groups (e.g. azide, chelated nickel, alkyne, and additional reactive chemical group identifiable by a skilled person), a biologically active compound (e.g. DNA, peptide, carbohydrate, and additional biologically active group identifiable by a skilled person) or a small molecule (e.g. cellular targeting compound, adjuvant, drug, and additional small molecules identifiable by a skilled person). In some embodiments, the functionalized amphipathic compound is a functionalized lipid compound. Functional groups that enhance the lipid solubility are referred to as hydrophobic or lipophilic functional groups. Functional groups that lack the ability to either ionize or form hydrogen bonds tend to impart a measure of lipid solubility to a drug molecule. The functional group can be attached to the lipid polar head through covalent or ionic bonds and “weak bonds” such as dipole-dipole interactions, the London dispersion force and hydrogen bonding, preferably covalent. Moreover, functionalization of the lipid can involve hydrophobic quantum dots embedded into the lipid bilayer. The following article is incorporated by reference in its entirety: R. A. Sperling, and W. J. Parak. “Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles”. Phil. Trans. R. Soc. A 28 Mar. 2010 vol. 368 no. 1915 1333-1383.
In some embodiments, functionalized amphipathic compounds can comprise one or more of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-((folate)amino)hexanoyl), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanyl), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(hexanoylamine), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanylamine), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl), 1,2-Dioleoyl-sn-Glycero-3-Phospho(Ethylene Glycol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carb oxy(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000], cholesterol modified oligonucleotides, cholesterol-PEG2000-azide, cholesterol-PEG2000-Dibenzocyclooctyl, cholesterol-PEG2000-maleimide, cholesterol-PEG2000-N-hydroxysuccinimide esters, cholesterol-PEG2000-thiol, cholesterol-azide, cholesterol-Dibenzocyclooctyl, cholesterol-maleimide, cholesterol-N-hydroxysuccinimide esters, cholesterol-thiol, C18 modified oligonucleotides, C18-PEG2000-azide, C18-PEG2000-Dibenzocyclooctyl, C18-PEG2000-maleimide, C18-PEG2000-N-hydroxysuccinimide esters, C18-PEG2000-thiol, C18-azide, C18-Dibenzocyclooctyl, C18-maleimide, C18-N-hydroxysuccinimide esters, C18-thiol.
In some embodiments, the functional group presented on the NLP is selected to bind and in particular to specifically bind one or more protein tags presented on one or more VP1 and/or one or more genetically engineered FMDV herein described.
The term “bind”, “binding”, “conjugation” as used herein indicates an attractive interaction between two elements which results in a stable association of the element in which the elements are in close proximity to each other. If each element is comprised in a molecule the result of binding is typically formation of a molecular complex. Attractive interactions in the sense of the present disclosure includes both non-covalent binding and, covalent binding. Non-covalent binding as used herein indicates a type of chemical bond, such as protein-protein interaction, that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions. Non-covalent bonding includes ionic bonds, hydrophobic interactions, electrostatic interactions, hydrogen bonds, and dipole-dipole bonds. Electrostatic interactions include association between two oppositely charged entities. An example of an electrostatic interaction includes using a charged lipid as the functional membrane lipid and binding an oppositely charged target molecule through electrostatic interactions:
Protein tag substrates and corresponding protein tag capable of binding through non-covalent binding include but are not limited to those listed in Table 1 below.
A covalent bond is instead a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds. In short, attraction-to-repulsion stability that forms between atoms when they share electrons is known as covalent bonding. Covalent bonding includes many kinds of interaction, including σ-bonding, π-bonding, metal to non-metal bonding, agostic interactions, and three-center two-electron bonds.
Protein tag substrates and corresponding functional groups on protein tags capable of binding through covalent binding include but are not limited to those listed in Table 2 below.
In some embodiments, the tag substrate can be a molecule chelating a bivalent metal ion and the protein tag can be a polyhistidine molecule. In some of those embodiments, the bivalent metal ion can be selected from the group consisting of Ni2+, Zn2+−, Co2+, and Cu2.
In some embodiments, the tag substrate can be a negatively charged moiety and the protein tag is a poly-arginine molecule.
In some embodiments, the tag substrate can be avidin or a derivative thereof and the protein tag is selected from the group consisting of Avi-Tag, SBP-tag and Strep-tag
In some embodiments, the tag substrate can be a thiol and the protein tag is selected from the group consisting of maleimide derivatives, haloacetamides, pyridyldithio-propionate and thiosulfates.
In some embodiments, the tag substrate can be selected from the group consisting of maleimide derivatives, haloacetamides, pyridyldithio-propionate and thiosulfates, and wherein the protein tag is a thiol presenting protein tag.
In some embodiments, the tag substrate can be an amine and the protein tag is selected from the group consisting of protein presenting active esters, activated carboxylic acids, isothiocyanates, sulfonyl chlorides, dichlorotriazines, aryl halides and acyl azides.
In some embodiments, the tag substrate can be selected from the group consisting of active esters, activated carboxylic acids, isothiocyanates, sulfonyl chlorides, dichlorotriazines, aryl halides and acyl azides, and wherein the protein tag is a protein presenting an amine group.
In some embodiments, the tag substrate can be an azide molecule and the protein tag is a protein presenting an acetylene group.
In some embodiments, the tag substrate can be an acetylene molecule and the protein tag is a protein presenting an azide molecule.
In some embodiments, the tag substrate can be is selected from the group consisting of hydrazines, hydroxylamines or aromatic amines and protein tag is a protein presenting an aldehyde or ketone group.
In some embodiments, the tag substrate can be an aldehyde or ketone molecule and the protein tag is selected from the group consisting of proteins presenting hydrazines, hydroxylamines and aromatic amines.
In some embodiments, the functional group presented on the NLP is selected to specifically bind one or more protein tags presented on one or more genetically engineered VP1 and/or one or more genetically engineered FMDV herein described.
The wording “specific” “specifically” or “specificity” as used herein with reference to the binding of a first molecule to second molecule refers to the recognition, contact and formation of a stable complex between the first molecule and the second molecule, together with substantially less to no recognition, contact and formation of a stable complex between each of the first molecule and the second molecule with other molecules that may be present. Exemplary specific bindings are antibody-antigen interaction, cellular receptor-ligand interactions, polynucleotide hybridization, enzyme substrate interactions etc. The term “specific” as used herein with reference to a molecular component of a complex, refers to the unique association of that component to the specific complex which the component is part of. The term “specific” as used herein with reference to a sequence of a polynucleotide refers to the unique association of the sequence with a single polynucleotide which is the complementary sequence. By “stable complex” is meant a complex that is detectable and does not require any arbitrary level of stability, although greater stability is generally preferred.
In some embodiments one or more functionalized amphipathic compounds are comprised together with non-functionalized membrane forming lipids in the lipid component of the NLP. In some embodiments functionalized amphipathic compounds can be functionalized membrane forming lipid. In some embodiments, one or more functionalized membrane forming lipids are added or replace the membrane forming lipids in the lipid component of the NLP herein described also comprising one or more cross-linking lipids. For example, cross-linking lipids comprise commercially available lipids (e.g., Avanti Lipids) that are known to those skilled in the art, as described in Bader et al. 1985 [36], such as diyne- or diacetylene-containing lipids.
