scholarly journals Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays

2020 ◽  
Author(s):  
Jennifer Mehalko ◽  
Matthew Drew ◽  
Kelly Snead ◽  
John-Paul Denson ◽  
Vanessa Wall ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Protein Production ◽  
High Sensitivity ◽  
Magnetic Bead ◽  
Binding Domain ◽  
List Type ◽  
Receptor Binding Domain ◽  
Dna Elements ◽  
Increased Sensitivity ◽  
Higher Sensitivity

AbstractThe receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.Highlights: Improved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parametersTwo versions of RBD show different sensitivity in serology assaysYields of greater than 50 mg/l obtained under optimal conditionsMagnetic bead purification technology improves throughput of protein production

scholarly journals 480 Preliminary evaluation of a novel coronavirus vaccine (CORVax) using electroporation of plasmid DNA encoding a stabilized prefusion SARS-CoV-2 spike protein alone or with transfection of plasmid IL-12

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A514-A514
Author(s):  
Shawn Jensen ◽  
Christopher Twitty ◽  
Christopher Paustian ◽  
Madelein Laws ◽  
Glenna McDonnell ◽  
...  
Keyword(s):  
Immune Responses ◽  
Receptor Binding ◽  
Binding Domain ◽  
Tumor Rejection ◽  
Spike Protein ◽  
Preliminary Evaluation ◽  
List Type ◽  
Receptor Binding Domain ◽  
Igg Antibodies ◽  
Viral Neutralization

BackgroundSARS-CoV-2 (CoV2) has precipitated a global pandemic and the effectiveness of standard vaccine strategies to induce potent and persistent immunity to CoV2 is in question, particularly for the elderly. This problem is not dissimilar to what we have struggled with in our quest to induce immunity to cancer antigens, where vaccine-induced anti-cancer immune responses can be weak. Here, we describe a novel vaccine approach which leverages electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax). As IL-12 has been shown to augment the efficacy of immunotherapy in aged mice,1 we have initiated studies to evaluate if plasmid IL-12 (TAVO™) can similarly augment anti-CoV2 immune responses in young mice and have planned studies in aged animals.MethodsA prefusion stabilized CoV2 spike plasmid expression vector was constructed, a master cell bank generated and clinical-grade plasmid manufactured. C57BL/6 and BALB/c were vaccinated via intramuscular (IM) and/or intradermal (ID) injection followed immediately by EP of plasmids encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12 on days 1 and 14 or 21. Mice were followed for >120 days to assess safety. Splenocytes and serum were harvested at different time points to interrogate virus-specific cellular responses as well anti-spike IgG1/IgG2 antibody titers. A surrogate viral neutralization test (sVNT) assessed serum blockade of soluble hACE2R binding to immobilized CoV2 spike.ResultsPreliminary data shows that EP of CORVax alone or combined with IL-12 was safe. EP of CORVax was able to elicit anti-Spike IgG antibodies (IC50 = 1/2112), as well as IgG antibodies targeting the receptor binding domain of the Spike protein (IC50 = 1/965) approximately 40 days after the booster vaccination. In 2 of 2 experiments, CORVax combined with IL-12 significantly (P<0.0001) increased the sVNT titers at 2 months, but this benefit was lost by 3 months.ConclusionsEarly preclinical data shows that EP of CORVax can induce IgG responses to CoV2 Spike and the receptor binding domain (RBD) as well as apparent viral neutralizing activity. The addition of IL-12, at least transiently, increased sVNT titer. We plan to investigate alternate vaccine boosting strategies while extending these studies into aged animals and initiate a clinical trial in the near future.ReferencesRuby CE, Weinberg AD. OX40-Enhanced tumor rejection and effector T cell differentiation decreases with age. J Immunol2009;182:1481–9. https://doi.org/10.4049/jimmunol.182.3.1481.

