Publications by authors named "Vineet D Menachery"

105 Publications

The N501Y spike substitution enhances SARS-CoV-2 infection and transmission.

Nature 2021 Nov 24. Epub 2021 Nov 24.

Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA.

Beginning in the summer of 2020, a variant of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the United Kingdom. This B.1.1.7 variant, also known as Alpha, increased rapidly in prevalence, attributed to an increase in infection and/or transmission efficiency. The Alpha variant has 19 nonsynonymous mutations across its viral genome, including 8 substitutions or deletions in the spike protein, which interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that, of the 8 individual spike protein substitutions, only N501Y exhibited consistent fitness gains for replication in the upper airway in the hamster model as well as primary human airway epithelial cells. The N501Y substitution recapitulated the phenotype of enhanced viral transmission seen with the combined 8 Alpha spike mutations, suggesting it is a major determinant of increased transmission of this variant. Mechanistically, the N501Y substitution improved the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil, South Africa, and elsewhere, our results indicate that N501Y substitution is a major adaptive spike mutation of major concern.
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http://dx.doi.org/10.1038/s41586-021-04245-0DOI Listing
November 2021

CCR2 Signaling Restricts SARS-CoV-2 Infection.

mBio 2021 Nov 9:e0274921. Epub 2021 Nov 9.

Center for Childhood Infections and Vaccines of Children's Healthcare of Atlanta, Department of Pediatrics, Emory Universitygrid.189967.8grid.471395.dgrid.189967.8grid.471395.dgrid.189967.8grid.471395.d School of Medicine, Atlanta, Georgia, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45 cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45 cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.
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http://dx.doi.org/10.1128/mBio.02749-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8576528PMC
November 2021

Mouse-adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge.

PLoS Biol 2021 11 4;19(11):e3001284. Epub 2021 Nov 4.

Departments of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America.

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2-infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease.
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http://dx.doi.org/10.1371/journal.pbio.3001284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594810PMC
November 2021

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis.

bioRxiv 2021 Oct 15. Epub 2021 Oct 15.

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in SARS-CoV-2 nucleocapsid protein. Recreating the alpha variant mutation in an early pandemic (WA-1) background, we found that the R203K/G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. Importantly, the R203K/G204R mutation increases nucleocapsid phosphorylation, providing a molecular basis for these phenotypes. Notably, an analogous alanine substitution mutant also increases SARS-CoV-2 fitness and phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are also key components in SARS-CoV-2's continued adaptation to human infection.

One-sentence Summary: A mutation in the nucleocapsid gene of the SARS-CoV-2 alpha variant is found to enhance replication, fitness, and pathogenesis.
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http://dx.doi.org/10.1101/2021.10.14.464390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8528077PMC
October 2021

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

Elife 2021 09 28;10. Epub 2021 Sep 28.

Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called , which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.
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http://dx.doi.org/10.7554/eLife.68479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478411PMC
September 2021

Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant.

bioRxiv 2021 Sep 5. Epub 2021 Sep 5.

SARS-CoV-2 Delta variant has rapidly replaced the Alpha variant around the world. The mechanism that drives this global replacement has not been defined. Here we report that Delta spike mutation P681R plays a key role in the Alpha-to-Delta variant replacement. In a replication competition assay, Delta SARS-CoV-2 efficiently outcompeted the Alpha variant in human lung epithelial cells and primary human airway tissues. Delta SARS-CoV-2 bearing the Alpha-spike glycoprotein replicated less efficiently than the wild-type Delta variant, suggesting the importance of Delta spike in enhancing viral replication. The Delta spike has accumulated mutation P681R located at a furin cleavage site that separates the spike 1 (S1) and S2 subunits. Reverting the P681R mutation to wild-type P681 significantly reduced the replication of Delta variant, to a level lower than the Alpha variant. Mechanistically, the Delta P681R mutation enhanced the cleavage of the full-length spike to S1 and S2, leading to increased infection via cell surface entry. In contrast, the Alpha spike also has a mutation at the same amino acid (P681H), but the spike cleavage from purified Alpha virions was reduced compared to the Delta spike. Collectively, our results indicate P681R as a key mutation in enhancing Delta variant replication via increased S1/S2 cleavage. Spike mutations that potentially affect furin cleavage efficiency must be closely monitored for future variant surveillance.
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http://dx.doi.org/10.1101/2021.08.12.456173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8404900PMC
September 2021

Binding and entering: COVID finds a new home.

