Specific-pathogen-free primary CEFs were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% heat-inactivated calf serum (Gibco Life Technologies, Grand Island, NY, USA), 5% Tryptose Phosphate Broth (Difco Laboratories, Detroit, MI, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. MRC-5 and Vero cells were grown in DMEM supplemented with 10% heat-inactivated calf serum, with 100 U/mL penicillin and 100 µg/mL streptomycin.
The plasmid prepared for in-vivo recombination contained the cPrME gene sequence of ZIKV (Fig. 1). This fragment included the whole envelope gene sequence (54.38 kDa; 504 amino acids), as well as the genes that encode for part of the capsid protein (~ 2 kDa; 18 amino acids), the membrane precursor (10.12 kDa; 92 amino acids), and the membrane protein (8.4 kDa; 76 amino acids) . In particular, the capsid hydrophobic tail is a signal peptide for the translocation of PrM to the endoplasmic reticulum , whereas PrM prevents the rearrangements of the envelope proteins in an acidic milieu, and their fusion with cell membranes during translocation through the secretory pathway.
The ZIKV RNA genome was obtained from the serum of a 2015 Brazilian patient (ZikaSPH2015 strain) and was supplied by the EVAg project through the courtesy of M.R. Capobianchi (National Institute for Infectious Diseases L. Spallanzani, I.N.M.I., Rome, Italy). It was retro-transcribed and amplified using the forward V436 (5′ CCG CGC CCG GGA AGC TTA TGG GCG CAG ATA CTA GTG TC 3′) primer and reverse V437 (5′ GGG GTA CCG CGG CCG CAT AAA AAT TAA GCA GAG ACG GCT GTG GA 3′) primer, to get the cPrME fragment. The primers were designed to include the SmaI/HindIII sites at the 5′ end, followed by the ATG sequence, and the NotI/KpnI sites at the 3′ end. These sites were needed to clone the ZIKV cPrME gene fragment into the pFP recombination plasmid. At the 3′ end, a T5NT sequence was also added, as an additional poxviral transcription termination signal.
RNA (10 ng) was retro-transcribed using Transcriptor One-Step RT-PCR kit (Roche Molecular Systems, Indianapolis, IN, USA) in 50 μL following the manufacturer instructions.
After deletion of the A27L gene from pFPA27L recombination plasmid [43, 44] and subcloning of the cPrME fragment, the resulting pFPcPrME18 clone was sequenced to exclude any possible mistakes due to the PCR amplification. A non-synonymous mutation at nucleotide 759, where the cytosine nucleotide had replaced the thymine giving origin to an alanine instead of a valine, was corrected by site-specific mutagenesis [45, 46].
The cPrME mutagenized fragment was thus inserted inside the 3-β-hydroxysteroid dehydrogenase 5-delta 4 isomerase gene, downstream of the Vaccinia virus H6 (H6) early/ late promoter . The sequence was aligned with the env gene of ZIKV (GenBank accession number KU991811) using Align Plus 2.0. This pFPcPrME recombination plasmid (10,274 bp) was finally designated as pFPzenv.
Recombinant fowlpox virus
The FPzenv viral recombinant putative vaccine was generated by in-vivo homologous recombination . Briefly, FPzenv was obtained on specific pathogen-free primary CEFs, using the recombination plasmid pFPzenv described above (62.5 µg) and the wild-type FP virus (5 PFU/cell). Recombinant plaques were identified by autoradiography after hybridization with the [32P]-labelled zenv probe. Recombinants were subjected to multiple cycles of plaque purification until one clone was selected for correct expression. The recombinant was amplified in CEFs, purified on discontinuous sucrose density gradients, and titrated essentially as described previously . Briefly, the cells were harvested, ultracentrifuged at 30,000 × g for 2 h at 4 °C, and the pellets were resuspended in 1 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4. After addition of trypsin (0.06% final concentration), the pellet was incubated for 5 min at 37 °C, and the virus was released from the cells by sonication. The supernatant was overlaid onto a discontinuous 30% to 45% (w/w) sucrose gradient, in the same buffer. After ultracentrifugation at 38,000 × g for 1 h, the viral band at the interface was recovered, diluted with 1 mM Tris–HCl, pH 9, and pelleted at 67,000 × g for 1 h. The purified virus was resuspended in Ca2+-free and Mg2+-free phosphate-buffered saline (PBS–), briefly sonicated, and then aliquoted and frozen at − 80 °C until use.
