Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome k -display library Elisa Beghetto 1 , Nicola Gargano 1 , Susanna Ricci 2 , Gabriella Garufi 3 , Samuele Peppoloni 4 , Francesca Montagnani 2 , Marco Oggioni 2 , Gianni Pozzi 2 & Franco Felici 1,3 1 Kenton Srl, Pomezia, Rome, Italy; 2 Department of Molecular Biology, University of Siena, Siena, Italy; 3 Department of Microbiology, Genetics, and Molecular Biology, University of Messina, Italy; 4 Department of Hygienic Microbiological and Biostatistical Sciences, University of Modena and Reggio Emilia, Italy. Correspondence: Franco Felici, Department of Microbiology, Genetics, and Molecular Biology, University of Messina, Salita Sperone, 31 - 98166 Messina, Italy. Tel.: 139 090 6765197; fax: 139 090 392733; e-mail: franco.felici@unime.it Received 2 March 2006; revised 11 May 2006; accepted 7 June 2006. First published online 6 July 2006. DOI:10.1111/j.1574-6968.2006.00360.x Editor: Tim Mitchell Keywords Streptococcus pneumoniae ; l phage display; expression library; pneumococcal antigen. Abstract Streptococcus pneumoniae is a causative agent of otitis media, pneumonia, meningitis and sepsis in humans. For the development of effective vaccines able to prevent pneumococcal infection, characterization of bacterial antigens involved in host immune response is crucial. In order to identify pneumococcal proteins recognized by host antibody response, we created an S. pneumoniae D39 genome library, displayed on l bacteriophage. The screening of such a library, with sera either from infected individuals or mice immunized with the S. pneumoniae D39 strain, allowed identification of phage clones carrying S. pneumoniae B-cell epitopes. Epitope-containing fragments within the families of the histidine-triad proteins (PhtE, PhtD), the choline-binding proteins (PspA, CbpD) and zinc metalloproteinase B (ZmpB) were identified. Moreover, library screening also allowed the isolation of phage clones carrying three distinct antigenic regions of a hypothetical pneumococcal protein, encoded by the ORF spr0075 in the R6 strain genome sequence. In this work, Spr0075 is first identified as an expressed S. pneumoniae gene product, having an antigenic function during infection. Introduction Streptococcus pneumoniae (pneumococcus) is a ubiquitous human pathogen that causes significant morbidity and mortality worldwide. It is a gram-positive bacterium that colonizes the upper respiratory tract of the host, causing various invasive diseases such as pneumonia, sepsis and meningitis (Fedson et al., 1999; Tuomanen, 2000). The disease rate is particularly high in children, the elderly and patients with chronic pathologies or immunosuppressive illnesses (Ejstrud et al., 1997; Bogaert et al., 2004a). In adults, pneumococcus is the most frequent cause of com- munity-acquired pneumonia; in children, it is the second most common cause of otitis media and meningitis after Haemophilus influenzae and Neisseria meningitidis (Dagan et al., 1994). In the last 20 years, at least three different vaccines using S. pneumoniae capsular polysaccharides have been devel- oped and commercialized (for a review, see Obaro, 2002). Such formulations have limited efficacy in children below 2 years and in immunocompromised individuals (Butler et al., 1999), due to the low immunogenicity of capsule polysaccharides and the lack of T-cell activation. Moreover, capsular polysaccharides are serotype-specific and thus the requirement for multivalent protection requires immuniza- tion with multiple polysaccharides. Conjugation of pneu- mococcal polysaccharides to carrier proteins can improve the host immune response and has been successfully intro- duced in clinical practice (Black et al., 2001), although conjugated vaccine production is still quite expensive. In the last decade, there has been great interest in using surface and secreted pneumococcal proteins conserved among a large number of serotypes as immunogens. Although many pneumococcal proteins, including pneumolysin, surface protein A (PspA), surface adhesin A (PsaA), surface protein C (PspC), neuraminidase and autolysin, have been proposed as potential vaccine candidates, PspA, PsaA and pneumoly- sin are currently the most promising (Briles et al., 2000; Bogaert et al., 2004b). This work focuses on the identification of pneumococcal protein domains containing B-cell epitopes, which are recognized by antibodies of infected humans. To this end, we utilized a powerful approach based on l phage display technology, which has already shown great efficacy in FEMS Microbiol Lett 262 (2006) 14–21 c  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved