Cell and viruses

BHK-21 cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS at 37 °C with 5% CO 2 . RABV strain CVS-24 (GenBank: ADR03123.1) was provided by the Institute of Animal Health, Guangdong Academy of Agricultural Sciences (Guangzhou, China). RABV strain BD06 (GenBank: ACB38373.1) was provided by the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences (Changchun, China). BD06 strain could cause 80% mortality in unvaccinated dogs after challenge [35] and is responsible for most rabies cases in humans and dogs in China [36].

Vaccines

mRNA vaccines were produced based on the Liverna Therapeutics platform (China patent ZL201911042634.2). The mRNA molecules included a 5’ cap structure, a 5’ UTR, an ORF, a 3’ UTR and a poly(A) tail. The ORF in this study encodes the glycoprotein (RABV-G) of the CTN-1 strain (GenBank: ACR39382.1), which has been used for production of human rabies vaccine in China and is Chinese domestic isolates [37, 38]. Three different mRNA constructs were developed: RABV G-A, RABV G-B, and RABV G-C. The RABV G-A sequence consisted of a 5’ UTR, the ORF of RABV-G, a 3’ UTR and a 64A + 36C + histone stem loop. RABV G-B was an optimized RABV G-A sequence with a different ORF. RABV G-C was identical to RABV G-B with the exception of its poly(A) tail. Unless specifically noted, RABV G-C was the sequence of our mRNA vaccine, named LVRNA001.

mRNAs were produced by in vitro transcription (IVT). DNA templates were linearized from plasmids containing the open reading frames flanked by 5’/ 3’UTR and Poly-A sequences. IVT reactions were performed using DNA templates, an optimized T7 RNA polymerase (Novoprotein Scientific Inc., China), NTP and Cap GAG m7G(5′)ppp(5′ (2′-OMeA)pG (Jiangsu Synthgene Biotechnology Co, China). The reaction was terminated by addition of DNase I (Novoprotein Scientific Inc., China). mRNAs were purified using Oligo-dT affinity column (Sepax Technologies, Inc., China) and Tangential Flow Filtration (TFF, Repligen Corporation, America). Microfluidic capillary electrophoresis (Fragment Analyzer systems 5200, Agilent) was used to assess RNA integrity, and the characterization including concentration, pH, residual DNA, proteins, and dsRNA impurities of the solution were also performed. To prepare the mRNA vaccines, purified mRNAs were encapsulated in LNPs according to a modified procedure wherein cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a polyethylene glycol-lipid and a cationic lipid was rapidly mixed with an aqueous solution containing mRNA. Then, the analytical characterization of product was carried out, including the determination of particle size and polydispersity, encapsulation, pH, endotoxin, and bioburden. The final products were lyophilized into powder in 2 ml glass vials. Sterile water (1 ml) was added to each vial to produce a 25 μg/ml (mRNA) solution before use. Appropriate volumes were taken from the vial for animal injection according to the experimental plan.

Inactivated vaccines were purchased from an animal hospital (Rabvac®, produced by Boehringer Ingelheim Vetmedica, Inc. Lot number 4150466A, labelled potency is 1 dose ≥ 2.0 IU) or a domestic vaccine manufacturer (rabies vaccine made from aGV strain for human use, freeze-dried, labelled potency is 1 dose ≥ 2.5 IU).

Protein expression

Protein expression was measured using flow cytometry analysis as previously described [39]. Briefly, HEK293 cells were transfected with either RABV-G mRNA or Luc mRNA (negative control) for 24 h. Cells were then collected and stained with a monoclonal mouse anti-rabies antibody (HyTest Ltd, Turku, Finland) and an FITC-labeled goat anti-mouse IgG (Life Technologies GmbH, Darmstadt, Germany). Flow cytometric analysis of FITC-positive cells confirmed protein expression.

Transmission electron microscopy (TEM)

The morphology of the nanoparticles was analyzed using TEM. Briefly, a drop of aqueous nanoparticle sample was dropped onto a carbon-coated copper grid. Subsequently, the grid was air-dried completely at room temperature. TEM micrograph images were captured at an accelerating voltage of 80 kV.

Mouse and dog immunization and viral challenge

BALB/c mice (~ 6 weeks old, 20–25 g) were purchased from the Institute of Animal Health, Guangdong Academy of Agricultural Sciences (Guangzhou, China). Dogs (~ 3 months old beagles, 5–6 kg) were obtained from the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences (Changchun, China). All experiments were approved by the Research Ethics Committee of the College of Animal Health, Guangdong Academy of Agricultural Sciences, and the Research Ethics Committee of the College of the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences (IACUC of AMMS-11–2021-19). Mouse immunizations were carried out according to the methods described previously [40], LVRNA001 (0.2–5 μg) or 0.1 dose (1 dose ≥ 2.0 IU) of Rabvac® were intramuscularly (i.m.) injected into the thigh of hindlimb once (0d) or twice (0d/7d); 14 days post immunization (dpi), blood samples were collected for antibody tests and mice were injected with viruses (strain CVS-24, 50-fold LD 50 pre-determined by the Institute of Animal Health, Guangdong Academy of Agricultural Sciences for mouse in this experiment) intracerebrally, followed by observation of animal survival and last round of blood sample collection on day 21 post challenge for neutralization antibody test.

