Protective Activity of Streptococcus pneumoniae Spr1875 Protein Fragments Identified Using a Phage Displayed Genomic Library Angela Cardaci1, Salvatore Papasergi1, Angelina Midiri1, Giuseppe Mancuso1, Maria Domina1, Veronica Lanza Cariccio1, Francesca Mandanici1, Roberta Galbo 2 , Carla Lo Passo2, Ida Pernice2, Paolo Donato3, Susanna Ricci4, Carmelo Biondo 1 , Giuseppe Teti1, Franco Felici5, Concetta Beninati1* 1 The Elie Metchnikoff Department, Universita` di Messina, Messina, Italy, 2 Dipartimento di Scienze della Vita M. Malpighi, Universita` di Messina, Messina, Italy, 3 Novartis Vaccines and Diagnostics, Messina, Italy, 4 Dipartimento di Biotecnologia, Universita` di Siena, Siena, Italy, 5 Dipartimento S.T.A.T., Universita` del Molise, Pesche (IS), Italy Abstract There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunopro- tection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine. Citation: Cardaci A, Papasergi S, Midiri A, Mancuso G, Domina M, et al. (2012) Protective Activity of Streptococcus pneumoniae Spr1875 Protein Fragments Identified Using a Phage Displayed Genomic Library. PLoS ONE 7(5): e36588. doi:10.1371/journal.pone.0036588 Editor: Gunnar F. Kaufmann, The Scripps Research Institute, United States of America Received November 19, 2011; Accepted April 3, 2012; Published May 3, 2012 Copyright: 2012 Cardaci et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants Progetti di Ricerca di Rilevante Interesse Nazionale 2005 (DM n. 287/2005) and 2008 (DM n. 1407/2008) from the Ministero dell’Istruzione, dell’Universita` e della Ricerca of Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: two authors declare a potential conflict of financial interest as employee (PD) or consultant (GT) of Novartis Vaccines and Diagnostics. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. The authors have no other competing financial interests. * E-mail: cbeninati@unime.it Introduction Streptococcus pneumoniae, or pneumococcus, is an extracellular human respiratory pathogen causing sinusitis, otitis media, pneumonia, sepsis and meningitis. Invasive pneumococcal infec- tions are an important cause of mortality and morbidity worldwide, especially among young children and the elderly [1]. Pneumococci cause at least 1 million deaths worldwide every year, mostly as a result of community-acquired pneumonia [2]. Moreover, antibiotic resistance is increasing at an alarming rate among pneumococcal clinical isolates. The 23-valent polysaccha- ride vaccine provides partial, serotype-specific protection in adults, but has limited efficacy in young children. Although conjugate vaccines are effective also in children, protection is limited to the serotypes present in vaccine formulation and serotype replacement is threatening to decrease vaccine efficiency. This is exemplified by the increase, in the 7-valent vaccine era, of invasive disease caused by strains of serotype 19A, a serotype not included in this vaccine [3,4]. Therefore, the development of serotype-independent vaccines targeting protein virulence factors is being actively pursued and several pneumococcal proteins have been proposed as potential vaccine candidates [5]. We have recently used a lambda phage displayed whole genome library to identify several antigenic pneumococcal fragments, based on their ability to bind to serum antibodies from patients convalescing from pneumococcal infection or from experimentally infected mice. This powerful approach allowed the identification of a large panel of B-cell epitopes within known virulence factors or protective antigens, including members of the choline-binding, histidine-triad and zinc metalloproteinase families [6]. Further- more, we identified new antigenic regions matching the sequence of a novel pneumococcal adhesin, which was designated as plasminogen and fibronectin binding protein B [7]. We report here on the identification of a novel fragment designated as R4, which is encoded by ORF spr1875 in the R6 genome. The spr1875 gene was found to be conserved amongst pneumococcal strains isolated from different geographical areas. In an experimental model of sepsis, a mutant strain devoid of Spr1875 was attenuated PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e36588