Human Cytomegalovirus Fcγ Binding Proteins gp34 and gp68 Antagonize Fcγ Receptors I, II and III

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.DFG grant He 2526/6-2.European Commission grants QLRT-2001-01112 and MRTN-CT-2005-019248.Helmholtz Association through VISTRIE VH-VI-242.UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

A novel assay for detecting virus-specific antibodies triggering activation of Fcγ receptors

IgG responses are crucial in antiviral defence and instrumental for the serodiagnosis of infections. Fcγ receptors (FcγRs), which recognize the Fc-part of IgG, differ regarding their IgG binding affinity, IgG subclass preference, cellular expression profile and pathogen elimination mechanisms elicited upon activation. Assessing their activation in vitro is of fundamental importance, but technically difficult. Therefore, a novel assay for measuring antiviral IgG antibodies triggering activation of individual host Fcγ receptors was established. The assay comprises the co-cultivation of virus-infected target cells with immune IgG antibodies and mouse BW5147 hybridoma cells stably expressing chimeric FcγR–CD3ζ chain molecules consisting of the extracellular domain of human FcγRIIIA, FcγRIIA or FcγRI, respectively, fused to the transmembrane and intracellular domains of the mouse CD3ζ chain. Triggering of the chimeric FcγR receptors by immune complexes formed on the surface of IgG-opsonized virus-infected target cells resulted in FcγR activation leading to IL-2 secretion by BW5147 cells, which was quantified as a surrogate marker in an ELISA. Target cells infected with various human pathogenic viruses including herpes simplex virus type 1 (HSV-1), Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), measles virus (MV), and respiratory syncytial virus (RSV) or displaying human immunodeficiency virus-1 (HIV-1) gp120 evoke dose-dependent IgG responses demonstrating the universal applicability of the assay. Taken together, a new reliable and simple tool for measuring antibodies triggering activation of Fcγ receptors was established. This assay will be instrumental for defining novel correlates of IgG immunity and the design of new therapeutic IgGs.UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

HCMV gp68 and gp34 inhibit FcγRIII activation by rituximab antibody isotypes.

<p>CD20 transfected 293T cells were infected for 16/cell of VACV wt or rVACV expressing gp68, gp34 or MULT-1 as a control. After opsonization with 1 µg/ml of each antibody isotype for 30 min. and removing of unbound antibody by washing, cells were co-cultivated with 1×10⁵ BW:FcγRIIIA-ζ reporter cells per well for 16 h before supernatants were collected and mIL-2 was determined by ELISA. Each value represents three replicates; means with standard deviations (error bars) are shown for 2 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

The presence of the vFcγRs on the surface of infected cells inhibits antibody dependent NK cell degranulation.

<p>(<b>A</b>) MRC-5 fibroblasts were infected with HSV-1F wt, ΔgE and ΔgE revertant for 24 h before cells were opsonized with HSV IgG positive and negative human sera. After 30 min of incubation, unbound antibodies were washed away and NK cells at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells was measured in FACS. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a-positive cells with the immune antibody opsonization - % of CD107a-positive cells with non-immune antibody). (<b>B</b>) MRC-5 fibroblasts were infected with HCMV HB5 wt, HB5Δgp68, HB5ΔIRLΔgp34 and HB5ΔIRLΔgp68/Δgp34 for 72 h before cells were opsonized with HCMV IgG positive and negative human sera. After 30 min of incubation, unbound antibodies were washed away and NK cells at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells was measured in FACS. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a-positive cells with the immune antibody opsonization - % of CD107a-positive cells with non-immune antibody) (<b>C</b>) MRC-5 fibroblasts were infected with HCMV HB5 wt and HB5ΔIRLΔgp68/Δgp34 for 72 h before opsonized with Cytotect or human HCMV-IgG negative sera at 1∶10 and 1∶100 dilutions. After 30 min of incubation, unbound antibodies were washed away and NK cells from six different donors at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells against HCMV-infected cells was measured. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a positive cells with the immune antibody opsonization - % of CD107a positive cells with non-immune antibody). Single measurements of CD107a cells are shown. One of three (A, B) or two (C) representative experiments is shown.</p

Soluble ectodomains of HCMV vFcγRs interfere with FcγR activation.

