Chisenga et al. BMC Public Health (2023) 23:152 BMC Public Health https://doi.org/10.1186/s12889-023-15051-w RESEARCH Open Access Assessment of the influence of ABO blood groups on oral cholera vaccine immunogenicity in a cholera endemic area in Zambia Caroline C. Chisenga1*, Samuel Bosomprah1,2, Obvious N. Chilyabanyama1, Peter Alabi1, Michelo Simuyandi1, John Mwaba1,3, Harriet Ng’ombe1,3, Natasha M. Laban1, Charlie C. Luchen1 and Roma Chilengi1,4 Abstract Background Histo-blood group antigens (HBGAs) which include the ABO and Lewis antigen systems have been known for determining predisposition to infections. For instance, blood group O individuals have a higher risk of severe illness due to V. cholerae compared to those with non-blood group O antigens. We set out to determine the influence that these HBGAs have on oral cholera vaccine immunogenicity and seroconversion in individuals residing within a cholera endemic area in Zambia. Methodology We conducted a longitudinal study nested under a clinical trial in which samples from a cohort of 223 adults who were vaccinated with two doses of Shanchol™ and followed up over 4 years were used. We measured serum vibriocidal geometric mean titers (GMTs) at Baseline, Day 28, Months 6, 12, 24, 30, 36 and 48 in response to the vaccine. Saliva obtained at 1 year post vaccination was tested for HBGA phenotypes and secretor status using an enzyme-linked immunosorbent assay (ELISA). Results Of the 133/223 participants included in the final analysis, the majority were above 34 years old (58%) and of these, 90% were males. Seroconversion rates to V. cholerae O1 Inaba with non-O (23%) and O (30%) blood types were comparable. The same pattern was observed against O1 Ogawa serotype between non-O (25%) and O (35%). This trend continued over the four-year follow-up period. Similarly, no significant differences were observed in seroconversion rates between the non-secretors (26%) and secretors (36%) against V. cholerae O1 Inaba. The same was observed for O1 Ogawa in non-secretors (22%) and the secretors (36%). Conclusion Our results do not support the idea that ABO blood grouping influence vaccine uptake and responses against cholera. Keywords Cholera, Histo-blood group antigens, Secretory status, Vaccine response, Zambia Introduction *Correspondence: Caroline C. Chisenga Zambia is among countries in sub-Saharan Africa Caroline.Chisenga@cidrz.org contributing to 2.9 million cases and 95,000 deaths 1 Centre for Infectious Disease Research in Zambia, Lusaka, Zambia 2 resulting from cholera globally [1]. Vibrio cholerae Department of Biostatistics, School of Public Health, University of Ghana, Accra, Ghana (V. cholerae), serogroups O1 and O139 are the causa- 3 Department of Biomedical Sciences, School of Health Sciences, tive agents for cholera. Of the two serogroups, O1 is University of Zambia, Lusaka, Zambia 4 responsible for most epidemics in developing coun- School of Medicine, University of Lusaka, Lusaka, Zambia tries and is further classified into serotypes Ogawa, © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/l icen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdomai n/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chisenga et al. BMC Public Health (2023) 23:152 Page 2 of 10 Inaba and Hikojima [2]. Ogawa and Inaba are the major to the new cholera vaccine, Shanchol™ among the Zam- serotypes, and these have been isolated in Zambia [3]. bian population. Transmission of cholera is mainly via consumption We thus set out to investigate the impact of HBGAs of contaminated water or food and in many endemic and secretor status among individuals residing in a chol- areas, children often suffer the highest disease burden era endemic area and assessed impact on immunogenic- [4]. ity to OCV. In 2016, Zambia for the first time deployed the oral cholera vaccine (OCV) Shanchol™ (Shantha Biotechnics Private Limited, Hyderabad, India) [5]. The vaccine was Methods WHO prequalified in 2011 [6]. While the vaccine has Study design and participants shown a protective efficacy of up to 66% in adults, very Using a longitudinal study, we used data from a clinical little is known about the factors that impact its immuno- trial (ClinicalTrials.gov with trial # NCT04423159) that genicity in cholera endemic areas. aimed at describing the longevity and waning of vibrioc- Oral vaccines are known for several distinct benefits idal antibodies (IgG and IgM) after receiving two doses of compared with parenteral vaccines in that (i) they can be OCV (Shanchol ™) [16]. Thus, for the longitudinal