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Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad-spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS-CoV, MERS-CoV, hCoV-229E, SARS-CoV-2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS-CoV-2. Thus, AT is an endogenous inhibitor of SARS-CoV-2 that may be involved in COVID-19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti-TMPRSS2 and anti-SARS-CoV-2 activity of AT, suggesting that repurposing of native and activated AT for COVID-19 treatment should be explored.

The COVID-19 pandemic caused by SARS-CoV-2 presents a global health emergency. Therapeutic options against SARS-CoV-2 are still very limited but urgently required. Molecular tweezers are supramolecular agents that destabilize the envelope of viruses resulting in a loss of viral infectivity. Here, we show that first-generation tweezers, CLR01 and CLR05, disrupt the SARS-CoV-2 envelope and abrogate viral infectivity. To increase the antiviral activity, a series of 34 advanced molecular tweezers were synthesized by insertion of aliphatic or aromatic ester groups on the phosphate moieties of the parent molecule CLR01. A structure-activity relationship study enabled the identification of tweezers with a markedly enhanced ability to destroy lipid bilayers and to suppress SARS-CoV-2 infection. Selected tweezer derivatives retain activity in airway mucus and inactivate the SARS-CoV-2 wildtype and variants of concern as well as respiratory syncytial, influenza, and measles viruses. Moreover, inhibitory activity of advanced tweezers against respiratory syncytial virus and SARS-CoV-2 was confirmed in mice. Thus, potentiated tweezers are broad-spectrum antiviral agents with great prospects for clinical development to combat highly pathogenic viruses. CA Sanchez-Garcia und CA Schrader und CA extern

The COVID-19 pandemic caused by the β-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax – a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD – induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

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.

Human semen contains various amyloidogenic peptides derived from Prostatic Acid Phosphatase (PAP) and Semenogelin proteins that are capable of enhancing HIV-1 infection when assembled into fibrils. The best characterized among them is a 39 amino acid peptide PAP(248–286), which forms amyloid fibrils termed SEVI (semen-derived enhancer of viral infection) that increase the infectivity of HIV-1 by orders of magnitude. Inhibiting amyloid formation by PAP(248–286) may mitigate the sexual transmission of HIV-1. Several vitamins have been shown to reduce the aggregation of amyloids such as Aβ, α-Synuclein, and Tau, which are associated with neurodegenerative diseases. Since ascorbic acid (AA, vitamin C) is the most abundant vitamin in semen with average concentrations of 0.4 mM, we here examined how AA affects PAP(248–286) aggregation in vitro. Using ThT binding assays, transmission electron microscopy, and circular dichroism spectroscopy, a dual and concentration-dependent behavior of AA in modulating PAP(248–286) fibril formation was observed. We found that low molar ratios of AA:PAP(248–286) promoted whereas high molar ratios inhibited PAP(248–286) fibril formation. Accordingly, PAP(248–286) aggregated in the presence of low amounts of AA enhanced HIV-1 infection, whereas excess amounts of AA during aggregation reduced the infectivity enhancing effect in cell culture. Collectively, this work provides a biophysical insight into the effect of AA, an important seminal component, on SEVI fibrillation which might impact amyloid formation kinetics, thereby modulating the biological activity of semen amyloids., Human semen contains various amyloidogenic peptides derived from Prostatic Acid Phosphatase (PAP) and Semenogelin proteins that are capable of enhancing HIV-1 infection when assembled into fibrils.

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.

