HBV specific T-cell responses in hepatitis B

Hepatitis B virus (HBV) infection is a global health problem with more than 400 million chronically infected people worldwide, of which one million die annually with complications like liver failure, cirrhosis and hepatocellular carcinoma.[1]Asians account for almost 75% of people with chronic HBV infection.[2] The available treatment options include oral antivirals and interferon.

HBV is a non-cytopathic virus. The varied manifestations and course of HBV infection in different individuals is determined by the immune response mounted by that individual against the virus. When HBV infection occurs in an adult, it usually manifests as an acute hepatitis which may be icteric or anicteric. It has been observed that acute hepatitis B which usually is self limiting is followed by clearance of hepatitis B surface antigen, an indicator of resolved hepatitis B infection. Some individuals fail to achieve HBsAg clearance leading to persistent infection. Many of them manage to suppress the virus to a certain extent remaining as inactive carriers. In a small subset of patients the infection may progress to develop chronic hepatitis and ultimately cirrhosis.

The difference between the immune response in acute and chronic hepatitis B has been of immense interest to virologists and immunologists. Various investigators have used different techniques including T cell proliferation assays, ELISPOT assays, tetramer staining, and cytokine release assays for demonstrating the differences between the immune responses in the two groups. Ferrari et al[3] had reported that HBV infected subjects who develop a self-limiting acute hepatitis show a vigorous peripheral blood mononuclear cells (PBMC) response to HBcAg and HBeAg, whereas patients with chronic HBV infection exhibit significantly lower level of T cell responses.There have been quite a few reports which suggest that impairment of innate and adaptive immune responses as well as enhanced liver immune tolerance synergistically lead to inadequate clearance of the virus and viral antigen proteins in an established chronic hepatitis B (CHB) patient.[4,5]

The available reports do not reveal the nature of immune responses in patients who are in different stages of chronic hepatitis B infection. Patients with cirrhosis represent a population of particular interest. Bacterial infections are more common in patients with cirrhosis than in general population.[6,7]

Complement deficiency and impaired macrophage and neutrophil functions in these patients may be the reason for the high incidence of infections.[8,9] They are predisposed to develop sepsis and subsequent organ failure and have an increased risk of sepsis related mortality.[10] However there are few studies which have investigated the immune anomalies in HBV cirrhotics especially related to the virus specific T cell responses.[11] Similarly there exists a paucity of data regarding the immune responses in inactive carriers. Hence we did the present study on HBV specific T cell proliferative response in acute hepatitis B, chronic hepatitis B, HBV related cirrhosis and inactive HBV carriers.

Methods

The study sample included patients who were diagnosed to have HBV infection and were registered to the liver clinic at PGIMER, Chandigarh. An informed consent was taken from all the patients. The study was approved by institute’s ethics committee. The study population comprised of five groups: Group A - 10 acute viral hepatitis B (AVH) patients, Group B - 19 chronic hepatitis B (CHB) patients, Group C - 10 HBV cirrhotics,Group D – 10 inactive carriers and Group E included ten age matched healthy controls.

Patients

Acute hepatitis B patients (7 males, 3 females; mean age 34 years) were characterized by typical clinical presentation and ALT >10 times of normal with HBsAg and IgM anti-HBc positivity. The patients who were also positive for IgM anti- HEV or IgM anti-HAV or those who had evidence of chronic liver disease were excluded.[12]

Chronic hepatitis B patients (16 males, 3 females; mean age 33 years) had elevated liver enzymes on evaluation. Only those HBeAg negative patients with HBV DNA greater than 2000 IU/ ml and HBeAg positive patients with HBV DNA greater than 20,000 IU/ml were included in this study.13 Cirrhosis and hepatocellular carcinoma were ruled out in these patients on the basis of ultrasound or CT imaging, gastroscopy and alphafeto protein (AFP) levels. HBV cirrhosis (5 males, 5 females; mean age 39 years) was diagnosed on the basis of USG or CT scans or on endoscopy which showed varices.14 HCC was excluded in these patients with dynamic CT scans and AFP levels.

Inactive carriers (6 males, 4 females; mean age 35 years) included HBsAg positive individuals who were HBeAg negative, had persistently normal liver enzymes for 6 monthsand whose DNA levels were less than 2000 IU/ ml.[13] HBsAg and HBeAg were assayed in the sera of patients by ELISA method (Intec Products, Inc.; Xiamen and Wantai Biological Pharmacy enterprise Co. Ltd., Beijing respectively). Healthy controls (8 males, 2 females; mean age 32 years) included 10 apparently healthy volunteers, who were HBsAg negative with normal liver functions and normal ultrasound. Any patient in whom chronic liver disease was suspected due to an etiology other than HBV was excluded. This included patients who were positive for HCV antibodies, autoimmune markers, or had low ceruloplasmin levels. Significant alcohol abuse was another exclusion criterion. Patients with end stage renal disease, uncontrolled diabetes, human immunodeficiency virus (HIV) positivity or those on immunosuppressive medications were also excluded.

