Today I got my first shot of the Pfizer/BioNTech coronavirus vaccine! I feel a bit uncomfortable sharing this, because I know that many people are not yet eligible for vaccines, and/or are in places where vaccines are not available. I wasn’t expecting to get vaccinated until May, but I just became eligible because of my job (essential in-person worker at an educational institution). I hope that seeing this might help some people feel more comfortable taking the vaccine. And, to go one step further, in addition to the vaccine selfie, I’m going to give you some more information about how mRNA vaccines work (both Pfizer/BioNTech & Moderna are this type). Although the bulk of the post will focus on mRNA vaccines, at the end I will give a brief overview of some of the other types you’ve likely been hearing about. 

update: if you would like a video form: http://bit.ly/mRNAvaccinebiochem on my blog & directly on YouTube here: https://youtu.be/dXmzKVBcuD0 

Much of this is an updated version of a post I made last November, with some more details and myth-busting. I hope that this post can help make you more comfortable getting the vaccine when you get the chance and equip you with some answers and arguments you can use to help convince your friends and family who might be hesitant. I think it’s important that we recognize there’s a big difference between never-vaxxers and vaccine-hesitant people. It’s normal (and good!) to think critically about new technologies, but if you don’t have a biochemistry and/or immunology background, it can be hard to understand what’s going on. So people tend to gloss over all the details and just say – it works, take it. Instead, I want to try to actually explain the biochemistry behind them, and the basic theory. And then hope you take it :). 

TLDR version: In response to viral infection, the immune system makes little proteins called antibodies which specifically bind viral parts and call for help to combat the infection. And then, some stick around for a while to keep watch in case the virus tries again. The point of a vaccine is to get people to make such watchmen antibodies without those people having to actually get the disease. Traditional vaccines have done this by using inactivated (“killed”) or severely weakened (live attenuated) versions of the virus – so your body sees “pre-made” viral parts. Another strategy is to introduce lab-grown and purified viral proteins (“recombinant proteins”). The idea with mRNA vaccines is that, instead of introducing premade viral pieces, you introduce genetic instructions for making some viral proteins (which are harmless on their own) and get the injected person’s body to make them. 

Basically, you take messenger RNA (mRNA), which are copies of the recipe for making a protein (in the case of the SARS-CoV-2 coronavirus, the recipes are of the Spike protein) and get those mRNAs into cells (often snuck in by encapsulation in a lipid coat). These mRNAs are then used (over and over again) by the cell’s ribosomes (protein-making complexes) to make lots of the corresponding protein, Spike, which then gets displayed to the immune system which learns to recognize it as foreign and produce an immune response against it. 

The technology behind it is largely the work of a scientist named Katalin Karikó – she believed in synthetic mRNA’s potential when few else did. Early attempts in the 90s & 2000s to use synthetic mRNA (mainly for replacing or supplementing damaged proteins) were faced with problems because the RNA was being recognized as foreign and triggering a generic immune response, leading to inflammation, destruction of the mRNA, etc.. Katalin Karikó figured out that if she modified the RNA so that it contained more of the features of human mRNA (such as pseudouridine) it wouldn’t cause that unwanted reaction, and it would even boost the amount of protein made. She did this groundbreaking work despite lacking support and funding, even taking a demotion to continue on with the project. She was still denied promotion to the tenure-track position she held before the demotion, so in 2013 she left to take a job as senior VP at BioNTech. There, she has spearheaded numerous projects and helped lead the development of the Pfizer/BioNTech mRNA vaccine. More about Karikó and her early path-paving work here: http://bit.ly/katalinkariko 

But for now let’s turn back to the mRNA vaccine technology…

SARS-CoV-2 is a single-stranded RNA virus – it holds all of its genetic information, including instructions for making viral proteins, in one strand of RNA. When the virus travels, it coats its RNA in proteins and a lipid (oily) coat. Embedded in that coat are proteins including the Spike protein, S, which is the one that juts out from the surface and docks onto ACE2 receptors on cell surfaces, then shape-shifts to fuse the viral membrane with the cell membrane and dump the viral RNA inside. https://bit.ly/coronavirusspike 

