WO2020018429A1 - Compositions et combinaisons de vaccins contre le rotavirus et leurs procédés de fabrication et d'utilisation - Google Patents

Compositions et combinaisons de vaccins contre le rotavirus et leurs procédés de fabrication et d'utilisation Download PDF

Info

Publication number
WO2020018429A1
WO2020018429A1 PCT/US2019/041819 US2019041819W WO2020018429A1 WO 2020018429 A1 WO2020018429 A1 WO 2020018429A1 US 2019041819 W US2019041819 W US 2019041819W WO 2020018429 A1 WO2020018429 A1 WO 2020018429A1
Authority
WO
WIPO (PCT)
Prior art keywords
rotavirus
composition
cells
bgm
group
Prior art date
Application number
PCT/US2019/041819
Other languages
English (en)
Inventor
Karen Brown
Michael Hall
Hailong Guo
Jeremy GRIFFEL
Peter Winter
Original Assignee
Phibro Animal Health Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Phibro Animal Health Corporation filed Critical Phibro Animal Health Corporation
Publication of WO2020018429A1 publication Critical patent/WO2020018429A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/15—Reoviridae, e.g. calf diarrhea virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12351—Methods of production or purification of viral material

Definitions

  • the present invention relates to rotavirus vaccine compositions and combinations and methods for making and using the compositions and combinations.
  • Rotavirus is the leading cause of viral gastroenteritis in neonates of all species.
  • Rotavirus is a genus within the Reoviridae family and classified as a double stranded RNA virus, with the rotavirus genome containing 11 double-stranded RNA segments.
  • Eight different serogroups, also sometimes referred to as serotypes (shortened to groups or species herein), have been identified and are typically referred to as groups A through H, although groups I and J have been proposed.
  • the groups are identified based on structural protein VP6.
  • Groups A, B and C are the most prominent for most animal species, with group A being the most prominent across all species.
  • groups E and H so far have only been found in swine, while groups D, F and G have, to date, been identified in birds.
  • Group A is the most commonly identified rotavirus in samples from diseased animals. However, this may be because it is the most easily isolated and grown in tissue culture.
  • Rotaviruses generally infect young animals of all species, including humans, and can cause diarrhea, dehydration, and death. Rotaviruses produce a profuse watery diarrhea resulting from villus atrophy leading to malabsorption and wasting. Death can occur quickly in the very young. Older animals usually demonstrate significant weight loss but the death loss is significantly less. In unweaned pigs, rotavirus infections usually involve a morbidity of 50-80% and a mortality of up to 15%.
  • a composition made by the method.
  • the method comprises contacting buffalo green monkey (BGM) cells with a rotavirus in a growth media.
  • BGM cells may be Buffalo Green Monkey cell line strain MH18 WCB 5-16-18 P05 (MH18).
  • the rotavirus may be of group A, B, C, D, E, F, G or H, and in some embodiments, is of groups B, C, D, E, F, G or H, or a non simian rotavirus of group A.
  • the growth media may comprise proteolytic enzyme, DEAE-dextran, DMSO, EDTA, D-glucosamine or any combination thereof.
  • the proteolytic enzyme may be any suitable proteolytic enzyme, such as trypsin, chymotrypsin, papain, amylase, pancreatin or a combination thereof.
  • the method may further comprise releasing rotavirus viral particles from the BGM cells by any suitable method, such as freezing and thawing the BGM cells.
  • the method may comprise separating the viral particles from BGM cell fragments and contacting fresh BGM cells with the viral particles.
  • the rotavirus may be passaged through BGM cells from 2 to 15 or more times, and/or the method may comprise allowing the rotavirus to incubate in the BGM cells until CPE is substantially complete.
  • Viral particles may be released from the BGM cells by any suitable method, such as freezing and thawing and/or adding a detergent, after CPE is substantially complete.
  • Suitable detergents include, but are not limited to, Triton-X-lOO, NP40 (nonyl phenoxypolyethoxylethanol), deoxycholate (sodium deoxycholate), polysorbate 80, polysorbate 20, sodium dodecyl sulfate (SDS) or a combination thereof.
  • the method may further comprise adding an inactivating agent, such as formalin, formaldehyde, binary ethyleneimine (BEI), beta propiolactone (BPL), Triton-X-lOO, NP40 (nonyl phenoxypolyethoxylethanol), deoxycholate (sodium deoxycholate), polysorbate 80, polysorbate 20, sodium dodecyl sulfate (SDS) or a combination thereof.
  • an inactivating agent such as formalin, formaldehyde, binary ethyleneimine (BEI), beta propiolactone (BPL), Triton-X-lOO, NP40 (nonyl phenoxypolyethoxylethanol), deoxycholate (sodium deoxycholate), polysorbate 80, polysorbate 20, sodium dodecyl sulfate (SDS) or a combination thereof.
  • an inactivating agent such as formalin, formaldehyde, binary ethyleneimine (BEI), beta propiolactone (BPL
  • the adjuvant may be an oil-in water, oil, saponin, pluronic polyol, nonionic block copolymer, water in oil, polymer, carbomer, microfluidized emulsion, polylactide glycolide adjuvant, or a combination thereof.
  • the adjuvant is Emulsigen ® -D, Emulsigen ® -DL, or a combination thereof.
  • the rotavirus may be passaged through one or more non-BGM cells prior to adding an inactivating agent and/or adjuvant.
  • non-BGM cells include, but are not limited to, Rhesis Monkey Kidney cells, MARC 145, MA104, Swine Testicle cells, Swine Kidney cells, Bovine Kidney cells (MDBK), Canine Kidney cells (MDCK or CLDK), Bovine Turbinate cells (BT), Feline Kidney cells, Hamster Kidney Cells (BHK), Mouse cells, Bovine lung cells, human lung cells, human rectal tumor cells, or a combination thereof.
  • the method comprises contacting a first portion of BGM cells with a rotavirus of group B, C, D, E , F, G or H, or a non-simian rotavirus of group A; allowing the rotavirus to grow on the cell for from 0.5 hours to 90 hours; freezing and thawing the first portion of BGM cell to release viral particles; contacting a second portion of BGM cells with the viral particles; releasing rotavirus antigens from the second portion of BGM by contacting the second portion of BGM cells with a detergent, freezing and thawing the second portion of BGM cells, or a combination thereof; contacting the rotavirus antigens with an inactivating agent to form inactivated antigens; and forming an inactivated vaccine composition comprising an adjuvant and the inactivated antigens.
  • the method comprises contacting a first portion of BGM cells with a rotavirus of group B, C, D, E , F, G or H, or a non-simian rotavirus of group A;
  • Forming the live, modified or attenuated vaccine composition may comprise adding an adjuvant.
  • the rotavirus may be a group A, B, C, E or H rotavirus.
  • the rotavirus is a porcine rotavirus, and may be a group A, B, or C porcine rotavirus.
  • the rotavirus is a porcine C rotavirus.
  • the rotavirus is a bovine rotavirus, and may be a bovine group A rotavirus.
  • the rotavirus is a human rotavirus, and may be a human group A rotavirus.
  • compositions comprising BGM cell fragments and antigens from a rotavirus of group A, B, C, D, E, F, G or H, such as group B, C, D, E, F, G or H or a non-simian group A rotavirus.
  • the composition may further comprise a detergent, an inactivating agent, a neutralization product, or a combination thereof.
  • the composition does not comprise formalin, or may comprise from 0% to 0.5% free formalin, such as from greater than zero to 5% free formalin.
  • the composition comprises antigens from one or more rotavirus strains, BGM cell fragments, at least one adjuvant, neutralized inactivating agent, and optionally may further comprise neutralizing agent (if used and/or if used in excess), DMSO, proteolytic enzyme and/or detergent.
  • a composition comprising rotavirus group A antigens may not comprise DMSO.
  • the composition may have a virus titer of at least 1 x 10 1 TCID /mL. In some
  • the composition is a modified live or attenuated vaccine composition having a vims titer of at least 1 x 10 4 TCID mL. Additionally, or alternatively, the composition may have an antigenic protein content sufficient to fully protect an animal to which it is administered. The antigenic protein content may be greater than zero when measured by an ELISA.
  • the composition may further comprise antigens from a second, different rotavirus.
  • the composition comprises antigens from rotavirus groups A, B, C, E, H or a combination thereof, such as antigens from rotavirus groups A, B, C, or a combination thereof.
  • the composition comprises antigens from rotavirus groups B, C, D, E, F, G, H or a combination thereof, but does not comprise antigens from rotavirus group A.
  • the composition comprises antigens from rotavirus group C.
  • the composition may comprise antigens from a porcine rotavirus, a bovine rotavirus, a human rotavirus, or a combination thereof.
  • composition may comprise antigens from one or more non-rotavirus diseases, such as a porcine, bovine, or human disease, or a combination thereof.
  • non-rotavirus diseases such as a porcine, bovine, or human disease, or a combination thereof.
  • a method comprising administering to an animal a vaccine composition prepared by contacting a BGM cell with a rotavirus of group A, B, C, D, E, F, G, or H.
  • Administering may comprise administering intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intravaginally, orally, topically, intrathecally, inhalationally, intranasally, transdermally, or rectally, and/or may comprise administering an immunologically effective amount of the vaccine composition.
  • the vaccine composition may be an inactivated vaccine composition or a live modified or attenuated vaccine composition.
  • the animal may be a mammal, such as a human, porcine or bovine. In some embodiments, the mammal is a non human mammal.
  • FIG. 1 is a digital image illustrating CPE observed on MH18 cells that are infected with a swine rotavirus A vims.
  • FIG. 2 is a digital image illustrating non- infected MH18 cells.
  • FIG. 3 is a digital image illustrating an immunofluorescence stain of rotavirus A in MH18 cells.
  • FIG. 4 is a digital image illustrating an immunofluorescence stain of the non-infected MH18 cells.
  • FIG. 5 is an electron micrograph illustrating entrance of rotavirus Group A into MH18 cells.
  • FIG. 6 is an electron micrograph illustrating growth and formation of a virosome by rotavirus Group A grown in MH18 cells
  • FIG. 7 is an electron micrograph illustrating entrance of rotavirus Group C into MH18 cells.
  • FIG. 8 is an electron micrograph illustrating growth of rotavirus Group C in the cytoplasm of MH18 cells.
  • FIG. 9 is an electron micrograph illustrating CPE of Rotavirus Group C in infected MH18 cells.
  • FIG. 10 is an electron micrograph illustrating non-infected MH18 cells.
  • FIG. 11 is an immunofluorescence image illustrating infection of MH18 cells by rotavirus Group C.
  • FIG. 12 is an immunofluorescence image illustrating the background fluorescence from non-infected MH18 cells.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • sequence Listing is submitted as an ASCII text file, created on July 9, 2019, 465 KB, which is incorporated by reference herein. In the accompanying sequence listing:
  • SEQ ID NOS: 1-11 are nucleotide sequences of Rotavirus A Strain RVA/Simian- tc/ZAF/SAl l-H96/l958/G3P5B[2]; serotype:G3.
  • SEQ ID NOS: 2-22 are nucleotide sequences of Porcine Rotavirus A Strain RVA/Pig- tc/THA/P343/l99l/Gl0P[5].
  • SEQ ID NOS: 23-33 are nucleotide sequences of Bovine Rotavirus A Strain RVA/Cow- wt/BRA/BRAl532/2009/G6P[5].
  • SEQ ID NOS: 34-44 are nucleotide sequences of Rotavirus A pheasant- wt/HUN/216/2015/G23P[37] Strain pheasant- wt/HUN/2l 6/20 l5/G23P[37] .
  • SEQ ID NOS: 45-55 are nucleotide sequences of Human rotavirus B Strain Bang373.
  • SEQ ID NOS: 56-66 are nucleotide sequences of : Rotavirus B Strain JN285.
  • SEQ ID NOS: 67-77 are nucleotide sequences of Rotavirus B Strain JN292.
  • SEQ ID NOS: 78-88 are nucleotide sequences of Rotavirus B Strain RVB/Pig- tc/US A/LS0001 l_Ohio/XXXX/GXP[X] .
  • SEQ ID NOS: 89-99 are nucleotide sequences of Porcine rotavirus C Strain RVC/Pig- wt/Ishi- 1/2015/G 13P[4] .
