Evaluation of humoral and cellular immune responses against Vibrio cholerae using oral immunization by multi-epitope-phage-based vaccine.

INTRODUCTION: Cholera is a severe gastrointestinal disease that manifests with rapid onset of diarrhea, vomiting, and high mortality rates. Due to its widespread occurrence in impoverished communities with poor water sanitation, there is an urgent demand for a cost-effective and highly efficient vaccine. Multi-epitope vaccines containing dominant immunological epitopes and adjuvant compounds have demonstrated potential in boosting the immune response. MATERIAL AND METHODS: B and T epitopes of OMPU, OMPW, TCPA, CTXA, and CTXB proteins were predicted using bioinformatics methods. Subsequently, highly antigenic multi-epitopes that are non-allergenic and non-toxic were synthesized. These multi-epitopes were then cloned into the pCOMB phagemid. A plasmid M13KO7ΔpIII containing all helper phage proteins except pIII was created to produce the recombinant phage. Female Balb/c mice were divided into three groups and immunized accordingly. The mice received the helper phage, recombinant phage or PBS via gavage feeding thrice within two weeks. Serum samples were collected before and after immunization for the ELISA test as well as evaluating immune system induction through ELISpot testing of spleen lymphocytes. RESULTS: The titer of the recombinant phage was determined to be 1011 PFU/ml. The presence of the recombinant phage was confirmed through differences in optical density between sample and control groups in the ELISA phage technique, as well as by observing transduction activity, which demonstrated successful production of a recombinant phage displaying the Vibrio multi-epitope on M13 phage pIII. ELISA results revealed significant differences in phage antibodies before and after inoculation, particularly notable in the negative control mice. Mice treated with multi-epitope phages exhibited antibodies against Vibrio cholerae lysate. Additionally, ELISpot results indicated activation of cellular immunity in mice receiving both Vibrio and helper phage. CONCLUSION: This study emphasizes the potential of multi-epitope on phage to enhance both cellular and humoral immunity in mice, demonstrating how phages can be used as adjuvants to stimulate mucosal immunity and act as promising candidates for oral vaccination.

Safety and immunogenicity of the Euvichol-S oral cholera vaccine for prevention of Vibrio cholerae O1 infection in Nepal: an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial.

