Antibody longevity and waning following COVID-19 vaccination in a 1-year longitudinal cohort in Bangladesh.

COVID-19 vaccines have been effective in preventing severe illness, hospitalization and death, however, the effectiveness diminishes with time. Here, we evaluated the longevity of antibodies generated by COIVD-19 vaccines and the risk of (re)infection in Bangladeshi population. Adults receiving two doses of AstraZeneca, Pfizer, Moderna or Sinopharm vaccines were enrolled at 2-4 weeks after second dosing and followed-up at 4-monthly interval for 1 year. Data on COVID-like symptoms, confirmed COVID-19 infection, co-morbidities, and receipt of booster dose were collected; blood was collected for measuring spike (S)- and nucleocapsid (N)-specific antibodies. S-specific antibody titers reduced by ~ 50% at 1st follow-up visit and continued to decline unless re-stimulated by booster vaccine dose or (re)infection. Individuals infected between follow-up visits showed significantly lower S-antibody titers at preceding visits compared to the uninfected individuals. Pre-enrolment infection between primary vaccination dosing exhibited 60% and 50% protection against reinfection at 5 and 9 months, respectively. mRNA vaccines provided highest odds of protection from (re)infection up to 5 months (Odds Ratio (OR) = 0.08), however, protection persisted for 9 months in AstraZeneca vaccine recipients (OR = 0.06). In conclusion, vaccine-mediated protection from (re)infection is partially linked to elevated levels of S-specific antibodies. AstraZeneca vaccine provided the longest protection.

High rates of placental inflammation among samples collected by the Multi-Omics for Mothers and Infants consortium.

BACKGROUND: The Multi-Omics for Mothers and Infants consortium aims to improve birth outcomes. Preterm birth is a major obstetrical complication globally and causes significant infant and childhood morbidity and mortality. OBJECTIVE: We analyzed placental samples (basal plate, placenta or chorionic villi, and the chorionic plate) collected by the 5 Multi-Omics for Mothers and Infants sites, namely The Alliance for Maternal and Newborn Health Improvement Bangladesh, The Alliance for Maternal and Newborn Health Improvement Pakistan, The Alliance for Maternal and Newborn Health Improvement Tanzania, The Global Alliance to Prevent Prematurity and Stillbirth Bangladesh, and The Global Alliance to Prevent Prematurity and Stillbirth Zambia. The goal was to analyze the morphology and gene expression of samples collected from preterm and uncomplicated term births. STUDY DESIGN: The teams provided biopsies from 166 singleton preterm (<37 weeks' gestation) and 175 term (≥37 weeks' gestation) deliveries. The samples were fixed in formalin and paraffin embedded. Tissue sections from these samples were stained with hematoxylin and eosin and subjected to morphologic analyses. Other placental biopsies (n=35 preterm, 21 term) were flash frozen, which enabled RNA purification for bulk transcriptomics. RESULTS: The morphologic analyses revealed a surprisingly high rate of inflammation that involved the basal plate, placenta or chorionic villi, and the chorionic plate. The rate of inflammation in chorionic villus samples, likely attributable to chronic villitis, ranged from 25% (Pakistan site) to 60% (Zambia site) of cases. Leukocyte infiltration in this location vs in the basal plate or chorionic plate correlated with preterm birth. Our transcriptomic analyses identified 267 genes that were differentially expressed between placentas from preterm vs those from term births (123 upregulated, 144 downregulated). Mapping the differentially expressed genes onto single-cell RNA sequencing data from human placentas suggested that all the component cell types, either singly or in subsets, contributed to the observed dysregulation. Consistent with the histopathologic findings, gene ontology analyses highlighted the presence of leukocyte infiltration or activation and inflammatory responses in both the fetal and maternal compartments. CONCLUSION: The relationship between placental inflammation and preterm birth is appreciated in developed countries. In this study, we showed that this link also exists in developing geographies. In addition, among the participating sites, we found geographic- and population-based differences in placental inflammation and preterm birth, suggesting the importance of local factors.

Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: a qualification analysis.

BACKGROUND: Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). METHODS: Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. FINDINGS: High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1-7·7% coefficient of variation for all three ELISAs, 3·8-19·5% for PNA, and 17·1-24·1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9-49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. INTERPRETATION: The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines. FUNDING: CEPI.

