Preclinical evaluation of a two-dose vaccination schedule of recombinant Hansenula Polymorpha hepatitis B vaccine in animals

Abstract

Aims: The current 3-dose regimen of hepatitis B vaccination for infants requiring over 6 mo period may pose the poor rate of compliance and later protection from hepatitis B virus (HBV) infection. This preclinical study is to investigate the feasibility of reducing the number of doses of hepatitis B (HB) vaccine.

Results: Eight groups of guinea pigs immunized with two doses of HP-HB vaccines at either 0 and 4 weeks or 0 and 8 weeks elicited geometric titers (GMT) of anti-HBs similar to that of four groups immunized with three doses of controls. The overall GMT of anti-HBs were not significantly different between the E- and C-groups (p > 0.05) of monkeys. Specifically, the anti-HBs titers in the C-group reached the peak of 24857 (938.3–104585) mIU/mL one week after the 3rd dose, which were statistically higher than those of the E-group. However, they were reduced to comparable levels of anti-HBs in the E-group during weeks 9–12, suggesting comparable immune response of both vaccination regimens.

Methods: Twelve groups of guinea pigs (four animals in each group) were immunized with 2 experimental recombinant yeast Hansenula Polymorpha derived HB vaccines (HP-HB vaccine) and 2 commercial recombinant yeast Saccharomyces Cerevisiae vaccines (Temrevac-HB) as controls at 0, 4 and 8 weeks, 0 and 4 weeks, and 0 and 8 weeks respectively. Each guinea pig received 2µg vaccine. Twelve Cynomolgus monkeys were randomly divided into two groups (six animals in each group). Animals in the experimental group (E-group) were injected with two doses of pilot produced 20 µg HP-HB vaccine. Animals in the control group (C-Group) were immunized with three doses of 10 µg Temrevac-HB. Both vaccines were administered at an interval of 3 weeks for monkeys.

Conclusions: The 2-dose regimen of the HP-HB vaccine has comparable HBV immune responses as the 3-dose regimen of Temrevac-HB vaccine in Cynomolgus monkeys.

Introduction

Hepatitis B is a common infection in the world.^1-3 Immunization with hepatitis B vaccine is the most effective method to control the transmission of hepatitis B virus (HBV). The regular HBV vaccination schedule requires 3 doses that are given over a course of 6 mo, which makes it difficult to cover all infants, particularly those in the remote areas of developing countries like China. It has been proposed that a 2-dose regimen may be adequate to protect against HBV infection.^4-13 Along with more vaccines being integrated into the Expanded Program on Immunization (EPI), increasing the number of doses of various vaccines may increase the potential side effects in neonates and infants and can affect compliance. If HBV vaccination can be reduced by one dose, this dose can be spared for newly integrated vaccine into the EPI. Therefore, direct experimental evidence is required to evaluate the efficacy of 2-dose strategy for hepatitis B vaccines (HB vaccine). In this report, we describe the effective immune response against HBV by two-dose vaccination of a proprietary recombinant yeast Hansenula Polymorpha (HP) derived HB vaccine (HP-HB vaccine) in guinea pigs and primate Cynomolgus monkeys.

Results

Anti-HBsAg antibody (Anti-HBs) levels of guinea pigs immunized with experimental recombinant Hansenula Polymorpha HB vaccines (HP-HB vaccine)

