The short version: Recently, multiple vaccine candidates for COVID-19 include mRNA vaccines, which are relatively new. They have been known since the 1990s, and while not in routine use for any disease, show promising signs of efficacy in clinical and preclinical trials. The major barrier to their use is probably that they are so hard to store- mRNA is a very unstable molecule and literally tears itself apart if exposed to heat or base (alkaline)- so they have to be stored at very cold temperatures. However, mRNA production can be accomplished extremely rapidly, and thus these candidates are an attractive option for pandemic vaccines. Once inside the cells, mRNA is used to generate a protein- in this case a piece of the spike protein of SARS-CoV-2- and then is rapidly degraded. mRNA only survives within the cell for a few hours at most (and it’s taken a lot of research to figure out how to make them last even that long) and the cell has machinery that actively degrades it. Concerns about mRNA vaccines causing autoimmune diseases are unfounded and when considered in context, avoiding an mRNA vaccine does not do anything to change your chances of developing an autoimmune disease.
The anti-vaccine disinformation machine is tireless and with everything happening with COVID-19, a reasonable person without specific knowledge of vaccines or the concepts underlying them could be convinced that they have a point (they never do- not to the extent that we would need to reconsider guidelines regarding vaccination). Most recently, I have observed a number of absurd claims regarding mRNA vaccines aiming to undermine confidence in any of the mRNA vaccine candidates. Let me be explicit about one thing though before I start to disentangle lies from truth:
At no point in this blog post will I make the claim that any of these vaccine candidates are safe or effective. CONFIRMATORY OR DISCONFIRMATORY FINDINGS REGARDING THAT CLAIM DO NOT EXIST YET (at the time of writing this)- not until we have, at a minimum, Phase 3 data demonstrating explicitly that.
However, the presumption that they are unsafe, as I will demonstrate, is a very poorly founded one and the claims put forth by the anti-vaccine lobby regarding the specific dangers posed are frankly laughable.
To the science!
I: An Introduction to RNA
The central dogma of molecular biology states that DNA serves as a template that produces RNA (this process is known as transcription) which serves as a template that produces proteins (this process is known as translation):
Proteins are typically thought of as the executors of biological function (a bit of an oversimplification, but indulge me). DNA is the fundamental instruction manual for the cell. What then, is the purpose of RNA? Well, the truth is that there are about 30 different kinds of RNA (it is far more interesting than DNA, in my humble opinion, and at one point I did help to do structural biology research on special kinds of RNA called riboswitches), all with different functions:
Broadly, RNA can be either coding or noncoding. Coding RNA refers to RNA that encodes a (protein) product- messenger RNA, or mRNA for short. The noncoding RNAs refer to everything else. Messenger RNA is exactly what it sounds like- it encodes a message that is then read and processed into a protein through a molecular machine called the ribosome (translation). Ribosomes can form structures called polyribosomes, or polysomes, wherein multiple ribosomes read the same mRNA at the same time (different parts though- kind of like sharing a scroll with a bunch of people and reading different parts), each one using it to synthesize the specified protein. This is really useful because in this way, the synthesis of a single mRNA leads to many molecules of a protein, which is called signal amplification. Our mRNA is made as a linear molecule, but proteins can bring the ends very close together to simulate a circle and enhance protein synthesis (as shown in the picture to the left). In doing this, ribosomes can go over the single mRNA multiple times to produce the encoded protein product.
II: How to make mRNA
The successful production of an mRNA is not a trivial thing and there are many quality control steps (for reasons that will become clear shortly). Firstly, eukaryotic (organisms with membrane-bound organelles- plants, animals, humans, fungi, etc. as distinct from prokaryotic- bacteria and archaea) mRNA is initially synthesized as something called pre-mRNA.
When mRNA is produced, first an RNA polymerase (a protein that makes RNA from nucleotides using a DNA template; RNA polymerase II makes mRNA in eukaryotes) finds the DNA sequence to use as a template and synthesizes a primary transcript (aka hnRNA for heterogeneous nuclear RNA). Immediately upon synthesis, the primary transcript undergoes 2 processes: capping and polyadenylation. We define one end of the primary transcript to be a 5'-end (pronounced “five prime”) and one end to be a 3'-end (pronounced “three prime”) and to the 5'-end we add a cap made of 7-methylguanosine triphosphate (this is very important for reasons that will be made clear shortly). To the 3' end we add a poly(A) tail, a sequence containing many A’s (RNA has 4 bases, A, U, C, and G while DNA has T instead of U). Both the cap and the tail help to protect the mRNA from degradation (RNA in general is a very fragile molecule and will degrade if you so much as look at it funny), and the cap is also what allows the mRNA chain to undergo elongation (the process of transcription comprises an initiation phase, followed by an elongation phase, and then a termination phase).
After this is accomplished a process called splicing happens. The process itself is fairly complicated but the outcome is that certain sequences are removed while others are retained. The removed sequences are known as introns, and the retained ones are exons. Why is this done?
