What was the breakthrough behind the “sudden” feasibility of mRNA vaccines in 2020?

Question

Several sources describe the initial failures in the realization of a successful mRNA vaccine. E.g., this 2017 article from Stat describes the following problem faced by Moderna while working on one of their mRNA vaccines:

The safe dose was too weak, and repeat injections of a dose strong enough to be effective had troubling effects on the liver in animal studies.

Another Stat article describes a similar challenge:

In animal studies, the ideal dose of their leading mRNA therapy was triggering dangerous immune reactions — the kind for which Karikó had improvised a major workaround under some conditions — but a lower dose had proved too weak to show any benefits.

The work by Karikó was conducted before Moderna and BioNTech were founded, so it does not seem to be the breakthrough that led to feasible mRNA vaccines. I am also aware that one of these companies, Moderna, is secretive about its technology.

However, I would like to learn, at least at a high level, the reason mRNA vaccines are a viable option against COVID-19, while earlier attempts to develop them against other diseases, such as Crigler–Najjar syndrome, were unfruitful.

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I am aware of Why weren't mRNA vaccines clinically tried earlier?, which was closed for being opinion-based. However, my question is specific to the biological mechanism behind the breakthrough that enabled feasible mRNA vaccines, so is objective and within the scope of this site.

Answer 1

Answering my own question after reading the 2018 Nature review article “mRNA vaccines — a new era in vaccinology”

The resources and motivation engendered by the COVID-19 pandemic are a major factor in the development of the first mRNA vaccines approved by national governments. However, before the COVID-19 pandemic, there were recent advances in mRNA vaccine pharmacology, which made everything possible.

Introduction

The Nature review points out that it was not a single breakthrough, but a lot of research that was conducted during the last couple of years.

Demonstrations of protective immune responses by mRNA vaccines against various infective pathogens were published in recent years. In the first one, published in 2012, direct injection of non-replicating mRNA vaccines was shown to be immunogenic against various influenza virus antigens in multiple animal models¹. Since then, several studies on animals, and in some cases, healthy human volunteers, have managed to induce protective immunity against rabies^2,3, HIV-1^4,5,6, Zika^7,8,9, H10N8 and H7N9 influenza¹⁰, and other viruses.

The authors of the review, which was written before the COVID-19 pandemic, believed that “mRNA vaccines have the potential to solve many of the challenges in vaccine development”. Therefore, had the pandemic not happened, it is likely that we still would have seen effective mRNA vaccines being developed, albeit at a slower pace.

Recent technological advancement has largely overcome the main challenges in the development of mRNA vaccines.

The Challenges

1. Instability

Protein expression after the vaccine is administered might be insufficient if, for instance, the half-life of the vaccine is too low, or if in vivo mRNA translation is insufficient^11,12.

2. Inefficient in vivo delivery

mRNA vaccine delivery is tricky. For instance, mRNA can aggregate with serum proteins and undergoes rapid extracellular degradation by RNases. Therefore, formulating mRNAs into carrier molecules is often necessary, and delivery formulations need to take into account factors such as the biodistribution of the vaccine after delivery, mRNA uptake, and protein translation rate^13,14.

3. Safety

The complexity of modulating the immunogenicity of the mRNA used in vaccines can potentially lead to unwanted stimulatory effects on the immune response^15,16,17.

The Recent Advances

1. Optimization of mRNA translation and stability

Sequence optimization techniques such as replacing rare codons with more frequently used synonymous codons¹⁸, as well as enrichment of G:C content¹⁶, have been examined for increasing in vivo protein expression.

2. Progress in mRNA vaccine delivery

There are numerous delivery methods for mRNA vaccines that have been examined in the literature. In recent years, the limitations of some of these, such as using physical methods (e.g., electroporation) to penetrate the cell membrane, were demonstrated¹⁹. On the other hand, progress was made toward the increased efficacy and reduced toxicity of other delivery methods such as cationic lipid and polymer-based delivery^13,16,20,21.

