Epitopes recognized by antibodies to denatured core protein of hepatitis B virus.

Particulate and denatured core protein as well as e-antigen (HBe) of hepatitis B virus (HBV) differ in part immunologically but this has not been studied in sufficient detail. Therefore, in this study the B-cell immune response to native and denatured HBV core protein which both can exhibit HBe-specific epitopes was examined using a panel of mouse MABs and rabbit polyclonal antibodies to native and denatured core protein and polyclonal anti-HBe/anti-HBc antibodies from sera of infected patients. Epitope mapping was performed using a set of partially overlapping synthetic HBc peptides, carboxy-terminally truncated HBc proteins and various HBc fusion proteins. A major immunogenic region between amino acids 134-140 and two less immunogenic regions, one spanning amino acids 2-10 and one with three partially overlapping epitopes between amino acid positions 138 and 154, were defined by mouse MABs. Polyclonal rabbit antibodies to denatured HBc, woodchuck and ground squirrel hepatitis core proteins (WHc and GSHc) recognized similar epitopes but in addition occasionally region 61-85, and the latter was also recognized on particulate HBc. Two antigenic regions (amino acid positions 2-10 and 138-145) were found to be exposed on HBe from human serum, and were recognized by mouse anti-HBe but not by anti-HBc antibodies from sera of infected patients. This study demonstrates a more complex pattern of HBc and HBe epitopes than detected previously and provides tools to study conformational changes which may take place during HBc/HBe processing, transport and core particle assembly.

Immunochemical structure of the carboxy-terminal part of hepatitis B e antigen: identification of internal and surface-exposed sequences.

The C-terminal region of hepatitis B virus (HBV) e antigen (HBeAg), amino acids (aa) 121 to 147, was characterized for reactivity with 15 monoclonal antibodies (MAbs) and sera from 16 chronic carriers on the HB surface antigen with anti-HBe. Recombinant proteins exposing fragments of the HBc/e polypeptide (aa 29 to 176, 60 to 176, 101 to 176, 121 to 176, 134 to 176, 138 to 176, 139 to 176, 140 to 176, 146 to 176 and 156 to 176) fused to the N terminus of the coat protein of RNA phage fr were constructed, as were two sets of synthetic peptides covering residues 121 to 136 and 130 to 147, where each residue was sequentially substituted by alanine. The MAbs were found to recognize overlapping epitopes in the fusion proteins within residues 121 to 176; however, none of the MAbs reacted with proteins covering residues 146 to 176 and 156 to 176. Using the synthetic peptides it was found that the MAbs recognized epitopes at residues 128-TPPAYR-133, 133-RPPNAP-138, 135-PNAPIL-140, 138-PILSTLPE-145 and 143-LPET-146. Only MAbs recognizing the epitope 128-TPPAYR-133 were found to react with both native HBeAg and denatured HBcAG, whereas MAbs recognizing epitopes located closer to the C terminus of HBeAg were reactive only with denatured HBcAg. The recognition sites for the human IgG1 overlapped with the epitopes of the MAbs recognizing native HBeAg. Our interpretation of these findings is that the region 124 to 133 is on the surface of native HBeAg and denatured HBcAg, and that the adjacent region 135 to 147 is not accessible on the surface of native HBeAg, but becomes exposed on denatured HBcAg.