  • SEQ ID NOS: 100-110 are nucleotide sequences of Bovine rotavirus C Strain Y/03.
  • SEQ ID NOS: 111-121 are nucleotide sequences of Rotavirus C Strain RVC/Dog- wt/HUN/ 174/2012/G 10P8.
  • SEQ ID NOS: 122-132 are nucleotide sequences of Rotavirus C Strain Bristol.
  • SEQ ID NOS: 133-143 are nucleotide sequences of Rotavirus D
  • SEQ ID NOS: 144-154 are nucleotide sequences of Rotavirus D isolate RotaD/D62/20l3.
  • SEQ ID NOS: 155-165 are nucleotide sequences of Rotavirus F
  • SEQ ID NOS: 166-176 are nucleotide sequences of Rotavirus G
  • SEQ ID NOS: 177-187 are nucleotide sequences of Rotavirus G strain RVG/turkey- wt/USA/Minnesota-2/20l6.
  • SEQ ID NOS: 188-198 are nucleotide sequences of Rotavirus G strain RVG/turkey- wt/USA/Minnesota- 1/2016.
  • SEQ ID NOS: 199-209 are nucleotide sequences of Adult diarrheal rotavirus strain J19.
  • SEQ ID NOS: 210-220 are nucleotide sequences of Rotavirus I strain KE135/2012.
  • the Buffalo Green Monkey Kidney cells have been deposited under conditions that assure that access to the cells will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. ⁇ 1.14 and 35 U.S.C. ⁇ 122.
  • the deposits represent a substantially pure sample of the deposited cells.
  • the cells are designated as Buffalo Green Monkey (BGM) cell line, strain MH18 WCB 5- 16-18 P05, deposited on July 18, 2018.
  • BGM Buffalo Green Monkey
  • the deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
  • Adjuvant refers to a component that is added to a vaccine antigen to accelerate, prolong and/or enhance the immune response to the antigen by an animal’s immune system, such as a mammalian immune system.
  • exemplary adjuvants include, but are not limited to, various types of oils, such as mineral oil or vegetable oil; aluminum salts, such as aluminum hydroxide or aluminum phosphate; oil-in-water based adjuvants, such as Emulsigen ® , Emulsigen ® -D, Emulsigen ® -DL90, Emulsigen ® -P, Emulsigen ® -BCL, Emulsimune ® , or TS6; saponin-based adjuvants, such as Quil A, QS-21 and saponin; pluronic polyols; nonionic block copolymers; Amphigen ® ; water-in-oil adjuvants, such as ISA 71 VG, ISA 35, ISA 50V, ISA
  • a vaccine and/or an adjuvant may include CpG oligonucleotides, muramyl dipeptide (MDP), bacterial exotoxins, E.
  • coli labile toxin tetanus toxoid
  • BCG sequences monophophoryl lipid A (MPLA), immunostimulatory complexes (ISCOMS), block copolymers such as L121, ethylene maleic anhydride, or combinations of any of these or other immunostimulating or immunomodulating or antigen presenting components that may act to stimulate an immune response.
  • MPLA monophophoryl lipid A
  • ISCOMS immunostimulatory complexes
  • block copolymers such as L121, ethylene maleic anhydride, or combinations of any of these or other immunostimulating or immunomodulating or antigen presenting components that may act to stimulate an immune response.
  • Antigen refers to a compound, composition, or substance that can stimulate the production of antibodies and/or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. Typically, an antigen reacts with the products of specific humoral or cellular immunity.
  • a rotavirus antigen may be a whole viral particle, one or more proteins from a whole viral particle, glycoprotein or a portion or fragment of such viral particles or an extract of such a viral particle that is recognized by an anti-rotavirus antibody.
  • the portion or fragment may be a peptide with an attached moiety, such as, but not limited to, a sugar moiety, a phosphate moiety, and/or a lipid moiety.
  • the portion or fragment may be a peptide without any attached non-peptide moieties.
  • Antigen mass refers to the amount of rotavirus antigens as described above that is required to produce an immune response in an animal, such as a mammal.
  • Antimicrobial agents are agents, such as antibiotics and/or antifungals, that are added to vaccine compositions to control contamination ⁇ They may be added during the growth process or added as preservatives to a final vaccine formulation.
  • Exemplary antimicrobial agents include, but are not limited to, Penicillin, Gentamicin, tetracyclines, Streptocycin, Neosporin, Polymyxin B, Mycostatin, Nystatin, Amphotericin B, Kanamycin, Fungistat, Enrofloxicin (Baytril or
  • Ciprofloxicin Tulathromycin (Draxxin), Erythromycin, or combinations thereof.
  • BGM cells refers to any cell line or lineage (whether modified or not) which were derived from the Almen L. Barron cloned continuous cell line of 1962 derived from African Green Monkey Kidney Cells and designated from that point forward as Buffalo Green Monkey cells (BGM).
  • Exemplary BGM cells include, but are not limited to, MH18, deposited with ATCC, and ECACC 90092601, which is available from the European Collection of Authenticated Cell Cultures (ECACC).
  • the BGM cells are MH18 cells.
  • “Combination” refers to a combination including two or more components that are administered such that the effective time period of at least one component overlaps with the effective time period of at least one other component.
  • a component may be a composition.
  • the effective time periods of all components administered overlap with each other.
  • the effective time period of the first component administered may overlap with the effective time periods of the second, third and fourth components, but the effective time periods of the second, third and fourth components independently may or may not overlap with one another.
  • the effective time period of the first component administered overlaps with the effective time period of the second component, but not that of the third or fourth; the effective time period of the second component overlaps with those of the first and third components; and the effective time period of the fourth component overlaps with that of the third component only.
  • a combination may be a composition comprising the components, a composition comprising one or more components and another separate component (or components) or composition(s) comprising the remaining component(s), or the combination may be two or more individual components.
  • the two or more components may comprise the same component administered at two or more different times, two or more different components administered substantially simultaneously or sequentially in any order, or a combination thereof.
  • CPE Cytopathic effect
  • administering refers to methods that may be used to enable delivery of compositions to the desired site of biological action. These methods include, but are not limited to, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intravaginally, orally, topically, intrathecally, inhalationally, intranasally, transdermally, rectally, and the like. Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The
  • an adjuvant is selected to facilitate administration to mucosal membranes, and/or stimulate a mucosal immune response.
  • the adjuvant may adhere to the mucosal membrane.
  • Mucosal immune responses typically comprise the production of IgA antibodies but may also stimulate IgG responses, which may be advantageous in certain disclosed embodiments.
  • Intranasal formulations may include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function.
  • Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not limited to antimicrobials such as gentamicin, penicillin, streptomycin, polymyxin B, nystatin and chemicals such as formaldehyde and thimerosal.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented dry in tablet form or a product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservative.
  • compositions provided herein can be made into aerosol formulations (e.g., they can be "nebulized") to be administered via inhalation.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • compositions can be formulated for parenteral administration, such as, for example, by intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, and subcutaneous routes.
  • parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
  • controlled-release parenteral dosage forms can be prepared.
  • Suitable materials for such administration include sterile water; saline solution; glucose solution; aqueous vehicles, such as sodium chloride injection, Ringer's Injection, Dextrose Injection, Dextrose, Sodium Chloride Injection, Lactated Ringer's Injection; alcohols and polyols, such as polyethylene glycol; non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, and isopropyl myristate; aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • aqueous and non-aqueous sterile suspensions that can
  • compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • parenteral administration, oral administration, and/or intravenous administration are the methods of administration.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules, bottles, and vials.
  • the terms“co-administration,”“administered in combination with,” and the like encompass administration of two or more antigenic agents to a single subject, and include treatment regimens in which the agents are administered by the same or different routes of administration or at the same or different times.
  • the one or more compositions described herein will be co-administered with other agents, including, but not limited to, other vaccines, antibiotics, or combinations thereof.
  • These terms encompass administration of two or more agents to the subject so that both agents and/or their metabolites are present in the subject at the same time. They include simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents are present.
  • the compositions described herein and the other agent(s) are administered in a single composition.
  • the compositions described herein and the other agent(s) are admixed in the composition.
  • Immunologically effective refers to an amount of an immunogenic rotavirus composition is the amount required to protect an animal from exposure to live rotaviruses of the same or a different group.
  • “protect” or“protection” refers to prevention of infection by the rotavirus, amelioration or reduction in clinical signs, such that substantially no clinical symptoms, such as diarrhea, intestinal lesions, and/or fecal shedding, are observed in the animal after challenge by a live rotavirus, and/or at least a four-fold increase of antibody titers in the animal compared to the antibody titers in an animal that is not administered the immunogenic composition.
  • Immuno response refers to a response of an immune system, such as in a subject, tissue sample, or cell, to a stimulus such as an antigen.
  • the immune response may be a response by an immune system cell, such as a B-cell, T-cell, macrophage or polymorphonucleocyte.
  • An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine.
  • an immune response includes, but is not limited to, an innate immune response or inflammation ⁇
  • a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection, such that reduced or substantially no clinical signs are observed), ameliorates the effects and/or symptoms of an infection, and/or treats an infection.
  • “Pharmaceutically acceptable carrier” refers to any media, coating, solvent, adjuvant, stabilizing agent, antibacterial agent, diluent, antifungal agent, isotonic agent, or adsorption agent. Acceptable carriers and pharmaceutically acceptable carriers are considered equivalent.
  • Vaccine and“vaccine composition” refer to a preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of disease, such as an infectious disease.
  • the immunogenic material may include, for example, antigenic proteins, peptides or nucleic acids (DNA and/or RNA) derived from them.
  • Vaccines may elicit prophylactic (preventative) and/or treatment responses.
  • Methods of administration vary according for the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration as discussed herein. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous or intramuscular.
  • Mucosally such as by intranasal, oral, vaginal or rectal. Topically such as by microneedle patches. Vaccines may be administered with an adjuvant to boost the immune response.
  • Group A rotaviruses have been propagated in several different cell lines. They typically require incorporation of proteolytic enzymes, DEAE-dextran, or a combination of both for propagation. Using increased virus inoculum volumes has also contributed to the success in growing some of the group A human rotavirus strains. Group A rotavirus diagnostic kits, and bovine and porcine group A rotavirus modified live virus vaccines, are commercially available.
