A novel high-throughput microwell outgrowth assay for HIV-infected cells.

Although antiretroviral therapy (ART) is effective at suppressing HIV replication, a viral reservoir persists that can reseed infection if ART is interrupted. Curing HIV will require elimination or containment of this reservoir, but the size of the HIV reservoir is highly variable between individuals. To evaluate the size of the HIV reservoir, several assays have been developed, including PCR-based assays for viral DNA, the intact proviral DNA assay, and the quantitative viral outgrowth assay (QVOA). QVOA is the gold standard assay for measuring inducible replication-competent proviruses, but this assay is technically challenging and time-consuming. To begin progress toward a more rapid and less laborious tool for quantifying cells infected with replication-competent HIV, we developed the Microwell Outgrowth Assay, in which infected CD4 T cells are co-cultured with an HIV-detecting reporter cell line in a polydimethylsiloxane (PDMS)/polystyrene array of nanoliter-sized wells. Transmission of HIV from infected cells to the reporter cell line induces fluorescent reporter protein expression that is detected by automated scanning across the array. Using this approach, we were able to detect HIV-infected cells from ART-naïve people with HIV (PWH) and from PWH on ART with large reservoirs. Furthermore, we demonstrate that infected cells can be recovered from individual rafts and used to analyze the diversity of viral sequences. Although additional development and optimization will be required for quantifying the reservoir in PWH with small latent reservoirs, this assay may be a useful prototype for microwell assays of infected cells.IMPORTANCEMeasuring the size of the HIV reservoir in people with HIV (PWH) will be important for determining the impact of HIV cure strategies. However, measuring this reservoir is challenging. We report a new method for quantifying HIV-infected cells that involves culturing cells from PWH in an array of microwells with a cell line that detects HIV infection. We show that this approach can detect rare HIV-infected cells and derive detailed virus sequence information for each infected cell.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

The Rashomon Importance Distribution: Getting RID of Unstable, Single Model-based Variable Importance.

Quantifying variable importance is essential for answering high-stakes questions in fields like genetics, public policy, and medicine. Current methods generally calculate variable importance for a given model trained on a given dataset. However, for a given dataset, there may be many models that explain the target outcome equally well; without accounting for all possible explanations, different researchers may arrive at many conflicting yet equally valid conclusions given the same data. Additionally, even when accounting for all possible explanations for a given dataset, these insights may not generalize because not all good explanations are stable across reasonable data perturbations. We propose a new variable importance framework that quantifies the importance of a variable across the set of all good models and is stable across the data distribution. Our framework is extremely flexible and can be integrated with most existing model classes and global variable importance metrics. We demonstrate through experiments that our framework recovers variable importance rankings for complex simulation setups where other methods fail. Further, we show that our framework accurately estimates the true importance of a variable for the underlying data distribution. We provide theoretical guarantees on the consistency and finite sample error rates for our estimator. Finally, we demonstrate its utility with a real-world case study exploring which genes are important for predicting HIV load in persons with HIV, highlighting an important gene that has not previously been studied in connection with HIV. Code is available at https://github.com/jdonnelly36/Rashomon_Importance_Distribution.

A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells.

A long-lived latent reservoir of HIV-1-infected CD4 T cells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 T cells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory T cells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy.

Antiretroviral therapy (ART) halts HIV replication; however, cellular / immue cell viral reservoirs persist despite ART. Understanding the interplay between the HIV reservoir, immune perturbations, and HIV-specific immune responses on ART may yield insights into HIV persistence. A cross-sectional study of peripheral blood samples from 115 people with HIV (PWH) on long-term ART was conducted. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and the intact proviral DNA assay (IPDA) were performed. Total and intact HIV DNA was positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A Leave-One-Covariate-Out (LOCO) inference approach identified specific HIV reservoir and clinical-demographic parameters that were particularly important in predicting select immunophenotypes. Dimension reduction revealed two main clusters of PWH with distinct reservoirs. Additionally, machine learning approaches identified specific combinations of immune and clinical-demographic parameters that predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA. The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology.

Automated microarray platform for single-cell sorting and collection of lymphocytes following HIV reactivation.

A promising strategy to cure HIV-infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5%, 11.2%, and 12.1% percentage of cells, respectively. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single-cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found.

New Concepts in Therapeutic Manipulation of HIV-1 Transcription and Latency: Latency Reversal versus Latency Prevention.

