Immune Response In Patients With Chronic Hepatitis B And The Efficacy Of Antiviral Therapy On Immunologic Function

The hepatitis B virus (HBV) is a noncytopathic DNA virus that causes both acute and chronic liver disease. Currently, there are about 350 million people infected with HBV around the world. About 10% of the infected population has developed to chronic infection when the HBV invades human body. The chronicity of HBV infection is the main contributing factor to hepatocirrhosis and hepatic carcinoma. Every year, about 1 million people die of chronic HBV. Therefore, the top priority is to conduct antiviral and immunologic treatment on patients infected with HBV.The pathogenesis of chronic hepatitis B (CHB) is evoked by the host's inappropriate immune response between the immune molecules and HBV-infected hepatocytes. The cellular immune response to the viruses has been described as a double-edged sword. On one hand, a strong and multi-specific T-cell response is helpful to viral clearance. On the other hand, the hepatocytes can be damaged when the organism is clearing the viruses. Therefore, constant immunologic response can result in liver damage if the viruses cannot be eliminated. Our focus is laid on immunologic response and immunologic function of the organism when the virus load decreases in the anti-viral course with nucleotide. Different human bodies have different immunologic responses to HBV, which necessarily results in the difference in clinical turnovers. More and more attention has been drawn to the illustration of the relationship between the features of immunologic response and clinical turnover. It may be a basic strategy in controlling HBV infections permanently to enhance or to recover the specific immunity of human body. The immunologic response to lamividine (LAM) therapy may have a close relation to its clinical efficacy. Therefore, this research observes the features of immunologic response in CHB patients and analyses the relationship between clinical efficacy and immunologic response in the CHB anti-viral course with lamividine. It is not only helpful for us to select an effective therapeutic timing and scheme and to make proper judgment on the clinical efficacy and prognosis, but also beneficial for us to find a new immunologic therapy.Objects: to investigate the effects of Lamivudine (LAM) treatment on IFNγ(Th1 type cytokine) and IL-4 (Th2 type cytokine) levels in patients with CHB, to observe the changes of CD4+CD25+regulatory T cells (Tregs) frequencies in the peripheral blood of patients infected with HBV, and to analyze their probable association with drug resistance and prognosis of antiviral therapy.Methods : 1. Serum levels of INF-r and IL-4 were measured with enzyme linked immunosorbent assay (ELISA): Ninety HBeAg positive CHB patients (male 70 and female 20,age 30±12.6 years) were selected in the study. Admitted criteria for our study were: detectable HBsAg, HBeAg, serum HBV DNA≥105copies/ml,ALT>80U/L,ALB>30g/L, PLT>100×1012/L. All patients were treated with LAM at a dose of 100mg/d for a year. Serum levels of INF-r and IL-4 were measured with enzyme linked immunosorbent assay (ELISA) at baseline and 3 and 12 months during the treatment. Serum indexes including liver function, HBsAg, HBsAb, HBeAg, HBeAb, HBcAb and HBV DNA were also measured regularly. Twenty healthy volunteers (male 12 and female 8, age 21-36 years) were served as normal control.2. The proportion of CD4+CD25+Tregs in the peripheral blood were measured with flow cytometry.Forty HBeAg positive CHB patients were selected ,including 10 HBeAg positive CHB group (male 5 and female 5, age 28±11.6 years), 10 HBVcarriers group ( male 4 and female 6, age 33±10.5 years) whose liver function are normal, 10 maintained response group (male 5 and female 5, age 30±10.6 years) after treated by LAM 100mg/d,10 breakthrough group (male 8 and female 2, age 59±16.5 years) during treated with LAM 100mg/d were selected. Simultaneously,10 healthy volunteers (male 5 and female 5, age 29±6.5years) were served as normal control. The frequencies of CD4+CD25+Tregs in CD4+ T cells were measured with flow cytometry. Other serum indexes were also measured as above.Results: 1. Among the ninety patients (all are HBeAg positive) with CHB, 15 (16.7%) patients had complete response, 47 (52.2%) patients had partial response, and 31.1% (28/90) were non-response at the end of one year's LAM treatment. 34(37.8%)patients showed partial response in three monthes, 48 patients had maintained response in one year, 14(15.5%)patients showed breakthrough (including virological and biochemical breakthrough), among which two came from the group having complete response and twelve came from the group having partial response(Five came from partial response within three months and seven came from partial response after three months).2.The changes of serum IFNγ,IL-4 levels:①In CHB group, the IFNγlevel was 18.83±7.35pg/ml and the IL-4 was 25.54±12.56pg/ml before the treatment. In control group, the values were 15.24±3.93pg/ml and 13.51±5.86pg/ml, respectively. There were statistical significance (P<0.05). In CHB group, the IFNγlevel was lower, the IL-4 level was higher, as a result, the ratio of IFNγ/IL-4 was lower apparently than control group (P<0.05) (Table 1).