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The Safety and Immunogenicity of Two Hepatitis B Vaccine Formulations (Thiomersal-free and Thiomersal-containing) in Healthy Vietnamese Infants

A Phase III, Prospective, Single-blinded, Randomized, Controlled Trial

Hieu, Nguyen Trong MD*; Sarnecki, Michal MD; Tolboom, Jeroen MSc

Author Information
The Pediatric Infectious Disease Journal: January 2015 - Volume 34 - Issue 1 - p 79-83
doi: 10.1097/INF.0000000000000479

Abstract

Hepatitis B is a serious disease that is caused by the hepatitis B virus (HBV), 1 of at least 7 hepatitis viruses.1,2 HBV is a hepadnavirus, a family of viruses that replicate by reverse transcription in the hepatocytes of their hosts. The World Health Organization (WHO) estimates that each year HBV causes approximately 4 million acute infections worldwide, and is associated with the deaths of an estimated 600,000 people.1 Following acute infection, 80–90% of infants, 30–50% of children and 5–10% of adults do not clear the virus and become chronic hepatitis B surface antigen (HBsAg) carriers.1,2 It has been estimated that in excess of 2 billion people have been infected by HBV and over 350 million people in the world are carriers.1,2 Chronically infected individuals represent the primary reservoir of the virus, and therefore pose a continuous threat to others in their environment. Long-term HBV infection can lead to chronic hepatitis, cirrhosis, hepatic failure and hepatocellular carcinoma (HCC), a fatal disease with very poor response to current chemotherapy.3 Over half of HCCs are attributable to chronic HBV infection; HCC is the third leading cause of cancer-related deaths worldwide.4

There is no effective cure for HBV infection. Currently, prophylactic vaccination against HBV infection offers the only method of protection against hepatitis B and the long-term effects caused by it. Vaccines against hepatitis B have been available since 1982.1 Hepatitis B vaccine is 95% effective in preventing HBV infection and its chronic consequences.1 Universal immunization beginning at birth and other successful hepatitis vaccination strategies have resulted in a dramatic reduction in HBV transmission in many countries with historically high endemicity. This has gradually resulted in a reduction of HBV-related chronic hepatitis, liver cirrhosis and HCC,5 which have caused major concerns for public health and the economy in these areas. As of 2012, 181 countries had incorporated hepatitis B vaccine as an integral part of their national infant immunization programs, and an estimated 79% of the 2012 global birth cohort received 3 doses of hepatitis B vaccine.6

Southeast Asia is a region of high endemicity with an HBsAg prevalence rate of at least 8%. In endemic areas, the high incidence of both vertical and horizontal transmission leads to infection occurring early in life.2 According to the WHO, Vietnam has a hepatitis B prevalence of 8%9 and the virus constitutes a major health problem.7,8 The Vietnamese Ministry of Health put in place universal infant hepatitis B vaccination in 2003 and, by 2006, 64.3% of neonates received a birth dose of hepatitis B vaccine.9 However, because of a series of widely publicized neonatal deaths that were wrongly attributed to vaccine, coverage in Vietnam fell to 20.4% in 2008 and has only recently begun to increase to pre-2006 levels.9

Hepavax-Gene is a recombinant hepatitis B vaccine using Hansenula polymorpha expression technology.10 Clinical studies and data gathered since its introduction in 1995 indicate that Hepavax-Gene is well tolerated and provides long-term protection against infection with HBV in all age groups studied.11,12 Hepavax-Gene is a WHO prequalified vaccine for active immunization against HBV. Since its launch in Korea in 1995, and subsequent international marketing since 1996, over 590 million doses of Hepavax-Gene have been distributed. Hepavax-Gene contains recombinant HBsAg derived from yeast cells. It confers immunity against HBV infection through the stimulation of a specific immune response as measured by the induction of anti-HBs antibodies. Hepavax-Gene itself contains thiomersal, an organomercurial, as a preservative (0.01%, w/v).13 In response to a move by some regulatory authorities to reduce the mercury content of vaccines on safety grounds and since the preservative is not essential in single-dose formulations, Crucell developed a thiomersal-free vaccine formulation, Hepavax-Gene TF. Both Hepavax-Gene vaccine formulations are indicated for active immunization against HBV infection in neonates, children and adults.

In a previous study comparing the immunogenicity and safety of Hepavax-Gene, Hepavax-Gene TF and Engerix-B (containing trace amounts of thiomersal) in a large healthy adult population (N = 770), it was shown that Hepavax-Gene TF was noninferior to Hepavax-Gene and Engerix-B with respect to seroprotection rates (≥10 IU/L) 1 month after the third vaccination using the 0–1–6-month schedule.14

The current study was conducted to evaluate the safety and immunogenicity of Hepavax-Gene TF compared with the thiomersal-containing formulation of Hepavax-Gene in healthy Vietnamese neonates.

