mRNA Vaccines Flashcards
What is the makeup of traditional vaccines?
-Viral or microbial components
—–Often proteins present on the outside of the pathogen
-Inactivated pathogen
-Many other methods
What is the method and manufacturing of traditional vaccines?
-Method: stimulates the immune system by introducing a foreign body
-Manufacturing:
-Slow and expensive
-Can often be stored at room temperature
What do mRNA and traditional vaccines have in common?
-Designed to teach your immune cells how to react to a future infection
—-Via identification of a foreign body and subsequent antibody production
-Tons of research to identify a useful antigen before vaccine production
What is the makeup of mRNA vaccines
-Made of mRNA and a lipid nanoparticle
-mRNA codes for a harmless viral protein like the COVID spike protein
What is the method of mRNA vaccines
Injected mRNA serves as a template for your body to produce antigens
What is the manufacturing of mRNA vaccines?
-Faster and cheaper to produce on a large scale
—–RNA is easier to synthesize than proteins
-Initial problem: must be stored at ultra cold temperatures or it goes bad quickly
How is eukaryotic mRNA unique:
-5’ cap
-3’ Poly(A) tail
-5’ and 3’ untranslated regions (UTRs)
-Introns
What method is used to make mRNA for vaccines and what are some characteristics of this?
-In virtro transcribed (IVT) mRNA
-Requires dsDNA template
-Uses T7 (phage) polymerase
-Tmplate needs:
-T7 polymerase promoter
-Poly(T) tail
-ORF
-UTRs
Why use a T7 (phage) promoter?
-T7 synthesizes RNA faster than other RNAPs
-Highly promoter specific
-Low error rate
-Terminates less frequently
How are IVT mRNA optimized for translatability and stability?
-5’ and 3’ untranslated reations improve stability, increase half life, and improve expression
-5’ cap is required for stability
-G:C enrichment: increases stead state levels of mRNA
-Optimal length of poly(A) tail (regulatory)
-Replace rare codons with more common synonymous codons
-Modification of nucleosides to modulate immune response
How is a 5’ cap added to IVT mRNA?
-Enzymatic capping reaction: Several reactions to add a cap to mRNA, results in a lot of lost mRNA in the process
-Co-transcription with cap analogues: Basically two nucleotides that trick polymerases into starting there
What are Toll-Like Receptors (TLRs) in mammalian cells?
A class of innate immunity proteins that recognize microbial-specific products (PAMP)
What do Toll-Like Receptors recognize and what do they activate?
-Unmethylated CpG on DNA activates TLR9; common motif in bacteria and viral genomes
-dSRNA can activate TLR3, frequently found in viruses
-Nucleic acids from bacteria and viruses have a unique ability to activate these TLR systems
What happens if RNA from IVT is injected in mammalian cells?
TLRs and a massive immune response, even with a 5’ cap and Poly(A) tail
What needs to be added to IVT mRNA to evade TLRs?
-Mutations
-Specifically, modified uridines
-N1-methylpseudouridine is the most commonly used in mRNA vaccines
What are membranes made up of and how does it act/exclude?
-Phospholipid bilayer, with hydrophilic head with a phosphate group and two hydrophobic fatty acid tails
-Highly dynamic and acts as a tight mesh
-Excludes large molecules (too large) and charged molecules (hydrophobic region)
Why don’t channels exist to bring nucleotides in and out of the cell?
-Nucleic acids in the environment are probably pathogen-derived
What are the two approaches of mRNA delivery for COVID vaccines?
-Loading into immune cells and transferring back into the patient
-Direct injection
—–Cost effective, rapid, less precise
What are Lipid Nanoparticles (LNPs) used for?
Transport mRNA into the cell
What are LPNs made of?
-Phospholipids - for structure
-Cholesterol - increases stability
-Lipid-linked PEG - increases half life on nanoparticles for delivery
-Cationic lipids - useful for releasing mRNA into cytoplasm
What is the mechanism for endosomal escape?
-The acidic lumen of the endosome protonates ionizable lipids, resulting in cationic lipids
-Cationic and anion lipids form ion pairs, resulting in a conical shape that promotes membrane fusion/disruption
-mRNA is released into the cytosol where it can be used by the cell
