The Journal of Infectious Diseases MAJOR ARTICLE

The Journal of Infectious Diseases MAJOR ARTICLE A Longitudinal Hepatitis B Vaccine Cohort Demonstrates Long-lasting Hepatitis B Virus (HBV) Cellular Immunity Despite Loss of Antibody Against HBV Surface Antigen Brenna C. Simons, 1,2,3 Philip R. Spradling, 4 Dana J. T. Bruden, 2 Carolyn Zanis, 2 Samantha Case, 2 Tammy L. Choromanski, 1 Minjun Apodaca, 5 Hazel D. Brogdon, 3 Gaelen Dwyer, 3 Mary Snowball, 1 Susan Negus, 1 Michael G. Bruce, 2 Chihiro Morishima, 5 Cindy Knall, 3 and Brian J. McMahon 1,2 1 Liver Disease and Hepatitis Program, Alaska Native Tribal Health Consortium, 2 Arctic Investigations Program, Division of Preparedness and Emerging Infections, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), and 3 WWAMI School of Medical Education, College of Health, University of Alaska Anchorage; 4 Division of Viral Hepatitis, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, CDC, Atlanta, Georgia; and 5 Department of Laboratory Medicine, University of Washington, Seattle Background. Long-lasting protection resulting from hepatitis B vaccine, despite loss of antibody against hepatitis B virus (HBV) surface antigen (anti-hbs), is undetermined. Methods. We recruited persons from a cohort vaccinated with plasma-derived hepatitis B vaccine in 1981 who have been followed periodically since. We performed serological testing for anti-hbs and microrna-155 and assessed HBV-specific T-cell responses by enzyme-linked immunospot and cytometric bead array. Study subgroups were defined 32 years after vaccination as having an anti-hbs level of either 10 miu/ml (group 1; n = 13) or <10 miu/ml (group 2; n = 31). Results. All 44 participants, regardless of anti-hbs level, tested positive for tumor necrosis factor α, interleukin 10, or interleukin 6 production by HBV surface antigen specific T cells. The frequency of natural killer T cells correlated with the level of anti-hbs (P =.008). The proportion of participants who demonstrated T-cell responses to HBV core antigen varied among the cytokines measured, suggesting some natural exposure to HBV in the study group. No participant had evidence of breakthrough HBV infection. Conclusions. Evidence of long-lasting cellular immunity, regardless of anti-hbs level, suggests that protection afforded by primary immunization with plasma-derived hepatitis B vaccine during childhood and adulthood lasts at least 32 years. Keywords. hepatitis B virus; vaccine; cellular immunity; booster vaccination; antibody against hepatitis B surface antigen; plasma-derived vaccine. Hepatitis B vaccination has had a significant impact on the reduction of morbidity and mortality due to hepatitis B virus (HBV) infection and the incidence of hepatocellular carcinoma, particularly in regions of previous endemicity, such as Alaska [1 7]. Although studies show a high response rate to primary vaccination in infants, children, and adults [5, 8, 9], indicated by an antibody to HBV surface antigen (anti-hbs) level of 10mIU/mL, the duration of protection by the vaccine is not completely understood. Longitudinal studies in populationbased cohorts indicate waning anti-hbs levels found in the blood over time, with a more rapid decline among persons vaccinated at birth compared to those vaccinated as children or adults [1, 6, 8 21]. Since implementation of universal hepatitis B vaccination in the United States, a significant proportion of persons vaccinated at birth lose anti-hbs by adulthood [1, 10, 11, 13, 14, 16]. Received 6 November 2015; accepted 1 April 2016; published online 7 April 2016. Presented in part: Immunology 2015, New Orleans, Louisiana, 8 12 May 2015. Correspondence: B. C. Simons, 3900 Ambassador Dr, Anchorage, AK 99508 (bcsimons@ anthc.org). The Journal of Infectious Diseases 2016;214:273 80 The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com. DOI: 10.1093/infdis/jiw142 Although the vast majority of individuals respond to booster vaccination (defined as an additional vaccine dose given after completion of the recommended 3-dose series) [1, 10, 11, 13, 14, 16], determination of persistent immunity has depended on measurement of anti-hbs before and after booster dose administration. The