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

Abstract

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.

Figure 1:

(A) Representative nanosight image for exosomes from vaccinated individuals with mean and median sizes (black thin line in the graph indicates the 3 measurements of the same sample and red line is the average of all 3 lines). (B) Transmission Electron Microscopy Images of SARS-CoV-2 spike antigen on exosomes from control exosomes from control and vaccinated individuals. Arrows indicate SARS-CoV-2 spike positive exosomes. Right side third image is the zoomed image of positive exosome from Vaccinated sample. We have used anti-coronavirus FIPV3-70 Ab as negative control for both the samples.

Figure 2:

(A) Levels of SARS-CoV-2 spike Ab in vaccinated healthy individuals at D0, D7 and 14 after first dose of vaccine, D14 after second dose of vaccine and 4 months after second dose of vaccine. (B) Western blot of exosome protein SARS-CoV-2 spike protein S2 at D0, D7 and D14 of first vaccination, D14–2 after second dose of vaccine and 4 months after second dose of vaccine. (C) Densitometry and statistical analysis of western blots. (D) Before and after line plot of levels of SARS-CoV-2 spike Ab in vaccinated healthy individuals at D14–1, D14–2 and 4M after second dose of vaccine (E) Before and after line plot of densitometry of western blots for SARS-CoV-2 spike protein in vaccinated healthy individuals at D14–1, D14–2 and 4M after second dose of vaccine. (F) Western blot of SARS-CoV-2 spike protein S2 in exosomes within 14 days after second dose of vaccine in healthy individuals. (G) Densitometry and statistical analysis of western blots. (H) Levels of SARS-CoV-2 spike Ab in vaccinated healthy individuals at 14 days after second dose of vaccine. (I) EliSPOT for cytokine development (TNFα and IFNγ) in response to SARS-CoV-2 spike protein. *All graphs are represented as scatter dot plots with mean and SD (vertical bar line). CD9 is used to normalize the all the blots. Western blots and Antibody development experiments were performed at least 3 times independently.

Figure 3:

(A) ELISA of serum SARS-CoV-2 spike after immunizing the mice with exosomes from controls, and vaccinated individuals at D15, D21 and D30. (B) EliSPOT for cytokine development (IL10, IL17, TNFα and IFNγ) in splenocytes in response to SARS-CoV-2 spike antigen after immunizing the mice with exosomes from controls, and vaccinated individuals at D30. Graphs are represented as bar graphs with mean and SD (vertical bar line). The mice experiments were performed at least 3 times independently and in each group there were n=3 or n=5 animals.