study, produced in large quantities at relatively low cost, (ii) are we included all the participant data from this trial and easy to administer and (iii) have the capacity to induce dropped those with incomplete data at analysis. mucosal immunity in the gut, thereby directly protect- The clinical trial was conducted in Lukanga Swamps ing vaccinated individuals against subsequent infection. of Central Province Zambia between October 2016 to In addition, by blocking onward transmission, there is October 2020. The trial enrolled 223 male and female enhanced herd immunity [7, 8]. adults aged 18 to 65 years, residing in Lukanga Swamps Notwithstanding all the potential benefits of oral vac- and who provided written informed consent to partici- cines, some genetic factors including histo-blood group pate in the study. Individuals were excluded from par- antigens (HBGAs) have been implicated in the mecha- ticipation if they had medical conditions such as hepatic nistic cellular invasion of the host by pathogens. Some disease, diarrhoea within the previous 7 days, had previ- enteric pathogens and pathogen-derived toxins use the ously received OCV or were pregnant. HBGAs as attachment factors [9]. For example, blood type O has been linked with an increased occurrence of paralytic poliomyelitis [10]. In the case of cholera, indi- Vaccination ™ viduals with blood group O are less likely to be colonized All participants received two doses of Shanchol (Shan- by V. cholerae [11], but once colonized, are more likely to tha Biotechnics Private Limited, Hyderabad, India), suffer severe symptoms [11, 12]. given 28 days apart. Participants were monitored for However, there isn’t sufficient evidence in literature on any adverse events following vaccination for 30 minutes vaccine immunogenicity effects of these blood groups. after which they were allowed to go home. Upon release, Intriguingly, blood group O frequencies do not differ participants were asked to return to the study site or to markedly in regions where oral cholera vaccines have contact any study staff if they felt ill after receipt of the underperformed compared to those in which a robust vaccine. All the vaccines were kept at 2-8 °C on site as vaccine response is described [13, 14]. recommended by the manufacturers. HBGAs may present on the surface of epithelial cells and in mucosal secretions such as saliva which is deter- Sample collection mined via fucosyl-transferase 2 (FUT2) gene. Individu- Participants who received both doses (at Day 0 and Day als with functional FUT2 gene facilitating secretion of 28) were followed up for 48 months (Fig.  1). A 10 ml HBGAs are referred to as secretors whereas those with venous blood sample was collected after provision of mutations in FUT2 that abrogates ability to secrete written informed consent but prior to immunization HBGAs as non-secretors respectively. In one report, on day 0. A second blood sample was collected before non-secretors were more likely to suffer from severe administering dose 2 at day 28. Thereafter, further blood cholera than secretors [15]. However, the extent to which samples were collected at months 6, 12, 24, 30, 36 and 48 the individual’s HBGA secretor status could influence accordingly. In addition, saliva was collected using Sal- vaccine immunogenicity remains unclear. ivaBio collection kit (Salimetrics LLC) at month 12. All Given the high prevalence of blood type O among biological samples were processed and stored at − 80 °C Zambians and the reports that individuals with blood pending sample processing. To avoid inter-assay varia- group O are less likely to suffer from cholera [11], we tions, all samples for each individual were processed at hypothesized that there might be poor immune response the same time. Chisenga et al. BMC Public Health (2023) 23:152 Page 3 of 10 Fig. 1 Schema for sample collection and vaccination time points Laboratory procedures at 37 °C. Color development was done using 3, 3′, 5, 5′ ABO blood typing Tetramethyl benzidine (TMB) (Sigma Aldrich) substrate Was done by direct typing and reverse typing. This test and stopped by addition of sulphuric acid before read- was carried out according to the manufacturer’s instruc- ing absorbance at 450 nm wavelength (BioTek). Through- tion (Biotec laboratories, UK). Briefly, one end of a slide out the assay, the microtiter plate wells were washed was labelled Anti-A, the middle one labelled Anti-D and five times with 0.05% weight/volume Tween 20 in PBS the other end Anti-B. A drop of Anti-A test serum was (PBST) in between