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
Condition: Hepatitis B Vaccines
MeSH terms: Hepatitis A; Hepatitis B; Hepatitis
Study type: Interventional
Phase: Phase 4

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): hepatitis B vaccine made by Recombinant DNA Techniques in Saccharomyces Cerevisiae
Intervention type(s): Biological
Intervention description(s): 20 μg HBsAg/1.0ml per dose, Shenzhen Kangtai Biological Products Co., Ltd., Shenzhen, Guangdong Province, China
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Prevention