An oral B subunit : whole cell vaccine against cholera J. Holmgren *~, A.-M. Svennerholm*, M. Jertborn*, J. Clemens +, D. A. Sack*, R. Salenstedt ++and H. Wigzell ++ During the last decade there has been a rapid progress in the development of new, much improved vaccines against cholera. These vaccines, which are given orally to stimulate the gut mucosal immune system, are based on either a combination of purified cholera toxin B (binding) subunit and killed cholera vibrios oflnaba and Ogawa serotypes and El Tor and classical biotypes (B subunit-whole cell vaccine, B WC) or on a live attenuated mutant strain o f V i b r i o c h o l e r a e producing the B subunit ( CVD 103-HgR). The safety of the oral B - W C cholera vaccine and the immunogenicity and protective efficacy of this' vaccine against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli have been extensively documented, e.g. in a large randomized, placebo-controlled field trial in 90000 persons living in a cholera endemic area. The potential for inexpensive large-scale manufacturing of the B - W C vaccine has recently been much facilitated by the introduction of recombinant DNA technology jbr production o f the B subunit component. This now gives promise that this" vaccine could become a useful, cost-effective tool in future strategies to control cholera both in endemic situations and in relation to acute epidemic outbreaks. Keywords: Cholera; B subunit whole cell vaccine; oral; field trials
INTRODUCTION There is a great need for an effective cholera vaccine. Cholera remains an important cause of illness and death in many parts of the world, especially in Asia but also in Africa and the Middle East and, since 1991, in South and Central America as well. It has been estimated that more than 150000 people, both children and adults, die from cholera each year. Although cholera can be treated simply by oral and intravenous rehydration, diarrhoea treatment centres are still scarce in areas endemic for cholera, and use of an effective vaccine may in many areas be the most promising possibility to control the disease. However, the parenteral whole-cell cholera vaccines that have existed for almost a century to prevent cholera are no longer regarded useful from a public health standpoint, mainly because of the short duration of protection they provide. Several field trials since 1960 have shown that these vaccines usually protect older people for only a few months and fail to protect many young children at all. The identification of the critical role of a toxin in the *Department of Medical Microbiology and Immunology, University of GSteborg, Guldhedsgatan 10, S-413 46 G6teborg, Sweden. *International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh. ~National Bacteriological Laboratory, S-105 21 Stockholm, Sweden. ~To whom correspondence should be addressed 0264-410X/92/130911-04 :i(' 1992Butterworth-HeinemannLtd
pathogenesis of cholera led to great expectations that a more effective cholera vaccine could be developed based on the use of a toxoid. However, a large field trial with a parenterally administered glutaraldehyde toxoid vaccine in Bangladesh in the mid-1970s failed to give any better protection than that achieved by previous parenteral vaccines. This did not come as too much of a surprise, however, since during the time it took to organize, execute and evaluate the trial, it had become more clearly apparent from animal studies that protective cholera immunity does not depend on serum antibodies, which are the type of antibodies mainly stimulated by the parenteral vaccination, but rather on mucosal secretory IgA antibodies produced locally in the gut, which are only inefficiently stimulated by parenteral antigen administration. Therefore, since the late 1970s, attention has turned instead to development of oral vaccines that could stimulate intestinal immunity more efficiently. The different 'modern' approaches towards development of cholera vaccines have all taken their departure from the new insights into the mechanisms of disease and immunity in cholera achieved during the 1970s and early 1980s (for specific reviews, see Refs 1 and 2). First, the clarification of the subunit structure of cholera toxin and of the role of different subunits in the toxin action immediately suggested a way to prepare a safe and highly immunogenic 'toxoid' consisting of the purified cholera B subunits. Studies showed that the
Vaccine, Vol. 10, Issue 13, 1992 911
Oral cholera vaccine: d. Holmgren et al.