In particular, the ratio between functionalized membrane forming lipid and membrane forming lipids is dependent on the identity of the functionalized membrane forming lipid, and it can be as low as 1% or even lower and as high as 100% as NLPs have been successfully formed with 100% functionalized membrane forming lipid such as DOGS-NTA-Ni (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)). This suggests that NLPs can be formed with any percentage of functionalized membrane forming lipid (from 0 to 100%), depending on the specific functionalized membrane forming lipid used.
In some embodiments, the ratio of functionalized amphipathic compounds can vary from 0.1 mol % to 95 mol % (relative to polymerizable lipid) depending on the functionalized amphipathic compounds. Functionalized amphipathic compounds that are lipids themselves, such as DOGS-NTA-Ni (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)) can be used at 95 mol %, with cross-linking lipid comprising at least 5 mol %. A preferred molar ratio of DOGS-NTA-Ni:polymerizable lipid:membrane forming lipid is 35:20:45. In contrast, functional amphipathic compounds that are less lipid like, such as cholesterol modified oligonucleotides, a lower mol % (0.1-10 mol %) is needed for successful NLP assembly.
In some embodiments, the nanolipoprotein particles herein described can further comprise other functional molecules embedded in the membrane lipid bilayer (e.g. interacting with the membrane lipid bilayer components through van der Waals forces), conjugated to a lipophilic anchor compound inserted into the membrane lipid bilayer (e.g. through hydrophobic-hydrophilic interactions) or conjugated through binding of a functional group with a corresponding functional group presented on functionalized membrane forming lipid of the membrane lipid bilayer. In some of those embodiments, the other functional molecules comprise small molecules and in particular cyclic or non-cyclic peptides and can be comprised in the NLP here described in an amount that varies from case to case, and that in general can range from 0.1-10 mol %. inorganic nanoparticles”. Phil. Trans. R. Soc. A 28 Mar. 2010 vol. 368 no. 1915 1333-1383.
In some embodiments herein described, the ratio of functionalized NLP to engineered FMDV is from 1:1 to 60:1. In some embodiments, the ratio of functionalized NLP to engineered FMDV is from 12:1 to 60:1. Exemplary data shown in the Table of
Methods and systems for production of FMDV functionalized nanolipoprotein particles are known to those skilled in the art. The membrane forming lipid and the protein components of the FMDV functionalized NLP are generally able to self-assemble in a biological (largely aqueous) environment according to the thermodynamics associated with water exclusion (increasing entropy) during hydrophobic association. For instance, exemplary FMDV functionalized nanolipoprotein particles disclosed herein can be made following the methods described in Blanchette et al. 2010 [37] and Blanchette et al. 2009 [38].
Alternatively, the amphipathic lipid and the protein components of the NLP can be provided and allowed to assembly in cell free expression systems known to those skilled in the art.
As used herein, the wording “cell free expression” refers to at least one compound or reagent that, when combined with a polynucleotide encoding a polypeptide of interest, allows in vitro translation of said polypeptide/protein of interest.
In some embodiments, one or more genetically modified FMD viruses herein described is attached to a substrate to provide an array.
The term “array” as described herein, alternatively known as a “lab-on-a-chip” or “microarray”, is a device configured for high-throughput screening and detection of biological molecular interactions, typically comprising a solid phase wherein a first molecule is attached to a substrate, and a liquid phase wherein a biological sample material is applied under suitable time and conditions to allow binding of a second molecule in the liquid phase to the first molecule attached to the solid phase. The solid phase can be comprised, for example of a glass slide, with immobilized molecules “spotted” onto the surface, or can comprise multi-well plates made of glass, plastic, or other materials known to those skilled in the art, with molecules attached to the inside surface of the well. As would be understood by those skilled in the art, different types of arrays can be used to detect a wide range of biological molecular interactions, such as DNA microarrays, RNA arrays, protein microarrays, peptide microarrays, tissue microarrays, chemical compound microarrays, and antibody microarrays, among others. Other arrays can comprise multi-well plates such as those used for ELISA, as described herein.
In some embodiments described herein, the engineered tagged FMDV can attach to an array or a plate via tag-substrate binding, wherein the substrate is coated onto the surface of the solid phase of the array or plate. For example, the mutant FMDV 2A6H and FMDV P16H described herein can bind directly to Ni-coated plates, such as those used for ELISA as described in the Example 8. As would be understood by those skilled in the art, this eliminates the need for virus-capturing biological molecules to attach the FMDV to the solid phase, such as FMDV strain-specific monoclonal antibodies or integrin receptor molecules.
In some embodiments, the array can be used for detection of antibody against one or more strains of FMD virus for investigation, research and/or diagnostic purposes. For example, an array can be used for the differentiation of animals vaccinated with a tagged FMDV from FMDV-infected animals, as described in Example 8. In addition, the ability to introduce a polynucleotide comprising a tagged VP1 protein into different FMDV strains by cloning into unique restriction sites as described herein (see
In some embodiments, a composition comprising one or more genetically modified FMD virus, one or more genetically modified VP1 and/or one or more FMD functionalized nanolipoprotein particles together with a suitable vehicle is provided. In some embodiments, the composition is a pharmaceutical composition comprising one or more genetically modified FMD virus, and/or one or more FMD functionalized nanolipoprotein particles together with a pharmaceutically acceptable vehicle.
The term “vehicle” as used herein indicates any of various media acting usually as solvents, carriers, binders or diluents of a one or more genetically modified FMD virus, and/or one or more FMD functionalized nanolipoprotein particles comprised in the composition as an active ingredient.
In some embodiments, one or more genetically modified FMD virus, and/or one or more FMD functionalized nanolipoprotein particles can be comprised in pharmaceutical compositions (e.g. a vaccine) together with an excipient or diluent. In particular, in some embodiments, pharmaceutical compositions are described which contain one or more genetically modified FMD virus, and/or one or more FMD functionalized nanolipoprotein particles, in combination with one or more compatible and pharmaceutically acceptable vehicle, and in particular with pharmaceutically acceptable diluents or excipients.
The term “excipient” as used herein indicates an inactive substance used as a carrier for the active ingredients of a medication. Suitable excipients for the pharmaceutical compositions herein disclosed include any substance that enhances the ability of the body of an individual to absorb the FMD functionalized nanolipoprotein particles. Suitable excipients also include any substance that can be used to bulk up formulations with NLP to allow for convenient and accurate dosage. In addition to their use in the single-dosage quantity, excipients can be used in the manufacturing process to aid in the handling of NLP. Depending on the route of administration, and form of medication, different excipients may be used. Exemplary excipients include but are not limited to antiadherents, binders, coatings disintegrants, fillers, flavors (such as sweeteners) and colors, glidants, lubricants, preservatives, sorbents.
The term “diluent” as used herein indicates a diluting agent which is issued to dilute or carry an active ingredient of a composition. Suitable diluents comprise any substance that can decrease the viscosity of a medicinal preparation.
In certain embodiments, compositions and, in particular, pharmaceutical compositions can be formulated for systemic administration, which includes parenteral administration and more particularly intravenous, intradermic, and intramuscular administration. In some embodiments, compositions and, in particular, pharmaceutical compositions can be formulated for non-parenteral administration and more particularly intranasal, intratracheal, vaginal, oral, and sublingual administration.
Exemplary compositions for parenteral administration include but are not limited to sterile aqueous solutions, injectable solutions or suspensions including FMD functionalized nanolipoprotein particles. In some embodiments, a composition for parenteral administration can be prepared at the time of use by dissolving a powdered composition, previously prepared in a freeze-dried lyophilized form, in a biologically compatible aqueous liquid (distilled water, physiological solution or other aqueous solution).