duration of protection remains unknown because the majority of booster vaccine responses quickly decrease to <10 miu/ml within a few weeks after booster receipt [1, 11, 18, 20 22]. This has direct implications for new healthcare professionals, as most would likely have undetectable anti-hbs in absence of booster vaccine [11, 12]. Depending on anti-hbs exclusively as a surrogate for long-lasting protection against HBV is potentially incomplete. There is need for more direct confirmation of long-lasting protection against HBV. Before introduction of hepatitis B vaccine, high rates of HBV infection in Alaska disproportionately affected Alaska Native people [3, 9]. The Alaska Vaccine Demonstration Cohort was initiated in 1981 and consisted of Alaska Native adults and children aged 6 months who were immunized with a 3-dose series of plasmaderived hepatitis B vaccine [3, 9, 20, 22 26]. Recently, Bruce et al demonstrated that 51% of these persons who were never given additional hepatitis B vaccine after initial series maintained anti-hbs levels of 10 miu/ml 30 years after primary vaccination, yet no participant was found to have evidence of HBV infection [19]. Lasting Protection Without Antibody JID 2016:214 (15 July) 273

Recent studies have identified markers of cellular immunity that correlate with anti-hbs vaccine response [27 34]. However, further definition and confirmation of these potential markers are needed in population-based cohorts that have extensive vaccination history and a significant proportion of persons who maintain anti-hbs over time. To understand the relationship between cellular immunity, amnestic response, and long-lasting protection against HBV, we studied 44 persons from the Alaska Vaccine Demonstration Cohort [9] 32 years after vaccination, to determine anti-hbs levels, HBV-specific T-cell responses, and serum-derived microrna-155 (mir-155) levels. METHODS Study Participants We recruited Anchorage-area residents who participated in the 30-year Alaska Vaccine Demonstration study in 2011[19] and had no history of immunosuppressive therapies or diabetes for at least 2 years prior to study entry. Participants were enrolled at the Alaska Native Medical Center. This study was approved by the Alaska Area (Indian Health Service), the Centers for Disease Control and Prevention (CDC), and the University of Alaska Anchorage institutional review boards. The study was approved by the Alaska Native Tribal Health Consortium and the Southcentral Foundation Board of Directors. All participants provided written informed consent. We performed this study 32 years after participants had completed a plasma-derived vaccine primary series [9, 19, 20, 22, 23, 25, 26]. We defined groups on the basis of anti-hbs level at 32 years after vaccination, with group 1 comprising 13 individuals with a level of <10 miu/ml, and group 2 comprising 31 individuals with a level of 10 miu/ml. Group 1 included those who either received a booster at the 22-year follow-up or the 30- year follow-up [19] but also 2 persons who never received a booster, owing to previous loss to follow-up. Group 2 included 3 participants who were boosted at the 30 year follow-up [19] and continued to maintain an anti-hbs level of 10 miu/ml at the time of this study. The remaining 28 participants never received a booster. At the beginning of the cohort, in 1981, some participants exhibited an anti-hbs level of <10 miu/ml following the initial 3-dose series and a subsequent second 3-dose series and were categorized as vaccine nonresponders. Two nonresponders were recruited but not included in final analysis. Anti-Hepatitis B Surface Antigen Antibody Measure Serologic specimens from participants of this follow-up study were tested for anti-hbs (ETI-AB-AUK PLUS and ABAU- STD-SET; DiaSorin) at the CDC Arctic Investigations Program [23, 25, 26, 35]. The lower limit of detection of this assay was defined as an anti-hbs value of 5 miu/ml. Determining Breakthrough Infections Serologic specimens from participants in the 30-year vaccine demonstration cohort [19] were tested for antibody to HBV core antigen (anti-hbc), as described previously [19, 26]. None of the 32-year study participants previously tested