Antiretroviral therapy (ART) has dramatically improved the prognosis for people living with HIV-1, but a cure remains elusive. The largest barrier to a cure is the presence of a long-lived latent reservoir that persists within a heterogenous mix of cell types and anatomical compartments. Efforts to eradicate the latent reservoir have primarily focused on latency reversal strategies. However, new work has demonstrated that the majority of the long-lived latent reservoir is established near the time of ART initiation, suggesting that it may be possible to pair an intervention with ART initiation to prevent the formation of a sizable fraction of the latent reservoir. Subsequent treatment with latency reversal agents, in combination with immune clearance agents, may then be a more tractable strategy for fully clearing the latent reservoir in people newly initiating ART. Here, we summarize molecular mechanisms of latency establishment and maintenance, ongoing efforts to develop effective latency reversal agents, and newer efforts to design latency prevention agents. An improved understanding of the molecular mechanisms involved in both the establishment and maintenance of latency will aid in the development of new latency prevention and reversal approaches to ultimately eradicate the latent reservoir.

Impact of Cannabis Use on Immune Cell Populations and the Viral Reservoir in People With HIV on Suppressive Antiretroviral Therapy.

BACKGROUND: Human immunodeficiency virus (HIV) infection remains incurable due to the persistence of a viral reservoir despite antiretroviral therapy (ART). Cannabis (CB) use is prevalent amongst people with HIV (PWH), but the impact of CB on the latent HIV reservoir has not been investigated. METHODS: Peripheral blood cells from a cohort of PWH who use CB and a matched cohort of PWH who do not use CB on ART were evaluated for expression of maturation/activation markers, HIV-specific T-cell responses, and intact proviral DNA. RESULTS: CB use was associated with increased abundance of naive T cells, reduced effector T cells, and reduced expression of activation markers. CB use was also associated with reduced levels of exhausted and senescent T cells compared to nonusing controls. HIV-specific T-cell responses were unaffected by CB use. CB use was not associated with intact or total HIV DNA frequency in CD4 T cells. CONCLUSIONS: This analysis is consistent with the hypothesis that CB use reduces activation, exhaustion, and senescence in the T cells of PWH, and does not impair HIV-specific CD8 T-cell responses. Longitudinal and interventional studies with evaluation of CB exposure are needed to fully evaluate the impact of CB use on the HIV reservoir.

Automated microarray for single-cell sorting and collection of lymphocytes following HIV reactivation.

A promising strategy to cure HIV infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of various LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5 %, 11.2 %, and 12.1 % percentage of cells respectively, reactivated similar to that observed in other experimental systems. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found. These results advance our conceptual understanding of HIV reactivation dynamics at the single-cell level toward a cure for HIV.

Depletion of HIV reservoir by activation of ISR signaling in resting CD4+T cells.

HIV reservoirs are extremely stable and pose a tremendous challenge to clear HIV infection. Here, we demonstrate that activation of ISR/ATF4 signaling reverses HIV latency, which also selectively eliminates HIV+ cells in primary CD4+T cell model of latency without effect on HIV-negative CD4+T cells. The reduction of HIV+ cells is associated with apoptosis enhancement, but surprisingly is largely seen in HIV-infected cells in which gag-pol RNA transcripts are detected in HIV RNA-induced ATF4/IFIT signaling. In resting CD4+ (rCD4+) T cells isolated from people living with HIV on antiretroviral therapy, induction of ISR/ATF4 signaling reduced HIV reservoirs by depletion of replication-competent HIV without global reduction in the rCD4+ T cell population. These findings suggest that compromised ISR/ATF4 signaling maintains stable and quiescent HIV reservoirs whereas activation of ISR/ATF4 signaling results in the disruption of latent HIV and clearance of persistently infected CD4+T cells.

A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency.

Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent-AZD5582, an activator of the non-canonical NFκB pathway-to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NFκB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.

Discovery and Optimization of Potent and Orally Available CTP Synthetase Inhibitors for Use in Treatment of Diseases Driven by Aberrant Immune Cell Proliferation.

Herein, we report the discovery of a first-in-class chemotype 2-(alkylsulfonamido)thiazol-4-yl)acetamides that act as pan-selective inhibitors of cytidine 5'-triphosphate synthetase (CTPS1/2), critical enzymes in the de novo pyrimidine synthesis pathway. Weak inhibitors identified from a high-throughput screening of 240K compounds have been optimized to a potent, orally active agent, compound 27, which has shown significant pharmacological responses at 10 mg/kg dose BID in a well-established animal model of inflammation.

