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RNA Vaccines And Their Lipids

So now that people (not enough of them!) are getting vaccinated in the US with the Pfizer/BioNTech and Moderna mRNA vaccines, let’s talk about some more details of what are in those injections and what happens once the shot is given. The workings of an mRNA vaccine touch on a lot of different cellular processes and a lot of drug-delivery issues, so we can Talk Corona while also talking drug discovery, biology, and chemistry at the same time. I want to start off by recommending this piece by Bert Hubert on the workings of the Pfizer/BioNTech vaccine – Bert goes into a lot of detail that I’m going to run through rather quickly in the next few paragraphs, and you’re probably going to have a better shot understanding it from him than you do from me!)

One theme that will show up many times in this post is that these vaccines were not invented from scratch. There’s a long list of things that had to be worked on in order for the field to be in the shape it was in at the beginning of 2020, and that’s why things ran so quickly. “RNA as a therapeutic agent” is an idea that has had billions of dollars of work poured into it over the last twenty or thirty years, so when you hear about these vaccines as something new, remember that’s only for certain definitions of “new”.

As all the world knows, these vaccines are based on messenger RNA (mRNA). That, of course, is the type that’s produced in a living cell by reading off a given stretch of DNA and assembling the matching RNA, after which it goes off on its own to be fed into a ribosome which will assemble proteins based on its code, reading off by three “codon” letters at a time. So messenger RNA has its feet in both worlds, if it had feet: it’s down there in the nucleus being put together next to an exposed and unwound strand of DNA, but afterwards it’s also present right in the middle of the ribosome machinery, as amino acids get brought in and spliced together into a growing protein strand. Genetic information gets turned into proteins (there’s the Central Dogma of molecular biology for you), and mRNA is how that happens.

Now, the specialists in the room will appreciate the huge number of details that go into both those processes. The concepts are pretty straightforward (read off DNA to make mRNA, read off mRNA to make protein), but the execution is something else again. It’s worth going into those in a little detail to explain why the mRNAs in the vaccines look the way that they do, and why designing a good one is a lot harder than it looks.

As a new mRNA strand is generated by the action of the RNA polymerase II machinery on a stretch of DNA, it gets a “cap” attached to the end that’s coming out from the DNA (the “5-prime” end), a special nucleotide (7-methylguanosine) that’s used just for that purpose. But don’t get the idea that the new mRNA strand is just waving in the nucleoplasmic breeze – at all points, the developing mRNA is associated with a whole mound of specialized RNA-binding proteins that keep it from balling up on itself like a long strand of packing tape, which is what it would certainly end up doing otherwise.

So the 5-prime end is capped, and then the other one (the “3-prime” end) undergoes some processing of its own. It has a certain number of residues scissored right back off, and then a stretch of “poly-A” (one adenosine residue after another) is added on – these processes are done by another big complex of enzymatic and scaffolding proteins working on that end of the molecule. By the time that’s finished, an mRNA can have a couple of hundred A residues tailing off its 3-prime end. This doesn’t get turned into protein, though – otherwise every protein that gets made would come out of the ribosome with a long tail of lysines on it, since the “AAA” codon under other circumstances means “Lys” to the translation machinery.

Then there’s another key step. In most organisms, the DNA doesn’t just read off the uninterrupted code for a whole protein. It has interruptions of other stretches of code (“introns”), and at this point those are clipped out and the actual mRNAs are spliced together by assembling their pieces (the “exons”) into their final form. That may seem like a rather weird process if you haven’t run into it, and it certainly was a surprise when it was discovered back in the late 1970s. This is done by yet another Death-Star-sized mass of proteins, the “spliceosome”, and it provides opportunities for “splice variants” along the way that will produce different proteins when a ribosome gets ahold of them. And that’s a big reason why we have a lot more different proteins in our bodies than we have different genes: many of them can be mixed-and-matched into these different variants back at the mRNA level.

I mentioned the poly-A tail, but there are also key regions at both the 5-prime and the 3-prime ends of an mRNA strand that also don’t get translated into protein. These contain important regulatory information for how that translation should go. There are “start” and “stop” codons that aren’t always associated with any particular amino acid (the start codon can also code for methionine, depending on the context), but rather convey those instructions to the ribosomes. The “leader” sequence at the beginning of the mRNA and sections at the other end as well can have profound effects on how readily it gets taken up by any given ribosome and how efficiently it moves through. Ribosomes themselves have at least two ways to feed an mRNA into their protein-making machinery: the normal way, which requires a “capped” mRNA and an “internal ribosome entry site” (IRES) that doesn’t care, and the use of these is also mediated by the untranslated RNA regions. It goes on and on! The last 30 or 40 years of biology have seen these details brought to light through vast amounts of effort in the lab (and similarly vast amounts of staring out windows trying to sort out mentally what’s going on), and that process is nowhere near at an end.

I’ve rambled on about all this to bring us back to the mRNA vaccines. You can see from that quick tour of the machinery that it would be a bit too hopeful just to produce a plain stretch of RNA that codes for the viral Spike protein and expect that to work right off the bat. No, you’re going to have to optimize both ends of it so that ribosomes are enthusiastic about it and zip right down the strand producing that Spike for you. (And remember, the vaccines we have are also producing a variation of the Spike that keeps it stable in its final active shape, the better to have antibodies recognizing that, so you’re not even coding for the “native” Spike from the very beginning).

And as you’ll know if you’ve read that article from Bert Hubert that I linked to at the beginning, the mRNA vaccines also feature a good deal more such engineering. The three-letter codons for amino acids have some redundancies in them, but not all of those are processed with the same alacrity. Ones that are heavier in C and G residues seem to be run through more efficiently, so the sequences are biased that way. There are also the modified bases like pseudouridine/1-methylpseudouridine that get read off at the ribosome like their native cousins (in this case, good ol’ uridine, U) but make the mRNA strand both more stable and less likely to set off an immune response against itself. So the sequences in the vaccines have human fingerprints all over them – see Bert’s article for more.

But all that engineering availeth one not if the mRNA doesn’t make it to the cells and inside the cells. And that takes us to the formulations, which are another essential part of the whole mRNA vaccine story. Cell and molecular biologists tend to think of RNA molecules in general as pretty fragile things, and that reputation has been earned. They’re intrinsically less stable than the corresponding DNA molecules, and the odds are further stacked against them in the body by our own immune system’s defenses against foreign RNAs from pathogens like the current coronavirus. Just for starters, there are plenty of “RNA-ase” enzymes out there ready to tear any wandering RNAs to bits – the body can use circulating RNA molecules as signals, but these things are under tight control. So if you just inject a naked RNA sequence into someone’s blood, it’ll get stripped down to nothing before it’s traveled very far.

What are your alternatives for a more suitably clothed RNA? Well, as mentioned earlier, mRNA vaccines are not a new idea, nor is the idea of therapeutic RNA in general (remember siRNA?). So there’s been a lot of work over the years to find suitable carriers (see this 2016 review for an overview). It was not obvious which of these possibilities (lipids, carrier proteins, synthetic polymers, and more) would work out, of course. The only way to find out was (and is) to spend the time, spend the money, and go run the experiments. One thing that many of these ideas have in common is the carrier molecules having numerous positive charges on them, though, because RNA (and DNA) have lots of negatively-charged phosphate groups, and these would match up together to form a stable complex. Results from those experiments have tended to elevate the idea of lipid nanoparticles as a carrier, because they can help out in two ways simultaneously: they protect the mRNA construct itself as it travels through the bloodstream, and they seem to help it cross cell membranes and get from the blood into its destination. That’s not something you can just assume is going to happen on its own.

That point deserves a quick elaboration, because one thing that you have likely noticed is that there’s been a lot more work during this pandemic on RNA vaccines as opposed to DNA ones, even though DNA has that stability advantage mentioned above. There are several reasons for that, but one big one is that an RNA payload just has to get into the cell to encounter its site of action (the ribosomes, which are all over the place). A DNA therapeutic, though, has to get into the nucleus to do anything, and that’s yet another membrane to cross (and one with its own set of properties and gatekeepers). There’s also the possibility for a DNA species to get mistakenly incorporated into a cell’s own genome, which for a vaccine you don’t want (as opposed to a gene therapy), and using RNA completely takes that off the table, but the “just get into the cytosol” advantage is a real one, too.

So what are these lipid formulations like? They’ve been investigated for many years themselves, because these sorts of carrier properties could of course be useful for a lot of other therapeutic agents beside RNA. Here’s a short article at STAT about them. There are a lot of variations on the lipid idea, and one kind involves a sort of spherical bubble of lipid (a liposome) – generally a bilayer, as with our own cell membranes, because lipid molecule just naturally stack up like this, with greasy interior layers and the polar parts facing the solvent on the outside (see above, illustration by SuperManu via Wikipedia). In this case, the “hydrophilic head” will tend to incorporate some sort of positively charged group (as mentioned above). The payload will be in that little blue area in the middle, safe and secure as it drifts along. The lipid nanoparticles being used now are more of a solid lump, with the RNA and the lipids mixed together into tiny masses. The cell membrane is largely made of phospholipid bilayer, with the outside hydrophilic part being negatively charged, so these positively charged nanoparticles have all the more reason to stick to them.

When that happens, it appears that endocytosis kicks in, the general process of importing larger particles into a cell. There are several varieties of endocytosis, but they tend to end up with the external particle emerging on the other side of the cell membrane wrapped in a new endosomal vesicle of its own (can’t be too careful, from a cellular perspective). A well-chosen lipid nanoparticle formulation can actually help the RNA payload escape such an endosomal compartment and finally make it into the cytosol itself, ready for action.

Now we get into a forest of picky details. There is also no way to be sure from first principles which of the many, many, many possible lipid nanoformulations is going to work out the best for carrying therapeutic mRNAs. Small amounts of various other lipid species present in the bilayer can affect their properties a great deal, so you have a lot of experimentation to do and lessons to learn, and years of work have already been spent on just that sort of thing. For example, one broad lesson has been that nanoparticles formed from lipids that have permanently charged head groups (like quaternary amines) don’t seem to perform as well as ones made from amines that are charged by having ionizable H atoms on them. You don’t want to have to discover all this on your own at the same time you’re working out the details of the RNA construct, so therapeutic development has almost invariably been through partnerships.

The Pfizer/BioNTech vaccine uses lipid nanoparticles developed by the Canadian company Acuitas, who have (under one name or another!) been working in this area for over a decade now, trying out countless variations on various lipid combinations. Back then, it was mostly for siRNA delivery, but the lessons learned from that work have been invaluable for mRNA vaccine delivery. Meanwhile, Moderna has been involved in a vigorous and long-running patent dispute with a smaller company called Arbutus, who have also been investigating lipid nanoparticle formulations and whose technology Moderna once licensed. Arbutus has been claiming that Moderna’s research programs (and indeed their now-launched vaccine) avail themselves of Arbutus’ intellectual property, while Moderna (naturally) disputes this with equal vigor. I Am Not a Patent Attorney, and a damn good thing, too, so I have no useful opinion about who’s in the right. If Arbutus has a case, I would expect them to eventually get a judgement giving them some royalties off the Moderna vaccine, but my only solid prediction is that a number of lawyers will have steady employment thanks to this issue for some time to come.

A closer look at the Pfizer/BioNTech vaccine shows that it has four lipid components, two of which appear to be proprietary to Acuitas. One of these is ALC-0315, and the other is ALC-0159. You’ll note that both of those are tertiary amines (protonated to a positive charge under physiological conditions) and not quaternary charged ones, for the reasons mentioned above. The other two lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC), which is a well-known phosphotidylcholine lipid (as evidenced by the number of references in that link) and cholesterol, which is rather better-known still. These four components are of course present in a specific ratio, which I would rather not try to exfoliate out of the patent filings. But that should give you some idea of what’s in a formulation like this and what the lipids themselves look like. The physical process by which you reliably prepare such nanoparticles is another thing that needs experimentation, of course, but they’re cranking out the vials as we speak.

So that’s a look under the hood, and as promised, there’s a lot in there. It’s all the more remarkable that these therapeutics came together as quickly as they did, but if it had not been for the years of prep work in all of these areas, we would still be waiting!

177 comments on “RNA Vaccines And Their Lipids”

  1. Alex Beribisky says:

    Hi Derek,

    While the “stop” codons are indeed not associated with any amino acids, the “start” codon is associated with the amino acid methionine.

    1. Paula Thompson says:

      Yes, but. There are a handful of interesting exceptions where there are different start and xstop codons.

    2. Googulator says:

      In fact, even the stop codon (specifically, the “UGA” one) can code for something other than “stop”: selenocysteine. This works using a “suffix” at the end of the mRNA, specifying how many selenocysteines are to be added. Subsequently, when a stop codon is hit, a selenocysteine is translated, until the specified number has been added. Then, on the next stop codon, translation is stopped.

  2. An Old Chemist says:

    Please correct me if I am wrong: Any peptide with a methionine residue is not long lived as methionine gets oxidized quickly and is susceptible to racemization.

    1. Alex Beribisky says:

      The N-terminal methionine is often cleaved as part of post-translational processing. That’s how you get diversity at the N-terminal end. But if a methionine occurs internally, it can remain stable for a while, as it can be buried in a protein’s secondary structure.

  3. DTI says:

    Thank you! That’s a wonderful explanation. My daughter and her boyfriend are both graduating microbiology majors and over the (socially-distanced) holidays they waxed enthusiastically about how much work has already been done on both tempering mRNA and tailoring lipids for delivery. They might be interviewing for PhD programs but I’m certainly not. Your posts make it possible for me to at least follow their conversations. Thanks again!

  4. ccm says:

    Nice post. One small thing: ‘There are “start” and “stop” codons that aren’t associated with any particular amino acid…’ The start codon is definitely associated with methionine, even if that methionine is often modified or removed in the final protein.

