Firoz Ahmed

Field trial of oral cholera vaccines in Bangladesh: results from three-year follow-up

The protective efficacy (PE) of B subunit killed whole-cell (BS-WC) and killed whole-cell-only (WC) oral cholera vaccines was assessed in a randomised double-blind field trial among children aged 2-15 years and women over 15 years in rural Bangladesh. Among the 62 285 subjects who received three doses of BS-WC, WC, or Escherichia coli K12 strain placebo, cumulative PE at 3 years of follow-up was 50% for BS-WC and 52% for WC. PE was similar against severe and non-severe cholera, but was significantly lower in children who were vaccinated at 2-5 years (26% for BS-WC; 23% for WC) than in older persons (63% for BS-WC; 68% for WC). Among persons vaccinated at 2-5 years, protection at 4-6 months of follow-up was similar to that for older persons, but rapidly waned thereafter and was not evident during the third year of follow-up. In contrast, persons vaccinated at older ages were protected even in the third year of follow-up (PE 40% for BS-WC; 62% for WC). PE was substantially higher against classical cholera (58% for BS-WC; 60% for WC) than against El Tor cholera (39% and 40%).

Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition

BackgroundDiarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.ResultsWe use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.ConclusionsOur findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

Supplementation with Zinc, but Not Vitamin A, Improves Seroconversion to Vibriocidal Antibody in Children Given an Oral Cholera Vaccine

To investigate whether micronutrient supplementation could improve the vibriocidal antibody response of children to a killed oral cholera vaccine, 2-5-year-old children were randomly assigned to receive vitamin A and zinc (AZ group), vitamin A and a placebo (A group), zinc and a placebo (Z group), or both placebos (P group). All children received 2 doses of the vaccine. The number of children who had a > or = 4-fold increase in vibriocidal antibody was significantly greater in the AZ group than in the P group (P = .025-.028). Factorial analysis suggested that the proportion of children with a > or = 4-fold increase in vibriocidal antibody titer was significantly greater in the zinc-supplemented groups than in the groups that did not receive zinc (P = .013-.048) and that vitamin A supplementation did not have a significant effect. Thus, supplementation with zinc improves seroconversion to vibriocidal antibody and, hence, has the potential to improve the efficacy of oral cholera vaccine in children.

Increased Levels of Inflammatory Mediators in Children and Adults Infected with Vibrio cholerae O1 and O139

ABSTRACT Investigations were carried out to study the production of factors associated with the innate immune response in the systemic and mucosal compartments in adults and children infected with Vibrio cholerae O1 and V. cholerae O139. The levels of nonspecific mediators of the innate defense system, i.e., prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and lactoferrin (Lf), as well as myeloperoxidase (MPO), were elevated at the acute stage of the disease in stools obtained from both O1- and O139-infected adults and children. In the systemic compartment, the levels of Lf were increased after onset of disease, which in children remained elevated up to convalescence compared to the healthy controls. Increased concentrations of C-reactive protein were seen in the sera of adult cholera patients at the acute stage of infection. Elevated levels of the nitric oxide (NO·) metabolites (nitrite and nitrate [NO2− and NO3−]) were detected in plasma but not in urine. The activity of the scavenger of reactive oxygen species, superoxide dismutase, was higher in the plasma of adults immediately after the onset of disease, suggesting that an active scavenging of reactive oxygen species was taking place. The concentration of 8-iso-prostaglandin F2α remained unchanged in the systemic and mucosal compartments in the study subjects. After the recovery of patients from cholera, the concentration of the majority of the metabolites decreased to baseline levels by day 30 after the onset of infection. Immunohistochemical staining showed increased tissue expression of MPO, Lf, and inducible nitric oxide synthase at the acute stage in the duodenal biopsies of adults and rectal biopsies obtained from children with cholera. Very little difference was seen in the levels of the different inflammatory mediators in patients infected with V. cholerae O1 or the encapsulated V. cholerae O139. In summary, these results suggest that elevated concentrations of Lf, MPO, PGE2, LTB4, and NO·, as well as other metabolites, during the acute stage of the disease indicate that the innate defense system, as well as the inflammatory process, is activated in both adults and pediatric patients infected with V. cholerae O1 and O139.

Antigen-Specific Immunoglobulin A Antibodies Secreted from Circulating B Cells Are an Effective Marker for Recent Local Immune Responses in Patients with Cholera: Comparison to Antibody-Secreting Cell Responses and Other Immunological Markers

ABSTRACT Gut-derived lymphocytes transiently migrate through the peripheral circulation before homing back to mucosal sites and can be detected using an ELISPOT-based antibody secreting cell (ASC) assay. Alternatively, transiently circulating lymphocytes may be cultured in vitro, and culture supernatants may be assayed for antigen-specific responses (antibody in lymphocyte supernatant [ALS] assay). The ALS assay has not been validated extensively in natural mucosal infection, nor has the ALS response been compared to the ASC assay and other cholera-specific immunological responses. Accordingly, we examined immune responses in 30 adult patients with acute cholera in Bangladesh, compared with 10 healthy controls, measuring ALS-immunoglobulin A (IgA), ASC-IgA, and serum and fecal IgA responses to two potent Vibrio cholerae immunogens, the nontoxic B subunit of cholera toxin (CtxB) and lipopolysaccharide (LPS) and a weaker V. cholerae immunogen, the mannose-sensitive hemagglutinin (MSHA). We found significant increases of anti-CtxB, anti-LPS, and anti-MSHA IgA in supernatants of lymphocytes cultured 7 days after onset of cholera using the ALS assay. We found that ALS and ASC responses correlated extremely well; both had comparable sensitivities as the vibriocidal responses, and both procedures were more sensitive than fecal IgA measurements. An advantage of the ALS assay for studying mucosal immune responses is the ability to freeze antibodies in supernatants for subsequent evaluation; like the ASC assay, the ALS assay can distinguish recent from remote mucosal infection, a distinction that may be difficult to make in endemic settings using other procedures.

