Clin. exp. Immunol. (1992) 90, 194-198

Immune response to hepatitis B virus surface antigen peptides during HBV infection A. MISHRA*, H. DURGAPAL*, V. MANIVELt, S. K. ACHARYAt, K. V. S. RAOt & S. K. PANDA* *Department of Pathology, and tDepartment of Gastroenterology, All India Institute of Medical Sciences, New Delhi, and tVirology Group, International Centre for Genetic Engineering and Biotechnology, Shaheed Jeet Singh Marg (NII Campus), New Delhi, India

(Acceptedfor publication 3 August 1992)

SUMMARY Antibody responses of patients with acute (n = 73), fulminant (n = 30) and chronic (n = 51) hepatitis B virus (HBV) infection as well as recovered individuals (n = 7) were studied against three synthetic peptides, Pre-SI amino acids (aa.12-32), Pre-S2 amino acids (aa.120-145), and S amino acids (aa.124-147) of the envelope region (HBsAg). T cell blastogenic response was investigated in a proportion of the patients (27 acute, nine fulminant, 13 chronic hepatitis and seven recovered individuals) along with seven HBV vaccinated and three normal individuals. The presence of T cell response against S peptide was observed in all the cases (9/9, 100%) during early acute hepatitis. This was suppressed during late stages (8/18, 44%) followed by partial reversal during recovery (5/7, 71 %). T cell response and antibodies to Pre-Sl and Pre-S2 peptides were present only in one-third of the patients throughout these periods. The T cell blastogenic response as well as antibody reactivity against these peptides were absent and minimal in chronic hepatitis. Immune response against envelope protein appears to play a major role in acute hepatic injury due to HBV infection and help in virus clearance. Keywords hepatitis B virus peptides T cell proliferative responses antibody reactivity acute and chronic hepatitis

INTRODUCTION Hepatitis B virus (HBV) is a major pathogen associated with acute and chronic hepatitis as well as having an asymptomatic carrier state. It is widely believed that pathogenicity is directly linked to the immune response generated against the viral antigens, particularly the nucleocapsid protein (core antigen) [1]. Very little information is available regarding T cell response to envelope antigen (HBsAg) because of limited immunogenicity of envelope proteins at T cell level [2] and suppression of immune response during chronic illness [3-5]. Some of the earlier studies of the peripheral cell mediated immune response to HBsAg using crude plasma-derived antigen had demonstrated its importance in virus clearance and pathogenesis [6-9]. However more recent investigations using highly purified antigens [10,11] have failed to demonstrate anti-HBsAg blastogenic response or lymphokine production during acute illness. In a prospective study, anti-Pre-S2 T cell response was demonstrated a month before the onset of clinical symptoms Correspondence: Dr S. K. Panda, Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-I 10 029, India.

followed by T cell response against S region [11]. However the T cell response to S region became undetectable during the clinical illness [11]. The responsiveness reappeared after recovery [11,12]. The antigen specific suppressor T cell response to HBsAg was undetectable during acute illness [11]. Idiotypic suppression was suggested [12] to explain this observation. A weak lymphocyte proliferative response and delayed hypersensitivity reaction were demonstrated during convalescence [12,13]. More recently, using transgenic mice, the immune response against S and Pre-S regions have been elegantly worked out in relation to the liver damage [14]. Both antibody and T cell mediated immune responses to S region have been shown to cause liver injury [14]. The cytotoxic T cell response was identified against peptide S (aa.21-40) in mice [14]. The CD4+ positive helper T cell response for surface antigen peptides as well as nucleocapsid antigens have been studied in acute and chronic hepatitis [15]. Demonstration of T cell recognition of small linear peptides from viral nucleocapsid that can drive conformational B cell responsiveness to native surface antigen in non-responsive conditions has clinical correlation among acute hepatitis B in vaccine non-responders [16]. T cell epitopes associated with MHC class II molecules which are adjacent to B cell epitopes have been identified on the envelope protein both in mice [16-18] and humans [19].

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Immune response to HBV In the present study we have analysed the T cell blastogenic and antibody response to various synthetic peptides of envelope protein, covering Pre-SI(aa. 12-32), Pre-S2 (aa. 120-145) and S (aa. 124-147) regions in patients of acute and chronic hepatitis B virus infection. MATERIALS AND METHODS Patients

Seventy-three patients with acute hepatitis, 51 patients with chronic hepatitis, 30 cases of fulminant hepatic failure and seven individuals recovered from acute hepatitis B attending Gastroenterology services of the All India Institute of Medical Sciences (AIIMS), were recruited for this study after obtaining informed consent. Only a proportion of them (27 acute hepatitis, 13 chronic hepatitis, nine fulminant and seven recovered cases) were studied for T cell blastogenic response. Seven individuals who had received plasma derived hepatitis B vaccine (Merck, Sharp and Dohme, USA) and three normal persons served as control. Acute hepatitis cases selected had a short course of illness preceded by a prodromal illness. The serum aspartate transaminase (AST) and alanine transaminases (ALT) were at least two and half times greater than normal values (AST < 40 IU/1, ALT 005), X2 test. t T cell blastogenic response to Pre-S peptides were not tested in one case.

