Received: 29 April 2023

Revised: 29 July 2023

Accepted: 15 August 2023

DOI: 10.1002/prca.202300048

R A P I D C O M M U N I C AT I O N

Detection of recombinant Spike protein in the blood ofindividuals vaccinated against SARS-CoV-2: Possible
molecular mechanisms
Carlo Brogna1Simone Cristoni2
Giuliano Marino3
Luigi Montano4
Valentina Viduto5Mark Fabrowski6
Gennaro Lettieri7
Marina Piscopo7
1

Department of Research, Craniomed Group
Facility Srl, Bresso, Italy

2

ISB–Ion Source & Biotechnologies Srl, Bresso,
Italy

3

Abstract
Purpose: The SARS-CoV-2 pandemic prompted the development and use of nextgeneration vaccines. Among these, mRNA-based vaccines consist of injectable solu-

Marsanconsulting Srl. Public HealthCompany, Napoli, Italy

tions of mRNA encoding for a recombinant Spike, which is distinguishable from the4wild-type protein due to specific amino acid variations introduced to maintain the

Andrology Unit and Service of LifeStyle
Medicine in Uro-Andrology, Local Health
Authority (ASL) Salerno, Salerno, Italy

5Long COVID-19 Foundation, Brookfield
Court, Garforth, UK

6

Department of Emergency Medicine, Royal
Sussex County Hospital, University Hospitals
Sussex, Brighton, UK

7

Department of Biology, University of Naples
Federico II, Napoli, Italy
Correspondence
Marina Piscopo, Department of Biology,
University of Naples Federico II, 80126 Napoli,
Italy.
Email: marina.piscopo@unina.it
Carlo Brogna, Department of Research,
Craniomed group facility Srl, Bresso, 20091,
Italy.
Email: dir.brogna@craniomed.it

protein in a prefused state. This work presents a proteomic approach to reveal thepresence of recombinant Spike protein in vaccinated subjects regardless of antibody
titer.
Experimental design: Mass spectrometry examination of biological samples was used
to detect the presence of specific fragments of recombinant Spike protein in subjects
who received mRNA-based vaccines.
Results: The specific PP-Spike fragment was found in 50% of the biological samples
analyzed, and its presence was independent of the SARS-CoV-2 IgG antibody titer. The
minimum and maximum time at which PP-Spike was detected after vaccination was 69
and 187 days, respectively.
Conclusions and clinical relevance: The presented method allows to evaluate thehalf-life of the Spike protein molecule “PP” and to consider the risks or benefits incontinuing to administer additional booster doses of the SARS-CoV-2 mRNA vaccine. This approach is of valuable support to complement antibody level monitoring
and represents the first proteomic detection of recombinant Spike in vaccinatedsubjects.
KEYWORDS

COVID-19, mass spectrometry, SARS-CoV-2, Spike protein, vaccine mRNA

Abbreviations: m1Ψ, methyl pseudouridine; HCoV, human coronaviruses; LNPs, lipid nanoparticles; mRNA, messenger ribonucleic acid; DBS, Dry Blood Spot; SARS-CoV-2, Severe acuterespiratory syndrome coronavirus 2; ACE2, Angiotensin-converting enzyme 2; S, Spike protein; PP, Double proline amino acid; wt, Wild type; IgG, Immunoglobulin G.
Carlo Brogna and Simone Cristoni contributed equally to this study.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2023 The Authors. Proteomics – Clinical Applications published by Wiley-VCH GmbH
Proteomics Clin. Appl. 2023;2300048.
https://doi.org/10.1002/prca.202300048

www.clinical.proteomics-journal.com

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1

RAPID COMMUNICATIONINTRODUCTION
Significance of the study

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is thecausative coronavirus of COVID-19 respiratory disease (coronavirus

Although the COVID-19 pandemic brought the whole world

disease 2019), responsible for the ongoing pandemic that holds theto its knees, it also allowed many scientists to develop ideas

entire world in check. Researchers around the world during these pan-and solutions against viruses. These include mRNA vaccines,

demic years have studied this virus, trying to understand its mechanism

which, due to their versatility in production, may representof action [1–11]. The RNA genome of SARS-CoV-2 consists of abouta new standard of vaccination. However, it is the duty of the

30,000 nucleotides and contains 11 major coding genes. From a struc-scientist not to neglect controls. Herein lies the importancetural point of view, SARS-CoV-2 is characterized by a large numberof monitoring vaccine-induced Spike protein “PP” after a timeof glycosylated Spike (S) proteins covering its surface and facilitating

period after vaccination in human biological samples. The

binding to the angiotensin-converting enzyme 2 (ACE2) receptor of thepresented method allows to assess the half-life of the Spikehost cell, mediating viral cell entry [12]. The Spike protein (S) is one“PP” protein molecule and to consider the risks or benefits inof the four major proteins of SARS-CoV-2 [13]. It enables recognition

continuing further booster doses of the SARS-CoV-2 mRNAof the host cell receptor and subsequent entry into the host cell. It

vaccine.

