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Presence of immune memory and immunity to hepatitis B virus in adults after neonatal hepatitis B vaccination
Vaccine 29 (2011) 7835–7841
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Presence of immune memory and immunity to hepatitis B virus in adults after neonatal hepatitis B vaccination Chang-Lin Zhu a,1 , Ping Liu a,1,2 , Taoyang Chen b , Zhengping Ni b , Ling-Ling Lu b , Fei Huang b , Jianhua Lu b , Zongtang Sun a,∗ , Chunfeng Qu a,∗∗ a State Key Laboratory of Molecular Oncology, Cancer Hospital/Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, 17 Panjiayuan Nanli, Beijing 100021, China b Qidong Liver Cancer Institute, Jiangsu Province 226200, China
a r t i c l e
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Article history: Received 10 April 2011 Received in revised form 11 July 2011 Accepted 20 July 2011 Available online 2 August 2011 Keywords: Hepatitis B Neonatal hepatitis B vaccination Immune memory Cellular immunity Booster
a b s t r a c t Neonatal vaccination against hepatitis B virus (HBV) infection was launched in the 1980s in Qidong, China, where HBV and hepatocellular carcinoma were highly prevalent. Presence of immune memory and immunity against HBV in adults needs to be clarified. From a cohort of 806 who received plasma-derived Hep-B-Vax as neonates and were consecutively followed at ages 5, 10, and 20 years, 402 twenty-fouryear-old adults were recruited for booster test. Among them 4 (1%) were found to be HBsAg(+), 27 (6.7%) were HBsAg(−)anti-HBc(+), 121 (30.2%) were HBsAg(−)anti-HBc(−)anti-HBs(+), and 252 (62.4%) were HBsAg(−)anti-HBc(−)anti-HBs(−). Of them, 141 subjects with HBsAg(−)anti-HBc(−) were boosted with 10-g recombinant HBV vaccine on day-0 and 1-month. The conversion rates of anti-HBs ≥10 mIU/ml on D10–12 and 1-month post-booster were 71.4% and 87.3% respectively in the vaccinees who were anti-HBs(+) at age 5, higher than in those who were anti-HBs(−) at age 5, 57.5% and 80.0% respectively, but no statistically significant. After the second dose of booster, all subjects with anti-HBs(+) at age 5 had anti-HBs >500 mIU/ml. However, 6/40 subjects, with anti-HBs(−) at age 5, had anti-HBs <10 mIU/ml, geometric mean concentration was 3.6 (95% CI 2.0–7.7). Of the subjects received booster, 44 subjects were determined the presence of T cell immunity on D10–12, 41 had HBsAg-specific T cells detectable, including 7/10 subjects whose anti-HBs were <10 mIU/ml 10–12 days post-booster. Among 27 HBsAg(−)anti-HBc(+) subjects, 19 had detectable serum HBV-DNA, and an “a” epitope mutation was found in 1/5 HBV isolates. One subject who was anti-HBc(+) at age 20 converted into HBsAg(+) 4 years later. The adults received neonatal HBV vaccination had immune memory and immunity against HBV infection. However, 31.9% of neonatal HBV vaccinees who responded weakly at an early age might be susceptible to HBV infection after childhood. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is the leading cause of illness and death in China. Each year, an estimated 263,000 people die from HBV-related liver cancer or cirrhosis, accounting for 37–50% of HBV-related deaths worldwide [1]. Universal hepatitis B (HB) vaccination is the best cost-effective and preventive strategy for controlling HBV [2,3]. HB vaccination of neonates and infants has proven to be highly effective in inducing protective antibodies to
∗ Corresponding author. Tel.: +86 10 6763 1675; fax: +86 10 6771 3917. ∗∗ Corresponding author. Tel.: +86 10 8778 3103; fax: +86 10 6771 3917. E-mail addresses: [email protected] (Z. Sun), [email protected] (C. Qu). 1 Theses authors contributed equally to this work. 2 Current address: Department of Immunology, Harbin Medical University, Harbin 150081, China. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.07.098
HBV surface antigen (anti-HBs) and in reducing the prevalence of HBV surface antigens (HBsAg) among children [2,3]. After HB vaccines were included in the Expanded Program on Immunization (EPI) in China, the HBsAg-positive rate of children decreased significantly from 10% to 1–2% [4]. Qidong was one of the areas in China with a high prevalence of HBV infection and hepatocellular carcinoma [5]. Neonatal vaccination against HBV infection was launched in the 1980s [2]. Although this national vaccination program has been very successful, clinical serological surveys conducted in different areas worldwide, including China, showed that the anti-HBs wane over time. Serum levels of anti-HBs ≥10 mIU/ml are considered to be protective against HBV infection [3]. However, the HB vaccine was considered to induce protective immunity, which was present even after the period when the anti-HBs had waned [6,7]. There was also some evidence that a natural boosting effect may occur from exposure to individuals with chronic HBV, and that this is a mechanism for long-term
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protection against the disease in areas where there is high HBV endemicity [8]. However, recent studies conducted in college students in Taiwan suggest that HBV breakthrough infections might occur in young adults, who received their primary immunization as children, who initially had low responses to the HB vaccine [9,10]. These reports provoked concerns about the long-term protection of neonatal HB vaccination against HBV infection in adults and its preventive effect on HBV-related primary hepatocellular carcinoma (HCC) [3,10]. Therefore, there is concern about what could happen if these vaccinated subjects begin to engage in behaviors that would put them at high-risk for HBV transmission in endemic areas. Booster immunizations for certain high-risk groups or individuals living in the endemic areas have been suggested by some researchers [9]. Currently, the HBsAg is still as high as 7.2% based on the seroepidemiological survey on HBV infection conducted in 2006 among the overall population [4]. The uncertainties about whether the immune memory and immunity, including humoral and cellular immunity, among young adults should be clarified. In this study, a total of 141 young adults confirmed to have received neonatal plasma-derived Hep-B Vax (manufactured and donated to the project by Merck & Co. through the WHO) were sampled from a long-term, consecutively followed cohort, and boosted with recombinant HB vaccine. Our results demonstrate that the neonatal HB vaccinees had immune memory as well as immunity against HBV infection. Natural boosters might play an important role in maintaining the immune memory. However, 31.9% of the neonatal HB vaccinees responded weakly after vaccination and might be susceptible to HBV infection after childhood. 2. Subjects and methods 2.1. Subjects The study was conducted from September of 2009 to June of 2010. Based on the records in our database, 806 subjects and their parents were informed by letters to participate in the study, who were born in 1985 and were vaccinated with 5 g of plasmaderived Hep-B Vax after birth and at 1 and 6 months, and were consecutively followed at ages 5, 10, and 20 years, respectively [2], after vaccination. At their age 24, 402 subjects were unable to be followed, a total of 404 subjects were recruited for booster test (Figs. 1 and 2), their serum samples were obtained. After general physical examination, the following subjects were excluded: 3 pregnant, 1 hyperthyroidism, 4 HBsAg(+), 27 HBsAg(−)anti-HBc(+), and 9 who reportedly received HBV boosters after age 20. Totally 141 subjects, 74 males and 67 females, participated in the booster test, the other 219 ones refused to attend the study. HBsAg and anti-HBc were measured twice within a 2-month interval. Signed informed consent was obtained from all participants.
