Hepatitis B Vaccines Clinical Trial
Background Hepatitis B virus (HBV) co-infection in individuals with hepatitis C virus (HCV)
can enhance the severity of hepatitis and the risks of liver cirrhosis and hepatocellular
carcinoma (HCC). Hepatitis B vaccine is an effective measure to prevent HBV infection.
Whether patients with HCV infection have non-protective antibody responses to hepatitis B
vaccination more frequently than healthy subjects is still controversial and studies about
cytokine response have been seldom reported.
Methods Not-in-treatment patients with chronic HCV infection and 1:2 community/gender matched
healthy control were obtained from a community-based screening. All participants received
three doses of hepatitis B vaccine (20 μg HBsAg/ml/dose) on 0, 1 and 6 months schedule.
Anti-HBs was tested 1 month after the third dose of vaccination and was compared between two
groups. Spot-forming cells (SFCs) of interferon-γ (IFN-γ), interleukin-2 (IL-2),
interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-6 (IL-6) produced by lymphocyte
were tested by enzyme-linked immunospot (ELISPOT) and were compared between two groups.
Study design and participants The study was conducted in three counties (Yucheng, Xintai and
Dongchangfu) of Shandong province, China, which had the highest reported HCV case numbers in
the province. Potential patients with chronic HCV infection were recruited by checking the
medical records of the hospitals or by inquiring of village doctors. Healthy individuals were
randomly selected by 3 to 5 times of potential patients with HCV infection in the same
county. Questionnaire investigation was made and blood samples were collected for each
potential patient and healthy individual in the same way.
Questionnaire survey We performed a questionnaire-based survey to collect the base line
information of the participants, including demographic information (age, sex, height and
weight), medical history (allergy, diagnosis and treatment of chronic hepatitis C,
vaccination, fever, and other acute diseases), and behavioral status (smoking, drinking,
pregnancy and lactation).
Hepatitis B vaccination and follow-up Three doses of hepatitis B vaccine made by Recombinant
DNA Techniques in Saccharomyces Cerevisiae (20 μg HBsAg/1.0ml per dose, Shenzhen Kangtai
Biological Products Co., Ltd., Shenzhen, Guangdong Province, China) were given
intramuscularly in the deltoid region to all participants at 0, 1 and 6 months respectively.
Blood samples from the participants were collected one month after the first and the third
dose of vaccination.
Laboratory assays and physical examination Screening test and physical examination HBsAg,
anti-HBs, HBeAg, anti-HBe, anti-HBc, anti-HCV and HCV RNA were assayed for all subjects at
inclusion visits; serum bilirubin, serum albumin, prothrombin time, plasma ALT and aspartate
aminotransferase (AST) were detected for HCV group. HBsAg, anti-HBs, HBeAg, anti-HBe and
anti-HBc were assayed by Abbott Chemiluminesent Microparticle ImmunoAssay (CMIA) (Abbott
Ireland Diagnostics Division, Sligo, Ireland). Anti-HCV was measured by ELISA with a
commercial kit (Intec products, INC, Xiamen, China). HCV RNA was measured by Quantitative
Real-time PCR with commercially available kits (QIAGEN, Shenzhen, China). Serum bilirubin,
albumin, prothrombin time, plasma ALT and AST were measured using standard reagent and
methods. In order to assess disease activity, each patient with HCV infection received
clinical examination, including interrogation, physical examination and ultrasonography.
Anti-HBs assay after vaccination Anti-HBs was detected using CMIA (Abbott Ireland Diagnostics
Division, Sligo, Ireland) one month after the first and the third dose of vaccination.
Although without serological HBV markers, subjects were defined as having a history of HBV
infection or hepatitis B vaccination if anti-HBs ≥100 mIU/ml one month after the first dose
of vaccination.
CMI assay Before the first dose of vaccination and one month after the third dose of
vaccination, 43 subjects from HCV group and 37 subjects from healthy control group were
selected randomly to isolated peripheral blood mononuclear cells (PBMCs) and tested CMI
function respectively, including interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4
(IL-4), interleukin-5 (IL-5) and interleukin-6 (IL-6). S28-39 polypeptide, MHC class I
polypeptide, MHC class II polypeptide and HBsAg were used as cell immunologic stimulant of
IFN-γ respectively. Hepatitis B virus surface antigen was used as cell immunologic stimulant
of IL-2, IL-4, IL-5 and IL-6. S28-39 polypeptide ，MHC class I polypeptide mixtures and MHC
class II polypeptide mixtures was synthetized in BO MAI JIE Technology Co. LTD. (Beijing,
China). HBsAg was supplied by Shenzhen Kangtai Biological Products Co., Ltd. (Shenzhen,
China).
PBMCs separated from EDTA-anticoagulated blood were adjusted to the concentration of 2 × 106
cells /mL. 100 μL 2 × 106 cells /mL PBMCs were added in the pre-coated PVDF 96-well plates
and 100 μL HBsAg S28-39 peptide (IPQSLDSWWTSL, final concentration: 10 μg/mL), 100 μL MHC
class I polypeptide mixtures, 100 μL MHC class II polypeptide mixture or 100 μL HBsAg
(80μg/mL) in each well. When it came to IL-2, IL-4, IL-5 and IL-6, plates were flooded with
100 μL 2 × 106 cells /mL PBMC and 100 μL HBsAg (80μg/mL) in each well. Then they were
incubated at 37 °C, in a 5% CO2 and humidified incubator for 20 h (IFN-γ, IL-2) or 40 h
(IL-4, IL-5 and IL-6). After incubation the plates were manipulated according to R&D ELISPOT
Kit (R&D Systems, Inc.) Instruction Manual. Spot-forming Cells (SFCs) were enumerated with
ImmunoSpotTM system (Cellular Technology Ltd.).
Safety assessment Participants were provided with diary cards to record the occurrence and
severity of solicited local reactions at the injection site (pain, induration, erythema,
edema, pruritus) during 7 days after vaccination, solicited systemic reactions (fever,
headache, fatigued, cough, myalgia, asthenia, vertigo, diarrhea), and any unsolicited adverse
during 29 days after vaccination.
Statistical analyses Data entry and database management were performed by EPIDATA 3.0 and
Microsoft excel 2010 respectively. Data analysis was performed by Stata 11.0. Differences
between HCV group and healthy control group in continuous and categorical variables were
examined using conditioned logistic regression. Subgroups of non- and low-responders versus
normal- and high-responders were evaluated in relation to demography characteristics,
biochemical indicators, liver disease statues and HCV RNA. A Student's t test or one-way
ANOVA were used to compare the average anti-HBs titer between different subgroups. Fisher's
exact test was used to compare the response rates. Nonparametric test was used to compare
immunodotting spots. Sperman rank correlation analysis was used to evaluate the correlation
between immunodotting spots and anti-HBs. For all these comparisons, a two-side P<0.05 is
considered statistically significant.
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