Since the genetic code is universal, our cells are able to read these instructions and make viral proteins. Viruses “know” this, so they’re able to hijack our cells and get us to do their bidding, including making their proteins for them. But, can we beat them at their game? If we could introduce viral protein-making “recipes” (genes) into our body, our body’s cells could make those proteins – but, since we’re only giving them parts of the viral cookbook, no active virus will get made. ⠀

It’s kinda like making a single car part. Enough to recognize as a car part, but not enough to actually drive. This way, since your cells are doing all the hard work, production can be easier, and, unlike vaccine strategies which consist of pre-made, purified, proteins grown “recombinantly,” the proteins your cells make are “perfect” – just like the real thing! Any “post-translational modifications” like glycosylation (sugar addition) get added where they normally would, which could be really important because the S protein is quite sugar-coated! (it’s a glycoprotein)

jargon watch 1: post-translational modifications are bells and whistles that get added onto the protein after it’s “written,” a bit like drawing a heart over an “i.” The writing process for proteins is called translation and it involves protein-making complexes called ribosomes linking together the amino acids (protein letters) specified by 3-RNA-letter “words” called codons present in the mRNA. There are 20 common genetically-encoded amino acids, each with a generic backbone part which allows them to link together and a unique “side chain” (aka R group) that sticks off like a charm on a charm bracelet. Once a protein’s translated, you can’t change the order of these charms, but you can add “extras” such as sugar chains and phosphates onto some of them and this offers an extra level of regulation, etc. 

jargon watch 2: practically speaking “recombinantly” means made in a lab – technically, it means that you’ve inserted the genetic instructions for making the protein into an easy-to-work-with piece of DNA called a vector which has instructions that tell easy-to-work-with cells (such as bacteria or insect cells) to make that protein. The cells will make mRNA copies from the DNA (in a process called transcription) and then make protein from that mRNA in translation. Since you’ve recombined pieces of DNA we call this recombinant DNA and we call the protein that gets made recombinant protein. http://bit.ly/t7rnap 

Recombinant expression is a strategy I use all the time to make proteins to purify and study, but the expression cells might add on different post-translational modifications since those modifications aren’t called for in the mRNA sequence and instead depend on the cellular machinery to know what to do. And different cells have different machinery so the insect cells might not add the right sugar chains, etc. which could cause the immune system to learn to recognize the wrong thing. https://bit.ly/sugarssci 

Another disadvantage to using recombinant proteins as a vaccine strategy is that you only get as many protein copies as you purify and put in. On the other hand, if you put in the genetic instructions, you can get lots of protein copies from each mRNA copy and you only have to purify the mRNA & not the protein. 

mRNA can get made commercially using a process called in-vitro transcription. Lot’s more on it here: http://bit.ly/t7rnap 

But the basic idea is that you use recombinant DNA to make mRNA (transcribe it), but you “stop” before the translation (protein-making) step. “in vitro” just means that we’re doing it basically “in a test tube” (or a vat or something, just not in an actual organism). So, “in vitro transcription” is a way to make lots of RNA from a DNA template, often using the RNA polymerase (RNA copier) of a bacteriophage (bacteria-infecting virus) called T7. 

Like other RNAPs, T7 RNAP “recognizes” (by selectively binding to) specific stretches of DNA called promoter sequences which are located upstream of the start of the gene that they copy. note: for those people like me who always have to think twice about upstream vs. downstream, promoters are in front! If you stick the T7 promoter in front of a complementary DNA version of the RNA you want made (either in double-stranded DNA (dsDNA) such as a linearized plasmid, or single-stranded DNA (ssDNA) with a double-stranded promoter region), you can get T7 RNAP to make lots of that RNA. 

Problem is, RNA is really unstable. So you need a way to get the instructions safely into cells – and keep it from degrading before you even inject it!

This is a major practical difference between mRNA vaccines and traditional vaccines made from inactivated or weakened viruses. In those traditional vaccines, the viral goodies have the benefit of being protected with the virus’ lipid coat. Then, when the virus gets swallowed up by a type of immune cell called antigen-presenting cells, those cells chop up the viral proteins and display pieces of them on their cell surface to serve as antigens (things the immune system can learn to make antibodies against through a trial-and-error approach of seeing what sticks).

