Clarifying Yesterday's Lipid Nanoparticle Post

Better, nonredacted information and more context to add to the discussion from yesterday.

Thank you Brian Mowrey for providing me with a report from Australia that provided information on some of the redacted portions of yesterday’s study (R-20-0072). Please check out his Substack. I have really appreciated his insights into Original Antigenic Sin which contrasts a lot of the ideas posted on Substack. It’s nice to know that so much discourse has been happening on Substack. It really provides for the best, open discussion that we would otherwise not get anywhere else!

Yesterday I provided a preliminary examination of the Lipid Nanoparticle Study R-20-0072 that has been released as part of the Pfizer/BioNTech Comirnaty documents released on Tuesday.

To my chagrin a lot of the information was redacted and so it was really hard to parse a lot of the information and provided just a basic analysis.

However, another Substacker Brian Mowrey provided me with a report from Australia’s government that actually provides more insights into the study and even includes some of the figures. Because of that I will try to clarify some additional information from yesterday’s post that I have missed. Also, please make sure to check out Brian’s Substack:

Correcting the R-20-0072 Preliminary Analysis

To start there were quite a few things I have missed yesterday which I probably should not have.

One of the most important factors was the modRNA which I overlooked. In order to mimic the genetic code for the spike protein BioNTech came up with different formulations for luciferase to see what would happen:

Yesterday I thought the modRNA referred to modifications made (such as additional sequences or swapped bases) to the luciferase gene. However, as we can see the modRNA refers to the nucleoside swap done to the gene. More importantly, it refers to the swap of the uridine for pseudouridine which would match the formulation deployed in the vaccine’s spike protein.

So all of the studies from yesterday utilized this luciferase gene containing pseudouridine. This is important, because when I mentioned that the modRNA produced an innate immune response I did not take into account the pseudouridine. As we know full well by now, the difference here means a longer lasting RNA which has been raising a lot of concerns recently.

Also, it’s important to remember that the study did not specify whether the response was due to the LNP or due to the modRNA. It may be a misreading of the summary, but I took it to mean that the only difference between LNP administrations is the formulation of LNPs themselves. Therefore, an assumption can be made that it was the LNP formulation rather than the modRNA that is eliciting an innate immune response as the modRNA used was the same across all administrations.

Lastly, I conflated the adaptive immune response when looking at the results for luciferase IgG and the T-cell response. As Brian pointed out in the comments, luciferase assays may not be able to detect an adaptive immune response when only measuring IgG levels, which represent B cell immunity and antibody production. The T-cell response measured by ELISpot likely creates a more accurate representation, in which case the study does indicate an adaptive immune response occurred against luciferase albeit without antibody production. It’s also important to keep in mind that the IgG measures were done with an ELISA¹ test using sera collected from the mice while the ELISpot test used splenocytes (cells from the spleen) and isolated T-cells from there, as this distinction may have played a role in the different results as well.

I apologize that I did not analyze the information with a greater discerning eye yesterday. Even with all of the redactions some of the information was still available. Also, it pays to look at the materials and methods of a study! Again, thank you to Brian for pointing out the adaptive immune response discrepancies and please check out his Substack!

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Adding Clarity to R-20-0072

Now I’ll add some more additional information from the report provided by Brian and the Australian government which can be found here. What’s interesting about this document is that it provides summaries for many of BioNTech’s studies so it may be worthwhile to keep this document on hand.

Examining the LNP Formulations

First, this report actually provides us with the formulation for BioNTech’s LNP. This part is a bit unnecessary but I think it just provides a bit more context, but feel free to skip if this is not interesting.

So we know that LNP8 is the one that BioNTech is using in their vaccines and I showed their structures yesterday, but LNP12 is the one from BioNTech’s own in-house formulation.

What’s important with the formulations indicated above is the additional information that comes with it.

We’ll look a little more close into BioNTech’s in-house LNP formulation (first row).

The DODMA refers to the lipid shown below:

The structure of DODMA. It contains two chains of 18:1 oleic acid and has been used often in drug delivery systems.

