Wild-type DENV-1 16007, DENV-2 16681, DENV-3 16562, and DENV-4 1036 viruses were obtained from the Division of Vector-Borne Diseases, Centers for Disease Control and Prevention (CDC), Fort Collins, CO. DENVs were grown in Vero cells in Dulbecco’s modified minimal essential medium (DMEM) containing penicillin-streptomycin.

The DENVax viruses consist of cDNA clone-derived DENV- 2 VV45R virus (based on genome of DENV-2 PDK-53), and the DENV-2VV45R-based chimeras expressing the prM and E genes of wild-type (wt) DENV-1 16007, DENV-3 16562 or DENV-4 1036. The construction and characterization of these viruses has been previously reported [12–15]. To complete preclinical and clinical development of DENVax, new viral stocks were generated by introducing RNAs transcribed from infectious cDNA clones (pD2-VV45R [15] and chimeric pD2/1V, /3V, and/4V plasmids [12]) into certified vaccine production Vero cells under Good Manufacturing Practice (GMP) conditions at Shantha Biotechnics, Ltd., Hyderabad, India. The resulting re-derived viruses were amplified, plaque purified, characterized and sequenced. Based on these analyses, a formal pre-master virus seed was chosen and then was amplified to generate the master virus seed for each DENVax serotype (Huang et al., manuscript in preparation). For the studies reported here we used either GMP quality master seed virus stocks or surrogate master seed prepared in our laboratory from individual pre-master cGMP seeds. These surrogate master seed viruses were grown in serum-free media under vaccine production conditions.

Three tetravalent vaccine formulations containing different ratios of the individual DENVax components were prepared. For each, surrogate vaccine preparations were mixed into a novel FTA diluent (final concentration 1% F127 polyoxyethylene–polyoxypropylene block copolymer), 15% tre-halose, and 0.1% human serum albumin) that has shown to improve the thermal stability of these DENVax vaccine viruses [22]. Formulation 1 (3333) contained 10³ plaque forming units (pfu) of each of the four DENVax viruses per dose. Formulation 2 (3355) was prepared to contain 10³ pfu each of DENVax-1 and DENVax-2, and 10⁵ pfu each of DENVax-3 and DENVax-4 per dose. Formulation 3 (5555) contained 10⁵ pfu of each of the four DENVax viruses per dose. To validate the virus concentrations, the titer of each DENVax virus was determined by an immunofocus assay described below.

For measurement of body temperature, animals were implanted subcutaneously with BMDS IPTT-300 transponder (chips), purchased from BioMedic Data Systems, Inc. (BMDS, Seaford, DE) two to three days prior to start of the study and were monitored for signs of infection or migration of the transponder. Chips were scanned using a DAS-6007 transponder reader (BMDS).

Treatment of animals was in accordance with the University of Wisconsin-Madison Standard Operating Procedures, which adhere to the regulations outlined in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (1996). All animal study protocols were approved by the UW Institutional Animal Care and Use Committee. AG129 mice (supplied by B & K Universal Ltd., UK) lack the IFN-α/β and IFN-γ receptor genes and have been successfully used as a mouse model for DENV studies [12,13,23]. Although they possess IFN receptor deficiencies, AG129 mice otherwise exhibit immunological competence and are able to mount antibody and cellular immunity to variety of pathogens [24].

Immunogenicity studies with monovalent DENVax vaccines were conducted in groups of 5–8 week-old AG129 mice (n = 6) by subcutaneously (SC) injecting a dose of 10⁵ pfu of each of the individual DENVax vaccines in 0.5 ml. Control mice were injected by the same route with PBS. All animals received primary and secondary immunizations on days 0 and 42, respectively. Mice were monitored daily for morbidity and mortality. Serum samples collected at days 0, 42, and 56 were tested for DENV serotype-specific neutralizing antibodies.

A subsequent study evaluated the protective capacity of each monovalent DENVax vaccine using non-GMP surrogate vaccine preparations. Groups of ten 8–9 week old AG129 mice received a SC injection of 0.1 ml containing 10⁵ pfu of each surrogate master seed virus. Control mice were inoculated via the same route with FTA (the virus diluent). At five weeks after the single vaccine administration each group was split into two sub-groups of five mice and were challenged by intraperitoneal (IP) administration of 10⁶ pfu of DENV-1 Mochizuki or DENV-2 New Guinea C viruses in 0.1 ml. Following challenge, all mice were monitored twice a day for five weeks for morbidity (weight loss, temperature, neurological signs), and survival rates were recorded.

