To investigate the epitopes recognized by the antibodies in such ineffective antisera, we chose two serum samples (P3B and P4B [Fig

To investigate the epitopes recognized by the antibodies in such ineffective antisera, we chose two serum samples (P3B and P4B [Fig.1]) and measured their anti-6B antibodies before and after preabsorbing the sera with C-PS or other PS (Fig.2). of its polysaccharide (PS) capsule (7).S. pneumoniaeis a major causative agent for pneumonia, meningitis, and sepsis among young children and older adults (4). Antibiotic treatment has become less effective since the prevalence of antibiotic-resistantS. pneumoniaehas become very high (1). Thus, there is a great need for pneumococcal vaccines effective among young children and older adults. Antibodies SERPINA3 to capsular PS provide protection againstS. pneumoniaeexpressing the homologous or cross-reactive capsular serotypes, and pneumococcal vaccines are designed to induce antibodies to the capsular PS. The currently available vaccines contain the capsular PSs from 23 common serotypes ofS. pneumoniae(12). Because many PSs GSK2330672 in the 23-valent vaccine are not immunogenic in young children, PS-protein conjugate vaccines made up of several serotypes (e.g., serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) are being developed (16). The development of new GSK2330672 pneumococcal vaccines would be simplified greatly if there was a simple serologic assay for vaccine-induced protective immunity. Previous studies suggested that this concentration of antibodies in the serum correlates with in vivo animal protection (8) and in vitro opsonic activity (14,17), the key step of protection in vivo. Yet, a recent epidemiological study suggested that this antibody concentrations may not predict vaccine efficacy against homologous serotypes (18). Also, the correlation between antibody concentration and opsonic activity can GSK2330672 be low (r= 0.5) (10,13). We have therefore examined the antigen-binding properties of several sera with less opsonic activity than expected on the basis of antibody concentration. (Part of this material has been submitted as an abstract for the 199 meeting of the Society of Pediatric Research in New Orleans.) == MATERIALS AND METHODS == == Human sera and bacteria. == Twenty-five healthy adults were immunized once with a 23-valent pneumococcal vaccine (PNU-IMMUNE 23) from Lederle Laboratories (Pearl River, N.Y.). Serum samples were collected before and 1 month after vaccination and were stored frozen at 20C until analysis. The following strains ofS. pneumoniaewere used: DS2214 (G. Carlone, Atlanta, Ga.), a serotype 14 strain; WU2 (Janet Yother, Birmingham, Ala.), a serotype 3 strain; JY1119 (David Briles, Birmingham, Ala.) and JD908 (Janet Yother), WU2 variants lacking PspA and the PS capsule, respectively; Tre-108 (David Briles), a variant of D39 (serotype 2) lacking PspC (3); and CSR-SCS2 (G. Schiffman, Brooklyn, N.Y.), a variant lacking the capsule (15). == ELISA. == The amount of anti-capsular PS antibody was determined by sandwich-type enzyme-linked immunosorbent assay (ELISA). Briefly, the wells of Maxisorb plates (Nunc, Roskilde, Denmark) were coated at 37C with 10 g of the capsular PS (6B serotype)/ml overnight in phosphate-buffered saline, which was prepared fresh with water from a Milli-Q UF water purification system (Millipore, Bedford, Mass.) to minimize the background signal. All pneumococcal capsular PSs were purchased from the American Type Culture Collection (Rockville MD). After being coated with GSK2330672 the antigen, the plates were washed and blocked with phosphate-buffered saline made up of 1% bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) and 0.05% Tween 20. A serum pool (89-SF) (11) from C. Frasch of the Food and Drug Administration (Bethesda, Md.) was used as the standard and was found to contain 16.9 g of immunoglobulin G (IgG) anti-6B as published previously (11). All samples were assimilated with 10 g of C-PS (purchased from Statens Seruminstitut, Copenhagen, Denmark) per 20 l of serum in a total volume of 1 ml of diluent for 30 min at room temperature. The samples were then added to the wells, serially diluted, and incubated for 2 h at room temperature. For the inhibition ELISA, inhibitors were added to each well before the test serum was added. The wells were washed and incubated with GSK2330672 alkaline phosphatase-conjugated goat antibody specific for human IgG (Sigma Chemical). The amount of the enzyme immobilized to the well was decided withpara-nitrophenyl phosphate substrate (Sigma.