Adaptive Immune Responses in Hepatitis AVirus and Hepatitis E Virus...

Adaptive Immune Responses in Hepatitis AVirusand Hepatitis E Virus Infections

Christopher M. Walker

Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s, Columbus, Ohio 43004

Correspondence: [email protected]

Both hepatitis A virus (HAV) and hepatitis E virus (HEV) cause self-limited infections inhumans that are preventable by vaccination. Progress in characterizing adaptive immuneresponses against these enteric hepatitis viruses, and how they contribute to resolution ofinfection or liver injury, has therefore remained largely frozen for the past two decades. HowHAV and HEV infections are so effectively controlled by B- and T-cell immunity, and whythey do not have the same propensity to persist as HBV and HCV infections, cannot yet beadequately explained. The objective of this review is to summarize our understanding of therelationship between patterns of virus replication, adaptive immune responses, and acuteliver injury in HAVandHEV infections. Gaps in knowledge, and recent studies that challengelong-held concepts of how antibodies and T cells contribute to control and pathogenesis ofHAV and HEV infections, are highlighted.

Hepatitis Avirus (HAV) and hepatitis E virus(HEV) cause self-limited infections that are

often clinically silent. Safe and relatively inex-pensive vaccines prevent infection with bothviruses through induction of antibodies thatprovide apparent sterilizing immunity. HEVvaccines are not yet commercially available inmost countries, but HAV incidence has declineddramatically in regions of the world where thatvaccine has been widely deployed. Despite thesegenerally positive circumstances, there is still animperative to improve our poor understandingof immunity and pathogenesis in HAV andHEV infections. Liver disease has long been sus-pected of having an immunopathogenic originin HAV and HEV infection, but host responsescausing hepatocellular injury remain undefined.There is still a need to address these gaps in

knowledge. Sporadic or epidemic outbreaks ofHAV still occur in many regions of the worldand liver disease is sometimes severe, especiallyamong adults who often no longer acquire pro-tective immunity during childhood because ofthe decreased prevalence of the virus. The scopeof liver disease caused by HEV is broad andpoorly understood from a mechanistic stand-point. HEV genotype (gt)1 and gt2 infectionsare transmitted primarily by contaminated wa-ter and household contact. The outcome of in-fection is sometimes catastrophic, especially forwomenwho are infected during the late stages ofpregnancy. HEV gt3 and gt4 infections are pre-dominately zoonotic andmuchmore likely to beclinically inapparent than those caused by gt1and gt2 viruses. These genotypes have not yetbeen associated with adverse outcomes in preg-

Editors: Stanley M. Lemon and Christopher WalkerAdditional Perspectives on Enteric Hepatitis Viruses available at www.perspectivesinmedicine.org

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nancy. Importantly, however, the gt3 and possi-bly gt4 viruses may be unique in their ability tocause persistent infections and rapidly progres-sive liver disease in humans with compromisedimmunity. Insight into the nature of HEV im-mune responses that cause disease and preventpersistent infection is limited.

The objective of this review is to describecharacteristics of B- and T-cell immunity elicit-ed by the enteric hepatitis viruses, their contri-bution to control of acute infection and liverdamage, and mechanisms of viral evasion. Sig-nificant gaps in knowledge about the role ofadaptive immune responses in the outcome ofHAV and HEV infections remain and are high-lighted.

HUMORAL IMMUNE RESPONSES

Humans are susceptible to infectionwith virusesthat group into three HAV and four HEV geno-types (Krain et al. 2014; Lemon et al. 2017).Despite this genetic diversity, only single HAVand HEV serotypes have been described. Anti-bodies capable of cross-genotype neutralizationare elicited by natural infection and vaccination(Krain et al. 2014; Lemon et al. 2017). Neutral-izing antibodies directed against the HAV andHEV capsid proteins provide protection frominfection (Krain et al. 2014; Lemon et al.2017), and at least in the case of HAV preventor blunt symptoms of acute hepatitis when ad-ministered within the first 2 weeks of the 3- to 4-week incubation phase of infection (Lemon et al.2017). Precisely how antibodies neutralize theseviruses and whether they contribute to resolu-tion of infection is not known. Until recently, itwas assumed that HAV and HEV existed inblood, liver, and feces as naked particles suscep-tible to antibody neutralization. The observationthat most if not all HAV and HEV particles cir-culating in blood are cloaked in host cell mem-branes, a state defined as quasi-envelopment(Feng et al. 2013; Yin et al. 2016), has rekindledinterest in virus spread in the liver and suscept-ibility to antibody neutralization (Feng et al.2013; Yin et al. 2016). Below, the characteristicsof antibody responses against these viruses arereviewed and their potential to limit spread in

liver during acute HAV and HEV infection isdiscussed.

The Hepatitis AVirus

Evidence that antibodies protect against acutehepatitis A first emerged from a remark-able series of experiments conducted 30 yearsbefore isolation of the virus. The rationale forthe first successful test of passive immunizationwith immune gammaglobulin was described byStokes and Neefe (1945):

Because the virus agent responsible for epidemicor infectious hepatitis is present in blood duringthe preicteric and early icteric phases of disease, itseemed reasonable to postulate that such neutral-izing antibodies in gamma globulin might be ef-fective in aborting or in attenuating this disease ifadministered during the incubation period orpreicteric stage.

To test this hypothesis, gammaglobulin fromconvalescent donors was transferred to childrenat high risk of infectious hepatitis because of asevere epidemic during summer camp. A dra-matic decline in overt hepatitis was reported inthe treatment group receiving gammaglobulinversus an untreated control group, suggestingthat this approach prevented infection and/orthe development of acute hepatitis (Stokes andNeefe 1945). Successful use of immune gamma-globulin to prevent infectious hepatitis was alsoreported in 1945 by Havens and Paul during anepidemic at a New Haven, CT, orphanage (Ha-vens and Paul 1945), and by Gillis and Stokes ina study of military personnel deployed in theMediterranean theater of operations duringWorld War II (Gellis et al. 1945). The modernera for analysis of antibody-mediated protectionagainst acute type A hepatitis did not arrive until1973, however, when Feinstone, Purcell, andKapikian used immune electron microscopy(IEM) to visualize viral antigen in stool samplesfrom infected humans (Feinstone et al. 1973;see Feinstone 2018 for details). IEM was alsoused to provide the first evidence of seroconver-sion to the presumptive viral capsid antigenusing paired serum samples from subjects ex-perimentally infected with HAV (Feinstoneet al. 1973).

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Kinetic and Durability of the AntibodyResponse in Acute and Relapsing HAVInfection

Immune electronmicroscopy was a cumbersomeapproach for characterization of antibodies thatbind the HAV capsid. Rapid development ofmore facile assays, including radioimmunoassay(RIA) and enzyme-linked immunosorbent as-says (ELISA), revealed that the acute phaseantibody response against the HAV capsid iscomprised of IgM, IgA, and IgG isotypes (Sta-pleton 1995). Anti-HAV IgM seroconversionoccurs approximately coincident with the onsetof jaundice and other physical signs of acutehepatitis (Fig. 1A) (Lemon et al. 2017). IgM an-tibody titers then typically decline to undetect-able levels within 4–6 months. Because of theearly and transient nature of the response,anti-HAV IgM has been described as an acute-phase antibody that is diagnostic of HAV infec-tion. Anti-HAV IgA appears in serum at aboutthe same time as the IgM response (Sikuler et al.1983; Stapleton et al. 1991), and HAV:anti-HAVIgA complexes are present in the stool of someindividuals during the later phases of acute in-fection (Locarnini et al. 1980). Anti-HAV IgGalso appears in serum during the acute sympto-matic phase of infection, but titers are initiallylower and increasemore slowly than those of theIgM isotype (Fig. 1A). The IgG response toHAVis remarkably durable, persisting for life in mostindividuals after convalescence from acute hep-atitis A (Lemon and Binn 1983). The IgG anti-body response targets major capsid proteinsVP1, VP2, and VP3 (Wang et al. 1996) as wellas nonstructural HAV proteins (Jia et al. 1992).Although probably not important for control ofinfection, antibodies against individual non-structural proteins like the 3Cpro proteinasecan be used to distinguish humoral responsesthat are a consequence of infection versus vacci-nation with inactivated HAV capsid particles(Stewart et al. 1997). The kinetic of the serumanti-HAVantibody responsematches the timingof hepatic antibody and B-cell gene up-regula-tion observed more recently in experimentallyinfected chimpanzees. Genes encoding the threeantibody heavy chain isotypes as well as numer-

ous other genes associated with B-cell activationand homing to liver are strongly up-regulated atabout 3–4weeks after challengewithHAV (Lan-ford et al. 2011). Factors that contribute to therobust and durable antibody response againstHAV are not defined, but it is noteworthy thatquasi-enveloped HAV efficiently activates plas-macytoid dendritic cells (pDCs), resulting inpDC migration to the liver during acute HAVinfection and production of cytokines, includinginterferon (IFN)-α (Feng et al. 2015), which isknown to promote B-cell differentiation intoplasma cells (Jego et al. 2003). These observa-tions are consistent with expansion of plasmablasts that secret IgM against the HAV capsid(Hong et al. 2013). Plasma cells also infiltrateliver, a finding that typifies hepatitis A and israre in other types of viral hepatitis.

Approximately 5%–10% of acute HAV casesrelapse, usually in older patients who may notsustain effective antiviral immune responses(Schiff 1992). Relapse is characterized by a re-surgence in HAV replication and symptoms ofhepatitis a fewweeks after control of the primaryinfection (Fig. 1B) (Sjogren et al. 1987). Thecourse of relapsing hepatitis A is sometimes pro-longed but the virus is ultimately cleared (Fig.1B). Whether the anti-HAV antibody responsedevelops with a typical kinetic in individualswith relapsing hepatitis A is largely unknown.Serum anti-HAV IgM antibodies persist in indi-viduals with relapsing infections for reasons thatare not yet understood (Fig. 1B) (Jacobson et al.1985; Cobden and James 1986; Glikson et al.1992; Schiff 1992). Relatively little is knownabout the timing and neutralizing function ofanti-HAV IgG antibodies, or their capacity toprevent resurgent HAV replication (Fig. 1B).

Antibody Neutralization of HAV

Adaptation of HAV for replication in culturedprimary and established cell lines facilitatedstudies of antibody-mediated neutralization ofinfectivity (Frosner et al. 1979; Daemer et al.1981; Gauss-Muller et al. 1981; Kojima et al.1981). Because HAV replication in these culturemodels was persistent and noncytopathic (Fros-ner et al. 1979; Daemer et al. 1981; Gauss-Muller

Adaptive Immune Responses in HAV and HEV Infections

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Are HAV-specific CD4+ and CD8+ T-cell responsessimilar to those in typical infections?

Is relapse caused by a failure to sustain CD4+ orCD8+ T cells in the convalescent period?

Why does the IgM antibody response persist inrelapsing HAV infection?

What is the kinetic of the anti-HAV IgG response, and isit generated or sustained in relapsing infections?

Relapsing HAV infection: unresolvedquestions in adaptive immunity

Relapsing hepatitis A (>10% of infections)

Convalescent

Relapse(weeks to months)

Icteric(weeks 4–7)

Incubation(weeks 1–4)

Icteric(weeks 4–7)

Incubation(weeks 1–4)

Typical hepatitis A (>90% of infections)A

B

How does passive or active immunization inthe first 10–14 days of infection prevent hepatitis?

What is the kinetic and functional profile ofHAV-specific CD4+ and CD8+ T cells?

Is there a temporal kinetic relationship betweenfunctional T-cell responses, viral control, andthe onset of hepatitis?

Are CD8+ T cells required for control of infectionor as a cause of acute hepatitis?

What is the major immune mechanism of liverinjury, and why is severity so variable?

Do anti-HAV IgG antibodies assist in clearanceof quasi-enveloped HAV viruses?

Typical HAV infection: unresolvedquestions in adaptive immunity

HAV-specific CD8+ T cellsInnate cytotoxic cells

Inflammatory Treg cells

Hepatitis

Hepatitis

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Figure 1. Adaptive immunity in hepatitis Avirus (HAV) infection. (A) Typical hepatitis A. The majority (>90%)of acuteHAV infections are characterized by an incubation phase of 3–4weeks followed by an icteric phase of 2–4weeks (see Shin and Jeong 2018). Hepatitis, which is thought to be immune mediated, is highly variable inseverity. It ranges from clinically inapparent in young children tomore severe in older children and adults. Serumanti-HAV IgM antibodies appear within a few days of the onset of hepatitis and generally become undetectablewithin 4months. Anti-HAV IgG antibody titers increase in the late symptomatic phase and remain detectable forlife. HAV-specific CD8+ T cells have been detected in blood and liver of patients during the icteric phase(Vallbracht et al. 1986, 1989; Fleischer and Vallbracht 1991; Schulte et al. 2011), and probably decline infrequency with resolution of the infection (Schulte et al. 2011). Recent studies have also described expansionof innate cytotoxic cells and T regulatory (Treg) cells that have converted to an inflammatory phenotype inindividuals with symptomatic infections (Choi et al. 2018b; Kim et al. 2018). CD4+ and CD8+ T-cell frequencyand function have been described in too few studies to superimpose kinetic curves on the profile of resolvingHAVinfections. Key unanswered questions about adaptive immune responses in acute hepatitis A are shown to theright. (B) Relapsing hepatitis A. Approximately 5%–10% of acute HAV infections relapse about 1–4months afterapparent resolution of symptoms and virus replication. Very little is known about adaptive immune responses inthis uncommon but fascinating atypical form of the infection. Anti-HAV IgM responses persist through therelapse phase, and therefore are sustained for a longer period of time when compared with typical infections thatdo not relapse. Little is known about the anti-HAV IgM response or the nature of T-cell immunity during theprimary or relapsing phases of infection. It is likely that failure to develop or sustain a virus-specific humoral orcellular immune response facilitates relapse of infection. It is important to emphasize that even relapsinginfections eventually resolve, and persistence of HAV has not been described under conditions that lead tochronic hepatitis E virus (HEV) infection.

