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.

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At least for the adjuvant I managed to avoid pharma prices by buying pure aluminum salts.

From having read what Stöcker wrote about his vaccine production it seems like the pharmaceutically sold aluminum salts are delivered in a form that clumps less together which in turn increases bioavailability (that's also why you aren't supposed to freeze them).  

Interesting, do you happen to have the original blog post on hand? It is possible to get other adjuvants as far as I know and I do also believe that on Stocker's blog he posted a link to a particular vendor as well.

(And as a matter of fact it seems you are the one who answered my question on LW with the post in which he recommended that vendor): Vorschlag für die LEGALE Herstellung eines (banalen) Peptid-Impfstoffes durch einen Arzt | Prof. Dr. Winfried Stöcker (winfried-stoecker.de)

That vendor seems to be a pharmaceutical vendor. In this post, he speaks about "Adiuvans-Mikrokristallen". 

That means it's very small crystals. If you look at table salt the size of the crystals you get when you buy it differs and that likely works the same for the aluminium salts.

Just wanted to thank you for trying this!

Minor note: would you be willing to change the name? two extremely similar projects with extremely similar names is asking for trouble.

+1, I started reading this because I thought it was about RadVac

Thanks!

Hm, I'm not opposed to it, but given that the project is dead and any future biohacking project I'll take on will get a different name anyway, I'm not sure if changing the name retroactively accomplishes anything. I doubt this experiment will have enough of a lasting impact to cause trouble (beyond the people who were confused by this post, for which I apologize).

edit: I've changed the title for now, that seems to accomplish most of what's needed.

Your approach seems very similar to Stocker's who did run a small "trial" in Germany before the authorities weren't happy with what he did. Based on your location I'm guessing you probably already heard about this. He actually posted the data for himself on his personal blog and you can find it all here, for those who aren't aware: 

The best vaccine against Covid-19 | Prof. Dr. Winfried Stöcker (winfried-stoecker.de)
Immunization against Covid 19 | Prof. Dr. Winfried Stöcker (winfried-stoecker.de)

Stocker happened to dose himself four times about 2 weeks apart, though he was able to see a positive response after a 2nd dose, unlike in your attempt. It also seems like you used a very similar adjuvant.

I am assuming you took your dose intramuscularly, is that correct? 

Yes, I originally planned to include a small section about Stöcker, but it seemed only tangentially related to the project itself and fuel for extensive political discussions.

tldr for the unintiated: Stöcker is the founder of Euroimmun, a company that makes lab chemicals and also happens to make Covid antibody tests. Through his contacts, he managed to get his hands on the spike protein DNA early and made his own recombinant vaccine candidate. He also gave this to several dozen volunteers and lab employees, which he argues falls under a loophole that allows doctors to use unlicensed medicine if no proper treatment exists. The PEI, a German medical regulatory body, however argues that this constitutes an unlicensed medical trial. He is AFAIK still facing a lawsuit, but is nonetheless occasionally vaccinating volunteers until the local police make him stop.

He was always somewhat nutty when it came to politics, though, and has gotten significantly worse during the pandemic, even reblogging "fan mail" about Bill Gates Covid Vaccine conspiracies, so I'm not sure how much faith I have in his data. It's another argument in favor of at least testing subunit vaccines, though, since his writing about the economics of protein production (i.e. supplying Germany many times over within months) is perfectly credible.

Another OT but curious side note: His audience appears to be predominantly anti-vax when it comes to the licensed vaccines yet extremely willing to take his vaccine candidate. This surprised me - sure, I had always assumed that a large aspect of conspiracy communities is opposing anything supported by the government, but not that the specifics really don't matter at all. Stöcker's vaccine has all the (faux) pitfalls the anti-vax community raves against - aluminum, untested, GMO, made with embryonic cells - yet none of this matters in the slightest as long as he's fighting The Man. Might make for an interesting post in its own right.

And yes, I took the vaccine IM with no noticeable side effects.

I see, thanks for doing this. I have been really interested in self-vaccination since the original RADVAC whitepaper came out, but I never really pulled the trigger on any method due to a lack of expertise in the area and (expected) expressions of concern from some people close to me. 

A few more Q's:

It does seem like doing the purification step may require or at least benefit from some level of lab experience, which I unfortunately don't have. How important do you think the purification step is for the safety of the final product (as taken IM)? If it really shouldn't be skipped, I may be better off with RADVAC.

I notice that a lot of RBD are sold with "(His-Tag)" at the back. Based on some cursory reading, it seems that this is for the purposes of making purification easier. But does this tag have to be removed to be used in a vaccine? Some comments on ResearchGate suggest the answer is no but I'm really not sure.

Secondly since it does seem like people who have been exposed to SARS-CoV-2, a vaccine, or even SARS-CoV-1 mount a stronger response when later given another vaccine for SARS-CoV-2, do you think that it might be possible that dosing the DIY one again (but say, with an Omicron RBD) after getting the commercial vaccines could demonstrate the efficacy of the DIY one if the titer from a commercial antibody test increases after injecting it?

Purification isn't necessary if you buy already purified protein; in my case it was just cheaper to get it in bulk and filter it myself.

Removing the his-tag reduces the low-ish risk of it interfering with the immune response, but not doing so doesn't strike me as dangerous, it's just a dangling chain of histidine after all (and biology doesn't quite work like Unsong, luckily).

As for using peptide vaccines as a booster, I'm mildly optimistic given the evidence. Boosting vector vaccines with mRNA seems stronger than vice-versa, but it's still better than only having n-1 vaccines. I could see the same being true for peptide vaccines.

FWIW, my first official and fourth overall vaccination pretty much knocked me out for two days, so perhaps my experiment wasn't entirely without effect.