purified B subunit portion of cholera toxin was entirely devoid of toxicity, yet it contained selectively the protective toxin epitopes against which neutralizing antitoxin antibodies were directed. Indeed, it was found that the B subunit is particularly well suited to oral immunization because it retains the ability to bind to the intestinal epithelium, which has been shown to be important for stimulating mucosal immunity in animals, including local immunological memory 1'2. Another important line of vaccine research has focused on the gut mucosal immune system and how it can be stimulated by immunogens. These studies have defined the importance of locally produced IgA antibodies and IgA immunological memory for protection against cholera, and they have shown that the oral route of vaccine administration is usually superior to the parenteral route in both priming and boosting the mucosal immune system 1'2. A third important observation guiding the design of the new cholera vaccines has concerned the synergistic co-operation between antitoxic and antibacterial immune mechanisms in cholera. Two main protective antibodies have been identified, one being directed against the Vibrio cholerae cell wall lipopolysaccharide (LPS) and the other against the cholera toxin B subunits. Either of these two types of antibody can confer protection against disease by inhibiting bacterial colonization and toxin binding, respectively, and when present together in the gut they can have a strongly synergistic protective effect 1'2. The intense vaccine development and research efforts during the last 10 years have now provided one licenced oral cholera vaccine based on either a combination of non-living bacteria and purified B subunit antigen, and an additional candidate vaccine consisting of live attenuated mutants of Vibrio cholerae 01 producing the B subunit 3. The oral B subunit-whole cell vaccine
The composition of this vaccine was designed to safely provide the key antigens for evoking protective antitoxic and antibacterial mucosal immunity against V. cholerae 01 of different serotypes (Inaba and Ogawa) and biotypes (classical and E1 Tor). As mentioned, the B subunit component is completely non-toxic and is an exceptionally potent oral immunogen because of its ability to bind to the intestinal mucosa. The whole-cell (WC) component provides protective LPS antigens as well as various heat-labile bacterial antigens that may add further to the antibacterial immunogenic effect. The B - W C vaccine is given orally together with an alkaline buffer (provided by an effervescent tablet) to protect the vaccine during passage through the stomach. The vaccine has been extensively tested in several clinical trials in Swedish, Bangladeshi and American volunteers. In these studies, the vaccine has proved to be completely safe with no adverse reactions, and after either two or three doses, it stimulates a gut mucosal IgA antitoxic and antibacterial immune response (including memory) comparable to that induced by cholera disease itselP '5. In American volunteers who received three oral immunizations with either the B - W C vaccine or the WC vaccine component alone, vaccination protected against challenge with a dose of live cholera vibrios that caused disease in 100% of concurrently tested unvaccinated controls 6.
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Vaccine, Vol. 10, Issue 13, 1992
The B - W C vaccine and the WC component without any B subunit (both vaccines produced by Institut M6rieux, France in collaboration with the National Bacteriological Laboratory of Sweden, SBL) have recently been evaluated in a large randomized placebocontrolled field trial involving 89 596 vaccinated adults and children in Bangladesh. A total of 63 000 people - - children aged 2-15 years and adult women received three oral doses, at 6 week intervals, of B WC vaccine, WC vaccine or a placebo and another 26000 participants took one or two doses of three agents. Each dose of B WC vaccine contained 1 x 1011 killed whole vibrios representing three different strains belonging to the Inaba and Ogawa serotypes and the classical and E1 Tor biotypes; the B WC vaccine also contained 1 mg of purified B subunit from cholera toxin. The main results after 3 years of follow-up are summarized in Table 1. Both vaccines have been found to protect against cholera for at least 3 