The immunogenicity in healthy infants and efficiency to prevent mother to child transmission of Hepatitis B virus of a 10 µg recombinant yeast-derived Hepatitis B vaccine (Hep-KSC)
Background and Aims: To evaluate immunogenicity and efficacy of a 10 µg recombinant Saccharomyces cerevisiae-derived hepatitis B vaccine (Kangtai Biological Products Co. Ltd, Shenzhen, China) (Hep-KSC) in newborns.
Methods: Overall 1197 infants born to mothers negative for HBV markers (NM group) and 534 born to HBsAg-positive mothers (PM Group) were enrolled. Infants in NM group were given 10 µg Hep-KSC, 10 µg Engerix-B or 5 µg Hep-KSC and those in PM group received 10 µg Hep-KSC or 10 µg Engerix-B at 0, 1 and 6 months, with an additional 200 IU HBIG at birth for the latter.
Results: For NM Group, 10 µg Hep-KSC paralleled 10 µg Engerix-B but outperformed 5 µg Hep-KSC regarding seroprotective rate (95.06% vs 94.83% vs 89.67%, p = 0.0077) and anti-HBs geometric mean concentration (GMC) (798.87 mIU/ml vs 790.16 mIU/ml vs 242.04 mIU/ml, p < 0.0001) at 7 months. The proportion of infants with anti-HBs greater than 1000 mIU/ml was higher in 10 µg Hep-KSC than 5 µg Hep-KSC group (45.77% vs 11.93%, p < 0.0001) at 7 and 12 months. For PM Group, the HBsAg positivity rate in 10 µg Hep-KSC and 10 µg Engerix-B group was 1.60% and 4.27% at 7 months, respectively. In 10 µg Hep-KSC group, 93.61% and 91.29% achieved seroprotection at 7 and 12 months, respectively, and cor- respondingly 90.24% and 86.96% in 10 µg Engerix-B group. The anti-HBs GMC was comparable between 10 µg Hep-KSC and 10 µg Engerix-B group at 7 and 12 months (575.31 mIU/ml vs 559.64 mIU/ml; 265.79 mIU/ml vs 264.48 mIU/ml). Conclusions: 10 µg Hep-KSC might be appropriate for neonatal immunization with good immunogenicity and efficacy, especially for infants born to HBsAg-positive mothers. 1. Introduction Chronic hepatitis B virus (HBV) infection is a serious global pub- lic health problem, and the main cause of endstage liver diseases, including cirrhosis and hepatocellular carcinoma with high mor- bidity and mortality. Hepatitis B vaccine has been proven to be safe and effective in preventing HBV infection. Initially derived from donated human blood plasma, plasma-derived vaccine had the potential risk of transmitting other blood-borne infections as well as limited use because of its high cost, which has now been grad- ually replaced by yeast-derived recombinant hepatitis B vaccines available in large quantity at low cost, making extensive immuniza- tion in general population possible [1]. In China, hepatitis B vaccine has been formally included in the national immunization program since 2002 that every infant was required to be promptly immunized with a 5 µg/dose yeast- derived recombinant hepatitis B vaccine on a schedule of 0, 1 and 6 months. According to the results of the latest HBV epidemiolog- ical investigation in 2006, the HBV infection rate in children has sharply declined compared with the prevalence in 1992 [2]. How- ever, the current immunization strategy with 5 µg/dose hepatitis B vaccine administered solely might not be the best for prevent- ing mother to child transmission of HBV given that about 10% of the infants born to HBsAg-positive mothers still develop chronic HBV infection despite immunization. Studies have attempted to further reduce perinatal transmission rate by increasing the dosage of recombinant hepatitis B vaccine from 5 µg to 10 µg, and it has been demonstrated to be well tolerated and safe for newborns [3–5]. The Shenzhen Kangtai Biological 10 µg hepatitis B vaccine was made by recombinant deoxyribonucleic acid techniques in Sac- charomyces cerevisiae Yeast (HepB-SCY). Kangtai 10 µg HepB-SCY (10 µg HepB-KSC) has been well applied for immunoprophylaxis in young people above 16 years of age and in adults with good pro- tective efficacy and safety. Our recent study also showed that 10 µg HepB-KSC was safe for healthy infants and no serious adverse event was reported during observation period [6].This study aims to evaluate the immunogenicity of 10 µg/dose HepB-KSC in healthy infants born to HBV-free mothers and explore its effect on blocking the mother-to-infant transmission of HBV. 2. Materials and methods 2.1. Vaccines and Hepatitis B immunoglobulin (HBIG) The trial hepatitis B vaccine was produced by Kangtai Biolog- ical Products (HepB-KSC, 10 µg/0.5 ml; lot number 20061215-1; Co. Ltd., Shenzhen, China). The control vaccine were 10 µg/0.5 ml GlaxoSmithKline (GSK) Engerix-B (lot number XHBVB293AA) and 5 µg/0.5 ml HepB-KSC (lot number 20060512-8).HBIG was provided by Chinese Medicine Health Care Products (200 IU/0.1 ml, lot number 2006070101, Beijing, China). All vaccines and HBIG were stored at 2–8 ◦C. Freezing was avoided. 