Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/12011—Reoviridae
  • C12N2720/12311—Rotavirus, e.g. rotavirus A
  • C12N2720/12321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/12011—Reoviridae
  • C12N2720/12311—Rotavirus, e.g. rotavirus A
  • C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/12011—Reoviridae
  • C12N2720/12311—Rotavirus, e.g. rotavirus A
  • C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/12011—Reoviridae
  • C12N2720/12311—Rotavirus, e.g. rotavirus A
  • C12N2720/12351—Methods of production or purification of viral material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/12011—Reoviridae
  • C12N2720/12311—Rotavirus, e.g. rotavirus A
  • C12N2720/12361—Methods of inactivation or attenuation
  • C12N2720/12362—Methods of inactivation or attenuation by genetic engineering
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/16011—Caliciviridae
  • C12N2770/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• compositions comprise a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript, wherein the recombinant rotavirus protein is an RIX4414 strain rotavirus protein.
• each of the polynucleotides in the collection comprises a sequence encoding at least one RIX4414 strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4. and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2. VP3, VP4, VP6, VP7, NSP1. NSP2, NSP3. NSP4, and NSP5 proteins, wherein each of the sequences encoding one rotavirus protein are operably linked to a promoter.
• infectious particles comprise a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript, wherein the recombinant rotavirus protein is an RIX4414 strain rotavirus protein.
• the infectious particles are made by transfecting a cell with a collection of polynucleotides, each of the polynucleotides in the collection comprising a sequence encoding at least one RIX4414 strain rotavirus protein selected from VP1, VP2, VP3. VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, wherein each of the sequences encoding one rotavirus protein are operably linked to a promoter.
• compositions comprise an infectious particle comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript, wherein the recombinant rotavirus protein is an RIX4414 strain rotavirus protein.
• the methods comprise administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript, wherein the recombinant rotavirus protein is an RIX4414 strain rotavirus protein to a subject to elicit an immune response in the subject to a pathogen or vaccinate the subject against one or more pathogens.
• methods of generating a rotavirus in vitro comprise: introducing a composition comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript, wherein the recombinant rotavirus protein is an RIX4414 strain rotavirus protein; allowing the cell to express one or more rotavirus proteins selected from VP1. VP2.
• systems for generating recombinant rotavims comprise: (a) a composition comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript, wherein the recombinant rotavims protein is an RIX4414 strain rotavims protein; and (b) cells capable of expressing the compositions of (a).
• the systems for generating recombinant rotavims comprise: (a) a comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript, wherein the recombinant rotavims protein is an RIX4414 strain rotavims protein; and (b) cells capable of expressing the collection of (a).
• FIG 1 shows a polyacrylamide gel demonstrating the size of genome segments of rotavirus isolates.
• Lane 1 recombinant RIX4414 (human G1P[8]).
• Lane 2 recombinant reassortant of RIX4414 & Odelia.
• Lane 3 recombinant reassortant of RIX4414 & Odelia.
• Lane 4 recombinant Odelia (human G4P[8]).
• RIX4414 segment 7 RNA can be re-engineered to express NSP3 and capsid proteins of other RNA viruses, including norovirus (NoV) and SARS-CoV-2.
• NoV norovirus
• SARS-CoV-2 SARS-CoV-2 sequences into RIX4414 segment 7 allows the expression of NoV P and VP1 capsid proteins, respectively.
• insertion of 0.8 and 2.1 kb SARS-CoV-2 sequences into RIX4414 segment 7 allows the expression of the RBD and SI domains of the SARS-CoV-2 spike protein, respectively.
• the inventors generated a novel reverse genetics system for producing recombinant attenuated rotavirus, e.g., the rotavirus strain RIX4414 (more commonly known as the strain found in ROTARIX live-attenuated rotavirus vaccine).
• the novel reverse genetics system can be used to generate recombinant rotavirus comprising heterologous polynucleotide sequences, e.g., polynucleotide sequences encoding additional antigens, e.g., norovirus antigens.
• the inventors believe that the disclosed compositions, phannaceutical compositions, infectious particles, methods, and systems may allow the combined vaccination against the two most common causes of severe gastroenteritis in children.
• the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
• the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that pennit the inclusion of additional components further to those components recited in the claims.
• the terms “consist” and “consisting of’ should be interpreted as being “closed” transitional tenns that do not permit the inclusion of additional components other than the components recited in the claims.
• the tenn “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject mater.
• the phrase "A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.” [00291 All language such as '‘up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1. 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.
• compositions, methods, and infectious particles comprise heterologous polynucleotides that encode for additional, non-rotaviral, proteins or peptides. Therefore, in some embodiments, a subject in need thereof may refer to a subject at risk of rotaviral infection and/or infection by another pathogen, wherein the heterologous polynucleotide encodes an antigen, e.g., protein or peptide, from the pathogen which is not a rotavirus.
• an antigen e.g., protein or peptide
• subject may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects.
• % sequence identity refers to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety).
