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WO2025137311A1 - Rotavirus porcins recombinés et méthodes et systèmes de production et d'utilisation de ceux-ci - Google Patents

Rotavirus porcins recombinés et méthodes et systèmes de production et d'utilisation de ceux-ci Download PDF

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Publication number
WO2025137311A1
WO2025137311A1 PCT/US2024/061075 US2024061075W WO2025137311A1 WO 2025137311 A1 WO2025137311 A1 WO 2025137311A1 US 2024061075 W US2024061075 W US 2024061075W WO 2025137311 A1 WO2025137311 A1 WO 2025137311A1
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Prior art keywords
protein
polynucleotide
rotavirus
virus
osu
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Inventor
Anthony J. SNYDER
Chantal A. AGBEMABIESE
John T. Patton
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Indiana University
Indiana University Bloomington
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Indiana University
Indiana University Bloomington
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C12N2720/12011—Reoviridae
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    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12341—Use of virus, viral particle or viral elements as a vector
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12351—Methods of production or purification of viral material
    • C12N2720/12352—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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    • 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

  • polynucleotides are provided.
  • the polynucleotides comprise a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • sequences encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4. and NSP5 comprise SEQ ID NOs: 1-11, respectively.
  • sequences encoding at least one rotavirus protein selected from VP1, VP2. VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, andNSP5 consist of SEQ ID NOs: 1-11, respectively.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein.
  • the heterologous polynucleotide encodes a reporter.
  • the peptide or protein comprises a microorganismal peptide or protein.
  • the peptide or protein comprises a viral peptide or protein.
  • the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein.
  • the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17.
  • the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site.
  • the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • the polynucleotide further comprises a heterologous polynucleotide.
  • the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF.
  • the heterologous polynucleotide encodes a peptide or protein.
  • the heterologous polynucleotide encodes a reporter.
  • the peptide or protein comprises a microorganismal peptide or protein.
  • the peptide or protein comprises a viral peptide or protein.
  • the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein.
  • the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence.
  • the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).
  • the polynucleotide comprises a sequence encoding a linker.
  • the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • the infectious particles are made by transfecting cells with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein.
  • the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein.
  • the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence.
  • the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).
  • the polynucleotide comprises a sequence encoding a linker.
  • the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • compositions comprise an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein.
  • the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea vims (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein.
  • the hepatitis E vims (HEV) protein comprises an HEV capsid protein.
  • the hepatitis E vims (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E vims (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence.
  • the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).
  • the polynucleotide comprises a sequence encoding a linker.
  • the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • a method comprising: administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript to a subject.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide compnses any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroententis virus (TGEV) protein, or a coronavirus protein.
  • the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • methods of eliciting an immune response to one or more microorganism in a subject comprise: administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript to a subject to elicit an immune response to the one or more microorganism.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the hepatitis E vims (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17.
  • the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site.
  • methods comprise administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript to a subject.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein.
  • methods comprise: administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript to a subject to elicit an immune response in the subject to a pathogen or vaccinate the subject against one or more pathogens.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the polynucleotide comprises an OSU strain NSP3 protein.
  • the polynucleotide further comprises a heterologous polynucleotide.
  • the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF.
  • the heterologous polynucleotide encodes a peptide or protein.
  • the heterologous polynucleotide encodes a reporter.
  • the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea vims (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E vims (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E vims (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E vims (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17.
  • the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site.
  • the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).
  • the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the subject is a pig. In some embodiments, the subject is a piglet, optionally, wherein the piglet is has not yet been weaned.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the polynucleotide comprises an OSU strain NSP3 protein.
  • the polynucleotide further comprises a heterologous polynucleotide.
  • the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF.
  • the heterologous polynucleotide encodes a peptide or protein.
  • the heterologous polynucleotide encodes a reporter.
  • the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17.
  • the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site.
  • the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).
  • the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the one or more pathogens comprises hepatitis E virus (HEV).
  • the cells comprise a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript or an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the polynucleotide comprises an OSU strain NSP3 protein.
  • the polynucleotide further comprises a heterologous polynucleotide.
  • the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF.
  • the heterologous polynucleotide encodes a peptide or protein.
  • the heterologous polynucleotide encodes a reporter.
  • the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17.
  • the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site.
  • the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • the cells comprise a collection of polynucleotides, wherein each of the polynucleotides in the collection comprises a sequence encoding at least one OSU strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP 1, 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.
  • sequences encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 comprise SEQ ID NOs: 1-11, respectively.
  • sequences encoding at least one rotavirus protein selected from VP 1 , VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, andNSP5 consist of SEQ ID NOs: 1-11, respectively.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein.
  • the heterologous polynucleotide encodes a reporter.
  • the peptide or protein comprises a microorganismal peptide or protein.
  • the peptide or protein comprises a viral peptide or protein.
  • the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein.
  • the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17.
  • the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site.
  • the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).
  • the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • the cell is an MA- 104 cell, a Vero cell or a BHK-1 cell. In some embodiments, the cell expresses a heterologous RNA polymerase. In some embodiments, the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.
  • methods of generating an OSU strain rotavirus in vitro comprise: introducing a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript into a cell; allowing the cell to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5; incubating the cells for a sufficient time to produce rotavirus; and harvesting virus produced by the cells to generate the rotavirus in vitro.
  • the recombinant OSU strain rotavirus protein is a G5P[7J genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the polynucleotide comprises an OSU strain NSP3 protein.
  • the polynucleotide further comprises a heterologous polynucleotide.
  • the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF.
  • the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • HEV hepatitis E virus
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • the cell is an MA- 104 cell, a Vero cell or a BHK-1 cell.
  • the cell expresses a heterologous RNA polymerase.
  • the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.
  • sequences encoding at least one rotavirus protein selected from VP1 , VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 comprise SEQ ID NOs: 1-11, respectively.
  • sequences encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4. and NSP5 consist of SEQ ID NOs: 1-11, respectively.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein.
  • the heterologous polynucleotide encodes a reporter.
  • the peptide or protein comprises a microorganismal peptide or protein.
  • the peptide or protein comprises a viral peptide or protein.
  • the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein.
  • the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17.
  • the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site.
  • the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).
  • the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the cell expresses a heterologous RNA polymerase.
  • the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.
  • the cell comprises T7 RNA polymerase and, optionally, comprising African sw ine fever virus capping enzyme.
