Here, the solution of the germinal center RNA mystery is revealed - but only after an even deeper dive into the mystery itself, to familiarize the lay reader with the 100 different moving parts that are involved in the solution.
Or, head to Spoiler / Summary. if all you are interested in is the conclusion, without all the exciting bits in between.
When you come right down to it, this new spin on an old antivaccine trope is nothing more than an appeal to “nature” as being somehow always superior to anything humans can do.
Naturally, Gorski’s confident predictions that Covid vaccine products (mRNA, and the spike protein it induces) would all be “completely gone after, at most, a few weeks” post-injection were an over-simplification.
Biology is complex, and our understanding of the immune system is still primitive. A rational attitude to take with experimental products is that we should be cautious to rule things out before, you know, the experiments are done. That includes allegedly unlikely findings.
So just how surprising is it that Covid vaccine mRNA and spike could still be detected in lymph node samples up to 60 days after the 2nd dose?2
As it turns out, the findings regarding spike are not surprising, nor do they violate Gorski’s confident prediction. They fall under a sort of fine print clause to the promises that the protein would vanish from the body within weeks. It has long been understood that Follicular Dendritic Cells (FDCs) in germinal centers likely preserve antigen as long as said germinal centers are in “on” mode.
These cells are one of the “bla bla blas” mentioned in Part 1. Their job is to hang onto and hold outward the antigen that germinal center B Cells are developing and honing receptors and antibodies against. It turns out, they like their job. A lot.
In 1996 (already several decades after long-term antigen retention had been observed and theorized about3), Bachmann, et al. “immunized” (presumably by injection4) some mice with VSV.5
VSV is a virus which replicates poorly in mice cells, and yet induces both strong early and late antibody responses. So, if you want to study how the immune system develops strong antibody responses in the absence of persistent or recurring infection, the VSV-mouse model is a perfect choice. By extension, it’s the perfect candidate to template a non-replicating Lipid Nanoparticle with mRNA for the spike protein inside, and what would happen if human immune systems were making a strong anti-spike response even if there wasn’t any more spike floating around freely in the body.
And so, they demonstrated:
VSV, similar to many vaccines, induces a memory B cell response in the absence of a long-term depot in adjuvants6 (also in the absence of detectable viral RNA; U Hoffmann, unpublished observation) and viral antigen persists only in its natural form in association with FDCs. […]
[Immunization with a subunit VSV vaccine produced similar findings.] Thus the induction of long-lived [germinal centers] was independent of the use of a replicating agent but most probably dependent on the presence of persisting FDC-associated antigen. This is supported by the findings (a) long-lived [germinal centers] are associated with persisting antigen, (b) that VSV-specific [germinal center B Cells7]are only found in lymphoid organs containing persistent antigen.
A much more recent paper by Yewdell, et al.,8 provides a succinct description of the current understanding of how this long-term FDC antigen retention works (line breaks and emphasis added; image from a separate referenced review):
How are viral antigens maintained for such long periods of time?
Follicular dendritic cells (FDCs) play a central role in GC B cell positive selection by presenting antigens to [light zone] B cells assessing their own [B Cell Receptors] post-[Somatic Hyper Mutation] […]
The ability of FDCs to internalize complement-coated immune complexes to nondegradative endosomal compartments, which can then undergo multiple rounds of surface recycling […] make them a prime candidate to store persistent viral antigens.
Heesters, et al. Graphical Abstract. Follicular Dendritic Cells cycle immune complexes in and out of their uniquely inert exosomes, allowing for long-term preservation of complex-attached antigens without degradation.
FDCs are generally thought to be able to retain intact antigen for weeks to many months, although experimentally validating the limits of FDC antigen retention has been a major challenge […] Our data, in conjunction with the current model for GC B cell positive selection, indicate that FDCs may retain native [influenza hemagglutinin protein] well past 100 days post-infection.
Foll Guy
So, long-term persistence of spike inside of germinal centers is what should be expected if spike is simply one of those proteins that, for whatever reason, lead to this type of “let me get this right” response on the part of the immune system.9 Spike would further be expected to appear in a nebulous pattern, reflecting the distribution of FDCs in the germinal center. But is long-term retention actually normal, or rare?
