First, I would like to congratulate Mulroney et al. for this important and fascinating paper. Second, I need to stress that this note describes a conjecture to try to make sense of the apparent logical contradiction in this paper.

On the one hand, Mulroney et al. highlight some highly negative aspects of their finding, for example: “The main in-frame mRNA-encoded product is unlikely to elicit an adaptive immune response, but presentation of +1 frameshifted products could activate T cells that target host cells.”

While the former part may need some more explanation, the authors clearly alert to the potential of host cells becoming the target of an immune attack. As has been extensively described elsewhere, this could lead to excessive inflammation, autoimmunity, and related, or additional, serious clinical complications.

The authors highlight these negative aspects in that they stress that

1. IFNγ ELISpot responses to +1 frameshifted products were physically observed, and notably, precisely to those individuals vaccinated with BNT162b2.
2. Their findings are new and were previously not studied (“At present, it is unclear which modified ribonucleotides affect mRNA translation fidelity,” “the evolution of antibody and cytolytic T cell responses against +1 frameshifted spike variants and peptides has not been systematically evaluated in humans”).
3. There are no established methods to evaluate and quantify the type and amount of off-target/unintended proteins and to differentiate the resulting immune response, or other clinical sequelae, in different tissue/organs and various pathophysiological states across the wide spectrum of the overall population (or even future generations). Since this paper is the first to raise this issue, the interesting method that was developed to identify these occurrences cannot target the entire universe of biological activity triggered by these unanticipated products. Specifically, the authors caution that “ELISpot responses obtained from pooled peptides may also underestimate T cell responses.”

In this light, the assertion that “there is no evidence that frameshifted products in humans generated from BNT162b2 vaccination are associated with adverse outcomes” cannot be aligned with the rest of the article. This assertion was further critiqued by Wisemann et al. in their comment below.

Here, I would like to raise the question of whether the statement of lack of evidence of adverse outcomes hinges upon some definitions/conceptions that were made that many of us may not be aware of.

Specifically, the notion of adverse outcomes following vaccination (AEFI) has long been debated and has been clearly defined before. In 2019, the World Health Organization (WHO) [1] published the second edition of a “guide to a systematic, standardized global causality assessment process” to determine if an AEFI was caused by a vaccine. Chapter 8 in my book [2] on the mRNA vaccines (published by Springer), details why these criteria, even for mere technical/logistical reasons alone do not apply to mRNA Covid-19 vaccines – under these criteria, and in this context, the only possible outcome is “no causation shown.” This, however, does not mean that there is no causation. It only means that we do not have the data, information, knowledge, methods, tools, criteria, definitions, etc., to determine a clear causal relationship.

Consequently, the conclusion that there is no evidence of adverse outcomes from a mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, as stated in the paper by Mulroney et al., may simply be a technical artifact, a direct result of the fact that the AEFI-WHO criteria in this context never can determine that an AEFI was indeed caused by the vaccine.

There are numerous reasons why an AEFI following BNT162b2 (or, likely, in this case also Moderna’s mRNA Covid vaccine) technically cannot be determined as being caused by these injections [2]. For instance, in the context of the Mulroney et al. paper, the following strong requirements of the WHO-AEFI manual are not fulfilled for technical reasons alone:

• Potential AEFIs in the context described in the paper by Mulroney et al. do not fulfill the required criterion that they are clearly classifiable (in fact, they are hardly known).
• There is no known (i.e., clearly established) single-factor-based causal association (the nature of the unanticipated mechanism – frameshifting - automatically results in a host of different products of different types and lengths, as was also demonstrated in the paper; the inherent lack of knowledge of the mRNA products as pharmaceutical drugs triggering complex biological activities [3] and numerous known/unknown challenges & unknowns [2] in the context of mass-vaccination, combined with production/quality control concerns resulting in significant product inhomogeneity [4], contaminants that can evoke biological/clinical sequelae [5], or other scaling issues [2], will make it sheer impossible to prove a single-factor causal relationship as required by the WHO-AEFI criteria).
• There may be other qualifying factors (in fact, many underappreciated clinical sequelae following the mRNA injection can be overlapping, which means that it would be technically next to impossible to merely identify one isolated cause, as would be required).
• There may be many alternative explanations for an observed AEFI, especially when it is new, not expected, not appreciated, not readily classifiable, and overlapping with numerous other biological activities (e.g., excessive T-cell responses or autoimmune conditions may have different causes).
• There is no known (i.e., clearly established) population-based evidence for causality (again, we do not have such a clearly established knowledge at the population level because the phenomena are new – to infer population-wide causality, we ideally would have long-term independent RCTs, which however is not the case here). Additionally, for the same reasons as above, it would also not be possible to demonstrate that “The vaccine is the only cause of the [AEFI] event that can be shown.”).
• As per [1], causality at the individual level requires that there is population-based evidence for causality. It is unclear how this WHO-AEFI criterion in this context could ever determine causality at the individual level. Given that it is technically impossible to infer population-based evidence, a causal association at the individual level is thereby automatically ruled out. Consequently, however, this presumed lack of causality at the individual level now supports an ongoing detrimental circular argument. Absence of presumed causality at the individual level engenders a lack of any statistically significant causal relationship -- which will then feed back to seemingly demonstrate lack of population-based evidence. The logical oddity should be obvious.