Two expression plasmids, pVAXgag/proM766 (here referred to as pVAXgp) and pVAXzenv, were used to prime the mice. pVAXgp contains the SIVmacM766 gag/pro gene , which was a kind gift from G. Franchini (National Cancer Institute, NIH, Bethesda, MD, USA), and was used as an irrelevant negative control. The zenv gene was excised from pFPzenv and inserted into the pVAX expression plasmid (Invitrogen Corp., San Diego, CA, USA), which contained the human CMV promoter and is approved for use in humans. Transformation was performed using JM109 competent bacteria, in the presence of 50 µg/mL kanamicin, as pVAX contains the kanamicin resistance gene. Briefly, the zenv gene was cut from pFPzenv with HindIII/NotI and inserted into the pVAXenvM766 plasmid, from where the envM766 gene had been previously removed using the HindIII/NotI/SalI restriction enzymes. Bacterial selection was performed by PCR amplification using the V438/V441 primers, and 2.5 mM MgCl2. Amplification was carried out starting from 1 μL of each bacterial colony in a final volume of 20 μL in a mixture containing 1 μM of each primer, 200 μM of each dNTP, 2.5 mM MgCl2, and 0.025 U/µL Taq DNA polymerase (Fermentas). The PCR conditions were: 94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 51 °C for 30 s, 72 °C for 45 s, and extension at 72 °C for 7 min (PTC-200 thermocycler; MJ Research, Waltham, MA, USA).
Vero cells were infected for 1 h at 37 °C with 0.4 PFU ZIKV (MR766 strain), a kind gift from M.R. Capobianchi (I.N.M.I.), and maintained in DMEM with 5% heat-inactivated calf serum. After 5 days, the cells were harvested, disrupted, and centrifuged at 400 × g for 5 min. The supernatant was then aliquoted and titered according to the Vero cells.
To determine whether the Env protein was expressed, replication nonpermissive Vero cells were infected for 1 h at 37 °C using FPzenv (10 PFU/cell). After overnight incubations, the samples were collected, run on 12.5% polyacrilamide gels, and examined by Western blotting, as described previously [51, 52]. The blotted nitrocellulose membranes were incubated overnight at 4 °C using the human polyclonal anti-ZIKV–specific serum (dilution, 1:200), and rabbit polyclonal antibodies or mouse monoclonal antibodies (always at dilution 1:500). The primary antibodies were followed by horseradish-peroxidase-conjugated secondary antibodies, as goat anti-human serum (dilution, 1:1,000; DakoCytomation, Carpinteria, CA, USA) or goat anti-rabbit (dilution, 1:2,000) or goat anti-mouse (dilution, 1:1,000). After a 1-h incubation and 2-h washes, the proteins were revealed using the ECL system (Western Lightning Plus-ECL; PerkinElmer, Waltham, MA, USA) followed by exposure of the nitrocellulose membranes to a hyperfilm for different times (Amersham Hyperfilm ECL; GE Healthcare, Buckinghamshire, UK). Cells infected with FP wild-type and with ZIKV were used as negative and positive controls, respectively.
