Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231’s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231’s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models. Importance: The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19), a pandemic that has claimed over 700,000 human lives. The only SARS-CoV-2 antiviral, for emergency use, is remdesivir, targeting the viral polymerase complex. PF-00835231 is a pre-clinical lead compound with an alternate target, the main SARS-CoV-2 protease 3CLpro (Mpro). Here, we perform a comparative analysis of PF-00835231 and remdesivir in A549+ACE2 cells, using isolates of two major SARS-CoV-2 clades. PF-00835231 is antiviral for both clades, and, in this assay, statistically more potent than remdesivir. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps and validates PF-00835231’s time of action. Both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 in human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective antiviral for SARS-CoV-2, addresses concerns from non-human in vitro models, and supports further studies with this compound.
Both SARS-CoV-2 infection and vaccination elicit potent immune responses. A number of studies have described immune responses to SARS-CoV-2 infection. However, beyond antibody production, immune responses to COVID-19 vaccines remain largely uncharacterized. Here, we performed multimodal single-cell sequencing on peripheral blood of patients with acute COVID-19 and healthy volunteers before and after receiving the SARS-CoV-2 BNT162b2 mRNA vaccine to compare the immune responses elicited by the virus and by this vaccine. Phenotypic and transcriptional profiling of immune cells, coupled with reconstruction of the B and T cell antigen receptor rearrangement of individual lymphocytes, enabled us to characterize and compare the host responses to the virus and to defined viral antigens. While both infection and vaccination induced robust innate and adaptive immune responses, our analysis revealed significant qualitative differences between the two types of immune challenges. In COVID-19 patients, immune responses were characterized by a highly augmented interferon response which was largely absent in vaccine recipients. Increased interferon signaling likely contributed to the observed dramatic upregulation of cytotoxic genes in the peripheral T cells and innate-like lymphocytes in patients but not in immunized subjects. Analysis of B and T cell receptor repertoires revealed that while the majority of clonal B and T cells in COVID-19 patients were effector cells, in vaccine recipients clonally expanded cells were primarily circulating memory cells. Importantly, the divergence in immune subsets engaged, the transcriptional differences in key immune populations, and the differences in maturation of adaptive immune cells revealed by our analysis have far-ranging implications for immunity to this novel pathogen.
Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high-resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. We investigated the response of oligo-conjugated antibodies towards four variables: concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations causes unnecessarily high background and amount of antibody used can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background, and reduces costs. Background signal can account for a major fraction of total sequencing and is primarily derived from antibodies used at high concentrations. This study provides new insight into titration response and background of oligo-conjugated antibodies and offers concrete guidelines to improve such panels.
Simultaneous measurement of surface proteins and gene expression within single cells offers highresolution snapshots of complex cell populations. These methods rely on staining cells with oligoconjugated antibodies analogous to staining for flow-and mass cytometry. Unlike flow-and mass cytometry, signal from oligo-conjugated antibodies is not hampered by spectral overlap or limited by the number of metal isotopes, making it a highly sensitive and scalable approach. Signal from oligoconjugated antibodies is quantified by counting reads from high-throughput sequencing. Consequently, cost of sequencing is strictly dependent on the signal intensities and background from the pool of antibodies used in analysis. Thus, considering the "cost-of-signal" as well as optimizing "signal-tonoise", makes titration of oligo-conjugated antibody panels more complex and even more important than for flow-and mass cytometry. In this study, we investigated the titration response of a panel of oligo-conjugated antibodies towards four variables: Antibody concentration, staining volume, cell number at staining, and tissue of origin. We find that staining with high antibody concentrations recommended by published protocols and commercial vendors cause unnecessarily high background signal and that concentrations of many antibodies can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting highly abundant epitopes used at low concentrations and can be counteracted by reducing cell numbers at staining. We find that background signal from empty droplets can account for a major fraction of the total sequencing reads and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background signal of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved.
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