Fig 1.
Zika polyprotein processing and strategy for VLP assembly.
The schematic depicts the Zika virus genome single open reading frame (ORF) that translates into a polyprotein comprising both structural and non-structural proteins, which arise via several proteolytic cleavages. (A) In the early stages, the complex viral protease NS3 with its cofactor NS2B (↓) self-cleaves before cleaving the capsid protein. Host cell signalase is also involved in the maturation of the polyprotein (◊). As a final step, cellular furin cleaves (▽) the pr portion from the M protein to uncover E protein fusion peptide. (B) The VLP assembly strategy relies on the co-expression of the structural protein CprME (ZO2) together with the non-structural protein NS2B/NS3Pro (ZO3). The viral NS3 protein is truncated maintaining only its N-terminal protease domain NS3Pro, which is kept as a single transcription unit with its cofactor NS2B. The origin of the sequence encoding these genes and the cloning strategy are described in Material and Methods.
Fig 2.
Analysis of protein expression and processing in lysates of transfected cells by Western blot.
Cell lysates (30μg total protein) of Expi-HEK293 transfected with plasmids ZO2 (CprME), or ZO3 (NS2B/NS3Pro) or both (ZO2/ZO3) were probed with anti-Zika protein specific polyclonal antibodies (See Material and Methods). Zika virus (ZIKV) infected and uninfected (mock) Vero cell were controls. (A) Anti-E specific antibody detected E protein expression and cleavage in ZO2/ZO3 and ZO2 (lesser amount) cell lysates but not in that of ZO3. E was also detected in ZIKV infected cells but not in the mock control. (B) The prM protein (~32KDa) was detected in ZO2/ZO3 transfected cells as well as in ZIKV infected cells, which also showed a small amount of M (~ 8KDa). (C) NS3Pro (~19KDa) was detected in ZO3 and ZO2/ZO3 transfected cells whereas the full-length protease/helicase NS3 (~69KDa) was detected in ZIKV infected cells. Neither was detected in ZO2 transfected cells or the mock control. (D) NS2B (~14KDa) was detected in ZO2 and ZO2/ZO3 transfected cells and ZIKV infected cells whereas ZO2 and mock transfected cells were negative.
Fig 3.
Analysis of purified VLPs and ZIKV by Coomassie blue stain and Western blot.
A total of 1.8 μg of purified VLPs or ZIKV were resolved by SDS-PAGE and protein content and identity analyzed by (A) Coomassie stain and (B) Western blot using Zika protein specific antibodies, anti-E upper panel and anti-M lower panel (C) Comparison of the size of the E protein of ZIKV MR-766; FSS-13025 and VLPs by Western blot.
Fig 4.
Examination of reactivity of native VLPs and native and inactivated ZIKV with monoclonal antibodies recognizing structural epitopes.
VLPs were produced and purified as described in material and methods. ZIKV MR-766 and FSS-13025 were grown in Vero cells, PEG-8000 precipitated and concentrated by centrifugation through a 20% sucrose cushion. Half of the virus sample was inactivated with formalin and then treated and untreated (native) virus samples were further purified via density gradient centrifugation. Dot blot analysis of purified VLPs and ZIKVs (0.2μg/μl, 3μl per dot of E protein content) were probed the MAbs, 4G2, ZV-48, ZV-64 and ZV-67, which recognize conformational epitopes in distinct domains of the E protein, showed that VLPs and native ZIKVs reacted with the MAbs, whereas the reactivity of formalin inactivated ZIKVs virus was greatly reduced as compared to native VLPs. This outcome suggests that formalin has a deleterious effect on the ZIKV conformational epitope recognized by these MAbs.
Fig 5.
Electron micrographs of negative staining and immunogold labeling of VLPs and ZIKV.
Purified VLPs and ZIKV were examined by transmission electron microscopy (TEM). Panels A and B show uranyl acetate negatively stained VLPs which exhibit particle sizes from 50nm to 65nm in diameter (mean 60nm) and a structure that resembles the morphology and surface appearance of wild type Zika virus which is shown in Panel E. Immunogold labeling with two antibodies, mouse anti-E protein MAb 4G2 and a human serum from a Zika patient served as primary and counterstained with anti-mouse or anti-human secondary antibody conjugated with gold beads of 6 nm and 10nm in diameter, respectively. Both antibodies, 4G2 panel C and human serum panel D, bind to the particle surfaces as revealed by the presence of gold beads. Panel F shows wild-type ZIKV probed with the 4G2 antibody, which also binds the virus surface as revealed by the detection of gold beads; in this case goat anti-mouse secondary antibody conjugated with 10 nm gold bead was applied. These studies demonstrate that a specific anti-E MAb and an anti-Zika polyclonal antibody reacts with the VLP surfaces indicating that the major Zika surface antigen, the E glycoprotein, is indeed displayed on the VLP surfaces.