PLoS Pathog 2021 08 30;17(8):e1009857. Epub 2021 Aug 30.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged as a virus with a pathogenicity closer to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and a transmissibility similar to common cold coronaviruses (CoVs). In this review, we briefly discuss the features of the receptor-binding domain (RBD) and protease cleavage of the SARS-CoV-2 spike protein that enable SARS-CoV-2 to be a pandemic virus.
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http://dx.doi.org/10.1371/journal.ppat.1009857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8404997PMC
August 2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity.

Elife 2021 08 3;10. Epub 2021 Aug 3.

Department of Immunology, School of Medicine, University of Washington, Seattle, United States.

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.
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http://dx.doi.org/10.7554/eLife.71047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357416PMC
August 2021

Coagulation and wound repair during COVID-19.

J Heart Lung Transplant 2021 10 12;40(10):1076-1081. Epub 2021 Jun 12.

Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina. Electronic address:

While COVID-19 is best known as a respiratory infection, SARS-CoV-2 causes systemic disease manifestations including coagulopathies. Both dysregulated extracellular matrix remodeling pathways and circulating coagulation proteins are hallmarks of severe COVID-19 and often continue after the resolution of acute infection. Coagulation proteins have proven effective as biomarkers for severe disease and anticoagulants are a mainstay of COVID-19 therapeutics in hospitalized patients. While much knowledge has been gained about the role of clotting pathway activation in COVID-19, much remains to be elucidated in this complex network of signaling pathways.
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http://dx.doi.org/10.1016/j.healun.2021.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195688PMC
October 2021

Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity.

J Immunol 2021 07 30;207(2):376-379. Epub 2021 Jun 30.

Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN;

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing Abs target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with a human adenovirus type 5 vector expressing the SARS-CoV-2 nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and K18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral Ags in SARS-CoV-2 vaccines, even if they are not a target of neutralizing Abs, to broaden epitope coverage and immune effector mechanisms.
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http://dx.doi.org/10.4049/jimmunol.2100421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8516699PMC
July 2021

SARS-CoV-2 RBD trimer protein adjuvanted with Alum-3M-052 protects from SARS-CoV-2 infection and immune pathology in the lung.

Nat Commun 2021 06 11;12(1):3587. Epub 2021 Jun 11.

Emory Vaccine Center, Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.

There is a great need for the development of vaccines that induce potent and long-lasting protective immunity against SARS-CoV-2. Multimeric display of the antigen combined with potent adjuvant can enhance the potency and longevity of the antibody response. The receptor binding domain (RBD) of the spike protein is a primary target of neutralizing antibodies. Here, we developed a trimeric form of the RBD and show that it induces a potent neutralizing antibody response against live virus with diverse effector functions and provides protection against SARS-CoV-2 challenge in mice and rhesus macaques. The trimeric form induces higher neutralizing antibody titer compared to monomer with as low as 1μg antigen dose. In mice, adjuvanting the protein with a TLR7/8 agonist formulation alum-3M-052 induces 100-fold higher neutralizing antibody titer and superior protection from infection compared to alum. SARS-CoV-2 infection causes significant loss of innate cells and pathology in the lung, and vaccination protects from changes in innate cells and lung pathology. These results demonstrate RBD trimer protein as a suitable candidate for vaccine against SARS-CoV-2.
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http://dx.doi.org/10.1038/s41467-021-23942-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196016PMC
June 2021

Nasal delivery of an IgM offers broad protection from SARS-CoV-2 variants.

Nature 2021 07 3;595(7869):718-723. Epub 2021 Jun 3.

Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, Houston, TX, USA.

Resistance represents a major challenge for antibody-based therapy for COVID-19. Here we engineered an immunoglobulin M (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulin G (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern-B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)-and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044 mg per kg body weight confers prophylactic efficacy and a single dose at 0.4 mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19.
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http://dx.doi.org/10.1038/s41586-021-03673-2DOI Listing
July 2021

Catch Me if You Can: Superspreading of COVID-19.

Trends Microbiol 2021 10 11;29(10):919-929. Epub 2021 May 11.

Department of Microbiology and Immunology, Institute for Human Infection and Immunity, World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch at Galveston, Galveston, TX, USA. Electronic address:

While significant insights have been gained concerning COVID-19, superspreading of coronaviruses remains a mystery. The vast majority of cases have been linked to a relatively small portion of infected individuals. Yet, the genetic sequence of the virus, severity of disease, and underlying host parameters, such as age, sex, and health conditions, are not clearly driving the superspreading phenomenon. In this commentary we discuss what is known and what is not known about coronavirus superspreader transmission and explore whether characteristics of the virion, the donor, or the environment contribute to this phenomenon.
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http://dx.doi.org/10.1016/j.tim.2021.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112283PMC
October 2021

Hypergraph models of biological networks to identify genes critical to pathogenic viral response.