Two experimental recombinant HP-HB vaccine samples produced geometric titers (GMT) of 25.8~860.5 mIU/mL of anti-HBs in all 4 guinea pig groups 4 weeks after the 1st dose of the vaccines, whether injected at 0, 4 and 8 weeks (0–4-8w), 0 and 4 weeks (0–4w) or 0 and 8 weeks (0–8w) schedule. The 2 control Temrevac-HB vaccines (SC-HB vaccine) produced 34.6~65.6 mIU/mL anti-bodies in 2 groups at the same timepoint (Fig. 1). All vaccines induced GMT antibodies greater than 50 mIU/mL in all groups of animals 2 weeks after the 2nd vaccination. The anti-HBs GMT in guinea pigs 2 weeks after the 3rd vaccination (week 10) for 2 experimental HP-HB vaccines and 2 control SC-HB vaccines (0–4-8w regimen) reached peaks GMT titers of 64813 and 156068 mIU/mL, 30762 and 15324 mIU/mL respectively, with peak antibody responses declining by week 12. The 2-dose regimen of current commercial Temrevac-HB (either 0–4w or 0–8w) induced lower than 1600 mIU/mL during the 12 weeks. One 2-dose regimen of HP-HB at 0–4w induced 1741 mIU/mL anti-HBs GMT in one group, another 2-dose regimen at 0–4w and two 2-dose regimen of HP-HB at 0–8w induced 3595~9475 mIU/mL peak responses of anti-HBs in other 3 groups at the end of the experiment. These antibody levels were comparable to 7869~8845 mIU/mL elicited by the 3-dose control SC-HB vaccines (Fig. 1). The experimental HP-HB vaccines induced 25.0%~50.0% anti-HBs response in 4 of 6 animal groups, 33.3%~75.0% in all 6 groups after 2 and 4 weeks with 1st dose respectively, compared with 25.0%, 25.0%~33.3% in one and 3 of 6 groups for the SC-HB vaccines (Table 1). All animals were injected with 2µg HBsAg vaccines. Three guinea pigs were removed from one 0–4-8w HP-HB vaccine, one 0–8w SC-HB group before the 4th week and one 0–8w HP-HB vaccine group before the 6th week by animal care staff and not available for test of anti-HBs subsequently (Table 1). The guinea pig test results demonstrated a trend that the 2-dose regimen of experimental HP-HB vaccine has similar immune effects than dose the regular 3-dose regimen of the current commercial SC-HB vaccine. The phenomenon of several irregular profiles of anti-HBs for HB-vaccines in guinea pigs, such as 2 non-responders in 2 groups of SC-HB vaccines with 0–8w regimen, one animal responded only at the end of test in extremely low (22.8 mIU/mL) antibody in one SC-HB group of 0–4-8w suggesting that the guinea pigs might not be the most suitable species for the assay of HP-HB vaccine antigenicity and it would be worth conforming the immune effect of the new HP-HB vaccine in primates.

Figure 1. GMT of anti-HBs levels (mIU/mL) in the sera of guinea pigs vaccinated with experimental recombinant yeast Hansenula Polymorpha HB vaccines (HP-HB vaccine) prepared in laboratory scale and those with control vaccines. HP1: group vaccinated with experimental HP-HB vaccine (Lot HP20081004B). HP2: group vaccinated with experimental HP-HB vaccine (Lot HP20081005B). SC1: group vaccinated with recombinant yeast Saccharomyces Cerevisiae HB vaccine (Lot 200804415). SC2: group vaccinated with recombinant yeast Saccharomyces Cerevisiae HB vaccine (Lot 200701143). 0–4-8w: vaccinated at 0, 4 and 8 weeks. 0–4w: vaccinated at 0 and 4 weeks. 0–8w: vaccinated at 0 and 8 w.

Table 1. Anti-HBs responses of guinea pigs immunized with HB vaccines (%)^a.

Groupsb	Immune schedulec	Weeks after initial immunization (week)
		2	4	6	8	10	12
HP1	0–4-8W	1/4 (25.0)	3/4 (75.0)	4/4 (100.0	4/4 (100.0)	4/4 (100.0)	4/4 (100.0)
HP2		0/4 (0.0)	1/3 (33.3) *	2/3 (66.7)	2/3 (66.7)	3/3 (100.0)	3/3 (100.0)
SC1		0/4 (0.0)	0/4 (0.0)	4/4 (100.0)	4/4 (100.0)	4/4 (100.0)	4/4 (100.0)
SC2		0/4 (0.0)	0/4 (0.0)	2/4 (50.0)	4/4 (100.0)	4/4 (100.0)	4/4 (100.0)
HP1	0–4W	0/4 (0.0)	1/4 (25.0)	2/4 (50.0)	2/4 (50.0)	4/4 (100.0)	4/4 (100.0)
HP2		0/4 (0.0)	1/4 (25.0)	3/4 (75.0)	3/4 (75.0)	4/4 (100.0)	4/4 (100.0)
SC1		1/4 (25.0)	1/4 (25.0)	2/4 (50.0)	2/4 (50.0)	3/4 (75.0)	3/4 (75.0)
SC2		0/4 (0.0)	0/4 (0.0)	1/4 (25.0)	1/4 (25.0)	3/4 (75.0)	4/4 (100.0)
HP1	0–8W	2/4 (50.0)	2/4 (50.0)	4/4 (100.0)	4/4 (100.0)	4/4 (100.0)	4/4 (100.0)
HP2		2/4 (50.0)	1/4 (25.0)	3/4 (75.0)	2/3 (66.7)*	2/3 (66.7)	2/3 (66.7)
SC1		2/4 (50.0)	1/3 (33.3)*	2/3 (66.7)	2/3 (66.7)	2/3 (66.7)	3/3 (100.0)
SC2		0/4 (0.0)	1/4 (25.0)	3/4 (75.0)	3/4 (75.0)	3/4 (75.0)	3/4 (75.0)

