Animals

All animal procedures were approved by the Institutional Animal Care and Use Committee of Beijing Institute of Biological Products Co., Ltd. Male mice aged 3–4 weeks were used for the experiments.

2BS cell culture

The human diploid cell line 2BS was derived from the lung tissue of an aborted 3-month-old foetus and was established in 1974. The cell line has a long life cycle and can be passaged for 66 generations. 2BS cells were cultured in DMEM supplemented with 10% foetal bovine serum (FBS) (HyClone, USA) for 6–8 days. Then, the cells were digested and passaged. The passaged cells were used for rabies virus culture.

Western blot analysis

Proteins were extracted from the rabies virus samples using ice-cold RIPA lysis buffer. The total protein concentrations of the samples were measured by a BCA assay (Pierce, USA). Proteins were then separated on SDS‒PAGE gels and dyed with Coomassie brilliant blue or transferred to PVDF membranes (Merck Millipore, Germany). The membranes were blocked with 3% w/v bovine serum albumin for 1 h and then incubated with primary antibodies against the rabies virus G protein (HyTest 3R7-4F1, China) overnight at 4 °C. After washing with 1× TBST, the membranes were incubated with the appropriate secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Biodragon, China). Immunoblots were visualized with enhanced chemiluminescence (ECL) reagent and evaluated using a ChemiDoc XRS + instrument (Bio-Rad, Hercules, CA, USA).

Coomassie bright blue staining

The SDS‒PAGE gel with proteins was put into a protein staining apparatus (eStain LI, Genscript). The gel was stained for 2 min with Coomassie blue for one cycle and eluted for 2.5 min with decolorizing fluid for two cycles.

Sequencing

Nucleotide sequencing was performed by allwegene Beijing (Beijing, China). Genomic RNA was extracted from the viruses at different passage numbers, and the nucleic acid concentration was determined by a Qubit instrument. A VAHTSTM Universal DNA Library Prep Kit for Illumina V3 was used for library construction. After the library was sequenced with a NextSeq550 instrument (Illumina, USA), clean reads were obtained after quality control, bwa software (v0.7.17-r1198-dirty) was used to conduct in-depth comparison analysis with the reference genome (aG strain (GQ412744.1)), and the comparison rate and coverage were calculated.

Enzyme-linked immunosorbent assay (ELISA)

We assessed antigen levels by ELISA. Anti-aG strain antibodies were extracted from mouse sera and used to coat the ELISA plate. The anti-aG strain antibody was added to the 96-well plate and incubated at 37 °C for 1–2 h, followed by incubation at 4 °C overnight. The next day, the 96-well plates were blocked with 3% BSA for 1 h at 37 °C. Subsequently, the cells were added and cultured for 1 h. After incubation with the enzyme-labelled antibody for 30 min at 37 °C, TMB one solution and termination solution were added sequentially. The absorbance (OD value) of each well was measured by a microplate reader at wavelengths of 450 nm and 630 nm, and the final OD value was determined by subtracting the 630 nm absorbance value from the 450 nm absorbance value.

Virus plaque formation

2BS cells were seeded on six-well plates, and the rabies virus adapted to the 2BS cells was added at the same time. Then, 2BS cells and the rabies virus were cocultured in an incubator at 37 °C with 5% CO2 overnight. The next day, the culture solution was discarded, 0.5% methylcellulose was added, and 2BS cells infected with rabies virus were cultured for another 6 days. When cytopathic or suspected lesions could be seen on the six-well plates, the lesion site was selected. The selected plaques were placed in 96-well plates with a 2BS cell suspension and cultured in an incubator at 37 °C with 5% CO2 for 6 days. The supernatant was then harvested and transferred to 24-well plates with a 2BS cell suspension. After culturing for 6 days, the supernatant was harvested, and the antigen content was detected. The viruses with high antigen content were selected for further culture with 2BS cells. The virus titre was detected after 3 generations of the rabies virus with high antigen content adapted on 2BS cells, and the virus with the highest titre was selected for further culture.

Mass spectrometry

Proteins were extracted from rabies virus samples, and the protein structures were disrupted by treatment with the surfactant RapigestTM SF (Waters Corporation, USA). Then, the disulfide bond was opened by DTT (EMD Millipore Corporation, USA), followed by blocking with IAM (EMD Millipore Corporation, USA). The proteins were finally digested with trypsin, and the reaction was terminated by formic acid. Peptide separation was carried out on an Acquity Pertide BeyC18 column (Waters Corporation, USA), and the samples were collected by a Thermo Scientific Q Exactive Plus mass spectrometer. The collected data were analysed with BioPharma Finder software. Samples with low bias (Delta ≤ 5 ppm), a confidence score of 100, and the detection of both parent and daughter ions were compared.

