Jumping at shadows

I've been directed to a rather rambling piece by Robert Malone, nominally about pseudouridine but it takes a while to get there as the first half is about his favourite subject: Robert Malone. I can see from that segment where the 'inventor calls mRNA gene therapy' trope probably comes from, because he does discuss the use of nucleic acid delivery systems in gene therapy. But this does not make RNA vaccines gene therapy: gene therapy involves the complementation or repair of defective host DNA genes, and the mRNA vaccines do not contain such genes, nor even any DNA.

Malone has legitimate concerns about DNA-based gene therapy's safety, and points out that genotoxicity studies are required by the FDA, as it is potentially carcinogenic. These studies were not required for RNA vaccines because RNA should not affect DNA, and RNA is normally degraded in a matter of hours. Of course, anyone who has been following along will be aware that a Swedish study has been touted by Malone, McCullough and acolytes as firm evidence that vaccine RNA can be converted into DNA ('reverse transcribed') and integrated in the genome. As my linked piece shows, however, this is unjustified. No genomic integration was shown in the Swedish study, and the mechanism is not vaccine-RNA specific anyway. The proposal is that a transposable element, LINE-1, that normally RTs its own RNA sequence, may grab a nearby RNA instead, and if that's vaccine RNA, that's bad. But, where such off-target RT occurs, it will grab any nearby RNA regardless where it comes from, be that vaccine, virus or native RNA. It's not the vaccine that 'changes the genome', but LINE-1. If it's a problem, it's a problem irrespective of vaccine. 

And so, at last, to pseudouridine. RNA is a polymer formed of strings of four monomers, denoted by the letters A, C, G and U. U is uridine, and pseudouridine is a variant form of that, denoted Ψ. In an organism, the sequence in DNA is first transcribed into RNA, and then specific enzymes chemically alter certain U residues to Ψ. This modification seems to play a structural role. It affects the local flexibility of the RNA chain, and can alter the base pairing interactions that, in RNA, underlie the folding of structural RNAs such as ribosomal and transfer RNAs (rRNA and tRNA). It has also been found in linear RNAs such as messenger RNA (mRNA), whose role is to specify the sequence of proteins. Even here, though, the substitutions may relate to structure, since they tend to be associated with splice sites - regions removed during post transcription processing - which can have a structural component.

The RNA in vaccines is effectively a mRNA specifying the protein sequence of the SARS-CoV-2 'spike' protein, which is then presented to the adaptive immune system for generation of antibody and cellular immune responses.

An important distinction, though, is that all the U's in the vaccine RNA are Ψ's. This modification serves to 'disguise' the vaccine RNA. A major challenge of RNA vaccination is to get the RNA to survive long enough to be translated into protein. RNAs are constantly being degraded in the cell, and the immune system itself may attack the RNA directly, a mechanism of protection against RNA viruses. Ψ substitution serves to slow down these processes. You don't want to slow it down too much - you don't need the RNA hanging round forever, and you do need the immune system to recognise the RNA as foreign as that helps initiate the immune cascade: RNA can function as an adjuvant. Overstimulate the immune response, on the other hand, and you risk allergic reaction and anaphylactic shock. By careful design - and these principles have been years in the making - you can aim for an optimum. Pfizer and Moderna appear to have minimised the usage of codons (the 3-base triplets that code for amino acids) that include U in order to limit the number of Ψ's. Those Ψ's that remain help prolong the 'shelf-life' of the RNA.

Malone quotes extensively from a 2020 review by Erin Borschardt et al of the state of knowledge on pseudouridine in nature. It's interesting, but not necessarily all that relevant, and it's quite hard to see where Malone is going with it all. The principal issues for vaccine RNA relate to any effect of Ψ on 

• protein synthesis 
• immune response 
• longevity in the body

As far as the first two of these are concerned, we can take an empirical view. If spike protein antibodies and cellular responses are observed, we can be confident both that the supplied sequence (despite or because of its modifications) is participating successfully in protein synthesis, and that the RNA is sufficiently immunogenic (in tandem with its lipid vehicle) to initiate the immune cascade. If no hyperactive immune response is observed, conversely, we can say that the modifications are benign in that regard.

Malone's main concern seems to be the last of these. He points to this paper, which has been quite enthusiastically received by vaccine advocates as it shows that the repertoire of antibodies after vaccination is broader, and better able to deal with variants, than that after infection (a counter to the frequent inistence on the superiority of 'natural immunity'). The immune response matures in germinal centres (GCs). In infection, GCs can be disrupted, whereas on immunisation there is a robust and long-lived GC response. This allows the immune system to get a 'good look' at the spike protein surface and generate antibodies all over it, which reduces the mutational landscape for immune evasion by new variants. Even though the vaccines were formulated against the old 'Wuhan' spike, this broad coverage provides better protection against other variants than infection, which tends to produce narrow, variant-specific antibodies.

Which all sounds good, except that Malone is concerned about this very feature. He notes that vaccine RNA can be recovered (from GCs only, and in small quantities) up to 60 days after vaccination, and likewise the spike proteins synthesised from it. He speculates that this may be due to the Ψ's - with rather more certainty than is warranted.

it appears to me that the extensive random incorporation of pseudouridine into the synthetic mRNA-like molecules used for the Pfizer/BioNTech and Moderna SARS-CoV-2 vaccines may well account for much or all of the observed immunosuppression, DNA virus reactivation, and remarkable persistence of the synthetic “mRNA” molecules observed in lymph node biopsy tissues by Katharina Röltgen et al. 

The last might be the case, or it might equally be that vaccinee GCs simply persist because they are not disrupted, unlike those of Covid patients. GCs have long been known to harbour RNAs (Fauci alert!). Immunosuppression, however, is a debatable claim, directly contradicted by the Roltgen paper, which reports a better response in vaccinees, inconsistent with immunosuppression. 

Either way: longevity in GCs looks more like a feature than a bug.