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    Infection of human immature myeloid dendritic cells by ChimeriVax™-Dengue (CYD) 1–4 chimeras and implication of dendritic cell-specific intercellular adhesion molecule 3–grabbing non-integrin (DC-SIGN). Infection was monitored by intracellular staining with Alexa 488-coupled monoclonal antibody 4G2 specific for envelope antigen of flaviviruses and flow cytometry analysis after viral exposure for 48 hours. A, Infection with CYD3 (multiplicity of infection [MOI] = 0.05 or 5) or mock infection. B, Infection by CYD1–4 (MOI = 0.5) or mock-infection after pre-incubation with DC-SIGN-blocking antibodies or isotype control (10 μg/mL). The results of four independent experiments are shown. Histograms correspond to medians. FSC = forward scatter.

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    Up-regulation of phenotypic markers induced by ChimeriVax™-Dengue 3 (CYD) or heat-inactivated ChimeriVax™-Dengue 3 in human immature myeloid dendritic cells. Cells were incubated with chimera for 48 hours (MOI = 0.5). Cells were then collected and double-stained with phycoerythrin-conjugated cell surface monoclonal antibody (MAb) and with Alexa 488-coupled MAb 4G2 specific for envelope antigen of flaviviruses for flow cytometry analysis. A, Gating was performed on 4G2-positive (infected) or 4G2-negative (bystander) cells. Results are expressed as fold induction of the cell surface expression markers with treated cells compared with mock infected cells. Each symbol represents one of n independent donors. Horizontal lines are median values. M.F.I. = mean fluorescence intensities. **significant differences compared with mock infection P < 0.01 (comparing raw data). B, Histograms showing expression of surface markers (MFI) on the cell surface of the total population (without segregating infected cells from surrounding uninfected cells). The results are representative of two independent experiments.

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    Relative mRNA expression (A), secretion in supernatant (B), and intracellular accumulation (C) of cytokine/chemokine in human immature myeloid dendritic cells exposed to ChimeriVax™-Dengue 3 (CYD). A, Total RNA was extracted after infection for 24 hours (MOI = 0.5). RNA was converted to cDNA and a real-time polymerase chain reaction was performed. Relative mRNA levels of interleukin-6 (IL-6), tumor necrosis factor-β (TNF- β), monocyte chemoattractant protein 1 (MCP-1), and interferon-β (IFN-β) were measured using the comparative threshold cycle method using mock-infected dendritic cells as a reference sample. B, Cell-free supernatants were collected after infection for 48 hours (MOI = 0.5) and assayed for cytokine/chemokine secretion by an enzyme-linked immunosorbent assay or a cytometric bead array. C, Intracellular accumulation of TNF-α was detected by flow cytometry after infection for 48 hours (MOI = 0.5) and incubation for 18 hours with Brefeldin A. Cells were stained with phycoerythrin (PE)-conjugated anti-cytokine monoclonal antibody (MAb) and with Alexa 488-coupled MAb 4G2 specific for envelope antigen of flaviviruses. Gating was performed on 4G2-positive (infected) or 4G2-negative (bystander) cells. Each symbol represents one of n independent donors. Horizontal lines represent median values. **Significant differences compared with mock infection (P < 0.01) (no statistical analysis were performed on mRNA expression). D, Representative dot plot of double-staining for TNF-αand flavivirus envelope antigen. The number of events is indicated in each quadrant (UL = upper left; UR = upper right; LL = lower left; LR = lower right).

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    Up-regulation of phenotypic markers induced by ChimeriVax™-Dengue 1–4 (CYD) in human immature myeloid dendritic cells. Cells were collected after infection for 48 hours (MOI = 0.5) and double-stained with phycoerythrin (PE)-conjugated cell surface marker monoclonal antibodies (MAbs) and Alexa 488-coupled MAb 4G2 specific for envelope antigen of flaviviruses; 200,000 gated events were analyzed by flow cytometry and 10% of the dot-plots are represented. Mean fluorescence intensities (for HLA-DR and CD86 markers) or the percentage of positive cells (for CD83 marker) of phenotypic markers are indicated for infected (I) cells, non-infected bystander (NI) cells, or for all cells for mock condition. Data from a single donor are shown and results are representative of four independent experiments.

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    Comparable up-regulation of phenotypic markers induced by ChimeriVax™-Dengue (CYD) and wild-type (WT) parental dengue (DEN) serotypes 2, 3, and 4 in human immature myeloid dendritic cells. Cells were collected after infection for 48 hours (MOI = 0.5) and double-stained with phycoerythrin-conjugated cell surface marker monoclonal antibodies (MAbs) and Alexa 488-coupled MAb 4G2 specific for envelope antigen of flaviviruses. Gating was performed on 4G2+ (infected) and 4G2- (bystander) cells. Four similar independent experiments were performed.

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    Cytokine/chemokine secretion in human immature myeloid dendritic cells infected with ChimeriVax™-Dengue (CYD) and wild-type (WT) parental dengue (DEN) serotypes 1–4. Cell-free supernatants were collected after infection for 48 hours (MOI = 0.5). Concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α ), monocyte chemoattractant protein 1 (MCP-1), and interferon-β (IFN-β) were determined by an enzyme-linked immunosorbent assay or a cytometric bead array. Each symbol represents one individual. Mean values for infection rates were CYD1 = 7%, WT DEN1 = 1%, CYD2 = 30%, WT DEN2 = 61%, CYD3 = 7%, WT DEN3 = 55%, CYD4 = 33%, WT DEN4 = 52%. A, Histograms show the median result. B, IL-6/TNF-α ratios were calculated for each of four individuals (one symbol per individual).

