- Email: [email protected]
Development of O-specific polysaccharide-protein conjugates is based upon the protective effect of serum vibriocidal antibodies against cholera
0
INSTITUT
Bull. Inst. Pasteur
PASTEUR/ELSEVIER
Paris 1995
1995, 93, 269-272
Development of O-specific polysaccharide-protein conjugates is based upon the protective effect of serum vibriocidal antibodies against cholera S.C. Szu, R. Gupta,
P. Kovli;,
D.N. Taylor
and J.B. Robbins
(*)
National Institutes of Health, Bethesda, MD 20892 and Walter Reed Army Institute for Research, Washington, DC, 20014 (USA)
Our vaccine development program for cholera is based upon epidemiologic, clinical and immunologic data : 1) The highest attack rate, morbidity and mortality of cholera is in children (Mosley, 1969 ; Mosley et aZ., 1970; Tauxe et aZ., 1994). A vaccine for cholera should be safe and effective in infancy and in young children. Widespread vaccination for cholera will require a product that can be included into the vaccine schedule for children ; 2) Cholera is considered a non-invasive disease because the causative agent is confined to the intestinal lumen and the symptoms are caused by a pro tein toxin. The infectious dose of Vibrio cholerae 01 for volunteers who have eaten recently or in volunteers who have had their gastric pH buffered deliberately is = lo3 (Cash et al., 1974; Levine et al, 1981). Vaccine-induced immunity will have to kill the entire inoculum since, it is probable, as few as 10 viable organisms in the small intestine may cause cholera ; 3) Despite the urgency of the problem and almost a century’s worth of study throughout the world, there is no consensus about what is the protective antigen or host factor that confers immunity to cholera. Surprisingly, serum vibriocidal antibodies represent the only immune moiety that has been correlated with resistance to cholera caused by V. cholerae 01: the attack rate varies inversely with the serum level and the disease occurs rarely in indi-
Received
October
viduals with a titre of 21:160 (Mosley, 1969; Mosley et al., 1970; Szu et al., 1994). Further, newborns and very young children, independent of breast feeding, have a lower attack rate of cholera than older children, suggesting that maternally-derived serum IgG may confer immunity (Clemens et al., 1990; Mosley, 1969). Because of the almost universal view of cholera as a “mucosal” disease, serum vibriocidal antibodies are viewed as a “marker” of immunity and are not involved directly in protection (Clemens et al., 1990a,b; Finkelstein, 1984; Glass et al., 1985 ; Migasena et al., 1989) ‘. 4) Most, if not all, serum vibriocidal activity from convalescent sera can be absorbed by LPS. Serum IgM and IgG antibodies lyse V. cholerae 0:l with complement (Ahmed et al., 1970; Finlcelstein, 1962, 1984; Gupta et al., 1992, 1996; Neoh and Rowky, 1970; Szu et al., 1994). 5) Although protective in some animal models, there is no experimental data that serum or secretory antitoxin or secretory anti-LPS has preventative activity against cholera in humans (Clemens et al., 1990a; Glass et al., 1985; Nor&i, 1977). On the basis of these data, we proposed that a critical level of vaccine-induced serum IgG vibriocidal antibodies could confer long-lived protective immunity to cholera, as well as to other enteric bacterial infections, and outlined how this may occur (Gupta et al., 1992, 1996; Robbins et al., 1992, 1995; Robbins and Schneerson, 1990; Szu et al., 1994).
6, 1995.
(*) For correspondence. (I) To date, there are no secretory
antibodies
of defined
specificity
or titre
suggested
as an estimated
“protective”
level.
