50% of individuals vaccinated with mRNA COVID-19 vaccines had specific fragments of recombinant S protein in their blood samples 2–6 months after vaccination

BNT162b2 (Pfizer- BioNTech) and mRNA 1273 (Moderna) vaccines were the first messenger RNA (mRNA)-based vaccines ever approved. In both vaccines, an mRNA sequence determines the structure and assembly of the immunogen, the SARS-CoV-2 spike (S) glycoprotein. This S protein differs from the wild-type protein by specific amino acid changes at positions 986 and 987 (K986P and V987P), i.e., the amino acids lysine and valine are both replaced by two proline amino acids to stabilize the S protein conformation in an inactive prefusion state. In this study, the authors from Italy and the United Kingdom used mass spectrometry analysis to investigate the presence of recombinant S protein, encoded by the mRNA vaccine, in blood samples from participants vaccinated with mRNA COVID-19 vaccines and unvaccinated individuals. 

Glycosylated S protein is one of the four major proteins of SARS-CoV-2. It enables recognition of the host cell receptor and subsequent viral entry into the host cell. Because of these reasons, it is one of the most important targets for the development of vaccines and therapeutic approaches against COVID-19. The researchers noted that it is possible to distinguish synthetic and natural S proteins because they produce different tryptic digestion products. When digested by an enzyme, trypsin, the wild-type SAR-CoV-2 protein produces two smaller fragments, namely LDK + VEAEVQIDR. The recombinant S protein encoded by the mRNA vaccine produces an LDPPEAEVQIDR fragment (PP-spike marker). 

A previous study confirmed the presence of circulating S protein in plasma samples from approximately 6o% of patients with postacute sequelae of coronavirus disease within one year after diagnosis of SARS-CoV-2 infection.


About the study

The study included 60 individuals, 20 vaccinated with the full cycle of mRNA vaccine, 20 unvaccinated controls who tested negative for COVID-19, and 20 unvaccinated controls who tested positive for COVID-19. The presence of specific fragments of recombinant S protein encoded by the mRNA vaccine (in this study called PP-spike) in biological samples was detected by mass spectrometry.

Approximately 50% of subjects who received mRNA-based vaccines had specific fragments of recombinant S protein. The minimum time when the PP-spike fragment was detected was 69 days after vaccination, whereas the maximum was 187 days. The presence of circulating recombinant S protein was independent of the anti-SARS-CoV-2 IgG antibody titer. 

All samples from 40 unvaccinated individuals (tested negative or positive for COVID-19) were negative for recombinant S protein.

Based on these results, the authors proposed three possible molecular mechanisms responsible for the persistence of the “PP spike fragment”. Firstly, the mRNA can be integrated or re-transcribed in certain cells, secondly, pseudo-uridines at a specific sequence position induce the formation of the S protein which is always constitutively active, and thirdly, nanoparticles containing the mRNA are taken up by the blood microbiota, normally present in the blood at the basal level.

Figure from the original article by Brogna C et al. Proteomics Clin. Appl. 2023


In conclusion, mass spectrometry examination of blood samples has shown that approximately 50% of subjects who received mRNA-based vaccines had specific fragments of recombinant S protein in their blood samples 2-6 months after vaccination. All blood samples from individuals who were not vaccinated were negative for recombinant S protein.

This study is the first proteomic detection of recombinant S protein in subjects who had been vaccinated. These results confirmed that this method allows the detection of circulating S protein encoded by an mRNA vaccine, and the evaluation of its half-life.

This article was published in Proteomics Clinical Application.

Journal Reference

Brogna C, Cristoni S et al. Detection of recombinant Spike protein in the blood of individuals vaccinated against SARS-CoV-2: Possible molecular mechanisms. Proteomics Clin. Appl. 2023;2300048. (Open Access)

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