Responses to hepatitis B vaccine in isolated anti-HBc positive adults

ABSTRACT

Immune responses of isolated anti-HBc subjects are not well characterized in populations in China. This study aimed to evaluate immune responses to hepatitis B vaccination in isolated anti-HBc positive subjects. A cohort of 608 subjects were selected and separated into isolated anti-HBc (negative for HBsAg and anti-HBs, positive for anti-HBc) and control (negative for HBsAg, anti-HBs, and anti-HBc) groups, who were matched by age and sex. All subjects received 3 doses of hepatitis B vaccine (20μg) at months 0, 1, and 3, followed by testing for serological responses 1 month after the third vaccination. The positive seroprotection rate and geometric mean titer (GMT) for hepatitis B surface antibody (anti-HBs) of isolated anti-HBc subjects were significantly lower than those in the control group(86.2% vs.92.1%, P = 0.02; 47.26 vs.97.81 mIU/mL, P < 0.001). When stratified by age, positive seroprotection rate in the isolated anti-HBc group were 92%, 88.5% and 79.4% in the 20–34, 35–49, and 50–60 y old subgroups, respectively (χ2 = 5.919, P = 0.04). Additionally, the GMT level for anti-HBs in the isolated anti-HBc group for different age subgroups were 104.43, 47.87 and 31.79 mIU/mL respectively (χ2 = 19.44, P < 0.001). The GMT level for anti-HBc before vaccination were negatively correlated with GMT for anti-HBs after 3 doses of hepatitis B vaccine (r = −0.165, P < 0.001). In conclusion, isolated anti-HBc positive subjects can achieve good immune responses after hepatitis B vaccination, and the positive seroprotection rate and GMT level for anti-HBs were lower than the control group. Better responses could be observed in young adults, and significant negative correlations were found between GMT of anti-HBc before vaccination and GMT of anti-HBs after vaccination.

KEYWORDS:

• hepatitis B virus
• isolated anti-HBc
• vaccine

Introduction

The presence of hepatitis B core antibody (anti-HBc) in the absence of hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs) was defined as “isolated anti-HBc.” Anti-HBc can be virtually detected in all individuals who had been previously infected with hepatitis B virus (HBV), irrespective of whether they had recovered or actively infected. The isolated anti-HBc was rarely found among members of low-risk populations, but more common among members of high-risk groups. Isolated anti-HBc may indicate a “window” phase that follows acute HBV infection—a period during HBsAg has diminished from the circulation without anti-HBs appearance; a chronic HBV carrier state, in which HBsAg level is below the detected limit of routine assay or it may indicate that anti-HBc persists longer in persons who have had HBV infection than HBsAg.^1-3 Another possibility is that anti-HBc findings represented false positive results or resulted from interactive antibody, and that such individuals had never been exposed to HBV.⁴ The prevalence of these cases can range from 0.1% to 32% in various surveys,^5-12 depending upon the prevalence of HBV infection and patients investigated.

However, whether isolated anti-HBc individual requires vaccination has remained a debate. Some surveys found that patients with isolated anti-HBc exhibited strong resistance to reinfection and did not require vaccination,¹³ while others suggested that a reasonable approach would be to recommend that such subjects be vaccinated against hepatitis B and then to assess their response to vaccination. Some studies had shown that preventive antibody levels can be achieved after HBV vaccination in most isolated anti-HBc individuals.¹⁴ However, isolated anti-HBc subjects and their immune responses have not been studied in populations in China. This study aimed to evaluate the responses to hepatitis B vaccination in isolated anti-HBc positive subjects compared with subjects who tested negative for HBsAg, anti-HBs, and anti-HBc.

Results

Baseline characteristics

A total of 608 subjects were recruited including 77 males (25.3%) in the isolated anti-HBc group and 78 males (25.7%) in the control group. The mean age of the isolated anti-HBc group was 45.1 ± 9.1 y old, while the control group was 44.3 ± 9.6 y old; there were no significant difference between the 2 groups (t = –1.008, P = 0.31).

Immune responses to the HBV vaccine

In Table 1, the positive seroprotection rate was 86.2% in the isolated anti-HBc group, and 92.1% in the control group (χ2 = 5.507, P = 0.02). At 1 month after the third vaccination, the anti-HBs GMT of subjects in the isolated anti-HBc group was 47.26 mIU/mL, compared with 97.81 mIU/mL in the control group. These difference between the 2 groups was statistically significant (Z = −4.088, P < 0.001).

