detection-sars-cov-2-receptor-binding-domain-antibody-using-hibit

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Ottawa Hospital Research Institute

University of Ottawa

Immune regulation of cellular metabolism can be responsible for successful responses to invading pathogens. Viruses alter their hosts' cellular metabolism to facilitate infection. Conversely, the innate antiviral responses of mammalian cells target these metabolic pathways to restrict viral propagation. We identified miR-130b and miR-185 as hepatic microRNAs (miRNAs) whose expression is stimulated by 25-hydroxycholesterol (25-HC), an antiviral oxysterol secreted by interferon-stimulated macrophages and dendritic cells, during hepatitis C virus (HCV) infection. However, 25-HC only directly stimulated miR-185 expression, whereas HCV regulated miR-130b expression. Independently, miR-130b and miR-185 inhibited HCV infection. In particular, miR-185 significantly restricted host metabolic pathways crucial to the HCV life cycle. Interestingly, HCV infection decreased miR-185 and miR-130b levels to promote lipid accumulation and counteract 25-HC's antiviral effect. Furthermore, miR-185 can inhibit other viruses through the regulation of immunometabolic pathways. These data establish these microRNAs as a key link between innate defenses and metabolism in the liver. 

MicroRNAs regulate the immunometabolic response to viral infection in the liver.

Cancer stem cells (CSCs) have the capacity to self-renew and differentiate to give rise to heterogenous cancer cell lineages in solid tumors. These CSC populations are associated with metastasis, tumor relapse, and resistance to conventional anticancer therapies. Here, we focus on the use of oncolytic viruses (OVs) to target CSCs as well as the OV-driven interferon production in the tumor microenvironment (TME) that can repress CSC properties. We explore the ability of OVs to deliver combinations of immune-modulating therapeutic transgenes, such as immune checkpoint inhibitor antibodies. In particular, we highlight the advantages of virally encoded bi-specific T cell engagers (BiTEs) to not only target cell-surface markers on CSCs, but also tumor-associated antigens on contributing components of the surrounding TME and other cancer cells. We also highlight the crucial role of combination anticancer treatments, evidenced by synergy of OV-delivered BiTEs and chimeric-antigen receptor T cell therapy. Stem Cells 2019;37:716-723.

Concise Review: Targeting Cancer Stem Cells and Their Supporting Niche Using Oncolytic Viruses.

Vaccinia virus (VACV) possesses a great safety record as a smallpox vaccine and has been intensively used as an oncolytic virus against various types of cancer over the past decade. Different strategies were developed to make VACV safe and selective to cancer cells. Leading clinical candidates, such as Pexa-Vec, are attenuated through deletion of the viral thymidine kinase (TK) gene, which limits virus growth to replicate in cancer tissue. However, tumors are not the only tissues whose metabolic activity can overcome the lack of viral TK. In this study, we sought to further increase the tumor-specific replication and oncolytic potential of Copenhagen strain VACV ΔTK. We show that deletion of the anti-apoptosis viral gene F1L not only increases the safety of the Copenhagen ΔTK virus but also improves its oncolytic activity in an aggressive glioblastoma model. The additional loss of F1L does not affect VACV replication capacity, yet its ability to induce cancer cell death is significantly increased. Our results also indicate that cell death induced by the Copenhagen ΔTK/F1L mutant releases more immunogenic signals, as indicated by increased levels of IL-1β production. A cytotoxicity screen in an NCI-60 panel shows that the ΔTK/F1L virus induces faster tumor cell death in different cancer types. Most importantly, we show that, compared to the TK-deleted virus, the ΔTK/F1L virus is attenuated in human normal cells and causes fewer pox lesions in murine models. Collectively, our findings describe a new oncolytic vaccinia deletion strain that improves safety and increases tumor cell killing.

Deletion of Apoptosis Inhibitor F1L in Vaccinia Virus Increases Safety and Oncolysis for Cancer Therapy.

Immunotherapy is a rapidly evolving area of cancer therapeutics aimed at driving a systemic immune response to fight cancer. Oncolytic viruses (OVs) are at the cutting-edge of innovation in the immunotherapy field. Successful OV platforms must be effective in reshaping the tumor microenvironment and controlling tumor burden, but also be highly specific to avoid off-target side effects. Large DNA viruses, like vaccinia virus (VACV), have a large coding capacity, enabling the encoding of multiple immunostimulatory transgenes to reshape the tumor immune microenvironment. VACV-based OVs have shown promising results in both pre-clinical and clinical studies, including safe and efficient intravenous delivery to metastatic tumors.

Engineering vaccinia virus as an immunotherapeutic battleship to overcome tumor heterogeneity.

The Hippo pathway plays a critical role in tissue and organ growth under normal physiological conditions, and its dysregulation in malignant growth has made it an attractive target for therapeutic intervention in the fight against cancer. To date, its complex signaling mechanisms have made it difficult to identify strong therapeutic candidates. Hippo signaling is largely carried out by two main activated signaling pathways involving receptor tyrosine kinases (RTKs)-the RTK/RAS/PI3K and the RTK-RAS-MAPK pathways. However, several RTKs have also been shown to regulate this pathway to engage downstream Hippo effectors and ultimately influence cell proliferation. In this text, we attempt to review the diverse RTK signaling pathways that influence Hippo signaling in the context of oncogenesis.

