In vitro HBV infection and neutralization were assayed using an anti-preS1 murine monoclonal antibody (1B3) and anti-preS2 (H69K) and anti-S (CS131A) murine-human chimeric antibodies. The 1B3 (IgG1) and H69K (IgG1) was constructed previously and the CS131A was constructed for this study by expressing stably the chimeric heavy and light chains in Chinese hamster ovary cells and purifying from the culture supernatant. Previous study showed that the H69K and CS131A recognize known virus-neutralizing epitopes, while the 1B3 does not. For the assays, adult human hepatocyte primary culture was infected with the adr or ayw subtype of HBV, and the infectivity and subsequent replication was confirmed both by measuring the kinetics of HB-sAg secretion by the infected cells and detecting the intermediate replicative form of HBV DNA in the cells. Next, the hepatocytes were infected with the adr or ayw subtype of the virus that had been preincubated with various concentrations of each of the antibodies and the neutralization of HBV was analyzed. The results showed that the anti-preS2 and anti-S chimeric antibodies exhibited neutralizing activity against both the adr and ayw subtypes of the virus, with approximately 1,000 and 2,000 times higher specific activity than polyclonal hepatitis B immune globulin, respectively, but the anti-preS1 antibody scarcely neutralized the infection. The neutralizing activities of the antibodies were consistent with their epitope specificity and antigenbinding affinity, suggesting that this neutralization assay is specific. The in vitro neutralization assay will be useful for evaluating the neutralizing activity of anti-HBV antibodies before in vivo testing in chimpanzees.

We have efficiently generated mouse monoclonal antibodies (MAbs), which bind specifically to amino acids 21-47 of the preS1 domain of hepatitis B virus (HBV) by immunizing mice with the preS1 peptide (amino acids, aa 1-56) conjugated to keyhole limpet hemocyanin. Hybridomas were screened by an indirect enzyme-linked immunosorbent assay (ELISA) using the purified preS1 peptide as a coated antigen. Eighteen positive hybridomas were selected and subjected to isotyping. Of these, 5 clones secreted immunoglobulin G (IgG) and 13 clones secreted IgM. Four (KR1, KR2, KR3, and KR4) of the 5 IgG MAbs bound to preS1 peptide (aa 21-47). Epitope mapping using bacterially expressed GST fusion proteins revealed that three clones (KR2, KR3, KR4) (IgG1, K) recognize aa 21-35, while KR1 (IgG2a, K) recognizes aa 35-47 of the preS1. These MAbs immunoprecipitated HBV particles, demonstrating that they bind to native HBV particles.

No abstract

Poly-γ-glutamic acid (PGA), a major component of the bacterial capsule, is known to confer hydrophilicity to bacterial surfaces and protect bacteria from interactions with blood cells. We tested whether applying a bacteriomimetic surface coating of PGA modulates interactions of nanomaterials with blood cells or affects their safety and photothermal antitumor efficacy. Amphiphilic PGA (APGA), prepared by grafting phenylalanine residues to PGA, was used to anchor PGA to reduced graphene oxide (rGO) nanosheets, a model of hydrophobic nanomaterials. Surface coating of rGO with bacterial capsule-like APGA yielded APGA-tethered rGO nanosheets (ArGO). ArGO nanosheets remained stable in serum over 4 weeks, whereas rGO in plain form precipitated in serum within 5 minutes. Moreover, ArGO did not interact with blood cells, whereas rGO in plain form or as a physical mixture with PGA formed aggregates with blood cells. Mice administered ArGO at a dose of 50 mg/kg showed 100% survival and no hepatic or renal toxicity. No mice survived exposure at the same dose of rGO or a PGA/rGO mixture. Following intravenous administration, ArGO showed a greater distribution to tumors and prolonged tumor retention compared with other nanosheet formulations. Irradiation with near-infrared light completely ablated tumors in mice treated with ArGO. Our results indicate that a bacteriomimetic surface modification of nanomaterials with bacterial capsule-like APGA improves the stability in blood, biocompatibility, tumor distribution, and photothermal antitumor efficacy of rGO. Although APGA was used here to coat the surfaces of rGO, it could be applicable to coat surfaces of other hydrophobic nanomaterials.

The anti-melanogenic effects of the extract of Angelica tenuissima (AT) root and the extract of AT root fermented by Aspergillus oryzae (FAT) were investigated. These effects were determined by measuring the inhibitory activity of AT and FAT on melanin production in B16F10 melanocytes and with in vitro tyrosinase activity assays. The AT extract inhibited melanin production at concentrations above 250 μg/ml, and this inhibitory effect was significantly enhanced by the fermentation process with A. oryzae. HPLC analysis resulted in the isolation of two active compounds from both the AT and FAT extracts. Their chemical structures were identified as decursin and Z-ligustilide through comparison with previously reported NMR data. The decursin and Z-ligustilide contents were increased in the FAT extract and could be responsible for its enhanced inhibitory effects on melanin production and tyrosinase activity compared with that of the AT extract.