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INNATE IMMUNE RESPONSES IN HUMAN DENDRITIC CELLS UPON INFECTION BY CHIMERIC YELLOW-FEVER DENGUE VACCINE SEROTYPES 1–4

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  • 1 Research Department, sanofi pasteur, Marcy l’Etoile, France

Dengue infection is an important public health issue worldwide. The ChimeriVax™-Dengue (CYD) vaccine uses yellow fever (YF) 17D vaccine as a live vector. Dendritic cells (DCs) play a key role in initiating immune responses and could be an important primary target of dengue infection. We investigated in vitro the consequences of CYD infection of DCs on their activation/maturation and cytokine production. In CYD-infected DCs, we observed an up-regulation of HLA-DR, CD80, CD86, and CD83. Cells exposed to CYD secreted type I interferons, monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL-2), interleukin-6 (IL-6), and low amounts of tumor necrosis factor-α (TNF-α), but no IL-10, IL-12, or IL-1α. Parental dengue viruses induced a similar array of cytokines, but more TNF-α, less IL-6, and less MCP-1/CCL-2 than induced by CYD. Chimeras thus induced DCs maturation and a controlled response accompanied by limited inflammatory cytokine production and consistent expression of anti-viral interferons, in agreement with clinical observations of safety and immunogenicity.

INTRODUCTION

Dengue viruses are mosquito-borne flaviviruses that circulate in tropical areas and cause human disease with significant morbidity and mortality. Each of the four serotypes of dengue (DEN1–4) can cause clinical manifestations ranging from self-limiting dengue fever to severe dengue hemorrhagic fever (DHF) and fatal dengue shock syndrome. Up to 80 million dengue infections and 24,000 deaths are estimated annually.1,2

Although no licensed vaccine is yet available, several candidates are being developed to provide long-lasting protection against all four serotypes. Novel approaches for developing live attenuated flavivirus vaccines include the ChimeriVax™ technology, based on the well-known, safe, and immunogenic yellow fever (YF) 17D virus vaccine. The genes encoding the premembrane and envelope proteins of YF-17D are replaced by those of other flaviviruses, such as Japanese encephalitis and dengue.3,4 Chimeras have been constructed for each of the four dengue serotypes (ChimeriVax™-DEN1–4; CYD1–4) from wild-type (WT) dengue virus and are currently undergoing phase I clinical trials.5,6

It is postulated that an inappropriate immune response, implicating both humoral and cellular components, could partially explain disease severity. Enhancing antibodies, low-affinity T cell response, excessive production of soluble pro-inflammatory mediators, and complement activation may play a role in the pathogenesis of DHF.2,79 It is thus critical to study the early events after dengue virus infection at the cellular level. Although the predominant target of dengue virus is believed to be blood monocytes and tissue macrophages,10,11 recent studies have demonstrated the permissiveness of immature myeloid dendritic cells (DCs) to dengue virus.1214 These observations suggest that DCs in the epidermis (Langerhans cells) could be the primary target of infection after a mosquito bite and would constitute the first line of defense.

Dendritic cells are key elements of both innate and adaptive immune responses to infection. In response to viral infection, DCs rapidly produce type I (α and β) interferons (IFNs) that act as inducers of a number of antiviral genes and as activators of DCs, lead to subsequent development of adaptive response.1517 Activated DCs produce inflammatory cytokines, up-regulate costimulatory and HLA molecules, and migrate to lymph nodes where they function as potent antigen-presenting cells for naive T lymphocytes to initiate adaptive immune responses.18 Thus, the interaction between dengue virus and DCs has attracted considerable interest. Enhanced expression of cell-surface activation/maturation markers on DCs has been demonstrated upon in vitro exposure to non-attenuated dengue viruses or to live vaccine strains.13,1921 Some investigators have described the maturation of the infected subpopulation as well as in the surrounding uninfected cells, and have observed stronger variations either in the latter20 or former cell populations.19 Others argue that dengue-infected DCs loose their ability to up-regulate phenotypic markers.21 It has also been reported that DCs produce tumor necrosis factor-α (TNF-α) and IFN-α but minimal amounts of interleukin-12 (IL-12) after exposure to dengue virus.13,20,21 Chemokines, another type of soluble mediator that are known to play an important role in viral pathogenesis and immunity,22,23 are also secreted in vitro by various human myeloid cells after infection with dengue virus.2428

The CYD1–4 chimeras replicate in human DCs in vitro.29 To explore viral replication and the initiation of immune response at the early stages of infection, we investigated the consequences of CYD1–4 infection of DCs on activation/ maturation and the secretion of pro- and anti-inflammatory cytokines, chemokines, and type I interferons.

MATERIALS AND METHODS

Viruses and vaccines.