②Before the treatment, the IFNγfor the group that had complete response, the group that had partial response, and the group that had no response after one year's treatment were 23.65±3.24pg/ml, 14.10±2.08pg/ml, 8.59±3.14pg/ml, showing significant differences (P<0.05). After the treatment, the values were 33.74±6.63pg/ml, 18.57±3.76 pg/ml, 8.59±3.14pg/ml respectively, showing significant differences (p<0.05). IL-4 level decreased compared with the pre-treatment level in the group having complete response, having statistical significance (p<0.05). In other two groups, the IL-4 level had no significant variation, having no statistical significance (p>0.05) (Table 2).③After the treatment the IFNγin the group having complete response increased and the IL-4 decreased. The IFNγ/IL-4 ratio rose to 1.16±0.51, similar to or higher than the control group. The difference had statistical significance. After the treatment the IFNγin the group having partial response increased a bit, but lower than that of the group having complete response, and the IL-4 decreased. The IFNγ/IL-4 ratio was 0.75±0.53. After the treatment the IFNγ/IL-4 ratio in the group having no response increased a bit. But the ratios for the group having partial response and the group having no response had no significant difference compared with pre-treatment value (p>0.05) (Table2).④The pre-treatment level of IFNγof the group having breakthrough was 10.72±3.76pg/ml and the post-treatment level was 15.54±7.82pg/ml. There was statistical significance (p<0.05). The pre-treatment level of IL-4 was 19.16±7.63 pg/ml and the post-treatment level was 19.93±8.17pg/ml. There was no statistical significance. The IFNγ/IL-4 ratio after the treatment was 0.70±0.21, showing no statistical significance (p>0.05) (Table2).⑤The pre-treatment IFNγlevel of the group having partial response within three months'treatment was 16.93±5.02pg/ml, higher than that of the group having partial response after three months'treatment, 13.72±4.81pg/m. there were statistical significance (p<0.05) (Table 3)3. The frequencies of CD4+CD25+Tregs in the peripheral blood were measured with flow cytometry:①The frequencies of CD4+CD25+Tregs for the CHB group, HBV carriers and the control group were: HBV (6.14±0.53)%, CHB (4.11±0.53)%, control group (2.86±0.63) %. There were significant differences among them( P<0.05).②The frequency of CD4+CD25+Tregs in CD4+T cells for the CHB group having maintained response after treatment with LAM is (2.3±0.41) %, lower than the CHB group without treatment, having statistical significance (p<0.05). Compared with the control group, it had no statistical significance (p>0.05).③The frequency of CD4+CD25+Tregs in breakthrough group was (4.7±1.26)%, which was lower than HBV carriers, but higher than normal control or the group having maintained response. There were statistical significance (P<0.05). The difference between CHB group and breakthrough group has no statistical significance (p>0.05).Conclusion: 1. In HBeAg positive CHB group, the IFNγlevel decreased and the IL-4 level increased. It displays that Th2 cells prevail and Th1/Th2 lose balance. Cellular immune function is obviously in disorder in patients with hepatitis B, which might be closely associated with the chronicity of HBV infection.2. Before the treatment, IFNγlevels were higher in patients whose complete response happened within three months than those after three months. It suggests the close relation between immune function and the efficiency of antiviral treatment. Sutiable immunity betokens a better antiviral response. Therefore, detecting the immune function before antiviral therapy will help us select therapy and judge the therapeutic efficacy and prognosis of antiviral therapy.3. IFNγlevels in breakthrough group were higher, but remained Th2 superior response. It indicates IFNγlevel plays an important role in the antiviral response. After antiviral therapy by LAM, the HBV load decreases and the cellular immunity will be restored but not enough to lead to maintained response.4. After LAM treatment complete response group showed Th1 superior response and Th1/Th2 balance recovered. However, partial response group and non-response group remained Th2 superior response. The response to LAM therapy is related to the balance of Th1/Th2.5. The frequency of CD4+CD25+Tregs in CHB group was lower than that of HBV carriers group, but the frequency of the two groups were higher than control group. It indicates that in CHB group the immune tolerance relatively weakened and the CD8+T cells'function was strengthened, therefore, several liver cells incurred degeneration, necrosis and even apoptosis, and meanwhile HBV were partly cleared.6. After LAM therapy, maintained response group showed normal frequency of CD4+CD25+Tregs, but obviously lower than HBV carriers group and CHB group that were not treated. It indicates that the existence of HBV-specific Tregs might be capable of inhibiting HBV-specific T cell response in infected patients. The suppression function of CD4+CD25+Tregs will be alleviated by the inhibition of HBV.7. Breakthrough group showed lower frequency of CD4+CD25+Tregs than HBV carriers, but higher than group having maintained response. It indicated that with the decrease of the HBV, the immune function was relatively restored. But it was still not enough to control the replication of HBV. In this case, HBV mutation might easily emerge during long-time treatment.