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Recombinant attenuated Vibrio cholerae strains used as live oral vaccines
~) INSTITUTPASTEUR/ELsEVIER Paris 1990
Res. MicrobioL 1990, 141, 901-906
R E C O M B I N A N T A T T E N U A T E D VIBRIO C H O L E R A E S T R A I N S U S E D AS L I V E O R A L VACCINES
J.B. Kaper and M.M. Levine The Center for Vaccine Development, Division of Geographic Medicine, Department of Medicine, University of Maryland School of Medicine, 10 South Pine St., Baltimore, MD 21201 (USA)
Summary. Although great strides have been made in the development of recombinant attenuated Vibrio cholerae vaccine strains, the task has not been as simple as once imagined. The initial vaccine candidates proved to be unexpectedly reactogenic but further derivatives, such as CVD103-HgR, are well-tolerated, immunogenic and protective after a single dose. In addition, this strain carries a selectable marker to distinguish it from wild strains and has been evaluated in a practical, lyophilized formulation (Levine et al., 1988b). While CVD103-HgR is being further evaluated in expanded trials, we are also investigating a new secretogenic factor which could possibly explain the diarrhoea seen with the earlier vaccine strains. Hopefully, these studies will achieve the long-sought goal of a safe and effective vaccine for the prevention of cholera. KEY-WORDS: Vibrio cholerae, Cholera, Live oral vaccine; Recombinant strains, Diarrhoea.
Introduction. The use of attenuated Vibrio cholerae strains as oral attenuated cholera vaccines is based on volunteer challenge studies demonstrating that an initial infection with virulent V. cholerae stimulates an immune response that provides solid protection against re-challenge in 90-100 °70 of subjects (Cash et al., 1974; Levine et al., 1979, 1980, 1981). An attenuated V. cholerae vaccine strain would mimic infeaion-derived immunity by colonizing the proximal small bowel and stimulate both antibacterial and antitoxic immunity. The feasibility of this approach was initially shown with the V. cholerae strain Texas Star-SR, which was attenuated by chemical mutagenesis (Honda and Finkelstein, 1979, Levine et al., 1984). We have constructed a series of recombinant attenuated V. cholerae strains by deleting sequences encoding the cholera enterotoxin (CT), the critical virulence factor of V. cholerae. The mutations constructed in vitro using cloned cholera toxin genes (ctx) are then recombined into the chromosome by allelic exchange.
902
J.B. KAPER
AND M.M. LEVINE
Construction of attenuated vaccine strains V. cholerae 3BK70 and CVD101.
The cholera enterotoxin consists of five B and one A subunits which are responsibl~ L~I the binding and enzymatic activities, respectively, of the holotoxin. The genes encoding both the A subunit (ctxA) and the B subunit (ctxB) are transcribed from a single promoter with ctxA proximal to the promoter. We have utilised cloned ctx genes to prepare attenuated strains by deleting the genes that encode only the A suhunit or both the A and B subunits. Similar attenuated strains have been constructed by Mekalanos et al., (1983). To construct the attenuated V. cholerae strain CVD101, Kaper et al., (1984) digested the cloned ctx genes with the restriction enzymes XbaI and Clal and removed a 550-bp fragment containing 94 o70 of the sequences encoding the A1 peptide, the component responsible for the ADP-ribosylating activity of the cholera enterotoxin (Gill et al., 1978). The remaining sequences were re-ligated and the resulting plasmid retained sequences encoding the A2 and B subunits as well as the promoter for the ctx operon. The mutated genes were introduced in a two-step procedure into the chromosome of classical Ogawa strain 395, a strain which had been extensively studied in volunteers and demonstrated to induce severe diarrhoea as well as protective immunity (Levine et al., 1979, 1980, 1981). In the first step, an insertion mutation was made by cloning a gene encoding tetracycline resistance into the ctx gene which was then recombined into the chromosome of 395. In the second step, the plasmid encoding the deletion mutation was introduced into the tetracycline-resistant V. cholerae strain. Homologous recombination of the ctx deletion into the chromosome was detected by screening for tetracycline sensitivity. Both ctx gene copies in V. cholerae 395 were mutated in this manner and the resulting strain, CVDI01, produced the B but not the A subunit of cholera enterotoxin (A-B+). CVDI01 and another candidate vaccine strain JBK70, an A - B - derivative of El Tor Inaba strain N16961 (Levine et al. 1981), were evaluated in man by Levine et al., (1988a). In a series of volunteer experiments conducted at the Center for Vaccine Development, these strains were evaluated for safety and immunogenicity in single doses ranging from 104 to I0 t0 viable organisms. Both strains colonized the intestiTte well and engendered excellent immune responses. After a single dose, 37 of 38 volunteer~ receiving JBK70 or CVDI01 exhibited a four-fold rise in vibriocidal antibody with titres comparable to those seen after infection with the toxinogenic parent strains. When antitoxin antibodies were measured for recipients of CVD101, 19 of 24 volunteers had a significant rise in titre. To assess protective immunity, 10 volunteers vaccinated with J BK70 were challenged with the toxigenic parent strain N 16961. Diarrhoea occurred in 7 of 8 controls, but in only I of 10 vaccinees (P < 0.003, 89 % vaccine efficacy), demonstrating that antibacterial immunity alone in the absence of antitoxic immunity can confer highly significant protection. Although these strains were highly immunogenic, they were unexpectedly reactogenic. More than one half of the vaccinees developed mild diarrhoea which in many cases was accompanied by abdominal cramps, anorexia and low grade fever (Levine et al., 1988a). Similar diarrhoea and other symptoms were also seen with individuals who ingested CVDI01, which was nonetheless greatly attenuated compared to the parent strain. However, due to the unacceptable adverse reactions observed, no further studies hav¢~ been conducted with JBK70 or CVD101.