ChimeriVax™-DEN1–4 were produced in Vero cells and purified at sanofi pasteur (Marcy l’Etoile, France): serotype 1: batch DEN1 Chim 0002 (107.40 50% tissue culture infectious doses [TCID50]/mL) and 05DEV001 CYD1-03, P13FT (108.90 TCID50/mL); serotype 2: batch DEN2 Chim 0002 (107.63TCID50/mL) and 05DEV001 CYD2-03, P12FT (107.20 TCID50/mL); serotype 3: batch 03REC023 (107.03TCID50/mL) and 04DEV006, R4080604 (107.70TCID50/mL); and serotype 4: batch DEN4 Chim 002 (108.00TCID50/mL) and 05DEV001 CYD4-03, P8FT (107.55TCID50/mL). The WT parental dengue viruses were produced on C6/36 mosquito cells and purified at Acambis (Cambridge, MA): serotype 1: FSM 11-5-04 (108.49 plaque-forming units [PFU]/mL); serotype 2: FSM 12/23/04 (108.81 PFU/mL); serotype 3: FSM 12/19/04 (107.43 PFU/mL); and serotype 4: FSM 12-2-04 (108.46 PFU/mL). These viruses correspond to the following isolates: serotype 1: PUO359, serotype 2: PUO218, serotype 3: PaH881/88, and serotype 4: 1228. Yellow fever vaccine 17D 204 (Stamaril®), bulk lot (107.60TCID50/mL) was produced by sanofi pasteur (Val de Reuil, France).

Culture medium and reagents.

Complete medium was RPMI 1640 medium (Gibco, Paisley, Scotland), supplemented with 2 mM glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT) and 100 units/mL of penicillin-streptomycin (Gibco). Recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-4 were obtained from PeproTech (Rocky Hill, NJ) and added to the complete medium at concentrations of 50 ng/mL and 10 ng/mL, respectively.

Human DCs.

Whole blood from healthy adult volunteers was collected in sodium citrate bags by the local transfusion center (Etablissement Français du Sang, Lyon, France). Peripheral blood mononuclear cells were isolated by density-gradient centrifugation using Ficoll-Hypaque (Nycomed, Roskilde, Denmark). Monocytes were purified by positive selection using CD14 microbeads and a magnetic cell separator according to the manufacturer’s specifications (Miltenyi Biotech Inc., Auburn, CA). The CD14+ cells were cultured in complete medium supplemented with GM-CSF and IL-4 at 37° C in an atmosphere of 5% CO2 for six days at a density of 1 × 106 cells/mL in T75 flasks. Fresh cytokines were added every two days. The appropriate phenotype of immature DCs (CD14, CD1a+, HLA-DR+, CD83) was confirmed by flow cytometry before each experiment.

Infection of human DCs with CYD1–4.

Immature myeloid DCs were infected with CYD1–4 viruses at a multiplicity of infection (MOI) of 0.5 in RPMI 1640 medium containing 2% heat-inactivated FBS (2 × 106 DCs/mL) and supplemented with GM-CSF (50 ng/mL) and IL-4 (10 ng/mL). Infection rates were found to be higher at 32°C than at 37°C, which is associated with similar changes in immune parameters (cytokines and phenotypic markers), as previously reported.19 Since results were more consistent and reproducible at 32°C, this temperature was chosen for all experiments described in this report. Cells were incubated with viruses for 48 hours at 32°C in an atmosphere of 5% CO2 without washing. Control experiments included mock infection (medium alone) and infection with heat-inactivated viruses (56°C for 1 hour). Cells were collected after 48 hours and plated in 96-well plates for immunostaining and flow cytometry analysis. Cell-free supernatants were collected and stored at -80°C for cytokine quantification. To evaluate whether DC-specific intercellular adhesion molecule 3–grabbing non-integrin (DC-SIGN) is involved in the infection of DCs by CYD, cells were incubated with 10 μg/mL of anti-human DC-SIGN antibody (clone 120612, R&D Systems, Minneapolis, MN) or isotype control (BD Biosciences, San Diego, CA) for 1.5 hours at 37°C before infection. For intracellular cytokine analysis, Brefeldin A (Sigma, St. Louis, MO) was added at a concentration of 10 μg/mL after 48 hours of infection for an additional incubation for 18 hours.

The CYD3 assays were performed on cells from 8–15 donors. Assays on the other CYD serotypes were performed on 4–6 donors. Cells from four donors were also infected with parental WT DEN1–4 viruses (at an MOI of 0.5/cell) using the same method as described above.

Extraction of RNA, reverse transcription, and real-time polymerase chain reaction (RT-PCR).

DCs were lysed and total cellular RNA was extracted using Nucleospin RNA II columns (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. The quantity and quality of total RNA were checked with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, DE) and with a Agilent 2100 bioanalyzer using RNA 6000 Nano chips (Agilent Technologies, Palo Alto, CA).

