THE IMMUNE ESCAPE VARIANT GLY145ARG EMERGING UNDER HBIG TREATMENT IN PATIENTS WITH RECURRENT HBV AFTER LIVER TRANSPLANTATION IS SELECTED IN A MIXED POPULATION WITH WILDTYPE VIRUS DESPITE ITS SECRETION DEFECT:

O245 Aims: Liver transplant recipients who develop recurrent hepatitis B virus (HBV) infection despite prophylaxis with Anti-HBs hyperimmunoglobuline (HBIG) are often found to have selected a specific immune escape variant which carries a Gly145Arg substitution in the small surface protein (S-protein) of HBV. Previously, we demonstrated that this variant has in vitro a severe secretion defect which may be responsible for a cytotoxic effect of the virus causing fibrosing cholestatic hepatitis by accumulation of viral proteins and DNA (Hepatology 2003). Here, we investigated how the secretion defective Gly145Arg variant can emerge as the dominant viral population in the serum of patients. Methods: To investigate the interaction between wildtype virus (wt) and the Gly145Arg variant several cotransfection experiments were performed using varying amounts of wt and mutant full-length HBV genomes. In addition, viral constructs expressing only mutant or wt small (S-) or large (L-) surface protein were used for the studies. After transfection of viral genomes in human hepatoma cells Northern, Western and Southern blot analysis were performed to study viral production and secretion. Results: A small amount of wt was able to rescue secretion of the defective Gly145Arg mutant. Notably, the virions secreted after cotransfection of wt with the Gly145Arg mutant contained the secretion defective mutant HBV DNA, but were encoated by wt surface proteins. Further experiments revealed that only wt S-protein, but not wt L-protein was important was rescuing secretion of the Gly145Arg variant. Additional cotransfection experiments using constructs expressing only mutant S-protein and wt revealed that selection of the secretion defective mutant viral DNA was further supported by the fact that mutant S-protein was able to suppress secretion of wt virions. Therefore mutant S-protein had a transdominant negative effect on the secretion of Wt. Conclusions: Despite its secretion defect the immune escape variant Gly145Arg can become the dominant viral population in the serum of patients if it emerges in a mixed population with wt.