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

Abstract

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.

Figure 1

Binding of monoclonal anti-Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) antibodies to wild-type (full) KCP (a), or recombinant cell surface glycophosphoinositol (GPI)-anchored forms lacking complement control protein 1 (CCP1) (b: GPI-CCP234), CCP4 (c: GPI-CCP123), or CCP3 and CCP4 (d: GPI-CCP12) as determined by flow cytometry analysis. Average mean cellular fluorescence values are shown following incubation of cells with increasing concentrations of each antibody (the standard deviations for samples measured in triplicate are also shown).

Figure 2

Binding of monoclonal anti-Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) antibodies E7 (a), F8 (b), H10 (c) and J11 (d) to recombinant soluble KCP-Fc immobilized to 96-well plates, as detected by enzyme-linked immunosorbent assay (ELISA) methods. Plates were coated with recombinant KCP containing all four CCP domains (KCP) or lacking the first CCP domain (Del1), or with recombinant forms in which KCP CCP domain 2, 3 or 4 was replaced (swap2, etc.) with an irrelevant matched CCP domain from CD21. Bound monoclonal antibody was detected by peroxidase-conjugated specific secondary antibody activity (420 nm), following incubation of coated plates with increasing concentrations of each monoclonal antibody. Each point was determined in duplicate, and the experiment was replicated on at least three separate occasions.

Figure 3

Inhibition of Kaposi's sarcoma-associated herpesvirus complement control protein (KCP)-Fc binding to immobilized heparan sulphate following preincubation with monoclonal anti-KCP antibodies. One representative experiment is shown; each point was determined in triplicate. Binding of wild-type KCP-Fc to heparan sulphate was detected with a peroxidase-conjugated secondary antibody specific for the human Immunoglobulin G1 (IgG1) portion of KCP-Fc, and results are shown as the extent of binding relative to KCP-Fc binding in the absence of preincubation with monoclonal antibodies. The antibody:KCP ratio represents the ratio of antibody relative to each molecule of KCP, taking into account that KCP-Fc is dimeric (i.e. the 1 : 1 ratio is two molecules of monoclonal antibody for each dimeric KCP-Fc molecule).

Figure 4

Inhibition of classical C3 convertase decay acceleration by preincubation of wild-type Kaposi's sarcoma-associated herpesvirus complement control protein (KCP)-Fc with monoclonal antibodies. Results of inhibition are shown as the relative decrease in the ability of KCP-Fc to protect target cells from lysis [i.e. KCP inhibition in the absence of antibody = 0%, and complete loss of KCP-mediated decay-accelerating activity (DAA) = 100%, resulting in maximal lysis of target cells], when KCP-Fc was preincubated with increasing molar ratios of monoclonal antibodies (1 : 10 to 10 : 1). Monoclonal antibodies included B6 (grey triangle), J11 (grey square), H10 (black circle), F8 (black triangle), and E7 (black square). All assays were performed in triplicate and the analysis was repeated at least twice. Error bars represent standard deviation.

Figure 5

Representative autoradiographs showing generation of (a) C3b cleavage products (46 and 43 kDa) or (b) C4b cleavage product (C4d: 46 kDa), in the absence of factor I (no FI), in the presence of Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) and FI (KCP + FI), or in the presence of FI and KCP following preincubation with a 10-fold molar excess of anti-KCP monoclonal antibody (B6, E7, F8, H10 or J11). (c) Summary of inhibition of KCP C3b or C4b cofactor activity (CFA) as determined by relative decrease of iC3b (46 kDa species) or C4d autoradiograph band (determined by densitometry) following preincubation of each monoclonal antibody (i.e. KCP inhibition in the absence of antibody = 0%, and complete loss of KCP-mediated CFA = 100%, resulting in no production of degradation bands). The graph represents the average decrease from all performed experiments (repeated at least three times) for a 10-fold molar excess of antibody compared with KCP-Fc, and standard deviation.

Figure 6

Schematic summary of the mapped epitopes for antibody binding relative to functional regions of Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) (including functional regions previously identified by domain deletion and site-directed mutagenesis18,26,45). The N-terminal part of KCP containing the four complement control protein (CCP) domains (numbering to the right) is shown, and the dashed line at the bottom of the figure indicates the alternatively spliced serine/threonine-rich region leading to the conserved transmembrane region. Binding sites for the monoclonal antibodies are shown to the left (B6 not shown but is suspected to bind a partially hidden epitope in CCP3). CCP domains required for heparin, C3b and C4b binding, and C3b and C4b cofactor activity (CFA), as well as classical C3 convertase decay-accelerating activity (DAA), are shown to the right. Predicted N-glycosylation sites on the CCP domains are also indicated (octagons).