With mRNA vaccines, you don’t have that viral coat. And if you were to just inject a bunch of “naked” RNA into someone, it’d quickly get chewed up by non-specific RNA chewers and cutters called RNases. If you’ve worked in a biochemistry or molecular biology lab, you might be all too familiar with RNases because they’re hardy, practically everywhere, and capable of ruining some of your experiments! RNases are so ever-present because they offer an easy, generic, method of offense against viruses. Cells, organisms, etc, can protect their own RNA by keeping it in membrane-bound areas, guarding it with proteins, etc. and then make and secrete RNases to degrade free RNA which could correspond to viral wannabe invaders. https://bit.ly/rnaseadepc 

One defense scientists can use to protect naked RNA is to store it at really really cold temperatures, where RNases are inactive and RNA is less likely to “break” for other reasons (like one of its -OH legs attacking the other). https://bit.ly/rnafragility 

In the lab, for example, we keep our RNA at -80°C and this is the temperature that Pfizer’s vaccine requires, which will likely prove to be a real impediment for getting the vaccine distributed in remote and rural locations, where, unlike in our lab, where we have 3 big -80°C freezers, some places will likely have to rely on dry ice. Even for places with -80°C freezers, dry ice will be critical in the shipping process as well. more on dry ice here: http://bit.ly/sublimedryicescience 

So, the super-cold-ness can protect the mRNA before it gets into cells. And then, once released inside cells, it’s protected by the same protections as cellular mRNAs (a 5’ cap at one end and 3’ poly-A tail at the other). http://bit.ly/mrnaprocessing 

But how do you actually get the mRNA into cells? Cells don’t just let in anything that comes their way, especially not RNA which, thanks to the negatively-charged phosphate (PO₄³⁻) groups in the RNA backbone, isn’t really compatible with the cell’s oily membrane…

Vaccine manufacturers can sneak it in by encasing it in its own oily membranes – in the form of  self-forming sphere things called Lipid NanoParticles (LNPs) which encompass the RNA in a blend of lipids. Now for a bit more jargon… 

We’ve been talking about lipids as “oily things” and they are – mostly… When I’ve talked about “oily” I’ve basically been talking about “hydrophobicity” which is when a molecule is excluded from water. Water is highly polar, meaning that, although it is neutral overall, it has partly positive regions (the hydrogens) and a partly negative region (the oxygen). Opposite charges attract, so water molecules can form extensive networks and they’ll only let other polar or charged things hang out with them (i.e. hydrophilic things). “Oils” are made up of long hydrocarbon chains (carbon and hydrogen based) which are uncharged, nonpolar, and therefore unattractive to water (i.e. hydrophobic). So if you stick oil and water together, they won’t mix. http://bit.ly/hydrophobesarenotafraid

But the lipids making up our cell membranes are “phospholipids” – they have phosphate-containing head groups on top of their oily (water-excluded) tails. And, as we just talked about, phosphate groups are negatively-charged. Since phospholipids have parts that water likes (the hydrophilic heads) and parts that water doesn’t want to hang out with (the hydrophobic tails), we call them “amphiphilic.”

As a result, if you stick them in water, they’ll assemble into phospholipid bilayers which are like phospholipid sandwiches where the tails (which are water-excluded) hang out together like the peanut butter and the head groups face towards the water, like the (now-soggy…) bread. This way you can make water-based compartments (like cells) inside water-based compartments (like bodies). https://bit.ly/lipidlove 

watery outside environment

*********

||||||||||||

||||||||||||

*********

watery inside environment

You can also get membrane-bound “sub-compartments” inside of cells, and one way these form is through a process called endocytosis. It’s kinda like a biochemical sinkhole -> basically the membrane sucks in a part of itself (including anything bound to it) and pinches it off (inside the cell) so that, although whatever was bound to it is now inside the cell, it’s surrounded by a membrane, like a little membrane-bound packet called an endosome. 

If we get our LNP to bind to the membrane, it can hitch a ride inside and then, once inside, the lipids in the LNP can destabilize the endosomal membrane, allowing the RNA to spill out into the cell (endosomal release). 

