Antibody response to revaccination among adult non-responders to primary Hepatitis B vaccination in China

Abstract

About 10% adult failed to develop antibody response after primary hepatitis B vaccination, and revaccination may be an option to improve immune response, but the antibody responses to revaccination in adult non-responders have not been fully examined. Adult non-responders to primary 3-dose hepatitis B vaccination were randomly divided into 2 groups and revaccinated with 20 μg hepatitis B vaccine (HepB) derived from Saccharomyces Cerevisiae (HepB-SC) or 20 μg HepB derived from Chinese hamster ovary cells (HepB-CHO), respectively, at 0-, 1-, 6- month. Seroconversion rate and titer of antibody against hepatitis B surface antigen (anti-HBs) was measured one month after the 1st and 3rd revaccination dose. Anti-HBs seroconversion rates significantly increased from 54.98% [95% confidence interval (CI) 48.60%–61.24%] after the 1st revaccination dose to 89.24% (95% CI: 84.74%–92.79%) after the 3rd revaccination dose (P < 0.001), and the geometric mean titer (GMT) of anti-HBs increased from 12.18mIU/ml (95%CI: 7.81–18.98 mIU/ml) to 208.31 mIU/ml (95% CI: 148.87–291.47 mIU/ml) (P = 0.008).Compared with those with anti-HBs titer <2 mIU/ml after primary vaccination, those with antibody titer ≥2 mIU/ml after primary vaccination had higher seroconversion rate after the 1st dose revaccination (38.36% vs. 78.10%, P < 0.001) and after the 3rd dose of revaccination (84.25% vs. 96.19%, P = 0.003), and had higher antibody titer after the 1st dose of revaccination (3.32mIU/ml vs. 74.21mIU/ml, P < 0.0001) and after the 3rd dose of revaccination (145.73mIU/ml vs. 342.34mIU/ml, P = 0.01). Anti-HBs titer was significantly higher in those revaccinated with HepB-CHO than those revaccinated with HepB-SC after the 3rd dose (131.46 mIU/ml vs. 313.38mIU/ml, P = 0.01). Revaccination on adult HepB non-responders increased the immune response to HepB and may confer further protection against hepatitis B virus infection. If possible, revaccination might be an option to HepB non-responders to secure more protection.

Keywords:

• adult non-responders
• antibody response
• hepatitis B vaccine
• revaccination

Introduction

Over the world, around 350 millions of people were chronically infected with hepatitis B virus (HBV),¹ and one quarter of them would die of chronic liver disease, cirrhosis or hepatocellular carcinoma.² HBV infection is still an endemic disease in China, and the prevalence of hepatitis B surface antigen (HBsAg) was 7.2% among the Chinese population aged 1–59 y and 8–12% in adults above 20 y³

Hepatitis B vaccine (HepB) is the most effective way available to prevent HBV infection, which was recommended to the infants by the Chinese government in 1992. A guideline for adult HepB immunization in China was released by Chinese Center for Disease Control and Prevention (CDC) and Chinese Prevention Medicine Association (CPMA) in 2011, and HepB was recommended to all unvaccinated adults, especially those at high risk for HBV infection.⁴ The full series of HepB primary vaccination are 3 doses on a 0-, 1-, and 6-month schedule for both infants and adults in China. HepB is available to infants and young children free of charge, but it is vaccinated to adults on a voluntary and self-paid basis, even for the adults at high risk.

Although one full series of hepatitis B vaccination could induce protective antibody among more than 95% infants,^5,6 around 5%–10% adults fail to respond or respond poorly to 3 doses of HepB.^7,8 In China, the adult non-responding rate after HepB primary vaccination is around 4.7%–14.22% with difference in the dosage, type of HepB, and the participants.^9–11 Efforts have been made to enhance immunogenicity among adults including the development of new vaccines,^12–14 but the option available now might be to revaccinate with the currently available HepB.¹⁵ Although the effectiveness of HepB revaccination has been documented in non-responders, the risk factors associated with the antibody response after HepB revaccination have not been fully understood, which might be due to the limited sample size of non-responders in previous studies.^16-18

We conducted this study to examine anti-HBs response after revaccination with different number of dose and with different type of HepB among adult non-responders in China and to identify the risk factors associated with the antibody response.