• NCBI National Center for Biotechnology Information
• BLAST Basic Local Alignment Search Tool
• NCBI National Center for Biotechnology Information
• the BLAST software suite includes various sequence analysis programs including “blastp,’' that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
• Nucleic acids, proteins, and/or other compositions described herein may be purified.
• purified means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight byweight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
• Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• nucleic acid and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides.
• Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodiester bonds.
• Nucleic acids include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) such as messenger RNA (mRNA), transfer RNA (tRNA), etc.
• DNA deoxyribonucleic acids
• RNA ribonucleic acids
• mRNA messenger RNA
• tRNA transfer RNA
• nucleic acid 7 refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides).
• nucleic acid refers to an oligonucleotide chain comprising three or more individual nucleotide residues.
• nucleic acid encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule.
• nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications.
• a nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated.
• a nucleic acid is or comprises natural nucleosides, (e.g.
• nucleoside analogs e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine.
• chemically modified bases e.g.,
• compositions comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript are disclosed herein.
• an “RIX4414 strain rotavirus protein” refers to a protein derived from the RIX4414 strain of rotavirus.
• promoter transcriptional regulatory' elements that are operably linked to a sequence are physically contiguous to the transcribed sequence, i.e., they are cis acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
• Exemplary' promoters include a T7 bacteriophage promoter (SEQ ID NO: 14) and a T3 bacteriophage promoter (SEQ ID NO: 15).
• a suitable promoter may be chosen by from promoters known in the art.
• the cells are mammalian cells and are selected from MA-104 cells, Vero cells and BHK-1 cells.
• compositions may comprise a polynucleotide comprising any one of SEQ ID NOs: 1-11, or functional variants thereof (e.g., nucleic acid sequence variants that encode the same amino acid due to the redundancy in the genetic code, for example, or variants that result in different amino acid sequence(s) but encode a protein or polypeptide having the same function), or variants having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%.
• functional variants thereof e.g., nucleic acid sequence variants that encode the same amino acid due to the redundancy in the genetic code, for example, or variants that result in different amino acid sequence(s) but encode a protein or polypeptide having the same function
• variants having at least about 80%, at least about 81%
• compositions comprise a polynucleotide comprising a sequence encoding NSP3, e.g., (SEQ ID NO: 9), and further comprises a heterologous polynucleotide sequence.
• the heterologous polynucleotide encodes a protein or peptide.
• the sequence encoding NSP3, e.g., SEQ ID NO: 9, further comprises the heterologous polynucleotide fused to the 3' end of the sequence such that the heterologous polynucleotide encodes a protein or peptide in frame with the NSP3 sequence, thereby allowing transcription of a single mRNA that encodes both NSP3 and the heterologous polynucleotide.
• the polynucleotide comprising a sequence encoding NSP3 and a heterologous polynucleotide comprise a sequence encoding a cleavage site.
• the cleavage site is a self-cleaving peptide, e.g., porcine teschovirus P2A element (SEQ ID NO: 13).
• the disclosed compositions compnse, from 5’ to 3’, a polynucleotide encoding NSP3, fused in-frame to a sequence encoding a self-cleaving peptide which is fused inframe to a heterologous polynucleotide sequence encoding a peptide or protein.
• a fusion protein comprising, from N- to C-terminus.
• a rotavirus NSP3 protein fused to a self-cleaving peptide e.g., SEQ ID NO: 13, which is fused to a peptide or protein encoded by the heterologous polynucleotide, in a cell; following translation, the fusion protein self-cleaves resulting in two separate proteins (1) a functional rotavirus NSP3 protein and (2) the protein or peptide encoded by the heterologous polynucleotide.
• compositions further comprise a sequence encoding a sequence encoding a linker, e.g., a flexible linker located 3’ to, and in frame with, the sequence encoding NSP3 protein and 5’ to a cleavage site.
• a linker e.g., a flexible linker located 3’ to, and in frame with, the sequence encoding NSP3 protein and 5’ to a cleavage site.
• sequence encoding a cleavage site encodes a protease cleavage site, e.g., a thrombin cleavage site, e.g., SEQ ID NO: 12.
• the heterologous polynucleotide sequence described above comprise sequences encoding proteins or peptides derived from infectious organisms, e.g., norovirus or SARS-CoV-2. Therefore, in some embodiments, the disclosed compositions comprise sequences encoding rotavirus NSP3 fused, in-frame, to a heterologous polynucleotide encoding a norovirus protein or peptide, e.g., norovirus VP1, or SARS-CoV-2 protein or peptide, e.g., SARS-CoV-2 surface glycoprotein.
• infectious particles comprise a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript.
• infectious particles refers to any particle capable of causing an infection of an organism or cell. Exemplary infectious particles include, but are not limited to, viral particles, or virions and the like. The terms “virus,” “viral particle,” and “virion” are used interchangeably herein.
• compositions comprising polynucleotides encoding rotavirus proteins operably linked to a promoter, e.g.. a T7 promoter (SEQ ID NO: 13).
