Critical Role of Perforin-dependent CD8+ T Cell Immunity for Rapid Protective Vaccination in a Murine Model for Human Smallpox

Vaccination is highly effective in preventing various infectious diseases, whereas the constant threat of new emerging pathogens necessitates the development of innovative vaccination principles that also confer rapid protection in a case of emergency. Although increasing evidence points to T cell immunity playing a critical role in vaccination against viral diseases, vaccine efficacy is mostly associated with the induction of antibody responses. Here we analyze the immunological mechanism(s) of rapidly protective vaccinia virus immunization using mousepox as surrogate model for human smallpox. We found that fast protection against lethal systemic poxvirus disease solely depended on CD4 and CD8 T cell responses induced by vaccination with highly attenuated modified vaccinia virus Ankara (MVA) or conventional vaccinia virus. Of note, CD4 T cells were critically required to allow for MVA induced CD8 T cell expansion and perforin-mediated cytotoxicity was a key mechanism of MVA induced protection. In contrast, selected components of the innate immune system and B cell-mediated responses were fully dispensable for prevention of fatal disease by immunization given two days before challenge. In conclusion, our data clearly demonstrate that perforin-dependent CD8 T cell immunity plays a key role in MVA conferred short term protection against lethal mousepox. Rapid induction of T cell immunity might serve as a new paradigm for treatments that need to fit into a scenario of protective emergency vaccination

Thogoto Virus Infection Induces Sustained Type I Interferon Responses That Depend on RIG-I-Like Helicase Signaling of Conventional Dendritic Cells▿

Type I interferon (IFN-α/β) induction upon viral infection contributes to the early antiviral host defense and ensures survival until the onset of adaptive immunity. Many viral infections lead to an acute, transient IFN expression which peaks a few hours after infection and reverts to initial levels after 24 to 36 h. Robust IFN expression often is conferred by specialized plasmacytoid dendritic cells (pDC) and may depend on positive-feedback amplification via the type I IFN receptor (IFNAR). Here, we show that mice infected with Thogoto virus (THOV), which is an influenza virus-like orthomyxovirus transmitted by ticks, mounted sustained IFN responses that persisted up to 72 h after infection. For this purpose, we used a variant of THOV lacking its IFN-antagonistic protein ML, an elongated version of the matrix (M) protein [THOV(ΔML)]. Of note, large amounts of type I IFN were also found in the serum of mice lacking the IFNAR. Early IFN-α expression seemed to depend on Toll-like receptor (TLR) signaling, whereas prolonged IFN-α responses strictly depended on RIG-I-like helicase (RLH) signaling. Unexpectedly, THOV(ΔML)-infected bone marrow-derived pDC (BM-pDC) produced only moderate IFN levels, whereas myeloid DC (BM-mDC) showed massive IFN induction that was IPS-1-dependent, suggesting that BM-mDC are involved in the massive, sustained IFN production in THOV(ΔML)-infected animals. Thus, our data are compatible with the model that THOV(ΔML) infection is sensed in the acute phase via TLR and RLH systems, whereas at later time points only RLH signaling is responsible for the induction of sustained IFN responses

Concomitant type I IFN receptor-triggering of T cells and of DC is required to promote maximal modified vaccinia virus Ankara-induced T-cell expansion

Virus-induced expansion of CD8(+) T cells may be promoted by type I IFN receptor (IFNAR)-triggering of T cells, depending on the pathogen tested. We studied modified vaccinia virus Ankara (MVA), a promising vaccine vector candidate, which was derived from conventional vaccinia virus (VAC!) by more than 570 consecutive in vitro passages. In adoptive transfer experiments, we verified that VACV expressing the gp33 epitope of lymphocytic choriomeningitis virus (VACV(gp33)) induced largely IFNAR-independent expansion of gp33-specific T cells. On the contrary, MVA(gp33)-induced T-cell expansion was IFNAR dependent. Interestingly, under the latter conditions, T-cell activation was IFNAR independent, whereas T-cell apoptosis was enhanced in the absence of IFNAR. To address whether MVA-induced T-cell expansion was solely affected by IFNAR-triggering of T cells, expansion of endogenous T cells was studied in conditional mice with a T-cell- or DC-specific IFNAR deletion. Interestingly, both mouse strains showed moderately reduced T-cell expansion, whereas mice with a combined T-cell- and DC-specific IFNAR ablation showed massively reduced T-cell expansion similar to that of IFNAR(-/-) mice. These results are compatible with the model that IFN-inducing viruses such as MVA confer virus-specific CD8(+) T-cell expansion by concomitant IFNAR-triggering of DC and of T cells

Vaccinia Virus-Mediated Inhibition of Type I Interferon Responses Is a Multifactorial Process Involving the Soluble Type I Interferon Receptor B18 and Intracellular Components▿

