An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics

Borhani, Shayan; Levine, Max Z.; Krumpe, Lauren R H; Wilson, Jennifer A.; Henrich, Curtis J.; O’Keefe, Barry R.; Lo, Donald; Sittampalam, G. Sitta; Godfrey, Alexander G.; Lunsford, R Dwayne; Mangalampalli, Venkata; Tao, Dingyin; LeClair, Christopher A.; Thole, Aaron; Frey, Douglas D.; Swartz, James R.; Rao, Govind

doi:10.1016/j.nbt.2023.04.003

Cited by 4 publications

(5 citation statements)

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“…The use of CFPS has even been posited for individualized vaccines and distributed medicines manufacturing, presenting new medical paradigms if regulation can be safely adapted to meet this potential (Kanter et al, 2007;Ogonah et al, 2017;Zawada et al, 2022). The ALiCE ® system has specifically been considered for the distributed manufacturing of the antiviral protein griffithsin, with equivalent bioactivity to griffithsin produced in E. coli CFPS (Borhani et al, 2023). We have also demonstrated the production of approximately 100,000 potential doses of the HBc VLP from a 1 L BYL reaction (Armero-Gimenez et al, 2023).…”

Section: Discussionmentioning

confidence: 98%

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

Gupta¹,

Flaskamp²,

Roentgen³

et al. 2023

Biotech & Bioengineering

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Eukaryotic cell‐free protein synthesis (CFPS) can accelerate expression and high‐throughput analysis of complex proteins with functionally relevant post‐translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing.Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY‐2 cell culture (BY‐2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N‐glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale‐up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre‐scale reaction in a rocking‐type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields.Production of multimeric virus‐like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult‐to‐express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS‐CoV‐2 receptor‐binding domain; a human growth factor; and a G protein‐coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in‐depth PTM characterization of purified proteins through disulfide bond and N‐glycan analysis.Taken together, BYL is a promising end‐to‐end R&D to manufacturing platform with the potential to significantly reduce the time‐to‐market for high value proteins and biologics.

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Section: Discussionmentioning

confidence: 98%

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

Gupta¹,

Flaskamp²,

Roentgen³

et al. 2023

Biotech & Bioengineering

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“…Before embarking on assessing whether Griffithsin could act as a suitable inhibitor of SARS-CoV-2 viral infection, we first verified that the M78Q mutation did not disrupt the native fold of WT-Grft. This was important because although the antiviral/prophylactic capability of recombinant Q-Grft has been verified to match WT-Grft in the literature, there does not yet appear to be published verification that recombinant production of M78Q Griffithsin in E. coli does indeed produce a protein that exhibits structural similarity to WT-Grft [29,40,70,73]. To do so, we produced 15 N-labeled WT-Grft and Q-Grft (as described in the Materials and Methods Section 2.2 above) and then assessed both proteins by NMR HSQC (Figure 1D).…”

Section: Wild-type Griffithsin and M78q Griffithsin Both Display Mode...mentioning

confidence: 99%

“…To our knowledge, with the exception of one recent paper, the current existing literature exploring Griffithsin's in vitro inhibitory capabilities against SARS-CoV-2 has solely utilized WT-Grft [35][36][37][38][39]. Although one study observed an approximately 60% stronger SARS-CoV-2 pseudoviral inhibition for M78Q-Grft when compared to WT-Grft, the two constructs used in the publication differed slightly, with the WT-Grft construct retaining a Hexa-histidine tag and an enterokinase cut site [40]. In this study, we first aimed to assess whether the M78Q mutation altered Griffithsin's ability to act as an anti-SARS-CoV-2 entry inhibitor.…”

Section: Introductionmentioning

confidence: 99%

The Antiviral Activity of the Lectin Griffithsin against SARS-CoV-2 Is Enhanced by the Presence of Structural Proteins

Bains,

Fischer,

Guan

et al. 2023

Viruses

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Although COVID-19 transmission has been reduced by the advent of vaccinations and a variety of rapid monitoring techniques, the SARS-CoV-2 virus itself has shown a remarkable ability to mutate and persist. With this long track record of immune escape, researchers are still exploring prophylactic treatments to curtail future SARS-CoV-2 variants. Specifically, much focus has been placed on the antiviral lectin Griffithsin in preventing spike protein-mediated infection via the hACE2 receptor (direct infection). However, an oft-overlooked aspect of SARS-CoV-2 infection is viral capture by attachment receptors such as DC-SIGN, which is thought to facilitate the initial stages of COVID-19 infection in the lung tissue (called trans-infection). In addition, while immune escape is dictated by mutations in the spike protein, coronaviral virions also incorporate M, N, and E structural proteins within the particle. In this paper, we explored how several structural facets of both the SARS-CoV-2 virion and the antiviral lectin Griffithsin can affect and attenuate the infectivity of SARS-CoV-2 pseudovirus. We found that Griffithsin was a better inhibitor of hACE2-mediated direct infection when the coronaviral M protein is present compared to when it is absent (possibly providing an explanation regarding why Griffithsin shows better inhibition against authentic SARS-CoV-2 as opposed to pseudotyped viruses, which generally do not contain M) and that Griffithsin was not an effective inhibitor of DC-SIGN-mediated trans-infection. Furthermore, we found that DC-SIGN appeared to mediate trans-infection exclusively via binding to the SARS-CoV-2 spike protein, with no significant effect observed when other viral proteins (M, N, and/or E) were present. These results provide etiological data that may help to direct the development of novel antiviral treatments, either by leveraging Griffithsin binding to the M protein as a novel strategy to prevent SARS-CoV-2 infection or by narrowing efforts to inhibit trans-infection to focus on DC-SIGN binding to SARS-CoV-2 spike protein.

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“…Here, we describe a cell-free platform for producing recombinant allergens that overcomes the shortcomings of traditional allergen extract preparations and endows several favorable features beyond the capabilities of cell-based recombinant methods. Cell-free protein synthesis (CFPS) − is a technique that has been applied to protein biologic discovery, , glycoprotein synthesis, − point-of-care manufacturing, − distribution of medicines to resource-limited settings, , small molecules, − diagnostics, − and education. − …”

Section: Introductionmentioning

confidence: 99%

A Cell-Free Protein Synthesis Platform to Produce a Clinically Relevant Allergen Panel

et al. 2023

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Allergens are used in the clinical diagnosis (e.g., skin tests) and treatment (e.g., immunotherapy) of allergic diseases. With growing interest in molecular allergy diagnostics and precision therapies, new tools are needed for producing allergen-based reagents. As a step to address this need, we demonstrate a cell-free protein synthesis approach for allergen production of a clinically relevant allergen panel composed of common allergens spanning a wide range of phylogenetic kingdoms. We show that allergens produced with this approach can be recognized by allergen-specific immunoglobulin E (IgE), either monoclonals or in patient sera. We also show that a cell-free expressed allergen can activate human cells such as peripheral blood basophils and CD34+ progenitor-derived mast cells in an IgE-dependent manner. We anticipate that this cell-free platform for allergen production will enable diagnostic and therapeutic technologies, providing useful tools and treatments for both the allergist and allergic patient.

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An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics

Cited by 4 publications

References 61 publications

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

The Antiviral Activity of the Lectin Griffithsin against SARS-CoV-2 Is Enhanced by the Presence of Structural Proteins

A Cell-Free Protein Synthesis Platform to Produce a Clinically Relevant Allergen Panel

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