Response to Hepatitis B Vaccine in Egyptian Chronic Hepatitis C Patients

Background and study aim: Egypt unfortunately has the highest worldwide prevalence of chronic hepatitis C (CHC). Patients with CHC are advised to be vaccinated against hepatitis B virus (HBV) infection. Response to hepatitis B vaccination and risk factors for a weak response are not clearly defined.. The aim of this study is to assess the response to hepatitis B vaccination in CHC patients and identify predictors of a weak response. Patients and Methods: This prospective study included 112 consecutive adult, treatment- naive, CHC patients (cases group) and 54 non-hepatitis C virus (HCV) subjects (control group). Demographic and laboratory variables including HCV-viral load, schistosomal antibody (Ab) titre, and histopathological examination of liver biopsy were assessed. Three intramuscular 20 µg doses (given at 0, 1 & 6 months) of HBV-vaccine (Euvax-B, Korea) were administered, and hepatitis B surface antibody (HBsAb) titres were evaluated 6 – 8 weeks after the 3rd dose. Results: Out of 112 CHC patients, five (4.5%) had HBsAb titres of 1000. In comparison, out of 54 controls, one (1.9%) had a titre of 1000 (P= 0.001). CHC patients had highly significant lower mean Ab titres than controls (P<0.001). In a univariate regression analysis, HBsAb titre was negatively associated with age (P<0.001), ALT (P=0.03), AST (P=0.03), FIB-4 score (P=0.008) and schistosomal Ab titre (P= 0.007) and positively associated with platelet count (P=0.01). There was no association with gender, BMI, viral load or other variables (including METAVIR grade or stage). A multivariate regression analysis in CHC patients showed that age (P= 0.02) and schistosomal Ab titre (P= 0.04) were independent predictors of HBsAb titre response. Conclusions: CHC patients, particularly of older age or with schistosomiasis, have a significantly weakened  response to the HBV-vaccine.


INTRODUCTION
The most common viral diseases causing chronic liver diseases (CLD) worldwide are hepatitis B virus (HBV) and hepatitis C virus (HCV) infections [1].The global prevalence of hepatitis B surface antigen (HbsAg) positivity varies greatly and studies in the Middle East have shown that Egypt lies in the zone of intermediate prevalence, with HBsAg seroprevalence ranging between 3% to 11%, and genotype D is the most prevalent [2,3].On the other hand, Egypt is cursed with the highest worldwide prevalence of chronic hepatitis C (CHC), with an overall anti-HCV Ab prevalence of 14.7% and it is estimated that 9.8% of Egyptians are chronically infected with HCV.The main genotype in Egypt is genotype 4 (G4), which is responsible for >90% of infections, while the remaining infections are attributable to genotype-1 [4].In patients with dual chronic HCV and HBV infections, disease outcomes, including the development of liver cirrhosis and HCC, are generally more severe than those in patients with monoinfection.In addition, the incidence of HCC in co-infected patients is higher than the incidence in monoinfected patients [5,6].
Both acute and chronic coinfection with HBV in CHC patients is preventable by HBV vaccination [7,8].However, responses to the HBV vaccine are variable among different patients.Response of CHC patients to the HBV vaccine, factors affecting this response, suitable doses and the interval between doses are active areas of research especially in Egypt where HCV-G4 predominates and schistosomiasis is present.

Aim of the study:
To assess the response to HBV-vaccination in CHC patients and identify predictors of a weak response.

