No abstract

Background and Aims: The HEV is a small positive–sense RNA virus that encodes a cytoplasmic form of the capsid protein (ORF2c), essential for virion structure, and a secreted glycosylated form (ORF2s) that accumulates at high titer in serum and can mask neutralizing epitopes. We explored the contribution of ORF2s to HEV replication and its role in generating antibodies against ORF2 in a nonhuman primate model. Approach and Results: We used a recombinant HEV genotype 3 variant that does not express ORF2s due to the introduction of stop codons (ORF2smut). Rhesus macaques (RMs) were given intrahepatic injections of infectious wildtype HEV (ORF2swt) RNA or a variant lacking ORF2s expression (ORF2smut). The replication of the ORF2smut virus was delayed by ~2 weeks compared with ORF2swt, and peak titers were nearly tenfold lower. Reversions of the 3 mutations that blocked ORF2s expression were not detected in the ORF2smut genomes, indicating genetic stability. However, serum antibodies against ORF2 were transiently detected in RMs infected with ORF2smut, whereas they were long-lasting in RMs infected with ORF2swt. Moreover, RMs infected with ORF2smut were more susceptible to reinfection, as evidenced by the viral RNA detected in fecal samples and the expansion of HEV-specific CD8+ T cells. Conclusions: These findings indicate that ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody-mediated responses that protect against HEV re-exposure.

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.