270
2nd FORUM IN THE BULLETIN
1) In most cases, inocula of V. cholerue 01 and other enteric pathogen are small (= IO5 with feeding and = lo3 in fasting state reaching the jejunum). 2) Serum antibodies (mostly IgG) exude into the intestine. It is likely that serum complement proteins are also present. 3) Intestinal walls are in contact and churning (peristalsis) : there is no functional “lumen”. 4) Serum antibodies lyse pathogens on intestinal surface. V. choler-m 01, salmonellae, shigellae, and E. coli 0157 are susceptible to antibody-mediated complement-dependent lysis. Routine vaccination for cholera in endemic and epidemic areas has not been implemented due to deficiencies in the existing vaccines : 1) Parenterally administered cellular vaccines (inactivated V. cholerue 01) cause side reactions, and provide limited protective immunity in adults of ~60% with a duration of = 6 months and confer a lesser efficacy in children (Mosley, 1969). Purified LPS, as an investigational vaccine, elicited slightly higher rates of protection. Recipients of these two vaccines, with vibriocidal titres 2 l/160, were highly resistant to cholera. Orally administered cellular vaccines are safe and confer a similar degree of protective immunity administered parenterally for =3 years including a lesser efficacy rate in children (Clemens et al., 1990b). To date, a correlation between vaccine-induced host immune factors and protection has not been shown. 2) An orally administered attenuated CVD 103HgR strain of V. cholerue is safe but its efficacy and immunogenicity in young children have not yet been studied (Migasena ef al., 1989). The proponents measured serum vibriocidal antibodies to satisfy the requirements of the FDA and, indeed, levels of > l/160 vibriocidal activity were achieved in most adults after one dose. 3) The above vaccines are not compatible for inclusion in the formulation of bacterial vaccine (DTP, Hib conjugate) given routinely to infants. We developed synthetic schemes for O-specific polysaccharide-protein conjugates to elicit serum vibriocidal antibodies because : 1) Conjugates are prepared with purified components and laboratory standardization for contamination with LPS or “pyrogen” has reliably predicted their safety (Robbins and Schneerson, 1990). 2) Conjugates can be standardized by unambiguous chemical and biologic reactions so that their immunologic properties can be predicted reliably. 3) Conjugates elicit protective levels of IgG antipolysaccharide antibodies in infants when administered concurrently with recommended bacterial vaccines. If shown to be effective, conjugates can be
DE L’INSTITUT
PASTEUR
administered routinely without additional visits for vaccination. Our objective is to prepare conjugates with the O-specific polysaccharide of V. cholerue 0: 1 in order to elicit long-lived serum IgG LPS antibodies with bactericidal (vibriocidal) activity. The immunogenicity of conjugates in mice prepared with LPS from V. cholerue 01 serotype Inaba, treated with hydrazine or acetic acid, bound to cholera toxin by either single or multipoint attachment was characterized (Gupta et al., 1992). The most immunogenic conjugate was prepared with hydrazine-treated cyanogen bromide-activated LPS bound to cholera toxin with a spacer molecule. Vibriocidal antibodies elicited in mice by these conjugates and cellular vaccines were specific for the LPS. On the basis of these results, we evaluated the safety and immunogenicity of our conjugates in healthy adults (phase 1) composed of cholera toxin conjugated to hydrazine-treated LPS (DeALPS) of V. cholerue 01, serotype Inaba (Gupta et al., 1996). Conjugates of DeALPS, from serotype Inaba bound to cholera toxin, were administered to adults. Controls were given either 25 pg DeALPS alone or the licensed cellular vaccine containing 4x 109/ml each of serotypes Inaba and Ogawa. No serious adverse reactions were observed. DeALPS alone elicited only