2.2. Study population Infants born to mothers negative for HBsAg/HBeAg/ HBeAb/HBcAb or HBsAg-positive mothers from Jiangsu province were enrolled in this study approved by the Ethic Committee of Jiangsu Provincial Center for Disease Control and Prevention. Informed consent was obtained from guardians of each infant. Clinical trial No: 2006L01434. 2.3. Study design This clinical trial was designed to be randomized, observer-blinded and parallel-controlled. Mothers negative for HBsAg/HBeAg/HBeAb/HBcAb (NM group) or with positivity for HBsAg (PM Group) were enrolled. All their newborns were recruited based on birth order and received trial vaccine or control vaccine at 0, 1, 6 months of age. This study observed the principle of Three-arm clinical trial. Infants of the NM group, received 10 µg trial vaccine (10 µg HepB-KSC), 10 µg control vaccine (10 µg Engerix-B) or 5 µg con- trol vaccine (5 µg HepB-KSC) at a ratio of 2:1:1. While infants of the PM group were further divided into HBeAg-positive (EP) and HBeAg-negative (EN) groups according to maternal HBeAg status, vaccinated with either 10 µg HepB-KSC, or 10 µg Engerix-B by the proportion of 2:1 and an additional 200 IU HBIG within 24 h of birth in the meantime. Randomized subjects allocation was achieved and strictly exe- cuted using a randomization table generated by computer software. The person responsible for knitting blind was demanded not to participate in any work relating to the clinical trial or reveal any information to those involved. The subjects, the study personnel administering the treatments as well as those assessing the out- comes were all blinded with respect to the allocation of vaccines.Blood samples of infants were collected at 7 and 12 months after birth for HBV serum markers detection. 2.4. Laboratory examinations Serum HBsAg and anti-HBs titers were determined using the Architect i2000SR analyzer (Abbott Diagnostic, Chicago, IL, USA), which was based on CMIA according to the manufacturer’s specifi- cations [7]. The detection range of HBsAg assay was 0.05–250 IU/ml and a titer no less than 0.05 IU/ml was considered positive. Anti-HBs was defined positive if the level was higher than 10 mIU/ml. 2.5. Statistical methods Geometric mean concentration (GMC) and the corresponding 95% confidence intervals (CI) were calculated for anti-HBs levels. The comparison of GMC between groups was assessed by Student’s t-test or ANOVA. Categorical variables were analyzed by Chi square or Fisher’s exact test. The statistical analyses were performed using SAS 9.1 (SAS Institute Inc., Cary, NC, USA.). All p values were two- tailed and a p value <0.05 was considered statistically significant. 3. Results 3.1. Enrollment and follow-up of the subjects As shown in Fig. 1, a total of 1918 infants were screened and eventually 1731 subjects were enrolled in the study, with 1197 from NM group and 534 from PM group (EP group: n = 183, EN group: n = 351). Of the 1731 subjects, 1677 (96.88%) (NM group: n = 1161, EP group: n = 178 and EN group: n = 338) completed the 3- dose HBV vaccination schedule. Blood samples were collected from 1566 subjects (NM group: n = 1089, EP group: n = 162 and EN group: n = 315) at 7 months of age and 1547 (NM group: n = 1076, EP group: n = 158 and EN group: n = 313) were followed up to 12 months old. There were no statistical differences between test group and con- trol group with regard to the gender constituent ratio (data not shown). Fig. 1. Enrollment and follow-up. A total of 1918 infants were screened and eventually 1731 subjects were enrolled in the study, with 1197 from NM group and 534 from PM group (EP group: n = 183, EN group: n = 351). Of the 1731 subjects, 1677 (96.88%) (NM group: n = 1161, EP group: n = 178 and EN group: n = 338) completed the 3-dose