consists of the distal S1 subunit, which is useful for recognition, andthe proximal S2 subunit, which is essential for fusion with the hostcell membrane [13]. During the development of the two most widelyused mRNA-based vaccines, Pfizer-BioNTech (BNT162b2- Comirnaty)

The study group, from southern Italy, was 40 subjects, 20 were vac-and Moderna (mRNA-1273), all uridine nitrogen bases were replaced

cinated with the full cycle of mRNA vaccine as of April 2022, being

with methyl pseudouridine (m1Ψ)—a less immunogenic [14] but morepart of the health sector, and 20 were unvaccinated with negativity

stable nitrogen base [15]. At the same time, mutations were madefor COVID-19 to nasopharyngeal test and with no titer of any antibod-

within the 4284 nucleotides constituting the Spike protein, at positions

ies. Other 20 unvaccinated persons were added that were positive for

K986P and V987P to make the protein produced after ribosomal read-

COVID-19. The specific PP-Spike fragment was found in 50% of theing stable prefusion form to stimulate increased production of humanbiological samples analyzed (Figures 1C–E and 2). This presence wasantibodies (Figure 1A) [15]. The S protein of SARS-CoV-2 is highly

independent of the SARS-CoV-2 IgG antibody titer. The antibody titersconserved among all human coronaviruses (HCoV) and is involved in

had a geometric mean of 629.86 BAU/mL (Figure 1E). The minimum

receptor recognition, viral attachment, and entry into the host cells.

time PP-Spike was detected was 69 days after vaccination, while the

For this reason, it represents, for a part of scientists, one of the

maximum time was 187 days. All controls (samples from unvaccinatedmost important targets for the development of vaccines and thera-

individuals) were negative. The control group (20 unvaccinated peo-

peutic approaches against COVID-19. Among the COVID-19 vaccinesple) was also tested after contracting COVID-19 and was negative fordeveloped and tested, those that have shown the most promising

PP-spike.

results in preventing COVID-19 infection are a novel class of vaccine
products composed of messenger ribonucleic acid (mRNA) filaments

Some studies [23] have observed the presence of the vaccine Spikeprotein immediately after injection.

encapsulated in lipid nanoparticles (LNPs). Two of them have received

According to the authors [15] and in general, the vaccine messen-“emergency use authorization” (from the Federal and Drug Admin-ger RNA nanoparticle molecules should be picked up by the immune

istration) and “conditional approval” (from the European Medicines

cells in the lymph nodes after injection into the muscle. Recently, other

Agency). Both consist of a recombinant mRNA to be inoculated as aauthors have isolated vaccine messenger RNA sequences from periph-

vaccine, which encodes for a recombinant SARS-CoV-2 Spike protein.

eral plasma after 28 days after injection [24]. The question of whether

Although the mRNAs are different, both encode for the same recombi-

or not the vaccine RNA can be integrated into the lymphocyte or othernant Spike protein (here called PP-Spike). This differs from the natural

body cells is much debated. Nonetheless, the observation of the pro-one (wt-Spike, Figure 1B) by a double amino acid change at position 986

tein produced, as presented in this manuscript, goes beyond the purely

and 987 (K986P and V987P, i.e., the amino acids lysine and valine are

cognitive aspect and defines a method to verify not only the persis-

both replaced by two proline amino acids) [16, 17], in order to stabi-tence of the vaccine RNA but the quantification of the product, that is,lize the Spike conformation in an inactive prefusion state (Figure 1A).

the protein that is supposed to induce antibody production, in order to

The introduced double amino acid variation abolishes a tryptic diges-

verify the correct half-life and a possible need to update the vaccine

tion site. As a result, it is possible to distinguish, by tryptic digestion

status. Using mass spectrometry examination of biological samples, we[18], followed by mass spectrometry analysis [19–22], synthetic Spikedetected the presence of specific fragments of recombinant Spike pro-

proteins originated from the translation of the mRNA vaccines fromtein in about 50% of subjects who received mRNA-based vaccines. In

natural Spike circulating in biological fluids. Here we present a method-some cases, we found the PP-Spike marker in vaccinated individuals

ological approach that can specifically detect the presence of PP-Spike

more than 30 days after the vaccine, indicating that it is possible toin biological fluids of human and animal organisms, such as blood, urine,

detect vaccine “Spike” protein even sometime after vaccination and insaliva, and bronchoalveolar lavage fluids.

