Study design
We conducted a series of controlled experiments to evaluate the efficacy and immunogenicity of our DTMUV mRNA vaccine. The study comprised three sequential phases: (1) immunization of SPF ducks (n = 10; 5 vaccinated, 5 controls) to assess protective efficacy against viral challenge, (2) comparative analysis in male breeder drakes (n = 20; 10 per group) to benchmark against a commercial live-attenuated vaccine (FX2010-180P), and (3) evaluation in laying ducks (n = 12; 6 vaccinated, 6 controls) to assess maternal antibody transfer. Blood samples were collected at indicated intervals for serological analyses, and egg yolks from vaccinated laying ducks were harvested for maternal antibody assessment.
mRNA vaccine design and construction
The mRNA vaccine encodes the pre-membrane (prM) and envelope (E) proteins of DTMUV. The both prM and E proteins are sourced from UniprotKB (Accession No. G1CRV4). The N-terminus of prM and E proteins is fused with a signal peptide from Aythya fuligula (Tufted duck) CD5 protein (UniprotKB Accession No. A0A6J3D041) to enhance the efficiency of protein expression and secretion. This design, inspired by the successful strategies of Zika and dengue mRNA vaccine15,16, endeavors to recapitulate the native viral structure. The mRNA construct incorporates optimized 5′ and 3′ untranslated regions, poly(A) tail of approximately 110 nucleotides to enhance mRNA stability and translation efficiency, a type 1 cap structure, and the duck CD5 signal peptide to enhance expression and secretion efficiency. For plasmid construction, the pUC57 vector (sourced from GenScript) was utilized to facilitate in vitro mRNA transcription. The T7 promoter element was additionally introduced into the pUC57 vector to enable efficient in vitro mRNA transcription.
mRNA in vitro transcription and purification
mRNA was transcribed from linearized plasmid DNA using the MEGAscript® T7 Transcription Kit, with CleanCap (Cat# N-7413, TriLink), a trinucleotide cap1 analog for co-transcription. After a 6-h in vitro transcription reaction, mRNA was selectively precipitated using lithium chloride. The resulting mRNA pellet was dissolved in nuclease- and endotoxin-free water, followed by quantification and quality control using a Nanodrop spectrophotometer and capillary electrophoresis, respectively. Finally, the purified mRNA was aliquoted and stored at −80 °C.
Western blot analysis
293 T cells, seeded at a density of 3.75 × 106 cells per T75 flask, underwent transient transfection with naked mRNA using a lipid nanoformulation named LNP325, which was previously optimized for effective mRNA delivery. At 48 h post-transfection, the cell supernatant was collected and concentrated using an ultrafiltration tube, while the cells were harvested for lysis and protein extraction using the RIPA method. The lysis buffer contained 20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 μg/mL aprotinin, and 1 mM phenylmethanesulfonyl fluoride. The protein concentrations of both the concentrated supernatant and the cell lysate were determined using the BCA method. Subsequently, 40 µg of total protein from each sample was subjected to SDS-PAGE separation and then transferred onto PVDF membranes for further analysis. The membranes were blocked overnight at 4 °C with 5% skim milk in a phosphate buffer (pH 7.2) containing 0.05% Tween-20. Then membranes were probed with a primary anti-DTMUV-E polyclonal antibody (mouse origin), which was diluted 1:200 in the same blocking solution. This antibody was generously provided by Dr. Qingtao Liu’s laboratory (Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture; Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing, China) and used at room temperature for one hour. After washing three times with PBST, the membranes were exposed to a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody, diluted 1:2000 in 5% milk-PBST, for an hour at room temperature. Post three additional washes, the protein bands were visualized using the ChemiDocTM system (Bio-Rad, USA) and Immun-StarTM HRP substrate (Bio-Rad, USA) for chemiluminescence detection.
mRNA-LNP production
Lipid nanoparticles encapsulating mRNA (mRNA-LNPs) were produced using microfluidic technology. Lipids were dissolved at a molar ratio of 50:10:38.5:1.5 (SM102: DSPC: cholesterol: DMG-PEG2000). mRNA was prepared at a concentration of 0.1 mg/mL in 20 mM sodium acetate buffer (pH 5.5). The lipid and mRNA solutions were then mixed using a microfluidic cartridge at a total flow rate of 6 mL/min, with an aqueous to organic phase flow rate ratio of 3:1, resulting in an N/P ratio of 6. The obtained mRNA-LNPs were dialyzed three times against 20 mM Tris-HCl buffer (pH 7.4) using a Slide-A-Lyzer dialysis cassette with a 3.5 kD molecular weight cut-off. The final mRNA-LNP solution was adjusted to a concentration of 100 μg/mL in 10% sucrose (w/v) and aliquoted for storage under 2–8 °C conditions.
