How safe is lysed E.coli as an adjuvant?

[Michael Flynn]

Often in vaccine development, in addition to target antigens that you want to raise antibodies against, an adjuvant, something that stimulates the immune system like lipopolysaccharide, must be added in order to generate an adaptive immune response against the antigen. E.coli is covered with lipopolysaccharide, which reacts strongly to complement (antibacterial factors in blood), and so would be a good candidate for an adjuvant. This line of thinking leads to an "easy vaccine" where you express the antigen in E.coli and thermally lyse the cells, dilute them to some concentration in water for injection, and there you have your vaccine. In an outbreak like coronavirus, making vaccines like this would seem to be "low risk, high reward" in that nothing bad seems likely to come of it, but you could potentially immunize against this bad outbreak.

Anyone have any actual literature on this?

[Dakota Hamill]

I'm no immunologist but I think that's how you'd give yourself toxic shock syndrome. 

On Fri, Mar 6, 2020 at 9:21 PM Michael Flynn <mfly...@gmail.com> wrote:

Often in vaccine development, in addition to target antigens that you want to raise antibodies against, an adjuvant, something that stimulates the immune system like lipopolysaccharide, must be added in order to generate an adaptive immune response against the antigen. E.coli is covered with lipopolysaccharide, which reacts strongly to complement (antibacterial factors in blood), and so would be a good candidate for an adjuvant. This line of thinking leads to an "easy vaccine" where you express the antigen in E.coli and thermally lyse the cells, dilute them to some concentration in water for injection, and there you have your vaccine. In an outbreak like coronavirus, making vaccines like this would seem to be "low risk, high reward" in that nothing bad seems likely to come of it, but you could potentially immunize against this bad outbreak.

Anyone have any actual literature on this?

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[Cathal Garvey]

LPS is a bit too much for use as an adjuvent, I would think. And standardising the dose would be super important and super hard at DIY scale.

Generally I would say DIY is never injectable, and the skills and resources needed to do safe biomedicine are best acquired the traditional way.

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[Michael Flynn]

This doesn't seem to be a new or crazy idea. A couple reviews:

Bacterial ghosts as adjuvants: mechanisms and potential: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482964/

Bacterial Ghosts as antigen delivery vehicles: https://www.ncbi.nlm.nih.gov/pubmed/15878634/

On Friday, March 6, 2020 at 11:30:34 PM UTC-8, Cathal Garvey wrote:

LPS is a bit too much for use as an adjuvent, I would think. And standardising the dose would be super important and super hard at DIY scale.

Generally I would say DIY is never injectable, and the skills and resources needed to do safe biomedicine are best acquired the traditional way.

On 7 March 2020 04:32:31 GMT+00:00, Dakota Hamill <dko...@gmail.com> wrote:

I'm no immunologist but I think that's how you'd give yourself toxic shock syndrome. 

On Fri, Mar 6, 2020 at 9:21 PM Michael Flynn <mfly...@gmail.com> wrote:

Often in vaccine development, in addition to target antigens that you want to raise antibodies against, an adjuvant, something that stimulates the immune system like lipopolysaccharide, must be added in order to generate an adaptive immune response against the antigen. E.coli is covered with lipopolysaccharide, which reacts strongly to complement (antibacterial factors in blood), and so would be a good candidate for an adjuvant. This line of thinking leads to an "easy vaccine" where you express the antigen in E.coli and thermally lyse the cells, dilute them to some concentration in water for injection, and there you have your vaccine. In an outbreak like coronavirus, making vaccines like this would seem to be "low risk, high reward" in that nothing bad seems likely to come of it, but you could potentially immunize against this bad outbreak.

Anyone have any actual literature on this?

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[Michael Flynn]

Two E. coli examples:

Bacterial Ghosts as an Oral Vaccine: a Single Dose of Escherichia coli O157:H7 Bacterial Ghosts Protects Mice against Lethal Challenge: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1201255/

New strategies for combination vaccines based on the extended recombinant bacterial ghost system. https://www.ncbi.nlm.nih.gov/pubmed/10194817

[Cathal Garvey]

I'm not saying it's new or crazy. I'm saying it's inappropriate for DIY and the sweet spot for LPS between "adjuvent" and "lethal" is likely to be dangerously narrow. The margin for error with DIY-grade methods and equipment is probably far too large to permit it.

