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Title:  Rotavirus subunit vaccine

United States Patent:  6,589,529

Issued:  July 8, 2003

Inventors:  Choi; Anthony (Park Hills, KY); Ward; Richard L. (Cincinnati, OH)

Assignee:  Children's Hospital Medical Center (Cincinnati, OH)

Appl. No.:  429801

Filed:  October 29, 1999

Abstract

The present invention is directed to the generation and use of recombinant rotavirus fusion proteins as immunogens to produce a protective immune response from immunized individuals. In one embodiment, the present invention contemplates a recombinant rotavirus fusion protein vaccine composition comprising a rotavirus subunit protein or immunogenic fragment thereof, and an adjuvant in combination with the recombinant rotavirus subunit fusion protein. In one aspect of this embodiment, the recombinant rotavirus fusion protein comprises a rotavirus subunit protein and a fusion partner protein in genetic association with the rotavirus subunit protein, wherein the fusion partner protein does not interfere with expression and immunogenicity of the rotavirus subunit protein, the fusion partner protein prevents complex formation by the rotavirus subunit protein, and the fusion partner protein facilitates purification of the recombinant rotavirus fusion protein. In another aspect of this embodiment, the rotavirus subunit protein is selected from the group consisting of VP1 VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 or NSP5. In yet another aspect of this embodiment, the rotavirus subunit protein is VP6.

SUMMARY OF THE INVENTION

The invention disclosed herein relates to compositions comprising various rotaviral proteins and methods of using these compositions to provide protection against rotaviral disease. One embodiment of the invention is a composition comprising a rotavirus VP6 protein or a fragment thereof, and an adjuvant in a pharmaceutical carrier, wherein said adjuvant is effective in generating a disease-reducing response to said VP6 protein.

Another embodiment of the invention encompasses a recombinant rotavirus fusion protein composition, comprising: a rotavirus subunit fusion protein or fragment thereof, a fusion protein partner in genetic association with said recombinant rotavirus subunit protein or fragment thereof, and an adjuvant in a pharmaceutical carrier, wherein said adjuvant is effective in stimulating a disease-reducing immunogenic response to said rotavirus fusion protein.

A full-length DNA copy of a gene encoding a recombinant rotavirus fusion protein, wherein said gene encoding said recombinant rotavirus protein comprising a rotavirus subunit protein or an immunogenic fragment thereof, and a fusion partner protein is contemplated in another embodiment of the invention disclosed herein.

A host cell comprising a DNA clone encoding recombinant rotavirus proteins is contemplated in another embodiment of the disclosed invention.

The invention disclosed also contemplates a computer readable medium having recorded thereon peptide sequences of rotavirus proteins selected from the group consisting of SEQ ID NOs:15-25.

A number of methods are disclosed as part of the invention. For example, one embodiment discloses a method of generating an immune response in a subject in need of rotavirus immunity comprising the steps of administering an immunogenic composition of comprising a rotavirus protein or fragment thereof, and an adjuvant in a pharmaceutically acceptable form to said subject and generating a disease-reducing immunogenic response in said subject.

Another embodiment of the invention is a method of immunizing a subject in need of rotavirus immunity comprising the steps of: immunizing with a rotavirus vaccine; and subsequently immunizing said subject with a recombinant rotavirus vaccine composition, whereby immunological protection of said individual against rotavirus infection is increased.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention relates to the generation of rotavirus subunit proteins for use in vaccines and methods of providing protective immunity to vertebrates, including humans, against rotavirus infection or disease. As one embodiment, the protective immunity generated by vaccines containing the recombinant rotavirus proteins of the present invention is a dominantly cell-mediated immune response. This immune response may interfere with the infectivity or activity of the rotavirus, or it may limit the spread or reproduction of the virus. The immune response resulting from vaccination with a vaccine containing the proteins of the present invention provides protection against subsequent challenge by a homologous or heterologous rotavirus.

The vaccines of the present invention are composed of a native recombinant rotavirus protein or immunogenic fragment(s) thereof, a rotavirus fusion protein, or immunogenic fragment(s) thereof, an adjuvant, and a pharmaceutically acceptable carrier. According to one embodiment of the present invention, a composition comprising a rotavirus protein or an immunogenic portion thereof is genetically associated with a fusion protein partner, and an adjuvant such as the A1 subunit, the B subunit of cholera toxin or E. coli heat-labile toxin present in a pharmaceutically acceptable carrier. This composition is administered to an individual in whom an immune response directed against the rotavirus subunit protein is sought and protection against rotavirus infection and disease is desired.