low levels of IgM LPS antibodies. Both the conjugates and cellular vaccine elicited IgM antiLPS (difference NS). The conjugates, in contrast, elicited the highest levels of IgG LPS antibodies. Both conjugates and the cellular vaccine elicited vibriocidal antibodies. The correlation coefficient between IgG anti-LPS (ELISA) and 2-mercaptoethanol-resistant vibriocidal antibodies (IgG) was 0.81 (p=O.O004) indicating that the two assays measured the same molecules. Convalescent sera from cholera patients in Mexico had mean vibriocida1 titre of 2,525 that was removed by treatment with 2-mercaptoethanol indicating that most of this activity was IgM. As expected, serum vibriocidal activities of all groups were mostly absorbed by LPS but not by either cholera toxin. Conjugates elicited IgG vibriocidal antibodies that persist longer: studies have shown that IgG has a higher penetration into the intestinal fluid than IgM. Evaluation of these conjugates in children is being planned. Emphasis has been directed towards development of attenuated strains of V. cholerue, as orally administered vaccine to stimulate secretory IgA is based largely upon the incomplete and short-lived immunity conferred by parenterally-administered inactivated cells or LPS (Mosley, 1969). But there is an explanation for the less-than-optimal immunologic properties of injected cellular cholera vaccines. Parenterally-administered inactivated Gram-negative bacteria, including cellular cholera vaccine, elicit high levels of mostly IgM anti-LPS (Baumgartner er al., 1991; DeMaria et al., 1988). Simi-
CHOLERA larly, most vibriocidal antibodies in patients are also IgM anti-LPS and their levels decline in about 6 months (Finkelstein, 1984 ; Mosley, 1969 ; Mosley et al., 1970). The immune response, confined largely to IgM anti-LPS, provides an explanation for the short-lived protection of parenterally administered cellular vaccines and LPS as well as recurrence of cholera in patients (Levine, 1980; Woodword, 1971). We hope that conjugate vaccines, with their T-helper cell component, will elicit IgG antiLPS and overcome this deficiency. The O-specific polysaccharides of Inaba and Ogawa serotypes are a linear homopolymer of a( 1 + 2)-D-perosamine whose amino groups are acylated by 3-deoxy-L-glycero-tetronic acid (Kenne et al., 1979). The structural basis for their crossreactivity appears to be that both serotypes have the same O-specific polysaccharide (identity) but that Ogawa has a methyl group on C-2 of the non-reducing end (Ito et al., 1994). The immunogenicity of polysaccharides in a conjugate is related to their molecular size and density on the carrier protein (Robbins and Schneerson, 1990). The O-specific polysaccharide of V. cholerae 01 consists of = 15 residues (Gupta et al, 1992). The methyl ol-glycosides of mono-, di- and trisaccharide of the O-specific polysaccharide of V. cholerae 01, serotypes Inaba and the analogous saccharide of Ogawa (up to the tetrasaccharide), have been synthesized. We have also prepared a crystalline functionalized hexasaccharide fragment of the O-specific polysaccharide of V. cholerae 01, serotype Inaba, suitable for conjugation (Gotoh and KovgE, 1995; Lei et al., 1995a,b). Our current endeavors are directed, inter alia, towards preparation of conjugates utilizing oligosaccharide analogs of the O-specific polysaccharide of V. cholerae 01 and use of both the Inaba and Ogawa LPS types. V. cholerae 0139 now causes a significant proportion of cholera (Bodhidatta et al., 1995 ; Tauxe et al, 1994). This serotype is highly similar to V. cholerue 01 except that its LPS lacks the O-specific polysaccharide and it has a capsular polysaccharide that confers resistance to fresh serum (Johnson et al., 1994; Waldor et al., 1994). It is likely that immunity to this variant will require a critical level of serum capsular antibodies and we plan to synthesize and evaluate protein conjugates of this saccharide.
