Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediateearly protein, and remained higher over the first 6 h of infection than p7.5-or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5-and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigenspecific CD8 T-cell responses.

Modified vaccinia Ankara (MVA) is a safe and promising viral vaccine vector that is currently investigated in several clinical and pre-clinical trials. In contrast to inactivated or sub-unit vaccines, MVA is able to induce strong humoral as well as cellular immune responses. In order to further improve its CD8 T cell inducing capacity, we genetically adjuvanted MVA with the coding sequence of murine CD40L, a member of the tumor necrosis factor superfamily. Immunization of mice with this new vector led to strongly enhanced primary and memory CD8 T cell responses. Concordant with the enhanced CD8 T cell response, we could detect stronger activation of dendritic cells and higher systemic levels of innate cytokines (including IL-12p70) early after immunization. Interestingly, acquisition of memory characteristics (i.e., IL-7R expression) was accelerated after immunization with MVA-CD40L in comparison to non-adjuvanted MVA. Furthermore, the generated cytotoxic T-lymphocytes (CTLs) also showed improved functionality as demonstrated by intracellular cytokine staining and in vivo killing activity. Importantly, the superior CTL response after a single MVA-CD40L immunization was able to protect B cell deficient mice against a fatal infection with ectromelia virus. Taken together, we show that genetic adjuvantation of MVA can change strength, quality, and functionality of innate and adaptive immune responses. These data should facilitate a rational vaccine design with a focus on rapid induction of large numbers of CD8 T cells able to protect against specific diseases.

Current prophylactic vaccines work via the induction of B and T cell mediated memory that effectively control further replication of the pathogen after entry. In the case of therapeutic or post-exposure vaccinations the situation is far more complex, because the pathogen has time to establish itself in the host, start producing immune-inhibitory molecules and spread into distant organs. So far it is unclear which immune parameters have to be activated in order to thwart an existing lethal infection. Using the mousepox model, we investigated the immunological mechanisms responsible for a successful post-exposure immunization with modified vaccinia Ankara (MVA). In contrast to intranasal application of MVA, we found that intravenous immunization fully protected mice infected with ectromelia virus (ECTV) when applied three days after infection. Intravenous MVA immunization induced strong innate and adaptive immune responses in lethally infected mice. By using various gene-targeted and transgenic mouse strains we show that NK cells, CD4 T cells, CD8 T cells and antibodies are essential for the clearance of ECTV after post-exposure immunization. Post-exposure immunization with MVA is an effective measure in a murine model of human smallpox. MVA activates innate and adaptive immune parameters and only a combination thereof is able to purge ECTV from its host. These data not only provide a basis for therapeutic vaccinations in the case of the deliberate release of pathogenic poxviruses but possibly also for the treatment of chronic infections and cancer.

The findings of Martinez et al.(1) that vaccinia virus and its DNA are potent inducers of plasmacytoid dendritic cell (pDC)-derived IFN-α in a Toll-like receptor (TLR)9-independent, exclusively TLR8-dependent way came to us as a great surprise (2). The data (1) contradict our results and those of others.Murine TLR8 gene-transfected cells show a strong induction of an NF-κB luciferease reporter construct after stimulation with CL075 (1). In contrast, others have shown that sole CL075 (also known as 3M-002) was able to trigger murine TLR7 but not murine TLR8-dependent reporter activity (3). This is consistent with our data showing stimulation of human but not murine TLR8 transfectants.The A-type CpG oligodeoxynucleotides (CpG-ODN) used (1) was fully phosphorothioated, which we found to be biologically inactive. Furthermore, the used concentration of 1 nM is below the range of activity for A-type ODN.Martinez et al. (1) showed that in vitro flt3 ligand-generated pDC (FL-pDC) produces large amounts of IFN-α in response to CL075, CL087, or CpG-ODN, but the response to CL075 was lost in TLR8-knockdown pDC. In contrast, we found that CL087 and CL075 induced only very little IFN-α in sorted wildtype (WT) FL-pDCs compared with large amounts induced by CpG-ODN or Sendai virus. Furthermore, TLR7-deficient FL-pDCs still produced large amounts of IFN-α, IFN-λ, IL-6, TNF-α, CC chemokine ligand (CCL)3, CCL4, and CCL5 on stimulation with CpG-ODN, but no cytokines were produced in response to Sendai virus, CL075, or CL087.Data (1) indicate that poly(A) and poly T ODNs were highly stimulatory for IFN-α production in WT but not in TLR8-knockdown pDCs. In our hands, poly A10 and two different poly T ODNs did not induce IFN-α or other cytokines in sorted FLpDCs or ex vivo-isolated pDCs.High levels of TLR7, TLR8, and TLR9 transcripts were detected in murine pDCs (1). Our analyses show high-level transcripts for TLR7 and TLR9 but not for TLR8 in murine pDC. In addition, our quantitative proteomic analysis of isolated splenic pDC (4) or sorted FL-pDC revealed the presence of TLR7 and TLR9 but not of TLR8 protein.A central part of the paper (1) is the observation that the i.v. injection of 10 7 pfu of vaccinia virus strain Western Reserve (VV-WR) induced large amounts of IFN-α in WT but not MyD88-deficient mice 48 h after infection. We and others (5) found that VV-WR is highly inhibitory for the production of IFN-α in vitro and in vivo, and we could not detect any IFN-α in the sera in time kinetics up to 48 h. In summary, our experiments are consistent with previously published work in the field of vaccinia infection and its recognition, CpG-ODN use, and the role of TLR7, TLR8, and TLR9 for the response of murine pDCs (2-5). However, they largely contradict the results of Martinez et al. (1), which leads us to question the conclusions drawn that VV and VV-DNA are major agonists for murine pDC-expressed TLR8.

Bacterial flagellin enhances innate and adaptive immune responses and is considered a promising adjuvant for the development of vaccines against infectious diseases and cancer. Antigen-presenting cells recognize flagellin with the extracellular TLR5 and the intracellular NLRC4 inflammasome-mediated pathway. The detailed cooperation of these innate pathways in the induction of the adaptive immune response following intranasal (i.n.) administration of a recombinant modified vaccinia virus Ankara (rMVA) vaccine encoding flagellin (rMVA-flagellin) is not known. rMVA-flagellin induced enhanced secretion of mucosal IL-1β and TNF-α resulting in elevated CTL and IgG2c antibody responses. Importantly, mucosal IgA responses were also significantly enhanced in both bronchoalveolar (BAL) and intestinal lavages accompanied by the increased migration of CD8+ T cells to the mesenteric lymph nodes (MLN). Nlrc4−/− rMVA-flagellin-immunized mice failed to enhance pulmonary CTL responses, IgG2c was lower, and IgA levels in the BAL or intestinal lavages were similar as those of control mice. Our results show the favorable adjuvant effect of rMVA-flagellin in the lung as well as the intestinal mucosa following i.n. administration with NLRC4 as the essential driver of this promising mucosal vaccine concept.