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Abstract SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, 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 ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host. 

Abstract SARS‐CoV‐2 has led to a worldwide pandemic, catastrophically impacting public health and the global economy. Herein, a new class of lipid‐modified polymer poly (β‐amino esters) (L‐PBAEs) is developed via enzyme‐catalyzed esterification and further formulation of the L‐PBAEs with poly(d,l‐lactide‐coglycolide)‐b‐poly(ethylene glycol) (PLGA‐PEG) leads to self‐assembly into a “particle‐in‐particle” (PNP) nanostructure for gene delivery. Out of 24 PNP candidates, the top‐performing PNP/C12‐PBAE nanoparticles efficiently deliver both DNA and mRNA in vitro and in vivo, presenting enhanced transfection efficacy, sustained gene release behavior, and excellent stability for at least 12 months of storage at −20 °C after lyophilization without loss of transfection efficacy. Encapsulated with spike encoded plasmid DNA and mRNA, the lipid‐modified polymeric PNP COVID‐19 vaccines successfully elicit spike‐specific antibodies and Th1‐biased T cell immune responses in immunized mice even after 12 months of lyophilized storage at −20 °C. This newly developed lipid‐polymer hybrid PNP nanoparticle system demonstrates a new strategy for both plasmid DNA and mRNA delivery with the capability of long‐term lyophilized storage.