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et al. 1981; Kojima et al. 1981), an alternative tothe classical plaque reduction assay was neededto measure neutralization. The immediate solu-tion was a radioimmune focus assay (RIFA) thatprovided autoradiographic images of HAV-in-fected cell foci that developed beneath an agaroseoverlay (Lemon and Binn 1983). Inhibition offocus formationbyserumantibodies fromacute-ly infected humans and nonhuman primatesprovided the first evidence of a neutralizing re-sponse against HAV (Lemon and Binn 1983).Using this approach, it was demonstrated thatserum-neutralizing antibodies developed about1 week before a transaminase elevation in exper-imentally infected owl monkeys (Lemon andBinn 1983). The appearance of neutralizing an-tibodies also correlated with reduced fecal HAVshedding in these animals. Analysis of serumsamples frommilitary personnel infected duringan outbreak of HAV revealed a similar neutral-izing response to the virus (Lemon and Binn1983). Rate zonal centrifugation of serum sam-ples documented HAV neutralizing activity inthe 7S (IgG) and 19S (IgM) fractions (Lemonand Binn 1983). Neutralizing activity in the19S fraction was early and transient, as expectedfrom the kinetic of the anti-HAV IgM response(Lemon and Binn 1983). Neutralizing activitywas not consistently detected in the 7S fractionuntil later in infection when anti-HAV IgG titersincreased. In general, however, serum antibodiescollected during the late or convalescent phase ofinfection providedmore robustHAVneutraliza-tion than serum samples collected in the earlyacute phase of infection (Lemon and Binn 1983;Zahn et al. 1984). This may indicate that therelative efficiency of neutralization is greater forthe late IgG versus early IgM antibody response.

Development of anti-capsidmonoclonal an-tibodies facilitated mapping of neutralizing epi-topes. An early study employed the RIFA assayand physicochemical approaches to partially lo-calize a neutralizing domain to the capsid pro-tein VP1 (Hughes et al. 1984). HAV neutraliza-tion escape variants selected with monoclonalantibodies in cell culturemodels accelerated epi-tope identification (Ping et al. 1988; Ping andLemon 1992). The studies revealed that a dom-inant conformational (discontinuous) epitope

formed from VP1 and VP3 is targeted by neu-tralizing antibodies (Stapleton and Lemon 1987;Ping et al. 1988; Ping and Lemon 1992). Recentvisualization of the HAV capsid structure by X-ray crystallography provided structural details ofthis epitope (Wang et al. 2015). Another confor-mational neutralizing epitope was recently lo-calized to amino acid residues of VP2 and VP3targeted by antibodies that destabilize the cap-sid, perhaps by acting as a receptor mimic(Wang et al. 2017). Conservation of these struc-tures across HAV genotypes that infect humansis consistent with neutralization studies thatdocumented a single serotype.

Antibody-Mediated Protection againstHAV Infection

Multiple lines of evidence indicate that antibod-ies generated by natural infection or vaccinationwith inactivated HAV particles are sufficient toprotect against infection. Lifelong protectionfrom HAV reinfection is strongly associatedwith neutralizing anti-HAV IgG antibodies elic-itedbynatural infection (LemonandBinn1983).The formalin-inactivatedHAV vaccine also pro-vides protective immunity as effective and dura-ble as that conferred by natural infection. Prim-ing and boosting with the formalin-inactivatedvaccine generates anti-HAV antibody titers sim-ilar to those measured after natural infection,and greatly exceed theminimum10 internation-al units (IU)/mL that is considered protective(Lemon et al. 1997). High titer anti-HAV anti-bodies present in the serum ofmost infected andvaccinated individuals may well provide steriliz-ing immunity against infection. In support ofthis view, monkeys (D’Hondt et al. 1995) andchimpanzees (Purcell et al. 1992) challengedwith HAV after immunization with the forma-lin-inactivated vaccine had no virological, sero-logical, or biochemical evidence of infection.Vaccine-induced antibodies may provide pro-tection even when titers are at or below the limitof detection by binding and neutralization as-says. This was shown in a study of chimpanzeeschallenged with HAV after transfer of immuneglobulin from vaccinated and convalescent hu-mans (Purcell et al. 1992). At the time of HAV



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challenge, serum titers of passively transferredantibodies were much lower than those typicallygenerated by infection or vaccination. All chim-panzeeswere infected after challenge, as assessedby fecal HAV shedding and a sharp increase inserum anti-HAV IgM antibodies. Unlike un-treated control animals, however, none of the re-cipients of passively transferred antibodies hadhistological or biochemical evidence of acutehepatitis A (Purcell et al. 1992). Together, thesestudies indicate that antibody titers generated byinfection or vaccination are usually sufficientto provide apparent sterilizing immunity. Evenwhen titers are too low to protect against break-through infection, anti-HAVantibodiesmay stillprevent the onset of acute hepatitis.

Postexposure Antibody Therapy andProtection from Acute Hepatitis

As noted above, prevention of symptomatic in-fectious hepatitis by passive immunization withimmune gammaglobulin from convalescent do-nors was documented decades before discoveryof HAV. Gammaglobulin was most effective ifadministered early in the incubation phase ofinfectious hepatitis, ideally 1 week or more priorto the onset of symptoms. Postexposure treat-ment of infected subjects also appeared to atten-uate liver disease, as jaundice lasted for 7 days orless in the few gammaglobulin-treated individ-uals who developed symptoms, versus 14 days ormore in the untreated group (Stokes and Neefe1945). Finally, passive immunization appearedto provide durable protection from infectioushepatitis for approximately 6 months (Stokeset al. 1951).

These parameters defining the timing, effec-tiveness, and duration of gammaglobulin pro-tection remained largely unchanged with morecontemporary studies of postexposure protec-tion by anti-HAV antibodies (Conrad andLemon 1987; Green andDotan 1988). Antibodiesthat bind the HAV capsid and neutralize infec-tivity are present in serum after passive immu-nization with commercial immune globulinpreparations (Lemon et al. 1997). In general,anti-capsid antibody titers measured approxi-mately 1 week after passive immunization are

similar to those generated by active immuniza-tion with the formalin-inactivated vaccine, butdecline over time with a kinetic that is predict-able based on the known half-life of serum im-munoglobulins (Lemon et al. 1997). The neu-tralization titer of anti-HAV antibodies may behigher immediately after passive immunizationthan those generated by single dose of the for-malin-inactivated vaccine (Lemon et al. 1997).This could reflect enhanced maturation and/oraffinity of antibodies generated during naturalHAV infection of gammaglobulin donors. Pre-cisely how postexposure administration of im-mune globulin prevents acute hepatitis in indi-viduals who are already infected is not known. Itis likely that anti-HAV antibodies restrict spreadof the virus within the liver, perhaps by neutral-izing infectivity of quasi-enveloped particles af-ter endosomal uptake. This antiviral effect isstrong, as the majority of individuals treatedwithin the first 2 weeks of HAV exposure donot generate a de novo anti-HAV antibody re-sponse despite the likelihood that some virusreplication and capsid production occurred inthe liver (Sonder et al. 2004; Whelan et al.2013). In some studies, a small but significantpercentage of immune globulin recipients sero-converted but were nonetheless protected fromhepatitis (Sonder et al. 2004). It is likely thatsufficient HAV antigen was produced to triggeran adaptive immune response, even if virus rep-lication and attendant hepatitis were profoundlysuppressed by passively transferred anti-HAVantibodies. Antibodies generated as a conse-quence of subclinical infection might be ex-pected to provide protection that outlasts thoseexpected from passive immunization alone.This concept, termed “passive–active immuni-zation,” was proposed in the earliest studies ofpostexposure gammaglobulin therapy to explaindurable, perhaps lifelong protection, observed insome study subjects (Stokes et al. 1951).

With successful development of the forma-lin-inactivated vaccine to prevent HAV in-fection, studies were undertaken to determinewhether active immunization in the postexpo-sure period could also prevent clinically appar-ent hepatitis A. Two studies in nonhuman pri-mates provided early support for this concept. In

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the first, two chimpanzees received the forma-lin-inactivated vaccine 1 or 3 days after challengewith HAV (Robertson et al. 1994). The animalthat received vaccine 3 days after challenge wasnot infected. Fecal shedding ofHAVwas also notdetected in the animal vaccinated 1 day afterchallenge, but a sharp boost in anti-HAV anti-bodies provided serologic evidence of infection(Robertson et al. 1994). Acute hepatitis in thischimpanzee was attenuated compared to twounvaccinated control animals that followed atypical course of acute hepatitis A after chal-lenge. Similar results were obtained in marmo-sets that were vaccinated after HAV challenge(D’Hondt et al. 1995). Vaccination of marmo-sets at 48 hours after challenge prevented fecalshedding of virus and transaminase elevationseven though the animals were clearly infected asdetermined by visualization of viral capsid anti-gen in liver (D’Hondt et al. 1995). Postexposurevaccination therefore limited HAV spread andacute inflammation within the liver, as well asHAV shedding that perpetuates sporadic andepidemic transmission of the virus. Studies ofpostexposure immunization undertaken in hu-mans confirmed that the formalin-inactivatedvaccine can modify the course of acute hepatitisA. One large field study compared passive versusactive immunization for protection against hep-atitis A in contacts of individualswith confirmedHAV infection (Victor et al. 2007). Gammaglob-ulin and the formalin-inactivated vaccine wereboth administered within an average of 10 daysof exposure to the virus. Rates of hepatitisamong contacts who received vaccine or im-mune globulin did not exceed 5%, indicatingthat both approaches are highly effective in pre-vention of disease (Victor et al. 2007). A verymodest protective advantage was observed forimmune globulin, but active vaccination hasother significant advantages including perceivedsafety and lifelong protection from HAV infec-tion in regions where the virus is endemic.

HAV Evasion of Neutralizing Antibodies

Mutational escape of capsid epitopes does notcontribute to immune evasion fromneutralizingantibodies elicited by infection or vaccination.

As noted above, the dominant capsid neutraliz-ing epitope is conserved in all HAV strainscirculating in humans and there appears to berobust negative selection pressure against emer-gence of genetically stable immune escape vari-ants (Lemon et al. 1990). One study did describesix HAV variants with amino acid substitutionsat VP1 residues in close proximity to thosecomprising the major neutralization epitope(Perez-Sautu et al. 2011). Introduction of theconservative and semiconservative amino acidsubstitutions into cell-culture-adapted virusesresulted in evasion of antibody neutralizationand successful competition against the more fitparental HAV strain (Perez-Sautu et al. 2011).Nonetheless, the genetic stability of these virusesin nature is questionable given their reducedreplicative fitness and the apparently rare emer-gence of escape variants in human populations.This concept is supported by studies of owlmonkeys experimentally infected with an anti-genic variant ofHAV thatwas highly adapted forreplication in cell culture and resistant to neu-tralization by monoclonal antibodies (Lemonet al. 1990). Resistance was conferred by a singleamino acid substitution in the VP3 protein ofthe HAV capsid. The mutation was not stable inthe infected animals as viruses with wild-typecapsid sequences dominated in feces and liverby about week 3 of infection when hepatitis de-veloped (Lemon et al. 1990).

The effectiveness of the neutralizing anti-body response in preventing spread of HAV inthe liver may be limited by cloaking of the HAVcapsid in host cell membranes. Efficient trans-mission of infection between humans is medi-ated by naked HAV particles that lack an enve-lope component when shed in stool. SerumIgM and IgG antibodies readily neutralize in-fectivity of this form of the virus. However,most HAV particles acquire a quasi-envelopeas they are released from hepatocytes intocirculation (Feng et al. 2013) and bile (Hirai-Yuki et al. 2016b). This envelope provides pro-tection from IgM or IgG neutralizing antibodiesthat block virus attachment or entry at the cellmembrane (Feng et al. 2013; Hirai-Yuki et al.2016b). Quasi-enveloped HAV is neutralizedby IgG after entry into the cell, quite possibly



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in the endosome where particle uncoating oc-curs (Feng et al. 2013; Hirai-Yuki et al. 2016b).Anti-HAV IgM antibodies do not mediate post-entry neutralization (Feng et al. 2013; Hirai-Yuki et al. 2016b). This may reflect restrictedaccess or structurally instability of large pen-tameric IgM molecules in the endolysosomalcompartment. Envelope cloaking may thereforeprovide an important mechanism for HAV toevade the IgM response that dominants the earlyphase of acute infection.

Anti-HAVAntibodies and HAV Trafficking

As noted above, HAV is complexed with anti-HAV IgA antibodies in the stool of some pa-tients after seroconversion. Antibody-mediatedneutralizing activity is nonetheless uncommonin fecal samples (Stapleton et al. 1991), and theneed for a secretory IgA response to control localenteric replication is questionable given limitedevidence for HAV infection of the gastrointesti-nal (GI) tract. Anti-HAV IgA antibodies couldserve an entirely different function by acting as achaperone for HAV trafficking between the GItract and liver. There is evidence that the effi-ciency of virus transit across the polarized epi-thelium of the gut is enhanced by antivectorialtranscytosis of HAV:anti-HAV IgA complexes(Dotzauer et al. 2005), a process that coulddeliver the virus to the bloodstream and ulti-mately the liver where it replicates. The obser-vation that HAV:anti-HAV IgA complexes alsobind the asialoglycoprotein receptor on hepato-cytes, thereby enhancing virus entry and repli-cation (Dotzauer et al. 2000), provides addition-al compelling evidence that IgA may regulateenterohepatic trafficking of HAV.More recently,it was demonstrated that IgA also enhancestranscytosis of HAV from the basal to apicalmembrane of hepatocytes and export into bilein a rodent model of antibody facilitated virustrafficking (Counihan and Anderson 2016). To-gether, these observations suggest that the nor-mal protective function of IgA may be co-optedby HAV to increase the efficiency of liver infec-tion and transmission of this enteric hepatitisvirus. However, the significance of these mech-anisms to the pathogenesis of acute HAV infec-

tion is not entirely clear given high levels of fecalvirus shedding that occur before seroconversion.One recent study in a murine model suggestedthat IgA-mediated enhancement of HAV trans-location from the gut might facilitate reinfectionof the liver, especially if the competing IgG an-tibody response is weak or delayed (Dotzaueret al. 2012). This mechanism may therefore berelevant to prolonged or relapsing HAV infec-tions where IgG production is delayed, althoughthis remains to be established.