PBMC isolation

Five ml of heparinized blood was obtained by sterile venipuncture from each subject, after informed consent. Thereafter peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. PBMCs were then suspended in RPMI-1640 (Sigma, USA)
supplemented with 10% heat inactivated fetal bovine serum and antibiotics (complete RPMI).T cell responses in the study groups were assessed by calculating the proliferative index to phytohemagglutinin (PHA, Sigma, USA) and HBcAg (Prosper Tary Technogene Ltd., USA) using MTT [3(4,5-dimethylthiazoyl- 2-y1) 2,5diphenyltetrazolium bromide] (Sigma, USA) assay.[3]

Proliferation assay

PBMC were seeded at a concentration of 50,000 cells/well in a 96 well flat bottomed plate containing complete RPMI. The cells were then stimulated with PHA (10 µg/ml) for 3 days, and HBV specific antigen, recombinant HBcAg 0.5 µg/ml for 6 days in a humidified incubator at 37°C with 5% CO2. At the end of the experiment 10 µl of 5 mg/ml MTT was added to each well and the plates were incubated for another four hours, following which the cells were lysed using isopropyl alcohol and hydrochloric acid. During the 4-hour incubation, living cells convert the yellow tetrazolium into a purple formazan product and upon lysis the color is released in the supernatant. The optical density (OD) of the color was measured using an ELISA reader (Perkin Elmer) at 570nm. The optical absorbance at 570 nm is proportional to the number of viable cells in the wells.[15,16] The proliferation index (PI) was calculated according to the formula

OD of SS - OD of USS
Proliferation index = ––––––––––––––––––
OD of USS
SS=stimulated sample; USS=unstimulated sample

Analysis and statistics

T cell proliferation assays were done in all the groups and the responses were represented as proliferation indices. The mean + SD and range of responses were obtained in each group. One way ANOVA was used for comparing the mean T cell responses in all the four groups followed by Bonferroni correction. Correlation between bilirubin, ALT and T cell responses in group A was analyzed using Pearson’s correlation. A p value less than 0.05 was taken as significant. The analysis was done using SPSS statistical software package v11.0.

Results

Comparison of response to PHA and HBcAg in each cohort The T cell proliferative responses of AVH (group A) patients to PHA and HBcAg are shown in Figure 1. As expected the proliferative response to HBcAg (186.48 + 116.37; range: 50.74 to 434.76) in this group was better than that of PHA (55.60 + 42.53; range: 0 to 125.2). The response to HBcAg was significantly higher than that of PHA (p=0.0005) suggesting that HBV specific responses in AVH group were intact. There was a strong linear correlation between ALT values in AVH patients with the T cell responses to HBcAg with an r value (correlation coefficient) of 0.648 (Figure 2).

Similarly in chronic hepatitis B patients, the response to HBcAg (137.9 + 134.3) was better than that to PHA (62.7 + 40.9), although the difference was not significant (Figure 3).

In the cirrhotic patients (group C) the lymphoproliferative response towards PHA (44.2+ 46.9; range: 0.0 to 145.1) was comparable to HBcAg (55.7+ 42.71; range: 0.0 to 151). The T cell response to PHA and HBcAg are shown in Figure 4. Interestingly inactive carriers (group D) responded better to PHA (76.73+ 27.8 range: 20.2 to 366.5) than to HBcAg (67.5+ 41.2 range: 4.6 to 136.7) as reflected by their PI values (Figure 5). We had to exclude a patient (P100) who was a clear outlier while analyzing the results.

Since the immune response in healthy individuals (group E) were presumed to be intact, the response to PHA was very high (133.24 + 58.09; range: 52.73 to 205.2) while it was low towards HBcAg (45.92 + 51.50; range: 0 to 169.18). This was quite expected as healthy individuals have no prior exposure to hepatitis B virus. T-cell responses to PHA and HBcAg in this group have been shown in Figure 6.

Comparison of the T-cell responses in different groups The mean response to PHA was highest in healthy controls (133.2 + 58.1) and lowest in cirrhotics (44.1 + 46.9) with all the other groups falling in between (Figure 7). There was a significant difference between the mean response of healthy controls and cirrhotics (p=0.017).

The AVH group had the highest mean PI to HBcAg (186.5 + 116.4) followed by CHB (137.9 +134.3), inactive carriers (63.2 + 41.2) and cirrhotics (55.7 + 42.7), as shown in Figure 8.

