The HEV vaccine developed based on
The HEV vaccine, developed based on p239 particles, is of good efficacy, immunogenicity and safety, and was licensed in China in 2012. p239 (aa 368–606) and E2 (aa 394–606) share a common region of ORF2, referred to as E2s (aa 459–606), which harbors the major antigenic determinants of the HEV vaccine and is responsible for virus-host interactions. Due to its intrinsic self-assembly property, p239 can form particles, making it an excellent immunogen for immune recognition and antibody elicitation. In this study, we attempted to design a non-particulate immunogen by fusing E2 to the minimized functional fragment of CRM197, and evaluated whether this immunogen maintained the good antigenicity and immunogenicity of p239. As expected, CRM197(A)-E2 mainly associated as a homodimer in solution and conferred high immunogenicity in mice. However, CRM197(A)-E2 showed 10-times stronger immunogenicity than that of p239 in the ED50 assay, and elicited a 10- to 1000-fold higher antibody titer at the same dosage (2 μg and 0.5 μg) as p239. Even at lower dosages (0.05 μg), CRM197(A)-E2 still induced an antibody titer as high as 108 that persisted at 106 for at least 28 weeks, compared with the relatively much lower immunogenicity of p239 at no more than 103 titer and less than 102 over the long term, coming to levels as low as that of E2, which is known to have poor immunogenicity. Taken together, the CRM197 A-fragment can enhance the immunogenicity of the E2 dimer better than p239, which particulates E2 via the N-terminal hydrophobic tail (Zhang et al., 2016).
Conflicts of interest
Introduction Ubiquitylation is a post-translational modification which impacts almost every biological process in the cell. Dysregulation of the ubiquitylation pathway is associated with several diseases, including cancer, neurodegenerative disorders, and immunological dysfunctions. Single ubiquitin moieties or polyubiquitin chains are added to the substrate by the combined action of three different Ellipticine of enzymes: the E1 activating enzymes, the E2s conjugating enzymes, and the E3 ligase enzymes (Pickart, 2001). In the first step, a single ubiquitin molecule is coupled to the active site of an E1 ubiquitin-activating enzyme in an ATP-dependent reaction. In the second step, the ubiquitin molecule is transferred from E1 to an E2 ubiquitin conjugating enzyme. In the final step, ubiquitin is transferred to the protein substrate in a process mediated by an E3 ubiquitin ligase, which provides a binding platform for ubiquitin-charged E2 and the substrate. Ubiquitin chain formation is highly specific and regulated by a plethora of different E2 conjugating enzymes and E3 ligases. The human genome encodes two ubiquitin-activating E1, >30 ubiquitin-specific E2, and 600–700 of E3 ligases (Kim et al., 2011). Thus, including about 100 deubiquitylating enzymes, approximately 800 ubiquitin enzymes regulate the dynamic ubiquitylation of a wide range of protein substrates (Kim et al., 2011). Within this complexity, E3 ligases are the most diverse class of enzymes in the ubiquitylation pathway as they play a central role in determining the selectivity of ubiquitin-mediated protein degradation and signaling. E3 ligases have been associated with a number of pathogenic mechanisms. Mutations in the E3 ligases MDM2, BRCA1, TRIMs, and Parkin have been linked to multiple cancers and neurodegenerative diseases (Fakharzadeh et al., 1991, Hatakeyama, 2011, Welcsh and King, 2001), and MDM2-p53 interaction inhibitors have already been developed as a potential anti-cancer treatment (Shangary and Wang, 2009). This highlights the potential of E2 enzymes and E3 ligases as drug targets. Although all E3 ligases are involved in the final step of covalent ubiquitylation of target proteins, they differ in both structure and mechanism and can be classified in three main families depending on the type of E3 ligases promoting ubiquitin-protein ligation and on the presence of characteristic domains. The RING ligases bring the ubiquitin-E2 complex into the molecular vicinity of the substrate and facilitate ubiquitin transfer directly from the E2 enzyme to the substrate protein. In contrast, homologous to the E6-AP C terminus family (HECTs) covalently bind the ubiquitin via a cysteine residue in their catalytic HECT domain before shuttling it onto the target molecule. RING between RINGs (RBRs) E3 ligases were shown to use both RING- and HECT-like mechanisms where ubiquitin is initially recruited on a RING domain (RING1) then transferred to the substrate through a conserved cysteine residue in a second RING domain. The vast majority of human E3 enzymes belong to the RING family, while only 28 belong to the HECT and 14 to the RBR family of E3 ligases (Chaugule and Walden, 2016).