Abstract:
A method of producing an effective immune response in an animal against a first rotavirus, by (1) identifying an animal susceptible to or having a first rotavirus infection; and (2) administering to the animal an isolated VP6 polypeptide of a second rotavirus sufficient to produce an effective immune response against the first rotavirus, the second rotavirus capable of infecting a different species than the first rotavirus.

Description:
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made with Government support under National Institutes of Health grant RO1AI39637. 
    
    
     BACKGROUND OF THE INVENTION 
     The invention relates to rotavirus vaccines. 
     Rotavirus infections are ubiquitous throughout mammalian and avian species. The viruses typically appear to be species-specific in the wild. Infection occurs after ingestion of viral particles and is restricted to the mature absorptive epithelial cells on the villi of the small intestine. Multiplication of rotaviruses within these cells results in lysis, and eventual loss of normal villous structure. Copious acute watery diarrhea occurs as a result of intestinal damage and replacement of absorptive cells by secreting cells from the villous crypts in the intestine. 
     Rotaviruses have a multi-shelled capsid and a segmented RNA genome. Among the rotavirus structural proteins are VP7, the most abundant outer capsid protein in the virus particle, and VP6, the major component of the inner capsid. 
     SUMMARY OF THE INVENTION 
     The invention is based on the discovery that an isolated VP6 polypeptide from a rotavirus which naturally infects one species (such as a cow) can produce an effective immune response against a rotavirus which naturally infects another species (such as a mouse or human), so that VP6 is an effective cross-protective rotavirus vaccine. An isolated VP6 polypeptide is a VP6 polypeptide that is in an environment other than its natural environment, e.g., by purification or simply as a result of enhancing the percentage of VP6 present (“spiking”) in naturally occurring mixtures such as viral or infected cell preparations. 
     In general, the invention features a method of producing an effective immune response in an animal (for example, cows, pigs, horses, and especially humans) against a first rotavirus by (1) identifying an animal susceptible to or having a first rotavirus infection; and (2) administering (e.g., orally) to the animal an isolated VP6 polypeptide of a second rotavirus sufficient to produce an effective immune response against the first rotavirus, the second rotavirus capable of infecting a different species than the first rotavirus. For example, a cow, pig, or horse rotavirus VP6 is administered to a human in order to elicit an effective immune response against a human rotavirus. Alternatively, a human, cow, or pig rotavirus VP6 is administered to a horse in order to elicit an effective immune response against a horse rotavirus. In addition, the VP6 polypeptide can cross-react with antibodies produced from the animal (e.g., a human) when infected with the first rotavirus (e.g., a human rotavirus). 
     In some embodiments of the above method, the animal is of a first species, and the second rotavirus is capable of infecting a second species different from the first species and incapable of infecting the first species. 
     An immune response against the first rotavirus is considered effective if viral loads or the symptoms of infection are reduced in the animal when the VP6 is administered to the animal as compared to when the VP6 is not administered. Preferably, such reductions are statistically significant, as shown in the Example section below. For example, an immune response that protects against viral infection is “effective.” 
     The isolated VP6 polypeptide of the second rotavirus can be administered to the animal via an expression vector (e.g., a DNA plasmid) comprising a sequence encoding the VP6 polypeptide of the second rotavirus. When the isolated VP6 is administered as a DNA plasmid, DNA delivery can be facilitated by encapsulating the DNA in a microparticle, e.g., one made of poly(lactide-co-glycolide) (PLG). 
     An expression vector encoding the isolated VP6 polypeptide is not limited to a DNA plasmid. For example, an expression vector can be a virus, such as a retrovirus. Whether in the form of a plasmid or virus, the expression vector can further contain a cytomegalovirus intron A transcriptional regulatory sequence and a cytomegalovirus enhacer and promoter sequence operably linked to the sequence encoding the VP6 polypeptide of the second rotavirus. 
     Unless otherwise defined, 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 invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 is a graph of days post-challenge versus challenge virus titer in mice immunized with PLG-encapsulated DNA vaccines. 
    
    
     DETAILED DESCRIPTION 
     The invention relates to the use of an isolated rotavirus VP6 polypeptide as a cross-protective rotavirus vaccine. 
     I. Production and Isolation of VP6 Polypeptides 
     A. Production 
     VP6 polypeptides can be isolated from any rotavirus that can be propagated and purified. Once the rotavirus is purified, the virus particles can be disrupted, and the VP6 isolated by one or more standard procedures, such as high performance liquid chromatography or differential salt precipitation. 
     Alternatively, the VP6 can be produced recombinantly. The VP6 open reading frame from a variety of rotavirus isolates are known. See, for example, the cloned VP6 polypeptides disclosed in U.S. Pat. No. 5,620,896. Other VP6 sequences include the following: 
     GenBank Accession No. X53667 (bovine) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:1) 
               
               
                   
               
             
          
           
               
                 MDVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIITMNGNE 
               
               
                   
               
               
                 FQTGGIGNLPIRNWNFDFGLLGTTLLNLDANYVETARNTIDYFVD 
               
               
                   
               
               
                 FVDNVCMDEMVRESQRNGIAPQSDSLRKLSGIKFKRINFDNSSEY 
               
               
                   
               
               
                 IENWNLQNRRQRTGFTFHKPNIFPYSASFTLNRSQPAHDNLMGTM 
               
               
                   
               
               
                 WLNAGSEIQVAGFDYSCAINAPANTQQFEHIVQLRRVLTTATITL 
               
               
                   
               
               
                 LPDAERFSFPRVINSADGATTWYFNPVILRPNNVEVEFLLNGQII 
               
               
                   
               
               
