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.

Dengue is a major public health problem that affects tens of millions of people annually in tropical and sub-tropical countries. This acute viral infection happens to be severe and even life threatening but there is still no available drug or vaccine. Previous studies have noted early higher viral burden in patients who develop more severe symptoms suggesting that administration of a potent and safe antiviral may prevent progression to severe dengue. To verify this hypothesis, we have conducted the first RCT directed towards reducing the viral burden in vivo by administrating chloroquine (CQ), a cheap and well-tolerated drug that inhibits DENV in vitro with concentrations achievable in vivo, to 307 Vietnamese adults with suspected dengue (257 of them were laboratory-confirmed cases). Unfortunately, we did not see an effect of CQ on the duration of infection. However in patients treated with CQ, we observed a trend towards a lower incidence of severe forms. We did not find any differences in the immune response that can explain this trend. We also found more adverse events, primarily vomiting, with CQ. In addition, we have explored the relationships between clinical features, antibody responses and virological markers in these patients. We found that the early magnitude of viremia is positively associated with disease severity and there are serotype dependent differences in infection kinetics. We found as well that DENV was cleared faster and earlier in patients with secondary infections. To complete this study, we have also evaluated 2 rapid lateral flow tests for the diagnosis of dengue in a panel of plasma samples from 245 RT-PCR confirmed dengue patients and 47 with other febrile illnesses. Our data suggest that the NS1 test component of these tests are highly specific and have similar levels of sensitivity (~60%). Both NS1 assays were significantly more sensitive for primary than secondary dengue. The IgM parameter in the SD Duo test improved overall test sensitivity without compromising specificity. All these findings are of major importance for further anti-viral drug testing.