    1. Derek Lowe says:

      I’ll make a note of that in the post – thanks, you’re right!

      1. Derek,
        Thank you immensely for this thorough explication on the mRNA vaccines’ lipid nanoparticles, a subject I’ve been striving to get a handle on, but this also ties into another conundrum with the vaccines that’s been raising great concern for me and lately, most of medical and biomedical colleagues: What exactly is the tissue tropism and cellular localization of these nanoparticles, and could this pose a side effect threat through errant MHC-I-mediated autoimmunity? Are they crossing the blood-brain barrier? Entering the parenchyma of vital organs (that even SARS-CoV-2 can’t access) and expressing the coronavirus spike protein on MHC-1 molecules, making these critical tissues targets of cytotoxic attack?

        Briefly, a major safety checkbox for the mRNA vaccine modality in general is the tissue localization of the lipid nanoparticle vehicles, for which you’ve done a wonderful job of explaining the composition of, since they are mechanistically fundamentally different from all other vaccines here. Most vaccines (what I called Type 1, e.g. subunit and inactivated virus) are essentially “MHC-2 only,” get taken up by dendritic cell phagocytosis and presented to CD4 T-cells in lymph nodes, so little to no CD8-mediated cytotoxicity against presenting cells. Attenuated virus vaccines like MMR (“Type 2”) do have some cytotoxicity mediated by CD8 lymphocytes attacking target cells presenting viral epitopes on MHC-1 cell-surface molecules, *but still limited to the tropism of the wild-type virus*. That’s where the lipid nanoparticles make mRNA vaccines elementally different from both, with a much broader tropism as far as I can tell. Even if they’re not omnitropic, it seems that they can enter a much broader tissue range compared to even attenuated virus vaccines, an impression confirmed from what you’ve written here. And since the mRNA vaccines would induce SARS-CoV-2 viral spike protein expression, that seems to mean that people who get the mRNA vaccines are going to have a much greater range of cells and tissues vulnerable to cytotoxic attack, since they’d be expressing the spike protein on MHC-I molecules. While this may prove to be more immunostimulatory, it also seems to indicate that the mRNA vaccines pose a much greater risk of systemic and critical tissue and organ damage than other vaccines, especially if multiple booster shots are needed, with side effects that may not manifest for years (with cumulative damage and chronic inflammation).

        This is where the picture gets aggravatingly murky, since it seems that neither Pfizer nor Moderna has posted up much of anything in the peer-reviewed literature about cellular and tissue localization from what many of us can tell, and this doesn’t seem to show up in the regulatory documents either, even though it would seem to be THE critical safety question for any proposed COVID-19 mass vaccination campaign. For the animal studies, it’d be as simple as using the companies’ nanoparticles to package GFP or equivalent indicator proteins, and seeing which tissues they go to and express the gene product. But there seems to be precious little published on this, and again from your descriptions here, it seems those tissue localizations would be quite broad. There have been published studies on LNP formulations in the past with varying tropisms but again, from what I can tell, the specific formulations in the actual vaccines are hard to get a handle on — perhaps because they’re considered trade secrets, and Pfizer and Moderna are worried about the other stealing the formula? — and even more crucially, we don’t know where in the body they’re going (through endocytosis, which leads to an MHC-1 expression pathway for viral antigens). And therefore, which tissues the vaccines’ mRNA payload is thus expressing the SARS-CoV-2 spike protein in, inviting cytotoxic attack.

        The nightmare scenario would be if e.g. the mRNA vaccines’ lipid nanoparticles are, indeed, crossing the BBB and getting endocytosed into critical glial cells, like oligodendrocytes, or even worse, into neurons themselves in the brain and spinal cord, putting a bullseye on these critical cells for cytotoxic CD8 lymphocytes. If so, we’d be setting the stage for a rash of multiple sclerosis and ALS-type clinical scenarios down the road with multiple boosters. My old medical colleagues have been getting especially concerned about this possibility, and I think this may be behind the recent sharp plunge in willingness among more and more healthcare workers to take the mRNA vaccines. in the absence of long-term safety or efficacy data, which is an unfortunate shortcoming given the pandemic’s urgency, we can only go with fragmented hints here and there about potential downstream issues, so we need a wealth of information with full transparency to make up for that shortcoming. And I think the recent reports of some severe adverse effects in the VAERS (esp. neurological issues, or the vaccinated Ob-Gyn physician in Miami dying suddenly of ITP that from early reports, seems to have been triggered by the vaccine) are causing cold feet among doctors and healthcare workers, esp. in the absence of tissue localization information. I used to work in gene therapy and recall how we’d obsess on tissue tropism for our vectors before considering clinical trials, so I’m bewildered that this information seems almost absent for an almost entirely new vaccine modality (for mass vaccination of healthy populations) here — it would go a long way toward reassuring fears and increasing uptake of the mRNA vaccines, both among the general public and medical professionals.

        As a corollary to your topic and excellent explanations here, do you happen to have information about the tissue tropism of the mRNA vaccines, specifically where the nanoparticles are going? If you’d like to do a Blog post on this, I’ll be enthusiastic to forward it on to my ex-colleagues, as there’s a massive question mark about this very issue that’s halting further vaccine adoption for a huge number of people. With better information about precisely where the mRNA vaccines are going, hopefully the concerns of health professionals especially can be reassured and boost confidence for the vaccine program.

        1. For further reference on these specific concerns in case helpful, here’s the link to my Rapid Response last month in BMJ (clickable from my comment name since looks like we can’t provide links within the comment text body). It lays out the mechanistic considerations regarding the mRNA vaccines’ tissue tropism and cellular localization, and a more detailed rundown of how precisely their cellular uptake, and the immunological processing of the SARS-CoV-2 viral epitopes expressed from the mRNA payload, would differ fundamentally from all previous vaccine modalities. Hopefully this won’t prove to be a factor in provoking toxicity, and would be enthused to hear any detailed rundowns from anyone in the know to shed light on the tissue tropism question!

          1. The vaccines are injected intramuscularly. Blood brain barrier or organ targeting are a non-issue because this route of administration is not systemic. The vaccine is primarily working in the draining lymphatics of the injection site. When working on gene therapy with e.g. AAV, tropism is very important because the viruses are often administered systemically and rely on the inherent or engineered tropism of the virus to induce expression in desired tissues. Trafficking studies have been done. One that is quite similar to what you describe above is linked in my name. One thing that may help in your search is to use terms like trafficking or localization instead of tropism, as tropism as a term is more used for cell subsets which viruses preferentially infect/replicate in, while those other two terms are more often used for non-viral delivery systems.

          2. There are a lot of unknowns with this platform. I have been trying for a while to have my comments considered for publication. The furthest I got with it was that one of the top immunology journals helped me edit it to fit their requirements, and after all the efforts of trimming down they decided to drop it because the “climate is not appropriate”. Here it is, uploaded to Preprints: https://www.preprints.org/manuscript/202012.0493/v2 (or by clicking on my name).

          3. Marko says:

            “…they decided to drop it because the “climate is not appropriate” ”

            I wonder if the CDC made a similar calculation. This was on an earlier version of a CDC page describing how mRNA vaccines work :

            “COVID-19 mRNA vaccines are given in the upper arm muscle. Once the instructions (mRNA) are inside the muscle cells, the cells use them to make the protein piece.”

            This is on the current version :

            “COVID-19 mRNA vaccines are given in the upper arm muscle. Once the instructions (mRNA) are inside the immune cells, the cells use them to make the protein piece.”

            https://www.cdc.gov/coronavirus/2019-ncov/vaccines/different-vaccines/mrna.html

            Maybe when the climate is appropriate they’ll tell us the whole truth, but with the CDC’s record on flip-flopping on all things coronavirus, how would we ever know?

          4. KN says:

            EMA (European Medicines Agency) has published online its Public Assessment Reports for all three approved vaccines in Europe – Comirnaty (Pfizer vaccine), Moderna and Astra Zeneca’s. There is some information in them regarding the issue of tissue tropism.
            Could you comment on viral vector vaccines having or not such issue?

          5. M says:

            https://pubmed.ncbi.nlm.nih.gov/33872783/

            Read! First postmortem study conducted on fully vaccinated man…looks like that spike protein was detected in all organs they sampled.

        2. S says:

          Good information. Yes, a recent paper has been published in Nature Neuroscience entitled “The S1 protein of SARS-CoV-2 crosses the blood–brain barrier in mice” to uncover its ability to cross Blood Brain Barrier. Which tissues will mRNA vaccine target exactly? A little alarming situation, this. Would be better if more information by vaccine companies are provided rather than getting anxious on trade secrets.

          1. S,
            Thanks for apprising me of that paper. It helps to illuminate and builds upon many of the concerns I’ve been hearing among colleagues about potential tissue trafficking and localization. If indeed the spike protein components (as translated from the transduced vaccine mRNA) are expressed at high enough levels, this seems to raise additional questions about the potential for the gene product to cross the blood-brain barrier through adsorptive transcytosis even if expressed outside of it. This on top of the still-murky picture about whether the nanoparticles themselves can cross the BBB (or other critical tissue compartments) and enter glial cells or neurons outright, leading to expression of the cytotoxicity-inducing spike protein in complex with MHC-I on the surface of sensitive cells.

            Cole J. Batty (sorry can’t respond directly due to comment nesting, the commenting software doesn’t have a “Reply” option by your comment):
            Many thanks for providing the link to that paper. I’ve been reading as much as I can about the biochemistry and in vivo behavior of the various lipid nanoparticle formulations, and this does shed a good deal of light. However, I have to raise questions about one of the statements in your post: “The vaccines are injected intramuscularly. Blood brain barrier or organ targeting are a non-issue because this route of administration is not systemic.”

            This strikes me as questionable because intramuscular injection very much does lead to systemic circulation of the injected payload. In fact one of the first things we learn in med school is that IM injection is favored precisely because of the substantial vascularization of large muscles, which carries the vaccine material systemically. From the link (with my name): “Intramuscular injection is the method of installing medications into the depth of the bulk of specifically selected muscles. The basis of this process is that the bulky muscles have good vascularity, and therefore the injected drug quickly reaches the systemic circulation.”

            Although I agree that the vaccine’s lipid nanoparticles would likely tend to collect extensively in local lymphatics, I’ve never heard of an IM injection that’s restricted exclusively to the injection site or nearby lymphatics alone. Every time I’ve been in the clinic, IM (esp. the deltoid) has been chosen precisely because the injected material inevitably goes systemic, through which it could reach peripheral tissues, vital organs, and the blood-brain barrier. It’s still unclear of course if the vaccine is reaching the BBB, or endocytosing within glial cells or neurons beyond it, and your linked paper does help to illuminate some of the issues with trafficking and cellular localization. Still, the most challenging aspect of following the lipid nanoparticle literature is that the LNPs’ chemistry and in vivo behavior can vary so substantially depending on the specific formulation. And if the vaccine manufacturers are invoking trade secrets as a basis for withholding further details about their LNPs (or allowing independent labs to test tissue localization with reporter genes), this leaves the public with a big doughnut hole in critical knowledge about the vaccines’ in vivo behavior.

            Even very recent related literature on trafficking other LNPs can’t answer this question for us, since again the different lipid recipes vary so much in their chemistries and in vivo localization that we won’t know which tissues the vaccines themselves are entering. We need to have precise information regarding where exactly *the vaccine manufacturers’ specific LNPs* are going, and where the SARS-CoV-2 S protein is expressed, since a non-selective tissue localization–especially if traversing the BBB–could lead to marked cytotoxic attack on MHC-I-spike protein complexes, and thus significant cumulative tissue damage, in a range of targets potentially well beyond the localization of the wild-type virus. If anything, the wide population cohort of intended vaccine recipients, coupled with the compressed timeframe and lack of long-term efficacy and safety data, makes it all the more important to acquire such data instead of leaving such questions to chance. Even more so given the rate of adverse events and even deaths that are cropping up lately in the VAERS database logs.

          2. Marko says:

            JW,

            We’re led to believe the LNPs are taken up by the muscle cells around the injection site, and spike expression occurs therein within hours of the injection. While I have no problem believing that’s mostly true, I doubt that it’s 100% true. The question then becomes how much – 20% , 1% , .01% ? – escapes into the systemic circulation.

            I believe the number of RNA payloads per dose is on the order several billion, so some unknown fraction of that constitutes the number of potential off-target cells that could be reached and subsequently destroyed. If that number is randomly distributed throughout the body, it’s probably not a big deal, but if there is some significant “tropism” to specific nervous system tissues, for example, it might be.

            More transparency on this would indeed be welcome.

          3. I agree it was overly dismissive to call this a non issue. Rather it is not the issue that it would be for a systemic administration as in the case of the gene therapies you referenced. Regarding the administration of drugs intramuscularly due to dense vascularization, that seems as though it was written with small molecule or non-particulate therapeutics in mind. Particulate antigens with diverse physicochemical properties have been shown to primarily drain through lymphatics or be taken up by phagocytic cells upon i.m. administration. They are a different beast from the drugs that gave rise to the principles disseminated in med school. My name links another relevant study. That being said, I am absolutely all for transparency and I believe trafficking studies should be performed and any data collected in this regard by Pfizer or Moderna should be disseminated to the public.

        3. Derek Lowe says:

          The tissue selectivity is a good question – my immediate assumption would be that these intramuscular doses are mostly going to end up in the lymphatic system, and any that makes it into general circulation would hit the local vascular endothelium and the hepatocytes, if they make it that far. But I’ll dig into it!