Table 1. Comparison of the positive seroprotection rate and geometric mean titer of anti-HBs.

Group^*	N	PSR(%)	GMT (Range) (mIU/ml)
Isolated anti-HBc	262	86.2	47.26 (17.29–190.60 )
Control	280	92.1	97.81 ( 27.23–507.55)
χ2/Z		5.507	−4.088
P		0.02	0.00

* Isolated anti-HBc group (negative for HBsAg and anti-HBs, positive for anti-HBc) and control group (negative for HBsAg, anti-HBs, and anti-HBc) ; n: number of anti-HBs positive seroprotection; PSR: positive seroprotection rate

After three doses of the vaccine, the anti-HBs positive seroprotection rates of low, normal and high responses in the isolated anti-HBc group were 48.7%, 30.3% and 7.2%, respectively, compared with 43.4%, 36.2% and 12.5% in the control group. There were significant differences in the distribution of anti-HBs level between the 2 groups(χ2 = 11.694, P = 0.009). Furthermore, we found that high responders in the control group showed higher positive seroprotection rates than those of the isolated anti-HBc group (χ2 = 4.734, P = 0.03). However, the positive seroprotection rates of the low and normal responders in the control and isolated anti-HBc group were not significantly different (χ2 = 1.695, P =0.19; χ2 = 2.402, P = 0.12, Table 2).

Table 2. Comparison of anti-HBs titer distribution after 3 doses of vaccination.

	N(%)
Group^*	Non-response	Low response	Normal response	High response
Isolated anti-HBc	42 (13.8)	148(48.7)	92(30.3)	22(7.2)
Control	24 (7.9)	132(43.4)	110(36.2)	38(12.5)
χ2	5.507	1.695	2.402	4.734
p	0.02	0.19	0.12	0.03

* Isolated anti-HBc group (negative for HBsAg and anti-HBs, positive for anti-HBc) and control group (negative for HBsAg, anti-HBs, and anti-HBc); N: number of anti-HBs positive seroprotection; %: positive seroprotection rate.

Comparison of the positive seroprotection rates and geometric mean titers by age

The positive seroprotection rate and GMT for anti-HBs in different age groups were shown in Table 3. There were no significant differences among different age groups in the control group for the anti-HBs positivity seroprotection rates (χ2 = 0.525, P = 0.77) or anti-HBs GMT (χ2 = 0.215, P = 0.90). However, in the isolated anti-HBc group, the positive seroprotection rate and GMT level for anti-HBs were higher in the youngest group. Positive seroprotection rates were 92%, 88.5% and 79.4% in the 20–34, 35–49, and 50–60 y old groups, respectively (χ2 = 5.919, P = 0.04). The GMT level of anti-HBs titers in the isolated anti-HBc group of different ages were 104.43, 47.87 and 31.79 mIU/mL respectively (χ2 = 19.44, P < 0.001). The nonparametric analysis also showed a significant differences between 35–49 and 50–60 age group (P = 0.013) and 20–34 and 50–60 age group (P = 0.012).

Table 3. Comparison of the positive seroprotection rate and geometric mean titer by age.

	Control			Isolated anti-HBc
Group	N	PSR(%)	GMT(Range)(mIU/ml)	n	PSR(%)	GMT(Range)(mIU/ml)
20–34	59	92.2	115.6 (28.5–548.68)	46	92.0	104.43 (36.67–575.27)^*
35–49	135	93.1	93.38 (25.64–453.19)	139	88.5	47.87 (20.78–239.91)^**
50–60	86	90.5	99.45 (28.95–534.07)	77	79.4	31.79 (11.41–113.10)^***
χ2		0.525	0.215		5.919	19.44
P		0.77	0.90		0.04	<0.001

n: number of anti-HBs positive seroprotection; PSR: positive seroprotection rate;

* the Mann-Whitney test to compare the geometric mean titer between age group 20–34 and 35–49 (P = 0.15);

** the Mann-Whitney test to compare the geometric mean titer between age group 35–49 and 50–60 (P < 0.017));

*** the Mann-Whitney test to compare the geometric mean titer between age group 20–34 and 50–60 (P < 0.017).

Correlations of GMT for the anti-HBc and anti-HBs

Using Spearman rank correlation analysis, we found that the anti-HBc GMT before vaccination was negatively correlated with the anti-HBs GMT after 3 doses of hepatitis B vaccine (r = –0.165, P < 0.001).

