Paul Ehrlich Institut

Children with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) continue to face relapse and treatment resistance, emphasizing the urgent need for novel therapeutic strategies. In this study, we developed and characterized a recombinant oncolytic measles virus (MV-Blina) engineered to locally secrete a bispecific T cell engager (bsTE) targeting CD19 and CD3 (secBlina) to enhance antitumor immunity while minimizing systemic toxicity. MV-Blina demonstrated superior replication kinetics and cytotoxicity in vitro compared to the parental MV-Edm strain. MV-Blina infected ALL cells, secreted functional secBlina capable of engaging and activating T cells, leading to selective leukemia cell death. In in vitro and in vivo models, including patient-derived xenografts, MV-Blina demonstrated an additive yet heterogeneous anti-leukemic effect, with significant survival benefits and reduced CNS leukemic burden in MV-Blina-treated mice. Importantly, MV-Blina did not induce either short- or long-term toxicity in in vitro neuronal models encompassing PBMCs, nor in immunocompromised CD46 transgenic mice, even under additional immunosuppression. Collectively, these findings support further investigations of MV-Blina as a potential treatment for patients with relapsed or refractory BCP-ALL.

The recent SARS-CoV-2 pandemic has demonstrated the importance of vaccines in controlling outbreaks and in preparing for emerging viruses. Platform technologies are highly relevant as they reduce the time taken for vaccine development, market authorisation and production. Here, we present a novel platform based on modified, membrane-permeable hepatitis B virus (HBV) capsids that can flexibly carry various antigens via an adapter. The SARS-CoV2 spike-derived receptor binding domain serves as the antigen. This study aims to identify the most robust system for producing stable carrier/cargo complexes by comparing various platforms. The thereby identified system was used for detailed characterization of the immune response including its capacity for enabling needle free immunization by oral/nasal application. A comparison of various adapter systems (StrepTag, AviTag, SpyTag and DogTag) for coupling the cargo antigen to the cell-permeable capsid as the carrier revealed that the DogCatcher/DogTag system outperforms the others with regard to the yield, assembly and stability of the particles, as well as antigen loading onto the carrier surface. Immunization of mice showed that antigens coupled to the carrier induce a much stronger immune response than free, uncoupled antigens. Loading the capsid interior with CpG as an adjuvant further increased the immune response. Notably, the antigen carrier's cell permeability enables oral/nasal immunization, resulting in significant titres of neutralizing IgG and IgA antibodies. Taken together, this novel platform can be used as a flexible base for quickly adapting vaccines against emerging viruses. Most interestingly, the platform's cell permeability allows for needle-free immunization via the oral/nasal route.

Chronic hepatitis B virus (HBV) infection is a major health problem, affecting around 254 million people worldwide. Current treatments have side effects and can lead to resistance. Therefore, natural compounds derived from plants are being studied as potential antivirals. Isochlorogenic acid A (ICAA), which is derived from caffeoylquinic acid, exerts antiviral and hepatoprotective effects. The antioxidant effect of ICAA, triggered by upregulation of heme oxygenase 1 (HO-1), has been suggested as potential antiviral mechanism. However, the underlying mechanisms remain enigmatic. Our aim is to elucidate the mode of action of the antiviral effect of ICAA on HBV. Stable or transient transfected cells expressing HBV as well as HBV-infected cells were instrumental. (Sub)viral particles were characterized by biophysical and biochemical methods. Subcellular distribution of viral proteins was studied using confocal laser scanning microscopy. Viral genomes and transcripts were quantified by qPCR. Treatment with ICAA decreased levels of HBV surface and e antigens (HBsAg and HBeAg), as well as viral transcripts, genomes and most important cccDNA. Furthermore, impaired virus assembly was evident from accumulation of naked capsids suggesting improper capsid formation and impaired envelopment. ICAA-dependent effects on HBV correlate with upregulation of HO-1 and modulation of intracellular ROS Our data indicate a possible link between changes in the intracellular ROS level and altered free -SH groups in viral structural proteins, possibly influencing proper disulphide bond formation and thereby assembly. In conclusion ICAA-dependent effects on HBV life cycle are based on several pillars as modulation of intracellular ROS and impaired morphogenesis and replication.

Chimeric antigen receptor (CAR)-T cell therapy has transformed the outcomes of patients with haematological malignancies, yet its use is limited by labour-intensive manufacturing, constrained production capacity and variable clinical performance. In vivo CAR-T cell engineering, in which CAR-T cells are generated directly inside the patient's body, seeks to overcome these challenges by eliminating the need for ex vivo cell processing and complex logistics, as well as improve clinical performance. Recent advances in virology, RNA medicines and nanotechnology have catalysed a radical overhaul of this approach, which uses targeted delivery systems such as lentiviral vectors and lipid nanoparticles to introduce CAR-encoding genetic material into endogenous T cells. Early clinical studies have shown efficient transduction, sustained CAR expression and initial signs of antitumour activity, establishing proof of concept. This Review explores the underlying technologies - including RNA delivered by lipid nanoparticles and engineered viral vectors - and discusses how they are being adapted to develop more broadly applicable, scalable, safe and effective CAR-T cell therapies. By removing the need for ex vivo manipulation and chemotherapeutic conditioning, this strategy could enable the wider application of CAR-T cell therapies not just to blood cancers but to autoimmune diseases for which ex vivo CAR-T cell therapies have shown strong promise, such as systemic lupus erythematosus.