          1. Thanks Derek, a lot of us are intensively curious about anything you could find! I too had hoped off the bat that the local lymphatics would be the principal gathering site for the LNP-borne vaccine material, but in just going through my old medical school textbooks and reference lists, everything I’ve found has been pointing to one of the mantras we were taught in Year 1: An intramuscular injection (esp. in the deltoids) inevitably attains substantial systemic circulation, due to the rich vascularization of the injected tissue, and may well bypass the liver and kidneys to reach the blood-brain barrier in high volume. To expand on the quote in that resource (linked above in the reply to S and Cole’s comments), “The basis of this process is that the bulky muscles have good vascularity, and therefore the injected drug quickly reaches the systemic circulation … bypassing the first-pass metabolism.”
            So my back-of-the-napkin tracing of the circulatory route for a deltoid IM vaccine injection is roughly:

            local venules/capillaries –> cephalic vein –> axillary vein –> subclavian vein –> brachiocephalic vein –> superior vena cava –> right atrium of heart –> RV –> lungs –> LA –> left ventricle –> ascending aorta –> systemic circulation

            And thus from the aorta into the viscera and inferior peripheral tissues (through descending thoracic and abdominal aorta), and superiorly into the neck and head, including the brain’s blood supply through the common carotid arteries and the spinal cord (branching off from the vertebral artery in the neck). Just boiling it down — it seems from first examination that there might be considerable direct mRNA vaccine delivery to central nervous system tissues via the first couple passes, bypassing most first-pass metabolism. So then the question arises of whether the vaccines’ lipid nanoparticles would be able to traverse the BBB once at the doorstep and if so, whether they’d endocytose into oligodendrocytes and CNS neurons to express the spike protein on MHC-I complexes, or e.g. into cranial nerve (via the external carotid artery) and other PNS neurons (which could perhaps explain the Bell’s Palsy findings if these are indeed beyond the background population rate — and so on for e.g. GI and other visceral tissues.) Your excellent detailing of the LNP chemistry and adsorptive mechanisms as laid out above seems to indicate this is possible mechanistically, so from there it’s about whatever empirical data is lurking about the granular behavior of the LNPs at the BBB and other tissue barriers, which I’ve been at a loss to find. Huge thanks in advance for anything you can dig up, for all of us inquiring minds out there!

          2. Ștefan Talpalaru says:

            > my immediate assumption would be that these intramuscular doses are mostly going to end up in the lymphatic system, and any that makes it into general circulation would hit the local vascular endothelium and the hepatocytes, if they make it that far

            They do, and they get past the liver. Let’s look at some studies:

            “Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses” (2017):

            “Given this innovative vaccine platform, we examined the bio-distribution of the mRNA vaccines for both routes of administration. Male CD-1 mice received 6mg formulated H10 mRNA either IM or ID. Following IM administration, the maximum concentration (Cmax) of the injection site muscle was 5,680 ng/mL, and the level declined with an estimated t1/2 of 18.8 hr (Table 1). Proximal lymph nodes had the second highest concentration at 2,120 ng/mL (tmax of 8 hr with a relatively long t1/2 of 25.4 hr), suggesting that H10 mRNA distributes from the injection site to systemic circulation through the lymphatic system. The spleen and liver had a mean Cmaxof86.9 ng/mL (area under the curve [AUC]0–264of 2,270 ng.hr/mL)and 47.2 ng/mL (AUC0–264of 276 ng.hr/mL), respectively. In the remaining tissues and plasma, H10 mRNA was found at 100- to1,000-fold lower levels.”

            “A potent branched-tail lipid nanoparticle enables multiplexed mRNA delivery and gene editing in vivo” (2020):

            “IM injections yielded expression at the injection site (68%), liver (12%), and kidneys (11%). ”

            “Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes” (2015):

            “When mRNA-LNPs were injected intramuscularly and intratracheally, similar to intravenous and intraperitoneal deliveries, a large portion of the luciferase activity was detectable in the liver, demonstrating systemic spread of the nanoparticles. Also similar to intravenous and intraperitoneal deliveries, the high levels of protein produced in the liver occurred over a short duration with the majority of translation ceasing at day 2 post-injection (Fig. 2). Interestingly, significant bioluminescent signal could be measured in the lungs and muscles, as well, with the latter lasting for up to 8 days post injection.”

        4. cynical1 says:

          If what you say is true, the Pfizer vaccine generated a more robust CD8 T cell response versus the Moderna vaccine which was mostly CD4. One would expect to see a difference if and when booster shots start being administered. Also, would you not also expect that enough of a CD8 T cell response was generated from the first dose of the Pfizer vaccine such that you would trigger such a demyelinating event from the second dose of vaccine and we would have seen this already assuming it crosses the BBB? And if these vaccines do penetrate the BBB, then one would expect a plethora of cell types to take up the mRNA and release Spike protein initiating the T-cell response and massive inflammation. I don’t see a reason why basically everyone would not start developing a MS or ALS type (and other) symptoms on the first booster shot if it is indeed a risk. Another question is that if we are still making robust T-cell response to the Spike protein, why do we need a booster? Alternatively, maybe our booster a year or two from now will have to be a deactivated or attenuated virus or we could even, god forbid, use a small molecule antiviral to take care of the virus when we get infected.

          The big risk from your hypothesis would be natural infection where the virus crosses the BBB. Now that would be ugly: an already primed T-cell response ready to invade the CNS. With that said, T-cell response to the CNS basically always happens whenever we are infected with anything. The CNS is not the immunoprivileged place that many were taught to believe. The T-cells dissipate after a few days if there is nothing there. It’s when there is something there where all hell breaks loose.

          1. Cynical,
            All interesting questions. As far as the pathophysiology of MS and ALS, there are still some missing puzzle pieces, but the classically accepted pattern is an insidious onset followed by progressive exacerbation that eventually breaches the threshold for clinical significance. Thus an “MS-like” or “ALS-like” presentation (the latter a broad-brushed description of progressive UMN and LMN loss, since neither familial nor sporadic ALS per se has an autoimmune etiology) likely wouldn’t present in an “acute fulminant” form, but involve cumulative damage and repeated cycles of inflammation that eventually break through into clinical visibility.

            For MS, for instance, the classical model involves an extended “clinically silent” period in which subclinical damage to the affected white matter tracts and oligodendrocytes, from autoreactive T-cells, accumulates over many years prior to clinical presentation, with gradual deterioration after symptoms become clinically manifest — either in the form of PPMS (primary progressive), or RRMS (relapsing-remitting) that may progress to SPMS (secondary progressive). Drilling down a bit, multiple sclerosis is often heralded by worsening episodes of conspicuous but nonspecific manifestations like vision disturbances (most commonly), facial paralysis (often indistinguishable from Bell’s palsy, which is a PNS disease), ataxia, weakness, Lhermitte’s sign, or mood shifts, and there’s a lot of evidence to support the insidious and cumulative model of its pathophysiology whereby demyelination has occurred for many years below the clinical threshold by the time these indications become fully apparent. Without deep-diving into it, the best indication for this is the presence of radiologically significant plaques and other lesions on MRI (strongly associated with symptomatically diagnosed MS) incidentally in patients long before a diagnosis is suspected from specific symptoms per se, and the predictive value of such lesions as a prognostic hallmark of a likely subsequent MS diagnosis [e.g. see Nakamura et al. (2014), DOI: 10.7224/1537-2073.2013-016 ]. There are obviously many etiological uncertainties even with classical PPMS, and there may be a variety of “initial hits” that kick off the process of oligodendrocyte loss and demyelination — which may in turn be coupled with a heterogeneous array of “secondary” or “tertiary” hits that advance it further. In any case, once started, it appears to culminate in a similar final common pathway that only becomes apparent after many years, long after the initial physiological insult that triggers the first stages of demyelination, and often for reasons (such as nonspecific inflammation in a damaged tissue corridor) that may differ from the initial physiological insult.

            Thus if some early oligodendrocyte loss (or upper/lower motor neuron damage) is occurring, it’s not clear that it would present with an MS or ALS-like clinical picture offhand (or perhaps some of the neurological adverse events being reported in the VAERS might be precursors), but may percolate for years as a progressive yet subclinical pathophysiological process prior to symptomatic breakthrough, set off by the initial insult. Of course, it is also possible that wild-type SARS-CoV-2 could itself engender such pathology, and there have been studies examining its potential to disrupt the BBB and infect neurons or glial cells (both of which may indeed involve the S protein itself), with reported neurological findings in some patients. However, SARS-CoV-2 as a rule doesn’t appear to breach the tight junctions of the BBB in most cases presenting with a positive nasal swab, and the biochemical constitutions of SARS-CoV-2 virions and lipid nanoparticles are quite different, so we couldn’t readily conclude that a putative capacity for a given LNP subtype to traverse the BBB en masse would imply the same for the wild-type virus. Without knowing more about the specific LNP formulations and their cellular and tissue trafficking patterns, we just can’t say much of anything with certainty.

          2. Debra says:

            As a lay person, can anyone here help me understand the potential impact of the mRNA vaccine on those who carry the C9 mutation for ALS but are presymptomatic? I am a C9 carrier and wonder (worry) about the mRNA vaccine possibly triggering ALS.

          3. Q says:

            I’m not a physician, but I am aware that mistakes are made by people: if the needle misses and hits some kind of blood vessel, does most of this dialogue apply? I assume that the answer is “the vessels in the muscle are small”, but if there is a substantial danger from introducing the vaccine into larger vessels, like if the radio distracts a nurse in the town pharmacy, that ought to be known and advertised. But maybe it is already? A quick search, anyway, for accidental intravenous injection indicates that it is not unheard of.

        5. Olga Mayer says:

          Would this also be an issue with adenoviral vectored vaccines? e.g. Astra Zeneca, J&J

          1. mr j peacocke says:

            DOCTOR JOHN CAMPBELL ,regular YouTube star, says he has evidence that rna injected into blood vessels “consistently” causes thrombocytopaenia with clotting in mice.
            NHS instructions to vaccinators is not to aspirate when jabbing.
            Could there be a link between mysterious occasional sinus clots,etc; and the risk posed by not checking for a strike on a vein?

        6. Doug H MD says:

          This would seem to argue against your hypothesis:
          https://www.nature.com/articles/s41551-019-0399-y/figures/1

          1. Marko says:

            I’m not so sure it does. They equate “vaccine” with “mRNA and/or antigen”, which is not the topic of discussion. The question is, which cells pick up the LNPs after administration, rather than what happens to the mRNA/antigen produced by those cells after those target cells have been destroyed by cytotoxic cells.

            The paper highlighted in your link…

            https://www.nature.com/articles/s41551-019-0378-3

            …had this to say :

            “A question that remains to be answered is the degree to which protein expression in the muscle is a determining factor in subsequent antigen-specific immune responses.”

            In other words, they didn’t investigate what happens in the muscle or other cells that take up the LNPs, instead only looking at the fate of the mRNA and the antigen subsequently produced.

          2. Marko says:

            Actually, the full paragraph is more revealing. They did observe antigen production in muscle cells. To me , this sounds like “Step 1” in the vaccine response. Recruitment of mRNAs and Ag to the LNs sounds like “Step 2″ :

            A question that remains to be answered is the degree to which protein expression in the muscle is a determining factor in subsequent antigen-specific immune responses. Producing an innate immune response in the muscle is beneficial due to the potent production of chemokines and cytokines that recruit immune cells, but it is possible that the primary consequence of this in terms of vaccine efficacy is increased antigen transport to LNs where antigen presentation to T helper cells and costimulatory molecule activation occurs. In this study, we observed prME expression in the muscle and general recruitment of APC immune cells to the injection site. In the LNs, professional APCs were the primary cell types containing labelled mRNA.”

        7. Jen Lum says:

          Hi Dr. Ulm,

          I really appreciate your comments and questions. I’m 37 years old and 22 weeks pregnant with my third child. I do not work on the front lines. In fact, I work from home and so does my husband. We have a nanny who comes in the house a few times per week and she also watches children for another family several times each week. My daughter attends pre-school on 3 half days per week where the children are fully masked except for 30 minutes during lunch time. We make every effort to keep our circle very tight, but there are still some loose ends.

          I signed up online to be put in a que for a vaccination appointment because based on what I had been reading about the mRNA type vaccines, I felt comfortable getting it. I also follow several pregnancy forums where I’ve seen thread after thread of pregnant physicians discussing their choice to get vaccinated. Consequently, it was on one of these threads that I read a comment by a pregnant biochemist who mentioned her concerns regarding the vaccines based on the same questions you have posed here about the BBB. She questioned why certain very simple histological studies were not performed when Moderna released a study of mRNA technology in 2017 that showed “the presence of the construct all over the body, including the brain” quoted from her post). This is what lead me to do a search online and I came across this article. I’m confident my search terms pulled this particular article because of the key language in your comments/questions.

          What I’m getting at here is this… if you were me, would you get vaccinated? I’m not asking for your medical advice. I’m just asking you to put yourself in my shoes and tell me what your choice would be for yourself.

          **I’m hoping Dr. Ulm sees this comment, but I’d also like to open this question up to any of the MDs, PHDs and generally brilliant people commenting on this thread. Thank you in advance.

          1. Ger Dempsey says:

            Jen Lum,
            Unfortunately I am neither an MD, a PhD holder nor a generally brilliant person but I have been following the work of Dr JW Ulm and other medics and scientists on this topic, particularly on the critical issue of NLP systemic trafficking/carrying and tissue localisation.

            To help you with your decision, might I suggest you follow the data from the CDC Vaccine Adverse Events Reporting System(VAERS) database which collates information on all adverse events from vaccine doses distributed thus far. I believe the database is updated and published fortnightly.
            https://vaers.hhs.gov/data/datasets.html?
            In particular, have a look at the CSV File (VAERS Symptoms)
            Note the references to Placenta praevia, Premature separation of placenta and Premature delivery

            From what I have read so far, the Pfizer vaccine is not recommended during pregnancy unless there is an over-riding risk to NOT taking it, eg a significant underlying health condition or the pregnant person is at significant high risk of contracting severe CoVid 19, in which case the balance of risks must be weighed up in consultation with your local physician and/or your OBGYN.