Towards a comprehensive evaluation of dimension reduction methods for transcriptomic data visualization.

Dimension reduction (DR) algorithms project data from high dimensions to lower dimensions to enable visualization of interesting high-dimensional structure. DR algorithms are widely used for analysis of single-cell transcriptomic data. Despite widespread use of DR algorithms such as t-SNE and UMAP, these algorithms have characteristics that lead to lack of trust: they do not preserve important aspects of high-dimensional structure and are sensitive to arbitrary user choices. Given the importance of gaining insights from DR, DR methods should be evaluated carefully before trusting their results. In this paper, we introduce and perform a systematic evaluation of popular DR methods, including t-SNE, art-SNE, UMAP, PaCMAP, TriMap and ForceAtlas2. Our evaluation considers five components: preservation of local structure, preservation of global structure, sensitivity to parameter choices, sensitivity to preprocessing choices, and computational efficiency. This evaluation can help us to choose DR tools that align with the scientific goals of the user.

Combined noncanonical NF-κB agonism and targeted BET bromodomain inhibition reverse HIV latency ex vivo.

Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.

Crotonylation sensitizes IAPi-induced disruption of latent HIV by enhancing p100 cleavage into p52.

The eradication of HIV infection is difficult to achieve because of stable viral reservoirs. Here, we show that crotonylation enhances AZD5582-induced noncanonical NF-κB (ncNF-κB) signaling, further augmenting HIV latency reversal in Jurkat and U1 cell line models of latency, HIV latently infected primary CD4+ T cells and resting CD4+ T cells isolated from people living with HIV. Crotonylation upregulated the levels of the active p52 subunit of NF-κB following AZD5582. Biochemical analyses suggest that the ubiquitin E3 ligase TRIM27 is involved in enhanced p100 cleavage to p52. When TRIM27 was depleted, AZD5582-induced HIV latency reversal was reduced. TRIM27 small interfering RNA (siRNA) knockdown reduced both p100 and p52 levels without inhibiting p100 transcription, indicating that TRIM27 not only acts on p100 cleavage but also may impact p100/p52 stability. These observations reveal the complexity of HIV transcriptional machinery, particularly of NF-κB.

Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors.

Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency.

Epitope spreading toward wild-type melanocyte-lineage antigens rescues suboptimal immune checkpoint blockade responses.

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8+ T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.

SARS-CoV-2 infection is effectively treated and prevented by EIDD-2801.

All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.

The Role of Toll-Like Receptors in Retroviral Infection.

Toll-like receptors (TLRs) are key pathogen sensing receptors that respond to diverse microbial ligands, and trigger both innate and adaptive immune responses to infection. Since their discovery, a growing body of evidence has pointed to an important role for TLRs in retroviral infection and pathogenesis. These data suggest that multiple TLRs contribute to the anti-retroviral response, and that TLR engagement by retroviruses can have complex and divergent outcomes for infection. Despite this progress, numerous questions remain about the role of TLRs in retroviral infection. In this review, I summarize existing evidence for TLR-retrovirus interactions and the functional roles these receptors play in immunity and pathogenesis, with particular focus on human immunodeficiency virus (HIV).

Acute SARS-CoV-2 Infection is Highly Cytopathic, Elicits a Robust Innate Immune Response and is Efficiently Prevented by EIDD-2801.

All known recently emerged human coronaviruses likely originated in bats. Here, we used a single experimental platform based on human lung-only mice (LoM) to demonstrate efficient in vivo replication of all recently emerged human coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2) and two highly relevant endogenous pre-pandemic SARS-like bat coronaviruses. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats harbor endogenous coronaviruses capable of direct transmission into humans. Further detailed analysis of pandemic SARS-CoV-2 in vivo infection of LoM human lung tissue showed predominant infection of human lung epithelial cells, including type II pneumocytes present in alveoli and ciliated airway cells. Acute SARS-CoV-2 infection was highly cytopathic and induced a robust and sustained Type I interferon and inflammatory cytokine/chemokine response. Finally, we evaluated a pre-exposure prophylaxis strategy for coronavirus infection. Our results show that prophylactic administration of EIDD-2801, an oral broad spectrum antiviral currently in phase II clinical trials for the treatment of COVID-19, dramatically prevented SARS-CoV-2 infection in vivo and thus has significant potential for the prevention and treatment of COVID-19.