The response of hepatitis B vaccination on seronegative adults with different vaccination schedules

Abstract

The purpose of this study was to compare the response of hepatitis B vaccination with different vaccination schedules among seronegative adults, and to provide suitable vaccination schedules for floating and fixed population. The study included adults aged 20 to 39 y without prior history of vaccination with hepatitis B vaccine. The serum samples were collected and tested for hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), and hepatitis B core antibody (anti-HBc) levels. Out of all, 686 adults who were negative for anti-HBs, anti-HBc and HBsAg were vaccinated with 10 ug hepatitis B vaccine at 0, 1 and 3, 6 or 12 month schedules, and their antibody titers were monitored. The rates of completion of the vaccination in floating and fixed population were 90.4% and 94.1% respectively (p = 0.061). The anti-HBs positive rates in adults vaccinated at 0, 1 and 3 ,6 or12 month were 83.9%, 88.2% and 94.2% respectively (P = 0.0003). The corresponding geometric mean titers (GMTs) were 61.19 (95%CI:47.10-81.23) mIU/mL, 214.04(95%CI:157.14-291.61) mIU/mL and 345.78(95%CI:251.25-475.77) mIU/mL, respectively ( P < 0.0001). Vaccination of hepatitis B with both 0–1–6 and 0–1–12 month schedules in adults result in better level of immune responses. Also, a longer vaccination schedule (0–1–12 month) may be more suitable for floating population and 0–1–6 month schedule is recommended for the fixed population.

Introduction

Hepatitis B virus (HBV) infection is a worldwide health problem.¹ It is estimated that 2 billion people have been infected and 350 million are chronic carriers of the virus. HBV related diseases lead to0.6 to 1.2 million death globally every year.^2-5 In 2006, hepatitis B surface antigen (HBsAg) carrier rate in the general Chinese population aged 1 to 59 was 7.18%.^6-7 This suggest that more than 93 million people were chronically infected with HBV in China.⁸ Currently, hepatitis B vaccination is regarded as the most economical and effective method for the prevention and control of hepatitis B infection.^9-12

The universal infantile hepatitis B vaccination program began in China in the year 1992. After more than 10 y of mass hepatitis B vaccination, a great success in the prevention and control of HBV infection had been achieved. From 1992 to 2006, the HBsAg positive rate among children younger than 15 y old decreased significantly from 10% to 2.08% respectively.¹³ As a consequence of that, adulthood hepatitis B vaccine immunization should receive more attention. A report from a study conducted at the United States of America (USA) showed that the highest proportion of new hepatitis B infections occur in the age group of 25 to 44 y¹⁴ Vaccination for high-risk adults had already begun in USA in 1998.¹⁵ In addition, there were vaccination programs for high-risk adults in Britain and Italy.^16,17

In China, some experts proposed the importance of strengthening the measures for prevention of HBV infection in the population aged over 25 y¹⁸ But, the hepatitis B vaccine vaccination for adults has not been universally carried out, and it is also not free. So, some experts¹⁹ suggested that the government should reinforce the capital investment in hepatitis B vaccine vaccination for adults. The Chinese National Seroepidemiological survey revealed that the rate of hepatitis B vaccination is 12.02%¹³(95%CI: 10.79%–13.25%) among the rural population aged 15–59 y It also mentioned that the compliance of the vaccination is poorer in adults as compared to infants. . Therefore, different measures are required to improve the compliance in adults.

The floating population is special in China and it is also called internal migration in other countries. There are annually 230 million floating population in China. Generally, they leave their hometowns to find jobs in other cities and change their jobs frequently. After working for 11 months, most of them return home and rest for one month to celebrate the spring festival. Of this population, about 80% is from the rural community, with inadequate knowledge of health care and relatively poor conditions due to low income. The current floating population in China has an average age of 28 y²⁰ Some of these people might have multiple sexual partners that might increase the risk of sexual transmission of HBV.^21,22 And more importantly, there are high chances of infants getting infected if women of child-bearing age gets infected with HBV. The standard vaccination schedule for hepatitis B vaccine is 0–1–6 month, and many adults decline to receive the second or third dose for the occupational reasons. The time span of each dose may influence the compliance of adult vaccination, thereby affecting the effect of immunization.