The Hepatitis E Virus

Seroconversion against the agent of enteric non-A, non-B hepatitis was demonstrated in a hu-man subject who developed acute hepatitisafter ingestion of pooled fecal material from pa-tients with enterically transmitted non-A, non-B hepatitis (Balayan et al. 1983). Using IEM,serum antibodies that developed during the pre-clinical and early postclinical phases of infectionwere used to visualize spherical virus particles ofabout 27–30 nm in fecal samples (Balayan et al.1983). Follow-up studies demonstrated that se-rum antibodies collected fromwell-documentedcases of enterically transmitted non-A, non-Bhepatitis from Asia, Africa, and North Americaalso reacted with these particles, providing earlyevidence that the causative viruses from dif-ferent regions of the world were serologicallyrelated (Bradley et al. 1988). Moreover, serocon-version was documented in macaques experi-mentally challenged with the infectious etio-logical agent (Bradley et al. 1988). Molecularcloning of the HEV genome from bile of oneexperimentally infected macaque facilitated ex-pression of recombinant open reading frame(ORF)2 capsid protein that could be used tocharacterize the antibody response in infectedhumans (Reyes et al. 1990).

Kinetic and Durability of the AntibodyResponse in Acute and Chronic HEV Infection

The relationship between seroconversion, trans-aminase elevation, and HEV viremia or fecalshedding appears to be very similar in acuteHEV and HAV infections. The profile for HEV

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emerged from multiple studies of populationsinfected during sporadic and epidemic spreadof HEV (Bryan et al. 1994; Clayson et al. 1995;Chandra et al. 2010; Huang et al. 2010), andindividuals who were infected experimentally(Chauhan et al. 1993) or accidentally (Sarkaret al. 2015) with the virus. Anti-HEV IgM is firstdetected in serum at about the time serum trans-aminases increase (Fig. 2A). The IgM antibodyresponse declines sharply just after convales-cence and is diagnostic of acute primary infec-tion. An anti-HEV IgA response is also com-monly detected during the early phase ofinfection (Chau et al. 1993), approximately co-incident with onset of the IgM response. It hastherefore been proposed that detection of IgAcan also be used to identify cases of acute prima-ry HEV infection (Takahashi et al. 2010). Serumanti-HEV IgG antibodies against the ORF2 cap-sid are detected at the later stages of acute hep-atitis E, and increase in titer (Bryan et al. 1994;Huang et al. 2010) and avidity (Zhang et al.2002) with convalescence (Fig. 2A). Follow-upof patients for 1–2 years after recovery fromacute hepatitis E indicated that IgG antibodytiters declinedwith time but nonetheless persist-ed in all individuals (Bryan et al. 1994; Huanget al. 2010). Evidence that serum antibodies per-sist for a very long time was obtained in a studyof subjects infected during a 1978 epidemic inKashmir. Anti-HEV seropositivity was not sta-tistically different when samples collected dur-ing the acute phase of infection and 14 yearsafter convalescence were compared (Khurooet al. 1993). Recurrent exposure to HEV didnot appear necessary to maintain antibody pos-itivity in this population (Khuroo et al. 1993).

Despite this evidence for durable and per-haps lifelong humoral immunity, there is also abody of literature supporting the controversialconcept of seroreversion after resolution ofHEVinfection (Krain et al. 2014). Early studies dem-onstrating seroreversion are difficult to interpretas they were undertaken with assays for anti-HEV antibodies that varied widely in sensitivityand specificity. International anti-HEV anti-body reference standards were not available un-til recently, further complicating comparison ofassays and study findings. One recent retrospec-

tive study with well-characterized detection as-says documented loss of anti-HEV IgG in ap-proximately half of subjects multiply transfusedfor treatment of genetic disorders (Servant-Del-mas et al. 2016). Seroreversion was observedapproximately 7 years after first detection ofanti-HEV IgG (Servant-Delmas et al. 2016).Further study in nontransfused populations isrequired to determine whether seroreversion isa common event. Finally, the broad outline ofhumoral responses presented here is basedmostly on studies of clinically apparent HEVgt1 infections during periods of endemic or ep-idemic virus transmission. Other recent studieshave established that the timing and isotype pro-file of the humoral response is similar in acuteresolving HEV gt3 or gt4 infections that arezoonotic or transfusion-related and more oftenclinically silent (Bendall et al. 2008; Takahashiet al. 2010; Vollmer et al. 2016).

Antibody responses against theORF2 capsidhave also been characterized in patients withchronic HEV gt3 infections. In overview, thekinetic of the anti-HEV IgM and IgG responsesvaries greatly among patients with chronic hep-atitis E, perhaps reflecting differences in under-lying disease and immune-suppressive regi-ments. Because of this individual variability,the kinetic of anti-HEV antibody responsesand HEV replication or outcome of the persis-tent infection are not readily compared (Fig. 2B).Persistence of HEV in the absence of serocon-version has been described in some patients,while others do develop detectable IgM and/orIgG responses (Dalton et al. 2009; Legrand-Abravanel et al. 2010; Pas et al. 2012; Suneethaet al. 2012; Kamar et al. 2013; Moal et al. 2013).Two representative studies demonstrated thatanti-HAV IgM seroconversion can be delayedfor months in persistently infected patientstreated with immune-suppressive drug regi-mens to prevent allograft rejection (Legrand-Abravanel et al. 2010; Choi et al. 2018a). More-over, the IgM response can persist throughoutchronic infection, far longer than responses inindividuals with typical acute resolving infec-tions (Fig. 2B) (Legrand-Abravanel et al. 2010;Choi et al. 2018a). A similar delay in generationof humoral immunity and cocirculation of per-



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Is recovery of CD4+ and/or CD8+ T-cell immunityrequired for resolution of persistent infection afterimmune suppression is reduced?

How does HEV continue to persist in about 70% ofindividuals even after a reduction in immunesuppression?

Does HEV persist in the presence of neutralizinganti-HEV IgG antibodies?

What adaptive immune responses are requiredto prevent HEV persistence (CD4+ and/or CD8+

T cells, neutralizing anti-HEV antibodies?)

Do anti-HEV neutralizing antibodies persist for lifeas in HAV infection, and under what conditions dothey fail to provide protection from reinfection?

What is the major immune mechanism of liverinjury; why is severity so variable especially inwomen at the late stages of pregnancy?

Do anti-HEV IgG antibodies assist in clearanceof quasi-enveloped HAV viruses?

Can passive or active immunization prevent hepatitisas described in HAV infection and, if not, why doantibodies fail to provide postexposure prophylaxis?

A

B

HEV-specific CD8+ T cells

Hepatitis

Incubation(4–6 weeks)

Icteric(4–5 weeks)

Convalescent

Typical HEV infection: unresolvedquestions in adaptive immunity

Typical hepatitis E (immune competent host)

Persistent hepatitis E (immune compromised host)

Persistent HEV infection: unresolvedquestions in adaptive immunity

Reduced immunesuppression

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Hepatitis

Figure 2. Adaptive immunity in hepatitis E virus (HEV) infection. (A) Typical hepatitis E. The course of acuteHEV infection is very similar to that described for hepatitis Avirus (HAV) infection. An incubation phase of 3–4weeks can be followed by a period of overt hepatitis that persists for about an additional 2–4 weeks (see Aggarwaland Goel 2018). However, many HEV infections in adults are clinically inapparent, especially those caused bygenotype 3 viruses. The anti-HEV antibody profile is also similar to HAV infection, with an early IgM responsethat transitions to an IgG isotype during the later stages of acute infection. The IgG response is typically durableafter convalescence, but there is evidence that titers fall below protective thresholds in some patients who aresusceptible to reinfection. How commonly seroreversion occurs and whether the severity and duration of secondinfections is reduced is not yet known. HEV-specific CD8+ T-cell activity has been detected in the blood ofinfected patients who had symptoms of acute infection (Suneetha et al. 2012; Brown et al. 2016). Whetherexpansion of functional cytotoxic CD8+ T cells is temporally associated with control of virus replication orthe timing and severity of liver disease remains to be determined. To date there has been no analysis of othercytotoxic cell populations during acute infection, including those of the innate lineage, as has been described forHAV. Importantly, it is not yet known whether antibodies contribute to resolution of infection with this quasi-enveloped virus and is somewhat difficult to predict because there is as yet no positive demonstration that passivetransfer of anti-HEV antibodies during the incubation phase of infection will prevent or temper hepatitis. Theminimum humoral or cellular responses required to prevent HEV persistence have not yet been identified. Keyunanswered questions about adaptive immune responses in acute hepatitis E are summarized to the right of thepanel. (B) Persistent hepatitis E. HEV infection persistence has been described in individuals with compromisedimmunity caused by hematological malignancies, organ allograft, and HIV coinfection. HEV-specific T-cellresponses are clearly reduced or undetectable in these patients (Suneetha et al. 2012; Brown et al. 2016), butappear to strengthen at least transiently with resolution of infection (Suneetha et al. 2012). The timing andmagnitude of anti-HEV IgM and IgG responses in chronically infected patients is highly variable and difficult togeneralize; persistence of HEV in the absence of seroconversion has been described in some patients, while othersdo develop detectable IgM and/or IgG responses (Pas et al. 2012; Kamar et al. 2013; Moal et al. 2013). Studies insolid organ transplant recipients demonstrated that IgM seroconversion can takemonths to develop, and the IgMresponse appears to persist throughout chronic infection (Legrand-Abravanel et al. 2010; Choi et al. 2018a).

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sistent virus with anti-HEV IgG was also de-scribed in macaques experimentally infectedwith an HEV gt3 isolate during treatment withimmune-suppressive drugs (Gardinali et al.2017). It could be concluded from these obser-vations that antibodies are of limited importancein virus control during chronic infection, butcomprehensive analysis of anti-HEV IgG titers,avidity, and neutralizing capacity has not yetbeen reported.

Antibody Neutralization of HEV

Adaptation of HEV for replication in culturedcells provides an experimental model to assessantibody-mediated neutralization of infectivity(Emerson et al. 2006; Tanaka et al. 2007, 2009;Shukla et al. 2011). Even with this advance, thepaucity of literature describing neutralizing an-tibody responses during acute and chronic HEVinfections and after preventive vaccination isstriking. This may reflect in part the technicaldifficulty in measuring replication of this non-cytopathic virus in cell culturemodels. Readoutsinclude PCR-based quantification of HEV RNAand visualization of infected cell foci by immunefluorescence assay that are semiquantitative orcumbersome and time consuming. In the verylimited number of settings where these assayswere used, direct evidence for HEV neutraliza-tion by acute and convalescent phase serum an-tibodies was obtained. As an example, serumcollected from one individual with an acuteHEV gt3 infection had high titers of ORF2-binding IgM and IgG antibodies that efficientlyneutralized infectivity of virus derived fromstool but not serum (Takahashi et al. 2010).Anti-capsid IgG but not IgM antibodies werepresent in a second serum sample collected al-most 6 years after convalescence. The capsid-binding titer was 10-fold lower titer than thatmeasured during acute infection, but the anti-bodies still neutralized fecal-derived virus (Ta-kahashi et al. 2010). In another study, convales-cent serum from macaques that resisted cross-genotype challenge with HEV (Purcell et al.2003) was shown to efficiently neutralize HEVinfectivity (Emerson et al. 2006). Surrogate neu-tralization assays have also been developed

based on mapping of neutralizing epitopes to adomain spanning ORF2 amino acids 456–606that form the outer shell of the capsid (Zhanget al. 2012). Panels of monoclonal antibodiesdirected against the ORF2 capsid were used todefine two dominant neutralizing epitopes inthis domain (Zhang et al. 2012).

A fragment of the ORF2 capsid proteinspanning amino acids 386–606 designatedp239 was the minimum unit required for self-assembly of virus-like particles (VLPs) and hasbeen used as a highly effective vaccine that pre-vents HEV infection (Zhang et al. 2012). Bind-ing of fluorophore-labeled VLPs formed byp239 to a hepatocyte cell line is quantifiable byhigh throughput flow cytometry and has beenused to assess antibody-mediated neutralization(Cai et al. 2016). Importantly, assays that mea-sured neutralization of VLP binding and neu-tralization of HEV cell culture infectivity yieldedhighly concordant results. The kinetic of theneutralizing antibody response was determinedin experimentally infected macaques using theVLP-binding assay. In general, serum neutraliz-ing titers obtained with this surrogate assay cor-related with anti-HEV IgG antibody titers (Caiet al. 2016). For instance, the neutralizing anti-body and anti-capsid IgG responses both peakedat the same time afterHEVchallenge, coincidentwith a significant decline in virus replication.Moreover, the assay was used to document asharp increase in neutralizing antibody titers af-ter priming and boosting of macaques with thep239 vaccine (Cai et al. 2016).

Antibody-Mediated Protection against HEVInfection and Liver Disease

Concepts of antibody-mediated protectionagainst HEV infection and liver disease are stillnot as finely detailed as those described abovefor HAV. In overview, there is a consensus thatanti-HEV antibodies elicited by vaccinationor prior exposure to the virus most commonlyprovide cross-genotype protection against infec-tion. HEV viremia, fecal shedding, and hepatitisare not detected in individuals with this appar-ent sterilizing immunity. However, there is anemerging view that secondary HEV infection



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characterized by limited virus replication andmild or inapparent hepatitis can develop in im-mune individuals with low preexisting antibodytiters. Studies in immune macaques providesupport for this concept. Experimental infec-tion (Huang et al. 2008) or vaccination of ani-mals with a recombinant ORF2 capsid protein(Tsarev et al. 1994; Purcell et al. 2003) generallyelicits a high titer antibody response and com-plete resistance to challenge with HEV geno-type-matched and mismatched viruses. It isnotable, however, that a small number of ma-caques with low titer anti-HEV antibodies afterprimary infectionwere susceptible to reinfection(Huang et al. 2008). HEV RNA was detected inblood and stool of animals with breakthroughinfections, but hepatitis was not observed(Huang et al. 2008). The possibility that antibod-ies protect against hepatitis when sterilizing im-munity fails is also supported by passive immu-nization studies. Transfer of immune serumfrom convalescent humans to macaques 2 daysbefore experimental HEV challenge did not pre-vent infection, but hepatitis was mild or absentwhen compared with untreated controls (Tsarevet al. 1994).