Chol is actually easy; it refers to cholesterol. Cholesterol is vital for our own cell membranes as it provides some structural integrity to our membranes, and so the same may be happening with the addition of cholesterol here.

DOPE has quite a long name (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), but it refers to the structure below:

DOPE. It has a similar structure to that of DODMA aside from the phosphoethanolamine group located on the right.

PEGcerC16 refers to a lipid containing polyethylene glycol and ceramide. The “C16” refers to the length of the lipid chain (16 carbons long). I could not find an exact image, but a similar one (PEGcerC14) suffices below, just remember that the lipid chains should be two carbons longer. Also, do not take the polyethylene subscript into consideration since the actual value may be different:

PEGcerC14. Aside from the two fewer carbon atoms and the unknown polyethylene count, the structure here should be similar to that of PEGcerC16.

Next is the ratio. If you look at the LNP12 formulation you can see the numbers 40:48:10:2. That value is a ratio and it refers to the ratio of DODMA:Chol:DOPE:PEGcerC16. Another way of reading this is that for every 40 DODMA molecules there are 48 cholesterol molecules, 10 DOPE molecules, and 2 PEGcerC16 molecules. Although this may not translate to the actual ratio of the lipid nanoparticle membranes that are formed, it provides a relative value and allows you to know the proportion of each molecule. If we are concerned about the LNPs that are used this would be important to keep in mind. Unfortunately this post does not indicate the ratio used within the Acuitas blend or the GMP-ready blend, which is even more unfortunate since this is the formulation that BioNTech has gone with.

A few more things to point out:

• RNA-EH190611-01c refers to the modified luciferase gene that contains the pseudouridine.

• FSU/FM may refer to the LNP batch used, although I cannot determine that one specifically.

• % encapsulation: if anyone was unsure how the mRNA got into the LNPs, imagine those bottles of micelle toners found in the skincare aisle of drug stores. When you shake those bottles you can see the tiny blobs of micelles² suspended within the liquid. It’s a simplified representation, but to encase the spike mRNA both LNPs and mRNA are combined into a suspension and shaken up. Afterwards, the LNPs come together and surround mRNA to form micelle structures which are then isolated. The % here refers to how many of the mRNA molecules become encapsulated within the LNPs. It’s comparable to a % yield value seen in chemical reactions. This is important in determining how many of the micelles may contain mRNA. Also, from a business perspective it would indicate how “wasteful” the formulation would be if it had trouble encapsulating the mRNA.

• Diameter is really interesting. Remember in my hypothesis post that I said it may be more pertinent to examine these vaccines through the lens of a “pseudovirus” with the entire process mimicking a “pseudoinfection”. As you can see with all the diameter values above they hover around the 70-100 nm size. For comparison, the diameter of of a SARS-COV2 virion is estimated to be 100 nm meaning that these micelle structures are either comparable or even smaller. This is an important comparison to keep in mind, especially when considering where the vaccines may disperse. Also keep in mind that it was a hypothesis and is based on conjecture.

• Temperature Storage: It’s strange that the storage temperature for BioNTech’s in-house formulation was kept at refrigeration while the other ones were kept at -80C. It may not provide for a comparative assessment but there may have been a stability assessment done beforehand.

So was all of this information necessary? Not really since we know that the LNP8 formulation was the one BioNTech went with, but I do think it is interesting to at least break down some aspects of that list and understand what different parts of it meant and see how they came up with the formulation. It also helps to understand its important in some of these studies as well.

Luciferase Studies

Here’s where the interesting stuff comes in (if the stuff above was not interesting). For the following results I’ll refer mostly to the Australian report because it is not so redacted but I may reference the redacted FDA documents for comparison.

Starting with the luciferase studies we can see that the 20-fold higher expression occurred within the mice given LNP8 (formulation in use) and LNP5 compared to the mice given LNP12 (BioNTech’s in-house formulation), which would at least suggest that expression of luciferase was higher in those two formulations compared to BioNTech’s.

