SARS-CoV-2 Induced Cellular Adaptations to Avoid Humoral Immunity Countermeasures

Viruses have developed multiple strategies of immune evasion whose molecular biology has been increasingly studied in the past years. There are mechanisms, however, that are effective because they promote changes in the overall architecture of the infected cells. While optical microscopy allows functional characterization by fluorescent probes, ultrastructural studies with scanning and transmission electron microscopy are instrumental to the identification of these architectural changes, often sized below the optical resolution. The extensive use of helium-ion microscopy (HeIM) is described in this chapter. HeIM is a specific modality of ultrastructural microscopy that avoids the artifacts of electroconductive coating required by scanning electron microscopy. Meaningful results can be obtained by combining HeIM with fluorescence and phase-contrast microscopy in a correlative way. A group of structural adaptations induced by SARS-CoV-2 infection on VeroE6 cells has been detected by HeIM/fluorescence/phase-contrast correlative microscopy. These adaptations provide an alternative viral transmission route via intracytoplasmic mechanisms that are totally shielded from antibody-mediated immune surveillance. Correlative microscopy performed on VeroE6 cells infected with mNeonGreen-expressing SARS-CoV-2 showed the presence of (1) long “tunneling nanotubes” that connect two or more host cells over submillimeter distances; (2) large-scale multiple “cell fusion” events (syncytia); and (3) abundant “extracellular vesicles” of various sizes. Taken together, these three ultrastructural features provide a shared intracytoplasmic (or intra-cytosolic) environment that allows viral transmission without the need of the well-known extra-cytoplasmic virion–ACE2 docking mechanism. Such intracytoplasmic route may justify the elusiveness of SARS-CoV-2 to survive antibody-based immune surveillance and the recurrence of infections in vaccinated individuals.