MAKING AND USING

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the earlier filing dates of U.S. Provisional Patent Application Nos. 62/701,471 and 62/701,476, both filed on July 20, 2018, both of which are incorporated herein by reference in their entirety.

FIELD

The present invention relates to rotavirus vaccine compositions and combinations and methods for making and using the compositions and combinations.

BACKGROUND

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.

Typically, 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. Of the other groups, 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%.

SUMMARY

Disclosed herein are embodiments of a method for propagating a rotavirus in cell culture. Also disclosed is a composition made by the method. The method comprises contacting buffalo green monkey (BGM) cells with a rotavirus in a growth media. The 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.

Additionally, or alternatively, 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.

In any embodiments, 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. Additionally, or alternatively, the method may comprise adding an adjuvant. 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. And in some embodiments, 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. Exemplary 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.

In a particular embodiment, 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.

In another particular embodiment, 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; freezing and thawing the second portion of BGM cells to release rota vims antigens; and forming a live, modified or attenuated vaccine composition comprising the rotavirus antigens. Forming the live, modified or attenuated vaccine composition may comprise adding an adjuvant.

In any embodiments of the method, the rotavirus may be a group A, B, C, E or H rotavirus. In other embodiments, the rotavirus is a porcine rotavirus, and may be a group A, B, or C porcine rotavirus. In certain embodiments, the rotavirus is a porcine C rotavirus. In alternative embodiments, the rotavirus is a bovine rotavirus, and may be a bovine group A rotavirus. In other embodiments, the rotavirus is a human rotavirus, and may be a human group A rotavirus.

Also disclosed is a composition 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. In some embodiments, 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. In certain embodiments, 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. In particular embodiments, 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

embodiments, 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. In some embodiments, 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. And in certain embodiments, 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.

Additionally or alternatively, the composition may comprise antigens from one or more non-rotavirus diseases, such as a porcine, bovine, or human disease, or a combination thereof.

Also disclosed are embodiments of 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. And the animal may be a mammal, such as a human, porcine or bovine. In some embodiments, the mammal is a non human mammal.

The foregoing and other objects, features, and advantages of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

SEQUENCE LISTING

The 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. The 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

chicken/05V0049/DEU/2005 Strain: 05V0049.

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

chicken/03V0568/DEU/2003.

SEQ ID NOS: 166-176 are nucleotide sequences of Rotavirus G

chicken/03 V0567/DEU/2003.

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.

DEPOSIT OF BIOLOGICAL MATERIAL

The following biological material has been deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia, 20110, (ATCC) and given the following deposit designation number: PTA-125126

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. 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. DETAILED DESCRIPTION

I. Terms

The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein,“comprising” means“including” and the singular forms“a” or“an” or “the” include plural references unless the context clearly dictates otherwise. The term“or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term“about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word“about” is recited.

“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 51 or IS 720; water-in-oil-in-water based adjuvants, such as ISA 201 or ISA 206; polymer-based adjuvants, such as Carbigen™ or

Polygen™; Carbomer-based adjuvants, such as those containing 934P or 971P; Freunds complete adjuvant; Freunds incomplete adjuvant; microfluidized emulsions, such as MF59; polylactide glycolide (PLGA); or a combination thereof. Additionally, 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.

“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. In some cases, 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. Alternatively, 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.

“Buffalo Green Monkey” (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). In certain embodiments, 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. In some embodiments, the effective time periods of all components administered overlap with each other. In an exemplary embodiment of a combination comprising four components, 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. In another exemplary embodiment of a combination comprising four components, 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. In some embodiments, 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.

“Cytopathic effect” (CPE) refers to the appearance of damage to tissue culture cells in rotavirus infected cells when compared with control cells that do not contain the virus.

“Administer,”“administering,”“administration,” and the like refer 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

Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa., which are incorporated herein by reference.

Certain methods of administration deliver the immunogenic composition to mucosal membranes. These include, but are not limited to, intranasal, oral, intravaginal, and rectal. In some embodiments, 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.

The compositions provided herein, alone or in combination with other suitable components, 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.

