Medical Microbiology & Immunology

Welcome to the Department of Medical Microbiology & Immunology. We are a research-intensive department focusing on advanced studies in Bacteriology, Immunology, Virology, and the interface between these fields. We hope you enjoy your visit and if you want to have a look inside, please click the 3D Virtual Tour logo in the links panel.
Dr. Matthias Gotte (Chair)

Dr. Matthias Götte
Chair of MMI


 Covid-19 papers from MMI-affiliated faculty & trainees

Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit
J. Infect Dis. 2021 Jun 10

Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit

Matthew D Hall 1James M Anderson 2Annaliesa Anderson 3David Baker 4Jay Bradner 5Kyle R Brimacombe 1Elizabeth A Campbell 6Kizzmekia S Corbett 7Kara Carter 8Sara Cherry 9Lillian Chiang 10Tomas Cihlar 11Emmie de Wit 12Mark Denison 13Matthew Disney 14Courtney V Fletcher 15Stephanie L Ford-Scheimer 1Matthias Götte 16Abigail C Grossman 1Frederick G Hayden 17Daria J Hazuda 18Charlotte A Lanteri 19Hilary Marston 20Andrew D Mesecar 21Stephanie Moore 22Jennifer O Nwankwo 23Jules O'Rear 24George Painter 25Kumar Singh Saikatendu 26Celia A Schiffer 27Timothy P Sheahan 28Pei-Yong Shi 29Hugh D Smyth 30Michael J Sofia 31Marla Weetall 32Sandra K Weller 33Richard Whitley 22Anthony S Fauci 20Christopher P Austin 1Francis S Collins 2Anthony J Conley 20Mindy I Davis 20

Abstract

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on November 6, 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for COVID-19, including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss SARS-CoV-2 targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

SARS-CoV-2 Nonstructural Protein 1 Inhibits the Interferon Response by Causing Depletion of Key Host Signaling Factors
J Virol. 2021 Jun 10;95(13):

SARS-CoV-2 Nonstructural Protein 1 Inhibits the Interferon Response by Causing Depletion of Key Host Signaling Factors

Anil Kumar # 1Ray Ishida # 2Tania Strilets # 2Jamie Cole 1Joaquin Lopez-Orozco 3Nawell Fayad 1Alberto Felix-Lopez 2Mohamed Elaish 1Danyel Evseev 4Katharine E Magor 4Lara K Mahal 5Les P Nagata 6David H Evans 2 7Tom C Hobman 1 2 7

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. While previous studies have shown that several SARS-CoV-2 proteins can antagonize the interferon (IFN) response, some of the mechanisms by which they do so are not well understood. In this study, we describe two novel mechanisms by which SARS-CoV-2 blocks the IFN pathway. Type I IFNs and IFN-stimulated genes (ISGs) were poorly induced during SARS-CoV-2 infection, and once infection was established, cells were highly resistant to ectopic induction of IFNs and ISGs. Levels of two key IFN signaling pathway components, Tyk2 and STAT2, were significantly lower in SARS-CoV-2-infected cells. Expression of nonstructural protein 1 (NSP1) or nucleocapsid in the absence of other viral proteins was sufficient to block IFN induction, but only NSP1 was able to inhibit IFN signaling. Mapping studies suggest that NSP1 prevents IFN induction in part by blocking IRF3 phosphorylation. In addition, NSP1-induced depletion of Tyk2 and STAT2 dampened ISG induction. Together, our data provide new insights into how SARS-CoV-2 successfully evades the IFN system to establish infection. IMPORTANCE SARS-CoV-2 is the causative agent of COVID-19, a serious disease that can have a myriad of symptoms from loss of taste and smell to pneumonia and hypercoagulation. The rapid spread of SARS-CoV-2 can be attributed in part to asymptomatic transmission, where infected individuals shed large amounts of virus before the onset of disease. This is likely due to the ability of SARS-CoV-2 to effectively suppress the innate immune system, including the IFN response. Indeed, we show that the IFN response is efficiently blocked during SARS-CoV-2 infection, a process that is mediated in large part by nonstructural protein 1 and nucleocapsid. Our study provides new insights on how SARS-CoV-2 evades the IFN response to successfully establish infection. These findings should be considered for the development and administration of therapeutics against SARS-CoV-2.