Not to mention that there are lots of other toll-activating goodies in dead bacteria, and people's severity of reaction to any given toll-activating antigen is probably strongly affected by genetics, so your dose response curve probably has variables outside your control or ability to factor.

So, again.. some things really belong in the pro-tier labs. Those labs should be more democratic, accountable, focused on public interest, and all.. but the basic idea that some experiments belong at that level of experience, resource, accountability, and group competence still seems sound to me.

[Andreas "Mega" Stuermer]

I think I read in a paper, long ago, that LPS contamination might contribute to DNA vaccine immunogenicity. 

I would assume that expressing the protein in coli is not ideal - you get so little protein vs so much LPS. It might just cause the innate immune system to go bonker. Our immune system is trained to respond to the tiniest ammounts of LPS. Also you will get a B-cell response this way, but not much T-cell response. De-novo synthesis of proteins and display of fragments thereof mediates ADCC activity and the lack thereof is (for example) the reason why Herpes vaccines have failed so far. 

I'm not often one to say this isn't DIY-ble but considering everything the danger seems very high compared to potential gains. Sure, given enough training you could turn your basement into a certified 50k$ lab that is suitable. May be easier to join a lab. 

As far as I can tell, live recombinant or live attenuated viruses are the new thing for effective vaccines - here they inserted an Ebola protein into a "harmless-to-human" virus that replicates https://www.nejm.org/doi/full/10.1056/NEJMoa1414216 

But then, there's a lot of regulation around that topic and it is not smart to play around with this in a DIY setting. 

Sometimes there's a reason why things are mostly done in academia.

[Michael Flynn]

For the record, I agree with you both that a random person shouldn't just try to "DIY it".

However, I think some of the risks you mentioned can be attacked. Humans are very sensitive to LPS, with a fatal dose ranging in 1 ug/kg, however people have survived (and ethics boards have cleared experiments using!) doses of 4 ng of *purified* LPS per kg, so around 140 ng of LPS (https://www.ncbi.nlm.nih.gov/pubmed/2835680/). Assuming a saturated culture is completely made up of E.coli, about .1 ul of that culture would come out to around 100 ng of E.coli and thus would be around that dose, assuming E.coli is entirely made up of LPS, which it is not. At the same time, .1 ul of saturated culture would probably contain enough E.coli to mount an adaptive response against. You could even dilute further down to .01 ul... if an e.coli is about 1 femtoliter then .01 ul still contains 10 million e.coli so...  seems like it would be enough. Also I'd like to reiterate that these would be dead E.coli that have been thermally lysed. 

Anyways, at that point you wouldn't be dying of LPS toxicity. And the idea would be to proceed by eliminating risks one by one until the risk/reward payoff is positive, even if the payoff is small (slight chance of becoming immune to virus that probably won't kill you). 

[Cathal Garvey]

Just to change direction here a bit, (because in principle I do want to see better knowledge-bases on how to do medicine far from privilege): what's wrong with using inorganic adjuvents, which probably have a safer dose/response curve, probably lower response variability, and might be easier to determine purity/sterility?

[Michael Flynn]

I would say the difficulty there would be purifying the antigen, in that case? You would need to express the antigen in large quantities and purify it. You would need some kind of kit.

[Michael Flynn]

Regarding generating a B cell response vs a T cell response - doesn't a B cell response create a T cell response? If I remember correctly, if an antigen binds to a B cell receptor, it gets endocytosed, proteolytically processed, and presented on the membrane to T cells. 

[Michael Flynn]

https://www.frontiersin.org/articles/10.3389/fimmu.2017.00319/full

[Zonie deep]

Hola it's very easy to highly purify expressed proteins using affinity tags and resins that bind the tag. And very easy to make relatively large quanities.
Express the a portion of the receptor binding domain of the spike protein. There's data on best epitopes.

[Michael Flynn]

Just to make a correction to myself from before, .1 ul E.coli would contain 100 ug of the bacterium, not 100 ng. You would want to dilute it 1:1000  before re-dilution for injection - yikes! Don't try at home folks!

[Zonie deep]

You express covid protein in E. Coli and highly purify it biochemically. You can get rid of almost all of the lysate ie lps this way. Some insulin preps etc for human use are made similarily. There are lots of adjuvant formulations. I thinks there's a standard one for most of the many vaccines that are now in use. Of course you need some expertise for a hacker vaccine to make sure it's sterile right dosage adjuvant. Some type of safety test.