the ELISA steps. All antibodies were added to the end marked Anti-A, followed by Anti-D and diluted in 5% fetal bovine serum (GE Healthcare Life Sci- a drop of Anti-B serum was added to the end marked ences, USA) in PBST. Secretor status was determined in Anti-B. One drop of whole blood was added to each end a similar manner by detection of Fucα1-2Gal-R known to of the slide and mixed thoroughly using separate wooden be present in secretors but not in non-secretors. Diluted toothpicks. The results were read directly from the slide saliva samples were coated to the microtiter plate and within 60 seconds. A participant was reported to have incubated overnight as described above. After blocking, blood type A if agglutination occurred with the Anti- the plate was incubated with peroxidase-conjugated Ulex A test serum; type B if agglutination occurred with the europaeus (UEA-1) Lectin (Sigma Aldrich), developed Anti-B test serum; type AB if agglutination occurred with with TMB substrate before addition of Sulphuric acid both test serums, and O if there was no agglutination in and absorbance read at 450 nm. either case. Blood group was also categorized as rhesus positive or negative if there was agglutination on Anti-D or not. Vibriocidal antibody testing Vibriocidal antibody assays was also run as previously described with some modifications [18]. Local Zambian Secretor status testing V. cholerae O1 Inaba (EDVRU/ZM/091–10) and Ogawa We determined salivary blood group ABO phenotypes (EDVRU/ZM/2016) were used. These strains were qual- and secretor status using an enzyme-linked immuno- ity checked at Johns Hopkins University in the United sorbent assay (ELISA) based on published methods States and their performance was comparable to stand- [17]. In brief, a 100 μL volume of saliva diluted to 1:100 ard strains Inaba (T19479) and Ogawa (X25049). in coating buffer (carbonate-bicarbonate buffer, p-H 9.6) Briefly, colonies from overnight cultures were inocu- was added to microtiter plate (Nunc-Immuno F96 Max- lated in Brain Heart Infusion (BHI) Broth and incubated isorp, Thermo Fisher Scientific) and incubated at 37 °C at 37 °C for about 4 hours before harvesting the cells. Heat for 2 hours followed by incubation at 4 °C overnight. Fol- inactivated serum, exogenous guinea pig complement lowing overnight incubation, the plate was blocked with (Sigma Aldrich S1639-5ML) and V. cholerae bacterial 3% weight/volume bovine serum albumin in phosphate cells were then placed in 96-well microtitre tissue culture buffered saline (PBS) and incubated at 37 °C for 1 hour. plates (Life sciences, Durham, USA) and incubated at The plate was then incubated with 1:5000 diluted α-A 37 °C. Vibriocidal titres were defined as the reciprocal of (ABO1 clone 9113D10), and α-B (ABO2 clone 9621A8) the highest serum dilution resulting in a 50% reduction in (Diagast, France) monoclonal antibodies at 37 °C for optical density read at 595 nm compared to positive con- 1.5 hours. Next, the plate was incubated with 1:7500 trol wells without serum. Seroconversion was defined as diluted peroxide-conjugated goat anti-mouse IgG (heavy a 4-fold or greater increase from the baseline vibriocidal and light chain) (Abcam, United Kingdom) for 1.5 hours titres. A standard monoclonal antibody (mAb) and a high Chisenga et al. BMC Public Health (2023) 23:152 Page 4 of 10 titre standard serum was used to normalize the results in vaccine uptake, with each adjusted for the other in case of inter-assay variations. addition to potential confounders such as socio-demo- graphic, and WASH characteristics. Level of statisti- Sample size calculation cal significance was set at a 2-tailed p-value of 0.05 or The participants were selected from a convenience sam- less. Data analysis was performed using Stata 17 MP8 ple. All individuals satisfying inclusion criteria were con- (StataCorp, College Station, TX, USA). sidered for participation. Previously published data [19], suggested that at least, 80% adults would seroconvert. Due to marginal transience noted in this population it Ethical approval was estimated that 20% of those participating will be lost Ethical approval was obtained from the University to follow-up. The sample size calculated with 95% confi- of Zambia Biomedical Research Ethics Committee dence (α = 0.05  (two  tailed)), 80% power (β = 0.2), a dif- (UNZABREC) reference number 007–12-16, while ference of 0.3 and conservative estimates of 0.5 variance the National Health Research Authority provided the for pre-vaccine and post-vaccine groups is 176. Adding authorization