European Review for Medical and Pharmacological Sciences

2023; 27: 13-19

Presence of viral spike protein and vaccinal
spike protein in the blood serum of patientswith long-COVID syndrome
K. DHULI1, M.C. MEDORI1, C. MICHELETTI1, K. DONATO2,3, F. FIORETTI4,
A. CALZONI4, A. PRADERIO4, M.G. DE ANGELIS4, G. ARABIA5, S. CRISTONI6,
S. NODARI4, M. BERTELLI1,2,3
MAGI’S LAB, Rovereto, Trento, Italy
MAGI EUREGIO, Bolzano, Italy
3
MAGISNAT, Atlanta Tech Park, Peachtree Corners, GA, USA
4
Department of Medical and Surgical Specialities, University and Spedali Civili of Brescia,
Radiological Sciences and Public Health, Brescia, Italy
5
Department of Medical and Surgical Specialties, Radiological Sciences and Public Health
Cardiology Unit, University of Brescia, Brescia, Italy
6
ISB Ion Source & Biotechnologies srl, Italy, Bresso, Milan, Italy
1
2

K. Dhuli and M.C. Medori contributed equally to this work

Abstract. – OBJECTIVE: COVID-19 patients

experience, in 10-20% of the cases, a prolonged
long-COVID syndrome, defined as the persistenceof symptoms for at least two months after the infection. The underlying biological mechanisms ofthis syndrome remain poorly understood. Several hypotheses have been proposed, among which
are the potential autoimmunity resulting from molecular mimicry between viral spike protein andhuman proteins, the reservoir and viral reproduction hypothesis, and the viral integration hypothesis. Although official data state that vaccinal
spike protein is harmless and remains at the siteof infection, several studies proposed spike protein toxicity and found it in blood circulation several months after the vaccination.
To search for the presence of viral and vaccine
spike protein in a cohort of long-COVID patients.
PATIENTS AND METHODS: In this study,
we employed a proteomic-based approach utilizing mass spectrometry to analyze the serum of 81 patients with long-COVID syndrome.
Moreover, viral integration in patients’ leukocytes was assessed with a preliminary study,
without further investigation.
RESULTS: We identified the presence of theviral spike protein in one patient after infection
clearance and negativity of COVID-19 test andthe vaccine spike protein in two patients twomonths after the vaccination.
CONCLUSIONS: This study, in agreementwith other published investigations, demonstrates that both natural and vaccine spike protein may still be present in long-COVID patients,
thus supporting the existence of a possible
mechanism that causes the persistence of spike

protein in the human body for much longer than
predicted by early studies. According to theseresults, all patients with long-COVID syndrome
should be analyzed for the presence of vaccinaland viral spike protein.
Key Words:
Viral Spike Protein, Vaccinal Spike Protein, SARSCoV-2, COVID-19, Long-COVID syndrome, Mass spectrometry, Viral reservoir, Viral integration.

Introduction
COronaVIrusDisease-2019 indicates the disease caused in humans by the SARS-CoV-2 virus,
characterized by fever, cough, breathing difficulties, severe acute respiratory syndrome, and evendeath1-4. 10-20% of COVID-19 patients manifest
long-COVID syndrome, defined as the persistenceof symptoms two months after the infection5-8. The
most common symptoms associated with long-COVID include fatigue, breathlessness and cognitive
dysfunction9,10. Notably, even seven months after
the initial infection, patients with long-COVID
continue to experience cardiovascular and neuralproblems, indicating a prolonged and complex
disease course, and highlighting the significant
impact of long-COVID on individuals’ health andquality of life11. Extensive research12-22 has been
conducted to elucidate the underlying mechanisms
and pathophysiology of long-COVID. However,Corresponding Author: Kristjana Dhuli, MSc; e-mail: kristjana.dhuli@assomagi.org