But first the LNP needs to bind the membrane and there seem to be multiple ways this can happen. The exact details depend on the various formulations and the cell types, but LNPs are often a mix of neutral, cationic (positively charged) and/or ionizable lipids (lipids which are neutral or near-neutral at some pHs but charged (ionized) at other pHs). The cationic group can help with binding similarly to the reason we often use cationic carriers such as PEI when doing DNA transfections in the lab (transfection is where you get a cell to take in nucleic acids (DNA or RNA) and the term’s typically used when we’re working in the lab but I guess it’s also what these vaccines are doing in bodies!) http://bit.ly/transfectionmethods 

The reason we use cationic carriers in transfection is that DNA & RNA have negatively-charged (anionic) backbones. And cell membranes have those negatively-charged head groups – and we know that like charges repel. So naked RNA, even if it did manage to evade RNases, wouldn’t want to get anywhere near the cell surface. In fact, that RNA wouldn’t even want to get near itself! But cationic molecules can counter-balance the negative charge making it less repulsive – and potentially even attractive! In our bodies, for example, RNA & DNA often hang out with positively-charged magnesium ions Mg²⁺. 

When we do transfection, we often do the charge-neutralizing with molecules called cationic lipids. They’re similar but opposite to phospholipids – they have a hydrophobic tail and a charged head, but cationic lipids have a positively-charged head. This allows them to glob onto the negatively-charged RNA to keep them from repulsing the membrane and helps them stick to the phospholipid groups on the membrane. It works great for transfection in the lab, but cationic lipids can cause problems in the body, such as binding non-specifically to (and getting stuck on) plasma proteins (proteins in the blood). 

So, instead of relying on “always-positive” lipids, LNPs for in-body use have turned towards “sometimes-positive lipids” called ionizable lipids. Whether they’re positive (and/or how positive) they are depends on the pH. Lower pH (more acidic conditions) means there are more protons (H⁺) available. The ionizable lipids have “protonatable groups” which are able to take some, becoming protonated and positively-charged (cationic). But at higher pH, when there are fewer protons available, they won’t be protonated and thus will be neutral and less plasma-protein-sticky. So, you can assemble the LNPs at a low pH, when the lipids are + charged and then raise the pH to and end up with a complex thing where you have ionizable-lipid-coated RNA inside of little pockets inside of a broader, neutral, lipid shell. https://bit.ly/3f0Nwo7 

These can dock onto cells in different ways, some of which rely on sticking to lipid-binding receptor proteins. Then, once swallowed inside but trapped in the endosome, the ionizable lipids get to take a starring role again because, as part of the endosomal maturation pathway, the endosome acidifies, so the ionizable lipids becoming protonated and positively-charged (cationic) again. This helps them interact with, squirm into, and destabilize the endosomal membrane.

At least that’s the general idea I think – I’m a little fuzzy on the actual details and it seems to vary depending on the formulation. https://bit.ly/3kyjJE6  DOI:10.1038/mt.2010.85 

But Pfizer’s mRNA vaccine uses an LNP formulation made by a Canadian company called Acuitas. https://acuitastx.com/technology/  And, at least in the past paper their website sends you to, they “contain an ionizable cationic lipid (proprietary to Acuitas), phosphatidylcholine, cholesterol and PEG-lipid.”  DOI:10.1038/nature21428 

Phosphatidylcholine & cholesterol are both components of cell membranes, and they can act as “helper lipids” forming that outer shell. PEG-lipids are lipids that have been had a PolyEthyleneGlycol group attached – PEG is a long hydrocarbon with lots of hydrophilic -OHs, and such PEGylation  is often done to add an extra layer of protection, stability, bulk, and immune system avoidance. https://bit.ly/3lvx2qg  

No matter what the formulation, the end result is that you (hopefully) set the mRNA loose in the cell, ribosomes make protein from it (remember you can get lots of protein copies made from each mRNA copy), and then the immune system makes antibodies against it, which I’m not going to get into in the current post. But I did try to in this post  https://bit.ly/coronavirusvaccinetypes 

A lot of people have asked me about what happens to the proteins that get made, and that’s way beyond my expertise, but from what I’ve gathered, a few scenarios can happen