2.3. Laboratory examinations HBsAg and anti-HBc was determined using the ELISA kits from Shanghai Kehua Bioengineering Co., Ltd (Shanghai, China). The lower limit of HBsAg detection is 0.5 ng/ml. Anti-HBs were quantified using reagents from Roche Diagnostic GmbH in Cobas e 601 (D-69298 Mannheim, Germany). The range of this assay is 2–1000 mIU/ml. During follow-up at ages 5, 10, and 20 years, HBsAg, anti-HBc, and anti-HBs were determined using the reagents from Abbott (IL, USA). Anti-HBs <10 mIU/ml were considered to be negative [anti-HBs(−)]. 2.4. HBV DNA extraction, amplification, and S gene sequencing HBV DNA was extracted from 200 l serum using QIAamp MiniElute Virus kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions from 4 subject who were HBsAg(+) and 27 ones who were anti-HBc(+) but HBsAg(−). Strict precautions were taken to avoid possible contamination [1]. Three negative controls using 200 l of fetal bovine serum were included. Serum HBV-DNA was quantified by TaqMan real-time PCR. The S gene in HBV-DNA was amplified and sequenced as described previously [13]. 2.5. Determination of HBsAg- and HBeAg-specific T cells in peripheral blood mononuclear cells (PBMCs) by ELISPOT assays PBMCs were isolated by Ficoll density gradient separation according to standard laboratory protocols. HBsAg-specific and HBeAg-specific IFN-␥-producing T cells in the PBMCs were determined by ELISPOT assays using the reagents from BD Pharmingen (San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, membrane-bottomed 96-well plates (MAHA, Millipore) were coated with monoclonal antibodies against IFN-␥ (5 g/ml, 100 l/well) in PBS (pH 7.2) overnight at 4 ◦ C. After washing and blocking, 5 × 105 /well freshly isolated PBMCs in RPMI 1640 containing 5% autologous human serum were added. In each well, 50 ng/well of recombinant HBsAg (Dalian Hissen Bio-pharm Inc, China) or 50 ng/well of purified HBeAg (kindly provided by Dr. Zhu AG at Beijing Hepatitis Research Institute) were added and incubated for 46–48 h at 37 ◦ C. The spots were analyzed using CTL (Cellular Technology Ltd) ImmunoSpot® V5.0 Professional software in an ImmunoSpot® Versa Analyzer (Beijing, China Office). 2.6. Statistics SPSS version 13.0 for Windows (SPSS, Chicago, IL, USA) was used for data analysis. Differences in frequency between groups were tested by chi-square test. Differences in antibody concentration, and spot-forming cells (SPF) in response to HBsAg or HBeAg were examined using t-tests. All P values were two-tailed and P < 0.05 was considered to indicate statistical significance.
2.2. Protocol for the booster and follow-up 3. Results Study protocol was approved by the Board Committee of Good Clinical Practice in the Cancer Institute/Hospital of CAMS, Beijing (protocol #09-59/354). The participants received 10 g Vecon recombinant Hepatitis B vaccine via intramuscular injection, a yeast product of Shenzhen Kangtai Biological Products Co., Ltd (Shenzhen, China). This recombinant HB vaccine had been reported to induce well anti-HBs response in infants [11,12]. Study flow chart is shown in Fig. 1. All subjects were followed up to 6–7 months after the booster. At 10–12 days after the booster, 44 subjects were randomly sampled and the specific T cell responses to recombinant HBsAg and purified HBeAg were determined using IFN-␥ ELISPOT assays.
3.1. Persistent immunity against chronic HBV infection present among 24-year-old adults who received neonatal HBV vaccinations The cohort originally consisted of 904 neonates born in 1985, and was established during the 1980s as previously reported after neonatal vaccination with Hep-B Vax [2,14,15]. To verify the presence of immunity against chronic HBV infection after vaccination, we determined their serum HBsAg and anti-HBs periodically. At their age 5, a total of 806 subjects who were HBsAg(−) were then determined the presence of anti-HBs. Among them, 549 (68.1%)
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806 targeted subjects who were followed up consecutively at ages 5, 10, 20 year-old Recruitment and screening
402 were unable to be followed 404 sera were obtained for determination of HBsAg, anti-HBs, and anti-HBc 219 HBsAg(-)anti-HBc(-) refused to attend the booster test
Excluded: 3 pregnant, 1 hyperthyroidism, 4 HBsAg(+), 27 HBsAg(-)anti-HBc(+), 9 received HB vaccination after age 20
141 subjects attended booster test 36 HBsAg(-)anti-HBc(-)anti-HBs>10mIU/ml; 105 HBsAg(-)anti-HBc(-)anti-HBs<10mIU/ml Booster with recombinant HBV vaccine and follow up
D0: 1st dose of 10ug HBV vaccine (recombinant) 10-12 days: 1 month:
2 withdraw
36 subjects, 103 subjects Serum anti-HBs antibodies were quantified respectively
1 Month: 2nd dose of 10ug HBV vaccine (recombinant)
13 withdraw
36 subjects 90 subjects Serum anti-HBs antibodies were quantified
6-7Months
Fig. 1. Study design and follow-up after booster with recombinant HBV vaccine.
were anti-HBs(+), 257 (31.9%) were anti-HBs(−). During the followup, none of the anti-HBs(+) subjects at age 5 became HBsAg(+). However, 2 subjects at age 10 and 1 at age 20 were found to be HBsAg(+) among those with anti-HBs(−) at age 5 (Fig. 2). These results demonstrate that persistent immunity against chronic HBV infection was present among adults who responded well to the vaccines at an early age. However, 31.9% of the subjects who failed to respond or showed only a weak response to the vaccines, being anti-HBs(−) at age 5, may be at risk of being infected with HBV at an older age. Nevertheless, the possibility of becoming chronically HBsAg(+) was significantly less after they reached
Neonates
Age 5
the ages of 10 and 20 years compared with those at age 5 (Fig. 2, P < 0.01). At their age 24, 404 subjects in the cohort were recruited for booster test, the other 402 were unable to be followed (Figs. 1 and 2). Among the recruited, 4 (1.0%) were found to be HBsAg(+), 27 (6.7%) were HBsAg(−) but anti-HBc(+), 121 (30.2%) were HBsAg(−)anti-HBc(−)anti-HBs(+), and 252 (62.4%) were HBsAg(−)anti-HBc(−)anti-HBs(−). Totally 141 subjects, 74 males and 67 females, participated in the booster test, the other 219 refused to attend the study. The distribution of anti-HBs(+) and anti-HBs(−) at their early ages in the 141 subjects who agreed
Age 10
Age 20
Age 24
167:anti-HBs(-)
904 207:anti-HBs(-)
Vaccination D0. and 1, 6 months after birth
1: HBsAg(+ HBsAg(+)* ) 39:anti-HBs(+)
257: HBsAg(-) anti-HBs(-)
2: HBsAg(+)* 30:anti-HBs(-) 30:anti HBs( ) 48:anti-HBs(+)
185: were followed 4: HBsAg(+) 34: anti-HBs(+) 12: anti-HBc(+) 72: dropped
18:anti-HBs(+)
806 29: dropped 28: HBsAg+ 41:HBsAg(-) with anti-HBs no-detection d t ti
219:anti-HBs(-) 296:anti-HBs(-) 549: HBsAg(-) g( ) anti-HBs(+)
0: HBsAg(+) HBsAg( )
77:anti-HBs(+) 0: HBsAg(+) 122:anti-HBs(-)
253:anti-HBs(+)
219: were followed 0: HBsAg(+) 70: anti-HBs(+) ( ) 15: anti-HBc(+) 330: dropped
131:anti-HBs(+)
Fig. 2. Natural history after neonatal HB vaccination before booster test 904 neonates received plasma-derived Hep-B Vax (manufactured and donated to the project by Merck & Co. through the WHO) within 24 h and at 1, 6 months after birth. At age 5 years, a total of 876 vaccinees were followed, the 28 subjects with serum HBsAg(+) and 41 with HBsAg(−) but without detection of anti-HBs were not included for further analysis in this study. The 806 subjects were followed individually at ages 10 and 20 years, respectively. By the booster study, 404 subjects were followed and their HBsAg, anti-HBs and anti-HBc were determined (*P < 0.01 tested by Chi-square, 29/876 at age 5 vs. 2/806 at age 10 and vs. 3/806 at age 20).
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Table 1 Distribution of anti-HBs at age 5 in the subjects of 141 attended, 219 not-attended and 402 dropped to booster test at their age 24.