            The FDA published a briefing document titled “Vaccines and Related Biological Products” to its Advisory Committee on December 10, 2020 for the Pfizer-BioNTech COVID-19 Vaccine
            https://www.fda.gov/media/144245/download

            Page 42 of that document has a paragraph on Pregnancies and the trial participants.

            A New York Times article article titled “FDA may recommend against pregnant women getting Pfizer vaccine”
            December 11, 2020
            https://nypost.com/2020/12/11/fda-may-recommend-against-pregnant-women-getting-pfizer-vaccine/

            Good luck with your decision and good luck with your pregnancy.

          2. eddie s says:

            Remember the saying, “an apple a day keeps the doctor away?” There are so many brilliant people in this thread, and I thank them for their input! The key to living a health happy life lies in the foundation of a plant based diet, practicing healthy perspectives, spiritual development, decrease media input, and increase love input. Avoid taking this “experimental vaccine” live your life, muster courage, and congratulations!

        8. JJ says:

          I would like more info on these please sounds like you know what you are talking about here.

  5. Toni says:

    thank you very much for this explanation. Perhaps a naive question: but how is the vaccine-mRNA actually produced? Chemically or via a plasmide-DNA that is transcribed into mRNA?

    1. Wilhelm Cody says:

      As far as I know the RNA construct and the lipid-nanoparticles are produced chemically. the subunits for the RNA construct are also chemical products. It is possible that the phosphatidyl choline and cholesterol are produced by extraction from some organism.

      Supplies of at least some of the modified bases and special lipids are limited. One of the reasons Pfizer agreed to commit an additional 100 million doses to the USA this year was commitment through operation Warp Speed to have the federal government use the defense appropriations act to get more of these specialty chemicals made and distributed to Pfizer/BioNTech.

    2. Derek Lowe says:

      The RNA is made off a DNA template with purified in vitro enzymes – so we’ve hijacked the cellular machinery, but it’s not being done in actual cells: https://www.nature.com/articles/nrd.2017.243

      1. Wilhelm Cody says:

        Than you Derek and Mantis for the clarification. Not in cells but of cells.

    3. Mantis Toboggan says:

      I believe that the DNA precursor of the RNA is produced chemically and then it is converted to the RNA component by a cell free in vitro transcription reaction supplying modified bases. No idea how the IVT product gets packaged/purified, etc. There might be more info in patent lit. somewhere.

      A quote from an article below, “Lavish funding has allowed Moderna to set up production facilities that can manufacture more than 1000 new, made-to-order mRNA a month. (“Moderna has probably made more RNA by in vitro transcription than all of humankind ever,” quips Edward Miracco, a senior scientist on its process innovation team.)”
      https://www.sciencemag.org/news/2017/02/mysterious-2-billion-biotech-revealing-secrets-behind-its-new-drugs-and-vaccines

      1. Pall Thordarson says:

        I gave a lay-person’s overview of the whole process in this twitter thread here, using the Moderna patents to “decode” some of the key steps. There are probably some errors/omissions here but maybe this also helps as an overview of how mRNA is manufactured: https://twitter.com/PalliThordarson/status/1326339931078225920?s=19

        1. Mantis Toboggan says:

          That was a great summary! I particularly enjoyed this part, “Add to this that chemists and biologists have “complementary” misunderstanding on DNA and RNA synthesis. Some chemists think that all DNA and RNA is made by chemical synthesis. Many biologists think it is all done with the aid of cellular expression systems or enzymes. 6/35.”

          Thanks for sharing. I saw you shared a link for the moderna lipids patent. If you have other links handy for other parts of the process would you mind sharing them? If not I can find them.

          1. Mantis Toboggan says:

            Some parts of the summary don’t seem quite correct to me though, but I could definitely be wrong. I’m not sure why you included a diagram of RNA synthesis instead of DNA synthesis since we are talking about DNA synthesis and then a PCR reaction to combine several DNA fragments.

            You then described then DNA plasmid being replicated in bacteria (thread 11)after a transfection. I’ve never heard of anyone transfecting bacteria and the link you shared specifically referred to eukaryotic cells, which is the context I am familiar with transfection being used for. I don’t know why that step wouldn’t be a transformation, which is super common in molecular biology. You could use chemically competent or electrocompetent cells.

          2. Marko says:

            ” I’ve never heard of anyone transfecting bacteria”

            This resembles the “variant ≠ strain” pedantry so prevalent right now. Yes, he should have said “transforming”, but we all got the gist, so who cares?

          3. Mantis Toboggan says:

            The author of the twitter thread (if you read the whole thing) specifically said, “And correct me where I am wrong!” I’m not on twitter otherwise I would have put the comment there. He also acknowledged that there may be some errors, so I was offering my point of view.

            There is a fundamental difference between transfection and transformation. Yes both are processes of introducing foreign DNA, but bacterial membranes =/= to mammalian cell membranes, so the processes are entirely different.Transformation in chemically competent bacterial cells requires incubating cells with DNA and heatshocking. Transfections for mammalian cells use cationic lipids, etc (see the link that was shared in thread 11). Since Moderna is presumably using bacterial cells for plasmid maintenance, maybe some people are interested in how its actually done You can argue it’s semantics if you want, but I pointed out specifically why I thought the explanation was flawed because the link provided describes mammalian transfection.

          4. Marko says:

            Fair enough. I was overly critical, perhaps because I’ve been sensitized by the variant/strain battles. My apologies for jumping the gun.

          5. Mantis Toboggan says:

            All good. I’ve not waded into the strain vs variant discussions. I also understand that my comments were a but picky and most general audiences might not care about the technical differences between transformation and transfection.

  6. Cole J. Batty says:

    This is generally a good explanation, but it’s worth noting the difference between liposomes (which you pictured) and lipid nanoparticles (LNPs), which are the particle type used in the Pfizer and Moderna vaccines as well as other candidates. Formation of an ionic complex between the RNA and ionizable lipid causes precipitation of the particles as a solid nano-sized chunk of lipid and RNA which has its surface decorated with phospholipid heads and PEG, rather than a bilayer liposome with an aqueous core. While the dosage forms are similar in composition they do have different physical properties, like flexibility, which may result in different effects in the body. In addition, liposome manufacture can often be more challenging than manufacturing LNPs.

    1. Derek Lowe says:

      That’s a good point – I originally had liposomes split off, but lost that in an edit. I’ll go back and clarify!

  7. Christian says:

    Another great post – thank you very much for the information that you compile in such a nice way. And thank you for bringing together and nurturing the rational science-led community with all the high-quality posts over the years! A much needed counterpart to all the irrational things surfacing these days.

    Can anyone help me with the follow-up questions that arise from your story? In particular, “what [actually] happens once the shot is given”:
    What happens to the spike proteins once they are being put together? And how does the immune system learn to build antibodies against those? Is it sufficient that the spike proteins are presented on the human cell surface? Or does the process require lysis of vaccinated cells? In any of those cases, how does the immune system recognize the spike proteins as being foreign and worth generating an immune response, while the other cell surface proteins are not?
    It would be great if someone who knows the answers could explain here!

    1. Wilhelm Cody says:

      The full answers are complex but here is a simple summary that may still misrepresent the details…well simple compared to even an approximately accurate description.

      Many human cells, especially immune system cells, will take up particles by a process call endocytosis. Basically, after bumping into a particle the cell membrane will envelop it in a sphere of lipid and take that sphere inside the outer cell membrane. Once inside, the cell has systems to then burst that bubble,, releasing the RNA. The RNA has been designed to be recognized as a good mRNA and gets bound to a protein-producing particle: a ribosome. Many other proteins and other RNA’s combine to start reading off the sequence, producing a string of amino acids: the spike protein. As the first ribosome moves down the RNA, an additional ribosome binds to the empty end and so on until there is a string of ribosomes each with a partially produced spike protein. As each ribosome reaches the end, it falls off and the spike protein is released.

      The protein sequence allows the spike protein to get through the cellular membrane and be exposed on the outside of the cell membrane. The following description of the immune response is grossly over simplified but can help give an idea of what is going on. Derek has some earlier articles explaining the process in much better detail and accuracy: https://blogs.sciencemag.org/pipeline/archives/2020/07/07/more-on-t-cells-antibody-levels-and-our-ignorance.

      Some cells of the immune system might be able to bind to this protein. Each of this type of cell has a different surface binding protein. Many millions of binding agents have been spun off, each in a different cell, just hanging around until one binds to something and stimulates the immune response. Some of these may bind to the spike protein. Those that do trigger a series of complex responses that, hopefully, leads to immunity, including antibody production and cells that can kill other cells with a viral infection.

      Cells that produced a protein that could have bound to a human protein were filtered out during gestation in the womb so it is unusual for an immune cell to have a response to a human protein or other antigen. Warning: not impossible, as autoimmune diseases are a real concern. Many autoimmune diseases may be stimulated by some earlier infection.

      Note that from my perspective the immune system is a very complex net of possible interactions near the limit of efficacy versus toxicity. Infectious disease is a strong driver of evolution: lots of organisms and viruses want to use you as a way of making more of themselves and most of them are small. The immune system has evolved so you to live long enough to raise a child to the point they can have a child. However, lots of stuff can go wrong. Thus the concern for caution in tickling it with a vaccine.

      1. Christian says:

        Thank you, Wilhelm, that is very helpful. In fact, with the point about gestation in the womb, this all makes a lot more sense to me.
        Looking forward to see how our understanding of the limit of efficacy vs toxicity advances in the coming years…

  8. Leak says:

    “Bert Huber” could use an additional “t” at the end…

  9. Kent Matlack says:

    First paragraph: “…you’re probably going to have a better shot…” A wonderful pun.

    1. Mike S. says:

      Also “it would be a bit too hopeful just to produce a plain stretch of RNA that codes for the viral Spike protein and expect that to work right off the bat.” ISWYDT

  10. Mandark says:

    Ideally, the spike proteins should end up in specialized antigen-presenting cells, but is there anything preventing these lipid nanoparticles from entering regular cells in various tissues, leading to expression of the spike protein and eventually its presentation with MHC class I on the cell surface, marking the cells for destruction? Isn’t there any danger associated with it? A fuller version of this question was posed in this rapid response to a BMJ articicle and I haven’t seen an answer yet:
    https://www.bmj.com/content/371/bmj.m4037/rr-19

    1. Thomas says:

      Isn’t that what a viral infection does?
      I thought that would just be the mechanism – sacrifice some cells as if they are infected. The good part being that the vaccine doesn’t replicate, limiting the damage.

  11. LSD says:

    Very nice post!!
    A short remark, only one (and not two) of the Biontech/Pfizer lipids has a tertiary amine in the structure. The other, ALC-0159, has an amide bond instead and is not ionizeable.

    1. JW Ulm, MD, PhD says:

      Yes, very good catch.

  12. SirWired says:

    As somebody whose understanding of biochemistry is that of a well-read citizen who passed AP Chem 25 years ago, all I can say, after getting into that simplified explanation as to how protein synthesis works is: “How are we even *alive* with how complex that mess is? The fact that we can make *useful* heads or tails of this is a *bleep!*-ing miracle!”

    The more I learn, the more I feel like I’m not much better off than a caveman who has just figured out how this whole “fire” thing works. All this stuff makes my field of IT look not any more complicated than banging two rocks together to make some pretty sparks.

  13. chemist says:

    Isn’t the central issue with a lipid-encased mRNA vaccine that it will basically enter every cell type without specificity, thus causing massive off-target effects? There’s certainly no way to target these things specifically to cells infected with a virus.

    1. Marko says:

      If you’re trying to target your vaccine to cells that are already infected with CoV2, you’ve badly missed the boat.

      1. I think Chemist’s point is that the lipid nanoparticles may have too broad a tropism, far broader even than attenuated virus vaccines (which are still limited to the tropism of the wild-type virus), and thus could pose a uniquely high safety hazard due to cytotoxic attack on the broad cellular range that uptakes the LNPs. Since the LNPs would enter cells via endocytosis, the SARS-CoV-2 epitopes would be expressed on MHC-1 molecules, making them targets of cytotoxic CD8 lymphocytes, attacking a much greater range of cells than any previous vaccine modality. This is concerning in general, but it’s a nightmare scenario if the vaccines are crossing the blood-brain barrier and endocytosing into e.g. oligodendrocytes (multiple sclerosis risk) or motor neurons (which could possibly cause an ALS-type picture). No other vaccine has this broad tropism. That is THE major safety concern here — been working on submitting a comprehensive question to Derek about this very topic.

        1. Marko says:

          If I was 30 yrs old and healthy, I wouldn’t take the new vaccines in any case, at least until sufficient millions were vaccinated and sufficient time had past to truly evaluate safety, simply because, at that age, my risk from a CoV2 would be disappearingly small.

          For those in at risk categories, however, it’s a different calculus altogether. I’d weigh the risk of CoV2 destroying my brain via a stroke, if not by invading and destroying brain tissues directly, as far greater than any similar risk arising from nervous tissue tropism of the vaccine, simply because we’ve seen overwhelming evidence of the former, and very little of the latter, at least so far.

          Still, I think your point is well-taken, and this question needs to be evaluated post-rollout. I was only replying to the second, illogical part of chemist’s comment, not the first part.

          1. I agree, the risk-benefit calculation will vary substantially as you’ve noted. More granular risk stratification is going to be essential for any recommendations going forward, serially updated as we get more data year-by-year.

          2. chemist says:

            Sorry, I meant to say cells that WOULD be targeted by the coronavirus, not already infected.

        2. Toni says:

          Dear Mr. Ulm, thank you for your considerations on potential dangers of mRNA/LNP vaccines. Since I am not an immunologist, I have a question on the subject: Assuming that the recombinant spike protein is really expressed in a certain number of non-immune cells, it will only be a transient expression, ,won’t it? Would one therefore expect cytotoxic attacks – if at all – only with a 2nd or booster injection? Could this be a more serious problem when using self-replicating RNA constructs as vaccines (as in the case of Arcturus, for example)?