Results

Demographic characteristics of the subjects

Of the 438 non-responders, there were 187 non-responders loss of contact or declined to participate, with 251 non-responders who were enrolled and completed 3 doses of HepB. There was no difference between the participants and non-participants in gender, anti-HBs titer after primary vaccination or HepB type received in primary vaccination (P = 0.76, 0.09 and 1.00, respectively), while there were more subjects aged <40 years in the non-participants than the participants (44.39% vs. 31.47%, P = 0.02). Among 251 participants, there were 124 males and 127 females with average age of 41.90 y (range: 18–49 years). There were 118 subjects revaccinated with 20 μg HepB derived from Saccharomyces Cerevisiae (HepB-SC) and 133 subjects revaccinated with 20 μg HepB derived from Chinese hamster ovary cells (HepB-CHO). There were no differences in age, gender, HepB type received for primary vaccination, and anti-HBs titer after primary vaccination between the 2 vaccine groups (P = 0.33, 0.86, 0.78 and 0.80, respectively), but there were more overweighed participants revaccinated with HepB-CHO (P = 0.02) (Table 1).

Table 1. Demographic characteristics of the adult non-responders receiving different vaccines in revaccination

	HepB-SC group, n(%)N = 118	HepB-CHO group, n (%) N = 133	P value
Age (years)			0.33
18–29	6(5.08)	11(8.27)
30–39	26(22.03)	36(27.08)
40–49	86(72.88)	86(64.66)
Gender			0.86
Male	59(50.00)	65(48.87)
Female	59(50.00)	68(51.13)
BMI
Normal	45(38.14)	41(30.83)	0.02
Overweighed	46(38.98)	74(55.64)
Obese	27(22.88)	18(13.53)
HepB type for primary vaccination		0.78
10 μg/ml HepB-SC	25(21.19)	34(25.56)
10 μg/ml HepB-HP	30(25.42)	35(26.32)
20 μg/ml HepB-SC	32(27.12)	30(22.56)
20 μg/ml HepB-CHO	31(26.27)	34(25.56)
Anti-HBs titer after primary vaccination
Low (<2.0 mIU/ml)	65(55.08)	78(56.85)	0.80
High (≥2.0 mIU/ml)	50(42.37)	56(42.10)

Seroconversion after HepB revaccination

The anti-HBs seroconversion rate was higher after the 3rd dose of revaccination 89.24% [95% confidence interval (CI): 84.74%–92.79%] than after 1st dose of revaccination 54.98% (95%CI: 48.60%–61.24%) (P < 0.001). The same trends were found in the participants with different gender, different body mass index (BMI) level and in those aged 30–39 y and 40–49 y (P ≤ 0.01), but no significant difference was found in anti-HBs seroconversion between the 1st and 3rd revaccination dose among the participants aged 18–29 y (P = 0.07) (Table 2).

Table 2. Anti-HBs seroconversion rate after the first and the third revaccination dose among adult non-responders to primary HepB vaccination

		After the first revaccination dose		After the third revaccination dose
	Number of participants	% (95%CI)	P value	% (95%CI)	P value	1st vs. 3rd doseP value
Overall	251	54.98(48.60–61.24)	NA	89.24(84.74–92.79)	NA	<0.001
Age in years			0.45		0.45
18–29	17	58.82(32.92–81.56)		88.23(63.56–98.54)	0.07
30–39	62	61.29(48.06–73.40)		93.55(84.30–98.21)		0.009
40–49	172	52.32(44.59–59.99)		87.79(81.94–92.28)		<0.001
Gender			0.29		0.89
Male	124	51.61(42.47–60.68)		89.52(82.74–94.30)		0.001
Female	127	58.27(49.19–66.95)		88.98(82.20–93.84)		<0.001
BMI			0.49		0.24
Normal	86	50.00(39.02–60.98)		93.02(85.43–97.40)		0.01
Overweighted	120	58.33(48.98–67.26)		85.83(78.29–91.53)		<0.001
Obese	45	55.56(40.00–70.36)		91.11(78.78–97.52)		0.02
Vaccine received in revaccination	0.12		0.59
HepB-SC	118	60.17(50.75–69.07)		88.14 (80.90–93.36)		<0.001
HepB-CHO	133	50.38(41.58–59.16)		90.22 (83.87–94.69)		<0.001
Anti-HBs titer after primary immunization	<0.001		0.003
Low(<2.0 mIU/ml)	146	38.36(30.44–46.76)		84.25(77.31–89.74)		<0.001
High(≥2.0IU/ml)	105	78.10(68.97–85.58)		96.19(90.53–98.95)		<0.001