  • the cell is selected from an MA-104 cell, a Vero cell, and a BHK-1 cell. The method of claim 50, wherein the cell is a BHK-1 cell comprising T7 RNA polymerase and, optionally, comprising African swine fever virus capping enzyme.
  • systems for generating recombinant rotavirus comprise: (a) a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript into a cell; and (b) cells capable of expressing the polynucleotide of (a).
  • the cells comprise a heterologous RNA polymerase and, optionally, comprising African swine fever virus capping enzy me.
  • the cells comprise a cells from a cell line selected from MA-104 cells, Vero cells, and BHK-1 cells.
  • the cells comprise BHK-1 cells comprising T7 RNA polymerase. In some embodiments, the cells comprise BHK-1 cells comprising T7 RNA polymerase, Vero cells, and MA-104 cells.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1- 14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein.
  • the polynucleotide further comprises a heterologous polynucleotide.
  • the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF.
  • the heterologous polynucleotide encodes a peptide or protein.
  • the heterologous polynucleotide encodes a reporter.
  • the peptide or protein comprises a microorganismal peptide or protein.
  • the peptide or protein comprises a viral peptide or protein.
  • the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein.
  • the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.
  • the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter.
  • the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18.
  • the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence.
  • the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).
  • the polynucleotide comprises a sequence encoding a linker.
  • the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • the systems for generating recombinant rotavirus comprise: (a) a collection of polynucleotides, wherein each of the polynucleotides in the collection comprises a sequence encoding at least one OSU strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4.
  • the cells comprise a heterologous RNA polymerase and. optionally, comprising African swine fever virus capping enzyme.
  • the cells comprise a cells from a cell line selected from MA-104 cells, Vero cells, and BHK-1 cells. In some embodiments, the cells comprise BHK-1 cells comprising T7 RNA polymerase.
  • the cells comprise BHK-1 cells comprising T7 RNA polymerase, Vero cells, and MA-104 cells.
  • the sequences encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4. VP6. VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 comprise SEQ ID NOs: 1-11, respectively.
  • the sequences encoding at least one rotavirus protein selected from VP 1 , VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, andNSP5 consist of SEQ ID NOs: 1-11, respectively.
  • the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.
  • the polynucleotide encodes a positive sense viral transcript.
  • the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14.
  • the polynucleotide comprises an OSU strain NSP3 protein.
  • the polynucleotide further comprises a heterologous polynucleotide.
  • the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF.
  • the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein.
  • HEV hepatitis E virus
  • the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.
  • the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site.
  • the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.
  • FIG. 1 shows a schematic of the vector pT7/OSU NSP3.
  • the full-length cDNA of rOSU gene segment 7 (encodes for NSP3) is positioned within a pT7 plasmid, which is ligated upstream with a promoter for T7 polymerase and downstream with a hepatitis delta virus (HDV) ribozyme.
  • HDV hepatitis delta virus
  • FIG. 2 shows a schematic of the vector pT7/OSU NSP3-2A.
  • the full-length cDNA of rOSU gene segment 7 (encodes for NSP3) is positioned within a pT7 plasmid, which is ligated upstream with a promoter for T7 polymerase and downstream with a hepatitis delta virus (HDV) ribozyme.
  • HDV hepatitis delta virus
  • the OSU pT7 plasmid produces a full- length gene segment 7 (+)RNA with authentic 5’ and 3‘ termini.
  • the porcine teschovirus 2A-like (2A) element is positioned at the 3‘ end of the NSP3 coding sequence.
  • the rOSU pT7 gene segment 7 plasmid that expresses NSP3-2A was generated with in-fusion cloning
  • FIG. 3 shows a schematic of the vector pT7/OSUNSP3-2A-fUnaG.
  • the full-length cDNA of rOSU gene segment 7 (encodes for NSP3) is positioned within a pT7 plasmid, which is ligated upstream with a promoter for T7 polymerase and downstream with a hepatitis delta virus (HDV) ribozyme.
  • HDV hepatitis delta virus
  • the porcine teschovirus 2A-like (2A) element is positioned at the 3’ end of the NSP3 coding sequence followed by the in-frame coding sequence of the FLAG-tagged UnaG (fUnaG). Due to the activity 7 of the 2A element, translation of the RNA produces two proteins. NSP3 with remnants of the 2A element and the fUnaG.
  • the rOSU pT7 gene segment 7 plasmid that expresses NSP3-2A-fUnaG was generated with in-fusion cloning
  • FIG. 4 shows a schematic of the vector pT7/OSU NSP3-2A-HEV CP.
  • the full-length cDNA of rOSU gene segment 7 (encodes for NSP3) is positioned within a pT7 plasmid, which is ligated upstream with a promoter for T7 polymerase and downstream with a hepatitis delta virus (HDV) riboz me.
  • HDV hepatitis delta virus
  • the porcine teschovirus 2A- like (2A) element is positioned at the 3’ end of the NSP3 coding sequence lol I ow ed by the inframe coding sequence of the Hepatitis E virus (HEV) capsid protein (CP). Due to the activity of the 2A element, translation of the RNA produces two proteins. NSP3 with remnants of the 2A element and the HEV CP.
  • FIG. 5 shows a polyacrylamide gel loaded with RNA demonstrating the genome profiles of rOSU, rOSU-2A, rOSU-2A-fUnaG, and rOSU-2A-HEV CP compared to rSAl l and MAI 04 cell culture-adapted OSU.
  • Viral dsRNA was recovered from MA104 cells infected with rSAl 1, MAI 04 cell culture-adapted OSU, rOSU, or modified rOSU isolates, resolved on a 10% polyacrylamide gel by electrophoresis, and detected by ethidium-bromide staining on a Bio-Rad ChemiDoc Imaging System.
  • the migration of rSAl 1 RNA segments is indicated on the left side of the panel and the migration of rOSU RNA segments is indicated on the right side of the panel.
  • the migration of modified rOSU gene segments 7 is indicated with red arrows.
  • FIG. 6 shows fluorescent images showing the expression of fUnaG.
  • MAI 04 cells were infected with rOSU, rOSU-2A, or rOSU-2A-fUnaG at a multiplicity of infection of 5 plaque forming units per cell.
  • the cells w ere imaged using a ZOE Fluorescent Cell Imager under the brightfield and green (excitation: 480, emission: 517) channels.
  • the scale bars represent 100 pm.
  • FIG. 7 shows Western blots showing the expression of fUnaG and the activity of the 2A translational stop-start element.
  • MA104 cells were infected with rOSU, rOSU-2A, or rOSU-2A- fUnaG at a multiplicity of infection of 5 plaque forming units per cell. At 9 hours post infection, the cells were lysed and resolved on a 10% polyacrylamide gel by electrophoresis.