<p>(<b>A</b>) SKOV-3 cells were opsonized with 100 ng/ml trastuzumab for 30 minutes and washed three times with D-MEM 10% FCS (vol/vol) before soluble proteins were added in graded concentrations concomitantly with BW:FcγRIIIA-ζ transfectants. mIL-2 was determined in supernatants (which were harvested after 16 h of co-cultivation of responder cells with target cells) by ELISA. (<b>B</b>) As in (A) but SKOV-3 cells were opsonized with 500 µg/ml trastuzumab for 30 minutes and washed three times with D-MEM 10% FCS (vol/vol) before soluble proteins were added in graded concentrations concomitantly with BW:FcγRI-ζ cells. (<b>C</b>) MRC-5 cells were infected with HCMV HB5ΔIRLΔgp68/Δgp34 (2 PFU/cell) for 72 h before soluble proteins were added in graded concentrations concomitantly with BW:FcγRIIIA-ζ responder cells. MRC-5 fibroblasts were opsonized with 1∶50 diluted Cytotect for 30 min. After removing of unbound antibodies by washing, soluble proteins and BW:FcγR-ζ transfectants were added and co-cultivated overnight. (<b>D</b>) as in (C), but HCMV HB5ΔIRLΔgp68/Δgp34 infected target cells were opsonized with 1∶10 diluted Cytotect and BW:FcγRIIA-ζ cells were used as responders. n = 3 replicates, means with standard deviations (error bars) are shown for 2 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

Viral FcγRs bind Fcγ on the cell surface and opsonizing IgG dependent FcγR activation is restricted to the late phase of HCMV but not HSV replication.

<p>(<b>A</b>) MRC-5 cells were infected with 2 PFU/cell for 72 hpi with HCMV HB5 wildtype, HB5Δgp68, HB5ΔIRLΔgp34 or HB5ΔIRLΔgp68/Δgp34 (left) or 24 hpi with HSV-1 (right). Cells were resuspended with PBS/2% (vol/vol) FCS containing 2 mM EDTA, stained with hFcγ-FITC and measured in a FACS Canto II. Dead cells were excluded by PI or DAPI-staining. (<b>B</b>) MRC-5 cells were infected with 2–3 PFU/cell of HCMV HB5 wt or (<b>C</b>) HSV-1 strain F. After centrifugation enhancement of infection, cells were incubated 3 h at 37°C at 5% CO₂. After washing once, PAA (250 µg/ml) in D-MEM 10% (vol/vol) FCS was added. 48 hpi for HSV or 72 hpi for HCMV, cells were opsonized with grading dilutions of Cytotect for 30 minutes, washed twice with D-MEM 10% (vol/vol) FCS and co-cultivated with 1×10⁵ BW:FcγR-ζ reporter cells per well. Measurement of mIL-2 in supernatants after 16 h of co-cultivation of reporter cells with targets was performed by ELISA. Values are presented in the graphic as OD 450 nm. 3 independent wells were measured, means are shown with standard deviations (error bars) for 2 independent experiments. Significance of results (Student's t-test) is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

Ectopic expression of HSV-1 gE, HCMV gp68 and HCMV gp34 inhibit IgG1 (trastuzumab) mediated activation of FcγRs.

<p>(<b>A</b>) SKOV-3 cells were infected for 24 h with 2 PFU/cell VACV wt and rVACV expressing gE or (<b>B</b>) rVACV expressing gp68 or gp34 before opsonized with trastuzumab at different concentrations for 30 min. After removing of unbound antibodies by repeated washing with D-MEM 10% (vol/vol) FCS, 1×10⁵ BW:FcγR-ζ transfectants per well were added and co-cultivated overnight. BW:FcγR-ζ activation was determined by measuring mIL-2 by ELISA. Three independent replicates were measured, means with standard deviations (error bars) are shown for 3 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

Soluble ectodomains of HCMV vFcγR interfere with antibody dependent NK cell degranulation.

