Yellow fever virus (YFV) infection can cause severe-to-fatal liver damage in humans, while immunisation with the attenuated 17D vaccine strain has an excellent safety record, priming protective host immunity in the absence of pathology. To investigate virus-host correlates associated with these differential clinical outcomes, we combined YFV genome-level evolutionary analyses with investigations of vaccine and virulent strain hepatotropism. Evolutionary analyses confirmed purifying selection is the dominant force driving global YFV genome divergence, with functional constraints associated with the YFV transmission cycle selecting against vaccine attenuating mutations in virulent strains. In immune deficient hepatoma cells, 17D exhibited enhanced early propagation, spreading and apoptosis induction when compared to virulent strains. Ex vivo infections performed in primary human hepatocytes (PHH) from multiple donors confirmed robust propagation of both 17D and virulent YFV strains. RNA-sequencing revealed consistent and shared induction of IFNB and IFNL1-4, modulating overlapping gene programs associated with antiviral responses, immunity, chemotaxis and inflammation, cell-death, metabolic reprogramming and protein translation. Subtle differences in virion production kinetics and the magnitude and tempo of PHH transcriptional responses were observed. Antiviral responses to 17D were activated earlier while responses to virulent YFV were delayed but enhanced, mirroring virus propagation kinetics. More broadly, cellular responses to YFV infection are likely dominated by paracrine IFN signalling, with enriched LRP1 expression coupled with impaired IFNalpha production in PHH contributing to YFVs robust hepatotropism. In summary, we demonstrate comparable propagation kinetics and PHH transcriptional responses to vaccine and virulent YFV strains, highlighting impaired hepatotropism is not a correlate of vaccine attenuation. These data imply that unknown barriers restrict liver access and associated organ pathology upon vaccination with 17D. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, 416701689 Bundesministerium für Forschung, Technologie und Raumfahrt, 01KI2106, 01KI2113 Bundesministerium für gesundheit, ZMVI1-2516FSB416 ERC Horizon Europe, DEFENDER

RNA editing by adenosine deaminases acting on RNA (ADARs) is an essential cellular process performed by three enzymes in mammals: ADAR1-p150, ADAR1-p110, and ADAR2, demonstrating different target specificity and selectivity. Here we describe TSniffer, a novel tool to analyze RNA editing in RNA-sequencing datasets. TSniffer uses a rolling window approach to identify editing sites and operates in two modes allowing identification and quantification in single samples, and quantification in predefined regions across multiple datasets. Using wild type and ADAR-deficient datasets, we provide strategies for identification of ADAR editing sites and verify the accuracy and biological relevance of our findings.

The recombinant measles virus (MeV) is among those vaccine platform technologies, which are tested for the fast generation of effective vaccines against other pathogens, such as emerging virus diseases. Different live-attenuated vaccine strains have become available as backbones, with few differences. Here, we analyze how minor differences of recombinant Moraten (MVvac2) or Schwarz (MVSchw) constructs have an impact on the performance of respective derived Middle East respiratory syndrome (MERS) vaccine candidates. MVvac2 or MVSchw viruses, both encoding the Spike glycoprotein (S) of the MERS-coronavirus (MERS-CoV) in an additional transcription unit (ATU) between the hemagglutinin gene and the polymerase gene L cassette, were tested. Both robustly expressed the additional gene. However, approximately twofold to threefold higher amounts of S proteins were observed in cells infected by the MVSchw-MERS-S(H) virus, correlating with the same relative ratio of S mRNA found in these cells. Exchange of the short genomic 3' UTR ATU sequence in MVvac2 against the sequence in MVSchw enhanced gene expression by about the same factor and demonstrates causality. MeV-susceptible IFNAR-/--CD46Ge mice vaccinated with either candidate reveal that higher relative S antigen expression by MVSchw-MERS-S(H) tended toward induction of higher specific antibody titers, but lower numbers of S antigen-specific T cells. In subsequent challenge experiments, both candidates were effective. Thus, minor differences in the genetic composition of recombinant MeV's ATU may modulate the gene expression pattern of additionally encoded proteins that are reflected by changes in the respective immunogenicities, but which were not decisive for protection in the chosen disease model. IMPORTANCE:In case of emerging or re-emerging infections, vaccine platform technologies are needed to rapidly develop effective vaccines to aid public healthcare in pandemics. Besides mRNA vaccines, also viral platform technologies, that is, the adenovirus-derived vaccines Vaxzevria and JCOVDEN, have proven to be of immense value during the COVID-19 pandemic. For future pandemics, it is crucial to understand the factors in vector design that modulate immunogenicity. This knowledge allows the tailoring of vaccine vectors to fit specific target product profiles, for example, to build vectors which trigger an accentuated T cell or, alternatively, antibody response against an antigen of interest. Our study using the live-attenuated measles vaccine backbone as a promising example is therefore crucial in demonstrating that very minor differences in the vaccine backbone can alter the antigen expression profile of the vector-antigen system and impact the relative induction of T-cell or antibody responses against the added target antigen.

