Detection by super-resolution microscopy of viral proteins inside bloodborne extracellular vesicles

Abstract

Aim: Extracellular vesicles (EVs) are small particles released by all cells, including virally infected cells, into the extracellular space. They play a role in various cellular processes, including intercellular communication, signaling, and immunity, and carry several biomolecules like proteins, lipids, and nucleic acids that can modulate cellular functions mostly by releasing their cargo inside the target cells via the endocytic pathway. One of the most exciting aspects of EV physiology is its potential in liquid biopsy as a diagnostic and prognostic marker. However, due to their extremely small size and lack of a molecular approach to examine intravesicular content or cargo, we cannot fully utilize their potential in healthcare.

Methods: Here, we present a novel approach that allows examining bloodborne EVs at a single-particle level with the ability to examine their cargo without disrupting structural integrity. Our technique utilizes super-resolution microscopy and a unique permeabilization process that maintains structural integrity while facilitating the examination of EV cargo. We used a mild-detergent-based permeabilization buffer that protects the integrity of EVs, minimizes background, and improves detection.

Results: Utilizing this approach, we were able to recognize viral proteins of SARS-CoV-2 virus in COVID-19 patients, including spike and nucleocapsid. Surprisingly, we found an almost equal amount of spike protein inside and on the surface of bloodborne EVs. This would have proven difficult to determine using other conventional methods.

Conclusion: To summarize, we have developed an easy-to-perform, sensitive, and highly efficient method that offers a mechanism to examine bloodborne EV cargo without disrupting their structural integrity.

Keywords

Extracellular vesicles / super-resolution microscopy / permeabilization / intravesicular cargo / SARS-CoV-2 / spike / COVID-19

References

[1] Raposo G.Extracellular vesicles: exosomes, microvesicles, and friends.J Cell Biol2013;200:373-83 PMCID:PMC3575529

[2] Santos MF,Karbanová J.HIV-1-induced nuclear invaginations mediated by VAP-A, ORP3, and Rab7 complex explain infection of activated T cells.Nat Commun2023;14:4588 PMCID:PMC10415338

[3] Santos MF,Karbanová J,Corbeil D.VAMP-associated protein-A and oxysterol-binding protein-related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum.J Biol Chem2018;293:13834-48 PMCID:PMC6130944

[4] Gould SJ,Hildreth JE.The Trojan exosome hypothesis.Proc Natl Acad Sci U S A2003;100:10592-7 PMCID:PMC196848

[5] Kalluri R.The biology, function, and biomedical applications of exosomes.Science2020;367:eaau6977

[6] Green TM,Barsky SH,Lorico A.Analogies between cancer-derived extracellular vesicles and enveloped viruses with an emphasis on human breast cancer.Curr Pathobiol Rep2016;4:169-79 PMCID:PMC7099913

[7] Nolte-'t Hoen E,Gallo RC.Extracellular vesicles and viruses: are they close relatives?.Proc Natl Acad Sci U S A2016;113:9155-61 PMCID:PMC4995926

[8] Lenassi M,Liao M.HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells.Traffic2010;11:110-22 PMCID:PMC2796297

[9] Chen L,Yue H.Exosomes derived from HIV-1-infected cells promote growth and progression of cancer via HIV TAR RNA.Nat Commun2018;9:4585 PMCID:PMC6214989

[10] Arakelyan A,Zicari S,Margolis L.Extracellular vesicles carry HIV Env and facilitate Hiv infection of human lymphoid tissue.Sci Rep2017;7:1695 PMCID:PMC5431974

[11] Testa JS,Comber JD.Exosome-driven antigen transfer for MHC class II presentation facilitated by the receptor binding activity of influenza hemagglutinin.J Immunol2010;185:6608-16 PMCID:PMC3673890

[12] Zicari S,Palomino RAÑ.Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins.Virology2018;524:97-105 PMCID:PMC6258833

[13] Izquierdo-Useros N,Archer J.Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway.Blood2009;113:2732-41 PMCID:PMC2661860

[14] Robbins PD.Regulation of immune responses by extracellular vesicles.Nat Rev Immunol2014;14:195-208 PMCID:PMC4350779