The immunogenicity and protective efficacy of tetravalent DEN-Vax formulations (Formulation 1, 2 and 3) were tested in groups of eight 5–8 week old AG129 mice. Each of the three formulations and the FTA diluent was administered SC on day 0 and 42. Viremia levels were measured in samples collected on day 3 post-primary vaccination, the peak of dengue virus replication in the AG129 mouse model [25]. Serum neutralizing antibody responses against all four serotypes were measured in individual blood samples collected on days 30 and 56 post-priming. On day 56 each group of eight mice was sub-divided into two groups of four mice and challenged IP with 10⁶ pfu of DENV-1 Mochizuki or DENV-2 New Guinea C virus. Animals were monitored daily for three weeks for clinical signs of disease and survival rates were recorded.

Virus plaque forming units were measured by plaque titration performed in double agarose overlays in six-well plates of conflu-ent Vero cells as previously described [12]. The plaque titration was performed for each monovalent DENVax virus. For tetravalent DENVax formulations and viremia measurements of animals vaccinated with tetravalent DENVax, we performed immune focus assay using serotype-specific monoclonal antibodies (MAbs), 1F1 (DENV-1), 3H5 (DENV-2), 8A-1 (DENV-3), and 1H10 (DENV-4) to differentiate titers for each DENVax serotype. The immune focus assay was conducted in confluent Vero cells in 6-well plates. Cells were adsorbed with serially diluted samples for 1.5 h at 37 °C and overlaid with 3 ml/well of YE-LAH medium [12] containing 0.7% high viscosity carboxymethylcellulose (CMC) (Sigma). After 7-day incubation at 37 °C with 5% CO₂, overlay was aspirated, and cell sheets were washed 3 times with PBS before they were fixed by adding cold 85% acetone or 100% methanol for 30 min at −20 °C. After fixation, plates were air dried, washed once with PBS, and blocked with a buffer containing 2.5% (w/v) nonfat dry milk, 0.5% Triton X-100, 0.05% Tween-20 in PBS at 37 °C for 30 min. After blocking, cells were incubated with diluted DENV serotype-specific MAbs in the blocking buffer at 37 °C for 1 h or 4 °C for overnight, washed 3 times with washing buffer (0.05% Tween-20 in PBS), and incubated with diluted alkaline phosphatase-conjugated affinity pure goat anti-mouse IgG (Jack-son Immuno Research Laboratories) at 37 °C for 45 min. Plates were washed three times again with washing buffers and stained with 0.4 ml/well of 1-Step NBT/BCIP plus suppressor (Pierce). Immune foci were visible after 20 min at room temperature and color development was stopped by washing the plates with water within an hour to avoid dark cell background. Immune foci were counted to calculate the immune focus units (ifu)/ml of the input samples.

Serum samples collected for neutralization assay were heated at 56 °C for 30 min to inactivate complement and possible adventitious agents. Heat-inactivated sera were tested for neutralizing antibodies by the 50% plaque reduction neutralization test (PRNT₅₀) as previously described [12]. The neutralizing antibody titer was identified as the highest serum dilution that reduced the input number of virus plaques by at least 50% (PRNT₅₀). All samples were assayed in duplicate. The input virus numbers were calculated by back titration with 2-fold serial dilutions of the input viruses in each assay. Neutralizing antibody titers of less than 10 were considered negative and were assigned an inverse titer of 1 for calculation purposes. Duplicate PRNT₅₀ values for each sample were geometrically averaged and geometric mean titers (GMT) were calculated for each group of animals.

Groups of AG129 mice were immunized with monovalent chimeric DENVax vaccines and monitored for clinical signs of infection, weight loss and temperature changes. After vaccination there was no mortality and all animals showed no evidence of abnormal elevation of body temperatures or weight loss as compared to the control groups. The mean body weights and temperature of mice vaccinated with each monovalent DENVax vaccine are presented in Tables 1 and 2, respectively.

Following vaccination with each monovalent DENVax, serum samples collected after the primary and secondary immunization were tested for serotype-specific neutralizing antibody responses against the four DENV serotypes. As shown in Table 3, monovalent vaccines elicited strong primary and secondary neutralizing antibody responses against their wild-type homologous virus strains. In addition, all monovalent vaccines elicited low levels of cross-neutralizing antibodies to the three other serotypes with the highest cross-neutralizing reactivity observed against DENV-3 in the DENVax-1, DENVax-2, and DENVax-4 immunization groups. A weak anti-DENV-3 neutralizing activity in the serum of PBS immunized mice, suggesting possible non-specific serum factors that might neutralize this virus.