Abstract Chronic hepatitis B virus (HBV) infection contributes to hepatocellular carcinoma by disrupting host transcription, cell-cycle control, and apoptotic signaling. Isochlorogenic acid A (ICAA), a natural compound with antiviral and hepatoprotective properties, was previously shown to inhibit HBV replication by interfering with multiple steps of the viral life cycle. Because chronic HBV often reflects an imbalance between proliferation and cell death, we investigated how ICAA affects gene expression related to these processes in the presence or absence of HBV. We performed transcriptome analysis using RNA sequencing (RNA-seq) in HepAD38 cells (a HepG2-derived stable HBV-expressing line) and HepG2 control cells (HBV-negative) treated with ICAA or DMSO. HBV caused major differences in gene expression in HepAD38 cells compared with HBV-negative HepG2 cells. Principal component analysis showed that ICAA significantly altered HBV-dependent expression patterns, resulting in 189 differentially expressed genes (DEGs) that were regulated in opposite directions by both HBV and ICAA. Functional enrichment analysis highlighted pathways in viral carcinogenesis, apoptosis, MAPK signaling, and p53 signaling. Annexin V/propidium iodide assays showed apoptotic cells in both treated and untreated HepAD38 cultures, with only minor pattern changes. Mechanistically, in untreated HBV-positive cells caspase-9 cleavage failed to activate PARP, suggesting that induction of intrinsic apoptosis is followed by blocked execution. In contrast, ICAA inhibits caspase-9 cleavage in a dose-dependent manner, while activating PARP. Consistent with this, ICAA treatment increased apoptotic DNA fragmentation in HepAD38, reflecting the proapoptotic potential of ICAA under these conditions facilitating the elimination of HBV-positive cells by apoptosis. These findings highlight the potential therapeutic relevance of this compound in processes associated with HBV pathogenesis, together with its antiviral effect. Graphical abstract

3'-nucleotidases/nucleases, distinct class I nucleases of protozoan parasites, play a pivotal role in extracellular purine salvage. As Leishmania are purine auxotrophs and lack de novo synthesis, ectoenzymes facilitating nucleotide and nucleic acid cleavage are indispensable for subsequent uptake. Employing quantitative proteomics, we characterized a class I nuclease p1/s1 cluster in L. major that comprises enzymes exhibiting dual 3'-nucleotidase and endonuclease activity. Expression of these enzymes is induced upon miltefosine or staurosporine treatment and was specifically detected in stationary-phase, but not in logarithmic-phase promastigotes. After confirming secretion of p1/s1, ecto-enzymatic activity was detected on parasites and in the culture supernatant. Viable null mutants deficient for the p1/s1 cluster were only obtained when a diCre-based inducible knockout system was applied, whereas direct deletion approaches were lethal. The viable knockout strains exhibited significantly reduced 3'-nucleotidase/nuclease activity. Notably, these parasites adapted by compensatory enrichment of various alternative purine salvage proteins at the proteomic level. Furthermore, both enzymatic functions implied mechanisms of host-pathogen interactions to facilitate infection establishment: Utilizing 3'-nucleotidase activity, Leishmania generate extracellular adenosine to suppress inflammatory cytokine secretion from macrophages and reduce lymphocyte proliferation in a human primary cell model. The presence of ecto-nucleases also allowed these parasites to degrade and survive neutrophil extracellular traps, a potent first-line innate immune mechanism in pathogen defense. In summary, our integrative approach combining proteomics, immunological and genome editing methods expands current knowledge about Leishmania major 3'-nucleotidases/nucleases. By offering new insights into the diverse involvements in host-pathogen interactions, we highlight p1/s1 as pivotal factor during infection and potential drug target.