For reverse transcription of RNA to cDNA, the high-capacity cDNA archive kit (Applied Biosystems, Foster, CA) was used according to the manufacturer’s instructions. Samples were stored at −20°C. The PCR was performed in triplicate in a 96-well reaction plate (MicroAmp Optical; Applied Biosystems) using TaqMan universal PCR master mixture, assay-on-demand mix containing primers and probes (Applied Biosystems), 5 ng of cDNA (replaced with water for a negative control) and water in a final volume of 25 μL. An initial incubation for two minutes min at 50°C for optimal activity of uracyl-N-glycosylase was followed by incubation for 10 minutes at 95°C to activate AmpliTaq gold hot start polymerase, then 40 cycles for 15 seconds at 95°C and one minute at 60°C). Based on preliminary experiments, in which RT-PCR was performed 1, 24, or 48 hours post-infection, 24 hours was selected for further studies because this delay provided the highest fold induction relative to mock infection for all considered genes. Data were captured and analyzed using ABI Prism 7000 sequence detection system and version 1.1 Software (Applied Biosystems). The baseline was determined manually between two and seven cycles for the 18S rRNA and automatically for other genes. Thresholds were determined manually for all genes. Gene expression was measured by the comparative threshold cycle (Ct) method. The parameter threshold cycle (Ct) was defined as the cycle number at which the reporter fluorescence generated by the cleavage of the probe passed a fixed threshold above baseline. Cytokine gene expression was normalized using the endogenous reference 18S rRNA gene and mock-infected DCs were used as reference sample (calibrator). Relative gene expression was determined for each donor, using the equation 2 ×e-[(Ctgene - Ct18S)infected - (Ctgene - Ct18S)mock] (User Bulletin #2; Applied Biosystems). Values > 2 were considered as significant up-regulations between infected and mock condition.

Antibodies for surface and intracellular immunostaining.

Phycoerythrin (PE)-conjugated monoclonal antibodies (MAbs) were obtained from BD Biosciences (anti-human CD83, anti-human CD86, anti-human IL-6, anti-human IL-12p40/70, anti-human TNF-α, anti-human HLA-DR, and anti-human CD80) or DakoCytomation (Carpinteria, CA) (anti-human CD1a MAb). Anti-flavivirus MAb (clone 4G2 lot HB112-011012) was obtained from Biotem (Le Rivier d’Apprieu, France) and conjugated to Alexa 488 using the A 488 kit (Molecular Probes, Eugene, OR).

Immunostaining and flow cytometry analysis.

Cells were collected in 96-well plates and washed with cold staining buffer (0.1% bovine serum albumin in phosphate-buffered saline). To analyze phenotypic marker expression, DCs were cell-surface labeled according to the manufacturer’s specifications (BD Biosciences). Cells were washed twice, then fixed with 4% formaldehyde (Sigma-Aldrich Corp., St. Louis, MO) in staining buffer for 10 minutes at room temperature and permeabilized with 0.1% saponin (Sigma) in staining buffer for five minutes at 4°C. After a saturation step, achieved by incubation for 15 minutes in permeabilization buffer with human IgG (10 μg/106 cells; Sigma), intracellular staining for virus was accomplished by adding Alexa 488-conjugated anti-flavivirus 4G2 MAb (1 μg/106 cells) and incubating for 20 minutes at 4°C in the dark. For intracellular cytokine analysis, PE-conjugated MAbs were added at the same time, according to the manufacturer’s specifications. Cells were washed twice, then resuspended in 250 μL of staining buffer prior to analysis with FACScan or FACScalibur flow cytometer (BD Biosciences) and the CellQuest software (Becton Dickinson, Franklin Lakes, NJ). In all experiments, 100,000–200,000 events were gated on the forward scatter/side scatter dot plot, according to the infection rate, to analyze a sufficient number of cells among infected (4G2+) and surrounding uninfected cells (4G2-).

Cytokine production in culture supernatants.

IL-8, IL-1β, IL-6, IL-10, TNF, and IL-12p70 were quantified with the cytometric bead array (CBA) inflammatory kit (BD Biosciences). Both IFN-α (multi-species) and IFN-β were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits from PBL Biomedical Laboratories (Piscataway, NJ) and Fujerebio Inc. (Tokyo, Japan), respectively. Monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL-2) and, if necessary, TNF-α and IL-6, were quantified with OptEIA kits (BD Biosciences). The ELISA and CBA assay were performed according to manufacturers’ instructions. Based on preliminary experiments, in which cy-tokines were measured 24, 48, and 72 hours post-infection, 48 hours was selected for further studies regarding protein secretion and cellular viability.

Statistical analysis.

For non-parametric paired data, a permutation test was used to provide the exact probability that the difference observed between two groups occurred by chance. Analyses were performed with StatXact 4 software (Cytel Software, Cambridge, MA) with a one-sided risk of 5% for comparisons with control, and a two-sided risk of 5% for comparisons between stimulations.

For comparisons between CYD vaccine candidates and WT DEN parental viruses, a mixed model was used with a fixed factor: type of stimulation (CYD/WT DEN) and a random donor effect. Analyses were performed with SAS version 8.2 software (SAS, Cary, NC) at a two-sided risk of 5%.

RESULTS

Infection of human DCs by CYD1–4 and role of DC-SIGN molecules.

Infection of DCs was observed for each serotype. In addition to intracellular detection of viral antigen (Figure 1A), productive infection of DCs was confirmed in supernatants by quantification of virus by plaque assay and quantitative RT-PCR. Infection rates varied between donors and increased with increasing MOI. For all further experiments, an MOI of infection of 0.5 was selected. No infection was observed with heat-inactivated CYD. When viral infection was performed with MAbs to DC-SIGN, the level of infected cells decreased for all four serotypes of CYD, suggesting the involvement of DC-SIGN molecules in infection of DCs by CYD1–4 (Figure 1B).

Immune changes induced by CYD3 in human DCs.

