**7. Epitope vaccine delivery**

One of the drawbacks of using peptide fragments as vaccine antigens is the weak immunogenicity generated compared to inactive and live-attenuated vaccines [24]. A formulation with the addition of adjuvant ingredients is needed to increase the immune response in the subjects. One example of adjuvants that have been used commercially for a long time in vaccine formulations is aluminum salts or "alum" [25]. Several publications report alum mechanisms such as creating a gel depot that prolonged exposure of the immune system to antigen, forming particulate structures that promote antigen uptake by APCs (Antigen Presenting Cells) via phagocytosis, and inducing inflammation and secretion of chemokines. However, its inability to induce Th1 cell-mediated immune responses also becomes a limitation for this adjuvant [26], Since the previous study indicated that the typhoid sub-unit vaccine-induced cellular immunity through TCD4+ [27].

The benefit of liposome as carrier and adjuvant for antigen has been known for quite a long time [28]. Liposomes can boost immune response due to its mechanism to create depot effect by causing antigen retention and slowly releasing them to the immune cells [29]. Liposome delivery of antigen is influenced by lipid bilayer components such as lipid choice and the role of cholesterol [30]. We can either choose neutral lipid or charged lipid as the membrane constructing unit. However, previous research showed that positively charged lipid like DDA (Dimethyldioctadecyl ammonium) has an advantage over neutral lipid [31–33]. Cationic DDA can effectively interact with the APC cell membrane and increase the number of antigens delivered into the cell [34]. To put it simply, the more antigens delivered, the more significant immune responses are. However, DDA could not produce a stable vesicle due to its positive charge due to electro-repulsive force. Therefore, previous publications mentioned that DDA should be formulated with phosphatidylcholine as a major lipid component and cholesterol to maintain membrane integrity [35, 36].