In some embodiments, compositions herein described comprise His-tagged FMDV capsid expressed as virus-like particle (VLP) for FMD vaccine developments produced in various biological systems such as E. coli, insect cells infected with recombinant baculoviruses or in mammalian cells with adenovirus vectors, or employing less virulent marker FMDV (FMDVLL3B3D) vaccine platforms with DIVA capabilities (allowing differentiation of infected from vaccinated animals) [35, 39, 40].
In one embodiment, a vaccine is provided, the vaccine comprising one or more genetically modified FMD virus, one or more nanolipoprotein particle comprising a scaffold protein and a functionalized membrane forming lipid presenting a protein tag substrate and/or one or more functionalized FMDV nanolipoprotein particles herein described together with a pharmaceutically acceptable vehicle.
The term “vaccine” as used herein indicates a composition, and in particular a biological preparation, that establishes or improves immunity to a particular external pathogenic assault in an animal. Vaccines in the sense of the present description can be prophylactic, or therapeutic. In vaccine herein described a modified FMDV alone or presented on an FMDV NLPS can be used as an antigen to immunize a mammal against FMD.
The term “antigen” or “immunogen” as used herein indicates a substance that prompts the generation of antibodies and/or can cause an immune response. In particular, antigens in the sense of the present disclosure encompass all substances that can be recognized by an adaptive immune system. Exemplary antigens include exogenous antigens and endogenous antigens. Exogenous antigens are antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection. By endocytosis or phagocytosis, these antigens are taken into the antigen-presenting cells (APCs) and processed into fragments. APCs then present the fragments to T helper cells (CD4+) by the use of class II histocompatibility molecules on their surface. Some T cells are specific for the peptide: MHC complex. They become activated and start to secrete cytokines. Cytokines are substances that can activate cytotoxic T lymphocytes (CTL), antibody-secreting B cells, macrophages, and other particles. Endogenous antigens are antigens that have been generated within the cell, as a result of normal cell metabolism, or because of viral or intracellular bacterial infection. The fragments are then presented on the cell surface in the complex with MHC class I molecules. If activated cytotoxic CD8+ T cells recognize them, the T cells begin to secrete various toxins that cause the lysis or apoptosis of the infected cell. In order to keep the cytotoxic cells from killing cells just for presenting self-proteins, self-reactive T cells are deleted from the repertoire as a result of tolerance (also known as negative selection). They include xenogenic (heterologous), autologous and idiotypic or allogenic (homologous) antigens. Antigens are also generated between normal cells.
Vaccines of the present disclosure can further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others. Non-limiting examples of adjuvants that can be used in the vaccine of the present disclosure include MPLA, Montanide ISA206, the RIM adjuvant system (Ribi Inc., Hamilton, Mont.), polyICLC (Oncovir Inc, DC), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, and Avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions useful in the vaccine of the disclosure include modified SEAM62 and SEAM 1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween® 80 detergent, 2.5% (v/v) ethanol, 100 μg/ml Quil A, and 50 μg/ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines.
In particular embodiments, rationally designed FMDV vaccines are provided displaying 6×-His tags or other protein tags that interact with cations such as Ni2+, Co2+, nanolipoprotein particles (NLP) presenting chelated nickel (NiNLP) and NiNLPs preferably further containing adjuvant molecules.
Examples of other ions capable of binding to histidine tags comprise Cu, Zn, Ca, Mg, Fe, Ga, Sc, Y, In, and Tc. In particular, binding of metal ions to the surface of the His-tagged FMDV capsid can facilitate detection by in vivo tomographic imaging or other detection methods known to those skilled in the art. Metal ions can comprise diagnostic radioisotopes of ions such as Y, In, Tc, Cu, or Ga.
In some embodiments, a mutant FMDV vaccine is provided, when used in a chemically-inactivated form is capable of protecting an animal from clinical FMD when challenged with virulent FMDV wherein said vaccine comprises FMDV modified to display 6×-His tags on surface-exposed regions of the FMD viral capsid, wherein the latter are capable of interacting with adjuvanted NiNLP nanolipoprotein particles.
The term “chemically-inactivated” as used herein refers to an inactivated vaccine comprising of a genetically modified FMDV that has been chemically treated to ‘kill’ the virion so that it cannot divide, but the virus capsid proteins maintain some of their integrity to be recognized by the immune system and evoke an adaptive immune response. Chemicals that can be used to inactivate virions comprise paraformaldehyde (PFA), binary ethylenimine (BEI), and others known to persons skilled in the art. Other methods to inactivate virions comprising vaccines comprise treating with heat, radiation, and others identifiable by a skilled person.
In particular, in some embodiments, genetically modified FMDV-NLP herein described present MPLA alone or in combination with additional adjuvants. MLPA is a well-established adjuvant that has been shown to induce both cellular and humoral immune responses. MPLA is a low toxicity derivative of a bacterial cell wall component, lipopolysaccharide (LPS).
In any of the above embodiments, one or more additional same or different adjuvant and/or antigen can be attached to the genetically modified FMDV-NLP through binding the anchor compound-anchor substrate compound and/or through incorporation of an amphipathic adjuvant into the nanoparticle during self-assembly.
In some embodiments, binding or conjugation of the adjuvant or other immunological agent can be performed by chelation of the immunological agent to a functional group presented by one or more functionalized lipids in the genetically modified FMDV-NLP herein described. The term “chelation” as used herein indicates the binding or complexation of a bi- or multidentate ligand with a single metal ion. In particular, in some embodiments, the bi- or multidentate ligand is part of the lipid and is capable of binding a metal ion. The ligands, which are often organic compounds, are called chelants, chelators, chelating agents, or sequestering agents. Chelating agents form multiple bonds with a single metal ion. The term “chelants” as used herein indicates a molecule that forms a stable complex with certain metal ions. Examples of chelating moieties include, but are not limited to, nitrilotriaceticacid (NTA), iminodiacetic acid (IDA), and diethylenetriamine penta-acetic acid (DTPA).
In some embodiments, an adjuvanted NiNLP nanoconstruct can be provided in combination and/or directly attached to the FMDV virus particle via the 6×-His sequence (or other tag) that has been introduced/engineered into the viral particle coat proteins. The adjuvanted NiNLPs bind directly to the viral capsid structure, (see the exemplary construct shown in
Successful binding of an immunological agent to the NLP can be readily verified and quantified through a range of techniques that include but are not limited to centrifugal filtration, size exclusion chromatography, fluorescence correlation spectroscopy, cantilever-based sensing, force spectroscopy, Fourier transform infrared spectroscopy, surface plasmon resonance, total internal reflection fluorescence, Raman spectroscopy and additional techniques identifiable by a skilled person. In addition, binding specifically to the surface can be verified using atomic force microscopy and transmission electron microscopy and additional techniques identifiable by a skilled person.
In some embodiments, the formation of genetically modified FMDV-NLP herein described is amenable to the incorporation of multiple adjuvants, including compounds directed to enhance immune response e.g. non-human lipoproteins, bacterial peptides, DNA (e.g. CpG motifs), chemokines, cytokines, pattern-recognition receptors (PRR), lipids, polysaccharides, lipopolysaccharides, and the like; in general, agonists and immune stimulatory molecules, synthetic or natural, (known or unknown at this time) can be assembled in or on NLPs, providing for enhanced, specific, rapid immune stimulation at the site of NLP/antigen inoculation and spreading systemically.
