Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination (original) (raw)

Ethics statement

Animals

The investigators adhered to the Guide for the Care and Use of Laboratory Animals by the Committee on Care of Laboratory Animal Resources Commission on Life Sciences, National Research Council. Mouse studies were conducted under protocols approved by the University of Pennsylvania (UPenn) IACUCs. Rhesus macaques (Macaca mulatta) were housed at Bioqual, Inc. (Rockville, MD). Macaque experiments were reviewed and approved by Bioqual and UPenn Animal Care and Use Committees. All animals were housed and cared for according to local, state and federal policies in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility.

Human cells

Research involving human cells complied with the Declaration of Helsinki. De-identified leukapheresis cells were obtained from the UPenn Immunology Core under their Institutional Review Board (IRB)-approved protocol, and were deemed exempt by the UPenn IRB.

Antibody reagents

The pan-flavivirus mouse monoclonal antibody 4G2, clone D1-4G2-4-15 (EMD Millipore MAB10216) was used to detect ZIKV E protein by western blot. The following antibodies were used for flow cytometry: anti-CD4 PerCP/Cy5.5 (clone GK1.5, Biolegend), anti-CD3 APC-Cy7 (clone 145-2C11, BD Biosciences), anti-CD27 PE (clone LG.3A10, BD Biosciences), anti-TNF PE-Cy7 (clone MP6-XT22, BD Biosciences), anti-IFNγ AF700 (clone XMG1.2, BD Biosciences), anti-IL-2 APC (clone JES6-5H4, BD Biosciences). The live/dead fixable aqua dead cell stain kit (Life Technologies) was used to discriminate dead cells and debris. The following antibodies were used for ELISA assays: goat anti-mouse IgG HRP (Sigma 4416), goat anti-monkey IgG HRP (Sigma 2054) and ZIKV E-protein-specific monoclonal antibody NR-4747 clone E19 (BEI Resources). ZIKV-neutralizing human monoclonal antibody Ab3594 was provided by the Duke University, Duke-NUS Graduate Medical School, National University of Singapore team of C.E.M, G.D.S., R.P., E. E. Ooi, B.F.H., M. A. Moody, S. Lok, and H.-X. Liao.

Protein reagents

Purified recombinant ZIKV E protein (Aalto Bioreagents AZ 6312) was used in ELISAs to detect E-protein-specific IgG, in western blots as a positive control and in mouse splenocyte stimulation.

mRNA production

mRNA was produced as previously described25 using T7 RNA polymerase on linearized plasmid (pTEV-ZIKVprM-E-A101) encoding codon-optimized26 ZIKV strain H/PF/2013 (Asian lineage, French Polynesia, 2013, GenBank: KJ776791) prM–E glycoproteins. mRNA was transcribed to contain a 101 nucleotide-long poly(A) tail. 1-methylpseudouridine-5′-triphosphate (TriLink) was used instead of UTP to generate modified nucleoside-containing mRNA. mRNA was capped using a m7G capping kit with 2′-_O_-methyltransferase to obtain cap1 and was purified by a fast protein liquid chromatography (FPLC) method, as described27. mRNA was analysed by agarose gel electrophoresis and stored frozen at −20 °C.

Cell culture

Human embryonic kidney (HEK)293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM l-glutamine (Life Technologies) and 10% fetal calf serum (FCS) (HyClone) (complete medium). The HEK293T cell line was tested for mycoplasma contamination after receipt from ATCC and before expansion and cryopreservation. Human dendritic cells were generated from monocytes, as described28, and grown in RPMI1640 medium containing 2 mM l-glutamine (Life Technologies) and 10% FCS (HyClone) (complete medium) supplemented with 50 μg ml−1 recombinant human GM-CSF and 100 μg ml−1 recombinant human IL-4 (R&D systems). Cells were maintained by adding fresh medium containing IL-4 and GM-CSF every 3 days and were used on day 7. Mouse dendritic cells were generated from bone marrow cells obtained from the femurs of animals and grown in complete medium supplemented with 50 μg ml−1 mouse GM-CSF (R&D systems). Cells were maintained by adding fresh medium containing mouse GM-CSF every 3 days and were used on day 7.