[15] Vincent-Schneider H,Lankar D.Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells.Int Immunol2002;14:713-22

[16] Utsugi-Kobukai S,Hotta C,Minami M.MHC class I-mediated exogenous antigen presentation by exosomes secreted from immature and mature bone marrow derived dendritic cells.Immunol Lett2003;89:125-31

[17] Théry C,Segura E,Lantz O.Indirect activation of naïve CD4+ T cells by dendritic cell-derived exosomes.Nat Immunol2002;3:1156-62

[18] Fu C,Loschko J.Plasmacytoid dendritic cells cross-prime naive CD8 T cells by transferring antigen to conventional dendritic cells through exosomes.Proc Natl Acad Sci U S A2020;117:23730-41 PMCID:PMC7519282

[19] Ye Y,Mohty M.Plasmacytoid dendritic cell biology and its role in immune-mediated diseases.Clin Transl Immunology2020;9:e1139 PMCID:PMC7248678

[20] Marcoux G,Hasse S.Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules.Blood2021;138:2607-20

[21] Pesce E,Cordiglieri C.Exosomes recovered from the plasma of COVID-19 patients expose SARS-CoV-2 spike-derived fragments and contribute to the adaptive immune response.Front Immunol2021;12:785941 PMCID:PMC8801440

[22] Troyer Z,Tabler CO.Extracellular vesicles carry SARS-CoV-2 spike protein and serve as decoys for neutralizing antibodies.J Extracell Vesicles2021;10:e12112 PMCID:PMC8213968

[23] DeMarino C,Cowen M.Detection of SARS-CoV-2 nucleocapsid and microvascular disease in the brain: a case report.Neurology2023;100:624-8 PMCID:PMC10065204

[24] Chu H,Yuen TT.Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study.Lancet Microbe2020;1:e14-23 PMCID:PMC7173822

[25] Mondal A,Phulpagar P,Shiras A.Effective visualization and easy tracking of extracellular vesicles in glioma cells.Biol Proced Online2019;21:4 PMCID:PMC6419365

[26] Hartjes TA,Jenster GW,van Royen ME.Extracellular vesicle quantification and characterization: common methods and emerging approaches.Bioengineering2019;6:7 PMCID:PMC6466085

[27] Rappa G,Green TM.Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.Oncotarget2017;8:14443-61 PMCID:PMC5362417

[28] Rappa G,Anzanello F,Lorico A.Biochemical and biological characterization of exosomes containing prominin-1/CD133.Mol Cancer2013;12:62 PMCID:PMC3698112

[29] Théry C,Aikawa E.Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the international society for extracellular vesicles and update of the MISEV2014 guidelines.J Extracell Vesicles2018;7:1535750 PMCID:PMC6322352

[30] Santos MF,Karbanová J.Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum.J Extracell Vesicles2021;10:e12132 PMCID:PMC8363911

[31] White ED,Yi H,Scherer NF.Volumetric microscopy of CD9 and CD63 reveals distinct subpopulations and novel structures of extracellular vesicles in situ in triple negative breast cancer cells.BioRxiv2022;11:08.515679

[32] Mao K,Ma Y.Proteomics of extracellular vesicles in plasma reveals the characteristics and residual traces of COVID-19 patients without underlying diseases after 3 months of recovery.Cell Death Dis2021;12:541 PMCID:PMC8146187

[33] Yim KHW,Chahwan R.Serum extracellular vesicles profiling is associated with COVID-19 progression and immune responses.J Extracell Biol2022;1:e37 PMCID:PMC9088353

[34] Craddock V,Spikes L.Persistent circulation of soluble and extracellular vesicle-linked Spike protein in individuals with postacute sequelae of COVID-19.J Med Virol2023;95:e28568 PMCID:PMC10048846

[35] Cheng L.Therapeutically harnessing extracellular vesicles.Nat Rev Drug Discov2022;21:379-99

[36] Pontelli MC,Martins RB.SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.J Mol Cell Biol2022;14:mjac021 PMCID:PMC9384834

[37] Xia B,Luo RH.Extracellular vesicles mediate antibody-resistant transmission of SARS-CoV-2.Cell Discov2023;9:2 PMCID:PMC9821354