A single immunization of AG129 mice with each of the monovalent DENVax vaccine formulations provided complete protection against lethal challenge with wt DENV-1 Mochizuki virus, administered IP at five weeks post priming (Fig. 1A). Similarly, monovalent chimeric DENVax-1 or DENVax-2 conferred 100% protection against lethal IP challenge with DENV-2 New Guinea C virus. Immunization with DENVax-3 provided significant protection (P = 0.0031 based on a Log-rank test) from challenge with DENV-2 (Fig. 1B). Interestingly, while vaccination with monovalent DENVax-4 did not protect against DENV-2 challenge, the median survival time was significantly longer (P = 0.0067 based on a Log-rank test) than that of non-vaccinated control animals (32 and 7.5 days, respectively). None of the vaccinated animals displayed any sign of morbidity. In contrast, control infected animals succumbed to infection after early signs of morbidity (ruffled coat and/or hunched posture) followed by paralysis and sudden death or humane euthanasia.

Mice were immunized with the three formulations of the four DENVax strains. In one study, the replication of DENVax viruses was assessed by measuring viremia in blood samples collected shortly after the primary immunization. The magnitude of viremia following SC immunization with DENVax Formulation 2 (10³, 10³, 10⁵, 10⁵ of DENVax-1, -2, -3 and -4, respectively) and DENVax Formulation 3 (10⁵ of each strain) varied between the four vaccine strains and were dependent on the ratio of component chimeric viruses. After administration of Formulation 3, the DENVax-2 showed the highest viremia with detectable levels in all 8 mice of average value 3.36 log₁₀ ifu/ml followed by the DENVax-1 (6 out of 8 mice), DENVax-3 (4 out of 8 mice) and DENVax-4 (1 out of 8 mice) with average values of 2.0, 1.3, and 0.3 log₁₀ ifu/ml, respectively (Fig. 2B). After administration of Formulation 2 there were only two mice that had detectable viremia for DENVax-2 (average value 0.62 log₁₀ ifu/ml), and 7 out of 8 mice for DENVax-3 (average value 2.78 log₁₀ ifu/ml) (Fig. 2A). No mice demonstrated detectable DENVax-1 or DENVax-4 viremia. Comparison of viremia levels between formulation 2 and 3 for each monovalent DENVax vaccine using one-way ANOVA test reveal significant differences for DENVax-1 (P = 0.0005), DENVax- 2 (P < 0.0001) and DENVax-3 (P = 0.0424) but not for DENVax-4 (P = 0.3343). Viremia after administration of DENVax Formulation 1 was not assessed due to insufficient quantity of available serum samples.

Immunization of AG129 mice with formulations containing different ratios of the four DENVax viruses (Formulations 1, 2 or 3) elicited neutralizing antibody responses against all four serotypes (Fig. 3). After the booster immunization the neutralizing antibody response induced by Formulation 3 was dominated by the anti-DENV-2 and DENV-1 response followed by the anti-DENV-3 and anti-DENV-4 response. In Formulation 2, the neutralizing antibody response was dominated by the anti-DENV-3 and followed by anti-DENV-1, anti-DENV-2, and anti-DENV-4 response. Finally, neutralizing antibody responses elicited by Formulation 1 were lower than those induced by Formulations 2 and 3. It is interesting to point out that following a booster injection with Formulation 1, there was no clear boosting effect on DENV-1 and DENV-3 neutralizing antibody titers on sampling day 56, which could be accounted to differences in the kinetics of antibody responses to these two serotypes. In all tested formulations, the DENVax-4 vaccine was the least immunogenic of the DENVax vaccine components (Fig. 3). Overall, Formulation 3 gave significantly higher neutralizing antibody responses to all four serotypes than formulations 1 and 2 (P < 0.05) for both sampling days using repeated measures by ANOVA test.

Two weeks after the second DENVax administration all tetravalent-vaccinated and control mice were subdivided into two groups and challenged with a lethal dose of DENV-1 Mochizuki (Fig. 4A) or DENV-2 New Guinea C viruses (Fig. 4B). All DENVax immunized mice survived infection while control animals succumbed to infection between 6 and 11 days after challenge. All vaccinated animals failed to show any clinical signs of disease following viral challenge independent of the DENVax formulation. In contrast, by day 5 after challenge all control animals showed clinical signs of illness (e.g. ruffled coat, hunched posture and hind limb paralysis). On day 6 post-challenge, one animal from each of the challenged groups died and, by day 11 post-challenge, all control animals had succumbed to infection or been euthanized (Fig. 4A and B).