scholarly journals Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant

2021 ◽  
Author(s):  
Nariko Ikemura ◽  
Shunta Taminishi ◽  
Tohru Inaba ◽  
Takao Arimori ◽  
Daisuke Motooka ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Mutational Analysis ◽  
High Sensitivity ◽  
Binding Domain ◽  
Spike Protein ◽  
The Novel ◽  
Receptor Binding Domain ◽  
Therapeutic Drugs ◽  
Viral Mutation ◽  
Therapeutic Value

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera of 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. However, some Delta patients have relatively preserved neutralization efficacy, comparable to 3-month double BNT162b2 vaccination. Domain-wise analysis using chimeric spike revealed that this efficient evasion was, at least in part, caused by multiple mutations in the N-terminal domain. Omicron escapes the therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective against Omicron. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from mutational escape. Deep mutational analysis demonstrated that, indeed, the engineered ACE2 overcomes every single-residue mutation in the receptor-binding domain, similar to immunized sera. Like previous SARS-CoV-2 variants, Omicron and some other sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

scholarly journals Y380Q novel mutation in receptor-binding domain of SARS-CoV-2 spike protein together with C379W interfere in the neutralizing antibodies interaction

2021 ◽  
Author(s):  
Ivaine Tais Sauthier Sartor ◽  
Fernanda Hammes Varela ◽  
Mariana Rost Meireles ◽  
Luciane Beatriz Kern ◽  
Thaís Raupp Azevedo ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Novel Mutation ◽  
Signs And Symptoms ◽  
Disease Diagnosis ◽  
Binding Domain ◽  
Spike Protein ◽  
Health Concern ◽  
List Type ◽  
Receptor Binding Domain

AbstractBackgroundThe emergence of SARS-CoV-2 variants is a current public health concern possibly impacting COVID-19 disease diagnosis, transmission patterns and vaccine effectiveness.ObjectivesTo describe the SARS-CoV-2 lineages circulating early pandemic among samples with S gene dropout and characterize a novel mutation in receptor-binding domain (RBD) of viral spike protein.Study designAdults and children older than 2 months with signs and symptoms of COVID-19 were prospectively enrolled from May to October 2020 in Porto Alegre, Brazil. All participants performed RT-PCR assays for diagnosing SARS-CoV-2, samples with S gene dropout and Ct < 30 (cycle threshold) were submitted to whole genome sequencing (WGS), and homology modeling and physicochemical properties analysis were performed.Results484/1,557 participants tested positive for SARS-CoV-2. The S gene dropout was detected in 7.4% (36/484) as early as May, and a peak was observed in early August. WGS was performed in 8 samples. The B.1.1.28, B.1.91 and B.1.1.33 lineages were circulating in early pandemic. The RBD novel mutation (Y380Q) was found in one sample occurring simultaneously with C379W and V395A, and the B.1.91 lineage in the spike protein.ConclusionMutations in the SARS-CoV-2 spike region were detected early in the COVID-19 pandemic in Southern Brazil, regarding the B.1.1.28, B.1.91 and B.1.1.33 lineages identified. The novel mutation (Y380Q) with C379W, modifies important RBD properties, which may interfere with the binding of neutralizing antibodies (CR3022, EY6A, H014, S304).HighlightsCharacterization of novel mutation (Y380Q) in RBD of SARS-CoV-2 spike proteinThe Y380Q and C379W modify important properties in the SARS-CoV-2 RBD regionThe RBD mutations may interfere with the binding of neutralizing antibodiesThe B.1.1.28, B.1.91 and B.1.1.33 lineages were circulating in early pandemic

scholarly journals Development of a Fast SARS-CoV-2 IgG ELISA, Based on Receptor-Binding Domain, and Its Comparative Evaluation Using Temporally Segregated Samples From RT-PCR Positive Individuals

2021 ◽  
Vol 11 ◽  
Author(s):  
Farha Mehdi ◽  
Souvick Chattopadhyay ◽  
Ramachandran Thiruvengadam ◽  
Sarla Yadav ◽  
Manjit Kumar ◽  
...  
Keyword(s):  
Antibody Response ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Elisa Test ◽  
Binding Domain ◽  
Antibody Detection ◽  
Receptor Binding Domain ◽  
Rt Pcr ◽  
Vaccine Response ◽  
Higher Sensitivity

