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

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.

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

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.

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

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.

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

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.

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

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.

scholarly journals High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Thomas J. Esparza ◽  
Negin P. Martin ◽  
George P. Anderson ◽  
Ellen R. Goldman ◽  
David L. Brody
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Hek293 Cells ◽  
Scale Production ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
High Affinity ◽  
Diagnostic Potential

AbstractThere are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12–15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1–5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

scholarly journals An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Yan Guo ◽  
Wenhui He ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Jing Chang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Binding Domain ◽  
Full Length ◽  
Vaccine Antigen ◽  
Receptor Binding Domain ◽  
Protein Vaccine ◽  
Wild Type ◽  
S Protein ◽  
Glycosylation Sites

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines. IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

scholarly journals An engineered receptor-binding domain improves the immunogenicity of multivalent SARS-CoV-2 vaccines

2020 ◽  
Author(s):  
Brian D. Quinlan ◽  
Wenhui He ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Yan Guo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Viral Entry ◽  
Binding Domain ◽  
Vaccine Antigen ◽  
Receptor Binding Domain ◽  
Protein Vaccine ◽  
Wild Type ◽  
S Protein ◽  
H Pylori

ABSTRACTThe SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD expresses inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) expresses markedly more efficiently, and generates a more potent neutralizing responses as a DNA vaccine antigen, than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers such as an H. pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.

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

2021 ◽  
Author(s):  
Neil C. Dalvie ◽  
Andrew M. Biedermann ◽  
Sergio A. Rodriguez‐Aponte ◽  
Christopher A. Naranjo ◽  
Harish D. Rao ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Komagataella Phaffii

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

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.

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

2021 ◽  
Author(s):  
A. Berndt ◽  
T. Smalley ◽  
B. Ren ◽  
A. Badary ◽  
A. Sproles ◽  
...  
Keyword(s):  
Green Algae ◽  
Receptor Binding ◽  
Large Scale ◽  
Low Cost ◽  
Binding Domain ◽  
Intact Protein ◽  
Receptor Binding Domain ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Recombinant Production

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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