Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

ABSTRACTRecombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated, lacking the amino terminus. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

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Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells

Scientific Reports ◽

10.1038/s41598-020-78711-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Keyword(s):

Pichia Pastoris ◽

Receptor Binding ◽

Mammalian Cells ◽

Neutralizing Antibodies ◽

Large Scale ◽

Binding Domain ◽

Size Exclusion ◽

Scale Production ◽

Receptor Binding Domain ◽

Serological Tests

AbstractThe yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

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Stabilization of the SARS-CoV-2 Spike receptor-binding domain using deep mutational scanning and structure-based design

10.1101/2021.05.15.444222 ◽

2021 ◽

Author(s):

Daniel Ellis ◽

Natalie Brunette ◽

Katherine H. D. Crawford ◽

Alexandra C. Walls ◽

Minh N. Pham ◽

...

Keyword(s):

Receptor Binding ◽

Large Scale ◽

Protein S ◽

Binding Pocket ◽

Binding Domain ◽

Effective Vaccine ◽

Receptor Binding Domain ◽

Wild Type ◽

Vaccine Candidates ◽

Generation Vaccine

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.

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A magnetic bead immunoassay to detect human IgG reactive to SARS-CoV-2 Spike S1 RBD produced in Escherichia coli

10.1101/2021.12.22.21268286 ◽

2021 ◽

Author(s):

Marcelo dos Santos Conzentino ◽

Ana C Goncalves ◽

NIgella M Paula ◽

Fabiane G Rego ◽

Dalila Zanette ◽

...

Keyword(s):

Escherichia Coli ◽

Receptor Binding ◽

High Throughput ◽

Low Cost ◽

Magnetic Bead ◽

Binding Domain ◽

Receptor Binding Domain ◽

Operating Characteristics ◽

Human Igg ◽

Bead Immunoassay

Immunological assays to detect SARS-CoV-2 Spike receptor binding domain antigen seroconversion in humans are important tools to monitor the levels of protecting antibodies in the population in response to infection and/or immunization. Here we describe a simple, low cost and high throughput Ni2+ magnetic bead immunoassay to detect human IgG reactive to Spike S1 RBD Receptor Binding Domain produced in Escherichia coli. A 6xHis tagged Spike S1 RBD was expressed in E. coli and purified by affinity chromatography. The protein was mobilized on the surface of Ni2+ magnetic beads and used to investigate the presence of reactive IgG in the serum obtained from pre-pandemic and COVID-19 confirmed cases. The method was validated with a cohort of 290 samples and an area under the receiver operating characteristics curve of 0.94 was obtained. The method operated with>82% sensitivity at 98% specificity and was also able to track human IgG raised in response to vaccination with Comirnaty with 85% sensitivity. The IgG signal obtained with the described method was well correlated with the signal obtained when pre fusion Spike produced in HEK cell lines were used as antigen. This novel low-cost and high throughput immunoassay may act as an important tool to investigate protecting IgG antibodies against SARS-CoV-2 in the human population.

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Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design

Frontiers in Immunology ◽

10.3389/fimmu.2021.710263 ◽

2021 ◽

Vol 12 ◽

Author(s):

Daniel Ellis ◽

Natalie Brunette ◽

Katharine H. D. Crawford ◽

Alexandra C. Walls ◽

Minh N. Pham ◽

...

Keyword(s):

Receptor Binding ◽

Large Scale ◽

Protein S ◽

Binding Pocket ◽

Binding Domain ◽

Effective Vaccine ◽

Receptor Binding Domain ◽

Wild Type ◽

Vaccine Candidates ◽

Generation Vaccine

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.

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Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii

10.1101/2021.04.15.440035 ◽

2021 ◽

Author(s):

Neil C Dalvie ◽

Andrew M Biedermann ◽

Sergio A Rodriguez-Aponte ◽

Christopher A Naranjo ◽

Harish D Rao ◽

...

Keyword(s):

Receptor Binding ◽

Large Scale ◽

Scale Up ◽

Binding Domain ◽

Vaccine Antigen ◽

Scale Production ◽

Receptor Binding Domain ◽

Methanol Induction ◽

Vaccine Candidates ◽

Komagataella Phaffii

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

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SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice

10.1101/2020.11.04.367359 ◽

2020 ◽

Cited By ~ 1

Author(s):

Jeroen Pollet ◽

Wen-Hsiang Chen ◽

Leroy Versteeg ◽

Brian Keegan ◽

Bin Zhan ◽

...

Keyword(s):

Recombinant Protein ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Low Cost ◽

Neutralizing Antibody ◽

Binding Domain ◽

Receptor Binding Domain ◽

Wild Type ◽

Cell Immunity

AbstractThere is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.

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Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells

10.1101/2020.09.17.300335 ◽

2020 ◽

Author(s):

◽

Claudia R. Arbeitman ◽

Gabriela Auge ◽

Matías Blaustein ◽

Luis Bredeston ◽

...

Keyword(s):

Pichia Pastoris ◽

Receptor Binding ◽

Mammalian Cells ◽

Neutralizing Antibodies ◽

Large Scale ◽

Binding Domain ◽

Size Exclusion ◽

Scale Production ◽

Receptor Binding Domain ◽

Serological Tests

AbstractThe yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by HPLC, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

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Destabilizing the Structural Integrity of SARS-CoV2 Receptor Proteins by Curcumin Along with Hydroxychloroquine: An Insilco Approach for a Combination Therapy

10.26434/chemrxiv.12090438.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Akhileshwar Srivastava ◽

Divya Singh

Keyword(s):

Binding Energy ◽

Combination Therapy ◽

Receptor Binding ◽

Structural Integrity ◽

Binding Domain ◽

Receptor Binding Domain ◽

Receptor Proteins ◽

Main Protease ◽

The Stability ◽

Therapeutic Activities

Presently, an emerging disease (COVID-19) has been spreading across the world due to coronavirus (SARS-CoV2). For treatment of SARS-CoV2 infection, currently hydroxychloroquine has been suggested by researchers, but it has not been found enough effective against this virus. The present study based on in silico approaches was designed to enhance the therapeutic activities of hydroxychloroquine by using curcumin as an adjunct drug against SARS-CoV2 receptor proteins: main-protease and S1 receptor binding domain (RBD). The webserver (ANCHOR) showed the higher protein stability for both receptors with disordered score (<0.5). The molecular docking analysis revealed that the binding energy (-24.58 kcal/mol) of hydroxychloroquine was higher than curcumin (-20.47 kcal/mol) for receptor main-protease, whereas binding energy of curcumin (<a>-38.84</a> kcal/mol) had greater than hydroxychloroquine<a> (-35.87</a> kcal/mol) in case of S1 receptor binding domain. Therefore, this study suggested that the curcumin could be used as combination therapy along with hydroxychloroquine for disrupting the stability of SARS-CoV2 receptor proteins

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