Immunofluorescence was carried out as already described , using CEFs and Vero and MRC-5 cells, to examine the expression and subcellular localization of the ZIKV Env protein. Briefly, the cells were seeded at a density of 5 × 105/35-mm-diameter dish on sterile glass coverslips. After infection with FPzenv (5 PFU/cell; except for CEFs, which were infected with 0.5 PFU) at 37 °C for 1 h, the cells were incubated overnight at 37 °C in DMEM supplemented with 2% fetal calf serum. The cells were then washed twice with PBS–, and fixed with 2% paraformaldehyde (Polysciences) in PBS– for 10 min at room temperature, followed by 100% cold acetone for 5 min at 4 °C. The samples were incubated with the 1:100-diluted human polyclonal anti-ZIKV serum, which was a kind gift from M.R. Capobianchi, or with the 1:50-diluted rabbit polyclonal anti-Env serum (GeneTex Int. Corp., Inc., Irvine, CA, USA), or with the 1:50-diluted mouse monoclonal anti-Env antibody (GeneTex). The primary antibody was followed by the 1:50-diluted FITC goat anti-human or sheep anti-rabbit or goat anti-mouse antiserum (Cappel, MP Biomedicals, Inc., Aurora, OH, USA). FPwt and ZIKV that were previously produced in our laboratory were used to infect the cells, as negative and positive controls, respectively. The samples were viewed under a fluorescence microscope (Axioskop; Zeiss).
Ultrastructural analysis by transmission electron microscopy
Confluent MRC-5 and Vero cells were infected with 6 or 4 or 2 or 1 PFU/cell FPzenv, and CEFs with 0.05 or 0.1 or 0.5 PFU/cell, for 1 h at 37 °C, and were collected 3 days post infection (p.i.). ZIKV was used at 1 PFU/cell as the positive control. Inclusion was performed as already described . Briefly, after centrifugation at 1,000 × g for 10 min, the cells were all fixed in 2.5% glutaraldehyde (Polysciences, Warrington, PA, USA) in 0.1 M Na cacodylate buffer, pH 7.4, for 1 h at 4 °C, and then rinsed twice and post-fixed in cacodylate-buffered 1% OsO4 at 4 °C for 1 h. The specimens were dehydrated through a series of graded ethanol solutions and propylene oxide, and embedded in Poly/Bed 812 epoxy resin mixture. Sectioning was performed with an ultramicrotome (MT2B; Sorvall, New York, NY, USA) equipped with a diamond knife. After staining with water-saturated uranyl acetate and 0.4% lead citrate in 0.1 M NaOH, ultra-thin sections were examined using an electron microscope (CM10; Philips, Eindhoven, The Netherlands).
Expression over time of viral RNA transcripts in Vero cells
Confluent replication-restrictive Vero cells (1.5 × 106 cells/Petri dish; diameter, 5 cm) were infected with FPzenv at 5 PFU/cell for 1 h at 37 °C. The cells were rinsed twice with PBS−, scraped from the Petri dishes with a rubber policeman every 3 days for 4 weeks, and centrifuged at 1500 × g for 5 min at room temperature. Cell lysis and RNA extraction were performed according to the QIagen RNeasy mini kit protocol, following the manufacturer instructions, with minor modifications. Briefly, 350 µL RLT lysis buffer was added to the cell pellets, which were resuspended before freezing at − 80 °C. When all of the samples were ready, RNA extractions started by adding to each sample one volume 75% ethanol. The RNA was transferred to the kit columns, which were then centrifuged for 15 s at 8000 × g at room temperature. The columns were washed four times with wash buffer, as indicated by the manufacturer. The DNase treatment, after the first wash/ centrifugation cycle with 500 µL RPE, was also performed using the DNaseI incubation mix (QIagen, RNase-free DNase sets; 10 µL DNaseI in 70 µL RDD buffer). After the last wash with 500 µL RPE, elution was performed with 60 µL RNase/DNase-free water, and the RNA concentrations were determined using a spectrophotometer (SmartSpec 3000; BioRad, Hercules, CA, USA). RT-PCR was performed using RT-PCR system