The disclosed compositions can be formulated for parenteral administration, such as, for example, by intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, and subcutaneous routes. Examples of 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. In addition, 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. In the practice of this disclosure, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. In an independent embodiment, 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.

As used herein, 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. In some embodiments, 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. Thus, in some embodiments, the compositions described herein and the other agent(s) are administered in a single composition. In some embodiments, 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. In some embodiments,“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.

“Immune 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· As used herein, 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.

II. Overview

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.

But to date, no inactivated Group A rotavirus vaccines are commercially available.

However, propagation of group C rotaviruses in cell culture, using the techniques used to grow group A rotaviruses, have been unsuccessful. Using primary cells that are made directly from tissues is not generally accepted for propagation of the virus to prepare vaccine compositions because primary cells are prone to contamination, which may not be detectable before being used to manufacture vaccines. Additionally, primary cells are not generally stable over numerous passages. A stable cell line is obtained when the cells demonstrate a stable chromosome number over at least 20 passages. The disclosed BGM cell line, MH18, has demonstrated a stable karyology over the 20 passages from the master cell.

Some replication of porcine group C rotavirus has been demonstrated in two types of cell cultures: Primary Pig Kidney (PK) and embryonic Rhesus Monkey Kidney (Ma-l04). However, none of the cell culture passes have been reported to contain virus titers higher than 5 x 10 ⁶ FFU/ml (Fluorescent Focus Units). Complete destruction of the cells (100% CPE) was not observed.

Additionally, Saif et al. (J. Clin. Microbiol. 1988, Vol. 26(7) pp 1277-1282) disclosed that passaging the rotavirus in MA-104 cells required that the rotavirus be passaged 9 times in PK cells first. And subsequent inoculation of pigs by cell-culture passaged rotavirus did not prevent diarrhea, lesions, and patterns of immunofluorescent staining in the intestines of the pigs that were similar to those observed in pigs that were challenged with virulent virus. Isolation of porcine group C rotavirus in MA 104 cell cultures directly from intestinal contents of infected pigs was unsuccessful.

Additionally, 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. However, 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. Additionally, 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. However, in general, group A rotaviruses grow well in many cell lines, and the BGM cells are homologous cells to the simian rotavirus species. In this context, homologous refers to cells that are from the same general animal species. In this particular case, 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.

III. BGM Cell Line

Buffalo Green Monkey kidney cells, referred to herein as Buffalo Green Monkey cells or 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

Authenticated Cell Cultures (ECACC).

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.

However, disclosed herein is the surprising result that 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. Also disclosed herein are embodiments of 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. To the inventors’ knowledge, 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).

To date, no 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. However, the inventors have surprisingly discovered that 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.

IV. Method for Making an Immunogenic Composition

Disclosed herein are embodiments of a method for making a composition comprising rotavirus antigens. The rotavirus may be any rotavirus, such as a rotavirus from rotavirus group A, B, C, D, E, F, G, or H. In some embodiments, the group A rotavirus is a non-simian group A rotavirus. In certain embodiments, 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.

Additionally, or alternatively, 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. In particular embodiments, 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. In some embodiments, 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%,

99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to any one of SEQ ID NOS: 1-220. In some embodiments, 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%,

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: 1-44. In other embodiments, 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. In particular embodiments, 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%,

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: 89-132.

Typically, 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).

Additionally, or alternatively, 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. Without being bound to a particular theory, contacting the cells with a cell disrupting agent and/or a proteolytic enzyme may improve the growth of the rotavirus on the BGM cells. In some embodiments, 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. In other embodiments, no enzyme is used. And in particular embodiments, no enzyme is used for group B rotaviruses.

The buffer and enzyme, if used, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, once observable CPE has developed, 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.

In other embodiments, such as embodiments where CPE is not observed, 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. In some examples, 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. Additionally, or alternatively, 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.

In other embodiments, such as embodiments where the rotavims is continuously passaged through the cells, and/or where CPE is not observed, 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.

