*4.2. The Neutralizing Antibody Analysis*

An evaluation method of virus neutralizing efficiency of serum samples is essential to assess the vaccine-elicited immune response against the virus. However, inefficient propagation of HEV in cell models has posed a challenge to the evaluation of neutralizing antibodies in sera. The propagation and production of HEV were attempted in primary hepatocytes. Based on this cell system, an in vitro neutralization assay was developed to evaluate the anti-HEV antibodies. However, the difficulty in culturing the cells has limited its application [60]. To date, at least two cell culture systems for HEV have been developed. They are the culture system in PLC/PRF/5 and A549 cells established by Tanaka et al. [61] and the culture system in HepG2/C3A cells established by Shukla et al. [62] (Table 2). These cell culture systems allow the virus to propagate enough authentic HEV virions to support the neutralizing analysis [63]. Based on these HEV cell culture systems, polyclonal antibodies in serum samples or mAbs derived from immunized animals showed neutralizing ability against different viruses (Table 2). In one study, the HEV genotype 3 strain Kernow was shown to have high replication efficiency, using HepG2/C3A as a cell substrate. The infectivity of HEV was inhibited efficiently by the serum samples from immunized individuals [64] (Table 2). Apart from polyclonal antibodies in serum, several mAbs showed neutralizing ability. The neutralizing activity of two represent mAbs (8G12 and H6225) was studied with different cell systems. Gu et al. [33] reported that 8G12 was able to inhibit the replication of HEV based on Huh7 cells. H6225 was able to neutralize a genotype 3 HEV strain (JE03-1760F) in PLC/PRF/5 cells [65] (Table 2). Although many improvements were made, there was still a lack of an efficient and reliable cell-culture system for an HEV virus neutralization assay. Therefore, finding a surrogate of native virions may be a good choice to develop cell-based assays as HEV serological assays for vaccine evaluation.

**Table 2.** The neutralization analysis of monoclonal or polyclonal antibodies after vaccination or infection.


"\*" Using similar cell lines and virus strain, a robust HEV infection and replication system was reported recently (Todt et al. Robust hepatitis E virus infection and transcriptional response in human hepatocytes. *Proc Natl Acad Sci* 2020) with high virus titres obtained consistently. It is conceivable that the use of this system should facilitate the development of a virus neutralization assay.

The vaccine antigen p239 was used as a surrogate for native HEV virions in a cell-based functional assay for antibodies. He et al. [70] used p239 to simulate native HEV for virus attachment onto hepatocytes. P239 could attach to and enter the cells of four susceptible cell lines, i.e., HepG2, Huh7, PLC/PRF5 and A549 [71]. When neutralizing mAbs were used, the cell attachment of p239 was effectively blocked. More recently, a "neutralizing-like" blocking assay based on HepG2 cells was developed by Cai et al. [69] for antibody functionality assessment (Table 2). The assay was based on biotin-conjugated p239 and staining with allophycocyanin-conjugated streptavidin to amplify the fluorescence signal. Using this assay, the p239-blocking activity of serum samples from HEV-infected and vaccinated macaques was quantitatively evaluated [69].

With a well-characterized murine mAb (8G12), a competitive ELISA was developed for functional antibody evaluation of serum samples. 8G12 could efficiently block the binding of polyclonal antibodies in immunized human and rhesus macaque serum to vaccine antigen. 8G12 was applied to develop a competitive ELISA assay to detect 8G12-like antibodies in mouse and human serum samples. The 8G12-like antibody was predominant among the vaccine-induced anti-HEV antibodies in both human and mouse sera. Therefore, 8G12-like antibodies might be a promising surrogate for neutralizing antibodies and have the potential to be used as an indicator of the neutralization ability of the hepatitis E vaccine [72]. In another study, the mAb 8C11 was also used in a competitive ELISA to evaluate 8C11-like antibodies in serum samples from immunized mice [73]. In a similar format but with another virus, Palivizumab-like antibodies against respiratory syncytial virus were tested via a competitive ELISA to reflect the neutralization antibodies after vaccination [74]. Similarly, in the case of human papillomavirus, the neutralizing antibody level of each human papillomavirus serotype elicited by

vaccination was assessed by a multiplex competitive Luminex immunoassay with functional and type-specific mAbs (such as H16.V5 for type 16) as a specific probe for each type [75].

Recently, some modifications of HEV were performed at the genetic level to quantitatively monitor the infection and replication of HEV. Swiss scientists claimed that HEV genomes harbouring a haemagglutinin epitope tag or a small luciferase were found to be fully functional. This approach could enable the efficient production of infectious viruses with specific tags for ease of virus quantitation [76]. Based on HepG2/C3A cells infected with the HEV genotype 3 strain, virus replication and infection were monitored efficiently. Since these viruses can be easily engineered, different genotypes of HEV strains should be prepared in parallel, facilitating evaluation of the cross-genotype virus neutralization efficiency of various serum samples.
