Evaluation of Peru-15, a New Live Oral Vaccine for Cholera, in Volunteers

Transcription

1 201 Evaluation of Peru-15, a New Live Oral Vaccine for Cholera, in Volunteers David A. Sack, R. Bradley Sack, Janet Shimko, George Gomes, Donna O Sullivan, Karen Metcalfe, and Dale Spriggs Vaccine Testing Unit, Department of International Health, Johns Hopkins University School of Hygiene and Public Health, and General Clinical Research Unit, Johns Hopkins Hospital, Baltimore, Maryland; Virus Research Institute, Cambridge Massachusetts A new live oral cholera vaccine, Peru-15, was studied for safety, immunogenicity, and excretion in 2 groups of healthy volunteers. Twelve inpatient volunteers received freshly harvested vaccine in doses of either 10 7 or 10 9 cfu. Subsequently 50 outpatient volunteers received freeze-dried vaccine in doses of 10 8 or 10 9 cfu or placebo in a three-cell, double-masked, placebo-controlled trial. The strain was well tolerated at all dose levels, and it stimulated high levels of vibriocidal antibodies in most inpatient volunteers and in all outpatient volunteers. Although antitoxin responses were less frequent and of lower magnitude than the vibriocidal responses, antitoxin responses were seen in ú60% of the outpatient volunteers. About 60% of the volunteers excreted the vaccine in their feces; however, fecal excretion did not correlate with serologic responses. It is concluded that Peru-15 is a safe and immunogenic oral vaccine for cholera. Cholera remains an important public health problem with moter, was inserted into the reca locus, yielding Peru-3. This cases occurring in nearly all developing countries, including insertion inactivated reca, causing Peru-3 to be defective in 21 countries in the Western Hemisphere [1]. While improved homologous DNA recombination. A nonmotile variant was water and sanitation may eventually control the transmission isolated and designated Peru-15 [7]. of Vibrio cholerae, development of an effective vaccine could Following initial studies by Kenner et al. [7], Peru-15 was be extremely helpful in limiting morbidity and mortality from tested further at the Vaccine Testing Unit, Johns Hopkins Uni- the disease. Both killed and live oral vaccines are being devel- versity. Since Peru-15 is a newly developed, live, oral vaccine oped for cholera [2 5], and each has attractive attributes. strain, the first study was conducted in an inpatient unit, using Among a group of mutant V. cholerae strains [6], one promising a freshly prepared vaccine. The initial inpatient study was live oral vaccine candidate is Peru-15 [7]. Derived from an El needed for careful monitoring of the volunteers and for decon- Tor Inaba isolate, this new live oral vaccine is similar to the tamination of their stools before disposal. After safety and current pandemic strain. stability of the vaccine in inpatient volunteers was demon- Peru-15 has several defined genetic mutations that contribute strated, an outpatient, placebo-controlled study was conducted to its attenuation. First, wild-type V. cholerae C6709 was deleted using a freeze-dried formulation to further evaluate its safety at what was formerly termed the cholera toxin genetic and immunogenicity. element [8], recently discovered to encode a filamentous bacteriophage, designated CTX-phage [9]. Next, the cholera toxin B subunit gene, regulated by a V. cholerae heat-shock pro- Materials and Methods Inpatient study. Twelve community volunteers, aged years, were recruited for the inpatient study using advertisements in a local newspaper. Inclusion criteria included being healthy, Received 5 December 1996; revised 27 February being willing to participate, and completing a training session Presented in part: 31st Joint Conference, U.S.-Japan Cooperative Medical designed to provide sufficient knowledge of the disease and the Science Program, Cholera Panel, Kiawah Island, South Carolina, 1 3 Decem- protocol to give informed consent. Exclusion criteria included the ber following: chronic illness, immunosuppressive condition, abnor- Informed consent was obtained from all subjects, and experimentation folmal stool pattern, human immunodeficiency virus antibody positivlowed the guidelines of the US Department of Health and Human Services and of Johns Hopkins University. Protocols were approved by the Joint Committee ity, hepatitis B surface antigen positivity, pregnancy by history or on Clinical Investigation of Johns Hopkins University and were done by a positive urine human chorionic gonadotropin test 2 days under an Investigational New Drug Application from the Food and Drug Ad- before the study began, travel to a cholera-endemic area within 5 ministration. years, receipt of cholera vaccine, history of cholera infection, pre- D.O., K.M., and