Vaccine 31 (2013) 1163–1170 Contents lists available at SciVerse ScienceDirect Vaccine jou rn al h om epa ge: www.elsevier.com/locate/vaccine Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein  A. Lundgren a,∗ , S. Leach a , J. Tobias a , N. Carlin b , B. Gustafsson b , M. Jertborn a , L. Bourgeois c , R. Walker c , J. Holmgren a , A.-M. Svennerholm a a Gothenburg University Vaccine Research Institute (GUVAX), Department of Microbiology and Immunology, University of Gothenburg, Box 435, 405 30 Gothenburg, Sweden b Etvax AB, Gunnar Asplunds allé, 171 63 Solna, Sweden c PATH, 455 Massachusetts Avenue, NW, Washington, DC 20001, USA a r t i c l e i n f o Article history: Received 2 October 2012 Received in revised form 18 December 2012 Accepted 26 December 2012 Available online 7 January 2013 Keywords: ETEC Vaccine Mucosa CFA/I LTB Secretory IgA a b s t r a c t We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine. The prototype vaccine was found to be safe and equally well-tolerated as the reference vaccine at either dosage tested. The prototype vaccine induced mucosal IgA (fecal secretory IgA and intestine-derived IgA antibody secreting cell) responses to both LTB and CFA/I, as well as serum IgA and IgG antibody responses to LTB. Immunization with LCTBA resulted in about twofold higher mucosal and systemic IgA responses against LTB than a comparable dose of CTB. The higher dose of the prototype vaccine induced significantly higher fecal and systemic IgA responses to LTB and fecal IgA responses to CFA/I than the reference vaccine. These results demonstrate that CF over-expression and inclusion of the LCTBA hybrid protein in an oral inactivated ETEC vaccine does not change the safety profile when compared to a previous generation of such a vaccine and that the prototype vaccine induces significant dose dependent mucosal immune responses against CFA/I and LTB. © 2013 Elsevier Ltd. All rights reserved. Abbreviations: AE, adverse event; ALS, antibodies in lymphocyte supernatants; ASC, antibody secreting cell; CF, colonization factor; CT, cholera toxin; CTB, cholera toxin binding subunit; dmLT, double mutant LT; ELISA, enzyme linked immunosor- bent assay; ELISPOT, enzyme linked immunospot assay; ETEC, enterotoxigenic Eschericia coli; GM, geometric mean; GMP, good manufacturing practice; HRP, horseradish peroxidase; LT, heat labile toxin; LTB, heat labile toxin binding subunit; PBMCs, peripheral blood mononuclear cells; PV, prototype vaccine; RV, reference vaccine; STa, heat stabile toxin; sIgA, secretory IgA.  EudraCT 2009-015741-23, ISRCTN23764070. ∗ Corresponding author. Tel.: +46 31 7866213; fax: +46 31 7866205. E-mail addresses: anna.lundgren@microbio.gu.se (A. Lundgren), susannah.leach@microbio.gu.se (S. Leach), joshua.tobias@microbio.gu.se (J. Tobias), nils.carlin@etvax.se (N. Carlin), bjorn.gustafsson@etvax.se (B. Gustafsson), marianne.jertborn@microbio.gu.se (M. Jertborn), lbourgeois@path.org (L. Bourgeois), rwalker@path.org (R. Walker), jan.holmgren@microbio.gu.se (J. Holmgren), ann-mari.svennerholm@microbio.gu.se (A.-M. Svennerholm). 1. Introduction Enterotoxigenic Escherichia coli (ETEC) remains the most com- mon cause of bacterial diarrhea in children in low-resource countries and in travelers to these countries [1,2]. Despite this, no effective vaccine for ETEC is available. ETEC causes disease by col- onizing the small intestine through colonization factors (CFs) and producing heat-labile (LT) and/or heat-stable (STa) enterotoxins. Preclinical studies, as well as studies in humans, have indicated that locally produced intestinal IgA antibodies against LT and CFs are protective [3,4]. We have previously developed an oral ETEC vaccine consist- ing of a combination of recombinantly produced cholera toxin B subunit (rCTB) and formalin-inactivated ETEC bacteria expressing major CFs [5,6]. Extensive clinical evaluation of this first generation 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.12.063