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Vaccine 29 (2011) 8285– 8292

Contents lists available at SciVerse ScienceDirect

Vaccine

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afety and immunogenicity study of a killed bivalent (O1 and O139) whole-cellral cholera vaccine Shanchol, in Bangladeshi adults and children as young as 1ear of age

mit Sahaa, Mohiul Islam Chowdhurya, Farhana Khanama, Md. Saruar Bhuiyana, Fahima Chowdhurya,shraful Islam Khana, Iqbal Ansary Khana, John Clemensb, Mohammad Alib,lejandro Craviotoa, Firdausi Qadria,∗

International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, BangladeshInternational Vaccine Institute, IVI, Seoul, Republic of Korea

r t i c l e i n f o

rticle history:eceived 29 June 2011eceived in revised form 22 August 2011ccepted 24 August 2011vailable online 9 September 2011

eywords:ral cholera vaccinehancholafetymmunogenicity

a b s t r a c t

Background: Safety and immunogenicity study of an oral, killed, bivalent whole-cell, cholera vaccine,Shanchol was carried out in Bangladeshi participants. This study was conducted prior to initiating afeasibility study in Bangladesh.Study participants: The double-blind, randomized placebo controlled study was carried out in adults(18–45 years), toddlers (2–5 years) and younger children (12–23 months). Two doses of the vac-cine/placebo were given 14 days apart.Results: Shanchol did not elicit major adverse events in any age group. Vibriocidal antibody responsesin adults were 60% against Vibrio cholerae O1 Inaba, 72% against V. cholerae O1 Ogawa and 21% againstV. cholerae O139. In toddlers, responses were 84%, 75% and 64% and in younger children it was 74%, 78%and 54% against Inaba, Ogawa and O139 serotypes. The responses in all ages were higher in vaccinees

angladeshi adults and children compared to pre-immune titers or to responses in placebo recipients (P < 0.001).Plasma IgA antibody response to O1 Inaba LPS was seen in 61%, 73% and 45% of adults, toddlers and

younger children, respectively.Conclusions: The safety and immunogenicity data for Shanchol is promising and warrants future use inlarge scale trial in cholera endemic areas, high risk Bangladeshi population and in other countries in theregion.

. Introduction

Cholera remains an important public health problem withncreases in the numbers of cholera patients seen in Bangladeshach year [1]. Multiple outbreaks in different parts of the world,amely Zimbabwe, Nigeria, Pakistan and most recently Haiti [1–3]as led to a more concerted effort to control the disease. It stillemains a dreaded disease in cholera endemic countries with newegions being included where epidemics are being witnessed [4,5].

Bangladesh remains endemic for cholera, which peaks biannu-

lly with further increases seen during floods and cyclones [1,6]. Itffects all age groups with the majority of fatal cases occurring inhildren [7–10]. Therefore, immunization against cholera remains

∗ Corresponding author at: Centre for Vaccine Sciences, icddr,b, GPO Box 128,haka 1000, Bangladesh. Tel.: +880 2 8811751/8811760x2431;

ax: +880 2 8823116/2 8826050.E-mail address: fqadri@icddrb.org (F. Qadri).

264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2011.08.108

© 2011 Elsevier Ltd. All rights reserved.

an important public health tool for preventing the spread of choleraand for control of the disease [4].

Killed oral cholera vaccines have been studied in large PhaseIII field trials in Bangladesh as well as other areas of the world[11,12]. In addition, Phase I–III trials with live oral cholera vaccineshave been conducted [13–17]. Efforts to develop a safe, effectiveand affordable vaccine especially for the resource-scarce endemiccountries have intensified recently [4].

In line with this need, an inactivated whole-cell, bivalent choleravaccine containing both Vibrio cholerae O1 and O139 serogroupshas been used in Vietnam since 1997 [18]. To address severaldeficiencies of the Vietnam vaccine, this vaccine has since beenreformulated at the International Vaccine Institute in Korea andproduced in Shantha Biotechnics in India and marketed under thetrade name Shanchol [4,19]. A Phase III trial with Shanchol in

Kolkata has shown efficacy of 67% at 2 years of follow up amongindividuals aged 1 year and older [12,19]. As the cholera vaccine hasnot been tested in Bangladesh earlier, our regulatory requirementsmade it mandatory that we go through the Phase I/II studies to

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valuate safety and immunogenicity before using it in a large scaleeasibility study to deliver the vaccine to a high risk population inangladesh.

