疫苗的热稳定性,特别是包膜病毒载体疫苗,在任何需要的地方对他们的交付仍然是一个挑战。病毒载体疫苗的冷冻干燥是一种有前途的方法,但由于从病毒的外部和内部去除水的过程仍然具有挑战性。在包膜病毒的情况下,冷冻干燥会导致信封上的应力增加,这通常会导致病毒的失活。在这项研究中,我们设计了一种冷冻干燥表达SARS-CoV-2刺突糖蛋白的重组水泡性口炎病毒(VSV)的方法。由于VSV的包膜由50%的脂质和50%的蛋白质组成,制剂研究集中在载体的蛋白质和脂质部分。制剂主要使用蔗糖制备,海藻糖,和山梨糖醇作为冷冻保护剂;甘露醇作为冻干保护剂;和组氨酸作为缓冲剂。最初,在不同的最终水分含量水平下,研究了rVSV-SARS-CoV-2的感染性和滤饼稳定性。在3-6%的水分含量下发现了感染性病毒滴度的高恢复(〜0.5至1log损失),冻干蛋糕没有变质。为了进一步减少感染性病毒滴度损失,研究了赋形剂的组成和浓度。冷冻保护剂和冻干保护剂从5%增加到10%,加上0.5%的明胶,导致提高的感染性病毒滴度的恢复和稳定的饼形成。此外,冷冻干燥过程的二次干燥温度对rVSV-SARS-CoV-2的感染性有显著影响。当温度升高到20°C以上时,载体的感染性急剧下降。在整个长期稳定性研究中,含有10%糖(蔗糖/海藻糖)的配方,10%甘露醇,0.5%明胶,和10mM组氨酸在2-8℃下显示令人满意的稳定性6个月。这种冷冻干燥工艺的开发和优化的配方最大限度地减少了对昂贵的冷链配送系统的需求。
The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.