  • the resolved lysates were transferred to a nitrocellulose membrane and probed with FLAG M2 antibody (F 1804, Sigma, 1:2,000), 2A antibody (NBP2-59627, Novus, 1: 1,000), rotavirus NSP3 antibody (lot 55068, 1 :2,000), rotavirus VP6 antibody (lot 53963, 1 :2,000), or P-actin antibody (8H10D10, Cell Signaling Technology, 1:2,000).
  • FLAG M2 antibody F 1804, Sigma, 1:2,000
  • 2A antibody NBP2-59627, Novus, 1: 1,000
  • rotavirus NSP3 antibody lot 55068, 1 :2,000
  • rotavirus VP6 antibody lot 53963, 1 :2,000
  • P-actin antibody 8H10D10, Cell Signaling Technology, 1:2,000.
  • HRP horseradish peroxidase
  • CST horseradish peroxidase
  • KPL goat anti-guinea pig IgG
  • CST horseradish peroxidase
  • HRP signals were developed using the Bio-Rad Clarity Western ECL substrate and developed using a Bio-Rad ChemiDoc imaging system.
  • NSP3-2A-fUnaG, fUnaG, NSP3-2A, VP6, and - actin are indicated on the left side of the panels.
  • FIGs. 8A and 8B show production of OSU G5P[7] porcine rotavirus.
  • Rotavirus reverse genetics system [29] Recombinant rotavirus was prepared by transfecting BHK-T7 cells with 11 T7 plasmids, which contain full-length cDNAs of rotavirus genome segments, and the CMV-NP868R plasmid, which encodes the African Swine Fever Virus capping enzyme [28], The BHK-T7 cells were overseeded 2 days post-transfection with MA104 cells to facilitate the spread and amplification of recombinant rotavirus.
  • recombinant virus in the cells lysates was amplified on MAI 04 cells.
  • the amplified virus was analyzed by RNA gel electrophoresis followed by plaque purification. Image was adapted from [29] (B) Recovery’ of recombinant SAI 1/OSU monoreassortants by reverse genetics.
  • Viral dsRNAs from rSAl 1, rOSU, and 11 rSAl 1/OSU monoreassortants were resolved by electrophoresis on an 8% polyacrylamide gel and stained with ethidium bromide.
  • the migrations of rOSU gene segments in rSAl 1/OSU monoreassortants are indicated with red arrows. Genome segments 1-11 of rSAl l are indicated on the left side of the panel. Genome segments 1-11 of rOSU are indicated on the right side of the panel.
  • FIG. 9A, 9B, and 9C show production of recombinant OSU that encodes a foreign protein.
  • A Modifications of rotavirus genome segment 7. The schematics indicate the nucleotide positions of the coding sequences for NSP3, the porcine teschovirus 2A element, 3X FLAG, and the fluorescent reporter UnaG (green). The red arrows indicate the positions of the 2A translational stop-restart elements, and the asterisks indicate the ends of the open reading frames.
  • B Recovery of recombinant OSU-2A-UnaG by reverse genetics.
  • Viral dsRNAs from OSU-tc_(MA104), rOSU, rOSU-2A, and rOSU-2A-UnaG were resolved by electrophoresis on an 8% polyacrylamide gel and stained with ethidium bromide. The migrations of modified genome segment 7 are indicated with red arrows. Genome segments 1-11 of rOSU are indicated on the left side of the panel.
  • C Genetic stability. rOSU-2A and rOSU-2A-UnaG were serially passaged on MA104 cells. Viral dsRNAs from a total of 5 passages (P) were resolved by electrophoresis on an 8% polyacrylamide gel and stained with ethidium bromide. The migrations of modified genome segment 7 are indicated with red arrow. Genome segments 1-11 of rOSU are indicated on the left side of the panels.
  • FIGs. 10A, 10B, 10C, 10D, 10E, and 10F show characterization of recombinant OSU that expresses a foreign protein.
  • a and B Production of infectious virus. MAI 04 cells were infected with the indicated viruses at an MOI of 5 PFU/cell. In panel A, titers were determined by plaque assay at the indicated times post infection. In panel B, titers were determined by plaque assay upon complete cytopathic effect (typically 3-5 days).
  • polynucleotides that encode a segment of the OSU strain of rotavirus and can be used in a reverse genetics system to generate recombinant OSU strain rotaviruses, and collections of polynucleotides. Also disclosed herein are methods of using the polynucleotides, or collections, to generate recombinant OSU strain rotaviruses, pharmaceutical compositions comprising the recombinant rotaviruses, and methods of using the same to elicit an immune response and/or to vaccinate a subject against porcine rotavirus strains, e.g.. OSU strain.
  • the NSP3 segment of the OSU strain rotavirus may further comprise a heterologous polynucleotide sequence, e.g., encoding a peptide or protein from another source, e.g., a pathogen.
  • the disclosed polynucleotides, collections, methods, recombinant rotaviruses, and pharmaceutical compositions may be useful to vaccinate piglets, e.g., pre-weaning piglets, against the OSU strain of rotavirus, as well as one or more additional pathogens, e.g., HEV, porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV), porcine enteric caliciviruses, pseudorabies, classical swine fever, bacterial pathogens, e.g., swine brucellosis, and/or a coronavirus.
  • HEV porcine epidemic diarrhea virus
  • PEDV porcine transmissible gastroenteritis virus
  • PRRSV porcine reproductive and respiratory syndrome virus
  • swine influenza virus African swine fever virus
  • ASFV African
  • polynucleotides comprising a sequence encoding a recombinant rotavirus protein are disclosed herein.
  • ‘'OSU” or “OSU strain” refers to a Rotavirus A strain with a G5P[7] genotype, the genotype associated with porcine rotaviruses that most frequently cause disease in piglets.
  • the instant disclosure provides “recombinant OSU” rotaviruses produced by the disclosed reverse genetics systems, which are distinguished from cell-culture adopted OSU strains isolated by traditional means from OSU-infected animals, i.e., by reproducing virus recovered from animals on suitable host cells.
  • an “OSU strain rotavirus protein” refers to a protein derived from the OSU strain of rotavirus.
  • the polynucleotides are operably linked to a promoter to allow the expression of said polynucleotides in a cell, e.g., a mammalian cell.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, this refers to the functional relationship of transcriptional regulatory element (promoter) to a transcribed sequence.
  • a promoter is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate cell.