. Participants, materials and methods

.1. Field site, study participants and eligibility

The study was conducted in the Mirpur area in Dhaka [20].ealthy adult males and non-pregnant females aged between 18nd 45 years of age; toddlers, 2–5 years of age and younger chil-ren between 12 and 23 months of age were included. Exclusionriteria included chronic illness; any recent illness or those with

history of diarrhea lasting for more than 2 weeks in the preced-ng 6 months. Subjects who had taken any live or killed bacterialnteric vaccine in the last 4 weeks were also excluded [13]. Writtennformed consent was obtained from the study participants for thedults and from guardian of participating children. The study waspproved by the Ethical Review Committee of the icddr,b and Insti-utional Review Board of the IVI. This study was registered withhe Clinical Trials Data Bank (http://clinicaltrials.gov/), Identifier:CT01042951.

.2. Study design

This was a double blind placebo controlled trial. The sampleize was estimated based on the assumption of a 10% diarrhealate among placebo and vaccine recipients alike to exclude aaccine–placebo difference in the rate of diarrhea of greater than0% (upper boundary of the 1-tailed 95% confidence interval) with

power of 0.9, the minimum number of subjects required for eachroup was 46. The sample size was calculated on the assumptionhat vaccinees resulted in plasma vibriocidal responses comparedo the placebo group. The response rate in the placebo group wasssumed to be 5% and that of the vaccinees to be 25% [21]. With

15% attrition rate, the number was 55 in each group. An inde-endent Data Safety Monitoring Board oversaw study progress

ncluding safety results and for permission to move from one phaseo another. The study was also monitored by an external monitorrom Shantha Biotechnics.

.3. Randomization and blinding

The randomization lists were generated by a statistician at IVIho was not involved in the study. The study agents were labeledsing different letter codes (4 each for each age group) at Shanthaiotechnics according to the randomization list and were allocatedo the participants. Fixed block length of 4 was employed to effec-ively balance the interventions.

.4. Study agents, allocation and administration

Each dose of the Shanchol (Shantha Biotechnics) vaccine in.5 ml contained heat killed and formalin killed whole cell bacte-ia consisting of 600 ELISA Units (EU) of lipopolysaccharide (LPS)f formalin-killed V. cholerae O1 Inaba, El Tor biotype (strain Phil973). It also contains 300 EU LPS of heat-killed V. cholerae O1gawa classical biotype (Cairo 50); 300 EU LPS of formalin killed V.holerae O1 Ogawa classical biotype (Cairo 50); 300 EU LPS of heat-illed V. cholerae O1 Inaba, classical biotype (Cairo 48) and 600 EUPS of formalin killed V. cholerae O139 (4260B) [12,19]. The placeboonsisted of a 1.5 ml of sugar–buffer solution, similar in appearance

o the vaccine (Shantha Biotechnics).

Subjects were randomized to receive two doses of the vacciner placebo and were administered orally in a 14-day interval. Thetudy participants were instructed not to eat for 1 h before and after

(2011) 8285– 8292

intake of study agents and were requested to wait at the field clinicfor half an hour to monitor the side-effects.

2.5. Administration

The field assistant rotated the vial gently to disperse the cellularcontents before opening. Following administration, the partici-pants were offered half a cup of water.

2.6. Follow-up for adverse events

Trained study nurse and physicians were present during vac-cine administration and for adverse event monitoring. Symptomsincluding diarrhea, vomiting, nausea and other local and systemicreactions were recorded. Participants or guardians for all threestudy groups were interviewed by research assistants for 3 con-secutive days after each dose and asked to recall symptoms. Allside-effects were also recorded when they attended the field clinicfor any medical assistance up to 28 days for both local and systemicreactions. All reported symptoms were graded as mild (no interfer-ence with daily activity), moderate (some interference with dailyactivity) or severe (significant, prevented daily activity).

