Abstract: Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

Abstract: Background In 2013, a stockpile of oral cholera vaccine (OCV) was created for use in outbreak response, but vaccine availability remains severely limited. Innovative strategies are needed to maximize the health impact and minimize the logistical barriers to using available vaccine. Here we ask under what conditions the use of one dose rather than the internationally licensed two-dose protocol may do both. Methods and Findings Using mathematical models we determined the minimum relative single-dose efficacy (MRSE) at which single-dose reactive campaigns are expected to be as or more effective than two-dose campaigns with the same amount of vaccine. Average one- and two-dose OCV effectiveness was estimated from published literature and compared to the MRSE. Results were applied to recent outbreaks in Haiti, Zimbabwe, and Guinea using stochastic simulations to illustrate the potential impact of one- and two-dose campaigns. At the start of an epidemic, a single dose must be 35%–56% as efficacious as two doses to avert the same number of cases with a fixed amount of vaccine (i.e., MRSE between 35% and 56%). This threshold decreases as vaccination is delayed. Short-term OCV effectiveness is estimated to be 77% (95% CI 57%–88%) for two doses and 44% (95% CI −27% to 76%) for one dose. This results in a one-dose relative efficacy estimate of 57% (interquartile range 13%–88%), which is above conservative MRSE estimates. Using our best estimates of one- and two-dose efficacy, we projected that a single-dose reactive campaign could have prevented 70,584 (95% prediction interval [PI] 55,943–86,205) cases in Zimbabwe, 78,317 (95% PI 57,435–100,150) in Port-au-Prince, Haiti, and 2,826 (95% PI 2,490–3,170) cases in Conakry, Guinea: 1.1 to 1.2 times as many as a two-dose campaign. While extensive sensitivity analyses were performed, our projections of cases averted in past epidemics are based on severely limited single-dose efficacy data and may not fully capture uncertainty due to imperfect surveillance data and uncertainty about the transmission dynamics of cholera in each setting. Conclusions Reactive vaccination campaigns using a single dose of OCV may avert more cases and deaths than a standard two-dose campaign when vaccine supplies are limited, while at the same time reducing logistical complexity. These findings should motivate consideration of the trade-offs between one- and two-dose campaigns in resource-constrained settings, though further field efficacy data are needed and should be a priority in any one-dose campaign.

Abstract: Background Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). Methodology Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization. Principle Findings Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model. Conclusion We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens.

Abstract: Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

Abstract: Efforts to develop vaccines for prevention of acute diarrhea have been going on for more than 40 y with partial success. The myriad of pathogens, more than 20, that have been identified as a cause of acute diarrhea throughout the years pose a significant challenge for selecting and further developing the most relevant vaccine candidates. Based on pathogen distribution as identified in epidemiological studies performed mostly in low-resource countries, rotavirus, Cryptosporidium, Shigella, diarrheogenic E. coli and V. cholerae are predominant, and thus the main targets for vaccine development and implementation. Vaccination against norovirus is most relevant in middle/high-income countries and possibly in resource-deprived countries, pending a more precise characterization of disease impact. Only a few licensed vaccines are currently available, of which rotavirus vaccines have been the most outstanding in demonstrating a significant impact in a short time period. This is a comprehensive review, divided int...

Abstract: Enterotoxigenic Escherichia coli (ETEC) organisms are a leading cause of infectious diarrhea in developing countries. A live, attenuated cholera strain that expresses high levels of the nontoxic B subunit of cholera toxin, which might also serve as an ETEC protective antigen, was evaluated for safety, excretion, and immunogenicity in healthy volunteers. We enrolled four inpatient dose-escalation cohorts of 15 to 16 eligible subjects to randomly (3:1) receive a single oral dose of vaccine or placebo (buffer alone), evaluating 1 ×107, 1 ×108, 1 ×109, and 1 ×1010 CFU of the vaccine. The vaccine was well tolerated, although some subjects experienced moderate diarrhea. The serum Inaba vibriocidal antibody response appeared to display a dose-response relationship with increasing dosages of vaccine, plateauing at the 109-CFU dosage. The serum antitoxin (cholera toxin and heat-labile enterotoxin) antibody seroconversion rate (4-fold increase over baseline) also appeared to display a dose-response relationship. The vaccine strain was excreted in stool cultures, displaying a dose-response relationship. A single oral dose of Peru-15 pCTB at dosages up to 1 ×1010 CFU was safe and immunogenic in this first-in-human trial. These encouraging data support the ongoing clinical development of this candidate combined cholera and ETEC vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT00654108.)