References:
[1] World Health Organization and Others (2019) Causality assessment of an adverse event following immunization (AEFI): user manual for the revised WHO classification.
[2] Mueller, S. (2023). Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2: A Multidisciplinary Perspective. Cham: Springer International Publishing.
[3] Cosentino, M., Ferrari, M., & Marino, F. (2021). Coronavirus disease-19 vaccines best reflect effective pharmaceuticals. Journal of Neuroimmune Pharmacology, 16(3), 517-518.
[5] Schmeling, M., Manniche, V. and Hansen, P.R. (2023), Batch-dependent safety of the BNT162b2 mRNA COVID-19 vaccine. Eur J Clin Invest, 53: e14102. https://doi.org/10.1111/eci...
[6] Speicher, D. J., Rose, J., Gutschi, L. M., Wiseman, D. M., PhD, & McKernan, K. (2023, October 19). DNA fragments detected in monovalent and bivalent Pfizer/BioNTech and Moderna modRNA COVID-19 vaccines from Ontario, Canada: Exploratory dose response relationship with serious adverse events. https://doi.org/10.31219/os...

Does anyone think the possibility that m1Ψ causes readthrough of the in-frame premature termination codons in the reporter system shown in this paper as described in the prior paper 10.1016/j.molcel.2022.11.011?

