A.C. Nguiakam Sipowe, R. Sahu, E.B. Norton, V. Tadjuidje, O. Forte, V.A. Dennis
Alabama State University,
United States
Keywords: major outer membrane protein (MOMP), PLA-PEG, dmLT, macrophages, immune response
Summary:
Chlamydia trachomatis (Ct) is an obligate intracellular gram-negative bacterium and the most encountered bacterial sexually transmitted infection (STI) worldwide. Often underdiagnosed, they can lead to serious complications in women’s reproductive tract, including pelvic inflammatory diseases (PID), ectopic pregnancy, and sterility. Antibiotics are effective but have limited effects on reinfections and persistent cases. This makes Ct a serious public health concern, and there is still no FDA-approved chlamydial vaccine. Biodegradable polymeric nanoparticles as carriers for vaccine development have emerged as an efficient means to protect and enhance antigen immunogenicity based on their encapsulation efficiency, targeted delivery, slow-release, and self-adjuvating properties. In our study, we developed and characterized a nanovaccine against chlamydia (named PPrM), using biodegradable polymeric poly-lactic acid poly-ethylene glycol (PLA-PEG) nanoparticles to encapsulate a Chlamydia muridarum (Cm) major outer membrane protein (MOMP). We aimed to evaluate the effects of PPrM and MOMP alone as well as admixed with the double mutant heat-labile toxin (dmLT) adjuvant on the expression levels of Th1 cytokines (IL-6, IL-12p40, and TNF-α), pathogen recognition receptor (TLR2), and co-stimulatory molecules (CD80, CD86, and CD40) in mouse J774 macrophages. Our hypothesis is that PPrM would induce a more potent immune response in macrophages than MOMP and that dmLT would potentiate immune responses critical for an efficacious chlamydial vaccine candidate. As such, dose responses and time-kinetic studies were conducted where macrophages were stimulated with various concentrations of PPrM, MOMP, and dmLT, either alone or admixed, for 6, 24, 48, and 72 hours. Cell-free supernatants were collected for cytokine-specific ELISAs and total RNA for gene expression using TaqMan qPCR. Our results showed that macrophages stimulated with PPrM induced an enhanced production of MOMP-specific IL-6, IL-12p40, and TNF-α in a dose-dependent and time-dependent fashion compared to MOMP, highlighting the self-adjuvanting property of PLA-PEG nanoparticles. We also observed more upregulation of TLR-2, CD40, and CD80 gene expression levels by PPrM than MOMP. More importantly, dmLT was nontoxic to cells and, when admixed with PPrM, markedly enhanced the PPrM-induced responses, suggesting the potentiating effects of the adjuvant. The control PPP (PLA-PEG-PBS) nanoparticles did not stimulate macrophages. Overall, adjuvanting the chlamydial MOMP nanovaccine with dmLT bolstered the MOMP-induced immune responses in macrophages by potentiating the expression of Th1 cytokines, TLR2, and co-stimulatory molecules, essential to fostering a Th1 adaptive response for protective immunity. The dmLT may be crucial in developing efficacious chlamydial vaccines.