Vaccine candidates CVD102, CVD104 and CVD10$.
The cause of the diarrhoea seen with V. cholerae JBK70 and CVDI01 was not apparent from the volunteer trials but two hypotheses to explain these results were proposed (Levine et al., 1988a). One was that mere colonization of the proximal small intestine by adherent vibrios somehow perturbed intestinal function, leading to net
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intestinal secretion and diarrhoea. The other hypothesis held that the V. cholerae vaccine strains produced additional secretogenic factors, distinct from cholera toxin, which were responsible for the mild diarrhoea. To examine these possibilities, additional vaccine strains were constructed and evaluated. CVDI02 (Levine et al., 1988a) is a thymine-dependent auxotrophic mutant of CVDI01 which was developed to determine whether reactogenicity would diminish if a live but non-proliferating V. cholerae strain was fed to volunteers. While no adverse reactions were seen in 5 volunteers fed CVD102 at a dose of 107, the immune response was very poor with only 2 of 5 volunteers exhibiting a vibriocidal response of very low titre, and none had antitoxin seroconversions (Levine et al., 1988a). These data indicate that the auxotrophic mutation was overattenuating. If a second secretogenic factor was the cause of the reactogenicity, then it should be possible to clone the gene encoding this factor and mutate it in a manner similar to that employed for cholera toxin. The initial possibility we investigated was the haemolysin/cytotoxin of V. cholerae, a factor previously shown to have secretogenic properties in animal assays (Spira et al., 1986; Ichinose et al., 1987). JBK70 and CVDI01 were mutated in the gene (hlyA) encoding this haemolysin/cytotoxin. The hlyA gene was cloned and mutated in vitro and the mutated gene recombined into the V. cholerae chromosome using methods similar to those employed to mutate the ctx genes (Kaper et al., 1988). The resulting strains, CVD104 and CVD105, derived from JBK70 and CVD101, respectively, were fed to volunteers in doses of 105 to 107. Although the strains engendered good immune responses, there was no significant diminution in adverse reactions (Levine et al., 1988a).
Vaccine candidates CVD103 and CVD103.HgR. In 1984, O'Brien et al. reported that most strains of V. cholerae including N16961 and 395 produced Shiga-like toxin, a factor previously shown to have secretogenic properties. However, one toxinogenic strain of V. cholerae, Inaba 569B, did not. This strain, then, was mutated in the ctxA gene in the same manner used to construct CVD101. The resulting strain, designated V. cholerae CVD103, was significantly less reactogenic than the previous vaccine candidates (Levine et al., 1988b). Of 52 volunteers who ingested 106 or l0 s organisms, only 6 experienced any diarrhoea and the total stool volume exceeded 400 ml in only one instance (958 ml). Furthermore, CVD103 did not produce the anorexia, abdominal cramps, malaise and low-grade fever which was usually seen in recipients of JBK70, CVD101 and the 395N1 strain constructed by Mekaianos et al. (Mekalanos et al., 1983; Herrington et al., 1988). Only 1 of 6 CVD103 vaccinees who developed diarrhoea experienced any of these other symptoms compared to 7 of 7 JBK70 recipients (p < 0.005). CVD103 also elicited an excellent immune response. Of 46 recipients of a single 10s dose of CVDI03, 98 % exhibited 4-fold or greater rises in vibriocidal antibody with a peak geometric mean titre of 1339 (Levine et al., 1988). This level is approximately 2/3 that seen ia volunteers after clinical cholera caused by 569B, the toxinogenic parent strain of CVDI03. The antitoxin response induced by CVDI03 was also excellent, with 93 % exhibiting a response equivalent to that seen in volunteers after challenge with 569B. To assess the protective efficacy conferred by CVD103, three separate challenge studies were carried out with a variety of homologous and heterologous challenge strains. Recipients of a single immunizing dose of the classical Inaba CVD103 were significantly protected against challenge with strains 569B (classical Inaba), 395 (classical Ogawa) and N16961 (El Tor Inaba) with protective efficacy values of 87 o70,82 °70 and 67 %, respectively. Complete protection was seen against severe diarrhoea (stool volume > 5.0 liters) (0 of 26 vaccinees vs. 5 of 25 controls, p = 0.023) and nearly complete protection (94 °70) was seen against moderate diarrhoea (total stool volume >2.0 liters) (1 of 26 vs. 15 of 25, p <0.0001).
