scholarly journals Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2

Vaccines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 986
Author(s):  
Larissa R. Gomes ◽  
Andressa M. Durans ◽  
Paloma Napoleão-Pêgo ◽  
Jessica A. Waterman ◽  
Mariana S. Freitas ◽  
...  
Keyword(s):  
High Performance ◽  
De Novo ◽  
Chimeric Protein ◽  
Emerging Diseases ◽  
High Reactivity ◽  
Diagnostic Methods ◽  
Spike Protein ◽  
Spot Synthesis ◽  
Serological Tests ◽  
Large Populations

The COVID-19 pandemic has exposed the extent of global connectivity and collective vulnerability to emerging diseases. From its suspected origins in Wuhan, China, it spread to all corners of the world in a matter of months. The absence of high-performance, rapid diagnostic methods that could identify asymptomatic carriers contributed to its worldwide transmission. Serological tests offer numerous benefits compared to other assay platforms to screen large populations. First-generation assays contain targets that represent proteins from SARS-CoV-2. While they could be quickly produced, each actually has a mixture of specific and non-specific epitopes that vary in their reactivity for antibodies. To generate the next generation of the assay, epitopes were identified in three SARS-Cov-2 proteins (S, N, and Orf3a) by SPOT synthesis analysis. After their similarity to other pathogen sequences was analyzed, 11 epitopes outside of the receptor-binding domain (RBD) of the spike protein that showed high reactivity and uniqueness to the virus. These were incorporated into a ß-barrel protein core to create a highly chimeric protein. Another de novo protein was designed that contained only epitopes in the RBD. In-house ELISAs suggest that both multiepitope proteins can serve as targets for high-performance diagnostic tests. Our approach to bioengineer chimeric proteins is highly amenable to other pathogens and immunological uses.

scholarly journals Performance assessment of a multi-epitope chimeric antigen for the serological diagnosis of acute Mayaro fever

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Paloma Napoleão-Pêgo ◽  
Flávia R. G. Carneiro ◽  
Andressa M. Durans ◽  
Larissa R. Gomes ◽  
Carlos M. Morel ◽  
...  
Keyword(s):  
High Performance ◽  
Clinical Symptoms ◽  
Chimeric Protein ◽  
Cross Reactivity ◽  
Effective Control ◽  
Antigenic Determinants ◽  
Gene Encoding ◽  
Accurate Identification ◽  
Serological Tests ◽  
Mayaro Virus

AbstractMayaro virus (MAYV), which causes mayaro fever, is endemic to limited regions of South America that may expand due to the possible involvement of Aedes spp. mosquitoes in its transmission. Its effective control will require the accurate identification of infected individuals, which has been restricted to nucleic acid-based tests due to similarities with other emerging members of the Alphavirus genus of the Togaviridae family; both in structure and clinical symptoms. Serological tests have a more significant potential to expand testing at a reasonable cost, and their performance primarily reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene encoding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily expressed as a stable chimeric protein in bacteria. Its serological performance as the target in ELISAs revealed a high accuracy for detecting anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for dengue, chikungunya, yellow fever, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.

scholarly journals Performance assessment of a multi-epitope chimeric antigen for the serological diagnosis of acute Mayaro fever

2021 ◽  
Author(s):  
Paloma Napoleão-Pêgo ◽  
Flávia R. G. Carneiro ◽  
Andressa M. Durans ◽  
Carlos M. Morel ◽  
David W. Provance ◽  
...  
Keyword(s):  
High Performance ◽  
Clinical Symptoms ◽  
Chimeric Protein ◽  
Cross Reactivity ◽  
Effective Control ◽  
Antigenic Determinants ◽  
Serological Tests ◽  
Molecular Approaches ◽  
Mayaro Virus ◽  
Sensitivity Specificity

Abstract Mayaro virus (MAYV) is endemic to limited regions of South America and the possible involvement of Aedes spp. mosquitoes in its transmission increase the risk that its geographical range will reach greater populations. MAYV shares clinical symptoms and structural similarities with other emerging members of the alphavirus family such as Chikungunya (CHIKV), Ross River (RRV), and O'nyong nyong (ONNV) viruses, which complicate diagnosis and is currently restricted to nucleic acid-based tests. Effective control of the spread of these diseases will require the identification of infected individuals as well as the development of preventative and prophylactic therapies. Serological tests have a greater potential to expand testing at a reasonable cost than molecular approaches. Their performance largely reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene coding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily produced a stable chimeric protein in bacteria. The serological performance (AUC, sensitivity, specificity, and confidence intervals) of this multi-epitope protein as the target in ELISAs revealed a high diagnostic accuracy for the detection of specific anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for CHIKV, yellow fever (vaccinated), dengue, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.

scholarly journals Advances in Plant Disease Detection and Monitoring: From Traditional Assays to In-Field Diagnostics