                 NTYQARFGTIIARNFDTIRLSFQLMRPPNMTPAVAALFPNAQPFE 
               
               
                   
               
               
                 HQATVGLTLRIESAVCESVLADASETMLANVTSVRQEYAIPVGPV 
               
               
                   
               
               
                 FPPGMNWTDLITNYSPSREDNLQRVFTVASIRSMLVK 
               
             
          
         
       
     
     GenBank Accession No. M88768 (bovine) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:2) 
               
               
                   
               
             
          
           
               
                 MDVLFSIAKTVSELKKRVVVGTIYTNVEDIIQQTNELIRTLNGST 
               
               
                   
               
               
                 FHTGGIGTQPQKDWVVQLPQLGTTLLNLDDNYVQSARGIIDYLAS 
               
               
                   
               
               
                 FIEAVCDDEMVREASRNGMQPQSPTLIALASSKFKTINFNNSSQS 
               
               
                   
               
               
                 IKNWSAQSRRENPVYEYKNPMVFEYRNSYILHRADQQFGNAMGLR 
               
               
                   
               
               
                 YYTTSNTCQIAAFDSTMAENAPNNTQRFIYHGRLKRPISNVLMKV 
               
               
                   
               
               
                 ERGAPNNNNPTILPDPTNQTTWLFNPVQVMNGTFTIEFYNNGQLV 
               
               
                   
               
               
                 DMVRNMGIATVRTFDSYRITIDMIRPAAMTQYVQQLFPVGGPYSH 
               
               
                   
               
               
                 QAAYMLTLSVLDATTESVLCDSHSVDYSIVANTRRDSAMPAGTVF 
               
               
                   
               
               
                 QPGFPWEQTLSNYTVAQEDNLERLLLVASVKRMVM 
               
             
          
         
       
     
     GenBank Accession No. K02086 (human) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:3) 
               
               
                   
               
             
          
           
               
                 MEVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIVTMNGND 
               
               
                   
               
               
                 FQTGGIGNLPVRNWTFDFGLLGTTLLNLDANYVENARTIIEYFID 
               
               
                   
               
               
                 FIDNVCMDEMARESQRNGVAPQSEALRKLAGIKFKRINFDNSSEY 
               
               
                   
               
               
                 IENWNLQNRRQRTGFVFHKPNIFPYSASFTLNRSQPMHDNLMGTM 
               
               
                   
               
               
                 WLNAGSEIQVAGFDYSCAINAPANIQQFEHIVQLRRALTTATITL 
               
               
                   
               
               
                 LPDAERFSFPRVINSADGATTWFFNPVILRPNNVEVEFLLNGQII 
               
               
                   
               
               
                 NTYQARFGTIIARNFDAIRLLFQLMRPPNMTPAVNALFPQAQPFQ 
               
               
                   
               
               
                 HHATVGLTLRIESAVCESVLADANETLLANVTAVRQEYAIPVGPV 
               
               
                   
               
               
                 FPPGMNWTELITNYSPSREDNLQRVFTVASIRSMLIK 
               
             
          
         
       
     
     GenBank Accession No. X59843 (human) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:4) 
               
               
                   
               
             
          
           
               
                 MDVLFSIAKTVSDLKKKVVVGTIYTNVEDVVQQTNELIRTLNGNI 
               
               
                   
               
               
                 FHTGGIGTQPQKEWNFQLPQLGTTLLNLDDNYVQSTRGIIDFLSS 
               
               
                   
               
               
                 FIEAVCDDEIVREASRNGMQPQSPALILLSSSKFKTINFNNSSQS 
               
               
                   
               
               
                 IKNWNAQSRRENPVYEYKNPMLFEYKNSYILQRANPQFGSVMGLR 
               
               
                   
               
               
                 YYTTSNTCQIAAFDSTLAENAPNNTQRFVYNGRLKRPISNVLMKI 
               
               
                   
               
               
                 EAGAPNISNPTILPDPNNQTTWLFNPVQLMNGTFTIEFYNNGQLI 
               
               
                   
               
               
                 DMVRNMGIVTVRTFDSYRITIDMIRPAAMTQYVQRIFPQGGPYHF 
               
               
                   
               
               
                 QATYMLTLSILDATTESVLCDSHSVEYSIVANVRRDSAMPAGTVF 
               
               
                   
               
               
                 QPGFPWEHTLSNYTVAQEDNLERLLLIASVKRMVM 
               
             
          
         
       
     
     GenBank Accession No. U82971 (porcine) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:5) 
               
               
                   
               
             
          
           
               
                 MEVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIVTMNGND 
               
               
                   
               
               
                 FQTGGIGNSPVRNWNFDFGLLGTTLLNLDANYVENARTTIEYFVD 
               
               
                   
               
               
                 FIDNVCMDEMTRESQRSGIAPQSEALRKQSGIKFKRINFDNSSDY 
               
               
                   
               
               
                 IENWNLQNRRQRTGFVFHKPNILPYSASFTLNRSQPAHDNLMGTM 
               
               
                   
               
               
                 WINAGSEIQVAGFDYSCAFNAPANIQQFEHVVPLRRALTTATITL 
               
               
                   
               
               
                 LPDAERFSFPRVINSADGTTTWYFNPVILRPSNVEVEFLLNGQII 
               
               
                   
               
               
                 NTYQARFGTIIARNFDTIRLSFQLVRPPNMTPAVANLFPQAPPFI 
               
               
                   
               
               
                 FHATVGLTLRIESAVCESVLADASETLLANVTSVRQEYAIPVGPV 
               
               
                   
               
               
                 FPPGMNWTELITNYSPSREDNLQRVFTVASIRSMLIK 
               
             
          