HBV vaccination schedule. Blood samples were collected from 1566 subjects (NM group: n = 1089, EP group: n = 162 and EN group: n = 315) at 7 months of age and 1547 (NM group: n = 1076, EP group: n = 158 and EN group: n = 313) were followed up to 12 months old.NM group: infants born to mothers negative for HBsAg/HBeAg/HBeAb/HBcAb; PM group: infants born to HBsAg-negative mothers; EP group: infants born to mothers positive for both HBsAg and HBeAg; EN group: infants born to HBsAg-positive mothers with negativity for HBeAg. Fig. 2. The constituent ratio of anti-HBs level in NM infants of the test group and the two control groups. (a) Comparison of the anti-HBs constituent ratio between test group and two control groups in 7-month-old infants of the NM group; (b) Comparison of the anti-HBs constituent ratio between test group and two control groups in 12-month-old infants of the NM group. 3.2. Immunogenicity of Hep-KSC in infants of the NM group The anti-HBs-positive rate in 7-month-old infants of the 10 µg Hep-KSC group (95.06%, 520/547) was similar with that of the 10 µg Engerix-B group (94.83%, 257/271) (p = 0.8871) but was evidently higher than that of the 5 µg Hep-KSC group (89.67%, 243/271) (p = 0.0037). The 10 µg Hep-KSC was comparable with 10 µg Engerix-B in terms of anti-HBs GMC (798.87 mIU/ml vs 790.16 mIU/ml, p = 0.9502) but more potent than 5 µg Hep-KSC (242.04 mIU/ml) (p < 0.0001) (Table 1). Among the 10 µg Hep-KSC group, 7.88% (41/520) of the infants only mounted a low level of anti-HBs (10–100 mIU/ml), medium (100–1000 mIU/ml) and high (>1000 mIU/ml) levels of anti-HBs were achieved in 46.35% (241/520) and 45.77% (238/520), respectively. Low, medium and high levels of anti-HBs were found in 8.56% (22/257), 49.03% (126/257) and 42.41% (109/257) of the infants in 10 µg Engerix-B group and 24.69% (60/243), 63.37% (154/243) and 11.93% (29/243) in 5 µg Hep-KSC group. No significant difference was revealed in constituent ratio of anti-HBs level between 10 µg Hep-KSC group and 10 µg Engerix-B group but it showed a striking contrast when compared with 5 µg Hep-KSC group (Fig. 2a).
When followed up to 12 months, anti-HBs was positive in 93.53% (506/541) of the 10 µg Hep-KSC group, which was similar with the 92.45% (245/265) in 10 µg Engerix-B group (p = 0.5687) but markedly higher than the 81.95% (218/266) in 5 µg Hep- KSC group (p < 0.0001). The 10 µg Hep-KSC group parelleled with 10 µg Engerix-B group in anti-HBs GMC (328.98 mIU/ml vs 292.02 mIU/ml, p = 0.2509) but obviously higher than 5 µg Hep-KSC group (94.46 mIU/ml) (p < 0.0001) (Table 1). Low levels of anti-HBs were observed in 18.77% (95/506) of the 10 µg Hep-KSC group, while anti-HBs in 63.24% (320/506) and 17.98% (91/506) of the infants reached medium and high levels, respectively. In compari- son, 18.37% (45/245) of their counterparts in 10 µg Engerix-B group had a low level of anti-HBs while the remaining 69.80% (171/245) and 11.84% (29/245) demonstrated medium and high levels of anti- bodies to the vaccine. In 5 µg Hep-KSC group, low, medium and high anti-HBs levels were shown in 51.38% (112/218), 46.79% (102/218) and 1.83% (4/218), respectively. No significant difference was found in anti-HBs constituent ratio between 10 µg Hep-KSC group and 10 µg Engerix-B group but anti-HBs in 5 µg Hep-KSC group showed a distinct feature with an absolute dominance of low and medium levels. But the rate of high anti-HBs levels in the 10 µg Hep-KSC group was significantly higher than that in 10 µg Engerix-B group and 5 µg Hep-KSC group (p < 0.0001) (Fig. 2b). 3.3. Efficacy and immunogenicity of Hep-KSC in infants of the PM group The HBsAg positive rate in 7-month-old infants born to HBsAg- positive mothers of the 10 µg Hep-KSC group resembled that in 10 µg Engerix-B group (1.60%, 5/313 vs 4.27%, 7/164, p = 0.1194). For infants born to HBeAg-positive mothers, no significant dif- ference was found between 10 µg Hep-KSC and 10 µg Engerix-B (3.77%, 4/106 vs 5.36%, 3/56, p = 0.6937). But for infants born to HBeAg-negative mothers, 10 µg Hep-KSC might provide better protection than 10 µg Engerix-B (0.48%, 1/207 vs 3.70% 4/108, p = 0.0488). As shown in Table 2, 93.61% (293/313) of the infants in 10 µg Hep-KSC group and 90.24% (148/164) in 10 µg Engerix-B group were