Serosurveillance among urban slum and non-slum populations immunized with COVID-19 vaccines in Bangladesh.

Using two rounds of serosurveillance, we aimed to observe the COVID-19 vaccination status and the dynamics of antibody responses to different vaccines among urban slum and non-slum populations of Bangladesh. Adults (>18 years) and children (10-17 years) were enrolled in March and October 2022. Data including COVID-19 vaccine types and dosage uptake were collected. SARS-CoV-2 spike (S)-specific antibodies were measured in blood. The proportion of vaccinated children was significantly lower among slum than non-slum populations. Two doses of vaccines showed an increase in the level of anti-S-antibodies up to 2 months, followed by reduced levels at 2-6 months and a resurgence at 6-12 months. Children showed significantly higher anti-S-antibodies after two doses of the Pfizer-BioNTech vaccine than adults; however, after 6 months, the level of antibodies declined in younger children (10 - < 12 years). In a mixed vaccine approach, mRNA vaccines contributed to the highest antibody response whether given as the first two doses or as the third dose. Our findings emphasized the need for increasing the coverage of COVID-19 vaccination among slum children and booster dosing among all children. The use of mRNA vaccines in the mixed vaccination approach was found to be useful in boosting the antibody response to SARS-CoV-2.

Factors Associated with Reported COVID-like Symptoms and Seroprevalence Data Matched with COVID-like Symptoms in Slums and Non-Slums of Two Major Cities in Bangladesh.

OBJECTIVES: To examine the levels and socio-demographic differentials of: (a) reported COVID-like symptoms; and (b) seroprevalence data matched with COVID-like symptoms. METHODS: Survey data of reported COVID-like symptoms and seroprevalence were assessed by Roche Elecsys® Anti-SARS-CoV-2 immunoassay. Survey data of 10,050 individuals for COVID-like symptoms and seroprevalence data of 3205 individuals matched with COVID-like symptoms were analyzed using bivariate and multivariate logistic analysis. RESULTS: The odds of COVID-like symptoms were significantly higher for Chattogram city, for non-slum, people having longer years of schooling, working class, income-affected households, while for households with higher income had lower odd. The odds of matched seroprevalence and COVID-like symptoms were higher for non-slum, people having longer years of schooling, and for working class. Out of the seropositive cases, 37.77% were symptomatic-seropositive, and 62.23% were asymptomatic, while out of seronegative cases, 68.96% had no COVID-like symptoms. CONCLUSIONS: Collecting community-based seroprevalence data is important to assess the extent of exposure and to initiate mitigation and awareness programs to reduce COVID-19 burden.

Antibody response to different COVID-19 vaccines among the migrant workers of Bangladesh.

Background: Due to the ongoing COVID-19 pandemic, various host countries such as Singapore, imposed entry requirements for migrant workers including pre-departure COVID-19 seroconversion proof. To combat COVID-19 worldwide, several vaccines have acquired conditional approval. This study sought to assess antibody levels after immunization with different COVID-19 vaccines among the migrant workers of Bangladesh. Methods: Venous blood samples were collected from migrant workers who were vaccinated with different COVID-19 vaccines (n=675). Antibodies to SARS-CoV-2 spike protein (S) and nucleocapsid protein (N) were determined using Roche Elecsys® Anti-SARS-CoV-2 S and N immunoassay, respectively. Results: All participants receiving COVID-19 vaccines showed antibodies to S-protein, while 91.36% were positive for N-specific antibodies. The highest anti-S antibody titers were found among the workers who completed booster doses (13327 U/mL), received mRNA vaccines Moderna/Spikevax (9459 U/mL) or Pfizer-BioNTech/Comirnaty (9181 U/mL), and reported SARS-CoV-2 infection in the last six months (8849 U/mL). The median anti-S antibody titers in the first month since the last vaccination was 8184 U/mL, which declined to 5094 U/mL at the end of six months. A strong correlation of anti-S antibodies was found with past SARS-CoV-2 infection (p < 0.001) and the type of vaccines received (p <0.001) in the workers.Conclusion: Bangladeshi migrant workers receiving booster doses of vaccine, vaccinated with mRNA vaccines, and having past SARS-CoV-2 infection, mounted higher antibody responses. However, antibody levels waned with time. These findings suggest a need for further booster doses, preferably with mRNA vaccines for migrant workers before reaching host countries.

