V. Cholera hypereexpressing recombinant cholera toxin B subunit showing dual immunogenicity

Abstract

Immunogenic compositions imparting dual protection against enterotoxigenic labile toxin (LT)-expressing Escherichia coli (ETEC) and Vibrio cholerae based on the hyperexpression of recombinant cholera toxin B antigen by attenuated, live bacterial vectors derived from Vibrio cholerae are disclosed. The compositions exhibit dual immunogenicity and retain the ability to colonize gastrointestinal mucosa-associated lymphoid tissues.

What is claimed is: 
     
         1 . An immunogenic composition for protecting against enterotoxigenic  Escherichia coli  (ETEC) comprising a live attenuated  V. cholerae  bacterium genetically altered to express a recombinant cholera toxin B (CT-B) antigen at a level at least 10-fold higher than the parental strain of  V. cholerae  from which the attenuated  V. cholerae  bacterium is prepared or of wildtype pathogenic  V. cholerae,  said immunogenic composition providing dual immunogenicity, against  V. cholerae  and ETEC infection. 
     
     
         2 . The immunogenic composition according to  claim 1 , wherein said  V. cholerae  bacterium is genetically altered by the introduction of an expression plasmid comprising a polynucleotide encoding a cholera toxin B (CT-B) antigen operably linked to a promoter operable in a  V. cholerae  host cell. 
     
     
         3 . The composition of  claim 2 , wherein the polynucleotide encoding CT-B antigen is operably linked to the promoter Ptrc. 
     
     
         4 . The immunogenic composition of  claim 3 , wherein said expression plasmid is a balanced-lethal plasmid, which further comprises a polynucleotide encoding a gene expression product complementing a corresponding deletion or disablement of the genetic locus encoding said gene expression product on the chromosome of said  V. cholerae  bacterium. 
     
     
         5 . The immunogenic composition of  claim 4 , wherein said genetic locus encodes glutamine synthetase (Glna). 
     
     
         6 . The immunogenic composition of  claim 1 , wherein said live attenuated  V. cholerae  bacterium belongs to the El Tor biotype. 
     
     
         7 . The immunogenic composition of  claim 6 , wherein said live attenuated  V. cholerae  bacterium belongs to the Inaba or Ogawa serotype. 
     
     
         8 . The immunogenic composition of  claim 6 , wherein said live attenuated  V. cholerae  bacterium is Peru-15 (ATCC 55635). 
     
     
         9 . The immunogenic composition of  claim 3 , wherein said expression plasmid is pMEG-2350. 
     
     
         10 . A method of protecting a susceptible mammalian subject against an infection of ETEC and/or  V. cholerae  comprising administering to said subject an amount of the immunogenic composition according to  claims 1  sufficient to elicit an immunoprotective response in said subject including vibriocidal antibody and anti-CT-B antibody cross-reactive with ETEC labile toxin (LT).