BACKGROUND: In October, 2017, WHO launched a strategy to eliminate cholera by 2030. A primary challenge in meeting this goal is the limited global supply capacity of oral cholera vaccine and the worsening of cholera outbreaks since 2021. To help address the current shortage of oral cholera vaccine, a WHO prequalified oral cholera vaccine, Euvichol-Plus was reformulated by reducing the number of components and inactivation methods. We aimed to evaluate the immunogenicity and safety of Euvichol-S (EuBiologics, Seoul, South Korea) compared with an active control vaccine, Shanchol (Sanofi Healthcare India, Telangana, India) in participants of various ages in Nepal. METHODS: We did an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial at four hospitals in Nepal. Eligible participants were healthy individuals aged 1-40 years without a history of cholera vaccination. Individuals with a history of hypersensitivity reactions to other preventive vaccines, severe chronic disease, previous cholera vaccination, receipt of blood or blood-derived products in the past 3 months or other vaccine within 4 weeks before enrolment, and pregnant or lactating women were excluded. Participants were randomly assigned (1:1:1:1) by block randomisation (block sizes of two, four, six, or eight) to one of four groups (groups A-D); groups C and D were stratified by age (1-5, 6-17, and 18-40 years). Participants in groups A-C were assigned to receive two 1·5 mL doses of Euvichol-S (three different lots) and participants in group D were assigned to receive the active control vaccine, Shanchol. All participants and site staff (with the exception of those who prepared and administered the study vaccines) were masked to group assignment. The primary immunogenicity endpoint was non-inferiority of immunogenicity of Euvichol-S (group C) versus Shanchol (group D) at 2 weeks after the second vaccine dose, measured by the seroconversion rate, defined as the proportion of participants who had achieved seroconversion (defined as ≥four-fold increase in V cholerae O1 Inaba and Ogawa titres compared with baseline). The primary immunogenicity endpoint was assessed in the per-protocol analysis set, which included all participants who received all their planned vaccine administrations, had no important protocol deviations, and who provided blood samples for all immunogenicity assessments. The primary safety endpoint was the number of solicited adverse events, unsolicited adverse events, and serious adverse events after each vaccine dose in all ages and each age stratum, assessed in all participants who received at least one dose of the Euvichol-S or Shanchol. Non-inferiority of Euvichol-S compared with Shanchol was shown if the lower limit of the 95% CI for the difference between the seroconversion rates in Euvichol-S group C versus Shanchol group D was above the predefined non-inferiority margin of -10%. The trial was registered at ClinicalTrials.gov, NCT04760236. FINDINGS: Between Oct 6, 2021, and Jan 19, 2022, 2529 healthy participants (1261 [49·9%] males; 1268 [50·1%] females), were randomly assigned to group A (n=330; Euvichol-S lot number ES-2002), group B (n=331; Euvichol-S ES-2003), group C (n=934; Euvichol-S ES-2004]), or group D (n=934; Shanchol). Non-inferiority of Euvichol-S versus Shanchol in seroconversion rate for both serotypes at 2 weeks after the second dose was confirmed in all ages (difference in seroconversion rate for V cholerae O1 Inaba -0·00 [95% CI -1·86 to 1·86]; for V cholerae O1 Ogawa -1·62 [-4·80 to 1·56]). Treatment-emergent adverse events were reported in 244 (9·7%) of 2529 participants in the safety analysis set, with a total of 403 events; 247 events were reported among 151 (9·5%) of 1595 Euvichol-S recipients and 156 events among 93 (10·0%) of 934 Shanchol recipients. Pyrexia was the most common adverse event in both groups (57 events among 56 [3·5%] of 1595 Euvichol-S recipients and 37 events among 35 [3·7%] of 934 Shanchol recipients). No serious adverse events were deemed to be vaccine-related. INTERPRETATION: A two-dose regimen of Euvichol-S vaccine was non-inferior to the active control vaccine, Shanchol, in terms of seroconversion rates 2 weeks after the second dose. The simplified formulation and production requirements of the Euvichol-S vaccine have the potential to increase the supply of oral cholera vaccine and reduce the gap between the current oral cholera vaccine supply and demand. FUNDING: The Bill & Melinda Gates Foundation. TRANSLATION: For the Nepali translation of the abstract see Supplementary Materials section.

Early use of oral cholera vaccines as a prime control measure during outbreaks: Necessary but not sufficient.

INTRODUCTION: Despite being a preventable and treatable disease, cholera remains a public health problem in Sudan. The objective of the outbreak investigation was to identify associated risk factors that would help institute appropriate control measures. MATERIAL AND METHODS: A case control study design was chosen to identify the risk factors for cholera in Gadarif State. RESULTS: Multi-variate analysis of identified two risk factors and three preventive factors for cholera in Gadarif City. RISK FACTORS: Buying foods or drinks from street vendors (OR = 71.36), 95 % CI: 16.58-307.14), living in an urban setting (Gadarif City) (OR = 5.38), 95 % CI: 2.10-13.81); and the preventive factors were: Washing hands with water after defecation but without soap (OR = 0.16), 95 % CI: 0.04-0.63) or with soap (OR = 0.01), 95 % CI: 0.00-0.03), washing hands before eating (OR = 0.15), 95 % CI: 0.05-0.51) and taking Oral Cholera Vaccine (OCV) (OR = 0.19, 95 % CI: 0.08-0.44). The effectiveness of OCV (VE) was (Unadjusted VE: 80 %, 95 % CI: 69 %-87 %) or (Adjusted VE = 81.0 %, 95 % CI: 56.0 %-92.0 %). DISCUSSION: Cholera outbreaks, especially in the setting of a complex humanitarian crises, can spread rapidly, resulting in many deaths, and quickly become a public health crisis. Implementation of a community-wide vaccination campaign using OCV as early as possible during the outbreak while implementing other control measures to target hotspots and at-risk populations would expedite halting outbreaks of cholera and save lives.