  • promoter transcriptional regulatory elements that are operably linked to a sequence are physically contiguous to the transcribed sequence, i.e., they are cis acting.
  • 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: 17) and a T3 bacteriophage promoter (SEQ ID NO: 18).
  • a suitable promoter may be chosen from promoters known in the art.
  • the cells are mammalian cells and are selected from MA-104 cells, Vero cells and BHK-1 cells.
  • the polynucleotides comprise a sequence encoding a rotaviral protein, i.e., are selected from a sequence encoding rotavirus VPI, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5.
  • the sequences encoding the OSU strain VPI, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins are provided herein as SEQ ID NOs: 1-11, respectively.
  • the disclosed polynucleotides may comprise a sequence 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%.
  • 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%, at least about 82%, at least about 83%, at least about 84%, at least about 85%.
  • the inventors discovered that a reverse genetics system can be used to generate OSU strain rotavirus expressing heterologous polynucleotide sequences by fusing the heterologous polynucleotide sequences to the sequence encoding the rotaviral NSP3 protein.
  • the disclosed polynucleotides comprise a sequence encoding NSP3, e.g.. (SEQ ID NO: 9), and farther comprises a heterologous polynucleotide sequence.
  • the heterologous polynucleotide may encode a viral protein or peptide, e.g., hepatitis E virus (HEV) protein, whose amino acid sequence is SEQ ID NO: 19, or a sequence with 85% similarity, 86% similarity, 87% similarity, 88% similarity, 89% similarity, 90% similarity. 91% similarity, 92% similarity, 93% similarity, 94% similarity, 95% similarity, 96% similarity.
  • HEV hepatitis E virus
  • SEQ ID NO: 19 which may be encoded by SEQ ID NO: 20, respectively, or a sequence with 85% similarity’, 86% similarity, 87% similarity, 88% similarity, 89% similarity, 90% similarity, 91% similarity, 92% similarity, 93% similarity, 94% similarity, 95% similarity, 96% similarity, 97% similarity. 98% similarity, or 99% similarity to SEQ ID NO: 19 (see Table 1 for full sequences).
  • Table 1 Exemplary heterologous amino acid sequences encoded by heterologous polynucleotides.
  • 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: 16).
  • the disclosed polynucleotides comprise, from 5‘ to 3’, a polynucleotide encoding NSP3, fused in-frame to a sequence encoding a self-cleaving peptide which is fused in-frame 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: 16, 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.
  • a self-cleaving peptide e.g., SEQ ID NO: 16
  • the polynucleotides further comprise 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.
  • GAG GAG n linker
  • GSG GSG linker
  • sequence encoding a cleavage site encodes a protease cleavage site, e.g., a thrombin cleavage site, e.g., SEQ ID NO: 15.
  • the heterologous polynucleotide sequence described above may comprise sequences encoding proteins or peptides derived from infectious organisms, e.g., HEV, porcine epidemic diarrhea virus (PEDV). porcine transmissible gastroenteritis virus (TGEV). porcine reproductive and respirator ⁇ ' syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV), or a coronavirus.
  • HEV porcine epidemic diarrhea virus
  • PEDV porcine transmissible gastroenteritis virus
  • TGEV porcine reproductive and respirator ⁇ ' syndrome virus
  • PRRSV porcine reproductive and respirator ⁇ ' syndrome virus
  • swine influenza virus swine influenza virus
  • African swine fever virus African swine fever virus
  • coronavirus e.g., African swine fever virus
  • the disclosed compositions comprise sequences encoding rotavirus NSP3 fused, in-frame, to a heterologous polynucleotide encoding a porcine hepatitis E virus (HEV) peptide or protein, porcine epidemic diarrhea virus (PEDV) peptide or protein, porcine transmissible gastroenteritis virus (TGEV) peptide or protein, porcine reproductive and respiratory syndrome virus (PRRSV) peptide or protein, swine influenza virus peptide or protein, African swine fever virus (ASFV) peptide or protein, or coronavirus peptide or protein.
  • HEV porcine hepatitis E virus
  • PEDV porcine epidemic diarrhea virus
  • TGEV porcine transmissible gastroenteritis virus
  • PRRSV porcine reproductive and respiratory syndrome virus
  • swine influenza virus peptide or protein African swine fever virus (ASFV) peptide or protein, or coronavirus peptide or protein.
  • the polynucleotides comprise a polynucleotide comprising a sequence encoding a recombinant rotavirus NSP3, e.g., SEQ ID NO: 9, wherein the polynucleotide further comprises a heterologous polynucleotide in frame with the sequence encoding a recombinant rotavirus NSP3, wherein the heterologous polynucleotide encodes a peptide or protein comprising an HEV peptide or protein, or a fragment thereof.
  • the polynucleotides further comprise a sequence encoding a self-cleaving peptide, e.g., a sequence encoding SEQ ID NO: 16, fused, in-frame, between the polynucleotide and the heterologous polynucleotide.
  • a self-cleaving peptide e.g., a sequence encoding SEQ ID NO: 16, fused, in-frame, between the polynucleotide and the heterologous polynucleotide.
  • transcription and translation of said composition results in, with reference to an HEV peptide being selected as the heterologous peptide encoded by the heterologous polynucleotide, from N- to C-terminus, a functional rotavirus NSP3 protein, selfcleaving peptide, e.g., SEQ ID NO: 16, and an HEV protein or peptide, e g., HEV capsid, e.g., SEQ ID NO: 19, or a fragment thereof.
  • an HEV peptide being selected as the heterologous peptide encoded by the heterologous polynucleotide, from N- to C-terminus, a functional rotavirus NSP3 protein, selfcleaving peptide, e.g., SEQ ID NO: 16, and an HEV protein or peptide, e g., HEV capsid, e.g., SEQ ID NO: 19, or a fragment thereof.
  • SEQ ID NO: 12 encodes NSP3 fused in-frame to a P2A sequence (SEQ ID NO: 16).
  • SEQ ID NO: 13 encodes NSP3 fused in-frame to a P2A sequence and the reporter fUnaG.
  • SEQ ID NO: 14 encodes NSP3 fused in-frame to a P2A sequence and HEV capsid protein.
  • the polynucleotides may comprise one of SEQ ID NOs: 12-14, or a sequence with at least 85% similarity, at least 86% similarity, at least 87% similarity, at least 88% similarity', at least 89% similarity, at least 90% similarity, at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, or at least 99% similarity’ to SEQ ID NOs: 12-14.
  • Sequences of the instant disclosure include sequences with either a particular amount of “similarity'’ or “identity ” to the disclosed sequences.