kit (Access; Promega, Madison, WI, USA). Briefly, 50 ng RNA was used in a final volume of 20 μL in the presence of 1 μM of each primer, 250 μM of each dNTP, 1 U Thermus filiformis DNA polymerase, 1 U Avian Myeloblastosis Virus reverse transcriptase, and 3 mM MgSO4. The ZIKV env-specific primers V438 and V441 were used to obtain a 661-bp fragment. RNAs from ZIKV-infected and noninfected Vero cells were used as positive and negative controls, respectively. The reverse transcriptase reaction was performed at 45 °C for 45 min, followed by 2 min at 94 °C. PCR amplification was carried out for 40 cycles at 94 °C for 30 s, 58 °C for 30 s, and 68 °C for 45 s, followed by a final incubation at 68 °C for 7 min. β-actin was amplified, which gave a band of 518 bp using 5 ng RNA in a final volume of 20 µL, under the conditions described above, except that 1 mM MgSO4 was used. Primers V84 (5′ CTG ACT ACC TCA TGA AGA TCC T 3′ nt 630–651) and V85 (5′ GCT GAT CCA CAT CTG CTG GAA 3′ nt 1147–1127) were used. The PCR products were run on 1% agarose gels, and gel images were acquired by Speedlight Platinum apparatus (Lightools Research, Encinitas, CA, USA).
Two groups of 7-week-old female BALB/c mice were used (Charles River Laboratories, Wilmington, MA, USA), as seven mice/group (Fig. 3a). For the control Group 1 (G1), we used the pVAXgp plasmid (10 + 50 µg/mouse), followed by FPgp (1 × 106 PFU/mouse), where both the plasmid and the viral recombinants contain the same irrelevant SIVmacM766 gag/pro gene, previously described. For the experimental Group 2 (G2), we used the pVAXzenv plasmid (10 + 50 µg/mouse), followed by FPzenv (1 × 106 PFU/mouse) where both the plasmid and the viral recombinants contain the same ZIKV zenv gene, previously described. Before each immunization, the mice were anesthetized by intramuscular (i.m.) injection of 30 µL of a mixture of 3.5 µL Rompun (stock, 20 mg/mL; Bayer SpA, Milan, Italy) plus 5.7 µL Zoletil 100 (Virbac Srl, Milan, Italy) and 35.7 µL PBS–. The vaccination course with pVAXgp or pVAXzenv consisted of 50 μg i.m. injection and 10 µg s.c. injection, followed by electroporation. For the electroporation, one 50-ms transcutaneous low-voltage electric pulse (amplitude, 100 V) was administered at the i.m. injection site via a multiple-needle electrode connected to the electroporation apparatus (ECM830, BTX i45-168, Holliston, MA, USA). Priming was followed by four boost administrations of FPzenv: two s.c., one i.n., and one both i.n. and s.c.. Challenge with ZIKV (1 × 105 PFU/mouse) was performed s.c. at 10 days after the last immunization. For 6 days before the ZIKV challenge and for 4 days after the ZIKV challenge, the mice were immune suppressed with dexamethasone (Soldesam, 4 mg/mL; LFM, Milan, Italy) intraperitoneally : 50 mg/kg on the first 2 days, and 25 mg/kg for the following days. Bleedings were performed from the retro-orbital eye plexus before the first immunization (Fig. 3a, T0), before each subsequent immunization (Fig. 3a, T1–T5), and at different intervals thereafter, as indicated. The plasma fractions were aliquoted and frozen at − 80 °C. Dexamethasone was withdrawn 4 days after the challenge (T9) and 4 days before the sacrifice (T10, T11).
The mice were also monitored during the whole treatment period for weight loss until euthanasia. The experimental group did not show any significantly differences in weight compared to the control mice, with the weight variations seen as < 15%, compared to the starting period. All of the mice were maintained according to the Italian National Guidelines and the EU Directive 2010/63/EU for animal experiments. They were observed for signs of disease, and provided with food and water ad libitum. Every effort was made to minimize their suffering. Approval for this study was granted by the Ethical Committee of the University of Milan.