positive for anti-hbc [19, 20, 26]. Medical chart review was performed for 32-year study participants for any evidence of detectable HBV surface antigen (HBsAg) or symptomatic hepatitis that would indicate a breakthrough HBV infection. Peripheral Blood Mononuclear Cell (PBMC) Staining Isolated PBMCs were resuspended in staining buffer (1X Dulbecco s phosphate-buffered saline/2% fetal calf serum [FCS]) at 10 7 PBMCs/mL. Background and isotype controls were included. Antibodies were purchased from Biolegend (BL; San Diego, California) or BD Biosciences (BD; San Jose, California), unless otherwise noted (Supplementary Figure 1). For B cells, αcd20- FITC (BD), αcd38-pe (BD), αcd19-percp (ebioscience), and αcd27-apc (BD) were used. For natural killer (NK) cells and NK T cells, αcd4-fitc (ebioscience), αcd8-pe (BD), αcd3- PerCP (BL), and αcd56-apc (BL) were used. For memory T cells, αcd4-fitc, αcd3-pe (BD), αcd45ro-percp (BL), and αccr7-apc (BL) were used. For regulatory T cells, αcd127- FITC (BD), αcd3-pe, αcd4-percp (BL), and αcd25-apc (BL) were used. For aging/exhaustion cells, αcd57-fitc (BL), αcd279-pe (BL), αcd3-percp, and αcd4-apc (ebioscience) were used. Stained PBMCs were acquired on a FACSCalibur (BD Biosciences). Raw data were analyzed by FCS Express v.4.07.0011 (DeNovo Software, Glendale, California). Interferon γ (IFN-γ) Enzyme-Linked Immunospot (ELISpot) Assay ELISpot assays were performed as previously described [36, 37]. Briefly, 96-well multiscreen filtration plates (EMD Millipore, Billerica, Massachusetts) were coated with 0.1 μg/ml of antihuman IFN-γ monoclonal antibody (Mabtech, Cincinnati, Ohio). Fresh or cryopreserved PBMCs were suspended in R10 medium, consisting of Roswell Park Memorial Institute 1640 medium (Mediatech, Manassas, Virginia) supplemented with FCS (10%; Gemini Bio-Products, Sacramento, California), HEPES buffer (10 mm; Mediatech), L-glutamine (2 mm; Mediatech), and penicillin-streptomycin (50 U/mL; Mediatech), at a concentration of 10 6 PBMCs/mL. Stimulants were added to PBMCsfor72hoursat37 Cin5%CO 2 and then developed [36]. Recombinant HBsAg (Virogen, Watertown, Massachusetts) and HBV core antigen (HBcAg; Virogen) were added to the appropriate wells at a final concentration of 1 μg/ml. Wells containing PBMCs and 5% dimethyl sulfoxide R10 medium with phytohemagglutinin (Sigma-Aldrich, St. Louis, Missouri) or without any peptide (unstimulated) were used as positive and negative controls, respectively, in triplicate. A positive value was defined as a value that was 3 times the value for the negative control [37]. Cytometric Bead Array Supernatants were collected from the described ELISpot plates before ELISpot development [38, 39] and stored at 70 C. 274 JID 2016:214 (15 July) Simons et al

Cytometric bead arrays (for interleukin 17a [IL-17a], tumor necrosis factor α [TNF-α], interleukin 10 [IL-10], interleukin 6 [IL-6], interleukin 4 [IL-4], and interleukin 2 [IL-2]) were then conducted per the manufacturer protocol (BD Biosciences) and acquired on a FACSCalibur (BD Biosciences). Raw data were analyzed by FCAP Array v3.0.1 software (BD Biosciences). Quantitative Polymerase Chain Reaction (qpcr) Analysis of mir-155 Serum was collected and stored immediately at 70 C. Extraction and testing were performed as previously described [40]. MicroRNAs were extracted from sera (Qiagen). qpcr for mir-155 was performed and normalized to values for a U6 control, using a 7900HT Fast Real-Time PCR System (Thermo- Fisher Scientific). Statistical Analysis Among participants in the 30-year Alaska Vaccine Demonstration cohort, we compared age, sex, and anti-hbs levels between those who were and those who were not recruited into this study. Among participants in the 32-year study, we compared characteristics and immunological outcomes between those in group 1 and those in group 2. For continuous outcomes, comparisons between groups were performed using the Wilcoxon rank sum test or the Student t test, where appropriate. For dichotomous outcomes, the Pearson χ 2 test or Fisher exact tests were used as necessitated by sample size. We additionally used Spearman rank correlation to examine the association between continuous immunological outcomes and the