PATIENTS AND METHODS
This study was designed as a prospective clinical cohort study to assess the immunogenicity of the HBV vaccine in patients with CHC in comparison to healthy control subjects.The study protocol was approved by the Ethics Committee of Benha Faculty of Medicine.All patients gave written informed consent before enrollment in the study.
One hundred and twelve adult treatment-naive patients with CHC (cases group) and 54 non-HCV subjects (control group) who gave informed consent were included in the study.The inclusion criteria were: age older than 18 years, and CHC (in the cases group) that was diagnosed by both HCV-Ab (by 4 th generation ELISA test) and HCV-RNA-PCR positivity for ≥6 months before inclusion in the study.We excluded patients (or controls) who were positive for HBs Ag or HBc Ab (total); underwent previous HBV-vaccination; were pregnant, diabetic; underwent haemodialysis, organ transplantation, or immunosuppressive therapy; or who demonstrated malignancy and/or decompensated cirrhosis with ascites and/or HCC.Non-HCV healthy controls were recruited from subjects who came for vaccination for preemployment and pre-marital purposes or contacting HbsAg-positive patients.
Demographic data for patients and controls including age, gender and body mass index (BMI) were collected.Laboratory data, including haemoglobin level, white blood cell count, platelet count, liver function profile (ALT, AST, total bilirubin, prothrombin concentration and serum albumin), HCV-viral load and schistosomal Ab titre, were collected for all cases.FIB-4 score was calculated for all cases according to the standard formula [9,10].Abdominal ultrasonography was performed to exclude the presence of ascites and/or hepatic focal lesions, and histopathological data comprising METAVIR necroinflammatory grade (A0-3) and fibrosis stage (F0-4) were reported for those cases who underwent liver biopsy before receiving antiviral treatment within the national project of the Egyptian Ministry of Health.
The Euvax B vaccine (Euvax B, LG Life Sciences, Korea) used in this study is a liquid vaccine consisting of highly purified, non-infectious HBsAg particles absorbed onto aluminium salts as an adjuvant and preserved with thimerosal.It is a recombinant DNA hepatitis B-vaccine derived from HBsAg produced by recombinant DNA technology in yeast cells (Saccharomyces cerevisiae).
Vaccination of both cases and controls was accomplished by administering 3 doses of the Euvax B vaccine, each dose containing 20 µg of the active ingredient, purified HbsAg in a 1-mL volume; the vaccine was intramuscularly injected into the deltoid muscle at 0, 1, and 6 months.The response to the vaccine was measured by quantitatively assessing HBsAb titres (by ELISA test, according to the manufacturer's instructions), 6 -8 weeks after the 3 rd vaccination dose.Nonresponders were defined as patients who had an HBs-Ab titre of less than 10 mIU/mL, poor responders were patients with an HBs-Ab titre between 10 and 100 mIU/mL, and good (robust) responders were those who had HBs-Ab titre of more than 100 mIU/mL.

Statistical Methods :
SPSS (version 21) was used for statistical analysis.Comparison of patients and control groups was performed by using a two tailed "t" test for continuous variables and a Chi square test for categorical or dichotomous variables.Non-parametric tests were used when indicated.Univariate regression analysis was performed to assess the association between continuous variables and HBs-Ab titre within CHC patients.Independent samples two-tailed "t" test was performad to assess the association between categorical or dichotomous variables and HBs-Ab titre.Significant variables associated with HBs-Ab titre in all univariate analyses were included in a multivariate regression analysis to identify independent predictors of the response.Pearson correlation test was performed to test the correlation between age and HBs-Ab titre.For all tests, 0.05 was set as the level of significance.

RESULTS
This study included 112 CHC patients and 54 healthy controls.Out of the 112 patients, 36 (32.1%) were males.Tables (1) and (2) show descriptive demographic, laboratory and histopathological data for CHC patients.Only 79 patients had schistosomal-Ab data, and 60 had histopathological data of liver biopsy.Table (3) shows the comparison between cases and control groups in terms of age, gender distribution and BMI.Regarding the response to hepatitis B vaccine, we found that CHC patients had significantly lower HBs-Ab titres than healthy controls and significantly more number of non responders (Table 4 and Figure 1).
Univariate regression analysis showed that there were significant associations between HBs-Ab titre levels and age (P<0.001),AST (P= 0.03), ALT (P= 0.03), FIB4 score (P= 0.008), and platelet count (P= 0.01).Age showed a significantly negative linear correlation with HBs Ab response (Figure 2).There was no statistically significant difference between males and females regarding HBs-Ab titres while there was a statistically significant negative association between the presence of schistosomal Ab and HBs-Ab titre (P= 0.007).
Variables that showed a significant association with HBs-Ab titre in univariate analysis were included in multivariate regression analysis to identify independent predictors of HBs-Ab titre response.The only independent predictors for HBs-Ab response were age and the presence of schistosomal Ab (Table 5).The novel finding of our study is the association between the presence of schisosomal Ab and low HBs-Ab titres after vaccination, indicating that schistosomiasis alters the immunologic response to HBV vaccine.
The presence of schistosomal Ab and older age were found to be independent predictors of the response to HBV vaccine in our studied CHC patients.

CONCLUSIONS
Patients with CHC, especially older patients and patients who are schistosomal Ab positive, demonstrate a lower response to HBV vaccination.
Funding: None.Conflicts of interest: None.Ethical approval:Approved .

Table ( 1
): Description of demographic and laboratory data of CHC patients.

Table ( 3
): Comparison of demographic variables between cases and controls.

Table ( 4
): Comparison of HBV vaccine responses (HBs Ab titres) between cases and controls.

Table ( 5
): Multivariate regression analysis for independent predictors of the response to HBV vaccination (HBs Ab titre).In their study of 3,596 Egyptian patients, Abdel-Rahman et al. [24] reported that 27.3% had both HCV-RNA and schistosomal Ab in their sera.Patients demonstrating schistosomiasis/ HCV coinfection have increased HCV morbidity and chronicity [25].Defects in immune response with altered IFN-γ and IL-5 serum levels (related to cell-mediated immunity) and IgE levels (humoral immunity) have been reported, with a relative shift from cellular to humoral immunity, which might play a role in the persistence and severity of both diseases and lower HCV clearance rates [26,27].