Discussion

Responses to hepatitis B vaccine in isolated anti-HBc subjects range from 56% to 100% in various studies.^2,6,7 Ural and Findik observed that almost half of isolated anti-HBc subjects developed a primary immune response when challenged with HBV vaccine.¹⁵ A study of health care workers in Korea reported that the isolated anti-HBc positive group showed a relatively high rate of anti-HBs seroprotection (89.5%).¹⁶ Another study in China also demonstrated that among isolated anti-HBc patients, 91.67% developed a primary anti-HBs response.¹⁷ In our present study, the positive seroprotection rate was 86.2% in the isolated anti-HBc group, which also showed that isolated anti-HBc positive subjects could achieve robust immune responses after hepatitis B vaccination.

In previous studies, most individuals who tested positive for isolated anti-HBc mounted a slow or primary, rather than a rapid or anamnestic response after hepatitis B vaccination,^18-21 which suggested false positive reactivity to anti-HBc; this possibility could not be excluded in our study. Occult HBV infection, appears to be less implicated because of the low rate of nonresponses following the completion of a vaccination schedule. However, neither HBV DNA nor Hepatitis Be Antigen (HBeAg) was tested in our study, so the hepatitis B vaccination alone is not adequate to categorize adult subjects with isolated anti-HBc.

Our present study showed that the positive seroprotection rate and anti-HBs GMT of the isolated anti-HBc group were significantly lower than those of the control group. Byung-Ho Kim, et al.⁶ reported that anti-HBs seroconversion was achieved in the anti-HBc positive subjects after full vaccination, and this rate was comparable with that of the control subjects (89.5% vs. 96.6%, P = 0.067) in a study of health care workers in Korea. However, the mean titer was significantly lower in the isolated anti-HBc positive group after 3 round of vaccination (392.9 ± 122.7 vs. 669.3 ± 38.1 mIU/mL, P < 0.001). These findings are in accord with those CY Chan et al.²² who studied a cohort of subjects in Taipei. All of these studies suggested that the geometric mean anti-HBs level was significantly lower in the isolated anti-HBc group, and that minor differences in the positive seroprotection rate could be attributed to subject age, epidemiologic differences (prevalence of HBV infection) or prior vaccination history. In addition, the percentage of individuals with low responses (10 ≤ anti-HBs < 100 mIU/ml) after 3 round of vaccination is high, while the anti-HBs GMT tested one month after third injection are low compared with the other studies, this may due to the schedule of vaccination (0, 1, 2). This finding is consistent with one study of 17 pairs of children who were vaccinated with recombinant hepatitis B vaccine at months 0, 1, and 2; the geometric mean anti-HBs titers were significantly lower in the isolated anti-HBc group( 80.4 ± 26.3 vs. 188.2 ± 92.1, P < 0.001).²³

Better responses were observed in young adults, and the positive seroprotection rate and GMT value of anti-HBs declined with age in the isolated anti-HBc group, while no significant difference associated with age was detected in the control group. There are several explanations for these findings. First, old age has been shown to be an important factor that affects immunogenicity, and in some other studies, age-based differences were obvious as the seroconversion rates were 93.55% and 88.3% in subjects aged 20–35 or 36–46 y old, respectively, among individuals who were negative for HBsAg (P < 0.05).²⁴ Similar findings were reported by Yao Jun et al, who found that the seroprotection rate and GMT value of anti-HBs were significantly higher in the 20–35 y old group than in the 36–46 y old group (P < 0.05).^14,25 The other explanation is that older subjects may have higher anti-HBc level before vaccination. In our present study, the anti-HBc GMT increased with age, and the anti-HBc GMT in 50–60 y old group was higher than that of the 20–34 and 35–49 y old groups. We found that GMT for anti-HBc had a significant negative correlation with anti-HBs GMT may be another possible explanation.

