Anti‐HBc‐nonreactive occult hepatitis B infections with HBV genotypes B and C in vaccinated immunocompetent adults

Abstract Absence of anti‐HBc reactivity with detectable anti‐HBs was observed in blood donors with occult hepatitis B virus (HBV) infection (OBI). The prevalence and mechanisms underlying this uncommon condition were investigated over time in Chinese blood donors with OBI. Isolated anti‐HBs OBI status was identified from 466,911 donors from Dalian, China, and monitored in follow‐up (range: 2.6–84.3 months). HBV vaccination status was documented, and infecting viral strains were characterized. Of 451 confirmed OBIs (1:1035), 43 (9.5%; 1:10,858) had isolated anti‐HBs as only serological marker. Isolated anti‐HBs OBIs differed from anti‐HBc‐reactive OBIs by significantly younger age (median 24 years), higher HBV DNA (median: 20 IU/ml) and anti‐HBs (median 60.5 IU/L) levels, paucity of mutations in HBV Core and S proteins, and high vaccination rate (72%). Vaccinated isolated anti‐HBs OBIs (n = 31) differed from unvaccinated (n = 11) by significantly younger age (22 vs 38 years), higher anti‐HBs level at index (48% vs 9% with anti‐HBs >100 IU/L) and higher frequency of anti‐HBs immune response (44% vs 20%). Of 15 vaccinated and 5 unvaccinated OBIs follow‐up, 65% (8 vaccinated and 5 unvaccinated) became HBV DNA negative suggesting aborted recent infection, while 35% (7 vaccinated) had low persistent viraemia 2 to 65 months post index. In conclusion, isolated anti‐HBs OBI in Chinese blood donors appears associated with young, vaccinated, adults exposed to HBV who predominantly develop low level aborted infection revealed by transient HBV DNA and immune anti‐HBs response. However, a subset of individuals still experienced low but persistent viral replication whose clinical outcome remains uncertain.

intermittently detectable. Unusual combinations of anti-HBc and anti-HBs profiles were observed in OBI carriers that may lead to uncertainty in the interpretation of results or suspicion of testing errors. A multiregional study reported anti-HBc-negative OBI in 0.6% of asymptomatic blood donors, 83% of whom carried anti-HBs as the only serological marker. 2 Significantly lower frequencies were observed in OBI blood donors from Europe (2.4%), the Mediterranean region (4.3%) and South Africa (6.2%). This difference in isolated anti-HBs prevalence remains unclear but may be related to regional differences in HBV endemicity, prophylaxis programs, natural history of HBV infection and circulating HBV genotypes.
Rare cases of anti-HBc-negative overt chronic HBV infection have been documented, mainly in immunocompromised patients experiencing HBV reactivation or subjects co-infected with HIV, patients infected with HBV variants carrying mutations that might impair HBcAg expression or detection, and infants born to HBsAgpositive mothers. [3][4][5][6] In China and South East Asia, isolated anti-HBs were reported in 6.3%-20.3% of immunocompetent blood donors with OBI. 2,[7][8][9] Isolated anti-HBs OBI has been also reported in immunized neonates born to HBsAg-positive mothers and children with HBsAg-positive parents. [10][11][12][13][14] Whenavailable,follow-upofvaccinated children in these studies was relatively limited in time and often showed low and transient HBV DNA levels. 11,13 In contrast, a Taiwanese cross-sectional study reported HBV viraemia in fully immunized individuals aged >18 years who were negative for anti-HBc. 10 Further, vaccinated blood donors sexually exposed to partners with chronic HBV developed anti-HBc-negative OBI reflecting an aborted form of infection. 15 The host and/or viral mechanisms leading to isolated anti-HBs OBI and the evolution of this condition and its potential clinical outcomeremainlargelyunknown.Weinvestigatedimmunocompetent

| Hepatitis B virus serological and molecular testing
Blooddonorswerepre-qualifiedwithanHBsAgrapidtest(95%limit of detection [LoD]: 5 IU/ml). HBV, HCV and HIV infection status in blood donations collected from HBsAg nonreactive donors was determined according to the serological and molecular screening and confirmatory testing algorithms implemented in the Dalian Blood Center. 16 HBVDNAquantificationwasperformedwithanin-house real-time qPCR assay (95% LoD: 20 IU/ml). 17 Refer to Appendix S1 for further details.
The study was conducted according to the ethical guidelines of the1975DeclarationofHelsinkiandwasapprovedbytheAcademic and Ethics Committee of Dalian Blood Center (EC-20190731-1226).
All participants provided signed informed consent at donation time and during follow-up.