3. Modulation of immunogenicity

Recent studies have demonstrated that the immunostimulatory profile of mRNA can be controlled more precisely using a variety of techniques. These include chromatographic purification to remove double-stranded RNA contaminants, the introduction of naturally-occurring modified nucleosides to prevent the activation of unwanted innate immune sensors, and complexing the mRNA with various carrier molecules (this includes novel approaches to adjuvants that take advantage of the intrinsic immunogenicity of mRNA)^15,17,22,33.

Apart from the advances in techniques such as purification and the introduction of nucleosides, there was also an improvement in the understanding of when these techniques should be used, based on factors such as the mRNA platform used, RNA sequence optimization, and the extent of mRNA purification under consideration^16,24.

References

1. Petsch, B. et al. Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection. Nat. Biotechnol. 30, 1210–1216 (2012).
2. Schnee, M. et al. An mRNA vaccine encoding rabies virus glycoprotein induces protection against lethal infection in mice and correlates of protection in adult and newborn pigs. PLoS Negl. Trop. Dis. 10, e0004746 (2016).
3. Alberer, M. et al. Safety and immunogenicity of a mRNA rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first‑in‑human phase 1 clinical trial. Lancet 390, 1511–1520 (2017).
4. Pollard, C. et al. Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines. Mol. Ther. 21, 251–259 (2013).
5. Zhao, M., Li, M., Zhang, Z., Gong, T. & Sun, X. Induction of HIV‑1 gag specific immune responses by cationic micelles mediated delivery of gag mRNA. Drug Deliv. 23, 2596–2607 (2016).
6. Li, M. et al. Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via intra- and paracellular pathways. J. Control. Release 228, 9–19 (2016).
7. Pardi, N. et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature 543, 248–251 (2017).
8. Richner, J. M. et al. Modified mRNA Vaccines protect against Zika virus infection. Cell 168, 1114–1125.e10 (2017).
9. Richner, J. M. et al. Vaccine mediated protection against Zika virus-induced congenital disease. Cell 170, 273–283.e12 (2017).
10. Bahl, K. et al. Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 influenza viruses. Mol. Ther. 25, 1316–1327 (2017).
11. Weissman, D. mRNA transcript therapy. Expert Rev. Vaccines 14, 265–281 (2015).
12. Sahin, U., Kariko, K. & Tureci, O. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014).
13. Kauffman, K. J., Webber, M. J. & Anderson, D. G. Materials for non-viral intracellular delivery of messenger RNA therapeutics. J. Control. Release 240, 227–234 (2016).
14. Guan, S. & Rosenecker, J. Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems. Gene Ther. 24, 133–143 (2017).
15. Kariko, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008).
16. Thess, A. et al. Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals. Mol. Ther. 23, 1456–1464 (2015).
17. Kariko, K., Muramatsu, H., Ludwig, J. & Weissman, D. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 39, e142 (2011).
18. Gustafsson, C., Govindarajan, S. & Minshull, J. Codon bias and heterologous protein expression. Trends Biotechnol. 22, 346–353 (2004).
19. Johansson, D. X., Ljungberg, K., Kakoulidou, M. & Liljestrom, P. Intradermal electroporation of naked replicon RNA elicits strong immune responses. PLoS ONE 7, e29732 (2012).
20. Schlake, T., Thess, A., Fotin-Mleczek, M. & Kallen, K. J. Developing mRNA-vaccine technologies. RNA Biol. 9, 1319–1330 (2012).
21. Reichmuth, A. M., Oberli, M. A., Jeklenec, A., Langer, R. & Blankschtein, D. mRNA vaccine delivery using lipid nanoparticles. Ther. Deliv. 7, 319–334 (2016).
22. Fotin-Mleczek, M. et al. Messenger RNA-based vaccines with dual activity induce balanced TLR‑7 dependent adaptive immune responses and provide antitumor activity. J. Immunother. 34, 1–15 (2011).
23. Rettig, L. et al. Particle size and activation threshold: a new dimension of danger signaling. Blood 115, 4533–4541 (2010).
24. Kauffman, K. J. et al. Efficacy and immunogenicity of unmodified and pseudouridine-modified mRNA delivered systemically with lipid nanoparticles in vivo. Biomaterials 109, 78–87 (2016).