References Ahmed, A., Bhattacharjee, A.K. & Mosley, W.H. (1970), Characteristics of the serum vibriocidal and agglutinating antibodies in cholera cases and in normal residents of the endemic and non-endemic cholera areas. J. Zmmunol., 105, 431-441. Baumgartner, J.D., Heumann, D., Calandra, T. & Glauser, M.P. (1991), Antibodies to lipopolysaccharide after
VACCINES
271
immunization of humans with rough mutant Escherichia coli. .I. infect. Dis., 163, 769-772. Bodhidatta, L., Echeverria, P., Hoge, C.W., Piarangsi, C., Serichantalergs, O., Henprasert-tae, N., Harikul, S. & Kitpoka, P. (1995). Vibrio cholerue 0139 in Thailand in 1994. Epidemiol. Infect., 114, 71-73. Cash, R.A., Music, S.I., Libonati, J.P., Synder, M.J., Wenzel, R.P. & Homick, R.B. (1974), Response of man to infection with Vibrio cholerue. I. Clinical, serologic, and bacteriologic responses to a known inoculum. J. Infect. Dis., 129, 45-52. Clemens, J.D., Sack, D.A., Chakraborty, J., Rao, M.R., Ahmed, F., Harris, J.R., van Leon, F., Khan, M.R., Yunis, M., Huda, S., Kay, B.A., Svennerholm, A.-M. & Holmgren, J. (199Oa), Field trial of oral cholera vaccines in Bangladesh: evaluation of anti-bacterial and anti-toxic breast milk immunity in response to ingestion of the vaccines. Vaccine, 8, 469-472. Clemens, J.D., Sack, D.A., Harris, J.R., van Loon, F., Chakraborty, J., Ahmed, F., Stanton, B.F., Kay, B.A., Walter, S., Eeckels, R., Svennerholm, A.-M. & Holmgren, J. (199Ob), Field trial of oral cholera vaccines in Bangladesh: results from a three-year follow-up. Luncet, 335, 270-273. DeMaria, A., Johns, M.A., Berbeerich, H. & McCabe, W.R. (1988), Immunization with rough mutants of Salmonella minnesota: initial studies in human subjects. J. Znfect. Dis., 158, 301-311. Finkelstein, R.A. (1962), Vibriocidal antibody inhibition (VAI) analysis: a technique for the identification of the predominant vibriocidal antibodies in serum and for the detection and identification of Vibrio cholerae antigens. J. Zmmunol., 264-271. Finkelstein, R.A. (1984), Cholera, in “Bacterial Vaccines”, R. Germanier (ed.) (pp. 107-136). Academic Press, Inc., New York. Glass, R.I., Svennerholm, A.-M., Khan, M.R., Huda, S., Huq, M.I. & Holmgren, J. (1985), Seroepidemiological studies of El Tor cholera in Bangladesh: association of serum antibody levels with protection. .I. Infect. Dis., 151, 236-242.
Gotoh, M. & Kov&$ P. (1995), Synthesis of specifically deoxygenated analogues of the methyl a-glycoside of the intracatenary monosaccharide repeating unit of the 0-polysaccharide of Vibrio cholerae 0: 1. Carbohydr. Res., 268, 73-84. Gupta, R.K., Szu, SC., Finkelstein, R.A. & Robbins, J.B. (1992), Synthesis, characterization and some immunological properties of conjugates composed of the detoxified lipopolysaccharide of Vibrio choIerue 01 serotype lnaba bound to cholera toxin. Infect. Zmmun., 60, 3201-3208. Gupta, R.K., Taylor, D.N., Bryla, D.A., Robbins, J.B. & Szu, SC. (1996), Evaluation in adult volunteers of Vibrio cholerae 01, serotype lnaba, polysaccharidecholera toxin conjugates. I. Znfect. Dis. (in press). lto, T., Higuchi, T., Hirobe, M., Hiramatsu, K. & Yokota, T. (1994), Identification of a novel sugar, 4-amino4,6-dideoxy-0-methylmannose in the lipopolysaccharide of Vibrio cholerae 01 serotype Ogawa. Carbohydr. Res., 256, 113-128. Johnson, J.A., Salles, C.A., Panigraphi, P., Albert, M.J., Wright, A.C., Johnson, R.J. & Morris, J.G., Jr. (1994), Vibrio cholerue 0139 synonym Bengal is closely related to Vibrio cholerue El Tor but has
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IN THE
BULLETIN
important differences. Infect. Immun., 62, 2108 2110. Kenne, L., Lindberg, B., Unger, P., Holme, T. & Holmgren, J. (1979). Structural studies of the Vibrio cholerue O-antigen. Curbohydr, Res., 68, C14-C16. Lei, P.S., Ogawa, Y., Flippen-Anderson, J.L. & Kov& P. (1995a). Synthesis and crystal structure of methyl 4,6-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-20-methyl-a-D-mannopyranoside, the methyl ol-glycoside of the terminal unit and presumed antigenic determination of the O-specific polysaccharide of Vibrio cholerue 0: 1, serotype Ogawa. Curbohydr. Res. (in press).
