- Email: [email protected]
A recombinant toxin B subunit-whole cell vaccine for prevention of cholera
Conference Abstracts
Construction of Vibrio cholerae strains with enhanced production of Cholera toxin B subunit N.I. Smirnova, L.F. Livanova, N.I. Davydova and T.S. Ilyina
Department of Genetics, All-Union Research Antiplague Institute 'Microbe ', Saratov 410071, USSR With a view to obtain a Cholera toxin B subunit-producing strain, a cointegrate plasmid plEM3 was derived from two plasmids, plEM1 and pcTA27. The former is a derivative of pOX38. The latter is a derivative plasmid of pBR322 carrying a cloned ctxB gene which determines the synthesis of B subunit. plEM3 was introduced into V. cholerae non-O1 C197 lacking the structural genes for cholera toxin. As a result of disruption of plEM3 cointegrate in transconjugant C197 (plEM3) cells
and the subsequent loss of its plEM 1 moiety, strain KM93 was formed containing only the multicopy plasmid, pCTA27. This strain produced and secreted 10-12/~g/ml cholera toxin B subunit. Introduction of plEM3 into toxinogenic isogenic V. cholerae strains 569B Inaba and K M 1801 Ogawa (derived from 569B) made it possible to obtain strains with enhanced production of B subunit (20 to 26 #g/ml).
Characterization of a novel protective antigen of cell-surface origin in a toxigenic non-O1 Vibrio cholerae strain Tapas K. Sengupta and Asoke C. Ghose
Department of Microbiology, Bose Institute, P-1/12, C.I.T. Scheme VIIM, Calcutta 700 054, India A 22 kD cell-surface protein was identified and shown to be responsible for the haemagglutination and intestinal adherence activities of a toxigenic non-O1 V. cholerae strain (10259). Antigenically related protein of subunit molecular weight 20.5 kD was also present in another toxigenic non-O1 strain (10325) but not in several other O1 and non-O1 strains. In analogy with the TcpA, this protein was predominantly expressed (probably in the piliated form) in enriched media
but not in the chemically defined 'T'-medium. However, it was shown to be different from the TcpA of V. cholerae O1 strains. Antiserum raised against this protein induced passive protection against challenge with the parent strain in the suckling mouse model. Similar protection was also noted against the non-O1 strain 10325 but not against several other O1 and non-O1 strains.
A recombinant toxin B subunit-whole cell vaccine for prevention of cholera Ma Qningjnn, Yn Xiuqin, Liu Chnangxuan, Zhon Jianguang and Xion Lingshuang
Biotechnology Institute, Academy of Military Medical Sciences, P.O. Box 130(8), Beijing, P.R. China An engineered E. coli strain produced B subunit of cholera toxin was constructed by using recombinant techniques. The B subunit could be secreted into the medium in large quantities and had the same antigenicity and immunogenicity as the natural B subunit. The engineered B subunit combined with killed Vibrio cholerae vaccine (rBS-WC) was evaluated in animals and adult volunteers. The rBS-WC vaccine provided significant protective efficacy of 80-90% in mice, rabbits and
guinea pigs. Through the immunization test in volunteers, this vaccine could produce vibriocidal and antitoxic antibodies in serum and intestine. The titre and responsive rate were the same as BS-WC vaccine came from natural purified CT toxin. The rBS-WC vaccine has the advantages of simple technological process, safe and low cost. Thus, it may be a good candidate for use in protection against Vibrio cholerae as well as enterotoxigenic E. coli.
CT- clinical isolates of Vibrio cholerae as potential oral vaccines for cholera U. Dasgupta, R.K. Bhadra, D.K. Panda, A. Deb and J. Das
Biophysics Division, Indian Institute of Chemical Biology, Calcutta 700032, India Vibrio cholerae strains used so far as oral vaccines had an attenuation in the gene coding for the A subunit of cholera toxin, keeping the B subunit intact. These vaccines were not successful because of undesirable side effects. In contrast, a combination of heat-killed whole vibrios and purified B subunit was relatively more effective. In order to have antibacterial as well as a high level of antitoxic immunity at the same time, we have taken the strategy of using nontoxinogenic ( C T - ) clinical
286
Vaccine, Vol. 10, Issue 4, 1992
isolates of V. cholerae in which the gene for the B subunit has been cloned in a multicopy plasmid. The strains used in this study do not secrete any toxin at all as detected by a sensitive bead-ELISA. However, the recombinant strains excreted significant amounts of subunit B. The strains are being tested in animal models for their ability to colonize the intestine and their potential as oral vaccines for cholera is being investigated.