Abstract: Cholera remains an important global cause of morbidity and mortality, which is capable of causing periodic epidemic disease. A number of mathematical models have been developed to help in understanding the dynamics of cholera outbreaks and for use as a tool in planning interventions, including vaccination campaigns. We have explored the utility of models in assessing the spread of cholera in the recent epidemics in Zimbabwe and Haiti. In both instances, a mathematical model was formulated and fitted to cumulative cholera cases to estimate the basic reproductive number ℛ0, and the partial reproductive numbers reflecting potential differences in environmental-to-human versus human-to-human transmission were quantified. In Zimbabwe, estimated ℛ0 for the epidemic using aggregated data at the national level was 1.15; in Haiti, it was 1.55. However, when calculated at a provincial/departmental level, estimated basic reproductive numbers were highly heterogeneous, with a range of 1.11 to 2.72 in Zimbabwe and 1.06 to 2.63 in Haiti. Our models suggest that the underlying patterns of cholera transmission varied widely from region to region, with a corresponding variation in the amenability of outbreaks to control measures such as immunization. These data underscore the heterogeneity of transmission dynamics, potentially linked to differences in environment, socio-economic conditions, and cultural practices. They also highlight the potential utility of these types of models in guiding development of public health intervention strategies.

Abstract: Coverage was comparable to or higher than that in other countries.

Abstract: INTRODUCTION: Since 2010 WHO has recommended oral cholera vaccines as an additional strategy for cholera control. During a cholera episode pregnant women are at high risk of complications and the risk of fetal death has been reported to be 2-36%. Due to a lack of safety data pregnant women have been excluded from most cholera vaccination campaigns. In 2012 reactive campaigns using the bivalent killed whole-cell oral cholera vaccine (BivWC) included all people living in the targeted areas aged >/= 1 year regardless of pregnancy status were implemented in Guinea. We aimed to determine whether there was a difference in pregnancy outcomes between vaccinated and non-vaccinated pregnant women. METHODS AND FINDINGS: From 11 November to 4 December 2013 we conducted a retrospective cohort study in Boffa prefecture among women who were pregnant in 2012 during or after the vaccination campaign. The primary outcome was pregnancy loss as reported by the mother and fetal malformations after clinical examination. Primary exposure was the intake of the BivWC vaccine (Shanchol) during pregnancy as determined by a vaccination card or oral history. We compared the risk of pregnancy loss between vaccinated and non-vaccinated women through binomial regression analysis. A total of 2494 pregnancies were included in the analysis. The crude incidence of pregnancy loss was 3.7% (95%CI 2.7-4.8) for fetuses exposed to BivWC vaccine and 2.6% (0.7-4.5) for non-exposed fetuses. The incidence of malformation was 0.6% (0.1-1.0) and 1.2% (0.0-2.5) in BivWC-exposed and non-exposed fetuses respectively. In both crude and adjusted analyses fetal exposure to BivWC was not significantly associated with pregnancy loss (adjusted risk ratio (aRR = 1.09 [95%CI: 0.5-2.25] p = 0.818) or malformations (aRR = 0.50 [95%CI: 0.13-1.91] p = 0.314). CONCLUSIONS: In this large retrospective cohort study we found no association between fetal exposure to BivWC and risk of pregnancy loss or malformation. Despite the weaknesses of a retrospective design we can conclude that if a risk exists it is very low. Additional prospective studies are warranted to add to the evidence base on OCV use during pregnancy. Pregnant women are particularly vulnerable during cholera episodes and should be included in vaccination campaigns when the risk of cholera is high such as during outbreaks.

