Virus, cells, and antibodies

Cultures of Vero cells (ATCC CCL-81) were maintained in VP-SFM media (Life Technologies, CA) supplemented with 2 mM L-Glutamine, 2 mM GlutaMAX Supplement (Life Technologies, CA), 1X Non-essential amino acids solution (Life Technologies, CA), 1X ITSE (InVitria) and 500 ng/ml rhEGF (Life Technologies, CA).

VLP production and purification

Expi293 cells in suspension culture were transfected with a 1:2 ratio of pcDNA3.4-NS2b/NS3 (ZO3) and pcDNA3.4-CprME (ZO2) at 37°C in a 5% CO₂ environment and agitated at 150 rpm. Transfected cells were harvested 72 h post-transfection and clarified via two successive centrifugations. The first clarification was performed at 400g for 10 min at 4°C followed by a second clarification at 10,000g for 10 min at 4°C. The remaining proteins in the supernatant were precipitated using 8% (w/v) of polyethylene glycol 8000 and incubated overnight at 4°C. A protein pellet was collected after centrifugation at 14,000g for 30 min at 4°C and loaded onto a 20% w/v sucrose cushion in TNE buffer (10 mM Tris-HCl, pH 8.0, 120 mM NaCl and 1 mM EDTA) and spun by ultracentrifugation at 150,000g for 2 h at 4°C. The pellet was resuspended in TNE buffer. Viruses and VLP were then purified by ultracentrifugation through a linear potassium tartrate 10–35% (w/v) / glycerol 7–30% (v/v) density gradient at 180,000g for 4 h at 4°C. Fractions were collected and analyzed by dot blot using 4G2 monoclonal mouse antibody. The 4G2 MAb reacts with dengue, Zika and other flavivirus [21] and has been shown to recognize a conformational epitope within domain II of the dengue E protein [17] and presumably recognizes a similar epitope in the Zika virus. Selected fractions were further purified and concentrated using Amicon Ultra Centrifugal Filter Unit (Millipore, MA).

Dot blot, Western blot and Coomassie blue stain

The cell protein content was analyzed after clarification of transfected Expi293 cells. The cell pellets were lysed with RIPA buffer (Pierce Thermo Fisher, MA) according to the vendor protocol. For Western blotting, cell lysates and concentrated culture supernatants were loaded onto a 4–12% Bis-Tris SDS-polyacrylamide gel (Life Technologies, CA). After electrophoretic separation, proteins were electro-transferred from the gel onto a 0.45 μm nitrocellulose membrane (Life Technologies LC2001). For dot blot analysis, 3 μl of sample was applied to a 0.45 μm nitrocellulose membrane and allowed to dry for 5 min. The nitrocellulose membranes were then blocked with 5% non-fat milk in TBST (10 mM Tris-HCl, pH 7.4, 130 mM NaCl, 2.7 mM KCl and 0.1% Tween-20) for 1 h at room temperature followed by overnight incubation at room temperature in primary antibody diluted with blocking buffer. Membranes were washed 3 times with 1X TBST and then incubated for 2 h with secondary antibody diluted in blocking solution. Finally, membranes were washed 3 times with 1X TBST and developed with ECL system (Life Technologies, CA).

The total protein concentration was determined using the Bradford method. The E protein content in the purified VLPs and inactivated ZIKV (In-ZIKV) was determined using densitometry analysis of Coomassie blue stained SDS-PAGE gels. Different concentrations of a BSA standard were loaded onto the same gel. The stained gel image was acquired with a FluorChem M imager instrument (Protein Simple, CA). The optical density of the bands was analyzed using Alphaview software. The BSA concentrations were used to establish standard curve and parameters of the linear regression curve were used to determine E protein concentration of each VLP vaccine and In-ZIKV. Dot blot were performed with purified and quantified VLP or ZIKV and the amount of sample applied is specified in the corresponding figure legend.

Negative staining and immuno-gold labeling TEM

VLP or Zika virus samples were prepared for TEM examination as follow: Samples for immunogold labeling TEM (2.5 μl) were loaded onto CF200-CU carbon film 200 mesh copper grid (product of Electron Microscopy Science) and incubated for 5 min at room temperature, then washed with PBS, blocked in 1% BSA in PBS for 5 min and incubated on the surface of a primary antibody drop for 30 min at room temperature. We use two antibodies, the mouse MAb 4G2 diluted 1:500 in PBS and a polyclonal human serum from a Zika virus infected patient diluted 1:20 dilution in PBS. The grids were then washed 6 times with PBS and incubated on the surface of a drop of secondary antibody conjugated with gold beads for 30 min. A goat anti-mouse antibody conjugated with 10 nm gold beads was used with the mouse MAb 4G2 and a goat anti-human antibody conjugated with 6 nm gold beads was used with the human serum. Grids were finally washed 6 times with PBS, and then fixed for 15 min with 4% paraformaldehyde in PBS, washed with PBS and subsequently washed twice with 0.2M Sodium cacodylate buffer and finally stained with 1% uranyl acetate solution.