  • infectious particles comprise a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, e.g., one of SEQ ID NOs 1-14, or a sequence with at least 85% similarity, at least 86% similarity, at least 87% similarity’, at least 88% similarity’, at least 89% similarity’, at least 90% similarity, at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, or at least 99% similarity to one of SEQ ID NOs: 1- 14.
  • the disclosed polynucleotides differ from naturally occurring OSU strain disclosed in, e.g., Guo et al. “Amino Acid Substitutions in Positions 385 and 393 of the Hydrophobic Region of VP4 May Be Associated with Rotavirus Attenuation and Cell Culture Adaptation.” Viruses 2020, 72(4), 408. which is incorporated herein by reference in its entirety. See Tables 2 and 3 below.
  • infectious particles refers to any particle capable of causing an infection of a cell or an organism.
  • infectious particles include, but are not limited to, viral particles, or virions and the like.
  • viruses viral particle
  • virion are used interchangeably herein.
  • the instant disclosure provides polynucleotides comprising sequences encoding rotavirus proteins operably linked to a promoter, e.g., a T7 promoter (SEQ ID NO: 16).
  • a promoter e.g., a T7 promoter (SEQ ID NO: 16).
  • the polynucleotides may be used in a reverse genetics approach to generate recombinant rotavirus, e.g., recombinant rotavirus strain OSU.
  • the recombinant rotavirus may comprise the disclosed polynucleotides.
  • the disclosed infectious particles may comprise one or more heterologous proteins or peptides, e.g., a porcine hepatitis E virus (HEV) peptide or protein, porcine epidemic diarrhea virus (PEDV) peptide or protein, porcine transmissible gastroenteritis virus (TGEV) peptide or protein, porcine reproductive and respiratory syndrome virus (PRRSV) peptide or protein, swine influenza virus peptide or protein, African swine fever virus (ASFV) peptide or protein, or coronavirus peptide or protein, as described above.
  • HEV porcine hepatitis E virus
  • PEDV porcine epidemic diarrhea virus
  • TGEV porcine transmissible gastroenteritis virus
  • PRRSV porcine reproductive and respiratory syndrome virus
  • swine influenza virus peptide or protein African swine fever virus (ASFV) peptide or protein, or coronavirus peptide or protein, as described above.
  • heterologous proteins or peptides may be encoded in the infectious particle, e.g., viral genome, and subsequently produced during viral replication.
  • infectious particles comprising a heterologous protein or peptide may be advantageous in eliciting an immune response in a subject or as a vaccine composition.
  • compositions comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.
  • pharmaceutical compositions comprise an infectious particle made by transfecting a cell with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.
  • 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 which 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 formulated 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., porcine hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV), or a coronavirus.
  • a pathogen e.g., porcine hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV), or a coronavirus.
  • a pathogen e.g., porcine hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV),
  • 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., porcine hepatitis E virus (HEV).
  • HEV porcine hepatitis E virus
  • porcine epidemic diarrhea virus PEDV
  • porcine transmissible gastroenteritis virus TGEV
  • porcine reproductive and respiratory syndrome virus PRRSV
  • swdne influenza virus African swine fever virus (ASFV)
  • ASFV African swine fever virus
  • coronavirus administration of the disclosed pharmaceutical compositions elicits a protective immune response or vaccinates against the pathogen.
  • a '‘subject” or a “subject in need thereof’ may refer to a porcine subject, e.g., an adult pig, an adolescent pig, a piglet, e.g., a piglet that has not yet been weaned.
  • 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., porcine hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respirator ⁇ ’ syndrome vims (PRRSV), sw ine influenza virus, African swine fever vims (ASFV), or a coronavims, or vaccinating against the pathogen.
  • a pathogen e.g., porcine hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respirator ⁇ ’ syndrome vims (PRRSV), sw ine
  • 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.
  • Oral administration is an illustrative route of administering the compositions and methods disclosed herein.
  • Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes.
  • the route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver.
  • suitable formulations include those that are suitable for more than one route of administration.
  • the formulation can be one that is suitable for both intrathecal and intracerebral administration.
  • suitable formulations include those that are suitable for only one route of administration as w ell as those that are suitable for one or more routes of administration, but not suitable for one or more other routes of administration.
  • the formulation can be one that is suitable for oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, and/or intrathecal administration but not suitable for intracerebral administration.
  • the inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The usual methods of formulation used in pharmaceutical science may be used here.
  • compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, troches, suppositories, transdennal patches, and suspensions.
  • compositions contain from about 0.5% to about 50% of the compound in total, depending on the desired doses and the type of composition to be used.
  • the amount of the compound is best defined as the “effective amount’", that is, the amount of the compound which provides the desired dose to the patient in need of such treatment.
  • the activity of the compounds employed in the compositions and methods disclosed herein are not believed to depend greatly on the nature of the composition, and, therefore, the compositions can be chosen and formulated primarily or solely for convenience and economy.
  • Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules.
  • suitable diluents include inert powdered substances (such as starches), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), grain flours, and similar edible powders.
  • Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants, and disintegrators (in addition to the compounds). 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.
  • Typical diluents include, for example, various types of starch, lactos
  • 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.
  • a lubricant can be used in the tablet formulation to prevent the tablet and punches from sticking in the die.
  • the lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.
  • Tablets can also contain disintegrators.
  • Disintegrators are substances that swell when wetted to break up the tablet and release the compound.
  • the ⁇ ’ include starches, clays, celluloses, algins, and gums.
  • 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.
  • Compositions can be formulated as enteric formulations, for example, to protect the active ingredient from the strongly acid contents of the stomach.
  • Such formulations can be created by coating a solid dosage form with a film of a polymer which is insoluble in acid environments and soluble in basic environments.
  • Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.
  • Transdermal patches can also be used to deliver the compounds.
  • Transdemial patches can include a resinous composition in which the compound will dissolve or partially dissolve; and a film which protects the composition, and which holds the resinous composition in contact with the skin.
  • Other, more complicated patch compositions can also be used, such as those having a membrane pierced with a plurality’ of pores through which the drugs are pumped by osmotic action.
  • the formulation can be prepared with materials ( ⁇ ?.g., actives excipients, carriers (such as cyclodextrins), diluents, etc.) having properties (e.g. , purity) that render the formulation suitable for administration to humans.
  • the formulation can be prepared with materials having purity and/or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.
  • methods of generating OSU strain rotavirus in vitro comprise introducing a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein; allowing the cell to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5; incubating the cells for a sufficient time to produce rotavirus; and harvesting virus produced by the cells to generate OSU strain rotavirus in vitro.