  • porcine group C rotavirus has been demonstrated in two types of cell cultures: Primary Pig Kidney (PK) and embryonic Rhesus Monkey Kidney (Ma-l04).
  • PK Primary Pig Kidney
  • Ma-l04 embryonic Rhesus Monkey Kidney
  • Welter et al. (U.S. Patent No. 5,147,639) propagates a swine rotavirus C in swine testicular cells.
  • Welter states that continuous cell lines like MA-104 are undesirable.
  • BGM cell lines are continuous cell lines and the present inventors have surprisingly discovered that BGM cell lines, such as BGM cell line MH18 WCB 5-16-18 P05 deposited with ATCC, are able to successfully propagate rotavirus strains of groups A, B, C, D, E, F, G and H.
  • BGM cells can propagate the rotavirus sufficiently to produce a vaccine composition, including an inactivated vaccine composition, that can provide a protective immune response.
  • a simian (monkey) group A rotavirus (SA11) has been grown in BGM cells and demonstrated some CPE and growth of the virus.
  • group A rotaviruses grow well in many cell lines, and the BGM cells are homologous cells to the simian rotavirus species.
  • homologous refers to cells that are from the same general animal species.
  • both the BGM cells and the simian rotavirus are derived from monkeys. This result therefore is not an indication that rotaviruses from other, non-simian animals or mammals could replicate well in the BGM cell line.
  • Groups B, C, D, E, F, G and H, and also the proposed I and J groups have not been successfully isolated or propagated to high titers or to show significant CPE in any tissue culture cell such that an inactivated vaccine composition could be produced using prior known techniques. Only a swine group C rotavirus has been isolated in a homologous swine cell and propagated enough to produce an attenuated vaccine composition. But no group C rotavirus has been isolated or propagated in a non-homologous cell line, such as porcine rotavirus in a BGM cell line.
  • Buffalo Green Monkey kidney cells referred to herein as Buffalo Green Monkey cells or BGM cells
  • BGM cells are kidney cells from the African Green Monkey.
  • a particular BGM cell line developed and reported herein is designated MH18 WCB 5-16-18 P05.
  • An alternative Buffalo Green Monkey cell line EC ACC 90092601 is available from the European Collection of
  • BGM cell line MH18 is a continuous cell line that has been shown to have stable karyology over at least 20 passages from the master cell. These cells are free of adventitious agents and have been approved for use in vaccine manufacture by USD A/C VB.
  • BGM cell lines have been mainly used to culture Chlamydia but have also been evaluated for their ability to grow various viruses.
  • BGM cells produce a cytopathic effect when inoculated with simian rotavirus SA11, but growth of a simian virus on simian cells is not surprising as that is a homologous cell to the virus.
  • BGM cells can be used to grow non simian rotaviruses, such as porcine rotaviruses, both for group A rotaviruses and rotaviruses other than group A rotaviruses, such as group C.
  • a rotavirus vaccine composition and a method for making the same, particularly a mammalian or avian rotavirus vaccine composition for immunizing birds and/or mammals such as pigs, calves, chickens or humans against the rotaviruses in one or more of groups A, B, C, D, E, F, G and H.
  • BGM cells have not been used previously to isolate or propagate rotaviruses for production of such vaccines, particularly mammalian vaccines without first isolating on species specific cells first (example: simian cells for simian rotaviruses).
  • rotavirus group except A has been successfully isolated or propagated in BGM cells. And with rotavirus A, only a simian rotavirus Group A has been successfully grown on a BGM cell line.
  • a BGM cell line is more receptive to the various other groups of rotaviruses than other cell lines, including other monkey kidney cell lines, and that it will allow better isolation and enhanced propagation of rotavirus groups B, C, D, E, F, G and H so that a sufficiently high antigen mass can be produced that is useful for making a vaccine.
  • the rotavirus may be any rotavirus, such as a rotavirus from rotavirus group A, B, C, D, E, F, G, or H.
  • the group A rotavirus is a non-simian group A rotavirus.
  • the rotavirus is from one of groups B, C, D, E, F, G or H, and in particular embodiments, the rotavirus is a group C rotavirus.
  • the rotavirus may be a rotavirus that infects an animal, including humans and non-human animals, and may be a mammal or bird.
  • the non-human animal may be an avian or a mammal, such as a simian, porcine, bovine, equine, canine, feline, or other mammalian species.
  • the avian may be a chicken, turkey, guinea fowl, pheasant, partridge, pigeon or other avian species.
  • the animal is a porcine.
  • the composition comprises an antigen mass obtained from propagating a rota vims on BGM cells, such as MH18.
  • the composition may be used to produce an attenuated or live, modified vaccine, such as a live vaccine that is substantially non-pathogenic, or an inactivated or killed vaccine.
  • the rotavirus may be from any suitable source, such as a laboratory, a commercial source, such as a culture collection, or a sample from an infected animal. Suitable samples from infected animals may include fecal material, intestinal contents, bodily fluids such as saliva and blood, tissue samples, vomitus, or a combination thereof.
  • a sample from an infected animal is used to produce an autogenous vaccine, which typically is an inactivated or killed vaccine.
  • the rotavirus may be propagated on the BGM cells using any suitable method. In some embodiments, moving cultures are used, such as by using roller tubes, but in other embodiments, stationary cultures are used.
  • the rotavirus may comprise a gene sequence having at least an 80% sequence identity (i.e., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,
  • the rotavirus is a group A rotavirus and may comprise a gene sequence having at least an 80% sequence identity (i.e., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • the rotavirus is a group B rotavirus and may comprise a gene sequence having at least an 80% sequence identity (i.e., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to any one of SEQ ID NOS: 45-88.
  • the rotavirus is a group C rotavirus and may comprise a gene sequence having at least an 80% sequence identity (i.e., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
  • the BGM cells are washed, such as with a suitable buffer solution, to remove any residual serum that was used to grow the cells.
  • suitable buffers include, but are not limited to, TRIS, BIS, HEPES, Phosphate Buffered Saline (PBS) or a combination thereof.
  • the buffer solution may also comprise one or more cell disrupting agents, such as calcium chloride, potassium chloride, sodium chloride and/or various detergents, such as Triton-X-lOO, NP40, deoxycholate, polysorbate 80 or 20, sodium dodecyl sulfate (SDS).
  • the BGM cells may be treated with one or more proteolytic enzymes.
  • Suitable proteolytic enzymes include, but are not limited to, trypsin, pancreatin, chymotrypsin, protease, and the like.
  • contacting the cells with a cell disrupting agent and/or a proteolytic enzyme may improve the growth of the rotavirus on the BGM cells.
  • the amount of enzyme added to the BGM cells is from greater than zero to 80 pg / mL, or more, such as from greater than zero to 80 pg/mL.
  • no enzyme is used.
  • no enzyme is used for group B rotaviruses.
  • the buffer and enzyme may be added to the BGM cells sequentially in any order, or substantially simultaneously. In some embodiments, the buffer and enzyme are added as a combination to the BGM cells.
  • the BGM cells are then inoculated with the rotavirus to be grown.
  • the buffer and/or enzyme may be removed before inoculation with the rotavirus, or one or both may be present when the rotavirus is introduced.
  • the BGM cells are incubated in a suitable growth media for a time period and at a temperature suitable to facilitate rotavirus growth.
  • the media may comprise a proteolytic enzyme; DEAE-dextran; DMSO; EDTA; D-glucosamine; L-glutamine; pyruvate, insulin, physiological buffer such as HEPES, glucose; an antimicrobial, such as gentamicin; or any combination thereof.
  • Suitable proteolytic enzymes include, but are not limited to, trypsin, chymotrypsin, papain, amylase, pancreatin or a combination thereof.
  • the temperature may be any temperature suitable to facilitate rotavirus growth.
  • the temperature is from 28 °C or less to 42 °C or more, such as from 32 °C to 40 °C or from 34 °C to 38 °C.
  • the BGM cells may be incubated from greater than zero to 10 days or more, such as from 30 minutes to 28 days, from 1 hour to 10 days, from 8 hours to 10 days, from 1 day to 9 days, from 3 days to 9 days or from 5 days to 8 days, and in certain embodiments the BGM cells are incubated for 7 days.
  • the rotavirus is removed (for example, the media containing viral particles is removed) after an incubation period of from 30 minutes to 8 hours or more. However, in other embodiments, the rotavirus is left on the cells.
  • Some rotaviruses produce a cytopathic effect (CPE) in BGM cells.
  • the CPE may initially appear as enlarged cells or cell fusion and the cells may subsequently develop holes in the cell sheet, typically caused by lysis of the cells. Passage of the rotavirus in fresh BGM cells may produce significant CPE, and may approach 100% CPE. However, in some embodiments, this subsequent passage does not produce CPE but the rotavirus is still able to propagate effectively on the BGM cells.
  • Rotaviruses that do not produce a CPE effect may be used to develop a persistently-infected cell line. Such a cell line may be very efficient in producing a high enough antigen mass to produce either a modified live or inactivated (killed) vaccine.
  • the vims is passaged onto fresh cells treated in the same manner as described and allowed to progress to complete (100%) CPE.
  • the culture can also be frozen and/or a detergent added, to release as much virus, such as viral particles, antigens, RNA or a combination thereof, from the cells as possible. After thawing the frozen culture, the vims can then be passaged onto fresh cells that have been washed as described above.
  • the rotavims propagates by continuous passage through the cells, releasing viral particles into the media.
  • the rotavims may be grown for from less than one day to several weeks, with the vims, such as viral particles, being harvested from the cells.
  • a bioreactor is used and new BGM cells can be provided to further extend the growing period.
  • the media containing the viral particles is removed also referred to as harvested and fresh media is added to the cells so that propagation continues.
  • Continuous passage can allow the virus to adapt and grow to a higher antigen mass. After an acceptable antigen mass is produced and harvested, the culture can be inactivated and optionally adjuvanted as described below.
  • the antigen mass may be determined by any suitable method, such as by a titer and reported as TCID50 (tissue culture infective dose). With certain adjuvants, for example Emulsigen-D or Emulsigen-DL90, an acceptable antigen mass may be a TCID50 of about 1 x 10 4 virus particles/mL or more.
  • the antigen mass can be obtained by propagating a rotavirus on BGM cells as described above. Then, the cells and media can be treated with a detergent and/or subjected to a freezing and thawing to release vims particles and peptides from the cells prior to or after complete CPE develops as part of the harvest procedure.
  • the detergent may be any detergent suitable to release, such as extract, the viral particles and/or peptides from the cells.
  • Suitable detergents include, but are not limited to Triton-X-lOO, NP40 (nonyl phenoxypolyethoxylethanol), deoxycholate (sodium deoxycholate), polysorbate 80, polysorbate 20, sodium dodecyl sulfate (SDS) or a combination thereof.
  • the addition of the detergent may be sufficient to inactivate the rotavirus.
  • the released vims and/or antigens one or more of the inactivating agents described herein may be added to ensure the released viral particles are inactivated.
  • An adjuvant also may be added to the antigen mass.
