Beneath the surface: Amino acid variation underlying two decades of dengue virus antigenic dynamics in Bangkok, Thailand

Neutralizing antibodies are important correlates of protection against dengue. Yet, determinants of variation in neutralization across strains within the four dengue virus serotypes (DENV1-4) is imperfectly understood. Studies focus on structural DENV proteins, especially the envelope (E), the primary target of anti-DENV antibodies. Although changes in immune recognition (antigenicity) are often attributed to variation in epitope residues, viral processes influencing conformation and epitope accessibility also affect neutralizability, suggesting possible modulating roles of nonstructural proteins. We estimated effects of residue changes in all 10 DENV proteins on antigenic distances between 348 DENV collected from individuals living in Bangkok Thailand (1994-2014). Antigenic distances were derived from response of each virus to a panel of twenty non-human primate antisera. Across 100 estimations, excluding 10% of virus pairs each time, 77 of 295 positions with residue variability in E consistently conferred antigenic effects; 52 were within 3 sites of known binding sites of neutralizing human monoclonal antibodies, exceeding expectations from random assignments of effects to sites (p = 0.037). Effects were also identified for 16 sites on the stem/anchor of E which were only recently shown to become exposed under physiological conditions. For all proteins, except nonstructural protein 2A (NS2A), root-mean-squared-error (RMSE) in predicting distances between pairs held out in each estimation did not outperform sequences of equal length derived from all proteins or E, suggesting that antigenic signals present were likely through linkage with E. Adjusted for E, we identified 62/219 sites embedding the excess signals in NS2A. Concatenating these sites to E additionally explained 3.4% to 4.0% of observed variance in antigenic distances from when E alone (50.5%. to 50.8%); RMSE outperformed concatenating E with sites from any protein of the virus (Delta RMSE, 95%IQR: 0.01, 0.05). Our results support examining antigenic determinants beyond the DENV surface. Author summary Dengue viruses, even of the same serotype, are differentially recognized by preexisting antibodies of individuals. With antibody levels being an important indicator of infection risk and pathogenicity, understanding mechanisms underlying these differences are crucial for vaccine design and development. Investigations have primarily targeted surface regions of the envelope protein (E) where virus-antibody interactions were thought to primarily occur. However, the roles of non-surface regions of the E protein as well as nonstructural proteins has been limited. We looked at the entire virus to identify associations between, specific changes in the protein sequence and differences in how viruses were recognized by antibodies. In addition to recovering known determinants on the surface, we found signals in other areas on the structural building blocks of the virus. We also identified additional signals on specific areas of a protein that does not form structures of the virus but orchestrate virus formation. Our results point towards broadening the frame of investigation to gain a more comprehensive understanding of mechanisms giving rise to antibody recognition of dengue viruses, and may aid the design and evaluation of vaccines and/or assays to characterize dengue immunity.

Automated summary

Neutralizing antibodies play a crucial role in protecting against dengue. However, the factors that contribute to variation in neutralization across different strains of dengue virus are not well understood. This study investigated the effects of changes in the protein sequence of all 10 dengue viral proteins on antigenic distances. The results showed that residue changes in the envelope protein and nonstructural protein 2A had significant effects on antigenic distances, suggesting that nonstructural proteins may modulate neutralizability. This study highlights the importance of considering antigenic determinants beyond the surface proteins in understanding antibody recognition of dengue viruses.