        3. dave says:

          How long will a cell (any cell) keep on presenting a protein fragment “of interest” to the immune system, if the mRNA responsible for it is only inside the cell an an import?

          Does any part of the immune system “decide” that a cell which has ONCE presented something bad should be recognized for all time as suspicious. In other words could there be continuing attacks against that cell?

          I ask as someone who knows very little of the details, obviously.

          1. Marko says:

            “Does any part of the immune system “decide” that a cell which has ONCE presented something bad should be recognized for all time as suspicious. In other words could there be continuing attacks against that cell?”

            Traitorous cells are executed. No second chance. Immune justice is swift, fair, and final. Even the elites aren’t spared.

        4. Deb says:

          Hi Dr. Ulm,
          I was reading one of your earlier posts on another site and am considering the Johnson & Johnson or AstraZeneca vaccines. But, will the genomes turn off? And what would this mean in terms of safety for those vaccines if the genomes don’t turn off? If Dr. Ulm isn’t available, I would appreciate input from any of this group of learned people. I had a serious and long-term reaction to to a medication in the past and am trying to do my due diligence on these vaccines. Thanks.

          1. Derek Lowe says:

            Yes, all of the vaccine effects in cells diminish over time – you’re not altering your own genome, and the proteins produced will eventually turn over and get degraded. The mRNA or adenovirus DNA introduced will also get demolished, on a time scale of days to weeks.

    2. Zoidberg says:

      Since this article was posted, the European Medicines Agency Assesment Reports for the Moderna and Pfizer vaccines have been published, which both find some biodistribution, including across the blood/brain barrier.

      The Moderna study used an intramuscular injection of the same LNP as the vaccine (but a different mRNA), and found that “low levels of mRNA could be detected in all examined tissues except the kidney [, which] included heart, lung, testis and also brain tissues, indicating that the mRNA/LNP platform crossed the blood/brain barrier, although to very low levels (2-4% of the plasma level)”, with rapid clearing from most tissues.

      I think the Pfizer results are consistent with that: with radiolabelled LNP they found that “distribution from the [IM] injection site to most tissues occurred”, but again disproportionately more to the liver.

      Full details can easily be found at the European Medicines Agency.

      1. B Duffy says:

        Worthwhile discussion. While mRNA vaccines are now known to be effective we do not yet know safety. Nobody appears to have tested if Phizer and Moderna LNPs deliver their payload to brain tissue. And, nobody apparently has looked for an immune response to brain tissue making spike protein.A billion people, and entire populations may be depending on this outcome.

        1. Doug H MD says:

          not sure what signal one would realistically be waiting for? Uptick in various and sundry brain diseases over the next 20 years? will likely never be noticed even if it happened

  14. Bertrand45 says:

    According to the FDA, the control dose when testing the Pfizer vaccine was just a saline solution. (See the paragraph 1 on page 2 of this media letter.)

    https://www.fda.gov/media/144412/download

    Why aren’t there two controls, one being just saline and the other being empty lipid nanoparticles along with whatever adjuvants and other additives are used in the actual vaccine dose? (See paragraph 2 on page 4 for a complete listing of ingredients.)

    Or, if there’s only one control dose for pragmatic reasons, why doesn’t that dose include the empty nanoparticles and adjuvants instead of just saline?

    1. Marko says:

      “Why aren’t there two controls, one being just saline and the other being empty lipid nanoparticles along with whatever adjuvants and other additives are used in the actual vaccine dose? ”

      The danger is that you might find that empty lipid nanoparticles provoke an innate immune response that provide significant protection against CoV2 infection. That would be very bad for Capitalism.

      1. Derek Lowe says:

        Or, since many of these lipids are proprietary compounds, not.

      2. Ken says:

        Not sure if you’re being sarcastic, but doing the similar control test with the viral vector vaccines would be bad, in that you’d risk immunizing your control group against the vaccine.

    2. mymagoogle says:

      I am many years removed from my earlier working life in vaccines, but believe me the topic of “What Control to Use” comes up early and often in every adjuvanted vaccine clinical trial, ad even moreso with novel adjuvants (or carriers, or formulations). =So Often!= The result of each time around it goes seems to be to re-review what data is in hand and then start a new animal in a new size animal to study saline vs vehicle vs adjuvant vs adjuvant+antigen one shot + adjuvant antigen two needles injected close to each other + + +. Then the team talks, and the their senior managers talk, then everyone talks with the FDA, and then a decision for this trial is made.

      Next trial, it all starts over again. So I don’t know why they chose saline, but I do know from experience that it was very well discussed all the way around, and around again.

      1. Thomas says:

        Trials are also to detect side effects.
        If new components are also used in the control arm, any adverse effects caused by them would go unnoticed.

  15. Orange says:

    “The cell membrane is largely made of phospholipid bilayer, with the outside hydrophilic part being negatively charged, so these positively charged nanoparticles have all the more reason to stick to them.”

    Does anyone have a reference for this? Once I tried to modify cell culture dishes by covalently binding poly lysine to promote cell adherence. Neurons didn’t like the new surface (or the old for that matter) and I was never able to find a good explanation why it was supposed to work or why it failed. Maybe i searched for the wrong terms.

    1. Marko says:

      I’d try the commercially-available dishes against your home-brew ones before I gave up on the idea. Did other cell types stick better to your dishes?

      1. Marko,
        Excellent summary above, and as far as the follow-up safety testing you’ve hit the nail on the head with this conclusion in particular (again couldn’t reply directly above due to comment nesting):
        “The question then becomes how much – 20% , 1% , .01% ? – escapes into the systemic circulation. I believe the number of RNA payloads per dose is on the order several billion, so some unknown fraction of that constitutes the number of potential off-target cells”

        That’s exactly it — couldn’t have said it better. For the longitudinal follow-up in the clinical trials and broader population, it’d be helpful to have a more quantitative handle on the non-local distribution of those lipid nanoparticles distant from the injection site. I agree it wouldn’t be a huge concern if there’s just (relatively) random dispersal of injected LNPs systemically, and a nontrivial amount of hepatorenal clearance with each pass — that’s the ideal scenario. Where things get iffy are three-fold:
        1. Because of the anatomy and circulatory trajectory from the deltoid and cephalic vein (essentially a straight shot into the SVC), if enough “spillover LNPs” are getting shuttled into the right atrium and transiting through the pulmonary circulation — which could be high, another reference here for the rich vasculature around IM injection — then one of their earliest stops on the map after exiting the heart would be in tissues serviced by branches of the common carotid and subclavian arteries (including the CNS), enhancing delivery to tissues behind the blood-brain barrier simply due to higher relative concentration at tissue corridors more proximal to the injection site.
        2. Even if initial transit through the BBB and into other sensitive tissue parenchyma is relatively low, there’d be a cumulative effect with each booster delivering more spillover LNPs to those non-local sites.
        3. Related to that point, the duration of immunity is still unclear, and there seems to be general agreement that while COVID-19 symptoms are reduced with the immunization, viral spread is not. If antibody and memory B/T-cell levels wane within a few months after vaccination, then we’d be looking at repeated boosters possibly multiple times a year given ongoing community dissemination. And since the development of many e.g. CNS disorders is gradual — with subclinical issues taking shape over years before clinical manifestations become apparent (as seen in MS and ALS) — such cumulative damage likely wouldn’t raise red flags at first, but could increase in likelihood with successive boosters.

        Plus there are the potential issues raised by the paper that S dug up, such that even small levels of BBB traversal by the LNPs could engender spike protein uptake by e.g. oligodendrocytes and neurons beyond the number of LNPs themselves, due to that adsorptive transcytosis of S proteins into bystander glial and neural cells. The LNPs really are an intriguing and promising technology and it’ll be wonderful if this modality turns out to work. But simply because the clinical trial process is being accelerated so fast beyond the normal decade-long timeline for vaccine development, it just seems prudent to dot every i and cross every t when it comes to the follow-up and knowledge of the LNPs’ tissue localization, even more so given all the unknowns about the proprietary LNP formulations. In addition to animal studies with reporter genes, might be helpful to have data from more extensive imaging studies in patient cohorts, especially things like T1 and T2-weighted MRI to assess (mainly) CNS myelination and the white matter milieu in general over varying temporal intervals (3 months, 6 months, one year and so on) after receiving the injections.

        1. chemist says:

          I know this is simply an anecdote, but I contracted COVID over 6 months ago, and since then I’ve been traveling frequently and among large crowds. No reinfection, multiple negative tests since I recovered. Seems like your best bet is to just get the virus and get it over with

          1. Lee H says:

            That’s easy for you to say. You survived. Your post would be quite different had you not.

          2. Wilhelm Cody says:

            Lee H: this is the drowning sailor-dolphin paradox. We hear about the drowning sailors pushed to shore by dolphins but somehow the sailors pushed further out to sea by the dolphins never give their report.

        2. confused says:

          >>while COVID-19 symptoms are reduced with the immunization, viral spread is not

          Is there really agreement on this? I had thought that efficacy against asymptomatic infection was simply *untested* and thus *unknown*, not *known to be absent*.

          (Would there be precedent for ~95% reduction of symptomatic infection but no reduction of overall infection?)

          I don’t think it’s necessarily beneficial to promulgate doubts about the vaccine without fairly solid evidence, at this point.

          1. JS says:

            The inactivated polio vaccine mostly fits that bill. Extremely efficient at reducing paralysis and death. Very poor at reducing infection and transmission.

            On the other hand Moderna tested people (PCR) when they showed up for the second shot and saw 14 positive in the vaccine group versus 38 in the placebo, if I recall correctly. Not 95% reduction, not great statistics and not a direct measure of transmission, but at least promising.

          2. Confused,
            Again very good questions. I’ll say straight out that I strongly suspect that vaccine-mediated COVID-19 symptom reduction (if used as the primary endpoint for gauging clinical efficacy) almost certainly should imply a concomitant reduction in overall transmissibility. If there’s one factor that’s common to both viral spread and severity, it’s viral load itself, thus symptom mitigation would likely correlate with reduced viral load, which in turn should reduce airborne person-to-person transmission. Also, with reduced infectious severity, there’s decreased likelihood of colonization of the lower respiratory tract after the nasal passages, which should also contribute to this. With that said, there are a lot of questions about just how much of a viral load reduction is needed to tangibly reduce person-to-person spread and the rate of disease contraction in real-world scenarios (particularly superspreader events), which the trials themselves could not demonstrate. On that note, there’s been an ongoing debate in the literature about just what should constitute efficacy in the clinical trials, and what the proper clinical metrics and endpoints should be to define it. This has been the thrust of the critiques by Peter Doshi, editor at the British Medical Journal (article linked in name field). The issue with symptom reduction as the principal endpoint doesn’t so much appear to be whether it’s correlated with viral loads per se (which is highly likely), but rather whether what was observed corresponds to the way infections overall occur out in the field, and whether either arm of the clinical trial is representative of the broader population (esp. since the placebo group had an infection rate well below the general population). It’s reasonable to be hopeful that viral transmission is tamped down enough to measurably bring down the R0, but from the trials so far, the virus still appears capable of spreading, thus the public health recommendations to continue masking and social distancing regardless of immunization status.

        3. confused says:

          >>If antibody and memory B/T-cell levels wane within a few months after vaccination

          Is there a reason to think this would happen, at least for B/T cells? Wasn’t there a paper mentioned on here several months back that showed people infected with SARS-1 back in the early 2000s still had a T-cell response?

        4. JSA says:

          This is a very interesting thread. Can I assume that the problem of tissue tropism/the issue related to crossing the BBB is unique to mRNA vaccines and that others (AstraZeneca, Johnson & Johnson) will not have this potential risk?

        5. Dr. B says:

          Very interesting article and discussion. I definitely agree there is unknown risk any time you inject something new into (many) millions of people, and more follow-up is of course crucial (and longer time before widespread administration would have been ideal, but obviously there’s a reason the risk/benefit calculation went the other way), but a few thoughts on your specific concerns:

          1. Escape into systemic circulation: Absolutely some of that happens. Would be nice to know how much is typical/range etc, but there will be occasional cases where it’s a lot just because sometimes at least part of the beveled tip of the needle will be in some sort of blood vessel (there seems to be some debate about whether or not people should try to aspirate blood before injecting vaccines, but in any case they certainly don’t always do so). I’m a physician and I do a lot of percutaneous procedures; it’s a non-event for a 25g needle to pierce a blood vessel, but if you do enough percutaneous procedures you will definitely pull back and get blood from time to time and have to adjust slightly lest you inject something intravascularly when you don’t want to.

          In terms of blood flow following that, that seems less concerning than you suggest. Before blood gets from the arm to the brain it will (barring a shunt) have to go through the pulmonary circulation. Would seem like many/most of the LNPs, if they are taken up nonspecifically, would be taken up there, particularly in the capillaries. After that they will exit via the aorta, but saying the CNS is one of the “first stops” of blood exiting the heart is misleading for this— the important point is what % of blood, and thus LNPs, would go through the brain each pass, not how many seconds earlier it hits the brain than say the kidneys. The LNPs would certainly be well-mixed in the blood by the time they hit the aorta; they would not be more concentrated in blood flowing to the internal carotid arteries than arterial blood going elsewhere (as every CT angiogram will show you). To achieve that you’d basically need a carotid injection (which is done with contrast, via catheter, all the time by certain specialists).

