A chimeric dengue virus vaccine using Japanese encephalitis virus vaccine strain SA14-14-2 as backbone is immunogenic and protective against either parental virus in mice and nonhuman primates

Abstract

The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.

Fig 1

Design and construction of a genomic-length chimeric JEV/DENV-2 (ChinDENV) cDNA clone. (A) Strategy for the construction of the infectious cDNA clone of ChinDENV. The JEV prM signal peptide (blank box) is underlined. The C-terminal 3 amino acids of JEV E protein (E3, gray box) and the prM-E of DENV-2 lacking the C-terminal 3 amino acids of the E protein (dark gray box) are indicated. (B) Alignment of flavivirus amino acid sequences around the C-prM and E-NS1 junctions. The prM signal peptides are boxed. The last and antepenultimate C-terminal amino acids of flavivirus E proteins are highlighted in gray. Sites of cleavage by the viral NS2B-3 protease and signal peptidase are indicated by the open arrow and solid arrows, respectively.

Fig 2

In vitro characterization of ChinDENV. (A) Immunostaining of JEV-, ChinDENV-, DENV-2-, or mock-infected BHK cells with specific anti-JEV E, DENV-2 E, and JEV NS1 antibodies. Cells were infected with viruses at an MOI of 0.01. At 48 h postinfection, JEV or DENV-2 antigen was detected with monoclonal antibody 4AD5F5D5D6 (for JEV E protein), 2B8 (for DENV-2 E protein), and JN1 (for JEV NS1 protein). (B) Plaque morphology of JEV, ChinDENV, and DENV-2 on BHK-21 cells grown in 6-well plates were infected with a 10-fold serial dilution of viruses. The plates were incubated at 37°C for 1 h. Supernatant was removed and cells were overlaid with 1% low-melting-point agarose in DMEM containing 2% FBS. After further incubation at 37°C for 4 days, the cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet to visualize the plaques. (C) Plaque morphology of ChinDENV passaged in PHK cells. The chimeric virus ChinDENV was passaged in PHK cells up to 15 times. The plaque phenotypes of passage 3, 9, and 15 viruses were examined on BHK-21 cells 4 days after infection by plaque assay. (D) Growth curves of the chimera ChinDENV and parental viruses JEV and DENV-2 in cell culture. Monolayers of Vero, C6/36, and PHK cells were infected with the indicated viruses at an MOI of 0.01. At each time point, the media were removed and virus titers in cell culture media were determined on BHK-21 cells by plaque assay.

Fig 3

Immunogenicity of ChinDENV in mice. Groups of five 4-week-old BALB/c mice were immunized with doses of 60,000 or 600,000 PFU of the chimera by the s.c. route. Serum was collected from animals at 14, 28, and 42 days postvaccination for determination of titers of IgG antibody (A) and neutralizing antibody (B) against DENV-2 by using IFA and PRNT₅₀, respectively. The dotted line represents the limit of detection of the assay for each serum.

Fig 4

Cross protection against lethal JEV challenge induced by the chimera ChinDENV in mice. Groups of 4-week-old BALB/c mice were immunized with 10⁵ PFU of JEV, ChinDENV, or PBS via the s.c. route. At 28 days postimmunization, serum was collected for determination of titers of neutralizing antibody by PRNT₅₀. The immunized mice were then challenged with a lethal dose of JEV. From groups of ChinDENV- and PBS-immunized mice, 3 were sacrificed and their spleens were isolated for testing CD8⁺ T cell responses. (A) Survival data of the immunized mice after inoculation with a lethal dose of JEV (n = 8). (B) Neutralizing antibody level of ChinDENV-, JEV-, or PBS-immunized mice on day 28 postimmunization. (C) Cellular immune responses in the ChinDENV- or PBS-immunized mice. Purified lymphocytes from the spleen were stimulated with JEV or DENV-2 and their corresponding replicons for 36 h. The production of IFN-γ and IL-2 was measured by ELISPOT assay and expressed as spot-forming cells per 10⁷ lymphocytes.