Anti-HEV antibodies generated in humansby infection or vaccination are also associatedwith protection fromHEV. One early field studyconducted during an HEV epidemic noted thatall patients hospitalized for hepatitis had anti-HEV IgM antibodies that define primary infec-tion and concluded that secondary infection inimmune individuals was therefore uncommonor clinically inapparent (Bryan et al. 1994).More recent analysis of sporadic and epidemicHEVoutbreaks identified small numbers of sub-jects with serologic evidence of reinfection. Inthese individuals, increasing titers of high avid-ity anti-HEV IgG antibodies in the absence of anIgM response was considered a reliable markerof reinfection when virus replication and hepa-titis were too transient or attenuated for reliabledetection (Seriwatana et al. 2002; Bendall et al.2008; Huang et al. 2010). This concept is sup-ported by observations in an immune chimpan-zee, where rechallenge with HEV 45 monthsafter resolution of primary infection caused asharp boost in anti-HEV IgG titer in the absence

of a robust IgM response (Yu et al. 2003). Sur-veillance to determine the long-term efficacy ofan HEVORF2 vaccine detected a similar shift inantibody profile in some recipients with an en-demic risk of HEV exposure (Zhang et al. 2015).A boost in high avidity IgG antibodies was alsoobserved in the few vaccine recipients who ac-quired HEV infection (Zhang et al. 2015). Final-ly, reinfection was also described in the settingof solid organ transplantation. Serum anti-HEVantibodies were detected in three subjects priorto transplantation but they failed to prevent re-infection (Abravanel et al. 2016). The secondaryinfection persisted in the patient with the lowestanti-HEV antibody titer (Abravanel et al. 2016).

Together, these studies provide evidencethat antibodies elicited by infection or vaccina-tion do not always confer sterilizing immunitybutmay sharply attenuate liver disease andHEVreplicationwhen breakthrough infections do oc-cur. Antibodies could modify the course of in-fection as described above for HAV but the lim-ited studies conducted to date do not supportthis possibility. Postexposure treatment with im-mune gammaglobulin from convalescent do-nors failed to prevent acute hepatitis E in recip-ients (Joshi et al. 1985; Khuroo and Dar 1992),although these very early studies are interpretedwith caution because anti-HEV antibodies inthe donor preparations have not been character-ized. It is notable, however, that active immuni-zation of macaques with the ORF2 capsid vac-cine after HEV challenge also failed to modifythe course of infection when compared with un-vaccinated controls (Tsarev et al. 1997). Exper-imental conditions were similar to those used tosuccessfully prevent acute hepatitis A in ma-caques by postexposureHAVvaccination. Theseobservations highlight the need for a better un-derstanding of antibody-mediated neutraliza-tion of HEV in the setting of vaccination andinfection, and mechanisms used by the virus tosubvert this immune response.

HEV Evasion of Neutralizing Antibodies

HEV exists as a single serotype and there is noevidence that the antibody response in infectedhumans or animals for zoonotic infections select

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for escape variants. As described for HAV,HEV particles that circulate in blood are quasi-enveloped and resistant to antibody-mediatedneutralization. Initial studies demonstratedthat HEV particles in blood were not associatedwith cocirculating anti-capsid antibodies (Taka-hashi et al. 2010). Because these particles had alighter buoyant density on sucrose gradientsthan those in stool and could not be precipitatedwith antibodies against the capsid unless firsttreated with detergent, it was concluded thatthey were associated with lipids (Takahashiet al. 2010). It is now clear that HEV particlesacquire a quasi-envelope during their releasefrom infected cells via the exosomal pathway(Yin et al. 2016; Chapuy-Regaud et al. 2017;Nagashima et al. 2017). Infection requires cellu-lar uptake and uncoating of quasi-envelopedHEV particles in the endolysosome (Yin et al.2016), and it is at this point that they becomesensitive to antibody-mediated neutralization.Antibodies of the IgM isotype that arise first inprimary HEV infection cannot access this cellu-lar compartment and are therefore predicted tohave limited impact on spread of quasi-envel-oped HCV particles via blood. Anti-HEV IgGantibodies that arise later in acute infection canmediate intracellular neutralization of theseHEV particles after uncoating as describedabove for HAV (Yin et al. 2016).

Very recent studies have demonstrated thatHEVmay have a second mechanism for evasionof antibody-mediated neutralization involvingproduction of ORF2 capsid protein variants(Montpellier et al. 2018; Yin et al. 2018). TheORF2 protein is expressed as two variants basedon translational initiation from different startcodons (Yin et al. 2018), perhaps with furtherposttranslational processing by host cell prote-ases (Montpellier et al. 2018). One form ofthe ORF2 protein is translated from the firstavailable start codon (Yin et al. 2018), containsa signal peptide sequence, and is directed co-translationally to the endoplasmic reticulumwhere it is glycosylated and targeted for secre-tion via the trans-Golgi pathway (Montpellieret al. 2018; Yin et al. 2018). These proteins ap-pear to exist as dimers and are not associatedwith HEV genomes. A second, shorter form of

the capsid protein is translated from a down-stream start codon and delivered to the virionassembly sites (Yin et al. 2018). Importantly,the glycosylated secreted long form of ORF2 ismassively overproduced when compared withthe shorter form destined for virus assembly(Montpellier et al. 2018; Yin et al. 2018). Asexpected, the long secreted form is present inserum at very high concentrations during acuteand chronic infection, suggestive of a role inimmune evasion. Consistent with this possibil-ity, secreted ORF2 capsid proteins can interferewith antibody-mediated neutralization of HEVin a cell culturemodel (Yin et al. 2018).WhethersecretedORF2 protein can serve as sink or decoyfor neutralizing antibodies, including those de-livered for postexposure therapy during theincubation phase of HEV infection, remains tobe determined. Additionally, a role in modula-tion of other innate and adaptive immune re-sponses also cannot be excluded and meritsfurther study. Importantly, the secreted solublelong form of the ORF2 protein does not com-pete with virions for receptor-mediated uptakeby cells.

CELLULAR IMMUNE RESPONSES

There have been remarkably few studies of T-cell immunity against HAV and HEV. This re-flects, in part, the historical emphasis on analysisof antibody responses that provide protectionagainst infection, and in the case of HAV, bluntliver disease and transmission of the virus whendeployed as postexposure prophylaxis. Progresshas also been slowed by reliance on expensivenonhuman primate models of HAV and HEVinfection to studymechanisms of immunity andpathogenesis in the liver. To date, there havebeen too few studies of adaptive cellular immu-nity in man or nonhuman primates to reliablysuperimpose the kinetic of CD4+ helper andCD8+ cytotoxic T-cell responses on acute infec-tionHAVandHEV profiles. As noted above, theimperative for HAV is to determine whether Tcells are a cause of hepatocellular injury giventhe increasing burden of severe liver disease as-sociated with a shift toward infection duringadulthood in many regions of the world (see



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Jacobsen 2018). For HEV, there is a similar in-terest in determining the role of adaptive T-cellimmunity in liver injury, especially for the HEVgt1 and gt2 viruses that are more frequently as-sociated with severe and even life-threateninginfections.With the description of HEV gt3 per-sistence, it is also important to test the common-ly held view that CD4+ and CD8+ T cells arerequired for successful resolution of infection.Finally, andmore generally, comparative studiesof T-cell immunity against hepatitis viruses thattypically do not persist (HAV and HEV) andthose that do (HBV and HCV) may provideinsight into factors influencing infection out-come.

The Hepatitis A virus

Studies to characterize cellular responses duringinfectious hepatitis were undertaken decades be-fore discovery of HAV. They ranged from a de-scription of changes in circulating leukocytepopulations in experimentally infected volun-teers (Havens and Marck 1946) to developmentof a delayed type hypersensitivity (DTH) skintest to identify convalescent individuals withimmunity to infectious hepatitis (Henle et al.1950a; Drake et al. 1952). Remarkably, theDTH test relied on skin challenge with prepara-tions generated by serial blind passage of infec-tious materials through eggs and cultured cells,and thought to contain antigens of the yet-to-be-identified hepatitis A virus (Henle et al.1950b).

HAV-specific T-cell immunity was first doc-umented with certainty by Vallbracht and col-leagues in a series of studies conducted morethan 40 years later. A cell culture model wasused to demonstrate the presence of circulatingHAV-specific cytotoxic T lymphocytes in sevenpatients with acute hepatitis A (Fig. 1A) (Vall-bracht et al. 1986). Primary skin fibroblast celllines established from these subjects supportedpersistent, noncytopathic replication of a hu-man HAV isolate (Vallbracht et al. 1986). Co-culture of HAV-infected, 51Cr-labeled fibro-blasts with peripheral blood mononuclear cells(PBMCs) from the same patient resulted in cy-tolysis of the targets as measured by release of

the radioisotope (Vallbracht et al. 1986). Cyto-toxicity peaked in blood 2–3 weeks after theonset of icterus in five patients with a typicalcourse of acute hepatitis A. The remaining twopatients had a protracted course of hepatitis thatdid not resolve for at least 5 months (Vallbrachtet al. 1986). In these individuals, the highestcytotoxic activity against autologous HAV-in-fected hepatocytes was detected 8–12 weeks af-ter the first appearance of hepatitis (Vallbrachtet al. 1986). PBMCs from two uninfected con-trol subjects did not kill virus-infected targetcells, indicating that the cytotoxic activity wasassociated with acute HAV infection. Furthercharacterization revealed that the circulatingCD8+ T cells also produced IFN-γ that en-hanced HLA class I expression and directly in-hibited HAV replication in cultured target cells(Maier et al. 1988). From these studies, it wasconcluded that CD8+ T cells terminate acuteHAV infection by lysis of infected hepatocytesand production of IFN-γ. Infiltration of theseCD8+ T cells into the liver was assessed in afollow-up study of two subjects with acute hep-atitis A (Vallbracht et al. 1989). Mononuclearcells isolated from core liver samples taken at12 or 38 days after the onset of hepatitis wereexpanded in culture with a T-cell mitogen andinterleukin (IL)-2 growth factor. T-cell linesgenerated by this approach expressed CD8 andkilled autologous HAV-infected fibroblasts.These cells fit the definition of classical cytotoxicT lymphocytes; they did not kill autologous fi-broblasts that were uninfected or infected withunrelated viruses, or HLA class I mismatchedfibroblasts infected with HAV. Liver functiontests had not normalized in the subject biopsiedat week 12, and so it was concluded that intra-hepatic CD8+ T cells expanded from the livertissue contributed to hepatocellular injury inacute hepatitis A (Vallbracht et al. 1989).

Since publication of these early studies, therehas been significant progress in understandingthe nature of antigen recognition by antiviral Tcells, their differentiation and function in re-sponse to infection, and in methods to charac-terize their phenotype, function, and frequencyin blood and tissues. Two more recent studieshave used these methods to better characterize

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HAV-specific T-cell immunity during acute in-fection. In the first study, a pool of 52 overlap-ping peptides spanning the HAV VP1 capsidprotein was used for in vitro stimulation ofPBMCs from patients with acute, postacute,and post-HAV infections (Schulte et al. 2011).IFN-γ producing CD8+ T-cell lines targeting 18of these VP1 peptides were expanded in culture,demonstrating a multitude of class I epitopes ina single structural protein of the virus. VP1 epi-topes presented by HLA-A11, HLA-B35, andHLA-B40 were identified using HLA class Ibinding algorithms and common patterns ofVP1 peptide recognition by an individual whoshared these alleles (Schulte et al. 2011). Anoth-er 11 candidate epitopes in HAV structural andnonstructural proteins were identified usingpredictive algorithms for high-affinity bindingto HLA-A2 class I molecules. Of the 11 predict-ed epitopes, five were capable of expandingvirus-specific CD8+ T cells from PBMCs ofHAV-infected subjects who were positive forthe HLA-A2 class I allele. These epitopes wereconserved in genotype 1A, IB, IIIA, and IIIBHAV isolates, with the exception of one HLA-A2 epitope that was not conserved in the geno-type III viruses. Importantly, CD8+ T cellstargeting a dominant epitope in nonstructuralprotein 3Dpol were directly visualized in theblood of six subjects using class I HLA-A2 tet-ramers. In one of these subjects, the tetrameranalysis revealed activation of the 3Dpol-specificCD8+ T cells during the acute phase of infection.This CD8+ T-cell population was still present inblood 20 months later, well after resolution ofinfection, but was no longer activated and hadtransitioned to a memory phenotype (Schulteet al. 2011).

The second contemporary study of antiviralT-cell immunity was conducted in two HAV-infected chimpanzees (Zhou et al. 2012). Use ofthe animal model facilitated a kinetic analysis ofHAV replication, ALTelevation, and antiviral T-cell immunity from the time of experimentalchallenge through resolution of infection. Theinfection followed a typical course in both ani-mals. Viremia cleared and ALT normalized be-tween 6 and 8 weeks of infection. Fecal sheddingwas terminated by week 14–16 postinfection

when assessed by a highly sensitive PCR assayfor HAV RNA. Reduced viremia and fecal shed-ding was kinetically associated with a circulatingCD4+ T helper (Th) cell response that targetedmultiple class II epitopes in structural and non-structural proteins and produced a full array ofTh1 cytokines, including IFN-γ, tumor necrosisfactor (TNF)-α, IL-2, and IL-21 required forgeneration of a cytotoxic CD8+ T-cell response(Zhou et al. 2012). Indeed, CD8+ T cells werevisualized in blood at week 4 when ALT levelsspiked sharply and reached a peak frequency atweek 5 when a multilog drop in viremia wasobserved. Importantly, however, the HAV-spe-cific CD8+ T cells visualized in blood during thecritical period of elevated ALT and virus controlappeared to lack effector functions, includingcytotoxicity and production of cytokines likeIFN-γ and TNF-α that have the potential to sup-press HAV replication (Zhou et al. 2012). Gainof effector functions and transition towardmem-ory by CD8+ T cells did not begin until virusreplication was substantially controlled. Thesedata suggested that the highly functional CD4+

T-cell and anti-HAV antibody responses werebetter correlated with control of viremia thanthe CD8+ T-cell response that did not developantiviral functions until after hepatitis had sub-sided and HAV replication was reduced (Zhouet al. 2012).Whether this strong, multifunctionalCD4+ T-cell response provided direct control ofHAV replication, perhaps through production ofcytokines like TNF-α and IFN-γ (Todt et al. 2016;Wang et al. 2016) remains to be determined.

Mechanisms of Liver Injury in AcuteHepatitis A

As noted above, wild-type HAV is not directlycytopathic for cultured cells (Frosner et al. 1979;Daemer et al. 1981; Gauss-Muller et al. 1981;Kojima et al. 1981). High levels of virus replica-tion 3–4weeks before the onset of acute hepatitisalso suggests that the developing host response,and not virus replication, causes liver disease.The onset of the anti-HAV IgM antibodyresponse does coincide with elevation of serumtransaminases. However, serum antibodies fromthe acute and convalescent phase of HAV infec-



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tion did not mediate lysis of cultured HAV-in-fected cells even in the presence of complement(Flehmig et al. 1984), suggesting that they arenot a cause of hepatitis.