Fig 6.
Evaluation of serum IgG response elicited by VLP and control vaccinations.
Serum antibody (total IgG) elicited by the Zika VLP vaccine (VLP), inactivated Zika virus (In-ZIKV) and negative control (Neg. Ctr.) were measured by ELISA. Groups of BALB/c mice (n = 8) were immunized on days 0 and 21 with a VLP-based Zika vaccine (VLP), or an inactivated Zika virus vaccine (In-ZIKV) at doses of 1ug or 4ug with or without adjuvant. Blood samples were collected three weeks after the booster immunization and total serum IgG was measured via ELISA using as antigens two different Zika viruses MR766 (Upper panel) and FSS-13025 (Lower panel). Mice that received low dose (1μg alone or plus adjuvant) or high dose (4μg alone or plus adjuvant) of either vaccine showed a strong antibody response to the MR-766 virus compared to the placebo group (Upper panel). Mice showed high titers of IgG antibodies against FSS-13025 Zika virus as well (Lower panel). The values shown represent geometric means of the reciprocal dilutions of each mouse serum and data point standard deviations.
Fig 7.
Serum neutralizing antibody responses by plaque reduction neutralization test (PRNT) against two Zika viruses.
The serum neutralizing activity was plotted as PRNT50. (Left Panel) PRNT with MR-766 showed that the VLP vaccines elicited significantly higher neutralizing titers than equivalent composition of In-ZIKV vaccine. The VLP 1 μg plus adjuvant elicits higher titers than the corresponding In-ZIKV vaccine but the difference was not significant. The VLP (4μg+ adj) showed titers 5 fold greater than the human serum and the 4 μg alone was within this range, however statistical analysis was not feasible with only one human sample. (Right Panel) Similarly, PRNT with FSS-13025 showed that VLP vaccines exhibit stronger neutralization than the corresponding In-ZIKV vaccine. Neutralizing titers elicited by the In-ZIKV vaccine against FSS-13025 were much lower than those seen with the MR-766 and this may be due to the fact the In-ZIK vaccine was prepared with the MR-766 virus, which elicited a higher neutralizing titer toward the analogous virus (MR-766) than to a more distant one (FSS-13025). Similarly, the human serum showed a higher titer against FSS-13025 than MR-766, presumably because it resulted from a human infection in Brazil with a virus antigenically related to the FSS-13025. P-Values indicates t-tests.
Table 1.
Comparison of the neutralizing antibody responses (PRNT50) elicited by the VLP vaccine and controls.
A summary of geometric mean titers of PRNT50 GMT for comparison between the two ZIKV strains, MR-766 and FSS 13025 for both vaccine and virus control groups. Neutralizing activity elicited by the VLP vaccines against the two viruses were comparable, except the VLP for 1μg alone, which had greater activity against MR766 than that of FSS (a p = 0.0033). The In-ZIK virus control 4 μg plus adjuvant also showed a difference between the two viruses (b p = 0.026). The statistical analysis was performed using t-test at confidence intervals of 95% with GraphPad Prism 4 software. Human serum isolated from a patient who recovered from a Zika infection was used to validate PRNT technique. However, no statistical analysis can be performed to compare the neutralization with two different Zika viruses due to the low sample size.
Fig 8.
Antibody dependent enhancement (ADE) of dengue virus infection by the anti-ZIKV response elicited by vaccination.
Serum from four VLP immunized mice (4ug +adj); inactivated ZIK (4ug+adj) and negative control (PBS+adj) diluted 1/500 were mixed with DENV-2 virus and added to U397 cells. The MAb 4G2 and virus alone were used as controls. After 96h incubation, DENV-2 virus titers were measured on the culture supernatant by plaque assay. Error bars indicate standard deviation of the four samples performed in duplicates. Asterisk (*) indicates that difference between 4G2 (positive control) and remaining samples is statistically significant (p < 0.05).