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Interventional studies targeting environment enteropathy (EE) are impeded by the lack of appropriate, validated, non-invasive biomarkers of EE. Thus, we aimed to validate the association of potential biomarkers for EE with enteric infections and nutritional status in a longitudinal birth cohort study. We measured endotoxin core antibody (EndoCab) and soluble CD14 (sCD14) in serum, and myeloperoxidase (MPO) in feces using commercially available enzyme-linked immunosorbent assay (ELISA) kits. We found that levels of serum EndoCab and sCD14 increase with the cumulative incidence of enteric infections. We observed a significant correlation between the fecal MPO level in the children at 24 months of age with the total number of bacterial and viral infections, the total number of parasitic infections, and the total number of diarrheal episodes and diarrheal duration. We observed that the levels of serum EndoCab, sCD14, and fecal MPO at 3 months of age were significantly associated with whether children were malnourished at 18 months of age or not. Biomarkers such as fecal MPO, serum EndoCab and sCD14 in children at an early age may be useful as a measure of cumulative burden of preceding enteric infections, which are predictive of subsequent malnutrition status and may be useful non-invasive biomarkers for EE., Competing Interests: The authors have declared that no competing interests exist.

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP's function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP's carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

After the large influx of Rohingya nationals (termed Forcibly Displaced Myanmar National; FDMN) from Rakhine State of Myanmar to Cox's Bazar in Bangladesh, it was apparent that outbreaks of cholera was very likely in this setting where people were living under adverse water and sanitation conditions. Large campaigns of oral cholera vaccine (OCV) were carried out as a preemptive measure to control cholera epidemics. The aim of the study was to evaluate the immune responses of healthy adults and children after administration of two doses of OCV at 14 days interval in FDMN population and compare with the response observed in Bangladeshi's vaccinated earlier. A cross-sectional immunogenicity study was conducted among FDMNs of three age cohort; in adults (18+years; n = 83), in older children (6-17 years; n = 63) and in younger children (1-5 years; n = 80). Capillary blood was collected at three time points to measure vibriocidal antibodies using either plasma or dried blood spot (DBS) specimens. There was a significant increase of responder frequency of vibriocidal antibody titer at day 14 in all groups for Vibrio cholerae O1 (Ogawa/Inaba: adults-64%/64%, older children-70%/89% and younger children-51%/75%). There was no overall difference of vibriocidal antibody titer between FDMN and Bangladeshi population at baseline (p = 0.07-0.08) and at day 14, day 28 in all age groups for both serotypes. The seroconversion rate and geometric mean titer (GMT) of either serotype were comparable using both plasma and DBS specimens. These results showed that OCV is capable of inducing robust immune responses in adults and children among the FDMN population which is comparable to that seen in Bangladeshi participants in different age groups or that reported from other cholera endemic countries. Our results also suggest that the displaced population were exposed to V. cholerae prior to seeking shelter in Bangladesh., Competing Interests: The authors have declared that no competing interests exist.

