2.1. Cell line: Vero cells at passage 131, provided by the Institute Pasteur de Tunis, (Tunis, Tunisia) and originally obtained from ATCC (CCL-81), were sub-cultivated and Vero cells at passage 141 used in this study. BHK-21 C13 cells at passage 77, provided by ATCC, USA for microtitration assay and CVS-11 strain production for challenge studies
2.2. Rabies Virus Strain: Rabies virus Pastor strain LP-2061 at passage 22, provided by Institute Pasteur de Tunis, (Tunis, Tunisia) was conducted the following tests: sterility, mycoplasma, identity (by using the immunofluorescence technique). CVS-11 strain at passage 11, provided by Animal and Plant Health Agency, the UK for challenge studies.
2.3. Cell adaptation to VP-SFM AGT and preparation of Master Cell Bank (MCB)
One vial of Vero cells cryopreserved in MEM+ 20% FBS + 10%DMSO, containing 20×106 cells was revitalized in serum-containing medium EMEM+ 10% FBS according to standard protocol and let to attach for 3 – 4 h at 37 °C, 5% CO2 on T-175 Flasks’ surface. Static cultures of cells adapted to VP-SFM were carried out in a seeding density of 6- 8×104 cells/cm2. The medium was then replaced by VP-SFM + Glutamax I CTS solution (Gibco, USA no:30018), and the cells were incubated at 37 °C, 5%CO2 until the confluency . VP-SFM must be supplemented with Glutamax I CTS solution (includes L-glutamine+ Epithelial growth factors) for cell growth and rabies virus production. Cells were expanded in VP-SFM over six passages to create a master cell bank. Cells were frozen in VP-SFM +10% DMSO + 0.1% methylcellulose, cell density was equal to 5 – 8 ×106 cells/vial.
2.4. Preparation of Working Cell Banks (WCB)
Vero cells (ATCC CCL 81) are maintained for four passages in SFM for cell adaptation to this medium. The cells are amplified through subcultures in T-flasks using animal origin-free recombinant trypsin enzyme, and SFM. Vero cells from these flasks were used to prepare the Master Cell Bank (MCB). The cell suspension is distributed in cryogenic vials with 10 × 106 cells/vial and stored overnight at −80°C, and subsequently in liquid nitrogen. Using VP-SFM +10%DMSO+ 0.1% methylcellulose as a freezing medium for Vero cells was found efficient in growing performance after thawing. Vials from this bank are taken for quality control tests and the MCB is considered certified only after it passed such tests. As described by Rourou et al a working cell bank of adapted Vero cells was established .
2.5. Cell dissociation
Cells were sub cultivated using recombinant trypsin (TrypLE Express, GIBCO, Denmark). Cell subcultivation was performed as described by the manufacturer Briefly the cells were washed twice with Ca++ and Mg++ free phosphate buffer saline (PBS, Sigma) including % 0.01 EDTA, 5–6 mL of recombinant trypsin solution was added to a T-175 flask and cells were incubated for 3 - 5 min at 37 °C. Dislodged cells were then centrifuged at 800 - 1000 rpm for 10 min. The supernatant was discarded and fresh VP-SFM was added to the falcons. Cells were counted by an automated cell counter (Vi-cell XR, Beckman Dickenson, UK).
Spinner flask and bioreactor cultures with microcarriers, the stirrer was stopped and let the microcarriers down, the medium was discarded and microcarriers were washed with PBS and PBS+EDTA solution twice. 5–6 mL of recombinant trypsin solution was added to 500 ml and 3L spinner flasks or 30 L bioreactor and after a contact time of 10 -12 min at 37 °C, cell detachment was completed and 0.4 g/l STI was added to spinner or bioreactor (Figure 1: A, B). Dislodged cells were then centrifuged and washed as mentioned above.
2.6. Cell counting
Five millilitres of Vero cell culture were washed three times with PBS then treated in 5mL of 0.1M citric acid (Sigma) containing 0.1% crystal violet (Sigma) and 0.1% Triton X-100 (USB, Cleveland, OH, USA) and incubated at least for 1 h at 37 °C. The released nuclei were counted using a hemacytometer (HBG, Germany). Dissociated cells from microcarriers were counted by the Vi-Cell XR cell counting machine (Beckman Dickenson, UK).
2.7. Monitoring of cell infection
Aliquots of 5 mL of infected microcarriers culture were washed three times with PBS and then resuspended in a final volume of 2 ml. 2× 50µL were put into a microscope slide and let dry at room temperature in a Class II B cabinet. The slide was then fixed with 80% acetone and stained with fluorescein-labelled specific nucleocapsid monoclonal antibodies DFA reagent kit (EMD Millipore, Canada, Cat. Ref No. 5100). Slides were inspected using a fluorescent microscope with 490 nm excitation and 515 nm emission peak filters. Rabies protein in infected cells will fluoresce bright apple green (Figure 2: A, B).