  • the protein gets made and then chopped up by the proteasome, and then pieces of the protein (peptides) get displayed from the surface of the cell where immune cells can learn to recognize them (note: this is something that happens even to normal proteins as a sort of way the immune system keeps an eye on what’s going on – if it recognizes the proteins as self, “nothing happens” but if it doesn’t recognize it, then the adaptive immune response kicks in to find T-cell receptors that will specifically bind it, giving you “cell-mediated immunity”)
  • the protein gets secreted, or is present in dead cell debris, and is swallowed by an immune cell – the cell then does a similar chop up and display thing, followed by generation of a specific immune response. If I understand correctly, this time the peptides are displayed on a different protein (MHC class II instead of MHC class I) and that leads to the generation of antibodies, including the superstar “neutralizing antibodies” which bind in such a way that they prevent the virus from getting into cells
  • the protein gets displayed from the surface of the cell like it does from the viral membrane, and antibodies get made against it that way

here is a good article: https://www.cas.org/blog/covid-mrna-vaccine 

and an FAQ: https://www.idsociety.org/covid-19-real-time-learning-network/vaccines/vaccines-information–faq/ 

The injected mRNA can either be taken up locally by the muscle cells it was injected into, or it can drain to the lymph nodes, where a bunch of immune cells hang out. Yes, a few cells will get killed in the process, but only the ones that took in the mRNA – the mRNA itself is temporary and it can’t get passed on from cells. So only the cells that take it in will be affected. And it’s not enough to actually cause any harm. Furthermore, I heard on a podcast episode (an episode of TWiV but I don’t remember which one) that most of the uptake takes place in those lymph nodes, so you will only be causing negligible muscle damage (and the little there is helps stimulate the immune system to come to the area (accompanied by a little soreness)). 

The mRNA does not get into your DNA – it doesn’t even get into the same intracellular compartment as your DNA. The DNA is stored in the membrane-bound nucleus. And the mRNA only gets as far as the cytoplasm. And it’s in RNA form, so even if it wanted to sneak into your DNA, it couldn’t. So don’t let that hoax scare you off – I didn’t! 

Another concern some people have is that mRNA vaccines have never been FDA-approved for human use before. But, in their defense, the technology to make them hasn’t been around that long. Now that it is, however, the technique shows great promise – not just for Moderna and Pfizer/BioNTech and their coronavirus vaccines, but potentially for a wide range of viruses. They have a few key things going for them… mRNA is much easier to manufacture than other vaccine types, and it allows for an “early start” –  all you need to know is the genetic sequence of the virus – you just need the recipe and you can go to work. Of course, they have to choose the right protein and maybe tweak it a bit, but it’s still much much easier that having to actually express and purify the protein! Plus, each mRNA copy can be used countless times by the ribosomes, so you can get a much greater return on investment than if you just put in premade protein. 

But (at least these ones) do have that freezer problem. Pfizer’s has to be stored at -70°C (-94°F) which is really really cold, much colder than a “normal” freezer”. Moderna’s just needs -20°C (-4°F) which is just like your home freezer. But still not ideal https://bit.ly/2IE9oJw 

It’s probably not that Pfizer’s *really* has to be stored that cold (it’s not like it falls apart at -69), but that’s what they used when they did all their testing and stuff, and put in their application for approval. So now, Pfizer is working on further testing to show how cold it really *needs* to be. And potentially tweaking the LNP formulation if need be to increase the stability at higher temps. 

Thankfully, mRNA vaccines are just one of many many types of vaccines being tried, and some of the other methods don’t require such a cold “cold chain”! And some of them also don’t require a second shot. These are really important factors when considering vaccination at the global level (which we need to be doing more of). 

If you want to learn more about different vaccine types: https://bit.ly/coronavirusvaccinetypes but here are a few you might hear about…

The main other type currently being used in the U.S. is a viral vector-based vaccine. These use other, harmless, viruses (in these cases adenoviruses) as vectors or “vehicles” to get the SARS-CoV-2 Spike protein into cells. Vaccines that use this approach include the Oxford/AstraZeneca vaccine, the Johnson & Johnson vaccine, & the CanSino vaccine, & the Sputnik V vaccine). You can learn more about those here: http://bit.ly/viralvectorvaccines 

Globally, there are also inactivated viral vaccines being used, such as the one from China’s Sinovac (called CoronaVac). These use a “dead” version of the virus, so they introduce the whole SARS-CoV-2 virus that isn’t able to actually infect you. This is a more traditional vaccine approach, but it requires a lot a lot of virus to be cultured.  