SARS-CoV-2 antibody detection assays are crucial for gathering seroepidemiological information and monitoring the sustainability of antibody response against the virus. The SARS-CoV-2 Spike protein’s receptor-binding domain (RBD) is a very specific target for anti-SARS-CoV-2 antibodies detection. Moreover, many neutralizing antibodies are mapped to this domain, linking antibody response to RBD with neutralizing potential. Detection of IgG antibodies, rather than IgM or total antibodies, against RBD is likely to play a larger role in understanding antibody-mediated protection and vaccine response. Here we describe a rapid and stable RBD-based IgG ELISA test obtained through extensive optimization of the assay components and conditions. The test showed a specificity of 99.79% (95% CI: 98.82–99.99%) in a panel of pre-pandemic samples (n = 470) from different groups, i.e., pregnancy, fever, HCV, HBV, and autoantibodies positive. Test sensitivity was evaluated using sera from SARS-CoV-2 RT-PCR positive individuals (n = 312) and found to be 53.33% (95% CI: 37.87–68.34%), 80.47% (95% CI: 72.53–86.94%), and 88.24% (95% CI: 82.05–92.88%) in panel 1 (days 0–13), panel 2 (days 14–20) and panel 3 (days 21–27), respectively. Higher sensitivity was achieved in symptomatic individuals and reached 92.14% (95% CI: 86.38–96.01%) for panel 3. Our test, with a shorter runtime, showed higher sensitivity than parallelly tested commercial ELISAs for SARS-CoV-2-IgG, i.e., Euroimmun and Zydus, even when equivocal results in the commercial ELISAs were considered positive. None of the tests, which are using different antigens, could detect anti-SARS-CoV-2 IgGs in 10.5% RT-PCR positive individuals by the fourth week, suggesting the lack of IgG response.

scholarly journals A magnetic bead immunoassay to detect human IgG reactive to SARS-CoV-2 Spike S1 RBD produced in Escherichia coli

2021 ◽  
Author(s):  
Marcelo dos Santos Conzentino ◽  
Ana C Goncalves ◽  
NIgella M Paula ◽  
Fabiane G Rego ◽  
Dalila Zanette ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Receptor Binding ◽  
High Throughput ◽  
Low Cost ◽  
Magnetic Bead ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Operating Characteristics ◽  
Human Igg ◽  
Bead Immunoassay

Immunological assays to detect SARS-CoV-2 Spike receptor binding domain antigen seroconversion in humans are important tools to monitor the levels of protecting antibodies in the population in response to infection and/or immunization. Here we describe a simple, low cost and high throughput Ni2+ magnetic bead immunoassay to detect human IgG reactive to Spike S1 RBD Receptor Binding Domain produced in Escherichia coli. A 6xHis tagged Spike S1 RBD was expressed in E. coli and purified by affinity chromatography. The protein was mobilized on the surface of Ni2+ magnetic beads and used to investigate the presence of reactive IgG in the serum obtained from pre-pandemic and COVID-19 confirmed cases. The method was validated with a cohort of 290 samples and an area under the receiver operating characteristics curve of 0.94 was obtained. The method operated with>82% sensitivity at 98% specificity and was also able to track human IgG raised in response to vaccination with Comirnaty with 85% sensitivity. The IgG signal obtained with the described method was well correlated with the signal obtained when pre fusion Spike produced in HEK cell lines were used as antigen. This novel low-cost and high throughput immunoassay may act as an important tool to investigate protecting IgG antibodies against SARS-CoV-2 in the human population.

scholarly journals Fluorescence signatures of SARS CoV-2 spike S1 proteins and an human ACE-2: excitation-emission maps and fluorescence lifetimes

2021 ◽  
Author(s):  
Jonas Grzesiak ◽  
Lea Fellner ◽  
Karin Grünewald ◽  
Christoph Kölbl ◽  
Arne Walter ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Virus Detection ◽  
High Sensitivity ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Fluorescence Lifetimes ◽  
Comprehensive Knowledge ◽  
Clean Environment ◽  
Complex Protein ◽  
One Year