Nodosome inhibition as a novel broad-spectrum antiviral strategy against arboviruses, enteroviruses and SARS-CoV-2

Nodosome inhibition as a novel broad-spectrum antiviral strategy against arboviruses, enteroviruses and SARS-CoV-2

Daniel Limonta 1 2Lovely Dyna-Dagman 3William Branton 4Valeria Mancinelli 1 2Tadashi Makio 1Richard W Wozniak 1 2Christopher Power 3 4 5Tom C Hobman 6 2 3 5

Abstract

In the present report, we describe two small molecules with broad-spectrum antiviral activity. These drugs block formation of the nodosome. The studies were prompted by the observation that infection of human fetal brain cells with Zika virus (ZIKV) induces expression of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a host factor that was found to promote ZIKV replication and spread. A drug that targets NOD2 was shown to have potent broad-spectrum antiviral activity against other flaviviruses, alphaviruses, enteroviruses, and SARS-CoV-2, the causative agent of COVID-19. Another drug that inhibits the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) which functions downstream of NOD2, also decreased replication of these pathogenic RNA viruses. The antiviral effect of this drug was particularly potent against enteroviruses. The broad-spectrum action of nodosome targeting drugs is mediated in part by enhancement of the interferon response. Together, these results suggest that further preclinical investigation of nodosome inhibitors as potential broad-spectrum antivirals is warranted.

Remdesivir for the treatment of Covid-19: the value of biochemical studies

Remdesivir for the treatment of Covid-19: the value of biochemical studies

Matthias Götte 1

Abstract

The nucleotide analogue prodrug remdesivir remains the only FDA-approved antiviral small molecule for the treatment of infection with SARS-CoV-2. Biochemical studies revealed that the active form of the drug targets the viral RNA-dependent RNA polymerase and causes delayed chain-termination. Delayed chain-termination is incomplete, but the continuation of RNA synthesis enables a partial escape from viral proofreading. Remdesivir becomes embedded in the copy of the RNA genome that later serves as a template. Incorporation of an incoming nucleotide triphosphate is now inhibited by the modified template. Knowledge on the mechanism of action matters. Enzymatic inhibition links to antiviral effects in cell cultures, animal models and viral load reduction in patients, which provides the logical chain that is expected for a direct acting antiviral. Hence, remdesivir also serves as a benchmark in current drug development efforts that will hopefully lead to orally available treatments to the benefit of a broader population.

Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template
J Biol Chem. 2021 May 11.

Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template

Calvin J Gordon 1Egor P Tchesnokov 1Raymond F Schinazi 2Matthias Götte 3

Abstract

The RNA-dependent RNA polymerase (RdRp) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important target in current drug development efforts for the treatment of coronavirus disease 2019 (COVID-19). Molnupiravir is a broad-spectrum antiviral that is an orally bioavailable prodrug of the nucleoside analogue β-D-N4-hydroxycytidine (NHC). Molnupiravir or NHC can increase G to A and C to U transition mutations in replicating coronaviruses. These increases in mutation frequencies can be linked to increases in antiviral effects; however, biochemical data of molnupiravir-induced mutagenesis have not been reported. Here we studied the effects of the active compound NHC 5'-triphosphate (NHC-TP) against the purified SARS-CoV-2 RdRp complex. The efficiency of incorporation of natural nucleotides over the efficiency of incorporation of NHC-TP into model RNA substrates followed the order GTP (12,841) > ATP (424) > UTP (171) > CTP (30), indicating that NHC-TP competes predominantly with CTP for incorporation. No significant inhibition of RNA synthesis was noted as a result of the incorporated monophosphate (NHC-MP) in the RNA primer strand. When embedded in the template strand, NHC-MP supported formation of both NHC:G and NHC:A base pairs with similar efficiencies. The extension of the NHC:G product was modestly inhibited, but higher nucleotide concentrations could overcome this blockage. In contrast, the NHC:A base pair led to the observed G to A (G:NHC:A) or C to U (C:G:NHC:A:U) mutations. Together, these biochemical data support a mechanism of action of molnupiravir that is primarily based on RNA mutagenesis mediated via the template strand.