The genetically modified FMDV-NLP or the vaccines or compositions herein described can also be administered to an animal alone or in combination with additional immunostimulatory agents to immunize the animal.
In particular, in some embodiments, genetically modified FMDV-NLPs herein described can be used in combination with NLPs comprising an adjuvant such as microbial derivatives (e.g. CpG derivatives, MPLA), muramyl dipeptide derivatives (e.g. muroctasin), and any peptide or protein adjuvants (e.g. flagellin) can be incorporated into NLP directly to create an adjuvant NLP that can be used as an adjuvant or as a platform for subunit vaccine development with enhanced potency.
In particular, an adjuvant NLP according to the present disclosure can comprise single or multiple adjuvants, such as CpGs, MPLA, and cytokines. In some embodiments, an adjuvant NLP can be customized by including selected adjuvants in view of the desired effect based on the ability of different adjuvants to target different toll-like receptors (TLR) for immunostimulation (e.g. MPLA targets TLR 4, CpGs target TLR9, and flagellin targets TLR5). In some of these embodiments, the customization is performed in view of a specific vaccine formulation to be used in combination with the adjuvant NLP. The customization can be made to combine in the NLP only the adjuvants that are effective for the vaccine formulation of choice, since in some vaccine formulations only certain adjuvants are successful at enhancing the efficacy of the vaccine.
Immunization can be effected by simple injection, for example intramuscular injection in either the shoulder area or in the gluteus maximus hind muscular region. Genetically modified FMDV vaccines can be delivered following solubilization in sterile normal saline solution, for example.
In some embodiments, the genetically modified FMDV-NLP is significantly more immunogenic than the antigen alone, and can be used as a vaccine to protect against FMDV infection when injected into an animal, with or without the aid or use of an adjuvant.
The term “immune response” for purposes of this disclosure means the production of antibodies and/or cells (such as T lymphocytes) that are directed against, or assist in the decomposition or inhibition of, a particular antigenic epitope or particular antigenic epitopes. The phrases “an effective immunoprotective response”, “immunoprotection”, and like terms, for purposes of the present disclosure, mean an immune response that is directed against one or more antigenic epitopes of a pathogen so as to protect against infection by the pathogen in a vaccinated animal. For purposes of the present disclosure, protection against infection by a pathogen includes not only the absolute prevention of infection, but also any detectable reduction in the degree or rate of infection by a pathogen, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the pathogen in the vaccinated animal as compared to an unvaccinated infected animal. An effective immunoprotective response can be induced in animals that have not previously been infected with the pathogen and/or are not infected with the pathogen at the time of vaccination. An effective immunoprotective response can also be induced in an animal already infected with the pathogen at the time of vaccination.
An “antigenic epitope” is, unless otherwise indicated, a molecule that is able to elicit an immune response in a particular animal or species. Antigenic epitopes are proteinaceous molecules, such as polypeptide sequences, optionally comprising non-protein groups such as carbohydrate moieties and/or lipid moieties.
The genetically modified FMD viruses encoded by the above-described isolated polynucleotide molecules are, in one embodiment, able to elicit an effective immunoprotective response against infection by a FMDV. Such genetically modified FMD viruses are preferably able to elicit an effective immunoprotective response against any strain of FMD viruses.
Determining suitable locations for a mutation or mutations that will encode a FMDV that remains able to elicit an effective immunoprotective response against infection by a FMDV and which can differentiate a naturally infected animal from a vaccinated animal can be made based on SEQ ID NO:1 and/or SEQ ID NO:3 provided herein. One of ordinary skill can refer to the sequence of the infectious cDNA clone of FMDV provided by this disclosure, make sequence changes which will result in a mutation inserting residues comprising protein tags displayed on the surface of the FMDV capsid and test the viruses encoded thereby for their abilities to produce FMD in cattle and swine, to elicit an effective immunoprotective response against infection by a FMDV, and to make possible the differentiation of infected vs. vaccinated animals. In so doing, one of ordinary skill can refer to techniques known in the art and also those described and/or exemplified herein.
Antigenic epitopes that are detectable, and the sequences that encode them, are known in the art. Techniques for detecting such antigenic epitopes are also known in the art and include serological detection of antibody specific to the heterologous antigenic epitope by means of, for example, Western blot, ELISA, or fluorescently labeled antibodies capable of binding to the antibodies specific to the heterologous antigenic epitope. Techniques for serological detection useful in practicing the present disclosure can be found in texts recognized in the art, such as Coligan, J. E., et al. (eds), 1998, Current Protocols in Immunology, John Willey & Sons, Inc., which is hereby incorporated by reference in its entirety. Alternatively, the antigenic epitope itself can be detected by, for example, contacting samples that potentially comprise the antigenic epitope with fluorescently-labeled antibodies or radioactively-labeled antibodies that specifically bind to the antigenic epitopes.
Vaccines of the present disclosure can be formulated following accepted convention to include acceptable carriers for animals, such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
Vaccines of the present disclosure can optionally be formulated for sustained release of the FMDV-NLPs, and/or a combination of engineered FMDV and NLPs herein described. Examples of such sustained release formulations include FMDV-NLPs, and/or a combination of engineered FMDV and NLPs in combination with composites of biocompatible polymers, such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including Domb et al. 1992 [41], which is incorporated herein by reference. Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in texts known in the art, for example M. Chasin and R. Langer (eds), 1990, “Biodegradable Polymers as Drug Delivery Systems” in: Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, NY, [42] which is also incorporated herein by reference. Alternatively, or additionally, the FMDV-NLPs, and/or a combination of engineered FMDV and NLPs can be microencapsulated to improve administration and efficacy. Methods for microencapsulating antigens are well-known in the art.
Liposomes can also be used to provide for the sustained release of FMDV-NLPs, and/or a combination of engineered FMDV and NLPs herein described. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Pat. Nos. 4,016,100; 4,452,747; 4,921,706; 4,927,637; 4,944,948; 5,008,050; and 5,009,956, all of which are incorporated herein by reference.
In one embodiment, a method of purification of genetically modified FMDV is provided, wherein the modification enables affinity purification of the genetically modified FMDV via binding on the 6×-His tag to divalent cations on columns, beads, and plates.
Polyhistidine-tags are often used for affinity purification of genetically modified proteins. Polyhistidine-tags can be used for affinity purification of polyhistidine-tagged recombinant proteins expressed in suitable host cells. For example, cells can be harvested via centrifugation and the resulting cell pellet lysed either by physical means or by means of detergents and enzymes such as lysozyme or any combination of these. At this stage, raw lysate contains the recombinant protein among many other proteins originating from the host cell. This mixture is incubated with an affinity resin containing bound bivalent nickel or cobalt ions, which are available commercially in different varieties. Nickel and cobalt have similar properties and as they are adjacent period 4 transition metals. These resins are generally sepharose/agarose functionalized with a chelator, such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxylmethylaspartate (Co-CMA) for cobalt, which the polyhistidine-tag binds with micromolar affinity. The resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion. With Ni-based methods, washing efficiency can be improved by the addition of 20 mM imidazole (proteins are usually eluted with 150-300 mM imidazole). Generally, nickel-based resins have higher binding capacity, while cobalt-based resins offer the highest purity. The purity and amount of protein can be assessed by SDS-PAGE and Western blotting, among others known to those skilled in the art. Various purification kits for histidine-tagged proteins are available from commercial vendors such as Qiagen, Sigma, Thermo Scientific, GE Healthcare, Macherey-Nagel, Clontech, Bio-Rad, and others.