         
       
     
     GenBank Accession No. U65988 (murine) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:6) 
               
               
                   
               
             
          
           
               
                 MDVLYSISRTLKDARDKIVEGTLYSNVSDLIQQFNQMLVTMNGNE 
               
               
                   
               
               
                 FQTGGIGNLPLRNWNFDFGLLGTTLLNLDANYVESARTTIDYFVD 
               
               
                   
               
               
                 FIDNVCMDEMMRESQRNGIAPQSDALRKLSGVKFRRINFNNSSEY 
               
               
                   
               
               
                 IENWNLQNRRQRTGFTFHKPNIFPYSASFTLNRSQPQHDNLMGTM 
               
               
                   
               
               
                 WLNAGSEIQVAGFDYSCAINAPANIQQFEHIVQLRRVLTTATITL 
               
               
                   
               
               
                 LPDAERFSFPRVINSADGATTWYFNPVILRPNNVEVEFLLNGQVI 
               
               
                   
               
               
                 NTYQARFGTIVARNFDTIRLSFQLMRPPNMTPAVTALFPNAQPFE 
               
               
                   
               
               
                 HHATVGLTLRIDSAICESVLADASETMLANVTSVRQEYAIPVGPV 
               
               
                   
               
               
                 FPPGMNWTDLITNYSPSREDNLQRVFTVASIRSMLVK 
               
             
          
         
       
     
     GenBank Accession No. D16329 (avian) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:7) 
               
               
                   
               
             
          
           
               
                 MDVLYSLAKTLKDARAKIVEGTLYTNVADIVQQVNQVINSINGST 
               
               
                   
               
               
                 FQTGGIGNLPVRNWTFDFGTLGTTLLNLDANYVENARTTIDYFID 
               
               
                   
               
               
                 FVDSVCVDEIVRESQRNGIAPQSDSLRQLSNAKYKRINYDNESEY 
               
               
                   
               
               
                 IENWNLQNRRQRTGYLLHKPNILPYNNSFTLTRSQPAHDNVCGTI 
               
               
                   
               
               
                 WLNNGSEIEIAGFDSECALNAPGNIQEFEHVVPMRRVLNNATVSL 
               
               
                   
               
               
                 LPYAPRLTQRAVIPTADGLNTWLFNPIILRPNNVQVEFLLNGQVI 
               
               
                   
               
               
                 TNYQARYGTLAARNFDSIRISFQLVRPPNMTPGVAALFPQAAPFP 
               
               
                   
               
               
                 NHATVGLTLKIESASCESVLSDANEPYLSIVTGLRQEYAIPVGPV 
               
               
                   
               
               
                 FPAGMNWTELLNNYSASREDNLQRIFTAASIRSMVIK 
               
             
          
         
       
     
     GenBank Accession No. D00323 (equine) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:8) 
               
               
                   
               
             
          
           
               
                 MEVLYSISKTLKDARDKIVEGTLYSNVSDIIQQFNQIIVTMNGNE 
               
               
                   
               
               
                 FQTGGIGTLPIRNWTFDFGLLGTTLLNLDANYVETARTTIEYFID 
               
               
                   
               
               
                 FIDNVCMDEMTRESQRNGIAPQSDALRKLSGIKFKRINFDNSSEY 
               
               
                   
               
               
                 IENWNLQNRRQRTGFVFHKPNIFPYSASFTLNRSQPLHNDLMGTM 
               
               
                   
               
               
                 WLNAGSEIQVAGFDYSCAINAPANTQQFEHIVQLRRALTTATITI 
               
               
                   
               
               
                 LPDAERFSFPRVINSADGATTWFFNPVILRPNNVEVEFLLNGQII 
               
               
                   
               
               
                 NTYQARFGTIIARNFDTIRLSFQLMRPPNMTPAVNALFPQAQPFQ 
               
               
                   
               
               
                 HHATVGLTLRIDSAVCESVLADSNETMLAAVTAVRQEYAVPVGPV 
               
               
                   
               
               
                 FPPGMNWTELITNYSPSREDNLQRVFTVASIRSMLIK 
               
             
          
         
       
     
     GenBank Accession No. L11595 (lamb) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:9) 
               
               
                   
               
             
          
           
               
                 MDVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIVTMNGNE 
               
               
                   
               
               
                 FQTGGIGNLPIRNWNFDFGLLGTTLLNLDANYVETARNTIDYFVD 
               
               
                   
               
               
                 FVDNVCMDEMVRESQRNGIAPQSESLRKLSGIKFKRINFDNSSEY 
               
               
                   
               
               
                 IENWNLQNRRQRTGFTFHKPNIFPYSASFTLNRSQPAHDNLMGTM 
               
               
                   
               
               
                 WLNAGSEIQVAGFDYSCAINAPANTQQFEHIVQLRRVLTTATITL 
               
               
                   
               
               
                 LPDAERFSFPRVINSADGATTWYFNPVILRPNNVEVEFLLNGQII 
               
               
                   
               
               
                 NTYQARFGTIIARNFDTIRLSFQLMRPPNMTPAVAALFPNAQPFE 
               
               
                   
               
               
                 HHATVGLTLRIESAVCESVLADASETMLANVTSVRQEYAIPVGPV 
               
               
                   
               
               
                 FPPGMNWTDLITNYSPSREDNLQRVFTVASIRSMLIK 
               
             
          
         
       
     
     GenBank Accession No. L33365 (simian) 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:10) 
               
               
                   
               
             
          
           
               
                 NDVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIITMNGNE 
               
               
                   
               
               
                 FQTGGIGNLPIRNWNFNFGLLGTTLLNLDANYVETARNTIDYFVD 
               
               
                   