Frustratingly, few modern experiments into long term germinal center responses actually verify persistence of antigen,10 which means that assuming this should apply to any random new spike protein is hazardous. This includes not only the paper quoted above, but studies which have observed evidence of long-term germinal center activity after infection or receipt of the Covid vaccines.11
However, we should take it as overall a likely prima facie explanation for the persistence of spike in the findings by Röltgen, et al., which would potentially let the Covid vaccines off the hook.
If it wasn’t for the RNA.
The District Attorney mulls the evidence collected by the detectives so far. The state’s case is shakier than it seemed at first, but the RNA feels likely to get them over the finish line. It’s got smoking gun written all over it. Remember that tiny detail from Bachmann, et al.?
VSV, similar to many vaccines, induces a memory B cell response in the absence of a long-term depot in adjuvants (also in the absence of detectable viral RNA […])
The DA turns to the detectives. “Bring him in.”
The scene fades out, as the officers are reading the Covid vaccine its rights…
Law and Order: FDC
A call from work takes you away from the movie. By the time you reenter the theatre, the trial is already at climax.
The state’s star witnesses, one after the other, have laid out the case for why Covid vaccine RNA, unlike spike protein itself, can’t be explained by means so innocent and rosy as long-term retention of antigen by Follicular Dendritic Cells. Ergo:
Covid vaccine RNA must be integrating into DNA in some fashion, and being recycled that way, with eventual export of future copies to lymph nodes. The state’s first expert pointed to a DNA integration study from Sweden.12
Or, Covid vaccine Lipid Nanoparticles are sequestering somewhere after injection, and not being taken up by cells until later release. The state’s second expert claimed that the extracellular matrix of tumors - which are adjacent to porous capillaries, making them easy vectors for LNP therapies by default - are an especially likely location. This would neatly explain why these particular lymph nodes had so much Covid vaccine RNA, but sequestering could occur at a low rate in these largely cell-free areas throughout the body, even next to muscle.
Well, just which expert is it that the jury is meant to believe? Is the state trying to have it both ways? It didn’t seem to matter. The faces behind the rail spelled doom for the accused. These are American jurors, after all; if a Scientist™ tells them to send someone to prison, they do what they are told and don’t ask questions.
Nonetheless, the defense was at least prepared for the first witness, as said witness represented the case the state was expected to argue. And so she crossed with gusto, peppering the State Paid Credentialed Witch Doctor with questions about the weaknesses of the DNA integration study from Sweden.
It was the second expert witness that caught the defense unprepared. Tumor extracellular matrix? Enhanced permeability and retention?! That’s a thing?!13 The defense, in cross, was off-foot and wavering, like a boxer trying to catch their bearings after an unforeseen jab. The expert witness was ostentatiously delicate in explaining to the defense why her questions were more akin to a gargle of pool water than a “Marco?” It was only by miraculous luck that the judge adjourned early, even though it would inconvenience the exalted expert; something about a fresh round of riots somewhere in the city.
Not that you know any of that. You have walked back in as the lawyer who you don’t even know is the defense is at her wits end, in a darkened office, surrounded by sprawling printouts of evidence and notes. What’s even going on? Wasn’t this a murder mystery? Why is this lawyer suddenly the protagonist? You didn’t pay 5 bucks for a mystery story just to be misled and confused!
The defense stands up from her chair, and walks across to another surface carpeted with documents. She holds up a particular photograph for re-inspection. You recognize this one. It’s the scene of the crime.
A thought seems to strike her. In a flash she is shuffling through the surrounding paperwork.
She lands on the full case report of the detectives who first encountered the crime scene: Röltgen, et al.
Ah, yes - good thinking. What, exactly, did they make of these results?
First, the detectives offered background (emphasis added):
analysis of a model RNA vaccine for yellow fever virus in a rhesus macaque at 16 h postvaccination showed that vaccine RNA in LN cell suspensions was detected predominantly in professional antigen-presenting cells including monocytes, classical dendritic cells, and B cells at this early time point (Lindsay et al., 2019). Data from follicular dendritic cells were not reported.