Cell-mediated cytotoxicity by virus-specificCD8+ T cells has been considered a more likelymechanism of hepatocellular injury in acutehepatitis A, but direct evidence supporting thishypothesis is lacking. As noted above, HAV-spe-cific CD8+ T cells were expanded from the blood(Schulte et al. 2011) and liver (Vallbracht et al.1989) of patients of with acute hepatitis A, andthey were directly visualized in the peripheralcirculation of humans (Schulte et al. 2011) andchimpanzees (Zhou et al. 2012) with acute hep-atitis A using class I tetramers (Fig. 1A). Thisevidence is largely circumstantial, however.There has been no direct ex vivo analysis of in-trahepatic CD8+ T-cell effector function and itsrelationship to the severity of hepatocellular in-jury in acute hepatitis A. The observation thatcirculating virus-specific CD8+ T cells in infect-ed chimpanzees do not acquire effector func-tions until after transaminases normalize hasled to uncertainty about their contribution toliver injury (Zhou et al. 2012).

A variety of natural killer (NK) cell types areresident in the liver. If activated during infection,they could mediate innate cytotoxic activityagainst HAV-infected hepatocytes or contributeto an inflammatory environment that leads tohepatocellular injury. This possibility was rec-ognized as early as 1985 when it was demon-strated that PBMCs cultured in the presence ofHAVparticles produced high levels of type I IFNthat in turn activated NK cells capable of killingvirus-infected target cells (Kurane et al. 1985).Attention has very recently turned again to al-ternative mechanisms of acute liver injury inHAV-infected humans (Kim et al. 2018). Thesestudies were undertaken during a large andprolonged HAV epidemic in South Korea thatdisproportionately affected adults, resulting ina substantial burden of serious acute liver dis-ease. Analysis of peripheral blood samples frompatients with acute hepatitis A revealed in-creased activation and proliferation of circulat-ing CD8+ T cells that expressed cytotoxic mole-cules, the NK-cell-activating receptors NKG2D

andNKp30 (Fig. 1A) (Kim et al. 2018). Remark-ably, an increased frequency of these CD8+ Tcells was strongly correlated with increased se-rum ALT levels at the time of admission. Anal-ysis of the activated CD8+ T cells revealed acomplex mixture of populations targeting notonly HAV but also multiple other unrelatedviruses, including cytomegalovirus, influenza vi-rus, respiratory syncytial virus, and vacciniavirus. Further study demonstrated that HAVinfection of cultured hepatocytes induces pro-duction of IL-15, a cytokine that can activateCD8+ T cells regardless of the virus they target(Kim et al. 2018). It is notable that IL-15 levelswere elevated in the serum of patients in thisstudy, suggesting that this cytokine and nonspe-cific CD8+ T-cell activation is associated withsevere liver disease. Finally, the authors demon-strated that activated CD8+ T cells killed targetcells through engagement of the NK cell recep-tors and not theT-cell receptor (Kim et al. 2018).Thus, hepatocellular injury in acute hepatitis Awas associated with innate-like cytotoxicity ofbystander CD8+ T cells after IL-15 activation.

Further study of the same cohort of SouthKorean patients revealed further complexity inthe pathogenesis of liver injury during acutehepatitis A (Choi et al. 2018b). Earlier studiesof this cohort (Choi et al. 2015) and others (Per-rella et al. 2008; Manangeeswaran et al. 2012)demonstrated that Treg cells, defined by expres-sion of the CD4 coreceptor and the FoxP3 tran-scription factor, had an impaired ability to sup-press effector T-cell proliferation and functionduring acute hepatitis A. In the follow-up study,functional alterations in circulating Treg cellswere associated with more severe liver disease(Fig. 1A) (Choi et al. 2018b). As noted in earlierstudies, the normal suppressive function ofTregs was reduced and they instead producedthe inflammatory cytokine TNF-α. Moreover,TNF-α secretion was associated with at leastpartial conversion to a Th17 phenotype definedby expression of the RORγt transcription factor,CCR6, and production of IL-17A (Choi et al.2018b). Whether this switch of Treg to a proin-flammatory posture contributes directly to liverdisease was not established. Only end-stage liverwas available for analysis, and the frequency of

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TNF-α-producing Treg cells was not elevated inthese samples when compared to control livertissue (Choi et al. 2018b). However, the frequen-cy of TNF-α-producing Treg cells in circulationwas directly correlated with transaminase eleva-tions. It is, therefore, likely that proinflamma-tory conversion of Tregs during acute hepatitisA is a marker, if not a direct cause, of seriousinflammatory liver disease in this infection.Mechanisms that underpin the loss of inhibitoryfunction by Tregs and their acquisition of aproinflammatory phenotype have not beenidentified. It may be a direct consequence ofother immunoregulatory events in the liverthat have yet to be defined. Binding of HAVparticles to T cells could modulate their func-tion. For example, it has been reported thatHAVimpairs the function of Treg cells that express theT-cell immunoglobulin and mucin 1 (TIM-1,CD365) protein. Increased FasL-induced celldeath, inhibition of T-cell receptor activation,and reduced expression of suppressive cytokinesIL-10 and TGF-β were observed when T cellswere cultured in the presence of HAV particles(Manangeeswaran et al. 2012). Quasi-envelopedHAV particles could be of greatest relevance tomodulation of T-cell function as this is the formthat circulates in blood. Quasi-enveloped HAVparticles were also recently shown to bindTIM-1(Das et al. 2017), probably through interactionwith phosphatidyl serine residues in the enve-lope as demonstrated for other viruses (Moller-Tank and Maury 2014).

Polymorphisms in the human TIM-1 im-munoregulatory protein may explain in partthe wide spectrum in liver disease observed inhumans with acute hepatitis A (Kim et al. 2011).Twovariants of the TIM-1 gene (HAVCR1) werefound in patients with acute HAV infection re-sulting from a sporadic outbreak in Argentina.Severe hepatitis was associated with carriage of aHAVCR1 gene encoding a 6-amino-acid inser-tion designated 157insMTTTVP in exon 4 thathad previously been linked to allergic diseasesand the pathogenesis of HIV infection (Kimet al. 2011). Expression of this results in along-form TIM-1 protein in human NKT cellsthat enhanced their cytolytic activity againstHAV-infected hepatocytes when comparedwith

NKT cells that expressed the short form of thegene (Kim et al. 2011).

In summary, the role of HAV-specific CD8+

T cells in the pathogenesis of acute hepatocellu-lar injury remains uncertain. They are present inthe blood and liver of patients with acute hepa-titis A. Whereas these antiviral CD8+ T cellscould contribute to liver damage by lysis of in-fected hepatocytes, severity of liver disease hasnot yet been correlated with the magnitude andkinetic of the response. The observation thatcirculating CD8+ T cells lack effector functionsuntil after virus replication subsides and ALTvalues normalize may argue against this possi-bility. The question of whether they are func-tional in the liver and are associated with cyto-toxic activity at the mild or severe end of thedisease spectrum requires further study. Othercytotoxic cell populations, including nonspecif-ically activated classical CD8+ T cells, NKT cells,and proinflammatory Treg cells, have been di-rectly associated with the severity of acute liverdisease in HAV infection. Finally, it is likely thatliver injury is multifactorial because HAV in-fection of mice resulted in IFN-independent ac-tivation of IFN response factor 3 (IRF3) and 7(IRF7) that mediated mitochondrial apoptosisin hepatocytes (Hirai-Yuki et al. 2016a, 2018).Importantly, antibody-mediated depletion ofCD4+ or CD8+ T cells, or NK and NKT cells,prior to virus challenge had no amelioratingeffect on the inflammatory hepatitis associatedwith infection in these murine studies. Whetherthe mechanisms reviewed here also account forfulminant hepatitis that is sometimes observedin HAV- and HEV-infected patients remains tobe determined.

The Hepatitis E Virus

There is a remarkable deficit in knowledge aboutthe contribution of adaptive T-cell immunity tothe control and pathogenesis of HEV infection.The relatively recent description of persistentHEV gt3 and gt4 infections in individuals withcompromised immunity has rekindled interestin the requirement for CD4+ and/or CD8+ T-cell responses in resolution of acute infectionand prevention of chronic HEV replication



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and liver disease. As noted above, long-termpersistence of HAV has yet to be described un-der the same conditions of immune suppressionthat facilitate chronic HEV infection. This maysuggest a fundamental difference between theseenteric hepatitis viruses in mechanisms of pro-tective immunity that favor acute resolution ver-sus persistence of infection.

As with HAV, HEV causes a noncytopathicinfection in cultured cells (Shukla et al. 2011;Okamoto 2013) and replication of the virus isrobust during the prodromal phase of infection,well before the onset of hepatitis. It is thereforealso likely that liver injury in acute hepatitis E isalso immune mediated, but involvement of Tcells or other innate cytotoxic populations hasnot yet been investigated. Apparent differencesin the severity of liver disease inHEVgt1 and gt2versus gt3 and gt4 infections, and the severity ofHEV gt1 infections in the late stages of pregnan-cy, has also highlighted the need for a betterunderstanding of adaptive cellular immunityand the potential of the response to cause liverpathology.

Most early studies of T-cell immunity elicit-ed by HEV gt1 viruses involved analysis of re-sponses in peripheral blood against recombi-nant ORF2 capsid protein. Using the IFN-γELISpot assay as a readout, an elevated frequen-cy of circulating ORF-2-specific T cells was re-ported in subjects with acute and resolved infec-tions when compared to uninfected controlswith no known history of exposure to HEV(Wu et al. 2008; Husain et al. 2011; Tripathyet al. 2012). The ELISpot assay does not provideinsight into the relative contribution of CD4+

versus CD8+ T cells to IFN-γ production. Useof the recombinant ORF2 protein almost cer-tainly favored detection of the CD4+ helper sub-set because large proteins are processed ineffi-ciently (if at all) for class I presentation to CD8+

T cells. An IFN-γ ELISpot response against arecombinant ORF2 protein was also describedin six chimpanzees after recovery from experi-mental challenge with an HEV gt1 isolate (Shataet al. 2007). An attempt to detect IFN-γ produc-tion by ORF2-stimulated CD4+ and CD8+ Tcells from patients with sporadic acute icterichepatitis E using flow cytometry failed for rea-

sons that are not clear (Srivastava et al. 2007).Thus, the T-cell response to HEV gt1 virusesremains very poorly characterized despite theenormous number of symptomatic infectionsit causes globally, and the potential for cata-strophic infection outcome in pregnant womenand others who develop fulminant hepatitis.

More detailed analyses of T-cell immunitywere undertaken in humans with acute, conva-lescent, and chronic HEV gt3 infections. Collec-tively, these studies assessed T-cell responsesafter stimulation of PBMCs with overlappingpeptides sets spanning the ORF1, ORF2, andORF3 domains, an approach that facilitates de-tection of CD4+ and CD8+ T-cell responses (Su-neetha et al. 2012; Brown et al. 2016; Gisa et al.2016; Al-Ayoubi et al. 2018). Common findingsfrom these studies can be summarized as fol-lows:

1. Typical acute resolving HEV infection. CD4+

and CD8+ T cells targeting all three HEVopen reading are present in the peripheralblood during the acute phase of HEV infec-tion, as judged by their capacity to proliferateand/or produce cytokines like MIP-1β, IFN-γ, and TNF-α (Fig. 2A) (Suneetha et al. 2012;Brown et al. 2016; Gisa et al. 2016; Al-Ayoubiet al. 2018). HEV-specific T cells are alsopresent in the peripheral blood of convales-cent subjects (Suneetha et al. 2012; Brownet al. 2016; Gisa et al. 2016; Al-Ayoubi et al.2018), but frequencies decline rapidly in themonths after resolution of infection (Brownet al. 2016). Frequencies were much higher inthe 12 months immediately after infectionwhen compared with time points later than1 year (Brown et al. 2016). Epitopes targetedby CD4+ and CD8+ T cells were mapped tomore conserved domains of the HEV prote-ome (Brown et al. 2016). Indeed, some T-cellpopulations elicited by HEV gt3 infectionrecognized epitopes that were conserved inHEV gt1 viruses, indicating priming ofcross-genotype immunity.

2. HEV in immune compromised patients. Asnoted above, HEV can persist when immuni-ty is profoundly impaired and the number ofcirculating CD3+ and CD4+ T cells is signifi-

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cantly reduced (Kamar et al. 2008, 2011).When compared with uncomplicated acutehepatitis E, virus-specific CD4+ and CD8+ Tcells are present at very low frequency andare generally difficult to detect in patientswith chronic hepatitis (Suneetha et al. 2012;Brown et al. 2016). It is noteworthy thatCD8+ T cells from chronically infected sub-jects regained antiviral effector functionswhen cultured in the presence of antibodiesagainst the PD-1 coinhibitory receptor (Su-neetha et al. 2012). Inhibitory PD-1 signalingis a major driver of T-cell exhaustion in otherchronic virus infections. This result suggeststhat loss of T-cell proliferation and effectorfunctions in chronic hepatitis E occurs by asimilar process to that described for otherpersistent viruses like hepatitis C virus(HCV) (Suneetha et al. 2012). One key ques-tion is whether T-cell immunity recovers af-ter successful treatment of chronic hepatitis Ewith ribavirin or reduced immune suppres-sion. Longitudinal analysis of chronically in-fected patients undergoing therapy did revealexpansion of HEV-specific T cells in bloodwithin 4 weeks of viral clearance (Suneethaet al. 2012). In a second study, a strong IFN-γ ELISpot response by ORF2-stimulated-PBMC was also associated with successfulcontrol of chronic HEV infection after anti-viral therapy or reduction of immune sup-pression (Abravanel et al. 2016). How longthese T cells persist in blood after terminationof infection has not been determined. T-cellimmunity was not detected in transplant pa-tients studied approximately 2 years aftercure of chronic hepatitis E, suggesting thatlong-term memory may not develop or thatfrequencies drop below the threshold for de-tection in blood (Brown et al. 2016). Finally,improvement in CD4+ T-cell numbers andimmune function after initiation of antiretro-viral therapy in patients coinfected with HIVand HEV might be expected to facilitate res-olution of the HEV infection. However, in atleast some individuals, HEV may continueto persist despite apparent reconstitution ofimmune function (Ingiliz et al. 2016). Thisobservation suggests that chronic HEV infec-

tion established during immune suppressioncan cause a pervasive and perhaps permanentblock on generation of anti-HEV antiviral re-sponses that persists well after general condi-tions of immune suppression are reversed.