13

K. Dhuli, M.C. Medori, C. Micheletti, K. Donato, et al

the exact cause of long-COVID and the factors
contributing to its diverse symptomatology arestill not fully understood. Several hypotheses havebeen proposed, among which potential autoimmunity resulting from molecular mimicry between
viral spike protein and human proteins12,13, thereservoir and viral reproduction hypothesis14-17,
and the viral integration hypothesis18-22. Finally, it
has also been proposed that the spike protein, theprimary antigen targeted by COVID-19 vaccines,
could have a potential toxicity that is linked to thedevelopment of long-COVID symptoms23-28.
The spike protein used in vaccines differs fromthe viral spike protein found in SARS-CoV-2 because it has been modified to enhance its stability
and immunogenicity through prefusion stabilization with a double proline substitution29. Both theviral and the vaccine spike protein are consideredharmless and are not expected to circulate freely in the bloodstream, this being one essential
aspect of vaccine safety as official data report30-32.
Indeed, the vaccine spike protein is synthesized
by cells, it remains bound to the cellular membrane, and it is presented on the cell surface to theimmune cells33,34. Moreover, as from official data,
the spike protein should remain in the vicinity of
the injection site and local lymph nodes, where
the immune response is initiated35, and it may
persist up to a few weeks after vaccination36,37.
The official data about spike protein have beenchallenged by recent studies38,39 that proposedthat the spike protein has inherent toxicity, acting
as an inflammagen and thus stimulating inflammation and blood hypercoagulation. Moreover,viral and vaccine spike proteins have been foundin the bloodstream of individuals even months
after infection clearance and vaccination40-45.
Considering the proposed persistence of spike protein in long-COVID syndrome, this study aimed to
specifically investigate the presence of viral and vaccine spike proteins in the blood serum of long-COVID syndrome patients using mass spectrometry
analysis46,47. Secondly, polymerase chain reaction(PCR) was used for a preliminary study to check for
SARS-CoV-2 integration in the long-COVID patients’ leukocytes18, without further investigation48,49.

Patients and Methods
Patient Recruitment
Patient recruitment was conducted based on clinical history and symptoms. We aimed to include a diverse cohort of 81 long-COVID syndrome patients,
14

ensuring representation across different age groups,
genders, and disease severity. Informed consent was
obtained from each participant, and ethical guidelines were strictly followed throughout the study. The
study was approved by the Ethics Committee of Brescia (Italy) Prot. No. NP4588. All research process
was conducted according to the ethical guidelinesof the Declaration of Helsinki. A written informed
consensus was obtained from all patients at the timeof enrollment, and each of them was anonymized.
Mass Spectrometry
Mass spectrometry analysis was performed on
the serum samples obtained from the recruited
long-COVID syndrome patients, with the aim ofdetecting and quantifying spike protein fragments
present in the samples. To achieve this, trypsin digestion was employed, generating specific tryptic
fragments for each spike protein variant. The distinct
tryptic fragments identified in the samples allowed
for discrimination between the vaccine spike protein
and the viral spike protein. The analysis was conducted using an HPLC Surveyor system (Thermofisher, Waltham, MA, USA) equipped with a Halo
Peptide ES-C18 column (2.1 x 50 mm, 2.7 µm). A
two-phase gradient was utilized, with Phase A consisting of H2O with 0.2% Formic Acid (HCOOH)
and Phase C consisting of acetonitrile (CH3CN).
A volume of 5 µL of the sample was injected foranalysis. Data acquisition was performed using a“SANIST” mass spectrometer, utilizing electrosprayionization (ESI) as the ionization source.
Data Analysis
Statistical analysis was not performed due tothe descriptive design of our study. Data analysis
was carried out to analyze the mass spectrometry data and draw meaningful conclusions. The
analysis was processed by SANIST Hb software using a database containing the glycoprotein
spike and other proteins randomly selected to increase accuracy. For the detection of the LDPPEAEVQIDR fragment, ion extractions of the child
ion fragments at m/z 577 of the ions at m/z 979.4
and m/z 830.3 (MS3 technique) were performed.

Results
Patient Recruitment and Clinical Data
Analysis
The study included a total of 81 patients withlong-COVID syndrome. Clinical data were available for 70 patients (Table I).

Viral and vaccinal spike protein detected in the blood of long-COVID patients

Mass Spectrometry Analysis
Out of the 81 long-COVID patients analyzed, fragments of the vaccine spike protein were found in 2 patients, while fragments of the viral spike protein were
found in 1 patient (Table II). Control samples from unvaccinated individuals were negative for spike protein.
The areas of the identified fragments were quantified

to assess the presence and abundance of the spike
protein. Table III provides the areas of the standard
and of the samples in which the vaccine protein was
identified, as well as the corresponding percentages.
The samples in which vaccine spike protein was
identified were collected at least two months after
the administration of the second dose (Table IV).