NA: Not applicable.

The seroconversion rates after the 1st revaccination dose were 60.17% (95%CI: 50.75%–69.07%) and 50.38% (95%CI: 41.58%–59.16%) among those received HepB-SC and HepB-CHO vaccine, respectively, and the corresponding seroconversion rates increased to 88.14% (95% CI: 80.90%–93.36%) and 90.22% (95%CI: 83.87%–94.69%), respectively, after the 3rd dose of revaccination. The differences in anti-HBs seroconversion rate between the 1st and 3rd dose were statistically significant in both vaccine groups (P < 0.001 for both), but no significant difference was found between the 2 vaccine groups after both the 1st and 3rd revaccination dose (P = 0.12, P = 0.59, respectively) (Table 2).

After the 1st revaccination dose, anti-HBs seroconversion rates were 38.36% (95% CI: 30.44%–46.76%) and 78.10% (95%CI: 68.97%–85.58%) among those with low (<2 mIU/ml) and high (≥2 mIU/ml) antibody titer after primary vaccination, and after the 3rd revaccination dose, the seroconversion rates increased to 84.25% (95% CI: 77.31%–89.74%) and 96.19% (95% CI: 90.53%–98.95%) respectively(Table 2). The anti-HBs conversion rate was significantly higher after the 3rd revaccination dose than after the 1st revaccination dose among those with low antibody titer after primary vaccination and those with high antibody titer after primary vaccination (P < 0.001 for both). The participants with high anti-HBs titer after primary vaccination achieved significantly higher anti-HBs conversion rate than those with low anti-HBs titer after primary vaccination after both the 1st and 3rd revaccination dose (P < 0.001, P = 0.003, respectively). After adjustment for other factors, only antibody titer after primary vaccination remained significant in multivariable logistic regression with high antibody titer had 5.4 times more likely to achieve seroconversion after 3 doses of revaccination (P = 0.003)(Table 4).

Table 3. Anti-HBs titer after the first and the third revaccination dose among adult non-responders to primary HepB vaccination

		After the first revaccination dose		After the third revaccination dose
	Number of participants	mIU/ml (95%CI)	P value	mIU/ml (95%CI)	P value	1st vs. 3rd doseP value
Overall	251	12.18(7.81–18.98)		208.31(148.87–291.47)	0.008
Age in years			0.06		0.41
18–29	17	25.57(5.09–128.39)		275.50(91.15–832.70)		<0.0001
30–39	62	27.29(10.72–69.49)		296.87(143.49–614.19)		<0.0001
40–49	172	8.46(4.97–14.38)		178.34(118.94–267.39)		<0.0001
Gender			0.24		0.64
Male	124	9.32(5.10–17.04)		192.40(124.03–298.47)		<0.0001
Female	127	15.79(8.21–30.40)		225.11(134.83–375.84)		<0.0001
BMI			0.93		0.70
Normal	86	12.06(6.08–23.94)		238.60(137.06–415.36)		<0.0001
Overweighted	120	11.46(5.64–23.31)		179.15(106.99–299.99)		<0.0001
Obese	45	14.54(5.40–39.20)		240.25(111.94–515.60)		<0.0001
Vaccine received in revaccination	0.26		0.01
HepB-SC	118	15.99(9.31–27.45)		131.46(78.59–219.91)		<0.0001
HepB-CHO	133	9.56(4.79–19.07)		313.38(202.97–483.86)		<0.0001
Anti-HBs titer after primary immunization	<0.0001		0.01
Low (<2.0 mIU/ml)	146	3.32(1.81–6.08)		145.73(88.00–241.34)		<0.0001
High (≥2.0 mIU/ml)	105	74.21(46.49–118.45)		342.34(234.02–500.81)		<0.0001

NA: Not applicable.