Abstract: Background A bivalent killed whole cell oral cholera vaccine has been found to be safe and efficacious for five years in the cholera endemic setting of Kolkata, India, when given in a two dose schedule, two weeks apart. A randomized controlled trial revealed that the immune response was not significantly increased following the second dose compared to that after the first dose. We aimed to evaluate the impact of an extended four week dosing schedule on vibriocidal response.

Abstract: The 2010 earthquake in Haiti and the subsequent cholera outbreak taught us multiple lessons on how we might better avert cholera outbreaks, beyond simply improving access to clean water and sanitation [1]. Post-earthquake Nepal is now a test of how well we learned these lessons (Fig 1). Earthquake-associated mortality generally progresses in well-defined phases that ultimately give way to longer-lasting periods of new and complex health needs. The first phase is caused by the immediate kinetic trauma. The second is a product of infectious complications from the bodily trauma. The third is linked to infectious disease outbreaks enabled by the destroyed infrastructure; many of these latter diseases are now vaccine preventable, including cholera.

Abstract: We evaluated immune responses following bivalent oral cholera vaccination (Shanchol [Shantha Biotechnics]; BivWC) in a cohort of 25 human immunodeficiency virus (HIV)-infected adults in Haiti. Compared with adults without HIV infection, vaccination in HIV-infected individuals resulted in lower vibriocidal responses against Vibrio cholerae O1, and there was a positive relationship between the CD4(+) T-cell count and vibriocidal responses following vaccination. Nevertheless, seroconversion occurred at a rate of 65% against the Ogawa serotype and 74% against the Inaba serotype in adults with HIV infection. These results suggest that the vaccine retains substantial immunogenicity in adults with HIV infection and may benefit this population by protecting against cholera.

Abstract: Background Service provider costs for vaccine delivery have been well documented; however, vaccine recipients’ costs have drawn less attention. This research explores the private household out-of-pocket and opportunity costs incurred to receive free oral cholera vaccine during a mass vaccination campaign in rural Odisha, India. Methods Following a government-driven oral cholera mass vaccination campaign targeting population over one year of age, a questionnaire-based cross-sectional survey was conducted to estimate private household costs among vaccine recipients. The questionnaire captured travel costs as well as time and wage loss for self and accompanying persons. The productivity loss was estimated using three methods: self-reported, government defined minimum daily wages and gross domestic product per capita in Odisha. Findings On average, families were located 282.7 (SD = 254.5) meters from the nearest vaccination booths. Most family members either walked or bicycled to the vaccination sites and spent on average 26.5 minutes on travel and 15.7 minutes on waiting. Depending upon the methodology, the estimated productivity loss due to potential foregone income ranged from $0.15 to $0.29 per dose of cholera vaccine received. The private household cost of receiving oral cholera vaccine constituted 24.6% to 38.0% of overall vaccine delivery costs. Interpretation The private household costs resulting from productivity loss for receiving a free oral cholera vaccine is a substantial proportion of overall vaccine delivery cost and may influence vaccine uptake. Policy makers and program managers need to recognize the importance of private costs and consider how to balance programmatic delivery costs with private household costs to receive vaccines.

