Role of innate signalling pathways in the immunogenicity of alphaviral replicon-based vaccines

<p>Abstract</p> <p>Background</p> <p>Alphaviral replicon-based vectors induce potent immune responses both when given as viral particles (VREP) or as DNA (DREP). It has been suggested that the strong immune stimulatory effect induced by these types of vectors is mediated by induction of danger signals and activation of innate signalling pathways due to the replicase activity. To investigate the innate signalling pathways involved, mice deficient in either toll-like receptors or downstream innate signalling molecules were immunized with DREP or VREP.</p> <p>Results</p> <p>We show that the induction of a CD8⁺T cell response did not require functional TLR3 or MyD88 signalling. However, IRF3, converging several innate signalling pathways and important for generation of pro-inflammatory cytokines and type I IFNs, was needed for obtaining a robust primary immune response. Interestingly, type I interferon (IFN), induced by most innate signalling pathways, had a suppressing effect on both the primary and memory T cell responses after DREP and VREP immunization.</p> <p>Conclusions</p> <p>We show that alphaviral replicon-based vectors activate multiple innate signalling pathways, which both activate and restrict the induced immune response. These results further show that there is a delicate balance in the strength of innate signalling and induction of adaptive immune responses that should be taken into consideration when innate signalling molecules, such as type I IFNs, are used as vaccine adjuvant.</p

Viral vectors, in innate and adaptive immunity

Recombinant viral vectors and DNA vectors induce strong immune responses in animal models. However, in clinical trials, the generation of immune responses is less robust, suggesting that further optimization and a deeper understanding of nucleic acid-based vaccines are required. Improvements, such as combining the vaccine vectors in heterologous prime boost regimens and/or using vectors that do not induce strong immune responses against the vector itself may enhance immune responses against the antigen-of-interest. In Paper I, we performed head-to-head comparisons of adenovirus, ALVAC and Semliki Forest virus (SFV) vectors, in homologous and heterologous prime boost regimens. The recombinant viral vectors were evaluated for their potency to generate T cell responses and to protect against a tumor challenge. We show that the memory T cell response induced by the different immunization regimens were distinctly different and that protection against a tumor challenge was more dependent on the quality of the response than the magnitude. The potency of the nucleic acid-based vaccines depends on the activation of innate signaling pathways. In Paper II-IV, we investigated innate signaling pathways activated by different viral vectors and the role of these pathways for induction of T cell responses. The CD8alpha+ DCs play a major role in cross-priming of anti-viral T cells. This dendritic cell subtype phagocytoses apoptotic bodies, expresses high levels of toll-like receptor (TLR) 3 and has a unique ability to cross-present exogenously derived cell-associated material. In Paper II, we investigated the role of TLR3 expression in the CD8alpha+ DCs and its relevance for cross-priming of T cells. We show that dsRNA activates CD8alpha+ DCs to cross-prime T cells via TLR3. In addition to TLRs, the cytoplasmic RNA receptors RIG-I and MDA5, expressed by most cell types, are detectors of viral infection. It was initially suggested that both RIG-I and MDA5 recognize double-stranded RNA (dsRNA) intermediates generated in the cytoplasm during viral infection in the host cell. However, negative-sense RNA viruses do not generate detectable levels of dsRNA in infected cells, thus these viruses may be recognized via alternative non-self signatures. In Paper III, we show that RIG-I is a receptor for single-stranded RNA molecules bearing 5 -phophates, illustrating one of the differences between RIG-I and MDA5 virus recognition. The alphavirus replicon-based DNA (DREP) vectors induce superior immune responses in comparison to conventional DNA (convDNA) vectors in animal models. We hypothesized that DREP vectors induce potent innate signaling pathways that account for the immunogenic properties of these vectors. In Paper IV, we investigated T cell responses in mice deficient in innate signaling pathways, including TLR3, TLR9, MyD88, IRF3 and the interferon alpha/beta receptor (IFN-AR1), after SFV viral and DNA based vector immunization. We show that IFN-AR1 and IRF3, but not detectably the other molecules, influence the T cell response induced by these vectors

MyD88 Expression Is Required for Efficient Cross-Presentation of Viral Antigens from Infected Cells

While virus-infected dendritic cells (DCs) in certain instances have the capacity to activate naïve T cells by direct priming, cross-priming by DCs via the uptake of antigens from infected cells has lately been recognized as another important pathway for the induction of antiviral immunity. During cross-priming, danger and stranger signals play important roles in modulating immune responses. Analogous to what has been shown for other microbial infections, virally infected cells may contain several pathogen-associated molecular patterns that are recognized by Toll-like receptors (TLRs). We analyzed whether the efficient presentation of antigens derived from infected cells requires the usage of MyD88, which is a common adaptor molecule used by all TLRs. For this study, we used murine DCs that were wild type or deficient in MyD88 expression and fibroblasts that were infected with an alphavirus replicon to answer this question. Our results show that when DCs are directly infected, they are able to activate antigen-specific CD8(+) T cells in a MyD88-independent manner. In contrast, a strict requirement of MyD88 for cross-priming was observed when virally infected cells were used as a source of antigen in vitro and in vivo. This indicates that the effects of innate immunity stimulation via the MyD88 pathway control the efficiency of cross-presentation, but not direct presentation or DC maturation, and have important implications in the development of cytotoxic T lymphocyte responses against alphaviral replicon infections

Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response

Toll-like receptor 3 promotes cross-priming to virus-infected cells

Cross-presentation of cell-associated antigens plays an important role in regulating CD8+ T cell responses to proteins that are not expressed by antigen-presenting cells (APCs). Dendritic cells are the principal cross-presenting APCs in vivo and much progress has been made in elucidating the pathways that allow dendritic cells to capture and process cellular material. However, little is known about the signals that determine whether such presentation ultimately results in a cytotoxic T cell (CTL) response (cross-priming) or in CD8+ T cell inactivation (cross-tolerance). Here we describe a mechanism that promotes cross-priming during viral infections. We show that murine CD8alpha+ dendritic cells are activated by double-stranded (ds)RNA present in virally infected cells but absent from uninfected cells. Dendritic cell activation requires phagocytosis of infected material, followed by signalling through the dsRNA receptor, toll-like receptor 3 (TLR3). Immunization with virus-infected cells or cells containing synthetic dsRNA leads to a striking increase in CTL cross-priming against cell-associated antigens, which is largely dependent on TLR3 expression by antigen-presenting cells. Thus, TLR3 may have evolved to permit cross-priming of CTLs against viruses that do not directly infect dendritic cells