Vaccine 30 (2012) 5856–5863 Contents lists available at SciVerse ScienceDirect Vaccine j ourna l ho me pag e: www.elsevier.com/locate/vaccine Chimeric flagellin as the self-adjuvanting antigen for the activation of immune response against Helicobacter pylori Jerneja Mori a , Tanja Vranac c , Boˇ stjan Smrekar c , Maja ˇ Cernilec c , Vladka ˇ Curin ˇ Serbec c,f , Simon Horvat a,d , Alojz Ihan e , Mojca Benˇ cina a,b , Roman Jerala a,b,f,∗ a Department of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia b EN-FIST Centre of Excellence, Ljubljana, Slovenia c Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia d Biotechnical Faculty, Ljubljana, Slovenia e Medical Faculty, Ljubljana, Slovenia f Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia a r t i c l e i n f o Article history: Received 19 April 2012 Received in revised form 19 June 2012 Accepted 8 July 2012 Available online 20 July 2012 Keywords: Flagellin Helicobacter pylori Innate immunity Toll-like receptor 5 Adjuvant Chimeric protein a b s t r a c t Helicobacter pylori infection can cause gastritis, peptic ulcer and can lead to gastric cancer. Lengthy antibi- otic therapy does not protect the host against reinfection. H. pylori evolved to evade the recognition of the immune response by modifying several of its components whose orthologous proteins from other bacteria activate the innate immune response. Flagella are essential for the H. pylori effective colonization of human duodenum and stomach. TLR5, a member of the Toll-like receptor family, recognizes flagellin of most bacteria, such as Escherichia coli, but does not recognize the flagellin FlaA of H. pylori. We restored the ability of FlaA for the recognition by TLR5 by engineering a chimeric flagellin, in which both termi- nal segments of H. pylori flagellin were replaced by the corresponding segments from TLR5-activating E. coli flagellin. Recombinant chimeric flagellin folded correctly and was able to activate TLR5. Significantly increased serum IgG and IgA antibody responses were determined in mice vaccinated with chimeric flagellin in comparison to mice vaccinated with a control protein (FlaA) or negative control. Antibody titers remained high even 8 months after the last immunization. Antibodies were able to bind native flagellin from H. pylori lysate. Vaccination with chimeric flagellin provided mice with significant protec- tion against H. pylori. The approach of chimeric flagellin can therefore generate effective immunogens that enable activation of innate and adaptive immune response and can be used to construct efficient vaccines against H. pylori or other flagellated bacteria that evade TLR5 recognition. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Helicobacter pylori (H. pylori) is a microaerophilic Gram-negative bacterium that infects half of the world’s population and has been identified as the first pathogen to be a type I carcinogen [1,2]. Although in most patients the infection is asymptomatic, chronic infection is associated with duodenal and gastric ulcers, gastric adenocarcinoma and MALT lymphoma [1,3,4]. The H. pylori infec- tion can persist lifelong if not treated and antibiotic therapy is often not successful [5]. H. pylori has developed mechanisms to avoid innate immune response by altering amino acid sequence of its flagellin with respect to most other flagellated bacteria. Flagellin is the principal component of bacterial flagellum, which ∗ Corresponding author at: National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. Tel.: +386 1 476 0335; fax: +386 1 476 0300. E-mail address: roman.jerala@ki.si (R. Jerala). is an important virulence factor for the invasion and coloniza- tion of the stomach and duodenal mucosa. Although H. pylori flagellin can elicit anti-flagellin antibodies in infected patients, it evades recognition by TLR5 [6,7]. TLR5, a member of Toll- like receptor family, is activated by flagellin of most bacteria, except flagellin of ˛ and ε Proteobacteria, which include human pathogens H. pylori, Campylobacter jejuni and Bartonella bacilliformis [8]. The three-dimensional molecular structure of flagellin has been determined for Salmonella flagellar filament [9,10]. How- ever, flagellar filament from C. jejuni, a close relative of H. pylori, differs in the packing of flagellin into the flagellar fil- aments, consisting of 7 instead of 11 protofilaments found in Salmonella [11]. Flagellin structure can be divided into four domains (D0, D1, D2 and D3). Mutagenesis, deletion and replacement studies mapped the region important for TLR5 recognition to the conserved N-terminal and C-terminal end of flagellin (D0, D1) whereas the central variable region (D2, D3) is exposed 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.07.011