Lei, P.S., Ogawa, Y., Flippen-Anderson, J.L. & Kova& P. (1995b), Synthesis of the methyl cz-glycosidesof a di-, tri- and a tetrasaccharide fragment mimicking the terminus of the 0-polysaccharide of Vibrio cholerue 0: 1, serotype Ogawa. Curbohydr. Res. (in press). Levine, M.M. (1980), Immunity to cholera as evaluated in volunteers. Cholera and Related Diarrheas (0. Ouchterlony and J. Holmgren) (pp. 195203). 43rd Nobel Symposium. Stockholm, 1978. Levine, M.M., Black, R.E., Clements, M.L., Ciscernos, I., Nalin, R. & Young, C.R. (1981), The quality and duration of infection-derived immunity to cholera. J. Znfect. Dis., 143, 818820. Migasena, S., Pitisuttitham, P., Prayurahong, B., Suntharasamai, P., Supanaranonod, W., Desakom, Y., Vongsthongsri, U., Tall, B., Ketley, J., Losonsky, G., Cryz, S., Kaper, J.B. & Levine, M.M. (1989), Preliminary assessmentof the safety and immunogenicity of live cholera vaccine CVD103-HgR in healthy Thai adults. infect. Immun., 57, 3261-3264. Mosley, W.H. (1%9), The role of immunity in cholera. A review of epidemiological and serological studies. Tex. Rep. Biol. Med., 27 (Suppl. 1). 227-241. Mosley, W.H., Woodward, W.E., &iz, KM.A., Rahman, A.S.M.M., Chowdhury, A.K.M.A., Ahmed, A. & Feeley, J.C. (1970). The 19681969 chol~a+accine field trial in rural East Pakistan. Effectiveness of monovalent Ogawa and Inaba vaccinesand a purified Inaba antigen, with comparative results of serological and animal protection tests.J. Z@ct. Dis., 121, Sl-S9.
DE L’INSTITUT
PASTEIJR
Neoh, S.H. & Rowley, D. (197D), The antigens of Vibrio cholerue involved in the vibriocidal action of antibody and complement. J. Infecf. Dis., 121, 505-513. Nor&i, HI (1977), Evaluation of toxoid field trial in the Philippines, in ‘$Proceedipgsof the 12th Joint Conference U.S.-Japan Cooperative Medjcal S&Ice Program”. Symposium op Cholera. Sapporo, 1976 (Fukimi, H. and Zinnaka, Y. Eda) (pp. 302-310). National Institute of Health, Tokyo, Japan. Robbins, J.B., Chu, C. C Schneerson,R. (1992), Hypothesis for vaccine development: protective immunity to enteric diseasescaused by nontyphoidal Salmonellae and Shigellae may be conferred by serum IgG antibodies to the O-specific p~ly+&nuide of their lipopolysaccharides. Clin. Znfict. Dis., 15, 346-361. R&bins, J.B., Schneerson, R. & Szu, $.C (1995), Perspeetive: Hypothesis: Serum IgG antibody is suffleient to confer vote&on against infectious diseases by inactivating tlte inoculum. J. In$ piq, 171, 13871398. Robbins, J.R. & Schneerson, R. (199fJ), Bolysa&~~protein conjugates: a new generation of vaccines. J. Znfect. Dis., 161, 821-832. Szu, SC., Gupta, R, ,& Robbins, J.B. (1994), Induction of ecfum vibriocidal antibodies by O-specific polysacchar&-prt$eiB conjugate vaccines for preventiop of cholera, in “Vibrio chql,epe and Cholera” (Wachsmuth, I.K., Blake, P.A. and Glsvjk, 0. eds.) (pp. 381-394). American Society for Microbiology; Washington, DC. Tauxe, R., Blake, P., Olsvik, 0. & Wachsmuth, I.K. (1994), The future of cholera: persistence, change, and expanding research agenda, in “Vibrio cholerue and cholera” (Wachsmuth, I.K., Blake, P.A. and Olsvik, 0. (eds.)) (pp. 443-453). American Society for Microbiology, DC. Waldor, M.K., Colwell, I& & Mekalanos, J.J. (1994), The Vibrio cholerue 0139 serogroup includes an O-antigen capsule and lipopolysaccharid,e virulence determinants. Proc. Nutl. Acud. Sci. USA, 91, 1138811393. Woodwatd, W.E. (1971), Cholera reinfection in man. J. Infect. Dis., 123, 61-66.