anti-hbs level at 32 years. Comparisons were performed with SAS, version 9.3 (SAS Institute, Cary, North Carolina). All reported P values are 2 tailed, and a P value of <.05 was considered significant. RESULTS Cohort Description The mean age of the study group (n = 44) was 43 years, compared with 45.3 years in the original 30-year follow-up cohort (n = 391) who were not included in this study (P =.10) [19]. All participants were at least aged one year at vaccination. The study group was 70% female as compared to 50% female in the original cohort (P =.01) [19], but there were no significant differences in primary immunological outcomes between males and females in this study. The anti-hbs levels 6 months after the 3-dose primary vaccine series did not differ (P =.77) between the study group and the original cohort [19], nor did anti-hbs levels at 30 years after vaccination (P =.58) [19]. Groups 1 and 2 were defined by the 32-year anti-hbs level, regardless of recent booster history. No significant differences in sex and mean age were determined between group 1 (63% female and 42.9 years, respectively) and group 2 (67% female and 44.4 years, respectively). None of the study participants were observed to ever have had breakthrough HBV infection. Increased Frequency of NK T and CD8 + TEMRA Lymphocytes Among Persons in Group 2 We assessed whether PBMC phenotype frequency corresponded with the level of anti-hbs by comparing the PBMC phenotype frequency between group 1 and group 2. There was a significant (Table 1; P =.01) increase in the frequency of NK T cells (CD3 + CD56 + ) among group 2. In addition, group 1 had a higher proportion of CD8 + T EMRA (CD3 + CD4 CD45RO CCR7 )cells (P =.03; Table 1). No other statistically significant difference in phenotype frequency was observed between the groups (Table 1). We compared the PBMC phenotype frequency with anti-hbs level at 32 years after vaccination. The proportion of NK T cells directly correlated with the 32-year anti-hbs level (P =.008; Table 1). No other statistically significant correlative relationship between anti-hbs level and phenotype frequency was observed (Table 1). HBsAg-Specific T-Cell Responses Are Detected in All Participants, Regardless of Anti-HBs Level To determine whether HBsAg-specific T-cell responses corresponded with anti-hbs, we measured HBsAg-specific IFN-γ producing T cells by ELISpot analysis. ELISpot analysis indicated that T cells in 52% of group 2 participants (16) released IFN-γ in response to HBsAg, compared with 46% of group 1 participants (6; P =.74; Table 2). The median magnitude of the IFN-γ response to HBsAg was 4.0 spot-forming cells (SFCs)/ 10 6 PBMCs (range, 0 334.7 SFCs/10 6 PBMCs)ingroup1and5.0 SFCs/10 6 PBMCs (range, 0 780 SFCs/10 6 PBMCs) in group 2 (Table 2; P =.44). The magnitude of the IFN-γ based T-cell response to HBsAg did not correlate with the anti-hbs level 32 years after vaccination (P =.72; Table 2). To expand detection of HBsAg-specific T cells, we measured HBsAg-specific T cells producing TNF-α, IL-10, IL-17, IL-4, IL- 6, or IL-2. All 44 participants tested positive for HBsAg-specific T cells producing TNF-α, IL-10,orIL-6(Table2). HBsAgspecific T cells producing IL-17, IL-4, and IL-2 producing T cells were detected in participants but at significantly lower proportions (Table 2). The median magnitude of the TNF-α response to HBsAg among T cells in group 1 was 564.3 pg/ml (range, 48.9 1877 pg/ml) as compared to 334.9 pg/ml (range, 7.85 2459 pg/ml) in group 2 (P =.78; Table 2). The median magnitude of the IL-10 response to HBsAg among T cells was 197.8 pg/ ml (range, 12 1647 pg/ml) in group 1 and 92.4 pg/ml (range, 6.5 832.2 pg/ml) in group 2 (P =.15; Table 2). The median magnitude of the IL-6 response among T cells was 6863.3 pg/ml (range, 3120 32 779 pg/ml) in group 1 and 6478 pg/ml (range, 215 25 069 pg/ml) in group 2 (P =.24; Table 2). Only 1 participant in each group had measurable IL-4 or IL-17 response among T cells after HBsAg stimulation (Table 2). The measurable IL-2 response to HBsAg stimulation among T cells was low in both groups (P =.13; Table 2). The magnitude of any cytokine T-cell response to HBsAg did not Lasting Protection Without Antibody JID 2016:214 (15 July) 275

