Vibrio cholerae strains VCUSM1 and VCUSM4, method of producing same, and vaccine derivatives thereof

Abstract

Metabolic auxotroph of Vibrio cholerae 0139 synonym Bengal which has a mutation in its hem A gene and which is not capable of synthesizing aminolevulinic acid (ALA) de novo and which is obtained from a parent strain originally isolated from a patient's coproculture having all the identifying characteristics of Vibrio cholerae 0139 synonym Bengal is described. In this strain the hem A gene is mutated by inserting a kanamycin resistant gene cassette. Another metabolic auxotroph of Vibrio cholerae 01 El Tor where the hem A gene is mutated by a frame shift mutation is disclosed. Methods of producing the strains are disclosed.

1 . A method of producing metabolic auxotroph of  V. cholerae  0139 synonym Bengal identified herein as VCUSM-1 which has a mutation in its hemA gene and is not capable of synthesizing aminolevulinic acid (ALA) de novo, and which strain is obtained from a parent strain originally isolated from a patient's coproculture having all the identifying characteristics of  V. cholerae  O139 synonym Bengal as comma-shape, highly motile, Gram negative, optimal growth at 37° C. between a pH of 6-9, agglutinable with anti 0139 antisera, and non-agglutinable with anti O1 antisera, has intact genes of ctx, ace and zot and produces functional cholera toxin, accessory cholera enterotoxin and zonula occludens toxin characterised in that hemA gene of VCUSM-1 is mutated by inserting a kanamycin resistant gene cassette.  
     
     
         2 . A method of producing metabolic auxotroph of VCUSM-1 as claimed in  claim 1  wherein growth medium is supplemented with 40 microgram/ml of ALA to yield VCUSM-1 capable of acquiring identical growth pattern as that of wild type  V. cholerae  0139.  
     
     
         3 . A metabolic auxotroph of  V. cholerae  0139 synonym Bengal identified herein as VCUSM-1 which has a mutation in its hemA gene and is not capable of synthesizing aminolevulinic acid (ALA) de novo, and which strain is obtained from a parent strain originally isolated from a patient's coproculture having all the identifying characteristic of  V. cholerae  O139 synonym Bengal as comma-shape, highly motile, Gram negative and optimal growth at 37° C. between a pH of 6-9 is agglutinable with anti 0139 antisera and non-agglutinable with anti O1 antisera, and has intact genes of ctx, ace and zot and produces functional cholera toxin, accessory cholera enterotoxin and zonula occludens toxin characterised in that the VCUSM-1 is aminolevulinic acid (ALA) auxotroph and is not capable of growing in the absence of exogenous ALA.  
     
     
         4 . A metabolic auxotroph according to  claim 3 , wherein VCUSM-1 is a live vaccine strain.  
     
     
         5 . A metabolic auxotroph according to  claim 3 , wherein VCUSM-1 is administered to the subject orally.  
     
     
         6 . A metabolic auxotroph according to  claim 3 , wherein VCUSM-1 is capable of eliciting high titers of IgG and IgA against cholera toxin.  
     
     
         7 . A metabolic auxotroph according to  claim 3 , wherein VCUSM-1 is capable of eliciting immune response against various surface antigens of  V. cholerae  O139.  
     
     
         8 . A metabolic auxotroph according to  claim 3 , wherein VCUSM-1 is capable of eliciting high titers of IgG and IgA against lipopolysaccharide (LPS) and capsular polysaccharide (CPS).  
     
     
         9 . A metabolic auxotroph according to  claim 3  wherein VCUSM-1 is capable of eliciting high titers of vibriocidal antibodies.  
     
     
         10 . A metabolic auxotroph according to  claim 3  wherein VCUSM-1 is capable of eliciting high immunological response due to the synergistic interplay of toxin and somatic antigens.  
     
     
         11 . A metabolic auxotroph according to  claim 3  wherein VCUSM-1 is capable of protecting the vaccinees from infection with  V. cholerae  0139 synonym Bengal.  
     
     
         12 . A metabolic auxotroph according to  claim 3  wherein VCUSM-1 is severely limited in its growth unless growth environment has exogenous ALA.  
     
     
         13 . A metabolic auxotroph according to  claim 3  wherein VCUSM-1 has no residual toxicity in the small intestine of the subjects.  
     