to conduct the study. Written informed 20% to account for anticipated attrition yields a total consent was also obtained from all enrolled partici- required sample size of 212 [20]. pants into the trial. All study procedures were con- ducted according to good clinical practice guidelines. Statistical analysis Participants’ socio-demographic and clinical charac- teristics were presented as frequencies (percentages). Results Vibriocidal antibody titres were expressed as geomet- Baseline characteristics of study participants ric mean titres (GMT), and sero-conversion rate was Of the 223 participants enrolled in the study, 13 were defined as the percentage of participants with at least recorded as lost to follow-up as we could not track a four-fold rise in serum vibriocidal antibody titres their way about and 77 were excluded due to incom- after vaccination on Day 28 HBGA phenotype was plete data. A total of 133 were included in the final categorized into blood group O and non-blood group analysis (Fig. 2). Of the 133 participants, 76 (58%) were O. Equally, participants were categorised by secretor above 34 years old, 120 (90%) were male, 92 (72%) were status as secretors or non-secretors. Seroconversion engaged in fishing activities, 95 (81%) used water from to Ogawa and Inaba were calculated for each back- the Swamps for drinking and 87 (74%) practiced open ground characteristic. Since the outcome was common defecation on land (bush) or in the Swamps. Overall, (i.e., greater than 10% in the sample), we used Poisson there was no significant differences observed by sero- regression with robust standard errors to determine conversion rates to V. cholerae O1 Ogawa and Inaba the effect of HBGA phenotype and secretor status on using the baseline characteristics (Table 1). Fig. 2 Flow diagram for study participant enrollments and follow-up C hisenga et al. BMC Public Health (2023) 23:152 Page 5 of 10 Table 1 Sero-conversion rates by socio-demographic characteristics Characteristics Inaba Ogawa Total n(%) n(%)Seroconverted p value Total n(%) n(%) Seroconverted p value Age (years)a 0.805 0.728 16–24 27(20.5) 8(29.6) 27(20.3) 8(29.6) 25–34 29(22.0) 8(27.6) 30(22.6) 8(26.7) > 34 76(57.6) 18(23.7) 76(57.1) 17(22.4) Sex Females 13(9.8) 1(7.7) 0.108 13(9.7) 2(15.4) 0.384 Males 120(90.2) 34(28.3) 121(90.3) 32(26.5) Occupationa Fishing 92(71.9) 23(25.0) 0.741 92(71.3) 24(26.1) 0.267 Fish Trading 16(12.5) 5(31.3) 17(13.2) 5(29.4) Othersb 20(15.6) 4(20.0) 20(15.5) 2(10.0) Source of drinking watera Borehole/well 23(19.5) 6(20.0) 0.935 23(19.5) 3(13.0) 0.211 Swamps 95(80.5) 24(80.0) 95(80.5) 24(25.3) Type of toilet facility Swamp/bush 87(74.4) 21(24.1) 0.526 87(74.4) 21(24.1) 0.643 Pit latrine/Toilet 30(24.6) 9(25.8) 30(24.6) 6(20.0) Total 133(100) 35(26.3) 134(100) 34(25.4) a Total not equal to 133 or 134 because of missing information b Chi square was used to compare sero-conversion by socio-demographic characteristics, blood group and secretory status Effects of blood group and secretory status 0.79–5.60, P = 0.138). Furthermore, the non-secretors on sero‑conversion against Inaba have a 41% higher chance of seroconverting against Inaba Of the 133 participants, 63 (47%) had blood type O compared to the secretors; however, association was also based on direct typing, whereas 70 (53%) had a non-O not statistically significant (Adjusted PR = 1.41, 95% CI: blood type. Non-secretors were 14 (18%) while 62 (82%) 0.52–3.79, P = 0.496) (Table 2). were secretors (Table 2). The proportion of participants that seroconverted were higher among those with blood Effects of blood group and secretory status group O (30.2%) compared to non-O (22.9%) and among on sero‑conversion against Ogawa the secretors (35.7%) compared to non-secretors (25.8%). The proportion of participants that sero-converted Participants with blood group O were about two times against Ogawa was the same among those with blood more likely to seroconvert against Inaba compared to group O and the non-O blood group (25.4%) but higher the non-O blood groups, although this association was among the secretors (35.7%) than the non-secretors not statistically significant (Adjusted PR = 2.10, 95% CI: (22.2%). The probability of seroconverting against Ogawa Table 2 Effects of blood group and secretor status on sero-conversion against Inaba Characteristics Total n (%) n(%) Seroconverted Inaba Crude PR (95% CI) p value Adjusted PR (95% CI)a p value Blood Group Non-O 70 (52.6) 16 (22.9) Ref Ref O 63 (47.4) 19 (30.2) 1.32 (0.74–2.34) 0.343 2.10 (0.79–5.60) 0.138 Secretor Statusb