SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID

Neutralization of SARS-CoV-2 Variants by rVSV-ΔG-Spike-Elicited Human Sera

The emergence of rapidly spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major challenge to the ability of vaccines and therapeutic antibodies to provide immunity. These variants contain mutations of specific amino acids that might impede vaccine efficacy. BriLife® (rVSV-ΔG-spike) is a newly developed SARS-CoV-2 vaccine candidate currently in phase II clinical trials. It is based on a replication-competent vesicular stomatitis virus (VSV) platform. The rVSV-ΔG-spike contains several spontaneously acquired spike mutations that correspond to SARS-CoV-2 variants’ mutations. We show that human sera from BriLife® vaccinees preserve comparable neutralization titers towards alpha, gamma, and delta variants and show less than a three-fold reduction in the neutralization capacity of beta and omicron compared to the original virus. Taken together, we show that human sera from BriLife® vaccinees overall maintain a neutralizing antibody response against all tested variants. We suggest that BriLife®-acquired mutations may prove advantageous against future SARS-CoV-2 VOCs

Discriminative Identification of SARS-CoV-2 Variants Based on Mass-Spectrometry Analysis

The spread of SARS-CoV-2 variants of concern (VOCs) is of great importance since genetic changes may increase transmissibility, disease severity and reduce vaccine effectiveness. Moreover, these changes may lead to failure of diagnostic measures. Therefore, variant-specific diagnostic methods are essential. To date, genetic sequencing is the gold-standard method to discriminate between variants. However, it is time-consuming (taking several days) and expensive. Therefore, the development of rapid diagnostic methods for SARS-CoV-2 in accordance with its genetic modification is of great importance. In this study we introduce a Mass Spectrometry (MS)-based methodology for the diagnosis of SARS-CoV-2 in propagated in cell-culture. This methodology enables the universal identification of SARS-CoV-2, as well as variant-specific discrimination. The universal identification of SARS-CoV-2 is based on conserved markers shared by all variants, while the identification of specific variants relies on variant-specific markers. Determining a specific set of peptides for a given variant consists of a multistep procedure, starting with an in-silico search for variant-specific tryptic peptides, followed by a tryptic digest of a cell-cultured SARS-CoV-2 variant, and identification of these markers by HR-LC-MS/MS analysis. As a proof of concept, this approach was demonstrated for four representative VOCs compared to the wild-type Wuhan reference strain. For each variant, at least two unique markers, derived mainly from the spike (S) and nucleocapsid (N) viral proteins, were identified. This methodology is specific, rapid, easy to perform and inexpensive. Therefore, it can be applied as a diagnostic tool for pathogenic variants

Insights from the Infection Cycle of VSV-ΔG-Spike Virus

Fundamental key processes in viral infection cycles generally occur in distinct cellular sites where both viral and host factors accumulate and interact. These sites are usually termed viral replication organelles, or viral factories (VF). The generation of VF is accompanied by the synthesis of viral proteins and genomes and involves the reorganization of cellular structure. Recently, rVSV-ΔG-spike (VSV-S), a recombinant VSV expressing the SARS-CoV-2 spike protein, was developed as a vaccine candidate against SARS-CoV-2. By combining transmission electron microscopy (TEM) tomography studies and immuno-labeling techniques, we investigated the infection cycle of VSV-S in Vero E6 cells. RT-real-time-PCR results show that viral RNA synthesis occurs 3–4 h post infection (PI), and accumulates as the infection proceeds. By 10–24 h PI, TEM electron tomography results show that VSV-S generates VF in multi-lamellar bodies located in the cytoplasm. The VF consists of virus particles with various morphologies. We demonstrate that VSV-S infection is associated with accumulation of cytoplasmatic viral proteins co-localized with dsRNA (marker for RNA replication) but not with ER membranes. Newly formed virus particles released from the multi-lamellar bodies containing VF, concentrate in a vacuole membrane, and the infection ends with the budding of particles after the fusion of the vacuole membrane with the plasma membrane. In summary, the current study describes detailed 3D imaging of key processes during the VSV-S infection cycle

Induction of Innate Immune Response by TLR3 Agonist Protects Mice against SARS-CoV-2 Infection

SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection

Modeling SARS-CoV-2 Infection in Mice Using Lentiviral hACE2 Vectors Infers Two Modes of Immune Responses to SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a severe global pandemic. Mice models are essential to investigate infection pathology, antiviral drugs, and vaccine development. However, wild-type mice lack the human angiotensin-converting enzyme 2 (hACE2) that mediates SARS-CoV-2 entry into human cells and consequently are not susceptible to SARS-CoV-2 infection. hACE2 transgenic mice could provide an efficient COVID-19 model, but are not always readily available, and practically restricted to specific strains. Therefore, there is a dearth of additional mouse models for SARS-CoV-2 infection. We applied lentiviral vectors to generate hACE2 expression in interferon receptor knock-out (IFNAR1−/−) mice. Lenti-hACE2 transduction supported SARS-CoV-2 replication in vivo, simulating mild acute lung disease. Gene expression analysis revealed two modes of immune responses to SARS-CoV-2 infection: one in response to the exposure of mouse lungs to SARS-CoV-2 particles in the absence of productive viral replication, and the second in response to productive SARS-CoV-2 infection. Our results infer that immune response to immunogenic elements on incoming virus or in productively infected cells stimulate diverse immune effectors, even in absence of type I IFN signaling. Our findings should contribute to a better understanding of the immune response triggered by SARS-CoV-2 and to further elucidate COVID-19

Thermophilic Filamentous Fungus C1-Cell-Cloned SARS-CoV-2-Spike-RBD-Subunit-Vaccine Adjuvanted with Aldydrogel®85 Protects K18-hACE2 Mice against Lethal Virus Challenge

SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptors in vitro. Alhydrogel®‘85’-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates excellent safety profile in repeat-dose GLP toxicity study. In summary, subcutaneous prime/boost DYAI-100A85 inoculation induces high titers of RBD-specific neutralizing antibodies and protection of hACE2-transgenic mice against lethal challenge with SARS-CoV-2. Given its demonstrated safety, efficacy, and low production cost, vaccine candidate DYAI-100 received regulatory approval to initiate a Phase 1 clinical trial to demonstrate its safety and efficacy in humans

Correction: Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations.

[This corrects the article DOI: 10.1371/journal.pone.0155711.]