Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans.

A major challenge in vaccinology is to prospectively determine vaccine efficacy. Here we have used a systems biology approach to identify early gene 'signatures' that predicted immune responses in humans vaccinated with yellow fever vaccine YF-17D. Vaccination induced genes that regulate virus innate sensing and type I interferon production. Computational analyses identified a gene signature, including complement protein C1qB and eukaryotic translation initiation factor 2 alpha kinase 4-an orchestrator of the integrated stress response-that correlated with and predicted YF-17D CD8(+) T cell responses with up to 90% accuracy in an independent, blinded trial. A distinct signature, including B cell growth factor TNFRS17, predicted the neutralizing antibody response with up to 100% accuracy. These data highlight the utility of systems biology approaches in predicting vaccine efficacy.

Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity.

The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines available, with a 65-yr history of use in >400 million people globally. Despite this efficacy, there is presently no information about the immunological mechanisms by which YF-17D acts. Here, we present data that suggest that YF-17D activates multiple Toll-like receptors (TLRs) on dendritic cells (DCs) to elicit a broad spectrum of innate and adaptive immune responses. Specifically, YF-17D activates multiple DC subsets via TLRs 2, 7, 8, and 9 to elicit the proinflammatory cytokines interleukin (IL)-12p40, IL-6, and interferon-alpha. Interestingly, the resulting adaptive immune responses are characterized by a mixed T helper cell (Th)1/Th2 cytokine profile and antigen-specific CD8+ T cells. Furthermore, distinct TLRs appear to differentially control the Th1/Th2 balance; thus, whilst MyD88-deficient mice show a profound impairment of Th1 cytokines, TLR2-deficient mice show greatly enhanced Th1 and Tc1 responses to YF-17D. Together, these data enhance our understanding of the molecular mechanism of action of YF-17D, and highlight the potential of vaccination strategies that use combinations of different TLR ligands to stimulate polyvalent immune responses.

Neutrophils, dendritic cells and Toxoplasma.

Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.

Toxoplasma gondii inhibits toll-like receptor 4 ligand-induced mobilization of intracellular tumor necrosis factor alpha to the surface of mouse peritoneal neutrophils.

Neutrophils are well-known to rapidly respond to infection through chemotactic infiltration at sites of inflammation, followed by rapid release of microbicidal molecules, chemokines, and proinflammatory cytokines. For tumor necrosis factor alpha (TNF-alpha), we recently found that neutrophils contain intracellular pools of the cytokine and display the capacity to upregulate transcriptional activity of the gene during lipopolysaccharide (LPS) stimulation. We now show that triggering of mouse peritoneal neutrophils with Toll-like receptor 2 (TLR2), TLR4, and TLR9 ligands, but not ligands of TLR3, induces upregulation of surface membrane TNF-alpha. However, neutrophils infected with the protozoan Toxoplasma gondii displayed an inability to respond fully in terms of TLR ligand-induced increases in membrane TNF-alpha expression. Infected neutrophils failed to display decreased levels of intracellular TNF-alpha upon LPS exposure. In contrast to intermediate inhibitory effects in nontreated neutrophils, T. gondii induced a complete blockade in LPS-induced surface TNF-alpha expression in the presence of the protein synthesis inhibitor cycloheximide. Despite these inhibitory effects, the parasite did not affect LPS-induced upregulation of TNF-alpha gene transcription. Collectively, the results show that Toxoplasma prevents TLR ligand-triggered mobilization of TNF-alpha to the neutrophil surface, revealing a novel immunosuppressive activity of the parasite.

Screening for Toxoplasma gondii-regulated transcriptional responses in lipopolysaccharide-activated macrophages.

Toxoplasma gondii-infected macrophages are blocked in production of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) upon activation with lipopolysaccharide (LPS). Here, we used pathway-focused cDNA arrays to identify additional T. gondii-regulated transcriptional responses. Parasite infection decreased 57 (inclusive of IL-12 and TNF-alpha) and increased expression of 7 of 77 LPS-activated cytokine and cytokine-related genes. Interestingly, we found that the LPS-induced transcriptional response of the anti-inflammatory cytokine IL-10 was synergistically increased by T. gondii, results that we validated by conventional reverse transcription-PCR and enzyme-linked immunosorbent assay. Importantly, although the parasite exerted disparate effects in LPS-signaling leading to TNF-alpha versus IL-10 production, both responses required functional Toll-like receptor 4. We suggest that these effects represent parasite defense mechanisms to avoid or delay induction of antimicrobial activity and/or T-cell-mediated immunity during Toxoplasma infection.

Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies.

Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.

Microbial antigen triggers rapid mobilization of TNF-alpha to the surface of mouse neutrophils transforming them into inducers of high-level dendritic cell TNF-alpha production.

