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. 2014 Dec 18:14:233.
doi: 10.1186/s12866-014-0233-3.

Tetravalent recombinant dengue virus-like particles as potential vaccine candidates: immunological properties

Yan Liu  1   2 , Junmei Zhou  3   4 , Zhizhun Yu  5   6 , Danyun Fang  7   8 , Chunyun Fu  9   10 , Xun Zhu  11   12 , Zhenjian He  13   14 , Huijun Yan  15   16 , Lifang Jiang  17   18
Affiliations

Tetravalent recombinant dengue virus-like particles as potential vaccine candidates: immunological properties

Yan Liu et al. BMC Microbiol. .

Abstract

Background: Currently, a licensed vaccine for Dengue Virus (DENV) is not yet available. Virus-like particles (VLP) have shown considerable promise for use as vaccines and have many advantages compared to many other types of viral vaccines. VLPs have been found to have high immunogenic potencies, providing protection against various pathogens.

Results: In the current study, four DENV-VLP serotypes were successfully expressed in Pichia pastoris, based on co-expression of the prM and E proteins. The effects of a tetravalent VLP vaccine were also examined. Immunization with purified, recombinant, tetravalent DENV1-4 VLPs induced specific antibodies against all DENV1-4 antigens in mice. The antibody titers were higher after immunization with the tetravalent VLP vaccine compared to titers after immunization with any of the dengue serotype VLPs alone. Indirect immunofluorescence assay (IFA) results indicated that sera from VLP immunized mice recognized the native viral antigens. TNF-α and IL-10 were significantly higher in mice immunized with tetravalent DENV-VLP compared to those mice received PBS. The tetravalent VLP appeared to stimulate neutralizing antibodies against each viral serotype, as shown by PRNT50 analysis (1:32 against DENV1 and 2, and 1:16 against DENV3 and 4). The highest titers with the tetravalent VLP vaccine were still a little lower than the monovalent VLP against the corresponding serotype. The protection rates of tetravalent DENV-VLP immune sera against challenges with DENV1 to 4 serotypes in suckling mice were 77, 92, 100, and 100%, respectively, indicating greater protective efficacy compared with monovalent immune sera.

Conclusions: Our results provide an important basis for the development of the dengue VLP as a promising non-infectious candidate vaccine for dengue infection.