Immune cell landscape in symptomatic and asymptomatic SARS-CoV-2 infected adults and children in urban Dhaka, Bangladesh.

OBJECTIVES: The study of cellular immunity to SARS-CoV-2 is crucial for evaluating the course of the COVID-19 disease and for improving vaccine development. We aimed to assess the phenotypic landscape of circulating lymphocytes and mononuclear cells in adults and children who were seropositive to SARS-CoV-2 in the past 6 months. METHODS: Blood samples (n = 350) were collected in a cross-sectional study in Dhaka, Bangladesh (Oct 2020-Feb 2021). Plasma antibody responses to SARS-CoV-2 were determined by an electrochemiluminescence immunoassay while lymphocyte and monocyte responses were assessed using flow cytometry including dimensionality reduction and clustering algorithms. RESULTS: SARS-CoV-2 seropositivity was observed in 52% of adults (18-65 years) and 56% of children (10-17 years). Seropositivity was associated with reduced CD3+T cells in both adults (beta(ß) = -2.86; 95% Confidence Interval (CI) = -5.98, 0.27) and children (ß = -8.78; 95% CI = -13.8, -3.78). The frequencies of T helper effector (CD4+TEFF) and effector memory cells (CD4+TEM) were increased in seropositive compared to seronegative children. In adults, seropositivity was associated with an elevated proportion of cytotoxic T central memory cells (CD8+TCM). Overall, diverse manifestations of immune cell dysregulations were more prominent in seropositive children compared to adults, who previously had COVID-like symptoms. These changes involved reduced frequencies of CD4+TEFF cells and CD163+CD64+ classical monocytes, but increased levels of intermediate or non-classical monocytes, as well as CD8+TEM cells in symptomatic children. CONCLUSION: Seropositive individuals in convalescence showed increased central and effector memory T cell phenotypes and pro-resolving/healing monocyte phenotypes compared to seronegative subjects. However, seropositive children with a previous history of COVID-like symptoms, displayed an ongoing innate inflammatory trait.

Dependency of sanitation infrastructure on the discharge of faecal coliform and SARS-CoV-2 viral RNA in wastewater from COVID and non-COVID hospitals in Dhaka, Bangladesh.

The detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA in wastewater can be used as an indicator of the presence of SARS-CoV-2 infection in specific catchment areas. We conducted a hospital-based study to explore wastewater management in healthcare facilities and analyzed SARS-CoV-2 RNA in the hospital wastewater in Dhaka city during the Coronavirus disease (COVID-19) outbreak between September 2020-January 2021. We selected three COVID-hospitals, two non-COVID-hospitals, and one non-COVID-hospital with COVID wards, conducted spot-checks of the sanitation systems (i.e., toilets, drainage, and septic-tank), and collected 90 untreated wastewater effluent samples (68 from COVID and 22 from non-COVID hospitals). E. coli was detected using a membrane filtration technique and reported as colony forming unit (CFU). SARS-CoV-2 RNA was detected using the iTaq Universal Probes One-Step kit for RT-qPCR amplification of the SARS-CoV-2 ORF1ab and N gene targets and quantified for SARS-CoV-2 genome equivalent copies (GEC) per mL of sample. None of the six hospitals had a primary wastewater treatment facility; two COVID hospitals had functional septic tanks, and the rest of the hospitals had either broken onsite systems or no containment of wastewater. Overall, 100 % of wastewater samples were positive with a high concentration of E. coli (mean = 7.0 log10 CFU/100 mL). Overall, 67 % (60/90) samples were positive for SARS-CoV-2. The highest SARS-CoV-2 concentrations (median: 141 GEC/mL; range: 13-18,214) were detected in wastewater from COVID-hospitals, and in non-COVID-hospitals, the median SARS-CoV-2 concentration was 108 GEC/mL (range: 30-1829). Our results indicate that high concentrations of E. coli and SARS-CoV-2 were discharged through the hospital wastewater (both COVID and non-COVID) without treatment into the ambient water bodies. Although there is no evidence for transmission of SARS-CoV-2 via wastewater, this study highlights the significant risk posed by wastewater from health care facilities in Dhaka for the many other diseases that are spread via faecal oral route. Hospitals in low-income settings could function as sentinel sites to monitor outbreaks through wastewater-based epidemiological surveillance systems. Hospitals should aim to adopt the appropriate wastewater treatment technologies to reduce the discharge of pathogens into the environment and mitigate environmental exposures.

