Sequence design
The template for the DNA candidate vaccine is a codon-optimized version of the HTNV 76–118 strain M segment, which encodes the HTNV envelope glycoprotein, including Gn and Gc, with a length of 3616 base pairs (Sequence ID Y00386.1). The mRNA vaccine candidate encodes a codon-optimized version of the HTNV 76-118 strain M segment. Besides, it includes 5′ and 3′ untranslated regions (UTR) as well as a poly (A) tail, collectively referred to as the UTR region (Table S1 and Fig. S1). The 5′ UTR and 3′ UTR sequences are derived from homo sapiens hemoglobin subunit beta (Sequence ID: NM_000518.5). The 5′ UTR sequence comprises a regulatory sequence followed by the 5′ UTR sequence of β-globin, while the 3′ UTR sequence consists of the concatenated sequences of two segments from β-globin’s 3′ UTR. Additionally, a 120-nt long poly (A) tail is included to enhance RNA stability and translation efficiency. A 3×Flag tag is appended to the C-terminus of the GP to facilitate subsequent identification of vaccine antigen expression.
Cell culture
HEK293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a 5% CO2 incubator.
Construction and identification of the recombinant plasmid
We commissioned TsingKe Biotech Ltd to construct the codon-optimized pVAX1-GP plasmid and pUC57-UTR plasmid, and confirmed the accuracy through sequencing and agarose gel electrophoresis. The pGEM-UTR plasmid was obtained by cloning the UTR sequence from pUC57-UTR into the pGEM-11Zf plasmid (Promega). The pGEM-UTR-GP plasmid was obtained by inserting the GP sequence obtained from double enzyme digestion of pVAX1-GP into the pGEM-UTR plasmid. We employed Plasmid Maxi Kits (TIANGEN) for plasmid purification. Subsequently, the recombinant plasmids underwent horizontal electrophoresis on a 1% agarose gel after digesting with the restriction enzymes and were visualized under UV light.
Lipid nanoparticle formulation of the DNA
Lipid-nanoparticle (LNP) formulations were prepared using a modified procedure, previously employed for siRNA68,69. In summary, lipids, including ionizable lipid (ALC-0315) (MCE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (MCE), cholesterol (MCE) and PEG-lipid (MCE), were dissolved in ethanol at a molar ratio of 55:10:32.5:2.5. The 10 mM lipid mixture was mixed with 10 mM citrate buffer containing DNA at a 1:3 ratio through a mixer. Subsequently, the formulations were transferred to an aqueous buffer system, filtered through a 0.22 μm mesh, and concentrated to the desired concentration via diafiltration using a 50 kD molecular weight cutoff microsep (Millipore) against 30 volumes of PBS. The vaccine candidates were diluted in PBS containing 60 mM sucrose and stored at −20 °C at a 0.5 mg/mL concentration.
Characterization of LNP
We performed dynamic light scattering measurements using the Malvern Zetasizer Nano-ZS (Malvern). Electron microscopy imaging was conducted with the HT7800 (HITACHI) by depositing the sample onto a porous carbon grid. We utilized the PicoGreen dsDNA Quantification Assay Kit (Solarbio) to assess the encapsulation efficiency of DNA. In essence, DNA-LNP were divided into two groups: one group was subjected to lysis using 2% Triton X-100, while the other remained untreated. Subsequently, the samples were processed with PicoGreen (Solarbio) following the manufacturer’s instructions. The amount of DNA in the samples was then quantified using a microplate reader. Excitation light was set at 480 nm, and emission light at 520 nm.
In vitro transcription and purification of RNA
The mRNA was produced in vitro through T7 RNA polymerase-mediated transcription, using the plasmid pGEM-UTR-GP linearized by the restriction enzyme Xba I as a template. The DNA template was purified and quantified using spectrophotometry, followed by in vitro transcription using T7 High Yield RNA Transcription kit (Novoprotein), with N1-methylpseudouridine-5’-triphosphate (m1ΨTP) substituting for uridine-5’-triphosphate. The RNA was then purified using lithium chloride, capped enzymatically with cap1 (Novoprotein), and further purified after capping reaction completion. Subsequently, the solution’s concentration was determined, and the integrity of the RNA was assessed through microfluidic capillary electrophoresis. Besides, we employed a double-antibody sandwich NovoFast dsRNA ELISA (Novoprotein) to quantify dsRNA residuals in the mRNA vaccine candidate.
Transfection of HEK293T cells
HEK293T cells were transfected with plasmid pVAX1-GP, mRNA, and transfection reagent lipofectamine 3000 (Thermo Fisher Scientific) separately, following the manufacturer’s instructions, or transfected with DNA-LNP vaccine candidates.
Western blot analysis of transfected cells
After 24 h post-transfection, the cell lysates were collected by centrifugation at 1000 × g using RIPA buffer (Beyotime). The collected lysates were then mixed with 5×SDS loading buffer and subjected to SDS-PAGE electrophoresis. The antigen proteins expressed by the cells were detected by western blotting with a flag antibody (Proteintech). Blots were developed with High-sig Western ECL Substrate (Tanon) and imaged with a Fusion FX Imager (Vilber) using the Image Lab software version 6.0.
RNA isolation and quantitative real-time PCR
The total RNA from transfected 293 T cells was isolated using TRIzol reagent (Sigma). cDNA was prepared using PrimeScript RT Master Mix (TaKaRa), and quantitative real-time PCR (qPCR) was performed using SYBR-green PCR Master Mix (Qiagen). The qPCR experiments were carried out on a Bio-Rad thermal cycler (Bio-Rad Laboratories Inc., CA, USA CFX96). The PCR conditions were as follows: an initial incubation at 50 °C for 30 min, followed by a denaturation step at 95 °C for 5 min. Subsequently, 40 cycles of amplification were performed, each consisting of a 20 s denaturation step at 95 °C and a 1-min annealing/extension step at 55 °C. For the detection of vaccine antigens, we designed two pairs of antigen-specific primers based on the Gn and Gc gene regions. The human beta-actin gene was employed as the reference gene. Gene expression levels were calculated using the comparative Ct method. All primers were synthesized by TsingKe Biotech Ltd (Table S2).
Immunofluorescence analysis of eukaryotic protein expression
The transfected HEK293T cells were fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. After blocking with 3% bovine serum albumin-PBS solution to prevent non-specific binding, the cells were incubated overnight at 4 °C with Flag antibody (1:2000, Proteintech). Subsequently, 594-conjugated goat anti-rabbit antibody (1:200, Proteintech) was used as the secondary antibody and incubated in the dark at 37 °C for 2 h. DNA was stained with DAPI (Solarbio). Images were obtained using a confocal microscope (Fluoview FV3000, Olympus, Japan).
Animals and immunization
Female BALB/c mice (6 weeks old) were procured from the Laboratory Animal Center of the Fourth Military Medical University and were assigned to six groups using a random number generator. All mice were in good health and had never participation in any research previously. They were acclimated for one week prior to immunization. Before all invasive procedures, mice were anesthetized with 2% isoflurane inhalation to ensure they remained pain-free during the operations. Each group received two injections of candidate vaccines, administered into the gastrocnemius muscle under isoflurane anesthesia, on day 0 and day 28, respectively. The six groups of candidate vaccines were as follows: PBS (n = 21), empty lipid nanoparticle (LNP) without encapsulated nucleic acid (n = 21), inactivated vaccine (Inact) (n = 21), endotoxin-free DNA plasmid pVAX1-Gp (n = 21), LNP-encapsulated endotoxin-free DNA-LNP (n = 21), and mRNA-lipojet vaccine (n = 21). The inactivated vaccine, referenced to the manufacturer’s instructions, consisted of 10 μg of inactivated bivalent HFRS vaccine (HANPUWEI) diluted in 0.9% NaCl, with an injection volume of 50 μL. Both DNA and DNA-LNP candidate vaccines contained 30 μg of DNA with an injection volume of 50 μL. The mRNA-lipojet candidate vaccine comprised 10 μg mRNA, which is delivered using in vivo mRNA transfection reagent (Polyplus). The injection volume is 100 μL, administered via intramuscular injection in two separate points. Each group of mice received a final booster vaccination on day 182 with the same dose as the initial two vaccinations. Serum samples were collected from mouse eye blood on days 52 and 206 after the initial immunization to detect neutralizing antibody responses. Additionally, serum samples were collected from mouse tail vein blood on day 0 (before the initial immunization) and on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 98, 182, 196, and 203 after the initial immunization to assess specific antibody responses. Spleen tissues from the immunized mice in each group were collected on days 52 and 206 after the initial immunization for the assessment of cellular immune responses using ELISpot and flow cytometry. After the experiment, all mice were anesthetized with 2% isoflurane inhalation. After confirming adequate anesthesia, trained personnel humanely euthanized the animals by cervical dislocation, following standard procedures.
Sera antibody titer evaluation by enzyme-linked immunosorbent assay (ELISA)
Determination of the titer of HTNV-specific IgG antibodies was performed by ELISA. In brief, ELISA plates (Costar) were coated with inactivated HTNV virus diluted in sodium carbonate buffer (100 µL/well) and incubated overnight at 4 °C. After coating, the plates were washed once with wash buffer (PBST, PBS with 0.05% Tween 20, 200 µL/well) and then blocked with 1% bovine serum albumin diluted in wash buffer (200 µL/well) for 40 min at 37 °C. Subsequently, mouse sera were serially diluted, starting from 1:100, and 100 µL of each dilution was added to the wells as the primary antibody and incubated for 1 h at 37 °C. The plates were then washed five times and each well received 100 µL of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG as the secondary antibody (1:2000, Proteintech), followed by an incubation of 30 min at 37 °C. After washing six times, the plates were incubated with TMB substrate (Solarbio) for 15 min at 37 °C. Finally, 50 µL of ELISA stop solution (Solarbio) was added to terminate the reaction, and the absorbance was measured at 450 nm using a microplate reader (TECAN).
Serum neutralization test
Serum samples were serially diluted in a 2-fold gradient, starting from 1:10 to 1:320. Subsequently, they were mixed with a viral solution at a dose of 100 PFUs, followed by incubation at 37 °C for 1 hour. This mixture was then introduced to a 96-well plate containing a monolayer of Vero E6 cells, which were allowed to adsorb for 2 hours in a cell culture incubator. After discarding the mixture, DMEM containing 1.6% carboxymethyl cellulose and 2% FBS was applied to each well and the plate was incubated at 37 °C in a 5% CO2 atmosphere for 5 days. The cells were subsequently fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. A homemade monoclonal antibody, mAb1A8 against HTNV-NP, was added and incubated overnight at 4 °C. Followed by incubation with HRP-conjugated goat anti-mouse antibody (1:2000, Proteintech) at room temperature for 1 hour. Color development was performed using a Maxi-Blue precipitating TMB substrate, and blue plaques were counted after drying. The neutralizing titers were determined and calculated according to the Karber method.
Synthesis and preparation of GP peptide
The peptide pool used for ELISpot (30 μg/mL of each peptide) and flow cytometry (1 mg/mL of each peptide) consists of 10 sets of 15-mer peptides, all of which were obtained from Shanghai Apeptide Co (Table S3).
Enzyme-linked immunospot assay (ELISpot)
The ELISpot experiment was conducted following the manufacturer’s protocol to evaluate the cellular immune response in vaccinated mice using the mouse IFN-γ/IL-4 ELISpot kit (BD Pharmingen). In essence, the plates were initially incubated with capture antibodies at 4 °C overnight, followed by blocking with RPMI 1640 medium containing 10% FBS for 2 h. Subsequently, we plated 1,000,000 cells/well and ex vivo reactivated immune mouse splenocytes using either a treatment group consisting of a 15-peptide mixture (each peptide at a final concentration of 0.1 μg/mL, with a total concentration of 2 mg/mL, as detailed in Table S3) or control groups (negative control: RPMI 1640 medium containing 10% FBS; positive control: Concanavalin A, 2 μg/mL (Sigma)). After incubation for 24 h (37 °C, 5% CO2), these cells were collected for subsequent transcriptome sequencing as described below. Then the plates were subjected to a washing step, followed by the addition of biotinylated anti-mouse IFN-γ or IL-4 antibodies, and incubation at room temperature for 2 h. After rinsing the plates thoroughly, avidin-horseradish peroxidase and 3-amino-9-ethylcarbazole peroxidase substrate kit were introduced. Following this, an automated ELISpot plate reader (CTL) was employed to read and count the spots on the air-dried plates. Finally, we calculated the number of spot-forming cells per 1 million splenocytes.
Cellular immune response by flow cytometry
To perform intracellular cytokine staining of T cells from immunized mice, 1,000,000 splenocytes were ex vivo stimulated for 4 h using a cell stimulation cocktail (Invitrogen). After two washes with staining buffer, splenocytes were stained for cell surface antigens using directly labeled antibodies, including CD3 (FITC) (BioLegend), CD4 (PE) (BioLegend), and CD8 (Pacific Blue) (BioLegend). Subsequently, cells were adequately fixed and permeabilized using Cytofix/Cytoperm solution (BD) and incubated in the dark at 4 °C for 20 min. The permeabilized cells were then washed twice and subjected to intracellular cytokine staining using antibodies specific for IL-2, IL-4, IFN-γ, and Granzyme B (APC) (BioLegend) within Perm/Wash buffer (BD). Incubation was carried out in the dark at 4 °C for 30 min. Afterward, cells were washed twice with the staining buffer and subsequently resuspended in the same buffer before being subjected to flow cytometric analysis. Data analysis was conducted using NovoExpress software. Gating strategy to identify cytokine secretion in the T cells is present in Fig. 3e (take the IL-2 release from CD4+ T cells for example). From all events, lymphocytes can be distinguished by their FSC/SSC properties. Single cells are then isolated by their relationship between FSC-H versus FSC-A. From single cells, CD4+ T cells are gated, from which IL-2+ CD4+ T cells can be identified.