INSTITUT
Bull. Inst. Pasteur
PASTEUR/ELSEVIER
Paris 1995
1995, 93, 269-272
Development of O-specific polysaccharide-protein conjugates is based upon the protective effect of serum vibriocidal antibodies against cholera S.C. Szu, R. Gupta,
P. Kovli;,
D.N. Taylor
and J.B. Robbins
(*)
National Institutes of Health, Bethesda, MD 20892 and Walter Reed Army Institute for Research, Washington, DC, 20014 (USA)
Our vaccine development program for cholera is based upon epidemiologic, clinical and immunologic data : 1) The highest attack rate, morbidity and mortality of cholera is in children (Mosley, 1969 ; Mosley et aZ., 1970; Tauxe et aZ., 1994). A vaccine for cholera should be safe and effective in infancy and in young children. Widespread vaccination for cholera will require a product that can be included into the vaccine schedule for children ; 2) Cholera is considered a non-invasive disease because the causative agent is confined to the intestinal lumen and the symptoms are caused by a pro tein toxin. The infectious dose of Vibrio cholerae 01 for volunteers who have eaten recently or in volunteers who have had their gastric pH buffered deliberately is = lo3 (Cash et al., 1974; Levine et al, 1981). Vaccine-induced immunity will have to kill the entire inoculum since, it is probable, as few as 10 viable organisms in the small intestine may cause cholera ; 3) Despite the urgency of the problem and almost a century’s worth of study throughout the world, there is no consensus about what is the protective antigen or host factor that confers immunity to cholera. Surprisingly, serum vibriocidal antibodies represent the only immune moiety that has been correlated with resistance to cholera caused by V. cholerae 01: the attack rate varies inversely with the serum level and the disease occurs rarely in indi-
Received
October
viduals with a titre of 21:160 (Mosley, 1969; Mosley et al., 1970; Szu et al., 1994). Further, newborns and very young children, independent of breast feeding, have a lower attack rate of cholera than older children, suggesting that maternally-derived serum IgG may confer immunity (Clemens et al., 1990; Mosley, 1969). Because of the almost universal view of cholera as a “mucosal” disease, serum vibriocidal antibodies are viewed as a “marker” of immunity and are not involved directly in protection (Clemens et al., 1990a,b; Finkelstein, 1984; Glass et al., 1985 ; Migasena et al., 1989) ‘. 4) Most, if not all, serum vibriocidal activity from convalescent sera can be absorbed by LPS. Serum IgM and IgG antibodies lyse V. cholerae 0:l with complement (Ahmed et al., 1970; Finlcelstein, 1962, 1984; Gupta et al., 1992, 1996; Neoh and Rowky, 1970; Szu et al., 1994). 5) Although protective in some animal models, there is no experimental data that serum or secretory antitoxin or secretory anti-LPS has preventative activity against cholera in humans (Clemens et al., 1990a; Glass et al., 1985; Nor&i, 1977). On the basis of these data, we proposed that a critical level of vaccine-induced serum IgG vibriocidal antibodies could confer long-lived protective immunity to cholera, as well as to other enteric bacterial infections, and outlined how this may occur (Gupta et al., 1992, 1996; Robbins et al., 1992, 1995; Robbins and Schneerson, 1990; Szu et al., 1994).
6, 1995.
(*) For correspondence. (I) To date, there are no secretory
antibodies
of defined
specificity
or titre
suggested
as an estimated
“protective”
level.
