Murine monoclonal antibodies against RBD of the SARS-CoV-2 spike protein as useful analytical tools for subunit vaccine development and clinical trials

Blanco, Omar R.; Dorta, Dayamí; Hernández, Carlos Martínez; Abreu, Daymí; Domínguez, Andy G.; Luna, Yaramis; Valdivia, Onel; Pérez-Bernal, Maylín; Tamayo, Celia; Lemos, Gilda; Pasarón, Ivis; Pérez, Joel Javier; Benítez, Liudmila; Bequet-Romero, Mónica; Quintero, Anitza Fraga; Cabrera, Yeosvany; Pérez, Enrique R.

doi:10.1016/j.jim.2021.113195

Cited by 6 publications

(7 citation statements)

References 42 publications

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“…To confirm antigenicity of C-RBD-H6 recognition by known sera and monoclonal antibodies, the binding of the murine anti-RBD monoclonal antibodies SS-1, SS-4, SS-7 and SS-8 to C-RBD-H6 PP and C-RBD-H6 HEK was first compared by ELISA. These mAbs were obtained by immunization with RBD-H6 (produced in HEK293T cells), and one, SS-8, competes with ACE2 for binding to the RBD with an IC50 of 122.7 pM [23] . As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Briefly, the wells of 96-well microtiter plates (Corning Costar, Acton, USA) were coated overnight at 4 °C with 0.25 µg of RBD-H6 (produced in HEK293T cells) in 0.1 M sodium carbonate buffer (pH 9.6). After blocking the plates with 2% skim milk (Oxoid, Basingstoke, UK), 0.05% Tween 20 (Merck, Darmstadt, Germany), serially diluted animal sera or control monoclonal antibodies SS-1, SS-4, SS-7 and SS-8 (CBSSRBD-S.1, CBSSRBD-S.4, CBSSRBD-S.7 and CBSSRBD-S.8; Center for Genetic Engineering and Biotechnology, Sancti Spiritus, Cuba) [23] , were added and incubated at 37 °C for 2 h in 0.2% skim milk, 0.05% Tween 20 in PBS, followed by six washes with PBS containing 0.05% Tween 20. Bound antibodies were detected with anti-human IgG conjugated to HRP (1:10,000; 109-035-098; Jackson ImmunoResearch, West Grove, PA, USA) at 37 °C for 1 h, followed by washing.…”

Section: Methodsmentioning

confidence: 99%

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An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

et al. 2022

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

et al. 2022

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“…In contrast, values of API absorbed in the vaccine, measured directly by ELISA, decreased to around 50% in samples stored at 45°C, and 100% at 60°C with respect to the 2-8°C storage conditions. Hence, authors hypothesize this decrease was produced by the loss of the recognition capacity of the CBSSRBD-S.8 mAb [21] specific for a RBD conformational epitope, as observed in samples stored at the highest temperatures (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…In parallel, samples and controls were incubated in a separate non-protein binding plate with humanFc-RBD-HRP diluted in 0.2% (w/v) non-fat dry milk powder in DPBS, for 1 h at 37°C. The CBSSRBD-S.8 mAb that inhibits and neutralizes SARS-CoV-2 [21] was used as a positive control in this assay. After the incubation period, 100 µL of the sample-conjugate mixtures were added per ACE2-coated well with two replicates per point, and additionally incubated for another 90 min at 37°C.…”

Section: Surrogate Virus Neutralization Test (Svnt-elisa) Performed T...mentioning