               
               
                 FVDNVCMDEMVRESQRNGIAPQSDSLRKLSAIKFKRINFDNSSEY 
               
               
                   
               
               
                 IENWNLQNRRQRTGFTFHKPNIFPYSASFTLNRSQPAHDNLMGTM 
               
               
                   
               
               
                 WLNAGSEIQVAGFDYSCAINAPANIQQFEHIVPLRRVLTTATITL 
               
               
                   
               
               
                 LPDAERFSFPRVINSADGATTWFFNPVILRPNNVEVEFLLNGQII 
               
               
                   
               
               
                 NTYQARFGTIVARNFDTIRLSFQLMRPPNMTPAVAVLFPNAQPFE 
               
               
                   
               
               
                 HHATVGLTLRIESAVCESVLADASETLLANVTSVRQEYAIPVGPV 
               
               
                   
               
               
                 FPPGMNWTDLITNYSPSREDNLQRVFTVASIRSMLIKMDVLYSLS 
               
               
                   
               
               
                 KTLKDARDKIVEGTLYSNVSDLIQQFNQMIITMNGNEFQTGGIGN 
               
               
                   
               
               
                 LPIRNWNFNFGLLGTTLLNLDANYVETARNTIDYFVDFVDNVCMD 
               
               
                   
               
               
                 EMVRESQRNGIAPQSDSLRKLSAIKFKRINFDNSSEYIENWNLQN 
               
               
                   
               
               
                 RRQRTGFTFHKPNIFPYSASFTLNRSQPAHDNLMGTMWLNAGSEI 
               
               
                   
               
               
                 QVAGFDYSCAINAPANIQQFEHIVPLRRVLTTATITLLPDAERFS 
               
               
                   
               
               
                 FPRVINSADGATTWFFNPVILRPNNVEVEFLLNGQIINTYQARFG 
               
               
                   
               
               
                 TIVARNFDTIRLSFQLMRPPNMTPAVAVLFPNAQPFEHHATVGLT 
               
               
                   
               
               
                 LRIESAVCESVLADASETLLANVTSVRQEYAIPVGPVFPPGMNWT 
               
               
                   
               
               
                 DLITNYSPSREDNLQRVFTVASIRSMLIK 
               
             
          
         
       