270
2nd FORUM IN THE BULLETIN
1) In most cases, inocula of V. cholerue 01 and other enteric pathogen are small (= IO5 with feeding and = lo3 in fasting state reaching the jejunum). 2) Serum antibodies (mostly IgG) exude into the intestine. It is likely that serum complement proteins are also present. 3) Intestinal walls are in contact and churning (peristalsis) : there is no functional “lumen”. 4) Serum antibodies lyse pathogens on intestinal surface. V. choler-m 01, salmonellae, shigellae, and E. coli 0157 are susceptible to antibody-mediated complement-dependent lysis. Routine vaccination for cholera in endemic and epidemic areas has not been implemented due to deficiencies in the existing vaccines : 1) Parenterally administered cellular vaccines (inactivated V. cholerue 01) cause side reactions, and provide limited protective immunity in adults of ~60% with a duration of = 6 months and confer a lesser efficacy in children (Mosley, 1969). Purified LPS, as an investigational vaccine, elicited slightly higher rates of protection. Recipients of these two vaccines, with vibriocidal titres 2 l/160, were highly resistant to cholera. Orally administered cellular vaccines are safe and confer a similar degree of protective immunity administered parenterally for =3 years including a lesser efficacy rate in children (Clemens et al., 1990b). To date, a correlation between vaccine-induced host immune factors and protection has not been shown. 2) An orally administered attenuated CVD 103HgR strain of V. cholerue is safe but its efficacy and immunogenicity in young children have not yet been studied (Migasena ef al., 1989). The proponents measured serum vibriocidal antibodies to satisfy the requirements of the FDA and, indeed, levels of > l/160 vibriocidal activity were achieved in most adults after one dose. 3) The above vaccines are not compatible for inclusion in the formulation of bacterial vaccine (DTP, Hib conjugate) given routinely to infants. We developed synthetic schemes for O-specific polysaccharide-protein conjugates to elicit serum vibriocidal antibodies because : 1) Conjugates are prepared with purified components and laboratory standardization for contamination with LPS or “pyrogen” has reliably predicted their safety (Robbins and Schneerson, 1990). 2) Conjugates can be standardized by unambiguous chemical and biologic reactions so that their immunologic properties can be predicted reliably. 3) Conjugates elicit protective levels of IgG antipolysaccharide antibodies in infants when administered concurrently with recommended bacterial vaccines. If shown to be effective, conjugates can be
DE L’INSTITUT
PASTEUR
administered routinely without additional visits for vaccination. Our objective is to prepare conjugates with the O-specific polysaccharide of V. cholerue 0: 1 in order to elicit long-lived serum IgG LPS antibodies with bactericidal (vibriocidal) activity. The immunogenicity of conjugates in mice prepared with LPS from V. cholerue 01 serotype Inaba, treated with hydrazine or acetic acid, bound to cholera toxin by either single or multipoint attachment was characterized (Gupta et al., 1992). The most immunogenic conjugate was prepared with hydrazine-treated cyanogen bromide-activated LPS bound to cholera toxin with a spacer molecule. Vibriocidal antibodies elicited in mice by these conjugates and cellular vaccines were specific for the LPS. On the basis of these results, we evaluated the safety and immunogenicity of our conjugates in healthy adults (phase 1) composed of cholera toxin conjugated to hydrazine-treated LPS (DeALPS) of V. cholerue 01, serotype Inaba (Gupta et al., 1996). Conjugates of DeALPS, from serotype Inaba bound to cholera toxin, were administered to adults. Controls were given either 25 pg DeALPS alone or the licensed cellular vaccine containing 4x 109/ml each of serotypes Inaba and Ogawa. No serious adverse reactions were observed. DeALPS alone elicited only low levels of IgM LPS antibodies. Both the conjugates and cellular vaccine elicited IgM antiLPS (difference NS). The conjugates, in contrast, elicited the highest levels of IgG LPS antibodies. Both conjugates