Establishment and Validation of Pathogenic CS17+ and CS19+ Enterotoxigenic Escherichia coli Challenge Models in the New World Primate Aotus nancymaae.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal illness in the military, travelers, and children living in low- to middle-income countries. Increased antibiotic resistance, the absence of a licensed vaccine, and the lack of broadly practical therapeutics perpetuate the significant health and financial burden resulting from ETEC infection. A critical step in the evaluation of vaccines and therapeutics is preclinical screening in a relevant animal disease model that closely replicates human disease. We previously developed a diarrheal model of class 5a colonization factor (CF) CFA/I-expressing ETEC in the New World owl monkey species Aotus nancymaae using ETEC strain H10407. In order to broaden the use of the model, we report here on the development of A. nancymaae models of ETEC expressing the class 5b CFs CS17 and CS19 with strains LSN03-016011/A and WS0115A, respectively. For both models, we observed diarrheal attack rates of ≥80% after oral inoculation with 5 × 1011 CFU of bacteria. These models will aid in assessing the efficacy of future ETEC vaccine candidates and therapeutics.

Zika and Dengue Interactions in the Context of a Large Dengue Vaccine Clinical Trial in Latin America.

A phase III dengue vaccine trial including 9- to 16-year-olds in Latin America (NCT01374516) was ongoing at the time of a Zika outbreak. We explored interactions between dengue and Zika, in the context of dengue vaccination. Symptomatic virologically confirmed Zika (VCZ) was evaluated using acute-phase sera from febrile participants (January 2013-March 2018). Neutralizing antibody geometric mean titers (GMTs) were evaluated pre- and post-Zika outbreak (months 25 and 72) in 2,000 randomly selected participants. Baseline dengue serostatus was determined using the plaque reduction neutralization test or inferred post hoc using nonstructural protein 1 IgG ELISA at M13 (case-cohort analysis). Vaccine efficacy against VCZ and serologically suspected Zika (SSZ) was estimated. Overall, 239/10,157 (2.4%) acute-phase samples were VCZ positive during the study. Dengue vaccine efficacy against VCZ was 27.8% (95% CI: 0.3; 47.7) among baseline dengue-seropositive participants. No vaccine effect was evident against SSZ. Zika antibody GMTs increased from pre- to post-Zika epidemic, with smaller increases observed for participants who were dengue seropositive at baseline than for those who were dengue seronegative: post-/pre-Zika GMT ratios for baseline dengue-seropositive participants were 21.5 (vaccine group) and 30.8 (placebo); and for dengue seronegatives, 88.1 and 89.5, respectively. Dengue antibody GMTs post-Zika were higher in dengue vaccine and placebo recipients with SSZ than those without SSZ in both dengue seropositives and seronegatives. Dengue vaccine did not enhance symptomatic Zika illness in dengue-seropositive individuals, rather it reduced the risk of VCZ. Zika infection boosted preexisting vaccine-induced or naturally occurring dengue-neutralizing antibodies.

Evaluation of dengue serological tests available in Puerto Rico for identification of prior dengue infection for prevaccination screening.

We assessed a dengue IgG rapid diagnostic test (RDT) and IgG enzyme-linked immunosorbent assay (ELISA) to determine their suitability for dengue prevaccination screening. Each exhibited ≥99% specificity. The RDT demonstrated no Zika cross-reactivity, while the ELISA displayed greater sensitivity. Both could safely guide vaccination in Puerto Rico pending availability of improved serotests.

Maternal vaccination with a fimbrial tip adhesin and passive protection of neonatal mice against lethal human enterotoxigenic Escherichia coli challenge.

Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10(7) bacteria resulted in 50% lethal doses (LD50) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD50 challenge with H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.

Detection of the CS20 colonization factor antigen in diffuse-adhering Escherichia coli strains.

We analyzed a randomly selected group of 30 diffusely adherent (DAEC), 30 enteropathogenic, 30 enteroaggregative, and five Shiga toxin-producing Escherichia coli strains isolated from children with diarrhea. Enterotoxigenic E. coli (ETEC) colonization factors (CFs) were evaluated by a dot-blot assay using 21 CF-specific monoclonal antibodies. Out of 95 non-ETEC strains, three DAEC were found to express coli surface antigen 20 (CS20). No other E. coli expressed CFs. We confirmed the three CS20-positive strains as ETEC-negative by repeat PCR and as toxin-negative by ganglioside-GM1-enzyme-linked immunosorbent assay. To our knowledge, this is the first study that has identified currently recognized CFs in non-ETEC diarrheagenic E. coli strains identified using molecular methods. CFs may be an unrecognized relevant adherence factor in other E. coli, which may then play a role in pathogenesis and the immune response of the host.

The New World primate, Aotus nancymae, as a model for examining the immunogenicity of a prototype enterotoxigenic Escherichia coli subunit vaccine.

The colonization factors (CF) of enterotoxigenic Escherichia coli (ETEC) are being targeted for inclusion in a multi-subunit ETEC vaccine. This study was designed to examine the preclinical safety and immunogenicity of CF CS6, encapsulated in a biodegradable poly(DL-lactide-co-glycolide) (meCS6), and administered in the presence or absence of a mutated heat-labile enterotoxin, LT(R192G), in the non-human primate, Aotus nancymae. A. nancymae were inoculated intranasally (IN) with meCS6 (200 microg; positive control), or intragastrically (IG) with meCS6 (200 or 1000 microg) with or without 2 microg LT(R192G) in three doses given at 2-week intervals. In a second experiment, A. nancymae were inoculated IG with 950 microg of meCS6 with or without 2 microg LT(R192G) in four doses given every 48 h. Blood was collected to assess anti-CS6 and -LT serum immunoglobulin G (IgG) and IgA responses and safety variables (complete blood count and chemistry). Safety parameters were unchanged from baseline following all vaccinations. In Experiment 1, a dose-related serologic response to CS6 was observed; 78.6 and 57.1% of monkeys given 1000 microg meCS6 (n = 14) had a serum IgG and IgA response, respectively, compared to only 28.6% of monkeys given 200 microg meCS6 (n = 14) with a serum IgG and IgA response. No significant effect on the number of responders or the magnitude of responses was observed with the addition of LT(R192G). The three-dose, 2-week regimen with 1000 microg meCS6 was more effective at eliciting an immune response than the four-dose, 48-h regimen with 950 microg meCS6. Results from this study indicate that A. nancymae provide a useful ETEC preclinical safety and immunogenicity model.