Mouse immunogenicity and efficacy study

The aim of the study was to determine the immunogenicity and efficacy of a VLP-based Zika vaccine and its capacity to elicit a strong neutralizing serum protective immune response. Experimental groups comprised eight mice (n = 8) in order to study pairs of subjects while being able to reject a null hypothesis with statistical power of >99% and a standard deviation of 19. The alpha (α) error probability associated with this test is 0.01.

Nine groups of 6 to 8-week old female BALB/c mice (n = 8) were inoculated twice (day 0 and day 24) via the intramuscular (IM) route with either VLP vaccine, formalin inactivated ZIKV MR-766 (In-ZIKV) control or PBS plus adjuvant negative control (Neg. Ctr.). Mice received doses of either 1 μg or 4 μg of total E protein content formulated alone or admixed in a 1:1 volume ratio with a squalene-based oil-in-water nano-emulsion AddaVax (InvivoGen, CA). Serum samples were collected from all animals at day 42.

Evaluation of serum antibody levels by ELISA

Serum IgG titers against Zika viruses were determined by ELISA. Briefly, assays were performed in 96-well plates coated with 50 μl/well of purified and inactivated Zika virus MR-766 or FSS-13025 (2 μg/ml total protein concentration) at 4°C overnight. Subsequently, plates were washed 3 times with PBS-T (0.05% Tween-20 in phosphate buffered saline) and then blocked with 100 μl of blocking solution (5% non-fat milk in PBS-T) for 1 h at room temperature (RT). Vaccine and control sera were diluted in fourfold series in blocking buffer and applied to each well in triplicate and incubated for 2 h at RT. After 6 washes with PBS-T, plates were incubated for 2 h at RT with 50 μl of HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody diluted (1:5000) in blocking buffer). Subsequently, plates were washed 6 times with PBS-T and developed by adding 50 μl per well of Ultra TMB solution (Thermo Scientific, MA) for 20 min of incubation and stopped with 100 μl of stop solution (2 M HCL solution). ELISA end-point titer for each group was calculated as the reciprocal of the highest dilution with OD value 2σ above the mean of the negative control wells.

Plaque reduction neutralization test (PRNT)

The plaque reduction neutralization test was carried out in Vero cells using sera from immunized mice and either MR-766, or FSS-13025 Zika viruses following the method described for the evaluation of flavivirus vaccine efficacy [22, 23]. Briefly, ZIKV was amplified and titrated in Vero cells using the same plaque visualization procedure described below. Vero cells were seeded in 24-well plates at a density of 6x10⁴ cells per well 24 h prior to the initiation of the test. Control and test sera were heat inactivated at 56°C for 30 min. Starting at a 1:20 dilution, the sera were two-fold serially diluted with cell culture media supplemented with penicillin and streptomycin. Equal volumes of diluted virus to form ~60 plaques per well were added to each serum dilution. The sera-virus mixtures were incubated for 1 h at 37°C in a 5% CO₂ environment. Afterwards, each dilution was applied in duplicated to wells of 85% confluent monolayer of Vero cells and incubated for 90 min at 37°C in a 5% CO₂ incubator. Thereafter, the inocula were removed and a 1 ml overlay of 1.8% carboxymethyl cellulose (CMC) in culture medium was added to each well. Plates were then incubated for 3 days at which time the CMC overlay was removed, cells washed with PBST (phosphate buffer saline plus 0.05% Tween 20) and fixed with cold 80% acetone for 10 min at -20°C. Subsequently, plates were washed with PBST and then incubated with blocking buffer (2.5% non-fat milk in 0.5% of Triton X-100 PBS solution) for 1 h in a 37°C incubator. After one wash, the primary antibody (MAb 4G2) diluted 1:500 in blocking buffer was applied for 2 h at RT, followed by two PBST washes and incubation for 1 h at RT with a goat anti-mouse AP conjugated secondary antibody diluted (1:2000) in blocking buffer. Finally, plates were washed twice with PBST and once with alkaline phosphate buffer (APB: 100 mM Tris-HCl pH9.0, 150 mM NaCl, 1 mM MgCl₂). Viral plaques were detected by adding the alkaline phosphate (AP) substrate nitro blue tetrazolium chloride (NTB) and 5-brome-4-chlore-3-indolyl phosphate (BCIP) prepared and used as follows: Combine 33 μl of NTB (50 mg/ml in 70% dimethylformamide) with 5 ml of APB mix well and then add 16.5 μl of BCIP (50 mg/ml in 100% dimethylformamide) and the mixture should be used within 1 h of preparation. Plaques were counted and PRNT50s were determined using the PROBIT method [24]. The neutralization power calculated is expressed as the reciprocal of the highest serum dilution that neutralizes 50% of the virus and the histograms represent the average neutralization 50% per group and their standard deviations.