D.S. are employees of and own stock in the company that owns the vaccine strain being studied. vious participation in a cholera or enterotoxigenic Escherichia coli Grant support: NIH (RR-00035, General Clinical Research Center Osler study, inability to pass the written examination on diarrhea, chol- 5 Adult, Johns Hopkins Hospital, Baltimore); Virus Research Institute, Cam- era, and cholera vaccines, significant abnormality in screening labbridge, Massachusetts. oratory hematology and chemistry tests, and use of antibiotics Reprints or correspondence: Dr. David A. Sack, Johns Hopkins University within 7 days of vaccination. Vaccine Testing Unit, 550 N. Broadway, Suite 1001, Baltimore, MD The volunteers were admitted to the General Clinical Research The Journal of Infectious Diseases 1997;176: by The University of Chicago. All rights reserved. Center, Johns Hopkins Hospital, and were randomized to receive /97/ $02.00 either 10 9 or 10 7 cfu of the vaccine strain in a double-masked

2 202 Sack et al. JID 1997;176 (July) manner. On the day of admission, the volunteers drank their as- consistency in 24 h. They also submitted fecal specimens on days signed dose of vaccine with 100 ml of buffer (2.5 g of sodium 3, 7, 10, 14, and 21 after immunization, using tubes of both PBS bicarbonate and 1.65 g of ascorbic acid). Volunteers fasted for 60 and alkaline-peptone-water (APW) as transport media for the stool min before and after the dose. All volunteers received active vaccine specimen. These media were found to preserve the vaccine strain (i.e., none received placebo), but the clinical and laboratory most effectively during the inpatient study and were superior to staff were masked as to the dose each volunteer received. Cary Blair transport medium for this purpose. Serum was obtained The volunteers were clinically monitored at least twice daily by before and 10 and 21 days after immunization. a study physician, vital signs were taken four times daily, and all Laboratory procedures. Freshly harvested bacteria were pre- stools were examined, graded, and weighed. Stool consistency was pared for the inpatient volunteers following a standard procedure. graded with a score from 1 to 5 as previously described [5]. Stool Briefly, a vial of frozen Peru-15 seed was thawed, 0.1 ml of the grades 1 and 2 were considered normal, while grades 3 5 were thawed culture was spread on a plate of Luria-Bertani agar, and considered loose or watery. Intake and output of fluids were measured. the plate was incubated for 18 h at 30 C. After incubation, the Diarrhea was defined as 2 stools of a consistency of 3 bacteria were harvested with 5 ml of 0.85% saline, and standard totaling ú400 g or a single stool with a consistency of 3 totaling dilutions were prepared in saline. The optical density of the suspen- 300 g in 24 h. The first 2 stool specimens of each day from sion was determined and adjusted to produce a stock suspension each volunteer were cultured to detect excretion of the vaccine having a predicted concentration of colony-forming units per milli- strain, using both quantitative and qualitative methods. liter. The vaccine inoculum dose was verified by standard dilution On day 6, all volunteers began taking doxycycline: 200 mg as and plate counts on mock vaccine doses that were made in parallel a first dose, followed by 100 mg twice daily for 3 more days. The to those administered to the volunteers. volunteers were discharged when their stools were negative for To identify the vaccine strain in the volunteer s feces during the vaccine strain. During the inpatient stay, volunteers were under the inpatient study, the first 2 stools passed daily were cultured enteric (contact) precautions, and all feces were passed into dispos- quantitatively and qualitatively for V. cholerae in the Vaccine able plastic containers for observation by study personnel. After Testing Unit laboratory. For qualitative cultures, fecal specimens being weighed and sampled, feces were decontaminated by being were inoculated in Cary Blair transport medium and transported soaked in disinfectant for 1 h, after which the stool was flushed to the laboratory. There they were plated directly onto TCBS (thio- down the toilet. sulfate, citrate, bile salts, sucrose) agar and also inoculated into Serum samples were collected before vaccination, on day 6 after APW for subsequent plating onto TCBS agar. For quantitative vaccination (before starting antibiotic), and on days 14 and 28 cultures, 10-fold dilutions of stool were prepared in PBS, 100 ml after vaccination. Stool samples were collected on days 14 and 28 was spread onto TCBS plates, and typical colonies were counted. during follow-up visits to evaluate the possibility of continued Plates were incubated at 