Table 4. Multivariable analyses on factors associated with seroconversion and antibody titer of anti-HBs after 3 doses of revaccination among adult non-responders

	Seroconversion rate		Antibody titer
Variable	OR (95%CI)	P value	β value (95%CI)	P value
Age (years)
18–29	Reference		Reference
30–39	2.03(0.32–12.96)	0.45	0.02(−0.60–0.64)	0.95
40–49	1.17(0.23–5.91)	0.85	−0.13(−0.71–0.45)	0.67
Gender
Male	Reference		Reference
Female	0.77(0.33–1.84)	0.56	0.04(−0.27–0.31)	0.90
BMI
Normal	Reference		Reference
Overweighed	0.39(0.14–1.08)	0.07	−0.19(−0.51–0.14)	0.26
Obese	0.74(0.19–2.89)	0.67	0.02(−0.40–0.44)	0.92
Anti-HBs titer after primary vaccination
Low (<2.0 mIU/ml)	Reference		Reference
High (≥2.0 mIU/ml)	5.39(1.75–16.61)	0.003	0.44 (0.14–0.73)	0.004
HepB received in revaccination
HepB-SC	Reference		Reference
HepB-CHO	1.82(0.77–4.30)	0.17	0.47 (0.18–0.77)	0.002

Anti-HBs titer after HepB revaccination

The geometric mean titers (GMTs) of anti-HBs were higher after the 3rd revaccination dose (208.31 mIU/ml) than after the 1st revaccination dose (12.18 mIU/ml) (P = 0.008) (Table 3).The same trends were found no matter of gender, age and BMI of the participants (P < 0.0001).

The GMTs after the 1st revaccination dose were 15.99mIU/ml (95%CI: 9.31–27.45 mIU/ml) and 9.56 mIU/ml (95%CI: 4.79–19.07 mIU/ml) among those who received HepB-SC and HepB-CHO vaccine respectively, and the GMTs of anti-HBs increased to 131.46 mIU/ml (95%CI: 78.59–219.91 mIU/ml) and 313.38 mIU/ml (95%CI: 202.97–483.86 mIU/ml), respectively, after the 3rd revaccination (Table 3). The significantly higher anti-HBs titer was found after the 3rd dose than after the 1st dose in both vaccine groups (P < 0.0001 for both). The titer was significantly higher in the participants revaccinated with HepB-CHO than in those revaccinated with HepB-SC after the 3rd revaccination dose (P = 0.01), but the same trend wasn't found after the 1st revaccination dose (P = 0.26) (Table 3).

The GMTs of anti-HBs were 3.32 mIU/ml (95%CI: 1.81–6.08 mIU/ml) and 74.21 mIU/ml (95%CI: 46.49–118.45 mIU/ml) among those with low and high titer level after primary vaccination respectively, but the GMTs significantly increased to 145.73 mIU/ml (95% CI: 88.00–241.34 mIU/ml) and 342.34 mIU/ml (95% CI: 234.02–500.81 mIU/ml), respectively, after the 3rd dose (P < 0.0001 for both) (Table 3). In addition, the anti-HBs titers after both the 1st and the 3rd revaccination dose were significantly higher among those with high anti-HBs titer after primary immunization than those with low anti-HBs titer after primary immunization (P < 0.0001, P = 0.01, respectively) (Table 3).

After adjustment for other factors, antibody titer after primary vaccination and HepB type received in revaccination remained significant in linear regression (P = 0.004 and 0.002, respectively)(Table 4).