The rotavirus native recombinant, or fusion proteins of the present invention may be composed of any rotavirus protein product or immunogenic fragment thereof. The rotavirus protein may be chosen from any of the structural or non-structural viral proteins encoded by the rotavirus genome. For example, in one embodiment, the rotavirus protein or immunogenic fragment thereof may be chosen by selecting from the group of rotavirus genome segments consisting of segment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, that encode rotavirus protein sequences. In another embodiment, the rotavirus protein or immunogenic fragment thereof may be selected from the group of rotavirus structural proteins consisting of VP1, VP2, VP3, VP4, VP6 and VP7. In another embodiment, the rotavirus protein may be selected from the group of rotavirus non-structural proteins consisting of NSP1, NSP2, NSP3, NSP4 and NSP5. In yet another embodiment, the rotavirus protein used in the fusion protein construct is VP6. In still another embodiment, the use of an immunogenic fragment of VP6 may be used in the present invention.

The rotavirus recombinant native or fusion proteins of the present invention may be used in a vaccine composition at a concentration effective to elicit an immune response from an immunized subject. The concentration of rotavirus proteins of the present invention may range from about 0.01 .mu.g/ml to 1 mg/ml. In another embodiment, the concentration of rotavirus proteins used in a vaccine composition may range from about 0.1 .mu.g/ml to 100 .mu.g/ml. In yet another embodiment, the concentration of rotavirus proteins used in a vaccine composition may range from about 1.0 .mu.g/ml to 10 .mu.g/ml. In still another embodiment, the concentration of rotavirus proteins used in a vaccine composition may be about 8.8 .mu.g/ml. These ranges are provided for the sake of guidance in practicing the present invention. It should be noted that other effective concentrations of recombinant rotavirus proteins may be determined by one of ordinary skill in the art using experimental techniques well known in that art.

The rotavirus fusion proteins contemplated by the present invention are composed of a suitable fusion protein partner in genetic association with a rotavirus protein or immunogenic fragment thereof. The term in genetic association refers to a contiguous sequence of amino acids produced from a mRNA produced from a gene containing codons for the amino acids of the rotavirus protein and the fusion protein partner. A suitable fusion protein partner consists of a protein that will either enhance or at least not diminish the recombinant expression of the rotavirus fusion protein product when the two are in genetic association. Further, a suitable fusion protein partner may actively prevent the assembly of the rotavirus fusion proteins into multimeric forms after the rotavirus fusion protein has been expressed. For example, the fusion protein partner should prevent the formation of dimers, trimers or virus-like structures that might spontaneously form if the rotavirus protein were recombinantly expressed in the absence of the fusion protein partner. Still further, a suitable fusion partner will facilitate the purification of the chimeric rotavirus fusion protein. A representative list of suitable fusion protein partners includes maltose binding protein, poly-histidine segments capable of binding metal ions, inteine, antigens to which antibodies bind, S-Tag, glutathione-S-transferase, thioredoxin, .beta.-galactosidase, nonapeptide epitope tag from influenza hemagglutinin, a 11-amino acid epitope tag from vesicular stomatitis virus, a 12-amino acid epitope from the heavy chain of human Protein C, green fluorescent protein, cholera holo toxin or its B subunit, E. coli heat-labile holotoxin or its B subunit, CTA1-DD, streptavidin and dihydrofolate reductase.

The invention is also directed toward producing rotavirus proteins for use in vaccines directed to protect immunized individuals from rotavirus infection and/or disease. Accordingly, the invention contemplates the use of an adjuvant, such as an immunogenic protein, effective to induce desirable immune responses from an immunized animal. Such a protein mast possess those biochemical characteristics required to facilitate the induction of a protective immune response from immunized vertebrates while simultaneously avoiding toxic effects to the immunized animal.

In one embodiment of the present invention, rotavirus recombinant native or fusion proteins are mixed with an adjuvant such as a bacterial toxin. The bacterial toxin may be a cholera toxin. Alternatively, the rotavirus fusion protein may be mixed with the B subunit of cholera toxin (CTB). In another embodiment, an E. coli toxin may be mixed with the rotavirus fusion protein. For example, the rotavirus fusion protein may be mixed with E. coli heat-labile toxin (LT). The rotavirus fusion proteins of the present invention may be mixed with the B subunit of E. coli heat-labile toxin (LTB) to form a vaccine composition. Other adjuvants such as cholera toxin, labile toxin, tetanus toxin or toxoid, poly[di(carboxylatophenoxy)phosphazene] (PCPP), saponins Quil A, QS-7, and QS-21, RIBI (HAMILTON, Mont.), monophosphoryl lipid A, immunostimulating complexes (ISCOM), Syntax, Titer Max, M59, CpG, dsRNA, and CTA1-DD (the cholera toxin A1 subunit (CTA1) fused to a dimer of the Ig-binding D-region of Staphylococcus aureus protein A (DD)), are also contemplated.