Most CAR-T therapies rely on genetic T cell engineering with integrating viral vectors that, although effective, are associated with prohibitive costs. Here we have generated TranspoCART19 cells, a fully functional 4-1BB second-generation CAR-T cell product targeting CD19, fused to a truncated version of the human EGFR (hEGFRt) as reporter gene and safety-switch, based on the Sleeping Beauty transposon delivery system. Our manufacturing protocol allowed generation of TranspoCART19 cells under GMP conditions, showing similar in vitro and in vivo antitumoral efficacy than conventional CAR-T cells generated with lentiviral vectors. Additionally, membrane expression of hEGFRt facilitated in vivo CAR-T cell elimination after cetuximab administration. Safety analyses showed that TranspoCART19 cells presented low vector copy numbers and close-to-random vector integration profiles. Moreover, final TranspoCART19 products lacked non-integrated genomic material used for the generation of CAR-T cells and were free from transposase protein. In vivo biodistribution analyses revealed that TranspoCART19 cells were mainly present in hematopoietic organs with no gender bias. Altogether, this study provides a cost-effective, GMP-compliant manufacturing process for the generation of CAR-T cells using non-viral vectors. These results have supported the approval of a clinical trial to evaluate TranspoCART19 cells in patients with relapsed/refractory lymphoma (NCT06378190) that is currently ongoing.

Background & Aims: Hepatitis C virus (HCV) has a narrow species tropism and cannot infect mice. To understand HCV species tropism and to develop better animal models, we adapted HCV to infect mouse cells deficient in innate immunity and with minimal human HCV host factors. Methods: HCV was adapted via passaging an HCV infectious virus clone several times in human hepatoma cells, mouse liver cells, and eventually primary mouse hepatocytes deficient in innate immunity and ectopically expressing human occludin and human CD81. Using RNAseq the sequence of the adapted virus was analyzed, and several clones were generated to study replication and infection kinetics as well as neutralization assays in several human/mouse cell lines and primary hepatocytes from human, mouse, and macaques. Results: Accumulation of 35 non-synonymous and 66 synonymous mutations correlated with >1,000-fold enhanced production of infectious progeny from primary mouse hepatocytes. These mutations did not confer drug resistance or evasion from innate immunity. They did not enhance fitness in human or macaque hepatocytes. We show that non-synonymous mutations are necessary and sufficient for adaptation, and that changes to the glycoproteins are not essential. Mutations outside of viral envelope proteins enhanced specific infectivity and facilitated viral spread in murine cells. Conclusions: This study reveals key viral factors governing HCV species tropism. The mouse-adapted HCV opens up possibilities for the development of animal models to analyze HCV pathogenesis, immune control, and vaccine development. (c) 2025 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

ABSTRACT Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and costeffective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.

The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications, including gene therapy. We discovered a hyperactive variant of the SB100X transposase, called SB200X. This mutant, resulting from a specific amino acid replacement (Q124C), showed an ∼2-fold increase in transposition activity in various human and murine cells. Other amino acid replacements in position 124 also led to a hyperactive phenotype. Position 124 is located at the very edge of the linker region that connects the DNA-binding and catalytic domains of the transposase. Consistent with a role of the linker in an autoregulatory mechanism called overproduction inhibition (OPI) in the monophyletic group of mariner transposases, we show that the hyperactivity of Q124C manifests at high concentrations of the transposase, suggesting a partial resistance of SB200X to OPI. We demonstrate that the hyperactive phenotype of Q124C can be combined with features of other useful mutations in the SB transposase. Namely, Q124C improves the transposition efficiency of the previously described K248R variant, while maintaining or even slightly improving its safer genome-wide integration profile. The SB200X transposase could enhance the utility of SB transposon-mediated genome engineering in preclinical and clinical applications.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.