years. The B WC vaccine had the advantage over the WC vaccine alone of a significantly higher initial efficacy level (85% versus 58%) for the initial 4 - 6 month period ~. After the first 8-12 months the efficacy of the two vaccines was similar, however 8. Overall, for the 3 years of follow-up the B - W C vaccine conferred 50% protection and the WC vaccine 52% protection against culture-proven cholera: protection after two doses was similar to that after three doses. For both vaccines, protective efficacy was of shorter duration in children vaccinated at the age of 2 - 5 years; after a high initial level of protection (100%) for B WC during the first 6 months of follow-up, protection declined and no protection was observed in this age group in the third year of follow-up. In contrast, the level of protection in persons aged > 5 years was about 65% for both vaccines and was sustained during the entire follow-up period. Protection was slightly higher against classical than E1 Tor cholera but did not differ with serotype (Inaba or Ogawa) 8. Assuming that protection in adult males would have equalled that seen in adult females the overall protective efficacy of two or three doses over 3 years of follow-up can be estimated to be 60% for the B - W C vaccine and 58% for the WC vaccine. The B - W C vaccine, through its B subunit component, also provided substantial protection, 67%, for a few Table 1 Results from randomized placebo-controlled field trial of oral B subunit-whole cell (B-WC) cholera vaccine and whole-cell (WC) vaccine alone given in three (or two) doses (ICDDR,B: Matlab, Bangladesh) Vaccine efficacy ( % ) B-WC After 6 months of follow-up All ages Persons > 5 years Children < 5 years
After 3 years of follow-up All ages
WC
85
58
77 100
62 53
50
52
Persons > 5 years Children < 5 years
63 26
68 23
Recipients of only two doses, all ages
64
39
Overall protection after two or three doses, all ages (including estimate for adult men)
60
58
Oral cholera vaccine: J, Holmgren et al.
months against diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) producing either heat-labile toxin (LT, that cross-reacts immunologically with cholera toxin) alone or LT together with heat-stable toxin (ST) 9. Protection against severe disease was 86% and against milder illness 54O/o9. A similar protective effect, ~70%, of two doses of the oral B-WC cholera vaccine against ETEC diarrhoea was recently reported by Peltola et al. 1° in Finnish travellers to Morocco. Furthermore, in Bangladesh, during the large field trial, both the B-WC and the WC vaccines substantially and in a sustained manner reduced the overall diarrhoea morbidity among the vaccinees. Thus, over the 3 year follow-up period there was a 25% reduction in admissions for 'all diarrhoea' and a 50% reduction in admissions for life-threatening diarrhoea in the vaccinated as compared with the placebo group (Ref. 11 and J. Clemens et al. unpublished). The results with the oral B-WC vaccines represent marked improvements as compared with those achieved previously using parenteral cholera vaccines. In a cholera endemic area immunization with an oral vaccine could decrease the number of episodes of life-threatening cholera by more than 50% over 3 years. In an area with adequate intravenous as well as oral rehydration treatment facilities, rarely found in rural developing country communities, this could decrease the number of admissions to hospital and in an area without adequate facilities it could provide a proportionate decrease in cholera mortality.
Further developments Work to facilitate further the production and use of the oral B WC cholera vaccine described is in progress. By utilizing recombinant DNA technology, it may be possible to produce the B and WC components in a single step. This could greatly simplify large-scale production of the B-WC vaccine, reduce costs and allow local production of this vaccine in developing countries. Thus, a modified B-WC vaccine might be based on two strains - - one of classical and the other of E1 Tor biotype and representing the Inaba and Ogawa serotypes - - which through genetic manipulations lack the ability to produce cholera toxin but instead produce large amounts of cholera B subunit. With this goal in mind we have recently constructed overexpression systems in which the gene encoding the B subunit of cholera toxin has been placed under the