Douglas Borges De Figueiredo

Pulmonary dry powder vaccine of pneumococcal antigen loaded nanoparticles

Pneumonia, caused by Streptococcus pneumoniae, mainly affects the immunocompromised, the very young and the old, and remains one of the leading causes of death. A steady rise in disease numbers from non-vaccine serotypes necessitates a new vaccine formulation that ideally has better antigen stability and integrity, does not require cold-chain and can be delivered non-invasively. In this study, a dry powder vaccine containing an important antigen of S. pneumoniae, pneumococcal surface protein A (PspA) that has shown cross-reactivity amongst serotypes to be delivered via the pulmonary route has been formulated. The formulation contains the antigen PspA adsorbed onto the surface of polymeric nanoparticles encapsulated in L-leucine microparticles that can be loaded into capsules and delivered via an inhaler. We have successfully synthesized particles of ∼150 nm and achieved ∼20 μg of PspA adsorption per mg of NPs. In addition, the spray-dried powders displayed a FPF of 74.31±1.32% and MMAD of 1.70±0.03 μm suggesting a broncho-alveolar lung deposition facilitating the uptake of the nanoparticles by dendritic cells. Also, the PspA released from the dry powders maintained antigen stability (SDS-PAGE), integrity (Circular dichroism) and activity (lactoferrin binding assay). Moreover, the released antigen also maintained its antigenicity as determined by ELISA.

Quantification of capsular polysaccharide of Streptococcus pneumoniae serotype 14 in culture broth samples

Streptococcus pneumoniae is a major cause of mortality in underdeveloped countries, where more than one million people die from pneumococcal disease every year. Vaccines are the most efficient method for preventing the infection and are based on the capsular polysaccharide (PS) protection. The serotype 14 is the most frequent in pediatric infections worldwide. This study aimed to establish a quantification protocol for PS present in culture broth samples of S. pneumoniae serotype 14 (PS14) and use this protocol for selection of the best PS14 producer strain. Phenol-sulfuric, HPSEC, competitive ELISA, and sandwich ELISA methods were tested for PS14 quantification. Sandwich ELISA was the method with the best reproducibility and sensitivity and the least susceptible to interferences. The quantification limit and detection limit of this method were 0.99 and 0.57 ng/mL, respectively. Statistical analysis was performed to calculate the coefficient of variation (CV) intraassay (1-3% intraplate and 2-6% interplate) and interassay (11-15%) and the reproducibility in different days (CV<20%). The sandwich ELISA allows us to select, among six strains evaluated, the strain 5287 as the best PS14 producer (11.68 mg PS14/biomass) and it was shown to be the best choice for measurement of pneumococcal polysaccharides in culture broth samples.

Mucosal immunization with PspA (Pneumococcal surface protein A)-adsorbed nanoparticles targeting the lungs for protection against pneumococcal infection

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-ω-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.