Development of a novel multiplex electrochemiluminescent-based immunoassay to aid enterotoxigenic Escherichia coli vaccine development and evaluations.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea both among children in low and middle income countries and in travelers to these regions. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. The most advanced ETEC vaccine candidates include multiple colonization factors along with the heat labile toxin B subunit. In the absence of known correlates of protection, and to understand the mechanism of protection, monitoring immune responses to a majority of the vaccine associated antigens using various types of samples is needed. Unfortunately, a traditional ELISA is time consuming, labor intensive and requires substantial amounts of antigens and sample volumes. To address these constraints, we developed and validated a novel high throughput electrochemiluminescent (ECL) - based multiplex immunoassay using Meso Scale Discovery (MSD) platform for analyzing immune responses to ETEC antigens. The ETEC multiplex ECL assay is an 8-plex assay which includes the ETEC colonization factor antigens (CFA/I, CS1, CS2, CS3, CS5 and CS6) along with the two subunits of heat labile toxin (LTA and LTB). Our data suggested that a single dilution of sample provides a quantifiable result for a wide range of sample titers. To compare ETEC multiplex ECL with ELISA, we carried out assays using the same antigens with the two immunoassay platforms using a common sample set of serum and ALS (antibodies in lymphocyte supernatant) specimens. The MSD platform achieved excellent correlations with ELISA for the antigens tested, consistently detecting comparable antibody levels in the samples. The ETEC multiplex ECL can serve as a fundamental platform in evaluating performances of candidate ETEC vaccines in future field trials.

Vibriocidal antibody responses to a bivalent killed whole-cell oral cholera vaccine in a phase III trial in Kolkata, India.

BACKGROUND: During the development of a vaccine, identification of the correlates of protection is of paramount importance for establishing an objective criterion for the protective performance of the vaccine. However, the ascertainment of correlates of immunity conferred by any vaccine is a difficult task. METHODS: While conducting a phase three double-blind, cluster-randomized, placebo-controlled trial of a bivalent killed whole-cell oral cholera vaccine in Kolkata, we evaluated the immunogenicity of the vaccine in a subset of participants. Randomly chosen participants (recipients of vaccine or placebo) were invited to provide blood samples at baseline, 14 days after the second dose and one year after the first dose. At these time points, serum geometric mean titers (GMT) of vibriocidal antibodies and seroconversion rates for vaccine and placebo arms were calculated and compared across the age strata (1 to 5 years, 5 to 15 years and more than 15 years) as well as for all age groups. RESULTS: Out of 137 subjects included in analysis, 69 were vaccinees and 68 received placebo. There were 5•7 and 5•8 geometric mean fold (GMF) rises in titers to Vibrio cholerae Inaba and Ogawa, respectively at 14 days after the second dose, with 57% and 61% of vaccinees showing a four-fold or greater titer rise, respectively. After one year, the titers to Inaba and Ogawa remained 1•7 and 2•8 fold higher, respectively, compared to baseline. Serum vibriocidal antibody response to V. cholerae O139 was much lower than that to Inaba or Ogawa. No significant differences in the GMF-rises were observed among the age groups. CONCLUSIONS: The reformulated oral cholera vaccine induced a statistically significant anti-O1 Inaba and O1 Ogawa vibriocidal antibody response 14 days after vaccination, which although declined after one year remained significantly higher than baseline. Despite this decline, the vaccine remained protective five years after vaccination.