2.7. Follow-up and test methods for determining immuneresponses

Venous blood samples (3 ml from children and 5 ml from adults)were obtained prior to immunization (day 0) and 7 days after eachdose of the vaccine or placebo (day 7 and day 21). Plasma specimenswere preserved at −20 ◦C for immunological analyses.

2.8. Vibriocidal antibody assay

To determine vibriocidal antibody responses, specific responsesagainst V. cholerae O1 Inaba (strain T19479) and Ogawa serotypes(strain X25049) were carried out as described previously by test-ing serial 2-fold dilution of plasma (1:10 starting dilution) andusing guinea pig complement diluted 1:30 on microtiter plates(Nunc, Roskilde, Denmark). For determining vibriocidal antibodyresponses against V. cholerae O139, a modified procedure describedearlier was used [22,23]. V. cholerae O139, strain CIRS 134 [23] wasused with guinea pig complement at a final dilution of 1:10. Titra-tion of plasma collected on study days 0, 7 and 21 was carried outin duplicates.

Optical density (OD) was measured at 595 nm in the MultiskanAscent Reader (Thermo Labsystems, Denmark). Vibriocidal anti-body titer was defined as the reciprocal of the highest dilutioncausing a ≥50% reduction of OD compared control wells not con-taining plasma [24,25]. An increase of ≥4-fold between pre- andpost-immunization responses signified seroconversion. A titer of 5was assigned when no vibriocidal activity was observed [24].

2.9. Antibody responses to LPS in plasma

Plasma antibody responses to LPS from V. cholerae O1 Ogawa(strain X25049), Inaba (strain T19479) [25] and O139 (strain4260B) were measured using previously described ELISA methods[26]. Briefly, 96-well microtiter plates were coated with purifiedLPS (250 ng/well) [25] and incubated with plasma (1:10 dilu-tion) with serial 3-fold dilutions thereafter. Plates were washed,and secondary antibody that is horseradish peroxidase-conjugatedanti-human IgA antibodies were applied (Jackson Laboratories, Bar

Harbor, Maine). Plates were developed using 0.1% ortho-phenylenediamine (Sigma, St. Louis, Missouri) and 0.1% hydrogen peroxide,and OD measured at 492 nm. The endpoint titer was defined asthe reciprocal of interpolated dilution giving an absorbance of 0.4

A. Saha et al. / Vaccine 29 (2011) 8285– 8292 8287

Table 1Baseline characteristics of study participants.

Characteristics Adults Toddlers Younger children

Vaccine (n = 55) Placebo (n = 55) Vaccine (n = 55) Placebo (n = 55) Vaccine (n = 55) Placebo (n = 55)

Male, no./total (%) 16/34 (47) 18/34 (53) 30/59 (51) 29/59 (49) 33/63 (52) 30/63 (48)Female, no./total (%) 39/76 (51) 37/76 (49) 25/51 (49) 26/51 (51) 22/47 (47) 25/47 (53)Age, mean (SD) months 379 (88) 392 (95) 46 (11) 41(11) 21 (2) 19 (4)Weight, mean (SD) kg 53 (11) 54 (11) 13 (2) 12 (2) 10 (1) 9 (2)Height, mean (SD) cm 154 (8) 154 (6) 95 (8) 90 (9) 79 (5) 79 (6)

Of the 330 participants, 110 were enrolled in each age group.

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bove background. The titer calculations were carried out using theomputer-based program in the Multiskan Ascent ELISA Reader. A-fold or higher antibody response was considered a significantesponse.

.10. LPS specific antibody in lymphocyte supernatant (ALS)

Peripheral blood mononuclear cells (PBMC) (1 × 107 per ml) sep-rated from blood were cultured in 96-well tissue culture plates

ipants in the study.

for 48 h in 5% CO2. The ALS specimens were stored at −70 ◦C andtested for LPS specific antibody responses [22,27]. Specimens werecollected on study days 0, 7 and 21 and were tested by ELISA todetermine responses to O1 Inaba LPS and titers determined usingELISA procedures described earlier [28].

Reference standards using pooled plasma obtained from cholerapatients at convalescence [29] from V. cholerae O1 or O139 infectedpatients [26] were included in duplicates on each plate to ensureassay validity for the vibriocidal and ELISA assays.