Abstract: The World Health Organization (WHO) recently established a global stockpile of oral cholera vaccine (OCV) to be preferentially used in epidemic response (reactive campaigns) with any vaccine remaining after 1 year allocated to endemic settings. Hence, the number of cholera cases or deaths prevented in an endemic setting represents the minimum utility of these doses, and the optimal risk-averse response to any reactive vaccination request (i.e. the minimax strategy) is one that allocates the remaining doses between the requested epidemic response and endemic use in order to ensure that at least this minimum utility is achieved. Using mathematical models, we find that the best minimax strategy is to allocate the majority of doses to reactive campaigns, unless the request came late in the targeted epidemic. As vaccine supplies dwindle, the case for reactive use of the remaining doses grows stronger. Our analysis provides a lower bound for the amount of OCV to keep in reserve when responding to any request. These results provide a strategic context for the fulfilment of requests to the stockpile, and define allocation strategies that minimize the number of OCV doses that are allocated to suboptimal situations.

Abstract: Background The bivalent killed oral cholera vaccine (OCV) provides 65% cumulative protection over five years. It remains unknown whether a boosting regimen can maintain protection in previously immunized populations. This study examines the immunogenicity and safety of an OCV regimen given five years following initial dosing.

Abstract: Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund’s Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund’s complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

Abstract: The development of new alternative platforms for subunit vaccine production is a priority in the biomedical field In this study, Ustilago maydis, the causal agent of common corn smut or ‘huitlacoche’has been genetically engineered to assess expression and immunogenicity of the B subunit of the cholera toxin (CTB), a relevant immunomodulatory agent in vaccinology An oligomeric CTB recombinant protein was expressed in corn smut galls at levels of up to 13 mg g-1 dry weight (08% of the total soluble protein) Mice orally immunized with ‘huitlacoche’-derived CTB showed significant humoral responses that were well-correlated with protection against challenge with the cholera toxin (CT) These findings demonstrate the feasibility of using edible corn smut as a safe, effective, and low-cost platform for production and delivery of a subunit oral vaccine The implications of this platform in the area of molecular pharming are discussed

Abstract: During the last four decades, Uganda has experienced repeated cholera outbreaks in communities; no cholera outbreaks have been reported in Ugandan health facilities. In October 2008, a unique cholera outbreak was confirmed in Butabika National Mental Referral Hospital (BNMRH), Uganda. This article describes actions taken to control the outbreak, challenges, and lessons learnt. We reviewed patient and hospital reports for clinical symptoms and signs, treatment and outcome, patient mental diagnosis, and challenges noted during management of patients and contacts. Out of 114 BNMRH patients on two affected wards, 18 cholera cases and five deaths were documented for an attack rate of 15.8% and a case fatality rate of 28%. Wards and surroundings were intensively disinfected and 96 contacts (psychiatric patients) in the affected wards received chemoprophylaxis with oral ciprofloxacin 500 mg twice daily until November 5, 2008. We documented a nosocomial cholera outbreak in BNMRH with a high case fatality of 28% compared with the national average of 2.4% for cholera outbreaks in communities. To avoid cholera outbreaks and potentially high mortality among patients in mental institutions, procedures for prompt diagnosis, treatment, disinfection, and prophylaxis are needed and preemptive use of oral cholera vaccines should be considered.

Abstract: We propose the ideal oral cholera vaccine (OCV) should be an inexpensive, single, oral dose that rapidly confers immunity for a long duration, and is well tolerated by individuals vulnerable to cholera. Vaccine trials in industrialized countries of a single oral dose of 5 × 108 colony forming units (CFU) of the live, attenuated cholera strain CVD 103-HgR have shown 88–97% serum vibriocidal antibody seroconversion rates, a correlate of protection and documented vaccine efficacy of ≥80% using volunteer challenge studies with wild-type cholera. For individuals of developing countries, a 5 × 109 CFU dose of CVD 103-HgR is necessary to elicit similar antibody responses. Presently, a reformulation of CVD 103-HgR is in late-stage clinical development for prospective US FDA licensure; making a cholera vaccine for US travelers potentially accessible in 2016. The availability of CVD 103-HgR should be a welcome addition to the currently available OCVs.