To date, there is very limited direct evidencethat CD4+ and/or CD8+ T cells suppress HEVreplication or cause immunopathology in theliver. It is reasonable to speculate that noncyto-toxic T-cell control of HEV replication is com-mon because most infections, especially thosecaused by gt3 and gt4 viruses, are clinically in-apparent. Replication of the virus in cell cultureis sensitive to treatment with T-cell-producedcytokines like IFN-γ (Todt et al. 2016) andTNF-α (Behrendt et al. 2017). Cytokine-medi-ated inhibition of HEV replication is supportedby one compelling case report of severe exacer-bation of chronic infection during treatmentwith a TNF-α inhibitor for psoriatic arthritis(Behrendt et al. 2017). Interestingly, HEV-spe-cific T-cell immunity was detected in this pa-tient during treatment with the TNF-α inhibi-tor, but virus replication increased sharply untiltreatment with the inhibitor was discontinuedand ribavirin was administered to control theinfection (Behrendt et al. 2017).

SUMMARY AND FUTURE DIRECTIONS: THENEED TO UPDATE MODELS OF ADAPTIVEIMMUNITY IN ENTERIC HEPATITIS VIRUSINFECTIONS

The importance of adaptive immune responsesto control of enteric hepatitis virus infectionshas been considered largely settled for manyyears. For example, the model of T-cell immu-nity that emerged from the first studies of acuteHAV infection approximately 30 years ago isstill widely accepted. It was proposed that con-trol of acute HAV infection requires a virus-spe-cific CD8+ cytotoxic T-cell response that clearsinfected hepatocytes and causes acute hepatitisas collateral damage (Maier et al. 1988; Fleischeret al. 1990; Vallbracht and Fleischer 1992). Thesamemodel of CD8+ T-cell-mediated virus con-trol and liver injury has been adopted for HEV,given the very similar timing and clinical profile



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of acute infection for both enteric hepatitis vi-ruses. It is important to emphasize, however,that a temporal kinetic relationship between ex-pansion of functional antiviral CD8+ T cells,control of viremia or fecal shedding, and acutehepatitis remains to be established for HAV orHEV infection.

Remarkably few studies of either virus havebeen undertaken since the emergence of power-ful technologies that permit direct visualizationof antiviral CD4+ and CD8+ T cells, assessmentof antiviral functions, and differentiation intoeffector and memory populations. The value incontinuing studies of adaptive immunity againstHAV and HEV is highlighted by recent studiesthat argue against a requirement for CD8+ Tcells in termination of infection or as a causeof acute hepatitis. As described in this review,CD8+ T cells did not acquire effector functionsuntil after viremia was substantially controlledin one contemporary study of acute HAV infec-tion in chimpanzees (Zhou et al. 2012). Thisunexpected finding suggested that functionalcytotoxic CD8+ T cells are not necessarily re-quired to control acute HAV infection, and per-haps, by extension, HEV infection although thisremains to be determined. A link between HLAclass I restricted CD8+ cytotoxic T-cell activityand acute hepatitis has also not yet been con-firmed. Compelling new studies indicate thathepatocellular injury may instead be multifacto-rial, involving cytotoxic activity by innate or by-stander T cells (Choi et al. 2018b; Kim et al.2018) and activation of certain type I IFN sig-naling pathways (Hirai-Yuki et al. 2016a).

With regard to humoral immune responses,the capacity of neutralizing antibodies generatedby natural infection or vaccination to provideapparent sterilizing immunity against HAV orHEV is well established. This protective immu-nity appears to be lifelong in the case of HAVinfection. Reinfection with HEV has been doc-umented in some individuals after resolution ofa primary infection or even after vaccination,but how commonly antibody-mediated protec-tion fails and under what conditions remains tobe determined.

A key unresolved question is whether anti-bodies contribute to control of acute HAV or

HEV infection. As noted above, T-cell immuni-ty has been considered of paramount impor-tance in resolution of infection but a role forantibodies cannot yet be excluded. As notedabove, two observations provide some supportfor this concept. First, at least for HAV, active orpassive immunization within 14 days of virusexposure provides direct evidence for anti-body-mediated inhibition of virus replicationand tempering of acute hepatitis. Second, initialsuppression of HAV or HEV replication duringacute infection occurs at roughly the same timeas seroconversion. The recent finding that HAVand HEV particles circulating in blood have aquasi-envelope is of significance when consid-ering how and where virus is neutralized by an-tibodies (Feng et al. 2013; Yin et al. 2016). Thisform of the virus is almost certainly responsiblefor spread of both HAV and HEV between he-patocytes after infection is established. Becauseneutralization is now thought to occur after deg-radation of the quasi-envelope ofHEVandHAVparticles within the endolysosome, a role for theearly pentameric IgM antibodies in containinginitial spread of the virus seems doubtful. A keyunresolved question is whether antiviral IgG an-tibodies, which arise later in infection and neu-tralize HAV and HEV particles after removal ofthe quasi-envelope in the endolysosome, accel-erate termination of viremia or fecal shedding.

With these studies, new models of that ex-plain how HAV and HEV infections are con-trolled by antibodies and T cells, and why theseverity of liver disease is often so variable, havebegun to emerge. Further efforts to sort out un-resolved details of adaptive immunity againstthe enteric hepatitis viruses is of importancegiven the large number of infections that stilloccur globally each year, and the substantialburden of liver disease that they cause. HAV andHEVare also of importance as they are examplesof small RNA viruses that are successfully con-trolled by intrahepatic immune responses. Asnoted above, certain HEV genotypes do persistin individuals with compromised immunity,but HAV does not for reasons that are poorlyunderstood. A resurgence in interest and prior-ity for research into adaptive immune responsesagainst HAV and HEV could provide a much

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better general understanding of how virus infec-tions are contained in the unique environmentof the liver, and why host defenses sometimesfail during infections with viruses like HBV andHCV that have a propensity to persist.

REFERENCES�Reference is also in this collection.

Abravanel F, Barrague H, Dorr G, Saune K, Peron JM, AlricL, Kamar N, Izopet J, Champagne E. 2016. Conventionaland innate lymphocytes response at the acute phase ofHEV infection in transplanted patients. J Infect 72: 723–730.

� Aggarwal R, Goel A. 2018. Natural history, clinical manifes-tations, and pathogenesis of hepatitis E virus genotype 1and 2 infections. Cold Spring Harb Perspect Med doi:10.1101/cshperspect.a032136.

Al-Ayoubi J, Behrendt P, Bremer B, Suneetha PV, Gisa A,Rinker F, Manns MP, Cornberg M, Wedemeyer H, KraftARM. 2018. Hepatitis E virus ORF 1 induces proliferativeand functional T-cell responses in patients with ongoingand resolved hepatitis E. Liver Int 38: 266–277.

Balayan MS, Andjaparidze AG, Savinskaya SS, Ketiladze ES,Braginsky DM, Savinov AP, Poleschuk VF. 1983. Evi-dence for a virus in non-A, non-B hepatitis transmittedvia the fecal–oral route. Intervirology 20: 23–31.

Behrendt P, Luth S, Dammermann W, Drave S, Brown RJ,Todt D, Schnoor U, Steinmann E,Wedemeyer H, PischkeS, et al. 2017. Exacerbation of hepatitis E virus infectionduring anti-TNFα treatment. Joint Bone Spine 84: 217–219.

Bendall R, Ellis V, Ijaz S, Thurairajah P, Dalton HR. 2008.Serological response to hepatitis E virus genotype 3 infec-tion: IgG quantitation, avidity, and IgM response. J MedVirol 80: 95–101.

Bradley D, Andjaparidze A, Cook EH Jr, McCaustland K,Balayan M, Stetler H, Velazquez O, Robertson B, Hum-phrey C, KaneM, et al. 1988. Aetiological agent of enteri-cally transmitted non-A, non-B hepatitis. J Gen Virol 69:731–738.

Brown A, Halliday JS, Swadling L, Madden RG, Bendall R,Hunter JG,Maggs J, Simmonds P, SmithDB, Vine L, et al.2016. Characterization of the specificity, functionality,and durability of host T-cell responses against the full-length hepatitis E virus. Hepatology 64: 1934–1950.

Bryan JP, Tsarev SA, Iqbal M, Ticehurst J, Emerson S, Ah-med A, Duncan J, Rafiqui AR, Malik IA, Purcell RH, et al.1994. Epidemic hepatitis E in Pakistan: Patterns of sero-logic response and evidence that antibody to hepatitis Evirus protects against disease. J Infect Dis 170: 517–521.

CaiW, Tang ZM,WenGP,Wang SL, JiWF, YangM, YingD,Zheng ZZ, Xia NS. 2016. A high-throughput neutralizingassay for antibodies and sera against hepatitis E virus. SciRep 6: 25141.

Chandra NS, Sharma A, Malhotra B, Rai RR. 2010. Dynam-ics of HEV viremia, fecal shedding and its relationshipwith transaminases and antibody response in patientswith sporadic acute hepatitis E. Virol J 7: 213.

Chapuy-Regaud S, Dubois M, Plisson-Chastang C, Bonne-fois T, Lhomme S, Bertrand-Michel J, You B, Simoneau S,Gleizes PE, Flan B, et al. 2017. Characterization of thelipid envelope of exosome encapsulated HEV particlesprotected from the immune response. Biochimie 141:70–79.

Chau KH, Dawson GJ, Bile KM, Magnius LO, Sjogren MH,Mushahwar IK. 1993. Detection of IgA class antibody tohepatitis E virus in serum samples from patients withhepatitis E virus infection. J Med Virol 40: 334–338.

Chauhan A, Jameel S, Dilawari JB, Chawla YK, Kaur U,Ganguly NK. 1993. Hepatitis E virus transmission to avolunteer. Lancet 341: 149–150.

Choi YS, Lee J, Lee HW, Chang DY, Sung PS, JungMK, ParkJY, Kim JK, Lee JI, ParkH, et al. 2015. Liver injury in acutehepatitis A is associated with decreased frequency of reg-ulatory T cells caused by Fas-mediated apoptosis.Gut 64:1303–1313.

Choi M, Hofmann J, Kohler A, Wang B, Bock CT, Schott E,Reinke P, Nickel P. 2018a. Prevalence and clinical corre-lates of chronic hepatitis E infection in german renaltransplant recipients with elevated liver enzymes. Trans-plant Direct 4: e341.

Choi YS, Jung MK, Lee J, Choi SJ, Choi SH, Lee HW, Lee JJ,Kim HJ, Ahn SH, Lee DH, et al. 2018b. Tumor necrosisfactor-producing T-regulatory cells are associated withsevere liver injury in patients with acute hepatitis A. Gas-troenterology 154: 1047–1060.

Clayson ET, Myint KS, Snitbhan R, Vaughn DW, Innis BL,Chan L, Cheung P, Shrestha MP. 1995. Viremia, fecalshedding, and IgM and IgG responses in patients withhepatitis E. J Infect Dis 172: 927–933.

Cobden I, James OF. 1986. A biphasic illness associated withacute hepatitis A virus infection. J Hepatol 2: 19–23.

ConradME, Lemon SM. 1987. Prevention of endemic ictericviral hepatitis by administration of immune serum gam-ma globulin. J Infect Dis 156: 56–63.

Counihan NA, Anderson DA. 2016. Specific IgA enhancesthe transcytosis and excretion of hepatitis Avirus. Sci Rep6: 21855.

Daemer RJ, Feinstone SM, Gust ID, Purcell RH. 1981. Prop-agation of human hepatitis Avirus in African greenmon-key kidney cell culture: Primary isolation and serial pas-sage. Infect Immun 32: 388–393.

Dalton HR, Bendall RP, Keane FE, Tedder RS, Ijaz S. 2009.Persistent carriage of hepatitis E virus in patients withHIV infection. N Engl J Med 361: 1025–1027.

Das A, Hirai-Yuki A, Gonzalez-Lopez O, Rhein B, Moller-Tank S, Brouillette R, Hensley L, Misumi I, Lovell W,Cullen JM, et al. 2017. TIM1 (HAVCR1) is not essentialfor cellular entry of either quasi-enveloped or naked hep-atitis A virions. MBio 8: e00969.

D’Hondt E, Purcell RH, Emerson SU,WongDC, ShapiroM,Govindarajan S. 1995. Efficacy of an inactivated hepatitisAvaccine in pre- and postexposure conditions inmarmo-sets. J Infect Dis 171: S40–S43.

Dotzauer A, Gebhardt U, Bieback K, Gottke U, Kracke A,Mages J, Lemon SM, Vallbracht A. 2000. Hepatitis Avirus-specific immunoglobulin A mediates infection ofhepatocytes with hepatitis A virus via the asialoglycopro-tein receptor. J Virol 74: 10950–10957.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Dotzauer A, Brenner M, Gebhardt U, Vallbracht A. 2005.IgA-coated particles of hepatitis Avirus are translocalizedantivectorially from the apical to the basolateral site ofpolarized epithelial cells via the polymeric immunoglob-ulin receptor. J Gen Virol 86: 2747–2751.

Dotzauer A, Heitmann A, Laue T, Kraemer L, Schwabe K,Paulmann D, Flehmig B, Vallbracht A. 2012. The role ofimmunoglobulin A in prolonged and relapsing hepatitisA virus infections. J Gen Virol 93: 754–760.

Drake ME, Ward C, Stokes J Jr, Henle W, Medairy GC,Mangold F, Henle G. 1952. Studies on the agent of infec-tious hepatitis. III: The effect of skin tests for infectioushepatitis on the incidence of the disease in a closed insti-tution. J Exp Med 95: 231–239.

Emerson SU, Clemente-Casares P, Moiduddin N, ArankalleVA, Torian U, Purcell RH. 2006. Putative neutralizationepitopes and broad cross-genotype neutralization of hep-atitis E virus confirmed by a quantitative cell-culture as-say. J Gen Virol 87: 697–704.

� Feinstone SM. 2018. History of the discovery of hepatitis Avirus. Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031740.

Feinstone SM, Kapikian AZ, Purceli RH. 1973. Hepatitis A:Detection by immune electron microscopy of a viruslikeantigen associated with acute illness. Science 182: 1026–1028.

Feng Z, Hensley L, McKnight KL, Hu F, Madden V, PingL, Jeong SH, Walker C, Lanford RE, Lemon SM. 2013.A pathogenic picornavirus acquires an envelope by hi-jacking cellular membranes. Nature 496: 367–371.