MATERIALS AND METHODS

Study Design

This was a single-blind, randomized controlled study in healthy Vietnamese infants conducted at the Hung Vuong Hospital in Ho Chi Minh City. The primary objective was to demonstrate equivalence between the 10 μg Hepavax-Gene thiomersal-free formulation and the 10 μg Hepavax-Gene thiomersal-containing formulation in healthy infants with regard to seroprotection at the ≥10 IU/L level 1 month after the third dose of vaccine. Secondary objectives were to make this assessment 1 month after the first and second doses and also to assess seroprotection using a ≥100 IU/L cutoff 1 month after all doses.

Healthy infants, born after a normal gestational period (37–42 weeks) following an uncomplicated pregnancy and labor, with a minimum birth weight of 2500 g and a 5-minute Apgar score exceeding 8, a serum ALT level below 40 IU/mL and whose mothers were HBsAg negative were enrolled into the study. Study participants were randomly allocated in a 1:1 ratio to receive either Hepavax-Gene TC or Hepavax-Gene TF. All vaccines were administered intramuscularly in the anterolateral region of the thigh at 0 (the day of birth or the day after), 1 and 6 months of age (standard 0–1–6-month schedule). Maternal and cord blood samples were obtained before vaccination; in addition, blood samples (2 mL) were obtained from all infants at months 1, 6 and 7.

Hepavax-Gene (Crucell, Berna Biotech Korea Corporation, Incheon, Korea) is a recombinant noninfectious subunit viral vaccine derived from synthetic HBsAg produced in yeast cells. The vaccines used in this trial were formulated for intramuscular injection only. Each vial contained 10 μg/mL of HBsAg adsorbed onto approximately 0.25 mg/mL adjuvant (aluminum hydroxide) in a final volume of 0.5 mL (1 dose/vial). The 2 vaccines were similar, except that the study vaccine (TF) was thiomersal free, whereas the comparator (TC) contained thiomersal. Doses were given using a 0–1–6-month schedule with windows of ±3 days for the second and ±5 days for the third vaccination.

The study protocols, informed consent documents and all recruiting material were approved by the Science and Ethical Committee of Hung Vuong Hospital in compliance with the Declaration of Helsinki, Good Clinical Practice Guidelines and local laws. Parents or legal guardians gave their informed consent before infants were enrolled into the study.

Serology

Antibodies against HBsAg were determined using a standard enzyme-linked immunosorbent assay (commercial BioRad test kits and apparatus, BioRad, Hercules, CA). For the primary endpoint, hepatitis B seroprotection was defined using a cutoff level of ≥10 IU/L anti-HBs antibodies.15 All blood samples were analyzed locally at the Hung Vuong Hospital.

Safety Assessment

Parents (or legal guardians) recorded solicited local (redness, pain and swelling) and systemic adverse events (skin rash, irritability, vomiting, lethargy, etc.) in a subject diary distributed to subjects’ parents to record any adverse event that occurred within the 4 weeks after each vaccination, together with body temperature (orally) and any concomitant medication. Subject diaries were collected at each subsequent visit, and the parents/guardians were asked nonleading questions about their baby’s health status. Additionally, study personnel monitored infants for 30 minutes immediately following each vaccination. The occurrence of any serious adverse events was reported, as required up to 1 month after the third dose.

Analysis Populations

The intention-to-treat (ITT) population comprised all subjects who were randomized and received at least 1 dose of study vaccine. In this study, the ITT population was used for all immunogenicity assessments.

The safety population comprised all subjects who received at least 1 dose of study vaccine and provided follow-up safety data.

Sample Size

The primary endpoint of the study was the seroprotection rate at the ≥10 IU/L level 1 month after the third dose of vaccine. Assuming a reference seroprotection rate of 97%, and using a noninferiority limit of 5% and a 2-sided 95% confidence interval (CI), a sample size of 184 subjects per group was required to demonstrate noninferiority of the thiomersal-free formulation compared with the thiomersal-containing formulation with 80% power. Noninferiority is demonstrated when the lower limit of the 2-sided 95% CI for the difference in proportions is above −5%. When adjusted for a 10% dropout rate, 408 participants altogether were required for the 2 groups.

Primary Efficacy Analysis

The primary efficacy null hypothesis was that the seroprotection rate for the thiomersal-free formulation is at least 5% lower than the seroprotection rate for the thiomersal-containing formulation. The aim of the study was to reject this null hypothesis based on the data 1 month after the third dose of vaccine in favor of the alternative hypothesis that the seroprotection rate for the thiomersal-free formulation is at most 5% lower than for the thiomersal-containing formulation, thereby showing noninferiority with a 5% margin. Exact 2-sided 95% CIs were calculated for the seroprotection rates for each vaccine group 1 month after the third dose. Two-sided 95% CIs (normal approximation) for the differences between formulations were calculated.

