High-Yield Production of Receptor Binding Domain of SARS-CoV-2 Linked to Bacterial Flagellin in Plants Using Self-Replicating Viral Vector pEff

The development of recombinant vaccines against SARS-CoV-2 is required to eliminate the COVID-19 pandemic. We reported the expression of a recombinant protein Flg-RBD comprising receptor binding domain of SARS-CoV-2 spike glycoprotein (RBD) fused to flagellin of Salmonella typhimurium (Flg), known as mucosal adjuvant, in Nicotiana benthamiana plants. The fusion protein, targeted to the cytosol, was transiently expressed using the self-replicating vector pEff based on potato virus X genome. The recombinant protein Flg-RBD was expressed at the level of about 110–140 μg per gram of fresh leaf tissue and was found to be insoluble. The fusion protein was purified using metal affinity chromatography under denaturing conditions. To increase the yield of Flg-RBD, the flow-through fraction obtained after loading of the protein sample on the Ni-NTA resin was re-loaded on the sorbent. The yield of Flg-RBD after purification reached about 100 μg per gram of fresh leaf tissue and the purified protein remained soluble after dialysis. The control flagellin was expressed in a soluble form and its yield after purification was about 300 μg per gram of fresh leaf biomass. Plant-produced Flg-RBD protein could be further used for the development of intranasal recombinant mucosal vaccines against COVID-19.

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Construction and immunogenic studies of a mFc fusion receptor binding domain (RBD) of spike protein as a subunit vaccine against SARS-CoV-2 infection

Chemical Communications ◽

10.1039/d0cc03263h ◽

2020 ◽

Vol 56 (61) ◽

pp. 8683-8686 ◽

Cited By ~ 5

Author(s):

Xiaoxiao Qi ◽

Bixia Ke ◽

Qian Feng ◽

Deying Yang ◽

Qinghai Lian ◽

...

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Humoral And Cellular Immunity ◽

A Subunit

Herein, we report that a recombinant fusion protein, containing a 457 amino acid SARS-CoV-2 receptor binding domain and a mouse IgG1 Fc domain, could induce highly potent neutralizing antibodies and stimulate humoral and cellular immunity in mice.

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Design, expression, purification and characterization of a YFP-tagged 2019-nCoV spike receptor-binding domain construct

10.1101/2020.09.29.318196 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tobias Bierig ◽

Gabriella Collu ◽

Alain Blanc ◽

Emiliya Poghosyan ◽

Roger. M. Benoit

Keyword(s):

Fusion Protein ◽

Receptor Binding ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Fluorescent Labeling ◽

Binding Domain ◽

Size Exclusion ◽

Antibody Detection ◽

Receptor Binding Domain ◽

Binding Studies

Abstract2019-nCoV is the causative agent of the serious, still ongoing, worldwide COVID-19 pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFNα2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA IMAC purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.

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Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques

Nature Communications ◽

10.1038/s41467-021-25382-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Romain Marlin ◽

Veronique Godot ◽

Sylvain Cardinaud ◽

Mathilde Galhaut ◽

Severin Coleon ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Viral Vector ◽

Binding Domain ◽

Long Term Memory ◽

High Dose ◽

Receptor Binding Domain ◽

Humanized Mouse Model ◽

Subunit Vaccines ◽

Antigen Presenting

AbstractAchieving sufficient worldwide vaccination coverage against SARS-CoV-2 will require additional approaches to currently approved viral vector and mRNA vaccines. Subunit vaccines may have distinct advantages when immunizing vulnerable individuals, children and pregnant women. Here, we present a new generation of subunit vaccines targeting viral antigens to CD40-expressing antigen-presenting cells. We demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with long-term memory phenotypes, in a humanized mouse model. Additionally, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Vaccine-elicited antibodies cross-neutralize different SARS-CoV-2 variants, including D614G, B1.1.7 and to a lesser extent B1.351. Such vaccination significantly improves protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals.

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Obtaining and Purification of Recombinant Domain III of Human Alpha-Fetoprotein

Biotekhnologiya ◽

10.21519/0234-2758-2021-37-4-32-42 ◽

2021 ◽

Vol 37 (4) ◽

pp. 32-42

Author(s):

M.D. Mollaev ◽

A.I. Zabolotskii ◽

D.A. Mazalev ◽

N.V. Gorokhovets ◽

M.B. Sokol ◽

...