And there it is. In a previous experiment, where monkeys were given an RNA vaccine, the RNA was taken up by Antigen Presenting Cells, the executive “bosses” who migrate from the site of immune challenge to the lymph nodes. Distinctly, these executive cells run germinal center activity from outside.
Ruffling through more papers, the defense finds the corresponding results from the monkey study. It all matches expectation. RNA (marked in blue) appears in the space outside of and adjacent to the germinal center “thumb-prints,” and is further confirmed to be found within Antigen Presenting Cells (including non-FDC Dendritic Cells14) by other tests (i.e., flow cytometry):
Lindsay, K. et al. Fig 7. Rhesus macaque lymph node at 28 hours after intramuscular injection of mRNA vaccine for Yellow Fever protein. Vaccine RNA (blue) is adjacent, but outside of germinal centers, suggesting localization in Antigen Presenting Cells - as would be expected at the initiation of novel immune responses.
That looks nothing like what Röltgen, et al.’s RNA probe found. Returning to the cartoon of lymph node structure:
Here, orange area corresponds to “new antigen,” navy areas to “antigen being worked on for any number of days, weeks, or months.” And so in the crime scene photos, it doesn’t really seem like the Covid vaccine RNA is being brought to the lymph nodes - but that it is residing in the same FDC network that let the Covid vaccine off the hook for the lingering spike protein before you walked out of the theatre.
The defense reads on. What did Röltgen, et al. make of this discrepancy? Regarding their own findings, the detectives remarked:
Our histological data from SARS-CoV-2 mRNA-vaccinated humans at considerably later time points [than the monkey study] (7–60 days post-second dose) show vaccine RNA almost entirely in GCs, distributed primarily between the nuclei of GC cells, similar to the pattern seen by immunostaining for follicular dendritic cell processes or B cell cytoplasm.
Note the negative space. Our detectives essentially had no explanation to offer for why the Covid vaccine RNA seems spatially aligned with FDCs and/or germinal center B Cells, and not with the executive cells outside of the germinal centers. And so in the “discussion” section of their experiment, they merely report this fact, without any discussing.
The defense sets the detectives’ notes down, and gazes at the crime scene photo once again. And what the detectives reported in their notes seems almost irrefutable. Covid vaccine RNA is exactly as “stuck” in germinal centers as the spike protein. It has exactly the same “nebulous,” oblong footprint, suggesting localization in FDCs:
And what are some of the effects, or concerns, with adding the polyethylene glycol, or PEG, to the Lipid Nanoparticles?
EXPERT
Well, there are so many. For one, it reduces initial detection by the immune system. So in this way it promotes dispersion throughout the body. We saw in the BioNTech distribution study that the LNPs with this formulation leave the injection site very quickly, and end up in the blood. That’s different from previous mRNA vaccine experiments, so I think we have to blame the PEG for that. The LNPs can’t be intercepted by the immune system and they protect the RNA until it goes into a cell. Before that happens, other molecules in the blood have to attach to them. And if you disperse through the whole body, in the blood, you are going to deliver to places where the vasculature is more “leaky.”
Another danger, sort of a contradictory one, is that the immune system may form antibodies against the PEG. Like any foreign molecule, this is a potential. And this may effect the reaction to the second dose or to boosters. We don’t know.
Is it “normal” to find RNA from vaccines or viruses in the germinal centers? Is that something that’s just part of the immune response?
EXPERT
No. That’s something that has only been associated with chronic disease. For example HIV, Epstein-Barr, Chikungunya virus…
PROSECUTOR
So why is it happening here?
EXPERT
Well, I think it’s pretty obvious that the Covid vaccine is causing a diseased immune response. It may be that when sequestered vaccine RNA is discovered by Antigen Presenting Cells and brought back to the lymph nodes, they are somehow passing it onto Follicular Helper T Cells.
PROSECUTOR
Is there a way to verify that?
EXPERT
Well, unfortunately, the detectives did not perform an analysis of the type of cells that were positive for RNA. All we know is where the RNA was. So it could actually be infiltrating the Follicular Helper T Cells or germinal center B Cells somehow. I think that, even though these germinal centers look very orderly, they are clearly in a state of disease of some type.
Her eyes suddenly go wide. She dives into another pile of paperwork. From deep within, she pulls out a study - one of the references mentioned by the Star Expert when discussing the possible resemblance to HIV (not that you would know that). It’s an old one.
