Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

Abstract

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.

Mecanismos de virulencia de Escherichia coli enteropatógena

“…Para el control de las cepas de ECEP, desde hace varios años, con algunas de estas moléculas más conservadas, entre éstas la intimina, EspB, EspA y BfpA se han adelantado estudios para el desarrollo de vacunas en diversos modelos. A pesar de estos avances, aún no existen productos disponibles para el control de la infección por ECEP 8,135,136,139,140 .…”

Genetically modified proteins: functional improvement and chimeragenesis

“…92,93 The bioinformatics analysis of immunogenic sequences of the tumor-associated antigens MUC1 and HER2 allowed combining it in the hybrid protein for using as the breast cancer antigens in ELISA for detection of antibodies against MUC1 or HER2 in human serum (Table 1). 92 The optimized chimeric gene composed of the structural subunits of colonization factor antigens (CFA), labile toxin subunit B and the binding subunit of heat-labile and heatstable toxoid, was designed to provide broad-spectrum protection against seven enterotoxigenic E. coli (ETEC) strains, the most common cause of bacterial diarrhea, inducing anti-CFA and antitoxin immunity (Table 1).…”

Current Progress in Developing Subunit Vaccines against Enterotoxigenic Escherichia coli-Associated Diarrhea

“…Mice immunized intraperitoneally with CFA/I/II/IV-2ϫSTa N12S -dmLT, a single polypeptide carrying the CFA MEFA, two copies of STa N12S , and the LT-A2 and LT B peptides, developed antibodies to all seven CFAs and both toxins (86). Serum from these immunized mice inhibited the in vitro adherence of E. coli strains expressing these seven CFA adhesins and also neutralized both toxins (86). This CFA/I/II/IV-2ϫSTa N12S -dmLT MEFA has been modified to carry three copies of STa N12S for further enhancement of anti-STa immunogenicity.…”

“…The new CFA/I/II/IV-3ϫSTa N12S -dmLT has been shown to possess stronger anti-STa immunogenicity, and antibodies induced by this fusion product showed greater neutralizing activity against STa. Although coadministration of the CFA MEFA and toxoid fusion 3ϫSTa N12S -dmLT induces equivalently protective antibodies against seven CFAs and both toxins (86), the single CFA/I/II/IV-3ϫSTa N12S -dmLT MEFA is more cost-effective to manufacture and administer. This CFA/ I/II/IV-3ϫSTa N12S -dmLT MEFA can potentially be considered the most promising antigen for ETEC subunit vaccine development.…”