Feng Z, Li Y, McKnight KL, Hensley L, Lanford RE, WalkerCM, Lemon SM. 2015. Human pDCs preferentially senseenveloped hepatitis A virions. J Clin Invest 125: 169–176.

Flehmig B, Zahn J, Vallbracht A. 1984. Levels of neutralizingand binding antibodies to hepatitis-A virus after onset oficterus: A comparison. J Infect Dis 150: 461.

Fleischer B, Vallbracht A. 1991. Demonstration of virus-spe-cific cytotoxic T lymphocytes in liver tissue in hepatitisA—A model for immunopathological reactions. BehringInst Mitt 89: 226–230.

Fleischer B, Fleischer S, Maier K, Wiedmann KH, Sacher M,Thaler H, Vallbracht A. 1990. Clonal analysis of infiltrat-ing T lymphocytes in liver tissue in viral hepatitis A.Immunology 69: 14–19.

Frosner GG, Deinhardt F, Scheid R, Gauss-Muller V,Holmes N, Messelberger V, Siegl G, Alexander JJ. 1979.Propagation of human hepatitis Avirus in a hepatomacellline. Infection 7: 303–305.

Gardinali NR, Guimaraes JR, Melgaco JG, Kevorkian YB,Bottino FO, Vieira YR, da Silva AC, Pinto DP, da FonsecaLB, Vilhena LS, et al. 2017. Cynomolgus monkeys aresuccessfully and persistently infected with hepatitis E vi-rus genotype 3 (HEV-3) after long-term immunosup-pressive therapy. PLoS ONE 12: e0174070.

Gauss-Muller V, Frosner GG, Deinhardt F. 1981. Propaga-tion of hepatitis A virus in human embryo fibroblasts. JMed Virol 7: 233–239.

Gellis SS, Stokes J, Brother GM, Hall WM, Gilmore HR,Beyer E. 1945. The use of immune serum globulin (gam-ma globulin) in infectious (epidemic) hepatitis in theMediterranean theater of operations. I: Studies on pro-

phylaxis in two epidemics of infectious hepatitis. J AmMed Assoc 128: 1062–1063.

Gisa A, Suneetha PV, Behrendt P, Pischke S, Bremer B, FalkCS, Manns MP, Cornberg M, Wedemeyer H, Kraft AR.2016. Cross-genotype-specific T-cell responses in acutehepatitis E virus (HEV) infection. J Viral Hepat 23:305–315.

GliksonM, Galun E, Oren R, Tur-Kaspa R, Shouval D. 1992.Relapsing hepatitis A. Review of 14 cases and literaturesurvey. Medicine (Baltimore) 71: 14–23.

Green MS, Dotan K. 1988. Efficacy of immune serum glob-ulin in an outbreak of hepatitis Avirus infection in adults.J Infect 17: 265–270.

Havens WP Jr, Marck RE. 1946. The leukocytic response ofpatients with experimentally induced infectious hepatitis.Am J Med Sci 212: 129–138.

HavensWP, Paul JR. 1945. Prevention of infectious hepatitiswith gamma globulin. J Am Med Assoc 129: 270–272.

Henle G, Drake M, Henle W, Stokes J Jr. 1950a. A skin testfor infectious hepatitis. Proc Soc Exp Biol Med 73: 603–605.

HenleW,Harris S, Henle G, Harris TN,DrakeME,MangoldF, Stokes J Jr. 1950b. Studies on the agent of infectioushepatitis; propagation of the agent in tissue culture and inthe embryonated hen’s egg. J Exp Med 92: 271–281.

Hirai-Yuki A, Hensley L, McGivern DR, Gonzalez-Lopez O,Das A, Feng H, Sun L, Wilson JE, Hu F, Feng Z, et al.2016a. MAVS-dependent host species range and patho-genicity of human hepatitis A virus. Science 353: 1541–1545.

Hirai-Yuki A, Hensley L, Whitmire JK, Lemon SM. 2016b.Biliary secretion of quasi-enveloped human hepatitis Avirus. MBio 7: e01998.

� Hirai-Yuki A,Whitmire JK, JoyceM, Tyrrell DL, Lemon SM.2018. Murine models of hepatitis A virus infection. ColdSpring Harb Perspect Med doi: 10.1101/cshperspect.a031674.

Hong S, Lee HW, Chang DY, You S, Kim J, Park JY, Ahn SH,Yong D, Han KH, Yoo OJ, et al. 2013. Antibody-secretingcells with a phenotype of Ki-67low, CD138high, CD31high,and CD38high secrete nonspecific IgM during primaryhepatitis A virus infection. J Immunol 191: 127–134.

HuangW, ZhangH, Harrison TJ, Lang S, HuangG,Wang Y.2008. Cross-protection of hepatitis E virus genotypes 1and 4 in rhesus macaques. J Med Virol 80: 824–832.

Huang S, ZhangX, JiangH, YanQ,Ai X,WangY, Cai J, JiangL, Wu T, Wang Z, et al. 2010. Profile of acute infectiousmarkers in sporadic hepatitis E. PLoS ONE 5: e13560.

Hughes JV, Stanton LW, Tomassini JE, Long WJ, ScolnickEM. 1984. Neutralizing monoclonal antibodies to hepa-titis Avirus: Partial localization of a neutralizing antigenicsite. J Virol 52: 465–473.

Husain MM, Aggarwal R, Kumar D, Jameel S, Naik S. 2011.Effector T cells immune reactivity among patients withacute hepatitis E. J Viral Hepat 18: e603–608.

Ingiliz P, Mayr C, Obermeier M, Herbst H, Polywka S,Pischke S. 2016. Persisting hepatitis E virus infection lead-ing to liver cirrhosis despite recovery of the immune sys-tem in an HIV-infected patient. Clin Res Hepatol Gastro-enterol 40: e23–e25.

C.M. Walker


ww

w.p

ersp

ecti

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nm

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org



� Jacobsen KH. 2018. Globalization and the changing epide-miology of hepatitis A virus. Cold Spring Harb PerspectMed doi: 10.1101/cshperspect.a031716.

Jacobson IM, Nath BJ, Dienstag JL. 1985. Relapsing viralhepatitis type A. J Med Virol 16: 163–169.

Jego G, Palucka AK, Blanck JP, Chalouni C, Pascual V, Ban-chereau J. 2003. Plasmacytoid dendritic cells induce plas-ma cell differentiation through type I interferon and in-terleukin 6. Immunity 19: 225–234.

Jia XY, Summers DF, Ehrenfeld E. 1992. Host antibody re-sponse to viral structural and nonstructural proteins afterhepatitis A virus infection. J Infect Dis 165: 273–280.

Joshi YK, Babu S, Sarin S, Tandon BN, Gandhi BM, Cha-turvedi VC. 1985. Immunoprophylaxis of epidemic non-A non-B hepatitis. Indian J Med Res 81: 18–19.

Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Gui-tard J, Cointault O, Esposito L, Abravanel F, Danjoux M,et al. 2008. Hepatitis E virus and chronic hepatitis inorgan-transplant recipients. N Engl J Med 358: 811–817.

Kamar N, Garrouste C, Haagsma EB, Garrigue V, Pischke S,Chauvet C, Dumortier J, Cannesson A, Cassuto-ViguierE, Thervet E, et al. 2011. Factors associated with chronichepatitis in patients with hepatitis E virus infection whohave received solid organ transplants. Gastroenterology140: 1481–1489.

Kamar N, Izopet J, Dalton HR. 2013. Chronic hepatitis Evirus infection and treatment. J Clin Exp Hepatol 3: 134–140.

Khuroo MS, Dar MY. 1992. Hepatitis E: Evidence for per-son-to-person transmission and inability of low dose im-mune serum globulin from an Indian source to prevent it.Indian J Gastroenterol 11: 113–116.

Khuroo MS, Kamili S, Dar MY, Moecklii R, Jameel S. 1993.Hepatitis E and long-term antibody status. Lancet 341:1355.

Kim HY, Eyheramonho MB, Pichavant M, Gonzalez Cam-baceres C, Matangkasombut P, Cervio G, Kuperman S,Moreiro R, Konduru K, Manangeeswaran M, et al. 2011.A polymorphism inTIM1 is associatedwith susceptibilityto severe hepatitis A virus infection in humans. J ClinInvest 121: 1111–1118.

Kim J, Chang DY, Lee HW, Lee H, Kim JH, Sung PS, KimKH, Hong SH, Kang W, Lee J, et al. 2018. Innate-likecytotoxic function of bystander-activated CD8+ T cellsis associated with liver injury in acute hepatitis A. Immu-nity 48: 161–173.

Kojima H, Shibayama T, Sato A, Suzuki S, Ichida F, HamadaC. 1981. Propagation of human hepatitis A virus in con-ventional cell lines. J Med Virol 7: 273–286.

Krain LJ, Nelson KE, Labrique AB. 2014. Host immune sta-tus and response to hepatitis E virus infection.ClinMicro-biol Rev 27: 139–165.

Kurane I, Binn LN, Bancroft WH, Ennis FA. 1985. Humanlymphocyte responses to hepatitis A virus-infected cells:Interferon production and lysis of infected cells. J Immu-nol 135: 2140–2144.

Lanford RE, Feng Z, Chavez D, Guerra B, Brasky KM, ZhouY, YamaneD, PerelsonAS,Walker CM, Lemon SM. 2011.Acute hepatitis A virus infection is associated with a lim-ited type I interferon response and persistence of intra-hepatic viral RNA. Proc Natl Acad Sci 108: 11223–11228.

Legrand-Abravanel F, Kamar N, Sandres-Saune K, Gar-rouste C, Dubois M, Mansuy JM, Muscari F, Sallusto F,Rostaing L, Izopet J. 2010. Characteristics of autochtho-nous hepatitis E virus infection in solid-organ transplantrecipients in France. J Infect Dis 202: 835–844.

Lemon SM, Binn LN. 1983. Serum neutralizing antibodyresponse to hepatitis Avirus. J Infect Dis 148: 1033–1039.

Lemon SM, Binn LN, Marchwicki R, Murphy PC, Ping LH,Jansen RW, Asher LV, Stapleton JT, Taylor DG, LeDucJW. 1990. In vivo replication and reversion to wild type ofa neutralization-resistant antigenic variant of hepatitis Avirus. J Infect Dis 161: 7–13.

Lemon SM, Murphy PC, Provost PJ, Chalikonda I, DavideJP, Schofield TL, Nalin DR, Lewis JA. 1997. Immunopre-cipitation and virus neutralization assays demonstratequalitative differences between protective antibody re-sponses to inactivated hepatitis A vaccine and passiveimmunization with immune globulin. J Infect Dis 176:9–19.

Lemon SM, Ott JJ, Van Damme P, Shouval D. 2017. Type Aviral hepatitis: A summary and update on the molecularvirology, epidemiology, pathogenesis and prevention.J Hepatol.

Locarnini SA, Coulepis AG, Kaldor J, Gust ID. 1980. Copro-antibodies in hepatitis A: Detection by enzyme-linkedimmunosorbent assay and immune electron microscopy.J Clin Microbiol 11: 710–716.

Maier K, Gabriel P, Koscielniak E, Stierhof YD, WiedmannKH, Flehmig B, Vallbracht A. 1988. Human γ interferonproduction by cytotoxic T lymphocytes sensitized duringhepatitis A virus infection. J Virol 62: 3756–3763.

Manangeeswaran M, Jacques J, Tami C, Konduru K, Am-harref N, Perrella O, Casasnovas JM, Umetsu DT, Dek-ruyff RH, Freeman GJ, et al. 2012. Binding of hepatitis Avirus to its cellular receptor 1 inhibits T-regulatorycell functions in humans. Gastroenterology 142: 1516–1525.e3.

Moal V, Motte A, KabaM, Gerolami R, Berland Y, Colson P.2013. Hepatitis E virus serological testing in kidney trans-plant recipients with elevated liver enzymes in 2007–2011in southeastern France. Diagn Microbiol Infect Dis 76:116–118.

Moller-Tank S, Maury W. 2014. Phosphatidylserine recep-tors: Enhancers of enveloped virus entry and infection.Virology 468–470: 565–580.

Montpellier C, Wychowski C, Sayed IM, Meunier JC, SaliouJM, Ankavay M, Bull A, Pillez A, Abravanel F, Helle F,et al. 2018. Hepatitis E virus lifecycle and identification of3 forms of the ORF2 capsid protein. Gastroenterology154: 211–223.e8.

Nagashima S, Takahashi M, Kobayashi T, Tanggis, Nishi-zawa T, Nishiyama T, Primadharsini PP, Okamoto H.2017. Characterization of the quasi-enveloped hepatitisE virus particles released by the cellular exosomal path-way. J Virol 91: e00822.

Okamoto H. 2013. Culture systems for hepatitis E virus. JGastroenterol 48: 147–158.

Pas SD, de Man RA, Mulders C, Balk AH, van Hal PT,Weimar W, Koopmans MP, Osterhaus AD, van der EijkAA. 2012. Hepatitis E virus infection among solid organtransplant recipients, the Netherlands. Emerg Infect Dis18: 869–872.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Perez-Sautu U, Costafreda MI, Cayla J, Tortajada C, Lite J,BoschA, Pinto RM. 2011. Hepatitis a virus vaccine escapevariants and potential new serotype emergence. EmergInfect Dis 17: 734–737.

Perrella A, Vitiello L, Atripaldi L, Sbreglia C, Grattacaso S,Bellopede P, Patarino T, Morelli G, Altamura S, RacioppiL, et al. 2008. Impaired function of CD4+/CD25+ T reg-ulatory lymphocytes characterizes the self-limited hepa-titis A virus infection. J Gastroenterol Hepatol 23: e105–e110.

Ping LH, Lemon SM. 1992. Antigenic structure of humanhepatitis A virus defined by analysis of escape mutantsselected against murine monoclonal antibodies. J Virol66: 2208–2216.

Ping LH, Jansen RW, Stapleton JT, Cohen JI, Lemon SM.1988. Identification of an immunodominant antigenicsite involving the capsid protein VP3 of hepatitis A virus.Proc Natl Acad Sci 85: 8281–8285.

Purcell RH, D’Hondt E, Bradbury R, Emerson SU, Govin-darajan S, Binn L. 1992. Inactivated hepatitis A vaccine:Active and passive immunoprophylaxis in chimpanzees.Vaccine 10: S148–151.

Purcell RH, Nguyen H, Shapiro M, Engle RE, GovindarajanS, Blackwelder WC, Wong DC, Prieels JP, Emerson SU.2003. Pre-clinical immunogenicity and efficacy trial of arecombinant hepatitis E vaccine. Vaccine 21: 2607–2615.