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We do not know if the reduced reactogenicity seen with CVD103 relative to earlier vaccine candidates is due to the absence of ShigoAike toxin in this strain. To prove this hypothesis would require the cloning and mutagenesis of genes encoding the Shigalike toxin in V. cholerae. Despite intensive efforts towards this goal, neither we nor other investigators have yet succeeded in cloning the genes encoding the Shiga-like toxin in V. cholerae. Nevertheless, we have continued to develop and evaluate further derivatives of CVD103 and have also investigated other possible secretogenic factors in this species (see below).
Considerations in the environmental release of live genetically engineered vaccine strains. The initial clinical evaluations of the attenuated V. cholerae strains were conducted in the closed environment of the Center for Vaccine Development Isolation Ward. In this setting, all stool specimens potentially containing the attenuated vaccine strain can be collected and disinfected. However, administration of the vaccine to individuals outside such a closed ward raises the very real possibility that the vaccine strain would be released into the environment. We have constructed further derivatives of CVDI03 to address concerns raised about the release of a genetically engineered live bacterial vaccine into the environment. The first concern was the potential for a non-toxinogenic vaccine strain to mate with a toxinogenic V. cholerae strain in the intestine or the environment, resulting in the vaccine strain acquiring toxin genes. Even if this highly unlikely event were to occur, the consequences would be negligible in an environment already harboring toxinogenic V. cho!erae. However, because of the debate about deliberate environmental release of recombinant organisms, we modified the recA gene of CVDI03 to diminish the ability of this strain to incorporate exogenous genetic material into the chromosome. The recA gene of V. cholerae was cloned and mutated in vitro and the mutated gene was introduced into the chromosome of CVD103 by allefic exchange (Ketley et al., 1990). When tested in volunteers, the recA derivative of CVD103 (CVDI03 RM~ was greatly diminished in its immunogenicity, to the extent that it could not be used as a one-dose vaccine (Ketley et al. 1990). Consequently, further work with CVD103 RM has been abandoned. A second mutation was introduced into CVDI03 to allow this strain to be readily differentiated from wild type strains. To avoid the introduction of an antibiotic resistance marker, we incorporated a gene encoding resistance to mercury (mer) into the chromosome of CVD103. This gene was introduced by homologous recombination into the hlyA locus encoding haemolysin, a site selected since this site could be disrupted with no adverse consequences on immunogenicity (Levine et ai., 1988a). This vaccine (CVD103-HgR) proved both safe and immunogenic in volunteers (Levine et aL, 1988b). Of 25 volunteers who received CVDI03-HgR, only I (4 %) experienced mild diarrhoea. A single dose led to significant rises in vibriocidal antibody titres in 96 % of volunteers and significant antitoxin responses in 88 %, with excellent titres seen for both measures (Levine et ai., 1988b). A single dose also led to significant protection against challenge with the heterologous biotype strain N16961 (Levine et al., 1988b). Interestingly, CVD103-HgR was significantly diminished in excretion compared to the parent CVD103 strain. Of 25 vaccinees, CVDI03HgR was recovered from the stools of only 28 % compared to 87 % of recipients of CVDI03. Despite this diminished excretion, this strain still appeared to interact with the intestinal immune system in a fashion that elicited serological responses comparable to those seen with the toxinogenic 569B strain or the JBK70 and CVD101 strains. The reason for the diminished excretion of CVD103-HgR (which may be an advantage in dealing with concerns about environmental release) are not known.
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Further studies on secretogenic factors. We have continued to study the possible mechanisms by which V. cholerae strains JBK70 and CVDIO1 cause diarrhoea in the absence of cholera toxin. We have examined culture supernatants of these strains in Ussing chambers and measured changes in short circuit current, tissue conductance and potential difference across rabbit ideal tissue. We found that these strains could induce a significant increase in tissue conductance and that this increase was accompanied by changes in the morphology of the epithelial tight junctions (zonula occludens) (Fasano et al., 1990). The "loosening" of the intestinal tight junctions could produce diarrhoea through the paracellular pathway (/.e. between epithelial cells) rather than the transcellular pathway involved in diarrhoea due te cholera toxin. We are continuing to characterize the factor responsible for this effect and the gene encoding this factor, which we have named zot for zonula occludens toxin.