You might have also heard of Novavax – it works by using recombinantly expressed & purified proteins. So, instead of giving the person mRNA and having the person make the Spike, they make Spike and give it to the person. They actually give it in the form of “nanoparticles” which are like little bouquets of full-length Spike proteins. If I understand correctly, because they’re full-length, they have the membrane-spanning part so you can have a sort of ball of lipid called a micelle with multiple Spikes sticking through, creating what’s sometimes classified as a virus-like particle (VLP), although I’ve also seen this vaccine classified as a subunit vaccine. In addition to Spike, the vaccine contains an adjuvant – an immune-system stimulator to make sure the body realizes that you’ve stuck something foreign in it and generates an adaptive immune response. https://www.nytimes.com/interactive/2020/health/novavax-covid-19-vaccine.html Here’s the technology they’re using (I believe based on their citation, but this specific paper is from an older OG SARS vaccine development): https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4058772/ 

Vaccine development has been portrayed as a bit of a race (didn’t help that Russia named their vaccine “Sputnik”…) but in reality, the more vaccines the better! Because we have a looooot of people to vaccinate – all over the world – in all sorts of living situations and nearness to high tech low-temp freezers. And hopefully the technology will advance to make mRNA vaccines more shelf stable – and freezers (and health care in general) more accessible to everyone. 

I recently found this article about the trials underway testing additional vaccines, many of which are designed to be easy to deploy in lower-income countries. https://www.nature.com/articles/d41587-021-00001-x 

Today was super emotional for me. On the drive to the mass vaccination site I almost started crying but then once there I was basically just in a state of stunned disbelief. My mind keeps replaying the past year as I followed the news of the vaccine development and hoped that this day would come. And now it has and I am so incredibly grateful. But this gratefulness comes with a sense of survivor’s guilt. So many people have lost their lives or lost loved ones or lost their jobs. I have been so incredibly fortunate and privileged and I wish I could give my dose to someone who needs it more (I mean, I can’t believe I’m, without jumping the line, getting vaccinated before my parents (who work from home)), but I can’t. So I’m just telling myself I’m doing my part to protect everyone around me. Getting vaccinated isn’t just about me, it’s about the colleagues I go to work with in the lab – vaccination protects not only me, but everyone from the amazing janitorial staff to the president of the lab. And it helps protect the wider community as well, as each of us goes separate ways at the end of the day. 

I really hope you all will get vaccinated too when you are fortunate to get the chance. Protect yourself. Protect your loved ones. Protect the world – we’re all interconnected and no place is safe until everywhere is. So, don’t just watch the stats for your country, watch those for the world. Don’t return to complacency once your country hits 100% vaccinated. Help advocate for global vaccination. Help inform people about the cause. I hope this post gives some reassurance and sense of agency to those of you who may have wondered about how these vaccines work. I know I probably didn’t change any minds (especially given my audience) but hopefully it will enable some of you to help change minds. 

Wishing you all health, happiness, and stabs in the arm. 

Sincerely,

the bumbling biochemist

4 Thoughts on “COVID-19 vaccine biochemistry, with a focus on mRNA vaccines”

  • Thanks for the very technical article. I can tell you know a lot. I also appreciate your website being called The Bumbling Biochemist because since there is so much “propaganda” on this subject it’s hard to know which sources are being sincere, so when I went though my Google search results, I figured anyone that calls themselves “bumbling” is probably not trying to sell you something, and you aren’t.

  • This article was so helpful thank you! It’s nice to read a scientific perspective rather than an opinion-based perspective. I was curious if you would
    mind speaking on a question. It seems that the science community is pretty confident there is a low risk of long-term side effects of mRNA vaccines. What contributes to this confidence? Just a lay person trying to understand. Thank you!

    • That’s great to hear – thank you and glad I could help! The mRNA is only temporarily in your body before it gets degraded. The mRNA does not get into your DNA or anything like that, so there’s not really any biological reason why a side-effect would pop up in the future. The mRNA you’re getting is the “same” mRNA you would get with a more traditional vaccine – or with the infection, except you don’t have the actual virus, just the single mRNA so it can’t make you sick like the virus does. For vaccines – any type of vaccine – the biggest risk of side effects is right after vaccination and I don’t know of any case in which a side-effect of a vaccine has popped up at a much later date. Hope that helps!

Leave a Reply to Brianna Bibel Cancel reply

Your email address will not be published.