Additionally, or alternatively, after at least first passaging on BGM cells, further passages may be made on BGM cells and/or other cell lines. Such passages are 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.

In some embodiments, 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. By isolating and then propagating the rotaviruses on the BGM cell line and then continuing passage on this cell line and/or passaging on one or more non-BGM cell lines, the virus can be adapted and grown to produce an antigen mass suitable for making either an attenuated or live, modified vaccine, or an inactivated or killed vaccine. 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. When making an inactivated or killed vaccine, 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).

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). 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. For example, 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. After the inactivation process is complete, the BEI may be neutralized with thiosulfate, such as sodium or potassium thiosulfate. Upon neutralization, 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. Upon neutralization, the formalin, or formaldehyde, together with the bisulfite may form a neutralization product, such as a formaldehyde-bisulfite adduct, which may have a structure H0-S(=0) ₂ 0 .

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.

Without being bound to a particular theory, 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. In some embodiments, the composition has no free or unreacted inactivating agent present after neutralization.

Additionally, 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. There are many 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. More specifically, 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) and Emulsigen ^® -P (containing with a proprietary immunostimulant), Emulsimune ^® , Carbigen™, Poly gen™, 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, monophosphoryl lipid A (MPLA), immunostimulatory complexes (ISCOMS), 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.

Usable concentrations would be expected to be acceptable for injection of animals including swine, bovines, equines, canines, felines as well as humans wherein minimal tissue damage is observed or felt. In some embodiments, 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).

Alternatively, 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. Typically, when administered to an animal, the limited replication in the animal will not cause infection or will cause only mild symptoms, but will produce a substantially protective immune response.

V. Vaccine Composition

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. For disclosed vaccine compositions, 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. For example, DMSO may be used in the growth media for the BGM cells and the resulting vaccine composition may comprise DMSO. In some embodiments, DMSO is not used for growing rotavirus group A in BGM cells. However, a vaccine produced for human use typically will be substantially free from cells or cell fragments and also may be free from DMSO.

For an inactivated or killed vaccine, 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. And an inactivated or killed vaccine typically will comprise an adjuvant.

For an attenuated or modified live vaccine, the composition may comprise rotavirus antigens and/or viral particles, an adjuvant, and BGM and/or non-BGM cells and/or cell fragments. Typically, an attenuated or live, modified vaccine will not comprise a detergent or inactivating agent and/or neutralizing agent. However, 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.

Additionally, 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. Such 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 pharmaceutically acceptable carriers known in the art.

In certain embodiments, a vaccine composition, such as a killed or inactivated vaccine composition, is a formalin- or formaldehyde-free vaccine composition. In some embodiments, a formalin- or formaldehyde-free composition is inactivated using an inactivating agent that does not comprise formalin or formaldehyde. In other embodiments, 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. In a formalin- or formaldehyde-free composition, 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.

In some embodiments, 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. A person of ordinary skill in the art understands that certain inactivating agents, such as BPL, may not require a neutralizing agent. In certain embodiments, a composition comprising rotavirus group A antigens may not comprise DMSO.

VI. Co-administration

In some embodiments, embodiments of the disclosed rotavirus composition may be administered as part of a combination of therapeutic agents. In addition to disclosed vaccines, 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. In some embodiments, 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. In other embodiments of the combination, 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. When administered sequentially, 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.

In any embodiments, 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. For example, 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. In some embodiments, 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. In other embodiments, 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.

Additionally, or alternatively, 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. For example, for swine, 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. difficile, Actinobacillus suis, Haemophilus parasuis, Streptococcus suis, Escherichiae coli spp, Salmonella spp, Pasteurella multocida types A and/or D, Bordetella bronchiseptica, Erysipelothrix rhusiopathiae,

Arcanobacterium pyogenes, porcine reproductive and respiratory syndrome vims (PRRSv), Swine influenza vims (Influenza A Virus of Swine or IAV-S), or any combination thereof. For bovines, 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.

icterohaemorrhagiae, Neospora caninum, and/or Trichomonas.

And for humans, 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.

