**1. Introduction**

Hepatitis E virus (HEV) belongs to the genus *Orthohepevirus* within the family *Hepeviridae*. HEV was first identified in the 1970s, and its genome was successfully sequenced a decade later [1]. One third of the population in the world may be infected with HEV during their lifetime [2]. HEV infection usually causes acute, self-limiting hepatitis and may cause chronic hepatitis among immunocompromised individuals and solid organ transplant recipients. Moreover, HEV infection poses a threat to pregnant women, with a mortality of 10–50% [3]. A study on the global burden of the disease estimated that approximately 20.1 million people were infected with HEV, leading annually to 3.4 million symptomatic cases, 70,000 deaths and 3000 stillbirths [4]. The burden of HEV-related disease is considerable in developing countries, especially in Africa and Asia [5]. Although there is no evidence of outbreaks of hepatitis E in developed countries, sporadic locally acquired cases of HEV infections have been reported in France [6], Germany [7], Switzerland [8], Australia [9], Japan [10] and so on. To date, eight

genotypes of HEV have been isolated, and at least five genotypes (genotypes 1–4 and 7) can cause human infection [11–13].

HEV contains a single-stranded, positive-sense RNA with a size of approximately 7.2 kb. The genome of HEV comprises three open reading frames (ORFs) [14]. ORF1 encodes a non-structural protein that is responsible for viral RNA replication [15]. Recently, a protein encoded by the newly discovered ORF4 (within ORF1) was shown to be able to stimulate the replication of genotype-1 HEV [16]. ORF3 encodes a phosphoprotein, which is involved in virus release from host cells [14]. HEV was recently found to be a quasi-enveloped virus. It exists as non-enveloped virions in faeces and urine for transmission, whereas its form could be predominantly enveloped in serum for evading neutralizing antibodies [17–19]. The existence of two different forms of virions might be related to the function of ORF3 [20]. Notably, the most well-studied ORF, ORF2, encodes the sole capsid protein (pORF2), which has the ability to self-assemble into viral capsids to package the viral RNA after each replication cycle. Therefore, the capsid protein pORF2 is a rational target for vaccine design [21].

Based on pORF2, different truncations using recombinant DNA technology were performed to yield various proteins with different assembly forms [22]. Some of the truncated proteins form into virus-like particles (VLPs) or subviral particles, presenting virion-like epitopes on the particle surface [23]. In this review, we use the term VLP in a loose term regardless of the size of the VLPs. Some VLPs could be more virion-like, including the size and the array of the epitope, while others could be smaller in size and less regular in particle formation, which are essential for effectively stimulating the immune response. Among these truncated pORF2 molecules, three vaccine candidates have been studied in clinical trials. One vaccine, with a trade name of Hecolin®, was licensed in China in 2011. However, it was not prequalified by the World Health Organization (WHO), which was necessary for introduction into countries where the disease burden was considerable [24]. The WHO issued a recommendation in 2018 to provide guidance to national regulatory authorities and manufacturers on the manufacturing process and on nonclinical and clinical aspects to assure the quality, safety and efficacy of recombinant hepatitis E vaccines [3]. Needless to say, quality assurance is of utmost importance for a licensed vaccine for widespread use. For the VLP-based vaccines, the presence of virion-like epitopes on the surface of VLPs is the structural basis to elicit protection against the specific pathogen [25]. The process comparability, analytical comparability, and the link of a change to the clinical outcome were considered as essential issues for a VLP-based vaccine. One A-vax case study discussed the risk assessment and control strategy, which supports comparability studies of vaccines [26]. Various methods have been established as a "toolbox" to evaluate the vaccine antigen for quality assurance during bioprocessing and for stability during storage and transportation. Among them, a large panel of monoclonal antibodies (mAbs) against the HEV capsid protein was developed, and assays based on these mAbs are used during bioprocessing and in lot release and stability testing [25]. In addition, immunization with a vaccine could induce a response against viral B cell epitopes in vaccine recipients [27]. The functional polyclonal antibodies in serum elicited by immunization can be analysed by a series of methods. In this review, we focus on epitope-specific antigenic analyses of hepatitis E vaccine antigen and evaluation assays of vaccine-elicited functional antibodies for its overall neutralization activity or against well-characterized epitopes.