This is a point worth emphasizing; the expression of luciferase does not exactly correlate to the movement of LNP within the mice. LNP may still be travelling but the modRNA may not be uptaken or not translated to allow for luciferase expression. A way to rectify this would have been to use radiolabeled LNPs so that they could be tracked that way (this is usually the way that drugs are tracked for metabolism studies). However, this study was not powered to do so, so our interpretations are limited to expression. We also have to keep in mind that luciferin³ needs to be provided in order to undergo enzymatic reaction and glow, and so the pharmacokinetics of luciferin itself is at play as well.

Higher injection luciferase expression was seen with LNP8 and LNP5 compared to LNP-C12 and control. Expression continued until day 9 where mice were sacrificed in order to collect splenocytes. Questions should be brought up as to how long luciferase expression would occur if mice were alive for longer periods.

One thing to note here is that luciferase expression still occurred nearly 9 days afterwards within the injection site. There should be questions as to why luciferase is still being expressed more than a week after injection. It could be that luciferase is not being expressed on the outer membrane and is not likely to be as easy of a target for removal. There’s a lot of factors to take into consideration, including how well this finding translates to spike proteins.

One of the most important things to keep in mind here is that LNP8, the formulation in use with these vaccines, showed luciferase expression in many parts of the mice as soon as 6 hours post LNP injection. Keep in mind that expression could only be measured starting at 6 hours.

Red circles indicate injection sites. The injection was halved and each hind leg was provided one injection each. Both LNP5 and LNP8 formulations show visible luciferase expression starting at 6 hours post injection. However, the LNP8 injection shows high expression in other parts of the body.

If you looked through the redacted paper from the FDA, you may notice that all but the portion on the bottom left was kept in. What’s frustrating is that even with the portion they kept in they cut out the imaging showing luciferase activity within the upper portion of the mice:

Why would the redacted information remove the image that shows high levels of luciferase activity outside of injection site? At this point we have very good evidence that these LNPs are in fact travelling, but the idea that BionNTech would cover up this information within their redacted documents is absolutely astounding!

Because of my lack of rodent anatomy I can’t assume which organ may be expressing luciferase, but take into account that luciferin was injected into the abdominal region, which suggests that a high level of LNP has made their way into that region, the modRNA was uptaken, and luciferase was expressed. Again, all things we were told would not happen!

The researchers suggest that this is an indication that LNP8 drains into the livers (which explains their protocol within the redacted study):

LNP8, the LNP formulation in use with Comirnaty, showed expression within the livers of mice. Expression declined 24 hours post injection, although the initial expression should provide some concerns. Further studies should have been conducted to explain this finding.

But again, we have to take into account the dynamics at play here between LNPs, modRNA that are long-lived, and luciferase expression. This could very well mean that LNPs along with modRNA have travelled to the liver, which is usually the main site of metabolism of exogenous substances, but luciferase expression would indicate that uptake of the modRNA has occurred. Also, we cannot leave out the possibility that free-roaming modRNA may have found their way to the liver, although there’s no way of examining that through the study presented.

Either way, there needs to be an explanation as to why this is occurring, and what this means for other parts of the body that may not be detectable.

Activation of Innate Immune System

I would argue this one is relatively unchanged. No figures were provided within the Australian report but the main findings are below:

Overall LNP8 formulation showed an increase in inflammatory markers with LNP5 showing an even greater level. A caveat here, and one I should have been careful with, is that there’s no way of determining if the innate activation came from the LNP itself or from the modRNA, however speculation from the researchers within the redacted summary suggest that the difference in formulation may attribute to the difference in innate immune activation.

Activation of the Adaptive Immune System

So here is where I botched my initial analysis. The two assays should be assessed independently, as Brian pointed out. Although the ELISA assay for IgG expression turned up negative (suggesting no B cell antibody production), the T cell analysis indicated sensitivity to luciferase, which suggests that an adaptive immune response has been developed against luciferase.

Isolation of T cells from splenocytes show a high level of T-cell activity against luciferase in both LNP5 and LNP8 relative to LNP-C12 and control. Lack of AH1 T-cell activity suggests that the T-cell response seen can be attributed to luciferase.

