**6. The Second Vaccine Generation Against HBV: Yeast-Derived Recombinant HBsAgS SVPs**

The identification of HBsAgS as the major HBV envelope protein of plasma-derived HBsAg SVPs and encoding the major antigenic determinants prompted the expression of HBsAgS in mammalian cell lines and yeast [13]. Yeast cell strains (*Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha*) were developed which express HBsAgS in high quantities. HBsAgS SVPs are isolated from yeast cell extracts with a sedimentation rate and buoyant density similar to particles from human samples or expressed in cell culture [138–140]. Yeast-derived SVPs have reduced antigenic reactivity compared to SVPs derived from human plasma but immunization studies established that yeast-derived SVPs induce anti-HBs antibody responses, which provide protection of immunized chimpanzees following a challenge with HBV. Importantly, the yeast-derived HBsAgS SVP vaccine of the "adw" subtype conferred protection against HBV subtypes "adr" and "ayw" [28]. Clinical trials using different age groups, healthy individuals, and special target populations confirmed that the yeast-derived vaccine is highly immunogenic, and generated qualitative and quantitative anti-HBs antibody responses with a protective efficacy similar to the plasma-derived vaccines [141]. The vaccines achieved 99% seroprotection rates in healthy children and adolescents but approximately 5%–7% of the adult population are non-responders and the rate can increase to 70% in elderly persons and in special risk groups [13,129,136,142]. Also, genetically determined resistance may contribute to non-responsiveness to HBsAgS SVP vaccines [143–145]. At the molecular level, yeast-derived HBsAg SVPs are not *N*-glycosylated at the N146 position within the S-domain, in contrast to SVPs produced in mammalian cell lines and harvested from the cell culture medium, or isolated from the blood of chronic hepatitis B patients. HBsAgS expressed in yeast generate SVPs [146], but experimental evidence indicates that the SVP are not formed within the yeast cell and generated during the down-stream purification procedures. HBsAgS expression in *Pichia pastoris* showed that HBsAgS assembles at the ER into multi-layered lamellar structures [147]. Monitoring SVP assembly during the purification procedure demonstrated that particulate structures are formed after eluting HBsAg bound to colloidal silica. Irregular SVP-like structures were visualized, and morphological changes observed after pH adjustment (colloidal silica eluate pH 10.8 to 8.0), ion-exchange, and size-exclusion chromatography. The monodispersity improved after potassium thiocyanate (KSCN) treatment, also the SVPs have a more-fine structured surface [148–150]. The correct disulfide bonding is the molecular basis for the formation of native epitopes as probed with an anti-HBs antibody [151]. Lipid-containing SVPs undergo KSCN-induced maturation by the formation of intra- and inter-molecular disulfide bonds to

generate fully disulfide-bonded SVPs, resulting in a decreased conformational flexibility of HBsAgS in the matured particles [149,152]. Restricted conformational flexibility is possibly required for the formation of native HBsAg-specific antigenic structures, which allows epitope recognition by anti-HBs antibodies, and importantly in eliciting neutralizing antibodies [149]. The particle size (diameter) of yeast-derived SVPs is consistent with the data obtained from mammalian-cell-derived SVPs, but the size distribution can vary depending on the SVP maturation level [148,153]. Yeast-derived SVPs have a high content of alpha-helical structures, and the movement of the HBsAgS proteins is restricted due to the tight association with the lipid membrane [102,154]. Similarly, the lipid composition is characterized by high levels of phospholipids, in particular, phosphatidylcholine consistent with mammalian cell-derived SVPs [154–156]. No significant differences in the anti-HBs response induced by the plasma-derived or yeast-derived hepatitis B vaccines was observed [123,157].

The widely distributed, yeast cell-derived hepatitis B vaccines, Engerix®-B (GlaxoSmithKline) and Recombivax HB™ (Merck Sharp and Dohme) use aluminium hydroxide or aluminium hydroxyphosphate sulfate as adjuvanting substances, respectively. Aluminium-based adjuvants are widely used and activate the inflammasome pathway [158–160]. The adsorption of HBsAgS SVPs on aluminium hydroxide is mediated by binding of the phosphate groups of the HBsAgS SVPs phospholipids with hydroxyl groups of aluminium hydroxide through a ligand-exchange mechanism [161,162]. After vaccine administration, the SVPs are eluted from the aluminium adjuvant upon contact with the interstitial fluid [163,164]. Adsorption of HBsAgS SVPs derived from *Hansenula polymorpha* on an aluminium gel followed by a mild desorption step using competing phosphate anions demonstrated that the conformation of the HBsAgS protein is retained, and consistently, no significant changes of the lipid core and lipid membrane surface of the SVPs were identified [165]. To improve immunization outcomes in adults at risk of a hepatitis B infection, HBsAgS SVPs synthesized in *Hansenula polymorpha* are formulated with a Toll-like receptor 9 (TLR 9) agonist, cytidine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) 1018 as an adjuvant (Heplisav-B™, Dynavax Technologies). TLR9 is a pattern recognition receptor of the innate immune system, which induces the production of cytokines such as interleukin-12 and interferon-alpha to stimulate the adaptive immune response [134,166]. Heplisav-B™ induced earlier seroprotection rates allowing a two-dose regimen compared to three doses required for the Engerix-B vaccine, but it caused more injection-site reactions [134].

The ability of HBsAgS subunits expressed in yeast cells to form SVPs during down-stream procedures, and to reproduce native antigenic structures, allowed the development of a highly successful preventative vaccine. The advancement of the vaccine adjuvant technology allowed new HBsAgS SVP formulations and demonstrated strategies to enhance the magnitude of the anti-HBs immune response with immediate practical applications
