6B)

6B). contamination of susceptible hepatoma cells (IC50=1.8 nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50=1.25 nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100-fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12-scFv internalization into cells. Together these data suggest G12-scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications. 1.?Introduction Current therapies for chronic hepatitis B (CHB), i.e. free or pegylated interferon (IFN-) and nucleos(t)ide analogues (NAs) (Trepo et al., 2014), only rarely achieve loss of HBsAg in the treated patients (3-7% for IFN-; ~1% per year for NAs) and suffer from adverse effects and unpredictable duration of treatment (Zeisel et al., 2015). Hence there is an urgent need for the development of new anti-HBV therapeutics, including antibodies (Salazar et al., 2017). Sera of HBV infected patients contain complete genome-containing virions, genome-free vacant virions (Hu and Liu, 2017) plus a huge extra (up to 105-fold) of BCR-ABL-IN-1 vacant envelopes termed subviral particles (SVPs). All these particles contain membrane embedded S, M, and L surface proteins, although the L protein content is usually highest in virions (Heermann et al., 1984). M and L are extended versions of S carrying an extra 55 amino acid (aa) PreS2 (M) or extra PreS1 (108, 118, or 119 aa) plus PreS2 region (L) on their N termini. The 226-aa S protein is the most abundant among the surface proteins. Between the second and third of its four proposed transmembrane domains and comprising approximately aa 99-169 lies the AGL. It is uncovered on the surface of virions and SVPs, and presents BCR-ABL-IN-1 the dominant linear and conformational epitopes for neutralizing antibodies (Zanetti et al., 2008). Within the AGL, aa from about position 120-150 form the highly conformational epitopes of the a determinant which is usually rich in disulfide-bond forming cysteine residues (Mangold et al., 1995; Wounderlich and Bruss, 1996). Furthermore, this region interacts with heparan sulfate proteoglycans, concentrating virions around the cell surface for subsequent high affinity interactions between PreS1 a part of L protein and the HBV receptor sodium-taurocholate cotransporting polypeptide (NTCP) (Sureau and Salisse, 2013; Verrier et al., 2016); hence beyond PreS1 the AGL is usually a pivotal viral determinant of HBV infectivity. Accordingly, many mutations and/or disruption of the disulfide-bond network in the AGL interfere with viral infectivity (Urban et al., 2014), and antibodies targeting the AGL, like those targeting PreS1 such as mAb MA18/7 (Glebe et al., 2003), can possess computer virus neutralizing activity (Urban et al., 2014), the basis of prophylactic vaccination with S protein. Egress of HB virions and SVPs remain mechanistically elusive but are believed to proceed through different routes. SVP assembly likely initiates in the post-endoplasmic reticulum/pre-Golgi compartment, with particle secretion via the general secretory pathway (Huovila et al., 1992; Patzer et al., 1984). Mature nucleocapsids filled with relaxed circular DNA (rcDNA), and likely also vacant capsids (Ning et al., 2017), are enveloped by interacting with the PreS parts of L around the cytosolic side of the ER membrane (Hu and Liu, 2017; Kluge et al., 2005; Pairan and Bruss, 2009), followed by budding into the multivesicular body (MVB) compartment of late endosomes (Kian Rabbit Polyclonal to MBTPS2 Chua et al., 2006; Lambert et al., 2007; Watanabe et al., 2007) and finally exiting BCR-ABL-IN-1 the cell via an exosomal pathway (Watanabe et al., 2007). Notably, the AGL is also pivotal for secretion of virions and SVPs as shown by the impaired secretion of naturally occurring AGL mutants (Chen et al., 2016; Khan et al., 2004; Kwei et al., 2013). Furthermore, high concentrations (100 g/ml) of exogenously added AGL-specific immunoglobulins (Igs)reportedly allowed internalization and interference with secretion of viral particles, BCR-ABL-IN-1 although the exact particle types were not clearly discriminated (Neumann et al., 2010; Schilling et al., 2003). Recently, we developed a human anti-HBs mAb IgG12 with very high affinity (Kd=7.56 nM). IgG12 recognizes a conformational epitope in the AGL, most likely the a determinant, and displayed >1,000-fold higher HBV neutralizing activity than HBIG (Wang et al., 2016). Moreover, a one-dose administration of IgG12 into HBV transgenic mice achieved a prolonged (144-day) suppression of serum HBsAg levels (Wang et al., 2016). This might be due to immune-modulatory functions of IgG, e.g. Fc-Fc receptor mediated BCR-ABL-IN-1 effects, yet also to the.