提高口蹄疫分子疫苗的产量,我们试图了解口蹄疫病毒(FMDV)2B病毒的影响,基于质粒,病毒样颗粒(VLP)疫苗。尽管独立的2B表达对细胞活力有负面影响,但将FMDV病毒传播蛋白2B包含到人腺病毒5载体的FMD疫苗中增强了转基因表达。评估野生型2B和具有破坏的病毒蛋白活性的突变体,我们证实,当独立表达时,病毒蛋白活性对整体转基因表达有害。然而,将2B掺入含有野生型FMDV3C蛋白酶的FMD分子疫苗构建体中,一种病毒编码的蛋白酶,负责加工结构蛋白,导致转基因表达增强,验证以前的观察结果。当使用FMDV3CL127P突变体时,转基因表达的这种益处被否定,减少了宿主细胞蛋白质的加工,由2B病毒传播活性引起的逆转。将2B纳入VLP生产构建体也对抗原提取产生不利影响。细胞膜2B依赖性重排的可能副作用。这些结果证明,当存在野生型3C蛋白酶时,包含2B增强了转基因表达,但是对于3CL127P突变体的转基因表达是有害的。这对未来的分子口蹄疫疫苗的构建,其可以利用突变型FMDV3C蛋白酶。
To improve the production of foot-and-mouth disease (FMD) molecular vaccines, we sought to understand the effects of the FMD virus (FMDV)
2B viroporin in an experimental, plasmid-based, virus-like particle (VLP) vaccine. Inclusion of the FMDV viroporin
2B into the human Adenovirus 5 vectored FMD vaccine enhanced transgene expression despite independent
2B expression negatively affecting cell viability. Evaluating both wildtype
2B and mutants with disrupted viroporin activity, we confirmed that viroporin activity is detrimental to overall transgene expression when expressed independently. However, the incorporation of 2B into an FMD molecular vaccine construct containing a wildtype FMDV 3C protease, a viral encoded protease responsible for processing structural proteins, resulted in enhancement of transgene expression, validating previous observations. This benefit to transgene expression was negated when using the FMDV 3CL127P mutant, which has reduced processing of host cellular proteins, a reversion resulting from 2B viroporin activity. Inclusion of 2B into VLP production constructs also adversely impacted antigen extraction, a possible side effect of
2B-dependent rearrangement of cellular membranes. These results demonstrate that inclusion of 2B enhanced transgene expression when a wildtype 3C protease is present but was detrimental to transgene expression with the 3CL127P mutant. This has implications for future molecular FMD vaccine constructs, which may utilize mutant FMDV 3C proteases.