Self-adjuvanted mRNA vaccines induce local innate immune responses that lead to a potent and boostable adaptive immunity.

mRNA represents a new platform for the development of therapeutic and prophylactic vaccines with high flexibility with respect to production and application. We have previously shown that our two component self-adjuvanted mRNA-based vaccines (termed RNActive® vaccines) induce balanced immune responses comprising both humoral and cellular effector as well as memory responses. Here, we evaluated the early events upon intradermal application to gain more detailed insights into the underlying mode of action of our mRNA-based vaccine. We showed that the vaccine is taken up in the skin by both non-leukocytic and leukocytic cells, the latter being mostly represented by antigen presenting cells (APCs). mRNA was then transported to the draining lymph nodes (dLNs) by migratory dendritic cells. Moreover, the encoded protein was expressed and efficiently presented by APCs within the dLNs as shown by T cell proliferation and immune cell activation, followed by the induction of the adaptive immunity. Importantly, the immunostimulation was limited to the injection site and lymphoid organs as no proinflammatory cytokines were detected in the sera of the immunized mice indicating a favorable safety profile of the mRNA-based vaccines. Notably, a substantial boostability of the immune responses was observed, indicating that mRNA can be used effectively in repetitive immunization schedules. The evaluation of the immunostimulation following prime and boost vaccination revealed no signs of exhaustion as demonstrated by comparable levels of cytokine production at the injection site and immune cell activation within dLNs. In summary, our data provide mechanistic insight into the mode of action and a rational for the use of mRNA-based vaccines as a promising immunization platform.

mRNA-based vaccines synergize with radiation therapy to eradicate established tumors.

BACKGROUND: The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of messenger RNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics. METHODS: The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The molecular mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells. RESULTS: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression analysis of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor associated factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors. CONCLUSION: Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

Highly potent mRNA based cancer vaccines represent an attractive platform for combination therapies supporting an improved therapeutic effect.

Direct vaccination with mRNA encoding tumor antigens is a novel and promising approach in cancer immunotherapy. CureVac's mRNA vaccines contain free and protamine-complexed mRNA. Such two-component mRNA vaccines support both antigen expression and immune stimulation. These self-adjuvanting RNA vaccines, administered intradermally without any additional adjuvant, induce a comprehensive balanced immune response, comprising antigen specific CD4+ T cells, CD8+ T cells and B cells. The balanced immune response results in a strong anti-tumor effect and complete protection against antigen positive tumor cells. This tumor inhibition elicited by mRNA vaccines is a result of the concerted action of different players. After just two intradermal vaccinations, we observe multiple changes at the tumor site, including the up-regulation of many genes connected to T and natural killer cell activation, as well as genes responsible for improved infiltration of immune cells into the tumor via chemotaxis. The two-component mRNA vaccines induce a very fast and boostable immune response. Therefore, the vaccination schedules can be adjusted to suit the clinical situation. Moreover, by combining the mRNA vaccines with therapies in clinical use (chemotherapy or anti-CTLA-4 antibody therapy), an even more effective anti-tumor response can be elicited. The first clinical data obtained from two separate Phase I/IIa trials conducted in PCA (prostate cancer) and NSCLC (non-small cell lung carcinoma) patients have shown that the two-component mRNA vaccines are safe, well tolerated and highly immunogenic in humans.

Regulation of triggering receptor expressed on myeloid cells 1 expression on mouse inflammatory monocytes.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is an activating receptor involved in inflammatory diseases and septic shock. The TREM-1 ligand(s) (TREM-1L) have not yet been identified. In this study, we performed a detailed analysis of the expression of mouse TREM-1 and its ligand(s). Our results demonstrate that TREM-1 is expressed on bone-marrow-derived dendritic cells (BMDC). On bone-marrow-derived macrophages (BMM) its expression is induced in vitro after stimulation by granulocyte-macrophage colony-stimulating factor, interleukin-3 or by myeloid differentiation primary response gene 88 (MyD88)-dependent Toll-like receptor (TLR) ligands. Under steady-state conditions mouse TREM-1 is detectable on a Gr-1(-) F4/80(+) monocyte subpopulation bearing markers of resident monocytes, but not on Gr-1(+) F4/80(+) inflammatory monocytes. During lipopolysaccharide (LPS)-induced endotoxaemia TREM-1 was also up-regulated on inflammatory Gr-1(+) F4/80(+) cells in vivo. In tumour-bearing mice, TREM-1 was up-regulated on Gr-1(+) F4/80(+) monocytes, which phenotypically and functionally resembled mononuclear myeloid-derived suppressor cells. Using a soluble TREM-1 fusion protein, we demonstrate that after intravenous injection of LPS TREM-1L was induced on Gr-1(+) granulocytes and monocytes but not on other cell populations in peripheral blood. This up-regulation on granulocytes was directly mediated by TLR ligands and required the adapter protein MyD88. In contrast to human, mouse platelets expressed TREM-1L neither under steady-state conditions nor after LPS injection in vivo. Our study reveals differential regulation of TREM-1 expression on mouse monocyte subpopulations and improves our understanding of the biological role of TREM-1 during disease.

Non-T cell activation linker (NTAL) negatively regulates TREM-1/DAP12-induced inflammatory cytokine production in myeloid cells.

The engagement of triggering receptor expressed on myeloid cells 1 (TREM-1) on macrophages and neutrophils leads to TNF-alpha and IL-8 production and enhances inflammatory responses to microbial products. For signal transduction, TREM-1 couples to the ITAM-containing adapter DNAX activation protein of 12 kDa (DAP12). In general, ITAM-mediated signals lead to cell activation, although DAP12 was recently implicated in inhibitory signaling in mouse macrophages and dendritic cells. To date, signals downstream of the TREM-1 and DAP12 complex in myeloid cells are poorly defined. By analyzing receptor-induced tyrosine phosphorylation patterns, we discovered that the ligation of TREM-1 leads to tyrosine phosphorylation of the non-T cell activation linker (NTAL; also called linker of activation in B cells or LAB) in a myelomonocytic cell line and primary human granulocytes. Using RNA interference to decrease the expression levels of NTAL, we demonstrate that in NTAL knockdown cell lines the phosphorylation of ERK1/2 is enhanced. In addition, low levels of NTAL are correlated with decreased and delayed mobilization of Ca(2+) after TREM-1 triggering. Most importantly, we demonstrate that NTAL acts as a negative regulator of TNF-alpha and IL-8 production after stimulation via TREM-1. Our results show that activation signals delivered via DAP12 can be counterbalanced by the adaptor NTAL, identifying NTAL as gatekeeper of TREM-1/DAP12-induced signaling in myeloid cells.