Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning (preprint)

The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we performed a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identified mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we showed that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.

Evidence of antigenic drift in the fusion machinery core of SARS-CoV-2 spike (preprint)

Antigenic drift of SARS-CoV-2 is typically defined by mutations in the N-terminal domain and receptor binding domain of spike protein. In contrast, whether antigenic drift occurs in the S2 domain remains largely elusive. Here, we perform a deep mutational scanning experiment to identify S2 mutations that affect binding of SARS-CoV-2 spike to three S2 apex public antibodies. Our results indicate that spatially diverse mutations, including D950N and Q954H, which are observed in Delta and Omicron variants, respectively, weaken the binding of spike to these antibodies. Although S2 apex antibodies are known to be non-neutralizing, we show that they confer partial protection in vivo. We further demonstrate that such in vivo protection activity is diminished by the natural mutation D950N. Overall, this study indicates that the S2 domain of SARS-CoV-2 spike can undergo antigenic drift, which represents a potential challenge for the development of more universal coronavirus vaccines.

The SARS-CoV-2 B.1.1.529 Omicron virus causes attenuated infection and disease in mice and hamsters (preprint)

Despite the development and deployment of antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. The recent emergence of B.1.1.529, the Omicron variant1,2, which has more than 30 mutations in the spike protein, has raised concerns for escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in pre-clinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of multiple B.1.1.529 Omicron isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2) expressing mice and hamsters. Despite modeling and binding data suggesting that B.1.1.529 spike can bind more avidly to murine ACE2, we observed attenuation of infection in 129, C57BL/6, and BALB/c mice as compared with previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. Although K18-hACE2 transgenic mice sustained infection in the lungs, these animals did not lose weight. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease, and pathology with B.1.1.529 also were milder compared to historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from multiple independent laboratories of the SAVE/NIAID network with several different B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

Intranasal immunization with a vaccinia virus vaccine vector expressing pre-fusion stabilized SARS-CoV-2 spike fully protected mice against lethal challenge with the heavily mutated mouse-adapted SARS2-N501YMA30 strain of SARS-CoV-2 (preprint)

The Omicron SARS-CoV-2 variant has been designated a variant of concern because its spike protein is heavily mutated. In particular, Omicron spike is mutated at 5 positions (K417, N440, E484, Q493 and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501Y MA30 , contains a spike that is also heavily mutated, with mutations at 4 of the 5 positions in Omicron spike associated with neutralizing antibody escape (K417, E484, Q493 and N501). In this manuscript we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against disease and death from SARS2-N501Y MA30 . Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus, administered without parenteral injection can fully protect against the heavily mutated mouse-adapted SARS2-N501Y MA30 .

Molecular switches regulating the potency and immune evasiveness of SARS-CoV-2 spike protein (preprint)

What enabled SARS-CoV-2, but not other coronaviruses, to cause a global pandemic? Here we investigated key structural determinants of the pandemic. Using SARS-CoV-1 and bat RaTG13-CoV as comparisons, we identified two molecular switches that regulate the conformations of SARS-CoV-2 spike protein: (i) a furin motif loop turns SARS-CoV-2 spike from a closed conformation to a mixture of open and closed conformations, and (ii) a K417V mutation turns SARS-CoV-2 spike from mixed conformations to an open conformation. We showed that the open conformation favors viral potency by exposing the RBD for receptor binding and viral entry, while the closed conformation supports viral immune evasion by hiding the RBD from neutralizing antibodies. Hence SARS-CoV-2 spike has evolved to reach a balance between potency and immune evasiveness, which contributes to the pandemic spread of SARS-CoV-2.The dynamics between viral potency and invasiveness is likely to further evolve, providing insights into future evolution of SARS-CoV-2.

Structure-Guided Design of Potent Inhibitors of SARS-CoV-2 3CL Protease: Structural, Biochemical, and Cell-Based Studies. (preprint)

We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe-Ingold effect, deuteration, and stereochemistry in the design of highly potent and non-toxic inhibitors of SARS-CoV-2 3CLpro to combat SARS-CoV-2 and emerging variants.

Structure-Guided Design of Conformationally-Constrained Cyclohexane Inhibitors of SARS-CoV-2 3CL Protease (preprint)

A series of non-deuterated and deuterated dipeptidyl aldehyde and masked aldehyde inhibitors that incorporate in their structure a conformationally-constrained cyclohexane moiety was synthesized and found to potently inhibit SARS-CoV-2 3CL protease in biochemical and cell-based assays.