For dog challenge studies, LVRNA001 (5 or 25 μg, 0d/7d or 0d/7d/21d) or an inactivated vaccine made for human use (1 dose, ≥ 2.5 IU, 0d/3d/7d/14d/28d) were i.m. injected into the lateral thigh of hind limb. Blood samples were collected on days 0, 7, 9, 11, 13, 35 post first injection for antibody tests. Viruses (strain BD06) were given to the test dogs on day 35 by i.m. injection to the biceps femoris of the hind limb at 50-fold LD 50 (pre-determined by the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences for dog in this experiment). Observation of animal survival continued through 3 months after challenge, when blood samples were collected from survived dogs for neutralization antibody test.

Post-exposure immunization against RABV in dogs

Dogs were intramuscularly injected with 50-fold LD 50 of virulent RABV-BD06 strain in the biceps femoris of the hind limb. Six hours after challenge infection, dogs were i.m. injected with LVRNA001 or inactivated vaccine. Immunization procedures and experimental steps were the same as pre-exposure protocols shown above.

Enzyme-linked immunosorbent assays (ELISA)

Spleen lymphocytes from mice in each immunization group were collected and resuspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS). Then, 4 × 106 cells were seeded into a 24-well flat-bottom tissue culture plate and incubated with 5 μg of a synthesized peptide library of RABV-G for 72 h at 37°C. The cell supernatants were collected, the interferon (IFN)-γ and interleukin (IL)-4 levels were measured using ELISA kits (Neo Bioscience, China) according to the manufacturer’s instructions.

Intracellular cytokine staining

TNF-α-producing CD3+/CD4+ or CD3+/CD8+ T cells from mouse spleen lymphocytes (collected 7 days after booster immunization) were analyzed using flow cytometry. Spleen lymphocytes (4 × 106 cells/ml) were seeded into a 24-well flat-bottom tissue culture plate and incubated with 5 μg of a synthesized peptide library of RABV-G for 72 h at 37°C. Cells were then stained with the following antibodies at 4°C for 20 min: CD8-FITC (1:200), CD3-PerCP-Cy5.5 (1:200), CD4-BV605 (1:200) (all from BD Biosciences, Heidelberg, Germany), and TNF-α-PE (1:100) (BioLegend, San Diego, CA, USA). The fluorescence signals from staining were measured by flow cytometry, and data analysis was conducted using FlowJo software (Tree Star, Inc., Ashland, USA).

RABV-G-specific immunoglobulin measurements

Mouse serum samples were collected and scanned for RABV-G-specific immunoglobulin using a commercially available RABV antibody detection kit (Synbiotics Corp, France) following the manufacturer’s instructions. A positive antibody titer was recognized as OD 450 > 0.2.

Serum neutralization assay

Viral neutralizing antibody (VNA) titers against RABV were determined by fluorescent antibody virus neutralization (FAVN) tests according to standards set forth by the World Organization for Animal Health. Serum samples were serially diluted into a 96-well plate along with WHO reference serum diluted to 0.5 IU/ml. A 50 μL suspension containing the 50% tissue culture infectious dose (TCID 50 ) of challenge virus standard strain 11 (CVS-11, obtained from Chinese National Institutes for Food and Drug Control) was added to each well, and the plates were incubated at 37°C for 1 h. BHK-21 cells were cultured with DMEM containing 10% FBS and then added to the wells, and the plates were incubated at 37°C in a humidified incubator with 5% CO 2 for 48 h. Afterward, the cells were washed and fixed in 80% cold acetone for 30 min. Last, the cells were covered with a RABV-specific monoclonal antibody (KPL, Gaithersburg, MD, USA) followed by FITC-conjugated goat anti-mouse IgG (KPL, Gaithersburg, MD, USA). Fluorescent signals in each well were detected with a fluorescence microscope.

Direct immunofluorescence detection of RABV in mouse brain

Mouse brain tissue was collected and fixed with 4% paraformaldehyde. Tissue samples were blocked with bovine serum albumin (BSA) and incubated with a RABV-specific primary antibody for 1 h. Samples were then washed with an FITC-conjugated secondary antibody and imaged with a fluorescence microscope.

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 6.0 software. Comparisons between groups were performed with one-way ANOVA followed by Tukey’s test. A p value less than 0.05 was considered statistically significant.