Background: Enterotoxigenic Escherichia coli causes diarrhoea, leading to substantial mortality and morbidity in children, but no specific vaccine exists. This trial tested an oral, inactivated, enterotoxigenic E coli vaccine (ETVAX), which has been previously shown to be safe and highly immuongenic in Swedish and Bangladeshi adults. We tested the safety and immunogenicity of ETVAX, consisting of four E coli strains overexpressing the most prevalent colonisation factors (CFA/I, CS3, CS5, and CS6) and a toxoid (LCTBA) administered with or without a double-mutant heat-labile enterotoxin (dmLT) as an adjuvant, in Bangladeshi children., Methods: We did a randomised, double-blind, placebo-controlled, dose-escalation, age-descending, phase 1/2 trial in Dhaka, Bangladesh. Healthy children in one of three age groups (24-59 months, 12-23 months, and 6-11 months) were eligible. Children were randomly assigned with block randomisation to receive either ETVAX, with or without dmLT, or placebo. ETVAX (half [5·5 × 10 10 cells], quarter [2·5 × 10 10 cells], or eighth [1·25 × 10 10 cells] adult dose), with or without dmLT adjuvant (2·5 μg, 5·0 μg, or 10·0 μg), or placebo were administered orally in two doses 2 weeks apart. Investigators and participants were masked to treatment allocation. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunisation, were assessed as secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02531802., Findings: Between Dec 7, 2015, and Jan 10, 2017, we screened 1500 children across the three age groups, of whom 430 were enrolled and randomly assigned to the different treatment groups (130 aged 24-59 months, 100 aged 12-23 months, and 200 aged 6-11 months). All participants received at least one dose of vaccine. No solicited adverse events occurred that were greater than moderate in severity, and most were mild. The most common solicited event was vomiting (ten [8%] of 130 patients aged 24-59 months, 13 [13%] of 100 aged 12-23 months, and 29 [15%] of 200 aged 6-11 months; mostly of mild severity), which appeared related to dose and age. The addition of dmLT did not modify the safety profile. Three serious adverse events occurred but they were not considered related to the study drug. Mucosal IgA antibody responses in lymphocyte secretions were detected against all primary vaccine antigens (CFA/I, CS3, CS5, CS6, and the LCTBA toxoid) in most participants in the two older age groups, whereas such responses to four of the five antigens were less frequent and of lower magnitude in infants aged 6-11 months than in older children. Faecal secretory IgA immune responses were recorded against all vaccine antigens in infants aged 6-11 months. 78 (56%) of 139 infants aged 6-11 months who were vaccinated developed mucosal responses against at least three of the vaccine antigens versus 14 (29%) of 49 of the infants given placebo. Addition of the adjuvant dmLT enhanced the magnitude, breadth, and kinetics (based on number of responders after the first dose of vaccine) of immune responses in infants., Interpretation: The encouraging safety and immunogenicity of ETVAX and benefit of dmLT adjuvant in young children support its further assessment for protective efficacy in children in enterotoxigenic E coli-endemic areas., Funding: PATH (Bill & Melinda Gates Foundation and the UK's Department for International Development), the Swedish Research Council, and The Swedish Foundation for Strategic Research., (Copyright © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The safety and immunogenicity of the second generation oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of inactivated recombinant E. coli strains over-expressing the colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the heat labile toxoid LCTBA, were evaluated in Bangladeshi volunteers. To enable analysis of antibody responses against multiple vaccine antigens for subsequent use in small sample volumes from children, a sensitive electrochemiluminescence (ECL) assay for analysis of intestine-derived antibody-secreting cell responses using the antibodies in lymphocyte secretions (ALS) assay was established using Meso Scale Discovery technology. Three groups of Bangladeshi adults (n = 15 per group) received two oral doses of ETVAX with or without double mutant LT (dmLT) adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. CF- and LTB-specific ALS and plasma IgA responses were analyzed by ECL and/or ELISA. ETVAX was safe and well tolerated in the adults. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated well (r = 0.85 to 0.98 for the five primary antigens, P < 0.001) and ECL was selected as the ALS readout method. ALS IgA responses against each of the primary antigens were detected in 87-100% of vaccinees after the first and in 100% after the second vaccine dose. Plasma IgA responses against different CFs and LTB were observed in 62-93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on antibody responses to any antigen was detected, but the overall antigenic breadth of the plasma IgA response tended to favor the adjuvanted vaccine when responses to 4 or more or 5 vaccine antigens were considered. Responses in placebo recipients were infrequent and mainly detected against single antigens. The promising results in adults supported testing ETVAX in descending age groups of children. ClinicalTrials.gov Identifier: NCT02531802., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Rotavirus (RV)-specific immunoglobulin A (IgA) responses following oral RV vaccination are impaired in low-income countries, where the utility of RV-IgA as a correlate of protection (CoP) remains unclear. In a monovalent oral RV vaccine (Rotarix) efficacy trial among infants in Dhaka, Bangladesh, we identified factors associated with poor RV-IgA responses and explored the utility of RV-IgA as a CoP., Methods: Infants were randomized to receive Rotarix or no Rotarix at 10 and 17 weeks of life and followed with active diarrheal surveillance. RV-IgA concentration, seroconversion, and seropositivity were determined at 18 weeks of life and analyzed for correlation(s) with rotavirus diarrhea (RVD) and for contribution to Rotarix vaccine effect., Results: Among vaccinated infants, overall RV-IgA geometric mean concentration was 21 U/mL; only 27% seroconverted and 32% were seropositive after vaccination. Increased RV-specific maternal antibodies significantly impaired immunogenicity. Seroconversion was associated with reduced risk of RVD through 1 year of life, but RV-IgA seropositivity only explained 7.8% of the vaccine effect demonstrated by the clinical endpoint (RVD)., Conclusions: RV-IgA responses were low among infants in Bangladesh and were significantly impaired by maternal antibodies. RV-IgA is a suboptimal CoP in this setting; an improved CoP for RV in low-income countries is needed., Clinical Trials Registration: NCT01375647.