  • the antigen mass may be obtained by removing the media from the cells and releasing the antigens from the cells, such as by freezing and thawing the cells and/or contacting the cells with a detergent.
  • the viral particles may be killed or inactivated by contacting the solution with a detergent and/or an inactivating agent described herein. Additionally, or alternatively, the separated media containing viral particles may be contacted with an inactivating agent and/or detergent, to produce a killed vaccine.
  • BGM cells and/or other cell lines may be made using viral particles that have not been inactivated by contact with an inactivating agent and/or detergent.
  • Suitable other cell lines may be any non-BGM cell line, and in particular embodiments, may be a cell line homologous with an animal species from which a rotavirus sample was obtained, and/or an immunogenic composition is being prepared for.
  • Suitable non-BGM cell lines include, but are not limited to, Rhesis Monkey Kidney cells, MARC 145, MA104, Swine Testicle cells, Swine Kidney cells such as PK, PK15 and PK2A, Bovine Kidney cells (MDBK), Canine Kidney cells (MDCK or CLDK), Bovine Turbinate cells (BT), Feline Kidney cells, Hamster Kidney Cells (BHK), Mouse cell lines including mouse kidney and mouse lung, Bovine and human lung cells and various types of tumor cells including HRTs.
  • the rotavirus is passaged through BGM cells at least once, more typically at least twice, such as 2, 3, 4, 5, 10, 15, 20 or more times, such as 2, 3, 4, 5, 10, or 15 times.
  • the cells may be passaged through a non-BGM cell line at least once, such as 1, 2, 3, 4, 5, 10, 15, 20 or more times, such as 2, 3, 4, 5, 10, or 15 times.
  • Inactivated or killed vaccines typically require higher antigen mass. Antigen mass can be measured by titer or by other methods such as ELISA, electrophoresis, Western Blot, PCR, Fluorescent Antibodies (FFU) or any other method usable to detect proteins, glycoproteins, RNA or DNA.
  • An antigen mass suitable for an inactivated or killed vaccine typically is at least 1 x 10 4 TCID5o/mL and may be from 1 x 10 4 TCIDso/mL to 1 x 10 15 TCID mL or more.
  • the infected cells may be treated prior to complete formation of CPE to release the vims and/or proteins and antigens from the cells.
  • Any suitable technique can be used, such as, but not limited to, freezing and thawing, and/or addition of detergents such as Triton X-100, NP40, deoxycholate, polysorbate 80 or 20, or sodium dodecyl sulfate (SDS).
  • detergents such as Triton X-100, NP40, deoxycholate, polysorbate 80 or 20, or sodium dodecyl sulfate (SDS).
  • Inactivating agents are generally used after the acceptable antigen mass is achieved.
  • Such inactivating agents include, but are not limited to, formalin, formaldehyde, binary ethyleneimine (BEI), and/or beta propiolactone (BPL).
  • BEI binary ethyleneimine
  • BPL beta propiolactone
  • One or more detergents such as one or more of the detergents disclosed herein, may be used as an inactivating agent, either alone, or in combination with one or more of the inactivating agents discloses herein.
  • the inactivating agents typically are used at concentrations known to persons of ordinary skill in the art.
  • BEI may be used at a concentration of from 0.001 M to 0.1 M or more, such as from 0.005 M to 0.05 M, and in some embodiments, 0.01 M is used.
  • the BEI is allowed to mix with the rotavirus antigens for a period of time effective to inactivate any viral particles, such as from greater than zero to at least 48 hours, such as from 1 to 48 hours, at room temperature, or for from greater than zero to seven days or more with cooling, for example, in a refrigerator, such as from 2-10 °C.
  • the BEI may be neutralized with thiosulfate, such as sodium or potassium thiosulfate.
  • the BEI and thiosulfate may form may form a neutralization product, such as an aminoethylthiosulfate that may have a structure H2NCH2CH2SSO3 .
  • Formalin typically is used at a concentration of from 0.01% to 0.1% (v/v). After the addition of the formalin, inactivation may take place for a period of time effective to inactivate any viral particles, such as from greater than zero to 48 hours or more, such as from 1 to 48 hours, at room temperature, or with cooling as noted above with respect to BPI for from greater than zero to seven days or more. Formalin may remain in the mixture without neutralization or may be neutralized with bisulfite, such as sodium or potassium bisulfite.
  • BPL may be used at a concentration of from 0.0001 M to 0.1 M, such as from 0.001 M to 0.01M. Inactivation progresses until the pH stabilizes after addition of the BPL.
  • the pH may be from 6 to 8, such as from pH of 6.5 to 7.5. Inactivation with BPL typically does not require neutralization.
  • a neutralizing agent may chemically react with the inactivating agent, such that, after neutralization, there is only a small amount, substantially none, or none, of the inactivating agent left in the composition.
  • the amount of inactivating agent left after neutralization may be from 0.5% to 0%, such as from 0.25% to 0%, from 0.1% to 0%, from 0.05% to 0% or from 0.01% to 0%. That is, the amount of neutralizing agent added is sufficient to neutralize from 99.5% to 100% of the inactivating agent, such as from 99.75% to 100%, from 99.9% to 100% from 99.95% to 100% or from 99.99 % to 100% of the inactivating agent.
  • the composition has no free or unreacted inactivating agent present after neutralization.
  • an adjuvant may be added to disclosed vaccines.
  • An adjuvant may be added for example, in order to accelerate, prolong and/or enhance the immune response produced by the antigen in the vaccine.
  • adjuvants known to persons of ordinary skill in the art any of which may be used, such as adjuvants based on formulations such as oil-in-water, polymer, water-in-oil, water-in-oil-in-water, saponins, liposomes, nanoparticles, aluminum and toll receptor stimulators, and combinations thereof.
  • suitable adjuvants include, but are not limited to, Emulsigen ® -based adjuvants including Emulsigen ® , Emulsigen ® -D (containing dimethyldioctadecylammonium bromide (DDA)), Emulsigen ® -DL90 (containing DDA),
  • Emulsigen ® -BCL containing a block copolymer immunostimulant
  • Emulsigen ® -P containing with a proprietary immunostimulant
  • Emulsimune ® , CarbigenTM, Poly genTM, TS6, Quil, QS21, ISA 71 VG, ISA 35, ISA 50V, ISA 52, ISA 51, ISA 720, ISA 201, ISA 206, Carbomer 934P, Carbomer 971P, Freunds complete, Freunds incomplete, microfluidized emulsions (MF59), polylactide glycolide (PLGA), CpG oligonucleotides, muramyl dipeptide (MDP), bacterial extotoxins or bacterial endotoxins, BCG, E.
  • coli labile toxin tetanus toxoid
  • MPLA monophosphoryl lipid A
  • ISCOMS immunostimulatory complexes
  • block copolymers such as L121, ethylene maleic anhydride or combinations of any of these.
  • Amounts and concentrations of such adjuvants and/or additives useful in the vaccines described in this invention can readily be determined by a person of ordinary skill in the art.
  • an amount of from 0.05% to 70% or more (v/v) of the adjuvant and/or additional component(s) is added to a vaccine composition, such as from 1% to 50%, from 1% to 30%, from 1% to 20%, or from 12 % to 20% (v/v).
  • propagating the rotavirus on BGM cells, passaging them multiple times on BGM cells and/or other, non-BGM cells can produce a rotavirus that is no longer pathogenic but replicates to some limited extent in the mammal.
  • This type of vaccine composition is typically referred to as an attenuated or modified live vaccine.
  • Inactivating agents typically are not added to an attenuated or modified live vaccine compositions, but an adjuvant optionally may be included.
  • the limited replication in the animal will not cause infection or will cause only mild symptoms, but will produce a substantially protective immune response.
  • a vaccine composition prepared from a rotavirus that is isolated and/or propagated on BGM cells contains a sufficient amount of rotavirus antigen required to provide protection to an animal, such as a mammal, when later exposed to a rotavirus.
  • the amount of rotavirus antigen is referred to as the antigen mass.
  • Antigen mass may be determined by any suitable method, including but not limited to, vims titer (TCID50 conducted prior to inactivation), Fluorescent Antibody staining of cultures (FA), Enzyme linked immunosorbent assay (ELISA), Dot Blot, Western Blot, Polymerase Chain Reaction (PCR), and/or Eli Spot.
  • the antigen mass can be at least 1 x 10 1 TCIDso/mL, such as from 1 x 10 1 TCID o/mL to 1 x 10 15 TCID mL or more, for modified live or attenuated antigens, or at least 1 x 10 4 TCIDso/mL, such as from 1 x 10 4
  • TCID5o/mL to 1 x 10 15 TCIDso/mL or more, for inactivated or killed vaccines.
  • the antigen mass may be produced by propagating the rotavirus on BGM cells until CPE is observed or until the CPE has destroyed all the cells (i.e., 100% CPE). Alternatively, CPE may not be observed and the rotavirus may continually release viral particles into the media. Such viral particles can be harvested by removing and replacing the media.
  • the vaccine composition may comprise whole culture that is harvested and/or viral particles that are removed during continuous propagation in BGM cells. The culture may include media that the cells were grown in, and/or cells or cell fragments that are produced by CPE and/or lysing the cells, such as by freezing and thawing the cells and/or contacting the cells with a detergent, to release the viral particles and/or antigens.
  • Disclosed vaccine compositions may comprise BGM cells or cell fragments. However, if the rotavirus is passaged through different cells subsequent to being passaged through the BGM cells, the composition may additionally or alternatively comprise cells and/or cell fragments from those subsequent cells. Exemplary cell types are provided herein.
  • the vaccine composition may also comprise one or more components from the media that is used to grow the BGM and/or non-BGM cells.
  • DMSO may be used in the growth media for the BGM cells and the resulting vaccine composition may comprise DMSO.
  • DMSO is not used for growing rotavirus group A in BGM cells.
  • a vaccine produced for human use typically will be substantially free from cells or cell fragments and also may be free from DMSO.
  • the composition may also comprise one or more inactivating agents, one or more neutralizing agents, and/or a compound or compounds that result from a reaction between a neutralizing agent and an inactivating agent.
  • An inactivated or killed vaccine may also comprise one or more detergents, such as one or more detergents disclosed herein.
  • an inactivated or killed vaccine typically will comprise an adjuvant.
  • the composition may comprise rotavirus antigens and/or viral particles, an adjuvant, and BGM and/or non-BGM cells and/or cell fragments.
  • an attenuated or live, modified vaccine will not comprise a detergent or inactivating agent and/or neutralizing agent.
  • an attenuated or live, modified vaccine may comprise a stabilizer, such as glycerol, sucrose, dextrose, skim milk, or a combination thereof. And/or Carbigen or Carbopol 934P or 971P could be added as a stabilizer as well.
  • An attenuated or live, modified vaccine may be frozen, such as lyophilized.
  • an immunogenic composition and/or vaccine such as an attenuated, live modified, inactivated or killed vaccine, as described herein may further comprise one or more acceptable carriers.
  • acceptable carriers include any media, coating, solvent, stabilizing agent, antibacterial agent, diluent, antifungal agent, isotonic agent, or adsorption agent.
  • Acceptable carriers and pharmaceutically acceptable carriers are considered equivalent.
  • Diluents may include, but are not limited to, phosphate buffered saline, saline, water, glycerol and combinations thereof.
  • Isotonic agents may include, but are not limited to, sodium chloride, dextrose, mannitol, sorbitol and lactose.
  • Stabilizers may include, but are not limited to, serum, albumin, glycerol sucrose, dextrose and similar ingredients known to a person of ordinary skill in the art.
  • a carrier may be any pharmaceutically acceptable carrier. Such a carrier may or may not be a water soluble material or mixture of materials.