Quality control of mRNA-LNP complex
To ensure the integrity and performance of the mRNA-LNP complexes, comprehensive quality control measures were implemented. The size and polydispersity index (PDI) of the mRNA-LNPs were measured using dynamic light scattering (DLS). The particles exhibited a mean diameter ranging from 60 to 200 nm, with a polydispersity index (PDI) consistently below 0.2, indicating a narrow size distribution and high uniformity. The encapsulation efficiency of the mRNA was determined using the RiboGreen assay, and the results demonstrated that the mRNA-LNPs achieved an encapsulation efficiency greater than 80%, ensuring effective protection of the mRNA within the lipid shell.
Ethics and bio-containment statement
The conducted animal experiments received authorization from the Institutional Animal Care and Use Committee at Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences. All procedures involving animals were in strict compliance with ARRIVE reporting guidelines26. Experiments involving the DTMUV were conducted at Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences within a bio-containment unit located in a BSL-2 laboratory to ensure safety and containment.
Vaccination and challenge experiments
All experimental procedures were conducted using Jinding ducks, a domesticated breed obtained from the Harbin Veterinary Research Institute in China. At the conclusion of each experiment, ducks were humanely euthanized by intravenous injection of sodium pentobarbital at a dose of 100 mg/kg of body weight in accordance with institutional and national guidelines for the ethical treatment of experimental animals.
For the immunization of SPF ducks, initial priming was conducted when ducks were 7 days old, followed by a booster dose at 28 days. Each vaccination involved a 5 µg dose of the formulated mRNA vaccine delivered via intramuscular injection. Sampling for immunological assays was scheduled at days 28, 35, 42, and 49. For the challenge experiments, ducks received exposure to the highly pathogenic DTMUV strain JS804 (GeneBank: JF895923) at day 49 post-prime vaccination, with an infection dosage of 105 TCID50 in a volume of 0.5 ml. Protection after challenge was then monitored.
To compare the efficacy of our mRNA vaccine with commercial live attenuated vaccine (strain FX2010-180P), male breeder drakes were vaccinated at day 0 (prime) and subsequently received a booster dose on day 21. Each dose consisted of 10 µg of the vaccine administered via intramuscular injection. Blood samples were collected on days 21 and 28 post-initial vaccination to measure the levels of specific and neutralizing antibodies.
For the immunization of laying ducks, initial priming was conducted at day 0, followed by a booster dose at day 14. Each vaccination involved a 5 µg dose of the formulated mRNA vaccine delivered via intramuscular injection. Blood samples were collected on days 0, 14, 21, and 28 to measure the levels of neutralizing antibodies. Neutralizing antibodies in egg yolks were also determined. The laying ducks used were Jinding Ducks (JDD), selected for their high egg production and suitability for vaccine efficacy studies. They were maintained under controlled breeding conditions with a balanced diet to ensure optimal health and consistent egg quality.
Determination of anti-DTMUV E protein IgY antibodies
Serum specific E protein IgY were detected using commercial ELISA kits (Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences) following the manufacturers’ protocol. According to the manufacturer’s instructions, The ELISA titer is the highest dilution factor at which serum sample is determined to be positive. Through linear regression analysis, there is a certain correlation between the logarithm of the reciprocal of the serum ELISA titer and the OD value of the serum at a 1:10 dilution. A standard curve is plotted using the OD value of serum at a 1:10 dilution as x-axis and log [1/ET] as y-axis (log [1/ET] = a + bx, a and b respectively represent the intercept and slope of the linear regression equation, and x represents the OD value of the serum to be tested). The titer of each serum can be calculated from the regression equation.
Neutralization assay
Serum samples were collected, inactivated in a 56 °C water bath for 30 min, and diluted two-fold in Minimum Essential Media (Thermo Fisher). Equal volumes of serum dilutions were mixed with 100-fold diluted 50% tissue culture infective dose (TCID50) of DTMUV and incubated in 96-well plates at 37 °C for one hour. BHK-21 cells were then seeded into each well and co-cultured with the mixture for five days. Neutralizing antibody titers were assessed by monitoring the cytopathic effect (CPE): neutralizing activity was confirmed when at least two out of three wells showed no CPE.
Statistical analysis
Data were analyzed using appropriate statistical methods where indicated. Differences between groups were considered significant at *P < 0.05. Graphical representations of antibody titers and protection rates were generated to visualize the results.