positive for anti-HBs at 7 month olds (p = 0.1862), with comparable anti-HBs GMC in two groups (575.31 mIU/ml vs 559.64 mIU/ml, p = 0.8567). Low, medium and high anti-HBs levels were achieved in 12.97% (38/293), 50.17% (147/293) and 36.86% (108/293) of the infants in 10 µg Hep-KSC group respectively. While in 10 µg Engerix-B group, 13.51% (20/148) of the infants were defined as hypo-responders, 50.66% (75/148) and 35.81% (53/148) possessed medium and high levels of anti-HBs respectively. There was no obvious distinction in the constituent ratio of anti-HBs level between the two groups (Fig. 3a). When taking maternal HBeAg status into consideration, no notable differences between 10 µg Hep-KSC group and 10 µg Engerix-B group in anti-HBs positive rate, anti-HBs GMC and constituent ratio of anti-HBs level were revealed. At the age of 12 months, anti-HBs remained positive in 91.29% (283/310) of the infants in 10 µg Hep-KSC group and 86.96% (140/161) in 10 µg Engerix-B group (p = 0.1403), with similar anti-HBs GMC in two groups (265.79 mIU/ml vs 264.48 mIU/ml, p = 0.9732). Low, medium and high levels of anti-HBs were observed in 24.38% (69/283), 61.13% (173/283) and 14.49% (41/283) of the 10 µg Hep-KSC group respectively. The corresponding percentages in 10 µg Engerix-B group were 22.86% (32/140), 61.43% (86/140) and 15.71% (22/140), demonstrating no significant differences in the constituent ratio of anti-HBs level between the two groups (Fig. 3b). When stratifying according to maternal HBeAg status, anti-HBs positive rate, anti-HBs GMC and constituent ratio of anti-HBs level were also not differentiated between 10 µg Hep-KSC group and 10 µg Engerix-B group. 4. Discussion Universal HBV vaccination has been proven to be the most effective means of comprehensively preventing against and controlling HBV transmission [8]. The immunogenicity, efficacy, safety, and cost of hepatitis B vaccine candidates were the main considerations in determining the feasibility of extensive use in the implemen- tation of universal vaccination. In China, an immune strategy of 3-dose 5 µg recombinant yeast-derived hepatitis B vaccine accord- ing to a 0, 1 and 6 month schedule is adopted in healthy newborns. However, low-dose recombinant hepatitis B vaccines might be less efficient in conferring potent and persistent protective immu- nity than high-dose ones. Goldfarb et al. [9] reported that 5 µg Engerix-B failed to induce as great an anti-HBs concentration as did 10 µg in 153 healthy infants receiving either 5 µg or 10 µg Engerix-B at 2, 4 and 6 months of age. Zhang et al. [10] com- pared the antibody response induced by 5 µg and 10 µg hepatitis B vaccine made by recombinant deoxyribonucleic acid techniques in Saccharomyces cerevisiae (HepB-SC) and Hansenula Polymorpha (HepB-HP) respectively in newborns immunized on a 0-1-6 sched- ule and found that better anti-HBs response could be achieved by primary immunization with 10 µg HepB-HP than with 5 µg HepB-SC. The Kangtai 10 µg trial vaccine (Hep-KSC) and GSK 10 µg control vaccine (Engerix-B) were both made by recombinant deoxyribonu- cleic acid techniques in Saccharomyces cerevisiae. Engerix-B had demonstrated good protective efficacy against HBsAg carriage in healthy infants and children as well as in high-risk neonates born to HBsAg-positive mothers [11]. In China, only 5 µg hepatitis B vac- cine was used in universal HBV vaccination of general newborns previously due to limited domestic vaccine production capacity. With the development of economy, 10 µg hepatitis B vaccine is now available in China but under the policy of widespread immu- nization internal market demand still exceeds the supply capacity of a sole manufacturer. The 10 µg Hep-KSC has been well applied for immunization in adolescents above 16 years of age and in adults with good protective efficacy and safety. Safety of the 10 µg HepB- KSC has also been verified among healthy infants with no serious adverse events reported during the observation period. In this large-scale randomized controlled trial study, we first evaluated the immunogenicity of 10 µg HepB-KSC in infants born to healthy mothers in comparison with 10 µg Engerix-B and 5 µg HepB-KSC. The results showed that the immunogenicity and pro- tective efficacy of 10 µg