Erythroid precursors and progenitors suppress adaptive immunity and get invaded by SARS-CoV-2
Stem Cell Reports 2021 May 11;16(5)

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

Shima Shahbaz 1Lai Xu 1Mohammed Osman 2Wendy Sligl 3Justin Shields 4Michael Joyce 4D Lorne Tyrrell 4Olaide Oyegbami 1Shokrollah Elahi 5

Abstract

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.

N-Terminal Finger Stabilizes the S1 Pocket for the Reversible Feline Drug GC376 in the SARS-CoV-2 M pro Dimer
J Mol Biol 2021 Apr 22;433(13)

N-Terminal Finger Stabilizes the S1 Pocket for the Reversible Feline Drug GC376 in the SARS-CoV-2 M pro Dimer

Elena Arutyunova 1Muhammad Bashir Khan 2Conrad Fischer 3Jimmy Lu 1Tess Lamer 3Wayne Vuong 3Marco J van Belkum 3Ryan T McKay 3D Lorne Tyrrell 4John C Vederas 3Howard S Young 5M Joanne Lemieux 6

Abstract

The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug's properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug's P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.

Antibodies to neutralising epitopes synergistically block the interaction of the receptor-binding domain of SARS-CoV-2 to ACE 2
Clin Transl Immunology 2021 Mar 7;10(3)

Antibodies to neutralising epitopes synergistically block the interaction of the receptor-binding domain of SARS-CoV-2 to ACE 2

Manisha Pandey 1Victoria Ozberk 1Sharareh Eskandari 1Ahmed O Shalash 2Michael A Joyce 3Holly A Saffran 3Christopher J Day 1Ailin Lepletier 1Belinda L Spillings 1Jamie-Lee Mills 1Ainslie Calcutt 1Fan Fan 4James T Williams 5Danielle I Stanisic 1Laetitia Hattingh 5John Gerrard 5Mariusz Skwarczynski 2Johnson Mak 1Michael P Jennings 1Istvan Toth 2D Lorne Tyrrell 3Michael F Good 1

Abstract

Objectives: A major COVID-19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2). These vaccines will also induce T-cell responses. However, concerns were raised that aberrant vaccine-induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology.

Methods: We procured a series of overlapping 20-amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID-19 convalescent patients. Identified epitopes were conjugated to diphtheria-toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2.

Results: Seven putative vaccine epitopes were identified. Memory B-cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains.

Conclusion: COVID-19 convalescent patients have SARS-CoV-2-specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope-specific antibodies synergistically block RBD-ACE2 interaction.

Extensive environmental contamination and prolonged severe acute respiratory coronavirus-2 (SARS CoV-2) viability in immunosuppressed recent heart transplant recipients with clinical and virologic benefit with remdesivir
Infext Control Hosp Epidemiol 2021 Mar 12;1-3

Extensive environmental contamination and prolonged severe acute respiratory coronavirus-2 (SARS CoV-2) viability in immunosuppressed recent heart transplant recipients with clinical and virologic benefit with remdesivir

Irina Rajakumar 1Debra L Isaac 1 2Nowell M Fine 1 2Brian Clarke 1 2Linda P Ward 1Rebecca J Malott 2Kanti Pabbaraju 3Kara Gill 3Byron M Berenger 2 3Yi-Chan Lin 4David H Evans # 4John M Conly # 1 2
Peginterferon lambda for the treatment of outpatients with COVID-19: a phase 2, placebo-controlled randomised trial
Lancet Respir  Med 2021 May;9(5)

Peginterferon lambda for the treatment of outpatients with COVID-19: a phase 2, placebo-controlled randomised trial

