Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Abstract

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII. Dengue viruses are mosquito-borne flaviviruses and the agents of dengue fever and dengue hemorrhagic fever. It has been widely assumed that antibodies that neutralize dengue bind to regions on the viral envelope (E) protein that are conserved within each serotype. However, few studies have explored how natural variation influences dengue-antibody interactions. Mouse antibodies that strongly neutralize dengue bind to a region on domain III of E protein. This region has been the focus of much recent work because it might be the target of protective human antibodies as well. We compared a large number of E protein sequences and discovered that the region on E protein domain III targeted by neutralizing antibodies was highly variable between strains of dengue serotype 3. Using a panel of antibodies, we experimentally demonstrate that natural strain variation in dengue serotype 3 has a strong influence on antibody binding and neutralization. Our results challenge the dogma that neutralizing antibody binding regions are conserved within each serotype. The results of this study are relevant to the current global effort to develop and evaluate dengue vaccines.