^a Four female guinea pigs in each group were immunized with HB vaccines accordingly. The positive response of anti-HBs was defined as equal to or higher than 10 mIU/mL; ^bHP1 and HP2 were 2 Lots of experimentally prepared HP-HB vaccines (Lot No: HP20081004B and HP20081005B). SC1 and SC2 were 2 Lots of commercial SC-HB vaccines (Temrevac-HB) (Lot No: 200804415 and 200701143) as controls; ^cThe 0–4-8w, 0–4w and 0–8w indicated the guinea pigs were initially injected with HB vaccines at the beginning of the experiment and boosted at 4 weeks intervals for 3 injections, boosted at 4 weeks interval for 2 injections, and boosted at 8 weeks interval for 2 injections of 2µg HBsAg dose of relevant HB vaccines respectively. *One animal was taken out from the group by animal care staff and was not available for subsequent blood sampling.

Weights and behaviors of monkeys immunized with pilot-produced HP-HB vaccine

The median weight for monkeys in the experimental vaccine group (E-group) immunized with 2 doses of HP-HB vaccines produced in pilot scale was not significantly different from that of control vaccine group (C-group) with 3 doses of SC-HB vaccines (p > 0.05) (Fig. 2). No disease or death of animals occurred during the study,nor were any vaccination associated adverse effects observed. All animals behaved normally and were in good spirit during the investigation.

Figure 2. Weights (kg) of Cynomolgus monkeys vaccinated with pilot recombinant yeast HP-HB vaccine and of those vaccinated with control vaccine. C-Group: six animals vaccinated with recombinant yeast Saccharomyces Cerevisiae HB vaccine (Lot No: 20101166–1); E-Group: six animals vaccinated with recombinant yeast Hansenula Polymorpha HB vaccine (Lot No: 20110109) produced in pilot scale. All p > 0.05.

Anti-HBs levels of monkeys immunized with pilot-produced HP-HB vaccine

The anti-HBs levels were lower than the detection limit of assay before vaccination (day 1) and one week (day 7) after the first vaccination with the pilot produced HP-HB vaccine in all animals of both groups. On day 14 (day 14) after the 1st inoculation, the GMT of anti-HBs was 149.4 mIU/mL detected in 1/6 and 185.9 mIU/mL in 2/6 animals of E-group and C-group, respectively. The number of anti-HBs positive animals of E-group and C-group were increased to 4/6 and 3/6 of animals with GMT of 151.7 mIU/mL and 609.7 mIU/mL, respectively, before the second inoculation (day 21). One week after the second inoculation (day 28), all animals in the E-group and C-group showed positive anti-HBs GMT of 1636 mIU/mL and 2383 mIU/mL, respectively, with substantially higher titers. One week after the third inoculation (day 49) for the C-Group with SC-HB vaccine only, the GMT titers of anti-HBs of C-group were higher than those of the E-group with 2 doses of HP-HB vaccine and reached the peak of 24857 mIU/mL on day 49 before dropping gradually to 2368 mIU/mL of week 11 (day 77) and 7603.8 mIU/mL of week 12 (day 84). These values were similar to the levels of the E-Group of 1656.4 mIU/mL and 1791.3 mIU/mL by the end of the experiment (Fig. 3 and Table 2). The overall antibody titer of the E-group remained high throughout the investigation. The GMTs of anti-HBs for the C-group with 3 doses of SC-HB vaccine was significantly higher than that of the E-group with 2 doses of HP-HB vaccine one week after the third injection on day 49 (p = 0.006), but the overall GMT of anti-HBs was not significantly different between the E- and C-groups (p > 0.05).

