Julia Thorn-Seshold

Cellular antioxidant systems dynamically regulate biological activity, yet their function is typically inferred from static measures. We develop chemical tools that dynamically report on Trx and GSH system activity in living cells. Multicolour activity probe panels can resolve context-dependent differences in redox activity as ratiometric “redox rainbow” signatures. Adapted probes can release therapeutics instead of reporters to selectively address redox-dysregulated tissues with redox prodrugs.

Vaccines represent a major public health intervention against infectious diseases and potentially cancer. Surrogate markers of vaccine efficacy usually rely on neutralizing antibody titers afflicted by high interindividual variabilities. Automated multiplexed T cell assays currently allow to test the clinical relevance of T lymphocyte responses during vaccine rollout. We examined cellular and/or humoral immune responses in five independent cohorts of health care workers, young healthy individuals and patients with cancer (melanoma or lung cancer) receiving various immunizing formulations (non-replicating, viral/tumoral, mRNA/peptides/cellular/viral particles). Here we show that about 20% of vaccinees to non-replicating formulations fail to mount protective antibody and Th1/Tc1 responses while 9% receiving a live vaccine were hyperresponders. Vaccine outliers could at least in part be attributed to gut dysbiosis at baseline, evaluated by shotgun metagenomics-based machine learning or the TOPOSCORE. These findings highlight the requirement of diagnostic tools to identify intestinal dysbiosis, as well as microbiota-centered interventions to optimize the efficiency of mass vaccinations.

Here, we develop a general design for high-quality fluorogenic activity probes to quantify biochemical processes within live cells, via the release of a fully cell-retained, bright fluorescent soluble product upon reaction. Live cell probes must be membrane-permeable to access intracellular biochemistry; but often that means their fluorophore products are similarly permeable resulting in rapid signal loss from the activating cell, which limits their cell-by-cell resolution as well as their sensitivity for quantifying low-turnover processes. Current strategies to retain fluorescent products within cells usually disrupt native biology e.g. by non-specific alkylation or solid precipitation. Here, scanning charge- and polarity-based approaches to swap from permeable to cell-retained states, we developed a bright fluorogenic rhodol-based platform text-decoration:underline"Trappable text-decoration:underline"Green (TraG), balancing all key requirements for signal integration (rapid probe entry, but effective product retention, across many cell lines) and being modular so it can be adapted to quantify many biochemical target types (examples shown here include probes for GSH, TrxR, and H₂O₂). The simple and rugged TraG scaffold can now permit straightforward implementation in a range of cell-retained enzyme activity probes, that will enable more accurate cell-resolved imaging as well as higher-sensitivity integration of low-turnover processes, without the drawbacks of alkylation or precipitation-based strategies.

The live-attenuated yellow fever vaccine 17D-204 (YF17D) activates robust innate immune responses followed by rapid induction of adaptive immunity resulting in long-lasting protection. YF17D triggers the production of type I interferons (IFNs) which have a dual role in antigen presenting cells regulating their infection and contributing to their activation. Infection with YF17D was detected in primary human blood monocytes and conventional dendritic cells (DCs) and in monocyte-derived DCs but was highly restricted by type I IFN. Blocking IFNAR signaling in YF17D-infected PBMC from vaccinated donors resulted in increased activation of YF17D-specific CD8 ⁺ T cells. Consistently, peak IFN-alpha plasma levels correlated inversely with the CD8 ⁺ T cells response in YF17D vaccinees. Loss of function experiments demonstrated a dominant role of retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and mitochondrial antiviral signaling protein (MAVS) for type I IFN induction and restriction of YF17D. The type I IFN response was mediated by 5’ tri- or diphosphate dsRNA intermediates that are formed during YF17D infection. In vivo proximity labelling (IPL) of RIG-I and next-generation sequencing confirmed interaction of RIG-I with YF17D-dsRNA in infected cells. Thus, YF17D-triggered RLR-signaling restricts viral replication through type I IFN and thus limits the production of viral antigens that can be presented to T cells.

HaloTag proteins spontaneously ligate onto any chemical reagent featuring a chloroalkane motif ( CA ). We introduce the conditional CHalo motif, which ligates to HaloTag only after uncaging by light or enzymes. (1) Photo-triggered CHalo fluorogenic reagents allow spatiotemporally-specific labeling; (2) photo-triggered CHalo heterodimerisers can photocontrol protein recruitment; and (3) enzyme-triggered CHalo reagents can durably record diverse enzyme activities, and multiplexing them should allow quantitative ratiometric recording of multiple activities in parallel. CHalo thus permits manifold extensions to the HaloTag technology. Abstract Figure:

