JJ and SJ performed experiments

JJ and SJ performed experiments. activation in a Ascomycin (FK520) similar manner as IVIG. Moreover, bIgG was able to mediate T cell activation and internalization of pathogens, which are prerequisites for inducing an adaptive viral response. Using mouse experiments, we showed that bIgG is able to bind the murine activating IgG Fc Receptors (FcR), but not the inhibiting FcRII. Intranasal administration of the monoclonal antibody palivizumab, but also of bIgG and IVIG prevented RSV infection in mice. The concentration of bIgG needed to prevent infection was ~5-fold higher compared to IVIG. In conclusion, the data presented here indicate that functionally active bIgG facilitates adaptive antiviral T cell responses and prevents RSV infection and were opsonized with 500 ug/ml IVIG or bIgG or without antibody in 100 l 1% bovine serum albumin (BSA)-RPMI1640 for 15 min on ice. Washed bacteria were incubated in an effector: target ratio of 1 1:100 with 1 10e5 bone marrow derived WT mouse macrophages in V bottom 96 well plates (Greiner) for 30 min on ice. Cells were washed Ascomycin (FK520) with 100 ul ice-cold 1% BSA medium and equally divided over 2 wells prior to addition of opsonized bacteria. One part was incubated at 37C for internalization, while the other part was stained directly. Extracellular immune complexes (IC) were stained with 200x diluted Alexa647 conjugated hIgG (Jackson) or bIgG (Jackson) on ice. A decrease in extracellular signal is considered as internalized IC. In addition, cells were washed, fixated with 1% PFA and analyzed by flow cytometry (BD Bioscience, Canto II and FACS Diva software). RSV Prophylactic Mouse Model Female FcRC/C C57BL/6 mice or wild-type female littermates of the same age were used. Mice were anesthetized (3C4% isoflurane) and administered intranasal with 50 l antibody diluted in PBS with a varying dosing (0.2C5 mg/kg) of bIgG or IVIG or with a fixed dose of 5 mg/kg bIgG or 1 mg/kg for a similar prophylactic effect on the viral load. Palivizumab was used at 0.05 mg/kg. Mice were intranasally infected with 3 10e6 PFU RSV-A2-RL-Line19F in 50 l PBS after 24 h. Mice were euthanized by intraperitoneal injection of sodium pentobarbital 5 days post infection. A bronchioalveolar lavage was performed, after inflating the lungs, with 1 ml PBS and used to determine the viral load, as described previously (24). Statistical Analysis Statistical analysis was performed using GraphPad Prism 6 software. An unpaired Student’s < 0.05, **< 0.01, ***< 0.001, ****< Ascomycin (FK520) 0.0001. All graphs represent mean SD of triplicate measurements, unless indicated otherwise. Results Bovine IgG Binding and Neutralization of RSV For this study we made use of RSV-A2 and the more pathogenic strain RSV-A2-RL-Line19F, to evaluate whether the binding of purified bovine colostrum IgG (bIgG) and purified human plasma IgG (IVIG) is equal between both strains. HEp-2 cells were infected with RSV and dose-dependent binding of bIgG and IVIG was analyzed. Binding was compared to the clinically used antibody palivizumab (human IgG1 against RSV F protein). bIgG bound to the RSV-A2 infected cells, as shown previously, starting Mouse monoclonal to MER from a concentration of 1 1.2 g/ml bIgG. Binding of bIgG was equal to cells infected with both RSV strains, similar to what was observed with IVIG and palivizumab (Figure 1A). Open in a separate window Figure 1 bIgG RSV binding and neutralization. Antibody binding of serial diluted palivizumab (Pali),.