RNA editing by adenosine deaminases acting on RNA (ADARs) is an evolutionarily conserved posttranscriptional modification essential for organismal development and normal cell function. Three catalytically active ADARs are conserved in mammals: two isoforms of ADAR1 referred to as ADAR1-p150 and ADAR1-p110, as well as ADAR2. All recognize and edit double-stranded RNA (dsRNA) structures but demonstrate target specificity and selectivity that dictate the unique essential biological functions of the three enzymes. The editing activity of ADAR1-p150 suppresses autoimmune responses against self-RNA structures, whereas ADAR1-p110 and ADAR2 have other primary functions. To better understand the mechanism of target selection by ADARs, we developed TSniffer, which allows accurate de novo identification of edited transcripts and quantification of the extent of editing within each transcript. We found that 17-40% of protein coding transcripts in mice, ferrets, and humans are edited by ADARs. Individual transcripts can harbor hundreds and thousands of editing sites, mostly within inverted retrotransposable elements. For human transcripts, we found differential editing by ADAR1 and ADAR2, aligning with a supportive role for ADAR2, while some targets were dominantly edited by ADAR1. Relying only on RNA-seq data and reference genome, TSniffer represents a novel tool to decipher the role of ADAR editing in different physiological states including disease models. Its unbiased approach is suitable for any organism. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACTThe use of any semi-randomly integrating gene vector in a therapeutic setting is associated with genotoxic risks. The two major mechanisms of genotoxicity are disruption of a coding sequence (loss-of-function) or transcriptional upregulation of genes (gain-of-function) in the cellular genome where the genetic modifications are executed. A third, less widely recognized genotoxic risk stems from splice sites and polyadenylation sites within the vector sequences. These transcriptional elements may drive aberrant splicing and/or polyadenylation between transgene-contained and genomic sequences. A widely used promoter/enhance element present in gene vectors to ensure high transgene expression levels in mammalian cells is composed of a hybrid EF1α/HTLV-1 LTR, in which the retroviral LTR contains an intron. We assessed aberrant splicing initiated from the splice donor (SD) element present in the HTLV-1 LTR in CAR-T cells that had been engineered by either lentiviral vector (LV) orSleeping Beauty(SB) transposon-mediated gene transfer. We establish that the vector-contained canonical SD site gives rise to aberrantly spliced RNA species and thereby can cause misexpression of host gene segments that are involved in various host cell functions. This, potentially genotoxic, effect could be abrogated by mutating or completely eliminating the SD (or the entire intron) from the HTLV-1 LTR segment. CAR-T cells generated by the modified vectors are equally potent in efficiency of CAR-T cell manufacturing and in functionality. The simple genetic modifications that we describe here affecting vector design therefore enhance genomic safety while maintaining efficacy of gene-modified therapeutic cells.

LebensmittelchemieVolume 77, Issue S1 p. S1-133-S1-133 Aritlce Entwicklung von neuen nukleinsäurebasierten Reagenzien zum spezifischen Nachweis von Allergenen L. Schäfer, L. Schäfer Paul-Ehrlich-Institut, Allergologie, 63225 Langen, DeutschlandSearch for more papers by this authorC. Miskey, C. Miskey Paul-Ehrlich-Institut, Hämatologie, 63225 Langen, DeutschlandSearch for more papers by this authorS. Hein, S. Hein Paul-Ehrlich-Institut, Virologie, 63225 Langen, DeutschlandSearch for more papers by this authorE. Völker, E. Völker Paul-Ehrlich-Institut, Allergologie, 63225 Langen, DeutschlandSearch for more papers by this authorG. Mayer, G. Mayer Rheinische Friedrich-Wilhelms-Universität, Chemische Biologie & Medizinische Chemie, 53115 Bonn, DeutschlandSearch for more papers by this authorT. Holzhauser, T. Holzhauser Paul-Ehrlich-Institut, Allergologie, 63225 Langen, DeutschlandSearch for more papers by this author L. Schäfer, L. Schäfer Paul-Ehrlich-Institut, Allergologie, 63225 Langen, DeutschlandSearch for more papers by this authorC. Miskey, C. Miskey Paul-Ehrlich-Institut, Hämatologie, 63225 Langen, DeutschlandSearch for more papers by this authorS. Hein, S. Hein Paul-Ehrlich-Institut, Virologie, 63225 Langen, DeutschlandSearch for more papers by this authorE. Völker, E. Völker Paul-Ehrlich-Institut, Allergologie, 63225 Langen, DeutschlandSearch for more papers by this authorG. Mayer, G. Mayer Rheinische Friedrich-Wilhelms-Universität, Chemische Biologie & Medizinische Chemie, 53115 Bonn, DeutschlandSearch for more papers by this authorT. Holzhauser, T. Holzhauser Paul-Ehrlich-Institut, Allergologie, 63225 Langen, DeutschlandSearch for more papers by this author First published: 01 March 2023 https://doi.org/10.1002/lemi.202352103AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume77, IssueS1Supplement: Abstracts der Vorträge der Regionalverbände und die der Posterflashtalks der AG JLCMarch 2023Pages S1-133-S1-133 RelatedInformation

