Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate

Abstract

Streptococcus pneumoniae is a bacterial pathogen exclusive to humans, responsible for respiratory and systemic diseases. Pneumococcal protein vaccines have been proposed as serotype-independent alternatives to currently used conjugated polysaccharide vaccines, which have presented limitations regarding their coverage. Previously in our group, pneumococcal surface protein A (PspA) and detoxified pneumolysin (PdT) were genetically fused and the hybrid protein protected mice against pneumococcal challenge, offered higher cross-protection against different strains and showed greater opsonophagocytosis rate than co-administered proteins. As juxtaposed fusion was unstable to upscale production of the protein, flexible (PspA-FL-PdT) and rigid (PspA-RL-PdT) molecular linkers were inserted between the antigens to increase stability. This work aimed to produce recombinant fusion proteins, evaluate their stability after linker insertion, both in silico and experimentally, and enable the production of two antigens in a single process. The two constructs with linkers were cloned into Escherichia coli and hybrid proteins were purified using chromatography; purity was evaluated by SDS-PAGE and stability by Western blot and high performance size exclusion chromatography. PspA-FL-PdT showed higher stability at −20°C and 4°C, without additional preservatives. In silico analyses also showed differences regarding stability of the fusion proteins, with molecule without linker presenting disallowed amino acid positions in Ramachandran plot and PspA-FL-PdT showing the best scores, in agreement with experimental results. Mice were immunized with three doses and different amounts of each protein. Both fusion proteins protected all groups of mice against intranasal lethal challenge. The results show the importance of hybrid protein structure on the stability of the products, which is essential for a successful bioprocess development.

Functionalized 3D Hydroxyapatite Scaffold by Fusion Peptides-Mediated Small Extracellular Vesicles of Stem Cells for Bone Tissue Regeneration

“…Fusion peptides have become an indispensable component of the functionalized H-P1-sEVs and H-P2-sEVs scaffolds in this study. In recent years, widespread interest has arisen in the use of peptides as biological activity molecules for emerging drug delivery systems and shows enormous application potential. − The role of targeting peptides in drug delivery systems targeting therapy has been emphasized for precision medicine. Previous research has demonstrated that a specific cancer-targeting drug delivery system with a targeting peptide was successfully used to improve the therapeutic efficacy of rapamycin .…”

Fusion of PspA to detoxified pneumolysin enhances pneumococcal vaccine coverage

“…For their important contributions to pneumococcal infections, PspA and Ply have been investigated as potential candidates in serotype-independent vaccines in several disease models [ 3 , 25 ] and clinical trials [ 26 ]. The combination of PspA and pneumolysin derivatives has been particularly effective against severe outcomes like pneumonia [ 27 ] and bacteremia [ 8 , 12 , 28 ].…”

Recent progress in pneumococcal protein vaccines

“…Additionally, immunization with a PspA fusion protein in combination with CpG oligonucleotides and aluminum hydroxide gel provided significant protection against pneumococcal challenge in mice ( 73 ). Moreover, intranasal immunization with a PspA-based vaccine, fused with a protein anchor to display PspA on the surface of bacterium-like particles or pneumolysin, efficiently induced both IgG in the serum and IgA in mucosal washes, ultimately providing complete protection against pneumococcal challenge in mice ( 74 , 75 ). Despite the challenge posed by the variability in PspA, it remains a robust candidate with decades of research backing its potential as a valuable vaccine component.…”