Construction and preclinical evaluation of recombinant Peru-15 expressing high levels of the cholera toxin B subunit as a vaccine against enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler's diarrhea. The heat-labile (LT) and heat-stable (ST) toxins mediate ETEC induced diarrhea. ETEC strains may express LT, ST, or both LT and ST, with LT-expressing strains accounting for ∼50-60% of ETEC-related traveler's diarrhea. Cholera toxin (CT) is >80% homologous to LT and vaccination with CT-B subunit (CT-B) -based vaccines elicit a protective immune response against LT-producing ETEC strains. Peru-15 is an oral, single-dose, live-attenuated cholera vaccine candidate that has been investigated in several clinical trials (n > 400 subjects) and was proven well tolerated, immunogenic, and efficacious. Peru-15 was genetically engineered to express and secrete high levels of CT-B by cloning ctxB onto a glnA balanced-lethal plasmid under the transcriptional control of a strong constitutive promoter, resulting in Peru-15pCTB. In vitro characterization demonstrated that Peru-15pCTB secreted ∼30-fold more CT-B than Peru-15 and CT-B was stably produced after 40 generations of growth and throughout simulated Seed Bank and FDP (Final Drug Product) production conditions. In preclinical studies, the geometric mean anti-CT-B IgG titer in the sera of mice inoculated intranasally with two doses of Peru-15pCTB was >32-fold higher than in mice inoculated with Peru-15. Similarly, rabbits orally inoculated with a single dose of Peru-15pCTB developed titers that were ∼30-fold higher than rabbits inoculated with a single dose of Peru-15. Sera from Peru-15pCTB vaccinated mice and rabbits neutralized LT toxicity in an in vitro assay. Peru-15pCTB has several promising characteristics of an oral, single-dose, bivalent cholera/ETEC vaccine and is advancing towards a Phase 1 clinical trial.