Reyes GR, Purdy MA, Kim JP, Luk KC, Young LM, Fry KE,Bradley DW. 1990. Isolation of a cDNA from the virusresponsible for enterically transmitted non-A, non-Bhepatitis. Science 247: 1335–1339.

Robertson BH, D’Hondt EH, Spelbring J, Tian H, Kraw-czynski K, Margolis HS. 1994. Effect of postexposure vac-cination in a chimpanzee model of hepatitis A virus in-fection. J Med Virol 43: 249–251.

Sarkar S, Rivera EM, Engle RE, Nguyen HT, Schechterly CA,Alter HJ, Liang TJ, Purcell RH, Hoofnagle JH, GhanyMG. 2015. An epidemiologic investigation of a case ofacute hepatitis E. J Clin Microbiol 53: 3547–3552.

Schiff ER. 1992. Atypical clinical manifestations of hepatitisA. Vaccine 10: S18–S20.

Schulte I, Hitziger T, Giugliano S, Timm J, Gold H, Heine-mann FM, Khudyakov Y, Strasser M, Konig C, Caster-mans E, et al. 2011. Characterization of CD8+ T-cell re-sponse in acute and resolved hepatitis A virus infection.J Hepatol 54: 201–208.

Seriwatana J, Shrestha MP, Scott RM, Tsarev SA, VaughnDW, Myint KS, Innis BL. 2002. Clinical and epidemio-logical relevance of quantitating hepatitis E virus-specificimmunoglobulin M. Clin Diagn Lab Immunol 9: 1072–1078.

Servant-Delmas A, Abravanel F, Lefrere JJ, Lionnet F, Ha-mon C, Izopet J, Laperche S. 2016. New insights into thenatural history of hepatitis E virus infection through alongitudinal study of multitransfused immunocompetentpatients in France. J Viral Hepat 23: 569–575.

Shata MT, Barrett A, Shire NJ, Abdelwahab SF, Sobhy M,Daef E, El-Kamary SS, HashemM, Engle RE, Purcell RH,et al. 2007. Characterization of hepatitis E-specific cell-mediated immune response using IFN-γ ELISPOT assay.J Immunol Methods 328: 152–161.

� Shin E-C, Jeong S-H. 2018. Natural history, clinical mani-festations, and pathogenesis of hepatitis A. Cold SpringHarb Perspect Med doi: 10.1101/cshperspect.a031708.

Shukla P, Nguyen HT, Torian U, Engle RE, Faulk K, DaltonHR, Bendall RP, Keane FE, Purcell RH, Emerson SU.2011. Cross-species infections of cultured cells by hepa-titis E virus and discovery of an infectious virus-host re-combinant. Proc Natl Acad Sci 108: 2438–2443.

Sikuler E, Keynan A, Hanuka N, Friedman MG, Sarov I.1983. Detection and persistence of specific IgA antibodiesin serum of patients with hepatitis A by capture radioim-munoassay. J Med Virol 11: 287–294.

Sjogren MH, Tanno H, Fay O, Sileoni S, Cohen BD, BurkeDS, Feighny RJ. 1987. Hepatitis A virus in stool duringclinical relapse. Ann Intern Med 106: 221–226.

Sonder GJ, van Steenbergen JE, Bovee LP, Peerbooms PG,Coutinho RA, van den Hoek A. 2004. Hepatitis A virusimmunity and seroconversion among contacts of acutehepatitis A patients in Amsterdam, 1996–2000: An eval-uation of current prevention policy. Am J Public Health94: 1620–1626.

Srivastava R, Aggarwal R, Jameel S, Puri P, Gupta VK, Ra-mesh VS, Bhatia S, Naik S. 2007. Cellular immune re-sponses in acute hepatitis E virus infection to the viralopen reading frame 2 protein. Viral Immunol 20: 56–65.

Stapleton JT. 1995. Host immune response to hepatitis Avirus. J Infect Dis 171: S9–S14.

Stapleton JT, Lemon SM. 1987. Neutralization escape mu-tants define a dominant immunogenic neutralization siteon hepatitis A virus. J Virol 61: 491–498.

Stapleton JT, Lange DK, LeDuc JW, Binn LN, Jansen RW,Lemon SM. 1991. The role of secretory immunity in hep-atitis A virus infection. J Infect Dis 163: 7–11.

Stewart DR, Morris TS, Purcell RH, Emerson SU. 1997. De-tection of antibodies to the nonstructural 3C proteinase ofhepatitis A virus. J Infect Dis 176: 593–601.

Stokes J, Neefe JR. 1945. The prevention and attenuation ofinfectious hepatitis by γ globulin. J Am Med Assoc 127:144–145.

Stokes J Jr, Farquhar JA, Drake ME, Capps RB, Ward CS Jr,Kitts AW. 1951. Infectious hepatitis; length of protectionby immune serum globulin (gamma globulin) duringepidemics. J Am Med Assoc 147: 714–719.

Suneetha PV, Pischke S, Schlaphoff V, Grabowski J, Fytili P,Gronert A, Bremer B, Markova A, Jaroszewicz J, Bara C,et al. 2012. Hepatitis E virus (HEV)-specific T-cell re-sponses are associated with control of HEV infection.Hepatology 55: 695–708.

Takahashi M, Tanaka T, Takahashi H, Hoshino Y, Naga-shima S, Jirintai, Mizuo H, Yazaki Y, Takagi T, AzumaM, et al. 2010. Hepatitis E virus (HEV) strains in serumsamples can replicate efficiently in cultured cells despitethe coexistence of HEV antibodies: Characterization ofHEV virions in blood circulation. J Clin Microbiol 48:1112–1125.

Tanaka T, Takahashi M, Kusano E, Okamoto H. 2007. De-velopment and evaluation of an efficient cell-culture sys-tem for Hepatitis E virus. J Gen Virol 88: 903–911.

Tanaka T, Takahashi M, Takahashi H, Ichiyama K, HoshinoY, Nagashima S, Mizuo H, Okamoto H. 2009. Develop-ment and characterization of a genotype 4 hepatitis E

C.M. Walker


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



virus cell culture system using a HE-JF5/15F strain recov-ered from a fulminant hepatitis patient. J Clin Microbiol47: 1906–1910.

Todt D, Francois C, Anggakusuma, Behrendt P, EngelmannM,Knegendorf L, VieyresG,WedemeyerH,HartmannR,Pietschmann T, et al. 2016. Antiviral activities of differentinterferon types and subtypes against hepatitis E virusreplication. Antimicrob Agents Chemother 60: 2132–2139.

Tripathy AS, Das R, Rathod SB, Arankalle VA. 2012. Cyto-kine profiles, CTL response and T cell frequencies in theperipheral blood of acute patients and individuals recov-ered from hepatitis E infection. PLoS ONE 7: e31822.

Tsarev SA, Tsareva TS, Emerson SU, Govindarajan S, Sha-piroM,Gerin JL, Purcell RH. 1994. Successful passive andactive immunization of cynomolgus monkeys againsthepatitis E. Proc Natl Acad Sci 91: 10198–10202.

Tsarev SA, Tsareva TS, Emerson SU, Govindarajan S, Sha-piro M, Gerin JL, Purcell RH. 1997. Recombinant vaccineagainst hepatitis E: Dose response and protection againstheterologous challenge. Vaccine 15: 1834–1838.

Vallbracht A, Fleischer B. 1992. Immune pathogenesis ofhepatitis A. Arch Virol Suppl 4: 3–4.

Vallbracht A, Gabriel P, Maier K, Hartmann F, SteinhardtHJ, Muller C, Wolf A, Manncke KH, Flehmig B. 1986.Cell-mediated cytotoxicity in hepatitis A virus infection.Hepatology 6: 1308–1314.

Vallbracht A, Maier K, Stierhof YD, Wiedmann KH, Fleh-mig B, Fleischer B. 1989. Liver-derived cytotoxic T cells inhepatitis A virus infection. J Infect Dis 160: 209–217.

Victor JC, Monto AS, Surdina TY, Suleimenova SZ,Vaughan G, Nainan OV, Favorov MO, Margolis HS,Bell BP. 2007. Hepatitis A vaccine versus immune globu-lin for postexposure prophylaxis.NEngl JMed 357: 1685–1694.

Vollmer T, Diekmann J, Eberhardt M, Knabbe C, Dreier J.2016. Hepatitis E in blood donors: Investigation of thenatural course of asymptomatic infection, Germany,2011. Euro Surveill 21: 30332.

Wang CH, Tschen SY, Heinricy U, Weber M, Flehmig B.1996. Immune response to hepatitis A virus capsid pro-teins after infection. J Clin Microbiol 34: 707–713.

Wang X, Ren J, Gao Q, Hu Z, Sun Y, Li X, Rowlands DJ, YinW,Wang J, Stuart DI, et al. 2015.Hepatitis Avirus and theorigins of picornaviruses. Nature 517: 85–88.

WangW, Xu L, Brandsma JH, Wang Y, HakimMS, Zhou X,Yin Y, Fuhler GM, van der Laan LJ, van der Woude CJ,

et al. 2016. Convergent transcription of interferon-stim-ulated genes by TNF-α and IFN-α augments antiviralactivity against HCV and HEV. Sci Rep 6: 25482.

WangX, ZhuL, DangM,HuZ,GaoQ, Yuan S, SunY, ZhangB, Ren J, Kotecha A, et al. 2017. Potent neutralization ofhepatitis Avirus reveals a receptormimicmechanism andthe receptor recognition site. ProcNatl Acad Sci 114: 770–775.

Whelan J, Sonder GJ, Bovee L, Speksnijder A, van den HoekA. 2013. Evaluation of hepatitis A vaccine in post-expo-sure prophylaxis, The Netherlands, 2004–2012. PLoSONE8: e78914.

WuT, Zhang J, Su ZJ, Liu JJ,WuXL,WuXL, Lin CX,Ou SH,Yan Q, Shih JW, et al. 2008. Specific cellular immuneresponse in hepatitis E patients. Intervirology 51: 322–327.

Yin X, Ambardekar C, Lu Y, Feng Z. 2016. Distinct entrymechanisms for nonenveloped and quasi-enveloped hep-atitis E viruses. J Virol 90: 4232–4242.

Yin XD, Y, Lhomme S, Tang Z, Walker C, Xia N, Zheng Z,Feng Z. 2018. Origin, antigenicity, and function of asecreted form of ORF2 in hepatitis E virus infection.Proc Natl Acad Sci doi: 10.1073/pnas.1721345115.

Yu C, Engle RE, Bryan JP, Emerson SU, Purcell RH. 2003.Detection of immunoglobulinM antibodies to hepatitis Evirus by class capture enzyme immunoassay. Clin DiagnLab Immunol 10: 579–586.

Zahn J, Vallbracht A, Flehmig B. 1984. Hepatitis A-virus incell culture. V. Neutralizing antibodies against hepatitisA-virus. Med Microbiol Immunol 173: 9–17.

Zhang JZ, Im SW, Lau SH, ChauTN, Lai ST, Ng SP, PeirisM,Tse C, Ng TK, Ng MH. 2002. Occurrence of hepatitis Evirus IgM, low avidity IgG serum antibodies, and viremiain sporadic cases of non-A, -B, and -C acute hepatitis.J Med Virol 66: 40–48.

Zhang J, Li SW, Wu T, Zhao Q, Ng MH, Xia NS. 2012.Hepatitis E virus: Neutralizing sites, diagnosis, and pro-tective immunity. Rev Med Virol 22: 339–349.

Zhang J, Shih JW, Xia NS. 2015. Long-term efficacy of ahepatitis E vaccine. N Engl J Med 372: 2265–2266.

Zhou Y, Callendret B, XuD, Brasky KM, Feng Z, Hensley LL,Guedj J, Perelson AS, Lemon SM, Lanford RE, et al. 2012.Dominance of the CD4+ T helper cell response duringacute resolving hepatitis A virus infection. J Exp Med209: 1481–1492.



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published online May 29, 2018Cold Spring Harb Perspect Med Christopher M. Walker InfectionsAdaptive Immune Responses in Hepatitis A Virus and Hepatitis E Virus

Subject Collection Enteric Hepatitis Viruses

Replication StrategyHepatitis A Virus Genome Organization and

Kevin L. McKnight and Stanley M. Lemon

Evolutionary Origins of Enteric Hepatitis Viruses

N. Lukashev, et al.Anna-Lena Sander, Victor Max Corman, Alexander

and Hepatitis E Virus InfectionsAdaptive Immune Responses in Hepatitis A Virus

Christopher M. Walkerand the Discovery of Hepatitis E VirusEnterically Transmitted Non-A, Non-B Hepatitis

Stanley M. Lemon and Christopher M. WalkerSmall Animal Models of Hepatitis E Virus Infection

Tian-Cheng Li and Takaji Wakita2 Infections

andPathogenesis of Hepatitis E Virus Genotype 1 Natural History, Clinical Manifestations, and

Rakesh Aggarwal and Amit Goel

and Treatmentand 4 Infection: Clinical Features, Pathogenesis, Acute and Persistent Hepatitis E Virus Genotype 3

Nassim Kamar and Sven PischkeHepatitis Virusesand Re-Emerging Enterically Transmitted Hepatitis A Virus and Hepatitis E Virus: Emerging

Stanley M. Lemon and Christopher M. Walker

Virus InfectionsEpidemiology of Genotype 1 and 2 Hepatitis E

KmushKenrad E. Nelson, Alain B. Labrique and Brittany L.

Hepatitis A Virus Capsid StructureDavid I. Stuart, Jingshan Ren, Xiangxi Wang, et al.

History of the Discovery of Hepatitis A VirusStephen M. Feinstone Hepatitis E Virus Infection

Comparative Pathology of Hepatitis A Virus and

John M. Cullen and Stanley M. Lemon

United StatesVirus and Hepatitis E Virus Infections in the Epidemiology and Transmission of Hepatitis A

Eyasu H. TeshaleMegan G. Hofmeister, Monique A. Foster and

Innate Immunity to Enteric Hepatitis VirusesZongdi Feng and Stanley M. Lemon

Virus InfectionDerived Culture Models of Hepatitis E−Stem Cell

RiceViet Loan Dao Thi, Xianfang Wu and Charles M.

and Hepatitis E Virus InfectionsNonhuman Primate Models of Hepatitis A Virus

Stanley M. LemonRobert E. Lanford, Christopher M. Walker and

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved


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