Sensors ◽  
2021 ◽  
Vol 21 (6) ◽  
pp. 2129 ◽  
Author(s):  
Ilaria Buja ◽  
Erika Sabella ◽  
Anna Grazia Monteduro ◽  
Maria Serena Chiriacò ◽  
Luigi De Bellis ◽  
...  
Keyword(s):  
Emerging Diseases ◽  
Diagnostic Methods ◽  
Preventive Actions ◽  
Initial Symptoms ◽  
New Frontier ◽  
Information And Communication ◽  
Instruments And Techniques ◽  
The Difference ◽  
Asymptomatic Individuals

Human activities significantly contribute to worldwide spread of phytopathological adversities. Pathogen-related food losses are today responsible for a reduction in quantity and quality of yield and decrease value and financial returns. As a result, “early detection” in combination with “fast, accurate, and cheap” diagnostics have also become the new mantra in plant pathology, especially for emerging diseases or challenging pathogens that spread thanks to asymptomatic individuals with subtle initial symptoms but are then difficult to face. Furthermore, in a globalized market sensitive to epidemics, innovative tools suitable for field-use represent the new frontier with respect to diagnostic laboratories, ensuring that the instruments and techniques used are suitable for the operational contexts. In this framework, portable systems and interconnection with Internet of Things (IoT) play a pivotal role. Here we review innovative diagnostic methods based on nanotechnologies and new perspectives concerning information and communication technology (ICT) in agriculture, resulting in an improvement in agricultural and rural development and in the ability to revolutionize the concept of “preventive actions”, making the difference in fighting against phytopathogens, all over the world.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

scholarly journals Application of Humanized Zebrafish Model in the Suppression of SARS-CoV-2 Spike Protein Induced Pathology by Tri-Herbal Medicine Coronil via Cytokine Modulation

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 5091
Author(s):  
Acharya Balkrishna ◽  
Siva Kumar Solleti ◽  
Sudeep Verma ◽  
Anurag Varshney
Keyword(s):  
Herbal Medicine ◽  
High Performance ◽  
Normal Skin ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Spike Protein ◽  
Protective Effects ◽  
Cell Degeneration ◽  
Zebrafish Model ◽  
Tnf Α

Zebrafish has been a reliable model system for studying human viral pathologies. SARS-CoV-2 viral infection has become a global chaos, affecting millions of people. There is an urgent need to contain the pandemic and develop reliable therapies. We report the use of a humanized zebrafish model, xeno-transplanted with human lung epithelial cells, A549, for studying the protective effects of a tri-herbal medicine Coronil. At human relevant doses of 12 and 58 µg/kg, Coronil inhibited SARS-CoV-2 spike protein, induced humanized zebrafish mortality, and rescued from behavioral fever. Morphological and cellular abnormalities along with granulocyte and macrophage accumulation in the swim bladder were restored to normal. Skin hemorrhage, renal cell degeneration, and necrosis were also significantly attenuated by Coronil treatment. Ultra-high-performance liquid chromatography (UHPLC) analysis identified ursolic acid, betulinic acid, withanone, withaferine A, withanoside IV–V, cordifolioside A, magnoflorine, rosmarinic acid, and palmatine as phyto-metabolites present in Coronil. In A549 cells, Coronil attenuated the IL-1β induced IL-6 and TNF-α cytokine secretions, and decreased TNF-α induced NF-κB/AP-1 transcriptional activity. Taken together, we show the disease modifying immunomodulatory properties of Coronil, at human equivalent doses, in rescuing the pathological features induced by the SARS-CoV-2 spike protein, suggesting its potential use in SARS-CoV-2 infectivity.

scholarly journals A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Cécile Beck ◽  
Philippe Desprès ◽  
Sylvie Paulous ◽  
Jessica Vanhomwegen ◽  
Steeve Lowenski ◽  
...  
Keyword(s):  
High Performance ◽  
Neurological Diseases ◽  
Expression System ◽  
Cross Reactivity ◽  
Encephalitis Virus ◽  
Serological Tests ◽  
Multiplex Immunoassay ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Negative Controls

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using theDrosophilaS2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.

scholarly journals Recent Advances in Non‐nucleophilic Mg Electrolytes

2021 ◽  
Vol 7 ◽  
Author(s):  
Qijie Wu ◽  
Kewei Shu ◽  
Lili Sun ◽  
Haihua Wang
Keyword(s):  
High Performance ◽  
Early Stage ◽  
Oxidation Stability ◽  
Short Review ◽  
High Reactivity ◽  
Inherent Safety ◽  
Practical Application ◽  
Safety Issues ◽  
Ion Structure ◽  
Recent Advances