Figure 3. GMT of anti-HB levels (mIU/mL) in the sera of Cynomolgus monkeys vaccinated with pilot produced recombinant yeast HP-HB vaccine and those with control vaccine. C-Group: six animals vaccinated with 3 doses of recombinant yeast Saccharomyces Cerevisiae HB vaccine (Lot No: 20101166–1); E-Group: six animals vaccinated with 2 doses of recombinant yeast Hansenula Polymorpha HB vaccine (Lot No: 20110109) produced in pilot scale. p ≤ 0.05: E-group vs C-groups on day 49.

Table 2. Response of monkey anti-HBs (%)^a.

Groups	Daysb
	0	7	14	21	28–84
E-Groupc	0/6 (0.0)	0/6 (0.0)	1/6 (16.7)	4/6 (66.7)	6/6 (100.0)
C-Groupd	0/6 (0.0)	0/6 (0.0)	2/6 (33.3)	3/6 (50.0)	6/6 (100.0)

^a The positive response of anti-HBs was defined as equal to or higher than 10 mIU/mL. ^bDays after the first vaccination of the hepatitis B vaccines. ^cE-Group consisted 6 Cynomolgus monkeys vaccinated with 2-dose pilot produced 20 µg HP-HB vaccine (Lot No: 20110109). ^dC-Group consisted 6 Cynomolgus monkeys vaccinated with 3-dose control 10 µg SC-HB vaccine (Lot No: 20101166–1).

Anti-HBs subclasses levels of monkeys immunized with pilot-produced HP-HB vaccine

The anti-HBs IgG1, IgG2a and IgM subclass titer were detectable in a single animal of the E-group on day 21 prior to 2nd vaccination as 2909 ng/mL, 239.6 ng/mL and 3022 ng/mL, respectively. These 3 subclasses GMTs were detected at 4705 ng/mL, 362.2 ng/mL and 5292 ng/mL, respectively, in 4/6 the E-group animals and 3115 ng/mL, 233.0 ng/mL and 4293 ng/mL, respectively, in 1/6 the C-group animal before the third inoculation on day 42. Three weeks after the third dose (day 63) for the C-group with SC-HB vaccine only, they were detected at 4575, 392.8 and 5153 ng/mL, respectively, in 4/6 animals of the E-group and 8948, 392.8 and 11517 ng/mL, respectively, in all 6 animals of C-group. As shown in Figure 4, the E-group and the C-group showed similar titers during weeks 9–12 for all three subclasses (p = 0.383). The average ratios of IgG1/IgG2a of these two groups were also not significantly different from each other at weeks 6 (day 42) and 9 (day 63). At the end of the experiment (week 12, day 84), only the IgG1/IgG2a ratios had changed while the other measurements remained the same. The earlier induction of IgG1, IgG2a and particularly IgM in the animals of the E-group suggests early prophylaxis of HP-HB vaccine against HBV (Fig. 4).

Figure 4. GMT of anti-HB subclasses (ng/mL) in the sera of Cynomolgus monkeys vaccinated with recombinant yeast derived HB vaccines. C-Group: six animals vaccinated with 3 doses of recombinant yeast Saccharomyces Cerevisiae HB vaccine (Lot No: 20101166–1); E-Group: six animals vaccinated with 2 doses of recombinant yeast Hansenula Polymorpha HB vaccine (Lot No: 20110109) produced in pilot scale.