For the detection of cell surface antigens, 500,000 mouse splenocytes were stimulated with a GP peptide pool (1 mg/mL for each peptide, see Table S3) for 6 h at 37 °C under 5% CO2. Following two washes with staining buffer, we applied fluorescently conjugated antibodies, including CD3 (APC-Cyanine7) (BioLegend), CD4 (FITC) (BioLegend), CD8 (FITC) (BioLegend), CD44 (PE) (BD Biosciences), and CD62L (APC) (BioLegend), to label the splenocytes, which were then incubated at 4 °C in the dark for 30 min. After two additional washes with a staining buffer, the samples were analyzed using a flow cytometer. Data analysis was performed using NovoExpress software. Gating strategy to identify effector memory T (Tem) cells in the spleen is present in Fig. 3g (take the CD4+ Tem cells in splenocytes for example). From all events, lymphocytes can be distinguished by their FSC/SSC properties. Single cells are then isolated by their relationship between FSC-H versus FSC-A. From single cells, CD4+ T cells are gated, from which Tem (CD44hi CD62Llo) cells can be identified.
Transcriptome analysis
We collected 5,000,000 mouse splenocytes from immunized mice in each group, which were stimulated with peptide pools for 24 h. Each sample was derived from three immunized mice and preserved in TRIzol reagent (Sigma). Subsequently, the samples were sent to Seqhealth Ltd (Wuhan, China) for transcriptome analysis.
HTNV challenge of mice
The challenge model using the HTNV 76–118 strain for viral infection has been extensively described32,52. Each group of candidate vaccines underwent immunization. Subsequently, on day 53, six mice in each group received the challenge or via intramuscular injection with 5 × 105 PFUs of HTNV 76–118 strain per mouse. Five days before the challenge, the mice were relocated from the immunization facility to a biosafety level 3 (BSL-3) animal facility. On the 3rd day post-challenge, all animals were euthanized, and major organs (heart, liver, spleen, lung, and kidney) were collected for subsequent analysis, including viral RNA level determination, histopathological examination, and immunofluorescence staining. All experiments were conducted under BSL-3 conditions and adhered to the international laboratory biosafety guidelines.
Quantification of viral RNA in challenged mice tissues
After the challenge, we conducted qPCR to detect viral RNA in the major organs of challenged mice. In brief, tissue samples were weighed, and total RNA was extracted using TRIzol reagent (Sigma). We quantified HTNV RNA using the S segment as the target and utilized 18S rRNA as an internal reference gene. All primers were synthesized by TsingKe Biotech Ltd (Table S2).
Immunofluorescence staining of challenged mice tissues
Tissue samples from each group were collected for paraffin embedding and sectioning. After slide preparation, the tissue sections were subjected to a series of processes including sealing, incubation with the primary antibody, incubation with the secondary antibody, and nuclear staining, as detailed in “Immunofluorescence analysis of eukaryotic protein expression”. The primary antibody was a homemade anti-HTNV-NP mouse antibody, and the secondary antibody employed was a FITC-labeled goat anti-mouse antibody. The results were observed and analyzed using a fluorescence microscope.
Hematoxylin and eosin (H&E) staining
In terms of histopathology, we collected samples from six major organs and muscle tissue on the 24th day following long-term immunity. These samples were initially fixed using a 4% paraformaldehyde solution, subsequently embedded in paraffin, and then sectioned and stained with hematoxylin and eosin (H&E). The slides are scanned and photographed by a slide scanner (Winmedic). The staining results are certified by more than two pathologists.
Statistical analysis
All data analyses were conducted using GraphPad Prism 9.0 software. Unless specified, experimental dates are presented as mean ± SEM. Statistical significance among different groups was assessed using one-way ANOVA with multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). And the data analysts were blinded to the experimental group assignments.
Animal ethics statement
All animal experiments were conducted strictly under the regulations of the Chinese Regulations of Laboratory Animals and Laboratory Animal-Requirements of Environment and Housing Facilities. The procedures for the care and use of animals were approved by the Animal Ethics Committee of the Fourth Military Medical University (No. FMMU-DWZX-20221201).