References. CASH,R.A., Music, S.I., LIBONATI,J.P., SNYDER,M.J., WENZEL, R.P. ~ HORNICK,R.B. (1974), Response of man to infection with Vibrio cholerae. Protection from illness afforded by previous disease and vaccine. J. infect. Dis., 130, 325. FASANO,A., BAUDRY,B., PUMPLIN,D.W., TALL, B.D., KETLEY,J.M. ~; KAPER,J.B. (1990), Production of a second diarrheagenic factor by V. cholerae. Abstracts of the European Society for Pediatric Gastroenterology and Nutrition, May, 1990, Amsterdam. GILL,D.M. & M~REN,R. (1978), ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. Proc. nat. Acad. $ci. (Wash.), 7S, 3050, HERRINGTON,D.A., HALL, R.Ho, LOSONSXY,G., M~XALANOS,J.J., TAYLOR,R.K. & LEVI~, M.M. (19~8), Toxin, toxin co-regulated pili and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. exp. Med., 168, 1487. HONDA,T. & FINKELSTEIN,R.A. (1979), Selection and characteristics of a Vibrio cholerae mutant lacking the A (ADP-ribosylating) portion of the cholera em:erotoxin. Proc. nat. Acad. Sci. (Wash.), 76, 2052-2056. ICmNOSE,Y., YAMAMOTO,K., N ~ . s o ~ , I'4., TANAee,M.J., TAKEDA,T., MiWATANI,T. & IWANAOA, M. (1987), Enterotoxicity of El Tot-like hemolysin of non-01 Vibrio cholerae. Infect. Immun., 55, 1090. KAPER,J.B., LOCKMAN,H., BALDINI,M.M. & LEVINE,M.M. (1984), Recombinant nontoxinogenie Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature (Lond.}, 308, 655. KAPER, J.B., LOCKMAN,H. nALDINI.M.M. & LEV,N~,M.M. (1984), A recombinant live oral cholera vaccine. Bio Technology, 2, 345. KAPER, J.B., MOeLEY,H.L.T., MICHALSKI,J.M., HERRINGTON,D.A. & LEVmE,M.M. (1~88), Recent advances in developing a safe and effective live oral attenuated Vibrio cholerae vaccine. Advances in Research on Cholera and Related Diarrheas, vol. 6. (N. Ohtomo and R.B. Sack) (p. 161). KTK Scientific Publishers, Tokyo. KETLEV,J., KAPER,J.B., HERmNOTON,D., LOSONSKY,G. & LEWNE,M.M. (1990), Diminished immunogenicity of a rec,~mbination-deficient derivative of Vibrio cholerae vaccine strain CVD!03. Infect. Immun., S8, 1481-1484. LEVINE,M.M., NALIN,D.R., CRAm, J.P., HOOVER,D., BERGQUIST,E.J., WATERMAN,D., HOLLEY, H.P. Jr, HORNICK,R.B., PIERCE,N.F. & LIBONATi,J.P. (1979), Immunity to cholera in man: relative role of antibacterial versus antitoxic immunity. Trans. roy. Soc. trop. Med. I-kyg., 73, 3. LEWNE,M.M. (1980), Immunity to cholera as evaluated in volunteers. Cholera and Related Diarrheas (O. Ouchterlony and J. Holmgren) (p. 195). S. Karger, Basel. LEVINE,M.M., BLACK,R.E., CLE~tewrs,M.L., NALIN,D.R., CISNEROS,L. & FINXeLSTEIN,R.A. 1981), Volunteer studies in development of vaccines against cholera and enterotoxgenie Escherichia coli: a review, Acute Enteric Infections in Children: New Prospects for Treatment and Prevention (T. Holme, J. Holmgren, M. Merson, and R. Mollby) (p. 443). Elsevier/North Holland, Amsterdam.
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AND M.M. LEVINE
LEVINV,M.M,, KA~ER,J.B.~ BLACK,R.E. & CLEMENTS,M.L. (1983), New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development. Microbiol. t?ev., 47, 510. LEVitE, M.M., BLACK,R.E., CLEMENTS,M.L. et at. (1984), Evaluation in bumans of attenuated Vibrio cholerae El Tot Ogawa strain Texas Star-SR as a live oral vaccine. Infec¢. Immun., 43, 515-522. LF-VINE,M.M,, KAPER,JOB., HERRINGTON,D., LOSONSKY,G., MORRIS,J.G., CLEMENTS,M.L., BLACK,R.E., TALL, B. & H/,LL, R. (1988a), Volunteer ~tudies of deletion mutants of Vibrio cholerac 01 prepared by recombinant techniques. Infect. lmmun., 56, 161. LEVITE,M.M., KAPER,J.IL, HERRINGTON,D.A., KETLEV,J., LOSONSKY,G., TACKET,C.O., TALL, B. & CRYZ, S. (1988b), Safety, immunogenicity, and efficacy of recombinant live oral cholera vaccines, CVDI03 and CVDI03-HgR. Lancet, II, 467. LOCUMAN,H. & KAPER,J.B. (1983), Nucleotide sequence analysis of the A2 and B subunits of Vibrio cholerae enterotoxin. J. biol. Chem., 258, 13722. LOCKMAN,H.A., GALEN,J,E. & KAPER,J.B. (1984), Further analysis of the Vibrio cholerae enterotoxin genes: nucleotide sequence analysis of the gene encoding the ADPribosylating subunit. J. Bact., 1S9, 1086. MEKALANOS,J.J., SWARTZ,D.J., PEARSON,G.D.N., HARFORD,N., GROYNE,F. & DEWILDE,M. (1983), Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature (Lond.), 306, 551. O'BRIEN, A.D., CHE~, M.E., HOLMES,R.K., KAPER,J. & LEVINE,M.M. (1984), Environmental and human isolates of Vibrio cholerae and Vibrio parahaemolyticus produce a Shigella dysenteriae I (Shiga)-like cytotoxin. Lancet, II, 77. SPIRA, W.M., SACKt D.A., FEDORKA-CRAY,P.J., SANYAL,S., MADDEN,J. & MCCARDELL,B. (1986), Description of a possible new extracellular virulence factor in non-toxigenic Vibrio choleraeOl. Advances in Research on Cholera and Related Diarrheas, vol. 3 (S. Kuwahara, and N.F. Pierce) (p. 263). Martinus Nijhoff, The Hague.
