Vaccinia virus (VACV) is being developed as a recombinant viral vaccine vector for several key pathogens. Dendritic cells (DCs) are specialised antigen presenting cells that are crucial for the initiation of primary immune responses; however, the mechanisms of uptake of VACV by these cells are unclear. Therefore we examined the binding and entry of both the intracellular mature virus (MV) and extracellular enveloped virus (EV) forms of VACV into vesicular compartments of monocyte-derived DCs. Using a panel of inhibitors, flow cytometry and confocal microscopy we have shown that neither MV nor EV binds to the highly expressed C-type lectin receptors on DCs that are responsible for capturing many other viruses. We also found that both forms of VACV enter DCs via a clathrin-, caveolin-, flotillin- and dynamin-independent pathway that is dependent on actin, intracellular calcium and host-cell cholesterol. Both MV and EV entry were inhibited by the macropinocytosis inhibitors rottlerin and dimethyl amiloride and depended on phosphotidylinositol-3-kinase (PI(3)K), and both colocalised with dextran but not transferrin. VACV was not delivered to the classical endolysosomal pathway, failing to colocalise with EEA1 or Lamp2. Finally, expression of early viral genes was not affected by bafilomycin A, indicating that the virus does not depend on low pH to deliver cores to the cytoplasm. From these collective results we conclude that VACV enters DCs via macropinocytosis. However, MV was consistently less sensitive to inhibition and is likely to utilise at least one other entry pathway. Definition and future manipulation of these pathways may assist in enhancing the activity of recombinant vaccinia vectors through effects on antigen presentation.
Vaccinia virus (VACV) is a relative of the smallpox virus and was used for many decades as a successful vaccine that contributed to the eradication of smallpox. Today, through genetic recombination technology, VACV shows potential as a modern vaccine for many unconquered diseases including HIV and cancer. Dendritic cells (DCs) are a specialised subset of immune cells that initiate adaptive immune responses and exploiting the interaction between VACV and DCs, which has not been well studied, may be a key to improving the efficacy of these vaccines. In this study we investigated the mechanisms by which VACV binds to and enters DCs. Here, we examined both the abundant mature virus form of VACV as well as the less common, poorly studied extracellular form. We found that VACV does not bind to the common pathogen-uptake C-type lectin receptors expressed on DCs and that the virus enters DCs via macropinocytosis—a fluid-phase uptake process. Furthermore, the virus is not delivered to the conventional endolysosomal antigen processing pathway in these cells. Our study provides new insights into VACV biology and into possible mechanisms of action of VACV as a recombinant viral vaccine vector which may assist in their rational design in the future.