Structure-function of Dengue 2 virus envelope protein domain III with neutralizing antibodies

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Dengue is the most significant arboviral disease affecting humans and therefore it is essential to produce effective countermeasures against this disease in the form of vaccines and antiviral drugs. Neutralizing antibodies are a critical component of immune-mediated protection from disease caused by the DENVs and the envelope (E) protein is the primary target. Potency of neutralization has been correlated with the particular domain on the E protein recognized by a neutralizing antibody, with domain III of the E protein (ED3) being the target of the most potent neutralizing antibodies. The objective of this dissertation, therefore, was to obtain fundamental information by way of physically and biologically mapping the epitopes of DENV ED3-specific neutralizing monoclonal antibodies (MAbs). Site-directed mutagenesis of a DENV-2 recombinant ED3 (rED3) was done in order to map functionally important residues for binding of a panel of 13 ED3-specific MAbs. Seven of these MAbs were DENV-2 type-specific and recognized a single antigenic site on ED3 that centered on two functionally critical residues (K305 and P384) for binding. The other six MAbs were DENV subcomplex-/complex-specific and recognized a single antigenic site that centered on the functionally critical residue K310 for binding. MAbs that recognized these antigenic sites had similar binding affinities for both the human endemic and the ancestral, sylvatic DENV-2 rED3s suggesting that these two antigenic sites are evolutionarily conserved. Interestingly, the neutralization efficiency of MAbs that recognized the DENV subcomplex-/complex-specific antigenic site was much lower than MAbs that recognized the DENV-2 type-specific antigenic site. In particular, the DENV subcomplex-/complex-specific MAbs required much higher relative occupancy levels on the virion, compared to DENV-2 type-specific MAbs, in order to neutralize virus infectivity. Furthermore, isolation of ED3-specific MAb neutralization resistant variants of DENV-2 revealed that residues that are functionally critical for MAb binding can be exploited in order to escape neutralization, providing that the mutated residue is not functionally critical for multiplication. Thus, ED3 residues that are functionally critical for binding of the most potent neutralizing MAbs and are also essential for virus multiplication represent attractive targets for vaccines and antiviral drugs.

sylvatic dengue 2, neutralization escape, neutralization, monoclonal antibodies, epitope mapping, envelope protein domain III, dengue, binding energy hot spot, antibody occupancy