Discussion

The 3-dose regimen is the routine vaccination schedule against HBV. In China, this regimen has been more widely used along with dramatic increases in HB vaccine coverage rates from 60.8% during 1992–1997 to 93.3% in 2002–2005.¹⁴ However, due to the long interval between the first and the third doses, HB vaccine coverage rates are significantly lower for migrant children either in China (45.6%)¹⁵ or in the world as a whole (70%).¹⁶

Some studies have suggested using a 2-dose regimen for HBV vaccination can provide effective protection against infection of HBV.^4-13 Wilson, et al. argued that most individuals should be immune to HBV infection six months after the priming vaccination even without the third booster dose.¹⁷ More importantly, 2-dose administration of HB vaccine should largely reduce the large medical and socioeconomic burden for many countries including China caused by the 3-dose regimen. Furthermore, less vaccine injections to infants are certainly better for their health, since the 3rd dose contributes to the overall side-effects.¹⁸

To evaluate the immune effects of a 2-dose regimen for the current Temrevac-HB (SC-HB vaccine) and the HP-HB vaccine produced and being developed by TIANTANBIO, we conducted a preliminary test in guinea pig. Four guinea pigs in each group were injected with 2 experimentally prepared HP-HB vaccines and 2 control SC-HB vaccines at 0–4-8w, 0–4w and 0–8w regimen with 3- and 2-doses of 2µg HBsAg vaccines. The detection of anti-HBs in the immunized guinea pigs showed that the experimental HB-HB vaccine could induce earlier antibody response and similar antibody levels to that of SC-HB vaccines. Although several non- or extremely low responding individual animals were observed, either 0–4w or 0–8w regimen of 2-dose HP-HB vaccines induced antibody responses at a level comparable to the 3-dose SC-HB vaccines (Fig. 1 and Table 1). The results of the guinea pigs demonstrated acceptable antigenicity of the experimental HP-HB vaccine. A higher HBsAg dose may reasonably increase the antigenicity of HB vaccine. In preparation of experimental HP-HB vaccines we found that the HBsAg production capacity of the recombinant HP 404 strain was much more robust than the current SC-HB vaccine, making it feasible and efficient to consider production of a high HBsAg dose HB vaccine. As this small animal trial gave relatively large variation in anti-HBs levels between different lots of the same species of HB vaccines and the existence of several non- or extremely low responders in small samples, a larger primate like monkeys were viewed as a better model to study the immunogenicity of 2-dose HP-HB regimen.

The current 3-dose regimen of 5µg HBsAg Temrevac-HB derived from recombinant SC in China is produced dominantly by the TIANTANBIO. The dosage was originally designed in early 1990s for over 16 million neonates annually, when HBsAg — the key component of HB vaccine — was a rare pharmaceutical resource, either from blood or recombinant cells, the 5µg dose design was reasonable given the limited supply of HBsAg and imperative prophylaxis of hepatitis B in the 1990s. HBV infection is highly endemic in China, placing a substantial burden of liver disease,^3,19,20 as transmission during the perinatal period and early childhood is the most prevalent mode of infection.¹⁹ The immunization of neonates, particularly those born to HBV carrier mothers, and infants with high HBsAg HB dose vaccine was a feasible strategy. The experience with 10 µg HBsAg recombinant yeast and 20 µg HBsAg recombinant Chinese hamster ovary cell derived HB vaccines in China gives evidence that the strategy is practical and effective,^19,21 though the HBsAg yields were limited. Several studies reported that the protective efficacy of higher HB vaccine dosage in newborn infants of hepatitis B e antigen-positive-hepatitis B surface antigen carrier mothers were significantly higher as compared with that of lower dosage.^21-24 A 3-dose 10µg HBsAg Temrevac-HB produced by TIANTANBIO was recently approved by the Chinese State Food and Drug Administration, but the higher HBsAg dosage formula may exhaust the limited manufacturing capacity of Temrevac-HB. The progress of methylotrophic yeast HP platform for biopharmaceutical applications is favorable due to fermentation characters including high cell density growth and high expression competency of foreign proteins,²⁵ thus it is possible to provide larger quantities of HBsAg to afford high HBsAg dose HB vaccine. The recombinant HB vaccine with 20 µg per dose has been developed specifically for the 2-dose vaccination regimen by TIANTANBIO from the yeast Hansenula Polymorpha, which can produce greater than 15 times more purified HBsAg than the current recombinant yeast Saccharomyces Cerevisiae used for Temrevac-HB. Freed, et al. employed monkeys to investigate the safety and immunogecinity of a 2-dose HB vaccine with novel CpG adjuvant,²⁶ while strongly immunogenic, even at low doses, the clinical development of a HB vaccine using CpG motifs was recently suspended in the USA.²⁷ This study is part of the preclinical evaluation of this alum adjuvanted HP-HB vaccine’s safety and efficacy using the nonhuman primate Cynomolgus monkeys.