mRNA transfection

Transfection of human and mouse dendritic cells and HEK293T cells was performed with TransIT-mRNA (Mirus Bio) according to the manufacturer’s instructions: mRNA (0.3 μg) was combined with TransIT-mRNA reagent (0.34 μl) and boost reagent (0.22 μl) in 17 μl of serum-free medium, and the complex was added to 2 × 105 cells in 183 μl complete medium. Supernatant was collected and cells were lysed for 1 h on ice in RIPA buffer (Sigma) at 18 h after transfection.

Western blot analysis of envelope protein expression

Whole-cell lysates and supernatants from cells transfected with ZIKV prM–E were assayed for ZIKV E protein by non-denaturing SDS–PAGE western blot. Samples were combined with 4 × Laemmli buffer (Bio-Rad) and separated on a 4–15% precast polyacrylamide Criterion TGX gel (Bio-Rad) for 45 min at 200 V. Transfer to PVDF membrane was performed using a semi-dry apparatus (Ellard Instrumentation, Ltd) at 10 V for 1 h. The membrane was blocked with 5% non-fat dry milk in TBS buffer containing 0.5% Tween-20. E protein was detected using 1:10,000 4G2 ascites for 1 h, followed by secondary goat anti-mouse IgG HRP 1:10,000 for 1 h. Antibody incubations were performed at room temperature in blocking buffer. Blots were developed using Luminata Forte substrate (Millipore) and a Kodak X-OMAT 1000A processor. At least 2 independent experiments were performed.

Characterization of E protein in supernatant

Supernatant from HEK293T cells transfected with ZIKV prM–E mRNA was tested for whether E protein could be pelleted and disrupted with detergent, consistent with subviral particles. Supernatant was incubated in PBS alone or PBS with 0.5% Triton X-100 for 1 h on ice. Samples were then spun at 42,000 r.p.m. for 2.5 h in a Beckman TLA-55 rotor. The supernatant was then removed from the pellet, which was resuspended in 50 μl of PBS. Equal volumes of the input, pellet and post-centrifugation supernatant fractions were then analysed by western blot, as described above.

Lipid-nanoparticle encapsulation of the mRNA

FPLC-purified mRNA and polycytidylic acid (poly(C) RNA) (Sigma) were encapsulated in LNPs using a self-assembly process in which an aqueous solution of mRNA at pH 4.0 is rapidly mixed with a solution of lipids dissolved in ethanol29. LNPs used in this study were similar in composition to those described previously29,30, which contain an ionizable cationic lipid (proprietary to Acuitas), phosphatidylcholine, cholesterol and PEG-lipid (with a ratio of 50:10:38.5:1.5 mol/mol) and were encapsulated at an RNA to total lipid ratio of around 0.05 (wt/wt). The LNPs had a diameter of around 80 nm as measured by dynamic light scattering using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd). RNA–LNP formulations were stored at −80 °C at a concentration of RNA of about 1 μg μl−1.

Administration of LNPs to mice and rhesus monkeys

Mice

Female BALB/c and C57BL/6 mice aged 8 weeks were purchased from Charles River Laboratories, and cages of mice were randomly allocated to groups. Power analysis was used to calculate the size of all animal groups to ensure statistically significant results. RNA–LNP was diluted in PBS and injected into animals intradermally with a 3/10cc 29½G insulin syringe (BD Biosciences). Four sites of injection (30 μl each) over the lower back were used.

Monkeys

Ketamine-anaesthetized animals were shaved on their back and injected with mRNA–LNP diluted in PBS. Ten sites of injection (60 μl each) were used. Animals of similar age and weight were allocated to each group.

Blood collection from mice and rhesus macaques

Mice

Blood was collected from the orbital sinus under isoflurane anaesthesia. Blood was centrifuged for 10 min at 13,000 r.p.m. and the serum was stored at −20 °C and used for ELISA and virus neutralization assays. EDTA-plasma was collected to isolate RNA for quantitative PCR with reverse transcription (qRT–PCR) analysis.