No one knows for sure but there are a few ideas. Some argue because it’s economical. It allows you to impart a lot of diversity within a single gene. Some cells or tissues may express alternative spliceforms of a protein which may have different functions than other spliceforms or differ in their interactions. The exons that appear in every spliceform are said to be constitutive (they are represented in green in the figure above).
The other major argument is that by having intron sequences in your genes, mutations that are meaningful are less likely to occur (a mutation in an intron, at least, the middle part, has no effect on the gene product).
The product of this process is a mature mRNA which can now exit the cell’s nucleus and be translated into a protein.
But there’s one critical wrinkle here:
mRNA is not intended to be long-lived, broadly speaking. In fact, there are multiple pathways by which mRNA undergoes decay. It can be cut up from the 5'-end with the enzyme XRN1, it can be cut up from the 3'-end with a protein complex called the exosome (this is different from the other kind of exosomes which cells secrete), and endonucleases can cut it up from the middle too. Taken together, the point is that to maintain production of a protein using mRNA, you need to keep synthesizing new mRNA. With that out of the way we can talk about how mRNA vaccines use the central dogma of molecular biology to produce an immune response- hopefully a protective one.
III: mRNA Vaccines
IIIa: Immune response
I have another post on my blog discussing the different types of vaccines but it does not include mRNA vaccines. I’ll use this part to fix that error.
mRNA vaccines are, at least in principle, an extremely effective strategy for the control of infectious disease (though they are better known for their use in cancer immunotherapy). There are a few reasons for this. Firstly, the purpose of a vaccine at the basic level is to simulate an infection without the dangers inherent to that process e.g. production of dangerous toxins, excessive tissue damage, etc. Often there is a tradeoff in the production of vaccines that we have to make between reactogenicity (the propensity for the vaccine to cause unpleasant adverse effects) and its efficacy, and to bridge this gap we often turn to adjuvants. The amazing thing about mRNA vaccines is that they are self-adjuvanting- the mRNA is the adjuvant for the vaccine (the protein it encodes is the antigen). The purpose of an adjuvant in essence is to supply the immune system with the danger signals necessary for it to recognize the antigen as a threat, and mRNA lacking appropriate checkpoints is a pathogen-associated molecular pattern (PAMP). All viruses will produce mRNA at some point in their replication cycle:
Bacteria also use it (though bacteria typically have other PAMPs that take precedence for the immune system). Thus, we have evolved complex mechanisms to detect foreign mRNA (summarized below) and react to it accordingly.
Though mRNA is self-adjuvanting, it can be combine with other adjuvants to enhance vaccine potency, such as MF59, an oil-in-water emulsion used in the influenza vaccine for the elderly, or by encoding proteins that the immune system ordinarily uses to enhance the immune response into the mRNA (TriMix).
The concept of an mRNA vaccine is as follows: mRNA encoding a protein antigen is supplied to a cell (typically by packaging inside a lipid nanoparticle) wherein it enters the cell’s cytosol or may be endocytosed as naked mRNA. Translation begins of the mRNA to yield the encoded antigen. The antigen is additionally given sequences that direct it to the appropriate subcellular compartment to ensure a proper immune response e.g. the cell membrane or into the secretory pathway. As this occurs, pattern recognition receptors (PRRs) inside the cell detect the foreign mRNA and initiate production of proteins called type 1 interferons. Type 1 interferons do many things (see your immediate left but also discussed in greater detail here and here), but in short they are a critical means of protecting the body against viral infection and can essentially rewire the cell’s metabolism to be unable to support productive viral infection. Some mechanisms by which this is accomplished are discussed in the figure to your immediate left. Interferon alpha and interferon beta are the major type 1 interferons in humans (though there are a few others) and can be used therapeutically in some diseases (chronic viral infections like hepatitis B and C where they interfere with viral disease processes and in multiple sclerosis because type 1 interferons inhibit the action of type 2 interferons which are critical for the pathogenesis of multiple sclerosis).
mRNA vaccines fall into a few classes:
- The simplest is conventional mRNA which contains just the sequence encoding the antigen of interest and 5' and 3' untranslated regions (part of the quality control system for making productive mRNAs). This contains just the instructions for the production of the protein antigen and nothing else. The mRNA itself is then rapidly degraded by cellular machinery.
- Self-amplifying mRNA (SAM) contains the antigen we want, as well as an RNA-dependent RNA polymerase (an enzyme that makes new mRNA from an RNA template). Thus, when the mRNA is being translated, the RNA-dependent RNA polymerases also replicate the mRNA to enhance production of the antigen.
- Dendritic cell (DC) mRNA vaccines are similar in principle to other dendritic cell vaccines. They involve transfecting a patient’s dendritic cells outside the body with the mRNA to activate them and prime them against the antigen of interest, and then returning them to the body where they can orchestrate immune responses.
Conventional mRNA vaccines are excellent for resource-limiting settings because of how cheap and easy they are to make. Self-amplifying candidates are a great option because they are very easy to use (you don’t need to take out the patient’s cells like for a DC vaccine) and they better simulate a real viral infection than conventional mRNA vaccines i.e. because they replicate, they form double stranded RNA (dsRNA) which is a foreign structure that does not ordinarily occur in human cells and can be detected by proteins like MDA5 to induce immune responses through interferons. DC vaccines are amazingly effective but unfortunately very labor intensive and costly to make.