This study aimed to evaluate the response of different vaccination schedules of hepatitis B on seronegative adults. Vaccinations were carried out at 0–1–3, 0–1–6 and 0–1–12 month schedules among adults aged 20 to 39 y It also analyzed the compliance in fixed and floating population, to provide suitable vaccination schedule for different population.

Results

Study subject characteristics: A total of 1651 adults aged between 20–39 y were initially screened for inclusion and exclusion criteria. Out of them, HBsAg was positive in 1.7%, anti-HBc in 39.0%, and anti-HBs in 43.8%, respectively. So, these patients were excluded from the study. The remaining 744 subjects who had negative HBsAg, anti-HBs, and anti-HBc (45.1%) were eligible for the study.

Out of 744 subjects, 374 were floating population and 370 were fixed population. Overall, these 744 subjects were divided into 3 groups; group I (vaccinated at 0–1–3 month schedule, 264), group II (vaccinated at 0–1–6 month schedule, 249), and group III (vaccinated at 0–1–12 month schedule, 231) respectively. The number of floating population in group I, II and III were 140, 123 and 111, respectively. Out of 744 adults, 58 adults did not receive the third dose of hepatitis B vaccination. Of them, there were 11, 17 and 8 floating populations in group I, II and III, respectively. The rates of completion of the vaccination schedule in group I-III were 94.3%, 92.0% and 90.0%(χ² = 1.025, p = 0.599), respectively. The rate of completion of the vaccination in floating and fixed population was 90.4% and 94.1%(χ² = 3.504, p = 0.061), respectively.

In the floating population, the rate of completion of the vaccination schedule in groups was 92.1%, 86.2% and 92.8%(χ² = 3.738, p = 0.154), respectively. In the fixed population, the rate of completion of the vaccination schedule in different groups was 96.8%, 97.6% and 87.5%(χ² = 13.721, P < 0.05), respectively. After multiple comparisons (α = 0.0167), the rates of completion in group I and II were higher than in group III (χ²_{I VS. III} = 7.305, χ²_{II VS. III} = 9.280, P < 0.01). In group II, the rate of completion in the fixed population was higher than that of the floating population(χ² = 11.027, P < 0.05). The rate of completion among different groups and different populations is shown in Table 1.

Table 1.

The rate of completion among different groups and different populations

	Fixed population		Floating population
Groupa	N	Completed	N	Completed	Coefficient value	p-value
Group I	124	120(96.8%)c	140	129(92.1%)d	χ2 = 2.632b	p = 0.105
Group II	126	123(97.6%)c	123	106(86.2%)d	χ2 = 11.027b	p < 0.05
Group III	120	105(87.5%)c	111	103(92.8%)d	χ2 = 1.802b	p = 0.180
Total	370	348(94.1%)	374	338(90.4%)	χ2 = 3.504b	p = 0.061

^aSubjects were assigned to one of 3 groups depending on the vaccination schedule they chosen. Group I: vaccinated at 0–1–3 month schedule; group II: vaccinated at 0–1-6 month schedule; group III: vaccinated at 0–1–12 month schedule;

^bThe Pearson Chi-Square test to compare the rate of completion between fixed population and floating population;

^cThe Pearson Chi-Square test to compare the rate of completion between group I, II and III in fixed population (χ² = 13.721, P < 0.05);

^dThe Pearson Chi-Square test to compare the rate of completion between group I, II and III in floating population (χ² = 3.738, p = 0.154).

