Identification of Protective Pneumococcal TH17T_{H}17 Antigens from the Soluble Fraction of a Killed Whole Cell Vaccine

Mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell antigen (WCA) with an adjuvant protects mice from colonization by a $T_{H}17$ CD4+ cell-mediated mechanism. Using preparative SDS gels, we separated the soluble proteins that compose the WCA in order to identify fractions that were immunogenic and protective. We screened these fractions for their ability to stimulate IL-17A secretion from splenocytes obtained from mice immunized with WCA and adjuvant. We identified 12 proteins within the stimulatory fractions by mass spectrometry; these proteins were then cloned, recombinantly expressed and purified using an Escherichia coli expression system. The ability of these proteins to induce IL-17A secretion was then evaluated by stimulation of mouse splenocytes. Of the four most stimulatory proteins, three were protective in a mouse pneumococcal serotype 6B colonization model. This work thus describes a method for identifying immunogenic proteins from the soluble fraction of pneumococcus and shows that several of the proteins identified protect mice from colonization when used as mucosal vaccines. We propose that, by providing protection against pneumococcal colonization, one or more of these proteins may serve as components of a multivalent pneumococcal vaccine

Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC

Stimulation of splenocytes from immune mice with purified recombinant proteins.

<p>Mice (n = 7–10) were intranasally immunized with WCC and cholera toxin as described. Splenocytes from immunized mice were stimulated with 10 μg/ml of the indicated recombinant protein for 3 days, after which supernatants were harvested and assayed for IL-17A concentration. Values are normalized to the DMEM stimulated response for each animal. Bars represent medians with interquartile ranges.</p

T_H17 cell antigens isolated from immunogenic fractions of soluble portion of RM200, predicted function, and primers used for expression in <i>E. coli.</i>

<p>T_H17 cell antigens isolated from immunogenic fractions of soluble portion of RM200, predicted function, and primers used for expression in <i>E. coli.</i></p

Immunization with individual proteins confers protection against nasopharyngeal carriage.

<p>Mice were intranasally immunized twice with 1 µg of cholera toxin alone (CT) or CT combined with the proteins as indicated. Four weeks after the second immunization, mice were intranasally challenged with strain 0603; density of colonization was determined one week later. Bars indicate median values and nasal colonization density was compared by the Mann-Whitney <i>U</i> test.</p

Size separation of fractions and stimulation of splenocytes.

<p>A. SDS-PAGE of fractions was performed and gel was silver-stained; shown here are the electrophoretic patterns of three fractions eluted into the same chamber during different transverse elutions. B. Results from stimulation of splenocytes from WCC immunized mice (n = 6) with equal concentrations of each fraction. Supernatants were collected after 3 days of incubation and IL-17A concentration in the supernatant was measured by ELISA. IL-17A values are shown here, normalized to the DMEM-stimulated response of each animal. Bars represent medians with interquartile range. WCC: chloroform-inactivated pneumococcal whole cell antigen, 10 µg protein/ml; WCCsup: soluble fraction of WCC, 7 µg protein/ml.</p