By comparison of anti-mouseP. poor compliance to drug treatment schedules, recurrent infections, and adverse side effects to the drugs are problems (9,19). Therefore, investigation of host-parasite interactions which could lead to new treatment methods is worthwhile. A growing body Rabbit polyclonal to CD47 of evidence suggests that anti-Pneumocystisantibody therapy may be an effective means of preventing and treating PCP. Hyperimmune sera from mice immunized withP. cariniiorganisms resolved existingP. cariniiinfections in SCID mice and decreased the hyperinflammatory reaction inimmune-reconstituted mice (23,24). An early study using an anti-mouseP. cariniiantibody demonstrated partial protection against development of PCP (1). Our previous studies demonstrated that SCID mice are also protected from the development of PCP by passive prophylaxis using the anti-P. cariniiimmunoglobulin M (IgM) monoclonal antibody (MAb) 4F11 or its IgG1 switch variant, MAb 4F11(G1) (2). MAb 4F11(G1) recognizes multiple, similar epitopes on the surface ofP. cariniiisolated from mice within at least two different antigens, kexin and cDNA clone A12 (13,29). MAb 4F11(G1) is also capable of recognizingP. cariniiderived from humans, rhesus macaques, rats, and ferrets (2,29). In this investigation, we set out to determine the mechanism of protection against development of PCP by MAb 4F11(G1). Specifically, we wanted to determine whether protection by MAb 4F11(G1) was mediated through antibody Fc region-dependent mechanisms, such as complement fixation and/or Fc receptor-mediated phagocytosis by macrophages or neutrophils, or whether it occurred in the absence of Fc region via binding and agglutination ofP. cariniiorganisms. == MATERIALS AND METHODS == == Antibodies. == Sabinene An IgG1 switch variant of IgM monoclonal antibody 4F11(G1) specific for mouseP. cariniiKex1 and cDNA clone A12 (2,13,29) and an anti-Haemophilus influenzaeIgG1 monoclonal antibody were purified from ascites fluid by passage over a protein A-Sepharose column (Pierce, Rockford, Ill.). == Mice. == Pathogen-free SCID (C.B-Igh-1b/IcrTac-Prkdcscid) mice Sabinene were obtained from Taconic Farms (Germantown, NY), housed in microisolator cages in the University of Rochester animal care facilities, and fed sterile food and water. All procedures performed were subject to University of Rochester Committee on Animal Resources approval. == Production, purification, and antigen binding analysis of F(ab)2 fragments of 4F11(G1). == Aliquots containing 1.5 mg Sabinene of purified 4F11(G1) in digest buffer (0.1 M sodium citrate, 5 mM EDTA, 1 mM cysteine, pH 6.0) were digested overnight into Sabinene F(ab)2 fragments and Fc fragments by using an immobilized Ficin chromatography column as described by the manufacturer (Pierce). Digested protein was eluted by application of 4 ml of binding buffer (0.1 M sodium citrate, 5 mM EDTA, pH 6.0) to the Ficin column, and Fc fragments and intact IgG molecules were removed by passage over a protein A affinity chromatography column. Purity of the F(ab)2 fragments in the protein A column flowthrough was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and silver staining. Protein concentrations of F(ab)2 preparations were determined by bicinchoninic acid assay as described by the manufacturer (Pierce). F(ab)2 fragments were adjusted to a concentration of 1 1 mg/ml and filter sterilized, and single-use aliquots were stored at 80C until use. Antigen binding capability of F(ab)2 fragments was determined by enzyme-linked immunosorbent assay (ELISA). Plates were coated with sonicatedP. carinii-infected mouse lung homogenates diluted to an approximate protein concentration of 1 1 to 10 g/ml in carbonate-bicarbonate buffer. The plates Sabinene were incubated with twofold dilutions of either 4F11(G1) or 4F11(G1) F(ab)2 fragments, starting at a concentration of 60 g/ml in triplicate wells. A goat anti-mouse IgG Fab-specific antibody conjugated to alkaline phosphatase was used as a secondary antibody. Control wells received secondary antibody alone. The optical density at 660 nm was determined after 20 min of color development using a 96-well plate reader (Bio-Rad, Hercules, CA). == Intranasal immunization of mice and cohousing. == Starting on day 1 of the experiment and continuing daily through day 16, groups of six to seven mice were given 50 l of a 1-mg/ml suspension of either 4F11(G1), 4F11(G1) F(ab)2, or anti-H. influenzaetype b monoclonal antibody intranasally under light ketamine-xylazine (experiment 1) or halothane (experiment 2) anesthesia. All mice in the different treatment groups were cohoused together with threeP. carinii-infected source mice to ensure equal exposure toP. cariniibeginning on day 1 and ending on day 14, at which time the three treatment groups were separated and removed from the source mice. Mice were sacrificed by intraperitoneal (i.p.) injection of sodium pentobarbital.