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TRR 60:  Interaktion von Viren mit Zellen des Immunsystems bei persistierenden Virusinfektionen: Grundlagen für Immuntherapie und Impfungen

Fachliche Zuordnung Medizin
Förderung Förderung von 2009 bis 2018
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 47100475
 
Erstellungsjahr 2018

Zusammenfassung der Projektergebnisse

1.1. Summary In the last 8 years the TRR60 studied the interaction of the immune system with several different viruses that establish chronic infections in their hosts. These studies provided new concepts of immune regulation and dysregulation in chronic virus infections that can now be utilized for therapeutic approaches and vaccine development. The focus was on HIV, HCV, and HBV because therapeutic cure was rarely achieved for these chronic infections. Novel immunotherapy options, especially combination therapies, were tested in preclinical models of infectious diseases. Some of the possible new targets for immunotherapy were first described to play a role in infectious diseases by PIs of the TRR60. In order to test new tools for immunotherapy against chronic infections sophisticated animal models are needed. It has therefore always been a focus of the TRR60 to develop and improve animal models for chronic virus infections. Basic findings on immune regulation from animal models also need to be confirmed in patient studies, which is, why we analysed patient samples from very defined large cohorts of HIV-, HCV, or HBV-infected individuals in almost all TRR60 projects. The overall aim of the TRR60 project was to develop novel immunotherapies or combination therapies to achieve improved control of chronic viral infections. The treatment for chronic HIV, HTLV-I, HBV or HBV/HDV infection is still far from being optimal and cure is only achieved in rare cases of HBV therapy. It is widely believed that components of the immune system have to be involved to develop more potent therapies against these viruses. We therefore studied basic cellular and molecular mechanisms of immunity and immune escape in retrovirus, HBV and HCV infections in the first two funding periods to define targets for novel approaches of immunotherapy. T cells are very important effector cells for the control of viral infections, but they become dysfunctional when a chronic infection develops. We therefore studied effector T cell responses in many projects of the TRR60. CD8+ T cells, but also CD4+ T cells, can control retroviral or HBV infection by cytotoxic killing of virus-infected cells or the release of anti-viral cytokines, like IFNγ or TNFα [Kefalakes et al., 2015a; Johnson et al. 2015; Joedicke et al., 2014a; Zelinskyy et al., 2011; Manzke et al., 2013]. However, a hallmark of chronic viral infections is T cell immune escape or T cell exhaustion [Akmetzyanova et al., 2015; Kefalakes et al., 2015b; Zelinskyy et al; 2009; Dietze et al., 2011]. Thus, the anti-viral activities of T cells have to be reactivated during chronic infections to achieve better control or even elimination of persistent virus. The role of B cells and antibodies in chronic infections is less well characterized, but especially the B cell repertoire [Budeus et al., 2015; Saeedghalati et. al., 2017] and the contribution of follicular helper cells to antibody responses [Schultz et al., 2016] were investigated in TRR60 projects. Figure 1: Mechanisms of Treg expansion in chronic retroviral infection - 1 - A – Academic Section Since a very potent therapy against chronic HCV infection with DAAs has been developed in the last few years, we will focus our future efforts on testing new treatment options against HIV and HBV (+HDV) infections. Our previous work defined many new targets and tools for the immunotherapy of chronic viral infections. Cellular targets are immune cell populations that suppress effector T cell responses and contribute to T cell exhaustion during chronic virus infection. Our work focused in several projects on the role of Tregs for the establishment of viral chronicity. In both, retrovirus [Zelinskyy et al., 2009; Dietze et al., 2011; Zelinskyy et al., 2013; Joedicke et al., 2016] and HBV infection [Dietze et al., 2016; Kosinska et al., 2017] Tregs expand in the infected organs and suppress effector CD8+ as well as CD4+ T cell [Manzke et al.; 2013; Nair et al., 2010] and NK cell responses [Littwitz-Salomon et.al., 2015]. Interestingly, while HBV infection only redistributes natural Tregs to the liver, retrovirus infection results in activation and proliferation of the Tregs in infected organs [Dietze et al.,2016; Myers et al., 2013; Joedicke et al., 2014b]. We defined several molecules and receptors that are involved in the expansion of