43:678-684. might be useful surrogates for antibody (and vaccine) efficacy. The goal of vaccination with pneumococcal capsular polysaccharide (PPS)-based vaccines is usually to elicit type-specific antibodies to PPS that confer protection against is unique among pneumococcal serotypes because it causes disease predominantly in adults (29). Efforts to prevent invasive pneumococcal disease in individuals who are at the highest risk for disease have been plagued by poor PPS immunogenicity and reduced PPS-based vaccine efficacy (12). In addition, the lack of serologic surrogates that are predictive of antibody-mediated protection has hampered efforts to understand the determinants of vaccine efficacy or failure. Historically, the gold standard for serum protection against the pneumococcus was mouse protection (58). However, a therapeutic antiserum to serotype 3 could not be developed (14), and associations among parameters, such as serotype-specific antibody concentration, serum opsonic activity in vitro, and protection in experimental models, have been unpredictable (19, 39). Efforts to JNJ-54175446 JNJ-54175446 understand PPS vaccine efficacy and failure have been limited by the use of polyclonal sera (15, 18), the lack of confirmed correlates of vaccine efficacy, and insufficient information regarding the characteristics of antibodies to PPS that mediate protection. Polyclonal sera often yield conflicting results with regard to JNJ-54175446 antibody function in vitro and in vivo, because they are composed of antibodies of multiple specificities and isotypes that collectively, or individually, may be protective, nonprotective, or deleterious in vivo (14). In the hope of identifying surrogates of antibody efficacy against based on structure-function associations, we have taken the approach of characterizing the molecular genetic structures and in vivo functional efficacies of defined, monospecific antibodies (MAbs) to PPS. In this study, we generated human MAbs to PPS-3 in a transgenic mouse strain that expresses human immunoglobulin genes, the XenoMouse mouse (37), and examined their molecular genetic structures and in vivo efficacies against serotype 3 serotype 3 strain 6303 (American Type Culture Collection [ATCC] Manassas, Va.) was produced in tryptic soy broth (Difco Laboratories, Detroit, Mich.) to mid-log phase at 37C in 5% CO2 as described previously (51). The PPS used for immunization was a conjugate that was produced using purified serotype 3 PPS (ATCC) and tetanus toxoid (TT) (PPS-3-TT) according to methods described for another TT conjugate produced by our group (21). Generation of PPS-3-specific human MAbs from XenoMouse mice. XenoMouse mice obtained from Abgenix (Fremont, Calif.) were vaccinated subcutaneously at the base of the tail with a total dose of 2.5 g of PPS-3-TT. Splenocytes were isolated on day 7 or day 14, and hybridomas were generated by fusion with the mouse myeloma cell line NSO as described previously (51) and propagated with a cell-cloning system, ClonaCell-HY (Stem Cell Technologies Inc., Vancouver, British Columbia, Canada) according to the manufacturer’s instructions. Isotypes and PPS specificities of MAbs Hybridoma cell lines were tested for the secretion of antibodies that reacted with serotype 3 JNJ-54175446 PPS as described previously (20). Briefly, hybridoma supernatants were adsorbed with purified pneumococcal cell wall polysaccharide (Statens Seruminstitut, Copenhagen, Denmark). Polystyrene enzyme-linked immunosorbent assay (ELISA) plates (Corning Glass Works, Corning, N.Y.) coated with 10 g of PPS-3 (ATCC 6303)/ml were incubated with serial dilutions of the hybridoma Rabbit Polyclonal to E2F6 supernatants, washed, and incubated at 37C for 1 h with alkaline phosphatase-conjugated goat anti-human reagents to immunoglobulin G (IgG), IgM, IgA, and kappa light chains and a goat anti-mouse reagent to lambda light chains (Fisher Biotech, Fisher Scientific, Pittsburgh, Pa.). After the plates were washed, antibody binding was detected by developing the plates with serotypes 3, 4, 6B, 8, 9V, 14, 19F, and 23F (ATCC) were determined by a PPS capture ELISA as described above. Second, a modification of this ELISA was used in inhibition assays with soluble PPS-3. For these studies, plates were coated with 10 g of PPS-3/ml and incubated with solutions consisting of 5-g/ml constant amounts of the MAb and various concentrations of soluble PPS-3 ranging from 0.1 to 100 g/ml in duplicate. After incubation at 37C for 1 h, the plates were washed and incubated with alkaline phosphatase-conjugated goat anti-human IgM (Southern Biotechnology, Birmingham, Ala.) and developed as described above. The relative apparent affinity constant (aKa) of each MAb for soluble PPS-3 was decided for the conversation of each MAb with soluble PPS-3 as described previously (40). The aKa was defined as the inverse mole concentration of soluble PPS-3 needed to reduce maximal MAb binding to solid-phase PPS-3 by 50%. Although it is usually recognized that this method.