From January 2016 to July 2018, 267 BALB/c mice (139 male and 128 female) of SPF grade aged three to eight weeks, with median (25% to 75%, interquartile range, IQR) age of 6 (4, 8) weeks, were bred, among whom 136 were housed at animal experiment center of affiliated hospital of Qingdao university in 2016 and 131 were housed at animal experiment center of medical college of Qingdao university, among whom 60 were raised in 2017 and 71 in 2018. All mice were allowed to acclimate the environment within the first week at the beginning of the study and all of them had standard feedstuff and drank purified water freely. The mice were exposed to a 12 hour light – 12 hour dark cycle at 22 ± 2 °C with relative humidity of 60% ± 5%. All mice were recorded sex, age, dose of HepB Vac, dose of hepatitis B immunoglobulin (HBIG), vaccination route and vaccination schedule before the first immunization. The mice and standard feedstuff were purchased from Qingdao Daren Fucheng Graziery co. LTD in China.
Materials and methods
Recombinant Hepatitis B Vaccine (Hansenula Polymorpha): specification, 10μg /0.5 ml, approval number: S20040016, produced by Dalian Hanxin Biological Pharmaceutical Co., LTD in China.
HBIG: specification, 200 IU/2ml, approval number: S10930001, produced by Shandong Taibang Biological Products Co., LTD in China.
ARCHITECT Anti-HBs Reagent Kit: produced by Abbott Ireland Diagnostics Division.
⑵ Laboratory methods
Anti-HBs were performed by a chemiluminescence microparticle immunoassay using ARCHITECT i 2000 full-automatic immune analysis system. The normal reference value was set as 0-10 mIU/mL. One test was carried out for each sample.
For the specimens of anti-HBs titer＞1000 mIU/mL, they were diluted to 10 times, 20 times or 30 times, and so on, until anti-HBs levels fell below the upper limit of detection (1000 mIU/mL).
Grouping and Immunization Schedule
All the mice were divided into two groups (group A and group B) according to the different doses of HepB Vac injected to mice. Group A was sub-divided into three subgroups: A1, A2, A3; and group B was sub-divided into three subgroups: B1, B2, B3.
⑵ Number and injection routes of each group
Each group included at least 40 mice: around 20 males and 20 females. HepB Vac was injected through two routes: intramuscular injection (quadriceps femoris, at least 10 males and 10 females) and hypodermic injection (scruff, at least 10 males and 10 females). HBIG was administrated through intraperitoneal injection.
⑶ Immunization schedule
The time when the experiment was started was defined as 0 week (w), the next as 1w, and so on.
Subgroups A1, A2, A3
A1: 25 IU HBIG and 2μg HepB Vac were administrated at 0 w, and then the second and third HepB Vac of the same dose were administrated at 4 w and 8 w respectively.
A2: 50 IU HBIG and 2μg HepB Vac were administrated at 0 w, and then the second and third HepB Vac of the same dose were administrated at 4 w and 8 w respectively.
A3: Three doses HepB Vac of 2μg were administrated at 0 w, 4 w and 8 w (0-4-8 w) respectively.
Subgroups B1, B2, B3
B1: 25 IU HBIG and 5μg HepB Vac were administrated at 0 w, and then the second and third HepB Vac of the same dose were administrated at 4 w and 8 w respectively.
B2: 50 IU HBIG and 5μg HepB Vac were administrated at 0 w, and then the second and third HepB Vac of the same dose were administrated at 4 w and 8 w respectively.
B3: Three doses HepB Vac of 5μg were administrated at 0-4-8 w respectively.
Definition of immunization outcome
Anti-HBs＜10 mIU/mL was considered as non-response; anti-HBs from 10 to 100mIU/mL was considered as low-response; anti-HBs>100 mIU/mL was considered as protective response; anti-HBs>1000 mIU/mL was considered as high-response[8, 11].
Sample collection in mice
Four weeks after the third dose of HepB Vac, blood was collected from the mice eye socket by vacuum tubes (no additives) . The mice were sacrificed immediately after the blood was drawn. The serum were separated within one hour, transferred into 1.5 centrifuge tube and stored in a -70 ℃ for anti-HBs testing.
x2 or fisher’s exact tests were used for categorical variables. For measurement data, normal distribution was first tested. If the result was of non-normal distribution, it was expressed in terms of median (25% to 75% interquartile range, IQR). The result of anti-HBs titer in this study did not obey normal distribution, and it presented normal distribution after taking logarithm. Thus the measurement data was analyzed by means of logarithms and t test or analysis of variance was used, expressed asx±SD. Logistic regression analysis was used to identify the risk factors. Variables assignments are as follows: Sex, Female=0, Male=1; HBIG dose, 0 IU=0, 25 IU=1, 50 IU=2; HepB Vac dose, 2μg=0, 5μg=1; Route of HepB Vac injection, hypodermic=0, intramuscular=1. And categorical variables (HBIG 0 IU, 25 IU, 50 IU) were transformed into dummy variables [(1), (2), (3)]. Variables with p value <0.1 in the univariate analysis were included in a multivariate logistic regression. Statisticalcal culations were performed using SPSS 22.0 software package with a p value <0.05 considered significantly.