8 ine 29 (2011) 8285– 8292

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Fig. 2. Vibriocidal antibody titer using V. cholerae O1 Inaba as target bacteria in dif-ferent groups receiving vaccine or placebo. The bars indicate geometric mean titersand lines, the standard errors of mean. *Indicates statistically significant differencesin responses in vibriocidal antibody titers prior to immunization (D0; ) and afterimmunization (D7; ) or (D21; ). #Indicates statistically significant differencesin responses in vibriocidal antibody titers between vaccine and placebo recipientsat day 7 and day 21. ˚Indicates statistically significant differences in responsesin vibriocidal antibody titers among vaccinee. The Wilcoxon Signed Rank test, the

with higher responses seen compared to placebo group (P = 0.017to <0.001) (Table 3).

Fig. 3. Vibriocidal antibody titer using V. cholerae O1 Ogawa as target bacteria in dif-ferent groups receiving vaccine or placebo. The bars indicate geometric mean titersand lines, the standard errors of mean. *Indicates statistically significant differencesin responses in vibriocidal antibody titers prior to immunization (D0; ) and afterimmunization (D7; ) or (D21; ). #Indicates statistically significant differences

288 A. Saha et al. / Vacc

.11. Data management and statistical analyses

The data was doubly entered into a computerized data manage-ent system. After data checking and verification, it was monitored

y an independent clinical monitor from Shantha Biotechnics. Afterreezing the data, analyses were carried out once the study un-linded.

Results were expressed as geometric mean titers. Response ratesnd magnitude of responses were measured. Analyses were carriedut using the SigmaStat program (Rafael, CA). Paired samples weressessed by the Wilcoxon Signed Rank test and non-paired samplesy the Mann–Whitney U-test. P-values ≤0.05 were considered toe significant. Proportions were compared using the �2 or Fisher’sxact test. Association between vibriocidal antibody and the LPSgA responses were determined by the Spearman rho correlationnalysis. GraphPad Prism 5 software was used to generate graphs.

. Results

.1. Adverse events associated with Shanchol

The study cohort comprised of 330 participants of whom10 were adults (male:female = 34:76); 110 were toddlersmale:female = 59:51) and 110 were between 12 and 23 months ofge (male:female = 63:47) (Table 1). Of the participants, 120 weref blood type O, 94 were type B, 87 were type A and 29 were bloodype AB. Only 8 were lost to follow up for the study (n = 5 for vac-ine group; n = 3 for placebo group) (Fig. 1). The vaccine or placeboas well tolerated by all recipients. Of the 330 participants, only

2 had mild symptoms (Table 2). Among the symptoms, the mostommonly reported adverse events were fever (n = 14), vomitingn = 4), abdominal pain, headache, gas and diarrhea (n = 1 for each).

ore than one adverse event was not observed in any participant.o severe adverse event was reported in the study. There was no

ignificant difference in the rates of adverse events between vaccinend placebo recipients (P = NS).

.2. Vibriocidal antibody responses to V. cholerae O1 and O139

The baseline antibody titer in the adult participants ranged from minimum of 5 to a maximum of 2560. In the toddlers and youngerhildren baseline antibody titer ranged from 5 to 5120.

Overall 73% of vaccinees responded with vibriocidal antibodieso V. cholerae O1 Inaba, while the response was seen in only 5.5%f placebo recipients (P < 0.001). The overall response to O1 Ogawaas also higher in the vaccinees (75%) than in the placebo recipients

6.7%; P < 0.001). There was significant O139 specific responses inaccinees (46%) compared with the placebo group (7%; P < 0.001).

Among vaccinees, the baseline geometric mean titers (GMT)o V. cholerae O1 Inaba were 55, 22 and 8 in adults, toddlers andounger children respectively and there was a 9-fold increase indults, 12-fold in toddlers and 7-fold increase in younger childrenithin 7 days of intake of a single dose of the vaccine. Adults showed

60% Inaba specific seroconversion rate, which was higher thanhat seen in placebo recipients (7%; P < 0.001). The toddlers showed4% while the younger children, showed 74% seroconversion rates.verall the vaccine recipients in the combined children group, 79%

howed response rates compared to only 4.5% in the placebo recip-ents (P < 0.001) (Fig. 2).

