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Streptococcus pneumoniae can cause diseases with high mortality and morbidity. The licensed vaccines are based on capsular polysaccharides and induce antibodies with low cross reactivity, leading to restricted coverage of serotypes. For surpassing this limitation, new pneumococcal vaccines are needed for induction of broader protection. One important candidate is the pneumococcal surface protein A (PspA), which can be classified in 6 clades and 3 families. We have reported an efficient process for production and purification of untagged recombinant PspA from clade 4 (PspA4Pro). We now aim to obtain a highly pure recombinant PspA from clade 1 (PspA1) to be included, together with PspA4Pro, in a vaccine formulation to broaden response against pneumococci. The vector pET28a-pspA1 was constructed and used to transform Escherichia coli BL21(DE3) strain. One clone with high production of PspA1 was selected and adapted to high-density fermentation (HDF) medium. After biomass production in 6 L HDF using a bioreactor, the purification was defined after testing 3 protocols. During the batch bioreactor cultivation, plasmid stability remained above 90% and acetate formation was not detected. The final protein purification process included treatment with a cationic detergent after lysis, anion exchange chromatography, cryoprecipitation, cation exchange chromatography, and multimodal chromatography. The final purification process showed PspA1 purity of 93% with low endotoxin content and an overall recovery above 20%. The novel established process can be easily scaled-up and proved to be efficient to obtain a highly pure untagged PspA1 for inclusion in vaccine formulations. Key points: • Purification strategy for recombinant PspA1 from Streptococcus pneumoniae • Downstream processing for untagged protein antigens, the case of PspA1 • Purification strategy for PspA variants relies on buried amino acids in their sequences [ABSTRACT FROM AUTHOR]

Pneumococcal disease remains a global burden, with current conjugated vaccines offering protection against the common serotype strains. However, there are over 100 serotype strains, and serotype replacement is now being observed, which reduces the effectiveness of the current vaccines. Pneumococcal surface protein A (PspA) has been investigated as a candidate for new serotype-independent pneumococcal vaccines, but requires adjuvants and/or delivery systems to improve protection. Polymeric nanoparticles (NPs) are biocompatible and, besides the antigen, can incorporate mucoadhesive and adjuvant substances such as chitosans, which improve antigen presentation at mucosal surfaces. This work aimed to define the optimal NP formulation to deliver PspA into the lungs and protect mice against lethal challenge. We prepared poly(glycerol-adipate-co-ω-pentadecalactone) (PGA-co-PDL) and poly(lactic-co-glycolic acid) (PLGA) NPs using an emulsion/solvent evaporation method, incorporating chitosan hydrochloride (HCl-CS) or carboxymethyl chitosan (CM-CS) as hybrid NPs with encapsulated or adsorbed PspA. We investigated the physicochemical properties of NPs, together with the PspA integrity and biological activity. Furthermore, their ability to activate dendritic cells in vitro was evaluated, followed by mucosal immunization targeting mouse lungs. PGA-co-PDL/HCl-CS (291 nm) or CM-CS (281 nm) NPs produced smaller sizes compared to PLGA/HCl-CS (310 nm) or CM-CS (299 nm) NPs. Moreover, NPs formulated with HCl-CS possessed a positive charge (PGA-co-PDL +17 mV, PLGA + 13 mV) compared to those formulated with CM-CS (PGA-co-PDL −20 mV, PLGA −40 mV). PspA released from NPs formulated with HCl-CS preserved the integrity and biological activity, but CM-CS affected PspA binding to lactoferrin and antibody recognition. PspA adsorbed in PGA-co-PDL/HCl-CS NPs stimulated CD80+ and CD86+ cells, but this was lower compared to when PspA was encapsulated in PLGA/HCl-CS NPs, which also stimulated CD40+ and MHC II (I-A/I-E)+ cells. Despite no differences in IgG being observed between immunized animals, PGA-co-PDL/HCl-CS/adsorbed-PspA protected 83% of mice after lethal pneumococcal challenge, while 100% of mice immunized with PLGA/HCl-CS/encapsulated-PspA were protected. Therefore, this formulation is a promising vaccine strategy, which has beneficial properties for mucosal immunization and could potentially provide serotype-independent protection. [ABSTRACT FROM AUTHOR]