          2. Definitely agree there seems to be some risk of LNPs being taken up in undesirable locations, making spike there, etc. Should be looked at, and several obvious ways to look at esp with animal models immediately come to mind. Would think some of those studies have already been done? However, I would think that any negative result would present itself pretty early, unlike e.g. MS or ALS, for the following reason: The expression of the spike protein from the mRNA will be short lived, correct? That part of any issue, at least, is not due to auto-Abs forming that would continue to attack non-spike-producing cells into the future. So yes you could get a 2nd impact with the booster, but also should fizzle out soon after the injected synthetic mRNA stops making spike protein. Doesn’t negate the risk of persistent autoimmune problem, but that’s nothing to do with this specific issue. So, I think any issue here should be known pretty quickly.

          3. In terms of using MRI etc to look—yes potentially, although one should be cautious about using MRI to check large #’s of asymptomatic people. You will undoubtedly find a bunch of things of unclear significance if you look hard enough. Doing MRI etc willy-nilly in the absence of any symptoms etc is generally not a great idea.

          4. While this is obviously being looked at, there was (limited) data from one of the two mRNA trials suggesting protection against asymptomatic infection/viral spread as well. So I don’t think there is general agreement that viral spread is not reduced, although AFAIK still an open question. But I think the general feeling is viral spread is likely reduced significantly.

          5. My personal bottom line: I got the vaccine and was very relieved to do so. There is definitely some unknown risk, but I think very unlikely to be higher than the known risk I face from covid-19. Would not yet give it to my kids (teenagers), even if it were approved for them. Different risk/benefit calculation.

          1. Dr. B,
            Excellent points all, and great food for thought. I should say first off that whatever the vicissitudes and question marks about the mRNA modalities per se, I feel like overall we have every reason to be optimistic about improving immunoprophylaxis in some form helping us to bring the R0 down to a manageable level as the year progresses. Even with lingering questions about the LNPs or spikes in the VAERS reports for the ones that currently have EUA, we have perhaps a dozen other new vaccine candidates on deck over the next few months, almost all of which AFAIK are based on more tried-and-true modalities like viral protein subunit or inactivated virus protocols. Even with a similar accelerated timeframe for those (presuming they pass muster for Phase 1-2-3), in such cases we’d be piggybacking on the decades of prior confirmation for at least the overall modality, and since their own cellular localization would be restricted essentially to APCs in peripheral tissues/lymph nodes (or the wild-type tropism for attenuated virus modalities), there’s reason to be hopeful that they could more readily achieve broad adoption even among those who might hesitate to try out the mRNA modality.

            If there’s one key pharmacokinetic parameter to ideally get a better handle on straightaway for the LNPs here, it’s indeed that issue of whether the pulmonary capillaries can act as a sink (sort of a “pseudo-first pass”) for whatever fraction of lipid nanoparticles are shifting into systemic venous return via the cephalic vein after deltoid IM injection, which I’d also pondered in my comment above. I’m inclined to agree that whatever penchant the LNPs might have to traffic across the blood-brain barrier, a lot of it should be filtered out in the transit through the pulmonary vasculature. That said, in this case we don’t have to speculate so much about this capacity for LNPs in general, since the available literature features examples of lipid nanoparticle injections that clearly do result in large-scale traversal of the BBB in abundance after in vivo introduction at distant sites — thereby passing through the pulmonary vasculature (and sometimes even the hepatic portal circulation) — for example the paper linked in the name field (Rodrigues et al., 2019). With the caveat that these were mouse tail-vein injections with the express intent of BBB traversal, it’s at least proof-of-concept that a “particle bolus” of LNPs, introduced into the peripheral circulation, can indeed enter the right atrium and emerge out the aorta in enough quantity to attain a clinically significant presence beyond the BBB, indicating that for at least some LNP recipes, the pulmonary capillary bed isn’t a significant hindrance to arterial delivery into the common carotids. That’s where the other questions about the actual quantification of such LNP spillover, crossing of the tight junctions in the BBB, and whatever the particles do beyond them in the CNS come into play. Unfortunately without more precise knowledge of the proprietary LNP formulations in the vaccines, and especially solid empirical data about their trafficking and localization, we’re stuck piling conjecture upon conjecture on what’s actually transpiring in vivo, which is concerning for a large-scale immunization effort.

            With that in mind, though, I’m basically on board with the crux of your points. If I had to bet money on it, honestly I’d say that even with the cloud of uncertainty hanging over the tissue localization and trafficking issues (or e.g. antibody-dependent enhancement, which from prior lurking on Derek’s blog have been discussed here a good deal), the mRNA vaccine protocols are still quite likely to be safe. Even if errant systemic tissue trafficking and/or ADE are occurring, I doubt these would be major factors on a population level, and for higher-risk population cohorts (e.g. nursing home residents w/ multiple comorbidities), almost certainly vastly safer than a bout with COVID-19. (Thus the unquestionable validity of your fifth point and the need for risk stratification as recommendations evolve.)

            I guess a lot of this just boils down to one’s set point in applying the Precautionary Principle as to where to make the leap with a modality that — at least in regard to in vivo injection, as opposed to ex vivo APC transduction (for mRNA vaccines as immunotherapy) — is so much in embryo as a vehicle for widespread inoculation. We’re sort of conditioned in gene therapy to obsess about all the not-yet-known unknowns and, in particular, to go all-out in pinning down the in vivo tissue localization and trafficking. A lot of this is a product of the scars from the tragic OTC trial at Penn in 1999 (which incidentally did likely involve ADE), which set back the field by 10-15 years. It was a somber reminder of the common “hype-letdown-cautious application” progression for exciting new modalities (which CRISPR is probably going through today, in the early stages), and a reminder that even if a new treatment or prophylaxis approach is intrinsically sound, missteps or cut corners in the early phases can undercut public confidence and bottle up a field for a long time before it resuscitates. (I distinctly remember that after the Penn trial, the IRBs would often demand a trove of data on the tissue localization for gene therapy vectors in animal studies, demonstrated independently in multiple labs, to merely consider kicking off a Phase I, even for clear compassionate use cases involving severe rare diseases.) This would be especially tragic in the case of mRNA-based immunizations since they could be such a flexible and powerful tool for rapidly-developed vaccination if they pan out. It just makes it all the more important to take out the guesswork on the basic science and, hopefully, to shed more light on questions like this.

          2. Marko says:

            When thinking about capillary filtration, it’s useful to consider the size of a LNP – about 100 nm, or about 70x smaller than the diameter of a RBC. Lots of empty space in that capillary for the LNP, relatively speaking. The question then becomes one of the tendency of the LNP to bind to the vascular endothelium that it would be repeatedly encountering during its travels.

          3. Melissa says:

            You’ve made the most sense on this entire blog!

          4. Nepomucemo Trujillo says:

            When more tried-and-true modalities are available I’ll be in line. Until then I won’t be attending any indoor activities.

        6. GD says:

          Given a choice, would you feel more comfortable with an adenovirus based vaccine or an inactivated vaccine over mRNA ones at this stage in that case?

          1. sgcox says:

            Not sure about inactivated virus yet, as none has been approved, but I would take any of adeno or mRNA vaccines at the moment I get a call. More than 10 M of both types have been administrated so far across the world and only few anecdotal stories of side effects. Earnestly, I was expecting more fuss in media given the whole world attention and sensationalism around. All these discussions about potential side effects of new technologies are interesting and useful but totally moot for me now.

        7. Mary says:

          This searches suggests that spike protein itself as well as components such as RBD cross the blood brain barrier by disrupting it.
          https://www.sciencedirect.com/science/article/pii/S096999612030406X
          I know that the vaccine encodes a modified form of Spike to keep it properly folded. I wonder if this modification is enough to prevent entering the BBB? The data in VAERS points to possible neural inflammation with frequent reports of headaches and hundreds of reports of Bell’s palsy and other cases of persistent muscle weakness, dizziness and fatigue, in addition to many cases of cranial nerve issues, and deaths. As an RN with high risk of exposure to COVID and significant fear of this, I am also very apprehensive about the vaccines. I would like more data, as soon as possible, about the ability of the LNP or spike itself crossing the BBB and the truth about the relationship between the events in VAERS and the vaccines. This post, and the comments that follow, helped to validate my concerns. Please reply with perspectives on my questions.

          1. sgcox says:

            Vaccines, either adeno or mRNA do not have Spike protein, only DNA/RNA code.
            Upon muscle injection they transfect nearby cells and make them present Spike on their surface and alert immune system. Vaccines cannot physically enter brain using the molecular entity they simply do not have 🙂
            If Spike indeed can facilitate passing brain barrier, that even more reasons to get vaccinated as free floating virus particle coated with thousands Spikes will happily multiply and kill neurons like any other cell it can get into.

    2. BB says:

      I thought our cell membranes were generally considered net neutral due to inclusion of other lipids within the membrane (e.g. sterols), is that not the case? Our own antimicrobial peptides are cationic which allow them to have some specificty for negatively charged bacterial surfaces.

    3. Juan says:

      I used plates of unknown material. They would explode plant protoplast and leave a green mark. Maybe there was so much charge that the cooperative links broke the membrane. If that is the case, then changing the charge concentration could aid, but better find the adequate material.

  16. Kaleberg says:

    The more I learn about how the virus and vaccines work and especially about the little hacks and tweaks biologists use to encourage biological activity in the body, the harder it is to believe that the virus was engineered. Anyone modifying a coronavirus to cause a plague would have wound up swapping some nucleotides and throwing in an enhancement sequence or two to increase the odds of infection and the level of harm. Someone might just know how to engineer a deadly virus by cutting and pasting even if no one understands why it works. No one has any idea of how to do it and then hide his or her handiwork.

    It’s like chemical synthesis where chemists add step after step to hide things from reactions and then peel off the masking tape to expose them in a later step. It takes a lot of hard work and experimentation to simplify the reaction and make it look almost “natural”. I suppose I’ve turned the usual intelligent design argument inside out here, but the mark of evolution shows the work of aeons which makes the mark of mankind look positively clunky in comparison.

    1. A Nonny Mouse says:

      The furin cleavage site was introduced into the original SARS virus in 2009 which showed it to be more infective. No reason why it can’t have been done in 2019 with an accidental escape.

      https://www.pnas.org/content/106/14/5871

  17. Jonathan says:

    One query. You mention that the vaccine mRNA doesn’t code for the native spike protein, but a modified protein aimed to improve stability.

    Is that still a membrane protein like the original spike protein? in which case the immune system will react against the cells bearing them. Or do small spike-containing vesicles bud off as if they were real viruses? I had always taken it that viral budding requires nucleocapsid proteins as well as envelope proteins. Or is the modified form secreted as a soluble protein?

    One assumption implicit in any of those possibilities is that the mRNA inludes code for a signal sequence directing its cellular synthesis to rough endoplasmic reticulum.

    And thanks Cole Batty for the clarification about lipid nanoparticles. I had been wondering how they got so good at loading liposomes.

    1. Barry says:

      It’s absolutely expected that an effective vaccine will elicit both B-cell (humoral/mucosal) immunity and T-cell immunity. Among those T-cell will be killer T-cells which will destroy the cells expressing SPIKE under the influence of an mRNA vaccine. Intramuscular injection into the triceps is expected to limit that destruction to myocytes. But yes, any vaccine that gets loose into general circulation could invite T-cell attack on a cell elsewhere.

  18. Derek says:

    In reading around, I’ve got one query about these two mRNA vaccine, namely will they be as effective in folks who have heterozygotic familial hypercholesterolemia?

    That is because various technology references I come across refer to the lipid nano particles essentially presenting as LDL (containing cholesterol and the appropriate apolipoprotein) so as to be endocytosed via the LDL receptor. I’ve not come across a direct reference to these two vaccines using that specific mechanism, but certainly to various companies offering delivery technology which uses that mechanism.

    So since the prevalence of that FH form is around 1:500, does that suggest that these people should really be being offered one of the other vaccines, and that any such people (especially undiagnosed) who get one of these two may well be given a false sense of security?

    1. Patti says:

      This is exactly the question I have been searching and of which this site popped up for me. Have you found an answer yet?

  19. Excellent overview, Derek (and happy and safe New Year).
    Easy to forget that given the unprecedented rate of progress of COVID-19 mRNA vaccines from a base of very narrow and limited clinical studies (largely ‘flu) to approval, just how much enabling technology is in the background, and the challenges that kept mRNA vaccine development in the shadow of DNA approaches.

  20. charlie says:

    I thought Moderna settled the delivery system claims prior to their IPO?

  21. Lipidz says:

    Minor quibble, apologies if someone has already pointed this out:
    The proprietary lipid ALC-0159 used in Moderna’s formulation appears to be a PEGylated amide, not a tertiary amine. ALC-0315 is the only tertiary amine. (Judging by their linked Wikipedia articles, at least.)

  22. Smokerr says:

    Once again I request a re-visit of the one vs two shot and the conflicting information I am hearing on 52% effective vs 80-90% (not long term need for second shot)

    Or split it to a in general and in depth.

    While the tech data is of interest, this is one of the few good sources I have come across and we desperately need that basic information to determine how to navigate this, both for our own needs as well as to support the one shot policy or to disagreement with it so support for the best option we have now in regards to the public good.

  23. Botond Igyarto says:

    Hi Derek,

    Thank you!