To the Ogawa serotype, the baseline GMT was 61, 39 and 9 indults, toddlers and younger children respectively. About 7-, 9- and

-fold rise in plasma antibody titers were observed after receipt of

single dose. Adult vaccinees showed a 72% seroconversion rateo O1 Ogawa, higher than that seen in the placebo recipients (9%;

< 0.001). The response in toddlers (75% vs 3.6%; P < 0.001) and in

Mann–Whitney U-test were used for comparisons. P ≤ 0.05 considered statisticallysignificant.

younger children (78% vs 7.4%; P < 0.001) was also higher amongvaccines than in the placebo recipients respectively (Fig. 3). To theO139 serogroup, the GMT was 20, 38 and 21 in adults, toddlers andyounger children respectively. Between 2- and 4-fold increase inantibody titers were observed after receipt of a single dose. Thevibriocidal antibody response to V. cholerae O139 in adults, tod-dlers and younger children were 21%, 64% and 54%, respectively

in responses in vibriocidal antibody titers between vaccine and placebo recipientsat day 7 and day 21. ˚Indicates statistically significant differences in responsesin vibriocidal antibody titers among vaccinee. The Wilcoxon Signed Rank test, theMann–Whitney U-test were used for comparisons. P ≤ 0.05 considered statisticallysignificant.

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Table 2Adverse events in study participants after intake of study agents.

Within 3 days after 1st dose Adults Toddlers Younger children

Vaccine (n = 55) Placebo (n = 55) Vaccine (n = 55) Placebo (n = 55) Vaccine (n = 55) Placebo (n = 55)

Fever 0 0 1 1 1 1Vomiting 0 1 0 1 0 0Diarrhea 0 0 0 0 0 0Nausea 0 0 0 0 0 0Abdominal pain 1 0 0 0 0 0Gas 1 0 0 0 0 0Loss of appetite 0 0 0 0 0 0Headache 1 0 0 0 0 0

Within 3 days after 2nd dose Adults Toddlers Younger children

Vaccine (n = 53) Placebo (n = 55) Vaccine (n = 55) Placebo (n = 54) Vaccine (n = 54) Placebo (n = 53)

Fever 1 0 0 1 2 1Vomiting 0 0 0 0 0 1Diarrhea 0 0 0 0 1 0Nausea 0 0 0 0 0 0Abdominal pain 0 0 0 0 0 0Gas 1 0 0 0 0 0Loss of appetite 0 0 0 0 0 0Headache 0 0 0 0 0 0

Total no of adverse events after 28 daysa 5 (9%) 2 (4%) 1 (2%) 5 (9%) 4 (7%) 5 (9%)

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Among adults, 21 of 53 vaccinees did not respond to V. cholerae1 Inaba. Of them 86% (18/21) had high baseline titers ranging

rom 80 to 1280. In toddlers, 9/55 were non-responders; of them4% (4/9) had titers ≥80. Among younger children, 14/54 vaccineesid not seroconvert, of whom only 14% had vibriocidal titers over0. A similar relationship of baseline antibody level and failure toespond to the vaccine was found in responses to O1 Ogawa (dataot shown).

The vibriocidal antibody responses in vaccinees after intake ofhe first and second dose to V. cholerae O1 Inaba were comparedFig. 2). The magnitude of the immune response was highest afterntake of the first dose of vaccine in the adults (P = 0.044) and theoddlers (P = 0.006); only in the younger children the frequencyf response was higher after intake of two doses (P = 0.005). Theesponse to V. cholerae O1 Ogawa (Fig. 3) was similar as that seeno O1 Inaba (data not shown).

The vibriocidal antibody responses in the vaccinees belongingo the different blood groups were 66%, 66%, 72% and 78% in the O,, A and AB groups, respectively. Higher frequency of respondersas seen in participants in the AB blood group compared to the O

lood group (P = 0.004; �2 test).

.3. LPS specific IgA antibody responses in plasma

About 64% of adult vaccinees responded with LPS–IgA antibod-es following ingestion of two doses of Shanchol; 67% of toddlerslso responded with LPS–IgA antibodies. The maximal LPS specific1 Ogawa and Inaba specific response was seen after intake of therst dose in the adults and toddlers to both V. cholerae O1 Inaband Ogawa (P = 0.001) (Table 4). Among younger children, 33% sero-onverted with LPS–IgA antibodies. The magnitude of the responseecreased from day 7 (P = 0.001) to day 21 (P = 0.021).

