DENV-2 New Guinea C (NGC) used as challenge virus in this study was generously provided by Dr. Steven Whitehead (National Institutes of Health, Bethesda, MD, USA). For neutralizing antibody assays, we used virus strains from which the prM and E genes of each live-attenuated dengue vaccine serotype were derived (DENV-1 16007, DENV-2 16681, DENV-3 16562, and DENV-4 1036). DENVs were grown in Vero cells or C6/36 cells in Dulbecco’s modified minimal essential medium (DMEM) containing 5% fetal bovine serum (FBS) and penicillin–streptomycin.

The four vaccine viruses were generated from cDNA clone-derived DENV-2 VV45R virus (based on the genome of DENV-2 PDK-53), and the DENV-2 PDK-53-based chimeras expressing the prM and E genes of DENV-1 16007, DENV-3 16562, or DENV-4 1036. The construction and characterization of these viruses has been previously reported (10, 19).

Twelve adult male, DENV seronegative cynomolgus macaques originating from Vietnam were used. The animals were placed in quarantine for 30 days prior to study start. The study was conducted at the Charmany Instructional Facility of the University of Wisconsin, Madison, WI, USA in compliance with the Animal Care Regulations.

In this study, groups of monkeys (n = 3) received the TDV formulated into either 0.5 ml for SC administration or 0.1 ml for ID delivery using the PhJ device. Each full dose of the tetravalent vaccine used in this study contained 2 × 10⁴ PFU of TDV-1, 5 × 10⁴ PFU of TDV-2, 1 × 10⁵ PFU of TDV-3, and 3 × 10⁵ PFU of TDV-4 vaccine viruses. This vaccine constitutes the clinical trial material used for two Phase 1 studies conducted in USA and Colombia, as well as Phase 2 studies currently ongoing in endemic areas.

Each animal in the first two groups received two injections on day 0, one in each arm ID or SC. A third group of animals was injected SC on day 0 and 53 using the PhJ device. Control animals received PBS via the ID route using PhJ. On day 90, all animals were challenged SC with 10⁵ PFU of DENV-2 (NGC strain) using N–S. Serum samples were collected on days 0, 3, 5, 7, 10, 12, 14, 53, 64, 67, and 88 post-primary immunization to analyze vaccine viremia, and days 91, 93, 95, 97, 99, 101, 102, and 104 to analyze DENV-2 NGC viremia after challenge. Serum samples also were collected on days 0, 30, 53, 75, 88, and 104 to determine neutralizing antibody titers to each serotype. PBMCs from group 2 and 4 were collected to measure T cell responses.

Viral RNA in serum samples was measured using a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) as follows. Viral RNA was extracted from 140 μl of each individual serum sample using a QIAamp viral RNA kit (Qiagen, Valencia, CA, USA) and eluted in 60 μl elution buffer. Viral RNA standards, used to create a standard curve in all qRT-PCR assays, were in vitro transcribed from cDNA clones and quantified as previously described (20). E-gene primers, TaqMan probes, and RNA standards were serotype specific (Table 1). Using a different fluorophore for each serotype specific probe (sequences available upon request), qRT-PCRs were performed in duplex: one reaction quantified TDV-1 and TDV-2 vaccine viruses while a separate one quantified TDV-3 and TDV-4 viruses RNA. Following DENV-2 NGC challenge, viral RNA was quantified in a singleplex qRT-PCR. All qRT-PCR reactions were performed in a final volume of 25 μl using the QuantiTect Virus +ROX Vial Kit (Qiagen, Valencia, CA, USA). The reactions contained 5 μl extracted RNA, 0.4 μM of each primer, and 0.2 μM probe. The reaction was conducted in the iQ5 iCycler system (Bio-Rad Laboratories) using the following cycle; 1 cycle of 50°C for 20 min at room temperature (RT), 1 cycle of 95°C for 5 min, and 50 cycles of 95°C for 15 s. Limit of detection for the qRT-PCR was determined for each viral RNA standard by creating a standard curve consisting of nine replicates per dilution. While the sensitivity reached 3.9 copies/reaction (~2.7 log₁₀ copies/ml), 3.6 log₁₀ copies/ml met the criteria of a 100% detection rate as well as a low ( ≤0.5) cycle threshold standard deviation of the replicates and was used as a cutoff for the assay.

Serum samples were incubated at 56°C for 30 min to inactivate complement and possible adventitious agents. Heat-inactivated serum samples then were tested for neutralizing activity using a viral immunofocus reduction microneutralization assay and analyzed by an AID ELISpot reader (San Diego, CA, USA). Briefly, 96-well tissue culture plates were seeded with Vero cells at a density of 1.3 × 10⁵ cells/ml in 100 μl/well. Cells were grown at 37°C in a 5% CO₂ incubator for 48 h. Twofold serial serum dilutions were prepared in a separate 96-well plate and then mixed with virus suspension containing 100 PFU followed by incubation at 4°C for 13–15 h. Culture medium was discarded from the Vero cell plates and then 30 μl of the serum–virus mixture was added to each well in triplicate followed by incubation at 37°C for 2 h. Control positive and negative serum samples were also included. An overlay medium with 1.2% carboxymethyl cellulose was added (100 μl/well) and cells were incubated as above for 2 days for DENV-4, 2.5 days for DENV-1 and -3, and 3 days for DENV-2. After incubation, the overlay was removed and cells were fixed with 85% cold acetone for 10 min at RT. Acetone was then discarded and plates were stored at −20°C until further use. Prior to staining, plates were equilibrated to RT, and washed three times with PBS to rehydrate the cells and to remove any residual overlay. Rabbit anti-DENV polyclonal antibody diluted (1:1000) in PBS-T containing 2.5% (w/v) dry milk powder was added, and plates were incubated at 37°C for 2 h. Plates were washed three times with PBS-T and incubated with anti-rabbit antibody conjugated with horse radish peroxidase (HRP) at 37°C for 2 h. Finally, plates were washed three times with PBS-T and incubated with the substrate (3-amino-9-ethylcarbozole) for 10–30 min or until plaques were visible. The plates were then washed with water and air-dried. The viral immunofoci were quantified on an ELISpot reader. Fifty percent of the average number of foci in the negative control serum defined the cutoff point. The serum dilution closest to the cutoff was recorded as the reciprocal neutralizing titer.