Enhanced cholera surveillance to improve vaccination campaign efficiency.

Systematic testing for Vibrio cholerae O1 is rare, which means that the world's limited supply of oral cholera vaccines (OCVs) may not be delivered to areas with the highest true cholera burden. Here we used a phenomenological model with subnational geographic targeting and fine-scale vaccine effects to model how expanding V. cholerae testing affected impact and cost-effectiveness for preventive vaccination campaigns across different bacteriological confirmation and vaccine targeting assumptions in 35 African countries. Systematic testing followed by OCV targeting based on confirmed cholera yielded higher efficiency and cost-effectiveness and slightly fewer averted cases than status quo scenarios targeting suspected cholera. Targeting vaccine to populations with an annual incidence rate greater than 10 per 10,000, the testing scenario averted 10.8 (95% prediction interval (PI) 9.4-12.6) cases per 1,000 fully vaccinated persons while the status quo scenario averted 6.9 (95% PI 6.0-7.8) cases per 1,000 fully vaccinated persons. In the testing scenario, testing costs increased by US$31 (95% PI 25-39) while vaccination costs reduced by US$248 (95% PI 176-326) per averted case compared to the status quo. Introduction of systematic testing into cholera surveillance could improve efficiency and reach of global OCV supply for preventive vaccination.

Cholera toxin and O-specific polysaccharide immune responses after oral cholera vaccination with Dukoral in different age groups of Bangladeshi participants.

Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Methods for the estimation of direct and indirect vaccination effects by combining data from individual- and cluster-randomized trials.

Both individually and cluster randomized study designs have been used for vaccine trials to assess the effects of vaccine on reducing the risk of disease or infection. The choice between individually and cluster randomized designs is often driven by the target estimand of interest (eg, direct versus total), statistical power, and, importantly, logistic feasibility. To combat emerging infectious disease threats, especially when the number of events from one single trial may not be adequate to obtain vaccine effect estimates with a desired level of precision, it may be necessary to combine information across multiple trials. In this article, we propose a model formulation to estimate the direct, indirect, total, and overall vaccine effects combining data from trials with two types of study designs: individual-randomization and cluster-randomization, based on a Cox proportional hazards model, where the hazard of infection depends on both vaccine status of the individual as well as the vaccine status of the other individuals in the same cluster. We illustrate the use of the proposed model and assess the potential efficiency gain from combining data from multiple trials, compared to using data from each individual trial alone, through two simulation studies, one of which is designed based on a cholera vaccine trial previously carried out in Matlab, Bangladesh.

World's cholera vaccine stash is empty-but relief is on its way.

A dramatic shortage of the oral vaccine may ease in the years ahead as more companies enter the market.

Oral killed cholera vaccines for preventing cholera.

