Covid-19 – the hunt for a vaccine, and some of the key candidates and strategies on the scene. When it comes to immunity from Covid-19, little proteins called antibodies are key. Typically produced in response to infection, they 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. But doing this is NOT a breeze… In addition to some of the “classic ways” of making a vaccine being tried out (e.g. dead or weakened coronavirus) there are new ways being tried involving just using pieces of the viruses’ genetic information (e.g. mRNA and DNA vaccines). Each way has pros and cons, and I hope I can help you understand what’s going on.

note: I am NOT an immunologist, so I’m not going to go into the immune response details, instead focussing on the vaccines themselves, but it’s important to have an overall idea of what these vaccines hope to accomplish, which means an overview of the immune system.

The immune system is an interconnected network of cells and organs in your body that is responsible for keeping you safe from infection. When your body is confronted with a virus, your immune system has 2 main strategies it deploys. First is a generic attack using the innate immune response. This hopefully successfully fends off the virus. But, if not, it at least buys time for the adaptive immune response to develop a targeted attack strategy specific for that virus.

The way the adaptive immune system is able to target its enemy specifically is through the use of antibodies, and it takes a while to make the right ones. Aka immunoglobulins (Ig’s), antibodies are little proteins that recognize the virus and call for help. 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.  

Antibodies have unique “variable regions” and generic “constant regions” that serve as adapters. When I say “recognize” all that means is that the antibodies bind to that viral piece, the antigen, because the protein letters in the antibody’s variable region present chemical binding opportunities that the antigen finds oh so attractive (opposite charges, complementary shapes, etc.). But it’s important to be attractive ONLY to the specific virus. If, for example, the antibody binds your own proteins, it can lead to your body attacking itself (e.g. autoimmune diseases). 

Thankfully, there are a lot of different variable regions to choose from – through a process called somatic recombination, a type of immune cell called immature B cells, can randomly select a variable region (with the limitation that, once chosen, they can ONLY make antibodies variable region – no re-choosing). When an antibody happens to bind the virus and not human cells, the cell making that antibody gets selected for, the antibody it makes potentially gets strengthened by a process called “somatic hypermutation” where minor tweaks to it are made, and the body starts making lots of it. 

The variable region can be hooked onto different adapters to give you different classes of antibody – like IgM antibodies which are made earlier on and IgG antibodies that aren’t made until later but also stick around for a while (but we don’t know how long). Antibodies are used to direct the immune system to where it’s needed, call for backup, etc. 

Once the threat is over (all the virus is killed), some of the antibodies stick around to keep watch. So, if you take the blood of someone who’s recovered from Covid-19 (usually they just take the cell-less part of the blood called the plasma) and give it to someone who doesn’t have the virus, that person might now have temporary protection from catching the virus (SARS-Cov-2). Or, if you give that plasma to someone who currently has the virus, it might help them fight it off. This is called convalescent plasma therapy. A related treatment is hyperimmune globulins, where the plasma from a bunch of recovered people is pooled and concentrated. Then its antibody strength is tested and doses are given accordingly. This way, batch-to-batch plasma variability is less of an issue. more here: https://bit.ly/covid19potentialtreatments 

These only provide “passive immunity” because they only protect the person while they’re taking them. They don’t train the patient’s body to make the antibodies themselves. That training is needed to provide “active immunity” and that’s what vaccines aim to do.

it’s like the “teach a man to fish” parable, immune-system style: Give a man some antibodies (like from convalescent plasma) and he has passive immunity (only protected until those antibodies degrade). Teach a man to make antibodies and he has active immunity (protected as long as those antibodies keep getting made). There are 2 main ways to “teach a person” to make antibodies – you can learn the hard way (infection) or the easy way (vaccine). 

But, whereas it’s easier from the person’s point of view, making a vaccine isn’t easy. And there isn’t “one way” to do it – there are a lot of different ways. Some of the main ones:

  • inactivated (killed) virus
  • weakened virus
  • viral proteins (aka subunit vaccines)
  • virus-like particles (VLPs)
  • viral vectors
  • DNA
  • mRNA

Looks overwhelming, but they all aim to do the same thing (introduce the body to some part(s) of the virus in a harmless form). And we can kinda clump them into a couple main groups. The “look what I made for you” group introduces pre-made viral parts, whereas the “make it yourself” group introduces the genetic instructions for making viral parts and relies on the body’s cells to actually make those parts. note: this is completely separate from passive vs. active immunity. These are all ways to develop ACTIVE immunity – in each case, the body has to make its own antibodies. The difference between these groups I’ve broken them into is whether the body *also* has to make the viral pieces.

