Novel immunoassay for the detection of hepatitis B surface ?escape? mutants and its application in liver transplant recipients

Abstract

Hepatitis B virus (HBV) strains with mutations in the surface gene are responsible for the failure of prophylaxis with hepatitis B immunoglobulin (HBIG) in a proportion of patients transplanted for HBsAg positive cirrhosis. So far, the emergence and evolution of these 'surface antibody escape' mutants have been studied by DNA sequencing. In this study the use of an immunoassay is described for diagnosis and characterisation of HBV recurrence after liver transplantation (OLT), based on a monoclonal antibody able to recognise both wild-type and mutant HBsAg. Pre- and post-transplant samples from 22 patients transplanted for HBsAg positive cirrhosis were studied: Group A: 12 patients who reinfected the graft despite receiving HBIG; Group B: 6 patients with no HBV recurrence with continuous HBIG; Group C: 4 patients with HBV recurrence without prophylaxis. By running the new assay in parallel with an immunoassay that is susceptible to HBsAg mutants, 4 of 12 cases were identified in Group A with HBV recurrence due to surface antibody escape mutants, whereas in 8 patients this was due to the wild-type HBV. The results from the immunoassays were confirmed in all cases by HBV DNA sequencing. The surface gene mutations in the 4 patients affected codons 144 and 145 and in one of these 4 patients HBV strains with mutations in both codons were detected before and after transplantation. The epitope recognised by the new monoclonal antibody that reacts with both wild-type and mutant HBsAg seems to remain stable in the HBIG-induced HBV mutants. This serological approach allows rapid and cost-effective screening for HBsAg escape mutants in the liver transplant setting and may be helpful in the selection of appropriate prophylaxis.

Endocytosis of Hepatitis B Immune Globulin into Hepatocytes Inhibits the Secretion of Hepatitis B Virus Surface Antigen and Virions

“…For the detection of HBsAg, the membrane was incubated for 1 h at RT with the mouse anti-HBs antibody P2D3 diluted 1:250 in PBS plus 1% MP. The P2D3 antibody is known to react with both wild-type and mutant G145R HBsAg (8). After three washes in PBS-1% Tween 20, the blot was incubated for 30 min at RT with HRPO-labeled rabbit anti-mouse IgG or swine anti-rabbit IgG (Dako) diluted 1:10,000 in PBS plus 1% MP.…”

“…The plates were washed five times with TS buffer (100 mM NaCl, 0.5% Tween 20) and incubated at RT for 4 h with 100 l of 125 I-labeled monoclonal mouse anti-HBs P2D3 (50 nCi diluted in 20 mM Tris buffer containing 2% bovine serum albumin and 20% normal human serum). The epitope recognized by P2D3 was mapped between amino acid positions 121 and 135 of HBsAg, and the antibody was previously shown to react with wild-type and G145R mutant protein (8). The wells were washed twice with TS buffer, and the radioactivity was monitored in a Gammacounter (NE 1600; Nuclear Enterprises, Edinburgh, Scotland).…”

“…As a control, we cultured the cells with 1.0 mg of IgG/ml containing a high titer of anti-HBc but no anti-HBs. The secretion of HBsAg into the culture supernatants was monitored by RIA using the HBs-specific monoclonal antibody P2D3, which detects both wild-type and G145R mutant HBsAg (8). Cells transfected with G145R mutant HBV produced larger amounts of HBsAg in the supernatants than the wild-type HBV.…”

Molecular, immunological and clinical properties of mutated hepatitis B viruses

“…These tests therefore may have a good chance to find a wildtype epitope. One attempt was made with a monoclonal antibody directed to the first loop of the a-determinant [254]. This test was shown to recognize HBV variants with mutations in the second loop of the a-determinant.…”

“…This test was shown to recognize HBV variants with mutations in the second loop of the a-determinant. Unfortunately the wildtype genotype D subtype ayw 3 could not be detected [254]. Other radioimmunoassays which recognized epitopes within the preS1 region, within the preS2 region, and within the S-region together [255] also were found to be efficient for the detection of mutations within the a-determinant [252].…”