(1a) Anti-HBs(+) Anti-HBs(−)
(1b) Anti-HBs(+) Anti-HBs(−)
Recruited (n = 404), No. (%)
Dropped (n = 402), No. (%)
2 , P value
277 (68.6%) 127 (31.4%)
272 (67.7%) 130 (32.3%)
2 = 0.08, P > 0.05
Attended (n = 141), No. (%)
Not-attended (n = 219), No. (%)
2 , P value
101 (71.6%) 40 (28.4%)
150 (68.5%) 69 (31.5%)
2 = 0.40, P > 0.05
to, 219 ones who refused to attend the study, and 402 ones who dropped at age 24 was similar (Table 1, P > 0.05). 3.2. Serum levels of anti-HBs after recombinant HB vaccine booster To confirm the presence of immune memory, 141 subjects aged 24 years received booster test, of which 36 (25.5%, 36/141) had anti-HBs ≥10 mIU/ml, and 105 (74.5%, 105/141) had anti-HBs(−) <10 mIU/ml (Fig. 1). Ten to twelve days after the booster, when anamnestic responses were expected, samples were obtained from 103 of the 105 subjects, 63 from subjects who had anti-HBs(+) and 40 who had anti-HBs(−) at age 5 (Table 2). Levels of anti-HBs ≥10 mIU/ml were found in 71.4% (45/63) of the subjects with antiHBs(+), and 57.5% (23/40) of those with anti-HBs(−) at age 5. One month after the booster, anti-HBs levels were reassessed significantly in 87.3% of the subjects who were anti-HBs(+) at age 5, and in 80% of those who were anti-HBs(−) at age 5 (Table 2, P < 0.001). However, 12.7% (8/63) of the subjects who were anti-HBs(+) at age 5 still had levels below 10 mIU/ml, and 20% (8/40) of the subjects who were anti-HBs(−) at age 5 (Table 2). The conversion rate of anti-HBs ≥10 mIU/ml after booster at the two time-points, 10–12 days and 1 month post-booster, was higher in those with anti-HBs(+) than in those with anti-HBs(−) at age 5 years, but the difference was not statistically significant (P > 0.05). However, 28.6% (18/63) of the subjects who responded well to the vaccine at age 5 years, being showed anti-HBs(+), failed to increase their anti-HBs within 10–12 days after booster. These individuals had probably lost their humoral memory after they reached adulthood. The subjects received a second dose of HBV vaccine and were followed for 6–7 months. Serum samples were obtained from 90 vaccinees of the 103 subjects who were anti-HBs <10 mIU/ml pre-booster. All subjects with anti-HBs(+) at age 5 showed increased anti-HBs ≥10 mIU/ml; however, 6 of the subjects, who were anti-HBs(−) at age 5 still had levels <10 mIU/ml; 3 had levels of 4–9 mIU/ml and 3 had no detectable anti-HBs antibodies (Table 2). The results suggest that approximately 5.8% (6/103) of the adults in the general population in this area might respond inefficiently to the currently used HBV vaccine.
but increased to anti-HBs ≥10 mIU/ml post-booster (G2), and 10 had anti-HBs <10 mIU/ml both pre-booster and post-booster (G3) quantified 10–12 days after vaccine injection (Fig. 3). In G1, the HBsAg-specific IFN-␥-producing T cells were detectable in all 13 subjects, but with large variations in the number of spot-forming cells (SFC), that reflected the specific T cell frequency in peripheral blood. The same results were found in 21 G2 subjects. However, HBsAg specific IFN-␥-producing T cells were also detectable in 6/10 (60%) of the 10 G3 subjects whose anti-HBs were <10 mIU/ml 10–12 days post-booster, including 2 subjects who had anti-HBs <10 mIU/ml determined 6–7 months after booster (Fig. 3A). These results suggest that specific T cell memory was present in the vaccinees, even though their anti-HBs antibodies did not increase promptly after HB booster.
3.4. Specific T cells to purified HBeAg determined by ELISPOT in PBMCs Some vaccinees who were naturally boosted from infection showed anti-HBs(+) after age 5, and regained anti-HBs after their anti-HBs waned (Fig. 2). The presence of T cell immunity against HBV infection was further confirmed by detecting specific IFN-␥-producing T cells in response to HBeAg (Fig. 3B), which is not included in any of the HB vaccines. All 13 subjects from G1, whose anti-HBs were ≥10 mIU/ml pre-booster, showed the presence of specific T cell responses to the purified HBeAg, but with large variations in SPF. Among the 21 G2 subjects whose anti-HBs <10 mIU/ml but were ≥10 mIU/ml postbooster, 19/21 had detectable HBeAg-specific T cells. In the 10 G3 subjects whose anti-HBs were <10 mIU/ml both pre- and post-booster, HBeAg-specific T cells were also detectable in 7/10 (70%), including 3 with anti-HBs <10 mIU/ml 6–7 months after booster.
3.3. Specific T cells to recombinant HBsAg determined by ELISPOT in PBMCs None of the vaccinees became HBsAg-positive, even those in whom anti-HBs humoral immune memory was undetectable, in the subjects with anti-HBs(+) at age 5 years (Fig. 2). To study whether the individuals had a specific T cell immunological memory, we determined the presence of HBsAg-specific T cells in the PBMCs of 44 subjects. Of these, 13 subjects had anti-HBs ≥10 mIU/ml in their sera pre-booster (G1), 21 had anti-HBs <10 mIU/ml pre-booster
Fig. 3. Responses of HBsAg- and HBeAg-specific T cells in subjects after receiving a booster dose of recombinant HB vaccines. (A) The frequency of HBsAg-specific and (B) HBeAg-specific IFN-␥ producing T cells in peripheral blood mononuclear cells (PBMCs) in the subjects determined 10–12 days post-booster. The G1 subjects had anti-HBs >10 mIU/ml pre- and post-booster. The G2 subjects had anti-HBs <10 mIU/ml pre-booster and anti-HBs >10 mIU/ml post-booster. The G3 subjects had anti-HBs <10 mIU/ml both pre- and post-booster.
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3.5. HBV-DNA was detectable in the HB vaccinees who were anti-HBc-positive
Abbreviation: GMC, geometric mean concentration; 95% CI: 95% confidential interval. a P < 0.001 between the GMC determined in the 1 month vs. D10–12 after booster. b The 8 subjects with anti-HBs <10 mIU/ml, 1 month after the first dose of vaccine were followed, but 10 subjects with anti-HBs ≥10 mIU/ml, 1 month afterward dropped the tested. c The 8 subjects with anti-HBs <10 mIU/ml, 1 month after the first dose of vaccine were followed, but 3 subjects with anti-HBs ≥10 mIU/ml, 1 month afterward dropped the tested. d 3 had anti-HBs 4–9 mIU/ml and 3 had no detectable anti-HBs.
P
2 = 2.116, P > 0.05
2 = 0.994, P > 0.05
0 6d /3.6 (2–7.7) 53b /859.5 (843–1000) 31c /823.7 (746–1000) 8b (12.7%)/5.6 (3.1–7.5) 8c (20.0%)/3.1 (2–3.8) 55 (87.3%)/556.2 (390.9–795.6)a 32 (80.0%)/526.9 (350.9–791.4)a 45 (71.4%)/152.3 (102.8–225.7)a 23 (57.5%)/183.2 (138.2–257.7)a
<10 mIU/ml ≥10 mIU/ml <10 mIU/ml ≥10 mIU/ml <10 mIU/ml
18 (28.6%)/3.5 (2.8–4.8) 17 (42.5%)/3.1 (2.2–3.8)
≥10 mIU/ml
Anti-HBs(+) at age 5, n = 63 Anti-HBs(−) at age 5, n = 40
1 month, No. (%)/GMC (95%CI) D10–12, No. (%)/GMC (95%CI)
Table 2 Anti-HBs responses after booster with recombinant HB vaccine.
6–7 months, No. (%)/GMC (95%CI)
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During the follow up (Fig. 2), we noticed that 67 (67/806, 8.31%) subjects were HBsAg(−), anti-HBc(+) at age 20 years. When the booster vaccination study began, they were followed for 4 years. One subject, who was anti-HBs(−) at age 5, converted into HBsAg(+) and 14 retained their anti-HBc(+) status. To check for the presence of HBV-DNA in their sera, fragments of HBV-DNA were amplified in the 14 samples above as well as another 13 sera who were HBsAg(−), anti-HBc(−), at age 20 years. Fragments of the HBV S gene were found in 19/27 by nested PCR amplification (Fig. 4A). After quantification of the serum viral load, HBV-DNA was below 5 × 103 copies/ml in all of them (copy numbers are labeled under each band in Fig. 4A). Sequencing of the S gene in 5 isolates showed that mutations at the “a” epitope (aa 120–150) was present in 1/5 of them (G145R; Fig. 4B). The 4 remaining isolates were identical to the local wild isolate, GU434374 [13].