Table 1. Comparison of Peripheral Blood Mononuclear Cell Phenotype Frequency Indicates a Significant Correlation Between Natural Killer (NK) T Cells (CD3 + CD56 + ) and Antibody to Hepatitis B Virus Surface Antigen (Anti-HBs) Level Percentage of Parent Population, Median (Range) P Value Parent Population Population Group 1 (n = 13) Group 2 (n = 31) a Group 2 b Group 1 vs 32-y Anti-HBs Level c NK T cells, NK cells Lymphocytes NK T cells (CD3 + CD56 + ) 1.0 (0.0 3.1) 2.0 (0.3 13.0) a.01.008 Lymphocytes NK cells (CD3 CD56 + ) 4.9 (0.3 27.9) 11.9 (2.2 32.4).22.11 B cells Lymphocytes Total B cells (CD20 + CD19 + ) 7.7 (0.0 23.7) 8.1 (0.3 13.3).68.40 T cells CD3 + lymphocytes CD4 + T cells (CD3 + CD4 + ) 58.8 (29.9 72.8) 50.2 (21.1 72.3).23.08 CD3 + lymphocytes CD8 + T cells (CD3 + CD8 + ) 36.7 (24.7 63.8) 41.7 (8.3 71.9).58.06 Memory T cells CD3 + CD4 + T cells CD4 + TEMRO (CD3 + CD4 + CD45RO + CCR7 ) 14.4 (3.2 49.0) 13.8 (2.1 75.5).97.24 CD3 + CD4 + T cells CD4 + TEMRA (CD3 + CD4 + CD45RO CCR7 ) 1.0 (0.0 38.2) 1.4 (0.0 16.4).93.96 CD3 + CD4 + T cells CD4 + central memory T cells (CD3 + CD4 + CD45RO + CCR7 + ) 31.2 (1.3 60.9) 27.8 (6.5 55.9).43.39 CD3 + CD4 T cells CD8 + TEMRO (CD3 + CD4 CD45RO + CCR7 ) 31.3 (7.5 50.0) 32.8 (5.6 75.8).59.40 CD3 + CD4 T cells CD8 + TEMRA (CD3 + CD4 CD45RO CCR7 ) 34.3 (14.7 67.7) 18.8 (0.3 65.9).03.20 CD3 + CD4 T cells CD8 + central memory T cells (CD3 + CD4 CD45RO + CCR7 + ) 9.2 (0 32.8) 11.7 (1.0 43.2).77.88 Aging/exhaustion cells CD3 + CD4 T cells CD8 + PD-1 + T cells (CD3 + CD4 CD279 + ) 4.3 (1.1 41.4) 6.4 (0.4 32.6).80.24 CD3 + CD4 T cells CD8 + CD57 + T cells (CD3 + CD4 CD57 + ) 31.0 (3.9 55.8) 26.6 (0.5 70.2).93.79 CD3 + CD4 + T cells CD4 + PD-1 + T cells (CD3 + CD4 + CD279 + ) 5.2 (0.8 45.2) 5.3 (0.0 97.0).93.52 CD3 + CD4 + T cells CD4 + CD57 + T cells (CD3 + CD4 + CD57 + ) 5.7 (3.0 33.1) 6.0 (0.0 30.5).72.54 Regulatory T cells CD3 + CD4 + T cells Regulatory T cells (CD3 + CD4 + CD25 hi CD127 ) 1.9 (0.0 3.4) 1.6 (0.0 9.7).41.41 Group 1 comprised individuals who lost anti-hbs (level, <10 miu/ml) at the 32-year follow-up, and group 2 comprised individuals who maintained anti-hbs (level, 10 miu/ml) at the 32-year follow-up. a For NK T cell analysis, n = 29. Two results were indeterminate. b By the parametric t test or the nonparametric Wilcoxon rank sum test, as appropriate. c By Spearman rank order correlation between the anti-hbs level and immunological outcome. correlate with the anti-hbs level 32 years after vaccination (Table 2). Evidence of HBcAg-Specific T-Cell Responses To determine evidence of exposure, we evaluated HBcAg-specific T-cell responses by ELISpot assays and cytometric bead arrays. In group 1, 15% (2 subjects) had a positive IFN-γ response to HBcAg among T cells, compared with 19% in group 2 (6 subjects; P = 1.00; Table 3). The median magnitude of the IFN-γ response to HBcAg was 0 SFCs/10 6 PBMCs (range, 0 94 SFCs/10 6 PBMCs) in group 1 and 0 SFCs/10 6 PBMCs (range, 0 43 SFCs/ 10 6 PBMCs) in group 2 (P =.79; Table 3). The magnitude of the IFN-γ response to HBcAg among T cells did not correlate with the anti-hbs level 32 years after vaccination (P =.34; Table 3). We next measured TNF-α, IL-10, IL-6, IL-17, IL-4, and IL-2 responses among HBcAg-specific T cells. The proportion of participants testing positive for HBcAg-specific T cells varied by cytokine, from 84% (group 2; IL-6) to 0% (group 1; IL-4; Table 3). TNF-α, IL-10, and IL-6 responses among HBcAgspecific T cells were observed in the greatest proportion of participants (Table 3), but no individual cytokine was yielded by HBcAg-specific T cells in all participants. Fewer participants tested positive for HBcAg-specific T cells producing IL-17, IL- 4, and IL-2 (Table 3). In comparing the proportion of positive responses by cytokine between groups, no significant differences were measured (Table 3). We observed median TNF-α responses among HBcAgspecific median T cells of 154.9 pg/ml (range, 0 1364 pg/ml) and 68.9 pg/ml (range, 0 565.2 pg/ml) in groups 1 and 2, respectively (P =.26; Table 3). Median IL-10 responses among HBcAg-specific T cells were lower, with values of 40.8 pg/ml (range, 0 3446 pg/ml) in group 1 and 7.0 pg/ml (range, 0 1233 pg/ml) in group 2 (P =.22; Table 3). In group 1, the median IL-6 response among HBcAg-specific T cells was 1906 pg/ ml (range, 0 21 000 pg/ml), and in group 2, the value was 1149 pg/ml (range, 0 16 690 pg/ml; P =.89; Table 3). IL-2 responses among HBcAg-specific T cells were low, with median values of 0 pg/ml (range, 0 5.7 pg/ml) in group 1 and 0 pg/ ml (range, 0 10.1 pg/ml) in group 2. The majority of participants did not exhibit a IL-17 or IL-4 response among HBcAgspecific T cells (Table 3). Seven of 44 participants (3 in group 1 and 4 in group 2) did not exhibit any HBcAg-specific cytokine 276 JID 2016:214 (15 July) Simons et al

Table 2. Release of Tumor Necrosis Factor α (TNF-α), Interleukin 10 (IL-10), and Interleukin 6 (IL-6) by Hepatitis B Virus (HBV) Surface Antigen Specific T Cells Was Detected in All Recipients of Hepatitis B Vaccine, Regardless of Antibody to HBV Surface Antigen (Anti-HBs) Level Test, Response, Cytokine Group 1 (n = 13) Group 2 (n = 31) Group 1 vs Group 2 32-y Anti-HBs Level ELISpot Positive, a persons, no. (%) 6 (46) 16 (52).74 b IFN-γ Magnitude, SFCs/10 6 PBMCs, median (range) IFN-γ 4.0 (0 334.7) 5.0 (0 780).44 c.72 d Cytometric bead array Positive, a persons, proportion (%) TNF-α 13 (100) 31 (100) IL-10 13 (100) 31 (100) IL-6 13 (100) 31 (100) IL-17 1 (8) 1 (3).51 b IL-4 1 (8) 1 (3).51 b IL-2 6 (46) 6 (19).13 b Magnitude, pg/ml, median (range) TNF-α 564.3 (48.9 1877) 334.9 (7.85 2459).78 c.56 d P Value IL-10 197.8 (12.1 1647) 92.4 (6.5 823.2).15 c.50 d IL-6 6863.3 (3120 32 779) 6478 (215 25 069).24 c.12 d IL-17 0 (0 16.7) 0 (0 2.0) IL-4 0 (0 1.1) 0 (0 1.1) IL-2 0 (0 9.2) 0 (0 22.1).13 c.14 d Group 1 comprised individuals who lost anti-hbs (level, <10 miu/ml) at the 32-year follow-up, and group 2 comprised individuals who maintained anti-hbs (level, 10 miu/ml) at the 32-year follow-up. Abbreviations: ELISpot, enzyme-linked immunospot; IFN-γ, interferon γ; IL-2, interleukin 2; IL-4, interleukin 4; IL-17, interleukin 17; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell. a Defined as a measurable response, 3 times the background level. b By the Pearson χ 2 or Fisher exact tests, as appropriate. c By the parametric t test or nonparametric Wilcoxon rank sum test, as appropriate. d By Spearman rank order correlation between the anti-hbs level and the immunological outcome. response when measured by cytometric bead array or ELISpot assay. Quantitative Levels of mir-155 Among Persons Vaccinated for Hepatitis B There was no significant difference (P = 1.00) in quantitative measurements of mir-155 between group 1 (median, 1.22; range, 0.2 9.3) and group 2 (median, 0.94; range, 0.3 4.07). No direct relationship between quantitative measurements of mir-155 and anti-hbs levels was observed at the time of the study (P =.53; data not shown). DISCUSSION Our study provides evidence of continued longevity of HBVspecific T-cell memory for 32 years despite the loss of anti- HBs levels and in the presence of evidence of natural HBV exposure. NK T cells are important components of innate and adaptive immune responses, especially in the direct support of B-cell activity, which is critical to development and maintenance of vaccine immunity [41, 42]. Our findings align with these known functions of NK T cells, as we observed a significantly higher frequency of NK T cells among persons who maintained anti-hbs levels of 10 miu/ml for 32 years. Recent findings indicate potential differences in T-cell memory development between HBV-specific responses derived from vaccination (HBsAgspecific only) and HBV-specific responses derived from exposure (HBcAg specific and HBV polymerase specific) [32]. This is important in evaluating what vaccine memory responses are protective, especially for persons found to have anti-hbs levels of <10 miu/ml and in areas where HBV was previously endemic. All persons maintained a measurable T-cell response to HBsAg when measured on the basis of HBsAg-specific TNFα, IL-10, or IL-6 production, which may provide insight as to how to evaluate long-term immunity. Most importantly, all persons exhibited cellular response to HBsAg 32 years after vaccination. This includes the 2 nonresponders we were unable to include in the final analysis; both exhibited HBsAg-specific T cells producing TNF-α, IL-10, and IL-6- (data not shown). There are 4 8 weeks between exposure to HBV and development of symptoms in unvaccinated persons, thus providing ample time for a vaccinated person who has lost anti-hbs to recruit a cellular-driven immune response to attenuate HBV and abort clinical icteric hepatitis and long-term chronic viremia. Our findings suggest that immune memory to HBsAg from Lasting Protection Without Antibody JID 2016:214 (15 July) 277

