We used minor modifications for the isolation of circulating exosomes using a commercial precipitation kit followed by 0.22 u filtration, the quality of exosomes isolated had a mean size of 158.7 + 5.4 nm, and the NanoSight data were provided in Fig. 1A. For the Western blot, we have used a commercial Ab specific to SARS-CoV-2 (mAb 1A9 from Thermo Fisher Scientific). We found exosomes with S2 on day 14 after the first and second dose, and finally 4 mo after the second dose and no further time points were taken.
The article by Ogata et al. (1) is very different from our article and an entirely different study, so comparing the two results is not appropriate. Ogata et al. (1) did not deal with circulating exosomes and they analyzed S1, whereas we detected S2 on exosomes. We developed an in-house ELISA for quantifying Abs specific to the spike and nucleocapsid proteins. The details of commercial reagents used (Abs from Thermo Fisher Scientific and proteins from Sino Biological) were provided in our article. Because we did not perform serial analysis starting from day 14 following the first and second dose of vaccine for circulating exosomes with S2, we cannot state how long these Ags persisted in the exosomes. As stated above, we analyzed Ab development and exosomes on day 14 after the first vaccine dose, day 14 after the second dose, and finally at 4 mo.
Our results showed that exosomes with S2 declined dramatically by 4 mo (Fig. 2E). We did not perform the analysis for exosomes with S2 between day 14 and 4 mo, following the second dose. Therefore, due to a lack of testing at regular time intervals between day 14 after the first and second dose to 4 mo after the second dose of vaccine, we cannot state that circulating exosomes generated after the first dose of vaccination persisted until 4 mo after the second dose of vaccine. We did detect a very low level of S2 at 4 mo postvaccination. As the Abs used in this analysis did not detect any S2 on day 0, we do not believe the slight amount of detection of S2 at the 4 mo point is a Western blot artifact. As the vaccine mRNA is degraded rapidly, we propose that the S2 detection at 4 mo reflects membrane-bound S2 protein that is still present in inoculated cells. S2 is quite stable and there are reports of S2 protein present several weeks after transfection (2). The Infectious Diseases Society of America estimates that the spike protein generated by COVID-19 vaccines lasts up to a few weeks. Therefore, we propose that exosomes derived from vaccinated S2-positive cells are the source of the detectable S2 at the 4 mo time point. This needs further investigation.
The quoted article by Ogata et al. (1) described serial analysis at alternate days followed by first dose of vaccination (days 0, 1, 3, 5, 7, 9, 14, 28, 29, 31, 33, 35, 42 and 56). However, we did not perform similar serial analysis for circulating exosomes with spike protein or Abs to spike protein. Therefore, we cannot state that the exosomes with spike protein induced after first vaccination persisted.