confidence: 99%

Untitled

2022

BPJ

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From a regulatory standpoint, vaccine stability must be demonstrated, along with the prediction of stability during temperature excursions, before a vaccine can be approved for use in humans.In this work, Abdala subunit vaccine thermostability was studied under thermal stress conditions (2-8°C [control], 25°C, 37 °C, 45°C, and 60°C) for 15 days. Molecular integrity of the vaccine active pharmaceutical ingredient was monitored by SDS-PAGE, immunoblotting, RP-HPLC, mass spectrometry, and circular dichroism spectroscopy analysis. While functionality was monitored by immunogenicity assay, inhibition of binding between receptor-binding domain (RBD) and receptor, angiotensin converting enzyme 2 (ACE2), and RBD/ACE2 binding assay.Results showed that no degradation, loss of disulfide bridges, nor modifications of secondary structure of the RBD molecule were detected at 25°C and 37 °C. Moreover, high titers (1:48,853 -1:427,849) of anti-RBD-specific mouse antibodies were detected with the ability to inhibit, to different degrees, the binding between RBD/ACE2.In conclusion, the Abdala subunit vaccine is stable under thermal stress and storage conditions, which has an advantage over non-subunit vaccines previously approved or currently in development against COVID-19. The demonstrated high stability of this vaccine is a key factor in ensuring vaccine effectiveness, extending immunization coverage with fewer production runs, simplifying immunization logistics, and reducing cold chain-associated costs.Demonstrating "Abdala" Subunit Vaccine Stability Under Thermal Stress Conditions Pichia pastoris yeast as the host cell. [9][10][11] Therefore, the strategy applied to produce the Abdala subunit vaccine as active pharmaceutical ingredient (API) against COVID-19 was also based on the production of the SARS-CoV-2 receptor-binding domain (RBD) in the same P. pastoris production and regulatory platforms to speed up the approval process for massive application in humans. Details on the RBD expression are described by Limonta et al. [12] A sequence coding for residues 331-530 of the spike protein of SARS-CoV-2 strain 156 Wuhan-Hu-1 (NCBI Acc. No. YP_009724390) with the appropriate Nand C-terminal 157 extensions was expressed in P. pastoris under the AOX1 promoter. After the nucleotide sequence verification, a representative clone was used to prepare the yeast seed bank and to inoculate a 3000 L fermenter.For such purposes, and considering the most updated regulatory issues, the demonstration of vaccine stability is mandatory because API and vaccine stability are critical factors impacting quality, potency, and distribution. Consequently, all immunization campaigns have the challenge of validating and maintaining the cold chain during distribution, delivery, and storage of the vaccines.The World Health Organization (WHO) and other regulatory agencies have issued guidelines to regulate how stability studies will be conducted under storage and thermal-stress conditions. To fulfill such requirements, the application of physico-chemical an...

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“…This provided a very promising vaccination method for SARS‐CoV‐2 using RBD and HR‐based nanoparticles. Blanco and his team also generated nine murine monoclonal antibodies against SARS‐CoV‐2 spike protein RBD using hybridoma technology, six of which showed specific recognition of different recombinant RBD 107 . It provided a new idea for quantifying recombinant RBD‐S antigen in SARS‐CoV‐2 research projects in preclinical and clinical trials.…”

Section: Treatment Of Sars‐cov‐2mentioning

confidence: 99%

A comprehensive overview on the transmission, pathogenesis, diagnosis, treatment, and prevention of SARS‐CoV‐2

Zhang

Clarke

et al. 2023

Journal of Medical Virology

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Severe acute respiratory syndrome coronavirus (SARS-CoV) is a single positivestrand RNA virus that is responsible for the current pandemic that the world has been facing since 2019. The primary route of transmission of SARS-CoV-2 is through respiratory tract transmission. However, other transmission routes such as fecal-oral, vertical transmission, and aerosol-eye also exist. In addition, it has been found that the pathogenesis of this virus involves the binding of the virus's S protein to its host cell surface receptor angiotensin-converting enzyme 2, which results in the subsequent membrane fusion that is required for SARS-CoV-2 to replicate and complete its entire life. The clinical symptoms of patients infected with SARS-CoV-2 can range from asymptomatic to severe. The most common symptoms seen include fever, dry cough, and fatigue. Once these symptoms are observed, a nucleic acid test is done using reverse transcription-polymerase chain reaction. This currently serves as the main confirmatory tool for COVID-19. Despite the fact that no cure has been found for SARS-CoV-2, prevention methods such as vaccines, specific facial mask, and social distancing have proven to be quite effective. It is imperative to have a complete understanding of the transmission and pathogenesis of this virus. To effectively develop new drugs as well as diagnostic tools, more knowledge about this virus would be needed.

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Murine monoclonal antibodies against RBD of the SARS-CoV-2 spike protein as useful analytical tools for subunit vaccine development and clinical trials

Cited by 6 publications

References 42 publications

An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

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A comprehensive overview on the transmission, pathogenesis, diagnosis, treatment, and prevention of SARS‐CoV‐2

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