Alumni Symposium 2022
Robert Koch Postdoc Prize Awardees
Christian Drosten, Mathias Hornef, Max Löhning, Andreas Radbruch, Sabine Timmermann and Katrin Moser
Alumni of the Robert Koch Post-doctoral Award, Robert Koch Awardees 2022, Board of Directors, Scientific Advisory Council Members, and Sponsor Representatives
Thursday, 10th of November 2022
Frequencies and activation state of circulating DC and monocyte subsets were analyzed by flow cytometry in a cohort of hospitalized COVID-19 patients and in outpatients with a mild course of the disease. Compared to YF17D vaccination, patients with more severe COVID-19 showed low expression of costimulatory molecule CD86 in the cDC2, DC3 and monocytes. In contrast, non-hospitalized patients with mild COVID-19 showed an upregulation of CD86 similar to what was observed in YF17D vaccinees. The downregulation of CD86 in severe COVID-19 was accompanied by upregulation of PD-L1. This altered phenotype of peripheral APCs coincided with a reduced capability of DC3 and monocytes isolated from the blood of COVID-19 patients to co-stimulate autologous T cell activation and proliferation in vitro. An increase of Ki67+ DCs alongside temporary reductions in cDC1 and cDC2 frequencies indicated a higher turnover of the blood DC compartment in both COVID-19 patients and YF17D vaccinees. Profound longer lasting depletion of circulating DCs was a characteristic feature of severe COVID-19 while the reduction of blood DCs was transient after YF17D vaccination. Functional impairment and delayed regeneration of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.
Intensive research of the last 20 years has enormously advanced our understanding of the IFN system. We now know that all nucleated cells express sensor proteins which are able to recognize virus-specific RNA structures and activate the signaling chain leading to IFN induction. However, it also got increasingly clear that viruses have evolved effective counterstrategies. Viral proteins, the so-called IFN antagonists, can disturb or even completely block all stages of the IFN response, e.g. IFN induction, IFN signaling, or expression or action of antiviral genes.
In our group, we investigate the interplay between IFN system and pathogenic RNA viruses, e.g. Rift Valley fever virus or SARS-coronaviruses. On one hand, we study how a cellular sensor protein can recognize an RNA structure as being viral, and on the other hand we are elucidating the astonishing variety of strategies by which viruses inhibit the IFN response. Insights obtained by such investigations can help to improve vaccines or antiviral therapies.
We found that pre-stimulation of primary human foreskin fibroblasts (HFF-1) with BMPs induces low levels of transcription of interferon-stimulated genes (ISGs), though without affecting HCMV replication. However, co-stimulation of HFF-1 with BMP9 and IFNβ prior to infection significantly enhanced the antiviral activity of IFNβ as compared to IFNβ pre-stimulation alone. Further experiments showed that BMP9 significantly enhances the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and transcription of interferon regulatory factor 9 (IRF9) and STAT2, which are critical components of IFN-induced signaling. Moreover, we can show that HCMV US18 and US20 specifically antagonize BMP-mediated, but not IFN-mediated signaling, and their expression during HCMV infection impedes the responsiveness of HFF-1 to BMP9 stimulation, thus circumventing the potential effect of BMP9 on the antiviral host response to infection.
Taken together, our data reveal a previously underappreciated role of BMP9 as an important modulator of innate immunity and type I IFN signaling during HCMV infection.
 Eddowes et al., 2018, Nature Microbiology
 Fielding et al., 2018, eLife
Beside a better understanding the mutual interaction between commensal bacteria and the host we aim at identifying age-specific differences in the antimicrobial host response and susceptibility to infection with enteropathogenic microorganisms such as enteric Salmonella, enteropathogenic E. coli (EPEC), Listeria monocytogenes, rotavirus, and Giardia lamblia. These pathogens cause a very significant morbidity and – particularly in the infant population – also a significant mortality worldwide.
We hope that a better understanding of the feto-neonatal transition of the intestinal mucosa, the postnatal establishment of host-microbial homeostasis and the protective immune response to infection will contribute to reduce childhood mortality and reduce long-term sequelae.