Jordan J Feld 1Christopher Kandel 2Mia J Biondi 3Robert A Kozak 4Muhammad Atif Zahoor 3Camille Lemieux 5Sergio M Borgia 6Andrea K Boggild 5Jeff Powis 7Janine McCready 7Darrell H S Tan 8Tiffany Chan 9Bryan Coburn 5Deepali Kumar 10Atul Humar 10Adrienne Chan 4Braden O'Neil 11Seham Noureldin 3Joshua Booth 3Rachel Hong 3David Smookler 3Wesam Aleyadeh 3Anjali Patel 3Bethany Barber 3Julia Casey 3Ryan Hiebert 4Henna Mistry 4Ingrid Choong 12Colin Hislop 12Deanna M Santer 13D Lorne Tyrrell 13Jeffrey S Glenn 14Adam J Gehring 3Harry L A Janssen 3Bettina E Hansen 15

Abstract

Background: To date, only monoclonal antibodies have been shown to be effective for outpatients with COVID-19. Interferon lambda-1 is a type III interferon involved in innate antiviral responses with activity against respiratory pathogens. We aimed to investigate the safety and efficacy of peginterferon lambda in the treatment of outpatients with mild-to-moderate COVID-19.

Methods: In this double-blind, placebo-controlled trial, outpatients with laboratory-confirmed COVID-19 were randomly assigned to a single subcutaneous injection of peginterferon lambda 180 μg or placebo within 7 days of symptom onset or first positive swab if asymptomatic. Participants were randomly assigned (1:1) using a computer-generated randomisation list created with a randomisation schedule in blocks of four. At the time of administration, study nurses received a sealed opaque envelope with the treatment allocation number. The primary endpoint was the proportion of patients who were negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on day 7 after the injection, analysed by a χ2 test following an intention-to-treat principle. Prespecified analysis of the primary endpoint, adjusted for baseline viral load, using bivariate logistic regression was done. The trial is now complete. This trial is registered with ClinicalTrials.gov, NCT04354259.

Findings: Between May 18, and Sept 4, 2020, we recruited 30 patients per group. The decline in SARS-CoV-2 RNA was greater in those treated with peginterferon lambda than placebo from day 3 onwards, with a difference of 2·42 log copies per mL at day 7 (p=0·0041). By day 7, 24 (80%) participants in the peginterferon lambda group had an undetectable viral load, compared with 19 (63%) in the placebo group (p=0·15). After controlling for baseline viral load, patients in the peginterferon lambda group were more likely to have undetectable virus by day 7 than were those in the placebo group (odds ratio [OR] 4·12 [95% CI 1·15-16·73; p=0·029). Of those with baseline viral load above 106 copies per mL, 15 (79%) of 19 patients in the peginterferon lambda group had undetectable virus on day 7, compared with six (38%) of 16 in the placebo group (OR 6·25 [95% CI 1·49-31·06]; p=0·012). Peginterferon lambda was well tolerated, and adverse events were similar between groups with mild and transient aminotransferase, concentration increases more frequently observed in the peginterferon lambda group. Two individuals met the threshold of grade 3 increase, one in each group, and no other grade 3 or 4 laboratory adverse events were reported.

Interpretation: Peginterferon lambda accelerated viral decline in outpatients with COVID-19, increasing the proportion of patients with viral clearance by day 7, particularly in those with high baseline viral load. Peginterferon lambda has potential to prevent clinical deterioration and shorten duration of viral shedding.

Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression
Sic Adv 2021 Feb 5;7(6)

Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression

Ke Zhang 1Lisa Miorin 2 3Tadashi Makio 4Ishmael Dehghan 5 6Shengyan Gao 1Yihu Xie 7Hualin Zhong 8Matthew Esparza 1Thomas Kehrer 2 9Anil Kumar 4Tom C Hobman 4Christopher Ptak 4Boning Gao 10John D Minna 10 11Zhijian Chen 5 6Adolfo García-Sastre 2 3 12 13Yi Ren 7Richard W Wozniak 4Beatriz M A Fontoura 14

Abstract

The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.

Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-
Viruses 2020 Dec 26;13(1)

Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-19

Isabelle Bernard 1Daniel Limonta 2 3Lara K Mahal 4Tom C Hobman 1 2 3 5

Abstract

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) poses a persistent threat to global public health. Although primarily a respiratory illness, extrapulmonary manifestations of COVID-19 include gastrointestinal, cardiovascular, renal and neurological diseases. Recent studies suggest that dysfunction of the endothelium during COVID-19 may exacerbate these deleterious events by inciting inflammatory and microvascular thrombotic processes. Although controversial, there is evidence that SARS-CoV-2 may infect endothelial cells by binding to the angiotensin-converting enzyme 2 (ACE2) cellular receptor using the viral Spike protein. In this review, we explore current insights into the relationship between SARS-CoV-2 infection, endothelial dysfunction due to ACE2 downregulation, and deleterious pulmonary and extra-pulmonary immunothrombotic complications in severe COVID-19. We also discuss preclinical and clinical development of therapeutic agents targeting SARS-CoV-2-mediated endothelial dysfunction. Finally, we present evidence of SARS-CoV-2 replication in primary human lung and cardiac microvascular endothelial cells. Accordingly, in striving to understand the parameters that lead to severe disease in COVID-19 patients, it is important to consider how direct infection of endothelial cells by SARS-CoV-2 may contribute to this process.

Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-19
Viruses. 2020 Dec 26;13(1)
Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-19
Isabelle Bernard 1Daniel Limonta 2 3Lara K Mahal 4Tom C Hobman 1 2 3 5

Abstract

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) poses a persistent threat to global public health. Although primarily a respiratory illness, extrapulmonary manifestations of COVID-19 include gastrointestinal, cardiovascular, renal and neurological diseases. Recent studies suggest that dysfunction of the endothelium during COVID-19 may exacerbate these deleterious events by inciting inflammatory and microvascular thrombotic processes. Although controversial, there is evidence that SARS-CoV-2 may infect endothelial cells by binding to the angiotensin-converting enzyme 2 (ACE2) cellular receptor using the viral Spike protein. In this review, we explore current insights into the relationship between SARS-CoV-2 infection, endothelial dysfunction due to ACE2 downregulation, and deleterious pulmonary and extra-pulmonary immunothrombotic complications in severe COVID-19. We also discuss preclinical and clinical development of therapeutic agents targeting SARS-CoV-2-mediated endothelial dysfunction. Finally, we present evidence of SARS-CoV-2 replication in primary human lung and cardiac microvascular endothelial cells. Accordingly, in striving to understand the parameters that lead to severe disease in COVID-19 patients, it is important to consider how direct infection of endothelial cells by SARS-CoV-2 may contribute to this process.

Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology
Anal. Chem. 2020 Nov 26.
Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology
Bo Pang 1Jingyang Xu 1Yanming Liu 1Hanyong Peng 1Wei Feng 1Yiren Cao 1Jinjun Wu 1Huyan Xiao 1Kanti Pabbaraju 2Graham Tipples 3 4Michael A Joyce 4Holly A Saffran 4D Lorne Tyrrell 4Hongquan Zhang 1X Chris Le 1

Abstract

We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30-39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.

Engineered ACE2 receptor traps potently neutralize SARS-CoV-2
Proc. Natl. Acad. Sci. U S A 2020 Nov 10;117(45):28046-28055.
Engineered ACE2 receptor traps potently neutralize SARS-CoV-2
Anum Glasgow 1Jeff Glasgow 2Daniel Limonta 3 4Paige Solomon 2Irene Lui 2Yang Zhang 1Matthew A Nix 5Nicholas J Rettko 2Shoshana Zha 6Rachel Yamin 7Kevin Kao 7Oren S Rosenberg 6Jeffrey V Ravetch 7Arun P Wiita 5Kevin K Leung 2Shion A Lim 2Xin X Zhou 2Tom C Hobman 3 4 8Tanja Kortemme 1James A Wells 9 10

