On-demand, cell-free biomanufacturing of conjugate vaccines at the point-of-care

Background of preprint

Conjugate vaccines are among the safest and most effective methods for preventing life-threatening bacterial infections. However, the lack of access to these vaccines, along with obstacles to the approval of new conjugate vaccines, has contributed to low global vaccination rates with conjugate vaccines. To mitigate these challenges, Stark et al. developed technologies based on cell-free protein synthesis (CFPS) in the hope of enhancing the development and promoting the distribution of these conjugate vaccines.

In their preprint, Stark et al. develop an in vitro bioconjugate vaccine expression (iVAX) platform built on cell-free reactions (Fig. 1).

Figure 1. The production process of bioconjugate vaccines. Reproduced from the original preprint by Stark et al. under a CC BY-NC-ND 4.0 licence.

The iVAX platform expedites development and allows for cold chain-independent biosynthesis of these conjugate vaccines, with the following five advantages:

• iVAX produces multiple individual doses of bioconjugates every hour.
• iVAX produces equivalent amounts of bioconjugates over a range of temperatures.
• iVAX’s flexibility means it can interchange carrier proteins, e.g. in the case of both licensed conjugate vaccines and conjugated polysaccharide antigens.
• Shelf-stable. iVAX is derived from freeze-dried cell-free reactions that can be activated by the addition of water.
• iVAX avoids the contamination of vaccines with high levels of endotoxin present in non-engineered E. coli manufacturing platforms.

Key findings of preprint

The key findings of this preprint can be categorised into three main groups: (A) synthesis, (B) safety and portability in vaccine production, and (C) validation of vaccine efficacy.

(A) Vaccine synthesis

Stark et al. first showed that cell-free bioconjugate vaccine production is possible. The authors produced eight carrier proteins in soluble conformations in vitro via CFPS, as opposed to relying on the expression of these proteins in living E. coli. This had the advantage of allowing the authors to modify the chemical and reaction environment, improving production of more complex carriers. Having modified the carrier proteins at their C-termini with an optimal bacterial glycosylation motif DQNAT to enable glycosylation, the authors could then enhance the properties of carrier proteins by making small modifications to the production process, such as increasing soluble expression, improving assembly, and minimising protease degradation.

Having synthesised these carrier proteins, Stark et al. focussed on Francisella tularensis, a highly infectious and lethal bacterium for which there are no licensed vaccines. Recently, a bioconjugate vaccine against F. tularensis was developed using protein-glycan coupling technology (PGCT). This vaccine comprised an F. tularensis lipopolysaccharide (LPS)—specifically F. tularensis O-antigen polysaccharide (FtO-PS)—conjugated to the Pseudomonas aeruginosa exotoxin A carrier protein. Inspired by the efficacy of this vaccine in rats, Stark et al. showed that the iVAX platform could also produce anti-F. tularensis bioconjugate vaccine conjugates on-demand over 1 hour and across temperatures ranging from 21 to 37 °C. Following this, Stark et al. then showed that FDA-approved carriers could be conjugated with FtO-PS in iVAX reactions, concluding that iVAX could prove advantageous over PGCT in cases where the conjugate vaccines comprise high molecular weight O-PS antigens conjugated to carrier proteins. Notably, the iVAX platform could yield multiple doses per mL of vaccine per hour.

(B) Safety and portability

The most concerning contaminant in using E. coli in biomanufacturing is the lipid A, an endotoxin that can cause lethal septic shock. The reliance of the iVAX reactions on lipid-associated components described in this preprint prohibits the use of standard detoxification approaches involving the removal of lipid A. Therefore, Stark et al. applied strain engineering to detoxify lipid A; resulting in a penta-acylated, monophosphorylated lipid A with significantly reduced toxicity.

To enhance the storage and distribution of these conjugate vaccines, Stark et al. compared two production methods of FtO-PS bioconjugates: (i) running the reaction immediately after priming the plasmid encoded with the target protein, and (ii) running the reaction after the same reaction mixture was lyophilised and rehydrated. Stark et al. found that glycosylation activities were preserved after lyophilisation and rehydration, suggesting that these reactions were amenable to ambient temperature storage, and allowing for a wider range of distribution.

(C) Validation of vaccine efficacy

Stark et al. validated the efficacy of iVAX-produced bioconjugate vaccines by comparing its ability to generate IgG in mice sera to that of PGCT, the latter of which serves as a positive control. The authors found that the bioconjugates produced by iVAX induced significantly higher IgG responses than both the negative and positive controls. This difference was attributed to the enhanced Th2-biased response, which led to the increase in production of IgG1 antibodies.

What I like about this preprint

As the anti-vaccination movement rears its ugly head in recent times, public support for vaccines is again in a precarious state. From this perspective, I chose to highlight this preprint by Stark et al. for its noble aim of improving access to vaccines through iVAX. The issue is mitigated in three ways. First, the modular nature of iVAX enhances the flexibility of vaccine production by allowing different LPS components to be coupled to various protein carriers. Second, iVAX is robust and inexpensive, which offers the opportunity to produce these bioconjugate vaccines on-demand by decoupling production from its conventional reliance on cold chain distribution. Third, the efficacy of iVAX-produced bioconjugate vaccines was shown to provoke an enhanced immunological response in mice.

Future directions

In their preprint, Stark et al. developed the iVAX platform to perform the synthesis of bioconjugate vaccines. The next step forward is to use the iVAX platform to synthesise a variety of bioconjugate vaccines with a variety of carrier proteins and different lipopolysaccharides.

At the same time, the nascence of this technology also means that the safety of these vaccines must also be substantiated with further evidence at a later stage before it can be rolled out fully. Due to their prophylactic (rather than curative) nature, vaccines need to possess a higher margin of safety before they can be made fully accessible to entire populations. Notwithstanding these vestigial challenges, this preprint by Stark et al. offers an exciting development of vaccines that could make a key impact on the field.

Tags: biomanufacturing, cell free protein synthesis, conjugate vaccines

doi: https://doi.org/10.1242/prelights.11698

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