We then explored the influence of viral infection on innate immunity, including cell surface expression of activation/ maturation markers and production of inflammatory, chemo-tactic or antiviral mediators. We used CYD3 serotype as a prototype CYD to explore immune changes, before repeating the experiments with the other serotypes.

Phenotypic changes.

Infection with CYD3 triggered significantly enhanced expression of the following markers compared with mock infection: major histocompatibility complex class II molecules HLA-DR, specific DC maturation marker CD83, and co-stimulatory molecules CD80 and CD86 (Figure 2A). In most cases, expression was enhanced predominantly in the infected subpopulation. The median fold induction compared with mock infection was in the range 2–5 for infected cells, and ≤ 2 for bystander cells. In some donors, however, similar up-regulations were observed in both sub-populations of cells. No phenotypic variations were observed between cells treated with heat-inactivated CYD3 and mock-infected cells (Figure 2B).

Cytokine/chemokine production.

In addition to phenotypic changes, DCs also responded to CYD3 infection by secreting soluble mediators. Real-time quantitative PCR analyses of cytokine/chemokine gene expression showed significantly increased mRNA expression of the pro-inflammatory cytokines IL-6 and TNF-α, the MCP-1/CCL-2 chemokine, and type I IFN-βFigure 3A). The expression of type I IFN-βwas particularly strong (relative gene expression approximately 2,000). In contrast, no variations were observed in the expression of IL-12p40, IL-10, IL-1β or TGF-β, or of other chemokines such as CCL3 and CCL5. The ELISA and CBA analysis of secreted protein in the supernatants after 48 hours of exposure to CYD3 showed similar results (Figure 3B). Increased, but low levels of TNF-α were detected upon CYD3 infection (median value = 217 pg/mL compared with 60 pg/ mL with mock infection). Increased production was also observed for IL-6 (944 pg/mL compared with 170 pg/mL) and MCP-1 (14,226 pg/mL compared with 2,335 pg/mL), as well as for type I IFN-β (156 units/mL compared with < 12.5 units/ mL) and IFN-α. Interleukin-1β, IL-10, and IL-12p70 were also quantified but were not consistently detected even when infection rates were high. Flow cytometry analyses of the intracellular production of inflammatory cytokines showed the expression of TNF-α by CYD3-infected DCs (Figure 3C). This expression was systematically restricted to the infected subpopulation (approximately 9% of infected cells were TNF+ compared with 1% of the bystander cells), which is consistent with the low amount of TNF-α this cytokine detected in supernatants. Intracellular expression of IL-12p40/ p70 was not detected in any subpopulation.

Immune changes induced by CYD1, 2, and 4.

As was the case with CYD3, an up-regulation of the phenotypic markers HLA-DR, CD80, CD83 and CD86 was seen. Figure 4 shows results obtained with one donor as an example. Furthermore, similar profiles of inflammatory cytokines, chemokine, and type I IFN were observed with each of the four serotypes.

Immune changes induced by YF 17D.

Experiments were repeated using the 17D vaccine strain (seven donors). After 48 hours of exposure, intracellular staining for flavivirus antigen showed no significant infection of mono-DCs. Immune parameters were thus monitored on the total population. Despite inapparent infection, exposure to YF 17D resulted in an up-regulation of phenotypic markers (mean ± SD-fold induction versus mock infection: HLA-DR = 1.65 ± 0.28, CD80 = 1.56 ± 0.26, CD86 = 2.63 ± 0.59; P < 0.01). Similar changes were observed for CD83 but were analyzed on only four donors (fold induction = 2.73 ± 0.58). Mono-DCs exposed to YF 17D secreted IL-6, IFN-β at levels that were not different (P > 0.05) from those induced by CYD3. MCP-1 was also secreted at comparable levels, but the number of analyzed donors was insufficient for statistical analysis. In contrast, higher levels of TNF-α (1,712 pg/mL) were induced by YF 17D than by CYD3 (P < 0.05). Finally, and again in contrast with the chimeras, YF 17D induced, low levels of IL-10 in some donors (mock and CYD3 = < 20 pg/mL, YF 17D = 99 ± 20 pg/mL) and IL-12p70 (mock and CYD3 = < 20 pg/mL, YF 17D = 42 ± 8 pg/mL).

Immune changes induced by WT parental viruses.

We compared immune changes induced by the CYD vaccine candidates to those induced by their parental dengue viruses in four additional donors. Infection rates were high with WT DEN2 (20–82%), WT DEN3 (28–73%) and WT DEN4 viruses (29–65%) but were low with WT DEN1 (from undetectable rates to 2%), which prevented an accurate analysis by flow cytometry with this latter serotype. The enhancement of cell surface expression of maturation markers CD83, HLA-DR, and costimulatory molecules CD80 and CD86 induced by WT viruses was within the same range as that induced by the CYD viruses in both cell subpopulations when all donors were considered (see for one donor in Figure 5). Up-regulation of phenotypic markers, notably in bystander cells, varied between donors. Nevertheless, in all four donors evaluated, activation/maturation of cells infected with WT DEN virus showed similar trends than after CYD infection. Similarly, WT DEN2–4 also triggered intracellular expression of

TNF-α, predominantly in infected cells, and no expression of IL-12p40/p70. Another WT DEN1 strain was tested (DEN1 16007 prepared in Vero cells), which was more infectious in DCs than the CYD1 parental WT strain. It induced similar immunologic changes regarding cytokines and phenotypic markers than CYD2–4 parental strains. Cytokine secretion was also followed in the supernatant by ELISA and CBA assay (Figure 6A). The WT DEN2–4 triggered the production of IFN-β, IL-6, and TNF-α, and weak expression of MCP-1, but not IL-10, IL-1β or IL-12p70. For parental WT DEN1, results were heterogeneous and different from those obtained with the other WT serotypes 2 to 4. This was most likely due to the low/absent infection levels observed with this former serotype; except for one donor, it triggered, as mock infection, 10–100 times lower secretion of IL-6, TNF-α or IFN-β than WT serotypes 2–4.