Monkeys

Blood was collected by femoral venipuncture under ketamine anaesthesia, and serum and EDTA–plasma were collected and stored at −80 °C for ELISA, neutralization analysis and to isolate RNA for qRT–PCR.

Stimulation and staining of splenocytes

Single-cell suspensions from spleens were made in complete medium. Splenocytes were washed once in PBS and resuspended in complete medium at 2 × 107 cells per ml. Subsequently, 2 × 106 cells (100 μl) per sample were stimulated for 6 h at 37 °C using 2 μg ml−1 purified, recombinant ZIKV E protein. GolgiPlug (brefeldin A, BD Biosciences) and GolgiStop (monensin, BD Biosciences) were diluted 1:100 and 1:143 in complete medium, respectively, and 20 μl from both diluted reagents was added to each sample to inhibit the secretion of intracellular cytokines after 1 h. An unstimulated sample for each animal was included. Samples stimulated with PMA (10 ng ml−1)–ionomycin (250 ng ml−1) (Sigma) were used as positive controls.

After stimulation, cells were washed in PBS and stained using the live/dead fixable aqua dead cell stain kit (Life Technologies) and then surface stained for CD4 and CD27. Antibodies were incubated with cells for 30 min at room temperature. Following surface staining, cells were washed in FACS buffer and fixed using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Following fixation, the cells were washed in the appropriate perm buffer and incubated with antibodies against CD3, TNF, IFNγ and IL-2 for 1 h at room temperature. Following staining, the cells were washed with the appropriate perm buffer, fixed (PBS containing 1% paraformaldehyde) and stored at 4 °C until analysis. Results are obtained from one technical replicate.

Flow cytometry

Splenocytes were analysed on a modified LSR II flow cytometer (BD Biosciences). One hundred thousand events were collected per sample. After the gates for each function were created, the Boolean gate platform was used to create the full array of possible combination of cytokines, equating to seven response patterns when testing three cytokines. Data were expressed by subtracting the per cent positive unstimulated cells from the per cent positive cells stimulated with E protein.

Enzyme-linked immunosorbent assays (ELISA) for ZIKV E-specific IgG

Immulon 4HXB ELISA plates were coated with 6 μg ml−1 purified, recombinant ZIKV E protein in 0.1 M sodium bicarbonate buffer overnight at 4 °C. The plate was blocked with 2% BSA in PBS for 1 h, and washed three times with wash buffer (PBS with 0.05% Tween-20). Mouse or rhesus macaque serum was diluted in blocking buffer and incubated on the plate for 1 h at room temperature, followed by four washes. Secondary HRP-conjugated antibody was diluted 1:10,000 in blocking buffer and incubated on the plate for 1 h, followed by four washes. TMB substrate (KPL) was applied to the plate and the reaction was stopped with 2 Normal sulfuric acid. The absorbance was measured at 450 nm using an MRX Revelation microplate reader. ZIKV E-protein-specific IgG was analysed in two ways: as an endpoint dilution titre, defined as the highest reciprocal dilution of serum to give an OD greater than the sum of the background plus 0.01 OD units; and as an estimate of the absolute IgG concentration, which was based on the mouse monoclonal antibody NR-4747 as a standard (applicable only to mouse samples). All samples were run in at least technical duplicates.

ZIKV MR-766 plaque reduction neutralization tests (PRNT)