Table 4. T cell proliferative response to S (aa. 124-127), Pre-SI (aa. 12-32) and Pre-S2(120-145) peptides in acute hepatitis B in relation to positivity for serum HBV DNA (positive/total)

HBV DNA-ve

HBV DNA+ve

Stage of HBV infection Acute hepatitis (26)

S

Pre-SI

Pre-S2

S

Pre-SI

Pre-S2

7/13* (53 8%)

4/13 (30 7%)

3/13 (23%)

10/13* (76-9%)

4/13 (30 7%)

3/13 (23 3%)

* Not significant (P> 0 05), x2 test.

for anti-HBsAg and anti-S (aa. 124-147) antibody. The normal controls were lacking in all these antibodies.

DISCUSSION Several hypotheses have been put forward to explain the pathogenicity of hepatitis B virus. They include: (i) HBV is not cytopathic to liver cells [1], (ii) immune responses against HBV are responsible for liver cell injury, (iii) cytotoxic (CD8)associated responses against nucleocapsid antigen is responsible for chronic liver disease [1]. The immune response during acute illness which leads to acute liver injury and subsequent virus clearance is not well understood. Immunologically two distinct modes of immune recognition dependent on restriction by class I and class II major histocompatibility complex (MHC) molecules exist [27]. The CD8 and CD4 surface molecules on lymphocyte subsets and antigen receptors of the T cells (TCR) recognize small peptides of 6-8 amino acids long. The CD4+ T cells in turn drive the B cells to elicit specific antibody response B cells [27]. Unlike earlier studies [11], we observed the presence of T cell proliferative responses to S region peptides in all the cases of early acute hepatitis (9/9). However in the late stage of illness the T cell blastogenic response diminished only to reappear again during the convalescence. The suppression of immune response during late phases may be due to (i) a high level of circulating antigen (HBsAg) in the serum, (ii) segregation of reactive T cells in the liver, (iii) appearance of suppressor T cells, or (iv) idiotypic suppressive regulation. There was no change in the T cell blastogenic response to either Pre-S1 (aa.12-32) or Pre-S2 (aa.120-145) peptides during different phases of illness. We were unable to confirm the previous observations [11] regarding the appearance of immune responsiveness to Pre-S

peptides early in diseases. We detected antibody responses to S and both Pre-S peptides in a small number of cases during all phases of illness. This was possibly due to the presence of HBsAg in the serum which competes and binds away most of the antibody. We did not observe an enhanced T cell responsiveness or increase in antibody titres to any of the peptides during fulminant hepatitis. The fall in T cell responsiveness during late acute phase of hepatitis may be due to immunosuppression [3,5] and persistence of the same may also be involved in the development of chronicity. The peptide (aa. 124-147) used here self-oligomerizes to form conformation dependent B cell epitopes [28] and at least two human T helper cells (CD4+) recognized epitopes (Misra et al., 1992, unpublished data). The presence of T cell response against S region uniformly in all the cases ofearly acute hepatitis suggests its possible involvement in the acute liver cell injury. Liver damage after transfer of HBsAg sensitized syngenic spleen cells to transgenic mice having S antigen expression on the liver further strengthens this evidence [14]. There is a marked increase in lymphokine release during acute illness [15], while the T cell response to nucleocapsid antigen is depressed [11]. CD8+ T cytotoxic cells against nucleocapsid protein (core antigen) have been shown to be responsible for liver damage in chronic liver disease [15]. Based on these observations, we propose two mechanisms for liver damage. One during the acute phase of illness is mediated by antibody and/or T cells against envelope protein (HBsAg). The other one for chronic liver damage is cytotoxic (CD8) cellmediated against core antigen, and is responsible for chronic liver damage. The clearance of the virus possibly depends upon the immune response against envelope antigen (HBsAg) which neutralizes the released virions and helps in virus clearance. Persistent suppression of immune response against HBsAg

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possibly leads to chronicity whereupon the cytotoxic response to nucleocapsid antigen perpetuates it. ACKNOWLEDGMENTS The work was carried out by the financial assistance from Indian Council of Medical Research, New Delhi, India and Overseas Development Administration, UK.

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Immune response to hepatitis B virus surface antigen peptides during HBV infection.

Antibody responses of patients with acute (n = 73), fulminant (n = 30) and chronic (n = 51) hepatitis B virus (HBV) infection as well as recovered ind...
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