The 3rd dose of conventional SC-HB vaccine did generate an immediate increase in anti-HBs levels among the immunized Cynomolgus monkeys. However, they reduced quickly to comparable levels as those of the 2-dose regimen of HP-HB vaccine. Specifically, the 2-dose of HP-HB vaccine induced higher than 175mIU/mL of anti-HBs (175.4~12,700 mIU/mL) one week after the 2nd dose, and the antibody levels were sustained at higher than 150 mIU/ml (151.2~7,444 mIU/mL) throughout the experiment (Fig. 3). These levels may be high enough to block HBV infection, since a person with as low as 10 mIU/mL has been reported to have virtually complete protection against HBV.^28-32 van der Sande et al. conducted a clinical study to compare the efficacy of 2-dose and 3-dose HB vaccine regimens in infants. They found that the percentage of anti-HBs non-responders was not higher for those who were vaccinated with 2-dose regimen, and that the 2-dose regimen protected the infants who responded to the vaccine from HBV infection, as effectively as the 3-dose regimen did.¹³

In this study, we also observed earlier induction of IgM, IgG1 and IgG2a of anti-HBs in monkeys, suggesting that the 2-dose administration of HP-HB vaccine may generate earlier protection than the 3-dose SC-HB vaccination (Fig. 4 and Table 3).

Table 3. Responses of monkey anti-HBs subclasses (%)^a.

Daysb	21	42	63	84
E-Groupc	1/6 (16.7)	4/6 (66.7)	4/6 (66.7)	4/6 (66.7)
C-Groupd	0/6 (0.0)	1/6 (16.7)	6/6 (100.0)	5/6 (83.3)

^a Anti-HBs subclasses include the IgG1, IgG2a and IgM. The positive responses of IgG1, IgG2a and IgM were defined as equal to or higher than 37.5, 2.5 and 37.5 ng/mL. ^bDays after the first vaccination of the hepatitis B vaccines. ^cE-Group consisted 6 Cynomolgus monkeys vaccinated with 2-dose pilot produced HP-HB vaccine. ^dC-Group consisted 6 Cynomolgus monkeys vaccinated with 3-dose control SC-HB vaccine.

In this study, the behaviors and weight gains of monkeys were not significantly different among different groups. We have previously performed the active systemic anaphylaxis test in guinea pigs, the acute toxicity test in mice and the abnormal toxicity test in guinea pigs and mice. All of these results confirmed the safety of the HP-HB vaccine.

The main purpose of developing the 2-dose regimen 20µg HBsAg HP-HB vaccine is to substitute the current 3-dose regimen SC-HB vaccine manufactured by TIANTANBIO. While the lack of a 2 doses Temrevec-HB group in the monkey experiment is a shortcoming of this study from a scientific perspective, its production in a less-efficient yeast system makes its exclusion one of a practical matter, that is, if clinical development of a 2-dose regimen is to be considered (and perhaps require a slightly higher dose of HBsAg), it makes sense to begin with the higher-yielding production system. Furthermore we note that the proper 2-dose SC-HB vaccine was tested in the precursor guinea pigs.

This primate study was unable to determine whether other qualities of the antibody response such as longevity, memory, avidity and protection are similar between the E- and C-groups, although the comparable levels of anti-HBs titers induced after 2 and 3 doses of relevant vaccine regimen in this relatively short-term study suggest the new candidate vaccine is comparable to the existing standard of care. The real immune effect of the 2-dose 20µg HBsAg HP-HB vaccine can only be evaluated in a clinical study comparing the routine 3-dose 5µg HBsAg SC-HB vaccine, with a control group planned carefully to observed the effects of 2-dose of latter vaccine with a 3rd dose provided should it be determined to be in keeping with ethics principles.

