antigens, in the development of effective vaccines based on pneumococcal proteins. Materials and Methods Selection of the R4 fragment from the phage display library The R4 fragment was selected from a genomic pneumococcal phage displayed library using previously described methods [6]. Pneumococcal strains The following strains were used: 1) the encapsulated serotype 2 D39 strain [19] and its rough derivative R6 [20]; 2) the unencapsulated mutants D-D39, D-Tigr4, D-23F-Spain-1, and D-19F-Taiwan-14, which were kindly provided by Vega Ma- signani (Novartis Vaccines and Diagnostics s.r.l., Siena, Italy). The D39 deletion mutants DpspA and Dspr1875 (deleted for, respec- tively, pspA (spr0121) and spr1875) were constructed using a previously described procedure [21–23]; see also Text S1 and Tables S2, S3, S4, and S5. Briefly, we first amplified by PCR an antibiotic resistance gene and the 39 and 59 genomic regions adjacent to the gene of interest. Next, these fragments were assembled by PCR and the resulting product was used to transform wild type D39 (relevant primers are reported in Table S4 in supplemental materials). The mutants were verified by sequencing (Table S5 in supplemental materials). All bacteria were grown at 37uC in Todd-Hewitt broth (THB, Oxoid). When necessary, chloramphenicol (3 mg/ml) and erythromycin (1 mg/ml) were added. Neither the DpspA, nor the Dspr1875 strain differed from the wild-type D39 strain in its ability to grow in Todd-Hewitt broth. Immunization and challenge To study the protective activity of recombinant proteins, CD1 mice (5 wk old) were immunized intraperitoneally with 50 mg of recombinant proteins fused to glutathione S-transferase (GST) in complete (first injection) or incomplete (second and third injections) Freund’s adjuvant emulsions (0.2 ml) on day 0, 14, and 28. Control animals received GST only. The use of complete Freund’s adjuvant in the first immunization was justified by our previous observations that high titered sera were more consistently obtained with this adjuvant, as compared to other less ‘‘inflam- matory’’ adjuvants such as alum. However, care was taken to minimize discomfort to the animals by injecting a low volume (0.1 ml containing 0.05 mg of mycobacteria) of the oily compo- nent of the emulsion and by using sterile solutions and techniques to prepare it. Under these conditions no significant abdominal distension or complications at the injection site were observed throughout the experimental period. Three weeks after the last immunization mice were challenged i.v. with an approximately 90% lethal dose of D39 S. pneumoniae strain (16105 CFU) and monitored for up to14 days. To determine the virulence of deletion mutants pneumococcal strains were grown to mid log phase (OD600 = 0.4). Bacteria were washed, resuspended in PBS and plated for colony counts. Eight-week old CD1 mice were inoculated intravenously with 0.1 ml of suspensions containing the indicated bacterial doses. Signs of disease (e.g. rough hair, decreased mobility, lethargy) and lethality were recorded daily for 14 days and animals showing signs of irreversible disease were humanely euthanized. Overwhelming S. pneumoniae infection was confirmed as the cause of death by culturing the organs of moribund animals. For the passive protection experiment, each 8-week-old CD1 mouse received i.p. 50 ml of pooled sera from R4-GST-immunized Spr1875, a Pneumococcal Antigen or GST-immunized mice. Four hours later, mice from both groups were challenged i.v. with the D39 strain and survival was monitored as described above. All in vivo experiments were conducted at the animal facilities of the Metchnikoff Department of the University of Messina according to the European Union guidelines for the handling of laboratory animals and were approved by the relevant local (Comitato Etico per la Sperimen- tazione Animale) and national (Istituto Superiore di Sanita` Permit Numbers: 121/2007 - B) authorities. Flow cytometry immunofluorescence and western blot analysis S. pneumoniae strains grown to the early-log phase (OD600 = 0.2) were harvested by centrifugation, washed three times with PBS and blocked for 20 min at 20uC with PBS containing 2% fetal calf serum (PBS-FCS). Mouse antisera were diluted 1 100 in PBS-FCS and incubated with bacterial cells for 40 min at 4uC. Phycoery- thrin-conjugated goat anti-mouse IgG (Jackson Immunoresearch), diluted 1 50 was then added to the cells and incubated at 4uC for additional 30 min. Bacteria were then washed, fixed by parafor- maldehyde and analyzed with an LSR Flow Cytometer using the CellQuest software (both from BD Biosciences). Western blots were performed using anti-R4 sera on bacterial cell lysates exactly as described [6]. Production of recombinant fragments and antisera The R4 recombinant fragment was amplified from a phage clone and subcloned into the bacterial expression vector pGEX- SN, a previously described expression vector [24] to produce pGEX-SNR4 that allows the expression of recombinant proteins as fusions to GST. Similarly, to produce the GST fusion fragments R1 (117aa; E24-S140), R2 (124aa; Q141-K264), R3 (116aa; S265- G380), and R5 (186aa; T195-G380) of the Spr1875 protein, or to produce the whole Spr1875 protein fused to GST, the corre- sponding DNA sequences were amplified from the R6 genome and cloned into pGEX-SN. After induction of the fusion proteins, recombinant fragments were purified from the soluble lysate of bacterial cells by affinity chromatography [6]. Recombinant GST, to be used as a control, was also produced using the same procedures. The whole Spr1875 protein was also produced as a polypeptide fused to a biotinylable peptide tag (BIO) in Escherichia coli JM109, using the PinPoint Xa-1 Vector (Promega) according to manufacturer’s instructions. After the induction of fusion protein, this was purified from the cytoplasm of bacterial cells by affinity chromatography using the PinPointXa protein purification system (Promega). Recombinant chloramphenicol acetyl transfer- ase fused to biotin (CAT-BIO), to be used as a negative control, was produced and purified using the same methods. Determination of R4-specific serum antibody titers Anti-R4 serum antibody titers in immunized mice were measured by a previously described ELISA method [25]. Briefly, wells of microtiter plates were sensitized with R4-GST fusion protein (5 mg/ml). Mouse sera were serially diluted with antibody buffer containing 25 mg/ml of GST to block non-specific anti- GST antibodies. Serial serum dilutions were reacted for 2 h at 37uC before the addition of a 1 5,000 dilution of goat anti-mouse polyvalent IgG conjugated to alkaline phosphatase (Sigma). Plates were then developed, as described [25]. Antibody titers in human sera were measured by a similar ELISA assay, except that sera were diluted in plain antibody buffer (i.e. without GST) and an anti-human, instead of an anti-mouse, anti-IgG conjugate was used. PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e36588