any organic tissue (data in preparation). Based on the results obtained,

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F I G U R E 1 Schematic diagram of PP Spike protein production. (A) Formation of Spike PP protein after injection of vaccine mRNA. (B) Exampleof wild-type, unmodified Spike protein. (C) Extraction and detection scheme (more details in the text). (D) quantification of the antibody titer of the
20 vaccinated cases. (E) Time from the day of vaccination completion to the discovery of vaccine mRNA-induced Spike protein “PP”.

hypotheses can be made for possible molecular mechanisms of persis-

by radiometric analyses suggested the existence of living microbial

tence of “Spike PP.” In particular, three hypotheses are possible and areforms in erythrocytes [25]. In addition, the observation of the PP-

shown in Figure 3.

Spike marker in individuals vaccinated more than 30 days after thevaccine in about 50% of subjects could also be explained by the wide

1. It is possible that the mRNA may be integrated or re-transcribed insome cells.

biodiversity of eukaryotic and prokaryotic microbiota identified inblood by next-generation sequencing technologies [25].

2. It is possible that pseudo-uridines at a particular sequence position,
as described in the article, induce the formation of a spike protein

In conclusion, the possibility of detecting the presence of specificthat is always constitutively active. But it seems very remote as a

fragments of recombinant Spike protein opens new scenarios for mon-hypothesis.

itoring the presence and half-life of vaccine Spike protein in vaccinated

3. It is possible that the mRNA-containing nanoparticle will be pickedindividuals. The hypotheses advanced need more and larger studies.

up by bacteria normally present at the basal level in the blood. In

At present, these initial observations are limited only to assessing thefact, the existence of blood microbiota in clinically healthy individ-

presence of the vaccine protein with the aim of wanting to provide aid

uals was proven during the last 50 years. Indeed, indirect evidence

in the individual’s decision to administer booster doses or temporize.

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RAPID COMMUNICATIONRAPID COMMUNICATIONF I G U R E 2 Molecular ion of the sequence LDPPEAEVQIDR- Spike “PP.” (A) Shows the m/z (molecular ion) ratio of the SEQ ID NO:1 detection.
Shows an example of the spectrum of the peptide zone marked by the enzymatic failure to cut the “PP” amino acids of the “Spike” protein induced
by mRNA vaccines. (B-E) Extraction mass chromatograms of (B) vaccinated subjects and (D) control samples were obtained by fragmenting the[M+3H]3+ion at m/z 461 and monitoring the singly charged signal at m/z 851. Also shown are the tandem mass spectra obtained by analyzing (C)the vaccinated subject and (E) the control subject. Five microliters of digested samples were injected, and the eluent flow rate was 250
microliter/min.

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FIGURE 3

2

Possible molecular mechanisms of persistence of “Spike PP”.

ASSOCIATED DATA

3.1

Pre-analytic

Statistical analysis: t-test with © 2023 GraphPad Software was per-Preclinical dry blood sample collection was performed in compliance

formed between the two groups, vaccinated, and not vaccinated forwith the scientific literature: the Dry Blood Spot (DBS) technique is com-the presence of the peptide of spike called PP. "P-value and statisti-

mon for the collection of small amounts of blood, especially from newborns

cal significance: The two-tailed P value is equal to 0.0010. Confidence

for screening application. This method was first published by Robert Guthrieinterval: The mean of group one minus group two is equal to 0.40, 95%

in the early 1960s and is based on sampling a small blood spot onto a fil-confidence interval of this difference: 0.17–0.63. Intermediate values

ter paper instead of drawing a few milliliters of blood into a vial. With theused in calculations: t = 35,590, df = 38, standard error of the differ-

DBS technique, sampling is easy and fast without the need for trained pro-ence = 0.112. The difference in titer antibody between the two groups

fessionals. However, the small amounts of blood drawn (∼20–40 µL) makeis not significant.

this approach more analytically challenging in terms of sample preparationand sensitivity compared with traditional blood sampling [26, 27]. Blooddrops were collected in sterile mode. In order to preserve the protein

3

EXPERIMENTAL PROCEDURES

structure, the dry blood filter was maintained at −20◦ C conditions until
the processing [28].