Improving the efficacy of existing cytotoxic chemotherapeutics requires increasing drug delivery to tumours while minimising systemic toxicity. Formulating these drugs as nanoparticles can reduce their exposure to healthy tissues, but broadly applicable strategies to enhance tumoral accumulation are lacking. Here, we show that co-administering small molecule vascular disrupting agents together with nanoparticle formulations (e.g. diagnostic reporters, or clinical drugs irinotecan and doxorubicin) increases their tumoral uptake by up to threefold, without raising systemic exposure. In a syngeneic mouse model of triple-negative breast cancer, this enhancement diminished when co-treatments were repeated, limiting its therapeutic benefit. However, since most solid tumour types are susceptible to vascular disrupting agents, this approach may be a broadly applicable strategy to improve the selectivity of drug delivery: with particular relevance for single dose use in diagnostic or research settings. Graphical

Here, we develop a general design for high-quality fluorogenic activity probes to quantify biological processes in live cells, by creating a scaffold that efficiently generates cell-retained bright fluorescent soluble products upon reaction with biochemical targets. Live cell probes must be designed to be membrane-permeable; but that often means that their fluorophore products are similarly permeable, resulting in rapid signal loss from the activating cell: which limits their cell-by-cell resolution as well as their sensitivity for quantifying low-turnover processes. Current strategies to retain fluorescent products within cells usually disrupt native biology: e.g. by non-specific alkylation or solid precipitation. Here, scanning charge- and polarity-based approaches to trigger cell retention, we developed a bright fluorogenic rhodol scaffold Tra ppable G reen ( TraG ) that balances all key requirements for signal integration (rapid probe entry, but effective product retention, across a variety of cell lines) and is easily adaptable to quantify many target types (shown here with probes for GSH, TrxR, and H ₂ O ₂ ). The simple and rugged TraG scaffold can now permit straightforward elaboration to a range of cell-retained enzyme activity probes, that enable more accurate cell-resolved imaging as well as higher-sensitivity integration of low-turnover processes, without the drawbacks of alkylation or precipitation-based strategies. Abstract Figure:

Selectively labeling cells with damaged membranes is needed not only for identifying dead cells in culture, but also for imaging membrane barrier dysfunction in pathologies in vivo. Most membrane permeability stains are permanently colored or fluorescent dyes that need washing to remove their non-uptaken extracellular background and reach good image contrast. Others are DNA-binding environment-dependent fluorophores, which lack design modularity, have potential toxicity, and can only detect permeabilization of cell volumes containing a nucleus (i.e., cannot delineate damaged volumes in vivo nor image non-nucleated cell types or compartments). Here, we develop modular fluorogenic probes that reveal the whole cytosolic volume of damaged cells, with near-zero background fluorescence so that no washing is needed. We identify a specific disulfonated fluorogenic probe type that only enters cells with damaged membranes, then is enzymatically activated and marks them. The esterase probe MDG1 is a reliable tool to reveal live cells that have been permeabilized by biological, biochemical, or physical membrane damage, and it can be used in multicolor microscopy. We confirm the modularity of this approach by also adapting it for improved hydrolytic stability, as the redox probe MDG2. We conclude by showing the unique performance of MDG probes in revealing axonal membrane damage (which DNA fluorogens cannot achieve) and in discriminating damage on a cell-by-cell basis in embryos in vivo. The MDG design thus provides powerful modular tools for wash-free in vivo imaging of membrane damage, and indicates how designs may be adapted for selective delivery of drug cargoes to these damaged cells: offering an outlook from selective diagnosis toward therapy of membrane-compromised cells in disease.

The yellow fever 17D vaccine (YF17D) is highly effective but is frequently administered to individuals with pre-existing cross-reactive immunity, potentially impacting their immune responses. Here, we investigate the impact of pre-existing flavivirus immunity induced by the tick-borne encephalitis virus (TBEV) vaccine on the response to YF17D vaccination in 250 individuals up to 28 days post-vaccination (pv) and 22 individuals sampled one-year pv. Our findings indicate that previous TBEV vaccination does not affect the early IgM-driven neutralizing response to YF17D. However, pre-vaccination sera enhance YF17D virus infection in vitro via antibody-dependent enhancement (ADE). Following YF17D vaccination, TBEV-pre-vaccinated individuals develop high amounts of cross-reactive IgG antibodies with poor neutralizing capacity. In contrast, TBEV-unvaccinated individuals elicit a non-cross-reacting neutralizing response. Using YF17D envelope protein mutants displaying different epitopes, we identify quaternary dimeric epitopes as the primary target of neutralizing antibodies. Additionally, TBEV-pre-vaccination skews the IgG response towards the pan-flavivirus fusion loop epitope (FLE), capable of mediating ADE of dengue and Zika virus infections in vitro. Together, we propose that YF17D vaccination conceals the FLE in individuals without prior flavivirus exposure but favors a cross-reactive IgG response in TBEV-pre-vaccinated recipients directed to the FLE with potential to enhance dengue virus infection.