The adjuvants discussed above may be used in a vaccine composition at a concentration effective to assist in the eliciting of an immune response against the recombinant rotavirus fusion proteins of the present invention from an immunized subject. The concentration of adjuvant included in the vaccine compositions of the present invention may range from about 0.01 .mu.g/ml to 1 mg/ml. In another embodiment, the concentration of adjuvant used in a vaccine composition may range from about 0.1 .mu.g/ml to 100 .mu.g/ml. In yet another embodiment, the concentration of adjuvant used in a vaccine composition may range from about 1.0 .mu.g/ml to 100 .mu.g/ml. In still another embodiment, the concentration of adjuvant used in a vaccine composition may be about 10.0 .mu.g/ml. These ranges are provided for the sake of guidance in practicing the present invention. It should be noted that other effective concentrations of adjuvants may be determined by one of ordinary skill in the art using experimental techniques well known in that art.

The invention also contemplates immunization with a rotavirus fusion protein, a recombinant native protein, or a fragment or fusion fragment, and a suitable adjuvant contained in a pharmaceutically acceptable composition. Such a composition should be sterile, isotonic, and provide a non-destabilizing environment for the rotavirus fusion protein and the adjuvant. Examples of this are buffers, tissue culture media, various transport media and solutions containing proteins (such as BSA), sugars (sucrose) or polysaccharides.

The vaccine compositions of the invention contain conventional pharmaceutical carriers. Suitable carriers are well known to those of skill in the art. These vaccine compositions may be prepared in liquid unit dose forms. Other optional components, e.g., stabilizers, buffers, preservatives, excipients and the like may be readily selected by one of skill in the art. However, the compositions may be lyophilized and reconstituted by the individual administering the vaccine prior to administration of the dose. Alternatively, the vaccine compositions may be prepared in any manner appropriate for the chosen mode of administration, e.g., intranasal administration, oral administration, etc. The preparation of a pharmaceutically acceptable vaccine, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.

The dosage regimen involved in a method for vaccination, including the timing, number and amounts of booster vaccines, will be determined considering various hosts and environmental factors, e.g., the age of the patient, time of administration and the geographical location and environment.

Also included in the present invention are methods of vaccinating humans against rotavirus infection and disease with the novel rotaviral proteins and vaccine compositions described above. The vaccine compositions, comprising a full-length rotavirus protein, a rotavirus fusion protein, a recombinant native protein or fragments and fusion fragments, mixtures of the above, and an adjuvant described herein may be administered by a variety of routes contemplated by the present invention. Such routes include intranasal, oral, rectal, vaginal, intramuscular, intradermal and subcutaneous administration.

Vaccine compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions, the protein vaccine, and an adjuvant as described herein. The composition may be in the form of a liquid, a slurry, or a sterile solid which can be dissolved in a sterile injectable medium before use. The parenteral administration is preferably intramuscular. Intramuscular inoculation involves injection via a syringe into the muscle. This injection can be via a syringe or comparable means. The vaccine composition may contain a pharmaceutically acceptable carrier. Alternatively, the present vaccine compositions may be administered via a mucosal route, in a suitable dose, and in a liquid form. For oral administration, the vaccine composition can be administered in liquid, or solid form with a suitable carrier.

Doses of the vaccine compositions may be administered based on the relationship between the concentration of the rotavirus fusion protein contained in the vaccine composition and that concentration of fusion protein required to elicit an immune response from an immunized host. The calculation of appropriate doses to elicit a protective immune response using the rotavirus fusion protein vaccine compositions of the present invention are well known to those of skill in the art.

A variety of immunization methods are contemplated by the invention to maximize the efficacy of the rotavirus protein vaccine compositions described herein. In one embodiment, females of offspring-bearing age are immunized with the vaccines of the invention. In this embodiment, immunized females develop a protective immune response directed against rotavirus infection or disease and then passively communicate this protection to an offspring by nursing. In another embodiment, newborns are immunized with the vaccine compositions of the invention and shortly thereafter the nursing mother is immunized with the same vaccine. This two tiered approach to vaccination provides the newborn with immediate exposure to viral epitopes that may themselves be protecting. Nevertheless, the passive immunity supplied by the mother would augment the protection enjoyed by the offspring. This method would therefore provide the offspring with both active and passive protection against rotavirus infection of disease.

In still another embodiment, an individual is immunized with the vaccine composition of the invention subsequent to immunization with a multivalent vaccine. The immunization of a subject with two different vaccines may synergistically act to increase the protection an immunized individual would enjoy over that obtained with only one vaccine formulation. One draw back of immunization with a multivalent live virus vaccine formulation is that booster immunizations with the same vaccine are usually not effective. In this embodiment of the invention, the vaccine compositions serve as such a booster to increase the protection of the immunized individual against rotaviral infection or disease.

Claim 1 of 7 Claims

What is claimed is:

1. A composition comprising a rotavirus VP6 protein or a COOH-terminal fragment thereof, and an adjuvant in a pharmaceutical carrier, wherein said adjuvant is effective in combination with said VP6 protein or COOH-terminal fragment thereof to generate a disease-reducing response to rotavirus infection in a mammal, wherein said VP6 protein or COOH-terminal fragment thereof is not assembled into a viral particle.



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