In accordance with applicable territorial regulations, case reports, caseseries, and observational retrospective studies do not require ethics
committee approval. Informed consent expressed by all participants,

3.2

Rationale

South of Italy, was obtained. Twenty human biological samples werecollected from vaccinated subjects with express, free, and informed

Trypsin [18] is an enzyme belonging to the class of hydrolases capa-

consent for collection and use. The geometric mean of their antibodies′ble of reducing proteins to smaller polypeptides by proteolytic cuts

titer versus spike protein was 629,86 BAU/mL after 60 days from vac-with specificity for arginine (R) and lysine (K). Synthetic Spike protein

cination. In addition, 20 human biological samples were collected fromand natural Spike protein can thus be distinguished as they produce

unvaccinated subjects (control group, negative) with express, free, anddifferent tryptic digestion products:

informed consent for collection and use. Candidates had not previously undergone COVID-19 and were negative for molecular testing– When digested by trypsin, the PP-Spike encoded by vac-by nasopharyngeal swab, and the antibodies titer was negative. Other

cine mRNA produces an LDPPEAEVQIDR fragment (PP-Spike

20 human biological samples from unvaccinated patients, who were illwith COVID-19, were obtained (geometric mean of antibodies titer vs.
spike protein was 187,89 BAU/mL later 105 days from disease).

marker) (Figure 2C).
– The wild-type SAR-CoV-2 protein, when digested by trypsin,
produces two smaller fragments, namely LDK + VEAEVQIDR.

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RAPID COMMUNICATIONRAPID COMMUNICATIONTA B L E 1

Chromatographic conditions of gradient elution.

potential of 0 V, a nebulizer gas pressure of 75 PSI, and a dry gas flow of
1.0 L/min were used. The dry gas temperature is at 320◦ C [19–22].

Time (min)%C

Flow (mL/min)

0

2%

0.250

2.5

2%

0.250

3

80%

0.250

thank Dr Aquilino Frongillo for the technical support. This research

7

80%

0.250

received no external funding.

8

2%

0.250

ACKNOWLEDGMENTS
We thank all the patients that have participated in our studies. We

CONFLICT OF INTEREST STATEMENT

Note: The percentage of eluent phase C (CH3CN) is reported along with theeluent flow rate at different gradient elution times.

The authors declare no conflict of interest.

More than 6,600,000 SARS-CoV-2 genomes have been sequenced,

Data available on request from the authors.

DATA AVAILABILITY STATEMENT
and it appears that none of them have K986P and V987P mutations,
including the Omicron variant [29].
Buffers used: Double distilled water (VWR); ammonium bicarbonate

ORCID
Marina Piscopo

https://orcid.org/0000-0003-0560-6952(NH4 HCO3 ) (Sigma Aldrich). Reagents: Trypsin from Promega.
Reagent preparation: Trypsin solution 25 ng/µL.
Preparation procedure: Resuspend, in a vial, 20 µg of solid trypsin in
800 µL of 50 mM NH4 HCO3 solution. Vortex the vial until the trypsin
is completely dissolved.
The standard peptide corresponding to the fragment LDPPEAEVQIDR (Sigma Aldrich, UK) has been diluted to a concentrationof 10 ng/mL in bi-distilled water. Ten microliters were injected into the
LC-MS apparatus for control purposes. The peptide detected in plasma
was characterized on the basis of EU regulations [30].

3.3

Enzymatic digestion

The enzymatic digestion procedure of blood drops is performed undera fume hood in order to minimize operator exposure to any form ofchemical biohazard. The following method was used for each blood
sample. Place 2 µL of capillary blood into a labeled Eppendorf tube; add
40 µL of trypsin (Promega, Italy) solubilized in a 50 mmoL NH4 HCO3
solution to the blood sample; vortex for 30 s; test the pH, which must
be between 7 and 8; transfer the Eppendorf tube to the thermoblock;incubate for 2 days at 37◦ C; add 40 µL of NH4 HCO3 50 mmoL. Collect
40 µL of the supernatant obtained after centrifugation at 13000 G for
10 min and transfer to an Eppendorf tube. Add 2 µL of pure formic acidand transfer the solution to an injection vial. The vial is inserted into
the autosampler coupled to the mass spectrometer, and 5 µL is injected
into the chromatographic column.

3.4

LC–SACI–MS instrumentation

The analysis was performed with a Surveyor MS HPLC (Thermofisher,
USA). The column used was a Kinetex 50 × 4.6 mm 2.6 µm. The analysis was performed using a two-phase gradient: Phase A (H2 O + 0.2 %formic acid (HCOOH)) and Phase C (CH3 CN) (Table 1). The chromatographic gradient used is shown in Table 1. The volume of the sample
injected is 5 µL. The ionization source used is a SACI-ESI. A surface

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How to cite this article: Brogna, C., Cristoni, S., Marino, G.,
Montano, L., Viduto, V., Fabrowski, M., Lettieri, G., & Piscopo,
M. (2023). Detection of recombinant Spike protein in the bloodof individuals vaccinated against SARS-CoV-2: Possible
molecular mechanisms. PROTEOMICS – Clinical Applications,
1–7. https://doi.org/10.1002/prca.202300048

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