Molecular characterization and immunostimulatory properties of extracellular membrane vesicles (EMVS) of streptococcus pneumoniae

Streptococcus pneumoniae causes invasive and non-invasive diseases such as pneumonia, sepsis, meningitis, and otitis media, especially in young children and the elderly. There are over 103 serotypes of S. pneumoniae, but current vaccines protect against only 24 serotypes. Therefore, universal vaccine candidates are necessary for alternative prevention. Recent research has revealed that gram-positive bacteria like S. pneumoniae can produce extracellular membrane vesicles (EMVs). The role of EMVs as carriers for bacterial proteins is well established, but their impact on cellular processes and immune system interactions remains poorly understood. This study investigate the molecular characterization and immunostimulatory properties of EMVs from different serotypes of S. pneumoniae. First, S. pneumoniae isolates were isolated from invasive and non-invasive sites. After that, these isolates were identified using gram stain, catalase test, optochin test and bile solubility test. Then, the serotypes of S. pneumoniae isolates were determined using multiplex PCR. In this study, the EMVs were extracted using ultracentrifugation, ultrafiltration, and iodixanol gradient fractionation methods. Subsequently, the EMVs were observed under the transmission electron microscope and the proteins in the EMVs of S. pneumoniae isolates were identified using liquid chromatography-mass spectrometry (LC-MS). VaxiJen v.2.0 was used to evaluate the protein antigenicity and the protein subcellular localization was determined using PSORTb v3.0.2. For cytotoxicity assessment, the A549 lung epithelial cells were treated with EMVs at concentrations of 25, 50, and 100 µg/mL. Lastly, the immune response in the A549 lung epithelial cell line upon treatment of the EMVs from various serotypes of S. pneumoniae (6A, 14, 19A, 19F, 23F) were elucidated using RT2 ProfilerTM PCR Array Human Innate and Adaptive Immune Response. These five serotypes were shown to release extracellular vesicles, albeit in varying sizes (22 nm -250 nm). The LC-MS/MS analysis of S. pneumoniae EMVs identified 61 proteins, including 12 highly antigenic candidates such as PspA, PavB, HtrA, and ZmpB, which are known virulence factors involved in immune evasion and bacterial pathogenesis. The presence of membrane, cell wall, and extracellular proteins suggests that EMVs could serve as antigen delivery vehicles, potentially eliciting protective immune responses. For the MTT assay, according to the ISO 10993-5:2009 cytotoxicity classification, EMVs at concentrations of 25 µg/mL and 50 µg/mL exhibit weak cytotoxicity (viability above 60%). At 100 µg/mL, EMVs show moderate cytotoxicity, with the lowest viability recorded at 64%. These results suggest that EMVs are not highly cytotoxic to A549 lung epithelial cells, as viability remains above 40% in all tested conditions From the PCR array analysis, this study demonstrates that EMVs derived from various serotypes of S. pneumoniae can induce both innate and adaptive immune responses in the A549 lung epithelial cell line. The activation of innate immune pathways not only initiates immediate defense mechanisms but also primes the adaptive immune system for a more tailored and sustained response. The release pattern of cytokines from the treated A549 lung epithelial cell line was altered by EMVs, suggesting a potential immune system interaction. These findings suggest that EMVs from S. pneumoniae have potential as a basis for novel vaccine strategies. However, further in vivo model is required to validate their immunogenicity and efficacy.