4. Discussion There are few populations in which infant vaccination history and documentation of immune response to vaccination are still available at the adults (more than 20 years). However, using unique household registration system of China, we were able to follow their responses after neonatal vaccination. In this 806 consecutively followed neonatal HB vaccinees (Fig. 2), 68.1% were HBsAg(−)antiHBs(+) and 31.9% were HBsAg(−)anti-HBs(−) at age 5. During the follow-up by age 20 years, none of the subjects with anti-HBs(+) at age 5 became HBsAg(+). However, 2 subjects with anti-HBs(−) at age 5 became chronically HBsAg(+) at ages 10 and one at age 20 years, respectively. These results demonstrate that persistent immunity against chronic HBV infection was present among adults who had responded well to the vaccine at an early age. However, subjects who failed to respond or responded weakly to the vaccine, being showed anti-HBs(−) at age 5, might not be fully protected, and may be at risk of infection with HBV at older ages. Nevertheless, the possibility of becoming chronic HBsAg carriers was significantly less after the ages of 10 and 20 years compared with age 5. HB vaccination before age 5 may be critical to prevent chronic HBsAg. Serum levels of anti-HBs ≥10 mIU/ml induced by the HBV vaccine are considered to be protective from HBV infection. Levels decline rapidly during the first year and then more gradually [3]. Nevertheless, the immune memory is maintained for at least 12 years despite the disappearance of circulating antibodies [16]. Only about 20.5% of 18- to 23-year-old college students in Taiwan kept humoral immune memory [10]. The current study, conducted in a rural area of China, showed that 71.4% of the 24-year-old subjects, who responded well to the vaccine at their early ages, being showed anti-HBs(+) at age 5 years, had humoral immunological memory. For those who responded weakly to the vaccine, being showed anti-HBs(−) at age 5 years, the humoral memory was still detectable at 57.5% of the subjects. This percentage is much higher than that reported in Taiwan [10]. The difference mostly due to lower coverage of HB vaccination in the rural area that put the vaccinees into higher rate exposed in HBV infection [2,3]. We conducted a specific T cell immunity test after booster vaccination. HBsAg-specific IFN-␥ producing T cells were detectable in 41/44 subjects, including 7/10 subjects whose anti-HBs were <10 mIU/ml 10 days after the first booster. In addition, HBeAgspecific IFN-␥-producing T cells were also detectable in 40 subjects including 2 subjects whose anti-HBs were <10 mIU/ml both 10 days and 6–7 months after the booster. HBeAg was not included in any form of HB vaccines, these results demonstrate that the natural
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Fig. 4. Determination and sequencing of HBV-DNA isolated in neonatal HB vaccinees. (A) S-gene fragments of HBV-DNA amplified by nested PCR. Serum HBV-DNA was quantified by TaqMan real-time PCR and the viral concentration is shown under each PCR product band. (B) Alignment of deduced HBV S proteins from 5 subjects with HBsAg(−)anti-HBc(+) and 1 subject with HBsAg(+) (case #8), who converted from HBsAg(−)anti-HBc(+) at age 20.
infection as a booster might play an important role in maintaining the immune memory and immunity (humoral and cellular) against HBV infection after they were fully vaccinated with HB vaccine as neonates and in infancy as suggested [8]. No correlation between the anti-HBs antibody levels with the frequency of specific T cells was observed, and similar results were found in a study of 18- to 20-year olds in Thailand after neonatal vaccination [17]. Our observations differ from those reported in Taiwan recently [9,10] where most of the college student participants had lost their humoral immune memory after they were immunized as neonates and infants. In this report, the anti-HBs conversion 7–10 days after HB booster was only 20.5% [10] among college students. The conversion rate in our enrolled subjects with the same serological markers was more than twice that. In addition, HBsAg- and or HBeAg-specific memory T cells were detected in 43/44 sampled subjects, which was much higher than that reported in the Taiwanese college students [9]. Specific T cells were still detectable in some subjects who showed no increase in antibody levels after the booster, which is similar to results reported by other investigators [18]. The natural history of anti-HBs and other HBV markers indicated that exposure to HBV might be an important natural booster to maintain the immunological memory against HBV in this area [8]. Whether it is necessary to boost young adults is still under debate. Investigations conducted in Thailand [17] and Taiwan [9,10] suggest that it is necessary to consider 1 or 2 booster doses, especially in high risk groups. In the current study, about 31.9% of the subjects who responded weakly at an early age and 28.6% of those who responded well to the vaccine (with anti-HBs (+) at age 5) failed to respond rapidly to one booster vaccination. These subjects might be exposed in a high risk environment and infected with HBV. Anti-HBc conversion has been studied to estimate the exposure but few studies estimated the risk of disease or viral persistent after the conversion. Recently, we reported the presence occult HBV infection in anti-HBs-positive young adults after neonatal HB vaccination [13]. The current study showed that 19/27 of the subjects who were anti-HBc-positive but HBsAg-negative had detectable HBV-DNA S gene fragments. Of these 19 subjects, 14 were HBsAg(−), anti-HBc(+) for 4 years. Of 5 randomly sequenced isolates, the “a” epitope mutation was found in only 1 subject, but their serum HBV-DNA was at low levels (Fig. 4). This finding is similar to our recent report in subjects who were HBsAg(−), anti-HBs(+), and anti-HBc(+) [13]. In addition, one anti-HBc-positive child born from an HBsAg-positive mother became HBsAg-positive when she was sampled at the age of 24 years. The chances of being infected
with HBV in adulthood increases via horizontal transmission like sexual activity, needle injection, and other social activities. Therefore, HB booster for those with a high risk of contact with HBV infection might be necessary before they achieve adulthood. However, as reported in our previous study, HBV-DNA without mutation of the “a” epitope was also detectable in some vaccinees who had detectable anti-HBs in their serum [13]. Therefore, the presence of anti-HBs might not fully protect them from HBV infection [13]. HB booster vaccination of adolescents who were fully vaccinated at an early age still needs to be carefully evaluated in the clinic.Studies of patients with self-limited HBV infections demonstrated that antiHBV specific CD8+ T cells play critical roles in terminating HBV infection [19]. A defect in specific T cell immunity against HBV antigens caused persistent chronic HBV infection [20]. Therefore, vaccine-conferred T cell immunity might play an important role in protecting the vaccinees from overt HBV infection. This hypothesis is supported by evidence that some vaccinees were anti-HBs negative at age 10–11 years and later converted into anti-HBs-positive at age 24 years without becoming HBsAg positive. Thus, new types of HB vaccines should be able to induce specific T cell responses, as well as stimulate specific neutralizing antibodies. In endemic areas like China, the primary goal of HB vaccination is to protect children from HBV infection and prevent chronic infection, which is seen as HBsAg carriers [2]. This was achieved after the HB vaccine was included into the EPI program in China [4]. The risk of becoming a chronic HBsAg carrier is far lower when the unprotected come into contact with HBV infection in adulthood [21]. Boosting with the current vaccine is not presently recommended in China. However, we found in this study that serum HBV-DNA was detectable in some vaccinees at low levels without a mutation in the “a” epitope. Occult HBV infection occurred in some vaccinees. The importance of occult HBV infection in some liver diseases, including in HCC, has been addressed by other researchers [22–24]. The significance of this infection in the pathogenesis of hepatitis and liver cirrhosis, even in HCC, still needs to be carefully monitored. Prolonged follow-up and surveillance of vaccinees who were immunized at an early age should be continued.
Acknowledgements This work was supported by a grant from State Key Projects Specialized for Infectious Diseases (2008 ZX10002-015) and National Natural Science Foundation of China, 30973387. The authors are grateful to Dr. Eskild Pertersen at the Department of Infectious Diseases, Aarhus University Hospital, Skejby, Denmark for their critical review and valuable comments to the manuscript.