control of tacP or other strong 'foreign' promoters in a wide host range multicopy plasmid (Ref. 12; M. Lebens and J. Holmgren, unpublished data). Recombinant non-toxigenic classical and E1 Tor V. cholerae strains of different serotypes harbouring such plasmids have been made to excrete more than 500 mg 1- x of B subunit and may therefore be useful killed oral vaccine strains. Indeed, the Swedish National Bacteriological Laboratory has already manufactured almost one million doses of B-WC cholera vaccine based on such recombinantly produced B subunit which are to be used in various studies in Latin America. The production strains and the conditions of cultivation and inactivation that will ultimately be chosen for preparing the vaccine would ideally also ensure the inclusion of two WC antigens that were missing in the original B-WC vaccine and that might further enhance
the protective immunogenicity: (a) TcpA, which appears to be an important adhesive pilus involved in the colonization of at least classical cholera vibrios in the human intestine 13 (this antigen was not known at the time of prepaling the original B-WC vaccine) and (b) the E1 Tor-associated cell-bound haemagglutinin (MSHA) which has also been implicated in cholera pathogenesis and immunity. Especially the latter antigen, which was recently shown to be of a fimbrial nature 14, is worth serious attention in this regard. MSHA is being found on all strains of E1 Tor V. cholerae, and specific antibodies against MSHA have been shown to protect animals against experimental cholera caused by V. cholerae of the E1 Tor but not the classical biotype xS. Yet another, practically important further development of the oral B-WC cholera vaccine would be to prepare a tablet or sachet formulation containing the vaccine in combination with alkaline buffer in a dry form. This would facilitate storage and distribution and probably also further increase the stability of these vaccines for use in cholera endemic areas. However, it should be mentioned that even in its liquid form the B-WC vaccine has proved to be exceptionally stable having shown intact immunogenicity in human volunteers after more than 8 years of storage (unpublished data ). ACKNOWLEDGEMENTS The authors are grateful to their many co-workers at the University of G6teborg, ICDDR,B, SBL, Institut M6rieux and ACO Ltd (Stockholm) for valuable contributions in essential parts of the studies. The basic research was supported by the Swedish Medical Research Council, the Swedish Agency for Research Co-operation with Developing Countries and the World Health Organization. REFERENCES 1 2 3 4
5
6
7
8
9
Holmgren, J. Actions of cholera toxin and the prevention and treatment of cholera. Nature 1981, 292, 413-417 Holmgren, J. and Svennerholm, A.-M. Cholera and the immune response. Progr. Allergy 1983, 33, 106-119 Holmgren, J., Clemens, J., Sack, D.A. and Svennerholm, A.-M. New cholera vaccines. Vaccine 1989, 7, 94-96 Svennerholm, A.-M., Jertborn, M., Gothefors, L., Karim, A.M.M.M, Sack, D.A. and Holmgren, J. Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine. J. Infect. Dis. 1984, 149, 884-893 Quiding, M., Nordstr6m, I., Kilander, A., Andersson, G., Hanson, L.-A., Holmgren, J. and Czerkinsky, C. Intestinal immune responses in humans. Oral cholera vaccination induces strong intestinal antibody responses, gamma-interferon production, and evokes local immunological memory. J. Clin. Invest. 1991, 88, 143-148 Black, R.E., Levine, M.M., Clements, M.L., Young, C.R., Svennerholm, A.-M. and Holmgren, J. Protective efficacy in man of killed whole vibrio oral cholera vaccine with and without the B subunit of cholera toxin. Infect. Imrnun. 1987, 77, 1116-1129 Clemens, J., Sack, D.A., Harris, J.R., Chakraborty, J., Khan, MR., Stanton, B.F. et al. Field trial of oral cholera vaccines in Bangladesh. Lancet 1986, i, 124-127 Clemens, J., Sack, D.A., Harris, J.R., van Loon, F., Chakraborty, J., Ahmed, F. et al. Field trial of oral cholera vaccines in Bangladesh: results from three-year follow-up. Lancet 1990, i, 270-273 Clemens, J., Sack, D.A., Harris, J.R., Chakraborty, J., Neogy, P.K., Stanton, B. et al. Cross-protection by B subunit-whole cell cholera vaccine against diarrhoea associated with heat-labile toxinproducing enterotoxigenic Escherichia coil: Results of a large-scale field trial. J. Infect. Dis. 1988, 158, 372-377
