Dipartimento di Farmacia e Biotecnologie - FaBiT, University of Bologna, Bologna, Italy.
GSK, Siena, Italy.
FASEB J. 2024 Jan;38(1):e23326. doi: 10.1096/fj.202202130R.
The identification and recombinant production of functional antigens and/or epitopes of pathogens represent a crucial step for the development of an effective protein-based vaccine. Many vaccine targets are outer membrane proteins anchored into the lipidic bilayer through an extended hydrophobic portion making their recombinant production challenging. Moreover, only the extracellular loops, and not the hydrophobic regions, are naturally exposed to the immune system. In this work, the Domain 3 (D3) from Group B Streptococcus (GBS) pilus 2a backbone protein has been identified and engineered to be used as a scaffold for the display of extracellular loops of two Neisseria gonorrhoeae membrane proteins (PorB.1b and OpaB). A computational structure-based approach has been applied to the design of both the scaffold and the model antigens. Once identified the best D3 engineerable site, several different chimeric D3 displaying PorB.1b and OpaB extracellular loops were produced as soluble proteins. Each molecule has been characterized in terms of solubility, stability, and ability to correctly display the foreign epitope. This antigen dissection strategy allowed the identification of most immunogenic extracellular loops of both PorB.1b and OpaB gonococcal antigens. The crystal structure of chimeric D3 displaying PorB.1b immunodominant loop has been obtained confirming that the engineerization did not alter the predicted native structure of this epitope. Taken together, the reported data suggest that D3 is a novel protein scaffold for epitope insertion and display, and a valid alternative to the production of whole membrane protein antigens. Finally, this work describes a generalized computational structure-based approach for the identification, design, and dissection of epitopes in target antigens through chimeric proteins.
病原体功能抗原和/或表位的鉴定和重组生产是开发有效蛋白质疫苗的关键步骤。许多疫苗靶标是通过延伸的疏水区锚定在脂质双层中的外膜蛋白，这使得它们的重组生产具有挑战性。此外，只有细胞外环，而不是疏水区，自然暴露于免疫系统。在这项工作中，已经鉴定和设计了 B 组链球菌（GBS）菌毛 2a 骨干蛋白的结构域 3（D3），用作两种淋病奈瑟菌膜蛋白（PorB.1b 和 OpaB）细胞外环展示的支架。应用基于计算结构的方法设计支架和模型抗原。一旦确定了最佳的可工程化 D3 位点，就生产了几种不同的展示 PorB.1b 和 OpaB 细胞外环的嵌合 D3。每种分子都在可溶性、稳定性和正确展示外来表位的能力方面进行了表征。这种抗原切割策略允许鉴定 PorB.1b 和 OpaB 淋病抗原大多数免疫原性的细胞外环。获得了展示 PorB.1b 免疫显性环的嵌合 D3 的晶体结构，证实了工程化没有改变该表位的预测天然结构。总之，报告的数据表明 D3 是一种新型的表位插入和展示蛋白支架，是生产整个膜蛋白抗原的有效替代品。最后，这项工作描述了一种通过嵌合蛋白鉴定、设计和切割靶抗原表位的通用基于计算结构的方法。