High-performance electrolyte is still a roadblock for the development of rechargeable magnesium (Mg) batteries. Grignard-type electrolytes were once the only choice in the early stage of rechargeable Mg batteries research. However, due to their nucleophilic nature and high reactivity, Grignard-type electrolytes have inherent safety issues and low oxidation stability, which restrict the development of rechargeable Mg batteries in terms of practical application. Recently, emerging novel Mg battery systems such as Mg-S, Mg-O2/air batteries also require non‐nucleophilic electrolytes with high oxidation stability. This short review summarizes recent advances in non‐nucleophilic Mg electrolytes and aims to provide insights into electrochemical properties and active Mg ion structure of such electrolytes.

scholarly journals Assessment of a recombinant protein from Leishmania infantum as a novel tool for Visceral Leishmaniasis (VL) diagnosis in VL/HIV co-infection cases

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0251861
Author(s):  
Rhaíssa E. M. Ramos ◽  
Wagner J. T. Santos ◽  
Franklin B. Magalhães ◽  
George T. N. Diniz ◽  
Carlos H. N. Costa ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Recombinant Protein ◽  
Indirect Elisa ◽  
Hiv Infections ◽  
Diagnostic Methods ◽  
Direct Agglutination Test ◽  
Serological Tests ◽  
Perfect Agreement ◽  
Significant Drop ◽  
Life Threatening

Visceral Leishmaniasis and HIV-AIDS coinfection (VL/HIV) is considered a life-threatening pathology when undiagnosed and untreated, due to the immunosuppression caused by both diseases. Serological tests largely used for the VL diagnosis include the direct agglutination test (DAT), ELISA and immunochromatographic (ICT) assays. For VL diagnosis in HIV infections, different studies have shown that the use of the DAT assay facilitates the VL diagnosis in co-infected patients, since the performance of the most widely used ELISA and ICT tests, based on the recombinant protein rK39, are much less efficient in HIV co-infections. In this scenario, alternative recombinant antigens may help the development of new serological diagnostic methods which may improve the VL diagnosis for the co-infection cases. This work aimed to evaluate the use of the recombinant Lci2 antigen, related to, but antigenically more diverse than rK39, for VL diagnosis in co-infected sera through ELISA assays. A direct comparison between recombinant Lci2 and rK39 was thus carried out. The two proteins were first tested using indirect ELISA with sera from VL afflicted individuals and healthy controls, with similar performances. They were then tested with two different sets of VL/HIV co-infected cases and a significant drop in performance, for one of these groups, was observed for rK39 (32% sensitivity), but not for Lci2 (98% sensitivity). In fact, an almost perfect agreement (Kappa: 0.93) between the Lci2 ELISA and DAT was observed for the coinfected VL/HIV patients. Lci2 then has the potential to be used as a new tool for the VL diagnosis of VL/HIV co-infections.

scholarly journals A parallel computational framework for ultra-large-scale sequence clustering analysis

2018 ◽  
Vol 35 (3) ◽  
pp. 380-388 ◽  
Author(s):  
Wei Zheng ◽  
Qi Mao ◽  
Robert J Genco ◽  
Jean Wactawski-Wende ◽  
Michael Buck ◽  
...  
Keyword(s):  
Parallel Computing ◽  
High Performance ◽  
Large Scale ◽  
De Novo ◽  
Rapid Development ◽  
Operational Taxonomic Unit ◽  
Supplementary Information ◽  
Computational Framework ◽  
Speed Up ◽  
Scale Sequence

Abstract Motivation The rapid development of sequencing technology has led to an explosive accumulation of genomic data. Clustering is often the first step to be performed in sequence analysis. However, existing methods scale poorly with respect to the unprecedented growth of input data size. As high-performance computing systems are becoming widely accessible, it is highly desired that a clustering method can easily scale to handle large-scale sequence datasets by leveraging the power of parallel computing. Results In this paper, we introduce SLAD (Separation via Landmark-based Active Divisive clustering), a generic computational framework that can be used to parallelize various de novo operational taxonomic unit (OTU) picking methods and comes with theoretical guarantees on both accuracy and efficiency. The proposed framework was implemented on Apache Spark, which allows for easy and efficient utilization of parallel computing resources. Experiments performed on various datasets demonstrated that SLAD can significantly speed up a number of popular de novo OTU picking methods and meanwhile maintains the same level of accuracy. In particular, the experiment on the Earth Microbiome Project dataset (∼2.2B reads, 437 GB) demonstrated the excellent scalability of the proposed method. Availability and implementation Open-source software for the proposed method is freely available at https://www.acsu.buffalo.edu/~yijunsun/lab/SLAD.html. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

Science ◽  
2020 ◽  
Vol 370 (6521) ◽  
pp. 1208-1214 ◽  
Author(s):  
Thomas W. Linsky ◽  
Renan Vergara ◽  
Nuria Codina ◽  
Jorgen W. Nelson ◽  
Matthew J. Walker ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
High Specificity ◽  
Spike Protein ◽  
Host Proteins ◽  
Angiotensin Converting Enzyme 2 ◽  
Cryo Electron Microscopy ◽  
De Novo Protein Design ◽  
Single Spike

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo–electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

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