Immune responses to oral enteric vaccines in children and infants may be influenced by factors such as age, previous priming with related microorganisms and breast feeding. In this study, we aimed to determine optimal time points to assess immune responses to oral enteric vaccines in different clinical specimens. This was done by investigating antibody secreting cell (ASC) and fecal antibody responses on different days after vaccination using the licensed oral cholera vaccine Dukoral, containing cholera toxin B-subunit (rCTB) and inactivated Vibrio cholerae bacteria, as a model vaccine. Two vaccine doses were given 2weeks apart to infants (6-11months), young children (12-18months), toddlers (19months-5years) and adults in a cholera endemic country (Bangladesh). IgA ASC responses, as determined by the antibodies in lymphocyte supernatant (ALS) assay, plasma IgA and IgG responses and secretory IgA (SIgA) responses in extracts of fecal samples were evaluated 4/5 and 7days after each vaccination. After the first vaccine dose, anti-CTB ALS IgA responses in adults and toddlers were high and comparable on day 5 and 7, while responses were low and infrequent in young children. After the second dose, highest ALS responses were detected on day 5 among the time points studied in all age groups and the responses declined until day 7. In contrast, plasma IgA and IgG anti-CTB responses were high both on day 5 and 7 after the second dose. Fecal SIgA responses in young children and infants were highest on day 7 after the second dose. Our results suggest that ASC/ALS responses to two doses of the oral cholera vaccine Dukoral and related oral vaccines should be analyzed earlier than previously recommended (day 7) at all ages. Fecal antibody responses should preferably be analyzed later than ASC/ALS responses to detect the highest antibody responses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Environmental enteropathy (EE) is a poorly understood condition that refers to chronic alterations in intestinal permeability, absorption, and inflammation, which mainly affects young children in resource-limited settings. Recently, EE has been linked to suboptimal oral vaccine responses in children, although immunological mechanisms are poorly defined. The objective of this study was to determine host factors associated with immune responses to an oral cholera vaccine (OCV). We measured antibody and memory T cell immune responses to cholera antigens, micronutrient markers in blood, and EE markers in blood and stool from 40 Bangladeshi children aged 3-14 years who received two doses of OCV given 14 days apart. EE markers included stool myeloperoxidase (MPO) and alpha anti-trypsin (AAT), and plasma endotoxin core antibody (EndoCab), intestinal fatty acid binding protein (i-FABP), and soluble CD14 (sCD14). We used multiple linear regression analysis with LASSO regularization to identify host factors, including EE markers, micronutrient (nutritional) status, age, and HAZ score, predictive for each response of interest. We found stool MPO to be positively associated with IgG antibody responses to the B subunit of cholera toxin (P = 0.03) and IgA responses to LPS (P = 0.02); plasma sCD14 to be positively associated with LPS IgG responses (P = 0.07); plasma i-FABP to be positively associated with LPS IgG responses (P = 0.01) and with memory T cell responses specific to cholera toxin (P = 0.01); stool AAT to be negatively associated with IL-10 (regulatory) T cell responses specific to cholera toxin (P = 0.02), and plasma EndoCab to be negatively associated with cholera toxin-specific memory T cell responses (P = 0.02). In summary, in a cohort of children 3-14 years old, we demonstrated that the majority of biomarkers of environmental enteropathy were positively associated with immune responses after vaccination with an OCV., Competing Interests: The authors have declared that no competing interests exist.