The results of a global survey on blood safety measures to prevent transfusion-transmitted infectious diseases (TTIDs) were analysed, focusing on TTID testing requirements, practices and associated donor actions (vigilance). Responses by country/region (C/R) were categorized by World Bank income (WBI) levels: low- and lower middle-income (LLMI), upper middle-income (UMI) and high-income (HI). Consensus responses were used for C/Rs with multiple survey responses. Regions within China and India were analysed separately. Survey questions on TTID testing and donor vigilance were compared across WBI levels. Responses from 74 C/Rs representing 65 countries and Hong Kong were analysed. All C/Rs reported mandatory human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) screening, with a few exceptions for syphilis. Most testing standards were set by national law, using antibody and/or antigen testing for HIV, HBV and HCV; antibody testing was common for syphilis. Nucleic acid testing (NAT) was less common in LLMI than in UMI and HI C/Rs. Confirmatory testing was reported by almost all (96%) C/Rs, often using the same screening test. All C/Rs provided educational material to donors, and deferred/notified donors based on reactive/positive TTID results. Most C/Rs reported reactive/positive results to a central entity, and 89% withdrew and destroyed in-date units from previous collections. All reporting C/Rs screened for HIV, HBV and HCV, with most using confirmatory testing. Advanced tests such as NAT were less common in LLMI C/Rs. Donor vigilance was consistent across income groups, with education, notification and deferral for TTID results, and most reporting withdrawal/destruction of previous collections.

The European Directorate for the Quality of Medicines & HealthCarehas been co-ordinating an external quality assessment programme for blood establishment (BE) laboratories since 2010. To further expand the study portfolio, a new bacterial blood proficiency testing scheme (B-PTS) for platelet components (PCs) has been developed and validated in a pilot study. Sterile samples and those containing a low-count quantity of bacteria were prepared in spiking devices. Suitability of storage and shipping conditions for the samples was evaluated under different environmental conditions. Participants received the spiking devices, prepared the potentially contaminated PCs on site and tested them according to their routine screening procedures. Humidity compromised the quality of the samples. Optimized storage of the samples by adding a desiccant ensured satisfactory quality. In the pilot study, 9 of the 11 participants correctly identified the positive samples as being bacterially contaminated. The newly developed bacterial B-PTS was successfully implemented in a pilot study. It enables an inter-laboratory comparison to determine the performance of BEs for the testing of bacterial contaminations in PCs.

Critically ill patients suffer from a wide variety of clinical events, most of them leading to pro-inflammatory states such as sepsis or simply as consequence of major surgery. Many of these patients develop forms of acute kidney injury, heart or acute liver failure during intensive care. Lipid signaling is critically involved in triggering systemic inflammation processes, pain and vascular tone. We therefore hypothesized that fatty-acid-derived lipid mediators might be regulated during inflammatory stages and other clinical events in critically ill patients and might serve as potential biomarker candidates. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we determined the levels of 53 lipid mediators in plasma from nine patients. These patients were hospitalized at Frankfurt University Hospital's intensive care unit (ICU) after cardiac surgery. Inflammatory stages were illustrated over time using clinically established biomarkers such as interleukin-6 (IL-6) and leukocyte count. Normal range values of the lipids were obtained from healthy volunteers. Plasma levels clearly outside the normal range were observed for 22 of 53 lipid mediators, of which 13 were increased (including ceramides Cer (d18:0/18:0), Cer (d18:1/16:0), Cer (d18:1/18:1), glucosyl-ceramide GluCer (d18:1/24:1), lactosylceramide LacCer (d18:1/18:0), and LacCer (d18:1/24:1), 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), 11,12- and 14,15-DHET and 1- and 2-arachidonoyl glycerol (1-AG and 2-AG), Sphingosine SPH (d18:1) and 20-HETE. Furthermore, nine lipids were decreased (Cer (d18:1/24:0), LacCer (d18:1/16:0), LacCer (d18:1/24:0), sphingosine-1-phosphate S1P (d18:1), S1P (d18:0), the lysophosphatidic acids LPA (16:0), LPA (18:0), LPA (18:1) and 9-HODE. Among increased lipids, the remarkable changes in 1-AG, 2-AG, and to a lower extent of 6-keto-PGF1-alpha plasma levels showed a certain agreement with inflammatory phases. Furthermore, 6-keto-PGF1alpha had its peak shortly before initiation of continuous veno-venous hemodialysis (at least in 5 of the observed patients), 2-AG was elevated in all our nine patients during (right) heart failure in the context of either re-opening patient's chest, implementation of veno-arterial ECMO or at least while significantly increasing the amount of catecholamines. In this pilot trial we identified several evaluated lipids in critically ill patients representing either potentially (patho-) physiologically relevant mediators of the pro-inflammatory processes and during heart failure or possible markers preceding veno-venous hemodialysis.

DNA transposons have emerged as promising tools in both gene therapy and functional genomics. In particular, the Sleeping Beauty (SB) DNA transposon has advanced into clinical trials due to its ability to stably integrate DNA sequences of choice into eukaryotic genomes. The efficiency of the DNA transposon system depends on the interaction between the transposon DNA and the transposase enzyme that facilitates gene transfer. In this study, we assess the DNA-binding capabilities of variants of the SB transposase and demonstrate that the structural stability of the primary DNA-recognition subdomain, PAI, affects SB DNA-binding affinity and transposition activity. This fundamental understanding of the structure-function relationship of the SB transposase will assist the design of improved transposases for gene therapy applications.