Novel nucleotide-packaging vaccine delivers antigen and poly(I:C) to dendritic cells and generate a potent antibody response in vivo.

An issue with many current vaccines is the dependency on broadly inflammatory adjuvants, such as aluminum hydroxide or aluminum salts that affect many immune- and non-immune cells. These adjuvants are not necessarily activating all antigen-presenting cells (APCs) that take up the antigen and most likely they also activate APCs with no antigen uptake, as well as many non-immune cells. Conjugation of antigen and adjuvant would enable the use of smaller amounts of adjuvant and avoid unnecessary tissue damage and activation of bystander cells. It would ensure that all APCs that take up the antigen would also become activated and avoid that immature and non-activated APCs present the antigen to T cells without a co-stimulatory signal, leading to tolerogenesis. We have developed a novel vaccine that co-deliver antigen and a nucleotide adjuvant to the same APC and lead to a strong activation response in dendritic cells and macrophages. The vaccine is constructed as a fusion-protein with an antigen fused to the DNA/RNA-binding domain from the Hc2 protein from Chlamydia trachomatis. We have found that the fusion protein is able to package polyinosinic:polycytidylic acid (poly(I:C)) or dsDNA into small particles. These particles were taken up by macrophages and dendritic cells and led to strong activation and maturation of these cells. Immunization of mice with the fusion protein packaged poly(I:C) led to a stronger antibody response compared to immunization with a combination of poly(I:C) and antigen without the Hc2 DNA/RNA-binding domain.

Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds.

The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.

CD11c-targeted Delivery of DNA to Dendritic Cells Leads to cGAS- and STING-dependent Maturation.

Immunotherapeutic activation of tumor-specific T cells has proven to be an interesting approach in anticancer treatment. Particularly, anti-CTLA-4 and anti-PD-1/PD-L1 treatment looks promising, and conceivably, even better clinical results might be obtained if such treatment could be combined with boosting the existing tumor-specific T-cell response. One way to achieve this could be by increasing the level of maturation of dendritic cells locally and in the draining lymph nodes. When exposed to cancer cells, dendritic cells may spontaneously mature because of danger-associated molecular patterns derived from the tumor cells. Double-stranded DNA play a particularly important role in the activation of the dendritic cells, through engagement of intracellular DNA-sensors, and signaling through the adaptor protein STING. In the present study, we have investigated the maturational response of human monocyte-derived dendritic cells (moDC) and human monocytic THP-1 cells to targeted and untargeted DNA. We used an anti-CD11c antibody conjugated with double-stranded DNA to analyze the maturation status of human moDCs, as well as maturation using a cGAS KO and STING KO THP-1 cell maturation model. We found that dendritic cells can mature after exposure to cytoplasmic double-stranded DNA delivered through CD11c-mediated endocytosis. Moreover, we show that THP-1 cells matured using IL-4, GM-CSF, and ionomycin upregulate DC-maturation markers after CD11c-targeted delivery of double-stranded DNA. This upregulation is completely abrogated in cGAS KO and STING KO cells.