Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic--Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARYSARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

Monitoring SARS-CoV-2 infection using a double reporter-expressing virus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the highly contagious agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. An essential requirement for understanding SARS-CoV-2 fundamental biology and the impact of anti-viral therapeutics are robust methods to detect for the presence of the virus in infected cells or animal models. Despite the development and successful generation of recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter genes, knowledge acquired from their use in in vitro assays and/or in live animals are limited to the properties of the fluorescent or luciferase reporter genes. Herein, for the first time, we engineered a replication-competent rSARS-CoV-2 that expresses both fluorescent (mCherry) and luciferase (Nluc) reporter genes (rSARS-CoV-2/mCherry-Nluc) to overcome limitations associated with the use of a single reporter gene. In cultured cells, rSARS-CoV-2/mCherry-Nluc displayed similar viral fitness as rSARS-CoV-2 expressing single reporter fluorescent and luciferase genes (rSARS-CoV-2/mCherry and rSARS-CoV-2/Nluc, respectively), or wild-type (WT) rSARS-CoV-2, while maintaining comparable expression levels of both reporter genes. In vivo, rSARS-CoV-2/mCherry-Nluc has similar pathogenicity in K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice than rSARS-CoV-2 expressing individual reporter genes, or WT rSARS-CoV-2. Importantly, rSARS-CoV-2/mCherry-Nluc facilitates the assessment of viral infection and transmission in golden Syrian hamsters using in vivo imaging systems (IVIS). Altogether, this study demonstrates the feasibility of using this novel bireporter-expressing rSARS-CoV-2 for the study SARS-CoV-2 in vitro and in vivo. IMPORTANCEDespite the availability of vaccines and antivirals, the coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to ravage health care institutions worldwide. Previously, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter proteins to track viral infection in vitro and/or in vivo. However, these rSARS-CoV-2 are restricted to express only a single fluorescent or a luciferase reporter gene, limiting or preventing their use to specific in vitro assays and/or in vivo studies. To overcome this limitation, we have engineered a rSARS-CoV-2 expressing both fluorescent (mCherry) and luciferase (Nluc) genes and demonstrated its feasibility to study the biology of SARS-CoV-2 in vitro and/or in vivo, including the identification and characterization of neutralizing antibodies and/or antivirals. Using rodent models, we visualize SARS-CoV-2 infection and transmission through in vivo imaging systems (IVIS).

Using a reverse genetics system to generate recombinant SARS-CoV-2 expressing robust levels of reporter genes

Reporter-expressing recombinant virus represents an excellent option and a powerful tool to investigate, among others, viral infection, pathogenicity, and transmission, as well as to identify therapeutic compounds that inhibit viral infection and prophylactic vaccines. To combat the still ongoing coronavirus disease 2019 (COVID-19) pandemic, we have established a robust bacterial artificial chromosome (BAC)-based reverse genetics (RG) system to rapidly generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) to study the contribution of viral proteins in viral pathogenesis. In addition, we have also engineered reporter-expressing recombinant viruses in which we place the reporter genes upstream of the viral nucleocapsid (N) gene to promote high levels of reporter gene expression that facilitates the study of SARS-CoV-2 in vitro and in vivo. Although successful, the genetic manipulation of the BAC containing the entire SARS-CoV-2 genome of [~]30,000 nucleotides, is challenging. Herein, we depict the technical details to engineer rSARS-CoV-2 expressing reporter genes using the BAC-based RG approach. We describe i) assembly of the full-length (FL) SARS-CoV-2 genome sequences into the empty pBeloBAC, ii) verification of the pBeloBAC-FL, iii) cloning of a Venus reporter gene into the pBeloBAC-FL, and iv) recovery of the Venus-expressing rSARS-CoV-2. By following this protocol, researchers with basic molecular biology and gene engineering techniques knowledge will be able to generate wild-type and reporter-expressing rSARS-CoV-2.