To compare responses induced by CYD vaccine candidates to those induced by their parental strains and get sufficient statistical power, levels of cytokines induced after WT DEN or CYD stimulation were considered whichever the serotype, to the exclusion of serotype 1 for the reasons exposed above. As shown in Table 1, CYD vaccine candidates induced significantly less TNF-α and more IL-6 than parental viruses. As a consequence, IL-6/TNF-α ratios were dramatically higher with CYD than WT DEN (see individual ratios in Figure 6B). Additionally, the enhancement of MCP-1 production was significantly greater with CYD than with the parental strains. Conversely, levels of IFN-β induced by vaccine or parental strain were similar.

DISCUSSION

Dendritic cells are key components of innate and adaptive immunity. Their location in peripheral tissues and their permissiveness to dengue virus in vitro and ex vivo make them an important potential primary target of dengue infection after a mosquito bite. Dengue infection of human skin DCs in vivo after vaccination with a tetravalent vaccine has also been reported.12 Our study was conducted to characterize in vitro the effect of infection with live attenuated chimeric YF-dengue vaccines (CYD1–4) on the maturation/activation states and cytokine/chemokine production of human monocyte-derived DCs. We found that all four live attenuated CYD vaccine viruses stimulated the maturation of human DCs and triggered the selective expression of certain inflammatory cyto-kines. Additionally we found that although infection with the CYD viruses induced a similar panel of cytokines to that induced by the parental WT dengue strains, there were some quantitative differences. A higher IL-6/TNF-α ratio and stronger MCP-1/CCL-2 chemokine production were induced by infection with the chimeras.

In preliminary experiments, we confirmed that CYD1–4, contrary to Stamaril YF 17D, were able to productively infect human DCs in vitro, and found that dengue infection was probably mediated by the cell-surface C-type lectin DC-SIGN molecules. This is consistent with previous observations for dengue viruses,30,31 and supports the key role in this respect of dengue envelope and glycosylation sites, mediating interaction with DC-SIGN molecules. The DC-SIGN was recently found to be unnecessary for the infection of DCs by YF 17D vaccine,32 but the undetectable infection observed here with Stamaril vaccine has been reported in other DC-SIGN-positive cells30 and may be due to a specific glycosylation pattern of this 17D 204 strain. However, it is also possible that the technique used to monitor infection was insufficiently sensitive.

Previous studies have described differential immune effects on infected and bystander cells after exposure to dengue viruses21 or vaccines.19 It was therefore important to analyze immune parameters in both subpopulations. In our study, we did this using flow cytometry to simultaneously monitor infection status and immune changes in parallel with RT-PCR and ELISA and CBA assays on the total population.

The chimeras triggered up-regulation of the cell surface expression of HLA-DR, as well as CD80 and CD86 co-stimulatory molecules, and enhanced the expression of the CD83 DC-specific activation marker. Expression levels were usually much higher in infected subpopulation than in the surrounding cells, in which variations were generally low. These phenotypic changes in infected DCs associated with the activation and maturation process may subsequently allow efficient viral antigen presentation. The parental dengue viruses also triggered the maturation of infected cells. This is consistent with previous observations in our laboratory with another dengue strain,19 but in contrast to a report that infection by another DEN2 strain impaired the maturation of DCs.21 These differences are possibly strain or even batch dependent and associated with virulence, as seen in our own present experiments with two different WT DEN1 strains. These findings suggest that the live attenuated CYD chimeras efficiently induced early innate cell maturation and activation. However, it remains unknown whether mature CYD-infected DCs efficiently stimulate T cells and induce an appropriate immune response. For instance, DCs infected with a wild-type DEN2 strain have been found to exhibit reduced T-cell stimulatory capacities that are associated with IL-10 production.21 Infected DCs have also been found to enhance the proliferation of interacting T cells, but these then secrete both Th1 and Th2 cytokines.33

An array of inflammatory mediators was detected after exposure to CYD1–4. Antiviral type I interferons were strongly induced and the production of MCP-1 chemokine and IL-6 was enhanced. In contrast, neither inflammatory IL-12p70 and IL-1βnor anti-inflammatory IL-10 cytokines were consistently detected. In addition, low amounts of TNF-α were expressed by the infected population only, supporting the view that activation and maturation occurs mostly in this sub-population with these viruses. Despite the apparent lack of productive infection by the YF vaccine strain, we observed an up-regulation of all phenotypic markers considered and the secretion of the same cytokines as those secreted in response to the chimeras. In some donors low levels of both IL-12p70 and IL-10 were also observed, in agreement with recent data reported by Querec and others.34 These investigators also showed that YF 17D activates DCs by surface binding to toll-like receptors; this is in agreement with our data obtained in absence of significant infection, since even non-infectious UV-irradiated YF 17D could activate DCs in these studies.34 Taken together, the similarities and differences observed in the responses induced by CYDs or 17D vaccine suggest that the overall effect of DC infection by CYD is a result of the specific interactions between dengue E and DCs, as well as the 17D replication machinery.