    Food for thoughts: https://www.preprints.org/manuscript/202012.0493/v2

    Best,
    Botond

    1. Dr. CB says:

      For easy access and read, here are the relevant parts copied from the article above:

      Immunological features of LNPs

      LNPs are ~100 nm size carriers that consist of phospholipids, cholesterol, PEGylated lipids and cationic or ionizable lipids. The phospholipids and cholesterol have structural and stabilizing roles, whereas the PEGylated lipids support prolonged circulation4. However, there is growing number of evidence that PEG can be immunogenic and repeat administration of PEG can induce anaphylactoid, complement activation-related pseudoallergy reaction5. Humans are likely developing PEG antibodies because exposure to everyday products containing PEG. Therefore, some of the immediate allergic reactions observed with the first shot of mRNA-LNP vaccines might be related to pre-existing PEG antibodies. Since these vaccines often require a booster shot, the formation of anti-PEG antibodies is expected after the first shot, and thus, the allergic events are likely to increase upon re-vaccination3. Cationic/ionizable lipids are included to allow complexation of the negatively charged mRNA molecules and to enable exit of the mRNA from the endosome to the cytosol for translation4. Although cationic lipid containing LNPs were considered by some to be immunologically inert6, other data support that many are indeed highly inflammatory and possibly cytotoxic4. In concordance with these, we observe that empty LNPs6 given intradermally to mice induce robust inflammatory responses, characterized by neutrophil infiltration, inflammatory cytokine production, activation of diverse inflammatory pathways and excessive cell death (unpublished observations). The presentation of self-antigens in this highly inflammatory environment might ultimately lead to a break in tolerance. Therefore, we believe more careful characterization of LNPs is needed, and suggest that only the inflammatory, but minimally cytotoxic lipids are approved as vaccine components (Figure 1). Some DC subsets at optimized antigen dose can induce protective antibody responses in the absence of inflammatory agents7,8. These data suggest that LNP-based vaccine platforms that lack inflammatory cationic/ionizable lipids, could be a viable option to induce protective antibody responses if targeted to certain DC subsets.

      Off-target effects of vaccine mRNA

      Based on the current mRNA-LNP vaccine design, LNPs can be taken up by almost any cell type, near or far from the site of injection, transfecting them with the antigen-encoding mRNA9. Moreover, the mRNA used in these vaccines are modified to increase its stability in vivo, allowing extended periods of mRNA translation10,11. Also, a significant portion of the
      mRNA can be re-packaged and expelled from transfected cells in extracellular vesicles12. These vesicles could reach cells far from the injection site, further increasing the number of cells translating the antigen and extending the duration of its expression. Long-term mRNA translation in non-professional APCs might lead to unanticipated cell killing. Similar to any other self-protein, synthesized vaccine proteins have access to antigen presentation on MHC
      class I molecules on any nucleated cells13. Thus, any cell presenting antigenic determinants from the vaccine could become a target of T cell-mediated killing. Furthermore, if vaccine-derived proteins become inserted into the plasma membrane or secreted and associated
      with cell membrane, these cells could become targets of antibody-dependent cellular cytotoxicity14. Both should become evident after an adaptive immune response has been generated and may be accentuated upon secondary immunization (Figure 2). In line with this, systemic adverse events from the mRNA-LNP based SARS-CoV-2 vaccines were indeed
      more common after the second vaccination, particularly with the highest dose3. Strategies that allow delivery of the mRNA exclusively to DCs with the use of DC-targeting antibodies or ligands may limit the possible off-target effects.

      1. Amanda Perkins says:

        Hello, have these concerns been addressed to the point one would feel safe getting vaccinated?

    2. debinski says:

      This preprint is a little scary to say the least but I don’t have the background to know if the theories presented are reasonable or not. Would love to have a critique from someone who does. This could explain the autoimmune platelet destruction that apparently occurred in the Miami MD who died a couple weeks after the vaccine, IF it was caused by the vaccine. But why haven’t we seen more serious AEs if the LNPs could be causing these problems?

  24. Peter Waldo says:

    This article’s excellent content and the rich discussions in the comments thread raise a lot of prescient mRNA questions & issues that are, to be frank, concerning! Potential cytotoxic effects and a lack of understanding about the vaccine platform’s predictability in vivo are just a few. Has the ’normalcy bias’ lulled us into irrational confidence about how mRNA will actually act in the real world (based on 21,000 patients)? If vaccine AE’s and anecdotal yet potentially significant immune system reactions are underplayed and not reported by the media it could make matters far worse. Instead of promoting a vaccination rollout “frenzy”, isn’t there a ethical obligation to the people given the facts here; a compelling case to limit/scale back the mRNA rollout to better identify, track and understand safety signals and issues to ensure no harm is done on a mass scale? And, dedicate time and resources toward accelerating the other vectored/attenuated virus vaccine candidates’ clinical trials since we are more familiar with their side effect and safety profiles should they offer good efficacy?
    Peter.
    “Remember, if you’re heading in the wrong direction, God allows U-Turns” (A. Bottke)
    “The greatest enemy of knowledge is not ignorance, it is the illusion of knowledge” (D.Boorstin).

  25. Larry Fitz says:

    Phenomenal post & comments. The underlying biology for tropism, lab work to show the actual synthesis of the spike protein in vivo, and how the immune system responds, off target risk, etc. are yet to be understood.

  26. Marko says:

    Relevant to the discussion in comments above:

    “Neuroinvasion of SARS-CoV-2 in human and mouse brain”

    https://rupress.org/jem/article/218/3/e20202135/211674/Neuroinvasion-of-SARS-CoV-2-in-human-and-mouse#.X_82GDeU_I8.twitter

    “Altogether, our study provides clear demonstration that neurons can become a target of SARS-CoV-2 infection, with devastating consequences of localized ischemia in the brain and cell death, highlighting SARS-CoV-2 neurotropism and guiding rational approaches to treatment of patients with neuronal disorders.”

  27. Tim Mackey MD MS says:

    Your article was a walk down memory lane for me. I finished my masters in molecular biology in 1981. I was encapsulating Vaccinia virus nucleic acid inside of Small unilamellar vesicles(SUV) and Small multilamellar vesicles (SML), varying the phospholipid composition to generate the most optimal liquid crystalline to solid crystalline make up and maximize the incorporation of the polyribonucleic acid. Your mRNA vaccine explanation was succinct and to the point. I am glad that you pointed out the ground work for this “new” technology started 45+ years ago.

  28. Tim Mackey MS MD says:

    The important parameter I forgot to mention that my assay was done on a cell line known to be resistant to Vaccinia. It did work, I was able to infect those cells as evidenced by cell death plaque assay. When my hospital offered the vaccine, I briefly thought about LNP cell targeting and on a quick search found nothing useful. Being in my late 60s with asthma, I figured my relevant risk benefit ratio to weigh in on the side of get the Pfizer vaccine. However I am not advocating at this time for any of my very healthy children and grandchildren to get a mRNA vaccine.

  29. Mperkins says:

    As a non medical person, digging for information on potential long term effects, this thread is exactly what I have been looking for. Has anyone found further answers to the questions posed here?

  30. Marty says:

    I didn’t see this reference yet mentioned on lipid nanoparticle penetration into the brain:

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5475249/

    The mRNA was flu not covid, but the lipid nanoparticles were presumably similar since this was from Moderna.

  31. Marko says:

    This looks like a well-done serosurvey. They used multiple ELISAs and went to great effort to ensure that their samples were representative, though since the sampling was voluntary, there could have been some sampling bias. I’d have preferred they put a gun to people’s heads :

    https://twitter.com/KSadtler/status/1355877878299320321

    They come up with just short of 6% seroprevalence by mid-July, which agrees fairly well with covid19-projections’ estimate of 6.7% for that date. Covid19-projections then puts us at ~23% prevalence at the end of Dec., and the CDC estimate for then is ~25%. All these figures sound plausible to me.

  32. _Gi says:

    Hi, I have a naïve layperson’s question.

    When the synthetic mRNA decays will the modified base (1-methyl-3′-pseudouridylyl) be recycled in the cells to make new mRNA?

  33. sadu says:

    Empowering reports about Pfizer’s immunization were trailed by comparable reports from Moderna’s antibody’s late stage preliminaries too. For More Please Click The link Below
    https://www.greenforexclub.com/2020/11/optimism-covid-vaccine-golds-weakeness.html

  34. Jotabe says:

    It´s not my field, but I love learning and trying to find answers so I was looking for information about LNP´s from mRNA vaccines and I´m so glad I ended up checking this site. This blog goes straight into my bookmarks, thank you for sharing your knowledge, guys. I´ll keep reading, informing myself better and I will eventually pop up and dare to write some questions for some enlightment from you.

    Thanks again

  35. AJason says:

    I’m confused the CDC says the, “mRNA from the vaccine never enters the nucleus of the cell and does not affect or interact with a person’s DNA.” https://www.cdc.gov/vaccines/covid-19/hcp/mrna-vaccine-basics.html However this article says, “mRNA has its feet in both worlds, if it had feet: it’s down there in the nucleus being put together next to an exposed and unwound strand of DNA”. So can mRNA get into the nucleus or not?

    1. Derek Lowe says:

      Cellular mRNA is produced in the nucleus and exported from it into the cytosol. But the vaccine mRNA gets into the cytosol first, and is not imported back into the nucleus.

  36. Flavio Kaplan says:

    This is a very interesting article and surprisingly approachable even for people with no formal instruction in microbiology.
    After reading this, I am persuaded not to get vaccinated by mRNA vaccine options at this time.
    I have not yet seen any response to similar inquiries; that is, are “traditional” vaccines safer (interested in the Johnson & Johnson in particular, as it is the only other one available in the US at this time), or would there be similar concerns with those?

    1. Derek Lowe says:

      I’ve had several people write to me saying that the J&J is a “traditional” vaccine, but as an adenovirus vector, it’s far from that.

      1. Flavio Kaplan says:

        Flattered that you took the time to respond to me, much appreciated. As I don’t know enough, would you mind expanding what makes an adenovirus vector vaccine non-traditional? But more importantly, being “traditional” in my limited lingo only indicates “tried and true” when it comes to long term safety. I understand every vaccine imposes an individual risk that is impossible to quantify in the abstract, so we resort to a comparison of general risks. So under this context, the answer I’m seeking is whether there is a “safe” alternative to this disease – it seems that mRNA vaccines may not be it, so I’m left with the J&J one in the deck.

        1. K24 says:

          I’m a medical novice and in the same boat as you, trying to choose btw Pfi and JJ. All my friends are rushing to get the Pfi vax based on higher efficacy data without considering these questions. From what little I understand, virus vector vax tech was used for Ebola and Zika vax as well and it’s the same tech in Sputnik and AstraZ vax as well, so long term side effects of the tech maybe better known at this point than mRNA vax but there may be specific components of even JJ vax that are new and insufficiently studied. It’s come to choosing the lesser evil bc unfortunately vax passports and permissions are coming, we won’t be able to escape a Vax. I wish someone cud shed light on evils of JJ vax in comparison, tropism etc. Thanks to anyone who cares to share thoughts!

        2. MB says:

          “Traditional” modality would be live attenuated vaccines or inactivated viruses. If that’s what you’re looking for, you’ll need to google those and find out which companies/governments are developing/offering them.

          1. Doug H MD says:

            If you are in the US, you will be wating a while if it ever comes to fruition. Trials are underway to test the Indian Whole Virus vaccine in the US.

  37. K24 says:

    What an amazing thread with views from such knowledgeable folks!

    I’m not a scientist or doctor. Did folks on this thread have a view that unknown long term side effects might be fewer with JJ adenovirus vax rather than mRNA vax, which poses all of the above unknowns?

    It appears as though the effectiveness/efficacy data is not super comparable since JJ trials were conducted later when more stubborn variants/ mutations had already presented themselves.

    Bottomline is, do the vastly knowledgeable folks here conclude J&J is the better vax for having lesser known objections than less tried and tested mRNA vax?

    Everyone I know is rushing to get Pfizer but it seems they aren’t thinking of these issues raised above. Thanks so much for helping me decide!

  38. Doug H MD says:

    what is needed to answer these questions is head to head trials.
    Why not do a pfizer/moderna/jamd j face off?

    Industry doesnt want it. Too much to lose and not nearly enough to gain!

  39. Cris Freer says:

    Any thoughts on Novavax’s COVID-19 vaccine or other vaccines (EA or in the pipeline) and their ability to cross the bbb?

    Any thoughts on receiving a different vaccine as a booster for variants than that which you initially received (say a Novavax booster a year or two after having received an initial Pfizer/Moderna vaccine course now)?

    Any thoughts on if it is safer to just receive one Pfizer/Moderna vaccine dose vs. the recommended two now that high efficacy has been shown, I understand, from one dose?

    1. Doug H MD says:

      all good questions

  40. Susan N says:

    I found this transcript very interesting an informative.

    Interview with Dr. Vanessa Schmidt-Kruger
    Hearing # 37 of German Corona Extra-Parliamentary Inquiry Committee
    30 January, 2021
    First half in English, second in German.

    http://enformtk.u-aizu.ac.jp/howard/gcep_dr_vanessa_schmidt_krueger/

    1. Thomas Storz says:

      yes, especially this part : “…..The problem that BioNTech had is that in the clinical phase the product, i.e. the RNA, was produced with completely different techniques to how it is being produced now. During the clinical phase they only needed small volumes of vaccine, they were able to use very expensive techniques that delivered highly purified end products. Now that they have entered mass production, that is no longer possible, they have had to switch to lower-cost processes, e.g. using huge quantities of DNA that functions as the substrate to be able to produce the RNA in an in-vitro transcription reaction. This is done via bacteria, via the fermentation of transformed bacteria that contain this DNA. The bacteria multiply the DNA in huge amounts, and this leads to new dangers or risks, particularly contamination. At the moment for instance the situation is that the DNA is transformed in the bacteria, it is multiplied, next the bacteria are opened and the DNA is extracted, then it is linearised via enzymes, and after that the linearised DNA undergoes in-vitro transcription to produce the RNA using various procedures. The EMA Committee made various requirements of the vaccine manufacturer, i.e. BioNTech. The applicant needs to now develop and introduce various analysis processes to ensure that the substrate is free of microbiological contaminants – they probably mean E Coli bacteria for example. There don’t seem to be any processes to ensure or monitor for that. They also need to ensure that all the buffers – those are the solvents that are used – are free of RNAses. RNAses are enzymes that degrade RNA. If there are any contaminants of these RNAse solvents, then RNA in the vaccine will be degraded and the vaccine won’t have any effect anymore. They also have to analyse how strong the activity of the enzymes is; that is very important because I explained that after that the RNA is transcribed from the DNA and then the DNA has to be eliminated, it is digested by enzymes: by DNAses. And if this DNA is not digested well enough, if residues are left, this harbours risks – I’ll come back to the risks from DNA residues, but the activity of the enzymes has to be monitored well and at the end you need to have a pure RNA without any more DNA. And that is not the case. BioNTech has admitted that there are DNA contaminants”

  41. Patricia says:

    Does anyone here know if the 2 lab-created lipids make their way “out” of the body or are they stored indefinitely?