In one embodiment, a method of detecting genetically modified FMDV is provided, wherein the modification enables recognition of 6×-His tags by antibodies that specifically recognize the 6×-His sequence. For example, the polyhistidine-tag can be used to detect the protein via anti-polyhistidine-tag antibodies or alternatively by in-gel staining (SDS-PAGE) with fluorescent probes bearing metal ions. This can be useful in subcellular localization, ELISA, western blotting or other immuno-analytical methods known to those skilled in the art. The polyhistidine-tag can also be successfully used for the immobilization of proteins on a surface such as on a nickel- or cobalt-coated microtiter plate or on a protein array.
In one embodiment, a marker vaccine is provided, which allows a serological distinction between vaccinated animals and animals infected with FMDV.
A marker vaccine allows for immunological differentiation (or segregation) of infected from vaccinated animals, and is also referred to as a DIVA (or SIVA) vaccine [Differentiation (or Segregation) of infected from vaccinated animals] in veterinary medicine. In practical terms, this is most often achieved by omitting an immunogenic antigen present in the pathogen being vaccinated against, thus creating a negative marker of vaccination. In contrast, vaccination with traditional vaccines containing the complete pathogen, either attenuated or inactivated, precludes the use of serology (e.g. analysis of specific antibodies in body fluids) in epidemiological surveys in vaccinated populations. Apart from the advantage of allowing continued serological monitoring of vaccinated individuals, cohorts or populations; the serological difference between vaccinated individuals and individuals that were exposed to the pathogen, and were contagious, can be used to continuously monitor the efficacy and safety of the vaccine.
An exemplary marker vaccine provided herein comprises a 6×His tagged FMDV, which elicits an immune response specific to the tag, enabling the detection of vaccinated animal, for example by ELISA, and also detection of the vaccine virus. Exemplary methods for serological detection of an animal vaccinated with a marker vaccine are disclosed herein. For example, a serum sample can be obtained from an animal, which can then be applied to an indirect sandwich ELISA performed with modifications to the protocol specified by World Health Organization for Animal Health (2012) Chapter 2.1.5 Foot and mouth disease. In: Manual of diagnostic tests and vaccines for terrestrial animals 2012. OIE, Paris, France, which is herein incorporated in its entirety.
In some embodiments, genetically modified FMD viruses and related nanolipoprotein and compositions, and in particular vaccine compositions, can be used in a method to treat or prevent FMD in a cloven-hoofed animal. The method comprises administering to the cloven-hoofed animal an effective amount of a pharmaceutical composition and/or vaccine herein described by direct intramuscular, subcutaneous, intradermal and transdermal routes using needle injection or needle-free devices or automated devices such as gene guns, patches or any available vaccine delivery system.
In a particular embodiment, a method is provided for delaying onset or severity of FMD in an animal by administering an effective amount of chemically-inactivated genetically engineered marker FMDV vaccine formulation. In one embodiment, a method for protecting an animal against FMD by administering an effective amount of chemically-inactivated genetically engineered marker FMDV vaccine is provided.
One method of detecting whether an adequate immune response has been achieved is to determine seroconversion and antibody titer in the animal after vaccination. The timing of vaccination and the number of boosters, if any, will preferably be determined by a doctor or veterinarian based on analysis of all relevant factors, some of which are described above, such as seroconversion data.
An effective amount of any of the above-described vaccines can be determined by conventional means, starting with a low dose of virus, plasmid or viral vector, and then increasing the dosage while monitoring the effects. An effective amount may be obtained after a single administration of a vaccine or after multiple administrations of a vaccine. Known factors can be taken into consideration when determining an optimal dose per animal. These include the species, size, age and general condition of the animal, the presence of other drugs in the animal, and the like. The actual dosage is preferably chosen after consideration of the results from other animal studies. For example, a titer of 1.5 log of serum neutralizing antibodies measured against a homologous vaccine strain or related heterologous strains can be indicative of protection.
The effective dose amount of virus, infectious RNA molecule, plasmid, or viral vector, of the present disclosure can be determined using known techniques, taking into account factors that can be determined by one of ordinary skill in the art such as the weight of the animal to be vaccinated. The dose amount of virus of the present disclosure in a vaccine of the present disclosure preferably ranges from about 101 to about 109 pfu (plaque forming units), more preferably from about 102 to about 108 pfu, and most preferably from about 103 to about 107 pfu. The dose amount of a plasmid of the present disclosure in a vaccine of the present disclosure preferably ranges from about 0.1 g to about 100 mg, more preferably from about 1 μg to about 10 mg, even more preferably from about 10 μg to about 1 mg. The dose amount of an infectious RNA molecule of the present disclosure in a vaccine of the present disclosure preferably ranges from about 0.1 μg to about 100 mg, more preferably from about 1 μg to about 10 mg, even more preferably from about 10 μg to about 1 mg. The dose amount of a viral vector of the present disclosure in a vaccine of the present disclosure preferably ranges from about 101 pfu to about 109 pfu, more preferably from about 102 pfu to about 108 pfu, and even more preferably from about 103 to about 107 pfu. A suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml.
In certain embodiments, one or more tags, one or more FMD virus and/or reagents to genetically modify an FMD virus can also be comprised in a system to provide a genetically modified FMDV according to methods herein described. In some embodiments, one or more genetically modified FMD virus, scaffold proteins and/or membrane forming lipids can be comprised in a system to provide an FMDV-NLP according to methods herein described
In some embodiments, genetically modified FMDV, genetically modified VP1 proteins, polynucleotide, FMDV-NLPs and related compositions, can also be comprised in a system to treat or prevent FMD in a cloven-hoofed animal according to methods herein described. In those embodiments, the system can also comprise the immunostimulatory particle herein described and an adjuvant, the immunostimulatory particle and the adjuvant to be administered to the animal to immunize the animal against FMDV.
The systems herein disclosed can be provided in the form of kits of parts. In kit of parts, the tags, the one or more FMD virus and/or reagents to genetically modify an FMD virus can be included in the kit as a protein alone or in the presence of lipids/detergents for transition into nano-particles. The FMDV and/or the scaffold protein can be included as a plasmid or PCR DNA product for transcription/translation. The indicator protein may be included as encoded RNA for translation. In kit of parts for the immunization of an individual the immunostimulatory FDMV-NLP can be comprised together with adjuvant and/or adjuvant NLPs and additional components identifiable by a skilled person.
In a kit of parts, a polynucleotide, amphipathic lipid, target protein and/or scaffold protein, FMDV-NLPs, adjuvants, adjuvant NLPs and additional reagents are comprised in the kit independently possibly included in a composition together with suitable vehicle carrier or auxiliary agents. For example, a polynucleotide can be included in one or more compositions alone and/or included in a suitable vector, and each polynucleotide- is in a composition together with a suitable vehicle carrier or auxiliary agent.
Additional components can include labeled polynucleotides, labeled antibodies, labels, microfluidic chip, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure. In particular, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here disclosed. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (e.g. wash buffers and the like).
Further details concerning the identification of the suitable carrier agent or auxiliary agent of the compositions, and generally manufacturing and packaging of the kit, can be identified by the person skilled in the art upon reading of the present disclosure.
