# Design of Outer Membrane Vesicles as Cancer Vaccines: A New Toolkit for Cancer Therapy
## 3 New OMV Applications: Tumor Vaccines
### Mechanisms of OMVs Design as Cancer Vaccines
[PERSON] and colleagues identified this process in nontypeable _Haenophilus influenzae_ and showed that OMVs colocalized with the endocytosis protein caveolin, indicating that internalization is mediated by caveolae, which are cholesterol-rich lipid raft domains [78]. Despite sluggish internalization speed (five-times slower than clathrin-mediated), it leads in the efficient delivery of cargo to the cytosol [73]. Lipid rafts are domains of the plasma membrane that are also enriched in sphingolipids and cholesterol [77]. It is hypothesized that clustering of cholesterol-rich regions causes curvature in the membrane, driving movement of molecules into the host cell via invagination (90 nm). Membrane fusion is another mechanism utilized by OMV for entry into host cells; this does not entirely depend on active, energetic processes and the exact mechanisms involved are still under investigation [49; 79]. Further, macropinocytosis is characterized by the formation of large(over 200 nm in diameter), actin-driven, ruffled protrusions from the cell membrane [80]. However, macropinocytosis is generally not a cargo-induced process; it allows internalization of endocytic vesicles up to 1 \(\upmu\)m in diameter; thus, entry via this route is thought not to be a deliberate OMV-driven event [81]. Therefore, the size of the OMV population is relevant when studying endocytic routes, as macropinocytosis allows internalization of vesicles of 1 \(\upmu\)m diameter, while clathrin-dependent endocytosis allows internalization of particles with a maximum diameter of 120 nm. Different isolation methods can introduce a bias towards particular sizes of OMVs, and therefore the purification method is also one of the most important considerations in the design of OMV vaccines. Figure 1: Design of mechanisms underlying the functions and immune modulation of outer membrane vesicles (OMVs) recombinant with heterologous antigens and the pathways through which they gain entry into host cells. (**A**) OMVs can also cross the mucus barrier in the gut and reach the intestinal epithelium, delivering bacterial antigens to the underlying macrophages, triggering intestinal inflammation. (**B**) Mechanisms of OMV entry. Pathogenic Gram-negative bacteria are thought to utilize OMVs to interact with host cells during infection. For example, bacteria can use OMVs to mediate the delivery of virulence factors, such as toxins, into host cells, including immune cells, and OMVs may enter host cells through various endocytic routes including clathrin-dependent, caveolin-mediated, lipid raft, and membrane fusion pathways. The most frequently reported mode of OMV entry into host cells involves lipid rafts as OMVs could fuse with lipid rafts to facilitate their entry into host cells. The pathways of cholesterol-independent and clathrin-mediated endocytosis are independent of lipid rafts. Moreover, OMVs can enter host cells via the mechanism of membrane fusion in a size-dependent manner. (**C**) A model for OMVs targeting cancer cells, epithelial cells, and immature dendritic cells (DCs) to mediate immune responses. OMVs can interact directly with epithelial cells and immune cells or they may interact with pattern recognition receptors, such as Toll-like receptor 4 (TLR4), to induce the production of cytokines and chemokines. OMV adjuvant delivered antigen could be recognized by DCs that led to the recruitment of immune cells and stimulated antigen-presenting cells (APC) through toll like receptor (TLR) recognized pathogen-associated molecular patterns (PAMPs). These process enhanced T helper cells production (including Th1 and Th2), and fully amplified cellular and humoral immunity. Furthermore, OMVs delivering antigens can also traffic into non-immune cells and load onto MHC class II molecules. Activated antigen-presenting cells express MHC class II molecules that interact with the T cell receptor (TCR) on CD4+ T cells to drive antigen-specific T cell responses, resulting in T helper cell proliferation, thereby generating antigen-specific antibodies in various tissues.