Neutrophils play a critical role in early immunity to many microbial pathogens, and this may in part be due to their ability to release immunoregulatory cytokines and chemokines during infection. Here, we demonstrate by flow cytometric analysis that mouse polymorphonuclear leukocytes (PMN) up-regulate surface expression of TNF-alpha within 10 min of stimulation with LPS, and that this is followed by gradual loss over a period of 18 h. Early increases in surface TNF-alpha expression correlated with loss of intracellular pools of preformed TNF-alpha. Nevertheless, extended incubation with LPS resulted in increased levels of TNF-alpha mRNA synthesis and replenishment of intracellular cytokine. After triggering with LPS, PMN acquired the ability to induce dendritic cell (DC) TNF-alpha and IL-12 production. Transwell assays demonstrated that high-level DC TNF-alpha production induced by LPS-triggered neutrophils was dependent upon cell-to-cell contact and neutrophil TNF-alpha, but neither was required for neutrophil instruction of DC IL-12 synthesis. The data suggest that microbial Ag-triggered mouse PMN acquire the capacity to deliver potent DC-activating signals through elaboration of cytokines and direct interactions at the cell surface.

Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection.

Type I inflammatory cytokines are essential for immunity to many microbial pathogens, including Toxoplasma gondii. Dendritic cells (DC) are key to initiating type 1 immunity, but neutrophils are also a source of chemokines and cytokines involved in Th1 response ignition. We found that T. gondii triggered neutrophil synthesis of CC chemokine ligand (CCL)3, CCL4, CCL5, and CCL20, chemokines that were strongly chemotactic for immature DC. Moreover, supernatants obtained from parasite-stimulated polymorphonuclear leukocytes induced DC IL-12(p40) and TNF-alpha production. Parasite-triggered neutrophils also released factors that induced DC CD40 and CD86 up-regulation, and this response was dependent upon parasite-triggered neutrophil TNF-alpha production. In vivo evidence that polymorphonuclear leukocytes exert an important influence on DC activation was obtained by examining splenic DC cytokine production following infection of neutrophil-depleted mice. These animals displayed severely curtailed splenic DC IL-12 and TNF-alpha production, as revealed by ex vivo flow cytometric analysis and in vitro culture assay. Our results reveal a previously unrecognized regulatory role for neutrophils in DC function during microbial infection, and suggest that cross-talk between these cell populations is an important component of the innate immune response to infection.

Cutting edge: MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells.

Host resistance to the intracellular protozoan Toxoplasma gondii is highly dependent on early IL-12 production by APC. We demonstrate here that both host resistance and T. gondii-induced IL-12 production are dramatically reduced in mice lacking the adaptor molecule MyD88, an important signaling element used by Toll-like receptor (TLR) family members. Infection of MyD88-deficient mice with T. gondii resulted in uncontrolled parasite replication and greatly reduced plasma IL-12 levels. Defective IL-12 responses to T. gondii Ags (soluble tachyzoite Ag (STAg)) were observed in MyD88(-/-) peritoneal macrophages, neutrophils, and splenic dendritic cells (DC). In contrast, DC from TLR2- or TLR4-deficient animals developed normal IL-12 responses to STAg. In vivo treatment with pertussis toxin abolished the residual IL-12 response displayed by STAg-stimulated DC from MyD88(-/-) mice. Taken together, these data suggest that the induction of IL-12 by T. gondii depends on a unique mechanism involving both MyD88 and G protein-coupled signaling pathways.

Toxoplasma gondii triggers myeloid differentiation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte chemoattractant protein 1) responses using distinct parasite molecules and host receptors.

Toll-like receptors (TLR) that signal through the common adaptor molecule myeloid differentiation factor 88 (MyD88) are essential in proinflammatory cytokine responses to many microbial pathogens. In this study we report that Toxoplasma gondii triggers neutrophil IL-12 and chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1) production in strict dependence upon functional MyD88. Nevertheless, the responses are distinct. Although we identify TLR2 as the receptor triggering CCL2 production, parasite-induced IL-12 release did not involve this TLR. The production of both IL-12 and CCL2 was increased after neutrophil activation with IFN-gamma. However, the synergistic effect of IFN-gamma on IL-12, but not CCL2, was dependent upon Stat1 signal transduction. Although IL-10 was a potent down-regulator of Toxoplasma-triggered neutrophil IL-12 release, the cytokine had no effect on parasite-induced CCL2 production. Soluble tachyzoite Ag fractionation demonstrated that CCL2- and IL-12 inducing activities are biochemically distinct. Importantly, Toxoplasma cyclophilin-18, a molecule previously shown to induce dendritic cell IL-12, was not involved in neutrophil IL-12 production. Our results show for the first time that T. gondii possesses multiple molecules triggering distinct MyD88-dependent signaling cascades, that these pathways are independently regulated, and that they lead to distinct profiles of cytokine production.