A novel method for synthetic vaccine construction based on protein assembly

Liu, Zhida; Zhou, Hang; Wang, Wenjun; Tan, Wenjie; Fu, Yang-Xin; Zhu, Mingzhao

Different methods have been developed to construct synthetic vaccines. The traditional way is genetic manipulation, which provides great flexibility. However, this de novo generation usually takes a long time, and it is occasionally difficult to express large molecules. Chemical conjugation offers an alternative, fast way to overcome the shortcomings mentioned above. However, the chemical conjugation process is usually not easily controlled, resulting in poor homogeneity in terms of the degree of conjugation and the number of conjugation sites. An isopeptide bond is an amide bond formed between the side-chain amine of lysine and the side-chain carboxyl group of either glutamate or aspartate. This bond has been shown to be very useful for protein-protein coupling and protein modification 28 . SpyTag/SpyCatcher was recently developed and found to be highly efficient for site-specific protein conjugation. Impelled by the limitations of current methods for vaccine construction and inspired by the concept of synthetic vaccinology, we hypothesized that this new technique might be useful for easily and precisely assembling different vaccine components at the protein level. This approach may possess several advantages: (1) In contrast to genetic manipulation, this system does not generate a whole vaccine de novo; instead, it produces different protein components and assembles them as needed. This method may thus significantly reduce the time spent and the cost, especially when many designs need to be tested. (2) In contrast to chemical conjugation-based approaches, this system allows quantitative and site-specific installation of antigens at a designated position in the antibody that does not influence the antibody-targeting ability. (3) SpyTag and SpyCatcher can efficiently react with each other to form a stable covalent bond under diverse conditions. (4) SpyTag is reactive no matter it is at the N-terminus, the C-terminus, or the internal site of a protein and therefore offers greater flexibility than other split protein-derived systems or intein/ sortase tag systems do 29, 30 . In addition, recent work by the Howarth group has optimized this system to potentially provide further advantages 31 : (1) SpyCatcher can be shortened to KTag, allowing SpyTag and KTag to form a peptide-peptide ligation. Thus, this new system could further reduce the influence of SpyCatcher's own immunogenicity, if any. (2) The SpyTag-and-KTag system allows cycles of conjugation, which enables assembly of a vaccine with multiple components, such as protein-based adjuvants and multivalent antigens. (3) The short KTag makes high-throughput screening of epitopes feasible because the short KTag-epitope fusion peptides can be easily prepared by total synthesis.