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. 2019 Feb;34(1):88-96.
doi: 10.1007/s12250-019-00090-8. Epub 2019 Feb 21.

Electroporation-Mediated Immunization of a Candidate DNA Vaccine Expressing Dengue Virus Serotype 4 prM-E Antigen Confers Long-Term Protection in Mice

Ziyang Sheng  1 , Hui Chen  1 , Kaihao Feng  1 , Na Gao  1 , Ran Wang  1 , Peigang Wang  1 , Dongying Fan  1 , Jing An  2   3
Affiliations

Electroporation-Mediated Immunization of a Candidate DNA Vaccine Expressing Dengue Virus Serotype 4 prM-E Antigen Confers Long-Term Protection in Mice

Ziyang Sheng et al. Virol Sin. 2019 Feb.

Abstract

Dengue fever, caused by dengue viruses (DENVs), is a widespread mosquito-borne zoonotic disease; however, there is no available anti-dengue vaccine for worldwide use. In the current study, a DNA vaccine candidate (pV-D4ME) expressing prM-E protein of DENV serotype 4 (DENV-4) was constructed, and its immunogenicity and protection were evaluated in immunocompetent BALB/c mice. The pV-D4ME candidate vaccine induced effective humoral and cellular immunity of mice against DENV-4 in vivo when administered both at 50 μg and 5 μg through electroporation. Two weeks after receiving three immunizations, both doses of pV-D4ME DNA were shown to confer effective protection against lethal DENV-4 challenge. Notably, at 6 months after the three immunizations, 50 μg, but not 5 μg, of pV-D4ME could provide stable protection (100% survival rate) against DENV-4 lethal challenge without any obvious clinical signs. These results suggest that immunization with 50 μg pV-D4ME through electroporation could confer effective and long-term protection against DENV-4, offering a promising approach for development of a novel DNA vaccine against DENVs.

Keywords: DNA vaccine; Dengue virus (DENV); Electroporation; Long-term protection; Mouse.

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Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

The whole study was approved by the Institutional Animal Care and Use Committee of Capital Medical University, China (Approval Number: AEEI-2015-066). All institutional and national guidelines for the care and use of laboratory animals were followed.

Figures

Fig. 1
Fig. 1
Expression of pV-D4ME in vitro and in vivo. (A) DENV-4 antigens expression in plasmid-transfected Vero cells. The pV-D4ME (D4)- or pV-transfected Vero cells were cultured separately for 48 h and then the expression was detected by IFA. Cells infected with DENV-4 served as a positive control. Bar = 100 µm. (B) DENV-4 antigens expression in the quadriceps femoris muscle of BALB/c mice immunized with 50 µg (D4-50) or 5 µg (D4-5) of pV-D4ME. Samples were harvested at 2 weeks after the last of the three immunizations and the antigen was detected by IHC. Bar = 50 µm.
Fig. 2
Fig. 2
DENV-4-specific antibody responses in sera of immunized BALB/c mice 2 weeks after three immunizations. Endpoint titers of (A) IgG and (B) NAb determined by ELISA and PRNT, respectively. The reciprocals of titers are represented as the mean ± SD. n = 6. N/D, not detected. Titers were log-transformed and analyzed by two-way ANOVA. *P < 0.05; **P < 0.01.
Fig. 3
Fig. 3
Levels of splenocyte-secreted cytokines in immunized BALB/c mice at 2 weeks after three immunizations detected by ELISPOT. (A) IFN-γ, (B) IL-2, (C) IL-4, and (D) IL-10 levels. The numbers of cytokine-positive cells are shown as the mean counts of spot-forming unit (SFU) ± SD per 1 × 106 splenocytes. n = 6. Results were analyzed by the t test. *P < 0.05; **P < 0.01.
Fig. 4
Fig. 4
Protective immunity elicited by pV-D4ME against DENV-4 challenge in BALB/c mice at 2 weeks after three immunizations. Mice were challenged with 104 PFU DENV-4 and monitored daily for 21 days. (A) Percentage of body weight change compared to day 0. (B) Scores of illness symptoms. (C) Survival rates. Data are presented as mean ± SD in (A) and (B). n = 6. The percentage of body weight and the scores of illness symptoms were analyzed by two-way ANOVA. The survival rates were analyzed by the Kaplan–Meier method. **P < 0.01.
Fig. 5
Fig. 5
Long-term protection elicited by pV-D4ME against DENV-4 infection in BALB/c mice at 6 months after three immunizations. Mice were challenged with 104 PFU DENV-4 at 6 months after three immunizations and monitored daily for 21 days. (A) Percentage of body weight change compared to day 0. (B) Scores of illness symptoms. (C) Survival rates. Data are presented as mean ± SD in (A) and (B). n = 5. The percentage of body weight and the scores of illness symptoms were analyzed by two-way ANOVA. The survival rates were analyzed by the Kaplan–Meier method. **P < 0.01.
Fig. 6
Fig. 6
DENV-4-specific antibody responses in sera of immunized BALB/c mice at 6 months after three immunizations. Endpoint titers of (A) IgG and (B) NAb determined by ELISA and PRNT, respectively, at 6 months after three immunizations. To identify the memory immunization, antibody titers were also determined in surviving mice at 21 days after DENV-4 challenge. The reciprocals of titers are presented as mean ± SD. n = 5. N/D, not detected. The dotted line refers to the titers of IgG or NAb of immunized mice at 2 weeks after three immunizations. Log-transformed titers were analyzed by two-way ANOVA. *P < 0.05; **P < 0.01.
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