35 C for h. Colonies suspected excretion of the vaccine strain. from the qualitative and quantitative plate cultures to be Peru-15 Outpatient study. Fifty healthy men and women, years were confirmed by oxidase and agglutination with V. cholerae O1 old, were recruited to participate in the outpatient study, designed antiserum, and representative colonies were tested for motility to extend the inpatient studies by evaluating the same vaccine using a motility agar plate. strain when prepared in a lyophilized form. Recruitment for the For the outpatient study, the two vials (PBS and APW) were outpatient study was primarily through University bulletins and both inoculated onto TCBS media (the APW after 6hofincuba- most volunteers were either students or employees of Johns Hopspecimens tion). Quantitative cultures were not possible with the outpatient kins University or Hospital. The same inclusions and exclusions because the fecal samples were collected in transport were used for the outpatient study, but since this was the first broth. Again, colonies typical of V. cholerae were confirmed as outpatient study with this strain, potential volunteers were also Peru-15 using oxidase and agglutination tests and motility agar. excluded if they were food handlers or had close contact with By testing normal stools, to which known numbers of Peru-15 children under age 5, immunosuppressed persons, or pregnant were added, these methods yielded positive results if the concentra- women. tion of bacteria was ú /g of stool. Volunteers were randomized to receive either a 10 9 or 10 8 cfu Serum specimens were tested for vibriocidal antibody titer using dose or a placebo in a double-masked manner. The placebo cona an initial dilution of 1:10 and serial 2-fold dilutions of serum in sisted of freeze-dried cryoprotectant buffer with 1% powdered standard microtiter plate [10]. The assay used an El Tor Inaba skim milk, identical to that in which the vaccine strain was prethe strain (N16961) as the target strain, and the titer was expressed as pared. At the time of vaccination the lyophilized bacteria (or plawell. reciprocal of the highest dilution yielding no growth in the cebo) were reconstituted in 1 ml of the buffer administration IgG antitoxin antibodies were measured by a microtiter solution. Then either 1 or 0.1 ml of the vaccine was added to 100 ELISA method [10] with 3-fold serial dilutions using 1:10 as the ml of the sodium bicarbonate ascorbic acid buffer, and this was initial dilution. The antitoxin titer shown is the reciprocal of the given to the volunteer to drink. Each volunteer swallowed the dose extrapolated dilution of serum that yielded 0.4 OD in the plate. while being observed by an investigator (J.S.). To protect the All serologic assays were carried out in a blinded manner, and the masked code, some volunteers were assigned to 1 or 0.1 ml of code to the study was not opened until after the laboratory assays placebo. Volunteers fasted for at least 1 h before and after immunization. were complete. After immunization, the volunteers kept a daily symptom diary Results for 7 days, including the number and consistency of their stools. Inpatient study. No serious adverse events were noted dur- Diarrhea for the outpatient study was defined as 3 stools of 3 ing the inpatient study. One volunteer experienced a fever to

3 JID 1997;176 (July) Evaluation of Peru Table 1. Number of subjects who noted symptoms during the 7 abdominal gas, but none of the individuals felt the symptoms days following immunization with Peru-15 cholera vaccine or placebo to be distressing, nor did they limit usual activities. There were in outpatient volunteers. no fevers in the outpatient volunteers. Gastrointestinal Serum vibriocidal responses ( 4-fold) were seen in all symptoms Upper (100%) of those receiving the vaccine, regardless of the dose, respiratory but none of the placebo recipients developed a significant vib- Days 0 3 Days 4 7 symptoms Headache riocidal response. The vibriocidal GMT increased in recipients of 10 9 and 10 8 cfu by 73- and 90-fold, respectively, but it (n Å 16) was unchanged in the placebo group (table 2). The highest vibriocidal titers were seen on day 10. (n Å 16) IgG serum antitoxin titers increased significantly ( 2-fold) Placebo (n Å 18) in many (23/32) of the volunteers receiving vaccine, including NOTE. None of these differences are significant. 