Dengue virus (DENV), a member of Flaviviriade family, is a mosquito-borne human viral pathogen, causing every year more than 50 million infections, some of which can lead to dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). It co-circulates as four serotypes. Humoral immunity plays a significant role in controlling flavivirus dissemination within an infected host. However, antibodies raised against DENV may be both protective and pathogenic, due to the phenomenon of antibody-dependent enhancement (ADE), which leads to a dramatic increase in the infection of cells bearing Fcy receptors. As such ADE is a major obstacle in the development of a successful vaccine against DENV. The main challenge in the field is to understand the mechanisms of neutralization and enhancement of DENV infection at the molecular level and identify antibody epitopes that minimize ADE. DENV envelope (E) and precursor membrane (prM) proteins are the main targets of antibodies. Each monomer of E consists of three domains (El, Ell and EIII), of which EIII contains critical neutralization determinants. PrM acts as a chaperone for the correct folding of E and undergoes proteolytic cleavage to soluble pr peptide and membrane-associated M during virus maturation. This thesis reports the functional and structural characterization of four types of antibodies in complexes with recombinant antigens, E and prM. The crystal structures of murine antibodies 3H5 and 2C8 complexed with EIII indicate a possible explanation of ADE mediated by strongly neutralizing antibodies specific to EIII. The proposed structural rearrangement of E induced by 3H5 binding, results in neutralization at a significantly lower occupancy of the antibody on the virus than that observed with 2C8 which can bind without distorting the DENV envelope. The occupancy required for neutralization is directly correlated with ADE as low density of antibodies bound at neutralization may fail to reach a threshold to drive efficient Fc-receptor-dependent uptake. The crystal structures of the fully cross-reactive murine antibody 2H12 in complex with EIII from DENV serotypes 1, 3 and 4 revealed that it recognizes a highly conserved epitope, which has limited accessibility on the mature virus and the ability of the antibody to bind the virus is serotype- dependent. 2H12 displayed high affinity to isolated antigen (EIII) yet the position of the epitope in the mature virus hindered efficient neutralization. The structural analyses of human mAbs recognizing El-Ell prove to be challenging. Whilst the Fab fragment of 30E2 formed a stable complex with recombinant E, and yielded crystals, other Fabs did not bind to recombinant E in a monomeric form indicating that they target epitopes on oligomeric forms of E present only in the virions. DENV cross-reactive human antibodies against prM do not neutralize the infection with DENV yet greatly promote ADE. They bind immature and partially mature yet infectious particles and recognize a discontinuous epitope spanning across pr peptide and M. Initial crystallographic studies of Fab- prM complexes provide a platform for further experiments aimed at the elucidation of the specificity of prM-specific antibodies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:558310 |
| Date | January 2011 |
| Creators | Flanagan, Aleksandra |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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