Coronavirus disease 2019 (COVID-19) is a systemic, potentially severe and
life-threatening disease, triggered by severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2). Since the first cases of SARSCoV-2 infection were officially diagnosed, in
December 2019, more than 280 million cases and 5 million deaths have been declared
worldwide. In response to this public health emergency the international scientific
community has made an enormous effort to understand the virus and to develop a safe
vaccine to prevent the spread of COVID-19. Just few days after the World Health
Organization declared the SarsCoV-2 outbreak a global pandemic, it was published the
structure of the Spike viral protein [1], which has provided critical clues to design
vaccines. Within less than 12 months several vaccines against SARS-CoV-2 have been
developed. The first vaccine that has been approved in Europe and that is already being
administered in Italy is the COVID-19 RNA vaccine, developed by BioNTech-Pfizer
(BNT162b2). Both this vaccine and the one developed by Moderna consist of a lipid
nanoparticle (LNP) that deliver a nucleoside-modified messenger RNA (mRNA) encoding the
precise genetic information of the immunogen (SARS-CoV-2 full-length spike protein) to
antigen presenting cells and elicits potent immune responses. mRNA is transiently
expressed, does not integrate into the genome, and is degraded by physiological
pathways.mRNA vaccines are molecularly well defined, synthesized efficiently from DNA
templates by in vitro transcription, which is cell- and animal-origin material-free. mRNA
production and LNP formulation are fast processes of high scalability, rendering this
technology suitable for rapid vaccine development and pandemic vaccine supply [2].

Both mRNA vaccines, administered with a two-dose regimen, have been shown to provide a
94-95% protection against Covid-19 in persons 16 years of age or older [3,4]. However,
the thromboinflammatory response toward these vaccines has never been explored as they
exploit a completely new technology. For instance, it was reported that mRNA vaccines are
highly reactogenic right after vaccine administration in particular in young adults [3],
but we do not know which cells drive the early immune response to LNP-mRNA vaccines in
humans and if platelets become activated as well. Moreover, it is not known if female,
who have a heightened immune response to other vaccines, are able to mount a faster
response to this new type of vaccines.

AIMS OF THE STUDY

Primary objective To characterize the platelet and immune response and the
platelet-immune cross-talk in subjects undergoing SARS-CoV-2 vaccination.

Secondary objective To study the short-term kinetics of the immune and the antibody
response in subjects undergoing SARS-CoV-2 vaccination.

STUDY PROTOCOL

We will enrol female and male (1:1) volunteers without signs of infection who will be
subjected to SARS-CoV-2 Vaccination.

Enrolled healthy volunteers will be asked to undergo venous blood withdrawals at the
following time-points:

- T0: 0-24 hours before the first dose of the vaccine;

- T1: 72±24 hours after the first dose of vaccine administration;

- T2: 10±2 days after first dose of vaccine administration;

- T3: 0-24 hours before the second dose of vaccine;

- T4: 72±24 hours after second dose of vaccine administration;

- T5: 10±2 days after second dose of vaccine administration.

A small aliquot of blood anticoagulated with sodium citrate will be used within 1 hour
from blood withdrawal to perform in vivo assays (see list below), the rest will be
centrifuged within 2 hours and plasma and serum samples will be stored at -80°C for
further analysis (see list below).

REFERENCES

1. Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS,McLellan
JS.Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science.
2020 Mar 13;367(6483):1260-1263. doi:10.1126/science.abb2507.

2. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines - a new era in vaccinology.
Nat Rev Drug Discov. 2018 Apr;17(4):261-279. doi:10.1038/nrd.2017.243.

3. Polack FP, Thomas SJ, Kitchin N, et al. Safety and Efficacy of the BNT162b2 mRNA
Covid-19 Vaccine. N Engl J Med. 2020;383(27):2603-2615. doi:10.1056/NEJMoa2034577

4. Anderson EJ, Rouphael NG, Widge AT, Jackson LA, et al.; mRNA-1273 Study Group.
Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. N Engl J
Med. 2020 Dec 17;383(25):2427-2438. doi: 10.1056/NEJMoa2028436.

5. Lanza GA, Barone L, Scalone G, et al.; Inflammation-related effects of adjuvant
influenza A vaccination on platelet activation and cardiac autonomic function. J
Intern Med. 2011 Jan;269(1):118-25. doi: 10.1111/j.1365-2796.2010.02285.x.

At baseline (T0) each adult enrolled will be asked to fill in a short questionnaire, to
provide information on baseline characteristics, previous relevant medical history (i.e.
chronic medical conditions, use of drugs with a particular focus on immunosuppressant
agents), smoking and exposure to SARS-CoV-2 (i.e. if a previous diagnosis of COVID-19 was
received).

At any further timepoint (T1-T4), each participant will be asked to fill in a short
questionnaire on symptoms related to vaccination, occurrence of COVID-19 infection, and
drugs used, to account for potential confounders.