<p>(<b>A</b>) Cytotect was coated to a plate in binding buffer (0.1 M Na₂HPO₄ pH 9.0) at a concentration of 0.5 mg/ml and incubated for 2.5 hours at 37°C. After blocking for 30 minutes and washing unbound antibodies, soluble proteins, rIL-2 pre-activated primary NK cells and α-CD107a-PECy5 antibody were added and incubated for 4 hours at 37°C. Duplicates were measured for CD107a surface expression after dead cell exclusion with DAPI staining in a FACS Canto II. Means are shown with standard deviations (error bars). Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001. (<b>B</b>) To compare the amounts of soluble proteins used in (A), SDS-PAGE and anti-V5 immunobotting was performed.</p

HSV-1 gE, HCMV gp68 and HCMV gp34 interfere with host FcγR activation upon opsonization of cells with polyclonal immune IgG.

<p>(<b>A</b>) The HSV vFcγR gE inhibits FcγRIIIA and FcγRIIA activation but fails to inhibit FcγRI. Human MRC-5 fibroblasts were infected with 2 PFU/cell of HSV-1 strain F wt and ΔgE for 24 h. Cells were opsonized with Cytotect at different concentrations for 30 min. After removing of unbound antibodies with D-MEM 10% (vol/vol) FCS, 1×10⁵ BW:FcγR-ζ transfectants per well were added and co-cultivated overnight. BW:FcγR activation was determined by measuring mIL-2 by ELISA. Three independent replicates were measured; means with standard deviations (error bars) are shown for 4 independent experiments. (<b>B</b>) HCMV vFcγR gp68 interferes with FcγRIIIA, FcγRIIA and FcγRI activation. MRC-5 cells were infected with HCMV HB5 wt virus or HB5Δgp68 (2 PFU/cell) for 72 h. Fibroblasts were opsonized with Cytotect at different concentrations for 30 min. After removing of unbound antibodies by washing, 1×10⁵ BW:FcγR-ζ transfectants were added per well. Measurement of mIL-2 in supernatants after 16 h of co-cultivation of reporter cells with targets was performed by ELISA. Values are presented in the graphic as OD 450 nm. Three independent wells were measured; means with standard deviations (error bars) are shown for 4 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001. (<b>C</b>) HCMV vFcγR gp34 interferes with FcγRIIIA, FcγRIIA and FcγRI activation. As in (B) but MRC-5 cells were infected with HCMV HB5ΔIRL, HB5ΔIRLΔgp34 or HB5ΔIRLΔgp68/Δgp34 (2 PFU/cell) for 72 h. (<b>D</b>) gp34 and gp68 interfere with FcγR activation in AD169varL infected cells. As in (B) but MRC-5 cells were infected with AD169varL wt, AD169varLΔgp68, AD169varLΔgp34 or AD169varLΔgp68/Δgp34.</p

The HCMV vFcγRs gp68 and gp34 bind antigen-IgG complexes.

<p>(<b>A</b>) Schematic representation of the ‘antibody bipolar bridging’ model. (<b>B</b>) Lysates of SKOV-3 cells containing the heterocomplex of vFcγR-FLAG, antibody and antigen are immunoprecipitated using an anti-FLAG agarose. SKOV-3 cells expressing HER2 antigen on the surface were infected with rVACV expressing the vFcγRs before opsonized with the trastuzumab antibody (bipolar bridging-antibody) (T) or an isotype control IgG1 antibody, palivizumab (P). Lysates were prepared after incubation of infected cells with antibody. An anti-Flag agarose IP was performed and retrieved antigens were detected in western blot with anti-ErbB2-specific mAb recognizing human HER2, anti-human IgG, and anti-Flag (M2, Sigma-Aldrich) detecting the Flag-tagged vFcγRs. Equal expression of HER2 in cell lysates was verified by western blot analysis with an anti-ErbB2-specific rabbit mAb which detects human HER2 (bottom).</p