Bursectomised Chickens, and Other Breakthroughs

The four primary nucleobases of DNA, represented by the letters A (adenine), G (guanine), C (cytosine) and T (thymine), are transcribed as A, G, C and U (uridine) to make messenger RNA (mRNA), which instructs molecular machines called ribosomes how to make proteins from their amino acid building blocks. The letters are decoded in strings of three: AUG, for instance, is read as the amino acid methionine, GCA encodes alanine, and so on. After the correct number of amino acids have been put together in the correct sequence there’s an order such as UAG: stop.

DNA letters make RNA letters make protein: this is how it’s taught – but life is more complicated than that. The bases undergo a whole range of chemical modifications, represented by other letters and numbers, such as I, m⁵C and m⁷G. One modification, changing uridine to pseudouridine (ψ), underpins the efficacy of mRNA vaccines. For their work on understanding and using these chemical modifications of RNA to make better vaccines, Katalin Karikó and Drew Weissman have been awarded this year’s Nobel Prize for Physiology or Medicine – the only surprise being that they didn’t get it last year. Your share of the prize has probably been deposited into your deltoid muscle, if at some point you got the Pfizer/BioNTech or Moderna Covid vaccine.

RNA is much more than a message. It’s fundamental to life in a way that DNA isn’t. Good guesses about the origins of life imagine an RNA world, before the much more stable DNA was co-opted as the genetic storage material of choice for everything apart from some viruses and viroids. The existence of viroids – virus-like entities that don’t even bother to make protein as they replicate themselves – supports the RNA-world hypothesis. These remarkable entities entice cellular machinery to replicate their RNA genomes using only the genomes themselves. (Viroids can cause disease in plants but are not human pathogens. There is a near-viroid human infection though, the agent of hepatitis delta, which produces just one protein. It piggybacks along with the hepatitis B virus, and is associated with more severe disease.) RNA’s chemical flexibility allows it to play many of the roles that proteins accomplish. It is also a key part of the ribosomes that decode mRNA and synthesise protein. RNA is central to life because it’s probably how life started – with self-replicating RNA.

The potential for RNA molecules to be self-replicating is a problem for our cells. Viruses such as influenza and Sars-CoV-2 have RNA genomes and co-opt cellular machinery to make their mRNA and proteins. It’s therefore important for cells to have ways to distinguish between its own RNA molecules and those of intruding viruses. Cells must also regulate RNA levels, target them to the correct locations and remove defective or decaying RNA. If mRNA didn’t have a structure at the beginning called a cap, formed from m⁷G, the cell would either quickly chew it up or raise the alarm about a possible viral infection. Successful viruses have evolved mechanisms to escape being chewed up and avoid raising cellular alarm signals for as long as possible. Influenza goes in for ‘cap-snatching’: it rips the heads off cellular mRNAs to manufacture its own.

Karikó and Weissmann started working together on possible chemical modifications to make a synthetic mRNA molecule more stable and less likely to set off a cell’s alarm mechanisms. They were particularly interested in dendritic cells, which play a crucial role in initiating and regulating an immune response. For mRNA to be useful as a therapeutic, it would need to be translated into protein for long enough to generate an immune response to that protein. Suspicious cells are much too quick to shut down protein production from unadorned RNA. If you try the brute force approach of chucking in a large amount of mRNA you run the risk of generating a serious inflammatory reaction.

In a landmark publication, cited by the Nobel Assembly, Karikó, Weissmann and colleagues showed the effects of different modifications of RNA – m⁵C, m⁶A, m⁵U, s²U and pseudouridine – on dendritic cell activation. A model mRNA with pseudouridine (or otherwise modified U) wasn’t recognised as dangerous by dendritic cell receptors. In the way of these things, the paper didn’t end up in one of the top three scientific journals (Nature, Science or Cell). The New York Times reported that ‘the study was eventually accepted by a niche publication called Immunity,’ a cause of some hilarity for immunologists who would on the whole be delighted to publish there.

The best example of a landmark paper being published in a truly niche journal was the discovery that led to our understanding of which cells produce antibodies. We now call them B cells, the B standing for bursa of Fabricius. This is an organ, situated at the wrong end of birds, that mammals lack: we produce B cells from our bone marrow. In a 1956 article entitled ‘The Bursa of Fabricius and Antibody Production’, Bruce Glick, Timothy Chang and George Jaap described the phenomenon whereby bursectomised chickens failed to raise an antibody response. Science rejected their paper and it was eventually published in Poultry Science – not a journal that many immunologists keep abreast of.

Karikó and Weissman followed up their finding published in Immunity with papers describing the role and utility of pseudouridine in Molecular Therapy and showed the importance of mRNA purification in Nucleic Acids Research: solid, unflashy stuff, also in time cited by the Nobel Assembly. But Karikó was demoted and then let go by her university: not productive enough, didn’t win enough big grants. We shouldn’t care where science is published – the whole business of scientific publishing is increasingly silly and in the end a scientific paper stands or falls on its merits – but we do care because careers depend on it.

Did pseudouridine save us from the Sars-CoV-2 pandemic? It played an important part. Further optimisation settled on a slightly modified N1-methylpseudo-uridine (m1ψ) for the successful mRNA vaccines. Alternative RNA vaccines that didn’t use it were much less effective. There were other useful vaccines though, notably the ChAdOx platform, which was a bit less individually effective but easier to deploy in many settings. The Pfizer/BioNTech and Moderna vaccines were also more effective because of a different discovery about the way viruses invade our cells. Many nasty viruses get in using a large sugar-protein complex that binds to target cells, persuades them to take the virus in, and then fuses the viral membrane with the cell membrane. This fusion event is triggered by a conformational rearrangement in the entry weapon, known as spike in coronaviruses.

Neutralising antibodies can block this if they target spike pre-fusion, but if they are directed against the post-fusion spike it’s too late. Virologists had worked out a way to lock spike into its pre-fusion conformation, meaning that all the antibodies generated would be against the dangerous target, not its spent remnant. The successful RNA vaccines employed this trick, and without it they would have been much less good at generating the right sort of antibody response. Similar molecular manipulation is allowing the manufacture of effective vaccines against other important viruses, most notably respiratory syncytial virus (RSV), the cause of severe respiratory infections in infants to which no successful vaccine had been developed despite decades of effort.

It’s very likely the mRNA vaccines, pseudouridines at the ready, will be effective for many other viruses and in many other settings. There’s hope they may be useful as cancer vaccines. The big prizes in science, of which the Nobel is the most important, celebrate individuals rather than teams. This doesn’t reflect the way that most science is done, though it’s beyond doubt that Karikó and Weissman deserve their award. In an interview recorded immediately after the announcement, Karikó alluded to her difficulties with her university, in obtaining funding and in gaining recognition. She mentioned the moral support she’d got from her mother, who always believed in her even if grant funders and university administrators didn’t. She also mentioned her pride in her daughter’s achievements as a member of the USA women’s rowing eight, winning gold medals at the Beijing 2008 and London 2012 Olympics. One day perhaps team science will receive as much recognition as team sport.