Res. MicrobioL 1990, 141, 901-906
R E C O M B I N A N T A T T E N U A T E D VIBRIO C H O L E R A E S T R A I N S U S E D AS L I V E O R A L VACCINES
J.B. Kaper and M.M. Levine The Center for Vaccine Development, Division of Geographic Medicine, Department of Medicine, University of Maryland School of Medicine, 10 South Pine St., Baltimore, MD 21201 (USA)
Summary. Although great strides have been made in the development of recombinant attenuated Vibrio cholerae vaccine strains, the task has not been as simple as once imagined. The initial vaccine candidates proved to be unexpectedly reactogenic but further derivatives, such as CVD103-HgR, are well-tolerated, immunogenic and protective after a single dose. In addition, this strain carries a selectable marker to distinguish it from wild strains and has been evaluated in a practical, lyophilized formulation (Levine et al., 1988b). While CVD103-HgR is being further evaluated in expanded trials, we are also investigating a new secretogenic factor which could possibly explain the diarrhoea seen with the earlier vaccine strains. Hopefully, these studies will achieve the long-sought goal of a safe and effective vaccine for the prevention of cholera. KEY-WORDS: Vibrio cholerae, Cholera, Live oral vaccine; Recombinant strains, Diarrhoea.
Introduction. The use of attenuated Vibrio cholerae strains as oral attenuated cholera vaccines is based on volunteer challenge studies demonstrating that an initial infection with virulent V. cholerae stimulates an immune response that provides solid protection against re-challenge in 90-100 °70 of subjects (Cash et al., 1974; Levine et al., 1979, 1980, 1981). An attenuated V. cholerae vaccine strain would mimic infeaion-derived immunity by colonizing the proximal small bowel and stimulate both antibacterial and antitoxic immunity. The feasibility of this approach was initially shown with the V. cholerae strain Texas Star-SR, which was attenuated by chemical mutagenesis (Honda and Finkelstein, 1979, Levine et al., 1984). We have constructed a series of recombinant attenuated V. cholerae strains by deleting sequences encoding the cholera enterotoxin (CT), the critical virulence factor of V. cholerae. The mutations constructed in vitro using cloned cholera toxin genes (ctx) are then recombined into the chromosome by allelic exchange.
902
J.B. KAPER
AND M.M. LEVINE
Construction of attenuated vaccine strains V. cholerae 3BK70 and CVD101.
The cholera enterotoxin consists of five B and one A subunits which are responsibl~ L~I the binding and enzymatic activities, respectively, of the holotoxin. The genes encoding both the A subunit (ctxA) and the B subunit (ctxB) are transcribed from a single promoter with ctxA proximal to the promoter. We have utilised cloned ctx genes to prepare attenuated strains by deleting the genes that encode only the A suhunit or both the A and B subunits. Similar attenuated strains have been constructed by Mekalanos et al., (1983). To construct the attenuated V. cholerae strain CVD101, Kaper et al., (1984) digested the cloned ctx genes with the restriction enzymes XbaI and Clal and removed a 550-bp fragment containing 94 o70 of the sequences encoding the A1 peptide, the component responsible for the ADP-ribosylating activity of the cholera enterotoxin (Gill et al., 1978). The remaining sequences were re-ligated and the resulting plasmid retained sequences encoding the A2 and B subunits as well as the promoter for the ctx operon. The mutated genes were introduced in a two-step procedure into the chromosome of classical Ogawa strain 395, a strain which had been extensively studied in volunteers and demonstrated to induce severe diarrhoea as well as protective immunity (Levine et al., 1979, 1980, 1981). In the first step, an insertion mutation was made by cloning a gene encoding tetracycline resistance into the ctx gene which was then recombined into the chromosome of 395. In the second step, the plasmid encoding the deletion mutation was introduced into the tetracycline-resistant V. cholerae strain. Homologous recombination of the ctx deletion into the chromosome was detected by screening for tetracycline sensitivity. Both ctx gene copies in V. cholerae 395 were mutated in this manner and the resulting strain, CVDI01, produced the B but not the A subunit of cholera enterotoxin (A-B+). CVDI01 and another candidate vaccine strain JBK70, an A - B - derivative of El Tor Inaba strain N16961 (Levine et al. 1981), were evaluated in man by Levine et al., (1988a). In a series of volunteer experiments conducted at the Center for Vaccine Development, these strains were evaluated for safety and immunogenicity in single doses ranging from 104 to I0 t0 viable organisms. Both strains colonized the intestiTte well and engendered excellent immune responses. After a single dose, 37 of 38 volunteer~ receiving JBK70 or CVDI01 exhibited a four-fold rise in vibriocidal antibody with titres comparable to those seen after infection with the toxinogenic parent strains. When antitoxin antibodies were measured for recipients of CVD101, 19 of 24 volunteers had a significant rise in titre. To assess protective immunity, 10 volunteers vaccinated with J BK70 were challenged with the toxigenic parent strain N 16961. Diarrhoea occurred in 7 of 8 controls, but in only I of 10 vaccinees (P < 0.003, 89 % vaccine efficacy), demonstrating that antibacterial immunity alone in the absence of antitoxic immunity can confer highly significant protection. Although these strains were highly immunogenic, they were unexpectedly reactogenic. More than one half of the vaccinees developed mild diarrhoea which in many cases was accompanied by abdominal cramps, anorexia and low grade fever (Levine et al., 1988a). Similar diarrhoea and other symptoms were also seen with individuals who ingested CVDI01, which was nonetheless greatly attenuated compared to the parent strain. However, due to the unacceptable adverse reactions observed, no further studies hav¢~ been conducted with JBK70 or CVD101.