Table 3. Hepatitis B Virus (HBV) Core Antigen Specific T-Cell Responses Provide Evidence of Natural Exposure to HBV Among Recipients of Hepatitis B Vaccine, by Study Group Test, Response, Cytokine Group 1 (n = 13) Group 2 (n = 31) Group 1 vs Group 2 32-y Anti-HBs Level ELISpot Positive, a persons, no. (%) 2 (15) 6 (19) 1.00 b IFN-γ Magnitude, SFCs/10 6 PBMCs, median (range) IFN-γ 0(0 94) 0 (0 43).79 c.34 d Cytometric bead array Positive, a persons, proportion (%) TNF-α 10 (77) 25 (81) 1.00 b IL-10 10 (77) 25 (81) 1.00 b IL-6 8 (62) 26 (84) 0.13 b IL-17 2 (15) 1 (3).20 b IL-4 0 (0) 2 (6) 1.00 b IL-2 6 (46) 9 (29).31 b Magnitude, SFCs/10 6 PBMCs, median (range) TNF-α 154.9 (0 1364) 68.9 (0 565.2).26 c.54 d P Value IL-10 40.8 (0 3446) 7.0 (0 1233).22 c.71 d IL-6 1906.3 (0 21 000) 1148.6 (0 16 690).89 c.66 d IL-17 0 (0 221.9) 0 (0.3.4) IL-4 0 (0 0) 0 (0 0.9) IL-2 0 (0 5.7) 0 (0 10.1).29 c.44 d Group 1 comprised individuals who lost anti-hbs (level, <10 miu/ml) at the 32-year follow-up, and group 2 comprised individuals who maintained anti-hbs (level, 10 miu/ml) at the 32-year follow-up. Abbreviations: anti-hbs, antibody to HBV surface antigen; ELISpot, enzyme-linked immunospot; IFN-γ, interferon γ; IL-2, interleukin 2; IL-4, interleukin 4; IL-6, interleukin 6; IL-10, interleukin 10; IL-17, interleukin 17; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell; TNF-α, tumor necrosis factor α. a Defined as a measurable response, 3 times the background level. b By the Pearson χ 2 or Fisher exact tests, as appropriate. c By the parametric t test or nonparametric Wilcoxon rank sum test, as appropriate. d By Spearman rank order correlation between the anti-hbs level and the immunological outcome. the original plasma-derived vaccine is still present in our study group, and, as we have shown previously, the majority of these persons respond to a recombinant vaccine during booster [9, 19, 20, 22, 23, 25]. These data contribute evidence that hepatitis B vaccination, even when anti-hbs levels have diminished, provides a reduced likelihood of HBV infection by theoretically allowing earlier recognition of HBV and rapider clearance. Further study is needed to determine whether this cellular recognition of HBsAg has any clinical benefit to future exposures to HBV. To explain the seemingly contradictory findings of a positive cellular response to HBcAg in the absence of anti-hbc, we posit the following possibilities. First, natural exposure accounted for this finding and the finding that levels of anti-hbc antibody in sera remained below a level that commercial assays could detect. Our previous work demonstrated that natural boosting, defined byrisesinanti-hbswithoutevidenceofanti-hbcoractual HBV infection, occurred during the first 10 years in 8% of participants [43]. In 1981, HBV infection was highly endemic in Western Alaska, where all participants lived at the time of the initial vaccination series [9, 44 46]. Second, study group participants may have been infected but aborted the infection before measurable anti-hbc was detectable by ELISA or when anti- HBc antibody was only transiently present. Over the first 15 years of this cohort project, persons were identified to be transiently positive for anti-hbc [22, 23, 25]. Previous findings have identified cellular immune responses to hepatitis B vaccination [28, 29, 31, 33, 34]. Although these studies each have multiple variables that cannot be compared, all showed some evidence of existing cellular immune responses even in the absence of anti-hbs. Our data support recent findings that HBVspecific T-cell responses may be more sensitive indicators than anti-hbs detection of the long-term immune response