BACKGROUND: Cholera causes acute watery diarrhoea and death if not properly treated. Outbreaks occur in areas with poor sanitation, including refugee camps. Several vaccines have been developed and tested over the last 50 years. This is an update of a Cochrane review, originally published in 1998, which explored the effects of all vaccines for preventing cholera. This review examines oral vaccines made from killed bacteria. OBJECTIVES: To assess the effectiveness and safety of the available World Health Organization (WHO)-prequalified oral killed cholera vaccines among children and adults. SEARCH METHODS: We searched the Cochrane Infectious Diseases Group Specialized Register; CENTRAL, MEDLINE; Embase; LILACS; and two trials registers (February 2023). SELECTION CRITERIA: We included randomized controlled trials (RCTs), including cluster-RCTs. There were no restrictions on the age and sex of the participants or the setting of the study. We considered any available WHO-prequalified oral killed cholera vaccine as an intervention. The control group was given a placebo, another vaccine, or no vaccine. The outcomes were related to vaccine effectiveness and safety. We included articles published in English only. DATA COLLECTION AND ANALYSIS: Two review authors independently applied the inclusion criteria and extracted data from included studies. We assessed the risk of bias using the Cochrane ROB 1 assessment tool. We used the generic inverse variance and a random-effects model meta-analysis to estimate the pooled effect of the interventions. We assessed the certainty of the evidence using the GRADE approach. For vaccine effectiveness (VE), we converted the overall risk ratio (RR) to vaccine effectiveness using the formula: VE = (1 - RR) x 100%. MAIN RESULTS: Five RCTs, reported in 12 records, with 462,754 participants, met the inclusion criteria. We identified trials on whole-cell plus recombinant vaccine (WC-rBS vaccine (Dukoral)) from Peru and trials on bivalent whole-cell vaccine (BivWC (Shanchol)) vaccine from India and Bangladesh. We did not identify any trials on other BivWC vaccines (Euvichol/Euvichol-Plus), or Hillchol. Two doses of Dukoral with or without a booster dose reduces cases of cholera at two-year follow-up in a general population of children and adults, and at five-month follow-up in an adult male population (overall VE 76%; RR 0.24, 95% confidence interval (CI) 0.08 to 0.65; 2 trials, 16,423 participants; high-certainty evidence). Two doses of Shanchol reduces cases of cholera at one-year follow-up (overall VE 37%; RR 0.63, 95% CI 0.47 to 0.85; 2 trials, 241,631 participants; high-certainty evidence), at two-year follow-up (overall VE 64%; RR 0.36, 95% CI 0.16 to 0.81; 2 trials, 168,540 participants; moderate-certainty evidence), and at five-year follow-up (overall VE 80%; RR 0.20, 95% CI 0.15 to 0.26; 1 trial, 54,519 participants; high-certainty evidence). A single dose of Shanchol reduces cases of cholera at six-month follow-up (overall VE 40%; RR 0.60, 95% CI 0.47 to 0.77; 1 trial, 204,700 participants; high-certainty evidence), and at two-year follow-up (overall VE 39%; RR 0.61, 95% CI 0.53 to 0.70; 1 trial, 204,700 participants; high-certainty evidence). A single dose of Shanchol also reduces cases of severe dehydrating cholera at six-month follow-up (overall VE 63%; RR 0.37, 95% CI 0.28 to 0.50; 1 trial, 204,700 participants; high-certainty evidence), and at two-year follow-up (overall VE 50%; RR 0.50, 95% CI 0.42 to 0.60; 1 trial, 204,700 participants; high-certainty evidence). We found no differences in the reporting of adverse events due to vaccination between the vaccine and control/placebo groups. AUTHORS' CONCLUSIONS: Two doses of Dukoral reduces cases of cholera at two-year follow-up. Two doses of Shanchol reduces cases of cholera at five-year follow-up, and a single dose of Shanchol reduces cases of cholera at two-year follow-up. Overall, the vaccines were safe and well-tolerated. We found no trials on other BivWC vaccines (Euvichol/Euvichol-Plus). However, BivWC products (Shanchol, Euvichol/Euvichol-Plus) are considered to produce comparable vibriocidal responses. Therefore, it is reasonable to apply the results from Shanchol trials to the other BivWC products (Euvichol/Euvichol-Plus).

Effectiveness of one dose of killed oral cholera vaccine in an endemic community in the Democratic Republic of the Congo: a matched case-control study.

