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. 2017 Nov 14;91(23):e01181-17.
doi: 10.1128/JVI.01181-17. Print 2017 Dec 1.

An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design

Akane Urakami  1 , Mya Myat Ngwe Tun  2 , Meng Ling Moi  2 , Atsuko Sakurai  1 , Momoko Ishikawa  1 , Sachiko Kuno  1 , Ryuji Ueno  1 , Kouichi Morita  2 , Wataru Akahata  3
Affiliations

An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design

Akane Urakami et al. J Virol. .

Abstract

Dengue viruses (DENV) infect 50 to 100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine by utilizing virus-like particles (VLPs). We created recombinant DENV1 to -4 (DENV1-4) VLPs by coexpressing precursor membrane (prM) and envelope (E) proteins, with an F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. For use as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody responses induced by the VLPs were significantly higher than those with DNA or recombinant E protein immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at a 1:10 serum dilution. We also demonstrated that the Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation into the ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in viral structures, and they describe the promising development of an effective tetravalent vaccine against the prevalent flavivirus.IMPORTANCE Dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We developed a novel tetravalent dengue vaccine by using virus-like particles (VLPs), which are noninfectious because they lack the viral genome. Previous attempts of other groups to use dengue VLPs resulted in generally poor yields. We found that a critical amino acid mutation in the envelope protein enhances the production of VLPs. Our tetravalent vaccine elicited potent neutralizing antibody responses against all four DENV serotypes. Our findings can also be applied to vaccine development against other flaviviruses, such as Zika virus or West Nile virus.

Keywords: DNA vaccine; VLP; Zika virus; dengue virus; flavivirus; neutralizing antibodies; vaccine; virus-like particle.