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Oral cholera vaccine: J. H o l m g r e n et al. 10
Peltola, H., Siitonen, A., KyrSnsepp&, H., Simula, I., Mattila, L., Oksanen, P. et al. Prevention of travellers' diarrhoea by oral B-subunit/whole cell cholera vaccine. Lancet 1991,338, 1285-1289 11 Clemens, J., Sack, D.A., Harris, J.R., Chakraborty, J., Khan, M.R., Stanton, B.F. et al. Impact of B subunit killed whole-cell and killed whole-cell-only oral vaccines against cholera upon treated diarrhoeal illness and mortality in an area endemic for cholera. Lancet 1988, i, 1375 1379 12 Sanchez, J. and Holmgren, J. Recombinant system for overexpression of cholera toxin B subunit in Vibrio cholerae as a basis for vaccine development. Proc. Natl Acad. Sci. USA 1989, 86,
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481 485 Herrington, D.A., Hale, R.H., Losonsky, G., Mekalanos, J.J., Taylor, R.K. and Levine, M.M. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. Exp. Med. 1988, 168, 1487-1492 14 Jonson, G., Holmgren, J. and Svennerholm, A.-M Identification of a mannose-binding pilus on Vibrio cholerae El Tor. Microb. Pathog. 1991, 11,433-441 15 Osek, J., Svennerholm, A.-M. and Holmgren, J. Protection against Vibrio cholerae El Tot infection by specific antibodies against mannose-binding hemagglutinin pili. Infect. Immun. in press
13
INTRODUCTION There is a great need for an effective cholera vaccine. Cholera remains an important cause of illness and death in many parts of the world, especially in Asia but also in Africa and the Middle East and, since 1991, in South and Central America as well. It has been estimated that more than 150000 people, both children and adults, die from cholera each year. Although cholera can be treated simply by oral and intravenous rehydration, diarrhoea treatment centres are still scarce in areas endemic for cholera, and use of an effective vaccine may in many areas be the most promising possibility to control the disease. However, the parenteral whole-cell cholera vaccines that have existed for almost a century to prevent cholera are no longer regarded useful from a public health standpoint, mainly because of the short duration of protection they provide. Several field trials since 1960 have shown that these vaccines usually protect older people for only a few months and fail to protect many young children at all. The identification of the critical role of a toxin in the *Department of Medical Microbiology and Immunology, University of GSteborg, Guldhedsgatan 10, S-413 46 G6teborg, Sweden. *International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh. ~National Bacteriological Laboratory, S-105 21 Stockholm, Sweden. ~To whom correspondence should be addressed 0264-410X/92/130911-04 :i(' 1992Butterworth-HeinemannLtd
pathogenesis of cholera led to great expectations that a more effective cholera vaccine could be developed based on the use of a toxoid. However, a large field trial with a parenterally administered glutaraldehyde toxoid vaccine in Bangladesh in the mid-1970s failed to give any better protection than that achieved by previous parenteral vaccines. This did not come as too much of a surprise, however, since during the time it took to organize, execute and evaluate the trial, it had become more clearly apparent from animal studies that protective cholera immunity does not depend on serum antibodies, which are the type of antibodies mainly stimulated by the parenteral vaccination, but rather on mucosal secretory IgA antibodies produced locally in the gut, which are only inefficiently stimulated by parenteral antigen administration. Therefore, since the late 1970s, attention has turned instead to development of oral vaccines that could stimulate intestinal immunity more efficiently. The different 'modern' approaches towards development of cholera vaccines have all taken their departure from the new insights into the mechanisms of disease and immunity in cholera achieved during the 1970s and early 1980s (for specific reviews, see Refs 1 and 2). First, the clarification of the subunit structure of cholera toxin and of the role of different subunits in the toxin action immediately suggested a way to prepare a safe and highly immunogenic 'toxoid' consisting of the purified cholera B subunits. Studies showed that the
Vaccine, Vol. 10, Issue 13, 1992 911
Oral cholera vaccine: d. Holmgren et al.
