Soluble ORF2 protein enhances HEV replication and induces long-lasting antibody response and protective immunity in vivo

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.

Hepatitis E Virus-induced antiviral response by plasmacytoid dendritic cells is modulated by the ORF2 protein

“…Many studies have demonstrated that glycosylation is a common factor affecting cell-cell adhesion (Gu et al, 2012; Ohtsubo & Marth, 2006) . When rhesus macaques were infected with HEV variant that did not express ORF2g/c or ORF2s, viral replication was attenuated and viral shedding in feces declined significantly as compared to infection with WT HEV (Ralfs et al, 2023) . Therefore, ORF2 g/c may not be essential for viral replication but it may be instrumental for efficient viral production.…”

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

“…Therefore, subunit vaccines are still the main direction for the design of HEV vaccines. 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 96‐98 . 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.…”