The isolated positivity of anti-HBc is a special HBV infection status. Although this status was often regarded as one of the important reasons for non-response to HepB vaccine, the correlation between anti-HBc positivity and response to HepB vaccine is still controversial. Our study evaluated the immune response to hepatitis B vaccination in isolated anti-HBc positive subjects in China, also compared the relation between the anti-HBc and anti-HBs GMT, which were meaningful in the vaccination of isolated anti-HBc positive subjects. In addition, the vaccination schedules should be further researched. There were several limitations to our present study. First, we failed to acquire data about anti-HBs after the first-dose vaccination, so we could not evaluate whether they represented an amnestic response. Additionally, because HBV DNA and HBeAg were not tested in our present study, we could not categorize the adult subjects with isolated anti-HBC.

In conclusion, isolated anti-HBc positive subjects can achieve good immune effects after hepatitis B vaccination, but the positive seroprotection rate and GMT values of anti-HBs titers were lower than those of the control group. Better responses could be observed in young adults, and significant negative correlations were detected between the GMT level of anti-HBc before vaccination and the GMT level of anti-HBs after vaccination.

Materials and methods

Study procedures

This study was carried out in Putuo and Xianju Counties in Zhejiang Province. Subjects who were 20–60 y old were recruited and asked to complete a questionnaire; all of these participates were tested for HBsAg, anti-HBs and anti-HBc. A total of 304 subjects whose HBsAg and anti-HBs titers were negative, but tested anti-HBc positive were recruited as included in an anti-HBc group. Another 304 subjects were enrolled as a control group tested negative for HBsAg, anti-HBs, and anti-HBc and matched by age and sex to those in the isolated anti-HBc group.

Exclusion criteria were as follows: organ transplantation, renal dialysis, subjects with hepatitis C and acquired immune deficiency syndrome, vaccination contraindication and previous vaccination against HBV. All study subjects provided signed informed consent according to the study protocol and were willing to receive the HBV vaccine.

All subjects were vaccinated with a recombinant hepatitis B vaccine (dosage 20μg, Kangtai Biotech, Shenzhen, China) at months 0, 1 and 3. 3 ml blood samples were collected from each subject at 1 month after the third vaccination and preserved for further testing.

Laboratory testing

Frozen separated serum samples were sent to ADICON Clinical Laboratories Inc. in Hang Zhou for HBsAg, anti-HBs and anti-HBc quantification by chemiluminescence microparticle immunoassay (CMIA). An Architect-i2000_SR (Abbott, US) analyzer was used to perform the CMIA. Samples with anti-HBs levels≥1000 mIU/ml were diluted for further testing, while samples with anti-HBs levels>15000mIU/ml were excluded from further analysis. The following signal-to-noise (S/N) ratios were considered to indicate positivity: HBsAg≥0.05IU/ml. The maximum of HBsAg was 250 IU/ml. Anti-HBc levels ≥1 S/CO were positive. Previous study suggested anti-HBs levels of 2–9.9 mIU/mL could not be considered negative in subjects with a history of vaccination or resolved infection.²⁶ In our study those who have been vaccinated against HBV were excluded, so anti-HBs≥10 mIU/mL was considered positive and defined as having protective effect against HBV infection. A low response was defined as follows: 10mIU/ml≤anti-HBs<100mIU/ml. A normal response was defined as follows: 100 mIU/ml≤anti-HBs<1000 mIU/ml. A high response was defined as follows: anti-HBs≥1000 mIU/ml after the third dose of vaccine.²⁷

Statistical evaluation

Statistical analyses were performed using SPSS version 19.0 (SPSS Inc. Chicago, IL, USA). Continuous variables were expressed as means standard deviation, and categorical variables as frequencies and proportions.

Comparisons between groups were made using the χ² test, t-test, or nonparametric analysis Associations between anti-HBc and anti-HBs geometric mean titers were assessed using Pearson's correlation coefficient. A P-value of 0.05 or less was considered to indicate a statistically significant difference.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the Pu Tuo Center for Disease Control and Prevention (CDC), Xian Ju CDC and other relevant personnel for their contributions to this study.

Funding

This project was supported by the National Scientific and Technological Major Project of China (No. 2011ZX10004-901,2013ZX10004-904, No. 2014ZX10004008).