Fast and reliable detection of infectious virus loads of the SARS CoV-2 virus is still an important issue even after more than one year of the pandemic's outbreak. The spike protein's S1 subunit (including its receptor-binding domain) and human angiotensin-converting enzyme 2 (hACE2) are known as key players in the reproduction mechanism of the SARS CoV-2 virus. Because of its high sensitivity and simple application, fluorescence spectroscopy is promising to meet the sensitivity requirements for a virus detection in a clean environment. In such highly complex protein systems, a comprehensive knowledge of fluorescence data is presumed in order to evaluate the specificity of the spectra with respect to a possible detection. This includes full featured information on the fluorescence process, i. e. wavelength and time-dependent data. In this work, fluorescence spectral excitation-emission maps of the involved proteins are presented, namely the S1 part of the spike protein and its receptor-binding domain as well as the hACE2 enzyme. In addition, measurements of fluorescence lifetimes of the proteins are presented and analyzed by a bi-exponential kinetic approach.

Destabilizing the Structural Integrity of SARS-CoV2 Receptor Proteins by Curcumin Along with Hydroxychloroquine: An Insilco Approach for a Combination Therapy

2020 ◽  
Author(s):  
Akhileshwar Srivastava ◽  
Divya Singh
Keyword(s):  
Binding Energy ◽  
Combination Therapy ◽  
Receptor Binding ◽  
Structural Integrity ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Receptor Proteins ◽  
Main Protease ◽  
The Stability ◽  
Therapeutic Activities

Presently, an emerging disease (COVID-19) has been spreading across the world due to coronavirus (SARS-CoV2). For treatment of SARS-CoV2 infection, currently hydroxychloroquine has been suggested by researchers, but it has not been found enough effective against this virus. The present study based on in silico approaches was designed to enhance the therapeutic activities of hydroxychloroquine by using curcumin as an adjunct drug against SARS-CoV2 receptor proteins: main-protease and S1 receptor binding domain (RBD). The webserver (ANCHOR) showed the higher protein stability for both receptors with disordered score (<0.5). The molecular docking analysis revealed that the binding energy (-24.58 kcal/mol) of hydroxychloroquine was higher than curcumin (-20.47 kcal/mol) for receptor main-protease, whereas binding energy of curcumin (<a>-38.84</a> kcal/mol) had greater than hydroxychloroquine<a> (-35.87</a> kcal/mol) in case of S1 receptor binding domain. Therefore, this study suggested that the curcumin could be used as combination therapy along with hydroxychloroquine for disrupting the stability of SARS-CoV2 receptor proteins

Role of key point Mutations in Receptor Binding Domain of SARS-CoV-2 Spike Glycoprotein

2020 ◽  
Vol 20 ◽  
Author(s):  
Bipin Singh
Keyword(s):  
Receptor Binding ◽  
Point Mutations ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
Angiotensin Converting Enzyme 2 ◽  
Recent Outbreak ◽  
Novel Coronavirus ◽  
Genomic Similarity

: The recent outbreak of novel coronavirus (SARS-CoV-2 or 2019-nCoV) and its worldwide spread is posing one of the major threats to human health and the world economy. It has been suggested that SARS-CoV-2 is similar to SARSCoV based on the comparison of the genome sequence. Despite the genomic similarity between SARS-CoV-2 and SARSCoV, the spike glycoprotein and receptor binding domain in SARS-CoV-2 shows the considerable difference compared to SARS-CoV, due to the presence of several point mutations. The analysis of receptor binding domain (RBD) from recently published 3D structures of spike glycoprotein of SARS-CoV-2 (Yan, R., et al. (2020); Wrapp, D., et al. (2020); Walls, A. C., et al. (2020)) highlights the contribution of a few key point mutations in RBD of spike glycoprotein and molecular basis of its efficient binding with human angiotensin-converting enzyme 2 (ACE2).

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