Inoculation by mosquito induces durable differences in serological profile in non-human primates infected with DENV1

Natural dengue virus (DENV) infections are delivered by mosquito bite but how the route of inoculation route could shape the humoral immune response is not well understood. Here, we serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection in which the animals were inoculated by mosquito (N=10) or subcutaneous injection (N=10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. We found that serological profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year. We found differences in sero-reactivity, as indicated by the median area under the curve (AUC) in the Envelope (E; residues 215-406; p < 0.08), and Nonstructural-3 (NS3; residues 549-615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339-384 in domain III accounted for >99% of the total AUC difference across residues 215-406. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III (EDIII) seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation. These findings could have implications for DENV diagnostics and evaluation of vaccines. IMPORTANCEDengue virus (DENV) infections are transmitted by mosquito bite, but how being infected by mosquito bite affects the immune response is not known. In this study, we analyzed antibodies produced by rhesus macaques infected with DENV using programmable phage display, a high-throughput method for characterizing what viral protein derived peptides serum antibodies bind to. We found that while there was no difference in antibody binding profiles at early timepoints post-infection, at one year post-infection, there were substantial differences in the antibody binding profiles of macaques who were infected by mosquito bite versus those that were infected by injection. In general, antibodies in the macaques inoculated by mosquito maintained higher levels of sero-reactivity, with a strong signal still present one year post-infection. The findings we report could have implications for DENV diagnostics and evaluation of DENV vaccines.