These results should override my interpretation from yesterday’s post as this provides a more clear picture that T-cell expression did in fact occur.

Conclusion and Final Remarks

Lastly, here’s the conclusion provided within the Australian Report:

Now, I have problems with the first conclusion. Although luciferase expression was much higher within the injection site, there should be many questions about the level of luciferase expression within the liver and possibly other organ sites. Remember, we were told it does not travel! As more people on Substack continue to sift through other papers we may see if this gets clarified in other studies, but remember to keep in mind that this formulation is the one that is in use.

That leaves us with a few takeaways and my own concluding remarks:

• The LNP formulation in use by BioNTech for their COVID vaccines are the LNP8 “GMP-ready” formulations.

• High expression of luciferase was seen within the injection site but also within the liver, especially for LNP8. What this means for the COVID vaccines itself is unknown although it raises questions as to how much of these LNPs are travelling to other parts of the body.

• BionNTech’s redacted document sent to the FDA left out the images which clearly show luciferase expression in the liver (or at least regions outside of the injection site). One has to wonder if the idea that these vaccines do not travel was pushed by the FDA based on these types of results, in which case I would argue that would be indicative of spreading misinformation.

• Either the LNPs, modRNA or possibly the luciferase enzyme may be causing an innate immune response. The researchers commented that the formulation rather than the payload may be at play, which would suggest innate immune activation may be derived from the LNPs. Discussions about the immunosuppressive nature of the LNPs should take this into consideration and examine the discrepancies between these two ideas.

• T-cell adaptive immune response occurred against luciferase, although no B-cell and IgG antibody production appears to have occurred. Several factors may be at play here that were not addressed within this study that would otherwise elaborate on this difference.

• Timing of the luciferase activity needs to be taken into account. Luciferin was provided 10 minutes before the 6 hour post injection mark, and so the movement of luciferase and LNP is unknown before hand. Also, the endpoint of 9 days occurred due to the sacrifice of the mice to collect their splenocytes. Since considerable expression of luciferase was still seen within the livers of LNP8 mice further studies should have been conducted to see how long this luciferase expression would have lasted. Considering that we now have evidence that spike mRNA and spike proteins itself may persist up to two months after injection the lack of information here is quite worrying.

• The analysis of these reports need to taken with care such that full parallels cannot be drawn between luciferase and spike proteins, especially considering that spike proteins may be transported to host cell membranes. Also, the cytotoxicity of the spike proteins produced by the vaccines are still up to debate.

Hopefully this provides more clarity and removes some of the confusion that may have arisen from yesterday’s post. I apologize for not viewing the reports with a more keen eye. Also, once again thank you to Brian Mowrey for providing me the Australian Report that helped to tie everything together! Please check out his Substack.

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1

ELISA stands for Enzyme-linked Immunosorbent Assay. The way this assay is conducted is that an enzyme is attached to a plate or well. Then, samples are added to these wells/plates, such as the isolated sera from these mice. If exposure to luciferase produced an adaptive immune response and produced IgG antibodies in the mice, the antibodies should adhere to the luciferase enzymes in the plate/well (hence the immunosorbent aspect) if there are any. Usually a fluorescent molecule is added that fluoresces when binding between the IgG antibody and the luciferase enzyme occurs. After incubation the plate is measured for fluorescence.

2

Micelles are comprised of amphiphilic (polar group attached to a nonpolar/lipid group) molecules that self-aggregate into either a monolayer or bilayer structure. A monolayer structure has the polar head groups pointed outwards while the nonpolar head groups point inward away from the polar suspension. Bilayer structures mimic those of our own cells. The idea with these vaccines is that the LNPs, when shaken, will start to aggregate together and form micelle structures (likely bilayers) that surround then mRNA then encapsulate them within the structure as they complete aggregation.

3. As per the protocol luciferin was added 10 minutes before measures of activity was taken. Luciferin was added peritoneally (within the abdominal region) and so the pharmacokinetics and dispersal of luciferin needs to be taken into account.