Homogenously-sized chlamydial nanovaccine efficiently activates dendritic cells by caveolin endocytosis to boost innate immune responses 2260757

Introduction Previously, we employed a heterogeneously-sized PLGA (poly D, L-lactide-co-glycolide) encapsulating chlamydial major outer membrane protein (MOMP) nanovaccine (CnV), which induced Th1 immune responses in bone marrow-derived dendritic cells (BMDCs). Herein, we employed a microfluidics-assisted fabrication technique to obtain homogenously-sized CnV-H to assess the effects of size and homogeneity on innate immune responses. We hypothesized that due to homogeneity, CnV-H will bolster Th1 immune responses in BMDCs as compared to CnV. Methods The characterization data revealed that CnV-H had an average size of 166 nm, a surface charge of -2 mV and exhibited homogeneity as evidenced by a low polydispersity index (PDI > 0.06) and as confirmed by scanning electron microscopy. Contrastingly, CnV had an average size of 180 nm, a surface charge of -12 mV and a PDI (> 0.1), indicating heterogeneity. To assess in-nate immune responses, BMDCs were stimulated for 24 hours with CnV-H and or CnV (0.1, 1, and 5 µg/mL of encapsulated-rMOMP). Supernatants were collected for cytokine-specific ELISAs, and cells were processed for gene-expression analysis using TaqMan qPCR or were stained with fluorochrome-conjugated antibodies specific for co-stimulatory and antigen-presentation molecules for flow cytometry assessment. Results The data showed that higher concentrations (1 and 5 µg/ml) led to activation of the caveolin en-docytosis pathway, enhanced expression of pathogen pattern recognition receptors (TLR-2), co-stimulatory molecules (CD40, CD80, CD86), antigen-presentation molecules (MHC-II), and significantly increased Th1-cytokines (IL-6, IL-12p40) production, particularly TNF-α, compared to the CnV-stimulated BMDCs. The negative control CnV or CnV-H did not induce any cytokine production. Conclusion In summary, homogeneously-sized CnV-H is more efficient at activating dendritic cells, potentially for robust antigen-presentation, likely due to the uniform activation of the caveolin endocytosis pathway. Funding Source This research was supported by the National Science Foundation (NSF)-HBCU-UP (HRD-1911660) and the NSF-EiR (2200529) grants to Dr. Vida A. Dennis at Alabama State University.