Although seroconversion was also seen to LPS in the IgM andgG antibody isotypes, these were of lower magnitude (data not

hown). The baseline LPS titer in both the IgM and IgG isotypesere higher than that seen for the IgA isotype.

The response to O139 LPS was poor in the adults (7%) and thehildren (14%) and not significantly different from that seen in thelacebo recipients.

ne of participants showed more than 1 symptom within 28 days study period.

3.4. LPS specific responses in antibody in lymphocyte supernatantspecimens

The O1 Inaba and Ogawa specific LPS IgA responses in ALSspecimens in adult vaccinees were 75% and 65%, respectively. Theresponse rates in the toddlers were 25% and 35% and in the youngerchildren it was 3.7% and 11% to O1 Inaba and Ogawa, respectively.Significant differences in magnitude and responder frequencieswere seen compared to the placebo recipients among adults andtoddlers (P < 0.001) after day 7 of intake of the vaccine.

3.5. Correlation between vibriocidal antibody response and LPSIgA

We used the Spearman rho correlation analysis to determinethe association between vibriocidal antibody and the LPS IgAresponses. In the adults neither day 7 nor day 21 antibody responsescorrelated with the LPS IgA responses to the O1 Inaba and Ogawaserotypes. Correlation was, however, observed in toddlers bothto Inaba and Ogawa serotypes between vibriocidal and LPS–IgAresponses (r > 0.5; P < 0.001). Furthermore a correlation was alsoseen in younger children (r > 0.4; P < .005).

4. Discussion

This is the first report of the testing of the oral cholera vaccineShanchol in Bangladeshi participants in a high-risk cholera endemicsetting. The vaccine was tested in participants in an age descend-ing order and was found to be safe in adults and in children 1–5years of age. No serious adverse events were observed during thestudy period and only a few reports of non-serious events werereported. Overall, the symptoms were similar in both the vaccineand placebo groups. Formulations of the killed bivalent oral choleravaccine have also shown similar safety profiles in Kolkata, India[19,30] as well as in Vietnam [31,32]. This study concurs with theprevious studies showing that Shanchol has good safety profile in

the different age groups.

This safety profile of Shanchol is similar to that seen for otheroral cholera vaccines e.g. Dukoral and Peru-15 tested in our settingsearlier [13,14,21,33,34]. These results demonstrate that the vaccine

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Table 3Vibriocidal antibody response using Vibrio cholerae O139 as target bacteria.

Vibriocidal antibody response Adults Toddlers Younger children dOverall response

Vaccine (n = 53) Placebo (n = 55) Vaccine (n = 55) Placebo (n = 55) Vaccine (n = 54) Placebo (n = 54) Vaccine Placebo

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 7 Day 21

GMTa 20 19 11 11 38 26 9 8 21 9 6 5Fold increaseb 2 2 1 1 4 3 1 1 4 2 1 1Responder frequencyc n (%) 10 (19) 10 (19) 3 (5) 2 (4) 32 (58) 26 (47) 5 (9) 5 (9) 28 (52) 17 (31) 1 (2) 0 (0) 46% 7%P <0.001 0.005 NS NS <0.001 <0.001 NS NS <0.001 <0.001 NS NS

The Wilcoxon Signed Rank test, the Mann–Whitney U-test were used for comparisons. P ≤ 0.05 considered statistically significant. NS indicates no differences between groups (P > 0.05).a Geometric mean of titers (GMT).b fold increase of GMT on day 7 and day 21 compared to pre-immune titers.c Responder frequency indicates numbers and % of participants responding with ≥4-fold increase in titer at day 7 and day 21 post immunization compared with pre-immune titers.d Indicates total vibriocidal response in all age group.

Table 4Lipopolysaccharide specific IgA antibody response in study participants.