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Presence of immune memory and immunity to hepatitis B virus in adults after neonatal hepatitis B vaccination Chang-Lin Zhu a,1 , Ping Liu a,1,2 , Taoyang Chen b , Zhengping Ni b , Ling-Ling Lu b , Fei Huang b , Jianhua Lu b , Zongtang Sun a,∗ , Chunfeng Qu a,∗∗ a State Key Laboratory of Molecular Oncology, Cancer Hospital/Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, 17 Panjiayuan Nanli, Beijing 100021, China b Qidong Liver Cancer Institute, Jiangsu Province 226200, China
a r t i c l e
i n f o
Article history: Received 10 April 2011 Received in revised form 11 July 2011 Accepted 20 July 2011 Available online 2 August 2011 Keywords: Hepatitis B Neonatal hepatitis B vaccination Immune memory Cellular immunity Booster
a b s t r a c t Neonatal vaccination against hepatitis B virus (HBV) infection was launched in the 1980s in Qidong, China, where HBV and hepatocellular carcinoma were highly prevalent. Presence of immune memory and immunity against HBV in adults needs to be clarified. From a cohort of 806 who received plasma-derived Hep-B-Vax as neonates and were consecutively followed at ages 5, 10, and 20 years, 402 twenty-fouryear-old adults were recruited for booster test. Among them 4 (1%) were found to be HBsAg(+), 27 (6.7%) were HBsAg(−)anti-HBc(+), 121 (30.2%) were HBsAg(−)anti-HBc(−)anti-HBs(+), and 252 (62.4%) were HBsAg(−)anti-HBc(−)anti-HBs(−). Of them, 141 subjects with HBsAg(−)anti-HBc(−) were boosted with 10-g recombinant HBV vaccine on day-0 and 1-month. The conversion rates of anti-HBs ≥10 mIU/ml on D10–12 and 1-month post-booster were 71.4% and 87.3% respectively in the vaccinees who were anti-HBs(+) at age 5, higher than in those who were anti-HBs(−) at age 5, 57.5% and 80.0% respectively, but no statistically significant. After the second dose of booster, all subjects with anti-HBs(+) at age 5 had anti-HBs >500 mIU/ml. However, 6/40 subjects, with anti-HBs(−) at age 5, had anti-HBs <10 mIU/ml, geometric mean concentration was 3.6 (95% CI 2.0–7.7). Of the subjects received booster, 44 subjects were determined the presence of T cell immunity on D10–12, 41 had HBsAg-specific T cells detectable, including 7/10 subjects whose anti-HBs were <10 mIU/ml 10–12 days post-booster. Among 27 HBsAg(−)anti-HBc(+) subjects, 19 had detectable serum HBV-DNA, and an “a” epitope mutation was found in 1/5 HBV isolates. One subject who was anti-HBc(+) at age 20 converted into HBsAg(+) 4 years later. The adults received neonatal HBV vaccination had immune memory and immunity against HBV infection. However, 31.9% of neonatal HBV vaccinees who responded weakly at an early age might be susceptible to HBV infection after childhood. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is the leading cause of illness and death in China. Each year, an estimated 263,000 people die from HBV-related liver cancer or cirrhosis, accounting for 37–50% of HBV-related deaths worldwide [1]. Universal hepatitis B (HB) vaccination is the best cost-effective and preventive strategy for controlling HBV [2,3]. HB vaccination of neonates and infants has proven to be highly effective in inducing protective antibodies to
∗ Corresponding author. Tel.: +86 10 6763 1675; fax: +86 10 6771 3917. ∗∗ Corresponding author. Tel.: +86 10 8778 3103; fax: +86 10 6771 3917. E-mail addresses: [email protected] (Z. Sun), [email protected] (C. Qu). 1 Theses authors contributed equally to this work. 2 Current address: Department of Immunology, Harbin Medical University, Harbin 150081, China. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.07.098
HBV surface antigen (anti-HBs) and in reducing the prevalence of HBV surface antigens (HBsAg) among children [2,3]. After HB vaccines were included in the Expanded Program on Immunization (EPI) in China, the HBsAg-positive rate of children decreased significantly from 10% to 1–2% [4]. Qidong was one of the areas in China with a high prevalence of HBV infection and hepatocellular carcinoma [5]. Neonatal vaccination against HBV infection was launched in the 1980s [2]. Although this national vaccination program has been very successful, clinical serological surveys conducted in different areas worldwide, including China, showed that the anti-HBs wane over time. Serum levels of anti-HBs ≥10 mIU/ml are considered to be protective against HBV infection [3]. However, the HB vaccine was considered to induce protective immunity, which was present even after the period when the anti-HBs had waned [6,7]. There was also some evidence that a natural boosting effect may occur from exposure to individuals with chronic HBV, and that this is a mechanism for long-term
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protection against the disease in areas where there is high HBV endemicity [8]. However, recent studies conducted in college students in Taiwan suggest that HBV breakthrough infections might occur in young adults, who received their primary immunization as children, who initially had low responses to the HB vaccine [9,10]. These reports provoked concerns about the long-term protection of neonatal HB vaccination against HBV infection in adults and its preventive effect on HBV-related primary hepatocellular carcinoma (HCC) [3,10]. Therefore, there is concern about what could happen if these vaccinated subjects begin to engage in behaviors that would put them at high-risk for HBV transmission in endemic areas. Booster immunizations for certain high-risk groups or individuals living in the endemic areas have been suggested by some researchers [9]. Currently, the HBsAg is still as high as 7.2% based on the seroepidemiological survey on HBV infection conducted in 2006 among the overall population [4]. The uncertainties about whether the immune memory and immunity, including humoral and cellular immunity, among young adults should be clarified. In this study, a total of 141 young adults confirmed to have received neonatal plasma-derived Hep-B Vax (manufactured and donated to the project by Merck & Co. through the WHO) were sampled from a long-term, consecutively followed cohort, and boosted with recombinant HB vaccine. Our results demonstrate that the neonatal HB vaccinees had immune memory as well as immunity against HBV infection. Natural boosters might play an important role in maintaining the immune memory. However, 31.9% of the neonatal HB vaccinees responded weakly after vaccination and might be susceptible to HBV infection after childhood. 2. Subjects and methods 2.1. Subjects The study was conducted from September of 2009 to June of 2010. Based on the records in our database, 806 subjects and their parents were informed by letters to participate in the study, who were born in 1985 and were vaccinated with 5 g of plasmaderived Hep-B Vax after birth and at 1 and 6 months, and were consecutively followed at ages 5, 10, and 20 years, respectively [2], after vaccination. At their age 24, 402 subjects were unable to be followed, a total of 404 subjects were recruited for booster test (Figs. 1 and 2), their serum samples were obtained. After general physical examination, the following subjects were excluded: 3 pregnant, 1 hyperthyroidism, 4 HBsAg(+), 27 HBsAg(−)anti-HBc(+), and 9 who reportedly received HBV boosters after age 20. Totally 141 subjects, 74 males and 67 females, participated in the booster test, the other 219 ones refused to attend the study. HBsAg and anti-HBc were measured twice within a 2-month interval. Signed informed consent was obtained from all participants.
