At least one early Pfizer study left a number of safety questions unanswered, questions that one might reasonably expect to be investigated as development of an mRNA vaccine progressed toward emergency authorization.

Beginning in April 2020, Pfizer, together with study sponsor BioNTech, conducted a Phase 1/2 clinical trial designed to identify preferred vaccine candidates and appropriate dose levels.

One of the candidates studied was BNT162b1. Although BNT162b1 was not ultimately selected as Pfizer’s final COVID-19 vaccine, research involving the candidate was included among documents submitted to the U.S. Food and Drug Administration (FDA) in connection with development of the Pfizer vaccine.

The BNT162b1 Phase 1/2 Study

One of those documents was a paper by Mulligan et al. (2020), published in Nature, describing results from the Phase 1/2 trial of BNT162b1.

Researchers administered BNT162b1 to adults over age 18 using three dosing regimens:

• 10 micrograms on days 1 and 21
• 30 micrograms on days 1 and 21
• 100 micrograms on day 1

The study examined both the immune response generated by the vaccine candidate and its safety and tolerability at different doses.

Questions About mRNA and DNA

Mulligan et al. state that, with RNA-based vaccines, “RNA is not incorporated into the host genome.”

Subsequent laboratory research has examined related questions. Zhang et al. (2021), for example, reported evidence that SARS-CoV-2 RNA could be reverse-transcribed under experimental conditions and investigated whether resulting sequences could become integrated into cellular DNA.

Another study, by Aldén et al. (2022), reported that Pfizer’s eventual vaccine, BNT162b2, underwent reverse transcription in a human liver-derived cell line in vitro, with intracellular DNA corresponding to BNT162b2 detected as early as six hours after exposure.

Importantly, Aldén et al. did not establish that this DNA became integrated into the cellular genome. Reverse transcription and genomic integration are separate biological events, and the authors stated that further research would be required to determine whether integration occurred.

N1-Methylpseudouridine and Lymphocyte Counts

The Mulligan paper raises additional questions. The researchers explain that the vaccine candidate incorporated N1-methylpseudouridine, which they wrote “dampens innate immune sensing and increases mRNA translation in vivo.”

Among participants who experienced changes in blood measurements following vaccination, one of the most notable findings was a temporary decrease in lymphocytes, white blood cells that play an important role in immune function.

Approximately half of participants receiving an initial 30- or 100-microgram dose experienced decreased lymphocyte counts.

This raises questions worthy of further investigation. What mechanism caused the observed lymphocyte reductions? Did the modified nucleoside play any role? And what, if anything, could these findings tell researchers about the persistence and biological activity of mRNA vaccine components?

Those questions cannot be answered from the Mulligan study alone.

Adverse Events Increased With Dose

Changes in blood-cell counts were not the only effects observed.

Phase 1 and Phase 1/2 studies are intended in part to evaluate safety and determine appropriate dosing. In the Mulligan study, increasing the dose from 10 to 30 to 100 micrograms was associated with greater reactogenicity.

Reported reactions included:

• Fever
• Fatigue
• Headache
• Chills
• Diarrhea
• Muscle pain
• Joint pain

DailyClout volunteer researchers in Team 5 subsequently analyzed the dose-related reactogenicity reported in the trial and concluded that the relationship was statistically significant.

Researchers Recommended Continued Development

Despite these findings, Mulligan et al. regarded the overall results as encouraging. They concluded:

“The clinical findings for the BNT162b1 RNA-based vaccine candidate are encouraging and strongly support accelerated clinical development…for the rapid production of a SARS-CoV-2 vaccine to prevent COVID-19.”

BNT162b1 itself was ultimately not the vaccine candidate that proceeded to authorization. Pfizer and BioNTech instead advanced BNT162b2, making it important not to treat every finding involving BNT162b1 as though it automatically applied to the subsequently authorized formulation.

Nevertheless, the early study raises legitimate questions about dose-dependent adverse reactions, transient changes in lymphocyte counts, modified mRNA technology, and the biological fate of vaccine mRNA. The critical question is therefore not simply whether these findings existed, but how thoroughly they were investigated during subsequent development of BNT162b2 and what later evidence showed about their clinical significance.

Before drawing conclusions about whether the FDA authorization process adequately resolved those concerns, the later preclinical, clinical, and regulatory evidence should be examined alongside this early study.