I talk about “viral pieces” but the main piece that most of these vaccines is focusing on is the spike protein (S protein), which is the one that juts out of the viral membrane and docks onto human cells.

Antibodies can specialize to bind “anything” (even things that aren’t really harmful, like a peanut protein). So, antibodies can be produced that target any of the virus’ proteins, but S is seen as the most promising for a couple reasons – it’s most easily visible to other cells and it’s the virus’ key to cell entry. Antibodies can specifically bind “anywhere” on a protein (but only that one place) and the really valuable antibodies are the so-called “neutralizing antibodies” – these are just normal antibodies, there’s nothing really “unique” about them, they just happen to bind to the part of the virus that does the cell docking, hiding that part and preventing the virus from getting into cells. 

Scientists can test for these antibodies in plaque reduction neutralization testing (PRNT), where they put virus & antibodies onto a layer of non-infected cells in a dish and see if the virus infects those cells. If the antibodies are neutralizing, the virus can’t get in, so the cells survive. But if the antibodies are not neutralizing, the virus will infect cells and, since these cells are just in a dish (no immune system backup), those cells will die – and they’ll infect nearby cells, so you have whole regions of cells dying, leaving dead-cell zones called plaques. The “better” the neutralizing antibodies (either stronger antibodies or higher concentration of them) the fewer plaques will be seen. 

This PRNT is one of the early ways scientists can test if a vaccine shows promise – immunize a lab animal and test that animal’s blood to see if neutralizing antibodies are made. This is far from proof a vaccine is safe & effective, but it’s an important first step before further testing. But I’m getting ahead of myself. Now that we know *what* these vaccines are trying to do – introduce this S protein (or other viral parts) into a person’s body and get them to produce neutralizing antibodies – let me explain *how* they’re trying to do it.

Before I go into some details, a REALLY GREAT resource is this graphical guide https://go.nature.com/2Ssv9hs I used it a lot to help me understand and come up with my own guide of sorts. Note: everything is to the best of my knowledge as of 5/3/20 and there are a lot more players, but I only list 1 or 2 per category

THE “LOOK WHAT I MADE FOR YOU” GROUP

inactivated virus: this is basically exactly what it sounds like – scientists take the virus and heat it up really hot or add chemicals such as formaldehyde tor beta-propiolactone to “kill it” by fatally messing up its RNA & proteins (but no so much that the proteins become “unrecognizable” because you still need them to look legit enough to induce an immune response!

  • PROS: 
    • these can induce a strong immune response because (even though they’re dead) they clearly look like a foreign invader to the immune system
    • this technology has been safely and effectively used for a number of other viruses. For example, the hepatitis A & some flu vaccines use inactivated viruses
  • CONS:
    • it can be hard to find the right balance of dead but not disfigured, so a lot of trial and error is needed, testing different chemical concentrations, etc. 
    • you need a lot of live virus to start off with because, since the virus can’t make more copies of itself once you inject it, you need to inject a lot
  • PLAYERS:
    • Sinovac Biotech in Beijing is in human-testing of this sort of vaccine, as is Beijing Institute of Biological Products/Wuhan Institute of Biological Products

weakened virus (live attenuated virus): this is a version of the virus that’s still “alive” so it can infect cells, make copies of itself, etc. but it can’t make you sick. Scientists usually make such vaccines by haviing the virus pass through cells in a dish over and over and over. The virus has to keep copying itself, so it will inevitably pick up mutations. Scientists select for weakened viral strains and use them. 

  • PROS: 
    • these types of vaccines often provide a strong, lasting, immunity 
    • proven safe and effective for smallpox, chickenpox, rotavirus, and MMR (Measles, Mumps, and Rubella) 
  • CONS:
    • since the virus is still “alive,” this type of vaccine is “riskier”
  • PLAYERS:
    • Codagenix in Farmingdale, NY working with the Serum Institute of India – vaccine manufacturer in Pune

viral proteins (aka subunit vaccines): this is where scientists inject pre-made viral proteins (or parts of proteins), which have been grown in the lab “recombinantly” in expression cells in a flask (commonly special insect cells). The main candidates are the spike protein or even just the part of the spike protein that binds the cell’s ACE2 receptor – the Receptor Binding Domain.