2.3. Laboratory examinations HBsAg and anti-HBc was determined using the ELISA kits from Shanghai Kehua Bioengineering Co., Ltd (Shanghai, China). The lower limit of HBsAg detection is 0.5 ng/ml. Anti-HBs were quantified using reagents from Roche Diagnostic GmbH in Cobas e 601 (D-69298 Mannheim, Germany). The range of this assay is 2–1000 mIU/ml. During follow-up at ages 5, 10, and 20 years, HBsAg, anti-HBc, and anti-HBs were determined using the reagents from Abbott (IL, USA). Anti-HBs <10 mIU/ml were considered to be negative [anti-HBs(−)]. 2.4. HBV DNA extraction, amplification, and S gene sequencing HBV DNA was extracted from 200 l serum using QIAamp MiniElute Virus kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions from 4 subject who were HBsAg(+) and 27 ones who were anti-HBc(+) but HBsAg(−). Strict precautions were taken to avoid possible contamination [1]. Three negative controls using 200 l of fetal bovine serum were included. Serum HBV-DNA was quantified by TaqMan real-time PCR. The S gene in HBV-DNA was amplified and sequenced as described previously [13]. 2.5. Determination of HBsAg- and HBeAg-specific T cells in peripheral blood mononuclear cells (PBMCs) by ELISPOT assays PBMCs were isolated by Ficoll density gradient separation according to standard laboratory protocols. HBsAg-specific and HBeAg-specific IFN-␥-producing T cells in the PBMCs were determined by ELISPOT assays using the reagents from BD Pharmingen (San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, membrane-bottomed 96-well plates (MAHA, Millipore) were coated with monoclonal antibodies against IFN-␥ (5 g/ml, 100 l/well) in PBS (pH 7.2) overnight at 4 ◦ C. After washing and blocking, 5 × 105 /well freshly isolated PBMCs in RPMI 1640 containing 5% autologous human serum were added. In each well, 50 ng/well of recombinant HBsAg (Dalian Hissen Bio-pharm Inc, China) or 50 ng/well of purified HBeAg (kindly provided by Dr. Zhu AG at Beijing Hepatitis Research Institute) were added and incubated for 46–48 h at 37 ◦ C. The spots were analyzed using CTL (Cellular Technology Ltd) ImmunoSpot® V5.0 Professional software in an ImmunoSpot® Versa Analyzer (Beijing, China Office). 2.6. Statistics SPSS version 13.0 for Windows (SPSS, Chicago, IL, USA) was used for data analysis. Differences in frequency between groups were tested by chi-square test. Differences in antibody concentration, and spot-forming cells (SPF) in response to HBsAg or HBeAg were examined using t-tests. All P values were two-tailed and P < 0.05 was considered to indicate statistical significance.
2.2. Protocol for the booster and follow-up 3. Results Study protocol was approved by the Board Committee of Good Clinical Practice in the Cancer Institute/Hospital of CAMS, Beijing (protocol #09-59/354). The participants received 10 g Vecon recombinant Hepatitis B vaccine via intramuscular injection, a yeast product of Shenzhen Kangtai Biological Products Co., Ltd (Shenzhen, China). This recombinant HB vaccine had been reported to induce well anti-HBs response in infants [11,12]. Study flow chart is shown in Fig. 1. All subjects were followed up to 6–7 months after the booster. At 10–12 days after the booster, 44 subjects were randomly sampled and the specific T cell responses to recombinant HBsAg and purified HBeAg were determined using IFN-␥ ELISPOT assays.
3.1. Persistent immunity against chronic HBV infection present among 24-year-old adults who received neonatal HBV vaccinations The cohort originally consisted of 904 neonates born in 1985, and was established during the 1980s as previously reported after neonatal vaccination with Hep-B Vax [2,14,15]. To verify the presence of immunity against chronic HBV infection after vaccination, we determined their serum HBsAg and anti-HBs periodically. At their age 5, a total of 806 subjects who were HBsAg(−) were then determined the presence of anti-HBs. Among them, 549 (68.1%)
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806 targeted subjects who were followed up consecutively at ages 5, 10, 20 year-old Recruitment and screening
402 were unable to be followed 404 sera were obtained for determination of HBsAg, anti-HBs, and anti-HBc 219 HBsAg(-)anti-HBc(-) refused to attend the booster test
Excluded: 3 pregnant, 1 hyperthyroidism, 4 HBsAg(+), 27 HBsAg(-)anti-HBc(+), 9 received HB vaccination after age 20
141 subjects attended booster test 36 HBsAg(-)anti-HBc(-)anti-HBs>10mIU/ml; 105 HBsAg(-)anti-HBc(-)anti-HBs<10mIU/ml Booster with recombinant HBV vaccine and follow up
D0: 1st dose of 10ug HBV vaccine (recombinant) 10-12 days: 1 month:
2 withdraw
36 subjects, 103 subjects Serum anti-HBs antibodies were quantified respectively
1 Month: 2nd dose of 10ug HBV vaccine (recombinant)
13 withdraw
36 subjects 90 subjects Serum anti-HBs antibodies were quantified
6-7Months
Fig. 1. Study design and follow-up after booster with recombinant HBV vaccine.
were anti-HBs(+), 257 (31.9%) were anti-HBs(−). During the followup, none of the anti-HBs(+) subjects at age 5 became HBsAg(+). However, 2 subjects at age 10 and 1 at age 20 were found to be HBsAg(+) among those with anti-HBs(−) at age 5 (Fig. 2). These results demonstrate that persistent immunity against chronic HBV infection was present among adults who responded well to the vaccines at an early age. However, 31.9% of the subjects who failed to respond or showed only a weak response to the vaccines, being anti-HBs(−) at age 5, may be at risk of being infected with HBV at an older age. Nevertheless, the possibility of becoming chronically HBsAg(+) was significantly less after they reached
Neonates
Age 5
the ages of 10 and 20 years compared with those at age 5 (Fig. 2, P < 0.01). At their age 24, 404 subjects in the cohort were recruited for booster test, the other 402 were unable to be followed (Figs. 1 and 2). Among the recruited, 4 (1.0%) were found to be HBsAg(+), 27 (6.7%) were HBsAg(−) but anti-HBc(+), 121 (30.2%) were HBsAg(−)anti-HBc(−)anti-HBs(+), and 252 (62.4%) were HBsAg(−)anti-HBc(−)anti-HBs(−). Totally 141 subjects, 74 males and 67 females, participated in the booster test, the other 219 refused to attend the study. The distribution of anti-HBs(+) and anti-HBs(−) at their early ages in the 141 subjects who agreed
Age 10
Age 20
Age 24
167:anti-HBs(-)
904 207:anti-HBs(-)
Vaccination D0. and 1, 6 months after birth
1: HBsAg(+ HBsAg(+)* ) 39:anti-HBs(+)
257: HBsAg(-) anti-HBs(-)
2: HBsAg(+)* 30:anti-HBs(-) 30:anti HBs( ) 48:anti-HBs(+)
185: were followed 4: HBsAg(+) 34: anti-HBs(+) 12: anti-HBc(+) 72: dropped
18:anti-HBs(+)
806 29: dropped 28: HBsAg+ 41:HBsAg(-) with anti-HBs no-detection d t ti
219:anti-HBs(-) 296:anti-HBs(-) 549: HBsAg(-) g( ) anti-HBs(+)
0: HBsAg(+) HBsAg( )
77:anti-HBs(+) 0: HBsAg(+) 122:anti-HBs(-)
253:anti-HBs(+)
219: were followed 0: HBsAg(+) 70: anti-HBs(+) ( ) 15: anti-HBc(+) 330: dropped
131:anti-HBs(+)
Fig. 2. Natural history after neonatal HB vaccination before booster test 904 neonates received plasma-derived Hep-B Vax (manufactured and donated to the project by Merck & Co. through the WHO) within 24 h and at 1, 6 months after birth. At age 5 years, a total of 876 vaccinees were followed, the 28 subjects with serum HBsAg(+) and 41 with HBsAg(−) but without detection of anti-HBs were not included for further analysis in this study. The 806 subjects were followed individually at ages 10 and 20 years, respectively. By the booster study, 404 subjects were followed and their HBsAg, anti-HBs and anti-HBc were determined (*P < 0.01 tested by Chi-square, 29/876 at age 5 vs. 2/806 at age 10 and vs. 3/806 at age 20).
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Table 1 Distribution of anti-HBs at age 5 in the subjects of 141 attended, 219 not-attended and 402 dropped to booster test at their age 24.