Abstract: Summary: Background : Cholera became an endemic disease in Iraq, strikes in epidemic form nearly every ten years, but with irregular outbreaks. Iraq is facing great disasters of destruction of the infrastructures with shortage of electricity and safe water for drinking especially in the poor districts and the refugee camps that helps for the appearance of water-borne diseases including cholera. Also the sewage disposal systems has greatly damaged or obstructed. Aim : It is to shed light on the epidemiology, the pattern of cholera cases that appeared suddenly during the thirty eighth week of October in Baghdad districts and spread to many other governorates. The outcome of cases was also mentioned. It is also a descriptive analysis for the additive cases on daily bases through addition or discarding of cases of suspected cholera according to the reference laboratory testing. Subjects, Methods, and Materials: The study is a descriptive analysis; is that the data (officially supplied from the Center of Disease Control/ Ministry of Health) is dynamic and every day and week there are new cases and discarded cases till at the end of month the final report was done. As Iraq is an endemic country for cholera, the case definition in the endemic countries according to the WHO is: (Acute watery diarrhea for more than three bowel motions with or without vomiting in a patient aged 5 years or more). Accordingly, cases with the above complaints who attended the hospitals and health facilities are examined for stool cultures and sero- typing for Vibrio cholerae in the Central Public Health Laboratory (CPHL). Those who are stool culture- positive will be reported here as cholera cases. Follow up of the hospitalized cases for their outcome are reported; diarrheal cases reported from all governorates of Iraq together, with the positive cases of cholera are mentioned also. Epidemiological curves for cholera and acute diarrheal diseases for the years 2013, 2014 and 2015 are presented for comparison. Results: The number of acute diarrhea cases together with cholera cases for the year 2015, their ages and gender are reported according to the distribution by weeks. The trend of cholera in Iraq since the year 1990- 2014 is reported. The total number of admitted cases of cholera from the 23 rd September till 6/11/2015 that were laboratory confirmed are 2651 cases comprising of 1477 males and 1174 females, with all age groups were included. The sero typing of the isolates show that the Vibrio cholera was O1 sero type Inaba with few cases only of Ogawa sero type. This final figure was corrected every day through the addition of new cases and discarding of others; the number includes all cholera cases in Baghdad and fourteen other governorates. There were only two cases died with CFR of 0.075%. It appeared, the trend of attack of cholera in Iraq was classical i.e. during the months of September and October and not in the hot months of Sumer as that appeared during the years of 1990 and 2007. Conclusion and Recommendation : Communicable diseases surveillance needs an immediate notification, daily, weekly and monthly reports, with Syndromes Surveillance when the situation is complex. Epi- info soft ware is to be used to collect surveillance data via fixed systems and submitted to the center by using the internet. Health education campaigns should be adapted to the local cultures and their habits; they should promote practices of personal hygiene with concentration on hand-washing with soap and detergents. The storage and preparation of food should be safe, together with encouraging of breastfeeding for lactating women. Innovative WASH interventions are needed to prevent cholera, together with the surveillance and diagnostics through laboratory net work; the use of rapid diagnostic tests (RDTs) is needed. Oral cholera vaccine (OCV) should always be used as an additional public health tool with mass vaccination campaigns to be launched for people especially in the camps or when facing disasters of mobilization or facing flood and sewage contamination.

Abstract: Considerable variation in antibody response (AR) was observed among recipients of an injectable typhoid vaccine and an oral cholera vaccine. We sought to find whether polymorphisms in genes of the immune system, both innate and adaptive, were associated with the observed variation in response. For both vaccines, we were able to discover and validate several polymorphisms that were significantly associated with immune response. For the typhoid vaccines, these polymorphisms were on genes that belonged to pathways of polysaccharide recognition, signal transduction, inhibition of T-cell proliferation, pro-inflammatory signalling and eventual production of antimicrobial peptides. For the cholera vaccine, the pathways included epithelial barrier integrity, intestinal homeostasis and leucocyte recruitment. Even though traditional wisdom indicates that both vaccines should act as T-cell-independent antigens, our findings reveal that the vaccines induce AR using different pathways.