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Figures

Figure 1
Figure 1
Secretion analysis of DENV4 VLPs. (A) SDS-PAGE analysis of different fractions from sucrose density gradient ultracentrifugation. Sucrose gradient sedimentation and SDS-PAGE analysis of DENV4 VLP secretions. The pGAPZaA-sprME-D4-X33 yeast lysates were centrifuged by sucrose density gradient ultracentrifugation and different fractions were collected and analyzed by SDS-PAGE. 1: Marker; 2: 10%; 3: 15%; 4: 20%; 5: 25%; 6: 30%; 7: 35%; 8: 40%; 9: 45%; 10: 50%. (B) Western Blot analysis of E protein expression. The 20 ~ 25% fractions collected from sucrose density gradient ultracentrifugation were analyzed for E protein expression using the MAb IH10-6 (anti-DENV4 E protein). Arrows indicate E antigen. The size of the molecular weight marker is shown in kDa.
Figure 2
Figure 2
Indirect ELISA analysis of Dengue Virus or VLP specific IgG antibody. BALB/c mice were intraperitoneally immunized with 25 μg monovalent DENV1-4 VLP, DENV1-4 virions, or tetravalent combination (25 μg of each serotype) a total of three times at two-week intervals. Sera were collected on days 0, 13, 27, and 34 and indirect ELISA was used to test for antigen specific IgG. Sera from monovalent VLP groups and the tetravalent VLP group reacted with DENV1-4 VLP antigens and sera from inactivated DENV groups reacted with dengue virus 1–4 antigens. Data are expressed as mean absorbance at OD450 nm with a standard error of the mean (SEM) bar. The baseline (dashed line) indicates negative values with normal mouse serum. A. Binding of sera from mice immunized with DENV1-VLP and inactivated DENV1 virions to respective antigens; B. Binding of sera from mice immunized with DENV2-VLP and inactivated DENV2 virions to respective antigens; C. Binding of sera from mice immunized with DENV3-VLP and inactivated DENV3 virions to respective antigens; D. Binding of sera from mice immunized with DENV4-VLP and inactivated DENV4 virions to respective antigens; 0045. Binding of sera from mice immunized with tetravalent VLP to respective antigens.
Figure 3
Figure 3
Indirect immunofluorescence detected that sera from tetravalent VLPs recognized DENV antigens. The C6/36 cells infected with DENV1-4 were fixed, incubated with a 1:40 dilution of antisera from the tetravalent VLP group and then stained for the virus (green). Evens blue dye staining was used to label cells (red). Magnification 400 × .
Figure 4
Figure 4
ELISPOT assay. The mice immunized with tetravalent DENV-VLP were euthanized 7 days after the 3rd immunization and the spleen cells were isolated and stimulated in vitro with inactivated virions of each DENV type. IFN-γ (A), TNF-α (B), and IL-10 (C) producing lymphocytes were enumerated by ELISPOT assay. The mean number of spot forming cells (SFCs)/2 × 105 (splenocytes) is shown as virions-stimulated with an SEM bar. *indicates statistical significance (*P < 0.05; **P < 0.01).
Figure 5
Figure 5
Detection of monovalent immune serum neutralizing antibody against DENV. Balb/c mice were immunized with 25 μg monovalent DENV VLP or virions a total of three times at a two week interval. On day 7 after the 3rd immunization, neutralizing antibodies against DENV1 (A), DENV2 (B), DENV3 (C), and DENV4 (D) were assessed using a PRNT50 assay. 150-200PFU DENV were incubated with serially diluted mouse antisera in 24-well plates, using BHK-21 cell lines. Data from each group is expressed as the mean percentage of plaque reduction with an SD bar (n = 3). The PRNT50 titers for each immune sera after immunization with each monovalent vaccine against the corresponding virus were 1:32, 1:64, 1:32, and 1:32, respectively. *indicates statistical significance (*P < 0.05; **P < 0.01).
Figure 6
Figure 6
Detection of tetravalent immune serum neutralizing antibody against DENV. Balb/c mice were immunized with 100 μg tetravalent DENV-VLP (total of 25 μg for each DENV-VLP serotype) a total of three times at a two week interval. Neutralizing antibodies against DENV1-4 were assessed seven days after the 3rd immunization, using the PRNT50 assay. 150-200PFU DENV were incubated with serially diluted mouse antisera in 24-well plates, using BHK-21 cell lines. Data from each group is expressed as the mean percentage of plaque reduction with an SD bar (n = 3). The PRNT50 titer of the tetravalent immune sera against the four DENV serotypes were 1:32, 1:32, 1:16, and 1:16, respectively. (A) the PRNT50 titer of tetravalent immune serum; (B) the plaque morphology.
Figure 7
Figure 7
Protective assay in suckling mice. Protective effects of monovalent mouse antisera were evaluated in suckling mice. Suckling mice were intracerebrally inoculated with DENV1, 2, 3, or 4, and clinical signs of infection, mainly hind leg paralysis, alterations in spinal column and mortality, were monitored for 21 days. (A) survival curve; (B) mobidity curve. Different letters mean statistically significant differences by the Log-rank test. *indicates statistical significance (*P < 0.05; **P < 0.01).
Figure 8
Figure 8
Anti-tetravalent VLP sera protective assay in suckling mice. Protective effects of mouse antisera from the tetravalent VLP group were evaluated in suckling mice. Mice were intracerebrally inoculated with DENV1, 2, 3 or 4 and clinical signs of infection, mainly hind leg paralysis, alterations in spinal column and mortality, were monitored for 21 days. (A) survival curve; (B) mobidity curve. Different letters mean statistically significant differences by the Log-rank test. *indicates statistical significance (*P < 0.05; ** P < 0.01).
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