  • the carrier may comprise one or more of the following: monosaccharides, disaccharides, polysaccharides, or other carbohydrates such as dextrose, mannitol, fructose, polyfructosan, polydextrose, dextrin, glucose, invert sugar, lactitol, lactose, isomalt, maltitol, maltose, maltodextrin, sorbitol, xylitol, sucrose, sucralose, mannose, galactose, hyaluronic acid, arabinose, fructose, glucosamine, glactosamine, rhamnose, N- acetylglucosamine, iduronate, monnuronate, manopyranose, alginate, cellulose, carrageenan, pectin and combinations of these and other
  • a vaccine composition such as a killed or inactivated vaccine composition
  • a formalin- or formaldehyde-free vaccine composition is inactivated using an inactivating agent that does not comprise formalin or formaldehyde.
  • the formalin or formaldehyde is substantially neutralized, such as by adding a bisulfite compound such as sodium bisulfite, such that there is substantially no free formalin or free formaldehyde present.
  • the amount of formalin and/or formaldehyde present in the composition is from 0.5% to 0%, such as from 0.25% to 0%, from 0.1% to 0%, from 0.05% to 0% or from 0.01% to 0%, and in some embodiments, the amount of formalin and/or formaldehyde is substantially zero, such as when formalin or formaldehyde is not used, or when it is fully neutralized.
  • the composition comprises antigens from at least one rotavirus strain, BGM cell fragments, at least one adjuvant, neutralized inactivating agent (neutralization product), and optionally may further comprise neutralizing agent (if used and if an excess is used), DMSO, proteolytic enzyme and/or detergent.
  • neutralizing agent if used and if an excess is used
  • DMSO methyl methacrylate
  • proteolytic enzyme if used and if an excess is used
  • a composition comprising rotavirus group A antigens may not comprise DMSO.
  • embodiments of the disclosed rotavirus composition may be administered as part of a combination of therapeutic agents.
  • the combination may comprise one or more additional vaccines, and/or additional components that facilitate an immune response, that are beneficially received by a subject receiving disclosed vaccines, or additional agent or agents useful for administration to address a condition disparate from rotavirus infection, such as a food supplement and/or one or more antibiotics.
  • the combination may comprise a composition that comprises the rotavirus antigens produced by the method disclosed herein, and additional antigens from one or more additional diseases.
  • the additional antigens may be administered as a vaccine composition separate from the disclosed rotavirus composition.
  • Such a separate vaccine composition may be administered substantially simultaneously with the disclosed rotavirus composition, or sequentially in any order.
  • the separate compositions typically are administered to a subject such that a second composition is administered within a time period after administration of the first composition such that the subject is still receiving a benefit from the first composition and therefore receives a benefit from the combination of the first and second compositions that are administered.
  • the additional antigens may be from a second rotavirus, such as from a rotavirus group that is different from the rotavirus group of the first antigens.
  • a combination composition may comprise antigens from two or more of the rotavirus groups A, B, C, D, E, F, G and H, such as two or more from A, B, C, E and H for swine.
  • a combination composition comprises rotavirus C antigens and additional antigens from one or more of groups A, B, D, E, F, G and H.
  • a particular combination composition may comprise antigens from each of the known rotavirus groups, such as antigens from each group A, B, C, D, E, F, G and H.
  • a particular combination comprises antigens from groups A, B and C, and such a composition may be useful for administering to swine.
  • a combination composition such as a composition for administration to swine, comprises group H antigens in combination with antigens from at least one other rotavirus group, such as group C.
  • Other exemplary combinations include, but are not limited to Groups A, B, C and H; Groups A and C; or Groups A, B and C.
  • the combination may comprise other, non-rotavirus antigens.
  • the non-rotavirus antigens may be selected to produce an immune response in a particular species to which the composition may be administered.
  • the combination may comprise antigens from one or more of rotavirus groups A, B, C, E and H, and additional antigens from one or more swine diseases, such as, but not limited to, Mycoplasma hyopneumoniae , Mycoplasma hyorhinis, Mycoplasma hyosynoviae, various Clostridial bacterins or toxoids including but not limited to C. perferingens types A and/or C, C.
  • the composition may comprise rotavirus antigens from any known group, such as at least rotavirus group A, and one or more antigens from any disease that infects bovines.
  • Exemplary bovine diseases include, but are not limited to, Mycoplasma bovis, Salmonella spp, Pasteurella types A and/or D, Clostridial bacterins such as C. perfringens types A, C and/or D, C. sordellii, C. chauvoei, C. septicum, C. haemolyticum, Escherichiae coli spp, Mannheimia haemolytica antigens, Leptospira spp. such as L. hardjo, L. Pomona, L. grippotyphosa, L.
  • Neospora caninum icterohaemorrhagiae
  • Trichomonas icterohaemorrhagiae
  • Neospora caninum icterohaemorrhagiae
  • Trichomonas Trichomonas
  • the composition may comprise rotavirus antigens from any known group, such as at least rotavirus group A, and one or more antigens from any disease that infects humans, such as influenza, measles, mumps, rubella, hepatitis, particularly, hep A and/or hep B, tetanus, diphtheria, pertussis, polio, whooping cough, Hib, pneumococcal pneumonia, HPV, varicella, meningococcal meningitis, tick-borne encephalitis, yellow fever, tuberculosis, typhoid fever, cholera, Japanese encephalitis vaccine, or a combination thereof.
  • rotavirus antigens from any known group such as at least rotavirus group A
  • one or more antigens from any disease that infects humans such as influenza, measles, mumps, rubella, hepatitis, particularly, hep A and/or hep B, t
  • compositions may be administered to a subject in need thereof by any suitable technique known to a person of ordinary skill in the art.
  • Suitable administration routes include, but are not limited to, oral, topical, intraperitoneal, intravesical, intrathecal, intravenous, intradermal, subcutaneous, intramuscular, intranasal, vaginal or rectal.
  • the administration route is oral, intranasal, intramuscular, or subcutaneous.
  • the composition may be administered to an animal, including humans and non-human animals, such as a non-human mammal or bird.
  • the non-human mammal may be a simian, porcine, bovine, equine, canine, feline, or other non-human mammalian species.
  • the bird may be chicken, turkey, guinea fowl, pheasant, partridge, pigeon or other avian species.
  • the animal is a porcine.
  • the composition may be administered to a subject at any age, and is some embodiment, the composition is administered to a young subject.
  • a rotavirus vaccine typically is administered before the age of two, preferably with a first dose being administered at around 6 weeks of age and subsequent doses being given at least one month apart.
  • the composition may be administered to animals at any age.
  • the composition may preferably be administered to neonate pigs, such as from birth to 8 weeks old or to older pigs that may be exposed.
  • the composition may be administered to a calf, such as from birth to 6 weeks or to older calves that may be exposed.
  • the composition may be administered to pregnant and/or nursing subjects or to subjects who will become pregnant.
  • the composition may be administered to pregnant sows or gilts to stimulate antibodies that are passed to their nursing baby pigs through the colostrum and milk, thus protecting the baby pigs from disease caused by such rotaviruses.
  • Such vaccines may also be used to vaccinate pregnant cows during gestation to protect newborn calves, and to cows that will become pregnant.
  • the composition may be administered to produce an immune response, such as a protective immune response, against a rotavirus group A, B, C, D, E, F, G and/or H infection.
  • an immune response and/or protection is against a homologous infection, i.e., an administered composition comprising group C antigens will produce an immune response that protects against a group C infection.
  • the composition provides cross-protection. That is, administration of a composition comprising antigens from a first rotavirus group provides an immune response and/or protection against a second infection from a rotavirus from a different group.
  • a subject is administered with a composition prepared from a rotavirus that was obtained from the same species.
  • a swine is administered with a composition comprising antigens obtained from a swine rotavirus infection.
  • a subject is administered with a composition comprising antigens obtained from a rotavirus from a different species from the subject.
  • the composition may be administered to a subject in an amount sufficient to produce a desired result in the subject.
  • the amount that is administered may vary depending on the species and the age, and/or weight of the subject. In some embodiments, from 1 mL or less to 5 mL or more is administered, such as from 1 mL to 5 mL, or from 1 mL to 3 mL. For example, for pigs less than 50 pounds 1 mL of a vaccine composition may be administered. However, for pigs over 50 pounds, typically older pigs including sows and gilts, 2 mL of a vaccine composition may be administered.
  • a single dose of the vaccine composition may be administered, or multiple doses may be administered, such as 2, 3, 4, 5, or more doses.
  • 2 doses are administered.
  • a first dose may be administered up to about 5 weeks prior to farrowing, and a second dose may be administered closer to farrowing, such as about 2 weeks prior to farrowing.
  • a first and a second dose may be administered at least one month apart, such as from about 6 weeks, and at least one month later but before the age of 2 years. VIII. Examples
  • a control sample contained MH18 cells with DMEM media containing 10 pg/mL trypsin and was also incubated for 30 minutes at 37 °C.
  • One hundred microliters of the sample and the control were inoculated into separate wells of a 48 well plate containing confluent MH18 cells. Prior to inoculation, the cells were washed twice with PBS buffer to remove any residual serum. The plate was incubated for 60 minutes at 37 °C after which the plate was placed into a 37 °C incubator and observed daily for morphological changes. CPE was observed by Day 2 and is shown in FIG. 1. By way of comparison, FIG. 2 illustrates the non-infected control sample.
  • FIG. 3 demonstrates the presence of the virus in the MH18 cell as observed by immunofluorescence, and for comparison, FIG. 4 illustrates the background fluorescence from the non-infected control.
  • This study compared the growth of a group A swine rotavirus in three different cell lines. Pigs with watery diarrhea typical of rotavirus were sampled. PCR confirmed that two different samples contained only group A rotavirus. The intestinal contents from each sample were processed separately. First, the contents were centrifuged to remove the solids. The supernatant was removed and filtered through a 0.45m Nalgene filter followed by a 0.2m Nalgene filter.
  • Gentamicin was added to the filtrate at a concentration of 60 mg/L.
  • One T25 flask of each of three cell lines was inoculated with filtrate from each isolate to determine which cell could grow the rotavirus group A.
  • the cell lines inoculated were African green monkey (MA-104), Swine Testicle (ST) and BGM (MH18).
  • DMEM Dulbecco's Modified Eagle Medium
  • HyClone #SH30285.09 high modified media
  • L-glutamine VWR #02-0109-1000
  • HEPES buffer 1M solution Fisher #BP299-l00
  • calf serum HyClone #SH30087.04
  • the media When confluent, the media was poured off from each of the flasks and 10 mL of fresh media was added, containing only HEPES, L-glutamine and gentamicin at concentrations listed above. This viral growth media was not supplemented with serum.
  • the media in each of the rotavirus growth flasks contained trypsin (HyClone #SV30037.0l) at a concentration of 10 units/mL.