Having now generally described this disclosure, the same will be better understood by reference to certain specific examples, which are included herein only to further illustrate the disclosure and are not intended to limit the scope of the disclosure as defined by the claims.
Two 6×-His-tagged mutant viruses exemplify the modified viral capsids. The particular placement of the 6×-His insertions enables new effective vaccine formulations, purification protocols through histidine binding of divalent cations and anti-histidine antibody recognition protocols. The design of these 6×-His-mutant viruses carrying unique restriction sites flanking the capsid coding regions allows for exchange of cassettes representing relevant capsid coding regions of other FMDV strains/serotypes/and subtypes of FMDV field isolates.
FMDV type A24 Cruzeiro was derived from the infectious cDNA clone, pA24Cru [denoted herein as A24 FMDV WT [3]. Baby hamster kidney strain 21, clone 13, cell line (BHK-21) was maintained in Eagle basal medium (BME; Life Technologies, Gaithersburg, Md.) supplemented with 10% calf serum (HyClone, South Logan, Utah), 10% tryptose phosphate broth, and antibiotic/antimycotic. Cells were grown at 37° C. with 5% CO2 in a humidified atmosphere.
The structural model of FMDV A24Cru capsid was generated by homology modeling using the crystal structure of FMDV O1 BFS serotype (PDB, 1FOD) (Logan et al., supra). The structure was visualized on Pymol Molecular Viewer and analyzed for surface exposed residues/stretches. The 6×-His tag was inserted in pA24Cru parental cDNA clone by overlap extension PCR in combination with infusion HD cloning. Primer pairs P2289 and P2290, for A24 FMDV 2A6H, and primer pairs P2350 and P2351 for A24 FMDV P16H, were used to insert 6×-His tag in A24 FMDV (
Full length infectious clone of FMDV A24 Cruzeiro [3] was used as a template to PCR amplify various His-tagged FMDV VP1 or 2A fragments that were used for overlap extension PCR amplification of A24 FMDV 6×-His-tag amplicons. For A24 FMDV 2A6H, PCR primer P2289_VP16H-KQ Forward primer, P2290_VP16H-KQ Reverse primer, & P2345. For infusion, Sac II, P2346_Rev infusion Nhe I (Table 3) were used to generate the insert containing SacII and Nhe I infusion cloning sites. The parental FMDV A24 Cruz clone digested with Sac II and Nhe I was used as a vector to clone the insert in the vector using infusion HD cloning kit (Clontech® Laboratories, Inc.) to create the mutant virus plasmid DNA containing 6H insert in the VP1 region at the VP1-2A junctions (
The nucleotide sequence of the A24FMDV 2A6H mutant virus spans VP1 and mutated 2A and encodes 8 extra amino acids (total amino acid residues 26) (SEQ ID NO:12,
Thus, the mutant viruses A24 FMDV 2A6H and A24 FMDV P16H are derived by adding extra nucleotides encoding extra amino acids, (they are derived via insertions). Alternative locations were attempted to place a His-tag in the form of substitutions on the FMDV capsid and resulted in non-viable viruses. One example is presented in
A pair of unique restriction enzyme recognition sites were identified herein (
In order to show the location of 6×-His tag on the surface of mutant FMD viruses a homology model of FMDV A24Cru was constructed. Using this monomeric model, a pentameric model was constructed. As shown in
After sequence verification of the full genome cDNA construct shown above (
The mutant viruses A24 FMDV 2A6H and A24 FMDV P16H produced a cytopathic effect (CPE) in the transfected cells. Sequence analysis confirmed that the mutant viruses maintained the 6×His-tags following 4-5 passages. Full genome sequence of the progeny virus indicated that no additional mutations were fixed in the virus following passages in tissue culture. Stocks of these viruses were titrated and their plaque morphologies were compared with the parental virus (
For virus growth curves, BHK-21 monolayers were infected with A24 FMDV WT, A24 FMDV 2A6H and A24 FMDV P16H at a multiplicity of infection (MOI) of 5 PFU/cell. After 1 h of adsorption at 37° C., the monolayers were rinsed once with MES buffer (morpholine ethanesulfonic acid [25 mM], 145 mM NaCl [pH 5.5]) followed by the addition of virus growth medium (BME, 1× anti-anti, 1%). At various times post infection, virus titers were determined by fifty percent tissue culture infectious dose TCID50 assays. Plates were fixed and then stained with crystal violet (0.3% in Histochoice; Amresco, Solon, Ohio), and the plaques were counted. Titers were expressed as TCID50/ml and determined in quadruplicates.
From the results shown in
Next the antigenic properties were examined of parental and mutant viruses against hyperimmune sera generated in cattle infected with the parental A24 virus. As shown in
6×-His tag expression in the mutant 6×-His tagged FMDVs was detected by ELISA assay that utilized a pre-blocked nickel-coated plate (Ni-NTA HisSorb Strips, Qiagen, Catalog No. 35023) for determination of the specificity of binding of 6×-his tagged FMDV A24 virus. The optimal concentrations of 6×-his tagged FMDV A24 was determined for binding to the plate. For this purpose, a serial dilution of 6×-His tagged FMDV A24 (A24 FMDV 2A6H and A24 FMD P16H) in total of 200 uL of PBS were bound to pre-blocked nickel-coated wells of a 96 well plate overnight at 4° C. After incubation, the plate was washed three times with PBS and primary antibody (Anti-His tag pAb, Abnova, Catalog No. PAB0862) was added at a 1:500 dilution in 1% BSA in PBS for 90 minutes at 37° C. Post-antibody incubation, plates were washed with PBS three times. Secondary antibody (Goat anti-rabbit IgG h+1 HRP conjugated, Bethyl Laboratories, Catalog No. A120-101P) was added at a 1:2000 dilution in 1% BSA in PBS and incubated for one hour at 37° C. Plates were washed four times in PBS. Substrate (SureBlue Reserve TMB Microwell Peroxidase Substrate (1-Component), KPL, Catalog No. 53-00-00) was added in a volume of 100 uL and incubated at room temperature until color development was noted (approximately 10 minutes). Stop solution (TMB BlueSTOP solution, KPL, Catalog No. 50-85-31) was added to inhibit continued substrate reaction in a volume of 100 uL and plates were read at 630 nM with brief 5 second shaking via a colorimetric plate reader. On the basis of this experiment, FMDV A24-6×-his amount corresponding to OD=1.0 at 630 nM was found to be optimal for coating FMDV A24 6×-his virus on Ni-NTA HisSorb Strips.
The specificity of the binding of His-tag proteins to the Ni-NTA plate was examined. For this purpose, FMDV 3Dpol-6×His was bound to the Ni-NTA-coated pre-blocked plates (Qiagen). The binding of 3Dpol-6×His was detected with anti-His antibody (Novus Biologicals) at 1:500 dilution using TMB Sure kit [4]. As shown in
Next the lysates from mutant A24 FMDV 2A6H- and A24 FMDV P16H-infected cells were applied to Ni-NTA-coated pre-blocked plates (Qiagen) and the detection of 6×-His tag was performed under identical condition as described in
Next, the mutant viruses A24 FMDV 2A6H and A24 FMDV P16H were characterized for expression of intact VP1 capsid protein by western blot assay (
Four distinctive methodologies were utilized to demonstrate the formation of a complex between mutant viruses and NiNLP-MPLA nanoconstructs, as following: (1) electron microscopic examination of virus alone or in complexes, (2) binding of 6×-His mutant FMDV A24 2A6H to NiNLP-MPLA as determined by Agaraose Gel Electrophoresis (AGE), (3) binding of NiNLP to mutant A24-FMDV2A6H as determined by competitive ELISA, and (4) binding of mutant FMDV A24 6×-His-tagged viruses to Co-NTA beads.
