RNA: a coming-of-age technology for viral vaccines

Listen to this article on the DDW Podcast: Dr Andy Lane, Commercial Director at The Native Antigen Company, discusses the development of a new generation of vaccine platforms that can be rapidly adapted to novel threats on-demand and at low cost. Vaccines are one of the most effective healthcare interventions for reducing the morbidity and mortality associated with infectious diseases. Immunity to disease not only protects populations from infection, but generates significant cost savings that translate into meaningful socioeconomic benefits1. Since public vaccination programmes began to gain global traction in the 20th century, a range of technologies have reached maturity. Attenuated, inactivated and subunit vaccines, have proved successful in conferring robust protection against many infectious diseases, with notable achievements including the eradication of smallpox and rinderpest in the wild, as well as drastic reductions in the incidence of polio and measles2. However, population growth, large-scale urban development, deforestation and changing climates have impacted human-animal relationships and the balance of ecosystems. This has facilitated the emergence of several zoonotic viruses in recent years, including the SARS, MERS, SARS-2 coronaviruses, as well as HIV, Zika, Ebola, and H1N1 and H3N2 influenza. Intensified by the interconnected global economy, emerging diseases have been able to spread considerable distances in short spaces of time, resulting in a series of health and economic crises. For vaccine developers, the changing paradigm has presented several challenges. While traditional approaches to vaccine design are amenable to zoonotic pathogens, their research, development, clinical testing and manufacture typically exceeds 10 years3. Combined with the substantial financial risks of development and commercialisation, this has created substantial hurdles to vaccine innovation in the emerging diseases space. Addressing this, there has been encouragement by public-private consortia to develop a new generation of vaccine platforms that can be rapidly adapted to novel threats on-demand and at low cost4. Following the successes of Covid-19, RNA platforms have emerged as a leading contender in their own right. The science of RNA vaccines RNAs are responsible for carrying out a diverse range of cellular functions. Messenger RNAs (mRNAs) represent a subset of RNAs, responsible for transmitting genetic information from DNA in the nucleus to the ribosomal machinery of the cytoplasm, to direct protein synthesis. The significance of RNA as a cellular messenger came to be understood towards the end of the 20th century, leading scientists to consider their application as vectors for expressing synthetic genes. In the early 1990s, mRNA’s potential was proved when researchers successfully delivered transcripts expressing a variety synthetic genes to mice5. While the results of early experiments that followed were modest at best, they paved the way for subsequent research and encouraged much-needed investment. From the 2000s, mRNA research went through a series of boom-and-bust cycles, marked by feverish peaks of expectation and troughs of inevitable disappointment. However, by the 2010s, many of the initial hurdles to mRNA’s clinical application had been surmounted, with multiple therapeutics and vaccines moving into human trials6. Fast forward to late 2019 and SARS-CoV-2 was beginning to emerge in China. The virus soon spread globally, infecting over a hundred million worldwide and causing substantial mortality. In a race to contain the virus and support ailing economies, hundreds of different groups began developing a diverse range of putative vaccine candidates. Among many of the legacy vaccine technologies, mRNA drew considerable interest for its rapid early development and novel immunological mechanism. By late 2020, both Pfizer/BioNTech’s and Moderna’s mRNA candidates received Emergency Use Authorizations in record-breaking time, marking the first mRNA vaccines to gain licensure7,8. Design, formulation and delivery The basic structure of an RNA vaccine is deceptively simple. A nucleotide sequence typically includes one or more open reading frames (ORFs), which are flanked by untranslated regions (UTRs), and a 5’ cap and 3’ poly(A) tail at the ends. The ORFs encode the antigen(s) of interest, while the UTRs modulate gene expression, and poly(A) tails protect the transcript against host-enzyme degradation. The amenability of RNA to rapid design and editing in silico also allows for many variations on this basic theme. A common addition to antigen-encoding ORFs are sequences encoding synthetic replication machinery, known as self-amplifying RNAs (saRNAs). By expressing this replication machinery within the target cell, transcripts can be amplified to boost to gene expression and minimise the required dosage9. Imperial College London, for example, is evaluating an saRNA that encodes the SARS-CoV-2 Spike protein, and which could prove to be effective at up to 1,000-times lower dosages than conventional mRNAs10. Unlike traditional vaccines, the expression of mRNA-encoded antigens ensures folding and post-translational modifications in situ to stimulate a more ‘natural’ infection11. As mRNA is considerably more