Research Group of M. Mach

Virology

Head of Institute:
Prof. Dr. med. Klaus Überla

Protective Immune Response against Cytomegalovirus

Human cytomegalovirus (HCMV) is an important, ubiquitously occurring, human pathogen in immunocompromised hosts.

The virus can cause severe disease in transplant recipients. In large parts of the world HCMV is also the most common viral infection acquired in utero. In the USA and Europe an estimated 0.2-1.2% of all live born infants are infected with HCMV and 5-15% of these babies develop long term sequelae resulting from congenital infection. As a consequence of the importance of congenital HCMV infection for public health, the Institute of Medicine at the National Institutes of Health (NIH), USA, has ranked the development of a HCMV vaccine as a top priority.

Figure 1. MCMV gB is targeted by monoclonal antibodies. (Top) Structural domains of MCMV gB displayed in different colors in analogy to the HCMV gB crystal structure. TM: transmembrane region. Numbers indicate the beginning of the domains. Antigenic regions (AD) corresponding to identified domains on HCMV gB are indicated. (Bottom) neutralizing (nt) and non-neutralizing (nnt) mabs belong to various IgG-subclasses (neutralization was assayed on MEF and concentrations of 50% neutralization of input virus are given in μg/ml). Mabs have target specificities comparable to the human situation as indicated in the 3D ribbon model of monomeric gB (bottom right).

A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective responses that should be induced by the vaccine. HCMV is a complex virus containing numerous antigens within the viral envelope that could be targets for protective antibodies. Glycoprotein B (gB) is an important target for neutralizing antibodies and hence an interesting molecule for intervention strategies such as vaccination or passive immunotherapy. We have used the murine model system of CMV (MCMV) to explore the potential of gB-specific monoclonal antibodies (mabs) in immunotherapy or prophylaxis. The mabs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB (see Figure 1). When these mabs were used in immunodeficient RAG-/- mice to limit an ongoing infection we observed a reduction in viral load both with mabs having potent neutralizing capacity in vitro as well as mabs classified as non-neutralizing (Figure 2). In a therapeutic setting, nt mabs more potently reduced the viral burden compared to nnt mabs. Efficacy was correlated with sustained concentration of virus neutralizing mabs in vivo rather than their nt capacity in vitro. Combinations of nt mabs further augmented the antiviral effect and were as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mabs before infection was also protective and both nt and nnt mabs equally prevented immune-deficient mice from the lethal course of infection. In summary, our data argue that therapeutic application of potently neutralizing mabs against gB represents a strategy to block CMV infection in immunodeficient hosts. When present before infection, both nt and nnt anti-gB mabs were equally protective.

Figure 2. gB-specific antibodies protect from MCMV-infection. (A+B) Viral load of RAG-/- mice after therapy with nt or nnt antibodies or combinations of both. Mice were infected with 105 pfu MCMV157luc and treated with a total of 250 μg of the indicated IgG(s) or 200 μl serum per animal at three days after infection. (A) In vivo imaging was performed at the indicated days post infection (dpi). (B) Viral load in aliquots of organ homogenates containing 30 μg protein was determined 10 days after infection by a luciferase based assay. RLU: relative light units. Statistics: One way ANOVA using Bonferroni´s multiple comparison test *: p<0.05., dotted line: detection limit. (C+D) Protective capacity of anti-gB mabs following prophylactic application. A total of 250 μg IgG(s) per mouse or 200 μl serum/PBS was injected one day before infection with 104 pfu of MCMV157luc. (C) Blood was taken at the indicated dpi and MCMV load determined by qPCR. n = 4 in antibody treated groups and n = 3 in the PBS treated group. Values represent mean (SEM) of all mice within one group and duplicate determinations per sample. MCMV genome copy number is given per 1 μg total DNA. Detection limit: 1 copy/50 ng total DNA. (D) Survival was monitored for 100 days p.i. Statistics: log-rank (Mantel-Cox) test: p <0.0001. Representative data from 2 independent experiments.

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♦ PI Reuter (IZKF, subproject J45) –  Modulation of PRC2 by HCMV IE2

Chromatin-based modifications of herpesviral genomes crucially dictate the outcome of infection. Host cell multiprotein complexes like PML bodies or the Polycomb repressive complex 2 (PRC2) have been identified as regulators of viral gene expression on the epigenetic level. We investigated the role of PRC2 and its related complex PRC1 for HCMV infection and analyzed the mechanisms HCMV has evolved to modulate PRC1/2 function for its own benefit.

Role of PRC1/2 during lytic HCMV replication.

To address the relevance of PRC1/2 activity for the productive life cycle of HCMV, we dissected the levels of PRC1/2 core components following HCMV infection. This revealed that all major PRC1 and 2 factors are massively upregulated upon infection and recruited into viral replication compartments (VRCs; sites of viral DNA amplification) as infection progresses. Interestingly, however, the repressive histone marks instituted by PRC1/2 turned out to be specifically excluded from these sites suggesting a role of both complexes in viral DNA synthesis independent of their repressor activity. Indeed, using primary human foreskin fibroblast (HFF) knockdown cells in which individual PRC1/2 core factors were depleted by expression of respective shRNAs, we could show that PcG proteins are required for an efficient HCMV genome amplification. This is in accordance with recent reports from literature that identified a novel role of PcG factors in regulating the normal progression of cellular DNA replication.

Since PRC1/2 have emerged as promising drug targets in cancer therapy, we were able to test a series of diverse PRC1/2 inhibitory substances (Figure 3, A and B). All of these agents inhibit the enzymatic activity of the targeted repressor complex. Importantly, some substances additionally induce a destabilization of the respective complex, which is known to go along with a downregulation of certain PRC core components. Intriguingly, only substances which negatively affected complex stability like DZNep, Wedelolactone (WDL) or PTC-209 were able to compromise HCMV genome synthesis, while inhibition of PRC1/2’s enzymatic activity alone (UNC1999, GSK126, A395 and PRT4165) had no effect. Taken together, this leads to the overall assumption that regulation of DNA amplification by PRC1/2, which is poorly defined yet, occurs in an enzymatic-independent manner (non-canonical mode of action).

Figure 3. Role of PRC1/2 for lytic HCMV infection. (A) Quantification of viral genome copies following treatment of infected HFFs with the indicated PRC-inhibitors or Ganciclovir (GCV). (B) List of PRC1/2 inhibitors, their characteristics and effect on HCMV DNA replication. (C) Scheme: PRC1/2 are recruited by the HCMV IE2 protein into VRCs for efficient genome amplification. Both PRCs function in a non-canonical manner as they can only be inhibited by compounds that target complex stability.

Regulation of PRC1/2 activity by the HCMV effector protein IE2p86 (IE2)

In former studies, we discovered an interaction between the HCMV transactivator protein IE2 and the PcG protein EED, which is a shared component of both complexes, PRC1 and 2. This suggests that HCMV has the capacity to regulate PRC1/2 activity for its own benefit (Figure 3C). To test this theory, we generated recombinant viruses lacking the EED interaction interface within IE2. Multi-step growth curve analysis revealed a severe growth defect of the EED interaction-deficient IE2 mutants in comparison to wildtype (wt) HCMV. In accordance with our hypothesis, we observed an impaired intracellular accumulation of newly synthesized viral DNA in case of the mutant viruses which resulted from an incomplete relocalization of PcG proteins into VRCs when compared to wt HCMV. In summary, we identified a novel interaction between IE2 and EED, which contributes to the recruitment of PcG proteins into VRCs for efficient HCMV DNA replication.

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