Secondary Efficacy Analysis

Exact 2-sided 95% CIs were provided for the seroprotection rates for each vaccine group by cutoff and time point. Geometric mean titers (GMTs) at each time point were calculated. The seroprotection rate of the TF group was considered as good as that of the TC group if the 95% CI for the difference in seroprotection rate was contained within −5% to +5%. Two-sided 95% CIs (normal approximation) for differences between formulations were computed.

Statistical Methods

Comparisons between the TF and TC groups were done either by calculating the 95% CI for the ratio in GMT and in fold increase of titers between the groups or by calculating the 95% CI for the difference in seroprotection between the TF and TC groups. The level of significance was 0.05.

Descriptive statistics were generated for the safety data.

RESULTS

Study Population

A total of 408 healthy infants (192 female, 216 male) whose mothers were HBsAg negative were enrolled in the study between August 2008 and November 2008 and randomly allocated in a 1:1 ratio to receive either the thiomersal free or the thiomersal containing formulation of Hepavax-Gene using a 0–1–6-month schedule (groups TF and TC, respectively; see Table, Supplemental Digital Content 1, https://links.lww.com/INF/B959). There were no significant differences in the infant’s gender or birth weight between the 2 groups.

No subjects discontinued from the study because of any adverse event. Two participants were lost to follow-up at month 1, 11 more at month 6 and a further 20 at month 7. In total, 33 out of 408 participants (8.1%) were lost to follow-up. The reason for subjects being lost to follow-up were that the family had moved to another province in Vietnam. Thus 375 subjects were included in the immunogenicity and safety analyses 1 month after the third vaccination.

Immunogenicity

Seroprotection

Seroprotection rates at the 10 IU/L cutoff level in the TF group at 1, 6 and 7 months were 95.6%, 98.0% and 97.9%, respectively. The corresponding rates in the TC group were 95.1%, 97.5% and 97.3%, respectively. The mean differences between the groups (TF−TC) were 0.5% at each time point, and the corresponding 95% CIs all fell within the range from −5% to +5% showing equivalence within the 5% margin. Seroprotection rates at the 100 IU/L cutoff level in the TF group at 1, 6 and 7 months were 88.2%, 97.5% and 97.8%, respectively. The corresponding rates in the TC group were 90.6%, 97.5% and 97.3%, respectively. The mean differences between the groups (TF−TC) were −2.5%, −0.0% and 0.5% at 1, 6 and 7 months respectively. The corresponding 95% CIs fell within the range from −5% to +5% at 6 and 7 months showing equivalence but not after 1 month where the lower bound of the 95% CI was −8.4%. For the primary and most secondary endpoints the immunogenicity of Hepavax-Gene TF was therefore noninferior to Hepavax-Gene TC (Table 1).

T1-17
TABLE 1:
Seroprotection Rates and Differences at 1, 6 and 7 Months of Age, by Vaccination Group; ITT Population

Anti-HBs Titers

The ratios of the geometric mean anti-HBs levels between 2 groups were close to 1 and not statistically significantly different from 1 (95% CIs included 1): month 1 GMT ratio (TF/TC) was 0.89 (95% CI: 0.74, 1.07); month 6 GMT ratio was 1.07 (95% CI: 0.93, 1.22) and month 7 GMT ratio was 1.06 (95% CI: 0.93, 1.22) (Table 2).

T2-17
TABLE 2:
Geometric Mean Titers at 1, 6 and 7 Months of Age and Geometric Mean Ratios From Month 1 to 6, and Month 6 to 7; ITT Population

There were considerable increases in anti-HBs titers from month 1 to month 6: a 4.14-fold increase was observed in the TF group, and a 3.31-fold increase in the TC group. The ratio in fold increase (TF/TC; month 6/month 1) was statistically significant: 1.25 (95% CI: 1.06, 1.48, not including 1). In comparison, the fold increases from month 6 to month 7 were similar in both groups: 1.85-fold (TF) versus 1.84-fold (TC) with a ratio 1.00 (95% CI: 0.91, 1.11 including 1).

The CIs for all ratios between groups, for both GMTs and geometric mean fold increases, fell within the range from 0.67 to 1.5 showing equivalence within a factor of 1.5.

Safety

Overall there were no significant differences in the numbers of adverse events reported between the 2 groups (see Table, Supplemental Digital Content 2, https://links.lww.com/INF/B960). All adverse events were mild and resolved either spontaneously or after short-term treatment. During the study period, a total of 90 adverse events (48.6% of all adverse events) were recorded in the TF group, and 95 (51.4%) in the TC group. Within 30 days of vaccination, 13 adverse events were reported considered to be vaccine related, 7 after the first dose, 5 after the second dose and 1 after the third dose; of all these events, 5 occurred in the TF group and 8 in the TC group.

