Production of CCR6 To obtain anti-pneumococcal immune sera, to be used as positive controls, a group of mice was immunized with a cho- line binding proteins-enriched fraction designated as CCR6. CCR6 was obtained from strain R6 cells grown to the early exponential phase (OD600 = 0.2), washed with phosphate buffered saline (PBS; pH 7.2), and incubated in the presence of 2% choline chloride (Sigma) at 20uC for 10 minutes. The supernatant was dialyzed, concentrated and used as described above to immunize mice using 50 mg (total protein content) for each immunization. Inhibition ELISA These experiments were performed to assess the ability of recombinant protein fragments to inhibit the reactivity of anti-Spr1875 sera against the homologous antigen. To this end, mouse sera raised against Spr1875-BIO were reacted against Spr1875-GST. An anti-Spr1875-BIO serum pool (diluted 1 200 in PBS-FCS) was added to microtiter wells sensized with Spr1875-GST (1 mg/ml in PBS) in the presence and in the absence of the indicated concentrations of inhibitors (i.e. R4- GST and R5-GST). After washing with PBS-FCS, alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) was added at a 1 5000 dilution followed by p-nitrophenyl phosphate disodium salt (Sigma). Percent of inhibition was calculated by comparing the absorbance value of wells with and without the inhibitors. Supporting Information Figure S1 Lack of Spr1875 in cell lysates from the Dspr1875 pneumococcal strain. D39 or Dspr1875 cell lysates were loaded on a polyacrylamide gel and developed with anti-R4 mouse serum (1 1,000). The arrow indicates the 40 kDa Spr1875 protein in the D39, but not in the Dspr1875,cell lysate. (TIF) Figure S2 Protection induced by R4 immunization is mediated by serum antibodies. Two groups of 16 mice each were given i.p. 50 ml of serum pools from animals immunized with, respectively, GST (anti-GST) or R4-GST (anti-R4-GST). After 4 h both groups of animals were challenged i.v., with D39 (16105 CFU). *, statistically different (p,0.05) from animals given References 1. Rehm SJ, Farley MM, File TM, Jr., Hall WJ, Hopkins R, et al. (2009) Higher pneumococcal disease vaccination rates needed to protect more at-risk US adults. Postgrad Med 121: 101–105. 2. File TM (2003) Community-acquired pneumonia. Lancet 362: 1991–2001. 3. Hicks LA, Harrison LH, Flannery B, Hadler JL, Schaffner W, et al. (2007) Incidence of pneumococcal disease due to non-pneumococcal conjugate vaccine (PCV7) serotypes in the United States during the era of widespread PCV7 vaccination, 1998–2004. J Infect Dis 196: 1346–1354. 4. Miller E, Andrews NJ, Waight PA, Slack MP, George RC (2011) Herd immunity and serotype replacement 4 years after seven-valent pneumococcal conjugate vaccination in England and Wales: an observational cohort study. Lancet Infect Dis 11: 760–8. 5. Bogaert D, Hermans PW, Adrian PV, Ru¨mke HC, de Groot R (2004) Pneumococcal vaccines: an update on current strategies. Vaccine 22: 2209–2220. 6. Beghetto E, Gargano N, Ricci S, Garufi G, Peppoloni S, et al. (2006) Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome l display library. FEMS Microbiol Lett 206: 14–21. 7. Papasergi S, Garibaldi M, Tuscano G, Signorino G, Ricci S (2010) Plasminogen- and fibronectin-binding protein B is involved in the adherence of Streptococcus pneumoniae to human epithelial cells. J Biol Chem 285: 7517–7524. 8. Tu AH, Fulgham RL, McCrory MA, Briles DE, Szalai AJ (1999) Pneumococcal surface protein A inhibits complement activation by Streptococcus pneumoniae. Infect Immun 67: 4720–4724. Spr1875, a Pneumococcal Antigen anti-GST serum as assessed by Kaplan-Meier estimator of survival. (TIF) Figure S3 Lack of protection induced by immunization with recombinant Spr1875 protein fused to a GST tag (Spr1875-GST). (A) Groups of CD1 mice were immunized with Spr1875-GST or with GST, used as a negative control, and challenged with the D39 strain (16105 CFU). Results represent data from one experiment involving 8 animals per group. (TIF) Table S1 Anti-R4 Elisa titers of sera from patients convalescing from pneumococcal infection. (DOC) Table S2 Alignment of 27 S. pneumoniae strain se- quences available in DNA database using the ClustalW software (http://www.ebi.ac.uk/Tools/clustalw2/ index.html). (DOC) Amino acid sequences of Spr1875 fragments. Table S3 (DOC) Table S4 Primers used in the construction of DpspA and Dspr1875 deletion mutants. (DOC) Table S5 Genomic replacement sequences for DpspA and Dspr1875 deletion mutants. (DOC) Supplemental Materials and Methods. 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Curr Opin Microbiol 9: 12–20. 16. Sta˚lhammar-Carlemalm M, Waldemarsson J, Johnsson E, Areschoug T, Lindahl G (2007) Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine. Cell Host Microbe 2: 427–434. PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e36588