In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

La, Espinosa; Ramos, Yassel; Andújar, Iván; Eo, Torres; Cabrera, Gleysin; Martín, Alejandro; González, Daniel H.; Chinea, Glay; M, Becquet; González, Ileana L.; Canaán-Haden, Camila; Nelson, Elías; Rojas, Gertrudis; Pérez-Massón, Beatriz; Pérez-Martínez, Dayana; Boggiano, Tammy; Palacio, Julio; Sl, Lozada-Chang; Hernández, Liliet Matech; Kr, de la Luz Hernández; Saloheimo, Markku; Marika,; Valdés-Balbín, Yury; Santana-Medero, Darielys; Dg, Rivera; Vérez-Bencomo,; Emalfarb, Mark; Tchelet, Ronen; Guillén, Gerardo; Limonta, Miladys; Pimentel, Eulogio; Ayala, Marta; Besada, Vladimir; González, Luis Javier

doi:10.1101/2021.05.10.443404

Cited by 3 publications

(1 citation statement)

References 63 publications

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“…The presence of eight cysteine residues linked by four intra-molecular disulfide bonds (C336-C361, C379-C432, C391-C525, and C480-C488) identical to those present in the native RBD of SARS-CoV-2 was confirmed by ESI-MS/MS analysis. The correct arrangement of disulfide bonds in the RBD is essential to obtain a correctly folded protein with the expected biological activity given by its ability to bind to the human ACE2 receptor and the production of neutralizing antibodies in the immunization of humans and animals [44].…”

Section: Discussionmentioning

confidence: 99%

Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate

et al. 2022

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COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host’s cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.

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Section: Discussionmentioning

confidence: 99%

Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate

et al. 2022

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A recombinant SARS-CoV-2 receptor-binding domain expressed in an engineered fungal strain of Thermothelomyces heterothallica induces a functional immune response in mice

Lazo

Bequet-Romero

Lemos

et al. 2022

Vaccine

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Since the beginning of the COVID-19 pandemic, the development of effective vaccines against this pathogen has been a priority for the scientific community. Several strategies have been developed including vaccines based on recombinant viral protein fragments. The receptor-binding domain (RBD) in the S1 subunit of S protein has been considered one of the main targets of neutralizing antibodies. In this study we assess the potential of a vaccine formulation based on the recombinant RBD domain of SARS-CoV-2 expressed in the thermophilic filamentous fungal strain Thermothelomyces heterothallica and the hepatitis B virus (HBV) core protein. Functional humoral and cellular immune responses were detected in mice. To our knowledge, this is the first report on the immune evaluation of a biomedical product obtained in the fungal strain T. heterothallica. These results together with the intrinsic advantages of this expression platform support its use for the development of biotechnology products for medical purpose.

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The SARS-CoV-2 receptor-binding domain expressed in Pichia pastoris as a candidate vaccine antigen

Limonta

Chinea-Santiago

Martín-Dunn

et al. 2021

Preprint

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The effort to develop vaccines based on economically accessible technological platforms available by developing countries vaccine manufacturers is essential to extend the immunization to the whole world population and to achieve the desired herd immunity, necessary to end the COVID–19 pandemic. Here we report on the development of a SARS–CoV–2 receptor–binding domain (RBD) protein, expressed in yeast Pichia pastoris. The RBD was modified with the addition of flexible N– and C–terminal amino acid extensions aimed to modulate the protein/protein interactions and facilitate protein purification. Fermentation with yeast extract culture medium yielded 30–40 mg/L. After purification by immobilized metal ion affinity chromatography and hydrophobic interaction chromatography, the RBD protein was characterized by mass–spectrometry, circular dichroism, and binding affinity to angiotensin–converting enzyme 2 (ACE2) receptor. The recombinant protein shows high antigenicity with convalescent human sera and also with sera from individuals vaccinated with the Pfizer–BioNTech mRNA or Sputnik V adenoviral–based vaccines. The RBD protein stimulates IFNγ, IL–2, IL–6, IL–4, and TNFα in mice secreting splenocytes from PBMC and lung, CD3+ enriched cells. Immunogenicity studies with 50 μg of the recombinant RBD formulated with alum, induce high levels of binding antibodies in mice and non–human primates, assessed by ELISA plates covered with RBD protein expressed in HEK293T cells. The mouse sera inhibited the RBD binding to ACE2 receptor in an in–vitro test and show neutralization of SARS–CoV–2 infection of Vero E6 cells. These data suggest that the RBD recombinant protein expressed in yeast P. pastoris is suitable as a vaccine candidate against COVID–19.

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In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

Cited by 3 publications

References 63 publications

Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate

Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate

A recombinant SARS-CoV-2 receptor-binding domain expressed in an engineered fungal strain of Thermothelomyces heterothallica induces a functional immune response in mice

The SARS-CoV-2 receptor-binding domain expressed in Pichia pastoris as a candidate vaccine antigen

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