HepB-KSC was similar with that of GSK 10 µg Engerix-B in 7-month-old infants. The anti-HBs positive rates of HepB-KSC group and 10 µg Engerix-B group were 95.06% and 94.83% respectively, which were markedly higher than the 89.67% of 5 µg HepB-KSC group. A similar trend was observed in terms of anti-HBs GMC with significantly higher levels in 10 µg test and control groups than in 5 µg control group (798.87 mIU/ml vs 790.16 mIU/ml vs 242.04 mIU/ml). Therefore, the anti-HBs positive rate and anti-HBs GMC in infants tended to rise in parallel with the increasing immunization dose, which was consistent with previous reports [9,10]. Similar trends were observed in 12-month-old infants, indicating that 10 µg HepB-KSC could provide a persistent protective immunity analogous to 10 µg Engerix-B but superior to 5 µg HepB-KSC group. Hence, 10 µg HepB-KSC might be a good choice for infants born to healthy mothers in universal vaccination program. Fig. 3. The constituent ratio of anti-HBs level in PM infants of the test group and the two control groups. (a) Comparison of the anti-HBs constituent ratio between test group and two control groups in 7-month-old infants of the PM group; (b) Comparison of the anti-HBs constituent ratio between test group and two control groups in 12-month-old infants of the PM group. Perinatal transmission is one of the major routes of acquiring HBV infection and often leads to worse outcomes in the long run. Infants born to HBeAg-positive mothers have a 85–90% chance of becoming chronic HBV carriers while in those born to HBsAg- positive mothers negative for HBeAg, chronicity rate may reach 30–40% if no prophylactic measures are taken [12]. Therefore, preventing perinatal transmission is an integral part of the compre- hensive strategy in the combat against HBV and its related diseases. Currently, combined immunization with HBIG and vaccines in neonates born to HBsAg-positive mothers is considered to be most efficacious in preventing mother-to-infant transmission. The immunization guide of hepatitis B recommends that infants born to mothers positive for HBsAg should be immunized with three-dose 10 µg recombinant yeast-derived hepatitis B vaccines within 12 h of birth and at 1 and 6 months of age. Meanwhile, HBIG ( 100 IU) should also be administered within 12 h after birth, ensuring a bet- ter protection effect against perinatal transmission of HBV [13]. Data from this study also confirmed the effect of 10 µg HepB-KSC combined with 200 IU HBIG in preventing perina- tal transmission of HBV in comparison with 10 µg Engerix-B combined with 200 IU HBIG. The HBsAg-positive rates in 7-month-old infants were similar in 10 µg HepB-KSC group and 10 µg Engerix-B group (1.60% vs 4.27%, p = 0.1194) and no differ- ences were found in anti-HBs positive rate, anti-HBs GMC and anti-HBs constituent ratio in two groups either at 7 or 12 months of age. When stratifying according to maternal HBeAg status, dis- parities did not exist between 10 µg HepB-KSC group and 10 µg Engerix-B group in anti-HBs positive rate, anti-HBs GMC and con- stituent ratio of anti-HBs level. These results indicated that 10 µg HepB-KSC had an equivalent effect with 10 µg Engerix-B in pre- venting mother-to-infant transmission and was proper for infants born to HBsAg-positive mothers. In this study, the anti-HBs positive rate and anti-HBs GMC declined when followed up to 12 months as reported by previous studies, implicating that further evaluation of long-term protec- tive effect was needed. Although in most cases antibody response could reach a protective level, non-responders that might require additional boosters still existed. And for those infants who were immunization failure also need to be studied further. 5. Conclusion The 10 µg HepB-KSC had a good immunogenicity in healthy newborns and could provide efficient protection against HBV perinatal transmission. Moreover, the national bidding price for 10 µg HepB-KSC was per capita 9.3 RMB and the retail price was 58 RMB/dose, which was much lower than the 78–118 RMB/dose for 10 µg Engerix-B. Considering its low production costs, it might be a good choice for application in the universal infant hepatitis B immunization program,TEPP-46 especially for those at high risk of infecting HBV.