The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Efficient Induction of Cytotoxic T Cells by Viral Vector Vaccination Requires STING-Dependent DC Functions.

Modified Vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus and currently under investigation as a promising vaccine vector against infectious diseases and cancer. MVA acquired mutations in host range and immunomodulatory genes, rendering the virus deficient for replication in most mammalian cells. MVA has a high safety profile and induces robust immune responses. However, the role of innate immune triggers for the induction of cytotoxic T cell responses after vaccination is incompletely understood. Stimulator of interferon genes (STING) is an adaptor protein which integrates signaling downstream of several DNA sensors and therefore mediates the induction of type I interferons and other cytokines or chemokines in response to various dsDNA viruses. Since the type I interferon response was entirely STING-dependent during MVA infection, we studied the effect of STING on primary and secondary cytotoxic T cell responses and memory T cell formation after MVA vaccination in STING KO mice. Moreover, we analyzed the impact of STING on the maturation of bone marrow-derived dendritic cells (BMDCs) and their functionality as antigen presenting cells for cytotoxic T cells during MVA infection in vitro. Our results show that STING has an impact on the antigen processing and presentation capacity of conventionel DCs and played a crucial role for DC maturation and type I interferon production. Importantly, STING was required for the induction of efficient cytotoxic T cell responses in vivo, since we observed significantly decreased short-lived effector and effector memory T cell responses after priming in STING KO mice. These findings indicate that STING probably integrates innate immune signaling downstream of different DNA sensors in DCs and shapes the cytotoxic T cell response via the DC maturation phenotype which strongly depends on type I interferons in this infection model. Understanding the detailed functions of innate immune triggers during MVA infection will contribute to the optimized design of MVA-based vaccines.

Immune Protection by a Cytomegalovirus Vaccine Vector Expressing a Single Low-Avidity Epitope.

Experimental CMV-based vaccine vectors expressing a single MHC class I-restricted high-avidity epitope provided strong, T cell-dependent protection against viruses or tumors. In this study we tested the low-avidity epitope KCSRNRQYL, and show that a mouse CMV (MCMV) vector provides complete immune control of recombinant vaccinia virus expressing the same epitope if KCSRNRQYL is expressed within the immediate-early MCMV gene ie2 The same epitope expressed within the early M45 gene provided no protection, although MCMV vectors expressing the high-avidity epitope SSIEFARL induced protective immunity irrespective of gene expression context. Immune protection was matched by Ag-induced, long-term expansion of effector memory CD8 T cells, regardless of epitope avidity. We explained this pattern by observing regularities in Ag competition, where responses to high-avidity epitopes outcompeted weaker ones expressed later in the replicative cycle of the virus. Conversely, robust and early expression of a low-avidity epitope compensated its weak intrinsic antigenicity, resulting in strong and sustained immunity and immune protection.