**9. Enhancement of Platform Immunogenicity through Biochemical Modifications**

Based on the importance of SVPs as medical tools and platforms for the presentation of native viral antigenic sequences, it is critical to understand their immunogenicity in relation to antigen structure in order to enhance or to modulate their immunogenicity. Depending on the SVP type, targeted biochemical modifications of the SVP subunits may allow the generation of SVP variants with enhanced immunogenicity. HBsAgS SVPs are glycosylated lipoprotein particles and are stabilized by extensive intra- and inter-molecular disulfide bonds, which allows targeted modifications of the glycan content and level of disulphide bonding.

Changing disulfide bonding impacts on antigen processing and epitope selection by modifying the conformational flexibility [217]. The three-dimensional structure guides processing and presentation of T helper (Th) and CTL epitopes, and subtle changes in antigen structure can modulate T cell responses due to qualitative and quantitative differences in protein processing [217–220]. Distinct Th cell epitope profiles emerged from human immunodeficiency virus type 1 (HIV-1) gp120 molecules after destabilizing the three-dimensional structure as a consequence of deleted cysteine residues [221]. In an attempt to enhance immunogenicity, HBsAgS SVPs with a reduced level of disulfide bonds were generated. The biochemically modified SVPs showed a higher protease sensitivity, potentially due to introducing structural changes associated with enhanced cellular immunogenicity [65]. Altering SVP structure may represent an attractive strategy to modulate proteolytic sensitivity to influence antigen processing and promoting an enhanced immune response and/or a changed hierarchy of epitope presentation [218,222].

Manipulation of protein glycosylation represents an alternative strategy to promote antigen internalization and antigen presentation via MHC class I and class II molecules to enhance the adaptive immune responses [223,224]. The glycosylation status and glycan density of the immunogen impacts on its interaction with antigen-presenting cells and recognition by lectins [224]. Glycan-mediated interactions with immunocompetent cells impact on protein uptake and can enhance or modulate cell-mediated and humoral immune responses [225–229]. Contrarily, glycans can shield protein epitopes to evade recognition by antibodies and can block antigen processing [230]. Mannosylation provided an efficient strategy to improve uptake and processing of a SVP derived from the rabbit hemorrhagic disease virus [225]. Consistently, mannosylated solid lipid nanoparticles loaded with HBsAg induced stronger cellular responses than nanoparticles devoid of mannose [229]. Mutant HBsAgS subunits with additional *N*-glycosylation sites assembled into hyperglycosylated SVP. Antigenic fingerprints indicated that additional glycans do not extensively shield HBsAg-specific antigenic sites. Immunization studies demonstrated that the hyperglycosylated SVPs induced earlier and longer-lasting antibody responses than hypoglycosylated SVPs or wild type SVPs [231]. The ability of biochemically modified SVPs to promote immune responses possibly due to differences in their glycosylation-related interaction with cells of the innate immune system illustrates approaches for the design of immunogens with superior immunological characteristics.