Comparison of the Immune Responses to COVID-19 Vaccines in Bangladeshi Population.

BACKGROUND: The adaptive immune response is a crucial component of the protective immunity against SARS-CoV-2, generated after infection or vaccination. METHODS: We studied antibody titers, neutralizing antibodies and cellular immune responses to four different COVID-19 vaccines, namely Pfizer-BioNTech, Moderna Spikevax, AstraZeneca and Sinopharm vaccines in the Bangladeshi population (n = 1780). RESULTS: mRNA vaccines Moderna (14,655 ± 11.3) and Pfizer (13,772 ± 11.5) elicited significantly higher anti-Spike (S) antibody titers compared to the Adenovector vaccine AstraZeneca (2443 ± 12.8) and inactivated vaccine Sinopharm (1150 ± 11.2). SARS-CoV-2-specific neutralizing antibodies as well as IFN-γ-secreting lymphocytes were more abundant in Pfizer and Moderna vaccine recipients compared to AstraZeneca and Sinopharm vaccine recipients. Participants previously infected with SARS-CoV-2 exhibited higher post-vaccine immune responses (S-specific and neutralizing antibodies, IFN-γ-secreting cells) compared to uninfected participants. Memory B (BMEM), total CD8+T, CD4+ central memory (CD4+CM) and T-regulatory (TREG) cells were more numerous in AstraZeneca vaccine recipients compared to other vaccine recipients. Plasmablasts, B-regulatory (BREG) and CD4+ effector (CD4+EFF) cells were more numerous in mRNA vaccine recipients. CONCLUSIONS: mRNA vaccines generated a higher antibody response, while a differential cellular response was observed for different vaccine types, suggesting that both cellular and humoral responses are important in immune monitoring of different types of vaccines.

Seroprevalence of SARS-CoV-2 infection and associated factors among Bangladeshi slum and non-slum dwellers in pre-COVID-19 vaccination era: October 2020 to February 2021.

BACKGROUND: Seroprevalence studies have been carried out in many developed and developing countries to evaluate ongoing and past infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data on this infection in marginalized populations in urban slums are limited, which may offer crucial information to update prevention and mitigation policies and strategies. We aimed to determine the seroprevalence of SARS-CoV-2 infection and factors associated with seropositivity in slum and non-slum communities in two large cities in Bangladesh. METHODS: A cross-sectional study was carried out among the target population in Dhaka and Chattogram cities between October 2020 and February 2021. Questionnaire-based data, anthropometric and blood pressure measurements and blood were obtained. SARS-CoV-2 serology was assessed by Roche Elecsys® Anti-SARS-CoV-2 immunoassay. RESULTS: Among the 3220 participants (2444 adults, ≥18 years; 776 children, 10-17 years), the overall weighted seroprevalence was 67.3% (95% confidence intervals (CI) = 65.2, 69.3) with 71.0% in slum (95% CI = 68.7, 72.2) and 62.2% in non-slum (95% CI = 58.5, 65.8). The weighted seroprevalence was 72.9% in Dhaka and 54.2% in Chattogram. Seroprevalence was positively associated with limited years of formal education (adjusted odds ratio [aOR] = 1.61; 95% CI = 1.43, 1.82), lower income (aOR = 1.23; 95% CI = 1.03, 1.46), overweight (aOR = 1.2835; 95% CI = 1.26, 1.97), diabetes (aOR = 1.67; 95% CI = 1.21, 2.32) and heart disease (aOR = 1.38; 95% CI = 1.03, 1.86). Contrarily, negative associations were found between seropositivity and regular wearing of masks and washing hands, and prior BCG vaccination. About 63% of the population had asymptomatic infection; only 33% slum and 49% non-slum population showed symptomatic infection. CONCLUSION: The estimated seroprevalence of SARS-CoV-2 was more prominent in impoverished informal settlements than in the adjacent middle-income non-slum areas. Additional factors associated with seropositivity included limited education, low income, overweight and pre-existing chronic conditions. Behavioral factors such as regular wearing of masks and washing hands were associated with lower probability of seropositivity.

