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. 2020 Mar 17:10:87.
doi: 10.3389/fcimb.2020.00087. eCollection 2020.

Long-Term Protection Elicited by a DNA Vaccine Candidate Expressing the prM-E Antigen of Dengue Virus Serotype 3 in Mice

Kaihao Feng  1 , Xiaoyan Zheng  2 , Ran Wang  1 , Na Gao  1 , Dongying Fan  1 , Ziyang Sheng  1 , Hongning Zhou  3 , Hui Chen  1 , Jing An  1   4
Affiliations

Long-Term Protection Elicited by a DNA Vaccine Candidate Expressing the prM-E Antigen of Dengue Virus Serotype 3 in Mice

Kaihao Feng et al. Front Cell Infect Microbiol. .

Abstract

Dengue virus (DENV) is the causative agent of dengue, and its incidence has increased 30-fold in the past five decades. Among the four cocirculating serotypes, DENV3 is associated with an increased number of severe infections and has become widespread. Vaccination is the mainstay of prevention in reducing disease burden. Previously, the protective efficacy of DNA vaccine candidates toward DENV1, 2, and 4 was confirmed in mice. In this study, a DNA vaccine candidate (pVAX1-D3ME) expressing the prM and E proteins of DENV3 was constructed, and then the immunogenicity and protection were assessed in mice to further develop a tetravalent dengue vaccine. Moreover, the cross-reactive immune responses against the other three serotypes were investigated. The results showed that three doses of 50 μg of pVAX1-D3ME were sufficient to induce strong antigen-specific T cell responses and robust and consistent neutralizing antibodies. Additionally, immunization with pVAX1-D3ME offered protective immunity against not only DENV3 but also the other three serotypes, which could be observed even after 12 months. This study shows great promise for the further evaluation of a dengue tetravalent DNA vaccine candidate in large animal models, including non-human primates.

Keywords: DNA vaccine; cross-protection; dengue virus; immunization; prM-E.

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Figures

Figure 1
Figure 1
The immunization and sample collection schedule, and in vitro and in vivo expression of the prM-E proteins. (A) Diagram of immunization, sample collection and challenge in BALB/c mice. The mice were vaccinated at weeks 0, 3, and 6. Splenocytes and muscle tissues were obtained at the seventh week, and sera were collected at the ninth week, the sixth month, and the 12th month. The mice were challenged with DENVs at the ninth week and the 12th month. (B) In vitro expression of the prM-E proteins in plasmid-transfected Vero cells detected by IFA. DENV3-infected Vero cells served as the positive control. (C) The cell lysate and the supernatant of plasmid-transfected Vero cells were collected at 72 h after transfection and in vitro expression of the E protein was detected by Western blotting. DENV3-infected Vero cells served as the positive control. GAPDH served as the loading control. (D) In vivo expression of the prM-E protein at the injection site in muscle tissues detected by IHC.
Figure 2
Figure 2
Humoral immune response against DENV3 in mouse sera. Sera were collected at the ninth week after the immunization. (A) Endpoint titers (n = 8) of DENV3-specific NAbs were detected by PRNT50 and recorded as GMT ± SD. The limit of detection (L.O.D.) depicted as a dotted line represents the lowest dilution that the experiment could detect. (B,C) In vitro neutralizing activity and passive protective effect of immunized sera on suckling mice (n = 13 in the pVAX1-D3ME group, n = 10 in the pVAX1 group). The pooled sera mixed with live DENV3 were transferred into 1-day-old suckling mice. The mice were monitored daily for 14 days. (B) Body weight change from day 0. (C) The survival rate is shown as the percentage of survivors. **p < 0.01; ***p < 0.001.
Figure 3
Figure 3
Cellular immune response against DENV3 in mouse splenocytes. Splenocytes were isolated 1 week after the final immunization. (A–D) The antigen-experienced CD8+ T cell response was assayed by flow cytometry (n = 8). (A) Representative images of CD44+ CD62L− T cells (gated on CD3e+ CD8+ T cells). (B) Quantification of the frequency of CD44+ CD62L− CD8+ T cells. The data are expressed as the mean percentage ± SD from three independent experiments. (C) Representative images of CD11a+ IFN-γ+ T cells (gated on CD3e+ CD8+ T cells). (D) Quantification of the frequency of CD11a+ IFN-γ+ CD8+ T cells. The data are expressed as the mean percentage ± SD from three independent experiments. (E,F) Splenocyte-secreted cytokines detected by ELISPOT assay (n = 8). The number of cytokine-positive cells is recorded as the mean spot-forming unit (SFU)/5 × 105 splenocytes ± SD. ***p < 0.001.
Figure 4
Figure 4
Short-term active protective immunity against DENV3 challenge (n = 8). Mice were challenged with DENV3 at the ninth week after the immunization and monitored daily for 27 days. (A) Pathological symptoms recorded as the mean clinical sign scores. (B) Percentage of body weight from day 0. (C) Survival rate shown as the percentage of survivors. Results are representative of three independent experiments. **p < 0.01; ***p < 0.001.
Figure 5
Figure 5
Long-term NAb response and active protective immunity against DENV3 challenge at the 12th month after the immunization (n = 6). (A) Endpoint titers of DENV3-specific NAbs in sera were detected by PRNT50 and recorded as GMT ± SD. The L.O.D. depicted as a dotted line represents the limit of detection of the assay. (B–D) Mice were challenged with DENV3 and monitored daily for 27 days. (B) Pathological symptoms recorded as the mean clinical sign scores. (C) Percentage of body weight from day 0. (D) Survival rate shown as the percentage of survivors. **p < 0.01; ***p < 0.001.
Figure 6
Figure 6
Short-term cross-reactive NAb response and protection against DENV1 (A–D), DENV2 (E–H), and DENV4 (I–L) at the ninth week after the immunization (n = 7). (A,E,I) Endpoint titers of cross-reactive NAbs in sera were detected by PRNT50 and recorded as GMT ± SD. The L.O.D. depicted as a dotted line represents the limit of detection of the assay. (B–D,F–H,J–L). Mice were challenged with DENVs and monitored daily for 27 days. (B,F,J) Pathological symptoms recorded as the mean clinical sign scores. (C,G,K) Percentage of body weight from day 0. (D,H,L) Survival rate shown as the percentage of survivors. Results are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7
Figure 7
Long-term cross-reactive NAb response and protection against DENV1 (A–D), DENV2 (E–H), and DENV4 (I–L) at the 12th month after the immunization. For DENV1, n = 7 in the pVAX1-D3ME group, n = 5 in the pVAX1 group. For DENV2, n = 6 in the pVAX1-D3ME group, n = 5 in the pVAX1 group. For DENV4, n = 7 in the pVAX1-D3ME group, n = 5 in the pVAX1 group. (A,E,I) Endpoint titers of cross-reactive NAbs in sera were detected by PRNT50 and recorded as GMT ± SD. The L.O.D. depicted as a dotted line represents the limit of detection of the assay. (B–D,F–H,J–L) Mice were challenged with DENVs and monitored daily for 27 days. (B,F,J) Pathological symptoms recorded as the mean clinical sign scores. (C,G,K) Percentage of body weight from day 0. (D,H,L) Survival rate shown as the percentage of survivors. *p < 0.05; **p < 0.01; ***p < 0.001.
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