Contribution of soluble ORF2 viral protein to viral replication and host immune response during acute Hepatitis E virus infection

Abstract

The hepatitis E virus (HEV) is a small, positive-stranded RNA virus that is a major cause of acute viral hepatitis globally. Acute HEV infection is typically asymptomatic and resolves within 8–10 weeks. HEV encodes 2 forms of capsid protein. A cytoplasmic form (ORF2c) is essential for virion structure. A secreted glycosylated form (ORF2s) accumulates at high titer in serum and can mask anti-ORF2 neutralizing antibodies. Here, we explored the contribution of ORF2s to HEV replicative fitness in vivo, and its role in generating anti-ORF2 antibodies (Abs). Rhesus Macaques (RM) were challenged by direct hepatic injection of infectious ORF2s+ and ORF2s− RNA. The replication of an HEV mutant lacking ORF2s expression was delayed by ~2 weeks when compared with wildtype virus and peak titers were nearly 10-fold lower for ORF2s−. No reversion of the 3 ORF2s silencing mutations was detected in the ORF2s− genomes, indicating genetic stability. The delay in replication and lower peak titer was unexpected as the viruses replicate similarly in cell culture. In addition, our data demonstrated that ORF2s has a significant and unexpected impact on generation of antibodies. Specifically, serum anti-ORF2 antibodies were only transiently detected in ORF2s− infected RM. As expected, anti-ORF2 titers were high and sustained in ORF2s+ infected RM. Furthermore, anti-ORF2 Ab response primed by ORF2s− infection differed in protection against reinfection when compared to ORF2s+. The ORF2s− challenged animals were re-infected upon second exposure to HEV infection. These findings indicate ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody response to mediate protection against re-exposure.

Efficient formation and maintenance of humoral and CD4 T cell immunity targeting the viral capsid in acute-resolving hepatitis E infection

“…Previous studies have shown that the viral capsid, encoded by ORF2, is an important target of the antiviral CD4 T cell [19] and antibody response [20], although it has not unanimously been identified as the dominant target of T cell immunity [21]. In HEV-infected macaques, the occurrence of ORF2-specific antibodies is closely linked to viral clearance suggesting that ORF2-specific antibodies are centrally involved in mediating immune control [7, 9]. It is unclear, however, how this control is facilitated as ORF2-specific antibodies cannot bind serum-derived eHEV [22].…”

“…Although ORF-2-specific antibodies cannot directly target or opsonize eHEV that circulates in the serum, they are central for prevention from productive HEV re-infection [9]. It is unclear however, how long sterilizing immunity persists as cases of re-infection have been described in seropositive individuals [24, 25].…”

“…Indeed, while data from a macaque model have suggested that neutralizing antibodies (nAbs) may facilitate viral clearance as CD8 T cell depletion only delayed viral clearance [7], recent data from humans with chronic infection clearly link the appearance of CD8 T cell responses to the clearance of chronic infection [8]. nAbs might not only be required to mediate viral clearance, but also to protect from re-infection [9]. However, it remains unclear how long they persist after acute HEV-infection and whether they only target the ORF2-derived capsid in humans.…”

Multifunctional ORF3 protein of hepatitis E virus

“…Since the ORF2 protein contains T-cell and B-cell epitopes and induces neutralizing antibodies, effector cells, and cytokines, it has become the target of the development of safe and effective vaccines. [96][97][98] The Hecolin ® vaccine, approved in 2012, is a prophylactic vaccine that utilizes capsid protein from genotype 1 HEV isolates, 99 but it is unclear whether it provides comprehensive protection against all human or animal zoonotic HEV infections. Hecolin ® has been found to protect rabbits from genotype 4 HEV, but the challenge virus was nHEV, which originated from a fecal sample.…”

Deciphering the role of soluble ORF2 protein in virus-host interaction in HEV infection

“…In the current issue of Hepatology , Ralfs et al 7 addressed these 2 important questions regarding the origin and role of ORFs in rhesus macaques, a nonhuman primate model to study HEV infection in vivo . They constructed a recombinant HEV genotype 3 variant that does not express ORF2s due to the mutation of its start codon as well as introduction of 3 stop codons into the signal peptide sequence that is unique to ORF2s.…”