Discussion

Similar to the findings in clinical trials,^17,19-23 in adult non-responders to primary hepatitis B primary vaccination, we found that more than half seroconverted after one dose of revaccination and around 90% seroconverted after 3 doses of revaccination, the high conversion rate further verified that the non-responders to primary vaccination would gain protection after revaccination. In addition, even the antibody levels increase after one and 3 doses of revaccination, the average of antibody level was still low after one dose of revaccination, much below the level of at least 100 mIU/ml to confer sufficient long-lasting protection.¹⁹ However, the antibody titer increased dramatically after the 3rd revaccination dose. The higher titer of anti-HBs after HepB vaccination is documented to be associated with the longer duration of protective immunity.^24,25 In countries with endemic HBV infection such as China, the pressure of HBV infection is still high for fair long duration of time in the future and it is important to achieve a long-lasting antibody protection against HBV infection. Therefore, 3 doses of revaccination might be more appropriate for any non-responders seeking for revaccination after primary vaccination in China, just as what was suggested by China CDC and CPMA.⁴

A recent study in Italy found that pre-booster antibody titer higher than 2 mIU/ml might be predictive of an anamnestic response after booster vaccination.³⁸ In our study, the anti-HBs seroconversion rate after HepB revaccination was significantly lower in those whose anti-HBs titer after primary vaccination was < 2 mIU/ml compared with those whose anti-HBs titer after primary vaccination was ≥2 mIU/ml. Our results indicated that a different immune or genetic mechanism about HepB non-response might exist in those whose anti-HBs titer after primary vaccination was very low or undetected. Further studies should be conducted about it.

Two types of HepB including 10 μg and 20 μg are approved to be used in adults by China Food and Drug Administration (CFDA). HepB with higher dosage of antigens is documented to induce higher titer of anti-HBs after primary vaccination,⁹ which could further induce higher anti-HBs seroconversion rate after revaccination among adult primary vaccination non-responders according to our study. Therefore, 20 μg HepB seems more appropriate to be used in adults rather than 10 μg HepB, especially in those with risk factors of non-response such as elder age and obesity.^27,28

HepB-CHO and HepB-SC are 2 types of HepB widely used in China. A higher antibody level after 3-dose revaccination was observed among HepB-CHO group compared with HepB-SC group in our study. The reason for different immunogenicity between these 2 types of HepB has not been fully understood. Previous studies showed that even if the HepB was encoded by the same gene sequence, the immunogenicity differed because of varying molecular size, molecular weight when derived from different expression system.^29,30 HepB-CHO has got a glycosylated structure and the composition is more similar to the wild virus,^31,32 which might play an important role in the different antibody responses. Although a higher titer of anti-HBs was observed after revaccination with HepB-CHO in our study, the seroconversion rate of anti-HBs after revaccination was similar between these 2 vaccine groups. The 20 μg HepB-CHO used in our study is produced domestically (About US$38 for 3 doses), which is much cheaper than the imported HepB-SC used in our study (About US$57 for 3 doses). So 20 μg HepB-CHO seems to be more cost-effective to be used in revaccination among the adult non-responders.

It is reported some demographic and behavioral factors are associated with non-response to HepB primary vaccination, such as increasing age, male gender, smoking, alcohol use, and obesity.^27,28 However, there are only a few studies examined the association between the above factors and the response to HepB revaccination among non-responders. Clemens et al reported that BMI affected the response to one-dose HepB revaccination among HepB non- and low- responders,¹⁹ while a study in Sweden failed to detect any significant association of the response rate after revaccination with different vaccination routes, gender, age, or smoking,³³ similar to the findings of our study. Therefore, the demographic factors associated with immune response to HBV vaccine remain controversial to date and further studies are needed.

There are some limitations in our study. Among 438 non-responsive adults, only 251 subjects (57.30%) completed 3-dose revaccination and were included in the final analysis, and there were more persons under 40 y old in the lost cohort. However, we did not find correlation between age and anti-HBs response after revaccination, which might decrease the adverse effect of the difference in age composition between the participants and non-participants.

