*4.1. The Binding Antibody Analysis*

While the clinical end point for vaccine efficacy in a clinical trial could be a disease or a pathological marker, the seroconversion induced by vaccines is always a quantitative index to be measured, owing to the vaccination with a test vaccine. The binding titre in serum samples in an ELISA is always a straightforward method for serological analysis [56]. In ELISAs for measuring titres, 3 recombinant proteins and VLPs were used as coating antigens. For the hepatitis E vaccine, the coating antigens are generally the same or similar proteins as the vaccine antigens derived from ORF2 proteins. For the p239-based vaccine, anti-HEV IgG in serum samples from vaccinated humans and rhesus monkeys was analysed via E2-coated ELISA [57] (Table 1). For another vaccine candidate based on p495, the total immunoglobulin in serum samples derived from cynomolgus monkeys after immunization was evaluated in a p495-coated ELISA. Anti-HEV antibodies in monkeys' serum samples after receiving two doses of p495-based vaccine were elicited with titres of 1:104, and monkeys developed neither hepatitis nor viremia when challenged with virus [58].

**Table 1.** Different antigens used in the ELISA methods for supporting the vaccine clinical trials or preclinical development.


\* E2 and p239 share most of the critical epitopes.

Since different vaccine developers use different coating antigens, standardization of the serological assay would facilitate cross-lab and cross-product comparison if the same coating antigen could be used in the assay. Wen et al. [59] investigated the immunogenicity differences between hepatitis E vaccines based on p239 and p179. HEV p166 (aa 452−617) was used as a coating antigen to evaluate anti-HEV IgG in the serum of mice and humans immunized with p179 or p239 [59]. However, p166 was composed of aa 452−617, which is much shorter than p239 and may exclude some important functional epitopes. With this caveat in mind, a p495-based VLP antigen was more virion-like and contained all functional epitopes of p179 and p239 (Figure 1A), making it a better coating antigen. Similarly, efforts are being made to standardize the human papillomavirus serological assays using a more native- or virion-like VLPs as coating antigen in the ELISAs. Although the cross-lab comparability of ELISAs for human papillomavirus−16 and −18 has been formed, international standards are still required for the additional types in Gardasil®9 [56]. In most cases, correlations were observed between the binding titres and neutralizing titres. This result supports the use of binding titre measurement to support the clinical trials while using a subset of the clinical samples to establish such a correlation.
