The Effect of Deuteration and Homologation of the Lactam Ring of Nirmatrelvir on Its Biochemical Properties and Oxidative Metabolism.

This study explores the relationship between structural alterations of nirmatrelvir, such as homologation and deuteration, and metabolic stability of newly synthesized derivatives. We developed a reliable synthetic protocol toward dideutero-nirmatrelvir and its homologated analogues with high isotopic incorporation. Deuteration of the primary metabolic site of nirmatrelvir provides a 3-fold improvement of its human microsomal stability but is accompanied by an increased metabolism rate at secondary sites. Homologation of the lactam ring allows the capping group modification to decrease and delocalize the molecule's lipophilicity, reducing the metabolic rate at secondary sites. The effect of deuteration was less pronounced for the 6-membered lactam than for its 5-membered analogue in human microsomes, but the trend is reversed in the case of mouse microsomes. X-ray data revealed that the homologation of the lactam ring favors the orientation of the drug's nitrile warhead for interaction with the catalytic sulfur of the SARS-CoV-2 Mpro, improving its binding. Comparable potency against SARS-CoV-2 Mpro from several variants of concern and selectivity over human cysteine proteases cathepsin B, L, and S was observed for the novel deuterated/homologated derivative and nirmatrelvir. Synthesized compounds displayed a large interspecies variability in hamster, rat, and human hepatocyte stability assays. Overall, we aimed to apply a rational approach in changing the physicochemical properties of the drug to refine its biochemical and biological parameters.

Analysis of SARS-CoV-2 isolates, namely the Wuhan strain, Delta variant, and Omicron variant, identifies differential immune profiles.

There is an urgent need to better understand the impact of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on immune response and disease dynamics to facilitate better intervention strategies. Here, we show that SARS-CoV-2 variants differentially affect host immune responses. The magnitude and quantity of cytokines and chemokines were comparable in those infected with the Wuhan strain and the Delta variant. However, individuals infected with the Omicron variant had significantly lower levels of these mediators. We also found an elevation of plasma galectins (Gal-3, Gal-8, and Gal-9) in infected individuals, in particular, in those with the original strain. Soluble galectins exert a proinflammatory role in COVID-19 pathogenesis. This was illustrated by their correlation with the plasma levels of sCD14, sCD163, enhanced TNF-α/IL-6 secretion, and increased SARS-CoV-2 infectivity in vitro. Moreover, we observed enhanced CD4+ and CD8+ T cell activation in Wuhan strain-infected individuals. Surprisingly, there was a more pronounced T cell activation in those infected with the Omicron in comparison to the Delta variant. In line with T cell activation status, we observed a more pronounced expansion of T cells expressing different co-inhibitory receptors in patients infected with the Wuhan strain, followed by the Omicron and Delta variants. Individuals infected with the Wuhan strain or the Omicron variant had a similar pattern of plasma soluble immune checkpoints. Our results imply that a milder innate immune response might be beneficial and protective in those infected with the Omicron variant. Our results provide a novel insight into the differential impact of SARS-CoV-2 variants on host immunity. IMPORTANCE There is a need to better understand how different SARS-CoV-2 variants influence the immune system and disease dynamics to facilitate the development of better vaccines and therapies. We compared immune responses in 140 SARS-CoV-2-infected individuals with the Wuhan strain, the Delta variant, or the Omicron variant. All these patients were admitted to the intensive care unit and were SARS-CoV-2 vaccination naïve. We found that SARS-CoV-2 variants differentially affect the host immune response. This was done by measuring soluble biomarkers in their plasma and examining different immune cells. Overall, we found that the magnitude of cytokine storm in individuals infected with the Wuhan strain or the Delta variant was greater than in those infected with the Omicron variant. In light of enhanced cytokine release syndrome in individuals infected with the Wuhan strain or the Delta variant, we believe that a milder innate immune response might be beneficial and protective in those infected with the Omicron variant.

Multiplex Assays Enable Simultaneous Detection and Identification of SARS-CoV-2 Variants of Concern in Clinical and Wastewater Samples.