Mistrial
Another plot point revealed during your phone call was that the defense, quite naturally, doesn’t actually believe in her client’s innocence.
The Covid vaccine will not be taking the stand, for example, as to do so would allow the prosecution to bring up the “exosomes study” in cross.15 There is ample reason to at least be worried that “sequestering” and long-term expression of Covid vaccine mRNA is occurring. She knows this. But her priority is only to convince the jury that this evidence - the study by Röltgen, et al. - does not actually prove the crime.
She will be successful. And her (resumed) cross of the star expert will be full of tension and flair.
First she will get the expert to agree that very little research has ever looked for long-term retention of vaccine or virus RNA in germinal centers. We don’t know if it only happens for chronic illness, or if it even happens all the time in infections that can lead to chronic illness. Even in HIV research, for example, the persistence of RNA in germinal centers has been largely neglected since its initial discovery.16 So the older research is still pretty much what we have to work with, when looking at RNA retention in germinal centers.
Then she will get the expert to agree that, setting his theory of a diseased germinal center condition aside, the distribution of RNA is what would be expected if the RNA was being retained by Follicular Dendritic Cells.
Further, that this would only be likely if the RNA was still in the LNPs - otherwise the RNA would be degraded over time, pseudouridine or not.17 Meanwhile, actual real-world evidence supports the notion that RNA can survive inside the LNPs at body temperature much longer than implied by the hyper-cautious storage conditions and shelf-life attached to the Covid vaccines by their manufacturers (obviously the expert agrees with this one, since he proposed it in his testimony).18
She will review the expert’s testimony about what happens when the LNPs enter a cell, normally. During entry, the LNP “bubble” gets surrounded in another bubble (the endosome). As the bubble becomes more acidic, the “ionizable” lipids gain a positive charge, which leads to eventual bubble-bubble fusion and release of the mRNA:
But there is one type of cell in which “ingestion” of the LNPs without release is likely. The expert will concede that Follicular Dendritic Cells immune complexes cycle in and out of specialized endosomes that are designed not to release their contents, and maintain only mildly acidic interiors. Importantly, these FDC recycling endosomes require that the “antigen” in question is contained within an immune complex in advance. And the immune complex requires an antibody that corresponds to the antigen:
She will get the expert to agree that Röltgen, et al. did not look at what was going on with germinal centers immediately after injection, so their comparison with the monkey study is limited in that respect. In fact, Röltgen, et al. are looking at germinal centers only after the 2nd dose. This means there is a previously-primed immune response to consider.
She will get the expert to agree that this previously-primed immune response could include anti-PEG antibodies (after all, the expert already proposed the same). The expert, seeing where this is going, will express extreme skepticism that an anti-PEG antibody could lead to the formation of an immune complex capable of causing the entire Lipid Nanoparticle to be effectively recycled in FDCs.
Nonetheless, the defense will ask the expert to propose what this would mean. If retention of spike in Follicular Dendritic Cells goes with honing the anti-spike antibody response, then retention of LNPs in Follicular Dendritic Cells goes with honing the anti-PEG antibody response. Once the process finishes, the germinal centers would degrade, and the LNPs and RNA would dissipate. The defense will ask if this would account for the drop-off in RNA detection after day 37?
The expert will begrudgingly acknowledge that it would.
She will then walk the expert through the findings by Eitner, et al., in 2000, which were briefly referenced during the expert’s original testimony as one of the very few previous examinations of lingering RNA in germinal centers.19 To quote (emphasis added):
A shift of HIV-2 distribution was demonstrable between day 10 and day 14 after HIV-2 infection.
Coincident with a marked reduction in individual HIV-2 RNA cells [as measured by in situ hybridization, the same method usedby Röltgen, et al., the defense will point out]by day 14 post infection, there was a dramatic increase in germinal center-associated HIV-2 RNA. High concentrations of HIV-2 RNA persisted in germinal centers in all animals by days 21 and 28 postinfection.
Thus, HIV-2 appears to go through an initial, highly disseminated cellular phase followed by localization in the follicular dendritic cell network with relatively few infected cells.