2.8. Preparation of the Virus Seed Banks
Vero cells cultivated in 225 cm2 T-flasks were inoculated with Rabies LP-2061 strain with a multiplicity of infection (MOI) of 0.25–0.3, and the cultures were maintained in VP-SFM at 34 - 35°C for 3 days. The viral culture supernatants were harvested in a class II cabinet on days 4 after infection. The Rabies virus supernatants obtained in the harvests are clarified through a 0.45 to 0.6 μm membrane filter, and samples are taken for in-process tests. Then, the clarified Rabies virus harvests are stored at −80°C ± 5°C. After the determination of viral titer (≥10 5.0 FFD50/mL), clarified harvests are used to prepare the Virus Master Seed lot.
The rabies virus Working Seed Lots were prepared from rabies virus Master Seed Lots obtained in a maximum of five virus passages. The harvests were clarified (0.45 to 0.6 μm filter), distributed in aliquots, and stored under −156°C ± 5°C. Samples of this bank were taken for quality control tests.
Advantageous agent analyses were carried out on 13 – 15 g weight of mice, suckling mice, and 350 – 450 g weight of guinea pigs according to European Pharmacopeia.
2.9. Production of experimental rabies vaccine
Cytodex 1 microcarriers from Cytiva (Sweden) were used at 3 – 4 g/L throughout this study. Size ranges included 90-110 µm. They were prepared with PBS and sterilized according to the manufacturers’ recommendations.
Cultures were carried out in 500 mL spinner flasks (Corning, USA) containing 500 mL of cultured cells in VP-SFM+ Glutamax I CTS, at 37 °C in a 5% CO2 incubator as described by Trabelsi et al. . The stirring speed was set up at 25 - 35 rpm with a slow speed magnetic stirrer (VWR, USA) in an incubator. The spinners were inoculated with 5– 7×105 cells/ml. Samples were taken daily to observe cell confluency and growth parameters on microcarriers by an inverted microscope (Olympus, Japan). 3L spinner flasks (Corning, USA) containing 3000 mL of cultured cells, at 37 °C in a 5% CO2 incubator as mentioned above. The culture was started with 7×105 cells/ml and was maintained at 25 - 30 rpm stirring speed. Samples were taken daily to perform microscopic analysis and off-line analysis. pH set at 7.1 - 7.3 (regulated by the addition of NaHCO3 at 88 g/L). 50% of the culture medium was replaced daily two days after cell inoculation. For the rabies virus production step, pH was maintained at 7.2 - 7.4, agitation rate at 30 rpm and temperature at 34 - 35 °C. Once the temperature reached 34 -35 °C, cells were washed with PBS twice, added fresh VP-SFM at % 30 volume of spinner flasks and then infected at an MOI of 0.3 with the Rabies virus LP-2061 strain. Two hours after cell infection, VP-SFM was completed to the final volume of spinner flasks. Samples were taken daily to determine the following parameters: cell count, microcarriers load, virus titer, and cell infection. Supernatants were harvested after 4 days of infection and then repeated every 48 – 62 hours for 5 additional harvests. Spinner flask culture was repeated three times.
30 L basket bioreactor (SARTORIUS, Biostat C) starting volume was equal to 30 L with VP-SFM + Glutamax I CTS. The culture was seeded with 5 - 7 ×105 cells/mL from WCB, cultivated at 36.5 - 37°C and was continuously agitated at 30 rpm until the cell’s confluency was completed on the microcarriers (3 – 4 g/L). During the cell culture proliferation step, pH is set at 7.1 – 7.4 (regulated by injection of CO2 or addition of NaHCO3 at 88 g/L), pO2 maintained at 45 – 75 % air-saturation or pure oxygen when required, the temperature at 37 °C and agitation rate at 30 – 50 rpm. Samples were taken daily to perform microscopic analysis and off-line analysis (Figure 3: A-D). 50% of the culture medium was replaced daily two days after cell inoculation. Because the viability and growth parameters of cells could be affected by the shear environment, we used Pluronic F-68 10% (Gibco, USA) in the cell culture medium in a concentration range of 0.1% [18,19]. The addition of shear protectants such as Pluronic F-68 to a serum-free medium is often needed to protect cells against shear forces [20, 21].
For the rabies virus production step, pH was maintained at 7.2 - 7.4, pO2 at 30 – 40 % air-saturation, agitation rate at 30 – 50 rpm and temperature at 34 °C. Once the temperature reached 34 °C, cells were washed with PBS twice, added fresh VP-SFM at % 30 volume of bioreactor and then infected at an MOI of 0.3 with the Rabies virus LP-2061 strain. Two hours after cell infection, VP-SFM was completed to a final volume of 30 L basket bioreactor. Supernatants from this culture are harvested as mentioned above. Samples of the harvests are used for offline analysis.