Wastewater surveillance of SARS-CoV-2 in Bangladesh: Opportunities and challenges.

The ongoing pandemic of the coronavirus disease 2019 (COVID-19) is a public health crisis of global concern. The progression of the COVID-19 pandemic has been monitored in the first place by testing symptomatic individuals for SARS-CoV-2 virus in the respiratory samples. Concurrently, wastewater carries feces, urine, and sputum that potentially contains SARS-CoV-2 intact virus or partially damaged viral genetic materials excreted by infected individuals. This brings significant opportunities for understanding the infection dynamics by environmental surveillance. It has advantages for the country, especially in densely populated areas where individual clinical testing is difficult. However, there are several challenges including: 1) establishing a sampling plan and schedule that is representative of the various catchment populations 2) development and validation of standardized protocols for the laboratory analysis 3) understanding hydraulic flows and virus transport in complex wastewater drainage systems and 4) collaborative efforts from government agencies, NGOs, public health units and academia.

Functional Antibodies and Innate Immune Responses to WRSS1, a Live Oral Shigella sonnei Vaccine Candidate, in Bangladeshi Adults and Children.

BACKGROUND: We demonstrated in a randomized placebo-controlled trial that WRSS1, a live oral Shigella sonnei vaccine candidate, is safe in Bangladeshi adults and children, and elicits antigen-specific antibodies. Here, we describe functional antibody and innate immune responses to WRSS1. METHODS: Adults (18-39 years) and children (5-9 years) received 3 doses of 3 × 105 or 3 × 106 colony forming units (CFU) of WRSS1 or placebo, 4 weeks apart; children additionally received 3 × 104 CFU. Blood and stool were collected at baseline and 7 days after each dose. Functional antibodies were measured using serum bactericidal antibody (SBA) assay. Cytokine/chemokine concentrations were measured in lymphocyte cultures. Host defense peptides LL-37, HBD-1, and HD-5 were analyzed in plasma and stool. RESULTS: Children showed increased SBA titers over baseline after the third dose of 3 × 106 CFU (P = .048). Significant increases of Th-17 and proinflammatory cytokines (TNF-α, G-CSF, MIP-1ß), and reduction of anti-inflammatory and Th2 cytokines (IL-10, IL-13, GM-CSF) were observed in children. Plasma HBD-1 and LL-37 decreased in children after vaccination but were increased/unchanged in adults. CONCLUSIONS: Functional antibodies and Th1/Th17 cytokine responses in children may serve as important indicators of immunogenicity and protective potential of WRSS1. Clinical Trials Registration: NCT01813071.

Slow radiological improvement and persistent low-grade inflammation after chemotherapy in tuberculosis patients with type 2 diabetes.