BMC Bioinformatics 2021 May 29;22(1):287. Epub 2021 May 29.

Computing and Analytics Division, Pacific Northwest National Laboratory, Seattle, WA, USA.

Background: Representing biological networks as graphs is a powerful approach to reveal underlying patterns, signatures, and critical components from high-throughput biomolecular data. However, graphs do not natively capture the multi-way relationships present among genes and proteins in biological systems. Hypergraphs are generalizations of graphs that naturally model multi-way relationships and have shown promise in modeling systems such as protein complexes and metabolic reactions. In this paper we seek to understand how hypergraphs can more faithfully identify, and potentially predict, important genes based on complex relationships inferred from genomic expression data sets.

Results: We compiled a novel data set of transcriptional host response to pathogenic viral infections and formulated relationships between genes as a hypergraph where hyperedges represent significantly perturbed genes, and vertices represent individual biological samples with specific experimental conditions. We find that hypergraph betweenness centrality is a superior method for identification of genes important to viral response when compared with graph centrality.

Conclusions: Our results demonstrate the utility of using hypergraphs to represent complex biological systems and highlight central important responses in common to a variety of highly pathogenic viruses.
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http://dx.doi.org/10.1186/s12859-021-04197-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8164482PMC
May 2021

TMEM41B is a host factor required for the replication of diverse coronaviruses including SARS-CoV-2.

PLoS Pathog 2021 05 27;17(5):e1009599. Epub 2021 May 27.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America.

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics.
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http://dx.doi.org/10.1371/journal.ppat.1009599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189496PMC
May 2021

Common Mechanism of SARS-CoV and SARS-CoV-2 Pathogenesis across Species.

bioRxiv 2021 May 14. Epub 2021 May 14.

Sarbecovirus (CoV) infections, including Severe Acute Respiratory CoV (SARS-CoV) and SARS-CoV-2, are considerable human threats. Human GWAS studies have recently identified loci associated with variation in SARS-CoV-2 susceptibility. However, genetically tractable models that reproduce human CoV disease outcomes are needed to mechanistically evaluate genetic determinants of CoV susceptibility. We used the Collaborative Cross (CC) and human GWAS datasets to elucidate host susceptibility loci that regulate CoV infections and to identify host quantitative trait loci that modulate severe CoV and pan-CoV disease outcomes including a major disease regulating loci including ablation resulted in enhanced titer, weight loss, respiratory dysfunction, mortality, and inflammation, providing mechanistic support in mitigating protection from severe SARS-CoV-2 pathogenesis across species. This study represents a comprehensive analysis of susceptibility loci for an entire genus of human pathogens conducted, identifies a large collection of susceptibility loci and candidate genes that regulate multiple aspects type-specific and cross-CoV pathogenesis, and also validates the paradigm of using the CC platform to identify common cross-species susceptibility loci and genes for newly emerging and pre-epidemic viruses.
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http://dx.doi.org/10.1101/2021.05.14.444205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132217PMC
May 2021

Mouse Adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge.

bioRxiv 2021 May 4. Epub 2021 May 4.

Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.

The emergence of SARS-CoV-2 has resulted in a worldwide pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of COVID-19 disease have been hampered by the lack of robust mouse models. To overcome this barrier, we utilized a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARSCoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant disease. Importantly, mouse-adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Utilizing this model, we demonstrate that SARS-CoV-2 infected mice are protected from lethal challenge with the original SARS-CoV, suggesting immunity from heterologous CoV strains. Together, the results highlight the utility of this mouse model for further study of SARS-CoV-2 infection and disease.
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http://dx.doi.org/10.1101/2021.05.03.442357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109199PMC
May 2021

CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection.

bioRxiv 2021 May 4. Epub 2021 May 4.

SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B. 1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2021.05.03.442538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109197PMC
May 2021

Evaluation of Cellular and Serological Responses to Acute SARS-CoV-2 Infection Demonstrates the Functional Importance of the Receptor-Binding Domain.

J Immunol 2021 06 5;206(11):2605-2613. Epub 2021 May 5.