Secretor 62 (81.6) 16 (35.7) Ref Ref Non-Secretor 14 (18.4) 5 (25.8) 1.38 (0.61–3.16) 0.440 1.41 (0.52–3.79) 0.496 a Adjusted for age, sex, education, occupation, source of drinking water & type of toilet facility, PR- Prevalence Ratio b Total not equal to 133 because of missing information Chisenga et al. BMC Public Health (2023) 23:152 Page 6 of 10 was 12% lower among blood group O participants com- Kinetics of vibriocidal GMTs against V. cholerae O1 Inaba pared to the non-O, although this association was not and O1 Ogawa overtime by blood group statistically significant (Adjusted PR = 0.78, 95% CI: Overall, we observed insignificant increases in vibriocidal 0.27–2.20, P = 0.633) while the non-secretors are about GMTs (95% CI) by both non-O and O blood group types two times more likely to seroconvert against Ogawa at baseline, [2.44 (2.18–2.70)] and [2.81 (2.48–3.14)] and compared to the secretors and this association was also day28, [3.12 (2.75–3.49)] and [3.05 (2.65–3.46)] against V. not statistically significant (Adjusted PR = 1.91, 95% CI: cholerae O1 Ogawa as shown in Fig. 3. Thereafter, titres 0.77–4.76, P = 0.166) (Table 3). dropped steadily up to month 24 after which a gradual Table 3 Effects of blood group and secretor status on sero-conversion against Ogawa Characteristics Total n (%) n(%) Seroconverted Ogawa Crude PR (95% CI) p value Adjusted PR (95% CI)a p value Blood Group Non-O 71 (53.0) 18 (25.4) Ref Ref O 63 (47.0) 16 (25.4) 1.00 (0.56–1.80) 0.995 0.78 (0.27–2.20) 0.633 Secretor statusb Secretor 63 (81.8) 14 (35.7) Ref Ref Non-secretor 14 (18.2) 5 (22.2) 0.48 (−0.55–1.50) 0.362 1.91 (0.77–4.76) 0.166 a Adjusted for age, sex, education, occupation, source of drinking water & type of toilet facility, PR- Prevalence Ratio b Total not equal to 134 because of missing information Fig. 3 Kinetics of vibriocidal GMTs at different time points (left) V. cholerae O1 Inaba (right) V. cholerae Ogawa for blood type O and non-O. There was a rise in antibody titres in both serotypes at day 28 and then started to decline close to baseline titres at month 12. A significant but comparable rise was observed at months 36 and 48 C hisenga et al. BMC Public Health (2023) 23:152 Page 7 of 10 increase was again observed from month 30 up to month were not statistically significant between the two groups. 48. Although non-O individuals showed a higher trend in The trend in vibriocidal GMT (95% CI) against V. chol- terms of recorded titres than blood group O starting at erae O1 Inaba between the two groups (non-secretors month 30, the differences in titres were not statistically and secretors) were similar with that observed for O1 significant between the two groups. The trend in vibrioc- Ogawa (Fig. 4). idal GMT (95% CI) against V. cholerae O1 Inaba between the two groups (non-O and O blood types) were similar Discussion with that observed for O1 Ogawa (Fig. 3). Our study found that ABO blood groups as well as secre- tor status do not impact OCV immunogenicity and sero- Kinetics of vibriocidal GMTs to V. cholerae O1 Inaba and O1 conversion in the studied population. Ogawa overtime by secretor status The GMT kinetics were similar in either group i.e. Furthermore, we also noted that baseline GMTs (95% CI) non-O and O as well as secretor and non-secretor. We were lower in both the non-secretors 2.43 (1.80–3.06) also found that in our study population, majority of our and the secretors 2.63 (2.27–2.99) but increased slightly participants were secretors similar to what is reported at Day 28 in both the non-secretors 3.00 (2.11–3.88) as by Arifuzzaman and colleagues [21]. Specifically, GMTs well as the secretors 3.15 (2.73–3.57) against V. chol- in all the vaccinees, either belonging to blood type O erae O1 Ogawa as shown in Fig. 4. Similar to blood type or non-O were comparable. This finding is similar to trends, after Day 28, GMTs started to drop until month what was reported by Ramamurthy and colleagues [22]. 24. Beyond 24 months, there was a constant increase Despite the supposed frequency that people with non-O observed from month 30 up to month 48. Although non- may suffer from cholera [11], and assuming that each secretor individuals showed a higher trend in terms of exposure boosts immunity against cholera, our results recorded titres than the secretors, the differences in titres demonstrate that both individuals with O blood type Fig. 4 Kinetics of vibriocidal GMTs at different time points (left) V. cholerae O1 Inaba (right) V. cholerae Ogawa for secretors and non-secretors. There was a rise in antibody titres in both serotypes at day 28 and then started to decline close to baseline titres at month 12. A significant but comparable rise was observed at months 36 and 48 Chisenga et al. BMC Public Health (2023) 23:152 Page 8 of 10 and the non-O blood type elicit similar vibriocidal anti- Vibriocidal antibody titers have been reported to be body responses to Shanchol™ in an area where cholera is a useful marker for measuring immunologic response endemic. While our study was underpowered to show a including seroconversion to cholera vaccines [29, 30]. statistical difference, the data shows no definite trend to With this assay we found comparable seroconversion distinguish O from non-O blood types in terms of vac- rates in our study population either by blood type or cine immunogenicity; as such vaccination campaigns secretor status. Seroconversion rates also did not dif- should target all individuals regardless of their blood fer by V. cholerae serotypes. Nonetheless, Charles et  al. type. This is contrary to what was found elsewhere found that individuals with blood type O had greater [23], as our vibriocidal antibody response did not dif- vibriocidal GMTs against serotype Inaba compared to fer between O and non-O individuals. The referenced those with non-O blood types [31]. Similar to our study, trial conducted in Bangladeshi on the role of ABO blood there was no significant difference observed for vibrioc- group and efficacy of an oral, killed cholera vaccine, idal response to the Ogawa serotype in the same popu- found considerably lower protection in recipients who lation [31]. Timing of the blood sampling in our study were blood group O [23]. The implication here is that was further away from the vaccination date compared inactivated oral vaccines may not necessarily provide less to Charles and colleagues that collected and measured protection to individuals with O blood-type. Further to GMTs 7 days after every dose [31]; this may be important this, another study conducted in Bangladeshi among chil- given that vibriocidal antibody titers are known to wane dren allocated to receiving a live, oral attenuated cholera quickly following vaccination as demonstrated by other vaccine, found higher frequency of serological respond- researchers [32, 33]. ers in A blood group compared to the O blood group in We further observed the trends in GMTs over time the studied children [24]. and found no differences either by blood type and secre- The study by Arifuzzaman and colleagues reported tor status. We found that vibriocidal GMTs increased by that non-secretor individuals were more susceptible to Day 28 and begun waning beyond that. Although similar V. cholerae O1 disease compared to the secretors [21]. trends in vibriocidal GMTs have been reported by Har- However, in our population, we could not report on sus- ris et al. these only go as far as 1 year [33], while we are ceptibility to V. cholerae O1 disease as we did not collect reporting here kinetics through 48 months of follow up. stool samples to screen for asymptomatic infections. Additionally, Harris and colleagues report that by 1 Nonetheless, we postulated that if there is similar year, vibriocidal antibodies return to baseline levels [33]. response to the vaccine in the non-secretors and the In our study, we observed a reduction in titer getting to secretors, it is unlikely that in our population non-secre- the lowest about 20 months post vaccination, but they tors would be more susceptible to cholera. Further, our remained slightly higher than baseline levels, and then study showed that non-secretors generally had higher began to rise beyond 30 months remaining above Day 28 GMTs against O1 Inaba when compared to the secretors. levels at month 48. Part explanation for observed differ- It is thus plausible that non-secretors were more suscep- ences could be that there was ongoing exposure in our tible to V. cholerae O1 Inaba serotype and could be why study population, and hence the raise we see beyond they generated higher titers. Consequentially, this result month 30 is actually a boost of vaccine response by sub- could then be seen as consistent with Arifuzzaman et al clinical natural exposure. A compelling environmental who reported non-secretors having an increased risk of consideration from our study population is that they live symptomatic cholera [21]. However, we add caution to on a swamp where open defecation is a norm, and with such interpretation as several factors can influence sus- limited clean water sources, residual exposure is very ceptibility to