BACKGROUND: Diabetes mellitus type 2 (DM) may impede immune responses in tuberculosis (TB) and thus contribute to enhanced disease severity. In this study, we aimed to evaluate DM-mediated alterations in clinical, radiological and immunological outcomes in TB disease. METHODS: Newly diagnosed pulmonary TB patients with or without DM (TB n = 40; TB-DM n = 40) were recruited in Dhaka, Bangladesh. Clinical symptoms, sputum smear and culture conversion as well as chest radiography were assessed. Peripheral blood and sputum samples were collected at the time of diagnosis (baseline) and after 1, 2 and 6 months of standard anti-TB treatment. Blood samples were also obtained from healthy controls (n = 20). mRNA expression of inflammatory markers in blood and sputum samples were quantified using real-time PCR. RESULTS: The majority of TB-DM patients had poor glycemic control (HbA1c > 8%) and displayed elevated pulmonary pathology (P = 0.039) particularly in the middle (P < 0.004) and lower lung zones (P < 0.02) throughout the treatment period. However, reduction of clinical symptoms and time to sputum smear and culture conversion did not differ between the groups. Transcripts levels of the pro-inflammatory cytokines IL-1ß (P = 0.003 at month-1 and P = 0.045 at month-2) and TNF-α (P = 0.005 at month-1) and the anti-inflammatory cytokine IL-10 (P = 0.005 at month-2) were higher in peripheral blood after anti-TB treatment in TB-DM compared to TB patients. Conversely in sputum, TB-DM patients had reduced CD4 (P < 0.009 at month-1) and IL-10 (P = 0.005 at month-1 and P = 0.006 at month-2) transcripts, whereas CD8 was elevated (P = 0.016 at month-2). At 1- and 2-month post-treatment, sputum IL-10 transcripts were inversely correlated with fasting blood glucose and HbA1c levels in all patients. CONCLUSION: Insufficient up-regulation of IL-10 in the lung may fuel persistent local inflammation thereby promoting lung pathology in TB-DM patients with poorly controlled DM.

Fatal Infection Caused by Chromobacterium violaceum: A Case Report from a Tertiary Care Hospital in Bangladesh.

Chromobacterium violaceum is a Gram-negative bacterium, found in tropical and subtropical regions. C. violaceum infection rarely occurs, but once occurs, it is associated with significant mortality due to severe systemic infection. Since the first human case from Malaysia in 1927, >150 cases of C. violaceum infection have been reported worldwide. We have described here a fatal case of C. violaceum infection in a tertiary care hospital in Dhaka, Bangladesh. To the best of our knowledge, this is the first case of C. violaceum infection in Bangladesh.

A phase I trial of WRSS1, a Shigella sonnei live oral vaccine in Bangladeshi adults and children.

Shigella sonnei live vaccine candidate, WRSS1, which was previously evaluated in US, Israeli and Thai volunteers, was administered orally to Bangladeshi adults and children to assess its safety, clinical tolerability and immunogenicity. In a randomized, placebo-controlled, dose-escalation, age-descending study, 39 adults (18-39 years) and 64 children (5-9 years) were enrolled. Each adult cohort (n = 13) received one dose of 3x104, or three doses of 3 × 105 or 3 × 106 colony forming unit (CFU) of WRSS1 (n = 10) or placebo (n = 3). Each child cohort (n = 16) received one dose of 3x103, or three doses of 3x104, 3x105, or 3 × 106 CFU WRSS1 (n = 12) or placebo (n = 4). WRSS1 elicited mostly mild and transient reactogenicity events in adults and children. In the 3 × 106 dose group, 50% of the adults shed the vaccine; no shedding was seen in children. At the highest dose, 100% of adults and 40% of children responded with a ≥ 4-fold increase of S. sonnei LPS-specific IgA antibody in lymphocyte supernatant (ALS). At the same dose, 63% of adults and 70% of children seroconverted with IgA to LPS, while in placebo, 33% of adults and 18% of children seroconverted. Both the vaccinees and placebos responded with fecal IgA to LPS, indicating persistent exposure to Shigella infections. In conclusion, WRSS1 was found safe up to 106 CFU dose and immunogenic in adults and children in Bangladesh. These data indicate that live, oral Shigella vaccine candidates, including WRSS1 can potentially be evaluated in toddlers and infants (<2 years of age), who comprise the target population in an endemic environment.

Treatment with Entinostat Heals Experimental Cholera by Affecting Physical and Chemical Barrier Functions of Intestinal Epithelia.