Bacterial adaptation strategies during long-term asymptomatic colonization include genomic decay and attenuation. In addition, the bacteria have also developed sophisticated mechanisms to actively manipulate the host in their favour. For example, asymptomatic bladder colonization of E. coli isolate 83972 can protect human carriers from superinfections by uropathogens. This strain actively modulates the host environment, down-regulating transcriptional activity, including that of pro-inflammatory genes. Uropathogenic E. coli can affect host gene expression by repressing the expression of key transcription factors in the host. At the level of chromatin dynamics, the host also responds differently to interaction with pathogens or asymptomatic colonizers. These observations point to previously unknown strategies for bacterium-host interaction at mucosal membranes, the underlying molecular mechanisms of which have not yet been fully elucidated.
During genome defense, CRISPR-Cas nucleases typically rely on CRISPR RNA (crRNA) guides encoded in repeat-spacer arrays associated with the system to recognize foreign genetic material. In Type II systems, another RNA component, the trans-activating crRNA (tracrRNA) hybridizes to the crRNAs to drive their processing and utilization by the Cas9 nuclease. While Cas9 typically binds and cleaves double-stranded DNA, we had uncovered that C. jejuni Cas9 (CjCas9) can also target endogenous RNAs in a crRNA-dependent manner based on a RIP-seq (co-immunoprecipitation combined with RNA-seq) approach . While analyzing CjCas9-RNA complexes from additional C. jejuni strains, we recently discovered that the tracrRNA does not only bind to the crRNAs, but can also hybridize to certain cellular RNAs, such as mRNAs, leading to the formation of “non-canonical” crRNAs (ncrRNAs) . While the function of these ncrRNAs in C. jejuni remains elusive, we demonstrated that these mRNA-derived fragments are capable of guiding DNA targeting by Cas9 in vitro and in vivo. Our discovery of ncrRNAs inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA-of-interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection) . LEOPARD can detect multiple RNAs of respiratory viruses in parallel and can distinguish different SARS-CoV-2 viral variants at single nucleotide resolution in patient samples. Overall, our study revealed crRNAs can originate from outside of CRISPR-Cas systems, and we have translated this finding into a multiplexable RNA detection platform.
 Jiao C, Sharma S*, Dugar G*, Peeck NL, Bischler T, Wimmer F, Yu Y, Barquist L, Schoen C, Kurzai O, Sharma CM#, Beisel CL# (2021) Non-canonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9. Science, (6545):941-948. *Equal contribution, #Corresponding authors
 Dugar G, Leenay RT, Eisenbart SK, Bischler T, Aul BU, Beisel CL#, Sharma CM# (2018). CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9. Molecular Cell 69, 893–905.
Nature invented a variety of different „lipid II binders“ and their antibiotic activities can vary substantially depending on the compounds physicochemical and membrane-targeting properties, the binding site on lipid II, as well as the ability to interact with structurally similar precursors of other cell wall polymers, such as wall teichoic acid, capsule or arabinogalactan.
Besides the primary contact events, which result from initial drug-target interaction and direct binding of lipid II, sequestration of the ultimate cell wall building block can trigger multifaceted cellular events that all contribute to killing. These secondary events originate from the unique cellular role of lipid II, functioning as a structural and regulatory focus that directly and/or indirectly contributes to the organization of diverse cell wall biosynthetic processes.
The most recent addition to the portfolio of lipid II binding natural product antibiotics are teixobactin and newly identified teixobactin-like antibiotics (TLA), isolated from previously uncultured soil bacteria. Compared to all other lipid II binders investigated previously, teixobactin and TLAs appear unique in their mechanism of action and proved most refractory to resistance development.
NK:target contacts. In contrast, cell death mediated by caspase-8 was slower and a result of late target cell engagements. This suggested a kinetic regulation of the two cytotoxic pathways during serial killing. We observed that NK cells switch from inducing GrzB-mediated cell death in their first killing events to a death receptor-mediated killing during subsequent tumor cell encounters. Investigating the use of the different granzymes we found that GrzB dominated most killing events of freshly isolated or activated human NK cells, while we also detected the activity of other granzymes. GrzK initiated target cell apoptosis was limited to freshly isolated NK cells. Additionally, we found individual killing events of activated NK cells where GrzA or GrzM activity was dominant. This demonstrates that granzyme and death receptor-mediated cytotoxicity are differentially regulated during NK cell serial killing.
Friday, 11th of November 2022
Virology, part 1
In vitro experiments using recombinant HCV and DENV capsid protein showed the ability of C10 to establish a covalent interaction, inducing the formation of dimers, trimers, and higher molecular weight species. These oligomers formed at concentrations below the IC50, and the effect increased in a dose-dependent manner.