Construction of Vibrio cholerae strains with enhanced production of Cholera toxin B subunit N.I. Smirnova, L.F. Livanova, N.I. Davydova and T.S. Ilyina
Department of Genetics, All-Union Research Antiplague Institute 'Microbe ', Saratov 410071, USSR With a view to obtain a Cholera toxin B subunit-producing strain, a cointegrate plasmid plEM3 was derived from two plasmids, plEM1 and pcTA27. The former is a derivative of pOX38. The latter is a derivative plasmid of pBR322 carrying a cloned ctxB gene which determines the synthesis of B subunit. plEM3 was introduced into V. cholerae non-O1 C197 lacking the structural genes for cholera toxin. As a result of disruption of plEM3 cointegrate in transconjugant C197 (plEM3) cells
and the subsequent loss of its plEM 1 moiety, strain KM93 was formed containing only the multicopy plasmid, pCTA27. This strain produced and secreted 10-12/~g/ml cholera toxin B subunit. Introduction of plEM3 into toxinogenic isogenic V. cholerae strains 569B Inaba and K M 1801 Ogawa (derived from 569B) made it possible to obtain strains with enhanced production of B subunit (20 to 26 #g/ml).
Characterization of a novel protective antigen of cell-surface origin in a toxigenic non-O1 Vibrio cholerae strain Tapas K. Sengupta and Asoke C. Ghose
Department of Microbiology, Bose Institute, P-1/12, C.I.T. Scheme VIIM, Calcutta 700 054, India A 22 kD cell-surface protein was identified and shown to be responsible for the haemagglutination and intestinal adherence activities of a toxigenic non-O1 V. cholerae strain (10259). Antigenically related protein of subunit molecular weight 20.5 kD was also present in another toxigenic non-O1 strain (10325) but not in several other O1 and non-O1 strains. In analogy with the TcpA, this protein was predominantly expressed (probably in the piliated form) in enriched media
but not in the chemically defined 'T'-medium. However, it was shown to be different from the TcpA of V. cholerae O1 strains. Antiserum raised against this protein induced passive protection against challenge with the parent strain in the suckling mouse model. Similar protection was also noted against the non-O1 strain 10325 but not against several other O1 and non-O1 strains.
A recombinant toxin B subunit-whole cell vaccine for prevention of cholera Ma Qningjnn, Yn Xiuqin, Liu Chnangxuan, Zhon Jianguang and Xion Lingshuang
Biotechnology Institute, Academy of Military Medical Sciences, P.O. Box 130(8), Beijing, P.R. China An engineered E. coli strain produced B subunit of cholera toxin was constructed by using recombinant techniques. The B subunit could be secreted into the medium in large quantities and had the same antigenicity and immunogenicity as the natural B subunit. The engineered B subunit combined with killed Vibrio cholerae vaccine (rBS-WC) was evaluated in animals and adult volunteers. The rBS-WC vaccine provided significant protective efficacy of 80-90% in mice, rabbits and
guinea pigs. Through the immunization test in volunteers, this vaccine could produce vibriocidal and antitoxic antibodies in serum and intestine. The titre and responsive rate were the same as BS-WC vaccine came from natural purified CT toxin. The rBS-WC vaccine has the advantages of simple technological process, safe and low cost. Thus, it may be a good candidate for use in protection against Vibrio cholerae as well as enterotoxigenic E. coli.
CT- clinical isolates of Vibrio cholerae as potential oral vaccines for cholera U. Dasgupta, R.K. Bhadra, D.K. Panda, A. Deb and J. Das
Biophysics Division, Indian Institute of Chemical Biology, Calcutta 700032, India Vibrio cholerae strains used so far as oral vaccines had an attenuation in the gene coding for the A subunit of cholera toxin, keeping the B subunit intact. These vaccines were not successful because of undesirable side effects. In contrast, a combination of heat-killed whole vibrios and purified B subunit was relatively more effective. In order to have antibacterial as well as a high level of antitoxic immunity at the same time, we have taken the strategy of using nontoxinogenic ( C T - ) clinical
286
Vaccine, Vol. 10, Issue 4, 1992
isolates of V. cholerae in which the gene for the B subunit has been cloned in a multicopy plasmid. The strains used in this study do not secrete any toxin at all as detected by a sensitive bead-ELISA. However, the recombinant strains excreted significant amounts of subunit B. The strains are being tested in animal models for their ability to colonize the intestine and their potential as oral vaccines for cholera is being investigated.