The viral suspensions obtained in the harvests were clarified by low-speed centrifugation (15 min 3500 x g) and filtration (0.45 μm) and then harvests were concentrated 10 – 20-fold and buffer exchanged by Tangential flow filtration using Sartorius benchtop System (Sartorius Stedim, Germany) at a flow rate of 27 mL/min equipped with Slice 200 cassette Hydrosart (cut off 100 kDa) (Sartorius Stedim, Germany), transmembrane pressure (TMP) was around 2.7 bar. Concentrated bulk was inactivated by β-propiolactone (Acros, Germany) (final dilution 1/4000) at 2 – 6 °C for 18 - 24 hours . The inactivated bulk was transferred to a sterile vessel and stored by slow speed stirring at 25 °C for 2 h. After this period, samples are taken for sterility and effective virus inactivation tests .
Inactivated bulk was purified by AKTÄ Avanth system (GE Healthcare Life Sciences, Uppsala, Sweden) using XK 16 20 chromatography column. 450cm/hr velocity was used. Capto Core 700 chromatography resin (GE Healthcare Life Sciences, Uppsala, Sweden) was equilibrated with 10 Column volume with 20 mM Tris, 150 mM NaCl. A fraction that includes purified virus was collected in the flow-through; the impurities were discarded using 30 Column volume with 20 mM Tris and 1 M NaCl  (Figure 4).
The purified and inactivated Rabies virus concentrates obtained in a bioreactor cycle are mixed, filtered (0.45 + 0.22-μm capsule filter) in a 50-L Flexibody bag, and stored at 2–8°C. Samples of this mixed concentrate are taken for the quality control tests mentioned in European Pharmacopeia.
The residual Vero host cell DNA was measured using a res DNA-SEQ quantitative VERO HOST CELL DNA kit (Applied Biosystems, Life Technology, Warrington, UK). The isolation of the Vero cell DNA was performed according to the manufacturer’s recommendations. DNA Quantitation kit was used to measure residual Vero host cell DNA in the purified bulk using the Roche LightCycler 480 instrument.
Residual host cell protein levels were quantified using the Vero Host Cell Proteins kit (Cygnus Technologies, Southport, USA) according to manufacturer recommendations. The results were read at 450 nm wavelength in an ELISA reader with a GEN-5 software interface (BIOTEK Epoch Agilent, USA).
The bulk rabies vaccine was adjusted by adding 0.01 M Phosphate Buffered Saline solution, glycine (Merck, Germany), sucrose (Merck, Germany), and human serum albumin 1% (Sigma, USA) to obtain a potency of a minimum of 3.0 IU/dose. Samples were taken for necessary quality control tests according to European Pharmacopeia.
The formulated product is filled according to vaccine presentation: after the buffer exchange and the formulation the samples were immediately frozen at –80 °C and lyophilized, and subsequently, the samples were analysed. 0.7-mL vials for use in the freeze-dried process. The protocol (Table 1) was established with some modifications related to lyophilization described by Severo et al. . Mean residual moisture obtained was given in Table 2 for the freeze-dried vaccine and the results were found according to WHO standards .
2.10. Transmission electron microscope (TEM) experiments
The samples were negatively stained using 2% uranyl acetate (UA). Inactivated samples were mounted on pyeloform-covered copper grids, stained with 2% uranyl acetate (UA), washed two times with sterile pure water, and monitored in a TEM (Jeol, Tokyo, Japan) .
2.11. Glycoprotein titration
Rabies glycoprotein content was determined by commercial rabies antigen ELISA kit (DRG-ELISA, Germany) using monoclonal antibodies as manufacturer recommendations. ELISA assay was defined by M. Chabaud-Riou et al. as can quantify the rabies viral G-protein with high specificity, linearity, accuracy, and precision . In the 2011 Ames international workshop (USA), worldwide rabies experts agreed on the need to replace the NIH test with an enzyme-linked immunosorbent assay (ELISA) . The amount of rabies glycoprotein was read at 450 nm wavelength in a microtiter plate reader with GEN-5 software interface (BIOTEK Epoch Agilent, USA).
2.12. Rabies virus titration
Virus titres were determined using the Vero cell line (ATCC; CCL-81) in microplates. Cells (5 x 104) were distributed in each well and infected with 50 µl of log10 dilutions of either virus samples or a PV-LP2061/VERO reference virus previously titrated by the plaque-forming technique. After 24 h, microplates were washed with PBS (phosphate buffer saline supplemented with Ca2+ and Mg2+) and fixed with cold 80% acetone. Cells were stained with fluorescein-labelled anti-rabies-nucleocapsid immunoglobulins (EMD Millipore, Canada, Cat. Ref No. 5100). Results were expressed in fluorescent focusing units per ml (FFU/ml) by comparison of the sample assays with the reference.