Enzyme-linked immunosorbent assay
The mouse plasma samples from T0 to T11 were assayed for antibodies against ZIKV Env-specific proteins using enzyme-linked immunosorbent assays (ELISAs). Vero cells (1.5 × 106) previously infected for 2 days with FPzenv (2 PFU/cell) were used as the antigen, after plating in 96-well microtiter plates (MaxiSorp; Nunc, Thermoscientific, Roskilde, Denmark). Briefly, after infection and washing with PBS–, the cells were freeze-thawed three times, harvested with a rubber policeman, passed through the needle of an insulin syringe (30 G × 8 mm), and centrifuged for 5 min at 800 × g. Following resuspension in 0.05 M carbonate-bicarbonate buffer, pH 9.6 (15 mM Na2CO3, 35 mM NaHCO3, 0.2% NaN3), 1 × 105 cells in 50 µL were added to the wells of 96-well plates. The antigen was incubated overnight at 4 °C. ELISAs were performed in duplicate, essentially as described previously , using serum from each animal of both groups of mice (G1, control group; G2, experimental group) from T0 to T11. The sera dilutions were 1:1,000. The reactions were revealed using goat anti-mouse horseradish-peroxidase-conjugated serum (dilution, 1:1,000; DakoCytomation, Glostrup, Denmark) and tetramethylbenzidine substrate (Sigma–Aldrich). The pre-immune mouse sera (T0) were used as the negative controls. The absorbance of each well was read at 450 nm using a microplate reader (550; Bio-Rad, Hercules, CA, USA). Inactivated ZIKV (4 × 105 PFU/well) and the recombinant ZIKV Env-specific protein (10–300 ng, ZIKV envelope domain III, European Virus Archive goes Global, EVAg, Marseille, France) were also used as antigens (serum dilution, 1:100).
Virus neutralization assays
The neutralizing activities of the mice sera were determined by measuring the extent of in-vitro inhibition of virus infectivity at T0 (pre-immune serum) and T6 (pre-challenge serum). The assays were performed as previously described , by pre-incubation of an equal volume of ZIKV with heat-inactivated mouse serum, used at different dilutions (1:50 to 1:1600, in DMEM without serum) in 48-well plates, for 1 h at 37 °C. Briefly, the viral titer was adjusted to provide approximately 80 PFU ZIKV in the assays. The infections were performed in duplicate on confluent Vero cells, and were allowed to proceed for 1 h at 37 °C. The same amount of virus incubated with DMEM was used as the control. Two days later, 5 mL medium was added to maintain the correct pH, and 5 days p.i. the cells were fixed in 3 mL methanol:acetic acid (3:1; v/v) for 1–3 h at room temperature. After removing the fixing solution and the agarose overlay, staining was performed using 1 mL 2% crystal violet dye in methanol. The neutralizing activity is expressed as the plaque reduction numbers and calculated by comparing the plaque numbers after incubating the virus with immune sera to the plaque numbers found after incubating the virus with no serum or with pre-immune sera.
Viral RNA amplification to determine ZIKV after the challenge
To determine whether ZIKV was present after the challenge in the vaccinated mice, the viral RNA was extracted from the sera obtained at T7-T11 from the control and experimental mice, using QIAamp viral RNA mini kit (QIagen), according to the manufacturer instructions. Amplifications were performed using 50, 70, 250, 280, 400 ng of each RNA. Sera of some individual mice were also tested using 800 ng RNA. RT-PCR was performed using RT-PCR system kit (Access; Promega), as described above, using primers V438/V441 and under essentially the same conditions, with 3 mM MgSO4 and 58 °C annealing temperature, which was the most suitable for ZIKV detection.
Statistical analyses were performed using parametric t-tests and areas under the curves (AUCs), using the GraphPad Prism version 2.0 software. Statistical significance was set as p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).