We have shown previously that oral treatment with sodium butyrate or phenylbutyrate in an experimental model of shigellosis improves clinical outcomes and induces the expression of the antimicrobial peptide CAP-18 in the large intestinal epithelia. In a subsequent study, we found that entinostat, an aroylated phenylenediamine compound, has similar therapeutic potential against shigellosis. In this study, we aimed to evaluate entinostat as a potential candidate for host-directed therapy against cholera in an experimental model. Vibrio cholerae-infected rabbits were treated with two different dose regimens of entinostat: either 0.5 mg twice daily for 2 days or 1 mg once daily for 2 days. The effects of treatment on clinical outcomes and V. cholerae shedding (CFU count in stool) were observed. Immunohistochemical analysis was carried out to assess CAP-18 expression in ileal and jejunal mucosae. The serum zonulin level was measured by an enzyme-linked immunosorbent assay (ELISA) to evaluate gut permeability. Infection of rabbits with V. cholerae downregulated CAP-18 expression in the ileal epithelium; the expression was replenished by oral treatment with entinostat at either dose regimen. The level of zonulin, a marker of gut permeability, in serum was upregulated after infection, and this upregulation was counteracted after treatment with entinostat. Entinostat treatment also led to recovery from cholera and a decline in the V. cholerae count in stool. In conclusion, the improved clinical outcome of cholera for rabbits treated with entinostat is associated with the induction of CAP-18 and the reduction of gut epithelial permeability.

Ciprofloxacin Affects Host Cells by Suppressing Expression of the Endogenous Antimicrobial Peptides Cathelicidins and Beta-Defensin-3 in Colon Epithelia.

Antibiotics exert several effects on host cells including regulation of immune components. Antimicrobial peptides (AMPs), e.g., cathelicidins and defensins display multiple functions in innate immunity. In colonic mucosa, cathelicidins are induced by butyrate, a bacterial fermentation product. Here, we investigated the effect of antibiotics on butyrate-induced expression of cathelicidins and beta-defensins in colon epithelial cells. Real-time PCR analysis revealed that ciprofloxacin and clindamycin reduce butyrate-induced transcription of the human cathelicidin LL-37 in the colonic epithelial cell line HT-29. Suppression of LL-37 peptide/protein by ciprofloxacin was confirmed by Western blot analysis. Immunohistochemical analysis demonstrated that ciprofloxacin suppresses the rabbit cathelicidin CAP-18 in rectal epithelia of healthy and butyrate-treated Shigella-infected rabbits. Ciprofloxacin also down-regulated butyrate-induced transcription of the human beta-defensin-3 in HT-29 cells. Microarray analysis of HT-29 cells revealed upregulation by butyrate with subsequent down-regulation by ciprofloxacin of additional genes encoding immune factors. Dephosphorylation of histone H3, an epigenetic event provided a possible mechanism of the suppressive effect of ciprofloxacin. Furthermore, LL-37 peptide inhibited Clostridium difficile growth in vitro. In conclusion, ciprofloxacin and clindamycin exert immunomodulatory function by down-regulating AMPs and other immune components in colonic epithelial cells. Suppression of AMPs may contribute to the overgrowth of C. difficile, causing antibiotic-associated diarrhea.

Treatment with phenylbutyrate in a pre-clinical trial reduces diarrhea due to enteropathogenic Escherichia coli: link to cathelicidin induction.

Treatment of shigellosis in rabbits with phenylbutyrate reduces clinical severity and counteracts down-regulation of cathelicidin (CAP-18) in the large intestinal epithelia. We aimed to further evaluate whether in a rabbit model of enteropathogenic Escherichia coli (EPEC) diarrhea, CAP-18 is down-regulated in the small intestine and if oral phenylbutyrate treatment affects CAP-18 expression, clinical recovery, shedding of EPEC in stool and virulence properties of the isolated colonies. EPEC-induced diarrhea down-regulated CAP-18 in the small intestinal epithelia as revealed by immunohistochemistry. Phenylbutyrate treatment reduced clinical illness, improved histological features of inflammation and up-regulated CAP-18 in the epithelia. Active CAP-18 peptide was also released in the stool as noted in Western blot analysis. Multiplex PCR analysis of total bacterial DNA in the stool showed absence of EPEC specific genes eae and bfpA. Treated rabbits shed rough strains still harboring eae and bfpA genes, which were less potent in binding to HeLa cells and induced delayed onset of diarrhea in new rabbits. In conclusion, EPEC-mediated down-regulation of CAP-18 in the small intestinal epithelia was restored by phenylbutyrate treatment. Upregulation of CAP-18 in the epithelia was accompanied by healing of the epithelial lining, reduced shedding and virulence of EPEC and recovery from diarrhea.

Oral intake of phenylbutyrate with or without vitamin D3 upregulates the cathelicidin LL-37 in human macrophages: a dose finding study for treatment of tuberculosis.