  • Two hundred microliters of the filtrate from each sample (Sample 1 and Sample 2) was inoculated onto separate flasks containing each of the cell lines noted above. This first inoculation was designated pass zero (P0). Flasks were incubated at 34-37 °C for 7 days, and were observed daily for CPE.
  • Table 1 shows the results of this experiment. The results provided by table 1 illustrate that all of the cell lines tested were susceptible to both group A rotaviruses and were able to effectively isolate the organism displaying strong CPE. The control flasks containing only trypsin remained normal with no cell disruption.
  • Gentamicin was added to each filtrate as noted above and they were inoculated onto T25 flasks of either confluent MA-104 cells, ST cells or MH18 cells in the presence of DMEM high media containing HEPES buffer, L-glutamine, gentamicin and serum as described in Example 2. This was a medium that was sufficient to grow the cells and the vims. Other media formulations such as E199 or supplements such as LAH and pyruvate could be added.
  • the cell sheets Prior to inoculating the filtrates onto the various cells, the cell sheets were washed twice with phosphate buffered saline (PBS) to remove any serum that remained from growing the cells.
  • PBS phosphate buffered saline
  • the media used for rotavims isolation from the samples contained trypsin at a concentration of 10 units/mL for passages P0 through P2.
  • the P3 passage contained only 4 units/mL of trypsin.
  • a control flask of each cell line contained all components except the sample inoculum. All flasks were incubated at between 34 and 38 °C for a period of 7 days. At that time, flasks were placed into a freezer at or below -40 °C for at least 24 hours. Then, each flask was thawed and 200 pL was removed to inoculate onto freshly prepared cells of the same cell line for the next passage. As described in Example 2, four passages of each sample on each cell line were
  • Table 2 The results of all passages are shown in Table 2. The results provided by Table 2 demonstrate that only the MH18 cell line displayed CPE by P2. At this passage, all cells were gone by 4 days whereas the trypsin control remained normal. Table 2 also shows that when the trypsin level was reduced to 4 units/mL in P3, the MH18 cells still produced complete CPE. Rotavims group C-specific PCR was conducted to confirm that the observed CPE on the MH18 cells at P2 was produced by group C rotavims. Table 2 demonstrates that only the MH18 cells were useful for growth of group C swine rotavirus to a high enough antigen mass to be able to produce a vaccine. All control flasks remained normal showing no signs of cell disruption.
  • Example 3 The study described in Example 3 is repeated using samples that each contain only a group B, group D, groups E, group F, group G or group H rotavirus. Again, intestinal contents are first identified as containing the specific rotavirus group by PCR or an equivalent method. Then, the samples are centrifuged at room temperature to separate out the solids. The supernatants are removed and filtered through a 0.45m Nalgene filter followed by a 0.2m Nalgene filter. In some cases it is more effective to filter through a final 0.1 m filter.
  • Gentamicin or another antimicrobial agent is added to each filtrate as noted above and they are then inoculated onto T25 flasks of either confluent BGM cells in the presence of DMEM high media containing HEPES buffer, L-glutamine, gentamicin and serum as noted in Example 2. This is a medium that is sufficient to grow the cells and the virus.
  • Other media formulations such as E199 or supplements such as lactalbumin hydrolysate (LAH) and pyruvate can be used as well.
  • LAH lactalbumin hydrolysate
  • pyruvate Prior to inoculating the filtrates onto the various cells, the cell sheets are washed from one to five times with a buffer to remove any residual serum.
  • Buffers may include phosphate buffered saline (PBS), TRIS or HEPES with calcium chloride, potassium phosphate, DEAE dextran, D-glucosamine, sodium chloride or potassium chloride added or another similar diluent to remove allow the cells to be more receptive for virus growth.
  • PBS phosphate buffered saline
  • HEPES phosphate buffered saline
  • the media used for rotavirus isolation from the samples does not contain trypsin or a similar proteolytic enzyme.
  • a control flask of each cell line contains all components except the sample inoculum. All flasks are incubated at between 34 and 38 °C for a period of 7 days with microscopic observations made periodically.
  • flasks are placed into a freezer at or below -40 °C for at least 24 hours. Then, each flask is thawed and 200 pL is removed to inoculate onto freshly prepared cells of the same cell line for the next passage.
  • CPE CPE will be observed in all rotavirus flasks with the BGM cell line whereas all control flasks will remain normal and show no signs of cell disruption. Additionally, it is expected that flasks with infected MA-104 and ST cells will show little or no CPE. However, if CPE is not shown, infection can be visualized using any suitable technique, such as fluorescent antibody staining (IF A) or PCR techniques.
  • IF A fluorescent antibody staining
  • Roller bottles are inoculated with BGM cells in DMEM high medium supplemented with HEPES buffer, L-glutamine, gentamicin and calf serum, the latter at a concentration of 5% to 10% by volume.
  • Cells are incubated at between 34 and 39 °C until the cells have become confluent. Once confluent, the medium is removed and the cells are washed twice with 50 mL of PBS to remove any residual serum. Fresh media containing the same supplements is added to the bottles, except that serum is omitted. Additionally, trypsin is added at a concentration of 1 to 12 units/mL.
  • the BGM cells are then inoculated with group C rotavirus and incubated at 37 to 39 °C until CPE is observed. This takes from 2 to 7 days post inoculation. Once CPE indicates an adequate antigen mass, the culture is harvested by transferring the contents to a separate vessel.
  • An inactivating agent is added.
  • Preferred inactivating agents include binary ethyleneimine (
  • BPL propiolactone
  • BEI or BPL are added at a concentration of from 0.001 to 0.1M to the harvest fluids in order to inactivate the rotavirus. Inactivation proceeds for from 1 to 48 hours after which the inactivating agent is neutralized with either sodium thiosulfate (BEI) or sodium bisulfite (formalin). Neutralization of BPL is not required as it breaks down in aqueous solutions during the inactivation time.
  • Antigen mass is determined by a suitable method, such as, but not limited to, virus titer (TCID50 conducted prior to inactivation), Fluorescent Antibody staining of cultures (FA), Enzyme linked immunosorbent assay (ELISA), Dot Blot, Western Blot, Polymerase Chain Reaction (PCR), and/or Eli Spot.
  • TCID50 virus titer
  • FA Fluorescent Antibody staining of cultures
  • ELISA Enzyme linked immunosorbent assay
  • Dot Blot Dot Blot
  • Western Blot Western Blot
  • Polymerase Chain Reaction PCR
  • Eli Spot Eli Spot.
  • proof of inactivation typically is demonstrated by at least three backpasses in BGM cells prepared to be susceptible to the vims.
  • the inactivated virus is adjuvanted with one of the adjuvants known to the art.
  • Preferred adjuvants include Emulsigen ® -D or Emulsigen ® -DL90.
  • Intestinal samples were obtained from pigs showing diarrhea typical of rotavirus. The sample was submitted for PCR testing and frozen. PCR indicated that the sample was positive for rotavirus A. After this confirmation, the intestinal sample was removed from the freezer and thawed. The intestines were placed into a sterile petri dish and cut into two pieces with lengths of approximately 5 inches. The contents of the intestines were squeezed out with forceps into a 50 mL tube prefilled with 20 mL of DMEM. This tube was vigorously vortexed to assure that the contents were well mixed in the media after which the tube was centrifuged at 4000g for 10 minutes. The supernatant was transferred into a new 50 mL sterile tube and then the contents were aliquoted into 6 microcentrifuge tubes, placing about 1.5 mL into each aliquot. The
  • microcentrifuge tubes were centrifuged at l5000g for 5 minutes after which the supernatants from all tubes were collected and pooled. This supernatant pool was filtered through consecutive 0.45m, 0.22m and 0.2m filters into a sterile tube for storage. Five hundred microliters of the filtered sample were submitted for PCR confirmation of rotavirus A.
  • sample was rotavirus Group A
  • trypsin by adding 100 pL of 0.1% Trypsin Stock solution into 9 mL of DMEM media in a 15 mL conical tube and adding 1 mL of the filtered sample. This mixture was incubated at 37 °C for 30 minutes.
  • a 490 cm 2 roller bottle containing confluent MH18 cells was rinsed twice with sterile DMEM to remove the residual serum from cell growth. After rinsing the roller bottle, the 10 mL pretreated sample was added to the roller bottle containing confluent MH18 cells.
  • the roller bottle was incubated for 60 minutes at 37 °C and then 90 mL of rotavirus maintenance media (RMM) was added and the roller bottle after which it was gently swirled to mix contents.
  • RMM contains DMEM high with IX Gentamicin, IX L-Glutamine, lOpg/mL Trypsin and 0.5% DMSO.
  • a 500 pL sample was removed for PCR confirmation of rotavirus Group A.
  • the roller bottle was then incubated at 37 °C until CPE was greater than 80% after which the roller bottle was placed into the freezer at -20°C. This bottle was labeled Pl seed.
  • P4 (passage 4) was the product that was used to prepare a vaccine. After growth, the vims was harvested by pooling the contents of all of the roller bottles. Binary ethyleneimine was added to a concentration of 0.01M. The vessel was held at room temperature for 48 hours and then the BEI was neutralized with sodium thiosulfate in a 1 : 1 ratio. Inactivation was confirmed by conducting an inactivation assurance test and then the inactivated virus was adjuvanted with 12% Emulsigen ® -D.
  • This vaccine was used to vaccinate a herd of swine from which the vims was isolated.
  • the veterinarian using the vaccine reported that vaccinated animals were healthier than with fewer signs of rotavims as compared with the disease in the herd prior to vaccination.
  • the C. perfringens vaccine was prepared using a Clostridium perfringens Type C isolated from the same herd and grown using art-known procedures. After inactivation, the C. perfringens antigen was also adjuvanted with 12% Emulsigen ® -D. The combination vaccine was provided to a different group of swine from the herd of origin. Once again, the Clostridium and rotavirus diseases found in the herd prior to vaccination were reduced by the combination vaccine.
  • FIG. 5 is an electron micrograph showing that rotavirus Group A entered the cells (white arrows).
  • a swine rotavirus Group A was isolated according to the procedure described in Example 6. After isolation, the isolate was pretreated with 10 pg of trypsin for 30 minutes at 37 °C, diluted 1:100,000 with DMEM and inoculated onto MH18 cells grown to confluency in 6 well microtiter plates. Each well was inoculated with 500 pL. The plate was placed on a plate rotator and incubated for 1 hour at 37 °C after 3.5 mL of RMM was added to each well and the plate was incubated at 37°C. About 50% CPE was observed two days after inoculation. At this time, the cells were fixed using 4 ml ./well of chilled Karnovsky’s fixative.
  • FIG. 5 is an electron micrograph showing that the rota vims Group A is entering a MH18 cell.
  • FIG. 6 is an electron micrograph showing that rotavirus Group A forms virosomes within the cell (white circled area, the arrows show vims particles).
  • FIG. 7 is an electron micrograph showing that rotavirus Group C is entering the cells (white arrows).
  • FIG. 8 is an electron micrograph showing that rotavirus Group C is identified in the cytoplasm (white arrow).
  • BGM cells are grown and infected by group C rotavirus as described in Example 5.
  • the supernatant is removed and the rotavirus antigens are released from the cells.
  • Releasing the antigens may be performed by any suitable method, such as by adding a detergent and/or freeze/thaw cycle(s).
  • Methods comprising using a detergent may comprise contacting the cell sheet with a solution containing a suitable detergent to extract the rotavirus and peptides within the cells and related to the rotavirus.