Antibody-dependent enhancement (ADE) assay of DENV-2 infection

Suspension cultures of U937 cells grown in RPMI supplemented with 10% of FBS were used in the ADE assay, which was performed according to Diamond et. at. [25]. Prior to the initiation of the experiment (72h), the U397 cells were differentiated toward the granulocyte or macrophage lineage by the addition of dimethyl sulfoxide (DMSO; 1.25%) [25]. DENV-2 virus (50μl) was mixed with diluted serum samples (four for each vaccine group) and controls (virus alone or 4G2 MAb-200ng) and then added to 2.5x10⁵ cells resulting in a multiplicity of infection of 3 (MOI: 3). The resulting mixture of cells, virus and serum was incubated for 2 h at 37°C. Subsequently, cells were washed four times to remove free virus, suspended in culture medium and then incubated for 96 h at 37°C. Thereafter, virus titers in the culture supernatants were determined by plaque assay as described above in the PRNT assay.

Statistics

All statistical calculations were carried out using GraphPad Prism 4 software. Tests between two groups used two-tailed Student’s t-test. The P-value was calculated for <0.05 level of significance.

Production and characterization of Zika virus-like particles (VLPs)

The recombinant production of Zika VLPs was carried out in suspension cultures of Expi-HEK293 cells following co-transfection of the plasmids ZO2 (CprME) and ZO3 (NS2B/NS3 Pro). The VLPs were harvested from the culture supernatant and purified as described in Material and Methods. The Zika virus grown in Vero cells was subjected to an analogous purification scheme as the one described for the VLPs. The purity and identity of the protein component of the purified VLP and Zika virus were assessed by Coomassie blue staining and Western blot analysis. As shown in Fig 3A, the E protein was readily detected as the predominant component of both the ZIKV and the VLP preparations. The identity of the E protein was verified by Western blot analysis and exactly correlated with the E protein visualized in the stained gel (Fig 3B). The migration difference of the E proteins detected in the cell lysates was also seen between the E protein of the purified ZIKV-MR-766 and VLPs (Fig 3A and 3B). This disparity, as noted above, is due to the lack of four amino acids including the glycosylation site N-154 in ZIKV-MR-766. These residues are present in ZIKV-FSS-13025 and H/PF/2013 and sequences of this strain were used to produce the VLPs. Examination by Western blot of the migration patterns of the E protein from ZIKV-MR766, FSS-13025 and VLPs (genes derived from H/PF/2013) showed that the MR-766 E protein migrated slightly faster than the E protein from ZIKV FSS-13025 and VLPs, which exhibit similar size (Fig 3C). Coomassie staining also showed minor unidentified proteins in the VLP preparation that were also present in the ZIKV albeit in lesser amounts (Fig 3A). The M protein, a major structural component of the Zika virus, was clearly identified by Western blot analysis in both the ZIKV and VLP preparations but it was not clearly seen in the stained gel, possibly due to its small size and low concentration, which precluded retention of sufficient amounts of bound dye for clear detection (Fig 3A). Both, E and M the major structural proteins were clearly identified by Western blot (Fig 3B).

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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.

Furthermore, to corroborate whether conformational neutralizing epitopes of the E protein were exhibited on the VLPs, we probed by dot blot purified VLPs and ZIKVs with the domain specific neutralizing MAbs, ZV-48, ZV-64 and ZV-67 [20] in addition to the mouse MAb 4G2. This examination showed that the VLPs were indeed recognized by these MAbs indicating that these neutralizing epitopes were properly displayed in the VLPs as they also were in the ZIKV MR-766 and FSS-13025 (Fig 4). We have also observed that the reactivity of Zika viruses with these MAbs is mostly abrogated after the viruses are inactivated with formaldehyde (Fig 4). The VLP sample remained untreated.

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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.

