An attenuated Zika virus NS4B protein mutant is a potent inducer of antiviral immune responses

Live attenuated vaccines (LAVs) are one of the most important strategies to control flavivirus diseases. The flavivirus nonstructural (NS) 4B proteins are a critical component of both the virus replication complex and evasion of host innate immunity. Here we have used site-directed mutagenesis of residues in the highly conserved N-terminal and central hydrophobic regions of Zika virus (ZIKV) NS4B protein to identify candidate attenuating mutations. Three single-site mutants were generated, of which the NS4B-C100S mutant was more attenuated than the other two mutants (NS4B-C100A and NS4B-P36A) in two immunocompromised mouse models of fatal ZIKV disease. The ZIKV NS4B-C100S mutant triggered stronger type 1 interferons and interleukin-6 production, and higher ZIKV-specific CD4+ and CD8+ T-cell responses, but induced similar titers of neutralization antibodies compared with the parent wild-type ZIKV strain and a previously reported candidate ZIKV LAV with a 10-nucleotide deletion in 3′-UTR (ZIKV-3′UTR-Δ10). Vaccination with ZIKV NS4B-C100S protected mice from subsequent WT ZIKV challenge. Furthermore, either passive immunization with ZIKV NS4B-C100S immune sera or active immunization with ZIKV NS4B-C100S followed by the depletion of T cells affords full protection from lethal WT ZIKV challenge. In summary, our results suggest that the ZIKV NS4B-C100S mutant may serve as a candidate ZIKV LAV due to its attenuated phenotype and high immunogenicity.

independent of MAVS signaling. BM-macrophages of WT and Mavs −/− mice were infected with ZIKV mutants (MOI =2). At day 4 pi, viral load was measured by Q-PCR (a) and FFA (b). Cytokine production was determined by using Q-PCR (c-e). Data are presented as the fold increase compared to mockinfected (n= 4 per group). Data are presented as means ± s.e.m. and are representative of 3 similar experiments. ** P < 0.01 or *P < 0.05 compared to WT group (Unpaired t test). ## P < 0.01 or # P < 0.05 compared to Δ10 group (Unpaired t test). Figure 3. The effect of NS4B on type 1 IFN production and signaling. For type 1 IFN production, HEK-293T cells were co-transfected with IFN-β promoter firefly luciferase reporters, renilla luciferase plasmid, ZIKV NS4B-encoding plasmid or empty control plasmid, and RIG-I-(2CARD) stimulating plasmid. Data were normalized first by Renilla luciferase values, and then normalized by none stimulated samples to obtain fold induction. Empty vector samples were set as 100% fold induction. For type-1 IFN signaling, HEK-293T cells were co-transfected with ISRE firefly luciferase reporter plasmid and NS4B expressing plasmid or empty plasmid vector. At 24 h p.i., the cells were treated with 1,000 units/ml of IFN-α for 12 h. The luciferase activities were measured to indicate the IFN-α signaling. Data were presented as mean ± SD. Results are representative of 3 independent experiments with each one in triplicate. No significance (n.s.) indicates P > 0.05 compared to WT NS4B (Unpaired t test).

Indirect immunofluorescence assay (IFA): Transfected cells were seeded in an 8-well Nunc
Lab-Tek II Chamber Slide (Thermo Fisher Scientific). At various time points, cells were washed three times with phosphate-buffered saline (PBS) and fixed by cold methanol for 30 min at -20℃. The cells were then washed and incubated in a blocking buffer containing 1% FBS in PBS at room temperature for 1 h. Cells were stained with mouse monoclonal antibody 4G2 (ATCC; HB-112; 1:40), followed with a secondary antibody (Alexa Fluor®488 conjugated goat anti-mouse IgG, Invitrogen, A11001; 1:1000).
The cells were washed three times with washing buffer and counterstained with DAPI (4',6-diamidino-2phenylindole). Images were acquired using an Olympus fluorescence microscope. The images were processed using ImageJ software (National Institutes of Health, MD).
Plaque assay: Vero cells (2×10 5 per well) were seeded in 24-well plates 1 day prior to viral infection. Ten-fold serial dilutions of virus samples were prepared in DMEM containing 2% FBS and 1% penicillin/streptomycin and inoculated (100 µl) into each well. After 1 h incubation at 37°C with 5% CO2, the inoculum was replaced with 500 µl overlay medium. Plaques were developed after incubating the plates at 37°C with 5% CO2 for 4 days. Following the incubation, methyl cellulose overlay was removed and 0.5 ml methanol-acetone (1:1) solution was added into each well and incubated at room temperature for 15 min. Fixation solution was aspirated and plates were allowed to air dry. Plates were washed three times with PBS and incubated in blocking buffer with 3% FBS, followed by 1 h incubation with ZIKVspecific hyper-immune ascitic fluid (World Reference Center of Emerging Virus and Arboviruses WRCEVA). Plates were washed three times with PBS followed by 1 h incubation with a secondary antibody conjugated to horseradish peroxidase (Seracare; 5220-0286;1: 2000). Detection of immunostained foci were developed with the addition of aminoethylcarbazole substrate (ENZO Life sciences) according to the vendor's instructions.

Stability studies of NS4B mutant viruses:
To examine the stability of NS4B mutants, we passaged the virus on Vero cells for five rounds. Briefly, 1.5×10 6 Vero cells were seeded into T-25 flask.
The virus derived from RNA-transfected cells (defined as P0) was used to infect naive Vero cells. At 5 day post-infection, culture fluid was transferred to a new T-25 flask containing Vero cells in 5 ml of culture medium. After five rounds of such passaging, viral RNAs were extracted from P5 culture fluids using QIAamp Viral RNA Kit (Qiagen) and amplified by RT-PCR using SuperScript III one-step RT-PCR kits (Invitrogen). The RT-PCR products were directed subjected to DNA sequencing.
Type 1 IFN induction and signaling reporter assays: WT and C100S mutant NS4B gene of ZIKV were cloned into expression vector pXJ (1). For the type-1 IFN production assay, HEK-293T cells (ATCC) were co-transfected (1×10 5 cells per well in 24-well plate) with 5 ng of RIG-I (2CARD) expressing plasmid, 15 ng of IFN-β promoter luciferase reporter plasmid, and 20 ng of NS4B expressing plasmid using X-tremeGENE™ 9 (Roche) with a ratio 1: 2. Empty pXJ vector was used to ensure the same total amount of plasmids in each well. At 36 h, the cells were assayed for dual-luciferase activities per manufacturer's instructions (Promega) For testing type-I IFN signaling, HEK-293T cells were cotransfected with 250 ng of ISRE firefly luciferase reporter plasmid, 20 ng of Rinella luciferase plasmid (as a transfection efficiency control), and 230 ng of NS4B expressing plasmid. At 24 h, 1,000 units/ml of IFN-α (Millipore) was added to the cells. After another 12 h incubation, the cells were assayed for luciferase activities.