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Figures

FIG 1
FIG 1
Development of DENV1 VLPs. (A) Schematic illustration of the DENV genome and DENV1 VLP expression vectors. C, capsid; ss, signal sequence; prM, precursor membrane protein; E, envelope. (B) Western blot analysis of DENV1 VLP-containing culture supernatant. 293F cells were transfected with the control vector or an expression plasmid encoding wild-type DENV1 prM-E (prM-E) or prM-E with the F108A mutation (prM-EF108A) and then cultured for 4 days. Secreted DENV1 VLPs were detected using a mouse anti-DENV1-4 E monoclonal antibody. The intensity of each band was measured, and the relative intensity was calculated by setting the intensity of prM-E to 1.0. An image representative of at least 3 independent experiments is shown.
FIG 2
FIG 2
Development of DENV2 VLPs. (A) Schematic illustration of DENV2 VLP expression vectors. prM-E, wild-type sequence of DENV2 prM-E; prM-EF108A, prM-E with the F108A mutation; chimeras 1 to 4, chimeric DENV2 E constructs with the F108A mutation and different lengths of DENV1 E in the C-terminal region; prM-E_2/1, DENV2 E protein aa 297 to 495 were replaced by the corresponding region of the DENV1 E protein; prM-EF108A_2/1, prM-E_2/1 with the F108A mutation. EDIII, envelope domain III; ST/TM, stem and transmembrane anchor. (B and C) Western blot analysis of DENV2 VLPs in culture supernatants. Expression vectors were transfected into 293F cells, and culture supernatants were tested for VLP production on day 4 by using a goat anti-DENV2 E polyclonal antibody. Images representative of at least 3 independent experiments are shown. (B) Effect of F108A mutation in prM-E or prM-E_2/1. The intensity of each band was measured, and the relative intensity was calculated by setting the intensity of prM-E_2/1 to 1.0. (C) Effects of replacing different lengths of the DENV2 C-terminal region on VLP production.
FIG 3
FIG 3
Development of DENV3, DENV4, and ZIKV VLPs. (A and B) (Top) Schematic illustrations of VLP expression vectors. (Bottom) Western blot analyses of DENV3 and DENV4 VLPs in culture supernatants. Expression vectors were transfected into 293F cells, and culture supernatants were tested for VLP production on day 4 by using antibodies against DENV1-4 E (A) or DENV4 E (B). The intensity of each band were measured, and the relative intensity was calculated by setting the intensity of the comparator VLPs to 1.0. Images representative of at least 3 replicates are shown. (C) (Left) Schematic illustration of prM-EF108A_ZIKV/JEV. ZIKV E protein aa 404 to 504 were replaced with the corresponding region of JEV, and the F108A mutation was introduced. (Right) Western blot analysis of the ZIKV VLP constructs. Culture supernatants were harvested at 4 days posttransfection, and E proteins were detected by use of a rabbit anti-ZIKV E polyclonal antibody. Images representative of at least 3 independent experiments are shown.
FIG 4
FIG 4
Optimization of DENV2 and DENV4 VLP constructs. (A) VLP production levels of the prM-EF108A_2/1 construct with two different DENV2 strains. Expression vectors were transfected into 293F cells, and culture supernatants were tested for VLP production on day 4 by using a goat anti-DENV2 E polyclonal antibody. (B) Effects of prM E90D mutation on DENV4 VLP production. Culture supernatants were harvested at 4 days posttransfection and assessed by Western blotting using an anti-DENV4 E monoclonal antibody. Images representative of at least 3 independent experiments are shown.
FIG 5
FIG 5
Characterization of DENV VLPs. (A) SDS-PAGE analysis of purified DENV1 to -4 VLPs. Separated proteins were stained with Coomassie blue dye. Images representative of at least 3 replicates are shown. (B) Transmission electron microscopy images of purified DENV1 to -4 VLPs. Representative images are shown.
FIG 6
FIG 6
Immunogenicity and neutralizing activity of monovalent DENV VLPs. Six-week-old female BALB/c mice (n = 4) were immunized intramuscularly with PBS, 20 μg of DENV VLPs, or recombinant dengue virus E protein (rE) together with alum three times at 3-week intervals. Sera were collected 2 weeks after the last immunization. (A) Serum anti-flavivirus IgG titers were determined by ELISA. Mean log10 ELISA titers (with SD) are shown. *, P < 0.05 (Mann-Whitney U test for homologous serotype of DENV rE versus VLPs). (B to E) Serum neutralization titers of rE- or DENV VLP-immunized mice against DENV1 to -4 and JEV. Each symbol represents the FRNT50 titer from an individual mouse, and the geometric mean titers are represented by horizontal bars. Open and filled symbols indicate DENV rE and VLP immunizations, respectively. *, P < 0.05, ns, not significant (Mann-Whitney U test for homologous serotype of DENV rE versus VLPs).
FIG 7
FIG 7
Immunogenicity and neutralizing activity of tetravalent DNA or VLP vaccines. Six-week-old female BALB/c mice (n = 4) were immunized with 80 μg (20 μg of each serotype) of tetravalent DNA or tetravalent DENV VLPs three times at 3-week intervals. DNAs were administered by intramuscular injection and electroporation, and VLPs were administered by regular intramuscular injection with alum. Sera were collected 2 weeks after the last immunization. DNA_wild type, DENV1-4 prM-E-encoding plasmids; DNA_F108A, DENV1-4 VLP-encoding plasmids; DENV VLP, purified DENV1-4 VLPs. (A) Serum anti-flavivirus IgG titers were determined by ELISA. Mean log10 ELISA titers (with SD) are shown. *, P < 0.05 (one-way analysis of variance [ANOVA] followed by Tukey's multiple-comparison test). (B) Serum neutralization titers against DENV1 to -4. Each symbol represents the FRNT50 titer from an individual mouse, and the geometric mean titers are represented by horizontal bars. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant (one-way ANOVA followed by Tukey's multiple-comparison test).
FIG 8
FIG 8
Antibody-dependent enhancement activity of sera from tetravalent DENV DNA- or VLP-immunized mice. DENV1 to -4 were incubated with serially diluted mouse sera or anti-flavivirus E monoclonal antibody and added to FcγR-expressing BHK cells. After 2 days, infected cell numbers were counted. (A to D) Data were shown as fold infection enhancement values by setting the mean number of infected cells in the absence of serum or antibody to 1.0. The mean value for at least 3 negative-control wells plus 3 SD (indicated as a dotted line) was used as the cutoff value to determine which samples had ADE activity.
FIG 9
FIG 9
Neutralizing activity of monovalent DENV VLPs in outbred mice. Six-week-old female NIH/Swiss mice (n = 5) were immunized intramuscularly with PBS, DENV3 VLPs (0.31, 1.25, 5, or 20 μg), or DENV4 VLPs (1.25, 5, 20, or 40 μg) with alum three times at 3-week intervals. Sera were collected 2 weeks after the last immunization. (A) Serum neutralization titers of DENV3 VLP-immunized mice against DENV3. (B) Serum neutralization titers of DENV4 VLP-immunized mice against DENV4. Each symbol represents the FRNT50 titer from an individual mouse, and the geometric mean titers are represented by horizontal bars.
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