| Genetic analysis of anti-HBs only OBI sequences
The BCP/PC sequences of four isolated anti-HBs OBI genotype B ( Figure S2).
Pre-S1,Pre-S2and/orSaasequencesof3isolatedanti-HBsOBI B and 22 isolated anti-HBs OBI C strains were compared to their respective anti-HBc+ OBIs and HBsAg+ counterparts ( p < .0001) ( Figure 3). As shown in  The present study focused on the selected group of 43 OBI blood donorswithanti-HBsasisolatedserologicalmarkerofinfection.
Absence or delayed anti-HBc reactivity in patients with overt HBV infection has been associated with immunosuppression, HIV co-infection, insufficient sensitivity of anti-HBc assays and rare HBcAg variants. [3][4][5][6] None of these conditions applied to the present OBI cases. All isolated anti-HBs OBI donors were apparently immunocompetent with no evidence of liver disease, medical treatment orknownviralco-infection.Anti-HBcfalsenegativeresultwasexcluded by using three distinct assays, one of which included antigenantibody complex dissociation pre-treatment. No specific mutations in BCP/PC, Core and HBx that could alter HBcAg production or anti-HBc detection were identified, consistent with studies of overt anti-HBc-negative HBV infection. 6,21 The minority OBI group with isolated anti-HBs differed from the other two OBI groups by a significantly larger percentage of females, younger age, higher (although still relatively low) levels of viralDNAandanti-HBs,andhigherfrequencyofHBVvaccination (Table 1). The 72% vaccination rate observed in isolated anti-HBs  (Tables S1 and Table 2). In addition to age, the 31 vaccinated isolated anti-HBs OBIs differed in several other aspects: mostly males (68%), higher level of anti-HBs at index, and more frequent, albeit not significantly, HBV genotype C (Table 2). However, both groupshadincommonanabsenceofDNAoranti-HBcinlookback samples, persistence of isolated anti-HBs status in follow-up, and absence or paucity of amino acid substitutions in the Core and S MHR, respectively (Table S1, Figures S2 and S4, and Table 3 Isolated anti-HBs OBI has been reported in vaccinated infants aged 6-98 months born to chronically infected mothers. [10][11][12]14,22 HBV DNA levels were consistently low, but often only transiently per-sistentbeyond1-4 yearsofagewhenfollow-upwasavailable. 11 L109R, L110R, S117Δ, T118Δ a , G119Δ, P120Δ a , G130N a , S136Y, A159V  (Table S1 and Figure 2). However, low level of HBV DNA fluctuating around the LoDs of assays has been observed in both anti-HBc+ and isolated anti-HBs OBIs sometimes over several years. 8,24,25 Therefore, long-term OBI carriage with intermittently detectable viraemia over a long period of time in fully immunized subjects cannot be totally ruled out but remains improbable.
Whiletheeffectivenessofvaccinationprogramsinreducingthe prevalence of CHB is well established, their impact on the prevalence of HBV immune escape variants remains unclear. 26 Mutations associated with evasion of vaccine-induced immunity have been localized frequently, but not exclusively, in the MHR of the viral surface protein. 27 However, 70% (23 [2 OBI B + 21OBI C ]/33) of the isolatedanti-HBsOBIstrainshadwild-typeMHRsequences.Only four vaccinated and two unvaccinated strains carried 1-3 mutations previously associated with immune escape as indicated in Table 3.
Similarly, few large studies reported wild-type MHR sequences in 75%-83% of vaccinated isolated anti-HBs OBI carriers infected with HBV B and HBV C . 26,28,29 Nevertheless, multiple substitutions were also present outside the MHR of single isolated anti-HBs OBI strains including several not found in anti-HBc+ OBI and non-OBI strains (Table 3). However, it would be highly speculative to associate these substitutions with impaired anti-HBs neutralization without conducting appropriate functional investigations.  (Table S2), which may suggest a shorter infection history. Considering that HBV infection occurs mainly at an early age in Asia, and despite the low level of HBV replication in OBI irrespective of anti-HBc status, mutations seem to accumulate over time in OBI virus genome as suggested in Figure 3  The persistence of the isolated anti-HBs OBI profile observed in 11 unvaccinated donors in the present study and in 9 Southeast

Long-term OBI carriage and breakthrough infection
Asian donors in a previous study remains puzzling. 8 It is possible that some of these donors, particularly the two younger than 30, might have been vaccinated without recollection or evidence provided. Further studies are needed to determine whether this inability to produce anti-HBc might be related to a selective defect in the host immune system. 6,32 Overall, the prevalence of isolated anti-HBs OBI carriage may be underestimated in vaccinated populations, especially in areas of high HBV endemicity, and these indi-

vidualsmaybeatriskforHBVreactivationifimmunosuppressed.
In addition, HBsAg-negative/anti-HBc-negative blood donors with barelydetectableHBVDNAmayparticipatetotheresidualriskof HBVtransfusion-transmission,althoughthisriskislikelymitigated by the presence of anti-HBs. 33 In conclusion, occurrence and persistence over time in rare cases of isolated anti-HBs OBI were confirmed in Chinese blood donors. Isolated anti-HBs OBI appears mainly associated with vaccinated adult blood donors exposed to HBV one-or two-decades post-vaccination who remain largely protected from full blown HBV breakthroughinfectionsanddeveloplowlevelabortedinfectionrevealed by transient HBV DNA and an immune anti-HBs response.
However, a subset of individuals still experienced low but persistent viraemia and the potential for transmission and long-term clinical outcome of this uncommon OBI condition remain uncertain. funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available inGenBankathttps://www.ncbi.nlm.nih.gov/nucle otide/, reference number OM471070-OM471500.