BACKGROUND: We earlier showed that 4-phenylbutyrate (PB) can induce cathelicidin LL-37 expression synergistically with 1,25-dihydroxyvitamin D3 in a lung epithelial cell line. We aimed to evaluate a therapeutic dose of PB alone or in combination with vitamin D3 for induction of LL-37 expression in immune cells and enhancement of antimycobacterial activity in monocyte-derived macrophages (MDM). METHODS: Healthy volunteers were enrolled in an 8-days open trial with three doses of PB [250 mg (Group-I), 500 mg (Group-II) or 1000 mg (Group-III)] twice daily (b.d.) together with vitamin D3 {5000 IU once daily (o.d.)}, PB (500 mg b.d.) (Group-IV) or vitamin D3 (5000 IU o.d.) (Group-V), given orally for 4 days. Blood was collected on day-0, day-4 and day-8; plasma was separated, peripheral blood mononuclear cells (PBMC), non-adherent lymphocytes (NAL) and MDM were cultured. LL-37 transcript in cells and peptide concentrations in supernatant were determined by qPCR and ELISA, respectively. In plasma, 25-hydorxyvitamin D3 levels were determined by ELISA. MDM-mediated killing of Mycobacterium tuberculosis (Mtb) (H37Rv) was performed by conventional culture method. RESULTS: MDM from Group-II had increased concentration of LL-37 peptide and transcript at day-4, while Group-I showed increased transcript at day-4 and day-8 compared to day-0 (p < 0.05). Both Group-I and -II exhibited higher levels of transcript on day-4 compared to Group-III and Group-V (p < 0.035). Increased induction of peptide was observed in lymphocytes from Group-II on day-4 compared to Group-I and Group-IV (p < 0.05), while Group-IV showed increased levels on day-8 compared to Group-I and Group-III (p < 0.04). Intracellular killing of Mtb on day-4 was significantly increased compared to day-0 in Group-I, -II and -V (p < 0.05). CONCLUSION: The results demonstrate that 500 mg b.d. PB with 5000 IU o.d. vitamin D3 is the optimal dose for the induction of LL-37 in macrophages and lymphocytes and intracellular killing of Mtb by macrophages. Hence, this dose has potential application in the treatment of TB and is now being used in a clinical trial of adults with active pulmonary TB (NCT01580007).

Efficacy of sodium butyrate adjunct therapy in shigellosis: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Treatment of shigellosis in rabbits with butyrate reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Here, we aimed to evaluate whether butyrate can be used as an adjunct to antibiotics in the treatment of shigellosis in patients. METHODS: A randomized, double-blind, placebo-controlled, parallel-group designed clinical trial was conducted. Eighty adult patients with shigellosis were randomized to either the Intervention group (butyrate, n = 40) or the Placebo group (normal saline, n = 40). The Intervention group was given an enema containing sodium butyrate (80 mM), twice daily for 3 days, while the Placebo group received the same dose of normal saline. The primary endpoint of the trial was to assess the efficacy of butyrate in improving clinical, endoscopic and histological features of shigellosis. The secondary endpoint was to study the effect of butyrate on the induction of antimicrobial peptides in the rectum. Clinical outcomes were assessed and concentrations of antimicrobial peptides (LL-37, human beta defensin1 [HBD-1] and human beta defensin 3 [HBD-3]) and pro-inflammatory cytokines (interleukin-1ß [IL-1ß] and interleukin-8 [IL-8]) were measured in the stool. Sigmoidoscopic and histopathological analyses, and immunostaining of LL-37 in the rectal mucosa were performed in a subgroup of patients. RESULTS: Compared with placebo, butyrate therapy led to the early reduction of macrophages, pus cells, IL-8 and IL-1ß in the stool and improvement in rectal histopathology. Butyrate treatment induced LL-37 expression in the rectal epithelia. Stool concentration of LL-37 remained significantly higher in the Intervention group on days 4 and 7. CONCLUSION: Adjunct therapy with butyrate during shigellosis led to early reduction of inflammation and enhanced LL-37 expression in the rectal epithelia with prolonged release of LL-37 in the stool. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00800930.