Robust Antibody and Cytokine Response to Hepatitis B Vaccine Among Not-in-treatment Patients With Chronic Hepatitis C：An Open-label Control Study in China
Robust Antibody and Cytokine Response to Hepatitis B Vaccine Among Not-in-treatment Patients With Chronic Hepatitis C：An Open-label Control Study in China
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.
Inclusion Criteria:

 -  Inclusion criteria for HCV group were as follows: (1) aged ≥ 22 years and Han
 nationality; (2) no history of hepatitis B vaccination; (3) not-in-treatment patients
 with anti-HCV and HCV RNA positive; (4) diagnosis of chronic hepatitis C on the basis
 of self-reported history of HCV infection (more than 6 months) and screening tests for
 inclusion; (5) negative for HBsAg, anti-HBs, hepatitis B e antigen (HBeAg), antibody
 to hepatitis B e antigen (anti-HBe) and antibody to hepatitis B core antigen
 (anti-HBc).

 Inclusion criteria for healthy control group were as follows: (1) aged ≥ 22 years and Han
 nationality; (2) no history of hepatitis B vaccination; (3) negative for anti-HCV and HCV
 RNA; (4) negative for HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc; (5) no self-reported
 acute and chronic diseases.

 Exclusion Criteria:

 -  (1) allergy to any vaccine component; (2) pregnancy or lactation; (3) axillary
 temperature ≥38℃ in the past three days, acute disease in the past seven days or
 vaccination history of any vaccine in the past four weeks; (4) suffering from diseases
 that may influence immune function, such as severe cirrhosis with Child-Pugh score >5,
 renal failure, bleeding diathesis, malignant tumor and HIV infection; (5) have
 received or being taking antiviral treatment; (6) chronic liver diseases except
 causing by HCV.