Although allotype-specific linkages have been described in other experimental systems using various PS antigens (20-23, 73), the 44.1-Id differs from these systems in several aspects. PPS14-protein conjugate exhibits minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all 4 IgG subclasses during both the primary and secondary responses. 44.1-Id usage was linked to the allotype (21). Individual lack of expression of a particular dominant Id does not typically affect the quantity of the Mmp17 Ag-specific Ig response, with the exception of the murine response to 1-3 dextran (22). Human primary Ig responses following immunization with isolated PS, despite being TI, exhibit somatic mutations (19, 24, 25), at a rate that is similar to that observed at least early in the response to conjugate vaccines (19, 25). To explain this surprising result, it has been proposed that vaccination of adults with isolated or protein-conjugated PS, reactivates PS-specific memory B cells induced in response to PS conjugated to protein cell wall components of extracellular bacteria to which the host had been previously exposed (25, 26). This PS is perhaps in a TD form via association with bacterial protein. In this regard, studies from our laboratory utilizing inactivated, intact Cinnarizine (Group B Streptococcus type III [GBS-III]) Cinnarizine expressing a capsular PS identical to PPS14 (29). These studies emphasize the importance of the subcapsular domain in influencing the associated PS-specific Ig response. In this report, we have extended our studies on the distinct humoral immune responses to different antigenic forms of PPS14, and identified an Id (44.1-Id) that dominates the PPS14-specific IgG response to intact Pn14 as well as isolated PPS14, but not to a soluble PPS14-protein conjugate. However, the usage of the 44.1-Id is partly restored in the responses to PPS14-protein conjugate coupled to particles. In addition, the 44.1-Id is elicited in mice expressing the mice (commonly referred to as C.B-17) were obtained from The Jackson Laboratory (Bar Harbor, Maine). Mice were used between 8-12 wks of age. BALB/c mice used in the production of B cell hybridomas were obtained from Simonsen Laboratories (Gilroy, CA). The experiments in this study Cinnarizine were conducted in accordance with the principles set forth in the Guide for Care and Use of Laboratory Animals (30), and approved by the Uniformed Services University of the Health Sciences and Children’s Hospital Oakland Research Institute Institutional Animal Use and Care Committees. Bacterial strains and antigens The (Pn) R36A strain, an unencapsulated mutant of D39 (serotype 2) was provided by Dr. David Briles (University of Alabama, Birmingham, AL). The R6-14 strain is an isogenic variant of R36A expressing PPS14 (13). Group B (GBS) type III COH1 and COH1-11 strains, that express the native and desialylated type III PS, respectively were provided by Dr. C. Rubens (Children’s Hospital, Seattle, WA) (31). Frozen bacterial stocks were thawed and subcultured on blood agar plates (VWR International, Radnor, PA). Isolated colonies were grown in Todd Hewitt broth (BD Biosciences, Sparks, MD) to mid-log phase, collected, and heat-inactivated by incubation at 60C for 1 h. Bacteria were frozen at ?80C until their use. Purified PPS14 was purchased from ATCC (Manassas, VA) and biotinylated by cyanogen bromide activation and coupling to biotin-LC-hydrazide (Pierce, Rockford, IL) (24). Biotinylation did not affect the antigenic structure of PPS14. A covalent conjugate of rPspA and PPS14 was synthesized as described (13). The rPspA in the conjugate contains the first 302 a.a. of the PspA expressed by the R6-14 strain. The molar ratio of PPS14 to rPspA in the conjugate is 1:24. Preparation of R36Acoated with PPS14-PspA PPS14-PspA was adsorbed to R36A as described (13). Briefly, the unencapsulated R36A strain was depleted of choline-binding proteins, including PspA, by treatment with 2% choline chloride (R36Acontained 60 ng native PspA/109 CFU. R36Awas then incubated for 24 h with PPS14-PspA in 30 mM acetate buffer, pH 5. Conjugate was stably attached to the R36Asurface. Free conjugate represented 0.4% of the total content of the R36Aallotype closer to BALB/c (type III variants expressing a PS structurally identical to PPS14Female BALB/c mice (n=7) were immunized with 1 109 CFU/mouse of strain R6-14 or Group B type III strain COH1-11 (expressing a PS structurally identical to PPS14). Fourteen days later every group was challenged with the same strain, except for a group of mice immunized at day 0 with R6-14 and challenged at day 14 with COH1-11 (R6-14; COH1-11). The content of PPS14-specific IgG, IgG1 and IgM expressing the 44.1-Id (top panels) in the sera collected prior to the immunizations (preimmune), 14 days after the first (primary response), and second immunization Cinnarizine (secondary response), was determined by inhibition ELISA using plates coated with PPS14. Statistical significance (p 0.05) of the PPS14-specific Ig responses of each group relative to mice.