The MCP-1 chemokine plays a pivotal role in anti-viral immune responses, such as the recruitment of immune cells or the polarization of immune responses. It would therefore participate in the induction of both effective innate responses and adaptive responses after CYD infection.23 It has been proposed that type I IFNs are essential in controlling primary dengue infection.35 Secretion of these cytokines would therefore limit CYD replication in vivo and control DC activation and cytokine/chemokine production. Both TNF-α and IL-10 are associated with severe dengue;3639 therefore, the low or inexistent expression of these cytokines by CYD-infected DCs is supportive of the safety of the CYD vaccines. The parental dengue viruses induced similar cytokine profiles to those induced by the chimeras although the latter induced weaker TNF-α and stronger IL-6 and MCP-1 responses. Again, this is in contrast to previous observations with other WT dengue strains that showed IL-10 production by DCs and marked TNF-α secretion by both infected and surrounding cells.19,21 Although the critical role of TNF-α in disease severity has been highlighted, a recent report showed the predominant role of IL-6 over TNF-α in the late evolution towards fatal DHF.40 However, as stated by the investigators, the kinetics of TNF-α production might be different from that of IL-6. An early predominant IL-6 secretion over TNF-α as seen in our assays could eventually result in a lower in vivo reactogenicity than induced by WT viruses, although at later time points the consequences of IL-6 production could be different.

In conclusion, the activation/maturation of DCs associated with a strong induction of type I interferons, a restricted expression of TNF-α, and no production of IL-10 suggests that the exposure of DCs to the ChimeriVax™-Dengue 1–4 chimera results in a tightly controlled inflammatory response and a potential adaptive immunity. Although these results are insufficient to predict the safety of the CYD1–4 vaccine candidates in humans, they are encouraging and, taken together with other in vitro and in vivo preclinical studies in mosquitoes and monkeys,41,42 they support their safety and immunogenicity, which is consistent with clinical observations with other ChimeriVax™ vaccines.4

Table 1

Comparison between levels of cytokines released by CYD- or WT DEN-stimulated dendritic cells*

IFN-β, U/mLMCP-1, pg/mLIL-6, pg/mLTNF-α, pg/mLIL-6: TNF-α
* CYD = ChimeriVax™, WT DEN = wildtype parental dengue viruses; IFN-β= interferon-β; MCP-1 = monocyte chemoattractant protein 1; IL-6 = interleukin-6; TNF-α = tumor necrosis factor-α. Results are expressed as geometric means (95% confidence intervals) of values induced by serotypes 2, 3, and 4 considered together.
CYD 2–4319 [186–546]11,630 [4,364–30,996]3,188 [1,324–7,675]628 [315–1,161]5.23 [1.86–14.71]
WT DEN 2–4464 [271–795]2,208 [829–5,884]464 [271–795]1,799 [904–3,579]0.74 [0.26–2.09]
P0.313< 0.00010.0110.035< 0.0001
Figure 1.
Figure 1.

Infection of human immature myeloid dendritic cells by ChimeriVax™-Dengue (CYD) 1–4 chimeras and implication of dendritic cell-specific intercellular adhesion molecule 3–grabbing non-integrin (DC-SIGN). Infection was monitored by intracellular staining with Alexa 488-coupled monoclonal antibody 4G2 specific for envelope antigen of flaviviruses and flow cytometry analysis after viral exposure for 48 hours. A, Infection with CYD3 (multiplicity of infection [MOI] = 0.05 or 5) or mock infection. B, Infection by CYD1–4 (MOI = 0.5) or mock-infection after pre-incubation with DC-SIGN-blocking antibodies or isotype control (10 μg/mL). The results of four independent experiments are shown. Histograms correspond to medians. FSC = forward scatter.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 1; 10.4269/ajtmh.2007.76.144

Figure 2.
Figure 2.

Up-regulation of phenotypic markers induced by ChimeriVax™-Dengue 3 (CYD) or heat-inactivated ChimeriVax™-Dengue 3 in human immature myeloid dendritic cells. Cells were incubated with chimera for 48 hours (MOI = 0.5). Cells were then collected and double-stained with phycoerythrin-conjugated cell surface monoclonal antibody (MAb) and with Alexa 488-coupled MAb 4G2 specific for envelope antigen of flaviviruses for flow cytometry analysis. A, Gating was performed on 4G2-positive (infected) or 4G2-negative (bystander) cells. Results are expressed as fold induction of the cell surface expression markers with treated cells compared with mock infected cells. Each symbol represents one of n independent donors. Horizontal lines are median values. M.F.I. = mean fluorescence intensities. **significant differences compared with mock infection P < 0.01 (comparing raw data). B, Histograms showing expression of surface markers (MFI) on the cell surface of the total population (without segregating infected cells from surrounding uninfected cells). The results are representative of two independent experiments.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 1; 10.4269/ajtmh.2007.76.144

Figure 3.
Figure 3.

