Separation of decay-accelerating and cofactor functional activities of Kaposi's sarcoma-associated herpesvirus complement control protein using monoclonal antibodies.

Complement is an essential part of the innate immune system, which clears pathogens without requirement for previous exposure, although it also greatly enhances the efficacy and response of the cellular and humoral immune systems. Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently identified human herpesvirus and the likely aetiological agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. We previously reported that the KSHV complement control protein (KCP) was expressed on infected cells and virions, and could inhibit complement through decay-accelerating activity (DAA) of the classical C3 convertase and cofactor activity (CFA) for factor I (FI)-mediated degradation of C4b and C3b, as well as acting as an attachment factor for binding to heparan sulphate on permissive cells. Here, we determined the ability of a panel of monoclonal anti-KCP antibodies to block KCP functions relative to their recognized epitopes, as determined through binding to recombinant KCP containing large (entire domain) or small (2-3 amino acid residue) alterations. One antibody recognizing complement control protein (CCP) domain 1 blocked heparin binding, DAA and C4b CFA, but was poor at blocking C3b CFA, while a second antibody recognizing CCP4 blocked C3b CFA and 80% DAA, but not C4b CFA or heparan sulphate binding. Two antibodies recognizing CCP2 and CCP3 were capable of blocking C3b and C4b CFA and heparan sulphate binding, but only one could inhibit DAA. These results show that, while KCP is a multifunctional protein, these activities do not completely overlap and can be isolated through incubation with monoclonal antibodies.

Functional activity of the complement regulator encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with Kaposi's sarcoma and certain B-cell lymphomas. The fourth open reading frame of the KSHV genome encodes a protein (KSHV complement control protein (KCP, previously termed ORF4)) predicted to have complement-regulating activity. Here, we show that soluble KCP strongly enhanced the decay of classical C3-convertase but not the alternative pathway C3-convertase, when compared with the host complement regulators: factor H, C4b-binding protein, and decay-accelerating factor. The equilibrium affinity constant (KD) of KCP for C3b and C4b was determined by surface plasmon resonance analysis to range between 0.47-10 microM and 0.025-6.1 microM, respectively, depending on NaCl concentration and cation presence. Soluble and cell-associated KCP acted as a cofactor for factor I (FI)-mediated cleavage of both C4b and C3b and induced the cleavage products C4d and iC3b, respectively. In the presence of KCP, FI further cleaved iC3b to C3d, which has never been described before as complement receptor 1 only mediates the production of C3dg by FI. KCP would enhance virus pathogenesis through evading complement attack, opsonization, and anaphylaxis but may also aid in targeting KSHV to one of its host reservoirs since C3d is a ligand for complement receptor 2 on B-cells.

Soluble recombinant coxsackievirus and adenovirus receptor abrogates coxsackievirus b3-mediated pancreatitis and myocarditis in mice.

BACKGROUND: Group B coxsackievirus infection can result in organ injury and inflammation. The coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF; CD55) have both been identified as receptors for coxsackievirus B3 (CVB3). We have shown elsewhere that early DAF-Fc treatment attenuates CVB3-induced myocarditis and virus replication. METHODS: CAR was synthesized as a soluble IgG1-Fc fusion protein (CAR-Fc). In vitro, CAR-Fc blocked infection by 2 different strains of CVB3. A/J mice were infected in vivo with CVB3 and were administered CAR-Fc either 3 days before infection, during infection, or 3 days after infection and were compared with mice infected with virus alone and control animals. RESULTS: All CAR-Fc treatment groups had reduced recoverable infectious virus in the heart. CAR-Fc treatment of mice, either preceding or concurrent with CVB3 infection, resulted in complete inhibition of myocardial lesion area, cell death and inflammation, and viral RNA. Early treatment also completely blocked inflammation and cell death in the pancreas, an organ that is normally very sensitive to infection. CONCLUSION: To our knowledge, CAR-Fc is the only protein that has been shown to block coxsackievirus infection of the pancreas. However, regardless of the efficacy of the test protein, target tissue cannot be rescued after day 3 of infection in the A/J mouse model.