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Description:
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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 . |