The current SARS-CoV-2 pandemic has triggered the development of various SARS-CoV-2 neutralization tests. A wild-type virus (using African green monkey VeroE6 cells), a pseudovirus (using human Caco-2 cells), and a surrogate neutralization test platform were applied to characterize the SARS-CoV-2 neutralization potential of a cohort of 111 convalescent plasma donors over a period of seven months after diagnosis. This allowed an in-depth validation and assay performance analysis of these platforms. More importantly, we found that SARS-CoV-2 neutralization titers were stable or even increased within the observation period, which contradicts earlier studies reporting a rapid waning of Ab titers after three to four months. Moreover, we observed a positive correlation of neutralization titers with increasing age, number of symptoms reported, and the presence of the Rhesus Ag RhD. Validation of the platforms revealed that highest assay performances were obtained with the wild-type virus and the surrogate neutralization platforms. However, our data also suggested that selection of cutoff titers had a strong impact on the evaluation of neutralization potency. When taking strong neutralization potency, as demonstrated by the wild-type virus platform as the gold standard, up to 55% of plasma products had low neutralization titers. However, a significant portion of these products were overrated in their potency when using the surrogate assay with the recommended cutoff titer. In summary, our study demonstrates that SARS-CoV-2 neutralization titers are stable for at least seven months after diagnosis and offers a testing strategy for rapid selection of high-titer convalescent plasma products in a biosafety level 1 environment.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the “phosphate tweezer” CLR01, a “carboxylate tweezer” CLR05, and a “phosphate clip” PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) restrict numerous viral pathogens and are thought to prevent infection by severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudoparticle infection of cells artificially overexpressing IFITMs. Here, we confirmed that overexpression of IFITMs blocks pseudoparticle infections mediated by the Spike proteins of β-coronaviruses including pandemic SARS-CoV-2. In striking contrast, however, endogenous IFITM expression promoted genuine SARS-CoV-2 infection in human lung cells both in the presence and absence of interferon. IFITM2 was most critical for efficient entry of SARS-CoV-2 and enhanced virus production from Calu-3 cells by several orders of magnitude. IFITMs are expressed and further induced by interferons in the lung representing the primary site of SARS-CoV-2 infection as well as in other relevant tissues. Our finding that IFITMs enhance SARS-CoV-2 infection under conditions approximating the in vivo situation shows that they may promote viral invasion during COVID-19. HIGHLIGHTS Overexpression of IFITM1, 2 and 3 restricts SARS-CoV-2 infection Endogenous IFITM1, 2 and 3 boost SARS-CoV-2 infection of human lung cells IFITM2 is critical for efficient entry of SARS-CoV-2 in Calu-3 cells

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 α1-antitrypsin (α1-AT) as specific inhibitor of SARS-CoV-2. α1-AT targets the viral spike protein and blocks SARS-CoV-2 infection of human airway epithelium at physiological concentrations. Our findings show that endogenous α1-AT restricts SARS-CoV-2 and repurposes α1-AT-based drugs for COVID-19 therapy.

Prokaryotic adaptive immunity is established against mobile genetic elements (MGEs) by ‘naïve adaptation’ when DNA fragments from a newly encountered MGE are integrated into CRISPR–Cas systems. In Escherichia coli, DNA integration catalyzed by Cas1–Cas2 integrase is well understood in mechanistic and structural detail but much less is known about events prior to integration that generate DNA for capture by Cas1–Cas2. Naïve adaptation in E. coli is thought to depend on the DNA helicase-nuclease RecBCD for generating DNA fragments for capture by Cas1–Cas2. The genetics presented here show that naïve adaptation does not require RecBCD nuclease activity but that helicase activity may be important. RecA loading by RecBCD inhibits adaptation explaining previously observed adaptation phenotypes that implicated RecBCD nuclease activity. Genetic analysis of other E. coli nucleases and naïve adaptation revealed that 5′ ssDNA tailed DNA molecules promote new spacer acquisition. We show that purified E. coli Cas1–Cas2 complex binds to and nicks 5′ ssDNA tailed duplexes and propose that E. coli Cas1–Cas2 nuclease activity on such DNA structures supports naïve adaptation.

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax—a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)—induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started., Graphical abstract, We report the development, characterization, and preclinical evaluation of COVID-eVax, a novel COVID-19 vaccine candidate with improved supply and logistics profiles. The technology is based on the electroporation of engineered, synthetic cDNA encoding a secreted monomeric form of the receptor-binding domain of the SARS-CoV-2 spike protein.