Opsonic Antibodies against E. faecalis Figure 1. Cross-reactivity of antibodies directed against enterococcal LTA. (A) Binding of rabbit IgG raised against LTA purified from E. faecalis strain 12030 to LTA extracted from E. faecalis strains of various CPS-serotypes. The coating antigens and serum dilutions are specified in the legend. Each point represents the average of two determinations. (B) Opsonophagocytic killing of various E. faecalis strains by the same anti-LTA rabbit antiserum. A serum dilution of 1:800 was used to assess killing activity. The respective target strains are indicated in the legend. Opsonophagocytic killing activity was compared to controls from which leukocytes were omitted. Each bar represents the mean of four determinations and the error bar the SEM. doi:10.1371/journal.pone.0017839.g001 Antibodies against diheteroglycan are opsonic to CPS-C and CPS-D strains We previously generated antisera by vaccination with heat- killed, proteinase K digested bacterial cells of E. faecalis type 2 (CPS-C) and type 5 (CPS-D) that opsonize CPS-C or CPS-D strains [4]. The antisera contained antibodies that bound to LTA but also antibodies that recognized enterococcal diheteroglycan (Fig. 3A). Using both antigens as inhibitors in the opsonophago- cytic killing assay, we assessed the specificity of opsonic antibodies of the antisera against E. faecalis type 2 and type 5 (Fig. 3B). In agreement with results obtained with antiserum against LTA showing no opsonic activity against type 2 and type 5 strains, purified LTA was also a poor inhibitor (i.e. ,10% inhibition) of opsonophagocytic killing mediated by the serotype-specific antiserum. Purified diheteroglycan, on the other hand, inhibited 98% of opsonic activity of antiserum against E. faecalis type 2 Table 1. E. faecalis strains used in the study. Strain E. faecalis 12030 E. faecalis 12107 E. faecalis OG1RF E. faecalis type 1 E. faecalis type 2 E. faecalis type 21 E. faecalis R19.001 E. faecalis V583 E. faecalis FA2-2 E. faecalis HG101 E. faecalis type 5 *for reference, see [21]. doi:10.1371/journal.pone.0017839.t001 Serotype CPS-A CPS-B CPS-B CPS-B CPS-C CPS-C CPS-C CPS-C CPS-C – CPS-D Source clinical clinical oral unknown urine infant/fecal fecal blood clinical urine cpsI mutant of FA2-2 (CPS-C) and 88% of opsonic killing of E. faecalis type 5 (CPS-D) by the respective antiserum indicating that the majority of opsonic antibodies raised by whole cell vaccination are directed against this antigen. To further explore the potential of diheteroglycan as candidate for an enterococcal vaccine, we immunized a rabbit with purified antigen from E. faecalis type 2 (CPS-C). Western blot analysis of this antiserum with whole cells lysates of the same strain as antigen confirmed the presence of antibodies against the high molecular weight band of diheteroglycan. In addition, we detected antibodies against a second, broad band that migrated around 20– 30 kDa, suggestive of antibodies against LTA (data not shown). Diheteroglycan – like many other bacterial polysaccharide antigens – was overall poorly immunogenic and induced only moderate levels of specific antibodies as quantified by ELISA (Fig. S5). We further investigated cross-reactivity of diheteroglycan- specific antibodies by the opsonophagocytic killing assay (Fig. 4). MLST* Synonym Reference 64 21 1 21 11 30 6 8 – 68 unknown MCTC 8727 MCTC 8796 MCTC 8746 ATCC700802 MCTC 8731 [22] [22] [45] [26] [26] [26] [46] [47] [48] [12] [26] PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17839