  • cells comprising the disclosed polynucleotides, 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 polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.
  • the cells are selected from MA-104 cells, Vero cells, and BHK-1 cells.
  • Rotavirus vaccine strains have traditionally been 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 cells further comprise T7 RNA polymerase or T3 RNA polymerase.
  • T7 RNA polymerase or T3 RNA polymerase such cells are referred to as, e.g., BHK-T7 cells, because they are derived from BHK-1 cells, but express the heterologous RNA polymerase T7 bacteriophage RNA polymerase.
  • BHK-T7 cells are BHK-1 cells that express the heterologous RNA polymerase T7 bacteriophage RNA polymerase.
  • the instant disclosure provides polynucleotides, methods of making the same, and infectious particles. Therefore, in another aspect of the current disclosure, methods of eliciting an immune response are provided.
  • the methods comprise administering a pharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.
  • the methods comprise administering a pharmaceutical composition comprising an infectious particle made by transfecting cells with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.
  • methods of eliciting an immune response comprise administering a pharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein. In some embodiments, methods of eliciting an immune response comprise administering a pharmaceutical composition comprising an infectious particle made by transfecting cells with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.
  • methods of vaccinating a subject against one or more pathogens comprise administering a pharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein. In some embodiments, the methods comprise administering a pharmaceutical composition comprising an infectious particle made by transfecting cells with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.
  • systems for generating recombinant rotavirus comprise: (a) a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein; and (b) cells capable of expressing the polynucleotides of (a).
  • 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 permit 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 terms that do not permit the inclusion of additional components other than the components recited in the claims.
  • the term “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 matter.
  • the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same.
  • the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
  • a “subject in need thereof’ as utilized herein may refer to a subject at risk for rotavirus infection.
  • the disclosed compositions, methods, and infectious particles comprise heterologous polynucleotides that encode for additional, non-rotaviral, proteins or peptides.
  • 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 tenns “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 presen e 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
  • the BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known ammo acid sequence with other amino acids sequences from a variety of databases.
  • BNP Basic Local Alignment Search Tool
  • Nucleic acids, proteins, and/or other compositions described herein may be purified. As used herein, “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 by weight 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.
  • polypeptide protein
  • peptide are used interchangeably and refer to a polymer of 3 or more amino acids.
  • a protein may include two proteins that are joined (fused) together.
  • a protein may refer to a portion or fragment of a protein, e.g., SARS-CoV-2 SI protein, which is a fragment of the SARS-CoV-2 surface glycoprotein or “S” protein.
  • 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 phosphodi ester 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 molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage.
  • nucleic acid 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.
  • a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or include non-naturally occurring nucleotides or nucleosides.
  • the terms "nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc.
  • 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, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemical
  • methylated bases methylated bases
  • intercalated bases modified sugars (e g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).
  • modified sugars e g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose
  • modified phosphate groups e.g., phosphorothioates and 5'-N-phosphoramidite linkages.
  • hybridization refers to the fonnation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions.
  • nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning-A Laboratory 7 Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).
  • Example 1- Development of a reverse genetics system for OSU porcine rotavirus and its potential application for the production of combination vaccines against other porcine viruses
  • Rotaviruses are a significant cause of severe potentially -life threatening gastroenteritis in the young of many economically important farm animals, including piglets.
  • porcine rotavirus vaccines have been developed, both live oral and killed varieties, existing vaccines are generally ineffective in preventing rotavirus gastroenteritis in piglets.
  • the establishment of more effective porcine rotavirus vaccines could be advanced through the application of reverse genetics technologies to create recombinant porcine rotavirus vaccine candidates that are superior in generating protective immunological responses.
  • porcine rotavirus As a vaccine vector, it may also be possible to generate combination vaccines capable of protecting pigs against diseases caused by other major pathogenic porcine viruses, including porcine hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV) and others.
  • porcine hepatitis E virus porcine epidemic diarrhea virus (PEDV)
  • TGEV porcine transmissible gastroenteritis virus
  • PRRSV porcine reproductive and respiratory syndrome virus
  • swine influenza virus African swine fever virus
  • ASFV African swine fever virus
  • Rotavirus A species with a G5P[7] genotype the genotype associated with porcine rotaviruses that most frequently cause disease in piglets.
  • the OSU reverse genetics system is similar to rotavirus reverse genetics systems that have been previously established for other Rotavirus A species, including those infecting non-human primates (SAI 1 and RRV), humans (KU, CDC-9, Odelia, and RIX4414-like), cattle (UK and RF), mice (rD6/2-2g), and birds (PO- 13).
  • SAI 1 and RRV infecting non-human primates
  • humans KU, CDC-9, Odelia, and RIX4414-like
  • cattle UK and RF
  • mice rD6/2-2g
  • PO- 13 birds
  • OSU reverse genetics system can be used to generate recombinant OSU rotaviruses that function as expression vectors of foreign proteins, including the capsid protein of porcine HEV.
  • HEV Hepatitis E virus
  • the OSU reverse genetics system was developed as follows. Sequences for the eleven genome segments w ere determined by Nanopore sequencing of the MAI 04 cell culture-adapted OSU strain (see Supplement 1). The sequences were used by Azenta, Inc. to construct eleven pUC19- based T7 transcription plasmids, each expressing one of the eleven OSU plus-sense RNAs. For the expression of foreign proteins, the pUC 19-based T7 transcription plasmid corresponding to NSP3 was modified by in-fusion cloning.
  • the porcine teschovirus 2A-like (2A) element was positioned at the 3' end of the NSP3 coding sequence followed by the in-frame coding sequence of the foreign protein; we modified viral segment 7 to express fUnaG (FLAG- tagged green fluorophore) and HEV CP (viral capsid protein) (FIGs. 1-4).
  • the OSU pT7 plasmids were then used to produce recombinant OSU following the reverse genetics protocols that we previously described (Philip AA, Patton JT. Expression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element. J Virol.
  • Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in the young of many economically important animals.
  • vaccines against porcine rotavirus exist, both live oral and inactivated, their effectiveness in preventing gastroenteritis is less than ideal.
  • the Ohio State University (OSU) rotavirus strain represents & Rotavirus A species with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets.
  • Rotavirus A strains including those that infect non-human primates (SA11 and RRV), humans (KU, CDC-9, Odelia, and RIX4414-like). cattle (UK and RF), mice (rD6/2-2g), and birds (PO-13) [1-9], These systems can be used for producing next-generation live oral vaccines, dual vaccine platforms that express foreign proteins, and diagnostic tools.
