Formulation enhanced the stability of Foot-and-mouth virus and prolonged vaccine storage

Baby Hamster Syrian Kidney (BHK-21cells) were cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM) supplemented with 10% FBS. FMDV strains, O/MYA98/BY/2010 and Asia1/JSL/ZK/06 investigated extensively as vaccine strains, were provided by Zhongnongweite biotechnology Co., Ltd. Each strain was propagated in BHK-21 cells at a multiplicity of infection of 0.001 and incubated in 5% CO₂ at 37 °C. A PrimeScriptTM RT reagent kit containing gDNA Eraser and SYBR Premix Ex TaqTM II (Tli RNaseH Plus) was purchased from TaKaRa (Dalian, China). MTS assay was available from Abcam (Cambridge, UK). The 50% tissue culture infectious dose (TCID₅₀) was measured with the Reed and Muench method. Trehalose, CuSO₄·5H₂O were purchased from sigma (MO, USA). All other chemicals were analytical grade reagents purchased from Beijing Chemical works (Beijing, China) and all solutions were prepared using Milli-Q grade water (Millipore,USA).

The thermal stabilization effect of trehalose, NaCl and CuSO₄·5H₂O on virus were evaluated by anti-aging test with high performance size exclusion chromatography (HPSEC) method [21]. Briefly, the samples of 100 ul were injected and eluted at 0.6 ml/min with 50 mM phosphate buffer at pH 7.2 containing 100 mM Na₂SO₄. The peak area at 259 nm was linearly proportional to 146S concentration with established standard curve. Therefore, 146S content could be calculated according to the peak area. In order to quickly assess the efficiency of stabilizers, formulated virus was incubated at 37 °C for 5 h to accelerate dissociation. Briefly, virus was supplemented with trehalose, NaCl and CuSO₄·5H₂O at various concentrations respectively, as detailed in Table 1. Each FMDV was then inactivated with 1 mM binary ethyleneimine (BEI) at 37 °C for 24 h [22]. Subsequently, the formulation was determined as a combination of trehalose, NaCl and CuSO₄·5H₂O with optimal concentration screened.

MTS assay was performed to measure the cytotoxicity of formulation. In brief, 5 × 10⁴ of BHK-21 cells in 100 μl of complete medium were seeded into each well of a 96-well plate. After incubated at 37 °C for 24 h, the cells were incubated with 100 μl of formulation at various concentrations (1%, 3%, 5%, 10%) for another 72 h. The control cells were treated with DMEM. Then, the supernatants were discarded and 100 μl of DMEM along with 20 μl MTS solution were added to each well for additional 3 h. The cell viability was determined by the percentage of absorbance of the treated cells at 490 nm to that of the control cells.

Infectivity assay were carried out to further confirm the thermal stabilization effect of formulation on virus. After exposed to formulation for 16 h, virus were incubated at room temperature for 3, 5, 10 and 15 h respectively and then used for cells infection. The control virus were incubated for the same periods, but without exposure to the formulation. Briefly, 4 × 10⁵ cells (BHK-21) in 100 μl complete medium were seeded into each well of a 96-well plate. On the following day, the supernatants were discarded, and the cells were washed three times with MEM. Then, the cells were infected with treated virus at serially dilution (eight wells for each concentration) for 1 h. After removing inoculum and adding MEM, the plates were incubated for an additional 24–48 h at 37 °C. Until the cytopathic effect (CPE) was observed, TCID₅₀ value was calculated with Reed and Muench method [23]. The supernatants of each well were collected and the viral mRNA were determined by real-time PCR. Briefly, the total RNA from the BHK-21 cells was extracted using TRizol reagent, and 1 µl of RNA was used in reverse transcription reaction using a PrimeScrip™ RT reagent kit containing gDNA Eraser, following the manufacturer’s instructions. The reaction mixture for real-time PCR comprised diluted cDNA (1 µl), 10 µl primers (3DF, 5′-ACTGGGTTTTACAAACCTGTGA-3′; 3DR, 5′-GCGAGTCCTGCCACGGA-3′) and 12.5 µl of SYBR Green Master Mix to a final volume of 25 µl. The amplification conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 56 °C for 30 s, and 72 °C for 30 s.

The inactivation rate of virus both in formulated and non-formulated virus were determined after incubation at 25 °C, 37 °C for 36 h and 10 h respectively. Virus were sampled at indicated time points to measure virus titers, referred to TCID₅₀ value. According to the log of TCID₅₀/ml value over time, the linear curve of inactivation on virus infectivity was established, and the time when the virus lost 50% infectivity was obtained and compared.

To validate if the formulated virus has stable antigen expression throughout multiple passage, the virus genomes of different passage were determined. The conserved VP1 gene was amplified with PCR. Briefly, the formulated virus was continuously passaged for 12 times, then the genomic RNA in every 3 generation were isolated for reverse transcription. The VP1 gene was amplified by PCR in a final volume of 50 μl, containing 1 μl of each primer, 5 μl of PCR buffer, 4 μl of dNTP, 0.25 μl of DNA polymerase, 1 μl of DNA, and 37.75 μl of RNase-free water. The thermocycler conditions were an initial denaturation at 95 °C for 3 min, denaturation with 35 cycles at 95 °C for 50 s.