ZIKV strain MR-766 (African lineage, Uganda, 1947, GenBank: AY632535) (UTMB Arbovirus Reference Collection) was produced in Vero cells (ATCC CCL-81) and 50 PFU were incubated with increasing dilutions of heat-inactivated serum in serum-free DMEM (Corning) medium for 1 h at 37 °C. The virus–serum mixture (200 μl) was added to a confluent monolayer of Vero cells in 6-well format and incubated for 1.5 h at 37 °C with intermittent rocking. Then, 3 ml of overlay, containing a final concentration of 0.5% methylcellulose (4,000 centipoise) (Sigma), 1 × DMEM (Gibco), 16 mM HEPES, 0.56% sodium bicarbonate, 1.6× GlutaMAX (Gibco), 1 × penicillin/streptomycin (Corning), and 4 μg ml−1 amphotericin B (Gibco), was added to each well, and plates were incubated for 5 days at 37 °C in 5% CO2. The overlay was aspirated and cells were fixed and stained with 0.5% crystal violet (Sigma) in 25% methanol, 75% deionized water. Wells were rinsed with deionized water to visualize plaques. Neutralization titres (EC50) were determined by plotting a line through the linear portion of the curve that crossed 50% inhibition and calculating the reciprocal dilution of serum required for 50% neutralization of infection. EC50 titres are reported as the mean of one or two technical replicates and values below the limit of detection are reported as half of the limit of detection.

ZIKV MEX I-44 focus reduction neutralization tests (FRNT)

ZIKV MEX I-44 (Asian lineage, Mexico, 2016, GenBank: KX856011) stocks were generated in Vero 76 cells (ATCC CRL-1587) and collected as clarified cell-culture lysate/supernatant. FRNT was performed by combining a standard dose of ZIKV with twofold serial dilutions of heat-inactivated serum for 1 h at 37 °C. Virus–serum mixtures (100 μl) were then inoculated onto Vero 76 monolayers, incubated at 37 °C for 1 h and overlayed with an Avicel (FMC Biopolymer)-containing growth medium. After 3 days of incubation, plates were formalin-fixed, permeabilized, blocked and stained by sequential incubation with a biotin-conjugated 4G2 monoclonal antibody (ATCC HB-112), streptavidin-HRP (BD Biosciences) and TrueBlue peroxidase substrate (KPL). Virus input was verified in parallel (acceptable range: 20–60 foci). FRNT50 (EC50 titres) are reported as the highest reciprocal dilution giving a focus count ≤ the 50% neutralization cutoff, and the geometric mean was computed for technical duplicates.

Reporter virus particle (RVP) production

Pseudo-infectious RVPs were produced by complementation of a GFP-expressing WNV sub-genomic replicon23,31 with a plasmid encoding the viral structural proteins (capsid-prM–E). Briefly, ZIKV MR-766 and ZIKV H/PF/2013 RVPs were produced via co-transfection of HEK293T cells with the structural gene and replicon plasmids (3:1 ratio by mass) using Lipofectamine 3000 per the manufacturer’s protocol (Invitrogen). Transfected cells were incubated at 30 °C and RVP-containing supernatants were collected on days 3–6. Stocks were passed through a 0.2-μm filter and aliquots were stored at −80 °C until use. Stock titres were determined by infecting Raji-DCSIGNR cells with serial dilutions of filtered RVP supernatants. GFP-positive cells were assessed by flow cytometry at 48 h after infection and RVP titres were calculated.

RVP neutralization assay

Previously titred RVPs were diluted to ensure antibody excess at informative points on the dose–response curves and were incubated with serial dilutions of mouse or macaque serum for 1 h at 37 °C to allow for steady-state binding. Raji-DCSIGNR cells were then infected with antibody–RVP complexes in two technical replicates. Infections were carried out at 37 °C and GFP-positive infected cells were detected by flow cytometry 24–48 h later. Neutralization results were analysed by nonlinear regression to estimate the reciprocal dilution of serum required for half-maximal neutralization of infection (EC50 titre) (Prism 6, GraphPad). The initial dilution of serum (on the basis of the final volume of RVPs, cells, and serum) was set as the limit of confidence of the assay. Titres for which nonlinear regression was predicted to be below this threshold were reported as a titre half the limit of confidence. Individual EC50 titres are reported as the geometric mean of at least 2 technical replicates.