     
     VP6 can be produced by transformation (transfection, transduction, or infection) of a host cell with a VP6-encoding DNA fragment in a suitable expression vehicle. Suitable expression vehicles include plasmids, viral particles, and phage. For insect cells, baculovirus expression vectors are suitable. The entire expression vehicle, or a part thereof, can be integrated into the host cell genome. In some circumstances, it is desirable to employ an inducible expression vector, e.g., the LACSWITCH™ Inducible Expression System (Stratagene; Lajolla, Calif.). 
     Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems can be used to provide the recombinant protein. The precise host cell used is not critical to the invention. 
     Proteins and polypeptides can also be produced by plant cells. For plant cells, viral expression vectors (e.g., cauliflower mosaic virus and tobacco mosaic virus) and plasmid expression vectors (e.g., Ti plasmid) are suitable. Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al.,  Current Protocols in Molecular Biology,  John Wiley &amp; Sons, New York, 1994). The methods of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al., Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York (1994); expression vehicles may be chosen from those provided, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual (1985), Supp. (1987). 
     The host cells harboring the expression vehicle can be cultured in conventional nutrient media adapted as need for activation or repression of a chosen gene, selection of transformants, or amplification of a chosen gene. 
     Specific initiation signals may be required for efficient translation of inserted nucleic acid sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire native VP6, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. In other cases, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription or translation enhancer elements, (e.g., ones disclosed in Bittner et al., Methods in Enzymol, 153:516 [1987]). 
     Preferred polypeptides are those which are soluble under normal physiological conditions. Also within the invention are fusion proteins in which a portion (e.g., one or more epitopes) of VP6 is fused to an unrelated protein or polypeptide (i.e., a fusion partner) to create a fusion protein. The fusion partner can be a moiety selected to facilitate purification, detection, or solubilization, or to provide some other function. Fusion proteins are generally produced by expressing a hybrid gene in which a nucleotide sequence encoding all or a portion of VP6 is joined in-frame to a nucleotide sequence encoding the fusion partner. For example, the expression vector PUR278 (Ruther et al., EMBO J 2:1791 [1983]), can be used to create lacZ fusion proteins. The PGEX vectors can be used to express foreign polypeptides as fusion proteins containing glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. 
     A fusion protein can be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described in Janknecht et al., Proc Natl Acad Sci USA 88:8972 (1981), allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene&#39;s open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2+  nitriloacetic acid-agarose columns, and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. The same procedure can be used for a bacterial culture. 
     Alternatively, VP6 or a portion thereof, can be fused to an immunoglobulin Fc domain. Such a fusion protein can be readily purified using an affinity column. 
     Polypeptides used in the methods of the invention, particularly short amino acid sequences containing a VP6 epitope, can also be produced by chemical synthesis (e.g., by the methods described in  Solid Phase Peptide Synthesis,  2nd ed., [1984] The Pierce Chemical Co., Rockford, Ill.). 
     VP6 polypeptides useful in the methods of the invention include any protein or polypeptide sharing an epitope with a naturally occurring VP6. Such functionally related VP6 polypeptides can have additions or substitutions of amino acid residues within the VP6 amino acid sequence compared to the wild type VP6 sequence. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. 
     For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. 
     VP6 variants with altered amino acid sequences can be created by making random mutations to VP6 DNA (which has been described in U.S. Pat. No. 5,620,896) using random mutagenesis techniques well known to those skilled in the art, and isolating the polypeptide in a way that yields highly antigenic VP6. Alternatively, site-directed mutations of the VP6 coding sequence can be engineered using techniques also well-known to those skilled in the art. 
     To design variant VP6 polypeptides, it is useful to distinguish between conserved positions and variable positions. To produce variants with increased antigenicity, it is preferable that conserved residues are not altered. Moreover, alteration of non-conserved residues are preferably conservative alterations, e.g., a basic amino acid is replaced by a different basic amino acid. 
     Other mutations to the VP6 coding sequence can be made to generate VP6 polypeptides that are better suited for expression, e.g., for scaled up production, in a selected host cell. 
     B. Isolation 
     Both naturally occurring and recombinant forms of VP6 can be produced as described above. Secreted forms can be isolated from the culture media, while non-secreted forms must be isolated from the host cells. Polypeptides can be isolated by affinity chromatography. In one example, an anti-VP6 antibody is attached to a column and used to isolate the VP6 polypeptide. Lysis and fractionation of VP6-harboring cells prior to affinity chromatography can be performed by standard methods (see, e.g., Ausubel et al., supra). Alternatively, a VP6 fusion protein can be constructed and used to isolate VP6. 
     Once isolated, the VP6 polypeptide can, if desired, be further purified and/or concentrated, as long as further processing does not impair antigenicity. A variety of methods for purification and concentration are well known in the art (see, e.g., Fisher,  Laboratory Techniques In Biochemistry And Molecular Biology,  eds., Work and Burdon, Elsevier 1980), including ultracentrifugation and/or precipitation (e.g., with ammonium sulfate), microfiltration (e.g., via 0.45 μm cellulose acetate filters), ultrafiltration (e.g., with the use of a sizing membrane and recirculation filtration), gel filtration (e.g., columns filled with Sepharose CL-6B, CL-4B, CL-2B, 6B, 4B or 2B, Sephacryl S-400 or S-300, Superose 6 or Ultrogel A2, A4, or A6; all available from Pharmacia), fast protein liquid chromatography (FPLC), and high performance liquid chromatography (HPLC). 
     II. Preparation and Administration of VP6 Polypeptides 
     The invention includes the use of vaccine compositions (e.g., oral vaccines) containing at least one VP6 polypeptide, and, optionally, a pharmaceutically acceptable carrier, such as the diluent phosphate buffered saline or a bicarbonate solution (e.g., 0.24 M NaHCO 3 ). The carriers used in the composition are selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington&#39;s Pharmaceutical Sciences. An adjuvant, e.g., a cholera toxin,  Escherichia coli  heat-labile enterotoxin (LT), liposome, or immune-stimulating complex (ISCOM), can also be included in the new vaccine composition. 
     The amount of vaccine administered will depend, for example, on the particular VP6 and its antigenicity, whether an adjuvant is co-administered with the VP6, the type of adjuvant co-administered, the mode and frequency of administration, and the desired effect (e.g., protection or treatment), as can be determined by one skilled in the art. In general, the new vaccine antigens are administered in amounts ranging between 1 μg and 100 mg per adult human dose. If adjuvants are administered with the vaccines, amounts ranging between 1 ng and 1 mg per adult human dose can generally be used. Administration is repeated as necessary, as can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly intervals. A booster shot can be given at 8 to 12 weeks after the first immunization, and a second booster can be given at 16 to 20 weeks, using the same formulation. Sera or T-cells can be taken from the individual for testing the immune response elicited by the vaccine against a rotavirus. Methods of assaying antibodies or cytotoxic T-cells against a specific antigen are well known in the art. Additional boosters can be given as needed. By varying the amount of VP6 or pharmaceutical composition, the immunization protocol can be optimized for eliciting a maximal immune response. 
