In the present study we used the human fH transgenic rat model to investigate the ability of JAR 41 given individually or in combination with other non-bactericidal anti-fHbp mAbs to confer passive protection against bacteremia caused by challenge with wild-type group B strain H44/76. Despite lack of bactericidal activity = 0.009). Open in a separate window Figure 6 JAR 41 augments passive protective activity of anti-fHbp mAb JAR 5 against bacteremia caused by group B strain H44/76 in human being fH transgenic infant rats.Panel a, Experiment 1, 8- to 9-day time older rats challenged IP with 4900?CFU/rat; Panel b, Experiment 2, 6- to 7-day time older rats challenged IP with 760?CFU/rat. 2 or 3 3). Binding of an anti-PorA mAb was related with both the wild-type and mutant (Number 4b), which is definitely evidence that related numbers of wild-type and mutant cells were tested for JAR 41 binding. Open in a separate window Number 4 Binding of mAbs to live strains as measured by circulation cytometry.Panel a, Assessment of JAR 41 binding (25 g/ml) to wild-type strain H44/76 (H44/76-WT, stable black collection) with fHbp ID 1 in variant group 1, or an isogenic mutant with reduce manifestation of fHbp ID 1 (H44/76-LE, blue dashed collection). Solid green collection, H44/76-WT incubated with 25 g/ml of a negative control IgG mAb (JAR 11, specific for fHbp in variant organizations 2 and 3); green-shaded area, H44/76-LE incubated with JAR 11. Panel b, binding of a control anti-PorA mAb. Orange collection, H44/76-WT; orange-shaded area, H44/76-LE. Panels c-f, Concentration-dependent binding of JAR 41 with strain H44/76-WT (panel c), mutant strain H44/76-LE (panel d), strain 8047 with fHbp in variant group 2 (panel e), or M1239 with fHbp in variant group 3 (panel f). Panels c, d and e, thick black collection, 25 g/ml of JAR 41; dark grey-shaded area, 5 g/ml; dashed black collection, 1 g/ml; RF9 thin black collection, 0.2 g/ml; light grey-shaded area, 25 g/ml JAR 41 with a negative control H44/76 fHbp knock-out mutant; solid blue collection, 25 g/ml of JAR 5, which is definitely specific for fHbp in variant group 1 and a negative control mAb for variants 2 and 3. Panel f, thick black collection, 50 g/ml of JAR 41 with wild-type strain M1239 with fHbp in variant group 3; dark shaded area, 5 g/ml of JAR 41 (lower concentrations of JAR 41 were not tested); blue collection, 50 g/ml of JAR 5 (specific for fHbp variant group 1); Light gray shade area, 50 g/ml of JAR 41 incubated with M1239 fHbp knock-out mutant. We next tested binding of JAR 41 to strains H44/76 and its isogenic mutant using a range of mAb concentrations (Numbers 4c and 4d, respectively). As little as 0.2 g/ml of JAR 41 showed significant binding to the H44/76-WT or -LE Comp mutants over that of background binding, which was determined by screening 25 g/ml of JAR 41 having a H44/76 fHbp knockout mutant (light gray shaded area). JAR 41 also bound to the surface of group B strains 8047 (fHbp ID 77, variant group 2) and M1239 (fHbp ID 28, variant group 3) (Numbers 4e and 4f, respectively). For each of these strains the respective JAR 41 binding was related when tested at the two highest concentrations (5 g/ml or 25 g/ml for 8047, or 5 and 50 g/ml for M1239 [grey-shaded area and solid lines, respectively]). Strain 8047 also was RF9 tested with lower concentrations of the mAb. Binding above that of the IgG bad control (an IgG anti-fHbp mAb specific for variant group 1) was recognized with as little as 0.2 g/ml of JAR 41. JAR 41 elicits human being complement-mediated bactericidal activity with second anti-fHbp mAbs JAR 41 was not bactericidal when tested separately at concentrations of up to 100 g/ml against any of the four test strains (H44/76-WT [Number 5a], the H44/76 LE mutant [Number 5b], 8047 [Number 5c] or M1239 [data not demonstrated]). Activation of the classical match pathway by IgG requires binding of two IgG mAbs to appropriately spaced epitopes such that the respective IgG Fc areas can participate C1q24. When an individual mAb binds to a sparse antigen, there may be insufficient immune complex created to activate bacteriolysis, whereas mixtures of more than one mAb may be adequate24. When tested in combination with additional non-bactericidal anti-fHbp mAbs specific for fHbp in variant group 1, JAR 41 elicited cooperative bactericidal activity against strains H44/76 (Number 5a) or the H44/76 LE mutant with lower fHbp manifestation RF9 (Number 5b)..