Keyword(s):

Drug Delivery ◽

Recombinant Protein ◽

Receptor Binding ◽

Targeted Delivery ◽

Inclusion Bodies ◽

Antitumor Agents ◽

Binding Domain ◽

Alpha Fetoprotein ◽

Receptor Binding Domain ◽

Purification Step

Previous studies have demonstrated the applicability of alpha-fetoprotein or its receptor-binding domain fused to the 6-histidine tag at the C-terminus (rAFP3D-His6) as vectors for targeted delivery of antitumor agents. This tag is undesirable for further preclinical trials. Therefore, we designed a recombinant protein rAFP3D without any affine tags, and accessed its functional activity. The protein was produced as inclusion bodies in Escherichia coli BL21 (DE3) cells. Optimal conditions for washing inclusion bodies and refolding the target protein were selected. We used a saturated solution of (NH4)2SO4 as the primary purification step to precipitate the main fraction of protein admixtures. The second purification step included hydrophobic chromatography using butyl-cellulose. The identity of the protein sequence was confirmed by tandem mass spectrometry. Circular dichroism demonstrated the authenticity of the secondary structure. Fluorescently labeled rAFP3D actively penetrated into MCF-7 tumor cells. These results indicate that rAFP3D can be used for targeted drug delivery. Key words: alpha-fetoprotein, receptor-binding domain, recombinant protein, MALDI-MS, HPLC, drug delivery system Funding - The project was supported with Russian Foundation for Basic Research (project no. 18-29-09022/20).

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Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate

Human Vaccines & Immunotherapeutics ◽

10.4161/hv.27464 ◽

2013 ◽

Vol 10 (3) ◽

pp. 648-658 ◽

Cited By ~ 46

Author(s):

Wen-Hsiang Chen ◽

Lanying Du ◽

Shivali M Chag ◽

Cuiqing Ma ◽

Nancy Tricoche ◽

...

Keyword(s):

Recombinant Protein ◽

Receptor Binding ◽

Vaccine Candidate ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain

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Fusion Protein of Rotavirus VP6 and SARS-CoV-2 Receptor Binding Domain Induces T Cell Responses

Vaccines ◽

10.3390/vaccines9070733 ◽

2021 ◽

Vol 9 (7) ◽

pp. 733

Author(s):

Kirsi Tamminen ◽

Suvi Heinimäki ◽

Stina Gröhn ◽

Vesna Blazevic

Keyword(s):

T Cell ◽

Fusion Protein ◽

Receptor Binding ◽

Recombinant Baculovirus ◽

Binding Domain ◽

Receptor Binding Domain ◽

T Cell Epitopes ◽

Experimental Conditions ◽

Rotavirus Vp6

Vaccines based on mRNA and viral vectors are currently used in the frontline to combat the ongoing pandemic caused by the novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). However, there is still an urgent need for alternative vaccine technologies inducing/boosting long-lasting and cross-reactive immunity in different populations. As a possible vaccine candidate, we employed the rotavirus VP6-protein platform to construct a fusion protein (FP) displaying receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S) at the N-terminus of VP6. The recombinant baculovirus-insect cell produced VP6-RBD FP was proven antigenic in vitro and bound to the human angiotensin-converting enzyme 2 (hACE2) receptor. The FP was used to immunize BALB/c mice, and humoral- and T cell-mediated immune responses were investigated. SARS-CoV-2 RBD-specific T cells were induced at a high quantity; however, no RBD or S-specific antibodies were detected. The results suggest that conformational B cell epitopes might be buried inside the VP6, while RBD-specific T cell epitopes are available for T cell recognition after the processing and presentation of FP by the antigen-presenting cells. Further immunogenicity studies are needed to confirm these findings and to assess whether, under different experimental conditions, the VP6 platform may present SARS-CoV-2 antigens to B cells as well.

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

10.1101/2021.06.29.21259605 ◽

2021 ◽

Author(s):

Miladys Limonta-Fernandez ◽

Glay Chinea-Santiago ◽

Alejandro Miguel Martin-Dunn ◽

Diamile Gonzalez-Roche ◽

Monica Bequet-Romero ◽

...

Keyword(s):

Pichia Pastoris ◽

Recombinant Protein ◽

Receptor Binding ◽

Metal Ion ◽

Binding Domain ◽

Vaccine Antigen ◽

World Population ◽

In Vitro Test ◽

Receptor Binding Domain

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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Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques

10.21203/rs.3.rs-244682/v1 ◽

2021 ◽

Author(s):

Romain Marlin ◽

Véronique Godot ◽

Sylvain Cardinaud ◽

Mathilde Galhaut ◽

Severin Coleon ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

High Efficiency ◽

Viral Vector ◽

Binding Domain ◽

Long Term Memory ◽

High Dose ◽

Human Populations ◽

Receptor Binding Domain ◽

Subunit Vaccines

Abstract Controlling the circulation of the recently emerged SARS-CoV-2 in the human populations requires massive vaccination campaigns. Achieving sufficient worldwide vaccination coverage will require additional approaches to first generation of approved viral vector and mRNA vaccines. Subunit vaccines have excellent safety and efficacy records and may have distinct advantages, in particular when immunizing individuals with vulnerabilities or when considering the vaccination of children and pregnant women.. We have developed a new generation of subunit vaccines with enhanced immunogenicity by the targeting of viral antigens to CD40-expressing antigen-presenting cells, thus harnessing their intrinsic immune-stimulant properties. Here, we demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with a long-term memory phenotype, in a humanized mouse model. In addition, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Such vaccination thus significantly improved protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals. Viral dynamics modelling showed the high efficiency of the vaccine at controlling the viral dissemination.

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