HBV Pre-C/C Variation: Geographical and Functional Aspects

Infection with hepatitis B virus (HBV) can lead to a spectrum of disease manifestations, from asymptomatic acute hepatitis to fulminant hepatitis. Why such a spectrum should occur is not clear but the immune response to HBV has often been implicated. Normally, after seroconversion to anti-HBe, HBV DNA levels fall and disease activity is minimal. However, a group of individuals from the Far East and the Mediterranean were identified as having continued exacerbations of disease activity even after seroconversion to anti-HBe. This thesis examines variation in HBcAg from sequential serum samples of these individuals, and analyses the distribution of amino acid substitutions in relation to B- and Thelper cell epitopes. In the first study, the distribution of HBcAg amino acid substitutions was compared in Chinese patients with different pre-core (pre-C) variants. One variant has a serine at amino acid (aa) 15 of pre-C, and the other is the common Aisse which introduces a stop codon at aa 28. The serine15 strain is shown here to produce antigenically normal amounts of HBeAg. Both variants have been shown to be mutually exclusive, probably due to sequence requirements for encapsidation, and are separate lineages. Sequential sequencing of HBcAg showed that in those individuals with Ai896 there are significantly more aa substitutions, and that these are associated with Th and B-cell epitopes. In the second study, sequential samples were examined from individuals from the Mediterranean who either seroconverted to anti-HBe or who were continually anti-HBe, the results showed that selection of Aisse is temporally associated with aa substitutions in HBcAg. In seroconverters went into remission, aa substitutions occurred in the Th epitope from aa 50 to 69 (p=0.00045), but in anti-HBe positive patients with ongoing disease these occurred in B-cell epitopes (p=0.0007 for aa 74 to 83). Analysis of the timing of these substitutions showed that they are not associated with multiple flares of hepatic activity. Comparison of the results of the two studies shows that the distribution of HBcAg substitutions differs in the Chinese and the Mediterranean groups. An overlapping peptide series, covering the entire HBcAg, was constructed to test the hypothesis that the observed variants which arose during infection represented immune escape; either humoral, Thelper cell escape or both. Several patients failed to react to their homologous peptide in lymphoproliferation assays suggesting that the lack of response is not due to variation in Thelper cell epitopes. The results from the lymphoproliferation studies defined the Thelper epitope - previously described at aa 1 to 20 - as lying between aa 14 and 23. Antibody binding and T-cell studies indicated that branched chain peptides work in both systems, and that they are more reactive in ELISA when compared to monomeric peptides. Several of the patients studied in the B-cell assay reacted to peptides spanning aa 1 to 43, possibly indicating a putative B-cell epitope in this region