Experimental dengue virus challenge of human subjects previously vaccinated with live attenuated tetravalent dengue vaccines.

BACKGROUND: Protection against dengue requires immunity against all 4 serotypes of dengue virus (DENV). Experimental challenge may be useful in evaluating vaccine-induced immunity. METHODS: Ten subjects previously vaccinated with a live attenuated tetravalent dengue vaccine (TDV) and 4 DENV-naive control subjects were challenged by subcutaneous inoculation of either 10(3) plaque-forming units (PFU) of DENV-1 or 10(5) PFU of DENV-3. Two additional subjects who did not develop DENV-3 neutralizing antibody (NAb) from TDV were revaccinated with 10(4) PFU of live attenuated DENV-3 vaccine to evaluate memory response. RESULTS: All 5 TDV recipients were protected against DENV-1 challenge. Of the 5 TDV recipients challenged with DENV-3, 2 were protected. All DENV-3-challenge subjects who developed viremia also developed elevated liver enzyme levels, and 2 had values that were >10 times greater than normal. Of the 2 subjects revaccinated with DENV-3 vaccine, 1 showed a secondary response to DENV-2, while neither showed such response to DENV-3. All 4 control subjects developed dengue fever from challenge. Protection was associated with presence of NAb, although 1 subject was protected despite a lack of measurable NAb at the time of DENV-1 challenge. CONCLUSIONS: Vaccination with TDV induced variable protection against subcutaneous challenge. DENV-3 experimental challenge was associated with transient but marked elevations of transaminases.

Potential role for Toll-like receptor 4 in mediating Escherichia coli maltose-binding protein activation of dendritic cells.

The Escherichia coli maltose-binding protein (MBP) is used to increase the stability and solubility of proteins in bacterial protein expression systems and is increasingly being used to facilitate the production and delivery of subunit vaccines against various pathogenic bacteria and viruses. The MBP tag is presumed inert, with minimum effects on the bioactivity of the tagged protein or its biodistribution. However, few studies have characterized the immunological attributes of MBP. Here, we analyze the phenotypic and functional outcomes of MBP-treated dendritic cells (DCs) and show that MBP induces DC activation and production of proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, IL-8, tumor necrosis factor alpha, and IL-12p70) within 24 h and strongly increases Ikappabeta phosphorylation in treated cells. Interestingly, phosphorylation of Ikappabeta was largely abrogated by the addition of anti-human Toll-like receptor 4 (TLR4) antibodies, indicating that MBP activates signaling for DC maturation via TLR4. Consistent with this hypothesis, MBP activated the TLR4-expressing cell line 293-hTLR4A but not control cultures to secrete IL-8. The observed data were independent of lipopolysaccharide contamination and support a role for TLR4 in mediating the effects of MBP. These results provide insight into a mechanism by which MBP might enhance immune responses to vaccine fusion proteins.

Restricted replication and lysosomal trafficking of yellow fever 17D vaccine virus in human dendritic cells.

The yellow fever virus attenuated 17D vaccine strain is a safe and effective vaccine and a valuable model system for evaluating immune responses against attenuated viral variants. This study compared the in vitro interactions of the commercially available yellow fever vaccine (YF-VAX), Dengue virus and the live-attenuated dengue vaccine PDK50 with dendritic cells (DCs), the main antigen-presenting cells at the initiation of immune responses. Similar to PDK50, infection with YF-VAX generated activated DCs; however, for YF-VAX, activation occurred with limited intracellular virus replication. The majority of internalized virus co-localized with endolysosomal markers within 90 min, suggesting that YF-VAX is processed rapidly in DCs. These results indicate that restricted virus replication and lysosomal compartmentalization may be important contributing factors to the success of the YF-VAX vaccine.

Modulation of dengue virus infection of dendritic cells by Aedes aegypti saliva.

Dengue virus (DV) is a flavivirus carried by the Aedes aegypti mosquito that causes a spectrum of illnesses in the tropics, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Dendritic cells (DCs) are professional antigen presenting cells recently shown to be permissive for DV, and implicated as the primary targets of initial DV infection. DV is transmitted to human host by infected mosquitoes during a blood meal, but it is currently unknown whether transmission is modified by vector saliva that is also deposited in the host's skin during feeding. Previous studies evaluated only the outcome of DV infection of DCs, and did not address the influence of mosquito saliva. To more fully characterize natural transmission of DV, we evaluated the effects of Ae. aegypti saliva on DV infection of human myeloid DCs. We found that saliva inhibited DV infection in DCs. Moreover, pre-sensitization of DCs with saliva, prior to DV infection, enhanced inhibition. In addition, enhanced production of IL-12p70 and TNF-alpha were detected in DV-infected DC cultures exposed to mosquito saliva. The proportion of dead cells was also significantly reduced in these cultures. These data contribute to the overall understanding of the natural pathogenesis of DV infection and suggest that there is a protective role for mosquito saliva that limits viral uptake by DCs.

Intraocular adenoviral vector-mediated gene transfer in proliferative retinopathies.

PURPOSE: The purpose of this study was to compare levels and patterns of expression of reporter genes achieved with an E1-deleted and partially E3-deleted type 5 adenoviral (Ad) vector after intravitreous or subretinal injections, or after intravitreous injections in mouse eyes with proliferative retinopathies. METHODS: Ad vectors containing reporter gene constructs were injected into the vitreous cavity or subretinal space of wild-type mice or mice with proliferative retinopathies, and quantitative comparisons were made of expression of transgenes. RESULTS: In normal eyes, peak Ad-mediated expression of luciferase, driven by a cytomegalovirus (CMV) promoter, occurred after injection of 10(7) to 10(8) viral particles and was 10 times greater after subretinal injections than after intravitreous injections. Intravitreous injections of Ad containing beta-galactosidase (LacZ) expression constructs (AdLacZ.10) resulted in strong expression of LacZ in epithelial cells of the iris and ciliary body and focal expression in the retina. Subretinal injections of AdLacZ.10 resulted in strong expression in RPE cells. Expression of LacZ after intravitreous injection of AdLacZ.10 was significantly greater in mice with two types of proliferative retinopathy (ischemic retinopathy or transgenic mice with retina-specific expression of platelet-derived growth factor (PDGF)-BB or PDGF-AB) than littermate control animals. Cells within epiretinal membranes and activated Müller cells were preferentially transduced in eyes with proliferative retinopathy. CONCLUSIONS: These data suggest that although higher intraocular expression levels can be achieved after subretinal injection of adenoviral vectors, intravitreous injections provide good transduction of cells lining the vitreous cavity. Compared with normal eyes, eyes with proliferative retinopathy showed increased transduction, which occurred preferentially in cells participating in the disease process. Intravitreous injection of adenoviral vectors containing appropriate expression constructs may provide a good strategy for acute treatment of proliferative retinopathies, such as diabetic retinopathy and proliferative vitreoretinopathy.