Preparation of challenge ZIKV virus

Mice

Challenge ZIKV strain PRVABC59 (Asian lineage, Puerto Rico, 2015, GenBank: KU501215) (BEI Resources NR-50240) was grown in Vero CCL81 cells. A T175 flask of cells at 75–90% confluency was inoculated with an MOI of 0.01 ZIKV in 10 ml of serum-free DMEM medium. The flask was incubated at 37 °C, 5% CO2 for 1.5 h with intermittent rocking, then warmed medium was added to a final concentration of 1.5% FCS, 1 × GlutaMAX (Gibco) and 1 × penicillin/streptomycin (Corning) in a final volume of 25 ml. The flask was incubated for 4 days or until cytopathic effects were visible. Then, the supernatant was collected, clarified by low-speed centrifugation and ultra-centrifuged at 20,000 r.p.m. for 1 h at 4 °C in a Sorvall SureSpin 630 rotor. The supernatant was removed and the pellet was resuspended in 1 ml of serum-free DMEM, aliquoted and stored at −80 °C. Before challenge, virus was thawed and diluted in PBS to 2,000 PFU per ml.

Monkeys

Challenge ZIKV strain PRVABC59 was grown in Vero 76 CRL-1587 cells. T150 flasks of cells at 80–85% confluency were used for propagation. Infection was performed with 100 μl stock virus diluted in 4 ml of fresh L-15 medium (Gibco) supplemented with 10% FCS (Gibco), 10% tryptose phosphate broth (Sigma Aldrich), 1 × penicillin/streptomycin (Gibco) and l-glutamine (Gibco) and adsorbed for 1 h at room temperature with gentle agitation every 15 min. Each flask received 7 ml of fresh L-15 medium after adsorption and was incubated for 4 days at 37 °C. Cellular debris was removed by centrifugation at 1,200 r.p.m. for 5 min at 4 °C in an Eppendorf A-4-62 rotor. Virus stocks were aliquoted and stored at −80 °C.

Zika virus challenge in mice and rhesus macaques

Mice

At 2 or 20 weeks after vaccination, mice were bled and then challenged intravenously with 200 PFU of ZIKV PRVABC59 in 100 μl of PBS. Blood was collected 3 and 7 days after the challenge to determine viral loads (ZIKV RNA copies per ml) in plasma.

Monkeys

Macaques were anaesthetized with ketamine and injected subcutaneously in the hind thigh with 104 TCID50 of ZIKV PRVABC59 in a volume of 1 ml in PBS. Blood was collected 1, 3, 5, and 7 days after the challenge to determine viral loads (ZIKV RNA copies per ml) in plasma.

Viral load quantification (qRT–PCR)

Using blinded samples, RNA was isolated from 200 μl (macaque) or 50 μl (mouse) plasma using the QIAamp MinElute Virus spin kit (Qiagen). Extracted RNA was used for amplification using the SensiFAST Probe Lo-ROX One-Step Kit (Bioline BIO-78005) on a 7500 Real-Time PCR system (Applied Biosystems). Primers and probe were designed to amplify a conserved region of the capsid gene from ZIKV BeH815744, as follows: fwd 5′-GGAAAAAAGAGGCTATGGAAATAATAAAG-3′; rev 5′-CTCCTTCCTAGCATTGATTATTCTCA-3′; probe 5′-AGTTCAAGAAAGATCTGGCTG-3′.

Primers and probe were used at a final concentration of 2 μM, and the following program was run: 48 °C for 30 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 1 min at 60 °C. Assay sensitivity was 50 copies per ml for macaque and 200 copies per ml for mouse samples. Results are calculated from at least two technical replicates.

Statistical analysis

No statistical methods were used to predetermine sample size, unless indicated. The investigators were not blinded to allocation during experiments and outcome assessment unless indicated (qRT–PCR). GraphPad Prism 5.0f was used to perform Mann–Whitney and Kruskal–Wallis (with Dunn’s correction) tests to compare immune responses in vaccinated and control mice and in different dose groups of macaques, respectively. SPICE 5.35 and Microsoft Excel software was used to perform Student’s _t_-tests to compare T cell responses in vaccinated and control mice.

Data availability

All data and full plasmid sequences are available upon request from the corresponding author.