     
         14 . A metabolic auxotroph according to  claim 3  wherein VCUSM-1 is not able to survive in environments containing no ALA.  
     
     
         15 . A metabolic auxotroph according to  claim 3  wherein VCUSM-1 is not capable of surviving in environmental waters.  
     
     
         16 . A method of producing metabolic auxotroph of  V. cholrae  O1 El Tor identified herein as VCUSM-4 has a mutation in its hemA gene and is not capable of synthesizing aminolevulinic acid (ALA) de novo, and is obtained from a parent strain originally isolated from a patient's coproculture having all the identifying characteristics of  V. cholerea  O1 El Tor as comma-shape, highly motile, Gram negative, optimal growth at 37° C. between a pH of 6-9, agglutinable with anti 01 antisera and non-agglutinable with anti O139 antisera and has intact genes of ctx, ace and zot and the said auxotrophic mutant produces functional cholera toxin, accessory cholera enterotoxin and zonula occludens toxin characterised in that hemA gene of VCUSM-4 is mutated by frame shift mutation.  
     
     
         17 . A method of producing metabolic auxotrophic of  V. cholerae  O1 El Tor as claimed in  claim 16  wherein growth medium is supplemented with 80 micrograms/ml of ALA to yield VCUSM-4 capable of identical growth patterns as of wild type  V. cholerae.    
     
     
         18 . A metabolic auxotroph of  V. cholerae  O1 El Tor identified herein a VCUSM-4 which has a mutation in its hemA gene and is not capable of synthesizing aminolevulnic acid (ALA) de novo, and is obtained from a parent strain originally isolated from a patient's coproculture having all the identifying characteristics of  V. cholerae  O1 El Tor as comma-shape, highly motile, Gram negative, optimal growth at 37° C. between a pH of 6-9, is agglutinable with anti O1 antisera and non-agglutinable with anti O139 antisera and has intact genes of ctx, ace and zot and the said auxotrophic mutant produces functional cholera toxin, accessory cholerae enterotoxin and zonula occludens toxin characterized in that VCUSM-4 is an aminolevulinic acid (ALA) auxotroph and is not capable of growing in the absence of exogenous ALA.  
     
     
         19 . A metabolic auxotroph according to  claim 18 , wherein VCUSM-4 is a live vaccine strain.  
     
     
         20 . A metabolic auxotroph according to  claim 18 , wherein VCUSM-4 is orally administrable.  
     
     
         21 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is capable of eliciting high titers of IgG and IgA against cholera toxin.  
     
     
         22 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is capable of eliciting immune response against various surface antigens of  V. cholerae  O1 El Tor.  
     
     
         23 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is capable of eliciting high titers of IgG and IgA against lipopolysaccharide (LPS) of  V. cholerae  O1 El Tor.  
     
     
         24 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is capable of eliciting high titers of vibriocidal antibodies.  
     
     
         25 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is capable of eliciting high immunological response due to the synergistic interplay of toxin and somatic antigens.  
     
     
         26 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is capable of protecting the vaccinees from infection with  V. cholerae O 1 El Tor.  
     
     
         27 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is severely limited in its growth unless growth environment has exogenous ALA.  
     
     
         28 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 has no residual toxicity in the small intestine of the subjects.  
     
     
         29 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is not able to survive in environments containing no ALA.  
     
     
         30 . A metabolic auxotroph according to  claim 18  wherein VCUSM-4 is not capable of surviving in environmental water.  
     
     
         31 . A metabolic auxotroph according to  claim 1  wherein VCUSM-1 and VCUSM-4 independently protect vaccine from acquiring infection from  V. cholerae  O139 synonym Bengal and O1 El Tor respectively.  
     
     
         32 . The use of VCUSM-1 and VCUSM-4 formulated as a bivalent vaccine to provide protection against  V. cholerae  O139 synonym Bengal and O1 El Tor.  
     
     
         33 . The use of VCUSM-1 and VCUSM-4 for oral consumption by lyophilizing and reconstituting with appropriate buffer.  
     
     
         34 . The use of VCUSM-1 derivative as a vaccine to provide protection against  V. cholerae  0139.  
     
     
         35 . The use of VCSUM-4 derivative as a vaccine to provide protection against  V. cholerae  O1 El Tor.