Polymer-Mediated Cryopreservation of Bacteriophages.

Huba L Marton,Antonia P Sagona,Matthew I Gibson,Peter Kilbride,Kathryn M Styles

doi:10.1021/acs.biomac.1c01187

Huba L Marton, Antonia P Sagona + Show 3 more

Open Access

https://doi.org/10.1021/acs.biomac.1c01187

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Abstract

Bacteriophages (phages, bacteria-specific viruses) have biotechnological and therapeutic potential. To apply phages as pure or heterogeneous mixtures, it is essential to have a robust mechanism for transport and storage, with different phages having very different stability profiles across storage conditions. For many biologics, cryopreservation is employed for long-term storage and cryoprotectants are essential to mitigate cold-induced damage. Here, we report that poly(ethylene glycol) can be used to protect phages from cold damage, functioning at just 10 mg·mL–1 (∼1 wt %) and outperforms glycerol in many cases, which is a currently used cryoprotectant. Protection is afforded at both −20 and −80 °C, the two most common temperatures for frozen storage in laboratory settings. Crucially, the concentration of the polymer required leads to frozen solutions at −20 °C, unlike 50% glycerol (which results in liquid solutions). Post-thaw recoveries close to 100% plaque-forming units were achieved even after 2 weeks of storage with this method and kill assays against their bacterial host confirmed the lytic function of the phages. Initial experiments with other hydrophilic polymers also showed cryoprotection, but at this stage, the exact mechanism of this protection cannot be concluded but does show that water-soluble polymers offer an alternative tool for phage storage. Ice recrystallization inhibiting polymers (poly(vinyl alcohol)) were found to provide no additional protection, in contrast to their ability to protect proteins and microorganisms which are damaged by recrystallization. PEG’s low cost, solubility, well-established low toxicity/immunogenicity, and that it is fit for human consumption at the concentrations used make it ideal to help translate new approaches for phage therapy.

Highlights

The use of biologic therapies is rapidly growing, but there remain challenges to delivering them intact and functional to a patient.[1−5] Bacteriophages or phages are viruses that target and infect bacteria and are the most abundant organisms on earth.[6]
Phages can be divided into virulent and temperate phages, the former carrying out a lytic replication cycle, where the phage uses the bacterial host to replicate by seizing the host’s molecular machinery and escaping the cell to find a fresh host, the latter integrating into and remaining dormant in the host genome as a “prophage” and replicating with the host genome in a lysogenic cycle.[9]
Our initial hypothesis was that addition of ice recrystallization inhibiting (IRI) polymers may mitigate cold-induced damage to the phage, in particular by reducing the stress during the thawing stage

Summary

Introduction

The use of biologic therapies (e.g., cells, proteins, viruses, vaccines) is rapidly growing, but there remain challenges to delivering them intact and functional to a patient.[1−5] Bacteriophages (literally “bacteria eater”) or phages are viruses that target and infect bacteria and are the most abundant organisms on earth.[6] Competition between these viral predators and their bacterial hosts plays an important role in the evolutionary adaptations and diversification seen in many bacteria today.[7,8] Generally, phages can be divided into virulent and temperate phages, the former carrying out a lytic replication cycle, where the phage uses the bacterial host to replicate by seizing the host’s molecular machinery and escaping the cell to find a fresh host, the latter integrating into and remaining dormant in the host genome as a “prophage” and replicating with the host genome in a lysogenic cycle.[9] Phages are ubiquitous, from the depth of the oceans to hospital effluents.[10] It is becoming increasingly clear that phages play a role in the gut microbiome of the human body.[11,12] In aquacultures (the farming of seafood), lytic phages have been used to alleviate pathogenic bacteria of a range of fish and shellfish.[13] Phages have been approved for use as a food additive in meat products to protect consumers against Listeria monocytogenes by the Food and Drug Agency (FDA).[14] Another use of lytic phages is to treat bacterial infections inside the human body (phage therapy).[10] One of the positive attributes of phage therapy is that they can largely be applied without disruptions to the gut microbiota.[15] The vast abundance of the phage in nature[16] means that there is almost an endless pipeline and so phages can be applied as “cocktails”, thereby reducing the chances of resistance developing to individual treatment.[17−19]

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