VII. Method for Using the Vaccine

Disclosed embodiments of the composition 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. In particular embodiments, 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. In particular embodiments, 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 person of ordinary skill in the art will appreciate that the absolute age of a young subject will depend on the species. For example, for humans, 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. For swine, 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. And for a bovine, the composition may be administered to a calf, such as from birth to 6 weeks or to older calves that may be exposed. Additionally, the composition may be administered to pregnant and/or nursing subjects or to subjects who will become pregnant. For example, for swine, 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. In some embodiments, the 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. However, in some embodiments, 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.

In some embodiments, a subject is administered with a composition prepared from a rotavirus that was obtained from the same species. For example, a swine is administered with a composition comprising antigens obtained from a swine rotavirus infection. However, in other embodiments, 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.

Additionally, 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. In some embodiments, 2 doses are administered. For example, for pregnant sows or gilts, 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. And for humans 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

Example 1

This study evaluated the kinetics of growth of a group A swine rotavirus in the MH18 cell line. Pigs with watery diarrhea typical of rotavirus were sampled. PCR confirmed that a sample contained only Group A rotavirus, no rotavirus Group C or B. 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 mg/L. Trypsin was then added to the purified sample at a concentration of 10 pg/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 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.

Two days after inoculation, supernatants from the MH18 cells were removed from some sample and control wells so that they could be evaluated by immunofluorescent staining. The cells in the wells were fixed with cold 80% acetone for 20 minutes after which the plate was air dried. The primary monoclonal antibody (Kerafast) was diluted 1:500 in PBS buffer and added into the cell culture wells at 200 pL /well. The plate was incubated overnight at room temperature. The plate was then washed twice with PBS buffer to remove the monoclonal antibody after which a goat anti-mouse 2 ^nd antibody (MilliporeSigma) containing FITC conjugate was added at a concentration of 200 pL/well. The plate was incubated for 2 hours at 37 °C. It was then washed twice with PBS buffer again. The plate was observed under a fluorescence microscope using the excitation wavelength of 495 nm. 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.

Example 2

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).

Cells were grown in T25 flasks containing Dulbecco's Modified Eagle Medium (DMEM) high modified media (HyClone #SH30285.09 containing 4500 mg/L of glucose and 110 mg/L of sodium pyruvate) supplemented with L-glutamine (VWR #02-0109-1000) at a concentration of 10 mL/L, gentamicin at a concentration of 60 pg/L, HEPES buffer (1M solution Fisher #BP299-l00) used at a concentration of 10 mL/L, and calf serum (HyClone #SH30087.04) at a concentration of 12%. Three flasks were prepared for each cell line; one for each isolate and one control flask. The control flask contained all of the same media ingredients except for the sample inoculum.

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. At the end of 7 days, all flasks were frozen. The contents of each flask were thawed and an aliquot of each sample was inoculated onto separate sets of flasks for each cell line with the media as described previously. This was defined as the Pl passage. Two additional passages were made on each cell line using the same procedure as defined for Pl except that for P3, the trypsin was reduced to 4 units/mL.

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.

Table 1. Isolation and propagation of group A swine rotavirus on various cell lines

Example 3

This study was a repeat of the study described in Example 2, except that PCR showed that the intestinal samples contained only group C swine rotavirus. The samples of intestinal contents gave a RT PCR reading of 17 cycles indicating that they had a high concentration of vims. These 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 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.

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. 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. As in Example 2, 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 conducted.

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.

Table 2. Isolation and propagation of group C swine rotavirus on various cell lines

Example 4

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. 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. The media used for rotavirus isolation from the samples does not contain trypsin or a similar proteolytic enzyme. As indicated above, 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. At that time, 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.

It is expected that 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.

Example 5

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 (BEI), beta

propiolactone (BPL) and/or formalin. Typically, 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. In order to prepare the vaccine composition, proof of inactivation typically is demonstrated by at least three backpasses in BGM cells prepared to be susceptible to the vims. Then the inactivated virus is adjuvanted with one of the adjuvants known to the art. Preferred adjuvants include Emulsigen ^® -D or Emulsigen ^® -DL90. Uses for the vaccine composition include vaccinating pregnant sows or gilts prior to farrowing, or neonatal or young pigs to protect them from infection by group C rotaviruses.

