mRNA vaccines, such as the BioNTech/Pfizer and Moderna COVID-19 vaccines developed in 2020, were key to mitigating the COVID-19 pandemic across the globe and have saved millions of lives since their debut.¹ However, these vaccines — as do all mRNA vaccines — require ultra-cold storage at -60℃ and cold chain technology for transit due to RNA’s highly degradation-prone nature.² This increases the price of transporting, storing, and administering these vaccines in technologically or financially insecure areas around the world. Reliance on electric freezers to maintain mRNA vaccines at temperature creates additional problems for areas without a consistent or reliable power supply, as one thaw cycle of the Pfizer-BioNTech or Moderna vaccines may render them unsuitable for administration to patients.

And this problem isn’t just hypothetical.

In our home city of Dallas, USA, a major snowstorm in 2021 knocked out power across the city. Our university health center alone lost over 1,000 vaccines due to the power failure, amounting to around $20,000 in total losses. As climate change accelerates and Texas’ power grid remains unstable, our team set out to develop mRNA vaccines with increased resistance to different temperatures that can reduce or eliminate the need for expensive, energy-intensive cold-chain and ultra-cold storage technology.

To accomplish this, we turned to the latest frontier of stress tolerance in biological systems — tardigrade proteins. The microscopic organisms within phylum Tardigrada contain multiple unique proteins implicated in their impressive tolerance against heat shock, lyophilization, dehydration, and irradiation. These proteins include mitochondrial, cytosolic, and secretory abundant heat shock proteins (MAHS, CAHS, and SAHS), damage suppressor proteins (Dsup) and mitochondria-localized late embryogenesis abundant proteins (LEAM), all of whose properties are predicted to result from intrinsically disordered regions of the proteins.³

These disordered regions are predicted to form protective aggregates, sometimes described as glass-like matrices, around cellular components in the presence of certain stressors. Expression of these proteins in eukaryotic systems has led to improved host cell resistance to stressors like mechanical shearing,⁴ and research in vitro indicates these proteins can bind to cell-free biomolecules like nucleic acids.⁵

However, little current research describes whether these protective proteins could impact the degradation of biomolecules in response to environmental stressors like heat, freeze-thaw cycles, or irradiation. Our team set out to establish whether tardigrade proteins could help stabilize mRNA against the biggest factors driving up mRNA vaccine storage, transit and maintenance costs — degradation resulting from high temperatures or multiple freeze-thaw cycles — with the aim to lay a foundation for improved vaccine design.

If tardigrade proteins are successful at protecting mRNA from stress damage, then new vaccine formulations incorporating these proteins that will be able to withstand far higher temperatures can be distributed around the globe at significantly reduced costs. Our student health center could stock vaccines that don’t become unusable after a power failure or a patient cancelling their appointment once the vaccine has been set out to thaw. Successful protection of cell-free systems with tardigrade proteins may even help reformulate traditional vaccines like the influenza shot, made from pathogens or antigens instead of mRNA coding for antigens, to become more stress-tolerant as well.

Research Question

Can tardigrade proteins implicated in biological stress tolerance protect mRNA from degradation caused by temperature stress?

Design

Both mRNA types were generated via in vitro transcription. The 4 successfully generated proteins, as well as a mock transfection negative control were run through nickel-NTA columns. Purified proteins were then combined with one of the two mRNAs in a 10:1 molar ratio in a salt-buffered and sucrose-stabilized solution to create a scaled-down approximation of the Pfizer-BioNTech COVID-19 vaccine.⁶ Each experimental group underwent representative stressors: heat shock at 4℃ , 25℃, or 50℃ for 1 hour, with the non-stress condition being storage at -80℃. After stress application, each experimental group’s degradation was initially assayed via agarose gel electrophoresis and quantified in ImageJ/FIJI. Further assays will involve mRNA transfection into HEK cells to determine the mRNA’s translational capacity post-stress. To help determine which proteins are most likely to form stress-protective aggregates with the mRNA, we modeled each protein’s interactions with mCherry3C mRNA in the presence of vaccine buffer salts in silico using AlphaFold-3.

