Optimized peptide nanofibrils as efficient transduction enhancers for in vitro and ex vivo gene transfer.

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications.

Cryo-EM structure and polymorphic maturation of a viral transduction enhancing amyloid fibril.

Amyloid fibrils have emerged as innovative tools to enhance the transduction efficiency of retroviral vectors in gene therapy strategies. In this study, we used cryo-electron microscopy to analyze the structure of a biotechnologically engineered peptide fibril that enhances retroviral infectivity. Our findings show that the peptide undergoes a time-dependent morphological maturation into polymorphic amyloid fibril structures. The fibrils consist of mated cross-ß sheets that interact by the hydrophobic residues of the amphipathic fibril-forming peptide. The now available structural data help to explain the mechanism of retroviral infectivity enhancement, provide insights into the molecular plasticity of amyloid structures and illuminate the thermodynamic basis of their morphological maturation.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

Negatively Charged Peptide Nanofibrils from Immunoglobulin Light Chain Sequester Viral Particles but Lack Cell-Binding and Viral Transduction-Enhancing Properties.

Positively charged naturally occurring or engineered peptide nanofibrils (PNF) are effective enhancers of lentiviral and retroviral transduction, an often rate-limiting step in gene transfer and gene therapy approaches. These polycationic PNF are thought to bridge the electrostatic repulsions between negatively charged membranes of virions and cells, thereby enhancing virion attachment to and infection of target cells. Here, we analyzed PNF, which are formed by the peptide AL1, that represents a fragment of an immunoglobulin light chain that causes systemic AL amyloidosis. We found that negatively charged AL1 PNF interact with viral particles to a comparable extent as positively charged PNF. However, AL1 PNF lacked cell-binding activity, and consequently, did not enhance retroviral infection. These findings show that virion capture and cell binding of PNF are mediated by different mechanisms, offering avenues for the design of advanced PNF with selective functions.

Carrageenan-containing over-the-counter nasal and oral sprays inhibit SARS-CoV-2 infection of airway epithelial cultures.

Pharmaceutical interventions are urgently needed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission. As SARS-CoV-2 infects and spreads via the nasopharyngeal airways, we analyzed the antiviral effect of selected nasal and oral sprays on virus infection in vitro. Two nose sprays showed virucidal activity but were cytotoxic precluding further analysis in cell culture. One nasal and one mouth spray suppressed SARS-CoV-2 infection of TMPRSS2-expressing Vero E6 cells and primary differentiated human airway epithelial cultures. The antiviral activity in both sprays could be attributed to polyanionic ι- and κ-carrageenans. Thus, application of carrageenan-containing nasal and mouth sprays may reduce the risk of acquiring SARS-CoV-2 infection and may limit viral spread, warranting further clinical evaluation.

Drug Inhibition of SARS-CoV-2 Replication in Human Pluripotent Stem Cell-Derived Intestinal Organoids.

BACKGROUND AND AIMS: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need. METHODS: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19. RESULTS: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology. CONCLUSIONS: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.

Peptide and peptide-based inhibitors of SARS-CoV-2 entry.

To date, no effective vaccines or therapies are available against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pandemic agent of the coronavirus disease 2019 (COVID-19). Due to their safety, efficacy and specificity, peptide inhibitors hold great promise for the treatment of newly emerging viral pathogens. Based on the known structures of viral proteins and their cellular targets, antiviral peptides can be rationally designed and optimized. The resulting peptides may be highly specific for their respective targets and particular viral pathogens or exert broad antiviral activity. Here, we summarize the current status of peptides inhibiting SARS-CoV-2 entry and outline the strategies used to design peptides targeting the ACE2 receptor or the viral spike protein and its activating proteases furin, transmembrane serine protease 2 (TMPRSS2), or cathepsin L. In addition, we present approaches used against related viruses such as SARS-CoV-1 that might be implemented for inhibition of SARS-CoV-2 infection.

An enzyme-based immunodetection assay to quantify SARS-CoV-2 infection.

SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These methods are laborious, time-consuming and expensive and therefore not suitable for a high sample throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. determine TCID50 of virus stocks, antiviral efficiencies (IC50 values) of drugs or neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and inhibition and may facilitate future research.

Distinct Dynamics of Stem and Progenitor Cells in Blood of Polytraumatized Patients.

Endogenously mobilized stem and progenitor cells (SPCs) or exogenously provided SPCs are thought to be beneficial for trauma therapy. However, still little is known about the synchronized dynamics of the number of SPCs in blood after severe injury and parameters like cytokine profiles that correlate with these numbers. We determined the number of hematopoietic stem cells, common myeloid progenitors, granulocyte-macrophage progenitors, and mesenchymal stem/stromal cells in peripheral blood (PB) 0 to 3, 8, 24, 48, and 120 h after polytrauma in individual patients (injury severity score ≥ 21). We found that the number of blood SPCs follows on average a synchronous, inverse bell-shaped distribution, with an increase at 0 to 3 h, followed by a strong decrease, with a nadir in SPC numbers in blood at 24 or 48 h. The change in numbers of SPCs in PB between 48 h and 120 h revealed two distinct patterns: Pattern 1 is characterized by an increase in the number of SPCs to a level higher than normal, pattern 2 is characterized by an almost absent increase in the number of SPCs compared to the nadir. Changes in the concentrations of the cytokines CK, MDC, IL-8, G-CSF Gro-α, VEGF, and MCP-1 correlated with changes in the number of SPCs in PB or were closely associated with Pattern 1 or Pattern 2. Our data provide novel rationale for investigations on the role of stem cell mobilization in polytraumatized patients and its likely positive impact on trauma outcome.

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation.

The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/ß-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/ß-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts.