Animals and ethics statement

C57BL/6 and ICR female mice at the age of 6 to 8-week-old were purchased from the Centers for Disease Control of Hubei Province, China. All mice were bred and maintained within a controlled specific pathogen-free (SPF) facility at the Laboratory Animal Center of Huazhong Agricultural University. Mice demonstrating a reduction in their initial body weight of ≥ 25% were euthanized in accordance with humane protocols and recorded as deceased. The experimental protocol underwent review and received approval from the Scientific Ethics Committee of Huazhong Agricultural University, bearing the approval number HZAUMO-2023-0040.

Cells, viruses, and reagents

HEK-293T, BSR and BHK-ACE2 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Merck, Cat. No# D5546) supplemented with 10% fetal bovine serum (FBS) (Gibco, Cat. No# 16000-044) and 1% penicillin–streptomycin (Gibco, Cat. No# 15140122), within a 5% CO2 atmosphere at 37 °C in an incubator. The virulent strain CVS-24 of RABV was preserved in our laboratory as previously outlined [63]. The licensed commercial inactivated rabies vaccine purchased from Intervert International B.V. (Boxmeer, Netherlands) was used as a reference control. For simplicity, this vaccine is referred to as ITV in the paper. A 0.1 dose of the ITV vaccine was used in mice as a control, as previously described [18]. The procedures for preparing SARS-CoV-2 pseudovirus were executed in accordance with previously published methodologies [64]. Briefly, HEK-293T cells were transfected with a plasmid expressing the SARS-CoV-2 S protein and infected with G*ΔG-VSV pseudotyped virus. After infection, the uncoated ΔG-VSV genome expressed all enzymes and structural proteins from the VSV genome except the G protein, which was replaced by a luciferase (Luc) reporter gene. Cell supernatants were collected 24 h after infection and transfection, divided into aliquots, and cryopreserved at − 80 °C.

The antibodies labeled directly with fluorescein for flow cytometric analyses were purchased from BioLegend (CA, USA). FITC anti-mouse CD4 antibody (Cat. No. 100510), PE anti-mouse CD279 (PD1) antibody (Cat. No. 135206), and APC anti-mouse CD185 (CXCR5) antibody (Cat. No. 145506) were employed for the identification of Tfh cells within the inguinal lymph nodes; FITC anti-mouse CD45R/B220 antibody (Cat. No.103206), 647 anti-mouse GL7 antibody (Cat. No. 144606), PE anti-mouse CD95 antibody (Cat. No. 152608) were employed for the identification of GC B cells within the inguinal lymph nodes; FITC anti-mouse CD45R/B220 antibody (Cat. No. 103206) and APC anti-mouse CD138 (Syndecan-1) antibody (Cat. No. 142506) were employed for the assessment of plasma cell numbers within the bone marrow (BMs); PE/Cy7 anti-mouse CD45R/B220 antibody (Cat. No. 103222), FITC anti-mouse IgD (Cat. No. 405703), PE anti-mouse CD273 (Cat. No. 107205) and APC anti-mouse CD38 antibody (Cat. No. 102712) were employed for the identification of memory B cells (MBCs) within the spleens. Anti-RABV G protein monoclonal antibody and RABV G protein was prepared following established protocols as previously outlined [61, 63]. The antibodies labeled with fluorescein isothiocyanate (FITC) targeting the RABV N protein were purchased from Fujirebio Diagnostics, Inc. (Malvern, PA). Anti-IL-7 antibody (Cat. No. ab84271) was purchased from Abcam (Cambridge, UK). Anti-JAK1 antibody (Cat. No. A18323), anti-STAT5 antibody (Cat. No. A5029) and anti-Phospho-STAT5 antibody (Cat. No. AP0887) were purchased from Abclonal (Wuhan, China). Anti-Phospho-Jak1 antibody (Cat. No. 3331) was purchased from Cell Signaling Technology (Danvers, MA, USA). The horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, IgG2a, and IgG2b antibodies for enzyme-linked immunosorbent assay (ELISA) were procured from Boster (Wuhan, China). Ionizable cationic lipid (SM102) (Cat. No. 06040008800), PEG-lipid (Cat. No. 06020112402), cholesterol (Cat. No. 06040010300), and phosphatidylcholine (DSPC) (Cat. No. 06030001100) were purchased from SINOPEG Biotechnology Co., Ltd. (Xiamen, China).

mRNA synthesis

mRNA was synthesized in vitro using the T7 High Yield RNA Transcription Kit (Novoprotein, Cat. No# E137), incorporating N1-methylpseudouridine as a substitute for uridine. The linear DNA template serving as a substrate comprises both the 5′ and 3′ untranslated regions (UTRs). The Cap 1 Capping System (Novoprotein, Cat. No# M082) and Poly(A) Polymerase (Novoprotein, Cat. No# M012) were employed to add the Cap1 structure and a poly(A) tail, respectively, to the 5′ and 3′ ends of mRNA. The purification of mRNA products entails mixing with lithium chloride and incubating at − 20 °C for a minimum of 30 min. Subsequently, centrifugation at 10,000×g at 4 °C for 15 min is performed to collect the precipitate. The pellet is then washed three times with pre-chilled 70% ethanol and resuspended in RNase-free water. Following resuspension, the precipitate is stored at − 80 °C until further utilization.

LNP Formulation of the mRNA

The LNP-mRNA vaccine utilizes LNPs formulation to encapsulate the mRNA. The LNPs employed in this study were formulated by dissolving an ionizable cationic lipid, phosphatidylcholine, cholesterol, and DSPE-PEG2k at a ratio of 50:10:38.5:1.5 in anhydrous ethanol [65]. Meanwhile, mRNA was dissolved in a citrate buffer with a pH of 4 and a concentration of 50 mM. The LNP and mRNA were mixed at a volume ratio of 1:3 within a microfluidic device, followed by dilution of the LNP-mRNA formulations with a 35-fold volume of 1 × PBS buffer (pH 7.4) and subsequent concentration to an mRNA concentration of 0.1 mg/mL using 30 kD Amicon Ultra Centrifugal Filters (Millipore, Cat. No# UFC903096).

Nanoparticle characterization

Particle size (Dynamic Light Scattering, DLS) measurements were performed using a Zeta sizer Nano ZS instrument (Malvern Instrument Co., Ltd.). Nanoparticles were added to 1 cm test dishes at appropriate concentrations, followed by analysis using a dynamic light scattering instrument to ascertain particle size. Transmission electron microscopy (TEM) was conducted following the dilution of LNP-mRNA nanoparticle. Specifically, 10 μL aliquots were placed onto copper mesh and allowed to stand for 10 min. Following the removal of excess liquid using filter paper, the morphology of LNPs was inspected utilizing a transmission electron microscope (HITACHI) equipped with a field emission gun operating at 80 kV.

mRNA transfection in vitro

HEK-293T cells were seeded at a density of 4 × 105 cells per well in 24-well plates and incubated at 37 °C in 5% CO2 atmosphere for 12 h. Subsequently, LNP-encapsulated mRNA was directly transfected into HEK-293T cells. At 24 h after transfection, cell lysates were collected using RIPA Lysis Buffer (Beyotime, Cat. No# P0013D), followed by a 30 min incubation on ice. After centrifugation at 10,000×g, the supernatant was combined with SDS-loading buffer.

Mouse vaccination and challenge

Mice aged 6–8 weeks were randomly allocated to specified groups. Mice were immunized via intramuscular (i.m.) injection with LNP-encapsulated G mRNA, G&IL-7 mRNA, S mRNA, S&IL-7 mRNA. The ITV vaccine and PBS were used as a control. A mouse challenge model using the virulent RABV strain CVS-24 has been previously described [61]. At either 3 weeks or 6 months post-immunization, mice were subjected to an intracranial challenge with 30 μL of 50 LD50 (median lethal doses) of CVS-24. Subsequently, their body weights and mortality were monitored and recorded daily.

RABV virus-neutralizing antibody measurement

RABV virus-neutralizing antibody (VNA) titers were measured using the fluorescent-antibody virus neutralization (FAVN) assay, following previously described methods [66]. In brief, blood was collected from mice at specific time points, and the serum was subsequently separated and inactivated for 30 min at 56 °C. Test serum and standard serum were serially diluted in 96-well microplates. 100 μL of DMEM was dispensed into each well of a 96-well plate, followed by adding 50 μL of either test serum or standard serum to the first column, and then serial three-fold dilutions were performed. Each sample was added to four adjacent wells for analysis. A suspension of RABV (CVS-11) was added to each well, and the plates were then incubated at 37 °C for 1 h. Subsequently, 2 × 104 BSR cells were added to each well, followed by further incubation at 37 °C for 72 h. Samples were then fixed with 80% ice-cold acetone for 30 min and stained with FITC-conjugated antibodies targeting the RABV N protein. Fluorescence was observed utilizing an Olympus IX51 fluorescence microscope (Olympus, Tokyo, Japan). Fluorescence values were compared with reference serum values acquired from the National Institute for Biological Standards and Control in Hertfordshire, UK. Following this, the results were standardized and quantified as international units per milliliter (IU/mL).

ELISA analysis of antibody titers

ELISAs were performed to determine antibody, following established protocols [67]. In brief, serum samples were collected and subsequently inactivated at 56 °C for 30 min. ELISA plates were incubated overnight at 4 °C with purified RABV virion, which were diluted to a concentration of 500 ng per well in a protein coating buffer (5 mM Na2CO3, pH 9.6). After incubating overnight at 4 °C, the plates were washed three times and then blocked for 1 h at 37 °C. The serum was subsequently diluted in PBST containing 5% (wt/vol) skim milk at dilutions of 1:2,000 for IgG, 1:100 for IgG1, and 1:200 for IgG2a and IgG2b. Subsequently, 100 μL of the diluted serum was dispensed into the plates and incubated at 37 °C for 1 h. After washing the plates three times with PBST, 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgG2b was added to each well and incubated for 45 min at 37 °C. After the incubation, the plates were washed three times. Subsequently, 100 μL of tetramethylbenzidine (TMB) substrate (Biotime Biotechnology, Shanghai, China) was added to each well to initiate a chromogenic reaction, and the plates were allowed to incubated at 37 °C for 5 min before the addition of 50 μL of 2 M H2SO4. Optical densities were measured at 450 nm using a SpectraMax 190 spectrophotometer (Molecular Devices, CA, USA).

The SARS-CoV-2 S-specific IgG, IgG1, and IgG2a antibody were measured by ELISA. ELISA plates were coated overnight at 4 °C with 100 μL of a solution containing 2 ng/μL of SARS-CoV-2 S protein (Vazyme, Cat. No# CG202-01) diluted in coating buffer. After blocking the plates for 1 h, the diluted serum was added and incubated for 1 h, followed by 3 washes with PBST. Subsequently, plates were treated with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG, IgG1, or IgG2a, and incubated at 37 °C for 60 min. The subsequent chromogenic steps followed the previously described protocol. The endpoint titers were defined as the highest reciprocal dilution of serum to yield an absorbance greater than 2.1-fold of the background values.

Pseudovirus neutralization assay

SARS-CoV-2 neutralizing antibody titers were assessed following established protocols [64]. In brief, the mouse serum was serially diluted and then mixed with a specific quantity (ranging from 325 to 1300 TCID50/mL) of pseudotyped virus for 1 h at 37 °C. After that, BHK-ACE2 cells were added to each well, followed by further incubation at 37 °C for 24 h. A negative control using DMEM was incorporated for comparative analysis. Subsequently, the supernatant was removed, and a luciferase substrate as dispensed into each well. The mixture was then left to incubate in the dark at room temperature for 2 min. Luciferase activity was quantified using a Spark® Multimode microplate reader (TECAN, Swiss). The pVNT50 was characterized as the dilution fold achieving over 50% inhibition of pseudotyped virus infection relative to the control group.

Flow cytometry (FCM) and ELISpot assay

T follicular helper (Tfh) cells, Germinal Center B (GC B) cells, memory B cells (MBCs), and plasma cells (PCs) obtained from inguinal lymph nodes (LNs), spleens or bone marrows (BMs) were examined using flow cytometry, following established procedures [61]. In brief, LNs, spleens and BMs from mice were harvested, and solid tissues were gently homogenized in pre-cooled PBS (pH 7.4). The cells were suspended in PBS containing 0.2% BSA (w/v), filtered through a 40-mm nylon filter into a tube, centrifuged, and then washed with PBS containing 0.2% BSA. After removing red blood cells using lysis buffer (catalog number 555899, BD Biosciences Inc., Franklin Lakes, NJ, USA), the cells were washed twice with PBS containing 0.2% BSA and then resuspended. Subsequently, the resuspended cells were counted, and 1 × 106 cells were stained with fluorescence-labeled antibodies. After incubation at 4 °C for 30 min, the cells were washed twice with PBS containing 0.2% BSA. Finally, data acquisition and analysis were conducted utilizing a BD FACSVerse flow cytometer (BD Biosciences, CA, USA) along with FlowJo software (TreeStar, CA, USA).

The ELISpot assay was conducted to evaluate the production of RABV-specific antibody-secreting cells in the inguinal lymph nodes [68]. Multiscreen HA ELISpot plates (Millipore, MA, USA) were coated with 500 ng of purified RABV virions per well and then incubated for 16 h at 4 °C. The coated plates were washed and subsequently blocked with RPMI 1640 supplemented with 10% FBS for 2 h at 37 °C. Cell suspensions from inguinal LNs were added to the blocked ELISpot plates and incubated at 37 °C for 24 h. Following this, the cells in the ELISpot plates underwent sequential incubation with biotin-conjugated mouse IgG antibody (Bethyl Laboratories, TX, USA) and streptavidin–alkaline phosphatase (Mabtech, Stockholm, Sweden), followed by color development using BCIP/NBT-plus (Mabtech, Stockholm, Sweden). The plates were scanned, and spots were quantified. Then, the plates underwent scanning and analysis using the Mabtech IRIS FluoroSpot/ELISpot reader, employing RAWspot technology for multiplexing at the single-cell level.

Statistical analyses

Statistical analyses were conducted using GraphPad Prism software version 9.0 (GraphPad Software, Inc., CA). For the survival rate assessments, survival curves were evaluated using the log-rank (Mantel-Cox) test. For other datasets, significant variances among groups were assessed using Student’s t-test and one-way ANOVA followed by post-hoc tests. The notations used to denote significant distinctions between groups were as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant difference.