     Alternatively, the VP6 vaccine compositions can be administered as ISCOMs. Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS as the delivery vehicle for antigens (Mowat et al., Immunology Today 12:383 [1991]). Doses of antigen as low as 1 μg encapsulated in ISCOMs have been found to produce class I mediated cytotoxic T cell responses (Takahashi et al., Nature 344:873 [1990]). Polypeptides are delivered into cells using ISCOMS in a manner and dosage similar to that described above for liposomes. 
     The VP6 vaccines can be administered via any appropriate route, e.g, intravenously, intraarterially, topically by injection, intraperitoneally, intrapleurally, orally, subcutaneously, intramuscularly, sublingually, intraepidermally, or rectally. 
     III. Preparation and Administration of DNA Encoding VP6 Polypeptides 
     Instead of administering the VP6 polypeptide directly, an expression vector, such as a DNA vector, encoding the VP6 can be used as the vaccine. 
     A DNA vector includes at least two components: (1) VP6 coding sequence, and (2) a transcriptional promoter operatively linked for expression of the VP6. VP6-encoding DNA can encode more than one antigen, such as another VP6 from a different rotavirus. A variety of cloned VP6 sequences are readily available, including human VP6 (GenBank Accession No. U04741) and bovine VP6 (Tarlow et al., Nucl Acids Res 18:4921 [1990]). 
     The DNA vector can optionally include additional sequences such as enhancer elements, splicing signals, termination and polyadenylation signals, viral replicons, and bacterial plasmid sequences. Such vectors can be produced by a number of known methods. For example, DNA encoding the desired VP6 can be inserted into any number of commercially available expression vectors (see, e.g., Invitrogen Catalog, 1998). 
     The DNA vector can be administered to an individual, or inoculated, in the presence of adjuvants or other substances that have the capability of promoting DNA uptake or recruiting immune system cells to the site of the inoculation. For example, DNA encapsulated in microparticles have been shown to promote expression of rotaviral proteins from DNA vectors in vivo (U.S. Pat. No. 5,620,896). 
     An animal can be inoculated with DNA through any parenteral route, e.g., via intravenous, intraperitoneal, intradermal, subcutaneous, inhalation, or intramuscular routes, oral, or by particle bombardment using a gene gun. Muscle is a useful site for the delivery and expression of VP6-encoding DNA because animals have a proportionately large muscle mass which is conveniently accessed by direct injection through the skin. A comparatively large dose of DNA can be deposited into muscle by multiple and/or repetitive injections, for example, to extend therapy over long periods of time. 
     In addition, an individual can be inoculated by a mucosal route. The VP6-encoding DNA can be administered to a mucosal surface by a variety of methods including DNA-containing nose-drops, inhalants, suppositories, or microspheres. An example of oral administration of microsphere-encapsulated DNA is shown below. 
     A. DNA Delivery by Encapsulation in PLG Microparticles and Oral Administration 
     A DNA vector containing the VP6 gene can be encapsulated in poly(lactide-co-glycolide) (PLG) microparticles by a solvent extraction technique, such as the ones described in Jones et al., Infect Immun 64:489 (1996) and Jones et al., Vaccine 15:814 (1997). For example, the DNA is emulsified with PLG dissolved in dichloromethane, and this water-in-oil emulsion is emulsified with aqueous polyvinyl alcohol (an emulsion stabilizer) to form a (water-in-oil)-in-water double emulsion. This double emulsion is added to a large quantity of water to dissipate the dichloromethane, which results in the microdroplets hardening to form microparticles. These microdroplets or microparticles are harvested by centrifugation, washed several times to remove the polyvinyl alcohol and residual solvent, and finally lyophilized. The microparticles containing DNA have a mean diameter of 0.5 μm. To test for DNA content, the microparticles are dissolved in 0.1 M NaOH at 100° C. for 10 minutes. The A 260  is measured and DNA calculated from a standard curve. Incorporation of DNA into microparticles is in the range of 1.76 g to 2.7 g DNA per mg PLG. 
     Microparticles containing about 1 to 100 μg of DNA are suspended in about 0.1 to 1 ml of 0.1 M sodium bicarbonate, pH 8.5, and orally administered to mice by gavage. 
     B. DNA Delivery by Particle Bombardment 
     Alternatively, intradermal administration of DNA by particle bombardment can be used to deliver DNA for expression of rotavirus VP6 in skin cells. The Acyl particle bombardment device (“gene gun”; Agracetus, Middleton, Wis.) can be employed to deliver DNA-coated gold beads to the epidermis of mice. 
     Plasmid DNA is affixed to gold particles by adding 10 mg of 0.95 μm gold powder (Degussa, South Plainfield, N.J.), and an appropriate amount of plasmid DNA, to a 1.5 ml centrifuge tube containing 50 μl of 0.1 M spermidine. Plasmid DNA and gold is co-precipitated by the addition of 50 μl of 2.5 M CaCl 2  during vortex mixing, after which the precipitate is allowed to settle and washed with absolute ethanol and resuspended in 2 ml of ethanol. The gold/DNA suspension is transferred to a capped vial and immersed in a sonicating water bath for 2 to 5 seconds to resolve clumps. 163 μl of the gold/DNA suspension is layered onto 1.8 cm×1.8 cm Mylar sheets and allowed to settle for several minutes after which the meniscus is broken and excess ethanol is removed by aspiration. Gold/DNA-coated mylar sheets are dried and stored under vacuum. The total amount of DNA per sheet is a function of the DNA/gold ratio and rang from 0.2 to 0.0002 μg per sheet. 
     Animals are anesthetized with 30 μl of Ketaset/Rompun (10:2). Abdominal target areas are shaved and treated with NAIR® (Alkaline hair remover Carter-Wallace, N.Y.) for two minutes to remove residual stubble and stratum corneum. Target areas are thoroughly rinsed with water prior to gene delivery. DNA-coated gold particles are delivered into abdominal skin with the Acyl instrument, which employs an electric spark discharge as the motive force. Each animal receives two nonoverlapping deliveries per immunization, at a discharge voltage of 17 kV. Particle bombardment technology is presented in the following articles: Yang et al., Proc Natl Acad Sci USA 87:9568 [1990]; Yang, CRC Crit Rev Biotechnol 12:335 [1992]; and Cheng et al., Proc Natl Acad Sci USA 90:4455 [1993]. 
     The beads deliver DNA into cells, where the DNA can be expressed (Yang et al., Proc Natl Acad Sci USA 88:2726 [1991]). Expression is transient, with most of the expression being lost within 2-3 days due to the normal sloughing of the epidermis (Williams et al., Proc Natl Acad Sci USA 88:2726 [1991]). 
     IV. Evaluating Vaccine Efficacy 
     Before administering the above vaccines in humans, toxicity and efficacy testing in animals are desirable. In an example of efficacy testing, mice (as described below) can be orally vaccinated. After the initial vaccination or after optional booster vaccinations, the mice (and corresponding control mice receiving mock vaccinations) are challenged with 100 ID 50  murine rotavirus. Efficacy is determined if mice receiving the VP6 vaccine significantly lowers the virus shed in stool. Rotavirus titers in stool can be measured using the ELISA method described in Herrmann et al., J Infect Dis 152:831 (1985). As an example, the adult mouse model as described in Ward et al., J Virol 64:5070 (1990) can be used for testing protection against rotavirus infection. 
     The adult mouse model is convenient in assessing protection against an infectious agent by increasing the time period within which to perform the study. In this model, adult BALB/c mice (6 weeks or older) inoculated with a rotavirus do not show disease symptoms but, instead, exhibit infection as viral shedding in the feces for approximately one week post-infection. Virus shedding in feces is conveniently measured and quantitated by an enzme-linked immunosorbent assay (ELISA). Studies involving quantitation of viral shedding in adult mice is preferred over studies in which disease symptoms are observed in infant mice because the latter studies are hindered by the short period in which infant mice are susceptible to rotavirus illness (from birth to 15 days of age). 
     The invention will be further described in the following example, which does not limit the scope of the invention as described in the claims. 
     EXAMPLE 
     The bovine rotavirus VP6 gene (Indiana strain) and the EDIM murine rotavirus VP6 gene were separately cloned into expression vector JW4303, which is described in Yasutomi et al., J Virol 70:678 (1996). The Indiana bovine rotavirus is described in Fernandez et al., Vaccine 14:1303 (1996) and Fernandez et al., Vaccine 16:507 (1998). The EDIM murine rotavirus VP6 sequence is available as GenBank Accession No. U36474 and has the following sequence: 
     