In conclusion, the 2-dose regimen of the novel recombinant Hansenula Polymorpha HBV vaccine resulted in comparable and comprehensive HBV immune responses as the routine 3-dose regimen of commercial SC-HB vaccine in Cynomolgus monkeys. The efficacy of this 2-dose regimen of the HP-HB vaccine in humans should be determined in future clinical trials to determine whether its more efficient production system enables the development of a replacement product with improved public health benifits.

Materials and Methods

Vaccines and adjuvant

A yeast Hansenula Polymorpha (HP) (ATCC34438) derived HU-11 strain³³ was used as host to construct a recombinant yeast Hansenula Polymorpha 404 strain, in which the genome includes a redesigned gene encoding the hepatitis B surface antigen (HBsAg) with high HP frequency DNA integrated to express the HBsAg. Two experimental recombinant HP HB vaccine (HP-HB vaccine) samples (Lot Nos: HP20081004B and HP20081005B) containing 20µg/mL HBsAg were prepared at laboratory scale. HBsAg was extracted from ruptured yeast cells (strain 404) by microfiltration and ultrafiltration and purified by chromatography. The purified HBsAg was adsorbed to an aluminum-hydroxide adjuvant. One pilot lot of HP-HB vaccine (Lot No. 20110109) was produced in pilot manufacture scale. The experimental and pilot HP-HB vaccines were prepared in the Vaccine Research Department II, Beijing Tiantan Biological Products Co., Ltd (TIANTANBIO), Beijing, China. The pilot vaccine was designed for the 2-dose vaccination regimen and formulated as 20 µg HBsAg/0.5mL/vial dosage. The virus like particle (VLP) size of the purified HBsAg is about 28nm,³⁴ the VLP form content of purified HBsAg is higher than 95% detected with HPLC and the residual host HP cell protein is much less than 0.5% assayed with ELISA.

Temrevac-HB (Lot Nos: 200804415, 200701143 and 20101166–1), a commercial recombinant HBV vaccine (SC-HB vaccine) derived from the recombinant yeast Saccharomyces Cerevisiae (SC) with free plasmid carrying primitive hepatitis viral HBsAg gene to express HBsAg,³⁵ was manufactured by TIANTANBIO. This vaccine contains 10 µg recombinant purified HBsAg per mL that was adsorbed on the aluminum-hydroxide adjuvant. The VLP size of the HBsAg purified from recombinant SC is about 22nm,³⁵ the residual host SC cell protein is less than 1% as assayed by HPLC. Its safety and efficacy have been reported previously.³⁶

The aluminum-hydroxide adjuvant contains 0.49 mg/mL aluminum.

Guinea pigs and immunization schedule

Forty eight female guinea pigs weighting approximately 350 g were divided into 12 groups, each group including 4 animals. The grouped animals were immunized separately with the 2 experimental HP-HB vaccines and 2 control SC-HB vaccines (Lot 200804415 and 200701143) at (1) 0, 4 and 8 weeks (0–4-8w regimen), (2) 0 and 8 weeks (0–8w regimen) and (3) 0 and 4 weeks (0–8w regimen) respectively. Each animal was injected in thigh muscle with 2µg HBsAg dose of relevant vaccine. The guinea pigs were housed and monitored by animal care staff at the Laboratory Department of TIANTANBIO.

Monkeys and immunization schedule

Twelve healthy 3–4 y old Macaca Fascicular were housed and monitored daily by animal care specialists at the Primate Utility, Joinn Laboratories, Beijing, China. The care of experimental animals was in accordance with institutional guidelines. Six males (2.59–5.29 kg of weight) and 6 females (2.11–2.76 kg) were randomly numbered in EXCEL and divided into 2 groups with each group consisting of 3 males and 3 females: the vaccine group (E-group) and the control group (C-group). The E-group monkeys were immunized muscularly at quadriceps femoris with 2 doses of 0.5 mL of pilot produced HP-HB vaccine (Lot No: 20110109) at 0 and 3 weeks. The C-group animals were similarly injected intramuscularly with 3 doses of 1 mL SC-HB vaccine (Lot 20101166–1) at 0, 3 and 6 weeks. Each monkey was weighed every two weeks. The behavior of each animal was monitored twice a day.

