Aggregation of recombinant hepatitis B surface antigen induced in vitro byoxidative stress

In order to examine whether oxygen radicals could be responsible for aggreg ation of recombinant hepatitis B surface antigen (HBsAg) during its assembl y in yeast, purified HBsAg was oxidized with ammonium peroxodisulphate (AP) and analyzed by non-denaturing and denaturing size exclusion chromatograph y, immunoassay and immunoelectron microscopy. As a result, peroxodisulphate radicals induced a reproducible aggregation of HBsAg. At 44 mM AP, the agg regation process rook a few hours and the resulting structures were large, branched and non-antigenic. During more gentle oxidation with 9 mM AP and 2 0-80 mu M Cu2+, a continuous structural modification to HBsAg delaying for tens of hours preceded the aggregation event. During this pre-aggregation p eriod, peroxidation of HBsAg lipids and covalent cross-linking of S protein chains occurred that led a complete loss of antigenicity of oxidized parti cles. In contrast, yeast-derived HBsAg aggregate is decomposed to S monomer s under reducing conditions and recognized by anti-HBsAg polyclonal and mon oclonal antibodies, suggesting that is has been assembled in vivo from anti genic and reversibly cross-linked particles. Based on these observations, w e conclude that oxidation, at least with respect to the specific molecular sites oxidized by AP, is not a primary event in HBsAg aggregate formation i n vivo. Since oxidized HBsAg was shown to be irreversibly cross-linked and non-antigenic, there are no suitable techniques for detection HBsAg oxidati on in biological samples. Hence, at present, the magnitude of the in-vivo o xidative damage to HBsAg cannot be evaluated and thus, whether the plasma-d erived HBsAg undergoes radical-induced oxidation in the course of viral hep atitis remains to be established. If this occurs, this process is expected to contribute to low HBsAg levels in chronic hepatitis B carriers, failure of the currently available immunoassays to identify HBsAg-positive blood do nors and inconsistency in the results provided by HBsAg- and anti-HBsAg-bas ed tests in several recent reports. (C) 1999 Elsevier Science B.V. All righ ts reserved.