  • Suitable detergents include, but are not limited to, Triton X-100, NP40, deoxycholate, polysorbate 80 or 20, or sodium dodecyl sulfate (SDS).
  • EDTA may be added to the detergent.
  • Freeze/thaw cycles comprise freezing the harvest material at -20 °C or below and then thawing the material in order to release the rotavirus antigen from cells. These methods help concentrate the virus particles. The antigen mass is then measured by methods as described in Example 5 after which the extract is optionally inactivated if it has not been inactivated by the releasing process.
  • Suitable inactivating agents include BEI, BPL and/or formalin, typically used at concentrations known to persons of ordinary skill in the art.
  • BEI may be used at a concentration of from 0.001 to 0.1 M, with one preferred concentration being 0.01M, and allowed to mix with the rotavirus antigen for from 1 to 48 hours at room temperature, or up to seven days or more under refrigeration (such as from 2-10 °C). After the inactivation process is complete, the BEI is neutralized with sodium or potassium thiosulfate.
  • formalin exemplary concentrations are typically between 0.01% and 0.1% (v/v).
  • inactivation may take place at room temperature for from 1 to 48 hours or under refrigeration as noted above with respect to BPI for up to seven days or more.
  • Formalin may remain in the mixture without neutralization or may be neutralized with sodium or potassium bisulfite.
  • BPL When using BPL as the inactivating agent, it may be used at concentrations between 0.0001 and 0.1M, such as between 0.001 M and 0.01M. Inactivation progresses until the pH stabilizes after addition of the BPL.
  • the pH may be from 6 to 8, such as from 6.5 to 7.5. Inactivation with BPL typically does not require neutralization.
  • the antigen solution may be use undiluted or it may be diluted to provide a specific antigen mass.
  • An adjuvant typically is added, such as Emulsigen ® -D or Emulsigen ® - DL90. These adjuvants may be added at concentrations of between 1 and 20% (v/v), such as from 12 to 20%, or as recommended by the manufacturer (MVP adjuvants ® ).
  • the adjuvanted antigen may be combined with other adjuvanted groups of rotaviruses such as A, B, D, E, F, G and/or H that have been grown and inactivated by the same methods as described in Examples 4 or 5.
  • the rotavirus group C may also be combined with other bacterial or viral antigens.
  • a typical combination composition may comprise rotavirus types A, B and C, optionally with C. perfringens types A and C and/or Escherichia coli.
  • Vaccine compositions prepared according to Examples 2 through 6 are used to vaccinate pigs, typically sows or gilts, but such vaccines can also be used to vaccinate older or neonatal pigs.
  • a typical dose of a vaccine composition is 1 mL for pigs less than 50 pounds, and 2 mL for older pigs, including sows and gilts.
  • two doses of vaccine are preferred, one administered at 5 weeks prior to farrowing and the other at 2 weeks prior.
  • antibody titers typically are measure in the colostmm and/or the milk at farrowing. A four-fold increase in titers compared with non- vaccinated controls indicates an immune response that may provide protection.
  • antibody titers are measured at 21 and 42 days after the initial vaccination. Again, titers are at least four- fold higher by 42 days than the original titers of the animals on day 0 (day of first vaccination).
  • Mucosal delivery of an inactivated vaccine can be advantageous, especially in young pigs.
  • Vaccinating pigs intranasally or orally with either live, modified live or even inactivated (killed) antigens can stimulate both IgA and IgG immune responses that can help protect mammals.
  • a mucoadhesive adjuvant such as CarbigenTM or another Carbopol or Carbomer-based adjuvant, may be used to prepare a vaccine composition that is intended for intranasal or oral administration. In such cases, an inactivated vaccine is prepared according to one or more of the previous examples.
  • a mucoadhesive adjuvant such as CarbigenTM
  • a suitable concentration such as from 1 to 50% (v/v), or from 10 to 30% added.
  • Pigs typically young pigs, such as from newborn to 4 weeks of age, are vaccinated by administering the adjuvanted vaccine into the nares using one of several devices known for such purposes, such as cannulas or nebulizers that spray the vaccine into the area of the nasal passage that allows uptake by the mucosal associated lymphoid tissue (MALT) so as to stimulate a strong IgA and IgG immune response.
  • MALT mucosal associated lymphoid tissue
  • Intestinal samples were received from a swine producer’s veterinarian and found to be positive for rotavirus group A using PCR. The samples were centrifuged at room temperature to separate out the solids. The supernatants were removed and filtered through a 0.45m Nalgene filter followed by a 0.2m Nalgene filter. Gentamicin was added to each filtrate and they were inoculated onto T25 flasks of confluent MH18 cells in the presence of DMEM high media containing HEPES buffer, L-glutamine, and gentamicin as described in Example 2. This was a medium that was sufficient to grow the virus. However, there was no serum present in the viral growth media as it counter acts with the trypsin that was present in the viral growth media.
  • the cell sheets Prior to inoculating the filtrates onto the various cells, the cell sheets were washed twice with phosphate buffered saline (PBS) to remove any serum that remained from growing the cells.
  • PBS phosphate buffered saline
  • the media used for rotavirus isolation from the samples contained trypsin at a concentration of 10 units/mL. All passages were accompanied by a control flask of cells with the same media to demonstrate that the trypsin-containing medium did not damage the cells. All flasks were incubated at between 34 and 38 °C for a period of 7 days. At that time, flasks were placed into a freezer at or below -40 °C for at least 24 hours.
  • each flask was thawed and 200 pL was removed to inoculate onto freshly prepared cells of the same cell line for the next passage. After four passages of the sample on MH18 cells, the flasks were frozen once again. Upon thawing, the culture was checked for vims titer using 96 well plates seeded with MH18 cells. Cells were washed twice with PBS prior to inoculation of the virus dilution. Serial dilutions of the thawed virus were prepared from 10 1 to 10 8 and 100 pL of the appropriate dilution was added to each well that contained 100 pl of virus growth media as described previously. The plates were incubated at 34 - 38 degrees C for 7 days after which the titer was read by fluorescent antibody staining. The titer was reported as 10 5 TCID o/mL.
  • roller bottles containing confluent MH18 cells are washed three times with PBS to remove the serum used for cell growth prior to inoculating them with the rotavirus seed.
  • 200- 300 pL seed is added to 300-350 mL of virus growth media for each roller bottle.
  • Media used is the same as described for use in Example 2.
  • Roller bottles are incubated at 34-38 degrees C during which CPE is observed. When the majority of the cells are infected and CPE is complete, the roller bottles are frozen at -20 degrees C for at least 24 hours.
  • the rotavirus culture is inactivated with 0.01M binary ethyleneimine (BEI) for 48 hours at room temperature. The BEI is neutralized with 0.02M sodium thiosulfate.
  • the inactivated culture is adjuvanted with 20% Emulsigen ® -D.
  • Ten thousand 2.0 cc doses of monovalent rotavirus A were produced according to the procedure provided in Example 6. They were provided to a swine producer to vaccinate sows prior to farrowing. Sows were vaccinated with 2 doses. Additionally, adjuvanted Rotavirus A was combined with adjuvanted SIV to produce a combination vaccine. Ten thousand doses of this combination vaccine were also used by the producer to vaccinate sows twice prior to farrowing.
  • This study demonstrates the growth of a group B swine rotavirus in BGM cells.
  • Fecal samples from pigs with watery diarrhea typical of rotavirus are sampled and confirmed as group B using PCR.
  • the intestinal contents from the sample are processed by centrifugation to remove the solids and then filtration of the supernatant though a 0.45m Nalgene filter followed by a 0.2m Nalgene filter.
  • Gentamicin is added to the filtrate at a concentration of 60 mg/L, to remove extraneous bacterial contamination.
  • This filtrate is then inoculated into either several wells of a 96- well plate or a 24-well plate to determine whether the BGM cells will support growth of group B rotaviruses.
  • the cells are grown as described previously. Once the cells are grown the media is removed and replaced by a fresh viral growth media.
  • This viral growth media is MEMH supplemented with serum at a concentration of 1-5%, HEPES buffer at a concentration of 0.22 M, L-glutamine at 10 mM, sodium pyruvate at 0.1 M) and gentamicin at a concentration of 30 pg/m.
  • Proteolytic enzymes are not used to grow Group B rotavirus. Instead, the cells are pretreated with DEAE dextran during the adsorption process. In some cases, the DEAE dextran is allowed to remain on the cells from 1 hour to overnight. This pretreatment increases the viral adsorption and penetration into the cells.
  • the vims Prior to vims inoculation the vims is pretreated by addition of a chelating agent such as EDTA at a 1 to 10 mM concentration. This is allowed to mix with the vims for from 1 to 30 minutes prior to adding the virus to the BGM cells. After addition of the virus to the BGM cells the vessels are incubated at from 35 to 38 degrees C and observed for the development of CPE. Within about 24 to 48 hours CPE is expected to appear indicating the growth of the vims. It is expected that the virus will grow to titers greater than 1 X 10 4 TCID o/mL. This is high enough to be usable for vaccine formulation with a strong adjuvant.
  • a chelating agent such as EDTA
  • the intestinal contents from the sample were processed by first centrifuging the contents to remove the solids. The supernatant was removed and filtered through a 0.45m Nalgene filter followed by a 0.2 m filter. Gentamicin was added to the filtrate at a concentration of 60 pg/L. Trypsin was then added to the purified sample at a concentration of 10 mg/mL and place into a 37 °C incubator for 30 minutes.
  • a control sample contained MH18 cells with DMEM media containing 10 pg/mL trypsin and was also incubated for 30 minutes at 37 °C.
  • One hundred microliters of the sample and the control were inoculated into separate wells of a 48 well plate containing confluent MH18 cells. Prior to inoculation, the cells were washed twice with PBS buffer to remove any residual serum. The plate was incubated for 60 minutes at 37 °C after which 700 pL of DMEM containing 0.5% DMSO and 2.5 pg/mL of trypsin was added to each well. The plate was placed into a 37° C incubator and observed daily for morphological changes. CPE was observed by Day 2 and is shown in FIG. 9. By way of comparison, FIG. 10 illustrates the non-infected control sample.
  • FIG. 11 demonstrates the presence of the virus in the MH18 cell as observed by immunofluorescence, and for comparison, FIG. 12 illustrates the background fluorescence from the non-infected control.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un procédé de propagation d'un rotavirus, tel qu'un rotavirus de groupe A, B, C, D, E, F, G ou H sur des cellules de rein de singe vert buffalo (BGM). Selon certains modes de réalisation, le rotavirus est un rotavirus de groupe B, C, D, E, F, G ou H, ou un rotavirus de groupe A non simien. Le procédé peut consister à faire passer le rotavirus à travers un second cellule de BGM et/ou une cellule de non-BGM. Un agent d'inactivation et/ou un adjuvant peuvent être ajoutés pour produire une composition de vaccin. L'invention concerne également une composition comprenant des antigènes de rotavirus produits par le passage du rotavirus à travers une cellule de BGM, et un procédé d'administration d'une telle composition.
PCT/US2019/041819 2018-07-20 2019-07-15 Compositions et combinaisons de vaccins contre le rotavirus et leurs procédés de fabrication et d'utilisation WO2020018429A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201862701476P 2018-07-20 2018-07-20
US201862701471P 2018-07-20 2018-07-20
US62/701,471 2018-07-20
US62/701,476 2018-07-20

