Interaction of Immunoglobulin with Cytomegalovirus-Infected Cells

Abstract

Intravenous immunoglobulin (IVIG) is used to treat or prevent severe viral infection, especially cytomegalovirus (CMV) infections. IVIG was characterized to understand its interaction with CMV-infected cells. IVIG retarded CMV spread and reduced virus yields depending on the neutralizing (NT) antibody titer. Immediate early protein synthesis was reduced by IVIG in 3 to 15 h, and IVIG specifically reduced the ratio of 66/68k protein synthesis among immediate early proteins in an NT antibody-dependent manner between 4 and 8 h after infection, indicating that antigenic modulation of CMV-infected cells by IVIG reduced viral protein synthesis and virus production. The half-life of antibody bound to CMV-infected cells was 3.8 h. NT antibody titers to varicella-zoster virus (VZV) and CMV in IVIG were dose dependently absorbed by cells infected with VZV and CMV, respectively, but the antibody titers to CMV and VZV, respectively, were not affected. NT antibody in 0.3 mL of IVIG (15 mg) was specifically absorbed by 10⁸ CMV-infected cells and 10⁷ VZV-infected cells, suggesting that the NT antibody in IVIG might be inactivated by one-tenth of a similar volume of CMV-infected or VZV-infected cells. Various antiviral activities of IVIG may contribute to control and alleviation of CMV infection.

Keywords: ADCC; antigenic modulation; cytomegalovirus; intravenous immunoglobulin; varicella-zoster virus.

FIG. 1.

Growth of CMV in the presence or absence of antiserum. (A) The final antibody titer used was 1:64 in the NT test, and the spread of CMV infection was assessed by an IFC assay. The solid line indicates the number of CMV infectious centers that grew in the absence of antiserum, and the dotted line indicates that of CMV infectious centers that grew in the presence of antiserum. (B) Effects of NT antibody titer on CMV replication at 72 h and spread of infection were assessed by a plaque assay (solid line) and IFC assay (dotted line), respectively. CMV, cytomegalovirus; IFC, infectious center.

FIG. 2.

Time course of CMV protein synthesis in the presence or absence of antiserum. Viral proteins were labeled with ³⁵S methionine in the presence or absence of antiserum at the indicated time period (h) after infection and immunoprecipitated with IVIG. (A) IVIG (−) indicates that the cells were grown and labeled in the absence of antiserum, and IVIG (+) indicates that the cells were grown and labeled in the presence of IVIG at NT antibody titer of 1:64. (B) Effects of antibody titer of IVIG on the viral protein synthesis in CMV-infected cells 4–8 h after infection. The 66k protein was reduced by the increase in anti-CMV antibody titers, but 68k protein was not influenced.

FIG. 3.

ADCC against CMV-infected cells (A) and VZV-infected cells (B). CMV-infected cells with 133 plaques (n = 6) and VZV-infected cells with 128 plaques (n = 6) were expressed as 100% as the residual infected cells, and the residual infected cells in the various treatments were expressed as the percentage + SD. ADCC assays were performed in the condition at the E:T ratio of 100:1 in both infected cells and no IVIG and its dilutions of 1:25 and 1:100 based on the results in the previous report (49). Common observations in CMV- and VZV infection were no effect of antibody alone and significant reduction of plaques by splenocytes, corresponding to NK cell activity. In contrast, splenocytes with IgG (ADCC) significantly reduced plaques in VZV-infected cells but not in CMV-infected cells, as previously reported (49). * and ** indicate a p-value less than 0.05 and 0.01, respectively. ADCC, antibody-dependent cellular cytotoxicity; IgG, immunoglobulin; VZV, varicella-zoster virus.

FIG. 4.

Fate of IgG bound to CMV-infected cells. Western blot analysis of heavy chain of IgG in the cellular fraction of CMV-infected and -uninfected cells shows the specific binding of IgG to CMV-infected cells. (A) The amount of IgG bound to CMV-infected cells, as quantitated by Fujifilm LAS-4000 imaging system, decreased with time. (B) The half-life of IgG bound to CMV-infected cells was 3.8 h.

FIG. 5.

VZV- or CMV-infected cells and IgG (15 mg) from IVIG were mixed, and the residual NT antibody titer was determined. (A) Addition of CMV-infected cells gradually reduced the NT antibody titer to undetectable levels, but the NT antibody titer to VZV did not change. Photograph shows the amounts of CMV-infected cell pellets used for absorption of IVIG, and the supernatants were used for the NT tests. Thus, CMV-infected cells similarly reduced the NT antibody titer in IgG (IVIG 15 mg) without effects on VZV NT titer, and 9.6 × 10⁷ infected cells absorbed the NT activity of IgG. (B) Addition of VZV-infected cells gradually reduced the NT antibody titer to undetectable levels, but anti-CMV NT antibody titer was not affected. Photograph shows the amounts of VZV-infected cell pellets used for absorption of IVIG, and the supernatants were used for the NT tests. The NT antibody titer of IgG (IVIG 1 mg) to VZV-infected cells was lost at 1.17 × 10⁷ infected cells.

FIG. 6.

Schematic presentation of the difference between ADCC against VZV- and CMV-infected cells. (A) ADCC was exhibited against VZV-infected cells through the bound IgG on the cell surface and with NK cells as the effector. (B) CMV-infected cells express vFcγR on the cell surface. Anti-CMV antibodies recognize viral glycoproteins expressed on the CMV-infected cells, but the vFcγR binds to the Fc portion of the antibody (IgG). Thus, the Fc portion of IgG bound to viral proteins was captured by the vFcγR, and the Fc portion lost the binding to Fc receptors of NK cells. Consequently, CMV-infected cells interfere with the interaction of the Fc portion of IgG with Fc receptors of NK cells, leading to ADCC by the vFcγR. vFcγR, viral Fcγ receptor.