In the interest of creating a low-cost protein expression system to mitigate any additional vaccine manufacturing expenses, our team also investigated how to avoid the expensive and time-consuming re-transfection process each time a new batch of proteins was needed. We are cloning the SAHS RV gene into the PiggyBac transposon system to generate a plasmid that, upon one transfection, will integrate the gene of interest into the host cell’s genome and constitutively express the protein throughout the cell line’s lifetime.

Application and Community

Demonstrating a capacity for tardigrade proteins to protect RNA from stressors would have multiple potential applications in cell-free systems. The immediate use case is in mRNA vaccine design, where proteins could be incorporated into vaccine formulations to create injections that could be frozen at -20℃, refrigerated, or held at room temperature for long-term storage, and withstand multiple freeze-thaw cycles, thereby cutting costs and reducing waste. Before these protein-protected formulations can be introduced into live animals such as mouse models, each gene would have to be redesigned with tailored amino acid substitutions to render the proteins incompatible with the animal’s major histocompatibility complexes (MHCs) to avoid an allergenic response to the formulation.

These protective proteins may have additional applications in stabilizing RNA molecules beyond vaccine uses — for instance, in laboratory settings where RNA must be stored or transported at suboptimal temperatures or for extended periods of time. This would require developing a purification protocol to separate the RNA from the proteins, come time for use, which pre-existing separation protocols, such whole-cell RNA isolation, already provide a solid foundation for. Further research may suggest that other biological molecules, like viral envelope proteins, may also be stress-protected by tardigrade proteins, allowing for traditional vaccines to be similarly reformulated in service of improving storage and transit temperature optimums, and similar improvements to laboratory storage and transit of these biomolecules.

Tardigrade proteins have also been shown in whole-cell systems to protect against mechanical stress, irradiation and dehydration, suggesting additional research areas for in vitro biomolecule protection — for example, creating radiotolerant systems that can resist UV radiation-induced mutagenesis.

On a community level, lessening the financial burden of storing and moving vaccines gives health centers, hospitals, and clinics leeway to stock more vaccines, operate more mobile vaccine clinics to better reach health deserts and underserved communities, and distribute their resources more effectively. The countries with the lowest percentages of vaccination against COVID-19 face numerous, compounding health disparities that hardier healthcare solutions, such as reformulated temperature-resistant vaccines, can make sweeping strides in addressing.

Summary

RNA is a sensitive biomolecule prone to degradation, resulting in mRNA-based vaccines like the BioNTech/Pfizer and Moderna COVID-19 vaccines requiring costly ultra-cold storage and cold-chain transport to remain usable. These expenses exacerbate health disparities for economically insecure and underserved communities both at home and worldwide; our home city alone has lost tens of thousands of dollars in vaccine stocks due to power failures caused by natural disasters. To stabilize vaccine mRNA against heat shock and freeze-thaw, the most common causes for vaccine wastage, we investigated the in vitro stress-protective properties of tardigrade proteins, which have been demonstrated in previous research to fortify tardigrades against heat stress, desiccation, freeze-drying, and irradiation, as well as protecting eukaryotic systems expressing these proteins against multiple similar stressors.

We cloned 10 synthetic tardigrade protein genes across five different major classes, expressed them in CHO and HEK cell lines, purified them via His-tag affinity chromatography, confirmed expression via Western blot, and combined each of the purified proteins with buffer salts and fluorescent protein mRNA to generate scaled-down “mini-vaccines.” Each “mini-vaccine” underwent one of multiple heat shock conditions and mRNA degradation was assayed via agarose gel electrophoresis. Alternative expression methods were investigated via cloning into host genome integration vectors, and mRNA-protein interactions in buffer solution were modeled in silico via AlphaFold 3 to determine which proteins were most likely to form protective aggregates around the mRNA.

Protecting mRNA molecules against representative stressors using tardigrade proteins has significant potential application in mRNA vaccine design, where reformulations containing tardigrade proteins could better resist temperature stress and therefore reduce or eliminate cold-storage and cold-chain needs for clinics and hospitals worldwide. Designing a low-cost, high-yield, and reliable protein expression system for these downstream reformulations will help ensure these reformulated vaccines are maximally accessible to the communities that need them most from benchtop to bedside.