  • PROS: 
    • similar vaccines against SARS (the original) worked in monkeys (but haven’t been tested in humans)
    • viral protein based vaccines have been tested, approved, and are currently use, for other viruses like shingles & hepatitis B
  • CONS: 
    • recombinantly-expressing proteins can be resource-intensive (speaking as a protein biochemist…) – the “recombination” part is fairly easy – you cut out the gene for the protein and “recombine” it with a piece of DNA optimized for protein making in the expression cells. Then you stick this into those cells, and let the cells make the protein, which you then purify. Depending on the protein, etc. the yield can be really low and taking care of the cells can be time consuming. Also, depending on the types of cells used, the proteins that are made might have different modifications and/or might not fold right 
    • since you’re only introducing a tiny bit of the virus, you might need to give your immune system more of a signal that this little protein you’re introducing is a real threat – these protein vaccines might require adjuvants (immune-stimulating molecular companions) and multiple doses. 
  • PLAYERS:
    • GSK & Sanofi are collaborating, each bringing what they do best: Sanofi making the spike protein and GSK making the adjuvant (the chemicals co-injected with the proteins to make them more “immunogenic” (i.e. get the immune system to see the proteins as foreign & start making antibodies against them))

virus-like particles (VLPs): these are basically “empty” viral particles. A coronavirus’ particle consists of a single strand of RNA containing the virus’ blueprint (i.e. the viral genome) coated with nucleocapsid proteins, surrounded by an oily (lipid) membrane embedded with proteins (those spike proteins and also membrane proteins & envelope proteins). VLPs are the protein-embedded membrane without the viral information inside. Without that viral information, there’s not really a virus – but it looks like there is.

  • PROS:
    • Gardasil – the HPV (Human Papilloma Virus) vaccine – works like this
  • CONS:
    • can be hard to make – scientists have to find the right balance of viral parts
  • PLAYERS:

THE “MAKE IT YOURSELF” GROUP

The instructions for making viral proteins are written in the virus’ RNA. And, 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 proteins grown “recombinantly,” the proteins your cells make are “perfect” – just like the real thing!

Problem is, RNA is really unstable. So you need a way to get the instructions safely into cells. One method is to sneak it in using a different (harmless) virus. This is the strategy used with viral vectors – they use a second, weakened, virus as a vehicle (vector) for getting a coronavirus protein recipe inside. 

viral vectors: One of the genes from the coronavirus is cut out of the coronavirus genome and pasted into the genome of a different virus that has been weakened so it can’t cause disease – such as measles or an adenovirus (a type of virus responsible for some colds). 

I’m going to go out of order and start with the players… 

PLAYERS: 

Oxford University’s Jenner Institute has a leg up on this because they had already developed a similar vaccine for related MERS (Middle Eastern Respiratory Syndrome) virus. They just had to swap out the MERS gene for the SARS-Cov-2 gene to makeChAdOx1 nCoV-19, so named because it uses a CHimpanzee ADenovirus to express the spike protein (S) of the Novel COronaVirus (discovered in 2019). This virus is non-replicating – one of the proteins it needs to replicate has been genetically deleted so the virus can’t make copies of itself once inside you (in order to make the vaccine they infect cells that have a copy of that missing gene and use those cells as virus-making factories). Speaking of factories, a number of companies have joined to help with production, including India’s Serum Institute and AstraZeneca. https://bit.ly/3d9RkRO 

This vaccine is considered a front-runner. It’s shown useful in Macaque monkeys & is currently in a large trial in the UK https://nyti.ms/2Wi8VQG  

But it’s not alone in the the viral vector route. Ad5-nCoV from CanSino also uses an adenovirus vector (but a human one), and also expresses the S protein. Like Oxford, they had a leg up because they already had an adenovirus-vector vaccine in Phase II trials, but for Ebola (Ad5-EBOV). https://bit.ly/3d9RkRO 

Johnson & Johnson (US-based) is also trying out this approach. 

Non-replicating viral vectors can be “safer” but you also need to inject more of it then if you let the injected person make more copies. So other companies are using replicating viral vectors, such as weakened measles virus. This is how the recently-approved Ebola vaccine works, and France’s Pasteur Institute is going this route: https://bit.ly/2WpzHGP 

PROS & CONS: 

These techniques sound awesome because you can just swap out the gene for any virus, right? Problem is, the body might develop an immunity for the vector as well as (and hopefully not instead of) for the virus. So if you give a person a second vaccine with the same vector, they might over-react and destroy the virus without getting a new immunity

DNA vaccines: instead of relying on a viral vector, these just introduce “bare” DNA (they use DNA instead of RNA because it’s more stable) often in the form of a circular piece of DNA called a plasmid. To convince the DNA to actually go inside the cells, they can use a technique called electroporation, where they inject DNA and then apply electric charge to open up temporary pores in cell membranes to allow DNA to slip inside. I’d only heard of electroporation being used in cells in a tube in the lab, but apparently it’s also done in living animals using electrode patches, etc.

  • PROS:
    • cells can make many mRNA copies from a single DNA copy, and each mRNA copy can be used over and over by the protein-making ribosomes, so you can get cells to make a lot of protein from them
  • CONS:
    • DNA vaccines have never been successfully used in humans before
  • PLAYERS:
    • Inovio Pharmaceuticals is doing a trial of this to express the S protein

This “final class,” mRNA vaccines, is the “simplest” (which gave it an early head start in the vaccine race). Like the DNA vaccines, this technique has never been successfully proven, but that hasn’t stopped it from getting a lot of buzz (and funding)

mRNA vaccines: When the DNA vaccine gets inside a cell, before it can make a viral protein from it, the cell has to make an RNA copy of it. This copy is called messenger RNA (mRNA) and it gets read by protein-making complexes called ribosomes. This also happens when our bodies want to make proteins of our own – an mRNA copy of our DNA genome is made (in a process called transcription) and protein made from it (in a process called translation). mRNA vaccines provide an RNA copy of the viral protein instructions (properly “formatted” with a 5’ cap and 3’ poly-A tail for those geeks like me out there…). Instead of using electroporation, these vaccines are often encased in oily membranes as Lipid NanoParticles (LNPs), which can get in through the cells’ own oily membrane. 

A key player here is Moderna, which is working with the NIH’s NIAID (National Institute of Allergy and Infectious Diseases) to develop and test mRNA-1273, instructions for the full-length spike protein (with a mutation that stabilizes the protein in the “prefusion” conformation – the “pose” the protein takes before it actually infects a cell. Moderna’s gearing up for a Phase II trial with 600 patients starting in May & Moderna signed a worldwide development agreement with the Swiss pharmaceutical manufacturer Lonza. https://bit.ly/3d9RkRO 

Most people probably hadn’t heard of Moderna before recently, but, as an RNA researcher, I had – I’ve even seen their workers presenting presenting research at conferences. I’m not saying this to like name-drop or anything, just to say that Moderna has been around (but only since 2010) and they’re known for work on mRNA therapeutics (we’re discussing vaccines now, but mRNA can also be (potentially) used to replace or supplement proteins) They have never brought a product to market or even to Phase III clinical trials. But the US government is really gung-ho about Moderna’s vaccine, and on April 16, the federal Biomedical Advanced Research and Development Authority (BARDA) gave them a big investment to help accelerate development & manufacturing https://cnn.it/2KYDbKQ 

Another company, BioNTech has deals with Fudan and Pfizer for mRNA vaccines. They’re trying a few different ones – a few have modified RNA letters thought to help make them  and a fourth is a “self-amplifying mRNA with has the gene for a replicase protein which makes more copies of the mRNA once inside the cell. https://bit.ly/3d9RkRO 

A benefit of DNA & mRNA vaccines (collectively referred to as nucleic acid vaccines) is that they’re a lot easier to make. But a MAJOR downside is that they’ve never actually been proven in humans. So there’s a lot less known about their efficacy than is the case for more traditional vaccines.

As far as I know at the time of writing (05/03/20) there are over 90 vaccines in development – here’s a link to the WHO’s list: https://bit.ly/2L5mrSB 

Sorry this post got really long – and there’s a lot more to say! So I plan to do a separate post on how vaccines are tested and some things scientists and doctors will be looking out for – including a rare phenomenon called antibody dependent enhancement, where certain types of antibodies can actually make it easier for a virus to get inside certain cells.  

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