Development of ASC specks as carriers / adjuvants for novel vaccine technology

Abstract

Pathogen associated molecular patterns (PAMPs) and danger-associated molec ular patterns (DAMPs) are sensed by nucleotide binding oligomerization domain-like receptor (NLR) family of proteins in the cytosol. Certain NLRs like NLRP3 and AIM2 induce formation of inflammasome complexes. The adaptor protein ASC has criti cal functions in inflammatory and pyroptotic signaling pathways by forming a bridge between pro-caspase-1 via CARD-CARD interactions and NLR’s via pyrin-pyrin in teractions and by activating caspase-1. In unstimulated cells, ASC is soluble in the cytosol; however, upon stimulation it forms globular speck structures, called as ‘ASC speck’. ASC specks can also be produced artificially via overexpression in HEK293FT cells. Our group has previously show the stability of artificially produced ASC specks in PBS at 37 oC for more than 30 days. In this thesis, we investigated the immune stimulatory role of ASC specks in vivo. In our preliminary studies, we managed to purify antigen-bound ASC specks with truncated form OVA and also with mCherry protein as a tracker for imaging systems. We have shown that purified ASC specks engulfed and degraded by macrophages. Moreover, we documented that stimulation of THP-1 macrophages with purified ASC specks induces secretion of inflammatory cytokines; IL-1β, IL-6 and TNF-α. Secondly, we have shown that tOVA-ASC speck injections cause secretion of type I and type II cytokines from splenocytes of the immunized wild type C57BL/6 mice. Thirdly, we showed that tOVA-ASC injections cause complete eradication or reduction in size of the EG7-OVA tumor in C57BL/6 mice. Lastly, we have shown that intraperitoneally injected mCherry-ASC specks goes to secondary lymph nodes.