       
         
               
             
               
             
           
               
                 (SEQ ID NO:11) 
               
               
                   
               
             
          
           
               
                 MDVLYSISRTLKDARDKIVEGTLYSNVSDLIQQFNQMLVTMNGNE 
               
               
                   
               
               
                 FQTGGIGNLPLRNWNFDFGLLGTTLLNLDANYVESARTTIDYFVD 
               
               
                   
               
               
                 FIDNVCMDEMVRESQRNGIAPQSDALRNVSGVKFRRINFNNSSEY 
               
               
                   
               
               
                 IENWNLQNRRQRTGFTFHKPNIFPYSASFTLNRSQPQHDNLMGTM 
               
               
                   
               
               
                 WLNAGSEIQVAGFDYSCAINAPANIQQFEHIVQLRRVLTTATITL 
               
               
                   
               
               
                 LPDAERFSFPRAINSADGATTWYFNPVILRPNNVEVEFLLNGQVI 
               
               
                   
               
               
                 NTYQARFGTIVARNFDTIRLSFQLMRPPNMTPAVTALFPNAQPFE 
               
               
                   
               
               
                 HHATVGLTLRIDSAICESVLADASETMLANVTSVRQEYAIPVGPV 
               
               
                   
               
               
                 FPPGMNWTDLITNYSPSREDNLQRVFTDASIRSMLVK 
               
             
          
         
       
     
     The JW4303 vector contains sequences from the cytomegalovirus immediate early promoter to drive transcription and sequences from bovine growth hormone genes to provide a polyadenylation signal. 
     To prepare the vector for cloning, the TPA leader sequence was removed by treatment with restriction endonucleases Hind III and Bam HI. The Hind III site was changed to a Bam HI site and the gene for VP6 was inserted as a Bam HI-Bam HI fragment. 
     The VP6 genes had been inserted in the Bam HI site of plasmid Bluescript KS- and was released by Bam HI digestion prior to insertion into plasmid JW4303. Newly constructed plasmids in the correct orientation were identified by restriction endonuclease digestion. Expression of rotavirus VP6 in transfected COS cells was confirmed by indirect immunofluorescent staining with monoclonal antibody to VP6. The monoclonal antibody had been prepared against a rotavirus SA-11 strain (Cukor et al., J Clin Microbiol 19:888 (1984). The control DNA vaccine was the plasmid without the viral cDNA insert. 
     To encapsulate the DNA in PLG microparticles, the DNA was emulsified with PLG dissolved in dichloromethane, and this water-in-oil emulsion was emulsified with aqueous polyvinyl alcohol (an emulsion stabilizer) to form a (water-in-oil)-in-water double emulsion. This double emulsion was added to a large quantity of water to dissipate the dichloromethane, which resulted in the microdroplets hardening to form microparticles. These were harvested by centrifugation, washed several times to remove the polyvinyl alcohol and residual solvent, and finally lyophilized. The microparticles containing DNA had a mean diameter of 0.5 μm. To test for DNA content, the microparticles were dissolved in 0.1 M NaOH at 100° C. for 10 minutes. The A 260  was measured and DNA calculated from a standard curve. Incorporation of DNA into microparticles was 1.76 g to 2.7 g DNA per mg PLG for the VP6 DNA vaccines and 1.75 g to 3.61 g per mg PLG for the plasmid control. 
     Microparticles containing about 50 μg of DNA were suspended in about 0.5 ml of 0.1 M sodium bicarbonate, pH 8.5, and orally administered to each BALB/c mouse by gavage. Five mice received the bovine VP6 vaccine and three mice received the murine VP6 vaccine. Four mice received the control vaccine (vector without VP6 insert, as described above). 
     For evaluating serum antibody responses, an indirect ELISA for total antibody (IgG, IgM, and IgA) against EDIM rotavirus was used. Intestinal IgA antibodies to EDIM virus were determined by use of IgA-specific peroxidase-labeled anti-Ig in an indirect ELISA. Both ELISA assays are described in Herrmann et al., Vaccine 15:899 (1997). Five percent (wt/vol) stool suspensions in 0.01 M phosphate buffered saline (pH 7.1) were further diluted 1:4 (final dilution of 1:80) and used for assays of fecal IgA. The level of serum antibodies against murine rotavirus was measured at various times post-immunization, and the results are shown below. 
     
       
         loq(1/ELISA titer) 
       
     
     
       
         
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Weeks post- 
                 Mice Receiving: 
               
             
          
           
               
                   
                 immunization 
                 Bovine VP6 
                 Murine VP6 
                 Control 
               