The expert will rejoinder that this supports the suggestion that long term retention of RNA in germinal centers is, vaguely, “disease”-y. But he will have to concede that in the HIV precedent, long-term RNA localized to Follicular Dendritic Cells. The FDCs, on day 21 and afterward, were “holding on” to the detected HIV RNA. They weren’t being delivered new RNA from outside.
She will ask the expert to read out from the discusion section of that paper, where the authors propose an account for in what way the HIV RNA is being housed in Follicular Dendritic Cells (emphasis added):
Alternately, the early movement of virus into the FDC network may be a protective event that ameliorates early dissemination of the virus and/or is a consequence of virus trapping by FDC of virus complexed to anti-HIV antibodies.20
She will ask if this is a plausible explanation for the retention of Covid vaccine RNA in germinal centers. That by the 2nd dose, anti-PEG antibodies are enabling entire LNPs to be preserved in the FDC recycling system.
The expert will concede that it is.
Having gotten the state’s Star Expert to concede the plausibility of Covid vaccine RNA being trapped in germinal centers simply due to said germinal centers working on the anti-PEG antibody response for a while, the defense has won her client a mistrial.
At the time, it was believed that adjuvants, namely alum, partially achieves its immune-response-boosting effect by sequestering antigen, as opposed to “because poison.” Bachmann, et al. are thus pointing out that long-term persistence of VSV antigen is likely not to depend on the “depot” effect. The depot myth has only recently been overturned (but not the “poison that the body has no way to cycle out is totally safe” myth).
In the absence of a definitive mechanism of action, alum has remained in constant clinical use for the past 80 yr, and throughout this period, the depot theory of alum adjuvant activity has persisted. However, no evidence exists in the literature to demonstrate the importance, or otherwise, of the antigen depot in the enhancement of antigen presentation and subsequent primary T-cell and B-cell responses by alum adjuvants.
Bachmann, et al. refer to germinal center B Cells as “antibody forming cells,” seemingly meaning that they are future Plasma Cells, rather than both future Plasma Cells and Memory B Cells (as in today’s use of the term) - but this may be a confusion on my part.
In the recent experiment by Yewdell, et al. (cited in footnote 8), influenza infection of mice was observed to be followed by germinal centers that were still producing new Memory B Cells against the hemagglutinin protein for up to 150 days. However, no samples were tested for actual presence of said protein. Instead, B Cell receptors specific to the protein were detecting by sticking protein to them, and persistent presence of the protein in FDCs was just assumed, bizarrely.
The authors do, however, attempt to gain insights on why the response is being prolonged under that assumption, and intimate that germinal centers may be “hanging” on certain proteins that degrade during prolonged FDC recycling, but potentially re-stimulate immune response via “dark” antigens (meaning simply internal structures that become interesting to the immune system once they are exposed). But how this would somehow circumvent the executive roll played by Antigen Presenting Cells which are outside of the germinal center, and already digested the same protein into bits before activating the Follicular Helper T Cell that should be managing the B Cell response, is unclear. Does this all have relevance for the SARS-CoV-2 spike protein, that alleged monster with a hundred different suspicious epitopes? Could normally internal epitopes be creating weird antibodies we don’t know about? Who knows. The relevant text begins at “While the presence.”
And if the spike protein is of the “let me get this right” variety of antigens, additional doses of the mRNA transfections beyond the 1st one are entirely superfluous to the (long term) immune response (the plasma spike evaluations of Röltgen, et al., which showed evidence that dose 1 antibodies effectively block detection of new spike induced by the second dose, reinforce this conclusion).
So not only the 3rd but all of the 2nd doses were pointless all along; just 1 dose could likely achieve durable severe efficacy. “Funny.”
Fine Needle Aspiration biopsies of lymph nodes were evaluated for spike-specific germinal center B Cells, finding positives up to 15 weeks (the last sample date). No attempt to detect presence of the spike protein was made.
This one is likely a tiny, morbid goldmine, no idea why I haven’t seen it before. Tiny because there were very few samples and infection times are only known for two; morbid because the samples were from deceased organ donors; a goldmine because the samples were measured for Resident T and B Cell persistence. I will be reviewing it.