Out of 744 adults, 686 finally completed the vaccination schedule. Their serum sample analyses were conducted after completion of all 3 vaccinations. Among these subjects, the gender ratio (male:female) was 1:1.33, and the average age was 30.05(range:20.01–39.98) years. The sex ratio or the average subject age among groups was not significantly different (all P > 0.05). The age and sex distribution of study participants is shown in Table 2.

Table 2.

Age and sex distribution of study subjects

		Age		Sex
Groupa	No.	Median	Range	Male	Female
Group I	249	30.23	20.01–39.76	100	149
Group II	229	29.42	20.01–39.92	111	118
Group III	208	30.25	20.10–39.98	84	124
Total	686	30.05	20.01–39.98	295	391
Parameter		1.3606b		4.1966c
p-vaule		0.5065		0.1227

^aSubjects were assigned to one of 3 groups depending on the vaccination schedule they chosen. Group I: vaccinated at 0–1–3 month schedule; group II: vaccinated at 0–1–6 month schedule; group III: vaccinated at 0–1–12 month schedule;

^bThe Kruskal-Wallis test to compare age between group I, II and III;

^cThe Pearson Chi-Square test to compare sex between group I, II and III.

Antibody response

After completion of the vaccination schedules, the anti-HBs positive rates (anti-HBs ≥ 10 mIU/mL) in the various groups were 83.9%, 88.2%, 94.2% (P < 0.05) respectively. Out of this, 43.8%, 66.8%, and 73.1% (P < 0.05) in group I, II and III were considered to be good responders (anti-HBs≥100 mIU/mL). Also, 7.6%, 27.1%, 36.1% (P < 0.05) in group I, II and III anti-HBs were considered to be hyper-responders (anti-HBs ≥ 1000 mIU/mL).

After multiple comparisons (Boferroni, α = 0.0167), we found that the anti-HBs positive rate in Group III was higher than that of group I (χ² _{I VS. III} = 11 .9117, p = 0.0006). But, the anti-HBs positive rates in group I-II and group II-III were not significantly different (χ²_{I VS. II} = 1 .808, χ² _{II VS. III} = 4.9251, P > 0.0167). The percentages of good responders in both group II and III were higher than group I (χ²_{I VS. II} = 25.1715, χ²_{I VS. III} = 39.7257, P < 0.0001), while the percentages of good responders in group II and group III were not significantly different (χ²_{II VS. III} = 2.029, p = 0.154). The percentages of anti-HBs ≥ 1000 mIU/mL in both group II and III were also higher than group I (χ²_{I VS. II} = 31.1834, χ²_{I VS. III} = 56.0574, P < 0.0001).

The anti-HBs GMTs were 61.19(95%CI: 46.10-81.23) mIU/mL, 214.04 (95%CI: 157.14-291.61) mIU/mL and 345.78 (95%CI: 251.25–475.77) mIU/mL in group I-III (P < 0.05), respectively. Anti-HBs titers of Group I were much lower than that of group II and III. The distribution of anti-HBs titers in different vaccination schedules is shown in Table 3.

Table 3.

Anti-HBs titer distribution 1 month post vaccination in 20–39 y adults

	Antibody titer after vaccination (mIU/mL)
Groupa	Total (N)	≥10 n(%)	≥100 n(%)	≥1000 n(%)	GMT	GMT95% CI
Group I	249	209(84.0)b	109(43.8)c	19(7.6)d	61.19e	46.10–81.23
Group II	229	202(88.2)b	153(66.8)c	62(27.1)d	214.04e	157.14–291.61
Group III	208	196(94.2)b	152(73.1)c	75(36.1)d	345.78e	251.25–475.77
Total	686	607(88.5)	414(60.4)	156(22.7)	157.14	130.92–188.63

^aSubjects were assigned to one of 3 groups depending on the vaccination schedule they chosen. Group I: vaccinated at 0–1–3 month schedule; group II: vaccinated at 0–1–6 month schedule; group III: vaccinated at 0–1–12 month schedule;

^bThe Pearson Chi-Square test to compare the anti-HBs-positive-rates between group I, II and III (χ² = 11.8132, p = 0.0027);

^cThe Pearson Chi-Square test to compare the rates of anti-HBs ≥100 mIU/mL between group I, II and III (χ² = 46.6638, P < 0.0001);

^dThe Pearson Chi-Square test to compare the rates of anti-HBs ≥1000 mIU/mL between group I, II and III (χ² = 55.8015, P < 0.0001);

^eThe Kruskal-Wallis test to compare anti-HBs between group I, II and III (χ² = 70.2304, P < 0.0001).