BACKGROUND: A global shortage of cholera vaccines has increased the use of single-dose regimens, rather than the standard two-dose regimen. There is sparse evidence on single-dose protection, particularly in children. In 2020, a mass vaccination campaign was conducted in Uvira, an endemic urban setting in eastern Democratic Republic of the Congo, resulting in largely single-dose coverage. We examined the effectiveness of a single-dose of the oral cholera vaccine Euvichol-Plus in this high-burden setting. METHODS: In this matched case-control study, we recruited individuals with medically attended confirmed cholera in the two cholera treatment facilities in the city of Uvira. The control group consisted of age-matched, sex-matched, and neighbourhood-matched community individuals. We recruited across two distinct periods: Oct 14, 2021, to March 10, 2022 (12-17 months after vaccination), and Nov 21, 2022, to Oct 18, 2023 (24-36 months after vaccination). Study staff administered structured questionnaires to all participants to capture demographics, household conditions, potential confounding variables, and vaccination status. The odds of vaccination for the case and control groups were contrasted in conditional logistic regression models to estimate unadjusted and adjusted vaccine effectiveness. FINDINGS: We enrolled 658 individuals with confirmed cholera and 2274 matched individuals for the control group. 99 (15·1%) individuals in the case group were younger than 5 years at the time of vaccination. The adjusted single-dose vaccine effectiveness was 52·7% (95% CI 31·4 to 67·4) 12-17 months after vaccination and 44·7% (24·8 to 59·4) 24-36 months after vaccination. Although protection in the first 12-17 months after vaccination was similar for children aged 1-4 years and older individuals, the estimate of protection in children aged 1-4 years appeared to wane during the third year after vaccination (adjusted vaccine effectiveness 32·9%, 95% CI -30·7 to 65·5), with CIs spanning the null. INTERPRETATION: A single dose of Euvichol-Plus provided substantial protection against medically attended cholera for at least 36 months after vaccination in this cholera-endemic setting. Although the evidence provides support for similar levels of protection in young children and others in the short term, protection among children younger than 5 years might wane significantly during the third year after vaccination. FUNDING: Wellcome Trust and Gavi, the Vaccine Alliance.

Effectiveness of the Euvichol® oral cholera vaccine at 2 years: A case-control and bias-indicator study in Haiti.

OBJECTIVES: The World Health Organization recommends the use of oral cholera vaccine (OCV) in cholera control efforts. Euvichol®, pre-qualified in 2015, is the leading component of the Global OCV stockpile, but data on its field effectiveness are limited. To evaluate Euvichol® vaccine effectiveness (VE), we conducted a case-control study between September 2018 to March 2020 following an OCV campaign in November 2017 in Haiti. METHODS: Cases were individuals with acute watery diarrhea. Stool samples were tested by culture and real-time polymerase chain reaction of the Vibrio cholerae ctxA gene. Cases were matched to four community controls without diarrhea by residence, enrollment time, age, and gender, and interviewed for sociodemographics, risk factors, and self-reported vaccination. Cholera cases were analyzed by conditional logistic regression in the VE study. Non-cholera diarrhea cases were analyzed in a bias-indicator study. RESULTS: We enrolled 15 cholera cases matched to 60 controls, and 63 non-cholera diarrhea cases matched to 249 controls. In the VE analysis, eight (53%) cases reported vaccination with any number of doses compared to 43 (72%) controls. Adjusted two-dose OCV VE was 69% (95% CI -71 to 94%). CONCLUSIONS: Between 10-27 months after vaccination, Euvichol® was effective and similar to Shanchol™, suggesting that it can serve as one component of multi-sectoral comprehensive cholera control.

Euvichol-plus vaccine campaign coverage during the 2017/2018 cholera outbreak in Lusaka district, Zambia: a cross-sectional descriptive study.