Table I. Summary of clinical data for 70 patients.
Characteristics		

Case subjects (n=70)

Sex

35 (50%)46 (65.71%)
52
26.1
51 (72.86%)
0 (0%)
34 (48.57%)
35 (50%)1 (1.43%)
7.8%
5.1%
4.4%
2.1%
27 (38.57%)
43 (61.43%)
31 (44.29%)
39 (55.71%)
16 (22.86%)
54 (77.14%)
35 (50%)13 (18.57%)
0 (0%)
22 (31.43%)
50 (71.43%)
13 (18.57%)
42 (60%)18 (25.71%)
23 (32.86%)
18 (25.71%)
44 (62.86%)

Male
FemaleAge (year)		
BMI		
Vaccine (YES)		
Severity score
Asymptomatic
Mild symptoms
Severe symptoms
Intensive care
Asthenia (during COVID)
Asthenia (long-COVID)Headache (during COVID)Headache (long-COVID)
Reinfection
Yes
No
Clinical data
Pneumonia (NO)Pneumonia (YES)
Fever (NO)Fever (YES)
Serology
Not done
Negative
Doubtful
Positive
Therapy
Paracetamol
Hydroxychloroquine
Antibiotics
Antivirals
Corticosteroids
Eparine
Ventilation
BMI: body mass index.
Table II. Spike analysis processing results.
ID

Viral Spike Protein

Vaccinal Spike Protein (PP)

1
8
37

N.D.
SignalN.D.

Low signal
N.D.
Low signal

N.D. = Not Detected.
Table III. Areas of the samples in which vaccine protein was identified and area of the standard.
ID

Area m/z 830.3

Area m/z 979.4

830.3%

979.4%

1
37
Std

12.42
15.94
6554

35.27
13.05
3385

26.04
45.02
65.94

73.96
54.98
34.06

15

K. Dhuli, M.C. Medori, C. Micheletti, K. Donato, et al
Table IV. Vaccine and sample data for patients
ID

Type ofvaccineDate of 2nd
vaccine dose

Date of sample
collection

Vaccine Spikeprotein

Viral Spikeprotein1
37

Pfizer
Pfizer

02/2021
02/202126/04/2021
30/04/2021

Yes
Yes

No
No

Discussions
This study employed mass spectrometry analysis to investigate the presence of viral and vaccine
spike proteins in the blood serum of patients withlong-COVID syndrome. As reported in Table II, themass spectrometry analysis revealed the presence ofboth viral and vaccine spike protein fragments in a
subset of patients with long-COVID syndrome eventwo months after vaccination or after infection clearance and negativity of the COVID-19 test (Table
IV). Official data sustain that the vaccine spike protein remains in the vicinity of the injection site andlocal lymph nodes and that it may persist in the bodyup to a few weeks after vaccination20-24. Our findings, in alignment with other studies and in contradiction with official data, show the presence of boththe vaccine and the viral spike protein in the bloodstream even after infection clearance and several
months after vaccination40-42,45-49. Furthermore, viral
integration in patients’ leukocytes was assessed witha preliminary study following the protocol of Merchant18, without further investigation (Supplementary Data). Having detected the vaccinal protein
in two subjects and the viral protein in one subject
in a cohort of 95 patients, this study has mainly adescriptive function. Nevertheless, these results arealigned with many other already published literature
performed on other independent cohorts. We conclude that considering the proposed toxicity of the spike
protein and that official data sustain that it should
not persist in blood circulation a few weeks after
vaccination, blood samples of long-COVID patientsshould be routinely tested for the presence of vaccine and viral spike protein. Future research should
focus on investigating the specific pathways andinteractions through which viral and vaccine spike
protein can circulate and persist in blood circulation
several months after viral clearance or vaccinationand on its possible negative effects.

Conclusions
This study, in accordance with other publishedinvestigations, shows the persistence in blood
16

circulation of viral spike protein in one patient
after infection clearance and the negativity of
the COVID-19 test, of vaccine spike protein
in two patients two months after vaccination.
This study underscores the importance of mass
spectrometry analysis of long-COVID patientsto detect spike protein persistence. Further research is needed to understand the underlying
mechanisms of spike protein persistence.
Availability of Data and Materials

The data are within the test or in the supplementary materials document.

Funding

This research was funded by the Provincia Autonoma diBolzano, in the framework of LP 14/2006.

Authors’ Contributions

Conceptualization, M.B.; Methodology, S.C.; Investigation,
F.F., A.C., A.P., M.G.D.A., G.A., and S.N.; Writing- original draft preparation, K.D., and M.C.M.; Writing, review
and editing, C.M., K.D., A.M., F.F., A.C., A.P., M.G.D.A.,
G.A., S.C., S.N.; Project administration, M.B.; Funding acquisition, M.B. All authors have read and agreed to the published version of the manuscript.

Ethics Approval

The study was approved on the 12th of January 2021 by the
Ethics Committee of the University of Brescia (Italy), Prot.
No. NP4588.

Informed Consent

A written informed consensus was obtained from all patientsat the time of enrollment, and each of them was anonymized.

Conflicts of Interest

The authors declare no conflict of interest.

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