Vaccine candidates CVD102, CVD104 and CVD10$.
The cause of the diarrhoea seen with V. cholerae JBK70 and CVDI01 was not apparent from the volunteer trials but two hypotheses to explain these results were proposed (Levine et al., 1988a). One was that mere colonization of the proximal small intestine by adherent vibrios somehow perturbed intestinal function, leading to net
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intestinal secretion and diarrhoea. The other hypothesis held that the V. cholerae vaccine strains produced additional secretogenic factors, distinct from cholera toxin, which were responsible for the mild diarrhoea. To examine these possibilities, additional vaccine strains were constructed and evaluated. CVDI02 (Levine et al., 1988a) is a thymine-dependent auxotrophic mutant of CVDI01 which was developed to determine whether reactogenicity would diminish if a live but non-proliferating V. cholerae strain was fed to volunteers. While no adverse reactions were seen in 5 volunteers fed CVD102 at a dose of 107, the immune response was very poor with only 2 of 5 volunteers exhibiting a vibriocidal response of very low titre, and none had antitoxin seroconversions (Levine et al., 1988a). These data indicate that the auxotrophic mutation was overattenuating. If a second secretogenic factor was the cause of the reactogenicity, then it should be possible to clone the gene encoding this factor and mutate it in a manner similar to that employed for cholera toxin. The initial possibility we investigated was the haemolysin/cytotoxin of V. cholerae, a factor previously shown to have secretogenic properties in animal assays (Spira et al., 1986; Ichinose et al., 1987). JBK70 and CVDI01 were mutated in the gene (hlyA) encoding this haemolysin/cytotoxin. The hlyA gene was cloned and mutated in vitro and the mutated gene recombined into the V. cholerae chromosome using methods similar to those employed to mutate the ctx genes (Kaper et al., 1988). The resulting strains, CVD104 and CVD105, derived from JBK70 and CVD101, respectively, were fed to volunteers in doses of 105 to 107. Although the strains engendered good immune responses, there was no significant diminution in adverse reactions (Levine et al., 1988a).
Vaccine candidates CVD103 and CVD103.HgR. In 1984, O'Brien et al. reported that most strains of V. cholerae including N16961 and 395 produced Shiga-like toxin, a factor previously shown to have secretogenic properties. However, one toxinogenic strain of V. cholerae, Inaba 569B, did not. This strain, then, was mutated in the ctxA gene in the same manner used to construct CVD101. The resulting strain, designated V. cholerae CVD103, was significantly less reactogenic than the previous vaccine candidates (Levine et al., 1988b). Of 52 volunteers who ingested 106 or l0 s organisms, only 6 experienced any diarrhoea and the total stool volume exceeded 400 ml in only one instance (958 ml). Furthermore, CVD103 did not produce the anorexia, abdominal cramps, malaise and low-grade fever which was usually seen in recipients of JBK70, CVD101 and the 395N1 strain constructed by Mekaianos et al. (Mekalanos et al., 1983; Herrington et al., 1988). Only 1 of 6 CVD103 vaccinees who developed diarrhoea experienced any of these other symptoms compared to 7 of 7 JBK70 recipients (p < 0.005). CVD103 also elicited an excellent immune response. Of 46 recipients of a single 10s dose of CVDI03, 98 % exhibited 4-fold or greater rises in vibriocidal antibody with a peak geometric mean titre of 1339 (Levine et al., 1988). This level is approximately 2/3 that seen ia volunteers after clinical cholera caused by 569B, the toxinogenic parent strain of CVDI03. The antitoxin response induced by CVDI03 was also excellent, with 93 % exhibiting a response equivalent to that seen in volunteers after challenge with 569B. To assess the protective efficacy conferred by CVD103, three separate challenge studies were carried out with a variety of homologous and heterologous challenge strains. Recipients of a single immunizing dose of the classical Inaba CVD103 were significantly protected against challenge with strains 569B (classical Inaba), 395 (classical Ogawa) and N16961 (El Tor Inaba) with protective efficacy values of 87 o70,82 °70 and 67 %, respectively. Complete protection was seen against severe diarrhoea (stool volume > 5.0 liters) (0 of 26 vaccinees vs. 5 of 25 controls, p = 0.023) and nearly complete protection (94 °70) was seen against moderate diarrhoea (total stool volume >2.0 liters) (1 of 26 vs. 15 of 25, p <0.0001).
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J.B. K A P E R A N D M . M . L E V I N E
We do not know if the reduced reactogenicity seen with CVD103 relative to earlier vaccine candidates is due to the absence of ShigoAike toxin in this strain. To prove this hypothesis would require the cloning and mutagenesis of genes encoding the Shigalike toxin in V. cholerae. Despite intensive efforts towards this goal, neither we nor other investigators have yet succeeded in cloning the genes encoding the Shiga-like toxin in V. cholerae. Nevertheless, we have continued to develop and evaluate further derivatives of CVD103 and have also investigated other possible secretogenic factors in this species (see below).
