THE TIME-HONOURED DISCOVERY THAT’S ENDING THE PANDEMIC

 

“The phrase Nobel Prize elicits images of individuals whose work has, without exaggeration, changed the world,” said J. Larry Jameson. The research published by Katalin Karikó and Drew Weissman in 2005 changed the world in 2020 during the COVID-19 Pandemic, giving them a Nobel Prize in 2023. The Coronavirus (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. The virus consists of a single-stranded positive-sense RNA (+ssRNA) packed by a nucleocapsid and an envelope. The Nobel Laureates discovered the key to using modified RNAs as a vaccine. The study observed that bacterial and mitochondrial RNA stimulated higher inflammatory responses in TLR (Toll-Like Receptors) presenting cells than total mammalian RNAs did. The mammalian RNAs are abundant in methylated or otherwise modified nucleotides, suppressing the RNA recognition by TLRs.

Katalin Karikó and Drew Weissman

TLRs are extracellular receptors that belong to the family of Pattern Recognition Receptors (PRRs) and are found in Antigen Presenting Cells (APCs) that play an important role in the first line of defense against pathogens. They identify pathogens by Pathogen Associated Molecular Patterns (PAMPs) and link Adaptive immunity to Innate Immunity initiating inflammatory responses i.e. the innate immune system utilizes TLRs to initiate inflammation. In humans, 10 different TLRs have been identified and each of them binds to a distinct exogenous biomolecule of whom most have been identified. For example, TLR 9 binds to unmethylated CpG motifs of DNA(linear sequence of DNA in which adjacent Cytosine and Guanine are bound by a phosphorous group). This in turn stimulates the secretion of interferons in the immune cell. 

One crucial difference that was overseen for several years in the chemical structure of nucleic acids in bacteria and mammals was the microheterogeneity which was responsible for the inflammatory responses against bacterial nucleic acids but not against mammalian nucleic acids. The mammalian DNA and RNA consist of numerous modified bases that suppress the immune system, especially RNA can undergo up to 100 different modifications. This also helps immune cells to discriminate bacterial nucleic acids from that of mammalian’s.

 

The extent and quality of RNA modifications depend on the subtype, the most abundant RNA, the rRNA in cells consists of 10 times more pseudouridine, similarly, tRNAs consist of 25% more modifications in mammals, and mammalian mRNAs have modified nucleosides such as 5-methylcytidine (m5C), N6-methyladenosine (m6A), inosine and many 2’-O-methylated nucleosides in addition to N7-methylguanosine (m7G), which is part of the 5’-terminal cap but bacterial mRNAs do not have any base modifications. Furthermore, RNA base modifications were also seen in disease-causing viruses such as Influenza, Herpes simplex, and Adenoviruses.

Their previous studies also investigated the immunomodulatory effects of RNA on Dendritic Cells (DCs) which showed that invitro-transcribed RNAs activated DCs by activating TLR 3 through its double-stranded regions. The DCs in turn secrete IL-12. However, in their Nobel-winning study, they investigated the immunomodulatory effects of natural modifications and attempted to modulate its immunostimulatory effects which ended up becoming mRNA vaccines against COVID-19 in 2020. 

Milestones of the Research

In this study, they showed that the immunogenicity differed in different natural modifications and the most potent ones were the ones with the least modifications. To prove this, they first established that human TLR 7 binds to RNA. Then they proved that modifying U, A, and C nucleotides suppresses the ability of RNA to activate cytokine-generated DCs by using RNA with modified bases such as m5C, m5U, s2U, m6A, Ψ, or 2’-O-methyl-U, which were all constituents of naturally modified RNA, and by measuring the amount of secretion of IL-12 and TNF-α and expression of CD80, CD83, CD86, HLA-DR by the DCs. They also observed that distinct TLRs responded differently to different modified bases such as RNA with m6A and s2U modifications did not activate TLR3, and those with m5C, m5U, s2U, m6A, or Ψ did not activate TLR 7 and TLR 8, but RNA without any modifications activated all those TLRs. Another interesting observation was that uridine modifications eliminated primary blood-derived DCs whereas the other modifications could not do that. They finally hypothesized that the immunogenicity of RNA was indirectly proportional to the number of RNA modifications and just a few base modifications were enough to suppress the immunogenicity.

 working of mRNA vaccine

They also discussed the evolutionary purpose of base modifications in their paper. The distinct modifications in specific nucleosides help the host cells to identify invaders, for example, Bacteria methylates its genome at selected sites which differentiates it from invading nucleic acids and secretes restriction enzymes to destroy invaders, similarly in mammalian cells the cytidine of CpG motifs are methylated which enables TLR 9 to recognize unmethylated CpG motifs of foreign DNA which then induces mammalian innate immunity. Such DNA methylation processes have been a key evolutionary tool in immune mechanism. In their words, “ Nucleoside modification is the foundation of the most ancient immune mechanism.”

The insights gained from this study gave birth to further research on how and where the viral RNAs could be modified to be used as vaccines. It also shined a light on other research areas such as the pathogenesis of Autoimmune diseases where nucleic acids play a major role. 

REFERENCES: 

The Nobel winning paper: 

• Karikó, K. et al. (2005) ‘Suppression of RNA recognition by toll-like receptors: The impact of nucleoside modification and the evolutionary origin of RNA’, Immunity, 23(2), pp. 165–175. doi:10.1016/j.immuni.2005.06.008. 

Also, check out the work that led to the Nobel: 

• Weissman D, Ni H, Scales D, Dude A, Capodici J, McGibney K, Abdool A, Isaacs SN, Cannon G, Karikó K. HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response. The Journal of Immunology. 2000 Oct 15;165(8):4710-7.
• Karikó K, Ni H, Capodici J, Lamphier M, Weissman D. mRNA is an endogenous ligand for Toll-like receptor 3. Journal of Biological Chemistry. 2004 Mar 26;279(13):12542-50.