The targeted screening and sequencing approaches for COVID-19 surveillance need to be adjusted to fit the evolving surveillance objectives which necessarily change over time. We present the development of variant screening assays that can be applied to new targets in a timely manner and enable multiplexing of targets for efficient implementation in the laboratory. By targeting the HV69/70 deletion for Alpha, K417N for Beta, K417T for Gamma, and HV69/70 deletion plus K417N for sub-variants BA.1, BA.3, BA.4, and BA.5 of Omicron, we achieved simultaneous detection and differentiation of Alpha, Beta, Gamma, and Omicron in a single assay. Targeting both T478K and P681R mutations enabled specific detection of the Delta variant. The multiplex assays used in combination, targeting K417N and T478K, specifically detected the Omicron sub-variant BA.2. The limits of detection for the five variants of concern were 4-16 copies of the viral RNA per reaction. Both assays achieved 100% clinical sensitivity and 100% specificity. Analyses of 377 clinical samples and 24 wastewater samples revealed the Delta variant in 100 clinical samples (nasopharyngeal and throat swab) collected in November 2021. Omicron BA.1 was detected in 79 nasopharyngeal swab samples collected in January 2022. Alpha, Beta, and Gamma variants were detected in 24 wastewater samples collected in May-June 2021 from two major cities of Alberta (Canada), and the results were consistent with the clinical cases of multiple variants reported in the community.

Challenge Inoculum for Hepatitis C Virus Controlled Human Infection Model.

For any controlled human infection model (CHIM), a safe, standardized, and biologically relevant challenge inoculum is necessary. For hepatitis C virus (HCV) CHIM, we propose that human-derived high-titer inocula of several viral genotypes with extensive virologic, serologic, and molecular characterizations should be the most appropriate approach. These inocula should first be tested in human volunteers in a step-wise manner to ensure safety, reproducibility, and curability prior to using them for testing the efficacy of candidate vaccines.

Characterization of Monoclonal Antibodies to Measure Cell Surface Protein Levels of Human Interferon-Lambda Receptor 1.

Type III interferons (IFN-lambdas, IFN-λs) are important antiviral cytokines that can also modulate immune responses by acting through a heterodimeric receptor composed of the specific and limited expressed IFN-λR1 chain and the ubiquitous IL-10R2 chain, which is shared with IL-10 family cytokines. Conflicting data have been reported regarding which cells express the IFN-λR1 subunit and directly respond to IFN-λs. This is, in part, owing to transcript levels of the IFN-λR1 gene, IFNLR1, not always correlating with cell surface protein levels. In this study, we tested a panel of novel monoclonal antibodies (mAbs) that specifically recognize human IFN-λR1. Initially, antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA), from which a subset of antibodies was selected for additional flow cytometry and neutralization assays. We further characterized two antibodies based on their strong ELISA binding activity (HLR1 and HLR14) and found only HLR14 could reliably detect cell surface IFN-λR1 protein on a variety of cell lines by flow cytometry. HLR14 could also detect IFN-λR1 protein on certain primary human blood cells, including plasmacytoid dendritic cells and B cells from peripheral blood. Availability of the HLR14 mAb will enable the quantification of IFN-λR1 protein levels on cells and better characterization of the cell specificity of the IFN-λ response.

Bovine Serum Albumin-Protected Gold Nanoclusters for Sensing of SARS-CoV-2 Antibodies and Virus.

An approach to assess severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (and past infection) was developed. For virus detection, the SARS-CoV-2 virus nucleocapsid protein (NP) was targeted. To detect the NP, antibodies were immobilized on magnetic beads to capture the NPs, which were subsequently detected using rabbit anti-SARS-CoV-2 nucleocapsid antibodies and alkaline phosphatase (AP)-conjugated anti-rabbit antibodies. A similar approach was used to assess SARS-CoV-2-neutralizing antibody levels by capturing spike receptor-binding domain (RBD)-specific antibodies utilizing RBD protein-modified magnetic beads and detecting them using AP-conjugated anti-human IgG antibodies. The sensing mechanism for both assays is based on cysteamine etching-induced fluorescence quenching of bovine serum albumin-protected gold nanoclusters where cysteamine is generated in proportion to the amount of either SARS-CoV-2 virus or anti-SARS-CoV-2 receptor-binding domain-specific immunoglobulin antibodies (anti-RBD IgG antibodies). High sensitivity can be achieved in 5 h 15 min for the anti-RBD IgG antibody detection and 6 h 15 min for virus detection, although the assay can be run in "rapid" mode, which takes 1 h 45 min for the anti-RBD IgG antibody detection and 3 h 15 min for the virus. By spiking the anti-RBD IgG antibodies and virus in serum and saliva, we demonstrate that the assay can detect the anti-RBD IgG antibodies with a limit of detection (LOD) of 4.0 and 2.0 ng/mL in serum and saliva, respectively. For the virus, we can achieve an LOD of 8.5 × 105 RNA copies/mL and 8.8 × 105 RNA copies/mL in serum and saliva, respectively. Interestingly, this assay can be easily modified to detect myriad analytes of interest.

Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection.

Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate the transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). While the sensitivity of human coronaviruses to IFNs has been characterized, antiviral roles of IRFs during human coronavirus infection are not fully understood. Type I or II IFN treatment protected MRC5 cells from human coronavirus 229E infection, but not OC43. Cells infected with 229E or OC43 upregulated ISGs, indicating that antiviral transcription is not suppressed. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of IRFs demonstrated that IRF1 and IRF3 have antiviral properties against OC43, while IRF3 and IRF7 are effective in restricting 229E infection. IRF3 activation effectively promotes transcription of antiviral genes during OC43 or 229E infection. Our study suggests that IRFs may be effective antiviral regulators against human coronavirus infection.

Utilization of a Glucometer Test Strip and Enzymatic Reactions to Quantify Anti-SARS-CoV-2 Spike RBD IgG Antibody and SARS-CoV-2 Virus in Saliva and Serum.

A sensor capable of quantifying both anti-SARS-CoV-2 spike receptor-binding domain (RBD) antibody levels and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva and serum was developed. This was accomplished by exploiting the enzymatic reaction of maltose and orthophosphate (PO43-) in the presence of maltose phosphorylase to generate an equivalent amount of glucose that was detected using a commercial glucometer test strip and a potentiostat. Important for this approach is the ability to generate PO43- in an amount that is directly related to the concentration of the analytes. RBD-modified magnetic microparticles were used to capture anti-SARS-CoV-2 spike RBD antibodies, while particles modified with anti-SARS-CoV-2 nucleocapsid antibodies were used to capture SARS-CoV-2 nucleocapsid protein from inactivated virus samples. A magnet was used to isolate and purify the magnetic microparticles (with analyte attached), and alkaline phosphatase-conjugated secondary antibodies were bound to the analytes attached to the respective magnetic microparticles. Finally, through enzymatic reactions, specific amounts of PO43- (and subsequently glucose) were generated in proportion to the analyte concentration, which was then quantified using a commercial glucometer test strip. Utilizing glucose test strips makes the sensor relatively inexpensive, with a cost per test of ∼US $7 and ∼US $12 for quantifying anti-SARS-CoV-2 spike RBD antibody and SARS-CoV-2, respectively. Our sensor exhibited a limit of detection of 0.42 ng/mL for anti-SARS-CoV-2 spike RBD antibody, which is sensitive enough to quantify typical concentrations of antibodies in COVID-19-infected or vaccinated individuals (>1 µg/mL). The limit of detection for the SARS-CoV-2 virus is 300 pfu/mL (5.4 × 106 RNA copies/mL), which exceeds the performance recommended by the WHO (500 pfu/mL). In addition, the sensor exhibited good selectivity when challenged with competing analytes and could be used to quantify analytes in saliva and serum matrices with an accuracy of >94% compared to RT-qPCR.

Interferon-λ treatment accelerates SARS-CoV-2 clearance despite age-related delays in the induction of T cell immunity.

Interferons induced early after SARS-CoV-2 infection are crucial for shaping immunity and preventing severe COVID-19. We previously demonstrated that injection of pegylated interferon-lambda accelerated viral clearance in COVID-19 patients (NCT04354259). To determine if the viral decline is mediated by enhanced immunity, we assess in vivo responses to interferon-lambda by single cell RNA sequencing and measure SARS-CoV-2-specific T cell and antibody responses between placebo and interferon-lambda-treated patients. Here we show that interferon-lambda treatment induces interferon stimulated genes in peripheral immune cells expressing IFNLR1, including plasmacytoid dendritic cells and B cells. Interferon-lambda does not affect SARS-CoV-2-specific antibody levels or the magnitude of virus-specific T cells. However, we identify delayed T cell responses in older adults, suggesting that interferon-lambda can overcome delays in adaptive immunity to accelerate viral clearance in high-risk patients. Altogether, interferon-lambda offers an early COVID-19 treatment option for outpatients to boost innate antiviral defenses without dampening peripheral adaptive immunity.