Abstract

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2-pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases
Proc. Natl. Acad. Sci. U S A 2020 Oct 27;117(43):26946-26954.
Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases
Michael K Lo 1César G Albariño 2Jason K Perry 3Silvia Chang 3Egor P Tchesnokov 4 5Lisa Guerrero 2Ayan Chakrabarti 2Punya Shrivastava-Ranjan 2Payel Chatterjee 2Laura K McMullan 2Ross Martin 3Robert Jordan 3Matthias Götte 4 5Joel M Montgomery 2Stuart T Nichol 2Mike Flint 2Danielle Porter 3Christina F Spiropoulou 1

Abstract

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.

The Interaction of Natural and Vaccine-Induced Immunity with Social Distancing Predicts the Evolution of the COVID-19 Pandemic
mBio 2020 Oct 23;11(5):e02617-20.
The Interaction of Natural and Vaccine-Induced Immunity with Social Distancing Predicts the Evolution of the COVID-19 Pandemic
Michael F Good 1 2Michael T Hawkes 3 4 5 6 7

Abstract

The existence and nature of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently unknown; however, neutralizing antibodies are thought to play the major role and data from studying other coronaviruses suggest that partial clinical immunity lasting up to 1 year will occur postinfection. We show how immunity, depending on its durability, may work with current social practices to limit the spread of the virus. We further show that a vaccine that is 50% effective and taken by 50% of the population will prevent further loss of life, providing that social distancing is still practiced and that immunity does not wane quickly.IMPORTANCE The ability of our society to function effectively moving forward will depend on how the spread of the SARS-CoV-2 virus is contained. Immunity to the virus will be critical to this equation.

Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor
Nat. Genet. 2020 Oct 19. Online ahead of print.
Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor
Olusegun O Onabajo 1A Rouf Banday 1Megan L Stanifer 2Wusheng Yan 1Adeola Obajemu 1Deanna M Santer 3Oscar Florez-Vargas 1Helen Piontkivska 4Joselin M Vargas 1Timothy J Ring 1Carmon Kee 5 6Patricio Doldan 5 6D Lorne Tyrrell 3Juan L Mendoza 7Steeve Boulant 5 6Ludmila Prokunina-Olsson 8

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, transcriptionally independent truncated isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In The Cancer Genome Atlas, the expression of dACE2 was enriched in squamous tumors of the respiratory, gastrointestinal and urogenital tracts. In vitro, dACE2, which lacks 356 amino-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, an inflammatory tumor microenvironment or viral co-infections is unlikely to increase the cellular entry of SARS-CoV-2 and promote infection.

Neonatal and Children's Immune System and COVID-19: Biased Immune Tolerance versus Resistance Strategy
J. Immunol. 2020 Oct 15;205(8):1990-1997.
Neonatal and Children's Immune System and COVID-19: Biased Immune Tolerance versus Resistance Strategy
Shokrollah Elahi 1 2 3 4

Abstract

The recent outbreak of COVID-19 has emerged as a major global health concern. Although susceptible to infection, recent evidence indicates mostly asymptomatic or mild presentation of the disease in infants, children, and adolescents. Similar observations were made for acute respiratory infections caused by other coronaviruses (severe acute respiratory syndrome and Middle East respiratory syndrome). These observations suggest that the immune system behaves differently in children than adults. Recent developments in the field demonstrated fundamental differences in the neonatal immune system as compared with adults, whereby infants respond to microorganisms through biased immune tolerance rather than resistance strategies. Similarly, more frequent/recent vaccinations in children and younger populations may result in trained immunity. Therefore, the physiological abundance of certain immunosuppressive cells, a tightly regulated immune system, and/or exposure to attenuated vaccines may enhance trained immunity to limit excessive immune reaction to COVID-19 in the young.

Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of action
J. Biol. Chem. 2020 Sep 23;jbc.AC120.015720.
Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of action
Egor P Tchesnokov 1Calvin J Gordon 1Emma Woolner 1Dana Kocincova 1Jason K Perry 2Joy Y Feng 3Danielle P Porter 4Matthias Gotte 5

Abstract

Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19). RDV targets the viral RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have previously shown that incorporation of the active triphosphate form of RDV (RDV-TP) at position i causes delayed chain-termination at position i+3. Here we demonstrate that the S861G mutation in RdRp eliminates chain-termination, which confirms the existence of a steric clash between S861 and the incorporated RDV-TP. With wild type RdRp, increasing concentrations of NTP pools cause a gradual decrease in termination and the resulting read-through increases full-length product formation. Hence, RDV residues could be embedded in copies of the first RNA strand that is later used as a template. We show that the efficiency of incorporation of the complementary UTP opposite template RDV is compromised, providing a second opportunity to inhibit replication. A structural model suggests that RDV, when serving as the template for the incoming UTP, is not properly positioned due to a significant clash with A558. The adjacent V557 is in direct contact with the template base, and the V557L mutation is implicated in low-level resistance to RDV. We further show that the V557L mutation in RdRp lowers the nucleotide concentration required to bypass this template-dependent inhibition. The collective data provide strong evidence to show that template-dependent inhibition of SARS-CoV-2 RdRp by RDV is biologically relevant.

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication
Nat. Commun. 2020 Aug 27;11(1):4282.
Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication
Wayne Vuong 1Muhammad Bashir Khan 2Conrad Fischer 1Elena Arutyunova 2Tess Lamer 1Justin Shields 3 4Holly A Saffran 3 4Ryan T McKay 1Marco J van Belkum 1Michael A Joyce 3 4Howard S Young 2D Lorne Tyrrell 5 6John C Vederas 7M Joanne Lemieux 8

Abstract

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.

Molecular Diagnosis of COVID-19: Challenges and Research Needs
Anal. Chem. 2020 Aug 4;92(15):10196-10209.
Molecular Diagnosis of COVID-19: Challenges and Research Needs
Wei Feng 1Ashley M Newbigging 1Connie Le 2Bo Pang 1Hanyong Peng 1Yiren Cao 1Jinjun Wu 1Ghulam Abbas 3Jin Song 3Dian-Bing Wang 3Mengmeng Cui 3Jeffrey Tao 1D Lorne Tyrrell 2Xian-En Zhang 3Hongquan Zhang 1X Chris Le 1

Abstract

Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.

Nonessential Research in the New Normal: The Impact of COVID-19
Am. J. Trop. Med. Hyg. 2020 Jun;102(6):1164-1165.
Nonessential Research in the New Normal: The Impact of COVID-19
Stephanie K Yanow 1 2Michael F Good 3 2
Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.
Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency
Calvin J Gordon 1Egor P Tchesnokov 1Emma Woolner 1Jason K Perry 2Joy Y Feng 2Danielle P Porter 2Matthias Götte 3 4

 

Abstract

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2'-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.

COVID-19 and emerging viral infections: The case for interferon lambda
J. Exp. Med. 2020 May 4;217(5):e20200653.
COVID-19 and emerging viral infections: The case for interferon lambda
Ludmila Prokunina-Olsson 1Noémie Alphonse 2 3Ruth E Dickenson # 2Joan E Durbin 4 5Jeffrey S Glenn # 6Rune Hartmann 7Sergei V Kotenko 5 8 9Helen M Lazear 10Thomas R O'Brien 11Charlotte Odendall 2Olusegun O Onabajo 1Helen Piontkivska 12Deanna M Santer 13Nancy C Reich 14Andreas Wack 3Ivan Zanoni 15

Abstract

With the first reports on coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the scientific community working in the field of type III IFNs (IFN-λ) realized that this class of IFNs could play an important role in this and other emerging viral infections. In this Viewpoint, we present our opinion on the benefits and potential limitations of using IFN-λ to prevent, limit, and treat these dangerous viral infections.

The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus
J. Biol. Chem 2020 Apr 10;295(15):4773-4779.
The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus
Calvin J Gordon 1Egor P Tchesnokov 1Joy Y Feng 2Danielle P Porter 2Matthias Götte 3 4

Abstract

Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.

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