MyD88-Dependent Immunity to a Natural Model of Vaccinia Virus Infection Does Not Involve Toll-Like Receptor 2.

UNLABELLED: Although the pattern recognition receptor Toll-like receptor 2 (TLR2) is typically thought to recognize bacterial components, it has been described to alter the induction of both innate and adaptive immunity to a number of viruses, including vaccinia virus (VACV). However, many pathogens that reportedly encode TLR2 agonists may actually be artifactually contaminated during preparation, possibly with cellular debris or merely with molecules that sensitize cells to be activated by authentic TLR2 agonists. In both humans and mice, the most relevant natural route of infection with VACV is through intradermal infection of the skin. Therefore, we examined the requirement for TLR2 and its signaling adaptor MyD88 in protective immunity to VACV after intradermal infection. We find that although TLR2 may recognize virus preparations in vitro and have a minor role in preventing dissemination of VACV following systemic infection with large doses of virus, it is wholly disposable in both control of virus replication and induction of adaptive immunity following intradermal infection. In contrast, MyD88 is required for efficient induction of CD4 T cell and B cell responses and for local control of virus replication following intradermal infection. However, even MyD88 is not required to induce local inflammation, inflammatory cytokine production, or recruitment of cells that restrict virus from spreading systemically after peripheral infection. Thus, an effective antiviral response does require MyD88, but TLR2 is not required for control of a peripheral VACV infection. These findings emphasize the importance of studying relevant routes of infection when examining innate sensing mechanisms. IMPORTANCE: Vaccinia virus (VACV) provides the backbone for some of the most widely used and successful viral vaccine vectors and is also related to the human pathogens Cantagalo virus and molluscum contagiosum virus that infect the skin of patients. Therefore, it is vital to understand the mechanisms that induce a strong innate immune response to the virus following dermal infection. Here, we compare the ability of the innate sensing molecule Toll-like receptor 2 (TLR2) and the signaling molecule MyD88 to influence the innate and adaptive immune response to VACV following systemic or dermal infection

Age-dependent susceptibility to a viral disease due to decreased natural killer cell numbers and trafficking

Age-related defects in natural killer cell numbers and function underlie the decreased resistance to mousepox infection in aging mice

Direct Presentation Is Sufficient for an Efficient Anti-Viral CD8+ T Cell Response

The extent to which direct- and cross-presentation (DP and CP) contribute to the priming of CD8+ T cell (TCD8+) responses to viruses is unclear mainly because of the difficulty in separating the two processes. Hence, while CP in the absence of DP has been clearly demonstrated, induction of an anti-viral TCD8+ response that excludes CP has never been purposely shown. Using vaccinia virus (VACV), which has been used as the vaccine to rid the world of smallpox and is proposed as a vector for many other vaccines, we show that DP is the main mechanism for the priming of an anti-viral TCD8+ response. These findings provide important insights to our understanding of how one of the most effective anti-viral vaccines induces immunity and should contribute to the development of novel vaccines

Antibody Inhibition of a Viral Type 1 Interferon Decoy Receptor Cures a Viral Disease by Restoring Interferon Signaling in the Liver

Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy

Sequential activation of two pathogen-sensing pathways required for type I interferon expression and resistance to an acute DNA virus infection

Toll-like receptor 9 (TLR9), its adaptor MyD88, the downstream transcription factor interferon regulatory factor 7 (IRF7), and type I interferons (IFN-I) are all required for resistance to infection with ectromelia virus (ECTV). However, it is not known how or in which cells these effectors function to promote survival. Here, we showed that after infection with ECTV, the TLR9-MyD88-IRF7 pathway was necessary in CD11c(+) cells for the expression of proinflammatory cytokines and the recruitment of inflammatory monocytes (iMos) to the draining lymph node (dLN). In the dLN, the major producers of IFN-I were infected iMos, which used the DNA sensor-adaptor STING to activate IRF7 and nuclear factor κB (NF-κB) signaling to induce the expression of IFN-α and IFN-β, respectively. Thus, in vivo, two pathways of DNA pathogen sensing act sequentially in two distinct cell types to orchestrate resistance to a viral disease

Secreted T1-IFNbp bind back to the cell surface of infected and uninfected cells <i>in vitro</i>.

<p>L929 cells were left uninfected (top panels), infected with ECTV-GFP (middle panels) or ECTV Δ166-GFP (lower panels). 6 h after infection, the presence of T1-IFNbp at the cell surface was detected using anti-T1IFNbp (left panels) or naïve control sera (right panel). The data are representative of two similar experiments.</p

Treatment with blocking 10G7 but not with non-blocking 10F3 decreases overall virus loads as determined by whole body imaging.

<p>BALB/c mice were infected with 300 pfu of ECTV-Luc in the footpad, at 5 dpi, the mice were treated with indicated mAbs IP and imaged 2 dpt for light emission using a Carestream <i>In vivo</i> instrument for bioluminescence detection (left) The mouse on the top-left is an uninfected control. The bar graph (right) shows the mean ± SEM quantitative luminescence intensity of the two groups. The experiment is representative of two similar experiments.</p

The ECTV T1-IFNbp inhibitory mAb 10G7 inhibits the biological activity of the T1-IFNbp from OPVs important for human health.

<p>A) Hela cells were incubated with 10 ng recombinant VARV T1-IFNbp,UV treated supernatants from cells infected with VACV WR, or gamma-irradiated supernatants of cells infected with MPXV USA or MPXV Republic of Congo (RoC) strains as indicated. After 30 min the cells were thoroughly washed, incubated with mAb 10G7 (blue line) or IC (red line) for 1 h followed by FITC-anti-mouse IgG and flow cytometry analysis. All data are representative of 2 or 3 independent experiments with similar results. B) Tissue culture media (TCM, RPMI 10% fetal calf serum), 10 ng of recombinant VARV T1-IFNbp in TCM, 100 µl of supernatant of insect cells expressing recombinant VARV T1-IFNbp in TCM, or the indicated irradiated TCM supernatant from cells that had been infected with the indicated viruses were pre-incubated with the indicated amounts of hIFN-α for 1 h. The cocktails were then added to Hela cells in 24 well plates. Following 24 h incubation at 37°C, the cells were infected with VSV at a MOI of 0.1 for 24 h, fixed, and stained with crystal violet. Left and right panels correspond to the same plate but were separated to facilitate labeling of the figure. C) 10 ng of recombinant VARV T1-IFNbp or 100 µl of irradiated supernatant from cells that had been infected with MPXV USA were incubated with 10 ng mAb 10G7. After 30 minutes, 10 U/ml hIFN-α were added to the mixture and incubated for 1 h at 37°C. The cocktail was then added to Hela cells in 24 well plates. Following 24 h incubation at 37°C, the cells were infected with VSV at a MOI of 0.1 for 24 h, fixed, and stained with crystal violet. All data are representative of 2 or 3 independent experiments with similar results.</p