2.13. Nucleic acid extraction and first-strand cDNA synthesis
Total nucleic acids were extracted from clarified viral supernatants using the Mini Pathogen Nucleic Acid Kit (Qiagen, Germany) according to the manufacturer's instructions and collected in 50 – 100 μL elution volumes. The viral RNA was reverse transcribed depending on Moloney murine leukaemia virus reverse transcriptase (Thermo Scientific, USA) according to the manufacturer’s recommendations. At first, reaction mixtures were incubated for 60 min at 42˚C, and then the reaction was completed by heating the mixture at 95˚C for 5 min. The mixtures were chilled on ice, immediately.
48-hour (R9.4) sequencing protocol was performed using MinION™ control software, MinKNOW™ version 0.46.1.9 (R9.4). Read data were obtained based on 1.2.2 rev 1.5 workflows and software Metrichor™ agent (version 0.16.37960).
After sequencing, the results obtained in fast5 format were converted to fastq format using guppy v4.4.1 software (base-calling and de-multiplexing). Barcode and adapter sequences were cleaned using Porechop v0.2.3 software, and universal primers and tags were also deleted by deleting 45 bases from both ends of the sequences. After clearing the sequences, reads less than 100 bp in length were filtered and excluded from the analysis.
Cleaned reads were analysed with a customized workflow using the Genius Prime 2021.0.3 platform. The sequences were mapped by comparing the Rabies lyssavirus - NC_001542.1 viral whole-genome in the NCBI database, and the consistency of the resulting contigs and the organisms they matched in the database were scanned by de novo alignment. In addition, the consensus sequences produced polymorphism results and statistical information of contigs are presented in the manuscript.
2.15. SDS-Page and Western Blotting
β-Propiolactone inactivated Rabies virus samples were separated using the 10% polyacrylamide gels described by Laemmli and visualized using the Coomassie Blue staining kit (BIO-RAD).
2.16. Quantitative Real-Time PCR
The qRT-PCR assay was performed using the 2x One-Step RT -qPCR Master Mix, (Techne, UK). The Rabies Techne qPCR test is a highly specific in vitro diagnostic quantitative real-time reverse transcriptase PCR (qRT-PCR) assay to quantify nucleic acid from the Rabies virus. The assay utilizes real-time technology targeting the 3’ leader and nucleoprotein region. Each 20-μL reaction contained 10 μL Lyophilised OneStep 2x Master Mix (Techne, UK), 1 μL 5 μmol/L probes, 20 μmol/L forward and reverse primer mix, 4 μL nuclease-free water, and 5 μL nucleic acid extract. Amplification was carried out on a ROCHE LightCycler 480 Real-Time PCR instrument (ROCHE). Real-time PCR conditions consisted of 10 min at 55°C for reverse transcription, 2 min at 95°C for enzyme activation, and 40 cycles of 10 s at 95°C and 60 s at 60°C for a polymerase chain reaction. Specific rabies virus nucleotide copy number/µl is calculated based on the standard curve by absolute quantification of Roche 480 original software.
2.17. Determination of LD50 of rabies virus CVS strain
Determination of the rabies virus LD50 (MIT – mouse inoculation test) was carried out in 8–10 g Balb/c mice [28, 29]. Intracerebral inoculation of mice was performed with 6.1 LD50/0.03 ml of the CVS-11 strain. Five different tenfold dilutions (10–3 to 10–7) were administered to five groups of five Balb/c mice each. The only mouse that died between the 5th and 14th days after the challenge was considered to have died from rabies. Monitoring of the animals continued for 21 days. Specific death (was verified by FAT detection of the rabies antigen on brain imprints in a fluorescent microscope and, then the LD50 of the challenge virus strain was calculated according to Reed and Muench (1938) .
2.18. Potency tests
The total protective efficiency of lyophilised inactivated rabies virus vaccine candidate was determined according to the NIH test in 13 - 15 g weight Balb/c mice. Balb/c mice were immunized at days 0 and 7 by IP (intraperitoneal) route and then challenged by the intracerebral (IC) route using the Challenge Virus Standard strain (CVS-11 strain) a week after the second dose. An international reference vaccine was used to calculate potency expressed in International Units/mL (IU/mL) [1,31]. The only mouse that died between the 5th and 14th days post-inoculation was considered to have died from rabies. Brain imprints of non-vaccinated animals and challenged at day 21 with CVS-11 strain of rabies virus showed green-apple cytoplasmic inclusions intracerebrally challenged with CVS-11 strain (Figure 5).