In conclusion, high seroconversion rate and titer of anti-HBs could be achieved after 3-dose HepB revaccination in adult HepB primary vaccination non-responders in China, and 3 doses of revaccination might be an option for those non-responders seeking for revaccination to achieve more protection against HBV infection.

Material and Methods

Screening and enrollment of non-responders

In July 2009, a total of 12,119 adults aged 18–49 years, without history of HBV infection, HepB vaccination, hypertension, and underlying medical conditions were recruited in a program of adult HepB vaccination free of charge after informed consent. All subjects with subclinical infection were excluded before enrollment after laboratory testing on HBsAg, anti-HBs and antibody against hepatitis B core antigen (Anti-HBc). The participants were randomly divided into 4 groups to receive 3-dose HepB primary vaccination on 0-, 1-, 6-month schedule with 4 types of hepatitis B vaccines [10 μg HepB-SC, 10 μg hepatitis B derived from Hansenula Polymorpha (HepB-HP), 20 μg HepB-SC and 20 μg HepB-CHO]. Blood samples were collected one month after the 3rd dose of primary HepB vaccination for anti-HBs testing. Non-responders were defined as those whose anti-HBs titer after HepB primary vaccination was less than 10 mIU/ml and a total of 438 (10.20%) non-responders were identified (Fig. 1).

Figure 1. Procedure of screening for non-responders.

HepB revaccination and demographic information collection

The available non-responders enrolled were randomly divided into 2 groups on the places they lived. Both groups were revaccinated with additional 3 doses of HepB at 3 months, 4 months and 9 months after the 3rd dose of HepB primary vaccination. One group received 20 µg HepB-SC (GSK, UK) and the other group received 20 µg HepB-CHO (North China Pharmaceutical Co., Ltd., Hebei, China). All participants were interviewed before revaccination by the study staff from local CDC to get their demographic information including age, gender, weight, height, and histories of HBV infection and HepB vaccination.

Specimen collection and laboratory testing

After the 1st and the 3rd dose of revaccination, 3ml blood samples were collected from each participant. Serum was isolated and was frozen at −20□ for testing. The specimens were tested for anti-HBs by Chemiluminescence Microparticle Imunoassay (Abbott, USA) at Shandong Provincial CDC. Samples with anti-HBs≥1000 mIU/ml were diluted for further testing.

Statistical analyses

The seroconversion rates after the 1st and 3rd dose of revaccination across different groups of age, gender, BMI, vaccine received in revaccination, and anti-HBs titer after primary vaccination were assessed with Pearson Chi-square test and between the 1st and 3rd dose of revaccination. The anti-HBs titers after HepB revaccination were log-transformed and were compared among different groups with student t-test or Analysis of Variance wherever appropriate, and with paired t-test between titers after the 1st and 3rd dose of revaccination. Multivariable logistic regression and linear regression models were built to assess the independent contribution from different risk factors to seroconversion or titers of anti-HBs after adjustment for potential confounders. The BMI was categorized into 3 levels (normal: BMI<24 kg/m²; overweighed: 24 kg/m²≤BMI<28 kg/m²; and obese: BMI≥28 kg/m²) based on the national data collected in China.³⁴ The titer of anti-HBs titer after primary vaccination was categorized to low primary titer group (those with anti-HBs <2 mIU/ml after primary vaccination) and high primary titer group (those with anti-HBs titer ≥2 mIU/ml after primary vaccination) as prior report.²⁶ Seroconversion after revaccination was defined as anti-HBs≥10 mIU/ml. All analyses were conducted with SPSS 13.0 and the P value < 0.05 was considered to be statistically significant.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Ethical Statement

Shandong CDC Ethics Committee approved the protocol and consent forms were signed before starting.

Acknowledgments

We thank our colleagues at Zhangqiu County CDC of Shandong Province for their help in data collection. We also thank Ms. Jing Wang for her assistance with word editing.

Funding

This study was supported by the grants from the Major Project of National Science and Technology (No. 2012ZX100022001, 2013ZX10004902) and from the Shandong Medical and Health Science and Technology Development Programs (No. 2009QZ017, 2014WS0373).