11 of 16 in the 10 9 cfu group and 12 of 16 in the 10 8 cfu group. The antitoxin GMTs increased by 4.1- and 4.6-fold for the 10 9 cfu and 10 8 cfu groups, respectively, on days 0, 10, 38.7 C the evening following the vaccination, and this was and 21 (table 2). The GMTs increased significantly in both associated with chilly feelings but with no localized symptoms vaccine groups, with the peak titer being seen on day 21, but or physical findings. A blood culture and a complete blood there was no apparent difference between the vaccine groups count the evening of the fever were negative. The volunteer in antibody response. None of the placebo recipients had a felt well the next day and remained well throughout the remainder significant rise in titer, and the GMT did not change in the of the study. None of the inpatient volunteers complained placebo group. of loose stools, and none complained of abdominal cramps, The vaccine strain was excreted in the feces of 66% (21/32) nausea, vomiting, or other intestinal symptoms. On inspection of the volunteers receiving the vaccine, but it was not isolated of the stools however, loose stools (grade 3) were observed from those receiving the placebo (table 3). There was a trend from 5 of the 12 volunteers, with amounts ranging from 261 for the vaccine to be excreted longer in those receiving 10 9 to 388 g in 24 h. None of these met a common definition of cfu, but this was not statistically significant. None of the fecal diarrhea ( 3 loose stools in 24 h). None of the volunteers had specimens were positive after day 10. a watery stool (grade 4 or 5). Examination of the relation between fecal shedding of the Four of 6 volunteers receiving 10 9 cfu and 2 of 6 receiving vaccine strain and antibody responses revealed no clear relationship cfu developed significant ( 4-fold) increases in vibriocidal Volunteers with negative fecal cultures had the same antibodies; the geometric mean titer (GMT) rose 36- and 5- frequency and magnitude of vibriocidal and antitoxin responses fold in the 10 9 and 10 7 cfu groups, respectively. Two volunteers as those with positive cultures. in each group had at least a 2-fold rise in serum IgG antitoxin antibodies; the GMT rose 1.7- and 1.2-fold in the 10 9 and 10 7 cfu groups, respectively. Discussion Four of 6 volunteers in the high-dose and 4 of 6 in the low- Peru-15 is a promising new live oral cholera vaccine. When dose group excreted the vaccine strain in stool. Those receiving a single dose of a reconstituted freeze-dried formulation of 10 9 cfu tended to have higher concentrations in the stool; all Peru-15 was administered in a manner similar to that appropriate volunteers receiving this dose had peak concentrations ú10 3 for routine office practice, it was safe and highly immuvolunteers cfu/g, while 2 of those receiving 10 7 had concentrations õ10 2 nogenic. The universal seroconversions among the outpatients cfu/g. However, these differences were not significant. Peak receiving Peru-15 and the magnitude (fold rises) of the vibrioci- concentrations were 10 6 cfu/g in both groups. Seven of the 8 dal responses were similar to those reported for CVD 103- volunteers who excreted vibrios still had positive stool cultures HgR [11 13], a live oral cholera vaccine licensed in Europe, on day 6 when the antibiotic therapy was started. All recovered Canada, and South America. A true comparison would require isolates were nonmotile. including the two vaccine strains in the same study. Without Outpatient study. Fifty volunteers received the vaccine or a challenge study, one cannot conclude that these serologic a placebo in the outpatient study: 16 received 10 9 cfu, 16 responses will be accompanied by high-grade long-lasting pro- received 10 8 cfu, and 18 received placebo. All volunteers com- tection; however, past experience has shown that the development pleted their symptom diaries, all completed their assigned visits, of vibriocidal antibodies has been accompanied by protecpleted and all submitted all of their fecal and serum specimens. tion [5], and the past trial with the vaccine using freshly Surveillance for adverse events revealed no attributable side prepared vaccine did suggest protection [7]. effects, though a few mild symptoms were reported by persons The current study evaluated the strain first in inpatient volunteers who received vaccine as well as placebo as shown in table 1. in a clinical research ward, where the volunteers could The gastrointestinal symptoms consisted of mild cramps or be closely observed and the feces could be decontaminated to

