Lung Transplant Recipients with SARS-CoV-2 Infection Induce Circulating Exosomes with SARS-CoV-2 Spike Protein S2 Which Are Immunogenic in Mice

PURPOSE: Exosomes are nanosized vesicles released by cells into body fluids. We have demonstrated the presence of circulating exosomes containing viral antigens in lung transplant recipients (LTxR) undergoing rejection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an important risk factor for LTxR undergoing immunosuppression. Our goal is to determine whether exosomes with SARS-CoV-2 spike protein are induced in LTxR with SARS-CoV-2 infection and exosomes are immunogenic in mice, inducing immune responses to the spike protein METHODS: We analyzed 67 patients with SARS-CoV-2 infection for the induction of circulating exosomes with SARS-CoV-2 spike protein. Exosomes were isolated from plasma by an exosome precipitation protocol followed by 0.2 micron filtration and size determination by NanoSight300. Exosomes were first analyzed by western blot with specific antibodies to SARS-CoV-2 spike and its nucleoprotein. Eluted proteins from the gel were analyzed by mass spectrometry. Exosomes were subjected to transmission electron microscopy (TEM) to detect spike and nucleocapsid antigens. To determine the immunogenicity of isolated exosomes, C57BL/6 mice were immunized with exosomes carrying SARS-CoV-2 spike protein. RESULTS: Exosomes from SARS-CoV-2 infected LTxR expressed SARS-CoV-2 spike protein S2 and increased levels of RNA related to SARS-CoV-2. Peptides specific for SARS-CoV-2 spike protein in exosomes were confirmed by mass spectroscopy. TEM also revealed the expression of spike protein and nucleocapsid antigens on the exosome surface. Mice immunized with exosomes carrying the spike protein, developed antibodies to SARS-CoV-2 spike antigens. Severe inflammation and lesions were also demonstrated in the lungs of mice immunized with exosomes carrying SARS-CoV-2 spike protein. Splenic lymphocytes from mice immunized with exosomes carrying SARS-CoV-2 spike antigen also demonstrated increased frequency of T-cells which are spike protein antigen specific and secreting IFN-γ and TNF-α . CONCLUSION: SARS-CoV-2 infected LTxR induce circulating exosomes with spike protein and nucleic acids related to SARS-CoV-2. Since the induced exosomes are highly immunogenic, we propose that the exosomes induced by SARS-CoV-2 will have immunological consequences relevant to the COVID19 disease process.