compact than the proteins it encodes, it also has the capacity to encode complex multi-subunit proteins, whether they be full-length, or tandem constructs composed of multiple linear epitopes. Moderna Therapeutics, for example, is currently evaluating a cytomegalovirus (CMV) vaccine formulation in Phase III clinical trials, which includes five mRNAs encoding CMV’s pentamer complex and one encoding glycoprotein B12. Cellular uptake of mRNA is dependent on the cell type and properties of the mRNA complex. To achieve cell-specific delivery and maximise transcript stability, mRNAs are typically formulated in a vector―most commonly, lipids, polymer nanoparticles, or viral vectors. To administer vaccines, intramuscular injection has long been the preferred route, as this confers good immunogenicity and minimises adverse reactions13. However, delivery to antigen-presenting cells in the lymph nodes has gained popularity in recent years for stimulating more potent responses14. Research in this area is still in its infancy, with a range of new targets likely to emerge over the coming years. Fine-tuning the response To induce lasting protection against a pathogen, potent and broad stimulation of the immune system is required. For intracellular pathogens such as viruses, both antibodies are needed to prevent the establishment of infection, as well as cytotoxic T-cells (CD8+) to eradicate cellular reservoirs and prevent persistence. Of these immune responses, T-cells have traditionally been the most challenging to stimulate, as antigen uptake and presentation can prove difficult to achieve15. However, as mRNA-encoded antigens are produced in situ, they can be readily processed for presentation to induce potent T-cell responses16. Conversely, the induction of relevant and lasting antibody responses has been a greater challenge for mRNA vaccines, though the use of signal peptides has shown to effectively direct antigens extracellularly for antibody recognition, in addition to the cytoplasm, intracellular compartments, or outer membrane17. One of the initial incentives for developing mRNA modalities was their improved safety profile as compared with DNA, which has been historically hindered by regulatory concerns around the risks of genome integration, long-term expression and the induction of harmful autoantibodies18. mRNA has also shown advantages over whole virus or vectored vaccines, which risk reversion to pathogenicity and the induction of potent vector-specific immune responses. However, mRNA vaccine development has not been without its challenges. Mammalian immune systems have evolved a variety of ways to detect invading RNA viruses. Our cells therefore contain an array of RNA-specific detection receptors (TLRs, PKRs, RIG-1) that trigger potent innate inflammatory responses19. While this ‘auto-adjuvant’ activity can be useful in stimulating durable, on-target immunity, it has more frequently caused unwarranted adverse events20. From the 1990s to early 2000s, RNA’s innate immunogenicity was a major impediment to research and development. Fortuitous research around the same time found that unlike viruses, mammalian RNAs were highly modified to distinguish them as self21. Therefore, the modification of transcripts with methyl groups, pseudobases or inosines is now commonly used to mask synthetic transcripts from recognition by the immune system and minimise these unwarranted effects22. Finding the right balance between immunogenicity and safety continues to be a challenge for vaccine developers, and as mRNA vaccines are still in their infancy, there is work needed to understand immune parameters and optimise their performance23. Development and manufacture Taking a vaccine from concept to market is complex and costly, requiring investment in research, development, clinical trial, manufacturing and distribution capabilities. End-to-end, a commercially-successful vaccine typically takes eight to 14 years to reach patients, involving tens of thousands of individuals and costing up to US$1 billion. Yet in spite of these costs, of the vaccines that make it to preclinical testing, only 6% go on to commercialisation25. In outbreak scenarios, the success rates are a little better as developers must move quickly, while the clinical relevance, long-term pathogen circulation and feasibility of development is still highly uncertain. These resource requirements have long acted as an impediment to vaccine R&D, reflected by the recent departure of multiple pharmaceutical companies from the industry26 and many diseases gaining ‘neglected tropical disease’ status27. To create financial incentives, accelerate R&D, and improve market access, a range of public-private initiatives have therefore been established in recent years. Partnerships such as The Coalition for Epidemic Preparedness Innovations focus on supporting the development of platform technologies to accelerate candidate R&D28. Alongside the development of pathogen-specific vaccines, multiple initiatives are seeking to establish general platform technologies. The idea is that once successfully developed and tested, such platforms can be leveraged to develop disease-specific candidates in outbreak scenarios, reducing the time, cost and compliance hurdles associated with vaccine development. Design and feasibility Of the platform