4 204 Sack et al. JID 1997;176 (July) Table 2. Serologic responses in outpatient volunteers receiving Peru-15 oral cholera vaccine or placebo. Vibriocidal antibody IgG antitoxin (ELISA) Geometric mean titer (95% confidence interval) Geometric mean titer (95% confidence interval) No. of No. of 4-fold rises Day 0 Day 10 Day 21 2-fold rises Day 0 Day 10 Day 21 (n Å 16) (5.5 17) 698 ( ) 494 ( ) (5.7 23) 38 (15 93) 48 (20 115) (n Å 16) (5.3 13) 761 ( ) 538 ( ) (8.5 17) 33 (17 64) 54 (31 96) Placebo (n Å 18) 0 12 (6.8 20) 12 (6.9 21) 11 (6.5 19) (5.8 13) 8.7 (5.6 13) 8.1 (5.3 12) prevent premature release of this new, genetically engineered unteers were generally associated with Johns Hopkins University, strain into the environment. Under these conditions, there were whereas the inpatient volunteers were recruited from the no serious adverse events, and the phenotype of the recovered community. Both groups were healthy as determined by a history, strains remained unchanged. The fever in 1 inpatient volunteer physical examination, and screening laboratory tests, but it remains unexplained and its relation to the vaccine is not is possible that differences existed between the groups. Similar known. proportions of volunteers excreted the vaccine strain in the After the vaccine was found to be safe and genetically stable inpatient and outpatient studies, suggesting that a difference in in volunteers, an outpatient study was carried out using a colonization does not account for the differences in immune freeze-dried formulation. The freeze-dried vaccine was stored responses. frozen at 020 C and was reconstituted immediately before use The antitoxin responses observed following immunization in the same buffer in which it was administered (bicarbonate were less impressive than the vibriocidal responses. However, ascorbic acid buffer). All of the outpatient volunteers who ú60% of the outpatient volunteers developed an antitoxin IgG received this formulation developed a vibriocidal response but response and the GMT increased ú4-fold in the outpatient showed no evidence of attributable adverse events. A few minor groups receiving vaccine. This magnitude of increase in serum gastrointestinal complaints were reported in all groups with GMT is similar to that observed after two or three doses of similar frequencies, including the placebo group, suggesting the killed oral whole cell/b subunit (WC/BS) vaccines, though either that the buffer caused mild discomfort or that a certain the rate of responses is somewhat less [10, 14]. The WC/BS frequency of mild symptoms exist as background symptoms vaccine does protect against heat-labile strains of enterotoxiamong persons who are encouraged to report all symptoms. genic E. coli by way of antitoxic immunity for a few months Reasons for the more consistent serologic response in the [15], so it is likely that Peru-15 may also protect to a degree outpatients compared with the inpatients is not known. The against this other enterotoxigenic infection. timing of collection of samples for serum was slightly different Our study did not include measures of local intestinal immuin the 2 groups. It is also possible that the freeze-dried form nity, such as intestinal lavage specimens or antibody secreting of the vaccine is more immunogenic, or alternatively, the vol- cells [16 18]. While it is expected that an oral cholera vaccine unteers who participated in the outpatient study represented a protects primarily by stimulating local intestinal immunity [19], different, more immune-responsive group. The outpatient vol- measures of the local immune response are imperfect and there is no clear definition of what constitutes a protective local immune response. Serum vibriocidal responses following live Table 3. Fecal shedding of Peru-15 vaccine among outpatient voleasily standardized. Similarly, the IgG antitoxin response fol- oral vaccine have correlated well with protection and are more unteers who received Peru-15 or placebo. lowing oral vaccine has correlated well with intestinal IgA Stool culture positive on antitoxin [20]; hence, it seems likely that these serum measures Ever cultureof immunity are accompanied by an intestinal immune response positive* Day 3 Day 7 Day 10 as well. In the outpatient study, there was no discernable dose-re- (n Å 16) sponse effect between the two doses. Both were equally immu- nogenic. Whether a lower dose could also be immunogenic (n Å 16) remains to be determined. Placebo (n Å 18) Questions about the use of live oral cholera vaccines have * Volunteer had at least 1 positive fecal culture. recently been raised by the discovery that the toxin virulence

