Biology subjects

Muench, J.

Publications and source records attributed to Muench, J..

10 recordsLinked to original sources

bioRxiv2020

A novel humanized mouse model to study mucosal HIV-1 transmission and prevention

Humanized mice have been critical for HIV-1 research, but are inefficient at mucosal HIV-1 transmission. We present a fetal tissue-independent model called CD34T+ with enhanced human leukocyte levels in the blood and improved T cell homing to the gut-associated lymphoid tissue. CD34T+ mice are highly permissive to intra-rectal HIV-1 infection, which can be prevented by infusion of broadly neutralizing antibodies. Therefore, CD34T+ mice provide a novel platform for mucosal HIV-1 transmission and prevention studies.

immunology↗

bioRxiv2020

An optimized derivative of an endogenous CXCR4 antagonist prevents atopic dermatitis and airway inflammation

BackgroundAberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes including chronic inflammatory diseases. Thus, the chemokine receptor CXCR4 is a promising target for the therapy of inflammatory disorders, such as atopic dermatitis or allergic asthma. A natural fragment of serum albumin, named EPI-X4, has previous been identified as endogenous peptide antagonist and inverse agonist of CXCR4. The endogenous CXCR4 antagonist provides a promising basis for the development of improved analogues for the therapy of inflammatory diseases. ObjectiveTo increase the anti-CXCR4 activity of EPI-X4 and to evaluate the therapeutic potential of optimized analogs in mouse models of atopic dermatitis and asthma. MethodsMolecular docking analysis of the interaction of EPI-X4 with CXCR4 was performed to define critical interaction motifs and to rationally design analogs with increased activity. EPI-X4 derivatives were synthesized and CXCR4 binding and antagonizing activity determined in assays for antibody competition, inhibition of CXCR4-mediated HIV-1 infection, CXCL12-dependent Ca2+ mobilization, ERK and AKT phosphorylation and cell migration. Toxicity of peptides was evaluated in zebrafish embryos. The therapeutic efficacy of the lead peptide EPI-X4 JM#21 was determined in mouse models of atopic dermatitis and asthma. ResultsDocking analysis identified key interaction motifs of EPI-X4/CXCR4. Rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4 and resulted in the generation of the lead analog JM#21, which bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. JM#21 did not exert toxic effects in zebrafish embryos and efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Moreover, EPI-X4 and its improved derivative suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model. ConclusionThe rationally designed EPI-X4 derivative JM#21 is a potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2020

IFITM proteins promote SARS-CoV-2 infection in human lung cells

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine 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 entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.

microbiology↗

bioRxiv2020

Microtiter plate-based antibody-competition assay to determine binding affinities and plasma/blood stability of CXCR4 ligands

C-X-C chemokine receptor type 4 (CXCR4) is involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis, asthma and pulmonary fibrosis. Thus, CXCR4 represents a promising drug target and several CXCR4 antagonizing agents are in preclinical or clinical development. Important parameters in drug lead evaluation are determination of binding affinities to the receptor and assessment of their stability and activity in plasma or blood of animals and humans. Here, we designed a microtiter plate-based CXCR4 antibody competition assay that enables to measure inhibitory concentrations (IC50 values) and affinity constants (Ki values) of CXCR4 targeting drugs. The assay is based on the observation that most if not all CXCR4 antagonists compete with binding of the fluorescence-tagged CXCR4 antibody 12G5 to the receptor. We demonstrate that this antibody-competition assay allows a convenient and cheap determination of binding affinities of various CXCR4 antagonists in living cells within just 3 hours. Moreover, the assay can be performed in the presence of high concentrations of physiologically relevant body fluids, and thus is a useful readout to evaluate stability (i.e. half-life) of CXCR4 ligands in serum/plasma, and even whole human and mouse blood ex vivo. Thus, this optimized 12G5 antibody competition assay allows a robust and convenient determination and calculation of various important pharmacological parameters of CXCR4 receptor-drug interaction and may not only foster future drug development but also animal welfare by reducing the number of experimental animals.

immunology↗

bioRxiv2020

A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases

Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once--enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.

synthetic biology↗

bioRxiv2020

Alpha-1 antitrypsin inhibits SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). To identify factors of the respiratory tract that suppress SARS-CoV-2, we screened a peptide/protein library derived from bronchoalveolar lavage, and identified 1-antitrypsin (1-AT) as specific inhibitor of SARS-CoV-2. 1-AT targets the viral spike protein and blocks SARS-CoV-2 infection of human airway epithelium at physiological concentrations. Our findings show that endogenous 1-AT restricts SARS-CoV-2 and repurposes 1-AT-based drugs for COVID-19 therapy.

microbiology↗

bioRxiv2020

Holder Pasteurization Inactivates SARS-CoV-2 in Human Breast Milk

SARS-CoV-2 RNA has been detected in the human breast milk of infected mothers, raising concerns regarding the safety of breastfeeding upon infection. We here show that holder pasteurization inactivates SARS-CoV-2 and provides an alternative and safe option for infected mothers to continue feeding breast milk to their infants.

microbiology↗

bioRxiv2020

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. HighlightsO_LIDetermination of SARS-CoV-2 infection by enzymatically quantifying the expression of viral spike protein in bulk cell cultures C_LIO_LITargeting a highly conserved region in the S2 subunit of the S protein allows broad detection of several SARS-CoV-2 isolates in different cell lines C_LIO_LIScreening of antivirals in microtiter format and determining the antiviral activity as inhibitory concentrations 50 (IC50) C_LI

microbiology↗

bioRxiv2020

Remdesivir but not famotidine inhibits SARS-CoV-2 replication in human pluripotent stem cell-derived intestinal organoids

Gastrointestinal symptoms in COVID-19 are associated with prolonged symptoms and increased severity. We employed human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) to analyze SARS-CoV-2 pathogenesis and to validate efficacy of specific drugs in the gut. Certain, but not all cell types in PSC-HIOs express SARS-CoV-2 entry factors ACE2 and TMPRSS2, rendering them susceptible to SARS-CoV-2 infection. Remdesivir, a promising drug to treat COVID-19, effectively suppressed SARS-CoV-2 infection of PSC-HIOs. In contrast, the histamine-2-blocker famotidine showed no effect. Thus, PSC-HIOs provide an interesting platform to study SARS-CoV-2 infection and to identify or validate drugs.

microbiology↗

bioRxiv2020

The Zinc Finger Antiviral Protein restricts SARS-CoV-2

Recent evidence shows that the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly sensitive to interferons (IFNs). However, the underlying antiviral effectors remain to be defined. Here, we show that Zinc finger antiviral protein (ZAP) that specifically targets CpG dinucleotides in viral RNA sequences restricts SARS-CoV-2. We demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Strikingly, SARS-CoV-2 and its closest relatives from bats show the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, knock-down of ZAP significantly increased SARS-CoV-2 production in lung cells, particularly upon treatment with IFN- or IFN-{gamma}. Thus, our results identify ZAP as an effector of the IFN response against SARS-CoV-2, although this pandemic pathogen may be preadapted to the low CpG environment in humans. HighlightsO_LISARS-CoV-2 and its closest bat relatives show strong CpG suppression C_LIO_LIIFN-{beta}, -{gamma} and -{lambda} inhibit SARS-CoV-2 with high efficiency C_LIO_LIZAP restricts SARS-CoV-2 and contributes to the antiviral effect of IFNs C_LI

microbiology↗