OBJECTIVE: To determine the coverage for the oral cholera vaccine (OCV) campaign conducted during the 2017/2018 cholera outbreak in Lusaka, Zambia. STUDY DESIGN: A descriptive cross-sectional study employing survey method conducted among 1691 respondents from 369 households following the second round of the 2018 OCV campaign. STUDY SETTING: Four primary healthcare facilities and their catchment areas in Lusaka city (Kanyama, Chawama, Chipata and Matero subdistricts). PARTICIPANTS: A total of 1691 respondents 12 months and older sampled from 369 households where the campaign was conducted. A satellite map-based sampling technique was used to randomly select households. DATA MANAGEMENT AND ANALYSIS: A pretested electronic questionnaire uploaded on an electronic tablet (ODK V.1.12.2) was used for data collection. Descriptive statistics were computed to summarise respondents' characteristics and OCV coverage per dose. Bivariate analysis (χ2 test) was conducted to stratify OCV coverage according to age and sex for each round (p<0.05). RESULTS: The overall coverage for the first, second and two doses were 81.3% (95% CI 79.24% to 83.36%), 72.1% (95% CI 69.58% to 74.62%) and 66% (95% CI 63.22% to 68.78%), respectively. The drop-out rate was 18.8% (95% CI 14.51% to 23.09%). Of the 81.3% who received the first dose, 58.8% were female. Among those who received the second dose, the majority (61.0%) were females aged between 5 and 14 years (42.6%) and 15 and 35 years (27.7%). Only 15.5% of the participants aged between 36 and 65 and 2.5% among those aged above 65 years received the second dose. CONCLUSION: These findings confirm the 2018 OCV campaign coverage and highlight the need for follow-up surveys to validate administrative coverage estimates using population-based methods. Reliance on health facility data alone may mask low coverage and prevent measures to improve programming. Future public health interventions should consider sociodemographic factors in order to achieve optimal vaccine coverage.

Vaccination of Rabbits with a Cholera Conjugate Vaccine Comprising O-Specific Polysaccharide and a Recombinant Fragment of Tetanus Toxin Heavy Chain Induces Protective Immune Responses against Vibrio cholerae O1.

There is a need for next-generation cholera vaccines that provide high-level and durable protection in young children in cholera-endemic areas. A cholera conjugate vaccine (CCV) is in development to address this need. This vaccine contains the O-specific polysaccharide (OSP) of Vibrio cholerae O1 conjugated via squaric acid chemistry to a recombinant fragment of the tetanus toxin heavy chain (OSP:rTTHc). This vaccine has been shown previously to be immunogenic and protective in mice and found to be safe in a recent preclinical toxicological analysis in rabbits. We took advantage of excess serum samples collected as part of the toxicological study and assessed the immunogenicity of CCV OSP:rTTHc in rabbits. We found that vaccination with CCV induced OSP-, lipopolysaccharide (LPS)-, and rTTHc-specific immune responses in rabbits, that immune responses were functional as assessed by vibriocidal activity, and that immune responses were protective against death in an established virulent challenge assay. CCV OSP:rTTHc immunogenicity in two animal model systems (mice and rabbits) is encouraging and supports further development of this vaccine for evaluation in humans.

Genomic associations with antibody response to an oral cholera vaccine.

Oral cholera vaccine is one of the key interventions used in our fight to end the longest pandemic of our time, cholera. The immune response conferred by the currently available cholera vaccines, as measured by serum antibody levels, is variable amongst its recipients. We undertook a genome wide association study (GWAS) on antibody response to the cholera vaccine; globally, the first GWAS on cholera vaccine response. We identified three clusters of bi-allelic SNPs, in high within-cluster linkage disequilibrium that were moderately (p < 5 × 10-6) associated with antibody response to the cholera vaccine and mapped to chromosomal regions 4p14, 4p16.1 and 6q23.3. Intronic SNPs of TBC1D1 comprised the cluster on 4p14, intronic SNPs of TBC1D14 comprised that on 4p16.1 and SNPs upstream of TNFAIP3 formed the cluster on 6q23.3. SNPs within and around these clusters have been implicated in immune cell function and immunological aspects of autoimmune or infectious diseases (e.g., diseases caused by Helicobacter pylori and malarial parasite). 6q23.3 is a prominent region harbouring many loci associated with immune related diseases, including multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus, as well as IL2 and INFα response to a smallpox vaccine. The gene clusters identified in this study play roles in vesicle-mediated pathway, autophagy and NF-κB signaling. No significant effect of O blood group on antibody response to the cholera vaccine was observed in this study.