  42. ben says:

    WC2N 5DU

  43. Ben says:

    Hello.
    Is it possible that the mRNA “vaccines” are actually responsible for producing the variants? I ask this because in Israel they’re finding those vaccinated with the Pfizer BioNTech vaccine are testing positive for the South African variant 8X more than unvaccinated people.

    This particular description of how mRNA works tweeked my curiosity:

    “Then there’s another key step. In most organisms, the DNA doesn’t just read off the uninterrupted code for a whole protein. It has interruptions of other stretches of code (“introns”), and at this point those are clipped out and the actual mRNAs are spliced together by assembling their pieces (the “exons”) into their final form. That may seem like a rather weird process if you haven’t run into it, and it certainly was a surprise when it was discovered back in the late 1970s. This is done by yet another Death-Star-sized mass of proteins, the “spliceosome”, and it provides opportunities for “splice variants” along the way
    >>>> that will produce different proteins <<<<
    when a ribosome gets ahold of them. And that’s a big reason why we have a lot more different proteins in our bodies than we have different genes: many of them can be mixed-and-matched into these different variants back at the mRNA level."

    Is the mRNA producing variants in the process of creating the SARS-COV2 spike protein?

    1. Derek Lowe says:

      No, splice variants are completely different beasts. The variant coronavirus strains are mostly collections of single-amino-acid changes, and these arise from mistakes made by the virus’s RNA polymerase enzyme. Some of these mistakes can cause a piece of a protein to drop out completely but it’s still a consequence of such copying mistakes.

      Splice variants take place after a protein has been “read off” and produced, and while it’s getting assembled. Viral proteins tend to get read straight through, but human ones come in sections, which then get spliced together to make full-length protein. Mistakes there look very different: whole stretches of protein get joined in the wrong order, or mix-and-matched with a long piece of some other protein entirely.

      None of the coronavirus variants are splice variants. They’re all from errors at the earlier stage.

      1. Druid says:

        I understood splicing was done in in the nucleus in transcription from DNA to mRNA. Perhaps you meant “protein sequence”. Multiple DNA segments –> single mRNA sequence –> correct protein. Multiple DNA segments + splice variant –> single new mRNA –> single new protein. Correct to say that viruses do not code in DNA segments, so they cannot benefit from this mechanism.

        1. Derek Lowe says:

          Sorry, didn’t mean to give the impression that it’s the protein itself being assembled. Just conceptually, that’s where the final protein sequence comes from. It is indeed handled from multiple nucleic acids.

          1. Jean-Luc says:

            Hello Derek,
            in one of your articles about mRNA-vaccine manufacturing you say that you would be surprised if the “mixing” of the lipids and the mRNA would be done without new specially-built microfluidics devices. I wonder if you have seen Biontech using a 50L steel container/autoclave to encase the mRNA in a bubble of lipids through a process that uses pure, pressurized ethanol. The device is shown in a video in TIME (https://time.com/5955247/inside-biontech-vaccine-facility/), where the reporters visit the Biontech facility in Marburg

    2. sgcox says:

      Looks like Ben get confused by the way the numbers can get presented (I am charitable here).
      I think my math is right but please correct.
      Suppose vaccine gives 90% protection against “normal” virus but 60% protection against new variant. I think these numbers are more or less in line what is know so far about existing variants of concern (SA, Brazil, India).
      Now, if circulation of the variant is 20% then assuming all other factors are similar, 20% infections will be by new variant.
      Now, for vaccinated people, there will be 0.1*800=80 infections of normal but 0.4*200=80 (!) infected by variant or 50% of total infections
      So overall vaccine efficiency will drop a bit to (1000-160)/1000 = 84% but at the same time vaccinated people will be da-dam ! – in the hand of antivaxxers – 2.5 times more likely to be infected by new variant than “normal people” and probably even bread it !!
      Cue antibody enhancing, virus splicing and all other crap.

      1. sgcox says:

        Sorry, forgot to mentioned that calculations were for hypothetical 1000 infections in general population to get numbers easy to illustrate

  44. Alex says:

    I didn’t have any issues after first dose of Pfizer. However, the day after second dose I had fever of 103 degrees and sever muscle aches. Three days later, I developed severe depression. It came seemingly from nowhere and hit me hard. It’s been almost three weeks, and I still feel mildly depressed. Many people I know who took Pfizer complained of brain fog lasting multiple weeks. It would be hard to explain without considering vaccine crossing brain-blood barrier.

    1. Fyodor says:

      Interesting, I know maybe 50 people that got Pfizer and nobody had any adverse effects lasting more then a day or two, while few Moderna recipients had muscle pain up to a week.
      Are you sure “Alex” that it was not a Sputnik with novichok or polonium residues that you got ? Or maybe it’s an AD due a cold borscht from a broken vending machine ?

      1. William Banks says:

        I don’t think that making a joke of someone’s 1st hand report of an adverse effect is very useful. I think we all need to remind ourselves that these mRNA Covid vaccines have never been tested on such a large scale before and so this mass human experiment could easily produce unexpected results. There are various medical papers showing that these lipid nano particles could potentially cross the blood brain barrier. IF that happened, I would hazard a guess that the brain would not be too happy.

  45. VikkiMcD says:

    Why not modify the mRNA further to increase its stability and make it more resistant to degradation by ribonucleases (for instance, altering the linkages in the mRNA’s backbone to something that endogenous RNAses can’t break down)?

    1. sgcox says:

      I am 100% sure many tricks have been tried for exactly this purpose but failed.
      First, long mRNA in vaccines is not made by organic chemists.
      It is synthesised in cell-free system by RNA polymerase who will only make precisely the same phosphodiesters which RNAes like to chew. It is basically the same chemical mechanism.
      Second, even if you make RNA with unnatural backbone in bulk, you need to fool ribosome to use it as as a template and that very large and complex enzymatic machine is no fool, it was around for ~3 billion years 🙂

      1. Derek Lowe says:

        Exactly – getting the ribosome to accept it is not easy! There have been some new linkages developed for antisense therapies, but those just have it sit on their partner sequences and gum things up. And they’re no fun to prepare, either.

  46. William Banks says:

    I have spent days trying to find the answer to a question – so I really hope someone here can help me!
    I am trying to find out what the expected lifespan of these Lipid Nano Particles is? (specifically the ones used in the Pfizer mRNA covid vaccine).
    After they have been injected into the subject. If they do not merge with the subject’s cells, How long could they exist for in their original shell state inside the body?
    Just to be clear: I am asking how long can these LNP’s containing mRNA exist for inside the human body before they are broken down by some other process (other than the process they are meant to carry out which is to enter the human cells)
    I read on some website that they can exist for up to 50 days, but that seems a lot to me.
    Is that possible? Or how many days or hours could they exist for?
    I assume the Lipid shell would eventually dissolve or be broken down by some chemical process after some time?

    1. Doug H MD says:

      half life of ALC 0315 is something like 6 weeks i heard

      1. Derek Lowe says:

        See the discussions starting on page 45 of this document. Clearance is strongly biphasic, with a rapid phase followed by a long slow one. In rates, complete clearance from the liver is estimated at 6 weeks.

        https://www.ema.europa.eu/en/documents/assessment-report/comirnaty-epar-public-assessment-report_en.pdf

    2. Derek Lowe says:

      Good question. The mRNA appears to still be active on a time scale of days, and the lipids may take several weeks to clear. But I think that the lipid clearance is almost certainly after the LNPs themselves no longer exist as such. That’s really rough answer, but it at least sets some possible bounds.

      1. William Banks says:

        Thanks for your reply Derek.
        The reason I ask is because I am looking into the possibility of the patient ‘shedding’ these LNP’s. If for example the patient happened to catch the flu or had allergies and was sneezing a lot immediately after receiving the Pfizer shot, do you think there is any possibility of both the LNP’s exiting the patients mouth/nose and also being absorbed into another person’s respiratory system? I would hazard a guess that this would be extremely unlikely, but potentially possible. Or not?

      2. Zozo1 says:

        Please see the recently published peer reviewed paper in the oxford university press entitled ‘Circulating SARS-CoV-2 Vaccine Antigen Detected in the Plasma of mRNA-1273 Vaccine Recipients’

        The conclusion is:. evidence of systemic detection of spike and S1 protein production from the mRNA-1273 vaccine is significant and has not yet been described in any vaccine study, likely due to limitations in assay sensitivity and timing assessment. The clinical relevance of this finding is unknown and should be further explored. These data show that S1 antigen production after the initial vaccination can be detected by day one and is present beyond the site of injection and the associated regional lymph nodes. Induction of IgG and IgA immune responses can be detected as early as day five post vaccination and are associated with clearance of spike and S1 antigen in the systemic circulation’’

        I’m interested to hear thoughts on this?

        Also the biodistribution study done in Japan on Pfizer and mice.. showing levels found in ovaries testes uterus etc have you seen that?

  47. R.D.L. says:

    Do we have any updates on the potential of the LNPs & mRNA crossing the blood brain barrier?
    Have either pfizer or moderna clarified their LNP formulation?

    What about the viral vector vaccines- would they have the same issue to some extent?

    1. CK1986 says:

      If you’ve carefully read all the discussion, you would have noticed that, although many have no problem theorizing about potential issues with the novel mrna vaccines, when asked no one has commented on which of the two types of covid vaccines they think would be safer long-term. Too many unknowns, the details of the LNP implementation for pfizer/moderna are still not provided, age restrictions in Europe for the AZ causing further confusion and fear mongering. Biden calls for full transparency which these Pharmas are opposing.
      At the end of the day we are faced with a dilemma that the current risk analysis is not equipped to solve due to the lack of studies/understanding. It’s a number’s game that does not sit well with human psychology. It’s a bit off topic I know but at the end of the day these public forums are also read by non-scientific people looking for a way out of this impossible dilema.

      1. Deb says:

        My sentiments too–it is a dilemma.

  48. smlr says:

    I have no training in chemistry and have tried to understand as much as possible what was discussed on this site especially since English is not my first language. Very informative. What about young adults who would like to have children? Any thoughts about whether there are concerns that the RNA Covid vaccins and the others could have an impact on fertility or on the development of the child?

  49. Zozo1 says:

    Please see the recently published peer reviewed paper in the oxford university press entitled ‘Circulating SARS-CoV-2 Vaccine Antigen Detected in the Plasma of mRNA-1273 Vaccine Recipients’

    The conclusion is:. evidence of systemic detection of spike and S1 protein production from the mRNA-1273 vaccine is significant and has not yet been described in any vaccine study, likely due to limitations in assay sensitivity and timing assessment. The clinical relevance of this finding is unknown and should be further explored. These data show that S1 antigen production after the initial vaccination can be detected by day one and is present beyond the site of injection and the associated regional lymph nodes. Induction of IgG and IgA immune responses can be detected as early as day five post vaccination and are associated with clearance of spike and S1 antigen in the systemic circulation’’

    I’m interested to hear thoughts on this?

  50. Belhou says:

    So i had my first Pfizer injection on May 21st and after reading this article and the answers i’m terrified and at the verge of a panic attack (neurological diseases terrifies me). Do you think it’s safer to stop there and only have one injection or to have the 2nd one (it’s on June 29th)? Or is it worse to only have one shot (because with one shot the immune response to the spike protein would be less effective with enhanced risks to break the balance of the immune system of the body, therefore enhancing the risks of inflammation if there is actually tropism)?

    1. Derek Lowe says:

      It looks like you really should have the second dose to get protection against variant strains. I continue to think that the likelihood of neurological damage from the mRNA vaccines is wildly overhyped by anti-vaccine activists.

      https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(21)01290-3/fulltext?utm_campaign=lancetcovid21&utm_content=168883563&utm_medium=social&utm_source=twitter&hss_channel=tw-27013292

      1. Zozo1 says:

        Hi Derek

        What are you’re thoughts on this peer reviewed study in the oxford university press on the moderna mRNA vaccine.. it shows the spike and S1 protein does travel beyond the injection site and into the plasma / blood stream. This has been one of the main questions on this thread that people have said we need the answer to.

        Thanks

        https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciab465/6279075

      2. Renate says:

        Hi Derek

        What are you’re thoughts on this peer reviewed study in the oxford university press on the moderna mRNA vaccine.. it shows the spike and S1 protein does travel beyond the injection site and into the plasma / blood stream. This has been one of the main questions on this thread that people have said we need the answer to.

        Thanks

        https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciab465/6279075

        I would like to know too. Thank you for all the interesting information.

      3. theasdgamer says:

        Maybe neurological damage is hyped, and maybe not.

        We have to look at normal levels of various coagulopathies and also at coagulopathies per million due to other vaccines.

        I haven’t yet seen a study do that.

        I have a little less agnosticism about the covid vaxxes now because of the swprs report about hospitalizations in the 80+ cohort being reduced markedly compared with younger cohorts after vaccination.

        https://swprs.org/the-latest-on-covid-vaccine-adverse-events/

        Starting to favor the vaxxes for those 70+.

        If the data were from the UK, where hospitalization is rejected a priori, the data would be meaningless.

  51. SJR says:

    Interesting reading, thank you for publishing and interesting to read all the comments, just a quick question, I am 36 male, would you say wait or its ok to have a pfizer or moderna vaccine, i’m a little hesitent, I appricate your responce.
    Many thanks

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