Direct fluorescent antibody test

A direct fluorescent antibody test was used to identify the rabies antigens of 2aG4-B40. BHK-21 cells were plated on 96-well plates and allowed to form a monolayer. Then, the cells were infected with the virus (diluted 5-fold series) for 24 h at 37 °C. Next, the cells were fixed with 80% ice-cold acetone and stained with isothiocyanate FITC-labelled N-protein. The plates were observed via fluorescence microscopy.

Virus titration

The virus titre was measured via intracerebral injection in mice as reported in the Chinese Pharmacopoeia. Briefly, the brains of NIH mice in different groups (6 mice for each group) weighing 11 ~ 13 g were inoculated with 0.03 ml of serial 10-fold dilutions of virus, and nonspecific deaths that occurred within three days after inoculation were excluded. The numbers of mice that developed clinical symptoms and died were scored after 14 days. The median lethal dose (LD50) and virus titres were calculated using the Reed and Muench method.

Rabies virus identification

The specific properties of the 2aG4-B40 strain were identified by an intracerebral neutralization test in mice. The virus was diluted 10-fold in series, and the appropriate dilutions of virus were mixed with rabies immunoglobulin (neutralization group) or negative serum (control group) in equal amounts. Groups of NIH mice (6 mice in each group) weighing 11 ~ 13 g were inoculated intracerebrally with 0.03 ml of the mixture at each dilution, and nonspecific death within three days after inoculation was excluded. The numbers of mice that developed clinical symptoms and died were scored after 14 days. The median lethal dose (LD50) and virus titres were calculated using the Reed and Muench method. The neutralization index is the antithesis of the control virus titre minus the neutralization virus titre.

Preparation of the test vaccine

The harvested 2aG4-B40 strains were first concentrated on 300 kDa ultrafiltration membranes (Millipore Pellicon 2, USA) and then purified on chromatographic columns. The purified virus was inactivated with β-propanolactone. The test vaccine was then formulated with the purified virus at an antigen concentration of 200 mIU/mL. The test vaccine was lyophilized with suitable protective agents in a vacuum freeze-drier (Shanghai Kyowa Vaccum Engineering Co., Ltd.) through a lyophilization process consisting of three stages: freezing at -40 °C, primary drying at -3 °C and secondary drying at 28 °C.

Immunogenicity studies

Preexposure prophylaxis (PrEP) and postexposure prophylaxis (PEP) vaccination are effective means to prevent rabies disease. Thus, the immunogenicity of the test vaccines was determined by PrEP and PEP vaccination.

The PrEP vaccination regimen was administered by intraperitoneal injection on D0 and D7. Specifically, NIH mice (12–14 g) were injected with 0.5 ml of test vaccines on Day 0 and Day 7. The negative control mice were injected with 0.5 ml of PBS in the same way. Both groups of mice were administered the standardized virus (CVS strain, 0.03 ml, 10 mice per dose) in serial 10-fold dilutions by intracerebral injection on Day 14. The numbers of mice that developed clinical symptoms and died were scored after 14 days. The median lethal dose (LD50) was calculated using the Reed and Muench method. The protection index was determined as the ratio of the LD50 between the vaccine group and the control group.

As recommended by the WHO, the five-dose regimen (Essen, 1-1-1-1-1) was one of the PEP IM regimens. To determine the rabies virus neutralizing antibody concentration (RVNA) in mice, the rapid fluorescent focus inhibition test (RFFIT) [24] was used to evaluate the serum samples that were collected at Days 0, 7, 14, 28 and 42.

Vaccine potency test

The NIH test (scheme) was selected as the detection method to evaluate the protective efficacy of the adapted vaccine against infection in mice and compare it to that of the national standard rabies vaccine. Different dilutions of the national standard and test vaccines were intraperitoneally injected into NIH mice (12–14 g, 0.5 ml for each mouse, 10 mice per dose) on Day 0 and Day 7. Then, all of the mice were challenged with standardized virus (CVS strain, 0.03 ml) via intracerebral injection on Day 14. Titration of the CVS strain was indispensable for this test. The median effective dose (ED50) was calculated using the Reed and Muench method. The relative potency of the test vaccines were obtained by comparison with the ED50 of the national standard.

Statistical analyses

Statistical data are presented as the mean ± SEM (standard error of the mean). GraphPad Prism version 9.0 was used for all the statistical analyses. Paired two-tailed Student’s t tests were used to compare two groups, and one-way analysis of variance (ANOVA) was used to compare differences among > 2 groups. All of the statistical experiments were repeated at least three times independently. P < 0.05 was considered to indicate statistical significance.