On-demand biomanufacturing of protective conjugate vaccines

Stark, Jessica C.; Jaroentomeechai, Thapakorn; Moeller, Tyler D.; Hershewe, Jasmine M.; Warfel, Katherine F.; Moricz, Bridget S; Martini, Anthony M; Dubner, Rachel S.; Hsu, Karen; Stevenson, Taylor C.; Jones, Bradley D.; DeLisa, Matthew P.; Jewett, Michael C.

doi:10.1126/sciadv.abe9444

Cited by 80 publications

(139 citation statements)

References 86 publications

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“…For the proof-of-concept studies performed herein, we selected the C. jejuni N-linked glycosylation system as a model because of the flexibility of CjPglB as a stand-alone, single-subunit OST 44 that has proven to be compatible with a diverse array of glycan donors and acceptor protein substrates including some with therapeutic potential. To date, CjPglB has been used to generate glycoproteins bearing bacterial 24,35,50 and smaller human-type glycans 24,[51][52][53][54] , and has enabled cell-free, one-pot systems for making N-and O-linked glycoproteins 24,55 as well as antibacterial conjugate vaccines 56 . While not directly demonstrated in this work, cell-free strategies such as the glycosylationon-a-chip platform described here could eventually provide access to glycoproteins that are modified with larger, complex-type N-glycans that mimic the structures commonly found on many human glycoprotein drugs such as monoclonal antibodies.…”

Section: Discussionmentioning

confidence: 99%

Glycosylation-on-a-chip: a flow-based microfluidic system for cell-free glycoprotein biosynthesis

Aquino

Manzer

Daniel

et al. 2021

Preprint

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In recent years, cell-free synthetic glycobiology technologies have emerged that enable production and remodeling of glycoproteins outside the confines of the cell. However, many of these systems combine multiple synthesis steps into one pot where there can be competing reactions and side products that ultimately lead to low yield of the desired product. In this work, we describe a microfluidic platform that integrates cell-free protein synthesis, glycosylation, and purification of a model glycoprotein in separate compartments where each step can be individually optimized. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, the ability to tether enzymes for reuse, and the potential for continuous manufacturing. Moreover, it affords an opportunity for spatiotemporal control of glycosylation reactions that is difficult to achieve with existing cell-based and cell-free glycosylation systems. In this work, we demonstrate a flow-based glycoprotein synthesis system that promotes enhanced cell-free protein synthesis, efficient protein glycosylation with an immobilized oligosaccharyltransferase, and enrichment of the protein product from cell-free lysate. Overall, this work represents a first-in-kind glycosylation-on-a-chip prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small batch, decentralized glycoprotein production.

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Section: Discussionmentioning

confidence: 99%

Glycosylation-on-a-chip: a flow-based microfluidic system for cell-free glycoprotein biosynthesis

Aquino

Manzer

Daniel

et al. 2021

Preprint

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“…To produce CRM197-FtO-PS glycoconjugate, E. coli strain CLM24 was transformed with plasmid pTrc99S-spDsbA-CRM197 4xDQNAT encoding the CRM197 carrier protein modified at its C-terminus with four tandemly repeated DQNAT glycosylation motifs (81), plasmid pGAB2 encoding the FtO-PS biosynthesis pathway (58), and plasmid pMAF10-PglB encoding the Campylobacter jejuni oligosaccharyltransferase PglB for transfer of the FtO-PS (82). Overnight cultures were subcultured 1:100 into fresh LB containing appropriate antibiotics.…”

Section: Strainsmentioning

confidence: 99%

A modular platform for on-demand vaccine self-assembly enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

Weyant

Liao²,

Jesus

et al. 2021

Preprint

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Engineered outer membrane vesicles (OMVs) derived from laboratory strains of bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. As mimics of the bacterial cell surface, OMVs offer a molecularly-defined architecture for programming repetitive, high-density display of heterologous antigens in conformations that elicit strong B and T cell immune responses. However, antigen display on the surface of OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. To address this shortcoming, we created a universal approach called AddVax (avidin-based dock-and-display for vaccine antigen cross (x)-linking) whereby virtually any antigen that is amenable to biotinylation can be linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen receptor (SNARE) comprised of an outer membrane scaffold protein fused to a member of the avidin family. We show that SNARE-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations were injected in wild-type BALB/c mice, strong antigen-specific antibody responses were observed that depended on the physical coupling between the antigen and SNARE-OMV delivery vehicle. Overall, these results demonstrate AddVax as a modular platform for rapid self-assembly of antigen-studded OMVs with the potential to accelerate vaccine generation, respond rapidly to pathogen threats in humans and animals, and simplify vaccine stockpiling.

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“…With a functional CRISPR-Cas12 diagnostic for Wolbachia detection in hand, we next wanted to assess the possibility of freeze-drying the system. Previous works have shown that cell-free systems, including CRISPR-based diagnostics, can be freeze-dried for increasing stability and portability 18,19 . Such features would be advantageous for preparation and delivery in the classroom setting.…”

Section: Characterization Of Freeze-dried Cas12 Diagnosticsmentioning

confidence: 99%

Development of a Freeze-Dried CRISPR-Cas12 Sensor for Detecting Wolbachia in the Secondary Science Classroom

Rybnicky

Dixon

Kuhn

et al. 2021

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Training the future synthetic biology workforce requires opportunity and exposure to biotechnology concepts and activities in secondary education. Detecting Wolbachia bacteria in arthropods using PCR has become a common way for secondary students to investigate and apply DNA technology in the science classroom. Despite this framework, cutting-edge biotechnologies like CRISPR-based diagnostics have yet to be widely implemented in the classroom. To address this gap, we present a freeze-dried CRISPR-Cas12 sensing reaction to complement traditional DNA technology education and teach synthetic biology concepts. The reactions accurately detect Wolbachia from arthropod-derived PCR samples in under 2 hours and can be stored at room temperature for over a month without appreciable degradation. The reactions are easy-to-use and cost less than $40 to implement for a classroom of 22 students including the cost of reusable equipment. We see this technology as an accessible way to incorporate synthetic biology education into existing biology curriculum, which will expand biology educational opportunities in science, technology, engineering, and mathematics (STEM) education.

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On-demand biomanufacturing of protective conjugate vaccines

Cited by 80 publications

References 86 publications

Glycosylation-on-a-chip: a flow-based microfluidic system for cell-free glycoprotein biosynthesis

Glycosylation-on-a-chip: a flow-based microfluidic system for cell-free glycoprotein biosynthesis

A modular platform for on-demand vaccine self-assembly enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

Development of a Freeze-Dried CRISPR-Cas12 Sensor for Detecting Wolbachia in the Secondary Science Classroom

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