I assume the use of Brewer's yeast and probiotic bacteria is to avoid any immune triggering by the VLP generating organism itself? I also assume that the VLPs will be purified away from any replication-competent expressing yeast/bacteria prior to being given to people (and hopefully away from the vector or gDNA that the VLP expression cassette is carried on)? Otherwise I think there would a risk that chronic VLP production in the gut could switch off any immune reaction against the VLP.
Ordinary beer yeast and yogurt bacteria don't persist in the human gut, but your point is well taken for the fancier boulardii and sakei probiotics I'm now thinking about. Those can at least sometimes persist for awhile in the gut, nose, or skin microbiome.
Fortunately, there are failsafes. In the yeast experiments, we temporarily induce VLP production with high concentrations of a common sugar called galactose. Then we break up (lyse) the yeast with a freeze-thaw and/or shaking with glass beads and/or a kitchen blender. Then we apply the broken up yeast lysate to the mouse's nose or skin. Oral delivery has so far been disappointingly ineffective, but there are a few more tricks we haven't yet tried. In the unlikely event that some yeast survive the lysis and try to set up shop in the microbiome, they would quickly stop producing VLPs because there wouldn't be enough galactose around.
For yogurt bacteria, we're developing bacterial viruses (phages) carrying a gene that drives VLP production. It's a lytic phage, so there's even less chance of persistence in the yogurt approach.
One thing to note is that the VLPs we're using are an especially potent type of vaccine antigen:
The VLPs can bind unique sugars on the surface of human cells, so there's an aspect of the story where the immune system is actively sorting the VLP away from all the other stuff in the yeast extract.
Although I don't see any need for purification, if the need were to arise we could theoretically use kitchen-friendly one-step gel filtration:
I have deep respect the view that it's important for us to game out the risks. I hope it doesn't stir up a whole separate discussion, I'll note that I've personally begun to reluctantly accept the high plausibility of the hypothesis that SARS-CoV-2 is a synthetic sequence that inadvertently escaped from Wuhan Institute of Virology. But the sobering example of the lab leak hypothesis involved known-known risks. There's also risk in declining explore new ways of fighting pathogens simply because we're afraid of some sort of vague unknown-unknown Henderson Monster
It takes money and effort to develop a pathway (e.g. CRISPR-Cas, exonuclease) for genome degradation, but once you've developed it it would be cheap to activate it. The rationale is just being as safe as possible with respect to escape. With a side effect of protecting your proprietary IP. You don't have to stop *any* research, just engineer in an additional control.
"To examine the effects of type III and NucC activation on bacterial and plasmid DNA in vivo, gDNA integrity and plasmid loss were examined over time after induction of plasmid targeting. Genomic DNA was degraded and plasmid DNA was purged from the wild-type host in less than 30 minutes post-induction compared with untargeted and repressed controls (Figures 3D and S3A). In contrast, genomic and plasmid DNA degradation was not detectable in the absence of NucC (Figures 3D and S3A)."
As stated above, I'm primarily concerned about the possibility of any ongoing VLP expression in the gut and the effect of this on the host human. But there are other issues if horizontal gene transmission occurs and your VLP machinery (or parts thereof) transfer to other people. The general public is *very* leery of vaccine transmission, or threats thereof. It would ease their concerns if you've already engineered in multiple ways to prevent that.
Engineering yeast to express recombinant nucleases sounds vaguely *more* scary than yeast carrying the type of standard expression plasmids that have demonstrably never turned into Henderson monsters for the past 50 years.
And proprietary IP? Anathema! The germ of this idea occurred to me while watching people swap sourdough cultures early in the pandemic. The deeper dream here is that if the system works then somebody could theoretically grow up some vaccine beer, induce some for themselves, and send a little side-culture along to their grandma. I guess the dream might sound vaguely scary, but it ought to be weighed against the concrete scariness of uncontrolled Covid turning the leader of the free world into a zombie. There's danger and then there's danger.
I've read anti-vaxxer and anti-vaxxer adjacent commentary where people are fearful of plant-based vaccines being secretly put into salad greens.
Unlike the VLPs, it's not like these nucleases aren't already present in the environment or in our guts (e.g. I-SceI, a host of Cas enzymes).
A Henderson problem is one thing, a chronic immune trigger is something else. Has anyone even bothered to check for horizontal transmission and maintenance of a plasmid in the gut? This might be more likely to happen if you use auxotrophic selection, as the additional gene-provided nucleotide, tRNA, or amino acid might actually be helpful for the uptaking organism in the modern human digestive system.
Most people never have an airbag deploy. We still require them in cars. I'm really just interested in scientists preparing ahead to reduce escape. We've done enough screwing around with ecology. And like our current model organisms, once you have a strain that inductively autophages, it's simple to keep using it as a base platform far into the future.
I assume the use of Brewer's yeast and probiotic bacteria is to avoid any immune triggering by the VLP generating organism itself? I also assume that the VLPs will be purified away from any replication-competent expressing yeast/bacteria prior to being given to people (and hopefully away from the vector or gDNA that the VLP expression cassette is carried on)? Otherwise I think there would a risk that chronic VLP production in the gut could switch off any immune reaction against the VLP.
