**2. The Design of Hepatitis E Vaccines**

#### *2.1. Molecular Structure of Di*ff*erent Truncated Versions of pORF2*

HEV ORF2 encodes a viral capsid protein containing 660 amino acids (aa). A recent study reported that a secreted form of pORF2 was observed in serum samples from both HEV-infected rhesus macaques and humans. Two different forms, pORF2<sup>C</sup> (capsid) and pORF2<sup>S</sup> (secreted), are two different translation products of the same viral ORF2 gene [28]. Compared to ORF2C, ORF2S contains an additional 15 aa. This 15-aa peptide segment could represent a signal sequence that drives ORF2S secretion [28]. The prolonged existence of ORF2<sup>S</sup> in the blood raises the possibility of

decoying, leading to partial or full depletion of neutralizing antibodies in the sera of convalescent patients. Thus, ORF2S antigen in HEV-infected patient serum could reduce the protective efficiency after vaccination. Further characterization of ORF2S, such as the complexing forms and kinetics in blood is of clinical importance [28].

To test the immunogenicity of the capsid protein, a series of truncated forms of pORF2 were prepared in different laboratories [22]. They include the nearly full-length p595 (aa 14−608) and p495 (aa 112–606), as well as much smaller particulate forms, such as p239 (aa 368–606) and p179 (aa 439−617), and the even shorter proteins, such as E2 (aa 394–606) and E2s (aa 459–606), that are amenable for crystallization (Figure 1A). The N- and C-terminally truncated version of pORF2, i.e., aa 14−608 (p595), can form T = 3 icosahedral VLPs that are highly analogous to native virions [29]. Additional highly virion-like VLPs were observed with p495, with a further truncation from p595 containing aa 112−606. P495 self-assembled into well-formed T = 1 icosahedral VLPs [30]. Through analysis of pORF2 using cryo-EM and X-ray crystallographic studies, three functional domains (S, P1 and P2) were identified [29]. The P2 domain, also named the E2s domain, harbours the major neutralizing epitopes on the viral capsid. The E2s domain, which has available high-resolution structural data, forms a tight homodimer with key interacting residues identified at the dimeric interface [31]. The E2s domain may play a key role in the interaction of HEV with host cells because antibodies directed towards this region tend to be neutralizing and functional. Moreover, the structure of the E2s domain in a complex with a potent neutralizing mAb, 8C11 [32], was determined. The epitopes recognized by the mAb 8C11 were identified to comprise three different peptide segments, namely, Asp496-Thr499, Val510-Leu514, and Asn573-Arg578. Among these, Arg512 was found to be the most crucial residue for 8C11 interaction with and neutralization of HEV. In addition, the complex crystal structure of 8G12 [33], another highly efficient neutralizing mAb, with E2s was also determined. Several important residues (Glu549, Lys554 and Gly591) were revealed that played an important role in 8G12 neutralization.

A 66-aa extension from the N terminus of E2s and could stabilize E2 (aa 394−606) to form hexamers in solution [34]. Further extended with another 26-aa extension on E2, p239 (aa 368–606) could self-assemble into a particulate form or VLPs with a 20–30 nm diameter [35]. Although there is a certain degree of particle size and particle regularity, the immunogenicity of the p239-based antigen was quite impressive. Notably, in experiments conducted in parallel, the immunogenicity of p239-based VLPs was shown to be approximately 240 times stronger than that of E2 in mice [35]. The enhanced immunogenicity is likely due to the multiplicity of arrayed virion-like epitopes on the VLP surface. More recently, p179 (aa 439–617) was found to self-assemble into VLPs with a diameter of approximately 20 nm [36]. This VLP form of the antigen also shares the major neutralization epitopes with p239. This antigen, along with the previously mentioned p239 and p495, was tested in clinical trials with the goal of human vaccine development (Figure 1B).