to HBV vaccine [28, 29, 32 34]. Recently Werner et al demonstrated cellular responses, sometimes in the absence of detectable anti-hbs, as well as evidence of natural exposure [31]. Collectively, these data indicate that the hepatitis B vaccine, albeit not sterilizing, prevents acute illness and chronic HBV infection, even after occupational exposure [32] or in areas of endemicity, regardless of anti-hbs level. Our findings bring new questions regarding mechanisms by which natural exposure may affect long-term immunity. Studies directly assessing whether natural exposure diminishes or boosts immune memory and response to new infection are needed. 278 JID 2016:214 (15 July) Simons et al

An alternative to anti-hbs testing would need to be easily obtained from a small sample, inexpensive, and require only a single clinic visit, without need for challenge doses and follow-up assays. MicroRNAs potentially offer accessible markers and have shown to be useful biomarkers of disease outcome, including hepatitis B [47 50]. Published reports thus far distinguish mir-155 as an immune activator [48]. Xiong et al showed a significant decrease in the level of mir-155 in persons with a successful hepatitis B vaccine response [50] within a cohort that vaccinated at college entry. In contrast, in a population-based cohort, we did not find a significant difference in mir-155 expression level between those who did and those who did not maintain anti-hbs. While mir-155 expression was found to correlate with response shortly after hepatitis B vaccination [50], further exploration is needed to determine whether this could be a potential long-term marker of hepatitis B vaccine response. Because laboratory testing required viable PBMCs, recruitment was limited to persons who resided in the Anchorage area; the majority (85%) of all participants in the Vaccination Demonstration Project cohort live in rural Alaska, inaccessible by road [19]; however, characteristics of the study group and the original cohort were similar. A second limitation was that the study was not conducted at the time of the 30-year cohort follow-up [19], so immediate cellular immunity data before and after booster receipt were not collected. This may influence responses, as there was varying booster history, but we were able to show universal cellular immunity in some of the assays we performed, regardless of booster history. Because seromarkers of HBV infection were not tested at the 32-year follow-up, there is a possibility these markers were present; however, chart reviews conducted in the present study did not reveal any breakthrough infections. We were only able to recruit 2 vaccine nonresponders, and further study is needed in this group, as well as in unvaccinated persons who have recovered from acute HBV infection [31, 32]. Finally, analyses of PBMC phenotype frequency were limited to a 4-color flow cytometer, which technically prevented assessing more-detailed PBMC subsets. General PBMC phenotype analysis in this study still provided insight about the direct relationship between NK T cells and CD8 + TEMRA lymphocytes and anti-hbs level. Development of new procedures that allow for mobile, small-scale assays will allow more-detailed immunological studies in Alaska. This study demonstrates long-lasting HBV-specific cellular immunity, despite loss of anti-hbs. When considered with evidence of a durable booster response [19] and a diminished incidence of HBV infection in areas of endemicity, these findings suggest that booster vaccination is not needed. Performance of similar studies among persons at a higher risk for earlier loss of anti-hbs, including those immunized starting at birth, are needed. Expanding our understanding of the long-term success of hepatitis B vaccine may help in the development of tools for confirming protection against HBV that help conserve healthcare resources. Supplementary Data Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. Notes Acknowledgments. We thank the Institute of Translational Health Sciences and the Functional Genomics Core Laboratory, University of Washington Seattle, and all study participants. Disclaimer. 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