• a promoter e.g. a T7 promoter (SEQ ID NO: 13).
• the compositions may be used in a reverse genetics approach to generate recombinant rotavirus, e.g., recombinant rotavirus strain RIX4414.
• the recombinant rotavirus may comprise the disclosed compositions.
• compositions useful in making recombinant rotavirus that may be suitable for administration to subjects. Therefore, in another aspect of the current disclosure, pharmaceutical compositions are provided.
• the pharmaceutical compositions comprise an infectious particle comprising a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript.
• the pharmaceutical compositions comprise an infectious particle made by transfecting a cell with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript.
• compositions and methods may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the disclosed compositions.
• Such compositions may take any physical form w hich is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions.
• Such pharmaceutical compositions contain an effective amount of a disclosed composition, which effective amount is related to the daily dose of the composition to be administered.
• Each dosage unit may contain the daily dose of a given composition or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose.
• the amount of each composition to be contained in each dosage unit can depend, in part, on the identity of the particular composition chosen for the therapy and other factors, such as the indication for which it is given.
• the disclosed pharmaceutical compositions may be fonnulated to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.
• compositions may be utilized in methods of eliciting an immune response or vaccinating against a pathogen, e.g., rotavirus, norovirus, SARS-CoV-2.
• pathogen e.g., rotavirus, norovirus, SARS-CoV-2.
• the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder.
• the methods disclosed herein encompass both therapeutic and prophylactic administration.
• a subject may be at risk for infection by a pathogen, e.g., rotavirus, norovirus, SARS-CoV-2, and administration of the disclosed pharmaceutical compositions elicits a protective immune response or vaccinates against the pathogen.
• a pathogen e.g., rotavirus, norovirus, SARS-CoV-2
• administration of the disclosed pharmaceutical compositions elicits a protective immune response or vaccinates against the pathogen.
• the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment.
• the disclosed methods may include administering an effective amount of the disclosed compounds (e.g, as present in a pharmaceutical composition) for eliciting an immune response to a pathogen, e.g., rotavirus, norovirus, SARS-CoV-2, or vaccinating against the pathogen.
• a pathogen e.g., rotavirus, norovirus, SARS-CoV-2
• an effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances.
• determining the effective amount or dose of composition administered a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular composition administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
• Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used.
• Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose, and the like). Natural and synthetic gums can also be used, including acacia, alginates, methylcellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.
• Tablets can be coated with sugar, e g., as a flavor enhancer and sealant.
• the compounds also may be formulated as chewable tablets, by using large amounts of pleasant-tasting substances, such as mannitol, in the formulation.
• Instantly dissolving tablet-like formulations can also be employed, for example, to assure that the patient consumes the dosage form and to avoid the difficulty that some patients experience in swallowing solid objects.
• Disintegrators are substances that swell when wetted to break up the tablet and release the compound. They include starches, clays, celluloses, algins, and gums. As further illustration, com and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose can be used.
• cells comprising the disclosed compositions, which may also be used in the disclosed methods and systems. Accordingly, in another aspect of the current disclosure, cells are provided.
• the cells comprise a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript.
• the cells are selected from MA- 104 cells, Vero cells, and BHK-1 cells.
• Rotavirus vaccine strains have been traditionally grown using Vero cells. This method of producing rotavirus has been found to be suitable for generation of rotavirus for administration to subjects. Therefore, in some embodiments, the cells are Vero cells.
• the methods comprise administering a pharmaceutical composition comprising an infectious particle made by transfecting cells with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript.
• the RIX4414 pT7 transcription plasmids and the pCMV-NP868R capping enzyme plasmid were transfected into BHK-T7 cells. Two days later, transfected BHK-T7 cells were overseeded with MA104 cells. Three days later, the BHK-T7/MA104 cell culture was overseeded with Vero cells (This is unique to RIX4414 reverse genetics system). Eight days later, the BHK-T7/MA104 cell mixture was freeze-thawed 3-times and used to amplify the recombinant viruses. When the cell culture reached complete infection, a lysate was prepared from the cells and rRIX4414 recovered by plaque isolation.
• the RIX4414 cDNA sequences were positioned downstream of a T7 promoter and upstream of a hepatitis D virus ribozyme sequence in the pT7 plasmids (9, 10).
• the pT7 plasmids were generated by Azenta Life Sciences using a pUC19 backbone.
• RNA genome profile of the rRIX4414-like virus is presented in Figure 1.
• the genome sequence of the rRIX4414-like isolate w as confirmed by Nanopore sequencing (14).
• Partial, Complete rotavirus cDNA sequence in T7 expression plasmid was generated by combining the partial RIX4414 segment sequence reported by the GenBank number with the 5' and/or 3' UTR sequences of other Wa-like rotaviruses.
• b VP2 protein encoded by T7 expression plasmid was engineered to contain residues common to other genotype Cl VP2 segments previously used in developing reverse genetics systems (7,8).

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