Having shown that the formulation could increase the thermostability of infectious FMDV, we speculated that the thermostability of inactivated vaccine would also be enhanced. To facilitate this, both non-formulated and formulated virus were inactivated and then emulsified with adjuvant ISA206 to produce vaccine. The 146S content in vaccine was monitored via HPSEC method during storage at 4 °C for up to 1 year. Samples were collected at indicated time points and then mixed with 1-pentanol in a 10 ml centrifuge tube at a ratio of 9:1. The mixture was shaken fully to break the emulsion. After being placed at 4 °C for 1 h, the upper oil phase was removed and the aqueous phase at the bottom was absorbed slowly for 146S determination.

This study was aimed to develop a formulation for FMD virus which has poor thermal stability. Trehalose, NaCl and CuSO₄·5H₂O were selected as protectant agents and the optimized concentrations were determined using anti-aging test by detecting residual 146S concentration. The test was performed at 37 °C to quickly screen reasonable concentration. As indicated in Table 1, both formulated and non-formulated FMDV 146S decreased significantly after incubation at 37 °C for 5 h. Whereas, the formulated virus exhibited relatively stable compared to non-formulated one. Among them, twenty trehalose, 500 mM NaCl and 3 mM CuSO4·5H2O was observed to protect virus against dissociation efficiently, with the residual 146S of 6.5 ± 0.3, 5.7 ± 0.3 and 5.8 ± 0.1 μg/ml respectively, thus, the formulated virus was determined to be a combination of twenty percentage trehalose, 500 mM NaCl and 3 mM CuSO₄·5H₂O.

Cytotoxicity assay were carried out to determine whether the formulation had toxic effects on BHK-21 cells and then attenuated virus infection. As is shown in Fig. 1, the formulation presented no cytotoxicity to cells. The cell viability was 95.3%, 94.8%, 97.2% and 94.2% at formulation concentrations of 1%, 3%, 5% and 10% (w/v) respectively.

To evaluate the thermal stabilization effect of formulation on virus, we firstly investigated the infectivity of the virus by monitoring the TCID₅₀ of formulated virus stock at different temperatures. As shown in Fig. 2a, the TCID₅₀ values of both formulated and non-formulated virus stock declined in a time-dependent manner during storage at room temperature, but the log₁₀TCID₅₀/ml values of formulated virus stock (6, 5.3, 3.8 and 1.9) were higher than the non-formulated one (4.2, 3.6, 1.7 and 0) after 3, 5, 10 and 15 h storage. The stable effect of formulation was then verified using real-time PCR analysis. Consistent with the TCID₅₀ value, the cell infected with formulated virus had higher relative mRNA level of 4.2%, 2.8%, 0.011% and 0.008%, compared to non-formulated virus (2.8%, 1.95%, 0.003% and 0.002%). Microscopy showed that after treatment at 37 °C for 10 h, the cells inocubated with formulated virus had a higher proportion of infected cells compared to non-formulated virus. Encouragingly, the formulation also show preserving activity for FMDV Asia1/JSL/ZK/06 strain, with log₁₀(TCID₅₀ /ml) values of 6.6, 5.7, 3.7 and 2.6 (formulated virus) as opposed to 4.2, 3.6, 1.7 and 0 (non-formulated virus). Similarly, the relative mRNA level is higher in formulated virus (5.1%, 3.05%, 0.0205% and 0.011%) than that of non-formulated virus (3.25%, 2.1%, 0.006% and 0.0035%) (Fig. 2b). Taken together, these results suggested that the formulation could stabilize the virus effectively and possess a potential broad-spectrum stabilization activity on other serotype of FMDV viruses.

The inactivation rate of virus both in formulated and non-formulated were determined after incubation at 25 °C, 37 °C for 36 h and 10 h respectively. Viruses were sampled at regular time points for virus titers detection, referred to TCID₅₀ value. As indicated in Fig. 3a, the formulated virus had a lower inactivation rate than the non-formulated virus, and the time against 50% loss in virus infectivity prolonged from 6 to 7 h at 25 °C, 0.9 h to 1.6 h at 37 °C. Also, the temperature required for complete inactivation of formulated virus increased to 64 °C, compared to 62 °C for non-formulated virus (Fig. 3b).

The stable expression of antigen gene was crucial for vaccine quality. Whether the formulation influence antigenic expression should be fully identified. It showed that the VP1 gene was consistent in continuous 12 passages and the amplification of fragment VP1 yielded 813 bp. In addition, the virus titer in different passages were maintained at relatively steady level (7.0 log10 TCID50/ml) (Additional file 1). These results demonstrated that the main protective antigenic site of FMDV remained stable during virus passage, ensuring the safe and efficiency of vaccine production.

Reducing dependence on cold storage and improving the stability of vaccine are two objectives of developing vaccine formulation. In order to further evaluate the efficiency of formulation on vaccine, we investigated the shelf life of vaccine stored at 4 °C. The 146S concentration indicates intact FMDV particles and therefore is a crucial parameter to assess vaccine quality. We applied HPSEC to monitor the 146S concentration considering its high accuracy, high sensitivity and requiring less time. As shown in Fig. 4a, the formulated vaccine exhibited excellent stability, half of the 146S remained even stored at 4 °C for 12 months, indicative of effective protection. However, the normal vaccine (prepared with inactivated virus for no formulation) resulted in a time-dependent loss in 146S concentration, only 10% remained. Furthermore, the dosage form of the formulated vaccine is W/O/W, rarely show any morphological abnormality (Fig. 4b). Collectively, these observations suggested the stabilizing effect of formulation on vaccine, allowing for its effective stockpiling.