  • Rotavirus (RV) accounts for a significant disease burden within important livestock, highlighting the need for effective animal rotavirus vaccines. Nonetheless, no reverse genetics systems currently exist for any porcine rotaviruses, including those with the G5P[7] genotype, which represents the most frequent cause of disease in porcine populations [10,11],
  • RV is a major cause of acute gastroenteritis in piglets [10,11], The vims is transmitted by the fecal-oral route and damages small intestinal enterocytes; as such, milk consumed by nursing piglets is not digested or absorbed into the intestines [12], Moreover, RV is resistant to environmental factors, such as temperature, pH, and common disinfectants, which creates a persistent risk of infection [13,14], Although RV -induced diarrhea is associated with low mortality and high morbidity, productivity losses create a significant economic burden on the global pork industry’. Cunent vaccines only control diarrhea among infected populations [10,11], Thus, aneed exists for vaccines that are more effective.
  • the virus can be used as an expression vector of foreign proteins [2,3,15-20]
  • the RV genome is composed of 11 segments of double-stranded (ds)RNA. Each segment contains the coding sequence for a single protein except for segment 11, which expresses two proteins [21],
  • the NSP3 open reading frame (ORF) in the segment 7 RNA is replaced with a modified ORF that encodes NSP3 fused to a foreign protein.
  • Insertion of the 2A translational stop- restart element allows for the modified segment 7 ORF to separately express NSP3 and the foreign protein [16-18.22-24].
  • This approach has been used to generate recombinant RV that express fluorescent reporters, such as UnaG (green), mRuby (red), and TagBFP (blue), and other viral proteins, such as the norovirus VP1 capsid protein and respirator ⁇ ' syndrome coronavirus 2 SI spike domain [3,15-18],
  • fluorescent reporters such as UnaG (green), mRuby (red), and TagBFP (blue
  • other viral proteins such as the norovirus VP1 capsid protein and respirator ⁇ ' syndrome coronavirus 2 SI spike domain [3,15-18]
  • Such recombinant viruses may be used to generate combination vaccines capable of inducing protective immune responses against RV and a second pathogenic virus.
  • Embryonic monkey kidney (MA104) cells were grown in Dulbecco’s modified eagle medium (DMEM) containing 5% fetal bovine serum (FBS, Gibco) and 1% penicillinstreptomycin at 37°C in a 5% CO2 incubator.
  • DMEM Dulbecco modified eagle medium
  • FBS fetal bovine serum
  • penicillinstreptomycin 1% penicillinstreptomycin at 37°C in a 5% CO2 incubator.
  • Baby hamster kidney cells that constitutively express the T7 RNA polymerase (BHK-T7) were provided by Dr.
  • GMEM Glasgow minimum essential medium
  • FBS penicillin-streptomycin
  • Gibco nonessential amino acids
  • glutamine 1% glutamine at 37°C in a 5% CO2 incubator.
  • BHK-T7 cells were grown in medium supplemented with 2% Geneticin (Invitrogen) in every other passage.
  • Virus was isolated from the clarified lysates by extraction with an equal volume of Vertrel-XF (TMC Industries) followed by ultracentrifugation at 100.000 xg for 2 h at 4°C.
  • the pelleted virus [OSU-tc(MA104)] virus was resuspended in 500 pL of Tris-buffered saline and stored at -80°C.
  • OSU-tc_(MA104) dsRNA was extracted from 250 pL of clarified infected cell lysate using a Zymo Research Direct-zol RNA Miniprep Kit following the manufacturer’s instructions. Prior to library preparation, the dsRNA was denatured with dimethylsulfoxide and poly(A)-tailed using New England Biolabs Escherichia coli Poly (A) polymerase following the manufacturer's instructions. The poly(A)-tailed RNA was subjected to library preparation using an Oxford Nanopore Technologies direct cDNA sequencing kit (SQK-DCS109) following the manufacturer’s instructions. The library’ w as sequenced using an Oxford Nanopore Technologies Mini ON sequencer. Sequence assemblies of the 11 genome segments of OSU-tc_(MA104) were prepared using Geneious Primer software version 2023.2.1.
  • Recombinant SA11 (rSAl l) viruses were prepared using the plasmids pT7/SAHVPl, pT7/SAl 1VP2. pT7/SAl 1VP3, pT7/SAl 1VP4, pT7/SAl 1VP6, pT7/SAl 1VP7, pT7/SAl 1NSP1. pT7/SAl 1NSP2. pT7/SAl 1NSP3, pT7/SAl 1NSP4, and pT7/SAl 1NSP5 and pCMV/NP868R (1. 28).
  • Recombinant OSU (rOSU) viruses were prepared using the plasmids pT7/OSUVPl, pT7/OSUVP2, pT7/OSUVP3, pT7/OSUVP4, pT7/OSUVP6, pT7/OSUVP7, pT7/OSUNSPl, pT7/OSUNSP2, pT7/OSUNSP3, pT7/OSUNSP4, and pT7/OSUNSP5 and pCMV/NP868R [28],
  • the pT7/OSU plasmids were made by Genewiz, Azenta Life Sciences, based on OSU sequences determined by Nanopore sequencing.
  • the plasmid pT7/SAl 1 NSP3-2A-UnaG was previously described [17], The plasmids pT7/OSU NSP3-2A and pT7/OSU NSP3-2A-UnaG were produced by fusing DNA frag-ments for 2 A or 2A-3xFLAG-UnaG, respectively, to the 3 '-end of the OSU NSP3 open reading frame of pT7/OSU NSP3 using the TaKaRa Bio In-Fusion cloning system. Primer synthesis and plasmid sequencing were performed by EuroFins Scientific (Table 2). [0135] Table 2. Primers used in constructing OSU segment 7 expression platforms
  • the activated lysates were diluted into 10 mL of serum-free DMEM and then used as inoculum to infect MAI 04 cells in T175 flasks.
  • the flasks were placed at 37°C in a 5% CO2 incubator for 1 h with rocking to ensure equal coverage of the inoculum over the monolayers.
  • the inoculum was removed and 25 mL of serum-free DMEM containing 0.5 pg/mL trypsin was added to each flask.
  • the flasks were returned to the incubator until all cells were lysed (typically 3-5 days).
