Quadruple mutation GCAC1809-1812TTCT acts as a biomarker in healthy European HBV carriers.

Many mutation analyses of the HBV genome have been performed in the search for new prognostic markers. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, the HBV core promoter/precore region was sequenced in serum samples from European inactive HBV carriers. Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels. In vitro GCAC1809-1812TTCT lead to drastically diminished synthesis of pregenomic RNA (pgRNA), precore mRNA, core, HBsAg, and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that was compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status was observed, BCP double mutation was strongly correlated with cirrhosis, but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.

A new approach for therapeutic vaccination against chronic HBV infections.

There are currently about 257 million people suffering from chronic HBV infection worldwide. In many cases, an insufficient Tcell response is causative for establishment of a chronic infection. To ensure a robust cellular immune response and induction of neutralizing antibodies a novel vaccine platform based on modified cell-permeable HBV capsids was utilized. Cell permeability was achieved by fusion of the membrane-permeable TLM-peptide to HBV core monomers, assembling the capsids. Insertion of a Strep-tagIII into the spike tip domain that protrudes from the capsid surface enables flexible loading with antigens that are fused to streptavidin. In this study, HBV surface antigen-derived PreS1PreS2 domain, fused to monomeric streptavidin, served as cargo antigen. Binding between antigen and capsids was characterized by surface plasmon resonance spectroscopy, electron microscopy and density gradient centrifugation. Confocal immunofluorescence microscopy and in vivo imaging of immunized mice demonstrated membrane permeability of cargo-loaded carriers and spread of antigen over the whole organism. Immunization experiments of mice revealed a robust induction of a specific cellular immune response, leading to destruction of HBV-positive cells and induction of HBV-specific neutralizing antibodies. Membrane permeability of these carriers allows needle-free application of antigen-loaded capsids as evidenced by induction of an HBV-specific CTL response and HBV-specific B cell response after oral or transdermal vaccination. These data indicate that cell-permeable antigen carriers, based on HBV capsids and loaded with HBV antigen, have the capacity to induce a cellular and a neutralizing humoral immune response. In addition, cell permeability of the vaccine platform enables antigen transfer across several cell layers, that could allow oral or transdermal immunization.

Formation of semi-enveloped particles as a unique feature of a hepatitis B virus PreS1 deletion mutant.

BACKGROUND: Naturally occurring variants with deletions or mutations in the C-terminal PreS1 domain from hepatitis B virus (HBV) chronically infected patients have been shown to promote HBsAg retention, inhibit HBsAg secretion and change the extracellular appearance of PreS1-containing HBV particles (filaments and virions). AIMS: To study the impact of N-terminal deletion in preS1 domain on viral secretion and morphogenesis. METHODS: An HBV mutant with 15 amino acids (aa 25-39) deletion in N-terminal preS1 was isolated. Intracellular and extracellular HBsAg were quantified by Western blot. Subcellular HBsAg distribution was analysed by confocal laser scanning microscopy. The viral morphology was characterised by sucrose density gradient ultracentrifugation, Western blot, electron microscopy, HBV mixed ELISA and HBV particle gel essay. RESULTS: Expression of this mutant genome released higher amounts of HBsAg in the form of shorter filaments. A significant fraction of semi-enveloped virions was observed in the supernatant that has been unprecedented so far. Stepwise insertion of aa 25-31, aa 32-39 and aa 25-39 increased the length of filaments. The rescue of aa 25-31 and aa 25-39 drastically reduced the amounts of extracellular HBsAg and semi-enveloped virions, while such effects could not be observed after insertion of aa 32-39, arguing against a simple spacer function of this region. The deletion and rescued mutants do not differ in subcellular HBsAg distribution and colocalisation with ER, Golgi and multivesicular bodies markers arguing against differences in release pathways. CONCLUSION: N-terminal PreS1-domain (aa 25-31) determines HBsAg secretion and triggers proper assembly of PreS1-containing particles.

HCV-induced oxidative stress by inhibition of Nrf2 triggers autophagy and favors release of viral particles.

Viruses are known to exploit the autophagic machinery for their own benefit. In case of the hepatitis C virus autophagy is induced. As autophagy serves as a degradation pathway to maintain cellular homeostasis, it is activated in response to cellular stress such as elevated levels of reactive oxygen species (ROS). Elevated levels of ROS trigger phosphorylation of the autophagic adaptor protein p62 on Ser349 (pS[349] p62) that is involved in the induction of autophagy. Consequently, pS[349] p62 binds with a higher affinity to Keap1 thereby releasing Nrf2 from the complex with Keap1. Although the released Nrf2 should induce as a heterodimer with the sMaf proteins the expression of Nrf2/ARE-dependent genes, in HCV-positive cells no activation of cytoprotective genes occurs even though elevated amounts of pS[349] p62 are present. In HCV-positive cells, free Nrf2 is trapped via delocalized sMaf proteins at the replicon complexes on the cytoplasmic face of the ER and is therefore prevented from its entry into the nucleus. Scavenging of ROS leads to decreased levels of pS[349] p62 and impaired induction of autophagy. Both, inhibition of autophagy and scavenging of ROS result in decreased amounts of released viral particles. Taken together, these data identify an intricate mechanism of HCV-dependent inhibition of Nrf2/ARE-mediated gene expression which counteracts pS[349] p62-induced activation of Nrf2. Thereby elevated ROS-levels are preserved that in turn activate autophagy to favor HCV particle release.