English Solomon, Fedosyuk Sofiya, Orliacq Francisco, Tem Vincent, Taylor William, Alam Nawsad, Xiang Zhi Q, Thorley Luke, López-Camacho César, Ertl Hildegund C, Douglas Alexander D
Jenner Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom.
Jenner Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom.
Vaccine. 2025 Jul 26;62:127541. doi: 10.1016/j.vaccine.2025.127541.
Current rabies vaccines require multiple doses and are relatively expensive, limiting their accessibility. Novel low-cost vaccines capable of inducing a protective antibody response against the rabies virus glycoprotein (RVG) are therefore desirable. Structure-guided engineering of the antigen may enhance its qualitative or quantitative immunogenicity, as may transgene cassette optimisation in the case of vectored vaccines. We investigated the potential of these approaches for the design of improved rabies vaccines. We evaluated twelve candidate cassette designs. While codon optimisation enhanced expression in vitro, it did not translate into improved immunogenicity. Co-expression or RVG with rabies matrix protein (RVM) did not detectably affect expression or immunogenicity. Inserting a C-terminal trimerisation domain was detrimental to expression in vitro and did not improve immunogenicity compared to the wild-type comparator. We screened 72 mutant constructs for in vitro expression and pre-fusion stabilisation. Several mutants enhanced expression and/or pre-fusion stability at low pH. Combination of the previously reported H270P mutation with the H419L substitution achieved enhanced stability. An L271Q + H419L double mutant achieved the greatest positive effect upon expression. Neither of double mutants improved immunogenicity compared to wild-type RVG when tested using an mRNA vaccine platform. These mutant constructs may be of value for protein subunit vaccines, but full length wild-type RVG may be sufficiently conformationally stable and well-expressed for optimal immunogenicity of adenovirus and mRNA vaccines in mice.
目前的狂犬病疫苗需要多剂接种且相对昂贵，限制了其可及性。因此，需要能够诱导针对狂犬病病毒糖蛋白（RVG）产生保护性抗体反应的新型低成本疫苗。对抗原进行结构导向工程设计可能会增强其定性或定量免疫原性，载体疫苗的转基因盒优化也可能有同样效果。我们研究了这些方法在设计改进型狂犬病疫苗方面的潜力。我们评估了12种候选盒式设计。虽然密码子优化在体外增强了表达，但并未转化为免疫原性的提高。RVG与狂犬病基质蛋白（RVM）共表达并未显著影响表达或免疫原性。插入C末端三聚化结构域不利于体外表达，与野生型对照相比也未提高免疫原性。我们筛选了72个突变体构建体用于体外表达和融合前稳定性研究。几个突变体在低pH条件下增强了表达和/或融合前稳定性。先前报道的H270P突变与H419L替换相结合实现了稳定性增强。L271Q + H419L双突变体对表达产生了最大的正向影响。当使用mRNA疫苗平台进行测试时，与野生型RVG相比，这两个双突变体均未提高免疫原性。这些突变体构建体可能对蛋白亚单位疫苗有价值，但全长野生型RVG在小鼠中对于腺病毒和mRNA疫苗的最佳免疫原性而言，可能具有足够的构象稳定性且表达良好。