Immunization with recombinant accessory protein-deficient SARS-CoV-2 protects against lethal challenge and viral transmission

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a worldwide Coronavirus Disease 2019 (COVID-19) pandemic. Despite high efficacy of the authorized vaccines, protection against the surging variants of concern (VoC) was less robust. Live-attenuated vaccines (LAV) have been shown to elicit robust and long-term protection by induction of host innate and adaptive immune responses. We sought to develop a COVID-19 LAV by generating 3 double open reading frame (ORF)-deficient recombinant (r)SARS-CoV-2 simultaneously lacking two accessory open reading frame (ORF) proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). Here, we report that these double ORF-deficient rSARS-CoV-2 have slower replication kinetics and reduced fitness in cultured cells as compared to their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2 showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against different SARS-CoV-2 VoC, and also activated viral component-specific T-cell responses. Notably, the double ORF-deficient rSARS-CoV-2 were able to protect, as determined by inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2. Collectively, our results demonstrate the feasibility to implement these double ORF-deficient rSARS-CoV-2 as safe, stable, immunogenic and protective LAV for the prevention of SARS-CoV-2 infection and associated COVID-19 disease.

Potent universal-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel {beta}-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human {beta}-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human {beta}-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for therapy

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

SARS-CoV-2 hijacks p38β/MAPK11 to promote viral protein translation

SARS-CoV-2, the causative agent of the COVID-19 pandemic, drastically modifies infected cells in an effort to optimize virus replication. Included is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammation and is a central driver of COVID-19 clinical presentations. Inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduces both cytokine production and viral replication. Here, we combined genetic screening with quantitative phosphoproteomics to better understand interactions between the p38/MAPK pathway and SARS-CoV-2. We found that several components of the p38/MAPK pathway impacted SARS-CoV-2 replication and that p38{beta} is a critical host factor for virus replication, and it prevents activation of the type-I interferon pathway. Quantitative phosphoproteomics uncovered several SARS-CoV-2 nucleocapsid phosphorylation sites near the N-terminus that were sensitive to p38 inhibition. Similar to p38{beta} depletion, mutation of these nucleocapsid residues was associated with reduced virus replication and increased activation of type-I interferon signaling. Taken together, this study reveals a unique proviral function for p38{beta} that is not shared with p38 and supports exploring p38{beta} inhibitor development as a strategy towards developing a new class of COVID-19 therapies. ImportanceSARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 mitogen-activated protein kinase (MAPK) pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.

A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 M against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, {beta}), Brazilian/Japanese variant P.1 (Gamma, {gamma}), and Californian (Epsilon, {varepsilon}), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

Visualization of SARS-CoV-2 infection dynamic

Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

4'-Fluorouridine is a broad-spectrum orally efficacious antiviral blocking respiratory syncytial virus and SARS-CoV-2 replication

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4-fluorouridine (4-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i+3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections. One-Sentence Summary4-Fluorouridine is an orally available ribonucleoside analog that efficiently treats RSV and SARS-CoV-2 infections in vivo.

SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy

SARS-CoV-2 variants of concern (VoCs) are impacting responses to the COVID-19 pandemic. Here we present a comparison of the SARS-CoV-2 USA-WA1/2020 (WA-1) strain with B.1.1.7 and B.1.351 VoCs and identify significant differences in viral propagation in vitro and pathogenicity in vivo using K18-hACE2 transgenic mice. Passive immunization with plasma from an early pandemic SARS-CoV-2 patient resulted in significant differences in the outcome of VoC-infected mice. WA-1-infected mice were protected by plasma, B.1.1.7-infected mice were partially protected, and B.1.351-infected mice were not protected. Serological correlates of disease were different between VoC-infected mice, with B.1.351 triggering significantly altered cytokine profiles than other strains. In this study, we defined infectivity and immune responses triggered by VoCs and observed that early 2020 SARS-CoV-2 human immune plasma was insufficient to protect against challenge with B.1.1.7 and B.1.351 in the mouse model.

Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF deficient ({Delta}ORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of {Delta}ORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the {Delta}ORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while {Delta}ORF7a, {Delta}ORF7b and {Delta}ORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCEDespite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.