               
                   
                   
               
               
                   
                 0 
                 &lt;2 
                 &lt;2 
                 &lt;2 
               
               
                   
                 6 
                 2.30 ± 0.00 
                 2.00 ± 0.00 
                 &lt;2 
               
               
                   
                 8 
                 2.15 ± 0.21 
                 2.60 ± 0.42 
                 &lt;2 
               
               
                   
                 10  
                 2.60 ± 0.00 
                 2.50 ± 0.46 
                 &lt;2 
               
               
                   
                 12  
                 2.30 ± 0.42 
                 2.78 ± 0.16 
                 &lt;2 
               
               
                   
                   
               
             
          
         
       
     
     The numerical values are average values for each set of mice with the 95% confidence interval as indicated. 
     The results indicate that the bovine VP6 DNA vaccine, as well as the murine VP6 vaccine, were capable of eliciting antibodies to murine rotavirus. 
     The mice described immediately above were then challenged at 12 weeks post-immunization with 100 ID 50  of EDIM murine rotavirus as described in Ward et al., J Virol 64:5070 (1990). The challenge virus was derived from epizootic diarrhea of infant mice. To monitoring viral antigen shedding in vaccinated mice feces, a monoclonal antibody-based ELISA was used as described in Herrmann et al., J Infect Dis 152:831 (1985). The viral titers are reported as A 450nm  values. 
     FIG. 1 shows the fecal viral loads after challenge as described above. (◯) represents viral loads in mice inoculated with murine PLG-VP6 DNA; (∇) represents viral loads in mice inoculated with bovine PLG-VP6 DNA; and () represents viral loads in mice inoculated with PLG control DNA. A positive ELISA titer is indicated if the A 450nm  value is at least 0.1. At three and four days post-challenge, viral titers for both the bovine and murine VP6 immunized mice were significantly lower as compared to the control immunized mice. Error bars represent one standard deviation from the mean. 
     Statistical analyses for all values shown above were performed using a non-parametric Wilcoxon 2-sample test for ranked data, analysis of variance, and the Student-Newman-Keuls test for multiple comparison of the differences among experimental groups. 
     
       
         
           
             11 
           
           
             1 
             397 
             PRT 
             Bovine rotavirus 
           
            1
Met Asp Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp
  1               5                  10                  15
Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln
             20                  25                  30
Gln Phe Asn Gln Met Ile Ile Thr Met Asn Gly Asn Glu Phe Gln Thr
         35                  40                  45
Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Asn Phe Asp Phe Gly
     50                  55                  60
Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr
 65                  70                  75                  80
Ala Arg Asn Thr Ile Asp Tyr Phe Val Asp Phe Val Asp Asn Val Cys
                 85                  90                  95
Met Asp Glu Met Val Arg Glu Ser Gln Arg Asn Gly Ile Ala Pro Gln
            100                 105                 110
Ser Asp Ser Leu Arg Lys Leu Ser Gly Ile Lys Phe Lys Arg Ile Asn
        115                 120                 125
Phe Asp Asn Ser Ser Glu Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg
    130                 135                 140
Arg Gln Arg Thr Gly Phe Thr Phe His Lys Pro Asn Ile Phe Pro Tyr
145                 150                 155                 160
Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Asp Asn Leu
                165                 170                 175
Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu Ile Gln Val Ala Gly
            180                 185                 190
Phe Asp Tyr Ser Cys Ala Ile Asn Ala Pro Ala Asn Thr Gln Gln Phe
        195                 200                 205
Glu His Ile Val Gln Leu Arg Arg Val Leu Thr Thr Ala Thr Ile Thr
    210                 215                 220
Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro Arg Val Ile Asn Ser
225                 230                 235                 240
Ala Asp Gly Ala Thr Thr Trp Tyr Phe Asn Pro Val Ile Leu Arg Pro
                245                 250                 255
Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln Ile Ile Asn Thr
            260                 265                 270
Tyr Gln Ala Arg Phe Gly Thr Ile Ile Ala Arg Asn Phe Asp Thr Ile
        275                 280                 285
Arg Leu Ser Phe Gln Leu Met Arg Pro Pro Asn Met Thr Pro Ala Val
    290                 295                 300
Ala Ala Leu Phe Pro Asn Ala Gln Pro Phe Glu His Gln Ala Thr Val
305                 310                 315                 320
Gly Leu Thr Leu Arg Ile Glu Ser Ala Val Cys Glu Ser Val Leu Ala
                325                 330                 335
Asp Ala Ser Glu Thr Met Leu Ala Asn Val Thr Ser Val Arg Gln Glu
            340                 345                 350
Tyr Ala Ile Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr
        355                 360                 365
Asp Leu Ile Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg
    370                 375                 380
Val Phe Thr Val Ala Ser Ile Arg Ser Met Leu Val Lys
385                 390                 395 
           
             2 
             395 
             PRT 
             Bovine rotavirus 
           
            2 
           
             3 
             397 
             PRT 
             Human rotavirus 
           
            3 
           
             4 
             395 
             PRT 
             Human rotavirus 
           
            4 
           
             5 
             397 
             PRT 
             Porcine rotavirus 
           
            5 
           
             6 
             397 
             PRT 
             Murine rotavirus 
           
            6 
           
             7 
             397 
             PRT 
             Avian rotavirus 
           
            7 
           
             8 
             397 
             PRT 
             Equine rotavirus 
           
            8 
           
             9 
             397 
             PRT 
             Bovidae ovis rotavirus 
           
            9 
           
             10 
             794 
             PRT 
             Simian rotavirus 
           
            10 
           
             11 
             397 
             PRT 
             Murine rotavirus 
           
            11