By structure-activity-relationship studies, a set of 45 C10-derivatives were designed, synthesized, and tested against HCV as well as DENV. In parallel, all compounds were evaluated in crosslinking assays. As a result, we observed a high correlation between the crosslinking ability of each compound and its activity in the cell-based assay. The activity and toxicity of C10 and two selected hit candidates (C45 and C46) were further characterized. IC50 values for different stages of HCV and DENV infection, as well as CC50 values, were determined. The calculated therapeutic index (Ti) showed that C45 is ~3-4 times better than C10 (Ti~330 HCV, ~870 DENV), being both very promising antiviral compounds. Moreover, toxicity of C10 and C45 were tested in vivo including zebrafish and mouse models, where different pharmacological parameters were evaluated. Finally, in vivo efficacy is tested in Flavivirus-infected mice. Altogether, C10 and C45 are promising molecules for further development as antiviral agents against virtually all members of the Flaviviridae family.
Methotrexate (MTX) has been used over decades for immunosuppression and cancer therapy. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleobases. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutically achievable concentrations, leading to up to 1000-fold reductions in virus progeny (Stegmann, Dickmanns et al., Virus Res 2021)
Inhibitors of Dihydroorotate dehydrogenase (DHODH) are in clinical use for similar purposes as MTX, and they interfere with the synthesis of pyrimidines. Our investigations revealed that DHODH inhibitors also reduce the replication of SARS-CoV-2. Moreover, these compounds strongly synergize with N4-Hydroxy-Cytidine, the active compound of the antiviral drug Molnupiravir (NHC). Mechanistically, inhibiting DHODH induces a lack of available pyrimidines, thus increasing the incorporation of NHC. The drugs also cooperated to alleviate COVID in animal models (Stegmann, Dickmanns et al., iScience 2022). We are now targeting CTP-Synthetase to further increase the efficacy of NHC and Molnupiravir. As an important caveat, however, we also found that NHC strongly enhances the occurrence of virus mutants, including escapers from neutralizing nanobodies.
Using inhibitors of nucleotide synthesis for treating SARS-CoV-2 infections still needs to be evaluated clinically, and the role of immunosuppression in disease progression awaits clarification. Within these limitations, however, our results are at least compatible with a possible use of such inhibitors in treating COVID-19. The seminar will raise the perspective of re-purposing cancer drugs to antagonize the spread of SARS-CoV-2.
Virology, part 2
We found that the human tetraspanin CD81 is a critical replication factor for Chikungunya virus in fibroblasts and hepatocytes, two major replication sites of the virus. CD81 co-localizes with virus replication sites at the plasma membrane. The protein is dispensable for virus entry and release and is thus a bona fide replication factor. The closely related tetraspanin CD9 can partially replace the function of CD81 in virus replication. Murine CD81 similar to human CD81 supports virus genome replication, indicating that CD81 may be a cross-species host factor for Chikungunya virus. CD81 is known to stabilize membrane microdomains through cholesterol binding. By mutating the cholesterol binding site of CD81, we could show that cholesterol binding is critical for the host factor function of CD81. Finally, we show that human pathogenic viruses from the same family also rely on CD81 for replication. Our work identifies the first human transmembrane protein hijacked by Chikungunya virus for its replication. We propose a model by which CD81 supports the formation of virus replication organelles through its cholesterol binding function. This work will spur future studies defining the minimum requirements for virus replication in human cells and may reveal targets for antiviral therapies.
Here, by transcriptomics and functional analysis of CD4+ T-cells from aviremic patients with HIV-1 and immortalized T-cell models of HIV-1 latency, we studied the impact of latency-reverting agents (LRAs) on the cellular milieu and viral reactivation. Our work reveals quantitative and qualitative heterogeneity of individual provirus-containing clones in terms of HIV-1 reactivation. Furthermore, selected LRAs used in the context of pharmacological “shock-and-kill” approaches modulate cell-intrinsic responses by impairing both basal and IFN-induced expression of the majority of IFN-stimulated genes, resulting in facilitated HIV-1 reactivation. However, this LRA-induced reprogramming may hamper cellular processes driving immune recognition and killing of reactivating cells. Finally, identification and analysis of individual, HIV-1 transcript-positive CD4+ T-cells enabled us to define a set of genes whose expression correlated with HIV-1 RNA abundance, representing yet-to-be-characterized potential biomarkers of HIV-1 transcriptional reactivation.