edit: Changed title from "Postmortem on RatVac" for clarity.

Note: We named the vaccine candidate "RatVac" as a tongue-in-cheek abbreviation for "Rationalist Vaccine". We have no association with the RaDVaC project and use a much simpler, almost trivial approach. I don't endorse making your own vaccine or taking anything nerdy people on the Internet send you. This is not medical advice.

tldr: In April of 2021 I assembled my own subunit vaccine against the Beta variant of SARS-Cov-2 ("SARS2"). Despite starting with the prior that this formulation should be somewhat effective, I could not demonstrate efficacy against the Alpha variant in antibody tests.

Introduction

I started getting interested in DIY vaccines soon after the Making Vaccine and We got what's needed for COVID-19 vaccination completely wrong posts were published. Particularly the idea of just making a standard subunit vaccine appealed to me, so I teamed up with some interested people from the rationalist diaspora and we ended up making our own vaccine candidate in early to mid 2021. Some contributed advice, many contributed funding (for which I am still extremely grateful), and while the project wasn't the clear success I had hoped for, maybe the true treasures are the friends we gained and the lab equipment we bought along the way.

Recombinant Vaccine ELI5

Subunit vaccines contain a subset of a single protein molecule. For viruses this will typically be a protein that contains the receptor-binding-domain (RBD), i.e. the part that actually binds with the host during infection.

Through the wonders of genetic engineering - see very short introduction below - we can produce these proteins and then simply introduce them into the human body.

While Yang et al have shown that an immune response occurs even with a vaccine purely containing viral RBD, the response can be enhanced by adding an immunologic adjuvant. Adjuvants are a very diverse class of chemicals, ranging from simple mineral salts to proteins or even small cells. The only thing they ostensibly have in common is that they're used to increase or modulate immune response, and that's why we're using them.

Genetic Engineering ELI5

For those curious about how we can manufacture proteins to begin with - because I definitely was - here is a very short and abridged intro. The process differs depending on the method used, but usually roughly follows these steps:

• Encode and synthesize the amino acid sequence of the protein as DNA.
• Add this DNA to an expression vector. These are typically either viruses or circular strings of DNA called plasmids.
• Introduce your expression vector into an expression host, often bacterial, yeast or mammalian cells. This is called transfection and can take several forms, from chemical and viral transfection to just shooting cells with a "gene gun" that fires microscopic gold particles coated in DNA.
• Select for the transfected cells. This is usually done by adding a gene for antibiotic resistance to the expression vector, so that you can then kill all non-transfected cells by said antibiotic.
• Grow and maintain your culture until sufficient amounts of protein have been produced, then extract and purify the protein.

The details will vary depending on protocol and I'm deliberately sticking to the basics and leaving out things like CRISPR, RNA plasmids and plant hosts.

More important is imo that while this may sound extraordinarily complicated, it's very much doable by a single individual or small team in a self-funded lab. DNA can be synthesized for hundreds of dollars and often already shipped as a customized plasmid. Host cells and growth media are also affordable (at least at the mg-µg scale we're aiming for, don't try making your own burger meat), which leaves purification. I'm not too happy with any of the DIY methods I've seen so far, but a jury-rigged column chromatography setup is very much possible for $1000, with $7k-15k for something I'd personally want to use.

RatVac in theory

With the data dump out of the way, let's return to the vaccine project.

The first question we tried to answer was whether it even made sense to target the SARS2 RBD as a vaccine candidate. Similar approaches were suggested for the first SARS and showed some promise. Luckily, this seemed true for SARS2 as well: Yang et al found in animal studies (on mice and primates) that a recombinant vaccine not only led to the production of antibodies, but also neutralized live SARS2 virus. This was promising.

The second question concerned which variant to target. Alpha appeared to be suitable for a vaccine, but the Beta variant was on the rise and inoculation against Alpha didn't necessarily imply inoculation against Beta. So, what about the inverse scenario? Would people vaccinated against Beta still get Alpha at proportional rates?

The diagram below from a Cele et al. study paints an optimistic picture: Beta antibodies provide far greater cross-variant immunity than Alpha antibodies. The sample size (n=19) is a bit small, but given this information, SARS2 Beta makes the better vaccine target.

Lastly, we had to pick an adjuvant. Aluminum salt ("alum") was the easiest option, since it's easy to acquire and easy to sterilize. It's not ideal, since it's biased towards a T-helper 2 response and we'd prefer a balanced response, but that's not a critical concern as long as the magnitude of the immune response is at a satisfactory level.

With these concerns at least somewhat abated, we arrived at a vaccine candidate containing 50µg of Beta Variant RBD, for three doses of a bit over 15µg each.

RatVac in practice

Unfortunately, Germany has some of the strictest GMO restrictions and violations can carry fines of up to €50k. Since I don't have a licensed lab at my disposal, I decided to outsource the protein expression and just import the protein from a large pharmaceutical company. Since it's not toxic, this isn't particularly difficult. At least for the adjuvant I managed to avoid pharma prices by buying pure aluminum salts.

The protein was purified in a column chromatograph, all glassware, the adjuvant and the vials were washed in alcohol twice to remove any macroscopic contaminants and then sterilized in a pressure cooker. Assembly of the vaccine happened in a clean room that I managed to "borrow" from a friendly biologist. While I don't think we actually operated at ISO 5 / WHO Grade A, working this way was clearly preferable to working in a home lab (and risking giving myself a pollen allergy, given the season).

Each dose was given two weeks apart, which, in hindsight, was almost certainly too short of an interval to maximize the immune response.

Successful response to the vaccine would result in antibodies against the viral RBD, so I bought and used at-home blood antibody tests, which returned negative. Since I wasn't sure if the antibody tests did in fact test for antibodies against RBD rather than other proteins, I also booked an official antibody test, which couldn't detect antibodies in my blood, either.

Unfortunately I was struggling with over a year of Covid isolation at that point, so I decided to just take my employer's offer of a speedy vaccination in June, rather than running more tests on myself.

Conclusion

In conclusion, meh. I couldn't run as many tests as I would've liked and my data is inconclusive.

Of course, this isn't a condemnation of subunit vaccines. Considering how easy it is to produce this type of vaccine, I do think that the current system for vaccine development is somewhat broken. While having pharmaceutical companies responsible for funding their own clinical trials removes the incentive to drain public funds by testing as many drug candidates as humanly possible, it also introduces a) an incentive for fraud and b) a necessity for the resulting drug to be either comprehensively protected by IP laws or so difficult to produce that the risk of legal copycats is negligible. Imo this is highly problematic for several reasons, mainly because we, as a society, would want the kind of drug that relies on well-established technology and is manufactured very, very easily, so that production can be up- and downscaled at will. Subunit (and to some extent DNA) vaccines check both of these boxes and I'm somewhat disappointed to see no real open-source vaccine receive the kind of government funding that would have been necessary. It might still have failed, of course, but compared to all the costs incurred by Covid, the expense of running several Phase II and a few Phase III studies would have been trivial.