Response to Comment on “Circulating Exosomes with COVID Spike Protein are Induced by BNT162b2 (Pfizer-BioNTech) Vaccination Prior to Development of Antibodies: Novel Mechanism for Immune Activation by mRNA Vaccines”

We used minor modifications for the isolation of circulating exosomes using a commercial precipitation kit followed by 0.2u filtration and the quality of exosomes isolated had a mean size of 158.7+5.4nm and the NanoSight data was provided in Figure 1A. For the western blot, we have used a commercial antibody specific to SARS-CoV-2 (monoclonal antibody 1A9 from Thermos Fisher Scientific). We found exosomes with S2 on day 14 after the first and second dose, and finally 4 months after the second dose and no further time points were taken. The article by Ogata et al, Clinical Infectious Disease 2021, is very different from our article and an entirely different study, so comparing the two results are not appropriate. Ogata et al is not dealing with circulating exosomes and they are analyzing S1, whereas we are detecting S2 on exosomes. We developed an in-house ELISA for quantifying antibodies specific to the spike and nucleocapsid proteins. The details of commercial reagents used (antibodies from Thermos Fisher Scientific and proteins from Sino Biologicals) are provided in our publication. Since 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 antigens persisted in the exosomes. As stated above, we analyzed day 14 after the first vaccine dose, day 14 after the second dose and finally at 4 months. Our results showed that exosomes with S2 declined dramatically by 4 months (Figure 2E). We did not perform the analysis for exosomes with S2 between day 14 and 4 months, 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 months after the second dose of vaccine, we cannot state that circulating exosomes generated after the first dose of vaccination persisted until 4 months after the second dose of vaccine. We did detect a very low level of S2 at 4 months post-vaccination. As the antibodies 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 month point is a western blot artifact. As the vaccine mRNA is degraded rapidly, we propose that the S2 detection at 4 months 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 (1). The Infectious Disease Society of American (IDSA) estimates that the spike protein that was generated by COVID-19 vaccines last 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 month time point. This needs further investigation.

The quoted article by Ogata et al, Clinical Infectious Disease 2021, has done serial analysis at alternate days followed by first dose of vaccination (D0, D1, 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 antibodies to spike protein. Therefore, we cannot state that the exosomes with spike protein induced after first vaccination persisted.