To assess the functional capability of TDV-elicited dengue-specific T cells, we performed intracellular cytokine staining (ICS) assays. For positive control, we used Staphylococcus Enterotoxin B (SEB) stimulation, for negative control, we used tissue culture medium devoid of added stimulatory peptides. Peptide arrays used in this study (Table 2) were obtained from the National Institute of Allergy and Infectious Diseases Biodefense and Emerging Infections Research Resources Repository (BEI Resources). Individual peptides were prepared as 10 mM stock solutions for NS1 and NS5 and 15 mM for NS5 peptides. An aliquot of 0.5–1.5 × 10⁶ PBMC in 200 μl total volume was incubated with peptides at 5 μM final concentration in the presence of anti-CD28 (clone L293), anti-CD49d (clone 9F10), and CD107a PE (clone H4A3) antibodies, and 1 μg per test of Brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) and Golgi Stop at 37°C in a 5% CO₂ incubator overnight. Cells were stained for the surface expression of CD3 (PE-CF594 clone SP34-2), CD4 (PerCP-Cy5.5-conjugated clone L200), CD8 (Pacific Blue-conjugated clone RPA-T8), and live/dead fixable Aqua Dead Cell stain (Invitrogen), washed twice with FACS buffer, and fixed with 2% paraformaldehyde. Cells were then permeabilized with 0.1% saponin buffer, intracellularly stained for IFN-γ (Alexa Fluor 700-conjugated clone 4S.B3), TNF-α (FITC-conjugated clone Mab11), and IL-2 (APC-conjugated clone MQ1-17H12), washed twice with saponin buffer, and fixed with 2% paraformaldehyde. All fluorescent-labeled antibodies and reagents were purchased from BD Biosciences except when mentioned. Sample data were acquired on a SORP BD LSR II equipped with a 50 mW 405 violet, a 100 mW 488 blue, and a 50 mW 640 red laser (BD Biosciences) using FACSDiva version 6.1 acquisition software. We collected approximately 150–300 thousand events in the lymphocyte gate defined by forward and side scatter parameters. Data were analyzed by FlowJo™ 9.4.2 software (Tree Star, Inc., Ashland, OR, USA). Background values from peptide stimulated values were subtracted. The frequency of cytokine-positive T cells was presented as the percentage of gated CD4⁺ or CD8⁺ T cells.

Following immunization, the presence of vaccine viral RNA in the serum was monitored by qRT-PCR of sequential bleeds collected over a period of 14 days post-primary immunization. TDV induced detectable TDV-2 virus replication from day 5 to 14 for animals injected SC, and day 7–12 for those injected ID (Table 3). No viral RNA from TDV-1, -3, and -4 vaccine viruses was detected in any of the groups on samples collected over a period of 14 days post-primary immunization.

The individual neutralizing antibody titers and kinetics of antibody responses elicited by TDV are shown in Table 4. Overall, administration of the vaccine by the ID or SC routes using the RIS (0, 0 vaccination schedule) induced comparable neutralizing antibody titers to all four serotypes. In all cases, the dominant neutralizing antibody response was to DEN-2, whereas TDV-4 was the least immunogenic component of the tetravalent vaccine formulation.

To determine the target proteins of the T cell response elicited by TDV, PBMCs from immunized animals (group 2) collected on day 53 post-priming were restimulated in vitro with pools of peptides encompassing the entire sequence of DENV-2 NS1, NS3, and NS5 proteins (Table 2). As shown in Figure 1, CD4⁺ T cells predominantly targeted the NS1 protein and to a lesser extent the NS3 and NS5 proteins, producing IFN-γ (a), IL-2 (b), and TNF-α (c). The vaccine also elicited CD8⁺ T cells mainly recognizing epitopes from the NS1 protein and to a lesser degree from NS3 and NS5 proteins (Figure 2). In particular, responses to the NS1 were characterized by the production of IFN-γ (a), IL-2 (b), TNF-α (c), and expression of CD107a⁺ marker (d). In contrast, T cell responses in PBS immunized animals (group 4) were comparatively very low (Figures 1 and 2). In addition, vaccine-specific CD8⁺ IFN-γ producing T cells were cross-reactive with epitopes from the NS3 and NS5 non-structural proteins of DENV-4 (Figure 3A) and were shown to express the CD107a⁺ marker (Figure 3B). A similar pattern of T cell responses recognizing predominantly the NS1 protein with no significant differences in frequencies of CD4⁺ and CD8⁺ T cells were also measured in group 3 (data not shown).

Since TDV-2 constitutes the backbone of TDV in this study, we examined the protective efficacy of this vaccine against challenge with DENV-2 NGC strain. Upon DENV-2 NGC challenge viral RNA was detected in the serum of all mock-immunized animals (Table 4). None of the vaccinated animals displayed DENV-2 NGC RNA. When the neutralizing antibody responses to DENV-2 were compared before and after challenge there was no significant increase in antibody titers in all vaccinated groups suggesting that DENVax elicits sterilizing immunity to DENV-2 (Table 5). In contrast, mock vaccinated animals developed a strong anti-DEN-2 neutralizing antibody response after challenge (Table 5).