Interferons are something of a double-edged sword with mRNA vaccines. They are needed to induce protective immunity, but they also terminate the production of antigen and in effect shorten the lifetime of mRNA inside the cell. There are many options available to modulate the residence time of mRNA inside the host however. For instance, TLR7 can recognize exogenous (derived from outside the cell) mRNA and set off interferon signaling, but incorporating “abnormal” (nonconventional) nucleotides like pseudouridine can prevent recognition by TLR7. Once the mRNA is translated into the protein antigen, the antigen can be directed to the plasma membrane or into the secretory pathway where it may become the target of antibodies or it will be processed for antigen presentation by T cells. The result is that mRNA vaccines are able to trigger both arms of the immune system: humoral and cell-mediated.
IIIb: Safety of mRNA vaccines
Much of the concern about mRNA vaccines has had to do with their novelty, but the reality is that this is a fairly extreme mischaracterization. The first instance of naked mRNA inducing an immune response was demonstrated in 1990 with mice and research into the concept has been ongoing since. Under ordinary circumstances it can take decades for a vaccine to make it from bench to bedside so it isn’t especially surprising that we do not have them in routine use yet. The major problem with nucleic acid vaccines in general has not been their safety, but their efficacy. It’s quite challenging to get any nucleic acid vaccine reliably in a cell and doing the things you want it to do, especially RNA. However, some studies have demonstrated very promising results. There has for instance been an mRNA vaccine tried that encoded rabies envelope glycoprotein which demonstrated protection against rabies in pigs upon challenge with the virus supplied directly to the brain and excellent antibody responses as well.
Another concern raised has been the idea that mRNA can somehow alter the host’s genome. That would actually be super cool and be huge for gene therapy (and I could finally give myself the giant bat wings I’ve always wanted) but this is not so. This is ordinarily impossible except if there is also a reverse transcriptase enzyme present that produces DNA from the RNA template, which is how retroviruses work. There is no such risk with any mRNA vaccine candidate. mRNA vaccines act entirely within the cytosol of the cell- they do not go near the nucleus where all the DNA is. That’s actually a major advantage of RNA-based vaccines over DNA ones.
Another concern I’ve heard raised is the possibility of long-term consequences that do not present until years after vaccination. This is the ever-present specter that the anti-vaccine movement likes to toss out with every vaccine and is honestly a mythical entity. While vaccines can vanishingly rarely produce conditions that become chronic (e.g. Guillain-Barre syndrome), these manifest soon after vaccination. As I’ve discussed before, Shoenfeld’s syndrome has no good evidence to support it. mRNA inside the cell is extremely short-lived. A great deal of work has been done to come up with new ways in mRNA pharmacology to make it last longer without having to give another dose and much progress has been made in that regard but nonetheless, mRNA is an incredibly unstable molecule. In fact it can degrade itself if exposed to even a little bit of heat or base and for that reason has to be stored at- 80 °C (which is why no mRNA vaccine candidate is likely going to be the fix for COVID-19 in lower and middle income nations).
Some individuals have also zeroed in on the importance of interferon responses in the action of mRNA vaccines and raised questions about autoimmunity. The fact of the matter is that interferons represent an extremely effective and ubiquitous mechanism to deal with viral infection and while aberrant interferon signaling does play a role in some autoimmune diseases one has to hold this in context: virtually every viral infection you encounter is going to elicit the production of interferons. This is how we evolved to deal with viruses. If somehow you find yourself in the position that you are on the brink of autoimmune disease and all that’s needed is some interferon to kick that into gear (and we have no way of knowing who such individuals are) then the reality is avoiding an mRNA vaccine is going to do essentially nothing to modify your risk of developing that autoimmune disease. You are bound to develop an infection at some point which will produce those interferons.
With COVID-19 specifically, there has been concern raised based on previous vaccines against coronaviruses for Th2(/Th17)-mediated immunopathology due to a vaccine. This is definitely a valid safety concern. The absolute worst thing that could happen from a vaccine is that it not only fails to prevent the disease but actually causes more severe disease upon encounter with the pathogen. But here there is excellent news: mRNA vaccines produce a highly-Th1 polarized immune response with minimal Th2 or Th17 activation. Thus given what we know about COVID-19 and vaccines against its related predecessors, it is quite rational to pursue an mRNA vaccine-based platform.
I do not know if any of the mRNA vaccine candidates for COVID-19 are safe or effective. No one does, and no one should be advocating for people to get (or not get) any particular candidate until those data exist. But the point is we have to stop poisoning the well and let that data be gathered. Thus far, there is virtually no cause for safety concerns with mRNA vaccines as a concept. The major problem I foresee is ensuring that they will be effective. It’s quite likely that the first-generation vaccines against COVID-19 will be leaky- enough to prevent serious complications but not enough to totally prevent disease or halt its spread. That would still be a tremendous victory. But the point is: let the scientists work. Their job is hard enough without people claiming that they are engineering an instrument of public harm.