In general, the incidence of individual adverse events was also similar between the 2 groups (Table 3). There were fewer events reported after the first dose compared with the other 2 doses for both formulations. Seventeen infants (8.4%) in the TF group and 14 in the TC group (6.9%) experienced fever. All instances of fever were reported after the second dose. Two infants in the TC group experienced local pain in total compared with no infants in the TF group.

T3-17
TABLE 3:
Solicited Adverse Events; Safety Population

No serious adverse events were reported during the study.

DISCUSSION

The development of Hepavax-Gene TF, a recombinant hepatitis B vaccine without the preservative thiomersal, is in line with the policy of removing or reducing thiomersal in vaccines,16 an important objective set by public-health policy makers.14 The results of this study clearly indicate that the thiomersal-free hepatitis B vaccine, Hepavax-Gene TF, can be used in place of the thiomersal-containing vaccine, Hepavax-Gene TC. Hepavax-Gene TF was shown to be noninferior to Hepavax-Gene TC regarding seroprotection following a full vaccination course using the standard 0–1–6-month vaccination regimen.

In comparison with our previously reported findings with Hepavax-Gene TC12 using the same standard vaccination regimen, both vaccine formulations in the current trial (thiomersal free and thiomersal containing) showed higher seroprotection rates (≥10 IU/L, 95.6% and 95.1%, respectively) and higher geometric mean anti-HBs titers (312.9 and 351.2 IU/L, respectively) at 1 month after the first vaccine dose compared with Hepavax-Gene TC at the same time point (seroprotection 62%, anti-HBs titer 158 IU/L) in the previous trial.12 These observed seroprotection rates and geometric meant anti-HBs titers at this time point are higher than previously reported in the literature. A possible explanation for this may be maternal transferred HBsAg antibodies in this endemic population, although this was not tested for in this study. Seroprotection rates 7 months after the first vaccine dose for both formulations are in line with our previous findings.

Two previous studies have shown Hepavax-Gene TC to be safe and immunogenic in neonates. In a Korean study, 100% seroprotection was found in 47 neonates vaccinated at 0, 1 and 6 months of age.11 In a Vietnamese study, 97% seroprotection was found in 124 neonates vaccinated at 0, 1 and 2 months.17 No safety concerns were noted in either study. In the current study in 408 neonates vaccinated at 0, 1 and 6 months of age, seroprotection rates at the ≥10 IU/L cutoff level were above 95% at all time points for both the TF and TC groups. Differences between the TF and TC formulations in terms of seroprotection were within 5%, predefined as being not clinically significant.

Although initial anti-HBs titers of ≥10 IU/L are regarded as seroprotective by the WHO, some authorities recommend a cutoff of 100 IU/L.18,19 Using this definition of seroprotection, Hepavax-Gene TF was again noninferior to Hepavax-Gene TC at any predefined study time point using the standard 0–1–6 vaccination schedule.

Both formulations were safe and well tolerated. No subjects discontinued from the study because of any adverse event. After a total of 1209 vaccinations, 185 adverse events were recorded (up to 1 month postvaccination), 90 after the TF formulation and 95 after the TC formulation. All adverse events were mild in severity, and only 13 were considered to be vaccine related. No infant in the TF group experienced a local reaction after injection, implying that the TF formulation may be associated with fewer local reactions compared with the TC vaccine, although the small sample size in this study precluded any definite conclusion. In our previous study,12 the majority of adverse events occurred after the first dose of Hepavax-Gene. In contrast, in the present trial both formulations produced fewer adverse events after the first dose compared with the other 2 doses. The general safety profile observed was similar to that seen in a study with Hepavax-Gene in Vietnamese neonates.12

CONCLUSIONS

This trial demonstrated the noninferiority of the thiomersal-free formulation of Hepavax-Gene compared with the thiomersal-containing formulation of Hepavax-Gene in terms of immunogenicity. Small differences in seroprotection were not clinically significant. Both vaccine formulations were well tolerated; local reactions were uncommon in subjects vaccinated with the thiomersal-containing vaccine and no reactions were observed after thiomersal-free vaccine injection.

ACKNOWLEDGMENTS

This study was sponsored by Crucell Switzerland AG. The authors thank Dr. Max Gough (Crucell Switzerland AG) for writing the article, and also all participants in the study.

Authors’ contributions: M.S. was involved in study analysis and critically reviewed the manuscript. J.T. was involved in analysis and critically reviewed the manuscript. N.T.H. was an investigator, and was involved in data collection and critical review of the manuscript. All authors approved the final version of the manuscript.

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Keywords:

hepatitis B; pediatric; vaccination; thiomersal; immunogenicity

Supplemental Digital Content

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