Hepatitis B from diagnosis towards prophylactic single domain antibodies

M.H. van Roosmalen

Research output: ThesisDoctoral thesis 1 (Research UU / Graduation UU)

Abstract

Diagnosis of the hepatitis B Virus (HBV) is important for treatment and prevention of further spread of the virus. Today, detection of hepatitis B virus surface antigen (HBsAg) is the method of choice for the screening and initial diagnosis of HBV. Genetic diversity and discovery of HBV variants with mutations in the immunological dominant region of HBsAg caused none reactivity in some diagnostic assays. Characterization of anti-HBs monoclonal antibodies lead to discovery of a unique monoclonal antibody revealing the true topology of the small s-protein. The knowledge of the antibody recognition spectra, lead to the development of a diagnostics assay showing for the first time detection of all HBsAg mutants based on three different epitope regions. The three epitope regions should in theory enable detection of all known HBsAg mutants, which was proven after testing HBsAg mutants in direct comparison to other diagnostic assay lacking complete mutant detection. The improved HBsAg screening assay permitted better and earlier diagnosis of HBV infections, leaving the diagnosed HBV patient for treatment. Today the main motivation for intervening in an on-going HBV infection is in case the liver is compromised. Depending on the status of the patient, antiviral therapy is started or in worse case when the liver stops to function, a liver transplantation is the final choice. Transplantation of the liver is preceded by antiviral therapy in combination with Hepatitis B immunoglobulin (HBIg) additions. In a quest for an alternative source for HBig we isolated and selected single-domain antibodies (VHHs) that recognize the major small s-protein of HBV. Testing of five VHH in an in-vitro neutralization experiment identified one VHH that could neutralize HBV comparable with good neutralizing anti-HBs reference antibodies. Pepscan analysis and amino acid substitution experiments suggested mutual recognition of the VHH of both the “a”-determinant and c-terminus of the s-protein, fixating the structure and preventing conformational changes needed during viral entry. Alternatively binding to a yet to be discovered HBV (co-) receptor is blocked by the VHH. Ab initio modeling of the dimer “a”-determinant sequence (aa100-155) with VHH S5 revealed a tempting structure that could explain our and published observations. Mainly the location of the cysteine residues in the newly found structure was striking, showing possible formation of inter and intra chain disulfide bridges. Separate analysed c-terminal hydrophobic region (155-226) showed structural homology with Arfaptin 2, a BAR domain protein. The homologous structure predicted importance of the region giving curvature to the aggregated s-proteins in the HBsAg particles. We conclude from the modelling data that the protruding spikes are dimers of the “a”-determinant with peripheral stabilized c-terminal s-protein fragments on the 3- and 5-fold axis.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Utrecht University
Supervisors/Advisors
  • Verrips, C.T., Primary supervisor
  • de Haard, J.J.W., Supervisor
Award date11 Jan 2012
Publisher
Print ISBNs978-90-8891-367-9
Publication statusPublished - 11 Jan 2012

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