Research

With a focus on reoviruses, adenoviruses, and herpesviruses, the following synergistic research lines build on Rob Hoeben’s expertise in virus-based strategies for cancer treatment and Maaike Ressing’s expertise in antiviral immunity. 

Biological questions that we address include: How does a cell detect viral infection? Once alerted, what cellular mechanisms are used to block viral infection? Are different mechanisms employed by different cell types? How do viruses evade or modulate innate sensing to permit replication or persistence? Which differences between normal cells and cancer cells are exploited by oncolytic viruses? We study Epstein-Barr virus, reovirus, and adenovirus with respect to virus-host cell interactions, the role of innate immunity and immune evasion, and oncolysis. 

Anti-viral immunity 

Our research aims at understanding how viruses achieve replication and persistence in immunocompetent individuals, including how they are detected by and modulate responses of the infected host. Thorough knowledge of the viral mechanisms used and the cellular processes affected should guide the development of strategies to manipulate these to improve human health. 

Viruses depend on host cells for their replication. To achieve successful infection, viruses exploit normal cellular processes and modulate anti-viral responses to their own benefit. Using state-of-the-art microscopy, we aim at elucidating the cytosolic transport process of viral particles from endosomal escape to nuclear import.

Cells of the human body have the capacity to inhibit viral infection by a repertoire of defense mechanisms. The innate immune response constitutes a host’s first line of defense. Activation of innate immunity occurs upon recognition of molecular patterns associated with pathogens by specific receptors. This results in the production of type I interferons and proinflammatory cytokines. Innate responses can exert or induce direct antiviral effects, for instance blocking viral replication. In addition, innate immunity plays a pivotal role in initiating tailored adaptive immune responses. Since it is important to raise an immune response of relevant strength and quality, we study what factors influence the quality and timing of TLR response induction in another project by implementing advanced microscopy and chemical immunology.

To effectively interfere with virus-specific immunity, evasion genes have been incorporated into the genomes of multiple viruses, and exemplary in this context are the herpesviruses. Both innate and adaptive arms of the immune system are indispensable to combat virus infection and, therefore, their constituents form ideal targets for modulation by viruses. Earlier, we found that the human gamma-herpesvirus Epstein-Barr virus (EBV) targets multiple steps along the pathways leading to the presentation of virus-derived peptides on HLA class I and class II molecules, by one or more viral gene products. As a result, HLA-restricted antigen presentation is reduced and, therefore, virus-infected cells can escape the recognition by and the effector functions of anti-viral T cells. 

Currently, we focus more on elucidating how EBV modulates innate immune responses, particularly those involving Toll-like receptors and cytosolic nucleic acid sensors. As an example, we found an EBV deubiquitinase (DUB) to block TLR signaling. More recently, we reported that human B cells have a dysfunctional cGAS-STING pathway for cytosolic DNA sensing, which would provide a niche for undetected EBV persistence. In a broader context, we investigate how DNA viruses - such as EBV and adenoviruses - and RNA viruses - in particular reoviruses - modulate cytosolic nucleic acid sensing to permit transit to the nucleus or local adaptations, respectively, to enable undetected genome replication. Thus, this line of research is dedicated to unraveling the fascinating strategies acquired by viruses to withstand anti-viral host responses.

Primary infection by persistent viruses, such as members of the herpesvirus family including EBV, leads to the induction of virus-specific T cells that keep these viruses under control. The effectiveness of such T cells becomes apparent from the substantial morbidity and mortality related to persistent viruses in immunosuppressed patients, for example undergoing transplantation or suffering from AIDS. Virus-specific T cells detect infected cells by virtue of surface display of viral protein fragments in the context of HLA molecules. Wouldn’t it be great to additionally redirect these activated virus-specific T cells to (virus-negative) cancer cells to exert cytolytic activity? In a collaborative research project, we explore this possibility of tumor targeting via specific antibodies. 


Virus-based strategies for cancer treatment 

It has been a long-standing observation that particular viruses have an intrinsic preference for lytic replication in tumor cells, while normal cells are usually left unaffected. One of the viruses for which this preference was noted is the mammalian reovirus. This propensity of reoviruses has facilitated the development of the wildtype reovirus T3D as an oncolytic agent for clinical use. Studies by others demonstrated that the use of reovirus T3D as oncolytic agent is safe with clear indications of antitumor efficacy. We embarked on studying these reoviruses. This was triggered in part by the wide host-range of the human reoviruses, which allows the reovirus to replicate efficiently in mouse cells. This facilitates studying the impact of reovirus replication in tumors in immune competent models. This should provide proper assessment of antitumor efficacy and the impact of antiviral immune responses on antitumor effects. 

Our efforts concentrated on the use of selection and genetic modification strategies to improve the reoviruses for their intended use as anticancer agents. To this end, we validated the relation between Ras-signaling and reovirus infection. In addition, we demonstrated that some tumors acquired resistance to reovirus infection by down-regulating the canonical reovirus receptor JAM-A. To remedy this, we started the selection of reovirus-mutants that can infect cells independent of JAM-A expression. These mutant viruses (designated as jin mutants) are capable of infecting JAM-A-negative tumor cells, while these cells fully resist the wild-type reovirus. We demonstrated that these viruses can use sialic acids as a their high-affinity receptor. These sialic acids are present on almost all tumor cells. These jin mutants also form the cornerstone for the creation of reoviruses that harbor a small foreign transgene for enhancing anti-tumor efficacy. In this way, we generated reoviruses that carry the GM-CSF gene to stimulate the immune responses against the virus and the tumor cells. Also we incorporated the adenovirus E4orf4 gene in an effort to increase the cytolytic potential of these viruses.

We evaluate the biological effects of our viruses in state-of-the-art models in close collaboration with more clinically oriented research groups. These studies focus on the evaluation of our viruses in preclinical models of pancreatic cancer, glioblastoma, prostate, and bladder cancer. In these studies, we try to answer key questions that relate to the immune responses against the viruses and the tumor cells, to the impact of pre-existing antibodies against the virus on anti-tumor efficacy, as well as to the identification of markers that may predict a patient’s response to oncolytic virus therapy. In addition, we intend to contribute to studies that compare the antitumor efficacy of different oncolytic viruses or oncolytic virus variants with the long-term prospect of matching the patient to a oncolytic virus. 

In conclusion, our research is dedicated to using insights in virus-host interactions for designing new treatment opportunities that can be clinically employed.

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