When the data in Table 3 were stratified by gender (Table 4). It was also evident from the study that there was no significant difference (χ² = 0.4644, 0.6150, 0.4890; p = 0.4956, 0.4329, 0.4844) between males and females in regard to anti-HBs positivity rates in the various groups. The anti-HBs GMTs in group I-III between male and female were not significantly different. The percentage of good responders in female was higher than male (χ² = 5.616, p = 0.018), and the anti-HBs GMTs in female was higher than male (F = 13.599, P < 0.018). The results are shown in Table 4.

Table 4.

Anti-HBs titer distribution 1 month post vaccination in 20–39 y adults stratified by gender

		Male						Female
		Antibody titer after vaccination(mIU/ml)						Antibody titer after vaccination(mIU/ml)
Groupa	Total (n)	≥10	≥100	≥1000	GMT	GMT95% CI	Total (n)	≥10	≥100	≥1000	GMT	GMT95% CI
Group I	100	82(82.0)	38(38.0)	7(7.0)	41.18	25.85–65.61	149	127(85.2)b	71(47.7)	12(8.1)	79.82e	56.05-113.66
Group II	111	96(86.5)	68(61.3)	22(19.8)	148.7	97.52–226.78	118	106(89.8)c	85(72.0)	40(33.9)	301.58e	192.93–471.41
Group III	84	78(92.9)	57(67.9)	25(29.7)	235.28	161.51–397.10	124	118(95.2)d	95(76.6)	50(40.3)	426.97e	274.60–663.90
Total	295	256(86.8)	163(55.3)	54(18.3)	111.97	85.57–146.55	391	351(89.8)	251(64.2)f	102(26.1)	202.91g	158.71–259.42

^aSubjects were assigned to one of 3 groups depending on the vaccination schedule they chosen. Group I: vaccinated at 0–1–3 month schedule; group II: vaccinated at 0–1–6 month schedule; group III: vaccinated at 0–1–12 month schedule;

^bThe Pearson Chi-Square test to compare the rate of anti-HBs ≥ 10 mIU/mL between male and female in group I (χ² = 0.4644, p = 0.4956);

^cThe Pearson Chi-Square test to compare the rate of anti-HBs ≥ 10 mIU/mL between male and female in group II (χ² = 0.6150, p = 0.4329);

^dThe Pearson Chi-Square test to compare the rate of anti-HBs ≥ 10 mIU/mL between male and female in group III (χ² = 0.4890, p = 0.4844);

^eThe Kruskal-Wallis test to compare anti-HBs GMT between male and female in group I, II and III (P > 0.05);

^fThe Pearson Chi-Square test to compare the rate of anti-HBs ≥ 100 mIU/mL between male and female (χ² = 5.616, p = 0.018);

^gThe One-way Analysis of Variance to compare anti-HBs GMT between male and female (F = 13.599, P < 0.018).

Discussion

Evaluating the vaccination schedules against hepatitis B is appealing because it may increase compliance and vaccination coverage. It will also provide protection for the floating or fixed population who are already in high risk groups and environment.