David Wiseman, L. Maria Gutschi, David J. Speicher, Jessica Rose, Kevin McKernan

We thank Mulroney et al. for this important contribution.
The paper provides evidence for the formation “off-target” or unintended proteins following vaccination with BNT162b2 due to frameshifting. Given the proposed mechanism, a similar problem is likely to exist for the Moderna product.
While the authors have not isolated samples of these proteins from vaccinated patients or animals, their existence is evidenced by the specific cellular immune responses elicited to frameshifted proteins the authors synthesized. It is not clear why B cell – antibody responses were not studied.
The authors state that “Although there is no evidence that frameshifted products in humans generated from BNT162b2 vaccination are associated with adverse outcomes.” It is unclear how it is possible to make this statement, given:
• The small number of vaccinated subjects (n=21) providing samples.
• This was not a controlled trial.
• None of these subjects had reported undue effects of vaccination. Accordingly, the sample is subject to selection bias.
• The toxicology of these unintended proteins must be studied.
• The authors acknowledge the misdirected immunity “has huge potential to be harmful.”
• These proteins may already have contributed to vaccine toxicity, which now must be the subject of investigation.
The full sequence of these proteins should be provided. Further, the homologies between the proposed frameshifted proteins and peptides and known proteins must be conducted using databases and tools such as BLAST. One of the proteins identified was characterized as a chimeric protein. McKernan et al. (1) showed how in theory, a chimeric viral-human protein might be formed that has a homology similarity to a human protein called gp130, which forms part of a receptor for IL-6.
The premise for the study reveals a developmental and regulatory failure to ask fundamental questions that could affect the safety and effectiveness of these products. This is no better exemplified by Pfizer’s retired head of vaccine R&D who was quoted in Nature as saying: “We flew the aeroplane while we were still building it.” (2)
According to WHO guidelines for mRNA vaccines, (3) manufacturers should provide details “unexpected ORFs,” (emphasis added).
“The complete annotated sequence identifying all ORFs (including any unexpected ORFs) and all other sequence elements (including their justification for use) should be provided. Justifications for the use of any specific noncoding sequence and of structural elements such as the chosen 5` cap structure should be provided. [..] The anticipated function and purpose of each gene sequence encoded in the mRNA should be indicated, as well as those of specific noncoding and structural elements, explaining their contribution to the overall mode- or mechanism-of-action.”
If Mulroney et al. were able to predict the existence of frameshifted proteins, why were Pfizer’s scientists unable to do so? The same question may be asked of regulators, especially in light of unresolved discrepancies and the specific obligation imposed by the European Medical Agency on BioNTech regarding the identities of the observed Western Blot (WB) bands obtained by in vitro expression assays.(4)
Documents disclosed under the FOIA (5) reveal that three categories of preclinical studies were not performed by Pfizer, relevant to the current findings: 1) secondary pharmacodynamics, 2) safety pharmacology and 3) pharmacodynamic drug interactions, In two of these categories, WHO guidelines were cited in justification (highlight added).
The package insert for COMIRNATY states (6):
“Each 0.3 mL dose of COMIRNATY (2023-2024 Formula) is formulated to contain 30 mcg of a nucleoside modified messenger RNA (modRNA) encoding the viral spike (S) glycoprotein of SARS-CoV-2 Omicron variant lineage XBB.1.5 (Omicron XBB.1.5).”
There is no mention of any other kind of protein.
The finding that unintended proteins may be produced as a result of vaccination is sufficient cause for regulators to conduct full risk assessments of past or future harms that may have ensued. We note that regulators have previously failed to insist on the study and assessment of risk of the pharmacology and toxicology of novel spike protein heterotrimers forming after injection of the bivalent COVID-19 modRNA vaccines.(7)
The paper was received by Nature on January 25, 2023, accepted for publication on October 31, 2023 and published today, December 6, 2023. This time frame appears rather protracted given the significance of these findings. The study was funded and conducted by agencies of the United Kingdom. The evidence for the formation of off-target proteins must surely be considered a reportable adverse event.
We must assume UK regulators, manufacturers, and international regulatory agencies, including FDA, were apprised of the data many months ago. We await their account of what steps they have taken to investigate why the formation of off-target proteins was not discovered sooner, what toxic effects they may have caused and what steps they are taken to prevent harm in the future and to inform the public of these findings.

We have prepared an expanded version of these comments.(8)
References
1. McKernan K, Kyriakopoulos, A. M., & McCullough, P. A. Differences in Vaccine and SARS-CoV-2 Replication Derived mRNA: Implications for Cell Biology and Future Disease. OSF Preprints 2022. Epub Feb 15 http://doi.org/doi.org/10.3...
2. Kingwell K. COVID vaccines: “We flew the aeroplane while we were still building it”. Nature Reviews Drug Disc 2022. Epub Nov 11 http://doi.org/doi.org/10.1...
3. WHO. Evaluation of the quality, safety and efficacy of messenger RNA vaccines for 10 the prevention of infectious diseases: regulatory considerations. WHO/BS/2021.2402. 2021. (Accessed June 16, 2022, at https://cdn.who.int/media/d...
https://cdn.who.int/media/d...
4. EMA. Assessment report: Comirnaty - Pfizer. 2021 19 Feb. at https://www.ema.europa.eu/e...
5. Pfizer. BNT162b2 Module 2.4. Nonclinical Overview. Document released pursuant to FOIA. 2021 Feb 8. at https://phmpt.org/wp-conten...
6. FDA. COMIRNATY Package Insert 2023-2024 Formula. 2023 Sept 11. at https://www.fda.gov/media/1...
7. Wiseman D. ACIP October 19-20-2022. BA4/5 bivalent quasi-vaccines in yet younger children: Further erosion of scientific and ethical standards. Written and Oral Comments. Research Gate 2022 Oct. Epub Oct 19 http://doi.org/10.13140/RG....
www.regulations.gov/comment...
8. Wiseman D, Gutschi, LM. Speicher, DJ. Rose, J, McKernan, K. Ribosomal frameshifting and misreading of mRNA in COVID-19 vaccines produces "off-target" proteins and immune responses eliciting safety concerns: Comment on UK study by Mulroney et al. Research Gate 2023. Epub Dec 6 http://doi.org/ResearchGate 10.13140/RG.2.2.36710.40005

My guess, the authors wanted to focus on demonstrating the effect and to avoid controversy. Their paper was published, so they seem to have made the right call. That said, a study should be done of the adverse effects this mechanism can produce as well as research into preventing frameshifting in future mRNA technologies.

Excellent response. Thank you.