The successful induction of T-cell and antibody responses by a recombinant measles virus-vectored tetravalent dengue vaccine provides partial protection against dengue-2 infection

Abstract

Dengue has a major impact on global public health, and the use of dengue vaccine is very limited. In this study, we evaluated the immunogenicity and protective efficacy of a dengue vaccine made from a recombinant measles virus (MV) that expresses envelope protein domain III (ED3) of dengue-1 to 4. Following immunization with the MV-vectored dengue vaccine, mice developed specific interferon-gamma and antibody responses against dengue virus and MV. Neutralizing antibodies against MV and dengue viruses were also induced, and protective levels of FRNT50 ≥ 10 to 4 serotypes of dengue viruses were detected in the MV-vectored dengue vaccine-immunized mice. In addition, specific interferon-gamma and antibody responses to dengue viruses were still induced by the MV-vectored dengue vaccine in mice that were pre-infected with MV. This finding suggests that the pre-existing immunity to MV did not block the initiation of immune responses. By contrast, mice that were pre-infected with dengue-3 exhibited no effect in terms of their antibody responses to MV and dengue viruses, but a dominant dengue-3-specific T-cell response was observed. After injection with dengue-2, a detectable but significantly lower viremia and a higher titer of anti-dengue-2 neutralizing antibodies were observed in MV-vectored dengue vaccine-immunized mice versus the vector control, suggesting that an anamnestic antibody response that provided partial protection against dengue-2 was elicited. Our results with regard to T-cell responses and the effect of pre-immunity to MV or dengue viruses provide clues for the future applications of an MV-vectored dengue vaccine.

Figure 1.

Preparation of the recombinant measles viral vector tetravalent dengue vaccine. (A) Schematic diagram of the infectious clone pMV containing the antigenomic cDNA of the Moraten MV strain is shown. The required elements including the T7 promoter (P_T7), an additional transcription unit (ATU), the delta ribozyme (δ) and the T7 polymerase terminator (ψ) are also indicated. The infectious clones of the recombinant virus, which carried either an EGFP reporter gene (pMV-EGFP) or tandem repeats of ED3 from DENV-1 and 3 (pMV-DV13) and DENV-2 and 4 (pMV-DV24) with a secretory signal (Ig) and linker (GGGGS x3) are shown at the bottom. (B) The presence of dengue ED3 protein in the cell lysate and culture supernatant of Vero cells infected with recombinant viruses were detected by Western blotting with an anti-ED3 monoclonal antibody and indicated by the arrow; the signal for the actin protein in the cell lysate is also shown at the bottom. (C) Vero cells were infected with the different recombinant viruses and cell lysate and supernatant were harvested to determine the virus titers by plaque assay. The growth kinetics of recombinant viruses are presented with the mean and standard deviation (SD) from 2 experiments. (D) Vero cells were infected with rMV-EGFP, and the merged image of rMV-EGFP-infected syncytial cells from bright field and fluorescent microscopy is shown. (E) The consensus amino acid sequence from 4 serotypes of ED3 was aligned and listed.

Figure 2.

The infection and replication of recombinant MV in CD46 transgenic mice. (A) Groups of human CD46 transgenic C57BL/6 mice (hCD46; n = 3) and wild-type C57BL/6 mice (B6; n = 2) were infected with 1 ×10⁶ pfu of rMV by ip injection. The mouse tissue and blood cells were harvested 9 days after infection, and the MV gene copy number was determined by quantitative RT-PCR and presented as MV RNA copies per ng total RNA. (B) Spleen cells from hCD46 or B6 mice were infected with rMV (MOI = 3) in vitro, and the rMV infected cells were detected by flow cytometry with FITC-conjugated anti-MV nucleoprotein monoclonal antibody. The mean and standard deviation of MV-infected cells in the CD3⁺ and B220⁺ cell populations from 3 experiments are shown.

Figure 3.

The induction of both MV- and DENV-specific T-cell responses by the MV-vectored dengue vaccine. Groups of hCD46 mice (n = 4) were immunized with 2 × 10⁶ pfu of rMV-EGFP or rMV-TDV (1 × 10⁶ pfu of rMV-DV13 and 1 × 10⁶ pfu of rMV-DV24) by ip injection. Spleen cells were harvested 9 days after a single immunization for the detection of IFN-γ (A) and IL-4 (B) production specific to the MV or ED3 pooled peptides of each serotype by ELISPOT assay. The results are presented as the mean and SD of spot forming cells (SFC) per million splenocytes. Mann-Whitney t-tests were used for statistical analyses. The dashed line indicates the cutting-off of 2 times the background (medium alone).

Figure 4.

The antibody responses elicited by the MV-vectored dengue vaccine. Groups of hCD46 mice (n = 4) were immunized with either 2 × 10⁶ pfu of rMV-EGFP or rMV-TDV by ip injection and boosted 4 weeks later (indicated by arrow). The reciprocal titers of specific IgG to MV (A) and DENV-1 to 4 (B-E) were determined by ELISA. The results are presented as the mean and SD of specific IgG titers. (F) The neutralizing antibody titers against parental MV and the 4 serotypes of DENV were determined by plaque reduction and FRNT, respectively. The reciprocal titer leading to a ≥50% reduction (PRNT₅₀ or FRNT₅₀) is shown. The detection limits for the IgG ELISA or neutralization assay are indicated with a dashed line. Mann-Whitney t-tests were used for statistical analyses, and the significance compared with the rMV-EGFP control is shown.

Figure 5.

The effect of pre-existing immunity on the immune responses induced by the MV-vectored dengue vaccine. The immunization schedule is shown at the top of the figure. Groups of hCD46 mice were pre-infected with 1 ×10⁶ pfu of rMV-EGFP, 1 ×10⁶ pfu of DENV-3 or a PBS-treated naïve control. After three weeks, all mice were immunized with rMV-TDV by ip injection and boosted 4 weeks later. (A) Specific T-cell responses to either MV or DENV were measured one week after a single immunization by ELISPOT assay and presented with the mean and SD of SFC per million spleen cells. The specific IgG titers to MV (B) or DENV-1 to 4 (C-F) were determined by ELISA. The detection limits for the ELISPOT assay or ELISA are indicated in a dashed line. Mann-Whitney t-tests were used for statistical analyses, and the significance compared with the naïve control is shown.

Figure 6.

The protective efficacy of the MV-vectored dengue vaccine was evaluated in terms of DENV-2 viremia in mice. The immunization and challenge schedule is shown at the top of the figure. Two groups of hCD46 mice (n = 3), with one pre-infected with 1 × 10⁶ pfu of rMV-EGFP at 3 weeks prior to vaccination and one naïve group, were immunized with rMV-TDV by ip injection. As a control, hCD46 mice (n = 3) were immunized with 2 × 10⁶ pfu of rMV-EGFP by ip injection. All the mice were boosted 4 weeks later with the same vaccine and introduced a viremia via an ip inoculation of 5 × 10⁷ DENV-2/16681-infected K562 cells at week 8. (A) Plasma viremia titers from individual mice were determined by viremia assay and are represented as the mean and SD. (B) Spleen cells were harvested 1 month post-viremia for the detection of DENV ED3-specific IFN-γ production by ELISPOT assay. The sera collected at 1 month post-viremia were used to detect the ED3-specific IgG responses by ELISA (C) and the neutralizing titers to DENV-1 to 4 by FRNT (D).