Tregs (CD25, TNFR2, CD74, HBsAg, endogenous retrovirus antigen;[Myers et al., 2013; Joedicke et al., 2014c], which can now be targeted with new immunotherapeutic approaches. Lately, we also defined MDSCs as cell population that responds to HBV or retroviral infections and suppresses effector T cell activity [Fang et al.,2015]. These cells can be manipulated with small molecules, which provides a new opportunity for the immunotherapy of chronic viral infections. Furthermore, effector cell functions are directly regulated by several receptors on T cells. These include inhibitory receptors, like PD-1, Tim-3 or Lag3, or co-stimulatory receptors, like TLR-2, TLR-3, NOD1, CD134/CD137 or CEACAM. Figure 2: Inhibitory ligands contribute to initial virus immune escape and subsequent T cell dysfunction The role of these molecules has been intensively studied in the TRR60 models, showing that they also play key roles in T and B cell function or dysfunction during chronic virus infections [Akhmetzyanova et al., 2015; Zhang et al., 2011; Zelinskyy et al., 2011,Wang et al., 2011a; Bengsch et al., 2010; Akhmetzyanova et al., 2016; Khairnar et al., 2015; Shaabani et al., 2016a; Zhang et al., 2012; Wu et al., 2014]. Since these receptors can be either blocked (inhibitory receptors) or activated by agonists (co-stimulatory receptors) they are currently prime targets for immunotherapies. - 2 - A – Academic Section Despite their direct effects on T cells immunomodulatory molecules can also influence the environment of T cells, which subsequently effects T cell activity and function [Liu et al., 2013]. Interestingly, several studies of the TRR60 suggested that combination therapy targeting several different immune checkpoints at the same time might be the most promising therapeutic approach against chronic infections [Dietze et al., 2013], maybe even in combination with DAA or therapeutic vaccines [Knuschke et al., 2016; Liu et al., 2014; Kosinska et al., 2013]. Thus, several TRR60 projects developed novel vaccine candidates that were tested as prophylactic and/or therapeutic vaccines. The most successful approach was a nanoparticle vaccine that contained T cell epitope antigens and TLR ligands in a biodegradable calcium phosphate shell [Knuschke et al., 2014a, Knuschke et al., 2014b]. This vaccine was very effective against chronic retroviral infection in a therapeutic immunization regiment (Figure 3). Figure. 3: Functionalized nanoparticle vaccines induce potent immunity in chronically retrovirus-infected mice A similar vaccine containing B cell epitopes could also induce potent antibody responses against HIV [Zilker et al., 2017]. In a hepatitis virus infection model of woodchucks a combination therapy with DAA, PD-1 blocking antibodies and a DNA vaccine was most effective and even cured some of the treated animals [Liu et al., 2014]. Enforced virus replication (Figure 4) was characterized as new immunological concept to initiate immune responses in viral infections and its role in augmenting vaccine responses in chronic viral infections was described [Honke et al., 2011; Shaabani et al., 2016b; Duhan et al., 2016]. Several new targets and tools to augment enforced virus replication were developed [Honke et al., 2013; Khairnar et al., 2015; Xu et al., 2015]. In addition, the concept of intrastructural help was defined and utilized to improve antibody responses against retroviral infections [Storcksdieck genannt Bonsmann et al., 2016; Storcksdieck genannt Bonsmann et al., 2015; Nabi et al., 2013]. Type I IFNs (Figure 5) might have the advantage that they stimulate the innate and adaptive immune system, but also induce directly acting antiviral enzymes. So they might combine effects that can otherwise only be achieved with DAA plus checkpoint blockers. IFNα2 is being used in current HBV therapy, but the success rate is not very convincing. In addition, it does not seem to be effective at all against HIV infection. The problem is that only one IFNα subtype (IFNα2) is used to treat patients in the clinic, but the IFNα gene family consists of 13 different subtypes. We have tested the antiviral activity of all subtypes against HIV/retroviruses and HBV and found that some are much more potent to suppress HIV or HBV replication in vitro and in vivo than IFNα2 [Lavender et al., 2016; Harper et al., 2015; Gibbert et al., 2012; Song et al., 2017]. We also char- - 3 - A – Academic Section acterized the immunomodulatory effects of the different subtypes in these studies. In addition, the molecular pathways of the antiviral activity of type I IFNs were characterized [Harper et al., 2013; Behrendt et al., 2013; Li et al., 2013]. With this knowledge we can now utilize the best subtypes for immunotherapy also