Pseudouridine, mRNA Vaccines & Spike Protein Persistence
The spike protein mRNA found in the Pfizer & Moderna vaccines differs in subtle but important ways from the mRNA found in the SARS-CoV-2 virus itself.
In M&M #58 I spoke to genomics expert Kevin McKernan, asking him a simple but important question: is the mRNA found in the Pfizer/BioNTech and Moderna COVID vaccines identical to the equivalent mRNA found in the SARS-CoV-2 virus?
The answer is, “No.”
The mRNA in these vaccines was engineered to be different from the natural viral mRNA in subtle but consequential ways. Let’s briefly review the basic molecular biology required to understand mRNA vaccines and SARS-CoV-2, examine how the vaccine mRNA differs from viral mRNA, and consider how this difference may relate to recent observations in the literature.
If you prefer to listen to Kevin’s explanation of this stuff, check out this clip:
Molecular biology 101 (skip this section as needed)
Our cells contain DNA, an elegant form of molecular information storage. The genes contained within our DNA encode the primary structure of proteins, the molecular machines which conduct the physiological business of life.
To go from DNA to protein, our cells use RNA as an intermediate. DNA gets turned into messenger RNA (mRNA), named for the fact that this mRNA relays the genetic the information contained within our genes (“the message”) to the molecular machines that manufacture proteins. The process of turning mRNA into protein is translation. For our purposes here, we will be focusing on how the properties of mRNA influence the process of protein synthesis (translation).
There are four molecular building blocks used to encode information in DNA, abbreviated as A, T, G, and C. Each letter stands for the name of a small molecule used a basic unit of our genome.
RNA is also composed of four building blocks, which are the same as those used in DNA with one difference: instead of “T,” there is a “U,” a molecule called uracil. Remember this bit about the letter “U”—it will be important as we go on.
Basic biology of SARS-CoV-2 infection (skip this section as needed)
SARS-CoV-2, the virus responsible for COVID-19, is a type of RNA virus. This means that each viral particle contains an RNA genome, not DNA. The outside of the virus is studded with spike proteins, which allow it to get inside of our cells. From there, it tricks our cellular machinery into using its viral RNA to make virus proteins, which are then assembled into new viral particles.
mRNA vaccines & mRNA metabolism (skip this section as needed)
Unlike traditional vaccines, which contain weakened versions of an entire virus, the Moderna and Pfizer/BioNTech (henceforth just “Pfizer”) vaccines contain only mRNA. More specifically, they contain the mRNA sequence encoding the SARS-CoV-2 spike protein.
Here’s the idea: by giving this mRNA molecule to our own cells, viral spike protein will be produced, but not a complete virus. This stimulates an immune response despite the fact that we are only exposed to a single SARS-CoV-2 protein.
Animal cells are really good at chopping up mRNA molecules with enzymes. In fact, the longevity of mRNA molecules is an important aspect of how our cells regulate gene expression. The more stable an mRNA molecule is, the longer it hangs around and the more protein that gets made from it.
Molecular biologists can use laboratory tricks to alter mRNA stability. Because mRNA is generally unstable, getting mRNA vaccine technology to work was challenging. It’s very easy to package mRNA into a vaccine and inject it into an animal, but the mRNA simply gets degraded by enzymes before any protein production takes place.
To get mRNA vaccines to work, scientists had to come up with a way to stabilize mRNA, allowing it to get into our cells and stick around long enough for protein synthesis to happen.
How is mRNA stability enhanced in mRNA vaccines?
Pfizer and Moderna solved this mRNA stability problem, in part, by making a subtle tweak to the mRNA encoding the SARS-CoV-2 spike protein. They swapped one of the standard letters in the RNA code, the “U” (uridine), a for slightly different molecule called N1-Methylpseudouridine.
Pseudouridine and uridine are very similar, differing by just a few atoms. Even though the vaccine mRNA is not identical to the native mRNA of the virus, it still encodes the SARS-CoV-2 spike protein. But even this slight difference makes a difference, which is why the engineering choice was made. Using pseudouridine has the effect of stabilizing the mRNA so that it is not immediately degraded when injected into an animal.
During an earlier R&D phase, scientists at Pfizer and Moderna presumably tried injecting regular mRNA into animals, which failed to stimulate an immune response. The stabilizing effect of pseudouridine allowed for robust spike protein production, thereby stimulating a potent immune response. (The spike protein mRNA in vaccines is also protected by lipid nanoparticles—small bubbles of fat.)
All of this makes perfect sense and seems to be a rational design decision. Two important questions spring to mind:
Can mRNA molecules be too stable and cause too much protein synthesis?
Beyond mRNA stability, are there any cellular consequences to swapping in pseudouridine for uridine?
The answer to both questions is, “Yes.”
Let’s investigate each in more detail, in light of some recent findings from the scientific literature and outstanding questions regarding mRNA vaccines.
mRNA stability & spike protein persistence
How long after vaccination do these pseudouridine-containing mRNA molecules stick around? This was something measured in a recent study comparing the immune responses to vaccination vs. SARS-CoV-2 infection.
To measure this, researchers biopsied the lymph nodes of people who had received two doses of the Pfizer vaccine. They measured the presence of vaccine mRNA in the lymph nodes as early as 7 days and as late as 60 days after the second vaccine dose.
Intriguingly, they were able to detect vaccine mRNA 60 days out. Vaccine mRNA seems to ramp up for a couple weeks, being detectable in most samples 16 days after the second shot. A similar pattern was seen for spike protein in vaccinated individuals. This suggests that a getting two shots will lead to spike protein expression for at least a couple of weeks in most individuals.
They also measured spike protein levels in samples taken from people who died from COVID, finding that spike antigen was present at lower levels in these samples compared to those taken from vaccinated individuals. This is quite remarkable, as it suggests that spike protein levels get to higher levels after two jabs of the Pfizer vaccine compared to people with severe COVID.
But most people who receive two shots of that vaccine (such as myself) are symptomatic for about a day and don’t experience any obvious side effects beyond that. So does it really matter that spike protein levels can persist for weeks following vaccination?
The answer is: we don’t know.
Let’s consider this issue of mRNA and spike protein persistence in the context of how long these things normally persist.
