RESUMO
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 classifed in 6 clades and 3 families. We have reported an efcient process for production and purifcation 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 purifcation was defned after testing 3 protocols. During the batch bioreactor cultivation, plasmid stability remained above 90% and acetate formation was not detected. The fnal protein purifcation process included treatment with a cationic detergent after lysis, anion exchange chromatography, cryoprecipitation, cation exchange chromatography, and multimodal chromatography. The fnal purifcation 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 efcient to obtain a highly pure untagged PspA1 for inclusion in vaccine formulations.
RESUMO
The importance of Streptococcus pneumoniae has been well established. These bacteria can colonize infants and adults without symptoms, but in some cases can spread, invade other tissues and cause disease with high morbidity and mortality. The development of pneumococcal conjugate vaccines (PCV) caused an enormous impact in invasive pneumococcal disease and protected unvaccinated people by herd effect. However, serotype replacement is a well-known phenomenon that has occurred after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) and has also been reported for other PCVs. Therefore, it is possible that serotype replacement will continue to occur even with higher valence formulations, but the development of serotype-independent vaccines might overcome this problem. Alternative vaccines are under development in order to improve cost effectiveness, either using proteins or the pneumococcal whole cell. These approaches can be used as a stand-alone strategy or together with polysaccharide vaccines. Looking ahead, the next generation of pneumococcal vaccines can be impacted by the new technologies recently approved for human use, such as mRNA vaccines and viral vectors. In this paper, we will review the advantages and disadvantages of the addition of new polysaccharides in the current PCVs, mainly for low- and middle-income countries, and we will also address future perspectives.
RESUMO
Widespread use of pneumococcal conjugate vaccines (PCVs) has led to substitution of vaccine-type (VT) strains by non-vaccine type (NVT) strains in nasopharyngeal carriage. We compared the efficacy of PCV13 and a nasal protein formulation containing pneumococcal surface protein A (PspA) adjuvanted with the whole-cell pertussis vaccine (wP) in the protection against co-colonization challenge models in mice with VT and NVT strains expressing different PspAs. Immunized mice were challenged with two different mixtures: i. VT4 (PspA3) + NVT33 (PspA1) and ii. VT23F (PspA2) + NVT15B/C (PspA4). Results from the first mixture showed a reduction in loads of VT4 strain in the nasopharynx of mice immunized with PCV13. A statistical difference between the loads of the VT and NVT strains was observed, indicating a competitive advantage for the NVT strain in PCV13-immunized animals. In the second mixture, no reduction was observed for the VT23F strain, probably due to low levels of anti-23F polysaccharide IgG induced by PCV13. Interestingly, a combination of the PspA formulation containing wP with PCV13 led to a reduction in colonization with both strains of the two mixtures tested, similar to the groups immunized nasally with wP or PspA plus wP. These results indicate that a combination of vaccines may be a useful strategy to overcome pneumococcal serotype replacement
RESUMO
Burden of pneumonia caused by Streptococcus pneumoniae remains high despite the availability of conjugate vaccines. Mucosal immunization targeting the lungs is an attractive alternative for the induction of local immune responses to improve protection against pneumonia. Our group had previously described the development of poly(glycerol adipate-co-ωpentadecalactone) (PGA-co-PDL) polymeric nanoparticles (NPs) adsorbed with Pneumococcal surface protein A from clade 4 (PspA4Pro) within L-leucine microcarriers (nanocomposite microparticles—NCMPs) for mucosal delivery targeting the lungs (NP/NCMP PspA4Pro). NP/NCMP PspA4Pro was now used for immunization of mice. Inoculation of this formulation induced anti-PspA4Pro IgG antibodies in serum and lungs. Analysis of binding of serum IgG to intact bacteria showed efficient binding to bacteria expressing PspA from clades 3, 4 and 5 (family 2), but no binding to bacteria expressing PspA from clades 1 and 2 (family 1) was observed. Both mucosal immunization with NP/NCMP PspA4Pro and subcutaneous injection of the protein elicited partial protection against intranasal lethal pneumococcal challenge with a serotype 3 strain expressing PspA from clade 5 (PspA5). Although similar survival levels were observed for mucosal immunization with NP/NCMP PspA4Pro and subcutaneous immunization with purified protein, NP/NCMP PspA4Pro induced earlier control of the infection. Conversely, neither immunization with NP/NCMP PspA4Pro nor subcutaneous immunization with purified protein reduced bacterial burden in the lungs after challenge with a serotype 19F strain expressing PspA from clade 1 (PspA1). Mucosal immunization with NP/NCMP PspA4Pro targeting the lungs is thus able to induce local and systemic antibodies, conferring protection only against a strain expressing PspA from the homologous family 2.
RESUMO
Burden of pneumonia caused by Streptococcus pneumoniae remains high despite the availability of conjugate vaccines. Mucosal immunization targeting the lungs is an attractive alternative for the induction of local immune responses to improve protection against pneumonia. Our group had previously described the development of poly(glycerol adipate-co-omega-pentadecalactone) (PGA-co-PDL) polymeric nanoparticles (NPs) adsorbed with Pneumococcal surface protein A from clade 4 (PspA4Pro) within L-leucine microcarriers (nanocomposite microparticles-NCMPs) for mucosal delivery targeting the lungs (NP/NCMP PspA4Pro). NP/NCMP PspA4Pro was now used for immunization of mice. Inoculation of this formulation induced anti-PspA4Pro IgG antibodies in serum and lungs. Analysis of binding of serum IgG to intact bacteria showed efficient binding to bacteria expressing PspA from clades 3, 4 and 5 (family 2), but no binding to bacteria expressing PspA from clades 1 and 2 (family 1) was observed. Both mucosal immunization with NP/NCMP PspA4Pro and subcutaneous injection of the protein elicited partial protection against intranasal lethal pneumococcal challenge with a serotype 3 strain expressing PspA from clade 5 (PspA5). Although similar survival levels were observed for mucosal immunization with NP/NCMP PspA4Pro and subcutaneous immunization with purified protein, NP/NCMP PspA4Pro induced earlier control of the infection. Conversely, neither immunization with NP/NCMP PspA4Pro nor subcutaneous immunization with purified protein reduced bacterial burden in the lungs after challenge with a serotype 19F strain expressing PspA from clade 1 (PspA1). Mucosal immunization with NP/NCMP PspA4Pro targeting the lungs is thus able to induce local and systemic antibodies, conferring protection only against a strain expressing PspA from the homologous family 2.