Discussion

A better understanding of the cellular mechanisms responsible for the clearance of hepatitis B virus, development of hepatocellular injury and evolution towards chronicity of hepatitis B infection requires an analysis of the lymphocyte response to Hepatitis B virus antigens in large populations of patients at different stages of HBV infection. In the present study we have addressed this issue by assessing the response to recombinant HBcAg in various groups of Hepatitis B virus infected patients. In our study we chose the MTT method for assaying T-cell proliferation. The MTT method has been shown to produce comparable results with tritiated thymidine method for assaying lymphocyte proliferation.[15,16] Moreover this method also avoids exposure to hazardous radioactive material. We used recombinant HBcAg for antigenic stimulation of Tcells, because earlier studies have shown it to be a better immunogenic agent than HBeAg.[3] HBsAg would have generated false positive responses in vaccinated individuals who had never been exposed to HBV. The AVH group showed maximum T-cell response to HBcAg which was significantly higher than the response to PHA. The fact that T-cell proliferation to HBcAg in the AVH group was more than that to PHA, which is a nonspecific T-cell mitogen, suggests that HBV specific cell mediated mechanisms are maximally active in this population. This efficient cell mediated response might be the reason why patients with acute viral hepatitis B behave differently from other patients. More than 90% of the infected adults clear HBV and become HBsAg negative.[17] An earlier study conducted by Lohr et al[12] in 1998 compared T-cell responses to HBcAg in acute and chronic hepatitis B patients.They studied nine AVH – B patients and thirteen patients with untreated CHB without evidence of cirrhosis and compared their T-cell proliferative response to HBV antigens using the tritiated thymidine assay. They reported that the lymphocyte proliferative response to HBcAg and HBeAg was much stronger during acute hepatitis B than in chronic hepatitis B virus infection with the difference being statistically significant [stimulation index (SI) in AVH 14.9 + 4.7 vs. 6.5 + 1.3; p=0.02).

Another study by Ferrari et al[3] in 1990 compared the T-cell proliferative responses in 21 AVH- B patients, 29 CHB patients and 13 asymptomatic carriers. They also reported that lymphocyte proliferative response to HBcAg and HBeAg was much stronger during acute hepatitis B than during chronic HBV infection and the difference between the mean stimulation index peaks in the two patient populations was highly significant. Our findings are similar to these earlier reports.

Jung et al[18] in 1999 used ELISPOT assays to determine the number of IFN-a and IL-4 producing cells recovered from PBMCs of patients with AVH and CHB infection in response to HBcAg stimulation in short-term cultures. The majority (61%) of patients with AVH showed a significant number of IFNgamma- producing cells in response to HBcAg (mean number of spots/2 × 105 cells: 28.2 + 35.3), whereas only 25% of the chronically infected patients responded to the viral antigen (mean number of spots/2 × 105 cells: 10 + 17; p < 0.0002).

In another study 20 AVH and 26 CHB patients were compared for T-cell proliferation response to HBcAg using the tritiated thymidine assay. The PBMCs of majority of patients with acute hepatitis (18/20) responded with significant proliferation to stimulation with HBcAg. Reactivity to HBcAg in terms of proliferation was much stronger during acute infection [mean stimulation index (SI) = 13] than in chronically infected individuals (mean SI = 4.8). High stimulation indices in patients with acute hepatitis were an almost constant phenomenon. In contrast to the frequent and strong HBcAgspecific response obtained with PBMCs of patients with acute hepatitis, only 23% of the chronically infected group demonstrated HBcAg-specific proliferation.[19]

In our study, CHB patients had lower responses to PHA as compared to healthy controls. This would suggest that CHB patients had diminished lymphocyte proliferation to nonspecific mitogens. This implies generalized T-cell hyporesponsiveness. Among the other groups, cirrhotics had the lowest mean PI with HBcAg. As expected, there was a significant difference between cirrhotics and the AVH group when the PIs to HBcAg were compared. However there was no significant differencebetween cirrhotics, CHB patients and inactive carriers. Cirrhotics also had the lowest proliferative response to PHA. These findings suggest that in HBV cirrhotics the immune hypo-responsiveness is not restricted to virus specific responses. There are no studies which look at T-cell immune responses in cirrhotics, but the fact that cirrhotics are more prone to recurrent infections suggests that there may be associated immune anomalies in them. This might explain the low T-cell response in cirrhotics to both HBcAg and PHA.

Inactive carriers had lower PI to HBcAg as compared to AVH patients. There was no statistically significant difference in HBV specific responses between inactive carriers and CHB patients or cirrhotics. In the study by Ferrari et al,3 when the PBMCs in inactive carriers were stimulated with HBcAg, the stimulation indices (SI) ranged from 0.5 to 10, while in CHB patients the SI ranged from 0.6 to 7. Our study also shows similar results suggesting that once HBV establishes persistent infection in an individual, the virus specific T-cell responses are low, irrespective of the stage of infection. Healthy volunteers had the highest proliferative responseto PHA. This is expected as these individuals who were apparently free from any disease would respond maximally to a nonspecific T-cell immunogen like PHA. They also had the lowest proliferative response to HBcAg. In the study by Ferrari et al,3 the mean stimulation index (SI) in response to HBcAg was 1.5 + 0.5 as compared to AVH patients who had very high SI values. This is no surprise considering the fact that these healthy individuals would never have been exposed to hepatitis B core antigen.

In conclusion we can say that HBV specific T-cell responses are enhanced in acute viral hepatitis patients. This is the reason why they are able to contain the infection at early stages. The finding that cirrhotics have the lowest T-cell response to HBcAg or PHA among all groups of chronic hepatitis B patients, suggest that their immune functions are seriously deranged. These findings suggest that lymphocyte proliferative responses to virus specific antigens reflect the clinical outcome of HBV infection and use of immunomodulators may help in the management of this disease better, when used as adjunct therapy.

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