Negative staining and immunogold labeling electron microscopy

To further characterize the structure and surface composition of the VLPs, we examined purified material by transmission electron microscopy (TEM). Negative staining studies revealed that the VLPs are fairly homogeneous spherical structures with an average diameter of 60 nm. The VLP surfaces appeared smooth without noticeable projection or rough features. Comparative analysis with purified ZIKV showed that both VLP and virus particles are similar in size, morphology and surface appearance (Fig 5A, 5B and 5E). This is in agreement with recent reports of the Zika virus structure [31, 32]. To better define the surface composition of the VLPs, we probed the recombinant particles by immunogold labeling with two distinct antibodies, 4G2, a MAb that recognizes a conformational loop in domain II of the E protein shared by some members of the flavivirus family including Zika [21], and a polyclonal human serum from a Zika patient. Subsequently these preparations were examined by negative staining TEM. These studies demonstrated the reactivity of the VLPs with both antibodies (Fig 5C and 5D) indicating not only that the E protein is displayed on the surface of the particles but that it also maintains its native conformation based on reactivity with the MAb 4G2 (Fig 5C). Examination of purified ZIKV also showed equivalent reactivity with the 4G2 antibody (Fig 5F) confirming that both the VLP and live ZIKV exhibit on their surfaces the conformational site recognized by 4G2 (Fig 5C and 5F). We further probed VLPs with the serum from a Zika patient and found that it also binds to the particles’ surface providing addition evidence that the ZIKV major surface glycoprotein E is indeed present on the VLP surface (Fig 5D).

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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.

VLP based Zika vaccine evaluation

We sought to assemble Zika VLPs with the main objective of developing a safe and effective vaccine to stem the rapidly spreading Zika epidemic. To establish immunogenicity and in-vitro efficacy of a VLP-based Zika vaccine, we designed a mouse study to assess the performance of a VLP vaccine and to compare the outcome to an equivalent formulation and dose of an inactivated Zika virus (In-ZIKV) control. Given the urgency of advancing a Zika vaccine and the lack, at the onset of the study, of well-established Zika animal models, we selected a mouse model to carry out this vaccine evaluation. Two groups of 6 to 8- week old female Balb/c mice (n = 8 each) were immunized via the intramuscular route (IM) twice, three weeks apart, with two difference doses, 1 μg and 4 μg of total E protein of VLP vaccine formulated with or without adjuvant (AddaVax, InVivoGen) a squalene-oil-in water nano-emulsion that stimulates a balanced immune response and has a formulation similar to MF59, a licensed adjuvant in flu vaccines in Europe [33, 34]. Similar groups of mice (n = 8 each) were immunized via the same route and schedule with a formalin inactivated Zika virus (In-ZIKV) control at the doses of either 1 μg or 4 μg of total E protein content also formulated with or without adjuvant. A negative control (Neg. Ctr.) group (n = 8) received phosphate buffered saline (PBS) plus adjuvant following the same immunization regimen as used for the vaccine groups. Three weeks after the booster immunization, animals in all groups were terminally bled and sera samples prepared for immunogenicity and efficacy evaluation.

Evaluation of immunogenicity by ELISA

We assessed the level of the total serum IgG response in all vaccinated animals by ELISA using as antigens two Zika virus strains, one isolated in 1947 (Zika virus MR-766) and a second more current strain isolated in Cambodia in 2013 (Zika virus, FSS 13025). The ELISA results demonstrated that mice vaccinated with the low dose (1 μg) or high dose (4 μg) of VLP vaccine stimulated the production of high levels of serum antibodies against both Zika virus strains (Fig 6 Upper and Lower panels). This response was enhanced when the VLP vaccines were formulated with adjuvant. The 1 μg VLP vaccine plus adjuvant elicited a markedly increased level of serum IgG as compared to VLP alone. This enhancement was even more significant using the 4 μg VLP vaccine dose formulated with adjuvant. Similar results were obtained with the two Zika virus antigens used in the ELISA studies (Fig 6 Upper and Lower panels). Mice vaccinated with the inactivated Zika virus (In-ZIKV) produced a serum IgG response that was quite comparable to the one triggered by the VLP vaccines.

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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.

Clearly, both the VLP and the inactivated Zika virus control compositions were capable of eliciting a high antibody response against two Zika viruses. In contrast, the negative control group did not demonstrate a specific IgG response against Zika.

We thank Walter Orenstein, MD and Katherine Frederick, MD for their encouragement and critical reading of the manuscript. We also thank Sulli Popilskis, DVM, Director of the New York Medical College Animal Facility and Kellie Elson and their team for their support with the animal study. We thank Dr. Diamond for generously providing the monoclonal antibodies FV-48, FV-64 and FV-67.