purified B subunit portion of cholera toxin was entirely devoid of toxicity, yet it contained selectively the protective toxin epitopes against which neutralizing antitoxin antibodies were directed. Indeed, it was found that the B subunit is particularly well suited to oral immunization because it retains the ability to bind to the intestinal epithelium, which has been shown to be important for stimulating mucosal immunity in animals, including local immunological memory 1'2. Another important line of vaccine research has focused on the gut mucosal immune system and how it can be stimulated by immunogens. These studies have defined the importance of locally produced IgA antibodies and IgA immunological memory for protection against cholera, and they have shown that the oral route of vaccine administration is usually superior to the parenteral route in both priming and boosting the mucosal immune system 1'2. A third important observation guiding the design of the new cholera vaccines has concerned the synergistic co-operation between antitoxic and antibacterial immune mechanisms in cholera. Two main protective antibodies have been identified, one being directed against the Vibrio cholerae cell wall lipopolysaccharide (LPS) and the other against the cholera toxin B subunits. Either of these two types of antibody can confer protection against disease by inhibiting bacterial colonization and toxin binding, respectively, and when present together in the gut they can have a strongly synergistic protective effect 1'2. The intense vaccine development and research efforts during the last 10 years have now provided one licenced oral cholera vaccine based on either a combination of non-living bacteria and purified B subunit antigen, and an additional candidate vaccine consisting of live attenuated mutants of Vibrio cholerae 01 producing the B subunit 3. The oral B subunit-whole cell vaccine
The composition of this vaccine was designed to safely provide the key antigens for evoking protective antitoxic and antibacterial mucosal immunity against V. cholerae 01 of different serotypes (Inaba and Ogawa) and biotypes (classical and E1 Tor). As mentioned, the B subunit component is completely non-toxic and is an exceptionally potent oral immunogen because of its ability to bind to the intestinal mucosa. The whole-cell (WC) component provides protective LPS antigens as well as various heat-labile bacterial antigens that may add further to the antibacterial immunogenic effect. The B - W C vaccine is given orally together with an alkaline buffer (provided by an effervescent tablet) to protect the vaccine during passage through the stomach. The vaccine has been extensively tested in several clinical trials in Swedish, Bangladeshi and American volunteers. In these studies, the vaccine has proved to be completely safe with no adverse reactions, and after either two or three doses, it stimulates a gut mucosal IgA antitoxic and antibacterial immune response (including memory) comparable to that induced by cholera disease itselP '5. In American volunteers who received three oral immunizations with either the B - W C vaccine or the WC vaccine component alone, vaccination protected against challenge with a dose of live cholera vibrios that caused disease in 100% of concurrently tested unvaccinated controls 6.
912
Vaccine, Vol. 10, Issue 13, 1992
The B - W C vaccine and the WC component without any B subunit (both vaccines produced by Institut M6rieux, France in collaboration with the National Bacteriological Laboratory of Sweden, SBL) have recently been evaluated in a large randomized placebocontrolled field trial involving 89 596 vaccinated adults and children in Bangladesh. A total of 63 000 people - - children aged 2-15 years and adult women received three oral doses, at 6 week intervals, of B WC vaccine, WC vaccine or a placebo and another 26000 participants took one or two doses of three agents. Each dose of B WC vaccine contained 1 x 1011 killed whole vibrios representing three different strains belonging to the Inaba and Ogawa serotypes and the classical and E1 Tor biotypes; the B WC vaccine also contained 1 mg of purified B subunit from cholera toxin. The main results after 3 years of follow-up are summarized in Table 1. Both vaccines have been found to protect against cholera for at least 3 years. The B WC vaccine had the advantage over the WC vaccine alone of a significantly higher initial efficacy level (85% versus 58%) for the initial 4 - 6 month period ~. After the first 8-12 months the efficacy of the two vaccines was similar, however 8. Overall, for the 3 years of follow-up the B - W C vaccine conferred 50% protection and the WC vaccine 52% protection against culture-proven cholera: protection after two doses was similar to that after three doses. For both vaccines, protective efficacy was of shorter duration in children vaccinated at the age of 2 - 5 years; after a high initial level of protection (100%) for B WC during the first 6 months of follow-up, protection declined and no protection was observed in this age group in the third year of follow-up. In contrast, the level of protection in persons aged > 5 years was about 65% for both vaccines and was sustained during the entire follow-up period. Protection was slightly higher against classical than E1 Tor cholera but did not differ with serotype (Inaba or Ogawa) 8. Assuming that protection in adult males would have equalled that seen in adult females the overall protective efficacy of two or three doses over 3 years of follow-up can be estimated to be 60% for the B - W C vaccine and 58% for the WC vaccine. The B - W C vaccine, through its B subunit component, also provided substantial protection, 67%, for a few Table 1 Results from randomized placebo-controlled field trial of oral B subunit-whole cell (B-WC) cholera vaccine and whole-cell (WC) vaccine alone given in three (or two) doses (ICDDR,B: Matlab, Bangladesh) Vaccine efficacy ( % ) B-WC After 6 months of follow-up All ages Persons > 5 years Children < 5 years
After 3 years of follow-up All ages
WC
85
58
77 100
62 53
50
52
Persons > 5 years Children < 5 years
63 26
68 23
Recipients of only two doses, all ages
64
39
Overall protection after two or three doses, all ages (including estimate for adult men)
60
58
Oral cholera vaccine: J, Holmgren et al.