Considerations in the environmental release of live genetically engineered vaccine strains. The initial clinical evaluations of the attenuated V. cholerae strains were conducted in the closed environment of the Center for Vaccine Development Isolation Ward. In this setting, all stool specimens potentially containing the attenuated vaccine strain can be collected and disinfected. However, administration of the vaccine to individuals outside such a closed ward raises the very real possibility that the vaccine strain would be released into the environment. We have constructed further derivatives of CVDI03 to address concerns raised about the release of a genetically engineered live bacterial vaccine into the environment. The first concern was the potential for a non-toxinogenic vaccine strain to mate with a toxinogenic V. cholerae strain in the intestine or the environment, resulting in the vaccine strain acquiring toxin genes. Even if this highly unlikely event were to occur, the consequences would be negligible in an environment already harboring toxinogenic V. cho!erae. However, because of the debate about deliberate environmental release of recombinant organisms, we modified the recA gene of CVDI03 to diminish the ability of this strain to incorporate exogenous genetic material into the chromosome. The recA gene of V. cholerae was cloned and mutated in vitro and the mutated gene was introduced into the chromosome of CVD103 by allefic exchange (Ketley et al., 1990). When tested in volunteers, the recA derivative of CVD103 (CVDI03 RM~ was greatly diminished in its immunogenicity, to the extent that it could not be used as a one-dose vaccine (Ketley et al. 1990). Consequently, further work with CVD103 RM has been abandoned. A second mutation was introduced into CVDI03 to allow this strain to be readily differentiated from wild type strains. To avoid the introduction of an antibiotic resistance marker, we incorporated a gene encoding resistance to mercury (mer) into the chromosome of CVD103. This gene was introduced by homologous recombination into the hlyA locus encoding haemolysin, a site selected since this site could be disrupted with no adverse consequences on immunogenicity (Levine et ai., 1988a). This vaccine (CVD103-HgR) proved both safe and immunogenic in volunteers (Levine et aL, 1988b). Of 25 volunteers who received CVDI03-HgR, only I (4 %) experienced mild diarrhoea. A single dose led to significant rises in vibriocidal antibody titres in 96 % of volunteers and significant antitoxin responses in 88 %, with excellent titres seen for both measures (Levine et ai., 1988b). A single dose also led to significant protection against challenge with the heterologous biotype strain N16961 (Levine et al., 1988b). Interestingly, CVD103-HgR was significantly diminished in excretion compared to the parent CVD103 strain. Of 25 vaccinees, CVDI03HgR was recovered from the stools of only 28 % compared to 87 % of recipients of CVDI03. Despite this diminished excretion, this strain still appeared to interact with the intestinal immune system in a fashion that elicited serological responses comparable to those seen with the toxinogenic 569B strain or the JBK70 and CVD101 strains. The reason for the diminished excretion of CVD103-HgR (which may be an advantage in dealing with concerns about environmental release) are not known.
RECOMBINANT
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Further studies on secretogenic factors. We have continued to study the possible mechanisms by which V. cholerae strains JBK70 and CVDIO1 cause diarrhoea in the absence of cholera toxin. We have examined culture supernatants of these strains in Ussing chambers and measured changes in short circuit current, tissue conductance and potential difference across rabbit ideal tissue. We found that these strains could induce a significant increase in tissue conductance and that this increase was accompanied by changes in the morphology of the epithelial tight junctions (zonula occludens) (Fasano et al., 1990). The "loosening" of the intestinal tight junctions could produce diarrhoea through the paracellular pathway (/.e. between epithelial cells) rather than the transcellular pathway involved in diarrhoea due te cholera toxin. We are continuing to characterize the factor responsible for this effect and the gene encoding this factor, which we have named zot for zonula occludens toxin.
