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Objectives: Dengue virus (DENV) infection is a significant risk to over a third of the human population that causes a wide spectrum of illness, ranging from sub-clinical disease to intermediate syndrome of vascular complications called dengue fever complicated (DFC) and severe, dengue hemorrhagic fever (DHF). Methods for discriminating outcomes will impact clinical trials and understanding disease pathophysiology., Study Design: We integrated a proteomics discovery pipeline with a heuristics approach to develop a molecular classifier to identify an intermediate phenotype of DENV-3 infectious outcome., Results: 121 differentially expressed proteins were identified in plasma from DHF vs dengue fever (DF), and informative candidates were selected using nonparametric statistics. These were combined with markers that measure complement activation, acute phase response, cellular leak, granulocyte differentiation and viral load. From this, we applied quantitative proteomics to select a 15 member panel of proteins that accurately predicted DF, DHF, and DFC using a random forest classifier. The classifier primarily relied on acute phase (A2M), complement (CFD), platelet counts and cellular leak (TPM4) to produce an 86% accuracy of prediction with an area under the receiver operating curve of >0.9 for DHF and DFC vs DF., Conclusions: Integrating discovery and heuristic approaches to sample distinct pathophysiological processes is a powerful approach in infectious disease. Early detection of intermediate outcomes of DENV-3 will speed clinical trials evaluating vaccines or drug interventions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dengue virus infection elicits a spectrum of clinical presentations ranging from asymptomatic to severe disease. The mechanisms leading to severe dengue are not known, however it has been reported that the complement system is hyper-activated in severe dengue. Screening of complement proteins demonstrated that C1q, a pattern recognition molecule, can bind directly to dengue virus envelope protein and to whole dengue virus serotype 2. Incubation of dengue virus serotype 2 with C1q prior to infection of THP-1 cells led to decreased virus infectivity and modulation of mRNA expression of immunoregulatory molecules suggesting reduced inflammatory responses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A dengue 2 plasmid DNA vaccine (pD2) expressing the pre-membrane and envelope proteins (preM-E) was modified by replacing the dengue transmembrane and cytoplasmic sequences with those of the mouse lysosome-associated membrane protein (pD2/LAMP). Immunofluorescence and confocal microscopy of human 293, NIH 3T3, and macrophage IC21 cell lines transfected with pD2/LAMP showed that the preM-E/LAMP protein chimera was present in vesicles containing endogenous LAMP and major histocompatability complex class II (MHC II), in contrast to the non-vesicular localization of native preM-E protein lacking the LAMP targeting sequence. Mice immunized with pD2 showed an antigen-specific immunoglobulin response but the neutralizing antibodies titers (plaque reduction neutralization test, PRNT(50)) elicited by the native protein were minimal. In contrast, vaccination with pD2/LAMP resulted in PRNT(50) of 270, 320 and 160 at approximately 1, 3 and 8 months after two immunizations with 50 microg DNA, and approached 100% neutralization at 1:20 dilution. Additional immunization with pD2/LAMP, after 8 months, increased the neutralizing antibody titers to >640. Comparable neutralizing antibody responses were induced by two vector backbones, pVR1012 and pVax-1, at 5 and 50 microg of DNA. The neutralizing responses to the pD2/LAMP chimera were greatly superior to those elicited by pD2 in all conditions. These results underscore the importance of MHC class II presentation of DNA-encoded dengue-virus envelope protein for production of neutralizing antibodies.