For electron microscopic (EM) analysis, A24 FMDV WT or 6×-His-tagged mutant FMDVs (A24 FMDV 2A6H or A24 FMDV P16H) chemically-inactivated with BEI were conjugated with NiNLP-MPLA in the presence of Tris-buffered-saline (TBS) pH=7.4 for 45 min at room temperature. Virus alone or complexes of NLP-MPLA mixed with Foot-and-Mouth Disease Viruses (A24 FMDV WT, A24 FMDV 2A6H and A24 FMDV P16H), NiNLP-MPLA alone, were adsorbed to 1% Alcian Blue-treated, formvar-carbon coated nickel grids (electron Microscopy Sciences (EMS), Hatfield, Pa.), stained with 2% uranyl acetate and imaged with a Hitachi T-7600 electron microscope operated at 80 KV. The dimensions of free NiNLP-MPLA and the diameters of FMDV particles and the FMDV-NiNLP-MPLA complexes were measured at 70,000× using Hitachi software that had been calibrated with a diffraction grating replica (EMS). The number of NiNLP-MPLA linked to FMDV particles was determined on micrographs at a magnification of 340,000×.
As seen in
The binding of 6×-His mutant A24 FMDV P16H to NiNLP-MPLA was determined by agarose gel electrophoresis. Native gel electrophoresis is a method of choice in the biochemical determination of protein-protein interaction; however, it is quite challenging when full virus particles are involved. Both the electrophoretic migration and the protein-protein interactions are affected by a number of parameters, such as: pH, ionic strength and buffering agent. Optimizing an agarose gel electrophoresis protocol as well as a passive protein transfer protocol enabled us to study the effect of NiNLP-MPLA on the electrophoretic migration of FMDV particles that contained 6×-His tag in their VP1 capsid protein. The mutant A24 FMDV P16H virus (1.5 ug-5 ug) was either mixed with increasing concentrations of antibodies: anti-FMDV A24 VP1 at 1:2000 dilution or anti-6×His at 1:500 dilution or with mixed, increasing concentrations of NiNLP-MPLA. The binding of virus with NiNLP-MPLA or antibodies was carried out in the presence of phosphate buffered saline pH=7.4 at room temperature for 45 min. After incubation, the reactions were mixed with 10% glycerol and 0.025% Bromophenol blue and loaded on 1% agarose gel. The gel was pre-run at 60 V for 4 hr @ 4° C. before electrophoresis of NiNLP-MPLA+virus reactions. The electrophoresis was carried out at 60 V for 2 hrs. After electrophoresis, the passive transfer of protein was carried out overnight. The protein bands were developed with 1:2000 dilution of rabbit anti-FMDV VP1 followed by 1:10,000 Goat anti-rabbit-HRP.
The binding of NiNLP-MPLA to mutant A24-FMDV2A6H was determined by competitive ELISA. The interaction of FMDV A24-6×-His with NiNLP-MPLA was determined by the interference to the binding of anti-6×-his antibody to FMDV A24-6×-his caused by Ni-NLP-MPLA. For this purpose, A24 FMDV 2A6H and A24 FMDV P16H corresponding to OD=1.0 at 630 nM were bound to the wells of Ni-NTA HisSorb Strips. After overnight binding at 4° C., the plate was washed three times with PBS and incubated with a series of concentrations of NiNLP-MPLA (1-100 ng). 0 ng Ni-NLP-MPLA was used as a control for no interference caused by NiNLP-MPLA; and the plate well coated with PBS, instead of virus, was used as negative control for reading the plate. After 45 min incubation at room temperature the plate washed three times with PBS and incubated with anti-6×-his (1:500 dilution) for 90 min at 37° C. The unbound antibody was washed by washing three times with PBS. The bound antibody was detected subsequently with 1:2000 dilution of secondary antibody (Goat anti-rabbit IgG h+1 HRP conjugated, Bethyl Laboratories, Catalog No. A120-101P), as described above. The cartoon diagram displayed in
Mock cell lysate or parental (WT)- or mutant 6×-His tagged FMDV A24-infected cell lysates (previously clarified) were mixed with 100 μl of Co2+-NTA resin [4] overnight at 4° C. with gentle rotation. After overnight incubation, the mixture was centrifuged at 760×g for 10 min. The supernatant was removed and the resin bound to proteins was washed with 50 mM Tris-HCl-500 mM NaCl pH 7.4 four times. The elution of bound protein was carried out using 300 mM imidazole-50 mM Tris-HCl-500 mM NaCl pH 7.4. The eluates were resolved on SDS-PAGE and transferred to nitrocellulose membrane. The protein bands were developed with 1:2000 Goat-anti rabbit VP1 (
The presence of surface-exposed 6×-His tags in mutant A24 FMDV 2A6H and A24 FMDV P16H viruses can potentially be exploited for one-step affinity purification (Co++ or Ni2+ affinity columns) and concentration of these viruses. After successful pull-downs of A24 FMDV P16H virus from crude cell lysates using Co2+-NTA beads, it was attempted to purify intact virus particles from infected crude cell lysates using 6×His affinity Co2+-NTA resin columns (G BIOSCIENCES). Co2+-NTA resin 3 ml spin columns were washed with 5 column volume of milliQ water and equilibrated with 5 column volume of either, binding buffer (50 mM Tris-HCl, pH=7.5, 500 mM NaCl, 25 mM imidazole) for elution with imidazole or binding buffer (50 mM Tris-HCl, pH=7.5, 500 mM NaCl) for EDTA elution. 25 ml of clarified A24 FMDV P16H-infected cell lysates were bound to the columns and the flow through (FT) were collected for further analysis. The columns were then washed with either 5 column volume of buffer A or B depending on the treatment and them the mutant FMDV particles were eluted with increasing concentrations of imidazole (E1 =100 mM Imidazole, E2=200 mM Imidazole, E3=300 mM Imidazole, E4=500 mM Imidazole, respectively in 50 mM Tris-HCl, pH=7.5, 500 mM NaCl) or with EDTA (E1 =10 mM EDTA, E2=25 mM EDTA, E3=50 mM EDTA, E4=100 mM EDTA, respectively in 50 mM Tris-HCl, pH=7.5, 500 mM NaCl. The elutions were carried out in 3 ml (1 column volume). An equivalent volume of the starting material (lysate prior column purification), unbound (FT) and different purified fractions were resolved by SDS-PAGE, transferred on to Nylon membrane and detected either by rabbit polyclonal anti-FMDV A24 VP1 (1:2,500) or monoclonal antibody against FMDV 3Dpol (1:200). In parallel virus titers were determined by plaque assays as described above for initial and treated samples (before purification, FT and different elution samples).
To determine the relative efficacy on recovery of infectious mutant virus and their protein contents (structural and non-structural proteins), fractions collected from pass through and eluates of Co2+-NTA resin columns, were examined by virus plaque assays and Western Blot analysis.