Centers for Childhood Infections and Vaccines, Children's Healthcare of Atlanta and Emory University, Department of Pediatrics, Atlanta, GA;

The factors that control the development of an effective immune response to the recently emerged SARS-CoV-2 virus are poorly understood. In this study, we provide a cross-sectional analysis of the dynamics of B cell responses to SARS-CoV-2 infection in hospitalized COVID-19 patients. We observe changes in B cell subsets consistent with a robust humoral immune response, including significant expansion of plasmablasts and activated receptor-binding domain (RBD)-specific memory B cell populations. We observe elevated titers of Abs to SARS-CoV-2 RBD, full-length Spike, and nucleoprotein over the course of infection, with higher levels of RBD-specific IgG correlating with increased serum neutralization. Depletion of RBD-specific Abs from serum removed a major portion of neutralizing activity in most individuals. Some donors did retain significant residual neutralization activity, suggesting a potential Ab subset targeting non-RBD epitopes. Taken together, these findings are instructive for future vaccine design and mAb strategies.
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http://dx.doi.org/10.4049/jimmunol.2001420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8482837PMC
June 2021

Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity.

bioRxiv 2021 Apr 27. Epub 2021 Apr 27.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.
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http://dx.doi.org/10.1101/2021.04.26.441518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095198PMC
April 2021

The variant gambit: COVID-19's next move.

Cell Host Microbe 2021 04 1;29(4):508-515. Epub 2021 Mar 1.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

More than a year after its emergence, COVID-19, the disease caused by SARS-CoV-2, continues to plague the world and dominate our daily lives. Even with the development of effective vaccines, this coronavirus pandemic continues to cause a fervor with the identification of major new variants hailing from the United Kingdom, South Africa, Brazil, and California. Coupled with worries over a distinct mink strain that has caused human infections and potential for further mutations, SARS-CoV-2 variants bring concerns for increased spread and escape from both vaccine and natural infection immunity. Here, we outline factors driving SARS-CoV-2 variant evolution, explore the potential impact of specific mutations, examine the risk of further mutations, and consider the experimental studies needed to understand the threat these variants pose. In this review, Plante et al. examine SARS-CoV-2 variants including B.1.1.7 (UK), B.1.351 (RSA), P.1 (Brazil), and B.1.429 (California). They focus on what factors contribute to variant emergence, mutations in and outside the spike protein, and studies needed to understand the impact of variants on infection, transmission, and vaccine efficacy.
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http://dx.doi.org/10.1016/j.chom.2021.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919536PMC
April 2021

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

bioRxiv 2021 Mar 11. Epub 2021 Mar 11.

Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX, USA.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ' '. Tiled-ClickSeq uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, obviating the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended using click-chemistry and a PCR reaction using Illumina adaptors generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.
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http://dx.doi.org/10.1101/2021.03.10.434828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987005PMC
March 2021

The N501Y spike substitution enhances SARS-CoV-2 transmission.

bioRxiv 2021 Mar 9. Epub 2021 Mar 9.

Beginning in the summer of 2020, a variant of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the United Kingdom (UK). This B.1.1.7 variant increased rapidly in prevalence among sequenced strains, attributed to an increase in infection and/or transmission efficiency. The UK variant has 19 nonsynonymous mutations across its viral genome including 8 substitutions or deletions in the spike protein, which interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that, of the 8 individual spike protein substitutions, only N501Y exhibited consistent fitness gains for replication in the upper airway in the hamster model as well as primary human airway epithelial cells. The N501Y substitution recapitulated the phenotype of enhanced viral transmission seen with the combined 8 UK spike mutations, suggesting it is a major determinant responsible for increased transmission of this variant. Mechanistically, the N501Y substitution improved the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil and South Africa, our results indicate that N501Y substitution is a major adaptive spike mutation of major concern.
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http://dx.doi.org/10.1101/2021.03.08.434499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986995PMC
March 2021

A trans-complementation system for SARS-CoV-2 recapitulates authentic viral replication without virulence.

Cell 2021 04 23;184(8):2229-2238.e13. Epub 2021 Feb 23.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
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http://dx.doi.org/10.1016/j.cell.2021.02.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901297PMC
April 2021

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

Immunity 2021 03 4;54(3):542-556.e9. Epub 2021 Feb 4.

Emory Vaccine Center, Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329, USA; Department of Pathology, Emory School of Medicine, Emory University, Atlanta, GA 30322, USA.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8 T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8 T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.
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http://dx.doi.org/10.1016/j.immuni.2021.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859620PMC
March 2021

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera.

Nat Med 2021 04 8;27(4):620-621. Epub 2021 Feb 8.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.

We engineered three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion + N501Y + D614G from UK; and E484K + N501Y + D614G from SA. Neutralization geometric mean titers (GMTs) of 20 BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses.
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http://dx.doi.org/10.1038/s41591-021-01270-4DOI Listing
April 2021
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