(1a) Anti-HBs(+) Anti-HBs(−)
(1b) Anti-HBs(+) Anti-HBs(−)
Recruited (n = 404), No. (%)
Dropped (n = 402), No. (%)
2 , P value
277 (68.6%) 127 (31.4%)
272 (67.7%) 130 (32.3%)
2 = 0.08, P > 0.05
Attended (n = 141), No. (%)
Not-attended (n = 219), No. (%)
2 , P value
101 (71.6%) 40 (28.4%)
150 (68.5%) 69 (31.5%)
2 = 0.40, P > 0.05
to, 219 ones who refused to attend the study, and 402 ones who dropped at age 24 was similar (Table 1, P > 0.05). 3.2. Serum levels of anti-HBs after recombinant HB vaccine booster To confirm the presence of immune memory, 141 subjects aged 24 years received booster test, of which 36 (25.5%, 36/141) had anti-HBs ≥10 mIU/ml, and 105 (74.5%, 105/141) had anti-HBs(−) <10 mIU/ml (Fig. 1). Ten to twelve days after the booster, when anamnestic responses were expected, samples were obtained from 103 of the 105 subjects, 63 from subjects who had anti-HBs(+) and 40 who had anti-HBs(−) at age 5 (Table 2). Levels of anti-HBs ≥10 mIU/ml were found in 71.4% (45/63) of the subjects with antiHBs(+), and 57.5% (23/40) of those with anti-HBs(−) at age 5. One month after the booster, anti-HBs levels were reassessed significantly in 87.3% of the subjects who were anti-HBs(+) at age 5, and in 80% of those who were anti-HBs(−) at age 5 (Table 2, P < 0.001). However, 12.7% (8/63) of the subjects who were anti-HBs(+) at age 5 still had levels below 10 mIU/ml, and 20% (8/40) of the subjects who were anti-HBs(−) at age 5 (Table 2). The conversion rate of anti-HBs ≥10 mIU/ml after booster at the two time-points, 10–12 days and 1 month post-booster, was higher in those with anti-HBs(+) than in those with anti-HBs(−) at age 5 years, but the difference was not statistically significant (P > 0.05). However, 28.6% (18/63) of the subjects who responded well to the vaccine at age 5 years, being showed anti-HBs(+), failed to increase their anti-HBs within 10–12 days after booster. These individuals had probably lost their humoral memory after they reached adulthood. The subjects received a second dose of HBV vaccine and were followed for 6–7 months. Serum samples were obtained from 90 vaccinees of the 103 subjects who were anti-HBs <10 mIU/ml pre-booster. All subjects with anti-HBs(+) at age 5 showed increased anti-HBs ≥10 mIU/ml; however, 6 of the subjects, who were anti-HBs(−) at age 5 still had levels <10 mIU/ml; 3 had levels of 4–9 mIU/ml and 3 had no detectable anti-HBs antibodies (Table 2). The results suggest that approximately 5.8% (6/103) of the adults in the general population in this area might respond inefficiently to the currently used HBV vaccine.
but increased to anti-HBs ≥10 mIU/ml post-booster (G2), and 10 had anti-HBs <10 mIU/ml both pre-booster and post-booster (G3) quantified 10–12 days after vaccine injection (Fig. 3). In G1, the HBsAg-specific IFN-␥-producing T cells were detectable in all 13 subjects, but with large variations in the number of spot-forming cells (SFC), that reflected the specific T cell frequency in peripheral blood. The same results were found in 21 G2 subjects. However, HBsAg specific IFN-␥-producing T cells were also detectable in 6/10 (60%) of the 10 G3 subjects whose anti-HBs were <10 mIU/ml 10–12 days post-booster, including 2 subjects who had anti-HBs <10 mIU/ml determined 6–7 months after booster (Fig. 3A). These results suggest that specific T cell memory was present in the vaccinees, even though their anti-HBs antibodies did not increase promptly after HB booster.
3.4. Specific T cells to purified HBeAg determined by ELISPOT in PBMCs Some vaccinees who were naturally boosted from infection showed anti-HBs(+) after age 5, and regained anti-HBs after their anti-HBs waned (Fig. 2). The presence of T cell immunity against HBV infection was further confirmed by detecting specific IFN-␥-producing T cells in response to HBeAg (Fig. 3B), which is not included in any of the HB vaccines. All 13 subjects from G1, whose anti-HBs were ≥10 mIU/ml pre-booster, showed the presence of specific T cell responses to the purified HBeAg, but with large variations in SPF. Among the 21 G2 subjects whose anti-HBs <10 mIU/ml but were ≥10 mIU/ml postbooster, 19/21 had detectable HBeAg-specific T cells. In the 10 G3 subjects whose anti-HBs were <10 mIU/ml both pre- and post-booster, HBeAg-specific T cells were also detectable in 7/10 (70%), including 3 with anti-HBs <10 mIU/ml 6–7 months after booster.
3.3. Specific T cells to recombinant HBsAg determined by ELISPOT in PBMCs None of the vaccinees became HBsAg-positive, even those in whom anti-HBs humoral immune memory was undetectable, in the subjects with anti-HBs(+) at age 5 years (Fig. 2). To study whether the individuals had a specific T cell immunological memory, we determined the presence of HBsAg-specific T cells in the PBMCs of 44 subjects. Of these, 13 subjects had anti-HBs ≥10 mIU/ml in their sera pre-booster (G1), 21 had anti-HBs <10 mIU/ml pre-booster
Fig. 3. Responses of HBsAg- and HBeAg-specific T cells in subjects after receiving a booster dose of recombinant HB vaccines. (A) The frequency of HBsAg-specific and (B) HBeAg-specific IFN-␥ producing T cells in peripheral blood mononuclear cells (PBMCs) in the subjects determined 10–12 days post-booster. The G1 subjects had anti-HBs >10 mIU/ml pre- and post-booster. The G2 subjects had anti-HBs <10 mIU/ml pre-booster and anti-HBs >10 mIU/ml post-booster. The G3 subjects had anti-HBs <10 mIU/ml both pre- and post-booster.
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3.5. HBV-DNA was detectable in the HB vaccinees who were anti-HBc-positive
Abbreviation: GMC, geometric mean concentration; 95% CI: 95% confidential interval. a P < 0.001 between the GMC determined in the 1 month vs. D10–12 after booster. b The 8 subjects with anti-HBs <10 mIU/ml, 1 month after the first dose of vaccine were followed, but 10 subjects with anti-HBs ≥10 mIU/ml, 1 month afterward dropped the tested. c The 8 subjects with anti-HBs <10 mIU/ml, 1 month after the first dose of vaccine were followed, but 3 subjects with anti-HBs ≥10 mIU/ml, 1 month afterward dropped the tested. d 3 had anti-HBs 4–9 mIU/ml and 3 had no detectable anti-HBs.
P
2 = 2.116, P > 0.05
2 = 0.994, P > 0.05
0 6d /3.6 (2–7.7) 53b /859.5 (843–1000) 31c /823.7 (746–1000) 8b (12.7%)/5.6 (3.1–7.5) 8c (20.0%)/3.1 (2–3.8) 55 (87.3%)/556.2 (390.9–795.6)a 32 (80.0%)/526.9 (350.9–791.4)a 45 (71.4%)/152.3 (102.8–225.7)a 23 (57.5%)/183.2 (138.2–257.7)a
<10 mIU/ml ≥10 mIU/ml <10 mIU/ml ≥10 mIU/ml <10 mIU/ml
18 (28.6%)/3.5 (2.8–4.8) 17 (42.5%)/3.1 (2.2–3.8)
≥10 mIU/ml
Anti-HBs(+) at age 5, n = 63 Anti-HBs(−) at age 5, n = 40
1 month, No. (%)/GMC (95%CI) D10–12, No. (%)/GMC (95%CI)
Table 2 Anti-HBs responses after booster with recombinant HB vaccine.
6–7 months, No. (%)/GMC (95%CI)
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During the follow up (Fig. 2), we noticed that 67 (67/806, 8.31%) subjects were HBsAg(−), anti-HBc(+) at age 20 years. When the booster vaccination study began, they were followed for 4 years. One subject, who was anti-HBs(−) at age 5, converted into HBsAg(+) and 14 retained their anti-HBc(+) status. To check for the presence of HBV-DNA in their sera, fragments of HBV-DNA were amplified in the 14 samples above as well as another 13 sera who were HBsAg(−), anti-HBc(−), at age 20 years. Fragments of the HBV S gene were found in 19/27 by nested PCR amplification (Fig. 4A). After quantification of the serum viral load, HBV-DNA was below 5 × 103 copies/ml in all of them (copy numbers are labeled under each band in Fig. 4A). Sequencing of the S gene in 5 isolates showed that mutations at the “a” epitope (aa 120–150) was present in 1/5 of them (G145R; Fig. 4B). The 4 remaining isolates were identical to the local wild isolate, GU434374 [13].
