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      Squalene in oil-based adjuvant improves the immunogenicity of SARS-CoV-2 RBD and confirms safety in animal models

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      1 , 1 , 1 , 1 , 1 , 1 , 1 , 2 , 1 , 2 , 1 , 1 , 1 , 1 , 2 , 1 , 1 , 1 , 1 , 1 , 2 , 1 , 1 , 1 , 1 , 2 , * , , for the COVID-19 Working Group in Perú
      PLoS ONE
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          Abstract

          COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus lead to the generation of new alternatives to improve the current sub-unit vaccines in development. In the present report, the immunogenicity of the Spike RBD of SARS-CoV-2 formulated with an oil-in-water emulsion and a water-in-oil emulsion with squalene was evaluated in mice and hamsters. The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions. In immunization assays, although the cellular immune response elicited by both adjuvants were similar, the formulation based in water-in-oil emulsion and squalene generated an earlier humoral response as determined by ELISA. Similarly, this formulation was able to stimulate neutralizing antibodies in hamsters. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results have shown the potential of this formulation vaccine to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection.

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          Most cited references44

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          A pneumonia outbreak associated with a new coronavirus of probable bat origin

          Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats 1–4 . Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans 5–7 . Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor—angiotensin converting enzyme II (ACE2)—as SARS-CoV.
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            Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

            Summary The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
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              Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

              A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from December 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an atomic level, we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural analysis identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analysed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Validation
                Role: Data curationRole: InvestigationRole: Methodology
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: Methodology
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: Investigation
                Role: Data curationRole: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: Formal analysisRole: Methodology
                Role: Formal analysisRole: Methodology
                Role: Formal analysisRole: Methodology
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: Formal analysis
                Role: ConceptualizationRole: InvestigationRole: Methodology
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Project administrationRole: Resources
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                23 August 2022
                2022
                23 August 2022
                : 17
                : 8
                : e0269823
                Affiliations
                [1 ] Laboratorios de investigación y desarrollo, FARVET SAC, Chincha, Ica, Perú
                [2 ] Laboratorio de Bioinformática, Biología Molecular y Desarrollos Tecnológicos, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru
                Instituto Butantan, BRAZIL
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                ¶ Membership of the COVID-19 Working Group in Peru is provided in the Acknowledgments.

                Author information
                https://orcid.org/0000-0002-9814-3821
                https://orcid.org/0000-0002-6881-8234
                https://orcid.org/0000-0002-2434-5244
                https://orcid.org/0000-0002-7203-8847
                Article
                PONE-D-21-36362
                10.1371/journal.pone.0269823
                9397949
                35998134
                b09338e2-9bd2-431d-82da-6e5df1d60272
                © 2022 Choque-Guevara et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 16 November 2021
                : 30 May 2022
                Page count
                Figures: 8, Tables: 0, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100010751, Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica;
                Award ID: 060-2020-FONDECYT
                Award Recipient :
                This study was funded/ supported by Laboratorios de Investigación y Desarrollo - FARVET and partially by Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica - FONDECYT ( https://www.fondecyt.gob.pe/) under the contract 060-2020-FONDECYT. MFD and MZ were granted by Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC). These funder supported salaries for RCG, RMM, APA, DRM, KGM, AM, SQG, MCM, AAAM, IRO, MCO, EHG, NPM, GIR, YSA and DVP and supplied materials for the study. The funders had no role in study desing, data collection and analysis, decision to publish, or preparation of manuscript.
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