V. cholerae including, but not limited to likely. While difficult to be conclusive, it is also plausi- lack of immunity on encountering the organism [11, 25], ble that vaccination campaign could have conferred herd nutritional deficiencies [26, 27] and human genetic poly- immunity and therefore the community remains rela- morphisms [28]. tively protected from a typical outbreak. Seeing that both the O1 Ogawa and the O1 Inaba Finally, we also noted that generally dose two was not cholera serotypes circulate in Zambia [3], our finding of as beneficial in that there was a reduction in immune higher GMTs against O1 Inaba serotype could also mean responses as seen in another similar study [34]. that during the period of study, O1 Inaba serotype was A major strength of this study is that it’s the first to the most circulating strain that people might have been measure genetic factors in individuals vaccinated against exposed to. However, reports by Mwape and colleagues oral cholera vaccine Shanchol™ in our region, and the indicate that in 2016, the most circulating cholera sero- longest follow up we have seen in literature. type isolated during the cholera outbreak was O1 Ogawa The major weaknesses for this study are first, the small [3]. sample size. The computed sample size was 212, but the C hisenga et al. BMC Public Health (2023) 23:152 Page 9 of 10 study ended up with only 133 included in this analysis Availability of data and materials and this could explain the wide confidence margins and Data will be made available to any interested researchers upon request. The CIDRZ Ethics and Compliance Committee is responsible for approving such potentially why the observed differences were not sta- request. To request data access, one must write to the Secretary to the Com- tistically significant. Secondly, we did not collect stools mittee/Head of Research Operations, Ms. Hope Chinganya (Hope.C hing anya@ to also assess for helminth infections which have been cidrz.o rg). Dataset request must include contact information, a research pro- ject title, and a description of the analysis being proposed as well as the for- found to impair the immune response to oral cholera vac- mat it is expected. The requested data should only be used for the purposes cine, particularly in non-O blood groups [35]. Thirdly, we related to the original research or study. The CIDRZ Ethics and Compliance imagine that our results may have been skewed because Committee will normally review all data requests within 48–72 hours (Monday - Friday), and provide notification if access has been granted or additional our target was vulnerable men and women involved in project information is needed, before access can be granted. fishing activities and it turned out that most of our par- ticipants were men. Fourth, because this was a nested Declarations study and used data for the clinical trial, we did not have our own eligibility criteria but relied on what the parent Ethics approval and consent to participate We obtained approval from the University of Zambia Biomedical Research Eth- study applied on enrolled participants. ics Committee (UNZABREC) reference number 007–12-16, while the National Health Research Authority provided the authorization to conduct the study. Written informed consent was also obtained from all enrolled participants Conclusion into the trial. All study procedures were conducted according to good clinical practice guidelines. Several factors that influence susceptibility to cholera may affect responses to cholera vaccines. Our study has Consent for publication failed to conclude that the ABO system or the secretor Not applicable. status do have an impact on vaccine response. Future Competing interests studies to address this question would be required to set- Authors declared no competing interests. tle the questions comprehensively and allow for result generalizability. Received: 14 July 2022 Accepted: 13 January 2023 In the absence of conclusive data, individuals in endemic areas at risk of cholera need to be vaccinated equally, regardless of the blood type or secretor status. Longevity of protection by the vaccine requires specific References research in order to inform policy on when to revaccinate 1. Ali M, Nelson AR, Lopez AL, Sack DA. Updated global burden of cholera in endemic countries. PLoS Negl Trop Dis. 2015;9:1–13. https:// doi. org/ in previously immunized communities which remain at 10.1 371/ journ al.p ntd.0 00383 2. risk of outbreaks. 2. Finkelstein RA. In: Baron S, editor. Medical Microbiology. 