Publications (1)

Publication Number Publication Date
WO2020018429A1 true WO2020018429A1 (fr) 2020-01-23

Family

ID=67480412

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/041819 WO2020018429A1 (fr) 2018-07-20 2019-07-15 Compositions et combinaisons de vaccins contre le rotavirus et leurs procédés de fabrication et d'utilisation

Country Status (1)

Country Link
WO (1) WO2020018429A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111978380A (zh) * 2020-09-02 2020-11-24 天康生物股份有限公司 野生型气肿疽梭菌细胞毒素a及其制备方法、应用和疫苗
CN113186170A (zh) * 2021-05-26 2021-07-30 西南民族大学 一株猪轮状病毒株及其制备的灭活疫苗和用途
CN117070475A (zh) * 2023-10-13 2023-11-17 金宇保灵生物药品有限公司 牛轮状病毒无血清悬浮培养方法及其产品和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007482A1 (fr) * 1989-11-13 1991-05-30 Ambico, Inc. Cultures de rotavirus du type b et applications de celles-ci
US5147639A (en) 1990-06-19 1992-09-15 Ambico, Inc. Type-c rotavirus cultures and uses therefor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007482A1 (fr) * 1989-11-13 1991-05-30 Ambico, Inc. Cultures de rotavirus du type b et applications de celles-ci
US5147639A (en) 1990-06-19 1992-09-15 Ambico, Inc. Type-c rotavirus cultures and uses therefor

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Microbiology", 1 November 2009, JOHN WILEY & SONS, INC., Hoboken, NJ, USA, ISBN: 978-0-471-72925-9, article MICHELLE ARNOLD ET AL: "Culturing, Storage, and Quantification of Rotaviruses", pages: 15C.3.1 - 15C.3.24, XP055368294, DOI: 10.1002/9780471729259.mc15c03s15 *
ADRIANA LUCHS ET AL: "Group A rotavirus gastroenteritis: post-vaccine era, genotypes and zoonotic transmission", EINSTEIN (S?O PAULO), vol. 14, no. 2, 1 June 2016 (2016-06-01), pages 278 - 287, XP055631029, ISSN: 1679-4508, DOI: 10.1590/S1679-45082016RB3582 *
CIARLET MAX ET AL: "VLA-2 (alpha2beta1) integrin promotes rotavirus entry into cells but is not necessary for rotavirus attachment", JOURNAL OF VIROLOGY, vol. 76, no. 3, February 2002 (2002-02-01), pages 1109 - 1123, XP009516572, ISSN: 0022-538X *
ESTES M K ET AL: "SIMIAN ROTAVIRUS SA-11 REPLICATION IN CELL CULTURES", JOURNAL OF VIROLOGY, vol. 31, no. 3, 1979, pages 810 - 815, XP055630685, ISSN: 0022-538X *
KOBAYASHI, N. ET AL.: "Diversity of G-type and P-type of human and animal rotaviruses and its genetic background.", VIRUS RESEARCH, 22 September 2014 (2014-09-22), Netherlands, pages 75 - 96, XP055545512, Retrieved from the Internet <URL:http://www.formatex.org/microbio/pdf/pages847-858.pdf> DOI: 10.1016/j.virusres.2014.06.016 *
PARIDA M M ET AL: "Studies on in vitro growth characteristics of human rotavirus serotype-3: A new substrate of buffalo green monkey kidney cells", BIOMEDICAL LETTERS, vol. 56, no. 221, 1997, pages 19 - 26, XP009516552, ISSN: 0961-088X *
SAIF ET AL., J. CLIN. MICROBIOL., vol. 26, no. 7, 1988, pages 1277 - 1282
T SANEKATA ET AL: "Isolation of group B porcine rotavirus in cell culture", JOURNAL OF CLINICAL MICROBIOLOGY, 1 March 1996 (1996-03-01), United States, pages 759 - 761, XP055535217, Retrieved from the Internet <URL:https://jcm.asm.org/content/jcm/34/3/759.full.pdf> *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111978380A (zh) * 2020-09-02 2020-11-24 天康生物股份有限公司 野生型气肿疽梭菌细胞毒素a及其制备方法、应用和疫苗
CN113186170A (zh) * 2021-05-26 2021-07-30 西南民族大学 一株猪轮状病毒株及其制备的灭活疫苗和用途
CN117070475A (zh) * 2023-10-13 2023-11-17 金宇保灵生物药品有限公司 牛轮状病毒无血清悬浮培养方法及其产品和应用
CN117070475B (zh) * 2023-10-13 2023-12-29 金宇保灵生物药品有限公司 牛轮状病毒无血清悬浮培养方法及其产品和应用

Similar Documents

Publication Publication Date Title
US7722884B2 (en) Vaccines and methods to treat canine influenza
US9364527B2 (en) Bovine vaccines and methods
US10918711B2 (en) Composition comprising antigens and a mucosal adjuvant and a method for using
US10280199B2 (en) Coronavirus proteins and antigens
US10434168B2 (en) Attenuated bovine coronavirus and related vaccines
WO2020018429A1 (fr) Compositions et combinaisons de vaccins contre le rotavirus et leurs procédés de fabrication et d&#39;utilisation
US20170340729A1 (en) Composition comprising pedv antigens and methods for making and using the composition
US20240197851A1 (en) A vaccine for protection against streptococcus suis serotype 9, sequence type 16
Kim et al. Coronavirus proteins and antigens
WO2021146399A1 (fr) Compositions comprenant un nouvel orthoréovirus de mammifère et leurs procédés de fabrication et d&#39;utilisation
Tan et al. Further studies on feline respiratory virus diseases: 1. Vaccination experiments
JP7349366B2 (ja) クロストリジウム類毒素を含むワクチン
RU2802072C2 (ru) Вакцина для защиты от streptococcus suis
US20220098558A1 (en) Novel pasteurella multocida strains and vaccines having hyac and nanp deletions
CA3197074A1 (fr) Virus de la diarrhee epidemique porcine attenuee
MXPA06010631A (en) Method of vaccination against testicular bvdv infection

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19746344

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19746344

Country of ref document: EP

Kind code of ref document: A1