The E484K mutation in the SARS-CoV-2 spike protein reduces but does not abolish neutralizing activity of human convalescent and post-vaccination sera.

One year in the coronavirus disease 2019 (COVID-19) pandemic, the first vaccines are being rolled out under emergency use authorizations. It is of great concern that newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can escape antibody-mediated protection induced by previous infection or vaccination through mutations in the spike protein. The glutamate (E) to Lysine (K) substitution at position 484 (E484K) in the receptor binding domain (RBD) of the spike protein is present in the rapidly spreading variants of concern belonging to the B.1.351 and P.1 lineages. We performed in vitro microneutralization assays with both the USA-WA1/2020 virus and a recombinant (r)SARS-CoV-2 virus that is identical to USA-WA1/2020 except for the E484K mutation introduced in the spike RBD. We selected 34 sera from study participants based on their SARS-CoV-2 spike ELISA antibody titer (negative [N=4] versus weak [N=8], moderate [N=11] or strong positive [N=11]). In addition, we included sera from five individuals who received two doses of the Pfizer SARS-CoV-2 vaccine BNT162b2. Serum neutralization efficiency was lower against the E484K rSARS-CoV-2 (vaccination samples: 3.4 fold; convalescent low IgG: 2.4 fold, moderate IgG: 4.2 fold and high IgG: 2.6 fold) compared to USA-WA1/2020. For some of the convalescent donor sera with low or moderate IgG against the SARS-CoV-2 spike, the drop in neutralization efficiency resulted in neutralization ID50 values similar to negative control samples, with low or even absence of neutralization of the E484K rSARS-CoV-2. However, human sera with high neutralization titers against the USA-WA1/2020 strain were still able to neutralize the E484K rSARS-CoV-2. Therefore, it is important to aim for the highest titers possible induced by vaccination to enhance protection against newly emerging SARS-CoV-2 variants. Two vaccine doses may be needed for induction of high antibody titers against SARS-CoV-2. Postponing the second vaccination is suggested by some public health authorities in order to provide more individuals with a primer vaccination. Our data suggests that this may leave vaccinees less protected against newly emerging variants.

Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab), but no vaccines, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. ImportanceSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

SARS-CoV-2 infection results in viral burden in the upper and lower respiratory tract, enabling transmission and often leading to substantial lung pathology. Delivering the antiviral treatment directly to the lungs has the potential to improve lung bioavailability and dosing efficiency. As the SARS-CoV-2 Receptor Binding Domain (RBD) of the Spike (S) is increasingly deemed to be a clinically validated target, RBD-specific B cells were isolated from patients following SARS-CoV-2 infection to derive a panel of fully human monoclonal antibodies (hmAbs) that potently neutralize SARS-CoV-2. The most potent hmAb, 1212C2 was derived from an IgM memory B cell, has high affinity for SARS-CoV-2 RBD which enables its direct inhibition of RBD binding to ACE2. The 1212C2 hmAb exhibits in vivo prophylactic and therapeutic activity against SARS-CoV-2 in hamsters when delivered intraperitoneally, achieving a meaningful reduction in upper and lower respiratory viral burden and lung pathology. Furthermore, liquid nebulized inhale treatment of SARS-CoV-2 infected hamsters with as low as 0.6 mg/kg of inhaled dose, corresponding to approximately 0.03 mg/kg of lung deposited dose, mediated a reduction in respiratory viral burden that is below the detection limit, and mitigated lung pathology. The therapeutic efficacy achieved at an exceedingly low-dose of inhaled 1212C2 supports the rationale for local lung delivery and achieving dose-sparing benefits as compared to the conventional parenteral route of administration. Taken together, these results warrant an accelerated clinical development of 1212C2 hmAb formulated and delivered via inhalation for the prevention and treatment of SARS-CoV-2 infection.

Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2

Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

Rescue of SARS-CoV-2 from a single bacterial artificial chromosome

An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.

Lethality of SARS-CoV-2 infection in K18 human angiotensin converting enzyme 2 transgenic mice

ABSTRACTVaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. Here, we show that transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2-transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2-transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 4. K18 hACE2-transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.