Live bacterial vaccines for viral infection prophylaxis or treatment

Abstract

The present invention provides a vaccine, method of use, and kit employing genetically isolated and stabilized, live attenuated bacterial strains including Salmonella that express one or more avian influenza antigens for use in live vaccine compositions that can be orally administered to an individual to protect against avian influenza. Genetic stabilization may be achieved through deletion of IS200 elements and bacteria phage and prophage elements. The bacterial strains may be genetically isolated from external phage infection by constitutive expression of a P22 phage repressor. Nucleic acid sequences encoding antigenic hemagglutinin and neuraminidase avian influenza proteins, having at least one modified codon for optimum expression when transferred into a prokaryotic microorganism for improved immunogenicity

1 . A live vaccine composition for protecting an animal against avian influenza infection, comprising a live attenuated  Salmonella  bacterium comprising:
 an attenuating mutation in a genetic locus of the chromosome of said bacterium that attenuates virulence of said bacterium;   an antigen-expressing DNA construct comprising:
 a nucleotide sequence coding for an immunogenic polypeptide comprising an avian influenza H or N antigen, 
 an immunogenic portion of said H or N antigen, or 
 a combination thereof, 
   wherein said nucleotide sequence is operably linked to a promoter that permits expression of said immunogenic polypeptide from said DNA construct; the gene encoding the immunogenic polypeptide has at least one codon optimized for bacterial expression, and said live vaccine composition elicits an immune response to at least one avian influenza antigen when administered orally to an animal.   
     
     
         2 . The live vaccine composition according to  claim 1 , wherein said live attenuated  Salmonella  bacterium is selected from the group consisting of  Salmonella enterica  serovar Typhimurium (S. typhimurium),  Salmonella enterica  serovar Typhi ( S. typhi ), 34  Salmonella enterica  serovar Paratyphi B ( S. paratyphi  B),  Salmonella enterica  serovar Paratyphi C(S. paratyphi Q,  Salmonella enterica  serovar Hadar (S. hadar),  Salmonella enterica  serovar Enteriditis (S. enteriditis),  Salmonella enterica  serovar Kentucky (S. kentucky),  Salmonella enterica  serovar Infantis (S. infantis),  Salmonella enterica  serovar Pullorum (S. pullorum),  Salmonella enterica  serovar Gallinarum ( S. gallinarum ),  Salmonella enterica  serovar Muenchen (S. muenchen),  Salmonella enterica  serovar Anatum (S. anatum),  Salmonella enterica  serovar Dublin (S. dublin),  Salmonella enterica  serovar Derby (S. derby), and  Salmonella enterica  serovar Choleraesuis var.  kunzendorf , and  Salmonella enterica  serovar Minnesota. 
     
     
         3 . The live vaccine composition according to  claim 2 , wherein said live attenuated  Salmonella  bacterium is  S. enterica  serovar Typhimurium (S. typhimurium). 
     
     
         4 . The live vaccine composition according to  claim 1 , wherein said attenuating mutation is in a genetic locus selected from the group consisting of phoP, phoQ, Mt, cya, crp, poxA, rpoS, htrA, nuoG, pmi, gale, pabA, pts, damA, purA, purB, purl, zwf, gua, cadA, rfic, rjb, rfa, ompR, Suwwan and combinations thereof 
     
     
         5 . The live vaccine composition according to  claim 4 , wherein said attenuating mutation is a deletion mutation. 
     
     
         6 . The live vaccine composition according to  claim 5 , wherein said attenuating mutation comprises at least a partial deletion mutation of phoP. 
     
     
         7 . The live vaccine composition according to  claim 1 , wherein said  Salmonella  bacterium comprises a lethal mutation, comprising a deletion in the asd gene, and said immunogenic polypeptide comprises a fusion protein comprising a V antigen or an immunogenic portion thereof, linked to an F1 antigen or an immunogenic portion thereof, encoded on an antigen-expressing, multi-copy plasmid. 
     
     
         8 . The live vaccine composition according to  claim 7 , wherein an origin of replication of said multi-copy plasmid is a ColE1, pUC, M15, or pBR322 plasmid origin of replication. 
     
     
         9 . The live vaccine composition according to  claim 1 , wherein said live vaccine composition comprises a plurality of live  Salmonella  serovars. 
     
     
         10 . The live vaccine composition according to  claim 1 , wherein said live attenuated  Salmonella  bacterium is genetically stabilized with respect to a wild type  Salmonella  of the same serovar. 
     
     
         11 . The live vaccine composition according to  claim 1 , wherein said live attenuated  Salmonella  bacterium is genetically stabilized against genetic exchange with other organisms with respect to a wild type  Salmonella  of the same serovar. 
     
     
         12 . A live vaccine composition comprising a  Salmonella  bacteria that expresses at least one of an avian influenza H or N antigen, and an immunogenic portion of said H or N antigen. 
     
     
         13 . The live vaccine composition according to  claim 12 , wherein the  Salmonella  bacterium comprises S. typhirmurium. 
     