4th ed. Galves- ton: University of Texas Medical Branch at Galveston; 1996. Available: Acknowledgments https:// www. ncbi. nlm.n ih. gov/ books/ NBK84 07/. We thank all the clinic staff at Waya Clinic, the volunteers that worked so tire- 3. Mwape K, Kwenda G, Kalonda A, Mwaba J, Lukwesa-Musyani C, Ngu- lessly in making this project a success as well as all the study participants for lube J, et al. Characterisation of Vibrio cholerae isolates from the 2009, their willingness to be a part of this program. We also thank the CIDRZ Enteric 2010 and 2016 cholera outbreaks in Lusaka province, Zambia. Pan Afr Disease and Vaccine Research Unit, our drivers Mr. Amon Kanganja and Mr. Med J. 2020;35:1–10. https:// doi.o rg/ 10. 11604/ pamj.2 020. 35.3 2.1 8853. Martin Lesa, our administrative team Ms. Annie Chikombo and Ms. Joyce Chil- 4. Deen JL, von Seidlein L, Sur D, Agtini M, Lucas MES, Lopez L, et al. The ekwa for managing all field related logistics. Furthermore, we would also like high burden of cholera in children: comparison of incidence from to thank the research nurses Ms. Fridah Madyabi and Ms. Margaret Chisambi endemic areas in Asia and Africa. PLoS Negl Trop Dis. 2008;2:e173. for managing the participants enrolments and blood draws. Finally, we extend https://d oi. org/1 0. 1371/j ourn al.p ntd.0 0001 73 Rao M, editor. Public our gratitude to Prof. David Sack, Dr. Amanda Debes and the team at Johns Library of Science. Hopkins University for all the various support and interactions on this work. 5. Poncin M, Zulu G, Voute C, Ferreras E, Muleya CM, Malama K, et al. 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Cooper PJ, Chico ME, Losonsky G, Sandoval C, Espinel I, Sridhara R, et al. of the vibriocidal antibody response in cholera due to Vibrio cholerae Albendazole treatment of children with ascariasis enhances the vibrioc- O139 bengal with the response in cholera due to Vibrio cholerae O1. Clin idal antibody response to the live attenuated oral cholera vaccine CVD Diagn Lab Immunol. 1995;2:685–8. https:// doi.o rg/1 0. 1128/ cdli.2. 6. 685- 103-HgR. J Infect Dis. 2000;182:1199–206. https:// doi.o rg/ 10. 1086/ 315837 688.1 995. United States. 19. Levine MM, Nalin DR, Rennels MB, Hornick RB, Sotman S, Van Blerk G, et al. Genetic susceptibility to cholera. Ann Hum Biol. 1979;6:369–74. https:// doi. org/ 10. 1080/ 030144 6790 00037 51 Taylor & Francis. Publisher’s Note 20. Fleckenstein JM, Kuhlmann FM, Santhanam S, Kumar P, Luo Q, Ciorba MA. 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Clin Vaccine Immunol. 2010;17:1232–7. https:// doi. org/ 10. 1128/ CVI. 00123- 10 American Society for Microbiology (ASM). 23. Clemens JD, Sack DA, Harris JR, Chakraborty J, Khan MR, Huda S, et al. ABO blood groups and Cholera: new observations on specificity of risk and modification of vaccine efficacy. J Infect Dis. 1989;159:770–3 Avail- able: http:// www. jstor. org/ stable/ 30137 111 Oxford University Press. 24. Qadri F, Chowdhury MI, Faruque SM, Salam MA, Ahmed T, Begum YA, et al. Peru-15 , a live attenuated oral cholera vaccine , is safe and immunogenic in Bangladeshi toddlers and infants. Vaccine. 2007;25:231–8. https://d oi. Ready to submit your research ? Choose BMC and benefit from: org/ 10.1 016/j. vaccin e. 2006. 08.0 31. • fast, convenient online submission 25. Harris JB, LaRocque RC, Chowdhury F, Khan AI, Logvinenko T, Faruque ASG, et al. Susceptibility to Vibrio cholerae infection in a cohort of house- • thorough peer review by experienced rese archers in your field hold contacts of patients with cholera in Bangladesh. PLoS Negl Trop Dis. • rapid publication on acceptance 2008;2:e221. https:// doi. org/ 10. 1371/j ourna l.p ntd. 000022 1. • support for research data, including large and complex data types 26. Albert MJ, Qadri F, Wahed MA, Ahmed T, Rahman ASMH, Ahmed F, et al. Supplementation with zinc, but not vitamin a, improves seroconversion • gold Open Access which fosters wider collaboration and increased citations to vibriocidal antibody in children given an oral cholera vaccine. J Infect • maximum visibility for your research: over 100M website views per year Dis. 2003;187:909–13. https:// doi. org/ 10.1 086/ 368132 United States. 27. Fischer Walker CL, Black RE. Micronutrients and diarrheal disease. Clin At BMC, research is always in progress. Infect Dis. 2007;45(Suppl 1):S73–7. https://d oi.o rg/1 0. 1086/5 18152. 28. Larocque RC, Sabeti P, Duggal P, Chowdhury F, Khan AI, Lebrun LM, et al. Learn more biomedcentral.com/submissions A variant in long palate, lung and nasal epithelium clone 1 is associated