technologies being considered for epidemic/pandemic vaccine development, mRNA modalities have proven themselves to be highly suited. Unlike most traditional platforms, at the early stages of feasibility testing, large arrays of mRNA sequences can be rationally designed, generated from basic reagents and screened in a highly parallelised manner. Combined with recent advances in immuno-informatics, 3D modelling and machine learning can then be used to predict the interactions of digitised transcripts, antigens and adjuvants with the host immune system, in vivo29. Candidates that pass initial screening then benefit from the extensive customisation that mRNA is amenable to, including codon, cap and UTR optimisation30. These options provide a means of overcoming many of the early design hurdles that traditional vaccines encounter, helping to accelerate their development and ultimate success. These benefits were exemplified by the pace at which Moderna Therapeutics’ produced its first batch of clinical samples of its now-licensed Covid-19 vaccine, mRNA-1273, only 42 days after the SARS-CoV-2 genome was published31. As vaccine design has become more rational in recent years, target product profiles (TPPs) have also become key documents in the vaccine development process. Often produced by regulatory agencies and NGOs, TPPs summarise the desired characteristics that a vaccine should have and provide a high-level roadmap of the requirements to prove safety and efficacy in target populations26. In using a TPP, developers have a clear idea of what regulators are looking for, and can undertake more structured approaches to vaccine design, in which the desired attributes of a vaccine are considered from the outset. As candidates make it down the pipeline to pre-clinical and clinical testing, those designed to a TPP specification may benefit from higher success rates, especially at Phases II–III when attrition is highest32. For the aforementioned reasons, mRNA is highly suited to this rational approach to TPP-direct candidate design. Scale manufacturing Following successful clinical trials and regulatory approval, a vaccine is ready to go to market. However, even the most promising vaccines can encounter stumbling blocks. In particular, meeting the demand of 21st century populations during epidemic/pandemics can pose significant challenges to rapid manufacturing. The emergences of multiple zoonoses in recent decades has shown how traditional vaccine manufacturing processes are ill-suited to these scenarios. Whether producing live virus or recombinant proteins, dedicated cell- or egg-culture and/or fermentation processes are required, necessitating specialised equipment and weeks of added time. For inactivated or attenuated vaccines, the growth of live viruses additionally requires high-biocontainment facilities and safety precautions. The combined requirements of resource-heavy infrastructure, extensive regulatory compliance and knowledge transfer can undermine financial incentives and act as a serious bottleneck to rapid vaccine production. In contrast, in vitro RNA production is entirely cell-free, excluding many biological contaminants and benefitting from incredible scalability9. After DNA templates are designed in silico, they can be used generate transcripts with the addition of polymerases via a simple chemical reaction. Therefore, what takes a week for cell- or egg-based vaccine production takes a matter of minutes for mRNA, and by one estimate, a single 5L bioreactor could produce enough mRNA for one billion vaccine doses within a year3. cAs a result, it is often the bottling and delivery that limit the speed of mRNA vaccination at the population-level. Yet, as RNA manufacture is relatively simple, more decentralised approaches to scale production may be able to overcome these factors. Decentralising multiple, smaller production facilities globally would be especially advantageous for developing countries that often lack established production capabilities. Indeed, researchers have begun to explore the use of off-the-shelf lab equipment for local RNA manufacture in order to improve accessibility and affordability in many parts of the developing world3. Rapid redesign Another roadblock to vaccine manufacturing in pandemic scenarios is that most facilities are limited to producing only one vaccine or technology-type. As a result, entirely new facilities and processes are often required to produce successful candidates, requiring further time and resource investments33. Many countries pre-emptively developed manufacturing capacity for specific Covid-19 candidates, for example, without a long-term view of their utility post-pandemic34. To maximise return on investment and facilitate tech transfer, more general-purpose manufacturing facilities are therefore needed. Here, RNA modalities again show clear advantages, as their manufacturing processes are largely independent of the antigen encoded, requiring only minimal adaptation to validation methods35. Another important consideration for viral vaccines is the propensity for the target pathogen to mutate. This is especially true of RNA viruses, which comprise a significant and growing proportion of epidemic/pandemic threats36. The influenza A viruses, for example, continuously mutate via genetic drift and shift, requiring continuous vaccine reformulation to match seasonal strains. However, as seasonal formulations don’t always match the predominating strains, and novel strains frequently emerge (H1N1, H5N1), flu vaccines are often limited in efficacy37,38. Fortunately, most RNA viruses aren’t as adept as influenza in their ability to mutate, though the risk that mutation poses is nonetheless an ongoing problem, as evidenced by novel SARS-CoV-2 variants. The fact that preparing new mRNA constructs is a relatively rapid process therefore makes them an ideal platform in this regard, as once a formulation has been evaluated, redesign and testing is relatively straightforward: the antigen’s gene sequence is modified in silico to match the novel strain, subtype or variant, inserted into the appropriate vector, manufactured and reformulated. While further testing is then required to ensure that safety and efficacy requirements are met, the ability to rely on established processes, provides significant time-savings and cost-reductions29. For an industry that typically dedicates years or decades to single-vaccine manufacture, modular, pathogen-agnostic facilities would provide considerable advantages in manufacturing agility. Future perspectives Progress in mRNA vaccine development over recent years has been prodigious, with the ongoing pandemic seeing multiple first-generation candidates over the finishing line. Emerging viral pathogens present themselves as compelling targets for this nascent technology, though there nevertheless remains room for improvement over the coming years. Comprised of single-stranded nucleic acids, mRNA is inherently unstable even at ambient temperatures. Therefore, even minor degradation can severely impede or stop proper translation of the target antigen, drastically reducing vaccine-shot efficacy. Storage temperatures as low as −80°C can ensure long-term stability, yet the −20°C standard of medical supply chains cannot accommodate this. Vaccine stability is a particular issue in developing economies and sparsely populated rural areas, where the lack of basic cold chain infrastructure limits pandemic responses and leaves populations susceptible to disease. Alternative methods now under investigation have shown promise, such as thermostable proteins and lipid nanoparticles, which allow mRNAs to be stored at ambient temperatures for over a week24. However, considerable research is still needed to standardise these methods and establish stability measures. Existing in vitro mRNA production methods could also be further optimised to increase speed, improve yields and recycle substrates. Further improvements in this domain could lower the costs per dose and better standardise pathways to market for emergency use authorisation. However, as manufacturing is optimised, downstream processing, filling, logistics and administration will likely become limiting factors. Various lines or research are now addressing this, such as prefilled syringes, intranasal sprays, micro-needles and “do-it-yourself” administration. However, many such methods are still in their infancy and need to surmount further technological and ethical hurdles for effective implementation. Finally, gaps in our understanding of the immune interactions of heterologous RNA, as well as its long-term safety and efficacy require further research. Innate immune sensing in particular, continues to be a rapidly developing field for RNA technologies, with many outstanding questions around the roles and interactions of host immune mediators. Volume 22, Issue 4 – Fall 2021 Figure 1: Schematic of mRNA vaccine mechanism of action. mRNA is delivered to target cells by a lipid or nanoparticle carrier, where it is translated into the target antigen. Target antigens are either exported for recognition by the humoural immune system, or processed by the immunoproteasome for MHC presentation. Figure 2: Schematic of basic mRNA vaccine design. Conventional mRNA vaccines encode central antigen(s), flanked by UTRs, 5’ cap and 3’ poly(A) tail. Differences 5’ caps, UTR secondary structures, codons, GC content and poly(A) tail length can be used to modulate stability and pharmokinetics. Figure 3: Pie chart showing break-down of mRNA vaccines in clinical trials by infectious disease. Adapted from Rosa and colleagues24. Figure 4: Schematic comparing development pipelines for conventional vaccines and mRNA platform technologies.Adapted from Kis and colleagues3 . Figure 5: Schematic of mRNA vaccine production and purification processes. About the author Dr Andy Lane is Commercial Director at The Native Antigen Company (now part of LGC’s Clinical Diagnostics business unit), a manufacturer of viral antigens and antibodies for vaccine research. Following his PhD in pathobiology, Lane joined the NHS and led a monoclonal antibody research group working in leukaemia and lymphoma diagnostics. He subsequently joined a major monoclonal antibody supplier to lead new product development and was executive director at Innova Biosciences. References “The Economic Value of Vaccination: Why prevention is Wealth”. J Mark Access Health Policy. August 2015. https://doi.org/10.1371/journal.ppat.1003001 Younger et al. “Childhood Vaccination: Implications for Global and Domestic Public Health”. Neurol Clin. 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