Example 6

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.

After confirmation that the sample was rotavirus Group A, it was pretreated with 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. In order to scale-up for production volumes, additional passages were made until the titer of the seed reached at least lxlO ⁵ TCIDso/mL by the same process as described for production of the 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.

Example 7

Part of the final adjuvanted vaccine from Example 6 was combined in equal parts with a Clostridium perfringens vaccine to produce a combination vaccine. 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.

Example 8

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. The cells were then washed twice with DMEM that contained no serum. Cells were fixed immediately using chilled Karnovsky’s fixative (4 mL/well). The plate was wrapped and stored at 4°C until further treatment and analysis. FIG. 5 is an electron micrograph showing that rotavirus Group A entered the cells (white arrows).

Example 9

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. The plate was wrapped and stored at 4 °C until it was prepared for electron microscopy. 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).

Example 10

A swine rotavirus Group C 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 rotator and incubated for 1 hour at 37 °C. The cells were then washed twice with DMEM media without addition of any serum. Cells were fixed immediately using 4 mL/well of chilled Kamovsky’s fixative. The plate was then wrapped and stored at 4 °C until further treatment and analysis. FIG. 7 is an electron micrograph showing that rotavirus Group C is entering the cells (white arrows).

Example 11

A swine rotavirus Group C 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 with gentle rotation. The cells were then supplemented with 3.5 mL of RMM and incubated at 37 °C for three days. No clear CPE was observed but the cells were fixed anyway using 4 ml ./well of chilled Kamovshy’s fixative. The plate was wrapped and stored at 4 °C until further treatment and analysis. FIG. 8 is an electron micrograph showing that rotavirus Group C is identified in the cytoplasm (white arrow).

Example 12

BGM cells are grown and infected by group C rotavirus as described in Example 5. In this experiment, as soon as CPE is observed, 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). Optionally 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. For example, 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.

However, for formalin exemplary concentrations are typically between 0.01% and 0.1% (v/v). After the addition of the formalin, 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.

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.

After inactivation, 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. Example 13

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. For pregnant sows or gilts, two doses of vaccine are preferred, one administered at 5 weeks prior to farrowing and the other at 2 weeks prior. In sows and gilts, 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.

In older pigs, 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).

Example 14

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 Carbigen™ 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 Carbigen™, is added at 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.

Example 15

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.

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. 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. 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. 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.

The seed from the previous passage is transferred into roller bottles for growth of the final vaccine product. 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. After thawing, 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. Prior to use for vaccination, 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.

Because this producer has such a problem with both Rotavirus group A and with SIV, a comparison will be made between the piglets farrowed to the vaccinated sows with those of non- vaccinated sows. It is expected that the piglets from vaccinated sows will perform better with fewer deaths, less morbidity and better weight gains.

Example 16

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. 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.

Example 17

This study evaluated the kinetics of growth of a group C swine rotavims in the MH18 cell line. Pigs with watery diarrhea typical of rotavims were sampled. PCR confirmed that a sample contained only Group C rotavims, no rotavims Group A or B. 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.

Two days after inoculation, supernatants from the MH18 cells were removed from some sample and control wells so that they could be evaluated by immunofluorescent staining. The cells in the wells were fixed with cold 80% acetone for 20 minutes after which the plate was air dried. The primary monoclonal antibody (Kerafast) was diluted 1:500 in PBS buffer and added into the cell culture wells at 200 pL /well. The plate was incubated overnight at room temperature. The plate was then washed twice with PBS buffer to remove the monoclonal antibody after which a goat anti-mouse 2 ^nd antibody (MilliporeSigma) containing FITC conjugate was added at a concentration of 200 pL/well. The plate was incubated for 2 hours at 37 °C. It was then washed twice with PBS buffer again. The plate was observed under a fluorescence microscope using the excitation wavelength of 495 nm. 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.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.