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

Oncolytic virus (OV) therapy is potentially a game-changing cancer treatment that has garnered significant interest due to its versatility and multi-modal approaches towards tumor eradication. In the field of cancer immunotherapy, the immunological phenotype of the tumor microenvironment (TME) is an important determinant of disease prognosis and therapeutic success. There is accumulating data that OVs are capable of dramatically altering the TME immune landscape, leading to improved antitumor activity alone or in combination with assorted immune modulators. Herein, we review how OVs disrupt the immunosuppressive TME and can be used strategically to create a "pro-immune" microenvironment that enables and promotes potent, long-lasting host antitumor immune responses.

Lighting a Fire in the Tumor Microenvironment Using Oncolytic Immunotherapy.

Oncolytic viruses (OVs) represent a rapidly advancing class of cancer immunotherapeutic that is helping to shift the treatment paradigm of cancer. Initially thought to only have direct oncolysis ability, the established anticancer mechanisms of action of OVs have rapidly expanded to incorporate a multitude of immune-activating properties. Specifically, the field is focused on harnessing OVs to train patients' immune systems to recognize and eradicate cancer. Recent scientific progress has enabled the shift in focus to clinical translation, and as such it has become increasingly important to utilize clinically relevant models in the development and testing of novel OVs. As a result of this need, some research groups have developed their own experimental models derived from clinical or surgical specimens at their home institutions. This chapter outlines methodology for the generation of three distinct patient-derived experimental models. This includes cell cultures derived from (1) malignant ascites of ovarian cancer patients, (2) direct excision of metastatic melanoma, and (3) core biopsies of pancreatic cancer. Each of these methodologies has been utilized to generate replicating cellular models and have been demonstrated as valuable tools in in vitro, in vivo, and ex vivo studies.

Generating Primary Models of Human Cancer to Aid in the Development of Clinically Relevant Oncolytic Viruses.

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.

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.

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
Lighting a Fire in the Tumor Microenvironment Using Oncolytic Immunotherapy. EBioMedicine\| PubMed ID: 29724655	2018
Generating Primary Models of Human Cancer to Aid in the Development of Clinically Relevant Oncolytic Viruses. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 31486045	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
Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction. International journal of molecular sciences\| PubMed ID: 33668756	2021
A High-Throughput NanoBiT-Based Serological Assay Detects SARS-CoV-2 Seroconversion. Nanomaterials (Basel, Switzerland)\| PubMed ID: 33809836	2021

Abera Surendran

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