Poxviruses such as virulent vaccinia virus (VACV) strain Western Reserve encode a broad range of immune modulators that interfere with host responses to infection. Upon more than 570 in vitro passages in chicken embryo fibroblasts (CEF), chorioallantois VACV Ankara (CVA) accumulated mutations that resulted in highly attenuated modified vaccinia virus Ankara (MVA). MVA infection of mice and of dendritic cells (DC) induced significant type I interferon (IFN) responses, whereas infection with VACV alone or in combination with MVA did not. These results implied that VACV expressed an IFN inhibitor(s) that was functionally deleted in MVA. To further characterize the IFN inhibitor(s), infection experiments were carried out with CVA strains isolated after 152 (CVA152) and 386 CEF passages (CVA386). Interestingly, neither CVA152 nor CVA386 induced IFN-α, whereas the latter variant did induce IFN-β. This pattern suggested a consecutive loss of inhibitors during MVA attenuation. Similar to supernatants of VACV- and CVA152-infected DC cultures, recombinantly expressed soluble IFN decoy receptor B18, which is encoded in the VACV genome, inhibited MVA-induced IFN-α but not IFN-β. In the same direction, a B18R-deficient VACV variant triggered only IFN-α, confirming B18 as the soluble IFN-α inhibitor. Interestingly, VACV infection inhibited IFN responses induced by a multitude of different stimuli, including oligodeoxynucleotides containing CpG motifs, poly(I:C), and vesicular stomatitis virus. Collectively, the data presented show that VACV-mediated IFN inhibition is a multistep process involving secreted factors such as B18 plus intracellular components that cooperate to efficiently shut off systemic IFN-α and IFN-β responses

Type I interferon receptor signaling delays Kupffer cell replenishment during acute fulminant viral hepatitis.

Virus-induced fulminant hepatitis is a major cause of acute liver failure. During acute viral hepatitis the impact of type I interferon (IFN-I) on myeloid cells, including liver-resident Kupffer cells (KC), is only partially understood. Herein, we dissected the impact of locally induced IFN-I responses on myeloid cell function and hepatocytes during acute liver inflammation. Two different DNA-encoded viruses, vaccinia virus (VACV) and murine cytomegalovirus (MCMV), were studied. In vivo imaging was applied to visualize local IFN-β induction and IFN-I receptor (IFNAR) triggering in VACV-infected reporter mice. Furthermore, mice with a cell type-selective IFNAR ablation were analyzed to dissect the role of IFNAR signaling in myeloid cells and hepatocytes. Experiments with Cx3cr1 VACV infection induced local IFN-β responses, which lead to IFNAR signaling primarily within the liver. IFNAR triggering was needed to control the infection and prevent fulminant hepatitis. The severity of liver inflammation was independent of IFNAR triggering of hepatocytes, whereas IFNAR triggering of myeloid cells protected from excessive inflammation. Upon VACV or MCMV infection KC disappeared, whereas infiltrating monocytes differentiated to KC afterwards. During IFNAR triggering such replenished monocyte-derived KC comprised more IFNAR-deficient than -competent cells in mixed bone marrow chimeric mice, whereas after the decline of IFNAR triggering both subsets showed an even distribution. Upon VACV infection IFNAR triggering of myeloid cells, but not of hepatocytes, critically modulates acute viral hepatitis. During infection with DNA-encoded viruses IFNAR triggering of liver-infiltrating blood monocytes delays the development of monocyte-derived KC, pointing towards new therapeutic strategies for acute viral hepatitis

Protective immunization depends on adaptive immune responses but not on the presence of B cells.

<p>(A) RAG-1^−/− mice lacking mature B and T cells, or (B) B cell-deficient μMT mice were i.n. immunized with MVA (○) two days before 3×LD₅₀ ECTV challenge. Mock-challenged (▪) and mock-vaccinated (▴) mice served as controls. In all experiments weight loss of individual mice was monitored daily (n = 3 to 6 per group). +indicate the individual time of death. Error bars indicate SEMs, and the numbers of surviving/total animals are given in parentheses. Data are representative of two or three similar experiments. Statistical significance of differences between groups is indicated by * for p-value<0.05, ** for p-value<0.01 and *** for p-value<0.001.</p

Unimpaired protective capacity of MVA immunization in mice lacking selected key adaptors of innate signaling pathways, or natural killer (NK) cells.

<p>(A) C57BL/6 mice (wt), (B) MyD88/Trif^−/−, (C) IPS^−/− knock-out mice, or (D) NK cell depleted C57/BL/6 mice were challenged with 3×LD₅₀ ECTV two days after MVA immunization (○), with mock-challenged (▪), and mock-vaccinated (▴) animals as controls. In all experiments weight loss of individual mice was monitored daily (n = 2 to 6 per group). +indicate the individual time of death. Error bars indicate SEMs, and the numbers of surviving/total animals are given in parentheses. Statistical significance of differences between groups is indicated by * for p-value<0.05, ** for p-value<0.01 and *** for p-value<0.001.</p

Intranasal vaccination of mice rapidly induces cellular and humoral adaptive immune responses in the respiratory tract.

<p>(A) Numbers of total CD3+ CD8+ (CD8+) and CD3+ CD4+ (CD4+) T cells in bronchoalveolar lavage (BAL) (mean pooled from three mice) at the indicated time points after inoculation with MVA (filled symbols) or mock vaccine (empty symbols). (B) Intracellular cytokine staining for gamma interferon (IFN-γ) in CD8+ CD62L^low spleen cells from individual mice (n = 6), or BAL cells pooled from 3 to 5 mice (mean ± SD from three independent experiments) stimulated with VACV specific B8R_20–27 or control peptides (LacZ₈₇₆) at 3, 5 and 7 days post MVA immunization. (C) Numbers of total B220+ CD3− B cells in BAL (mean pooled from three mice) at indicated time points after inoculation with MVA (filled symbol) or mock (empty symbol) vaccine. (D) MVA specific IgG levels in BAL fluids pooled from three mice at indicated time points after inoculation with MVA (filled bars) or mock (empty bars) vaccine. The graph depicts the OD 490 nm measured by ELISA at 1∶10 dilution of BAL fluid. All BAL data represent the results from two independent experiments.</p