Screening and formulation determination
This study aimed to develop a formulation for FMD virus possessing with thermal stability properties. Trehalose, NaCl and CuSO4·5H2O were selected as protectant agents and the concentration was determined by anti-aging test for detecting residual 146s concentration. The assay was performed at 37 ℃ to quickly screen reasonable concentration. As indicated in Table 1, the stability of virus treated with and without protectant were declined after being incubated at 37 ℃ for 5 h. Whereas, the preserved virus exhibited relatively stable compared to naked one. Among them, 20% trehalose, 500 mM NaCl and 3 mM CuSO4·5H2O was observed to protect virus against dissociation dramatically, with the residual 146s of 6.5, 5.7 and 5.8 respectively, thus, the confirmed formulation was determined to be a combination of trehalose, NaCl and CuSO4·5H2O with the optimal concentration.
Cytotoxicity assay was carried out to determine whether the formulation had toxic effects on BHK-21 cell and then attenuated viral infection. As is shown in Figure 1, the formulation presented no cytotoxicity to cells. The cell viability were 95.3%, 94.8%, 97.2% and 94.2% when treated with 1%, 3%, 5% and 10% (w/v) formulation, respectively. These data indicated that the formulation had no influence on viral infection efficacy.
Heating-resistance investigation
To evaluate the thermal stable effect of formulation on virus, we firstly investigated the virus-infecting capacity using the results of infectivity assay. Figure 2a illustrated that viral infectious dose, indicated as TCID50 values, was decreased in a time-dependent manner during storage at room temperature. Whereas, compared to control one (no exposure to formulation), the preserved virus showed relatively stable, with TCID50 values of 6, 5.3, 3.8, and 1.9 at 3, 5, 10 and 15 h, respectively, higher than the control sample (4.2, 3.6, 1.7 and 0). The stable effect of formulation was then verified using q-PCR analyses on infected cells. Consistent with the TCID50 values, the cell infected with preserved virus results in increased relative mRNA level of 4.2, 2.8, 0.011 and 0.008, compared to naked virus (2.8, 1.95, 0.003 and 0.002). Microscopy showed that more cells were infected with formulated virus after treatment at 37℃ for 10 h (Figure 2c). Interestingly, the formulation also display active against another FMDV strain, Asia1/JSL/ZK/06, with TCID50 values of 6.6, 5.7, 3.7 and 2.6 detected at the same time point mentioned above (Figure 2b). Taken together, these results suggested that the formulation could stabilize the virus effectively and possessed a potential broad-spectrum stabilize activity against other serotype of FMDV viruses.
Inactivation dynamics study
The inactivation rate of virus both in formulated and naked was determined after incubation at 25℃, 37℃ for 36 h and 10 h respectively. Virus were sampled at regular time points for virus titers detection, referred to TCID50 values. As indicated in Figure3, the formulated virus had a lower inactivation rate compared to naked virus, the time against 50% lose in virus infectivity prolonged from 6 h to 7 h at 25℃, 0.9 to 1.6 h at 37℃. Similarly, the temperature required for complete inactivation of formulated virus increased to 64℃, compared to 62℃ for naked virus.
Identification for viral genetic stability
The stable expression of antigen genesis was crucial for vaccine quality. Whether the formulation influence antigenic expression should be fully identified. As shown in Figure 4a, the VP1 genes were consistent in continuous 12 passages and the amplification of fragment VP1 yielded 813 bp (Figure 4a). Analysis of amino acid sequence in different viral passages showed that the homogeneity of VP1 were 97.5%. In addition, the virus titers in different passages were maintained at relatively steady level (Figure 4b). These results demonstrated that the main protective antigenic site of FMDV remained stable during viruses passages, ensuring the safe and efficiency of vaccine production.
Evaluation of vaccine stability on storage
Reducing the dependence of cold storage and improving the stability of vaccine are destination when deriving vaccine formulation. In order to further evaluate the efficiency of formulation on vaccine, we investigated the shelf life of vaccine stored at 4℃. 146s concentration, indicating the FMDV intact particles, is an crucial parameter to assess the vaccine quality. HPSEC has been demonstrated to be a more accurate, sensitive and rapid method in measuring the FMDV 146s content, therefore, in this study, we adopted the approach to monitor the 146s concentration. As shown in Figure 5a, the formulated vaccine exhibited excellent stability, half of the 146s remained even stored at 4℃ for 12 months, indicative of effective protection. However, the normal vaccine resulted in a time-dependent loss in 146s concentration, only 10% remained. Furthermore, compared with normal vaccine, the dosage form of the formulated vaccine is W/O/W, rarely show any morphological abnormality (Figure 5b). These observation suggested the stability effect of formulation on vaccine and may allow for its effective stockpiling.