Target bacteria (Vibrio cholerae) Response to LPS Adults Toddlers Younger children

Vaccine (n = 53) Placebo (n = 55) Vaccine (n = 55) Placebo (n = 55) Vaccine (n = 54) Placebo (n = 54)

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 7 Day 21

O1 InabaGMTa 171 119 73 84 37 22 8 7 13 13 7 10Responder frequencyb n (%) 27 (51) 19 (36) 3 (6) 5 (9) 37 (67) 30 (55) 14 (26) 9 (17) 18 (33) 19 (35) 12 (22) 10 (19)

O1 OgawaGMTa 202 152 97 93 59 35 10 10 18 19 14 13Responder frequencyb n (%) 22 (42) 19 (36) 2 (4) 4 (7) 35 (64) 30 (55) 7 (13) 8 (15) 15 (28) 23 (43) 9 (17) 11 (20)

The Wilcoxon Signed Rank test, the Mann–Whitney U-test were used for comparisons. P ≤ 0.05 considered statistically significant.a Geometric mean of titers (GMT) on day 7 and day 21 compared to pre-immune titers.b Responder frequency indicates numbers and % of participants responding with ≥2-fold increase in titer at day 7 and day 21 post immunization compared with pre-immune titers.

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an be used in all age groups above 1 year of age in future studiesn Bangladesh.

The immunogenicity of the Shanchol vaccine in the studyarticipants was determined using the vibriocidal antibody mea-urements, which is used as an indirect measure of protection [35].verall, the vibriocidal antibody response to V. cholerae O1 Inabaas 73% is similar to that seen in the Kolkata study, in which an

verall 67% response was seen. However, the baseline average vib-iocidal antibody titers was higher in the adults in Kolkata [19]ompared to those in Dhaka (GMT of 251 compared to 55 respec-ively to Inaba serotype). Among children the baseline GMTs wereomparable in the two settings.

We also found that vaccinees in Bangladesh, especially adultsith high pre-immune antibody titer (≥80) did not respond after

accination with O1 specific vibriocidal antibodies. About 86% ofdults who had vibriocidal titers of ≥80 did not seroconvert. How-ver, in non-responders among children and infants, this inverseelationship was less indicative. Among toddlers and infants only4% and 14% of those not showing an increase in vibriocidal anti-odies to V. cholerae O1 had titers that were ≥80. Thus otheractor(s) including age, micronutrient deficiency or the inability toespond to B cell independent carbohydrate antigens [8,36] maylay a role in this lowered response rate.

There appeared to be an age related lowering of the antibodyesponse to Shanchol. The baseline vibriocidal titers in toddlerss well as in the younger children were lower compared to thatn adults. This is probably due to prior exposure to V. choleraend the presence of preformed antibodies in adults living in aholera endemic country. The post-vaccination titers were alsoower going from adults to the youngest children showing ange related response rate in those receiving the vaccine. Similaresults have been observed in Bangladeshi adults and children whoeceived the Peru-15 vaccine [13,14]. We carried out a comparisonf responses of children who received Shanchol to data from a pre-ious study on children given Peru-15 and found that in all casesignificantly higher magnitude of responses were determined afterntake of even a single dose of Shanchol.

Analyses were also carried out to determine vibriocidal antibodyesponses to the Inaba serotype of V. cholerae O1 and results showedomparable responses obtained against the Ogawa serotype. In ear-ier studies with Peru-15, which is a strain of the Inaba serotype,he vibriocidal responses to V. cholerae O1 Ogawa was lower thato the Inaba serotype [13]. This was especially low in the youngestge group. We also observed in the earlier study on Peru-15 thathe magnitude was lower in infants than that seen in toddlers anddults, and only 50% of the infants who responded to the Inabalso responded to the Ogawa serotype. In studies with CVD103-gR, the live oral cholera vaccine, which is an attenuated strainf V. cholerae O1 Inaba serotype of the classical biotype, vacci-ation of adult Peruvians led to a higher response to the Inaba76%) than the Ogawa serotype (45%). The vibriocidal response inaccinees given Dukoral has been found to both the Ogawa andnaba serotypes [37]. Patients with natural cholera disease withgawa or Inaba strains are known to induce antibody responses

o both serotypes and even children responded to both serotypesfter natural infection with one or the other serotype [8]. It may beentioned, however, that over 90% of patients hospitalized with

holera mount a strong vibriocidal response to both serotypes of V.holerae [36]. When compared to vaccinees, higher magnitude ofesponses is seen in patients with cholera [8]. Responses to bothive and inactivated oral cholera vaccinees are less robust thanhat seen in cholera patients [36]. This may be also a reason for

he shorter duration of protection seen after vaccination than afteratural disease.