Relative mRNA expression (A), secretion in supernatant (B), and intracellular accumulation (C) of cytokine/chemokine in human immature myeloid dendritic cells exposed to ChimeriVax™-Dengue 3 (CYD). A, Total RNA was extracted after infection for 24 hours (MOI = 0.5). RNA was converted to cDNA and a real-time polymerase chain reaction was performed. Relative mRNA levels of interleukin-6 (IL-6), tumor necrosis factor-β (TNF- β), monocyte chemoattractant protein 1 (MCP-1), and interferon-β (IFN-β) were measured using the comparative threshold cycle method using mock-infected dendritic cells as a reference sample. B, Cell-free supernatants were collected after infection for 48 hours (MOI = 0.5) and assayed for cytokine/chemokine secretion by an enzyme-linked immunosorbent assay or a cytometric bead array. C, Intracellular accumulation of TNF-α was detected by flow cytometry after infection for 48 hours (MOI = 0.5) and incubation for 18 hours with Brefeldin A. Cells were stained with phycoerythrin (PE)-conjugated anti-cytokine monoclonal antibody (MAb) and with Alexa 488-coupled MAb 4G2 specific for envelope antigen of flaviviruses. Gating was performed on 4G2-positive (infected) or 4G2-negative (bystander) cells. Each symbol represents one of n independent donors. Horizontal lines represent median values. **Significant differences compared with mock infection (P < 0.01) (no statistical analysis were performed on mRNA expression). D, Representative dot plot of double-staining for TNF-αand flavivirus envelope antigen. The number of events is indicated in each quadrant (UL = upper left; UR = upper right; LL = lower left; LR = lower right).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 1; 10.4269/ajtmh.2007.76.144

Figure 4.
Figure 4.

Up-regulation of phenotypic markers induced by ChimeriVax™-Dengue 1–4 (CYD) in human immature myeloid dendritic cells. Cells were collected after infection for 48 hours (MOI = 0.5) and double-stained with phycoerythrin (PE)-conjugated cell surface marker monoclonal antibodies (MAbs) and Alexa 488-coupled MAb 4G2 specific for envelope antigen of flaviviruses; 200,000 gated events were analyzed by flow cytometry and 10% of the dot-plots are represented. Mean fluorescence intensities (for HLA-DR and CD86 markers) or the percentage of positive cells (for CD83 marker) of phenotypic markers are indicated for infected (I) cells, non-infected bystander (NI) cells, or for all cells for mock condition. Data from a single donor are shown and results are representative of four independent experiments.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 1; 10.4269/ajtmh.2007.76.144

Figure 5.
Figure 5.

Comparable up-regulation of phenotypic markers induced by ChimeriVax™-Dengue (CYD) and wild-type (WT) parental dengue (DEN) serotypes 2, 3, and 4 in human immature myeloid dendritic cells. Cells were collected after infection for 48 hours (MOI = 0.5) and double-stained with phycoerythrin-conjugated cell surface marker monoclonal antibodies (MAbs) and Alexa 488-coupled MAb 4G2 specific for envelope antigen of flaviviruses. Gating was performed on 4G2+ (infected) and 4G2- (bystander) cells. Four similar independent experiments were performed.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 1; 10.4269/ajtmh.2007.76.144

Figure 6.
Figure 6.

Cytokine/chemokine secretion in human immature myeloid dendritic cells infected with ChimeriVax™-Dengue (CYD) and wild-type (WT) parental dengue (DEN) serotypes 1–4. Cell-free supernatants were collected after infection for 48 hours (MOI = 0.5). Concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α ), monocyte chemoattractant protein 1 (MCP-1), and interferon-β (IFN-β) were determined by an enzyme-linked immunosorbent assay or a cytometric bead array. Each symbol represents one individual. Mean values for infection rates were CYD1 = 7%, WT DEN1 = 1%, CYD2 = 30%, WT DEN2 = 61%, CYD3 = 7%, WT DEN3 = 55%, CYD4 = 33%, WT DEN4 = 52%. A, Histograms show the median result. B, IL-6/TNF-α ratios were calculated for each of four individuals (one symbol per individual).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 1; 10.4269/ajtmh.2007.76.144

*

Address correspondence to Bruno Guy, Research Department, sanofi pasteur, Campus Merieux, 1541 Avenue Marcel Merieux, 69280 Marcy l’Etoile, France. E-mail: bruno.guy@sanofipasteur.com

Authors’ address: Florence Deauvieau, Violette Sanchez, Claire Balas, Audrey Kennel, Aymeric de Montfort, Jean Lang, and Bruno Guy, Research Department, sanofi pasteur, Campus Merieux, 1541 Avenue Marcel Merieux, 69280 Marcy l’Etoile, France, E-mail: bruno.guy@sanofipasteur.com.

Acknowledgments: We thank G. Marsh for critical help in the preparation of this manuscript, N. Burdin for constant support and helpful discussions, C. Fournier and C. Droy for providing the CYDs vaccines, V. Barban and F. Pradezynski for performing the viral quantifications in cell supernatants and for helpful discussions, and F. Guirakhoo (Acambis, Cambridge, MA) for providing the parental dengue virus strains.

Financial support: This study was supported by sanofi pasteur.

Disclosure: The authors wish to report that their company has an exclusive license to develop the ChimeriVax™-dengue vaccines initially developed by Acambis (Cambridge, MA) that are used in this study. This statement is made in the interest of full disclosure and not because the authors consider this to be a conflict of interest.

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