4. Discussion There are few populations in which infant vaccination history and documentation of immune response to vaccination are still available at the adults (more than 20 years). However, using unique household registration system of China, we were able to follow their responses after neonatal vaccination. In this 806 consecutively followed neonatal HB vaccinees (Fig. 2), 68.1% were HBsAg(−)antiHBs(+) and 31.9% were HBsAg(−)anti-HBs(−) at age 5. During the follow-up by age 20 years, none of the subjects with anti-HBs(+) at age 5 became HBsAg(+). However, 2 subjects with anti-HBs(−) at age 5 became chronically HBsAg(+) at ages 10 and one at age 20 years, respectively. These results demonstrate that persistent immunity against chronic HBV infection was present among adults who had responded well to the vaccine at an early age. However, subjects who failed to respond or responded weakly to the vaccine, being showed anti-HBs(−) at age 5, might not be fully protected, and may be at risk of infection with HBV at older ages. Nevertheless, the possibility of becoming chronic HBsAg carriers was significantly less after the ages of 10 and 20 years compared with age 5. HB vaccination before age 5 may be critical to prevent chronic HBsAg. Serum levels of anti-HBs ≥10 mIU/ml induced by the HBV vaccine are considered to be protective from HBV infection. Levels decline rapidly during the first year and then more gradually [3]. Nevertheless, the immune memory is maintained for at least 12 years despite the disappearance of circulating antibodies [16]. Only about 20.5% of 18- to 23-year-old college students in Taiwan kept humoral immune memory [10]. The current study, conducted in a rural area of China, showed that 71.4% of the 24-year-old subjects, who responded well to the vaccine at their early ages, being showed anti-HBs(+) at age 5 years, had humoral immunological memory. For those who responded weakly to the vaccine, being showed anti-HBs(−) at age 5 years, the humoral memory was still detectable at 57.5% of the subjects. This percentage is much higher than that reported in Taiwan [10]. The difference mostly due to lower coverage of HB vaccination in the rural area that put the vaccinees into higher rate exposed in HBV infection [2,3]. We conducted a specific T cell immunity test after booster vaccination. HBsAg-specific IFN-␥ producing T cells were detectable in 41/44 subjects, including 7/10 subjects whose anti-HBs were <10 mIU/ml 10 days after the first booster. In addition, HBeAgspecific IFN-␥-producing T cells were also detectable in 40 subjects including 2 subjects whose anti-HBs were <10 mIU/ml both 10 days and 6–7 months after the booster. HBeAg was not included in any form of HB vaccines, these results demonstrate that the natural
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Fig. 4. Determination and sequencing of HBV-DNA isolated in neonatal HB vaccinees. (A) S-gene fragments of HBV-DNA amplified by nested PCR. Serum HBV-DNA was quantified by TaqMan real-time PCR and the viral concentration is shown under each PCR product band. (B) Alignment of deduced HBV S proteins from 5 subjects with HBsAg(−)anti-HBc(+) and 1 subject with HBsAg(+) (case #8), who converted from HBsAg(−)anti-HBc(+) at age 20.
infection as a booster might play an important role in maintaining the immune memory and immunity (humoral and cellular) against HBV infection after they were fully vaccinated with HB vaccine as neonates and in infancy as suggested [8]. No correlation between the anti-HBs antibody levels with the frequency of specific T cells was observed, and similar results were found in a study of 18- to 20-year olds in Thailand after neonatal vaccination [17]. Our observations differ from those reported in Taiwan recently [9,10] where most of the college student participants had lost their humoral immune memory after they were immunized as neonates and infants. In this report, the anti-HBs conversion 7–10 days after HB booster was only 20.5% [10] among college students. The conversion rate in our enrolled subjects with the same serological markers was more than twice that. In addition, HBsAg- and or HBeAg-specific memory T cells were detected in 43/44 sampled subjects, which was much higher than that reported in the Taiwanese college students [9]. Specific T cells were still detectable in some subjects who showed no increase in antibody levels after the booster, which is similar to results reported by other investigators [18]. The natural history of anti-HBs and other HBV markers indicated that exposure to HBV might be an important natural booster to maintain the immunological memory against HBV in this area [8]. Whether it is necessary to boost young adults is still under debate. Investigations conducted in Thailand [17] and Taiwan [9,10] suggest that it is necessary to consider 1 or 2 booster doses, especially in high risk groups. In the current study, about 31.9% of the subjects who responded weakly at an early age and 28.6% of those who responded well to the vaccine (with anti-HBs (+) at age 5) failed to respond rapidly to one booster vaccination. These subjects might be exposed in a high risk environment and infected with HBV. Anti-HBc conversion has been studied to estimate the exposure but few studies estimated the risk of disease or viral persistent after the conversion. Recently, we reported the presence occult HBV infection in anti-HBs-positive young adults after neonatal HB vaccination [13]. The current study showed that 19/27 of the subjects who were anti-HBc-positive but HBsAg-negative had detectable HBV-DNA S gene fragments. Of these 19 subjects, 14 were HBsAg(−), anti-HBc(+) for 4 years. Of 5 randomly sequenced isolates, the “a” epitope mutation was found in only 1 subject, but their serum HBV-DNA was at low levels (Fig. 4). This finding is similar to our recent report in subjects who were HBsAg(−), anti-HBs(+), and anti-HBc(+) [13]. In addition, one anti-HBc-positive child born from an HBsAg-positive mother became HBsAg-positive when she was sampled at the age of 24 years. The chances of being infected
with HBV in adulthood increases via horizontal transmission like sexual activity, needle injection, and other social activities. Therefore, HB booster for those with a high risk of contact with HBV infection might be necessary before they achieve adulthood. However, as reported in our previous study, HBV-DNA without mutation of the “a” epitope was also detectable in some vaccinees who had detectable anti-HBs in their serum [13]. Therefore, the presence of anti-HBs might not fully protect them from HBV infection [13]. HB booster vaccination of adolescents who were fully vaccinated at an early age still needs to be carefully evaluated in the clinic.Studies of patients with self-limited HBV infections demonstrated that antiHBV specific CD8+ T cells play critical roles in terminating HBV infection [19]. A defect in specific T cell immunity against HBV antigens caused persistent chronic HBV infection [20]. Therefore, vaccine-conferred T cell immunity might play an important role in protecting the vaccinees from overt HBV infection. This hypothesis is supported by evidence that some vaccinees were anti-HBs negative at age 10–11 years and later converted into anti-HBs-positive at age 24 years without becoming HBsAg positive. Thus, new types of HB vaccines should be able to induce specific T cell responses, as well as stimulate specific neutralizing antibodies. In endemic areas like China, the primary goal of HB vaccination is to protect children from HBV infection and prevent chronic infection, which is seen as HBsAg carriers [2]. This was achieved after the HB vaccine was included into the EPI program in China [4]. The risk of becoming a chronic HBsAg carrier is far lower when the unprotected come into contact with HBV infection in adulthood [21]. Boosting with the current vaccine is not presently recommended in China. However, we found in this study that serum HBV-DNA was detectable in some vaccinees at low levels without a mutation in the “a” epitope. Occult HBV infection occurred in some vaccinees. The importance of occult HBV infection in some liver diseases, including in HCC, has been addressed by other researchers [22–24]. The significance of this infection in the pathogenesis of hepatitis and liver cirrhosis, even in HCC, still needs to be carefully monitored. Prolonged follow-up and surveillance of vaccinees who were immunized at an early age should be continued.
Acknowledgements This work was supported by a grant from State Key Projects Specialized for Infectious Diseases (2008 ZX10002-015) and National Natural Science Foundation of China, 30973387. The authors are grateful to Dr. Eskild Pertersen at the Department of Infectious Diseases, Aarhus University Hospital, Skejby, Denmark for their critical review and valuable comments to the manuscript.
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