Vaccine 19 (2001) 788–795 DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC) Ada M.B. Alves a,b , Ma ´rcio O. La ´saro a , Darcy F. Almeida a , Luı ´s C.S. Ferreira a, * a Laborato ´rio de Fisiologia Celular, Instituto de Biofı ´sica Carlos Chagas Filho, UFRJ -CCS, Cidade Uniersita ´ria, Rio de Janeiro, RJ 21941 -590, Brazil b Laborato ´rio de Tecnologia Recombinante, Instituto de Tecnologia em Imunobiolo ´gicos, Fundac ¸a ˜o Oswaldo Cruz, A. Brasil, 4365, Rio de Janeiro, RJ 21045 -900, Brazil Received 2 December 1999; received in revised form 10 July 219; accepted 18 July 2000 Abstract The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes. The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests. An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC. pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits. BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass. pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I. Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells. These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens. © 2000 Published by Elsevier Science Ltd. Keywords: DNA vaccines; ETEC; CFA/I fimbriae; Diarrheal disease www.elsevier.com/locate/vaccine 1. Introduction Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of acute diarrhea in children in developing countries and in travelers who visit those areas [1]. In fact, ETEC has recently been recognized by the World Health Organization (WHO) as a priority target to be controlled by vaccination [2]. The two most important events in ETEC pathogenesis are adherence of bacteria to the small intestine epithelial surface, a host-specific process mediated by colonization factor antigens (CFs or CFAs), and delivery of enterotoxins. Some experimental approaches, such as the ability to hemagglutinate group A human red blood cells or attachment to Caco-2 cells, have been designed to evaluate the binding properties of different ETEC strains [3]. CFA/I was the first CF described in ETEC and CFA/I-expressing strains are widely distributed in endemic areas as South America and Asia [4–6]. It is a fimbrial adhesin composed of a single 15 kDa subunit endowed with structural and functional (adhesion) properties [4 – 6]. Significant amino acid homology has been found at the N-terminal region of different ETEC fimbra, such as CFA/I, CS1, CS2, CS4 and PCFO166, but antibodies raised against conformational epitopes of intact CFA/I are usually specific to strains expressing this fimbriae [7,8]. Oral administration of purified CFA/I fimbriae can confer partial protection against homologous strains, * Corresponding author. Present address: Departamento de Micro- biologia-ICB II, USP, Av. Prof. Lineu Prestes, 1374, 005508-900, Sa ˜o Paulo, SP, Brazil. E-mail address: lcsf@usp.br (L.C.S. Ferreira). 0264-410X/00/$ - see front matter © 2000 Published by Elsevier Science Ltd. PII:S0264-410X(00)00244-9