Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate.

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.

Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate

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.

Anti-flavivirus vaccines: review of the present situation and perspectives of subunit vaccines produced in escherichia coli

This article aims to review the present status of anti-flavivirus subunit vaccines, both those at the experimental stage and those already available for clinical use. Aspects regarding development of vaccines to Yellow Fever virus, (YFV), Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) are highlighted, with particular emphasis on purified recombinant proteins generated in bacterial cells. Currently licensed anti-flavivirus vaccines are based on inactivated, attenuated, or virus-vector vaccines. However, technological advances in the generation of recombinant antigens with preserved structural and immunological determinants reveal new possibilities for the development of recombinant protein-based vaccine formulations for clinical testing. Furthermore, novel proposals for multi-epitope vaccines and the discovery of new adjuvants and delivery systems that enhance and/or modulate immune responses can pave the way for the development of successful subunit vaccines. Nonetheless, advances in this field require high investments that will probably not raise interest from private pharmaceutical companies and, therefore, will require support by international philanthropic organizations and governments of the countries more severely stricken by these viruses