in combination with other anti-virals to aim for cure or functional cure of chronic HIV or HBV infection. Figure.4: Enforced virus replication effects viral immunity and the establishment of chronic infection Chronic viruses often counteract the IFN system with viral proteins that inhibit its anti-viral activity [Chen et al., 2015; Chen et al., 2013; Trilling et al., 2011; Chen et al., 2014; Han et al. 2015]. Several viruses do that by inducing degradation of IFN signaling components. As an example for utilizing this knowledge therapeutically, we tested a neddylation inhibitor that can block this immune evasion pathway and efficiently restricts replication of retroviruses and hepatitis viruses [Le-Trilling et al., 2016]. In the current funding period as well as in future experiments cell culture models will be very important to investigate new antiviral concepts. Classical cell culture is a useful process in which immortalized cells can be grown under controlled conditions in vitro. Reproducibility is a great advantage of clonal cell lines, unfortunately, after a period of continuous growth, cell characteristics may greatly differ from those determined in the initial population. Hence, preparation and culture of primary cells are important improvements and more closely resemble the native state. Primary cell culture models that support virus infection are valuable tools to investigate virus host interactions in vitro. In the TRR60 many groups use different primary cell culture models, which were established during the last funding periods partly within the service project Z3. The Lamina Propria Aggregate Culture (LPAC) model is a versatile, robust and physiologically relevant system to study immune responses against HIV-1 in vitro. To set-up the LPAC model, gut samples from patients undergoing elective abdominal surgery are obtained through the Westdeutsche Biobank. These samples are judged as macroscopically normal by a pathologist. The Lamina Propria Mononuclear Cells (LPMC) contain all relevant immune cell subsets including various T cell subsets, NK cells, dendritic cells and macrophages. In contrast to conventional PBMC cultures, the LPAC model does not require exogenous mitogen stimulation for reproducible HIV- 1 infection, as >90% of the LPMC CD4+ T cells are already activated and express CCR5 [Harper et al., 2015]. Up to date, we cryopreserved LPMCs and PBMCs from more than 50 different patients that can be used for HIV infection experiments. - 4 - A – Academic Section Figure 5 The distinct anti-viral effects of IFNα subtypes Primary liver cell preparation from human tissues, obtained after liver resection, has been improved in the last TRR60 funding period, resulting in high purity of parenchymal and non-parenchymal liver cells with functional activity in vitro [Werner et al., 2015]. Primary human hepatocytes (PHH) can be cultured for at least 10 days. This PHH long-term culture model has been proven to support HBV infection. Hepadnaviral replication in the PHH infection model is sensitive to antiviral treatment and represents an important tool for the molecular characterization of virus host interactions. To perform therapeutic experiments pre-clinical animal models are absolutely essential. Therefore, development and improvement of animal models for chronic viral infections were of upmost importance during the first two funding periods. Basic aspects and new molecules were defined in mouse models of chronic virus infection. Mice were infected with either Lymphocytic choriomeningitis virus (LCMV) or Friend Retrovirus (FV) to study different aspects of viral chronicity in a natural virus-host interaction. The advantage of the FV model is that the virus belongs to the family of retroviruses and many features of the interaction between the virus and the immune system of the host are very similar to those in HIV-infected humans [Nair et al., 2011]. These model systems helped tremendously to discover new cell populations and molecules of the immune system that are critically involved in the maintenance of chronic infections. However, more applied animal models were also developed or established. These include the hydrodynamic injection of HBV plasmids into mice and the use of HBV transgenic mouse models [Meng et al., 2014; Song et al., 2014]. Both models are no bona fide infections of mice with HBV, because this is still not possible, but allow analyzing several aspects of the interaction of HBV with the immune system. Experiments on immunotherapy and therapeutic vaccination were successfully performed in these models. A real persistent infection with a hepadnavirus is found in chronic carrier woodchucks infected with the woodchuck hepatitis virus (WHV). We utilize