months against diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) producing either heat-labile toxin (LT, that cross-reacts immunologically with cholera toxin) alone or LT together with heat-stable toxin (ST) 9. Protection against severe disease was 86% and against milder illness 54O/o9. A similar protective effect, ~70%, of two doses of the oral B-WC cholera vaccine against ETEC diarrhoea was recently reported by Peltola et al. 1° in Finnish travellers to Morocco. Furthermore, in Bangladesh, during the large field trial, both the B-WC and the WC vaccines substantially and in a sustained manner reduced the overall diarrhoea morbidity among the vaccinees. Thus, over the 3 year follow-up period there was a 25% reduction in admissions for 'all diarrhoea' and a 50% reduction in admissions for life-threatening diarrhoea in the vaccinated as compared with the placebo group (Ref. 11 and J. Clemens et al. unpublished). The results with the oral B-WC vaccines represent marked improvements as compared with those achieved previously using parenteral cholera vaccines. In a cholera endemic area immunization with an oral vaccine could decrease the number of episodes of life-threatening cholera by more than 50% over 3 years. In an area with adequate intravenous as well as oral rehydration treatment facilities, rarely found in rural developing country communities, this could decrease the number of admissions to hospital and in an area without adequate facilities it could provide a proportionate decrease in cholera mortality.
Further developments Work to facilitate further the production and use of the oral B WC cholera vaccine described is in progress. By utilizing recombinant DNA technology, it may be possible to produce the B and WC components in a single step. This could greatly simplify large-scale production of the B-WC vaccine, reduce costs and allow local production of this vaccine in developing countries. Thus, a modified B-WC vaccine might be based on two strains - - one of classical and the other of E1 Tor biotype and representing the Inaba and Ogawa serotypes - - which through genetic manipulations lack the ability to produce cholera toxin but instead produce large amounts of cholera B subunit. With this goal in mind we have recently constructed overexpression systems in which the gene encoding the B subunit of cholera toxin has been placed under the control of tacP or other strong 'foreign' promoters in a wide host range multicopy plasmid (Ref. 12; M. Lebens and J. Holmgren, unpublished data). Recombinant non-toxigenic classical and E1 Tor V. cholerae strains of different serotypes harbouring such plasmids have been made to excrete more than 500 mg 1- x of B subunit and may therefore be useful killed oral vaccine strains. Indeed, the Swedish National Bacteriological Laboratory has already manufactured almost one million doses of B-WC cholera vaccine based on such recombinantly produced B subunit which are to be used in various studies in Latin America. The production strains and the conditions of cultivation and inactivation that will ultimately be chosen for preparing the vaccine would ideally also ensure the inclusion of two WC antigens that were missing in the original B-WC vaccine and that might further enhance
