Date Published: May 15, 2014
Publisher: Public Library of Science
Author(s): Eugenia Corrales-Aguilar, Mirko Trilling, Katja Hunold, Manuela Fiedler, Vu Thuy Khanh Le, Henrike Reinhard, Katrin Ehrhardt, Eva Mercé-Maldonado, Enver Aliyev, Albert Zimmermann, David C. Johnson, Hartmut Hengel, Paul Lehner.
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.
Human cytomegalovirus (HCMV) constitutes the prototypical human pathogenic β-herpesvirus found worldwide with high immunoglobulin G (IgG) seroprevalence rates of 50–98% . Despite the expression of a very large antigenic proteome of approximately 750 translational products , HCMV avoids sterile immunity and invariably persists lifelong in the human host in a latent state with periodic phases of reactivation and virus shedding. While infection of immune competent individuals is usually subclinical, HCMV causes severe symptoms in immunocompromised individuals and congenitally infected newborns , . Cytomegalovirus immune control is organized in a hierarchical as well as redundant manner, with crucial roles for natural killer (NK) cells as well as T lymphocytes . HCMV expresses a large set of immune evasion genes that impair recognition of infected cells by CD8+, CD4+ and NK effector cells and thus facilitate virus persistence, spread and superinfection – while cellular immune responses are nevertheless indispensable for CMV immune surveillance. Experimental and clinical evidence suggest that cytomegalovirus can persist for the lifetime by effectively defending itself from both cellular and humoral immunity. In the absence of either viral immune evasion genes or subsets of immune cells, the balance of pathogenesis versus clearance of the virus can be tilted. For example, B cell deficient mice exhibit a much higher susceptibility during recurrent mouse cytomegalovirus (MCMV) infection compared to control mice, reflected by 100–1,000-fold increased titers in the absence of CMV-specific IgG . Moreover, adoptive transfer of memory B cells into naïve Rag−/− mice is sufficient for long term protection from lethal MCMV disease , and passive immunization with immune IgG reduces MCMV-induced pathology in newborn mice . In clinical settings, HCMV-immune IgG preparations are used with varying degrees of success. Human intravenous hyperimmune immunoglobulin against HCMV (e.g. Cytotect) significantly lowers the risk of congenital CMV infection and disease at birth when given to primary HCMV-infected pregnant women . Nevertheless, meta-analyses of clinical studies with solid organ transplant recipients as well as patients undergoing hematopoietic stem cell transplantation document little if any benefit of IgG prophylaxis against HCMV disease –.
Here we identified various members of the human FcγR family, i.e. FcγRI/CD64, FcγRII/CD32A and FcγRIII/CD16A, to be targeted by the HCMV FcγRs gp34 and gp68 which act as antagonists of ligand induced FcγR responses. This ability enables HCMV to evade from IgG effector responses and should have direct proviral effects in scenarios of post-acute and recurrent infection when glycoprotein-specific IgG antibodies are synthesized . Several independent experimental approaches support this conclusion: i) HCMV HB5-derived mutants with deletions of the gp34 and gp68 coding genes, TRL11/IRL11 and UL118-119, respectively, showed significantly increased activation of host FcγRs upon opsonization of infected cells with polyclonal HCMV immune IgG using different types of responder cells (i.e. BW5147 transfectants expressing FcγR-ζ chain chimeras and primary human NK cells); ii) this HCMV phenotype was reproduced with targeted RL11 and UL118-119 mutants of the AD169varL and TB40/E strain and iii) fully reversed by retransfer of the responsible genes into the RL11- and UL118-119-deficient TB40/E genomes; iv) a gain-of-function approach based on ectopic VACV-based expression of gp34 and gp68 which allowed functional analysis of well characterized therapeutic human monoclonal antibodies and different IgG isotypes thereof and v) functional testing of recombinant soluble ectodomains of both HCMV inhibitors using BW5147:FcγR reporter cells as well as primary human NK cells. Importantly, the inhibitory effect of gp34 and gp68 was demonstrated at physiological concentrations of polyclonal HCMV immune IgG, i.e. within an extended concentration range of human serum and ten to fifty fold lower.