The rate of completion of the vaccination in 3 schedules in the study were quite high. The rates of completion of the vaccination in floating population and fixed population were similar. It may be explained that the researchers had taken enough measures to improve the compliance, and the compliance was more as the vaccination in this study was free. Dunzhe's¹⁹ previous study had shown that 94.35% subjects were willing to get vaccinated if it was for free. In the fixed population, the rates of completion of the vaccination at 0–1–3 and 0–1–6 months were higher than 0–1–12 months schedule. There was, however, no significant difference between the schedules in the floating population. This may be possibly due to the fact that a long vaccination schedule may cause decrease of compliance in fixed population. Therefore, to compare the immunological effect of different schedules and find a suitable schedule to cater for the populationis necessary.

This study showed that 20–39 year-old adults vaccinated at 0–1–6 or 0–1–12 month with 10 μg hepatitis B vaccine had a good immune response, which is similar to Zhangwei's ²³ conventional hepatitis B immunization (0–1–6 month schedule,88.8%) with the CHO hepatitis B vaccine and Pamela Rendi-Wagner's²⁴ hepatitis B immunization (0–1–6 to 12 month schedule; 88.6%). But the immune effect of 0–1–3 month schedule was not as good as 0–1–6 or 12month schedule in adults aged 20–39 y Both anti-HBs positive rates and the anti-HBs titers vaccinated at 0, 1, 3 month were lower and are in accordance with a previous study,.²⁵ The possible explanation for these results may be that, a shorter vaccination interval benefits from generating anti-HBs rapidly while a longer vaccination interval is good for generating anti-HBs both slowly but in high titer.^25,26 A study in Turkey²⁷ enrolled 120 subjects and got them vaccinated according to the accelerated (0–1–2 month) or the more accelerated (0–10–21 day) vaccination schedule also provided the same conclusion.

It is generally assumed higher the anti-HB titers after vaccination, the longer is the period of protection.^28,29 We then categorized adults in the study according to the American Academy of Immunization Practices standards³⁰ (anti-HBs titers 10–99 mIU/mL as hypo-responders, 100 -999 mIU/mL as good-responders , and ≥ 1000 mIU/mL as hyper-responders). The percentage of good-responders vaccinated at 0, 1 and 6 or 12month was nearly 1.5 times higher than that of good-responders vaccinated at 0, 1 and 3 months. The percentage of hyper- responders was nearly 4 times than that of good-responders vaccinated at 0, 1 and 3 months. It could be concluded that the hepatitis B immunization of 0,1 and 6 or 12 month may be the appropriate schedule that provides longer protection than that of vaccination at 0, 1 and 3 month. However, it requires a longer observation.

In contrast to earlier findings, a study in Cuba³¹ confirmed good immune response and rapid priming for the hepatitis B vaccines (Heberbiovac- HB®, Euvax-B®, Hepavax-Gene® and Engerix-B® vaccine ;containing 20 ug HBsAg) in the course of an accelerated 0–1–2 month schedule . The higher dosage of hepatitis B vaccine had resulted in better immune response. Rebedea-I³² also compared immunogenicity and safety of Hepavax-Gene®, Hepavax-Gene® TF and Engerix-B® (containing 20 ug HBsAg) vaccine in adults with 2 vaccination schedules. They had used the priming 0–1–2 month or the standard 0–1–6 month schedule. They observed that 63–70% adults were good-responder after the 0–1–2 month schedule while 94–96% adults were good-responder after the 0–1–6 month schedule.³² A higher titer was elicited and a longer protection was achieved using the standard schedule compared with the accelerated schedule.³² However, longer intervals between the last 2 doses resulted in higher final antibody levels but might increase the risk of HBV infection among individuals who have a delayed response to vaccination.³⁰ Hence, the best vaccination schedule for adults still merits further investigation.

Usually, males were thought to have a reduced responsiveness to conventional hepatitis B vaccines.^24,31 In the present study, the percentage of good responder in female was higher than male, and the anti-HBs GMTs in female was higher than male. When the respective groups were compared, there was no significant difference. The findings coincide with a previous study.³³

There were several limitations of this study. One was that we failed to acquire the data of body mass index (BMI) and the smoking status of the study subjects. Therefore, we do not know whether these factors were evenly distributed within the 3 groups, and whether the negative results had any association with these factors.^34,35 Besides, the generalizability of these results is limited as a result of possible volunteer bias in this study.