Differential Impact of SARS-CoV-2 Isolates, Namely, the Wuhan Strain, Delta, and Omicron Variants on Erythropoiesis.

SARS-CoV-2 variants exhibit different viral transmissibility and disease severity. However, their impact on erythropoiesis has not been investigated. Here, we show SARS-CoV-2 variants differentially affect erythropoiesis. This is illustrated by the abundance of CD71+ erythroid cells (CECs) in the blood circulation of COVID-19 patients infected with the original Wuhan strain followed by the Delta and Omicron variants. We observed the CD45+CECs are the dominant subpopulation of CECs expressing the receptor, ACE2, and coreceptor, TMPRSS2, and thus, can be targeted by SARS-CoV-2. Also, we found CECs exhibit immunosuppressive properties, specifically CD45+CECs are the dominant immunosuppressive cells and via reactive oxygen species (ROS) and arginase I expression can impair CD8+ T cell functions. In agreement, we observed CECs suppress CD8+ T cell effector (e.g., Granzyme B expression and degranulation capacity [CD107]), which was partially but significantly reversed with l-arginine supplementation. In light of the enriched frequency of CECs, in particular, CD45+CECs in patients infected with the original (Wuhan) strain, we believe this strain has a more prominent impact on hematopoiesis compared with the Delta and Omicron variants. Therefore, our study provides an important insight into the differential impact of SARS-CoV-2 variants on erythropoiesis in COVID-19 patients. IMPORTANCE Silent hypoxia has been the hallmark of SARS-CoV-2 infection. Red blood cells (RBCs) work as gas cargo delivering oxygen to different tissues. However, their immature counterparts reside in the bone marrow and normally absent in the blood circulation. We show SARS-CoV-2 infection is associated with the emergence of immature RBCs so called CD71+ erythroid cells (CECs) in the blood. In particular, we found these cells were more prevalent in the blood of those infected with the SARS-CoV-2 original strain (Wuhan) followed by the Delta and Omicron variants. This suggests SARS-CoV-2 directly or indirectly impacts RBC production. In agreement, we observed immature RBCs express the receptor (ACE2) and coreceptor (TMPRSS2) for SARS-CoV-2. CECs suppress T cells functions (e.g., Granzyme B and degranulation capacity) in vitro. Therefore, our study provides a novel insight into the differential impact of SARS-CoV-2 variants on erythropoiesis and subsequently the hypoxia commonly observed in COVID-19 patients.

Peptidomimetic α-Acyloxymethylketone Warheads with Six-Membered Lactam P1 Glutamine Mimic: SARS-CoV-2 3CL Protease Inhibition, Coronavirus Antiviral Activity, and in Vitro Biological Stability.

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e, 15h, and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.

On-Site Viral Inactivation and RNA Preservation of Gargle and Saliva Samples Combined with Direct Analysis of SARS-CoV-2 RNA on Magnetic Beads.

Samples of nasopharyngeal swabs (NPS) are commonly used for the detection of SARS-CoV-2 and diagnosis of COVID-19. As an alternative, self-collection of saliva and gargle samples minimizes transmission to healthcare workers and relieves the pressure of resources and healthcare personnel during the pandemic. This study aimed to develop an enhanced method enabling simultaneous viral inactivation and RNA preservation during on-site self-collection of saliva and gargle samples. Our method involves the addition of saliva or gargle samples to a newly formulated viral inactivation and RNA preservation (VIP) buffer, concentration of the viral RNA on magnetic beads, and detection of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction directly from the magnetic beads. This method has a limit of detection of 25 RNA copies per 200 µL of gargle or saliva sample and 9-111 times higher sensitivity than the viral RNA preparation kit recommended by the United States Centers for Disease Control and Prevention. The integrated method was successfully used to analyze more than 200 gargle and saliva samples, including the detection of SARS-CoV-2 in 123 gargle and saliva samples collected daily from two NPS-confirmed positive SARS-CoV-2 patients throughout the course of their infection and recovery. The VIP buffer is stable at room temperature for at least 6 months. SARS-CoV-2 RNA (65 copies/200 µL sample) is stable in the VIP buffer at room temperature for at least 3 weeks. The on-site inactivation of SARS-CoV-2 and preservation of the viral RNA enables self-collection of samples, reduces risks associated with SARS-CoV-2 transmission, and maintains the stability of the target analyte.

