The safety and effectiveness of conventional inactivated FMD vaccine has been widely recognized, while, the structural instability of vaccine antigens is one of the biggest challenges affecting the quality of vaccine. Thus, it is urgent to find an suitable solution for stabilizing the antigen and then prolonging the shelf life of vaccine.
Several studies on acid stability of FMDV had shown that a cluster of His residual located on the petameric interface were more likely protonation on low pH, inducing the dissociation of 146s (Ellard et al. 1999; Hong et al. 2017; Vazquez-Calvo et al. 2014), therefore, in this study, all experiments were carried out at pH 8.0 (Caridi et al. 2015). By quantitative analysis of 146s with HPSEC method, the thermal stability was performed, and the results exhibited that FMDV antigen was sensitive to temperature, with residual 146s of 2.5 µg/ml at 37℃ for one month, lower than that at 4℃ (19.3 µg/ml). Moreover, the vaccine was prone to dissociation easily than antigen stored at same temperature. This result was consistent with previous studies demonstrated that emulsification with Freund adjuvant can induce the denaturation of β-lactamase and then decrease the stability (Shimizu et al. 1993; Van de Weert et al. 2000). Hence, the interaction between solvent and water interface may be a factor influencing the stability of 146s, but the mechanism was not sure.
Many strategies have been used to improve the stability of vaccine. Targeted mutations of viral structural proteins have been proven effectively, but would possibly reduce viral infection, hinder viral propagation and even alter the antigenicity of the virus, leading vaccination less effective (Biswal et al. 2016; Lopez-Arguello et al. 2019). Considering the safe, reliable and application for large-scale production, a novel solution buffer might be employed. Initially, different solution buffers were screened as the basic buffer, and the results showed that PBS could improve the stability effectively, with residual 146s of 10.2 µg/ml, higher than the Tris·Cl (2.8 µg/ml) and the combination (4.7 µg/ml), thus, PBS was determined to be the basal buffer.
Excipients such as sucrose, arginine, calcium chloride, BSA, cysteine, ascorbic acid, trehalose and tween has been reported to be potential stabilizers for FMDV (Harmsen et al. 2015). In this study, we firstly evaluated the effect of excipients respectively, and the results showed that 5% trehalose exhibited effectively, while, tween was indistinctively with a PD value of nearly 90%, others showed moderate effect. Based on these results, orthogonal experiment was preformed to further optimize the combination of excipients for novel solution buffer. Although sucrose has been reported to increase the transition temperature related to 146s, our findings revealed that the novel solution buffer with only 5% sucrose could also improved the stability. Besides, as supplement in vaccine, the excipients with low concentration could maintain vaccine viscosity, easily absorbed by animal body. These results indicated that the novel solution buffer could be applied to vaccine preparation and application. Therefore, we achieved a novel solution buffer against FMDV dissociation.
To further confirm the stable effect on vaccine, the changes of 146s content in vaccine was monitored, the results revealed that the half shelf life of vaccine could prolonged from 10 d to 20 d at 37℃and the shelf life at 4℃was also prolonged to 300 d, suggesting the enhanced stability on inactivated vaccine. Meanwhile, the analysis of particle size distribution showed that supplement of excipients could not change the particle size, the diameter of most vaccine droplet were about 220 nm, consistent with normal one, thus, the novel solution buffer has no impact on vaccine physical properties, available for large-scale production.
Adjuvant, a key component of vaccine, has a very important effect on the stability of vaccine. In this study, we found that emulsification with ISA206 would stimulate the dissociation of 146s, while, the situation about other adjuvant such as ISA201 was not clear, thus, future investigation should be required to assess the stability in vaccine prepared with ISA201. In addition, antibody titer is an important parameter to evaluate the vaccine quantity. The problems whether the excipients could influence the immunogenicity and could stabilized vaccine contribute to antibody titer are the two significant aspects we should fully considered, therefore, further study should also take immune response in animals into account. Overall, these studies would provide a new insight into improvement the stability of inactivated vaccine.
In conclusion, our results demonstrated that the novel solution could increase the stability of vaccine antigen as well as prolong the shelf life of vaccine, suggesting the potential application of this solution in vaccine production regimens for FMD prevention.