Llama-derived single-domain intrabodies against the Hepatitis B Virus surface and core antigen (2009)

Hepatitis B virus (HBV) infections represent a global health problem with 2 billion people infected worldwide and 350 million suffering from a chronic infection. Today’s therapeutic approach for chronic HBV infection consists of antiviral treatment with interferon and/or nucleoside analogues but these have major limitations. Therefore, there is a need for novel strategies to tackle chronic HBV infections. Gene therapy is a novel approach with great potential that raises intense research interest. With clinical trials already in process, it will definitely make a valuable contribution to the cure of various diseases. In this thesis the potential of intrabody gene therapy to inhibit HBV replication was evaluated. Intrabodies are antibody fragments that are expressed and operate inside the cell. They affect protein function by several mechanisms and induce a downstream alteration of the targeted process. In the past, single-chain variable fragment intrabodies (scFv) have already shown potential to inhibit HBV replication in vitro. However, in vivo data about antiviral intrabodies have not been reported yet. We examined VHH fragments as intrabodies, which represent the antigen-binding domain of heavy-chain antibodies of camelids. Two protein structures of HBV were targeted, the Hepatitis B surface antigen (HBsAg) and the Hepatitis B core antigen (HBcAg).\nFirst, we examined five VHH-molecules (S1-S5), directed against the most abundant HBV envelope protein domain S. We observed a low affinity of these VHH-molecules in several binding assays. When expressed and retained inside the endoplasmic reticulum of hepatoma cells, these molecules were capable of retaining HBsAg particles inside the cell leading to an accumulation of intracellular HBsAg. This was a first indication of the antiviral capacity of these VHH-intrabodies. Next, we examined the VHHs in a hydrodynamics-based HBV mouse model. A decline in secreted HBsAg particles was observed, confirming the in vitro data. More importantly, a very strong suppression in the secretion of viral particles was detected. For VHH S5, the level of secreted virions was reduced more than 100-fold compared to the negative control groups. Our hypothesis is that the ER-retained VHHintrabodies act in the ER as a multivalent layer, capable of binding HBsAg with very high avidity.\nNext, we investigated six HBcAg-specific VHH-molecules (C1-C6). In different binding assays three of these VHHs (C2, C4 and C6) bind very strongly to HBcAg of two virus strains, one with subtype ayw and the other with subtype adw. Study of these three VHHs as intrabodies with a nuclear localization in HBV-transfected hepatoma cells revealed an increased intracellular accumulation of HBeAg and a vanishing of intracellular HBcAg. These phenomena still need to be elucidated. \nTaken together, these results provide proof of principle for the use of VHH-intrabodies to inhibit viral secretion in vivo. Further exploration of these VHH-intrabodies in a chronic HBV mouse model and an appropriate gene delivery system (viral or non-viral) will help to evaluate these compounds as a feasible treatment for HBV.