Animal experiments were performed in the high-containment facilities of the Plum Island Animal Disease Center, conducted in compliance with the Animal Welfare Act (AWA), the 2011 Guide for Care and Use of Laboratory Animals, 2002 PHS Policy for the Humane Care and Use of Laboratory Animals, and U.S. Government Principles for Utilization and Care of Vertebrates Animal Used in Testing, Research and Training (IRAC 1985), as well as an specific animal protocols reviewed and approved by the Institutional Animals Care and Use Committee (IACUC) of Plum Island Animal Disease Center (USDA/APHIS/AC Certificate number: 21-F-0001).
A FMD mouse model system has demonstrated that certain strains of adult mice, including C57BL/6, are susceptible to FMDV serotype C when infected subcutaneously (s.c.) in the footpad, developing a significant viremia and dying within a few days of infection. Furthermore, immunization with commercial vaccine protected the animals against homologous challenge with FMDV serotype C. Diaz San-Segundo et al. [43] confirmed that the model is an efficient tool for other FMDV serotypes more relevant in the field. C57BL/6 female mice, 6-7 weeks old, were purchased from Jackson Laboratories (Bar Harbor, Me.) and were acclimated for one week. Groups of 6 C57BL/6 mice were intraperitoneally (i.p.) vaccinated with different formulations of BEI-inactivated FMDV A24 vaccine as indicated in Table 4. Seven days after vaccination, animals were challenged with a lethal dose (1×105 pfu) of FMDV serotype A24 [43]. To perform the challenge, animals were anesthetized with isofluorane (Webster Veterinary, Devens, Mass.) and immediately infected subcutaneously (s.c.) in the left/right rear footpad with 105 pfu FMDV A24 WT in 50 ul. Animals were monitored for 8 days and blood taken at 0 days and on alternate days for the first seven days after challenge and then weekly until the end of the experiment.
7 dpvb
1 × 105 pfuc A24d
aIntraperitoneal inoculation (200 μl/mouse)
bDays postvaccination
cPlaque forming units
dChallenge subcutaneous in rear foot-pad (50 μl/mouse)
To confirm that commercial vaccine can protect C57BL/6 mice against FMDV serotype A, groups of 6 mice each were vaccinated with different doses of BEI-inactivated FMDV serotype A formulated with or without adjuvant Montanide ISA206 (Seppic Inc.) (Table 4). Seven days after vaccination, animals were challenged with a lethal dose of FMDV serotype A24 and disease was followed for 7 dpc. As previously described, all control animals died in two or three days after challenge (
To understand the nature of protection observed in these animals, the humoral immune response induced by the different doses and formulations of inactivated vaccines was analyzed. As observed in
Once the mouse model was established to be successful for the BEI-inactivated A24 Cru vaccine, it was used to test other FMD vaccine candidates against lethal challenge. A new experiment was performed to test efficacy of 6×-His tags A24 FMDV P16H mutant virus combined with NiNLP-MPLA nanoconstructs. Different concentrations (doses) of BEI-inactivated FMDV A24 FMDV P16H were tested, formulated with or without NLPs. Two control groups were included, one to test the effect of the NiNLP-MPLA alone in FMDV lethal challenge and another control group inoculated with PBS (Table 5).
7 dpvb
1 × 105 pfuc
A24d
aIntraperitoneal inoculation (200 μl/mouse)
bDays postvaccination
cPlaque forming units
dChallenge subcutaneous in rear foot-pad (50 μl/mouse)
After challenge at 7 dpv, all animals in the two control groups died within 48-72 hours after the challenge (
The vaccinated animals developed neutralizing antibodies at 7 dpv, and as expected a boost in the antibody titers was observed after virus challenge. Antibody titers in the group receiving 0.025 mg of BEI inactivated vaccine with NLPs, was significantly higher than those without NLPs (P<0.05) (
To study the immune response to the 6×His tag in animals, sera collected from mice following aerosol inoculation with live A24 FMDV WT (mice IDs 6.1, 6.2, 6.3, 6.4 SER pooled together) and A24 FMDV P16H (mice IDs R15-74, 75, 76, 77, 78 pooled together) were examined in an indirect sandwich ELISA (IS-ELISA) performed with modifications to the protocol specified by WHO (World Organization for Animal Health. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2012. OIE, Paris, France, Chapter 2.1.5. Foot and Mouth Disease). Briefly, 6×-His tagged recombinant human poly(rC) binding protein 2 (PCBP-2) was diluted in PBS to obtain 5 ng and 10 ng/μ1 concentrations that were bound to pre-blocked Nickel coated plates (Ni-NTA HisSorb Strips, Qiagen, Catalog No. 35023). After overnight incubation at 4°, the plates were washed four times with PBS. 1 hr blocking was performed at 37° C. with 1% bovine serum albumin (BSA) in PBS. 40 μl of test sera/well (1/6 in PBS) were applied to the plates, followed by incubation at 37° C. for 2 h. After four washes, 100 μl of peroxidase labeled goat antibody to mouse-IgG (H+L) (KPL)/well diluted 1:5,000 in (1% BSA in PBS) was added, followed by incubation for 1 h at 37° C. After four additional washes, the antigen-antibody complexes were detected by the addition of 100 μl/well of SureBlue Reserve (KPL) and stopped in 5 min with 100 μl of TMB BlueSTOP solution (KPL)/well. The optical density (OD) was determined at 630 nm on an automated ELISA plate reader.
In addition to easy one step purification and vaccine formulations consisting of Ni-doped nanolipoprotein and adjuvant, and other similar His-tag-specific formulations as described in the Examples above, chemical labeling of His-tagged virus utilizing fluorescent molecules is an attractive technique for detection of virus movement inside infected culture cells via affinity labeling (
In summary, provided herein are genetically engineered Foot and Mouth Disease Viruses (FMDV) modified by the strategic insertion of engineered Foot and Mouth Disease Virus (FMDV) and related engineered proteins and polynucleotides, nanolipoprotein particles, compositions, methods and systems. the genetically engineered FMDV modified by the strategic insertion of a protein tag into select regions of the FMDV genome which encode viral proteins that are exposed on the surface of the FMDV viral capsid. The inserted protein tag is displayed as a decoration or attachment on the viral capsid surface. In particular, the protein tag can be a HIS tag, two 6×his-marked mutant viruses A24FMDV 2A6H and A24FMDV P16H exemplify the modified viral capsids. The particular placement of the 6×-His-tag insertions enables new effective vaccine formulations, purification protocols through histidine binding of divalent cations and anti-histidine antibody recognition protocols. The design of these 6×his-mutant viruses allows the exchange of cassettes representing relevant capsid coding regions of other FMDV strains/serotypes/ and subtypes of FMDV field isolates.
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the materials, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Those skilled in the art will recognize how to adapt the features of the exemplified NLPs and related uses to additional NLPs formed by other membrane forming lipids, polymerizable lipids scaffold proteins and possibly functionalized amphipathic compounds and membrane proteins according to various embodiments and scope of the claims.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence. Further, the computer readable form of the sequence listing of the ASCII text file P1945-US-Seq-List-ST25 is incorporated herein by reference in its entirety.
The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified may be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein may be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the disclosure and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods may include a large number of optional composition and processing elements and steps.
In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.
The United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC, for the operation of Lawrence Livermore National Laboratory.
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| Number | Date | Country | |
|---|---|---|---|
| 20180326038 A1 | Nov 2018 | US |
