mRNA vaccines may make unintended proteins, but there’s no evidence of harm

Even after the billions of doses given during the pandemic, messenger RNA (mRNA) vaccines still hold surprises. A study out today reveals they may unexpectedly prompt cells to produce small amounts of unintended proteins. There is no evidence that these mistakes compromise the safety of the COVID-19 vaccines, which saved millions of lives, and the researchers have already proposed a fix that may help make future vaccines or drugs based on mRNAs safer and even more effective.

Other scientists say there is nothing alarming about the new work, reported in Nature, and agree that it could help improve the design of mRNA treatments still under development. The “landmark study” provides fresh insights into how cells make proteins from both natural and artificial mRNA, virologist Stephen Griffin at the University of Leeds told the U.K. Science Media Centre. He added that “some of the future wider therapeutic uses for RNA technology beyond vaccines may involve higher and more frequent dosing, so any and all possible issues need to be addressed.”

Strands of mRNA convey the blueprints encoded in cells’ genes to their proteinmaking machinery called ribosomes. The ribosomes read mRNA three bases at a time, with each of these so-called codons specifying an amino acid in the protein. Potential mRNA vaccines and therapies consist of artificial mRNA that carries the recipe for a specific protein. For vaccines, the goal is to generate an immune response to the protein—the spike protein of the pandemic coronavirus, for example. mRNA therapeutics aim to coax a person’s own cells to produce a protein that can treat a disease.

But because foreign RNA usually means a virus is attacking, cells tend to recognize and destroy it. Katalin Karikó and Drew Weissman of the University of Pennsylvania decades ago discovered that if they altered one of mRNA’s bases, replacing uridine with pseudouridine, the modified strand could elude destruction long enough for cells to make the intended protein. The research, for which they will receive Nobel Prizes on Sunday in Sweden, helped accelerate the development of potential mRNA vaccines and therapies.

However, the new work finds that the RNA modification strategy has a catch. University of Cambridge toxicologists Anne Willis and James Thaventhiran and their colleagues wondered what effect the pseudouridine might have on the proteinmaking process. Ribosomes sometimes translate natural mRNAs incorrectly, and the researchers wanted to know whether such mistakes are more common when altered bases are inserted. They designed mRNAs that would prompt cells in a dish to produce a fluorescent protein only if a ribosome “slips” and starts to read the three-letter codons incorrectly, a mistake known as a frameshift. With natural mRNAs, this typically produces inactive proteins that are broken down by the cell.

The researchers found that mRNAs containing the form of pseudouridine used in Pfizer’s and Moderna’s COVID-19 vaccines were much more likely to induce frameshifted proteins than mRNAs with normal uridines. In their experiments, roughly 8% of the proteins produced from their experimental mRNAs were frameshifted.

To test whether the same effect happens in the body, the team compared the immune reactions of mice that were vaccinated either with the COVID-19 mRNA vaccine made by Pfizer or with a less widely used DNA-based vaccine from AstraZeneca. They found that the Pfizer-vaccinated mice generated antibodies to frameshift-derived proteins, whereas animals vaccinated with the AstraZeneca vaccine did not.

The researchers then examined immune responses in 20 people who got the AstraZeneca vaccine and 21 who got the Pfizer vaccine. Blood samples from about one-third of the Pfizer recipients showed an immune reaction to the frameshifted proteins, whereas none of the samples from AstraZeneca recipients did. None of the people reported any side effects from their particular vaccine, and there is no evidence, the researchers say, that the frameshifted protein is harmful.

The Cambridge team emphasized at a press briefing that its work doesn’t indicate COVID-19 vaccines are unsafe. Indeed, Karikó notes that frameshifts frequently occur naturally when infected cells make viral proteins. These frameshifted proteins can naturally broaden the body’s immune response, she says, so it’s possible that the spike-targeting COVID-19 vaccines may even get a boost from the misreading. When asked about the new paper, Pfizer did not directly comment on its findings but in a statement said its vaccines had a “positive benefit-risk profile.” A Moderna spokesperson declined to comment.

But Thaventhiran says frameshifting might be more important in other applications of mRNA technology such as cancer vaccines, designed to trigger immune responses to tumor-specific proteins. Cancer patients are likely to have received other treatments that alter their immunity, so they might be more vulnerable to the mistaken proteins and any immune reactions they could trigger.

Further experiments by the Cambridge team suggested a reason for the frameshifts—and a possible way to avoid them. The group found that ribosomes slow down when they encounter the pseudouridine, especially when the sequence contains several of them in a row. That’s likely because the altered bases “aren’t as good a fit” to the ribosomes as the standard ones, which raises the chance of frameshifts, Willis says. But there’s a simple solution, the researchers say. Because mRNA can use several codons for a given amino acid, the molecules can be designed to avoid the slip-prone codons. For example, mRNA designers can use the sequence UUC (two pseudouridines and a cytidine) instead of UUU (three pseudouridines). Both code for the same amino acid, but the former should produce fewer errors. It’s a bit like writing mom instead of mum, Willis says. “It means the same thing.”

The researchers have developed an algorithm that identifies the potentially problematic sequences and replaces them with ones less likely to cause slips. Because of the synonymous codons, the same protein still gets made, just more accurately, Willis says. “It’s such an easy fix, that’s what’s so exciting about it.”

Rolf Marschalek, a molecular biologist at Goethe University Frankfurt, says he would like to see more evidence before he is convinced that frameshifts are a significant issue for modified mRNAs. He agrees, however, that the new work is not a reason to worry about the safety of the mRNA COVID-19 vaccines. “It’s a much bigger problem that people are underestimating the Omicron variant and are not getting the updated booster,” he says.

By Gretchen Vogel