the protective immunogenicity: (a) TcpA, which appears to be an important adhesive pilus involved in the colonization of at least classical cholera vibrios in the human intestine 13 (this antigen was not known at the time of prepaling the original B-WC vaccine) and (b) the E1 Tor-associated cell-bound haemagglutinin (MSHA) which has also been implicated in cholera pathogenesis and immunity. Especially the latter antigen, which was recently shown to be of a fimbrial nature 14, is worth serious attention in this regard. MSHA is being found on all strains of E1 Tor V. cholerae, and specific antibodies against MSHA have been shown to protect animals against experimental cholera caused by V. cholerae of the E1 Tor but not the classical biotype xS. Yet another, practically important further development of the oral B-WC cholera vaccine would be to prepare a tablet or sachet formulation containing the vaccine in combination with alkaline buffer in a dry form. This would facilitate storage and distribution and probably also further increase the stability of these vaccines for use in cholera endemic areas. However, it should be mentioned that even in its liquid form the B-WC vaccine has proved to be exceptionally stable having shown intact immunogenicity in human volunteers after more than 8 years of storage (unpublished data ). ACKNOWLEDGEMENTS The authors are grateful to their many co-workers at the University of G6teborg, ICDDR,B, SBL, Institut M6rieux and ACO Ltd (Stockholm) for valuable contributions in essential parts of the studies. The basic research was supported by the Swedish Medical Research Council, the Swedish Agency for Research Co-operation with Developing Countries and the World Health Organization. REFERENCES 1 2 3 4
5
6
7
8
9
Holmgren, J. Actions of cholera toxin and the prevention and treatment of cholera. Nature 1981, 292, 413-417 Holmgren, J. and Svennerholm, A.-M. Cholera and the immune response. Progr. Allergy 1983, 33, 106-119 Holmgren, J., Clemens, J., Sack, D.A. and Svennerholm, A.-M. New cholera vaccines. Vaccine 1989, 7, 94-96 Svennerholm, A.-M., Jertborn, M., Gothefors, L., Karim, A.M.M.M, Sack, D.A. and Holmgren, J. Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine. J. Infect. Dis. 1984, 149, 884-893 Quiding, M., Nordstr6m, I., Kilander, A., Andersson, G., Hanson, L.-A., Holmgren, J. and Czerkinsky, C. Intestinal immune responses in humans. Oral cholera vaccination induces strong intestinal antibody responses, gamma-interferon production, and evokes local immunological memory. J. Clin. Invest. 1991, 88, 143-148 Black, R.E., Levine, M.M., Clements, M.L., Young, C.R., Svennerholm, A.-M. and Holmgren, J. Protective efficacy in man of killed whole vibrio oral cholera vaccine with and without the B subunit of cholera toxin. Infect. Imrnun. 1987, 77, 1116-1129 Clemens, J., Sack, D.A., Harris, J.R., Chakraborty, J., Khan, MR., Stanton, B.F. et al. Field trial of oral cholera vaccines in Bangladesh. Lancet 1986, i, 124-127 Clemens, J., Sack, D.A., Harris, J.R., van Loon, F., Chakraborty, J., Ahmed, F. et al. Field trial of oral cholera vaccines in Bangladesh: results from three-year follow-up. Lancet 1990, i, 270-273 Clemens, J., Sack, D.A., Harris, J.R., Chakraborty, J., Neogy, P.K., Stanton, B. et al. Cross-protection by B subunit-whole cell cholera vaccine against diarrhoea associated with heat-labile toxinproducing enterotoxigenic Escherichia coil: Results of a large-scale field trial. J. Infect. Dis. 1988, 158, 372-377
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481 485 Herrington, D.A., Hale, R.H., Losonsky, G., Mekalanos, J.J., Taylor, R.K. and Levine, M.M. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. Exp. Med. 1988, 168, 1487-1492 14 Jonson, G., Holmgren, J. and Svennerholm, A.-M Identification of a mannose-binding pilus on Vibrio cholerae El Tor. Microb. Pathog. 1991, 11,433-441 15 Osek, J., Svennerholm, A.-M. and Holmgren, J. Protection against Vibrio cholerae El Tot infection by specific antibodies against mannose-binding hemagglutinin pili. Infect. Immun. in press
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