All samples were tested for spike-specific immune cells; no attempt to detect presence of the spike protein was made.
Solid tumors have two properties that favor drug/particulate accumulation and retention in tumors, i.e., leaky tumor blood vessels allow large molecules to extravasate whereas defective lymphatic drainage decreases the clearance of compounds from tumor interstitium, referred to as the enhanced permeability and retention (EPR) effect [47,48].
For EPR, retention plays a larger role relative to extravasation. EPR is predominant for compounds with molecular weights larger than 40 KD but negligible for smaller molecules that readily redistribute to blood circulation via diffusion and/or convection.
EPR is affected by the tumor size with a greater EPR in smaller tumors [47]. This is likely because of the greater vessel density allowing for more extravasation in smaller tumors compared to larger tumors with greater fractions of avascular regions. […]
Two major components of a solid tumor are tumor cells and ECM [extracellular matrix]. Both constitute significant barriers to interstitial transport [5,9–14].
ECM comprises fibrous proteins (e.g., collagen, elastin) and polysaccharides (e.g., hyaluronan, glycosaminoglycan) [18]. These proteins are a source of physical resistance to diffusional transport and are associated with lower hydraulic conductivity and lower convective flow in interstitium. Collagen appears to contribute more to transport resistance compared to glycosaminoglycan or hyaluronan, e.g., diffusion coefficient of IgG is inversely related to the collagen content in a tumor.
However likely (or not) it may be that this leads to long-term sequestering of Covid vaccine LNPs in real life, it serves as a poor explanation for the RNA distribution observed by Röltgen, et al., due to the reasons articulated by the defense in “the movie.”
Although sharing the name, Follicular Dendritic Cells are considered to be unrelated to other Dendritic Cells, and to not derive from the hematopoietic stem cells that give rise to all other immune system cells (as well as to Red Blood Cells and platelets). They reside in the lymph node follicles that convert into germinal centers, and partake in what is essentially the most menial task of the immune system; whereas all other (hematopoietic-derived) Dendritic Cells wouldn’t be caught dead in a germinal center (it would be a waste of their management talent).
Caveat: As far as I can tell. When Heesters, et al. parlayed their FDC research into examining HIV, they did not even cite the earlier work by Eitner, et al., apparently unaware that the mechanism for retention they were setting out to validate had been proposed over a decade before (see footnotes 19 and 20).
Caveat: Germinal center B Cells seem to have some interesting RNA-expression prolonging traits, but I think this is unlikely to be relevant. Too 3D-chess-y.
Aside from RNase (which may still end up in LNPs in trace amounts during manufacture, because RNase is ubiquitous in all environments), RNA is degraded by water. And yet Roesler, et al., in one of the only real-world attempts to test the limits of RNA storage, demonstrated that room temperature degradation of RNA is not all that fast.
Further, LNPs store RNA in a mix of lipids and water, with the lipids potentially shielding the RNA molecule, or the physical dynamics of water-RNA interactions potentially different than in a pure aqueous solution (akin to how air-exposed water surface behaves as a film). At all events it seems unlikely that the LNP interior makes matters worse than pure water storage.
So as long as the LNPs are intact, the RNA within is probably just fine at body temperature for quite a while. So why not just store it at room temp before injection; and why insist on throwing out product after a few hours in the refrigerator?
The mRNA Covid vaccine manufacturers may simply be imagining that expression efficacy will still be significantly safeguarded/improved by cold storage (obviously they could not have real-world data on long term performance of non-frozen product in advance of licensing/distribution, since they didn’t have any long term data on anything).
Eitner, et al.’s theory for immune-complex retention of HIV virons was finally validated in 2015 (caveat: as far as I know; but there may have been other papers), by Heesters, et al. (who are responsible for the 2013 FDC recycling study).
Well of course there’s no reason anyone should think “nature” in any way superior, after all, “nature” has only been tinkering for a billion years, we’ve been at it for a few hundred, and we’re super duper smart.
Well of course there’s no reason anyone should think “nature” in any way superior, after all, “nature” has only been tinkering for a billion years, we’ve been at it for a few hundred, and we’re super duper smart.
Reading this will be my 'Sunday night fights'....I so appreciate your quality/frequency ratio.