5 JID 1997;176 (July) Evaluation of Peru genes of V. cholerae (which are chromosomal) can be transattenuated 6. Taylor DN, Killeen KP, Hack DC, et al. Development of a live, oral, vaccine against El Tor cholera. J Infect Dis 1994;170:1518 ferred among strains by way of a filamentous bacteriophage 23. [9]. For Peru-15, however, the possibility of reacquisition of 7. Kenner JR, Coster TS, Taylor DN, et al. Peru-15, an improved live attenuthese toxin genes has been effectively eliminated by the nature ated oral vaccine candidate for Vibrio cholerae O1. J Infect Dis 1995; of the gene deletions engineered into this strain: the reca gene 172: and the att RS1 integration sites [6, 7]. Both of these are 8. Pearson GD, Woods A, Chiang SL, Mekalanos JJ. CTX genetic element encodes a site-specific recombination system and an intestinal colonizarequired for successful integration and maintenance of the toxin tion factor. Proc Natl Acad Sci USA 1993;90: gene cassette. 9. Waldor MK, Mekalanos JJ. Lysogenic conversion by a filamentous phage It is also important to note that bacterial virulence is a com- encoding cholera toxin. Science 1996;272: plex phenomenon and that there is no a priori reason to assume 10. Sack DA, Clemens JD, Huda S, et al. Antibody responses after immuniza- that reacquisition of one virulence determinant would be suffiin tion with killed oral cholera vaccines during the 1985 vaccine field trial Bangladesh. J Infect Dis 1991;164: cient to convert an attenuated strain into a fully virulent organ- 11. Cryz SJ Jr, Levine MM, Kaper JB, Furer E, Althaus B. Randomized ism. For example, a toxigenic V. cholerae strain that was unable double-blind placebo controlled trial to evaluate the safety and immunoto colonize the small intestine would not be as virulent as a genicity of the live oral cholera vaccine strain CVD 103-HgR in Swiss wild-type strain. Peru-15 has other mutations, such as reca adults. Vaccine 1990;8: deletion and nonmotility, that add to the margin of safety of 12. Lagos R, Avendano A, Prado V, et al. Attenuated live cholera vaccine strain CVD 103-HgR elicits significantly higher serum vibriocidal antithe strain. body titers in persons of blood group O. Infect Immun 1995;63:707 In conclusion, lyophilized Peru-15, when given with buffer, 9. was safe and immunogenic. Further studies are needed to inves- 13. Kotloff KL, Wasserman SS, O Donnell S, Losonsky GA, Cryz SJ, Levine tigate other dose levels and other buffer systems to optimize MM. Safety and immunogenicity in North Americans of a single dose the administration of the vaccine and to further document proplacebo-controlled, of live oral cholera vaccine CVD 103-HgR: results of a randomized, double-blind crossover trial. Infect Immun 1992; tection in volunteers and in the field. 60: Concha A, Giraldo A, Castaneda E, et al. Safety and immunogenicity of oral killed whole cell recombinant B subunit cholera vaccine in Acknowledgments Barranquilla, Colombia. Bull Pan Am Health Organ 1995;29: We thank the volunteers who took part in these studies; Wendy 15. Clemens JD, Sack DA, Harris JR, et al. Cross-protection by B subunit Luther, Kathy MacLeod, and Carol Agrawal for their technical whole cell cholera vaccine against diarrhea associated with heat-labile toxin producing enterotoxigenic Escherichia coli: results of a largeassistance; and the staff of the General Clinical Research Center, scale field trial. J Infect Dis 1988;158: Johns Hopkins Hospital. 16. Svennerholm AM, Jertborn M, Gothefors L, Karim AM, Sack DA, Holmgren J. Mucosal antitoxic and antibacterial immunity after cholera disease References and after immunization with a combined B subunit whole cell vaccine. J Infect Dis 1984;149: Centers for Disease Control and Prevention. Update: Vibrio cholerae O1 17. Czerkinsky C, Svennerholm AM, Quiding M, Jonsson R, Holmgren J. Western Hemisphere, , and V. cholerae O139 Asia, Antibody-producing cells in peripheral blood and salivary glands after JAMA 1995;273:1169. oral cholera vaccination of humans. Infect Immun 1991;59: Sack DA, Freij L, Holmgren J, eds. From the Swedish Agency for Research 18. Losonsky GA, Tacket CO, Wasserman SS, Kaper JB, Levine MM. Second- Cooperation with Developing Countries. Prospects for public health ary Vibrio cholerae specific cellular antibody responses following benefits in developing countries from new vaccines against enteric infec- wild-type homologous challenge in people vaccinated with CVD 103- tions. J Infect Dis 1991;163: HgR live oral cholera vaccine: changes with time and lack of correlation 3. World Health Organization. Development of vaccines against cholera and with protection. Infect Immun 1993;61: diarrhoea due to enterotoxigenic Escherichia coli: memorandum from 19. Czerkinsky C, Quiding M, Eriksson K, et al. Induction of specific immua WHO meeting. Bull World Health Organ 1990;68: nity at mucosal surfaces: prospects for vaccine development. Adv Exp 4. Clemens JD, Sack DA, Harris JR, et al. Field trial of oral cholera vaccines Med Biol 1995;371B: in Bangladesh. Lancet 1986;2: Jertborn M, Svennerholm AM, Holmgren J. Saliva, breast milk, and serum 5. Levine MM, Kaper JB, Herrington DA, et al. Safety, immunogenicity, antibody responses as indirect measures of intestinal immunity after and efficacy of recombinant live oral cholera vaccines, CVD 103 and oral cholera vaccination or natural disease. J Clin Microbiol 1986;24: CVD 103-HgR. Lancet 1988;2: