# 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  
OMVs from Gram-negative bacteria are gaining increasing attention as vaccine candidates for their potential use as carriers of heterologous antigens, presenting the potential for highly effective and easy to produce multi-valent vaccines. Since [PERSON] and [PERSON] first demonstrated the incorporation of heterologous expression of OM and periplasmic proteins in bacterial vesicles [18], several studies have used a variety of strategies to construct recombinant OMVs from different bacteria. Some of the most widely used bacteria for constructing functional OMVs are _Salmonella_ and _E. coli. Salmonella_ have been utilized to produce OMVs, which contain pneumococcal surface protein A (PspA) to protect against _Streptococcus pneumoniae_ infection; whereas _E. coli_ OMVs containing recombinant HtrA were used in chlamydia vaccine development, and both these strategies were directing target antigens to the lumen of OMVs [70; 71]. However, OMVs delivering tumor antigen as cancer vaccines are still in the infancy stage. We aim to explore the preliminary mechanism of OMVs as cancer vaccines for anti-tumor treatment so that more researchers can realize the potential of OMVs as novel cancer vaccines. The mechanisms involved in the design of recombinant OMVs and underlying the functions of OMVs recombinant with heterologous antigens and pathways through which they gain entry in host cells in vitro have been clarified (Figure 1). The mode of OMV transport to the site of the desired immune response has an important impact on potency. Generally, vaccines are injected subcutaneously or intramuscularly; therefore, transport of antigens through the lymphatic system from the peripheral tissues to the lymphoid organs must be considered in vaccine design. When fluids and serum components circulate between blood capillaries and the interstitial space, peripheral immune cells and antigens or pathogens can enter the lymph vessels. Initial lymph vessels are 10-60 \(\upmu\)m in diameter, whereas larger lymphatic vessels can be up to 2 mm in diameter. Bacteria must be carried into the lymphatic system by specialized cells, such as DCs, which can squeeze through openings between overlapping endothelial cells, whereas OMVs, with the size of 20-200 nm, have an intrinsic advantage in transport efficiency [42]. Furthermore, delivery of an OMV-associated antigen in a sulfatase-dependent manner was recently reported, where OMVs were found to traverse the gut mucosal barrier, accessing the gut epithelial cells and underlying intestinal macrophages in a sulfatase-dependent manner, and thus, triggering intestinal inflammation (Figure 1A) [72]. Furthermore, the uptake mechanism of OMVs should be identified and internalization of OMVs can be achieved by five different pathways: clathrin-, caveolin-, and lipid raft-mediated endocytosis; membrane fusion pathways; andropinocytosis (Figure 1B) [49]. Clathrin-mediated endocytosis utilizes dynamin for budding off, and endosomal trafficking routes for vesicle entry; its cargo can then either be returned to the cell surface or targeted to lysosomes for degradation [73]. Based on our understanding of virulence factor transport, it is reasonable to infer that OMVs can utilize toxin-receptor interactions to facilitate their cargo delivery via clathrin dependent endocytosis. The size limit for clathrin-mediated endocytosis is 120 nm (diameter), and therefore, several studies have identified this process as a route for OMV entry, via receptor-ligand binding [74; 75]. Since _Helicobacter pylori_ OMVs are known to transport vacuolating toxin VacA, an important cytotoxic virulence factor during infection, they have been shown to facilitate their cargo delivery via clathrin-mediated endocytosis [76]. Caveolin-mediated endocytosis occurs via invagination (80 nm) of membranes rich in cholesterol, sphingolipids, and caveolin [77].