Effective Protection Induced by a Monovalent DNA Vaccine against Dengue Virus (DV) Serotype 1 and a Bivalent DNA Vaccine against DV1 and DV2 in Mice

Abstract

Dengue virus (DV) is the causal pathogen of dengue fever, which is one of the most rapidly spread mosquito-borne disease worldwide and has become a severe public health problem. Currently, there is no specific treatment for dengue; thus, a vaccine would be an effective countermeasure to reduce the morbidity and mortality. Although, the chimeric Yellow fever dengue tetravalent vaccine has been approved in some countries, it is still necessary to develop safer, more effective, and less costly vaccines. In this study, a DNA vaccine candidate pVAX1-D1ME expressing the prME protein of DV1 was inoculated in BALB/c mice via intramuscular injection or electroporation, and the immunogenicity and protection were evaluated. Compared with traditional intramuscular injection, administration with 50 μg pVAX1-D1ME via electroporation with three immunizations induced persistent humoral and cellular immune responses and effectively protected mice against lethal DV1 challenge. In addition, immunization with a bivalent vaccine consisting of pVAX1-D1ME and pVAX1-D2ME via electroporation generated a balanced IgG response and neutralizing antibodies against DV1 and DV2 and could protect mice from lethal challenge with DV1 and DV2. This study sheds new light on developing a dengue tetravalent DNA vaccine.

Keywords: DNA vaccine; bivalent vaccine; dengue; dengue virus; electroporation; monovalent vaccine.

Figure 1

Determination of prME protein expression. (A) In vitro expression of the prME protein in DNA-transfected BHK cells by IFA (fluorescence microscopy, × 400). (B) In vivo expression of the prME protein in tissues by IHC. The muscle tissues were collected from mice sacrificed two weeks after the third immunization with 50 μg DNA (light microscopy, × 200).

Figure 2

DV1-specific antibody responses in mice sera. (A) Dynamics of the IgG responses detected by ELISA. The mice were immunized three times at two-week intervals. Sera were collected at weeks 0, 2, 4, and 6. All samples were diluted at 1:800 (n = 6). (B) End-point titers of anti-DV1 antibodies assayed by ELISA (n = 6). Sera were collected two weeks after the third immunization. The results were expressed as GMT + standard deviation (SD). (C) The anti-DV1 IgG subclasses in mice sera determined by ELISA. Sera were collected two weeks after the third immunization. Values of IgG2a and IgG1 were reported as the mean OD + SD at 492 nm at a serum dilution of 1:100 (n = 8). (D) Serum NAb responses assayed by PRNT₅₀ (n = 8). Sera were collected two weeks after the third immunization. NAb titers were recorded as GMT + SD. ^*p < 0.05, ^**p < 0.01.

Figure 3

The levels of splenocyte-secreted IFN-γ (A), IL-2 (B), IL-4 (C), and IL-10 (D) cytokines by ELISPOT assays (n = 6). Splenocytes were isolated two weeks after the third immunization. The numbers of cytokine-positive cells were recorded as the mean SFU/1 × 10⁶ splenocytes + SD. ^*p < 0.05; ^**p < 0.01.

Figure 4

DV1-specific cell-mediated immune responses in DNA-immunized mice. Splenocytes were isolated two weeks after the third immunization. (A) DV1-specific CTL response (n = 6). Percentages of specific lysis + SD were shown at different E:T ratios. (B) Lymphocyte proliferation measured with CCK-8 (n = 6). The results were recorded as the mean SI + SD. ^*p < 0.05, ^**p < 0.01.

Figure 5

Protective immunity elicited by the pVAX1-D1ME against DV1 infection (n = 10). (A) Pathological symptoms recorded as the mean sign scores. (B) The body weight reported as percentages compared to day 0. (C) The survival rate shown as the percentage of survivors. ^*p < 0.05, ^**p < 0.01.

Figure 6

End-point titers of anti-DV1 antibodies and protect immunity elicited by the pVAX1-D1ME under different times of vaccination (n = 6). (A) End-point titers of anti-DV1 antibodies. (B) Pathological symptoms recorded as the mean sign scores. (C) Body weight calculated as percentages compared with day 0. ^*p < 0.05, ^**p < 0.01.

Figure 7

DV1- and DV2-specific antibody responses in mice immunized with the bivalent vaccine (n = 5). Sera were collected two weeks after the third immunization. (A) End-point titers of anti-DV1 and anti-DV2 IgG assayed by ELISA. The results were recorded as GMT + SD. (B) End-point titers of anti-DV1 and anti-DV2 NAb assayed by PRNT₅₀. The results were recorded as GMT + SD. ^*p < 0.05, ^**p < 0.01.

Figure 8

Protective immunity elicited by the bivalent DNA vaccine against DV1 and DV2 infection (n = 5). (A,C) Pathological symptoms recorded as the mean sign scores. (B,D) The body weight reported as percentages compared to day 0. ^*p < 0.05, ^**p < 0.01.