  • lysates were resolved by electrophoresis on 10% polyacrylamide gels in Tris-glycine buffer and transferred to nitrocellulose membranes. After blocking with PBS containing 0.1% Tween-20 and 5% nonfat dry milk, the blots were probed with FLAG M2 antibody (F 1804, Sigma Aldrich, 1:2,000), 2A antibody (NBP2-59627, Novus, 1: 1,000), rotavirus NSP3 antibody (lot 55068, 1:2,000), rotavirus VP6 antibody (lot 53963, 1:2,000), or [Lactin antibody (D6A8, Cell Signaling Technology, 1:2000).
  • FLAG M2 antibody F 1804, Sigma Aldrich, 1:2,000
  • 2A antibody NBP2-59627, Novus, 1: 1,000
  • rotavirus NSP3 antibody lot 55068, 1:2,000
  • rotavirus VP6 antibody lot 53963, 1:2,000
  • [Lactin antibody D6A8, Cell Signaling Technology, 1:2000
  • Rotavirus infections were performed as previously described [29], Briefly, MAI 04 cells in 6-well plates were infected with 5 PFU/cell of the indicated viruses. Following adsorption, the infected cells were washed thrice with PBS and incubated in serum-free DMEM containing 0.5 pg/rnL trypsin at 37°C in a 5% CO2 incubator. At the indicated times post infection, the infected cells were freeze-thawed thrice, clarified by low-speed centrifugation at 800xg for 5 min at 4°C, and analyzed by plaque assay. Statistically significant differences in titer were determined using ANOVA.
  • Genotype G5P[7] is representative of most rotaviruses (RV) that cause acute gastroenteritis in suckling and weaned pigs, which leads to economic losses that plague the global pork industry' [10,11], Current treatments are generally ineffective at preventing disease [31-33]; thus, aneed exists for robust molecular tools to develop next-generation porcine rotavirus vaccines.
  • Reverse genetics systems exist for several Rotavirus A strains [1-9]; however, no such system is available for a porcine RV.
  • OSU-tc_(MA104) Laboratory-adapted OSU was grown in MA104 cells, and viral dsRNA was extracted from infected cell lysates. The isolated RNA was then processed for Nanopore sequencing to obtain the complete sequences of all 11 genome segments (deposited in GenBank).
  • Nanopore sequences for 7 OSU- tc_(MAl 04) genome segments were identical to the virulent (RVA/Pig-tc/USA/1975/OSU/G5P7/virulent) and attenuated (RVA/Pig- tc/USA/1975/OSU/G5P7/attenuated) strains.
  • VP1, VP4. and NSP1 were >99% identical, whereas NSP4 was >95% identical (nucleotide and amino acid sequence comparisons) [27],
  • the OSU-tc_(MA104) sequencing information was used to construct plasmids for reverse genetics experiments.
  • T7 plasmids Full-length cDNAs of each genome segment were positioned within T7 plasmids in between of an upstream T7 polymerase promoter and a downstream hepatitis delta virus ribozyme. In the presence of the T7 RNA polymerase, the OSU T7 plasmids produce full- length, positive sense RNA with authentic 5’ and 3’ termini.
  • SAI 1 represents the prototype strain of simian RV [34-36]
  • Reverse genetics experiments were perfonned as previously described (FIG 1A) [1,29], Briefly.
  • 1 OSU T7 expression plasmid, 10 SAI 1 T7 expression plasmids, and pCMV- NP868R were transfected into BHK-T7 cells, which were subsequently overseeded with MA 104 cells.
  • Recombinant viruses generated in the transfected cells were amplified and their dsRNA profiles analyzed by gel electrophoresis.
  • RV can be modified to express fluorescent reporters, such as UnaG (green), mRuby (red), and TagBFP (blue), and other viral proteins, such as norovirus VP1 and SARS-COV-2 S 1 [2,3,15- 20]).
  • fluorescent reporters such as UnaG (green), mRuby (red), and TagBFP (blue)
  • other viral proteins such as norovirus VP1 and SARS-COV-2 S 1 [2,3,15- 20].
  • the recombinant viruses generated in this work were derived from sequencing information of the laboratory-adapted strain. To detennine if these viruses exhibit similar growth characteristics, we compared their growth kinetics and plaque morphologies. The analysis showed that rOSU was a well growing virus, reaching peak titers of ⁇ 10 7 in MA104 cells. However, the peak titer produced reached by rOSU w as ⁇ 0.5 log less than that reached by the OSU-tc_(MAl 04) virus. Moreover, single-step grow th experiments indicated that rOSU grew slow er than the OSU- tc (MAI 04) virus.
  • the vector must express a foreign protein.
  • the protein products made by rOSU-2A-UnaG following infection were probed by immunoblot with an anti-FLAG antibody (FIG. 10E).
  • This assay revealed high levels of cleaved FLAG-UnaG ( ⁇ 18 kDa); rOSU-2A-UnaG directed expression of the foreign protein and a functional 2A element.
  • Probing with an anti-2A antibody revealed a protein product that migrated at the expected molecular weight for NSP3 linked to the remnant residues of the 2A peptide (—38 kDa) (FIG. 10E).
  • Viral protein VP6 and host protein 0- actin were detected under all infection conditions; however, NSP3-2A-UnaG, FLAG-UnaG, and NSP3-2A were not present in rOSU infected cell lysates.
  • fluorescence microscopy to determine if the expressed and cleaved FLAG-UnaG was functional. In contrast to rOSU, rOSU-2A-UnaG produced high levels of fluorescent signal within live cells (FIG. 10F).

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Abstract

L'invention concerne des polynucléotides, et des collections de polynucléotides, codant pour des segments de nouveaux rotavirus de souche OSU recombinés et leurs méthodes de fabrication, leurs systèmes de génération, ainsi que leurs méthodes d'utilisation pour déclencher une réponse immunitaire chez un sujet ou pour vacciner un sujet contre une infection à rotavirus et une infection.
PCT/US2024/061075 2023-12-20 2024-12-19 Rotavirus porcins recombinés et méthodes et systèmes de production et d'utilisation de ceux-ci Pending WO2025137311A1 (fr)

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US20180155411A1 (en) * 2015-07-08 2018-06-07 The Board Of Trustees Of The Leland Stanford Junior University Heterotypic antibodies specific for human rotavirus

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US20180155411A1 (en) * 2015-07-08 2018-06-07 The Board Of Trustees Of The Leland Stanford Junior University Heterotypic antibodies specific for human rotavirus

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PHILIP ASHA A., PATTON JOHN T.: "Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins", JOURNAL OF VIROLOGY, vol. 96, no. 22, 23 November 2022 (2022-11-23), US , pages 1 - 18, XP093332004, ISSN: 0022-538X, DOI: 10.1128/jvi.01262-22 *

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