Peptidomimetic nitrile warheads as SARS-CoV-2 3CL protease inhibitors.

Tragically, the death toll from the COVID-19 pandemic continues to rise, and with variants being observed around the globe new therapeutics, particularly direct-acting antivirals that are easily administered, are desperately needed. Studies targeting the SARS-CoV-2 3CL protease, which is critical for viral replication, with different peptidomimetics and warheads is an active area of research for development of potential drugs. To date, however, only a few publications have evaluated the nitrile warhead as a viral 3CL protease inhibitor, with only modest activity reported. This article describes our investigation of P3 4-methoxyindole peptidomimetic analogs with select P1 and P2 groups with a nitrile warhead that are potent inhibitors of SARS-CoV-2 3CL protease and demonstrate in vitro SARS-CoV-2 antiviral activity. A selectivity for SARS-CoV-2 3CL protease over human cathepsins B, S and L was also observed with the nitrile warhead, which was superior to that with the aldehyde warhead. A co-crystal structure with SARS-CoV-2 3CL protease and a reversibility study indicate that a reversible, thioimidate adduct is formed when the catalytic sulfur forms a covalent bond with the carbon of the nitrile. This effort also identified efflux as a property limiting antiviral activity of these compounds, and together with the positive attributes described these results provide insight for further drug development of novel nitrile peptidomimetics targeting SARS-CoV-2 3CL protease.

Computational determination of toxicity risks associated with a selection of approved drugs having demonstrated activity against COVID-19.

BACKGROUND: The emergence and rapid spread of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in thelate 2019 has caused a devastating global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19). Although vaccines have been and are being developed, they are not accessible to everyone and not everyone can receive these vaccines. Also, it typically takes more than 10 years until a new therapeutic agent is approved for usage. Therefore, repurposing of known drugs can lend itself well as a key approach for significantly expediting the development of new therapies for COVID-19. METHODS: We have incorporated machine learning-based computational tools and in silico models into the drug discovery process to predict Adsorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiles of 90 potential drugs for COVID-19 treatment identified from two independent studies mainly with the purpose of mitigating late-phase failures because of inferior pharmacokinetics and toxicity. RESULTS: Here, we summarize the cardiotoxicity and general toxicity profiles of 90 potential drugs for COVID-19 treatment and outline the risks of repurposing and propose a stratification of patients accordingly. We shortlist a total of five compounds based on their non-toxic properties. CONCLUSION: In summary, this manuscript aims to provide a potentially useful source of essential knowledge on toxicity assessment of 90 compounds for healthcare practitioners and researchers to find off-label alternatives for the treatment for COVID-19. The majority of the molecules discussed in this manuscript have already moved into clinical trials and thus their known pharmacological and human safety profiles are expected to facilitate a fast track preclinical and clinical assessment for treating COVID-19.

SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1.

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.

Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA.

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

CRISPR Technique Incorporated with Single-Cell RNA Sequencing for Studying Hepatitis B Infection.

Single-cell RNA sequencing (scRNA-seq) provides rich transcriptomic information for studying molecular events and cell heterogeneity at the single-cell level. However, it is challenging to obtain sequence information from rare or low-abundance genes in the presence of other highly abundant genes. We report here a CRISPR-Cas9 technique for the depletion of high-abundance transcripts, resulting in preferential enrichment of rare transcripts. We demonstrate an application of this CRISPR-mediated enrichment technique to scRNA-seq of liver cells infected with hepatitis B virus (HBV). Direct sequencing without the CRISPR-mediated enrichment detected HBV RNA in only 0.6% of the cells. The CRISPR-mediated depletion of the three most abundant transcripts resulted in selective enrichment of the HBV transcript and successful sequencing of HBV RNA in more than 74% of the cells. The improvement enabled a study of HBV infection and interferon treatment of a liver cell model. Gene clusters between the control and HBV-infected Huh7.5-NTCP cells were similar, suggesting that HBV infection did not significantly alter gene expression of the host cells. The treatment with interferon alpha dramatically changed the gene expression of Huh7.5-NTCP cells. These results from the single cell RNA-seq analysis of 7370 cells are consistent with those of bulk experiments, suggesting that HBV is a "stealth virus".

CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR-Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR-Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR-Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR-Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR-Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR-Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.

Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies.

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.

Erythroid precursors and progenitors suppress adaptive immunity and get invaded by SARS-CoV-2.

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71+ erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45+ CD71+ cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.