Impact of B subunit killed whole-cell and killed whole-cell-only oral vaccines against cholera upon treated diarrhoeal illness and mortality in an area endemic for cholera.

The impact of B subunit killed whole-cell (BS-WC) and killed whole-cell-only (WC) oral cholera vaccines was assessed in a randomised double-blind trial in rural Bangladesh. 62,285 children aged 2-15 years and women aged over 15 ingested three doses of one of the vaccines or placebo. During the first year of follow-up there was a 26% reduction of all visits for treatment of diarrhoea in the BS-WC group and a 22% reduction in the WC group. The reduction of all admissions for fatal or severely dehydrating diarrhoea was 48% in the BS-WC group and 33% in the WC group. Overall mortality rates were 26% lower in the BS-WC group and 23% lower in the WC group during the first year, and reductions of mortality were observed only in women vaccinated at ages over 15 years. However, no differences in cumulative mortality were evident at the end of the second year of surveillance.

Safety and immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi adults and children.

We have compared the B cell responses evoked in Bangladeshi, adults (n=11, median age 25 years) and children (n=21, median age 4.5 years), 7 days after intake of each of two doses of an oral, inactivated enterotoxigenic Escherichia coli (ETEC) vaccine composed of formalin-killed ETEC strains expressing the colonization factors, CFA/I, CFA/II and CFA/IV together with 1 mg of recombinant cholera toxin B-subunit (rCTB). The vaccine was well tolerated and only gave rise to negligible side effects. Peak antibody-secreting cell (ASC) response of the IgA isotype were seen 7 days after the first dose of the vaccine. The ASC responses to the different colonization factors (CFs) increased from a 29- to 46-fold (responder frequency 90-100%) in the adults and 13- to 24-fold (responder frequency 67-90%) in the children. The IgA-ASC response to rCTB also peaked after the first dose in the adults (426-fold, responder frequency 100%) and the children (46-fold, responder frequency 95%). Increased IgA antibody levels against CFA/I as well as IgA and IgG antibody levels to rCTB were seen in plasma after immunisation. About 86% of the children and 80% of the adults responded with faecal antibodies to rCTB, whereas about 67% of both groups responded to CFA/I. These results show that a single dose of the ETEC vaccine may elicit significant mucosal immune responses in both children and adults residing in an ETEC-endemic country such as Bangladesh.

Safety and immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi children 18–36 months of age

A phase II safety and immunogenicity study of an oral-formalin inactivated enterotoxigenic Escherichia coli (ETEC) vaccine containing six colonization factors (CFA/I, CS1, CS2, CS3, CS4, CS5) and 1mg of recombinant cholera toxin B subunit (the CF-BS-ETEC vaccine) was carried out in an urban slum of Dhaka city in Bangladesh. The study was carried out in a double blinded, placebo controlled design in 158 children, 18-36 months of age. Children were given two doses of the CF-BS-ETEC vaccine or the placebo which consisted of E. coli K12. The vaccine was well tolerated. The immune response was studied in 60 children (30 each in the placebo and vaccine group). Significant vaccine specific IgA antibody-secreting cell (ASC) responses were seen 7 days after ingestion of the first and second dose of the vaccine. The responses to CFA/I (P<or=0.001), CS2 (P=0.021), CS4 (P=0.009) and rCTB (P<or=0.001) were elevated in the vaccines in comparison to the pre-immune values and in comparison to those seen in the placebo recipients (P=0.018 to <0.001). Vaccines but not placebo recipients also showed significantly increased IgM ASC responses to all three CF antigens that were tested (P=0.012 to <0.001) and IgG-ASCs to rCTB (P<0.001). Peak ASC levels were reached after one dose of the vaccine with no further increase or decrease after the second dose. The vaccine recipients also responded with IgA plasma antibodies to CFA/I, CS1, CS2, CS4 and rCTB after one or two doses of the vaccine (P=0.01 to <0.001). Subjects in the placebo group failed to mount responses to any of the antigens. The vaccine also induced responses in mucosal IgA antibodies in feces to CFA/I, CS2 and rCTB (61, 88 and 69% responder frequency, respectively) and the magnitude of the response was elevated in comparison to the pre-immune levels (P=0.031 to <0.001) and to the levels of the control group (P=0.003 to <0.001). This study thus shows that the CF-BS-ETEC vaccine is well tolerated in children, 18-36 months of age and gives rise to significant systemic and mucosal IgA antibody responses.

Biotype as determinant of natural immunising effect of cholera

To test the hypothesis that clinical Vibrio cholerae O1 infections protect against recurrent cholera, treated cholera episodes in a rural Bangladesh population of 188,153 people who were followed between 1985 and 1988 were analysed. Of the 2214 people with initial episodes of cholera, 7 had a second episode. The incidence of cholera was 61% lower in subjects who had had an earlier episode than in those without such an episode. Whereas initial episodes of classical cholera were associated with complete protection against subsequent cholera, initial episodes of El Tor cholera were associated with negligible protection.

Mucosal and Systemic Immune Responses in Patients with Diarrhea Due to CS6-Expressing Enterotoxigenic Escherichia coli

ABSTRACT Colonization factor CS6 expressed by enterotoxigenic Escherichia coli (ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (n = 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.