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Dengue, which is the most prevalent mosquito-borne viral disease afflicting human populations, causes a spectrum of clinical symptoms that include fever, muscle and joint pain, maculopapular skin rash, and hemorrhagic manifestations. Patients infected with dengue develop a broad antigen-specific T lymphocyte response, but the phenotype and functional properties of these cells are only partially understood. We show that natural infection induces dengue-specific CD8(+) T lymphocytes that are highly activated and proliferating, exhibit antiviral effector functions, and express CXCR3, CCR5, and the skin-homing marker cutaneous lymphocyte-associated antigen (CLA). In the same patients, bystander human cytomegalovirus -specific CD8(+) T cells are also activated during acute dengue infection but do not express the same tissue-homing phenotype. We show that CLA expression by circulating dengue-specific CD4(+) and CD8(+) T cells correlates with their in vivo ability to traffic to the skin during dengue infection. The juxtaposition of dengue-specific T cells with virus-permissive cell types at sites of possible dengue exposure represents a previously uncharacterized form of immune surveillance for this virus. These findings suggest that vaccination strategies may need to induce dengue-specific T cells with similar homing properties to provide durable protection against dengue viruses., (Copyright © 2015, American Association for the Advancement of Science.)

Viruses must evade the host innate defenses for replication and dengue is no exception. During secondary infection with a heterologous dengue virus (DENV) serotype, DENV is opsonized with sub- or nonneutralizing antibodies that enhance infection of monocytes, macrophages, and dendritic cells via the Fc-gamma receptor (FcγR), a process termed antibody-dependent enhancement of DENV infection. However, this enhancement of DENV infection is curious as cross-linking of activating FcγRs signals an early antiviral response by inducing the type-I IFN-stimulated genes (ISGs). Entry through activating FcγR would thus place DENV in an intracellular environment unfavorable for enhanced replication. Here we demonstrate that, to escape this antiviral response, antibody-opsonized DENV coligates leukocyte Ig-like receptor-B1 (LILRB1) to inhibit FcγR signaling for ISG expression. This immunoreceptor tyrosine-based inhibition motif-bearing receptor recruits Src homology phosphatase-1 to dephosphorylate spleen tyrosine kinase (Syk). As Syk is a key intermediate of FcγR signaling, LILRB1 coligation resulted in reduced ISG expression for enhanced DENV replication. Our findings suggest a unique mechanism for DENV to evade an early antiviral response for enhanced infection.

Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4(+) and CD8(+) T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4(+) and 21 are CD8(+) T cell epitopes. We observe that whereas CD8(+) T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4(+) epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4(+) T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4(+) and CD8(+) T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.

The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes.

DENV (dengue virus) induces UPR (unfolded protein response) in the host cell, which strikes a balance between pro-survival and pro-apoptotic signals. We previously showed that Salubrinal, a drug that targets the UPR, inhibits DENV replication. Here, we examine the impact on UPR after direct or ADE (antibody-dependent enhanced) infection of cells with DENV clinical isolates. THP-1 cells in the presence of subneutralizing concentration of humanized antibody 4G2 (cross-reactive with flavivirus envelope protein) or HEK-293 cells (human embryonic kidney 293 cells) were infected with DENV-1-4 serotypes. UPR gene expression was monitored under these infection conditions using real-time RT-PCR (reverse transcription-PCR) and Western blots to analyse serotype-dependent variations. Subsequently, in a blinded study, strain-specific differences were compared between DENV-2 clinical isolates obtained from a single epidemic. Results showed that THP-1 cells were infected efficiently and equally by DENV-1-4 in the ADE mode. At 48 hpi (h post infection), DENV-1 and -3 showed a higher replication rate and induced higher expression of several UPR genes such as BiP (immunoglobulin heavy-chain-binding protein), GADD34 (growth arrest DNA damage-inducible protein 34) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein]. The ADE infection of THP-1 cells with epidemic DENV-2 high-UPR-gene-expressing strains appears to correlate with severe disease; however, no such correlation could be made when the same viruses were used to infect HEK-293 cells. Our finding that UPR gene expression in THP-1 cells during ADE infection correlates with dengue disease severity is consistent with a previous study [Morens, Marchette, Chu and Halstead (1991) Am. J. Trop. Med. Hyg. 45, 644-651] that showed that the growth of DENV 2 isolates in human peripheral blood leucocytes correlated with severe and mild dengue diseases.