this model in close collaboration with our Chinese partners for pre-clinical testing of new therapeutic approaches [Wang - 5 - A – Academic Section et al., 2011b; Meng et al., 2014]. Over the years, many new immunological tools for this model have been developed together in Essen and Wuhan [Zhang et al., 2011, Fan et al., 2012; Yan et al., 2016; Liu et al., 2017]. A new mouse model for HIV was developed during a sabbatical of PI Dittmer in the laboratory of Dr. Kim Hasenkrug (Rocky Mountain Lab, NIAID, NIH, USA). Humanized triple-knockout (C57BL/6 Rag2-/-γc-/- CD47-/- mice) BLT mice present with a highly functional human immune system and can be persistently infected with HIV [Lavender et al., 2013]. They develop all signs of an HIV-induced immunodeficiency, but unlike other humanized mice they do not develop graft versus host disease. In collaboration with Dr. Hasenkrug we tested first concepts of immunotherapy against HIV in these mice [Lavender et al., 2013] and we will initiate innovative HIV cure projects in this animal model in the next funding period. These mice are a perfect model to test treatment options that target the HIV reservoir, because HIV replication can be suppressed by ART, but virus rapidly rebounds from the reservoir when therapy is discontinued. For antibody studies transgenic mice with a human B cell receptor repertoire are available for experiments in Erlangen (PI Überla). To discover new mechanisms of immunity in persistent viral infections and to verify immunological features of chronic infections that were discovered in animal models clinical samples from well-defined cohorts of HIV-, HBV- or HDV-infected patients are required. This is an important element for translational research within this TRR60. Such samples are available via various partners of the consortium: Samples from HIV+ individuals are available through PI Streeck and Dr. Stefan Esser (UK Essen). They collected samples from 1700 HIV+ patients that are currently being treated in the HIV out-patient clinic in Essen. Baseline samples from 800 of those individuals are cryopreserved (serum, plasma, PBMC) and clinical data collected in a well-managed database. The RV464 Berlin cohort provides cryopreserved longitudinal samples from 150 acutely infected and 100 non-treated chronically infected HIV patients. The study is designed to capture individuals early in acute HIV infection and to characterize HIV infection in Germany for potential future cure and/or interventional studies. The study is linked to the Deutsche Zentrum für Infektionsforschung (DZIF) TOPHIV study for potential synergistic approaches. The BRAHMS study is a vaccine preparedness study in subtype B-prevalent regions (Germany: Ruhr Area, Berlin, Munich, Hamburg, Cologne and Frankfurt), which will start to roll out at nine sites in fall 2017. The study is coordinated by PI Streeck in Essen and involves various research activities of the DZIF, Robert Koch Institute, BMG, other universities and activist groups across Germany. It is the first study ever to closely assess incidences and transmission of 9 STDs including HIV. It is expected to provide interesting samples for further studies. This will be followed by a phase 1 clinical trial for testing HIV vaccine antigens and adjuvants (RV509), planned to begin at the end of 2017/2018 in Essen with support from the US Military HIV Research Program. Samples from these studies will also be available for research within the TRR60. Patient samples are also coming from the HIDIT-1 (n=90) and HIDIT-2 (n=120) cohorts. These are prospective investigator-initiated treatment trials exploring the efficacy of PEG-IFNa-2a for 48 weeks (HIDIT-1) or 96 weeks (HIDIT-2) in hepatitis delta with or without adding a nucleotide analogue against HBV. Informed consent has been obtained from each patient to study immune responses and genetic factors. Serum samples are available from all patients. DNA for SNP analysis is available for approx. 150 patients. PBMC have been collected before, during and after treatment for patients treated at the Hannover Medical School (approx. 40 patients). In addition, PBMC have been collected from all patients with hepatitis delta followed at Hannover Medical School since 2010. PBMC from more than 60 patients are available for immunological studies. Patient samples from the HepNet cohort (n= 400) are being used in the TRR60 network. These are serum and DNA samples from HBV or HBV/HDV co-infected patients collected by the Kompetenznetz Hepatitis and stored in Essen. Informed consent has been obtained from each patient to study immune responses and genetic factors. Clinical data from all patients are also available.

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