Although reading the study by Bansal et al. (1) was compelling and intriguing, some of the results presented raise some issues, which I highlight herein.

1) Fig. 1: There is a discrepancy between the size of the extracellular vesicle (EV) distribution by a quantitative method (NanoSight, Fig. 1A) compared with the micrograph images presented in Fig. 1B. An analysis of Fig. 1A reporting the distribution of EVs by their size indicates that the vast majority of EVs have a size between 100 and 200 nm. However, the micrograph images presented in Fig. 1B show a very different pattern between controls (unvaccinated individuals) and vaccinated individuals in terms of size. For instance, the EVs represented for the control appear representative of the expected size reported in Fig. 1A (i.e., the 100–200 nm range). In contrast, the micrograph image representative of a vaccinated group shows distinct features as marked by a paucity of EV counts per field, but also an abnormally large size (i.e., 400–500 nm, using the scale bar as a reference). In particular, the micrograph image presented in Fig. 1B for a vaccinated individual stained against SARS-CoV-2 spike (upper panel) significantly differs from the micrograph stained against anti-feline coronavirus (feline infectious peritonitis virus) nucleocapsid (bottom panel).

2) Fig. 1B: The transmission electron microscopy (TEM) micrographs of EVs using Abs against spike were, in my opinion, not able to reproduce similar experiments reported in the literature. Coronaviruses under TEM typically represent an osmiophilic “crown” at low resolution and distinguishable spike morphometric structures at a resolution of 100 nm [e.g., see Laue et al. (2) for a representative TEM].

3) Fig. 2B: The authors performed an immunoblot analysis for the presence of spike-laden EVs in vaccinated patients. My concern is that the blots presented in this figure do not follow common practice (3), as they do not present spike protein levels over time within the same blot (2). These data are normally accompanied by a semiquantitative graph (spike/CD9 OD ratio) with individual values and adequate statistical analysis (one-way ANOVA), because the data, as presented, suggest a very important interindividual variability.

1
Bansal
,
S.
,
S.
Perincheri
,
T.
Fleming
,
C.
Poulson
,
B.
Tiffany
,
R. M.
Bremner
,
T.
Mohanakumar
.
2021
.
Cutting edge: circulating exosomes with COVID spike protein are induced by BNT162b2 (Pfizer-BioNTech) vaccination prior to development of antibodies: a novel mechanism for immune activation by mRNA vaccines
.
J. Immunol.
207
:
2405
2410
.
2
Laue
,
M.
,
A.
Kauter
,
T.
Hoffmann
,
L.
Möller
,
J.
Michel
,
A.
Nitsche
.
2021
.
Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures
.
Sci. Rep.
11
:
3515
.
3
Tie
,
L.
,
H.
Xiao
,
D. L.
Wu
,
Y.
Yang
,
P.
Wang
.
2021
.
A brief guide to good practices in pharmacological experiments: Western blotting
.
Acta Pharmacol. Sin.
42
:
1015
1017
.