Effective interferon (IFN)-lambda treatment regimen to control lethal MERS-CoV infection in mice (preprint)

Effective broad-spectrum antivirals are critical to prevent and control emerging human coronavirus (hCoV) infections. Despite considerable progress made towards identifying and evaluating several synthetic broad-spectrum antivirals against hCoV infections, a narrow therapeutic window has limited their success. Enhancing the endogenous interferon (IFN) and interferon-stimulated gene (ISG) response is another antiviral strategy known for decades. However, the side effects of pegylated type-I IFNs (IFN-Is) and the pro-inflammatory response detected after delayed IFN-I therapy have discouraged their clinical use. In contrast to IFN-Is, IFN-λ, a dominant IFN at the epithelial surface, is shown to be less pro-inflammatory. Consequently, we evaluated the prophylactic and therapeutic efficacy of IFN-λ in hCoV infected airway epithelial cells and mice. Human primary airway epithelial cells treated with a single dose of IFN-I (IFN-a) and IFN-λ showed similar ISG expression, whereas cells treated with two doses of IFN-λ expressed elevated levels of ISG compared to IFN-a treated cells. Similarly, mice treated with two dose IFN-λ were better protected compared to mice receiving a single dose, and a combination of prophylactic and delayed therapeutic regimens completely protected mice from lethal MERS-CoV- infection. A two dose IFN-λ regimen significantly reduced lung viral RNA and inflammatory cytokine levels with marked improvement in lung inflammation. Collectively, we identify an ideal regimen for IFN-λ use and demonstrate the protective efficacy of IFN-λ in MERS-CoV infected mice.

Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19 (preprint)

ABSTRACT Coronavirus disease 2019 (COVID-19) is especially severe in aged populations 1 . Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility 2 . The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC) 2 . Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD 2 ) and of a phospholipase, PLA 2 G2D, contributed to poor outcomes in aged mice 3,4 . Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD 2 receptor, or PLA 2 G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA 2 G2D-PGD 2 /DP1 pathway is a useful target for therapeutic interventions. (Words: 254)

Exponential increase in neutralizing and spike specific antibodies following vaccination of COVID-19 convalescent plasma donors (preprint)

BackgroundWith the recent approval of COVID-19 vaccines, recovered COVID-19 subjects who are vaccinated may be ideal candidates to donate COVID-19 convalescent plasma (CCP). Case SeriesThree recovered COVID-19 patients were screened to donate CCP. All had molecularly confirmed COVID-19, and all were antibody positive by chemiluminescence immunoassay (DiaSorin) prior to vaccination. All were tested again for antibodies 11 to 21 days after they received the first dose of the vaccine (Pfizer). All showed dramatic increases ([~]50 fold) in spike-specific antibody levels and had at least a 20-fold increase in the IC50 neutralizing antibody titer based on plaque reduction neutralization testing (PRNT). The spike-specific antibody levels following vaccination were significantly higher than those seen in any non-vaccinated COVID-19 subjects tested to date at our facility. ConclusionSpike-specific and neutralizing antibodies demonstrated dramatic increases following a single vaccination post COVID-19 infection which significantly exceeded values seen with COVID-19 infection alone. Recovered COVID-19 subjects who are vaccinated may make ideal candidates for CCP donation.

Post-infection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and was proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 hr post infection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

Development of a saliva-optimized RT-LAMP assay for SARS-CoV-2 (preprint)

Conventional reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology has struggled to fulfill the unprecedented need for diagnostic testing created by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Complexity and cost hinder access to testing, and long turnaround-time decreases its utility. To ameliorate these issues, we focus on saliva and introduce several advances to colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technology; RT-LAMP offers a minimal equipment alternative to RT-qPCR. First, we validated the use of the novel dye LAMPShade Violet (LSV), which improves the visual clarity and contrast of the colorimetric readout. Second, we compared different inactivation conditions on infectivity and RNA yield from saliva. Third, we developed a ten-minute RNA purification protocol from saliva. We call this magnetic bead protocol SalivaBeads. Finally, we developed a magnetic stick, StickLAMP, which provides reliable bead-based RNA purification as well as simple and low-cost access to scalable testing from saliva.

The Development of a Novel Nanobody Therapeutic for SARS-CoV-2 (preprint)

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD with a Kd of 15.7picomolar (~3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND50 of 0.16microgram/milliliter (~6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic.