     
         14 . The live vaccine composition according to  claim 1 , produced from a kit comprising (a) a first container comprising a bacterial expression codon optimized antigen from a pathogenic avian influenza virus strain containing unique genetically engineered restriction sites contained within at least one of a bacterial protein expression plasmid or a bacterial chromosomal protein expression vector which allows rapid exchange of small segments; and (b) a second container comprising bacterial flagellar vectors having at least one bacterial flagellar antigens, wherein the  Salmonella  bacterium comprises multiple unique chromosomal localization vectors targeting at least IS200s, phage elements and metabolic genes for insertion of the expression codon. 
     
     
         15 . A method of immunizing an animal against avian influenza, comprising administering a live vaccine composition comprising a  Salmonella  bacteria that expresses at least one of an avian influenza H or N antigen, and an immunogenic portion of said H or N antigen. 
     
     
         16 . The method according to  claim 15 , wherein the live vaccine composition is adapted to protect an animal against avian influenza infection, wherein the  Salmonella  bacterium comprises an attenuating mutation in a genetic locus of the chromosome of said bacterium that attenuates virulence of said bacterium; an antigen-expressing DNA construct comprising a nucleotide sequence coding for an immunogenic polypeptide comprising an avian influenza H or N antigen, an immunogenic portion of said H or N antigen, or a combination thereof, wherein said nucleotide sequence is operably linked to a promoter that permits expression of said immunogenic polypeptide from said DNA construct; the gene encoding the immunogenic polypeptide has at least one codon optimized for bacterial expression, and said live vaccine composition elicits an immune response to at least one avian influenza antigen when administered orally to an animal. 
     
     
         17 . The method according to  claim 16 , wherein the  Salmonella  bacterium is selected from the group consisting of  Salmonella enterica  serovar Typhimurium (S. typhimurium),  Salmonella enterica  serovar Typhi ( S. typhi ), 34  Salmonella enterica  serovar Paratyphi B ( S. paratyphi  B),  Salmonella enterica  serovar Paratyphi C(S. paratyphi Q,  Salmonella enterica  serovar Hadar (S. hadar),  Salmonella enterica  serovar Enteriditis (S. enteriditis),  Salmonella enterica  serovar Kentucky (S. kentucky),  Salmonella enterica  serovar Infantis (S. infantis),  Salmonella enterica  serovar Pullorum (S. pullorum),  Salmonella enterica  serovar Gallinarum ( S. gallinarum ),  Salmonella enterica  serovar Muenchen (S. muenchen),  Salmonella enterica  serovar Anatum (S. anatum),  Salmonella enterica  serovar Dublin (S. dublin),  Salmonella enterica  serovar Derby (S. derby), and  Salmonella enterica  serovar Choleraesuis var.  kunzendorf , and  Salmonella enterica  serovar Minnesota. 
     
     
         18 . The method according to  claim 17 , wherein the  Salmonella  bacterium comprises  S. enterica  serovar Typhimurium (S. typhimurium). 
     
     
         19 . The method according to  claim 16 , wherein the attenuating mutation is in a genetic locus selected from the group consisting of phoP, phoQ, Mt, cya, crp, poxA, rpoS, htrA, nuoG, pmi, gale, pabA, pts, damA, purA, purB, purl, zwf, gua, cadA, rfic, rjb, rfa, ompR, Suwwan and combinations thereof 
     
     
         20 . The method according to  claim 19 , wherein the attenuating mutation comprises at least a partial deletion mutation of phoP. 
     
     
         21 . The method according to  claim 16 , wherein the  Salmonella  bacterium comprises a lethal mutation, comprising a deletion in the asd gene, and the immunogenic polypeptide comprises a fusion protein comprising a V antigen or an immunogenic portion thereof, linked to an F1 antigen or an immunogenic portion thereof, encoded on an antigen-expressing, multi-copy plasmid. 
     
     
         22 . The method according to  claim 16 , wherein the  Salmonella  bacterium is genetically stabilized with respect to a wild type  Salmonella  of the same serovar. 
     
     
         23 . The method according to  claim 16 , wherein the live attenuated  Salmonella  bacterium is genetic stabilized through deletion of IS200 elements and bacteria phage and prophage elements, and genetically isolated from external phage infection by a constitutive expression of a P22 phage repressor. 
     
     
         24 . A kit adapted to be used to produce a live vaccine composition comprising a  Salmonella  bacteria that expresses at least one of an avian influenza H or N antigen, and an immunogenic portion of said H or N antigen, comprising (a) a first container comprising a bacterial expression codon optimized antigen from a pathogenic avian influenza virus strain containing unique genetically engineered restriction sites contained within at least one of a bacterial protein expression plasmid or a bacterial chromosomal protein expression vector; and
 (b) a second container comprising bacterial flagellar vectors having at least one bacterial flagellar antigens.   
     
     
         25 . The kit according to  claim 23 , further comprising a bacterial strain; wherein the bacterial expression codon allows rapid exchange of small segments, wherein the bacterial strain comprises multiple unique chromosomal localization vectors targeting at least IS200s, phage elements and metabolic genes for insertion of the expression codon.