Transposons are nature’s gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random manner, which carries a risk of potential genotoxic effects associated with transposon integration. Here, we evaluated an experimental strategy to manipulate SB’s target site distribution by preferentially compartmentalizing the SB transposase to the nucleolus, which contains repetitive ribosomal RNA (rRNA) genes. We generated a fusion protein composed of the nucleolar protein nucleophosmin (B23) and the SB100X transposase, which was found to retain almost full transposition activity as compared to unfused transposase and to be predominantly localized to nucleoli in transfected human cells. Analysis of transposon integration sites generated by B23-SB100X revealed a significant enrichment into the p-arms of chromosomes containing nucleolus organizing regions (NORs), with preferential integration into the p13 and p11.2 cytobands directly neighboring the NORs. This bias in the integration pattern was accompanied by an enrichment of insertions into nucleolus-associated chromatin domains (NADs) at the periphery of nucleolar DNA and into lamina-associated domains (LADs). Finally, sub-nuclear targeting of the transposase resulted in preferential integration into chromosomal domains associated with the Upstream Binding Transcription Factor (UBTF) that plays a critical role in the transcription of 47S rDNA gene repeats of the NORs by RNA Pol I. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

ABSTRACT Lung immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for the ability to deal with infection. Using light sheet fluorescence microscopy of hamster lung slices in combination with virological, immunohistochemical, and RNA sequencing analyses, we show a wave of monocyte-derived macrophage (MDM) infiltration and the antiviral response which follows the spread of SARS-CoV-2 through the lungs and lead to virus elimination. These innate immune processes are related to the onset of necrotizing inflammatory and remodeling responses, which manifest as extensive cell death, vascular damage, and cell proliferation. We show that MDM appear in parallel with virus clearance and endothelial injury. Prothrombotic factor upregulation, tissue repair, and alveolar cell proliferation result in tissue remodeling, which is followed by fibrosis despite a decrease in inflammatory and antiviral activities. Although the lung tissue integrity is repaired, longer-term alterations of the lung arise as an outcome of concurrent tissue damage and regeneration processes. IMPORTANCE We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue. An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establishing persistence. Author Summary HCV infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms which underly persistence are incompletely defined. We utilized a long-term cell culture adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR T cell activity to prevent incoming graft rejection. Transient expression of an anti-CD117 CAR by mRNA conferred T cells the ability to eliminate CD117+ targets in vitro and in vivo. As an alternative approach, we used a Sleeping Beauty transposon vector for the generation of CAR T cells incorporating an inducible Caspase 9 safety switch. Stable CAR expression was associated with high proportion of T memory stem cells, low levels of exhaustion markers, and potent cellular cytotoxicity. Anti-CD117 CAR T cells mediated depletion of leukemic cells and healthy hematopoietic stem cells in NSG mice reconstituted with human leukemia or CD34+ cord blood cells, respectively, and could be terminated in vivo. The use of a non-viral technology to control CAR T cell pharmacokinetic properties is attractive for a first-in-human study in patients with acute myeloid leukemia prior to hematopoietic stem cell transplantation.