References. CASH,R.A., Music, S.I., LIBONATI,J.P., SNYDER,M.J., WENZEL, R.P. ~ HORNICK,R.B. (1974), Response of man to infection with Vibrio cholerae. Protection from illness afforded by previous disease and vaccine. J. infect. Dis., 130, 325. FASANO,A., BAUDRY,B., PUMPLIN,D.W., TALL, B.D., KETLEY,J.M. ~; KAPER,J.B. (1990), Production of a second diarrheagenic factor by V. cholerae. Abstracts of the European Society for Pediatric Gastroenterology and Nutrition, May, 1990, Amsterdam. GILL,D.M. & M~REN,R. (1978), ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. Proc. nat. Acad. $ci. (Wash.), 7S, 3050, HERRINGTON,D.A., HALL, R.Ho, LOSONSXY,G., M~XALANOS,J.J., TAYLOR,R.K. & LEVI~, M.M. (19~8), Toxin, toxin co-regulated pili and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. exp. Med., 168, 1487. HONDA,T. & FINKELSTEIN,R.A. (1979), Selection and characteristics of a Vibrio cholerae mutant lacking the A (ADP-ribosylating) portion of the cholera em:erotoxin. Proc. nat. Acad. Sci. (Wash.), 76, 2052-2056. ICmNOSE,Y., YAMAMOTO,K., N ~ . s o ~ , I'4., TANAee,M.J., TAKEDA,T., MiWATANI,T. & IWANAOA, M. (1987), Enterotoxicity of El Tot-like hemolysin of non-01 Vibrio cholerae. Infect. Immun., 55, 1090. KAPER,J.B., LOCKMAN,H., BALDINI,M.M. & LEVINE,M.M. (1984), Recombinant nontoxinogenie Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature (Lond.}, 308, 655. KAPER, J.B., LOCKMAN,H. nALDINI.M.M. & LEV,N~,M.M. (1984), A recombinant live oral cholera vaccine. Bio Technology, 2, 345. KAPER, J.B., MOeLEY,H.L.T., MICHALSKI,J.M., HERRINGTON,D.A. & LEVmE,M.M. (1~88), Recent advances in developing a safe and effective live oral attenuated Vibrio cholerae vaccine. Advances in Research on Cholera and Related Diarrheas, vol. 6. (N. Ohtomo and R.B. Sack) (p. 161). KTK Scientific Publishers, Tokyo. KETLEV,J., KAPER,J.B., HERmNOTON,D., LOSONSKY,G. & LEWNE,M.M. (1990), Diminished immunogenicity of a rec,~mbination-deficient derivative of Vibrio cholerae vaccine strain CVD!03. Infect. Immun., S8, 1481-1484. LEVINE,M.M., NALIN,D.R., CRAm, J.P., HOOVER,D., BERGQUIST,E.J., WATERMAN,D., HOLLEY, H.P. Jr, HORNICK,R.B., PIERCE,N.F. & LIBONATi,J.P. (1979), Immunity to cholera in man: relative role of antibacterial versus antitoxic immunity. Trans. roy. Soc. trop. Med. I-kyg., 73, 3. LEWNE,M.M. (1980), Immunity to cholera as evaluated in volunteers. Cholera and Related Diarrheas (O. Ouchterlony and J. Holmgren) (p. 195). S. Karger, Basel. LEVINE,M.M., BLACK,R.E., CLE~tewrs,M.L., NALIN,D.R., CISNEROS,L. & FINXeLSTEIN,R.A. 1981), Volunteer studies in development of vaccines against cholera and enterotoxgenie Escherichia coli: a review, Acute Enteric Infections in Children: New Prospects for Treatment and Prevention (T. Holme, J. Holmgren, M. Merson, and R. Mollby) (p. 443). Elsevier/North Holland, Amsterdam.
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LEVINV,M.M,, KA~ER,J.B.~ BLACK,R.E. & CLEMENTS,M.L. (1983), New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development. Microbiol. t?ev., 47, 510. LEVitE, M.M., BLACK,R.E., CLEMENTS,M.L. et at. (1984), Evaluation in bumans of attenuated Vibrio cholerae El Tot Ogawa strain Texas Star-SR as a live oral vaccine. Infec¢. Immun., 43, 515-522. LF-VINE,M.M,, KAPER,JOB., HERRINGTON,D., LOSONSKY,G., MORRIS,J.G., CLEMENTS,M.L., BLACK,R.E., TALL, B. & H/,LL, R. (1988a), Volunteer ~tudies of deletion mutants of Vibrio cholerac 01 prepared by recombinant techniques. Infect. lmmun., 56, 161. LEVITE,M.M., KAPER,J.IL, HERRINGTON,D.A., KETLEV,J., LOSONSKY,G., TACKET,C.O., TALL, B. & CRYZ, S. (1988b), Safety, immunogenicity, and efficacy of recombinant live oral cholera vaccines, CVDI03 and CVDI03-HgR. Lancet, II, 467. LOCUMAN,H. & KAPER,J.B. (1983), Nucleotide sequence analysis of the A2 and B subunits of Vibrio cholerae enterotoxin. J. biol. Chem., 258, 13722. LOCKMAN,H.A., GALEN,J,E. & KAPER,J.B. (1984), Further analysis of the Vibrio cholerae enterotoxin genes: nucleotide sequence analysis of the gene encoding the ADPribosylating subunit. J. Bact., 1S9, 1086. MEKALANOS,J.J., SWARTZ,D.J., PEARSON,G.D.N., HARFORD,N., GROYNE,F. & DEWILDE,M. (1983), Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature (Lond.), 306, 551. O'BRIEN, A.D., CHE~, M.E., HOLMES,R.K., KAPER,J. & LEVINE,M.M. (1984), Environmental and human isolates of Vibrio cholerae and Vibrio parahaemolyticus produce a Shigella dysenteriae I (Shiga)-like cytotoxin. Lancet, II, 77. SPIRA, W.M., SACKt D.A., FEDORKA-CRAY,P.J., SANYAL,S., MADDEN,J. & MCCARDELL,B. (1986), Description of a possible new extracellular virulence factor in non-toxigenic Vibrio choleraeOl. Advances in Research on Cholera and Related Diarrheas, vol. 3 (S. Kuwahara, and N.F. Pierce) (p. 263). Martinus Nijhoff, The Hague.