Hippo Signaling Pathway as a Central Mediator of Receptors Tyrosine Kinases (RTKs) in Tumorigenesis.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presents an urgent need for an effective vaccine. Molecular characterization of SARS-CoV-2 is critical to the development of effective vaccine and therapeutic strategies. In the present study, we show that the fusion of the SARS-CoV-2 spike protein receptor-binding domain to its transmembrane domain is sufficient to mediate trimerization. Our findings may have implications for vaccine development and therapeutic drug design strategies targeting spike trimerization. As global efforts for developing SARS-CoV-2 vaccines are rapidly underway, we believe this observation is an important consideration for identifying crucial epitopes of SARS-CoV-2.

Implications for SARS-CoV-2 Vaccine Design: Fusion of Spike Glycoprotein Transmembrane Domain to Receptor-Binding Domain Induces Trimerization.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.

Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction.

As the COVID-19 pandemic continues, there is an imminent need for rapid diagnostic tools and effective antivirals targeting SARS-CoV-2. We have developed a novel bioluminescence-based biosensor to probe a key host-virus interaction during viral entry: the binding of SARS-CoV-2 viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2). Derived from Nanoluciferase binary technology (NanoBiT), the biosensor is composed of Nanoluciferase split into two complementary subunits, Large BiT and Small BiT, fused to the Spike S1 domain of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. The ACE2-S1 interaction results in reassembly of functional Nanoluciferase, which catalyzes a bioluminescent reaction that can be assayed in a highly sensitive and specific manner. We demonstrate the biosensor's large dynamic range, enhanced thermostability and pH tolerance. In addition, we show the biosensor's versatility towards the high-throughput screening of drugs which disrupt the ACE2-S1 interaction, as well as its ability to act as a surrogate virus neutralization assay. Results obtained with our biosensor correlate well with those obtained with a Spike-pseudotyped lentivirus assay. This rapid in vitro tool does not require infectious virus and should enable the timely development of antiviral modalities targeting SARS-CoV-2 entry.

SARS-CoV-2 S1 NanoBiT: A nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.

High-throughput detection strategies for antibodies against SARS-CoV-2 in patients recovering from COVID-19, or in vaccinated individuals, are urgently required during this ongoing pandemic. Serological assays are the most widely used method to measure antibody responses in patients. However, most of the current methods lack the speed, stability, sensitivity, and specificity to be selected as a test for worldwide serosurveys. Here, we demonstrate a novel NanoBiT-based serological assay for fast and sensitive detection of SARS-CoV-2 RBD-specific antibodies in sera of COVID-19 patients. This assay can be done in high-throughput manner at 384 samples per hour and only requires a minimum of 5 μL of serum or 10 ng of antibody. The stability of our NanoBiT reporter in various temperatures (4-42 °C) and pH (4-12) settings suggests the assay will be able to withstand imperfect shipping and handling conditions for worldwide seroepidemiologic surveillance in the post-vaccination period of the pandemic. Our newly developed rapid assay is highly accessible and may facilitate a more cost-effective solution for seroconversion screening as vaccination efforts progress.

Article	Year
MicroRNAs regulate the immunometabolic response to viral infection in the liver. Nature chemical biology\| PubMed ID: 26479438	2015
Concise Review: Targeting Cancer Stem Cells and Their Supporting Niche Using Oncolytic Viruses. Stem cells (Dayton, Ohio)\| PubMed ID: 30875126	2019
Deletion of Apoptosis Inhibitor F1L in Vaccinia Virus Increases Safety and Oncolysis for Cancer Therapy. Molecular therapy oncolytics\| PubMed ID: 31428674	2019
Engineering vaccinia virus as an immunotherapeutic battleship to overcome tumor heterogeneity. Expert opinion on biological therapy\| PubMed ID: 32297534	2020
Hippo Signaling Pathway as a Central Mediator of Receptors Tyrosine Kinases (RTKs) in Tumorigenesis. Cancers\| PubMed ID: 32722184	2020
Implications for SARS-CoV-2 Vaccine Design: Fusion of Spike Glycoprotein Transmembrane Domain to Receptor-Binding Domain Induces Trimerization. Membranes\| PubMed ID: 32872641	2020
Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 33578036	2021
Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction. International journal of molecular sciences\| PubMed ID: 33668756	2021
SARS-CoV-2 S1 NanoBiT: A nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition. Biosensors & bioelectronics\| PubMed ID: 33706157	2021
A High-Throughput NanoBiT-Based Serological Assay Detects SARS-CoV-2 Seroconversion. Nanomaterials (Basel, Switzerland)\| PubMed ID: 33809836	2021

Ragunath Singaravelu

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