Construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis B virus surface antigen

Background: Virus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Due to their repetitive structure, VLPs are extensively used for protein engineering and generation of chimeric VLPs with inserted foreign epitopes. Hamster polyomavirus VP1 represents a promising epitope carrier. However, insertion of large sized protein sequences may interfere with its self-assembly competence. The co-expression of polyomavirus capsid protein VP1 with minor capsid protein VP2 or its fusion protein may result in pseudotype VLPs where an intact VP1 protein mediates VLP formation. In the current study, the capacity of VP1 protein to self-assemble to VLPs and interact with the modified VP2 protein has been exploited to generate pseudotype VLPs displaying large-sized antibody molecules. Results: Polyomavirus-derived pseudotype VLPs harbouring a surface-exposed functionally active neutralizing antibody specific to hepatitis B virus (HBV) surface antigen (HBsAg) have been generated. The pseudotype VLPs consisting of an intact hamster polyomavirus (HaPyV) major capsid protein VP1 and minor capsid protein VP2 fused with the anti-HBsAg molecule were efficiently produced in yeast Saccharomyces cerevisiae and purified by density-gradient centrifugation. Formation of VLPs was confirmed by electron microscopy. Two types of pseudotype VLPs were generated harbouring either the single-chain fragment variable (scFv) or Fc-engineered scFv on the VLP surface. The antigen-binding activity of the purified pseudotype VLPs was evaluated by ELISA and virus-neutralization assay on HBV-susceptible primary hepatocytes from Tupaia belangeri. Both types of the pseudotype VLPs were functionally active and showed a potent HBV-neutralizing activity comparable to that of the parental monoclonal antibody. The VP2-fused scFv molecules were incorporated into the VLPs with higher efficiency as compared to the VP2-fused Fc-scFv. However, the pseudotype VLPs with displayed VP2-fused Fc-scFv molecule showed higher antigen-binding activity and HBV-neutralizing capacity that might be explained by a better accessibility of the Fc-engineered scFv of the VLP surface. Conclusions: Polyomavirus-derived pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability may represent a novel platform for developing therapeutic tools with a potential application for post-exposure or therapeutic treatment of viral infections

Quantification of large and middle proteins of hepatitis B virus surface antigen (HBsAg) as a novel tool for the identification of inactive HBV carriers

Objective Among individuals with chronic hepatitis B, those with hepatitis B e-antigen (HBeAg)-negative chronic hepatitis (CHB) can be difficult to distinguish from those with HBeAg-negative chronic HBV infection, also referred to as inactive HBV carriers (ICs), but both require different medical management. The level of HBV surface antigen (HBsAg) has been proposed as a marker to discriminate between chronic infection and hepatitis stages. HBsAg consists of large, middle and small HBs. The aim of this study was to determine whether the composition of HBsAg improved the identification of ICs among HBsAg-positive subjects with different phases of HBV infections. Design HBV large surface proteins (LHBs) and HBV middle surface proteins (MHBs) were quantified in serum samples from 183 clinically well-characterised untreated patients with acute (n=14) HBV infection, ICs (n=44), CHBs (n=46), chronic HBeAg-positive phase (n=68) and hepatitis delta coinfection (n=11) using an ELISA, with well-defined monoclonal antibodies against the preS1 domain (LHBs) and the preS2-domain (MHBs). A Western blot analysis was used to verify the quantitation of the components of HBsAg. Total HBsAg was quantified using a modified commercially available assay (HBsAg V. 6.0, Enzygnost, Siemens, Erlangen). Results The composition of HBsAg showed specific patterns across different phases of hepatitis B. Individuals in the IC phase had significantly lower proportions of LHBs and MHBs than patients in acute or chronic phases irrespective of their HBV e-antigen status (p< 0.0001) or HBsAg level. Both LHBs and MHBs ratios better predicted the IC phase than total HBsAg levels. Conclusion Quantification of MHBs, particularly LHBs represents a novel tool for the identification of the IC stage

Integration of the End Cap TEC+ of the CMS Silicon Strip Tracker

The silicon strip tracker of the CMS experiment has been completed and inserted into the CMS detector in late 2007. The largest sub-system of the tracker is its end cap system, comprising two large end caps (TEC) each containing 3200 silicon strip modules. To ease construction, the end caps feature a modular design: groups of about 20 silicon modules are placed on sub-assemblies called petals and these self-contained elements are then mounted into the TEC support structures. Each end cap consists of 144 petals, and the insertion of these petals into the end cap structure is referred to as TEC integration. The two end caps were integrated independently in Aachen (TEC+) and at CERN (TEC--). This note deals with the integration of TEC+, describing procedures for end cap integration and for quality control during testing of integrated sections of the end cap and presenting results from the testing