and the cellular vaccine elicited vibriocidal antibodies. The correlation coefficient between IgG anti-LPS (ELISA) and 2-mercaptoethanol-resistant vibriocidal antibodies (IgG) was 0.81 (p=O.O004) indicating that the two assays measured the same molecules. Convalescent sera from cholera patients in Mexico had mean vibriocida1 titre of 2,525 that was removed by treatment with 2-mercaptoethanol indicating that most of this activity was IgM. As expected, serum vibriocidal activities of all groups were mostly absorbed by LPS but not by either cholera toxin. Conjugates elicited IgG vibriocidal antibodies that persist longer: studies have shown that IgG has a higher penetration into the intestinal fluid than IgM. Evaluation of these conjugates in children is being planned. Emphasis has been directed towards development of attenuated strains of V. cholerue, as orally administered vaccine to stimulate secretory IgA is based largely upon the incomplete and short-lived immunity conferred by parenterally-administered inactivated cells or LPS (Mosley, 1969). But there is an explanation for the less-than-optimal immunologic properties of injected cellular cholera vaccines. Parenterally-administered inactivated Gram-negative bacteria, including cellular cholera vaccine, elicit high levels of mostly IgM anti-LPS (Baumgartner er al., 1991; DeMaria et al., 1988). Simi-
CHOLERA larly, most vibriocidal antibodies in patients are also IgM anti-LPS and their levels decline in about 6 months (Finkelstein, 1984 ; Mosley, 1969 ; Mosley et al., 1970). The immune response, confined largely to IgM anti-LPS, provides an explanation for the short-lived protection of parenterally administered cellular vaccines and LPS as well as recurrence of cholera in patients (Levine, 1980; Woodword, 1971). We hope that conjugate vaccines, with their T-helper cell component, will elicit IgG antiLPS and overcome this deficiency. The O-specific polysaccharides of Inaba and Ogawa serotypes are a linear homopolymer of a( 1 + 2)-D-perosamine whose amino groups are acylated by 3-deoxy-L-glycero-tetronic acid (Kenne et al., 1979). The structural basis for their crossreactivity appears to be that both serotypes have the same O-specific polysaccharide (identity) but that Ogawa has a methyl group on C-2 of the non-reducing end (Ito et al., 1994). The immunogenicity of polysaccharides in a conjugate is related to their molecular size and density on the carrier protein (Robbins and Schneerson, 1990). The O-specific polysaccharide of V. cholerae 01 consists of = 15 residues (Gupta et al, 1992). The methyl ol-glycosides of mono-, di- and trisaccharide of the O-specific polysaccharide of V. cholerae 01, serotypes Inaba and the analogous saccharide of Ogawa (up to the tetrasaccharide), have been synthesized. We have also prepared a crystalline functionalized hexasaccharide fragment of the O-specific polysaccharide of V. cholerae 01, serotype Inaba, suitable for conjugation (Gotoh and KovgE, 1995; Lei et al., 1995a,b). Our current endeavors are directed, inter alia, towards preparation of conjugates utilizing oligosaccharide analogs of the O-specific polysaccharide of V. cholerae 01 and use of both the Inaba and Ogawa LPS types. V. cholerae 0139 now causes a significant proportion of cholera (Bodhidatta et al., 1995 ; Tauxe et al, 1994). This serotype is highly similar to V. cholerue 01 except that its LPS lacks the O-specific polysaccharide and it has a capsular polysaccharide that confers resistance to fresh serum (Johnson et al., 1994; Waldor et al., 1994). It is likely that immunity to this variant will require a critical level of serum capsular antibodies and we plan to synthesize and evaluate protein conjugates of this saccharide.
References Ahmed, A., Bhattacharjee, A.K. & Mosley, W.H. (1970), Characteristics of the serum vibriocidal and agglutinating antibodies in cholera cases and in normal residents of the endemic and non-endemic cholera areas. J. Zmmunol., 105, 431-441. Baumgartner, J.D., Heumann, D., Calandra, T. & Glauser, M.P. (1991), Antibodies to lipopolysaccharide after
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