In conclusion, 0–1–6 or 0–1–12 month schedule in adults provides a better level of immune responses and should be recommended. Also, for the floating population in China, who return to hometown and take about one month break to celebrate the spring festival, a longer vaccination schedule of 0–1–12 month may be more suitable. And among the fixed population, 0–1–6 month schedule is recommended.

Materials and Methods

Study participants

This study was performed in Tong Xiang County in Zhejiang Province. The participants were 20 to 39 year-old males and females. They were interviewed using the pre-designed questionnaire “Research Questionnaire on Adult Immunization Strategy for hepatitis B.” The same was designed to obtain the demographic details. A total of 3 ml of blood was collected from each participant. Each participant underwent serological screening. Only those who were negative for HBsAg, hepatitis B surface antibody (anti-HBs), and hepatitis B core antibody (anti-HBc) were included in the study. The eligible subjects belonged to either floating or fixed population. The eligible subjects were informed about the study vaccination schedules and other requirements. They received 3 doses vaccination against Hepatitis B virus according to the vaccination schedule that they had chosen. This study was approved by the Institutional Ethics Committee of the Zhejiang provincial Center for Disease Control and Prevention (Hangzhou, China). All the participants had provided written informed consent for the study.

Vaccines and vaccination

The subjects were assigned to group I, II and III depending on the vaccination schedule (lot Nos.2010010106; dosage: 10 μg; produced by the NCPC GeneTech Biotechnology development Co.,Ltd, China). The Group I participants were vaccinated at 0–1–3 month schedule; group II, 0–1–6 month schedule; and group III, 0–1–12 month schedule respectively. Every participant received 3 dosages of hepatitis B vaccine by intramuscular injection in the upper arm deltoid muscle according to the vaccination schedule.

Specimen collection and analysis

One month after the vaccination , 3 ml blood samples were collected from each subject and preserved for anti-HBs quantificational test. Serum was obtained from all blood samples and frozen at −20°C. Samples were sent to ADICON Clinical Laboratories Inc. in Hangzhou for HBsAg (test reagent lot number, 86040LF00), anti-HBs (test reagent lot number, 82521M100) and anti-HBc (test reagent lot number, 85276M500) quantification by chemiluminescence immunoassay (CLIA) using an Architect-i2000 (Abbott, US) analyzer. In cases of non-concordance between the HBsAg and anti-HBs results, samples were re-analyzed. Samples with an anti-HBs titer exceeding 15,000 mIU/mL were excluded from further dilution testing. The following signal-to-noise ratios (S/N) were considered to indicate test positivity: HBsAg ≥0 .05. Anti-HBs antibody ≥10 mIU/mL was positive and defined as having protective effect against HBV infection. Anti-HBc antibody levels ≥1 mIU/mL was positive.

Data collation and analysis

Data were analyzed using SAS 9.1 (Cary, NC). Geometric mean titers (GMTs) were calculated for anti-HBs titers. Because we were unable to detect anti-HBs antibody titer levels less than 0.01 mIU/mL, we assigned a value of 0.005 mIU/mL to these subjects when calculating GMT of anti-HBs,and samples with anti-HBs titer exceeding 15,000 mIU/mL were excluded from further dilution test. We assigned a value of 15000 mIU/mL to these subjects when calculating GMT of anti-HBs. The Chi-square test was used to compare categorical variables .The Kruskal–Wallis test was used to compare GMTs between groups. A two-tailed probability in statistical tests was used. Ap-value of less than 0.05 was considered to be significant.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This project was supported by the National Scientific and Technological Major Project of China (No. 2013ZX10004-904) and Scientific Research Fund of Medical Technology and Education in Zhejiang Province (No. 2013C25114).