K18-hACE2 Mice for Studies of COVID-19 Treatments and Pathogenesis Including Anosmia (preprint)

The ongoing COVID-19 pandemic is associated with substantial morbidity and mortality. While much has been learned in the first months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation and many patients with this finding show no or only minor respiratory signs. Studies in animals experimentally infected with SARS-CoV-2, the cause of COVID-19, provide opportunities to study aspects of the disease not easily investigated in human patients. COVID-19 severity ranges from asymptomatic to lethal. Most experimental infections provide insights into mild disease. Here, using K18-hACE2 mice that we originally developed for SARS studies, we show that infection with SARS-CoV-2 causes severe disease in the lung, and in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Further, we show that infusion of convalescent plasma (CP) from a recovered COVID-19 patient provided protection against lethal disease. Mice developed anosmia at early times after infection. Notably, while treatment with CP prevented significant clinical disease, it did not prevent anosmia. Thus K18-hACE2 mice provide a useful model for studying the pathological underpinnings of both mild and lethal COVID-19 and for assessing therapeutic interventions.

β-Coronaviruses use lysosomal organelles for cellular egress. (preprint)

{beta}-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). While much is known regarding their cellular entry and replication pathways, their mode of egress remains uncertain; however, this is assumed to be via the biosynthetic secretory pathway by analogy to other enveloped viruses. Using imaging methodologies in combination with virus-specific reporters, we demonstrate that {beta}-Coronaviruses utilize lysosomal trafficking for egress from cells. This pathway is regulated by the Arf-like small GTPase Arl8b; thus, virus egress is insensitive to inhibitors of the biosynthetic secretory pathway. Coronavirus infection results in lysosome deacidification, inactivation of lysosomal degradation and disruption of antigen presentation pathways. This coronavirus-induced exploitation of lysosomes provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

β-Coronaviruses Use Lysosomal Organelles for Cellular Egress (preprint)

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). While much is known regarding their cellular entry and replication pathways, their mode of egress remains uncertain; however, this is assumed to be via the biosynthetic secretory pathway by analogy to other enveloped viruses. Using imaging methodologies in combination with virus-specific reporters, we demonstrate that β-Coronaviruses utilize lysosomal trafficking for egress from cells. This pathway is regulated by the Arf-like small GTPase Arl8b; thus, virus egress is insensitive to inhibitors of the biosynthetic secretory pathway. Coronavirus infection results in lysosome deacidification, inactivation of lysosomal degradation and disruption of antigen presentation pathways. This coronavirus-induced exploitation of lysosomes provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.Funding: NAB, SG, TDR, EP, QQ, MF and CB were supported with NHLBI/NIH; GAB and SRA were supported with NCI/NIH intramural funds. PMT was supported by NIH R01 A1091985-05; SP by NIH R01 NS36592 and AF by F32-AI113973; VH by NIH R37GM058615; GW by NIH R01AI35270.Conflict of Interest: None.

Coronavirus Infection and PARP Expression Dysregulate the NAD Metabolome: A Potentially Actionable Component of Innate Immunity (preprint)

ABSTRACTInnate immune responses are critical to control of viruses. Coronaviruses (CoVs) are positive strand RNA viruses with double-stranded RNA replication intermediates that are recognized by the innate immune system. Upon recognition, infected cells secrete interferon, which induces a set of interferon-stimulated genes that inhibit viral replication. Here we show that SARS-CoV-2 infection strikingly dysregulates the set of genes involved in consumption and biosynthesis of nicotinamide adenine dinucleotide (NAD). Highly induced genes include those encoding noncanonical poly(ADP-ribose) polymerase (PARP) family members known to function as mono ADP-ribosyltransferases but not known to deplete cellular NAD and genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR). We demonstrate that overexpression of PARP10 is sufficient to depress NAD levels and that the enzymatic activities of PARP10, PARP12 and PARP14 are limited by and can be enhanced by pharmacological activation of NAM salvage. We further showed that infection with the β-coronavirus murine hepatitis virus (MHV) induces a severe attack on host cell NAD+ and NADP+. Finally we show that NAMPT activation, NAM and NR dramatically decrease replication in a MHV infection model that is sensitive to PARP activity. The data show that the antiviral activities of noncanonical PARP isozyme activities are limited by their own consumption of cellular NAD and that nutritional and pharmacological interventions to enhance NAD-based defenses may boost innate immunity.Competing Interest StatementCB is chief scientific adviser of ChromaDex and owns shares of ChromaDex stock. CB, SAJT, SP and ARF applied for a patent on uses of NAD boosting compounds to protect against CoVs. Others declare no competing interests.View Full Text