Serum anti-HBs antibody assay

Heart blood was sampled and separated for serum from each guinea pig once just before the first injection of vaccine and every 2 weeks thereafter for 12 weeks. The blood samples of monkeys were collected from the subcutaneous veins of limbs of monkeys once before vaccination and weekly thereafter until the 12th week. The serum was separated and assayed for the antibody against HBsAg (anti-HBs) with a commercial quantification ELISA kit (Wantai Biological Pharmacy, Beijing, China) following the manufacturer’s manual (http://www.ystwt.com/wantai_english/IFU/anti-HBs_q_IFU.pdf). The sensitivity of the kit is 5 mIU/mL of anti-HBs, and its linear range is 5~160 mIU/mL of anti-HBs. The serum was diluted by 10–100,000 folds with phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA) and assayed in duplicates. The OD values at 450 nm of samples and of the kit standard curve samples were read with the MULTISKAN MK3 (THERMO ELECTRON CORPOTATION, Shanghai, China). The crude anti-HBs concentration was deduced from the plot of sample OD data that were in the range of 0.085–2.230 and from the plot of the standard curve data. The crude anti-HBs data were multiplied by the dilution factor to obtain the adjusted anti-HBs data; when more than one diluted samples from same serum fall in standards range, the most diluted one was selected to calculate the adjusted anti-HBs. The mIU/mL of final anti-HBs was calculated as the mean of the duplicates-adjusted anti-HBs data. Animal is defined as anti-HBs responder when the antibody assayed equal to or higher than 10 mIU/mL in its serum.

Monkey anti-HBs subclass assay

The serum samples of monkeys that were collected before vaccination and 3, 6, 9 and 12 weeks post-vaccination were analyzed for IgG1, IgG2a and IgM levels with corresponding ELISA kits (Shanghai Yuanye Biotechnology Co., Ltd, Shanghai, China). The 96-well plate was coated with 100 µL/well of coating buffer containing 5 µg/mL purified HBsAg, incubated at 37°C for 2 h, rinsed and then blocked with 300 µL/well of 3% BSA blocking buffer at 37°C for 2 h. The curve standard samples of the kit for IgG1, IgG2a and IgM were diluted to 37.5–1200, 2.5–80 and 37.5–1200 ng/mL, respectively. Individual samples with anti-HBs subclass level no higher than the lowest point of the standard curve were defined as non-responders. The serum specimen was diluted by 50 folds. 50 µL of 50-fold diluted animal serum specimens and standard samples were added in duplicates to the wells of plates, incubated at 37°C for 30 min and rinsed. 50 µL of enzyme conjugate reagent was added, incubated at 37°C for 30 min and rinsed. 50 µL of color developer A and B were added to the wells of plates, incubated in darkness at 37°C for 15 min, followed by adding 50 µL stop reagent. The OD of 450 nm was read at the MULTISKAN MK3 of THERMO; the values of IgG1, IgG2a and IgM were deduced from the standard curves of relevant subclass antibodies; and the GMT data were calculated.

Statistical analysis

The median weights of cynomolgus monkeys in the E-group and the C-Group that were measured once every two weeks were compared and tested with the rank sum test. The geometric mean titers (GMT) of monkey anti-HBs and those of anti-HBs subtypes for the E-Group and the C-Group at each test time were analyzed with the F-test.

Glossary

Abbreviations:

HBV

hepatitis B virus

HB

hepatitis B

HBsAg

hepatitis B surface antigen

Anti-HBs

antibody against hepatitis surface antigen

E-group

experimental vaccine group

C-group

control vaccine group

SC-HB vaccine

Saccharomyces Cerevisiae derived recombinant hepatitis B vaccine

HP-HB vaccine

Hansenula Polymorpha derived recombinant hepatitis B vaccine

Temrevac-HB

commercial name for Saccharomyces Cerevisiae derived recombinant hepatitis B vaccine

Submitted

09/21/12

Revised

12/05/12

Accepted

12/12/12

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.