Monoclonal antibodies targeting nonstructural viral antigens can activate ADCC against human cytomegalovirus
Virginia-Maria Vlahava, Isa Murrell, Lihui Zhuang, Rebecca J. Aicheler, Eleanor Lim, Kelly L. Miners, Kristin Ladell, Nicolás M. Suárez, David A. Price, Andrew J. Davison, Gavin W.G. Wilkinson, Mark R. Wills, Michael P. Weekes, Eddie C.Y. Wang, Richard J. Stanton
Virginia-Maria Vlahava, Isa Murrell, Lihui Zhuang, Rebecca J. Aicheler, Eleanor Lim, Kelly L. Miners, Kristin Ladell, Nicolás M. Suárez, David A. Price, Andrew J. Davison, Gavin W.G. Wilkinson, Mark R. Wills, Michael P. Weekes, Eddie C.Y. Wang, Richard J. Stanton
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Research Article Immunology Virology

Monoclonal antibodies targeting nonstructural viral antigens can activate ADCC against human cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe disease following congenital infection and in immunocompromised individuals. No vaccines are licensed, and there are limited treatment options. We now show that the addition of anti-HCMV antibodies (Abs) can activate NK cells prior to the production of new virions, through Ab-dependent cellular cytotoxicity (ADCC), overcoming viral immune evasins. Quantitative proteomics defined the most abundant HCMV proteins on the cell surface, and we screened these targets to identify the viral antigens responsible for activating ADCC. Surprisingly, these were not structural glycoproteins; instead, the immune evasins US28, RL11, UL5, UL141, and UL16 each individually primed ADCC. We isolated human monoclonal Abs (mAbs) specific for UL16 or UL141 from a seropositive donor and optimized them for ADCC. Cloned Abs targeting a single antigen (UL141) were sufficient to mediate ADCC against HCMV-infected cells, even at low concentrations. Collectively, these findings validated an unbiased methodological approach to the identification of immunodominant viral antigens, providing a pathway toward an immunotherapeutic strategy against HCMV and potentially other pathogens.

Authors

Virginia-Maria Vlahava, Isa Murrell, Lihui Zhuang, Rebecca J. Aicheler, Eleanor Lim, Kelly L. Miners, Kristin Ladell, Nicolás M. Suárez, David A. Price, Andrew J. Davison, Gavin W.G. Wilkinson, Mark R. Wills, Michael P. Weekes, Eddie C.Y. Wang, Richard J. Stanton

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Figure 7

Anti-UL141–optimized Abs mediate efficient killing of HCMV-infected cells.

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Anti-UL141–optimized Abs mediate efficient killing of HCMV-infected cell...
(A–C) 51Cr release into the supernatant was used as a measure of the ability of NK cells to kill target cells. Targets were mixed with ex vivo–purified NK cells as effectors at a an E/T ratio of 20:1, and 51Cr release was measured 4 hours later. Seronegative IgG (50 μg/mL), Cytotect (50 μg/mL), or a mixture of 5 Fc-engineered (modified) UL141-specific mAbs were included as indicated. Targets were HF-CARs infected with RAd vectors expressing UL141 (RAd-UL141), or lacking a transgene (RAd Ctrl) (A); HFFF mock infected or infected with wild-type HCMV (HCMV) or HCMV lacking the viral FcRs (ΔFc) (B); or ARPE19 mock infected or infected with wild-type HCMV (C). For ARPE19 infection, cells were infected by coculturing with purified fibroblasts for 24 hours and then sorted to purity. All experiments were performed 48 hours after infection. (D and E) HCMV expressing mCherry linked to an immediate early gene (UL36), and EGFP linked to a late gene (UL32) were used to infect SFs at a low MOI. Autologous NK cells were then added alone or together with a control mAb or the mixture of 5 modified anti-UL141 mAbs (each at 1 μg/mL). Eight to 10 days later, the percentage of infected cells demonstrating expression of immediate early (D) or late (E) viral proteins were measured by flow cytometry for mCherry or EGFP, respectively, and normalized to the percentage of infected cells in the absence of NK cells. Results are representative of at least 2 experiments. Data are shown as the mean ± SD of triplicate samples. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA.