mRNA vaccines go into humans

The most recent epidemics of Ebola, Zika, influenza and many others have contributed to raise awareness of the need for a platform allowing the rapid implementation of new vaccines and antivirals. For vaccines, this platform may have already been identified in the form of mRNA vaccines. While more studies will be necessary to fully understand the safety and efficacy of mRNA vaccines in humans, in the almost 30 years from the first test of mRNA expression in vivo in 1990, scientists have made huge progress in understanding and ameliorating cellular responses, delivery routes, and manufacturing strategies. Pardi and colleagues summarise this progress in an excellent review.

What are mRNA vaccines?

mRNA vaccines are vaccines where the immunogen is delivered into cells in its mRNA form. Once inside the cell, capped and poly-A tagged as any other cellular messenger, this exogenous sequence is translated by the cellular transcriptional machinery, producing high yields of the encoded antigen, before being natural disposed of as all other cellular mRNA.

There are two types of RNA vaccines:

1) the non-replicating ones, where the antigen of choice is administered flanked only by the 5’ and 3’ UTRs, and its expression terminates once the cells disposes of the mRNA after expression; and

2) the self-amplifying (or replicon) ones, generally virus-derived, where the antigen is administered together with a virus replication machinery, which guarantees sustained expression over multiple rounds of replication. The amount of protein produced is very high, but the half-life of this mRNA harder to account for

Both formulations are easily prepared in vitro. The staring point is to clone the antigen of interest into DNA, together with the two flanking UTRs. Next, the DNA is linearised and in vitro transcribed using a T7, T3, or SP6 phage RNA polymerase. Finally, the RNA product is capped and poly-A-tailed to resemble the fully processed mature mRNA. The UTRs are important for stability and translation, and they increase the half-life of the mRNA. The 5’ capping, often added by vaccinia virus capping enzyme, is required for efficient protein production. Finally the poly-A tail, encoded in the DNA template or added by a poly-A polymerase, is important for translation and stability.

Once inside the cells and perfectly disguised as cellular mRNA, the transcript is rapidly translated and the antigen post-translationally modified and folded in a fully physiological context.

Pros and cons

It is not surprising that the approach has won the attention, the investments, and the effort of many players in recent years. One of the main problems of antigen administration as a recombinant protein is the production and purification of the antigen itself, a costly process that requires optimisation for each new product, given the diversity and complexity of different proteins. While mRNA may also require some optimisation (as illustrated below) it not only has a much simpler chemistry, but it is also reproducibly produced in vitro at very high yields (exceeding 2g/l) using fairly simple and commercially available kits. DNA is also much simpler than a protein, but while DNA vaccine are also being investigated, the risk of recombination, long and difficult to control half-life, and often low expression levels in vivo score an important point in favour of RNA vaccines. These advantages over the more obvious protein and DNA options, together with rapid expression after uptake, and considerable improvements in preproduction and delivery methods have encouraged the application of this technology not only to vaccines against infectious disease, but also to cancer vaccines, as well as mRNA encoding antibodies.

Of course nothing is ever so easy, and also mRNA comes with its own problem. The first is stability. mRNA is not that sturdy, and even the smallest RNAse contaminant is likely to degrade it very rapidly.

The second, also related to instability, is the inefficient delivery in vivo, as the extracellular space is full of RNAses and as such mRNA should be deliver directly into the target cells.

The third is its high immunogenicity, which while potentially advantageous in vaccine settings, can be problematic if we don’t know how to control it. Accustomed to RNA virus invasions, cells have evolved an arsenal of sensors to detect different flavours of mRNA that don’t quite look like cellular. For instance, small contaminations with double stranded RNA are potent triggers of an interferon response, which in only few hours activates a number of proteins entirely dedicated at chopping down the mRNA itself. Even more problematic is the fact that our understanding of how innate immunity modulates adaptive immunity is not quite complete, and while the innate immunity is well known to be crucial for the establishment of an effective cellular response, it has also been shown to trigger immune-regulatory mechanisms that can shut the immune response down. What this depends on is not quite clear, and for this reason, hard to control.

Troubleshooting the cons

Replacement of rare codons and G:C enrichment seem to be winning tricks to improve expression and steady state mRNA levels in vivo, although not every study seems to agree with this conclusion. This is likely to depend on the antigen encoded, as altering the nucleotide composition has the potential to change the RNA secondary conformations, impacting on translation and folding accuracy, as well as generating new T cell epitopes in alternative reading frames.

Increased stability can also be achieved by improving delivery into target cells. mRNA transfection into dendritic cells ex vivo followed by re-implantation has been used mostly for cancer vaccines and is a promising strategy. Also, intra-muscular or intradermal routes prolong stability and expression compared to systemic administration. Alternative approaches have delivered the mRNA in stabilising nanoparticles, including gold particles (gene gun) and lipid or polymer-based nanoparticles. Among these, protamines provide a good protection against RNAses but also associate too tightly with the mRNA to allow satisfactory levels of expression, while cationic lipid and polymer-based delivery has so far worked better in vitro than in vivo. Lipid nanoparticles are somehow more promising. Ionizable cationic lipids that self-assemble into 100nm particles can be internalised similarly to a virus and be released from endosomes to the cytoplasm. The exact mechanism of internalisation and release is not quite clear, but worth investigating, as this strategy could be optimised further. Lipids linked to PEG, cholesterol, and naturally occurring phospholipids also have a stabilising effect.

Extensive purification from dsRNA and other contaminants after in vitro transcription has been recommended to avoid unnecessarily high activation of innate immunity, and can be achieved through reverse-phase fast protein liquid chromatography or high performance liquid chromatography. Incorporation of pseudouridine and 1-methylpseudouridine into the mRNA have been shown to prevent activation of the RNA sensors TLR7 and TLR8 in dendritic cells. Conversely, activation of innate immunity can be achieved by using a variety of antigen formulations, including cationic nanoemulsions based on Novartis MF59, Trimix (which combines activators of CD70, CD40,and TLR4), or the CureVac AG vaccine platform, where naked sequence-optimised mRNA is administered together with some co-delivered RNA/protamine mix, acting through TLR7.

Although more work is definitely necessary to fully understand the consequences of activating different types and magnitudes of immune response, these data suggest that at least we have some ways to tweak it.

mRNA vaccines against viruses

Studies in small animal models have shown promising results following administration of mRNA vaccines, including a sustained CD8+ and CD4+ response, as well as high levels of neutralising antibodies already after 1 or 2 administrations.

Self-amplifying vaccines based on alphavirus genome, where the antigen of interest replaces the structural proteins, has been tried for a variety of viral antigens (RSV, CMV, rabies, HIV, EBOV, ZIKV, and also some bacteria) due to the high amount of protein produced from the administration of as little as 100ng in mouse. Administration of mRNA vaccines into dendritic cells has also been tried for HIV, but in spite of a significant CD4+ and CD8+ response, it led to no clinical benefit. Intradermal injection of uncomplexed mRNA encoding flu antigens, mixed with protamine-complexed RNA was immunogenic and protective in mice, but efficacy in human has been hard to prove. When between 80 and 640 microgams were administered 3 times intradermally or intramuscularly, with or without needle in healthy volunteers, a moderate site or systemic reaction was observed in almost every case. However only the formulation administered needle-free induced antibody production. Still, variability was very high and even the highest antibody titres started fading after 1 year. Some promising results were seen for a nucleoside-modified mRNA vaccine encoding Zika virus structural proteins in macaques, suggesting that the approach can also work in higher species. A number of studies have now reached Phase I or Phase II of clinical trial, although the results are not yet known. With one exception: the influenza H10N8-HA mRNA formulation, administered at 100 micrograms intramuscularly. 43 days later, good levels of neutralising antibodies were measured above the expected protective threshold, although still lower than what could have been anticipated following animal experiments.

Outlook

Significant investment has been made to support the development of a mRNA vaccine platform against infectious disease, and many years of study and optimisation have proven that this is a technology with great potential. The most concerning aspect is probably the limited effectiveness in human when compared to animal preclinical studies, although the same is true for many other biomedical approaches. In light of this limitation it is important persevere with clinical trials, and also with the systematic investigation of the effects that different modifications may have, particularly in the context of immune modulation. Some of the current discrepancies are likely to originate from the use of different cellular systems, and this is why a higher level of standardisation and the use of more physiological cell models are necessary. In the context of viral infection, different types of immune responses may be necessary to confer protection. On one hand this may compromise the concept of a universal vaccine platform, on the other a deeper understanding how to manipulate the system can be fundamental to rapidly provide a panel of testable options.

At Virology Research Services we have experience with a number of RNA viruses, as well as with techniques or RNA manipulation and expression systems. Contact us to find out how we can help by testing and developing new vaccines.