Thanks for taking the time to answer. When you say purified protein, what's the standard for "pure enough"? I see some listings that say things like ">95% by SDS-PAGE".

Stöcker is the founder of Euroimmun, a company that makes lab chemicals and also happens to make Covid antibody tests. Through his contacts, he managed to get his hands on the spike protein DNA early and made his own recombinant vaccine candidate. 

To be exact, part of creating the Covid antibody tests was creating the spike protein domains so his company was already creating those for reasons unrelated to making the vaccine. 

That side note is interesting and seems worth expanding on.  Would seem to suggest that people aren't so much distrustful of vaccines and medicine as they are distrustful of anything the government wants to stick in their arm for free with penalties for non-compliance.  That might even be a reasonable heuristic if it wasn't the start and end of any reasoning done.  

It would also suggest that the way to get the last stuborn 10%/20%/30% of the population vaccinated is to have someone loudly critical of the government make and promote a different vaccine (maybe license and rebrand one from a different not too friendly country), then make people pay for it.  Bonus points if the establishment is seen objecting that you shouldn't go pay for that vaccine when the govt has perfectly good free ones.

Curated. For most of the Covid pandemic, I've heard Rationalists say that we should have (or to have) invested in making our vaccines given how slow release of the mainstream ones was. It's very cool that caffemacchiavelli actually worked on this! I wasn't aware of the project until now, so major kudos. I hope going forward more people follow suite. Also, cool to know that this project was inspired by other LessWrong posts–fantastic to see them having that impact.

Has your experience with this project given you any insights into bioterrorism risk?

Suppose that, rather than synthesizing a vaccine, you'd wanted to synthesize a new pandemic.  Would that have been remotely possible?  Do you think the current safeguards will be enough to prevent that sort of thing as the technology develops over the next decade or so?

Not really, was concerned about biological X-risks before and continue to be.

I don't currently see any plausible defense against them - even if we somehow got a sufficient number of nations to stop/moderate gain-of-function research and think twice about what information to publish, genetic engineering will continue to become easier and cheaper over time. As a result, I can see us temporarily offsetting the decline in minimum IQ*money*tech_level needed to destroy humanity but not stop it, and that's already in a geopolitically optimistic scenario.

Luckily there are some intimidatingly smart people working on the problem and I hope they can leverage the pandemic to get at least some of the funding the subject deserves.

If you know of someone working on a solution such that think we're lucky rather than doomed, I'm curious whose work gives you hope?

I'm pretty hopeless on the subject, not because it appears technically hard, but because the political economy of the coordination problem seems insurmountable. Many scientists seem highly opposed to the kinds of things that seem like they would naively be adequate to prevent the risk.

If I'm missing something, and smart people are on the job in a way that gives you hope, that would be happy news :-)

Hm, most of the people I'm thinking of are rather technical, e.g. Kevin Esvelt's research on distributed secure research.

Coordination and incentive problems are of another nature and I only manage to be prescriptively optimistic. I've been interested in algorithms for decentralized economic planning for a while, plan to specialize in that area and am working with a local left-acc group to organize a think tank that works on these questions. Thanks to mechanism design taking off as a discipline and crypto hype fueling a lot of work on trustless computing, there's actually a surprising amount of relevant research.

I can respect consciously prescriptive optimism <3

(I'd personally be more respectful to someone who was strong and sane enough to carry out a relatively simple plan to put dangerous mad scientists in Safety Level 5 facilities while they do their research behind a causal buffer (and also put rogue scientists permanently in jail if they do dangerous research outside of an SL5)... though I could also respect someone who found an obviously better path than this. I'm not committed to this, its just that when I grind out the math I don't see much hope for any other option.)

If you want to synthesize a new pandemic you would need to know what proteins to add. That's very hard. It's much easier to 

It seems the South African's for example put older variants together in the lab with antibodies against the spike protein to test how soon it evolves to get immune evasion. That's the kind of research with the potential to produce new pandemics waves like Omicron. 

How big of a subunit were you able to get?  Last I looked at mail-order dna, the affordable stuff was only a few hundred bases.

Only about 300 bases as well. I remember a study on SARS1 that showed higher immune response to an RBD vaccine than a full S1 subunit vaccine in mice. And while I usually trust studies on mice as far as I can throw them, it served as a good enough excuse to be a cheapskate. (And after all, I can throw mice at least moderately far...)

Thanks for sharing this post-mortem!

I'm curious about the difference between the approach you described here and the DNA vaccines that were open sourced in Project MacAffee.  This was the one done as a course by Zayner and co.

My amateur take at the differences:

  • Different peptide/subunit target
  • Vaccine given as protein rather than plasmid to produce the protein

Oo, I wasn't even aware of that, thanks for the link!

That is a DNA vaccine, so it's more similar to the mRNA vaccines we have now in that it contains genetic data of the virus that is then built by the body itself. This one seems to contain the entire S and N proteins, not just a subunit of the S protein.

DNA vaccines are more complicated than recombinant vaccines to get right and can cause serious damage if done wrong. That and the fact that the more complex a project, the more likely I'm going to procrastinate and let it die, made me stick with the simpler recombinant approach.

At least the ELISA approach to antibody testing is one I could have borrowed, though, and in hindsight I'm a bit disappointed I didn't think of it myself.

Does a similarly-structured resource to the ones I linked exist for recombinant vaccines?  I'd be curious for learning more about what would be needed to do this kind of DIY-ish project in the future.

Not as far as I know, butThought Emporium on Youtube has a lot of tutorial videos on genetic engineering. (FWIW, Stöcker himself failed to express the protein in bacteria and iirc used CHO instead. I don't see any intrinsic reason why E.Coli shouldn't work, but I'd probably use HEK or CHO myself given the choice)

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