2.1. Cells and Viruses
cells (Invitrogen, Carlsbad, CA, USA) were propagated in Schneider’s medium supplemented with 10% FCS and 1% penicillin/streptomycin at 28 °C. Vero E6 (DSMZ, Braunschweig, Germany) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS and 1% penicillin/streptomycin at 37 °C with 5% CO2
. K562 cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium added with 10% FCS, 2 mM Glutamine and 1% penicillin/streptomycin at 37 °C with 5% CO2
. All viruses were grown in Vero E6 cells. ZIKV (Padova; Dominican Republic/2016/PD1; GenBank KU853012, kindly provided by Luisa Barzon, Padova University, Padova, Italy) was purified as previously described [25
]. After ultracentrifugation, viral pellets were resuspended in PBS containing 10% (w/v) sucrose. The purified virus was stored in aliquots at −80 °C until use. DENV-1 to -4 (DENV-1 isolate 2522/10, DENV-2 isolate 3229/11, DENV-3 isolate 3140/09, DENV-4 isolate 3274/0, kindly provided by Jonas Schmidt-Chanasit (Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany) were purified from virus containing cell culture supernatants 2–6 days post infection (dpi) by ultracentrifugation for 3 h at 4 °C. Viral pellets were resuspended in PBS with 10% (w/v) sucrose and stored in aliquots at −80 °C until use.
ZIKV titers were determined via the tissue culture infectious dose 50 (TCID50) assay based on endpoint dilutions. Briefly, 10-fold dilutions of viral stocks were incubated on Vero E6 cell monolayers and the cytopathic effect (CPE) was monitored 5 dpi. The 50% infectious dose was calculated using the Reed–Muench method [26
DENV-1 to -4 titers were determined by a focus forming assay. Viruses were serially diluted on Vero E6 cell monolayers in 96-well plates. After 1 h, the supernatant was removed and cells were overlaid with 1% methylcellulose in DMEM with 2% FCS and 1% penicillin/streptomycin. Two days later, the cells were fixed with 1% formaldehyde in PBS, permeabilized and blocked with Perm-Wash buffer (0.1% Saponin and 0.1% BSA in PBS). After the incubation of the primary flavivirus antibody 4G2 (Absolute antibody, Oxford, UK, 1:2000), several wash steps were applied with Perm-Wash buffer and followed by the incubation with an anti-mouse IgG HRP-conjugated secondary antibody (Dako, Glostrup, Denmark, 1:2000). For the detection of focus forming units, TrueBlue peroxidase substrate (Seracare Life Sciences, Milford, MA, USA) was used and the spots were counted automatically with an ELISpot reader (AID Diagnostika, Straßberg, Germany).
For ELISA measurements, ZIKV and DENV-1 to -4 were inactivated by treatment with 0.3% H2O2 in PBS at 37 °C overnight. Subsequently, the samples were dialyzed against PBS and stored in aliquots at −80 °C until further use.
2.3. Animal Experiments
All animal experiments were carried out in accordance with the EU Directive 2010/63/EU for animal experiments and were approved by local authorities (No.: TVA 02/18).
Groups of 6 (n = 6) nine weeks old, female BALB/c mice were immunized three times in 4-week intervals by intramuscular injection (hind leg) with 15 µg of either ZIKV Equad or ZIKV Ewt with the adjuvant 2% Alhydrogel (Invivogen, San Diego, CA, USA) at a ratio 1:1 (v/v). Control mice were not vaccinated. Serum samples were collected from blood before the first injection (prime) and thereafter before the boost immunizations by retrobulbar bleeding. Final blood sampling was performed 4 weeks after the second boost. The samples were inactivated for 30 min at 56 °C and stored in aliquots at −20 °C until use.
2.4. ELISA Analysis
Mouse sera were tested on microtiter plates coated either with the recombinant proteins ZIKV Equad, ZIKV Ewt, DENV Ewt (respectively 150 ng per well), WNV Ewt (200 ng per well) or inactivated viral particles in 100 µL buffer (15 mM Na2CO3, 7 mM NaHCO3 pH 9.6) overnight at 4 °C. The plates were then washed three times with PBS-0.05% Tween and blocked with 5% non-fat milk powder in PBS for 2 h at room temperature (RT). After another wash step, the incubation started with diluted mouse sera for 1.5 h at RT. Following a third wash step, the HRP-conjugated secondary anti mouse IgG antibody (Dako, Glostrup, Denmark, 1:3000) was added for 1 h at RT. After a final wash step, TMB substrate (Biozol, Eching, Germany) was added and incubated for 30 min at RT, before the reaction was stopped with 1 M H2SO4. The plates were analyzed at 450 nm with 520 nm as reference in a microplate reader (Infinite M200, Tecan, Männedorf, Switzerland). The ELISA assays on ZIKV proteins were performed with 1:1000 diluted sera, on DENV and WNV proteins serial diluted sera were used to determine endpoint titers. These were defined as the reciprocal of the highest dilution with signals were above the cut-off based on the control group which were calculated as the mean plus 8 standard deviations (SD) of sera from the control group. For the ELISA assay on inactivated viral particles, sera were diluted 1:100. Human sera were diluted 1:100 for ELISA assays on ZIKV Equad and Ewt and the secondary HRP-conjugated antibody anti human IgG was diluted 1:20,000 (Dianova, Hamburg, Germany). The human sera were kindly provided by Luisa Barzon (Padova University, Italy) and Jonas Schmidt-Chanasit (Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany). Ethical approvals were obtained from the respective local authorities for all samples. The 4G2 antibody (Absolute antibody, Oxford, UK) was diluted 1:1000 and the secondary mouse-antibody (Dako, Glostrup, Denmark) was diluted 1:3000. Tests were performed in duplicates and in two independent experiments.
Statistical analysis was performed using GraphPad Prism6 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical tests were calculated as ordinary one-way unpaired ANOVA or unpaired t-test, with * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.