The responses induced by Shanchol to both the O1 serotypes;naba and Ogawa are encouraging since either serotype can cause

(2011) 8285– 8292 8291

severe disease during endemic and epidemic cholera outbreaks.The recent outbreak in Haiti was due to V. cholerae O1 Ogawa[2] while that in Zimbabwe was due to both serotypes [4]. InBangladesh and the surrounding region serotype fluctuation is evi-dent from one season to another.

In our study we have measured the immune response to theShanchol vaccine, 7 days after intake of each dose. This was done todetermine the optimal responses that were generated by the vac-cine in each of the age groups. We have also measured responsesto all the different serotypes and serogroups of V. cholerae compo-nents in the vaccine. We have measured not only the vibriocidalresponses but also the LPS specific IgA responses and the analy-ses carried out here are more comprehensive than other publishedinformation available [19,30].

Our results show that a single dose of Shanchol elicits a highmagnitude of vibriocidal and LPS specific IgA responses in the adultsand children up to 2 years of age. This is very encouraging and futureprotective efficacy studies will be able to demonstrate if the vac-cine can be used in a single dose formulation in cholera endemiccountries in these age groups. Studies on Shanchol in India do sug-gest that a single dose satisfactory vibriocidal antibody responsesin adults and children [30]. Analyses have also been carried outrecently to determine the memory response after intake of oneor two doses of an oral cholera vaccine, Dukoral and it appearsthat a single dose can elicit a comparable response as two doses[36]. A single dose formulation would help in further facilitatingthe delivery and uptake of the vaccine and minimizing other asso-ciated public health costs of immunization and is being seriouslyvalidated in different field settings.

We also determined the responses to V. cholerae O139. We foundan overall vibriocidal antibody response rate of 46% with childrenresponding better than adults. In previous studies with this vac-cine in India [19] or with the cholera vaccine from Vietnam, poorresponses have been documented with response rates of around11% [18]. The reason for this difference may be the procedures usedfor determining the vibriocidal antibody responses in this study.We have optimized a technique that better measures the vibrioci-dal antibodies using a less capsulated strain of V. cholerae O139 aswell as lower concentrations of complement [26]. Thus this modi-fied method gives a better estimate of the seroconversion rate forO139 specific vibriocidal antibodies.

We measured IgA antibody responses to both O1 Inaba andOgawa LPS. The responses seen in the Shanchol vaccinees in dif-ferent age groups were similar to that seen in Dukoral recipients[28,37]. There was a lower LPS specific seroconversion rate in chil-dren among the vaccine recipients as has been shown earlier toinactivated [38,39] or to live oral vaccines [13,14].

In this study, significant correlation was not seen betweenvibriocidal responses and LPS IgA responses in adults. However,there was a positive correlation in responses observed in tod-dlers and younger children. Although both assays measure theresponse to the ‘O’ polysaccharide antigen of the LPS moiety,the vibriocidal is a functional assay measuring the bacterici-dal capacity of serum/plasma while the antigen specific ELISAmeasures the antibodies elicited to the antigen in the differentisotypes.

This is the first report of the safety and immunogenicity of thevaccine in Bangladesh. The results of the study are very promis-ing showing the vaccine to be safe with satisfactory immuneresponses in the Bangladeshi participants in all ages. The resultssupport the continued study of Shanchol in the Bangladeshi popu-lation as it clearly offers a potential tool for public health decision

makers in the protection of high-risk groups against cholera inendemic settings and evidence to support international organiza-tions to recommend its use as well as to stockpile for emergencies[40].

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cknowledgements

Informed consent was obtained from the study participants. Theesearch was approved by the Ethical Review Committee of thenternational Centre for Diarrhoeal Disease Research, Bangladeshnd IRB of the International Vaccine Institute. The study is regis-ered in the Clinical Trials Data Bank (http://clinicaltrials.gov).

Conflicts of interest statement: There were no conflicts of interestf any of the authors.

Funding: This work was funded by the Bill and Melinda Gatesoundation (Grant no. OPP50419).

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