Our preliminary epitope mapping studies and experiments measuring serum binding to bacterial surfaces suggest that vaccination can also expand the epitope repertoire of anti-PhtD antibodies (data not shown). Collectively, these results (S)-Metolachor showed that human anti-PhtD antibodies, whether naturally acquired or induced by the PhtD candidate vaccine, are functional. with an ED50 of 1679 ELISA units/ml (95% confidence interval, 14201946). Sera from subjects injected with aluminum-adjuvanted PhtD in a phase I trial had similar activity per unit of antibody (S)-Metolachor (ED50 = 1331 ELISA units/ml [95% confidence interval, 7622038]). Vaccine-induced activity in the passive protection model was blocked by pre-incubation with recombinant PhtD but not by a controlS. pneumoniaeantigen (LytB). These results show that human anti-PhtD antibodies, whether naturally acquired or induced by the PhtD candidate vaccine, are functional. This supports the development of the PhtD candidate as part of a broadly protective pneumococcal vaccine. Keywords:antibody, passive protection, pneumococcal histidine triad protein D,Streptococcus pneumoniae, vaccine == Abbreviations == colony forming units confidence interval dose providing 50% survival enzyme-linked immunosorbent assay ELISA units pneumococcal histidine triad protein D == == Each year,Streptococcus pneumoniaecauses more than 800,000 deaths worldwide in children under 5 years of age.1Currently marketedS. pneumoniaevaccines, which are based on polysaccharide capsular antigens from the most common strains, have substantially reduced pneumococcal disease rates.2However, because serotypes can vary between countries or regions, coverage may be incomplete in some cases.3Moreover, serotype replacement might eventually render these vaccines (S)-Metolachor less effective.4,5To provide broader, more diverse, and possibly infection stage-specific protection, vaccines based on conserved proteins are being investigated.2,6,7 Pneumococcal histidine triad protein D (PhtD) is a conserved surface protein that mediates attachment to respiratory epithelial cells6,7and can elicit a protective immune response.8-11In mice, intranasal immunization with PhtD generates robust serum antibody and CD4 Th1-biased immune memory responses and confers protection against pneumococcal colonization.12A second study in mice showed that vaccination with PhtD protects against nasopharyngeal and lung colonization.13In a primate study, vaccination with Rabbit Polyclonal to LFA3 PhtD and chemically detoxified pneumolysin induced high levels of antibodies and protected against a challenge withS. pneumoniaeserotype 19F.14A phase I trial in adults 1850 years of age showed that a aluminum phosphate-adjuvanted PhtD vaccine candidate was well tolerated, immunogenic, and could be boosted by a second vaccine dose.15During development of an enzyme-linked immunosorbent assay (ELISA) to measure antibody responses in the phase I trial, we found that individual and pooled serum from unimmunized healthy adults contained substantial PhtD-binding antibody (data not shown). To further investigate the immune response elicited by a PhtD-based pneumococcal vaccine, we developed a murine passive protection sepsis model for assessing the functional activity of human anti-PhtD antibodies. Naturally occurring human PhtD-binding antibody was purified from a commercial pooled serum (obtained from approximately 200 healthy individuals; Sigma, St. Louis, MO). The concentration of anti-PhtD antibody was determined by ELISA, and its purity and specificity were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and competition with recombinant PhtD (data not shown). The purified PhtD-binding antibodies were passively transferred by intraperitoneal injection (200 l) to 6- to 8-week-old female CBA/CaHN-Btk xid /J (CBA/N) mice (Jackson Laboratories, Bar Harbor, ME). After 1 h, the mice were challenged by intravenous injection with a lethal dose (50 colony forming units [cfu] in 200 l) ofS. pneumoniaestrain A66.1 (serotype 3) (obtained from D. Briles, University of Alabama-Birmingham). The proportion of mice surviving at 14 days post-challenge increased with the concentration of anti-PhtD antibody (Fig. 1A). The dose providing 50% survival (ED50) was estimated to be 1679 ELISA units (EU)/ml (95% confidence interval [CI], 14201946) by logistic regression with probit link. == Figure 1. == Dose-response of antibody activity in the passive protection model. 6- to 8-week-old female nave CBA/N mice (n = 5/group) received an intraperitoneal injection of 200 l test sample or PBS. Control mice received PBS. After 1 h, mice were challenged intravenously with a lethal dose of S. pneumoniae strain A66.1 (serotype 3), and survival was monitored for 14 days. Data were analyzed using logistic regression with probit link under PROC GLIMMIX in SAS version 8.2 to determine the ED50. In each plot, circles indicate survival data for individual samples, and the best fit regression is shown as a green line, with the upper and lower limits of the 95% confidence interval shown as yellow and red lines, respectively. The horizontal line indicates a median response of 50% survival. In (A), mice were injected with 2.827.5 EU (110 g) of purified anti-PhtD antibody or PBS. Survival data were from 4 passive protection experiments, which included 13 survival points. In five of the 13 cases (206, 275, 687, 1375, 2063, and 2750 EU/ml), the passive protection experiment was performed twice, and for 2 of these (275 and 2750 EU/ml), the points overlap and appear as a single data point. Protection experiments for 3 cases (43, 435, and 2178 EU/ml) were not repeated and were included to provide a more appropriate range of antibody concentrations. In (B), mice were injected with 1:20 to 1 1:60 post-immune sera. To account for the baseline levels of protection seen in.