Human Cytomegalovirus Escapes a Naturally Occurring Neutralizing Antibody by Incorporating It into Assembling Virions

SummaryHuman cytomegalovirus (CMV) is a common but difficult to treat infection of immunocompromised patients. MSL-109 is a human monoclonal IgG isolated from a CMV seropositive individual that recognizes the viral glycoprotein H (gH) surface antigen complexes that mediate entry. Although MSL-109 blocks CMV infection in vitro, it lacked sufficient efficacy in human trials, and CMV isolated from treated patients suggested the evolution of MSL-109 resistance. To understand how CMV escapes MSL-109, we characterized a MSL-109-resistant CMV strain. Our results elucidate a nongenetic escape mechanism in which the antibody is selectively taken up by infected cells and incorporated into assembling virions in a dose-dependent manner. The resistant virus then utilizes the Fc domain of the incorporated antibody to infect naive nonimmune cells. This resistance mechanism may explain the clinical failure of MSL-109, illustrate a general mechanism of viral antibody escape, and inform antiviral vaccine and therapeutic development

The Glycoprotein B Disintegrin-Like Domain Binds Beta 1 Integrin To Mediate Cytomegalovirus Entry ▿

Cellular integrins were identified as human cytomegalovirus (HCMV) entry receptors and signaling mediators in both fibroblasts and endothelial cells. The goal of these studies was to determine the mechanism by which HCMV binds to cellular integrins to mediate virus entry. HCMV envelope glycoprotein B (gB) has sequence similarity to the integrin-binding disintegrin-like domain found in the ADAM (a disintegrin and metalloprotease) family of proteins. To test the ability of this region to bind to cellular integrins, we generated a recombinant soluble version of the gB disintegrin-like domain (gB-DLD). The gB-DLD protein bound to human fibroblasts in a specific, dose-dependent and saturable manner that required the expression of an intact β1 integrin ectodomain. Furthermore, a physical association between gB-DLD and β1 integrin was demonstrated through in vitro pull-down assays. The function of this interaction was shown by the ability of cell-bound gB-DLD to efficiently block HCMV entry and the infectivity of multiple in vivo target cells. Additionally, rabbit polyclonal antibodies raised against gB-DLD neutralized HCMV infection. Mimicry of the ADAM family disintegrin-like domain by HCMV gB represents a novel mechanism for integrin engagement by a virus and reveals a unique therapeutic target for HCMV neutralization. The strong conservation of the DLD across beta- and gammaherpesviruses suggests that integrin recognition and utilization may be a more broadly conserved feature throughout the Herpesviridae

Differential Initiation of Innate Immune Responses Induced by Human Cytomegalovirus Entry into Fibroblast Cells

Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway (alphabeta IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-kappaB activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with beta(1) integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection

From Target Selection to the Minimum Acceptable Biological Effect Level for Human Study: Use of Mechanism-based PK/PD Modeling to Design Safe and Efficacious Biologics

In this paper, two applications of mechanism-based modeling are presented with their utility from candidate selection to first-in-human dosage selection. The first example is for a monoclonal antibody against a cytomegalovirus glycoprotein complex, which involves an antibody binding model and a viral load model. The model was used as part of a feasibility analysis prior to antibody generation, setting the specifications for the affinity needed to achieve a desired level of clinical efficacy. The second example is a pharmacokinetic–pharmacodynamic model based on a single-dose pharmacology study in cynomolgus monkey using data on pharmacokinetics, receptor occupancy, and the dynamics of target cell depletion and recovery. The model was used to estimate the MABEL, here defined as the minimum acceptable biological effect level against which a dose is selected for a first-in-human study. From these applications, we demonstrate that mechanism-based PK/PD binding models are useful for predicting human response to biologics compounds. Especially, such models have the ability to integrate preclinical and clinical, in vitro and in vivo information and facilitate rational decision making during various stages of drug discovery and translational research