Ordinary beer yeast and yogurt bacteria don't persist in the human gut, but your point is well taken for the fancier boulardii and sakei probiotics I'm now thinking about. Those can at least sometimes persist for awhile in the gut, nose, or skin microbiome.
Fortunately, there are failsafes. In the yeast experiments, we temporarily induce VLP production with high concentrations of a common sugar called galactose. Then we break up (lyse) the yeast with a freeze-thaw and/or shaking with glass beads and/or a kitchen blender. Then we apply the broken up yeast lysate to the mouse's nose or skin. Oral delivery has so far been disappointingly ineffective, but there are a few more tricks we haven't yet tried. In the unlikely event that some yeast survive the lysis and try to set up shop in the microbiome, they would quickly stop producing VLPs because there wouldn't be enough galactose around.
For yogurt bacteria, we're developing bacterial viruses (phages) carrying a gene that drives VLP production. It's a lytic phage, so there's even less chance of persistence in the yogurt approach.
One thing to note is that the VLPs we're using are an especially potent type of vaccine antigen:
https://cbuck.substack.com/p/extraordinary-evidence-requires-extraordinary
The VLPs can bind unique sugars on the surface of human cells, so there's an aspect of the story where the immune system is actively sorting the VLP away from all the other stuff in the yeast extract.
Although I don't see any need for purification, if the need were to arise we could theoretically use kitchen-friendly one-step gel filtration:
https://ccrod.cancer.gov/confluence/display/LCOTF/GelFiltration
Might want to autodigest the genomes following induction while you're at it, just to be on the safe side for horizontal gene transmission.
These are all remote issues, but if this becomes prevalent then you'll be dealing with hundreds of millions to billions of replicates per year.
I have deep respect the view that it's important for us to game out the risks. I hope it doesn't stir up a whole separate discussion, I'll note that I've personally begun to reluctantly accept the high plausibility of the hypothesis that SARS-CoV-2 is a synthetic sequence that inadvertently escaped from Wuhan Institute of Virology. But the sobering example of the lab leak hypothesis involved known-known risks. There's also risk in declining explore new ways of fighting pathogens simply because we're afraid of some sort of vague unknown-unknown Henderson Monster
https://youtu.be/PUsYNtLSfx8?si=cs6BToAxUEyZvP2H&t=4794
I don't autodigest genomes in beer and yogurt - I'd need a more concrete rationale for doing it for vaccine-beer and vaccine-yogurt.
It takes money and effort to develop a pathway (e.g. CRISPR-Cas, exonuclease) for genome degradation, but once you've developed it it would be cheap to activate it. The rationale is just being as safe as possible with respect to escape. With a side effect of protecting your proprietary IP. You don't have to stop *any* research, just engineer in an additional control.
https://www.sciencedirect.com/science/article/pii/S0092867421002415
"Elevated rates of horizontal gene transfer in the industrialized human microbiome"
And you wouldn't have to do a lot of research to develop the degradation pathway: https://www.cell.com/molecular-cell/pdf/S1097-2765(22)01056-5.pdf
"To examine the effects of type III and NucC activation on bacterial and plasmid DNA in vivo, gDNA integrity and plasmid loss were examined over time after induction of plasmid targeting. Genomic DNA was degraded and plasmid DNA was purged from the wild-type host in less than 30 minutes post-induction compared with untargeted and repressed controls (Figures 3D and S3A). In contrast, genomic and plasmid DNA degradation was not detectable in the absence of NucC (Figures 3D and S3A)."
As stated above, I'm primarily concerned about the possibility of any ongoing VLP expression in the gut and the effect of this on the host human. But there are other issues if horizontal gene transmission occurs and your VLP machinery (or parts thereof) transfer to other people. The general public is *very* leery of vaccine transmission, or threats thereof. It would ease their concerns if you've already engineered in multiple ways to prevent that.
Engineering yeast to express recombinant nucleases sounds vaguely *more* scary than yeast carrying the type of standard expression plasmids that have demonstrably never turned into Henderson monsters for the past 50 years.
And proprietary IP? Anathema! The germ of this idea occurred to me while watching people swap sourdough cultures early in the pandemic. The deeper dream here is that if the system works then somebody could theoretically grow up some vaccine beer, induce some for themselves, and send a little side-culture along to their grandma. I guess the dream might sound vaguely scary, but it ought to be weighed against the concrete scariness of uncontrolled Covid turning the leader of the free world into a zombie. There's danger and then there's danger.
I've read anti-vaxxer and anti-vaxxer adjacent commentary where people are fearful of plant-based vaccines being secretly put into salad greens.
Unlike the VLPs, it's not like these nucleases aren't already present in the environment or in our guts (e.g. I-SceI, a host of Cas enzymes).
A Henderson problem is one thing, a chronic immune trigger is something else. Has anyone even bothered to check for horizontal transmission and maintenance of a plasmid in the gut? This might be more likely to happen if you use auxotrophic selection, as the additional gene-provided nucleotide, tRNA, or amino acid might actually be helpful for the uptaking organism in the modern human digestive system.
Most people never have an airbag deploy. We still require them in cars. I'm really just interested in scientists preparing ahead to reduce escape. We've done enough screwing around with ecology. And like our current model organisms, once you have a strain that inductively autophages, it's simple to keep using it as a base platform far into the future.