River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host-parasite relationship.

Bacterial components are recognized by the immune system through activation of the inflammasome, eventually causing processing of the proinflammatory cytokine interleukin-1? (IL-1?), a pleiotropic cytokine and one of the most important mediators of inflammation, through the protease caspase-1. Synthesis of the precursor protein and processing into its bioactive form are tightly regulated, given that disturbed control of IL-1? release can cause severe autoinflammatory diseases or contribute to cancer development. We show that the bacterial Pasteurella multocida toxin (PMT) triggers Il1b gene transcription in macrophages independently of Toll-like receptor signaling through RhoA/Rho-kinase-mediated NF-?? activation. Furthermore, PMT mediates signal transducer and activator of transcription (STAT) protein-controlled granzyme A (a serine protease) expression in macrophages. The exocytosed granzyme A enters target cells and mediates IL-1? maturation independently of caspase-1 and without inducing cytotoxicity. These findings show that macrophages can induce an IL-1?-initiated immune response independently of inflammasome activity.

Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. Although sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides that are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability, and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian information criterion-driven error-tolerant peptide search (BICEPS) and offer an open source implementation based on this statistical criterion to automatically balance the information of each single spectrum and the database, while limiting the run time. We show that BICEPS performs as well as current database search algorithms when such algorithms are applied to sequenced organisms, whereas BICEPS only uses a remotely related organism database. For instance, we use a chicken instead of a human database corresponding to an evolutionary distance of more than 300 million years (International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695-716). We demonstrate the successful application to cross-species proteomics with a 33% increase in the number of identified proteins for a filarial nematode sample of Litomosoides sigmodontis.

Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. While sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides which are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian Information Criterion-driven Error-tolerant Peptide Search (BICEPS) and offer an open-source implementation based on this statistical criterion to

Granzyme (gzm) A and B, proteases of NK cells and T killer cells, mediate cell death, but also cleave extracellular matrices, inactivate intracellular pathogens, and induce cytokines. Moreover, macrophages, Th2 cells, regulatory T cells, mast cells, and B cells can express gzms. We recently reported gzm induction in human filarial infection. In this study, we show that in rodent filarial infection with Litomosoides sigmodontis, worm loads were significantly reduced in gzmA × B and gzmB knockout mice during the whole course of infection, but enhanced only early in gzmA knockout compared with wild-type mice. GzmA/B deficiency was associated with a defense-promoting Th2 cytokine and Ab shift, enhanced early inflammatory gene expression, and a trend of reduced alternatively activated macrophage induction, whereas gzmA deficiency was linked with reduced inflammation and a trend toward increased alternatively activated macrophages. This suggests a novel and divergent role for gzms in helminth infection, with gzmA contributing to resistance and gzmB promoting susceptibility.

Th2-biased inflammation with eosinophilia and IgE production is a hallmark of helminth infections. It is pronounced in hyperreactive onchocerciasis patients ('sowda' or 'local form'), who efficiently kill microfilariae resulting in severe dermatitis and lymphadenitis. In contrast, hyporeactive patients ('generalised form') tolerate high microfilarial loads. This is thought to be mediated by regulatory CD4+ T cells and macrophages producing suppressive cytokines such as IL-10 and transforming growth factor-beta (TGF-β). We investigated whether hyperreactivity was reflected by lower local TGF-β production, analysing stable latent TGF-β1 expression in onchocercomas, lymph nodes and skin from hyperreactive and hyporeactive patients by immunohistochemistry. TGF-β expression was compared with that of IgE, IgG1, IgG4, and the antigen-presenting, CD4+ T cell-inducing MHC class II molecule HLA-DR. TGF-β was weakly and less frequently expressed by various cell types in onchocercomas, skin and lymph nodes from hyperreactive compared to hyporeactive patients. This applied to reactions around living and dead adult worms as well as dead microfilariae. Antigen-presenting cells strongly expressed HLA-DR in both forms, but their numbers were reduced in hyperreactive nodules. Plasma cells produced more IgE and IgG1, but less of the anti-inflammatory antibody IgG4 in hyperreactive onchocercomas. In conclusion, hyperreactivity is linked with reduced local expression of TGF-β, HLA-DR and IgG4, which might contribute to the insufficient down-regulation of inflammation via TGF-β- and HLA-DR-induced regulatory lymphocytes.

Transforming growth factor-beta (TGF-β) is a key cytokine in immune regulation, cell differentiation, development, wound healing, and tissue remodelling. It mediates immunosuppression in filarial infections facilitating parasite persistence, while attenuating immunopathology, which is induced by migrating microfilariae. Immunosuppression rises with parasite burden, but it remains unknown whether filariae elicit local release of immunosuppressive cytokines. Therefore, using immunohistology, we investigated the expression of stable, released latent TGF-β1 in subcutaneous nodules from highly infected, hyporeactive onchocerciasis patients, harbouring adult Onchocerca volvulus. Since many cell types produce TGF-β, we elucidated the cellular source, distribution and dependency on the worms’ sex, productivity and vitality. We found TGF-β1 to be abundantly expressed by T cells, plasma/B cells, macrophages, mast cells, fibrocytes, and vascular endothelial cells, particularly in onchocercomas with productive or previously productive females, damaged, dead and resorbed adult worms or microfilariae. We conclude TGF-β to be antigen induced by the filariae since expression was scarce around subcutaneous arthropods or cholesterol crystals in onchocercomas. Enhanced expression after ivermectin or endobacteria-depleting doxycycline treatment indicates induction to depend on filariae and not on Wolbachia endobacteria. TGF-β+ cells were reduced in HIV co-infection. This finding of local and sustained TGF-β induction by vital and dead filariae, untreated and after treatment, adds new aspects to immunomodulation by helminths.

Ectopic secondary lymph follicles emerge in patients with autoimmune or infectious diseases, e.g. in the synovium in rheumatoid arthritis or the skin in Borrelia burgdorferi infection, but ectopic localisations in the skin are rarely described for helminth infections. We investigated the cellular composition of secondary lymph follicles in subcutaneous nodules from eight patients with hyperreactive onchocerciasis (synonymous "localised" form or sowda) using immunohistology. CD3- and CD45RO-positive T cells and CD20-positive B cells were present in the mantle zone. The germinal centre was characterised by many B cells and CD35-positive follicular dendritic cells, which formed a network of attached IgE- and CD23-positive cells with the low-affinity IgE (epsilon) receptor. Few of the B cells were labelled for IgG1, IgG2 and IgG4, whereas in other zones of the nodule IgG1 was expressed by plasma cells and IgG1-coated dead microfilariae. B cells and few macrophages expressed the MHC class II molecule HLA-DR. Mature CD68-positive tingible body macrophages with phagocytosed leukocytes and CD57-positive lymphocytes occurred in the germinal centre. Macrophages in the germinal centre only weakly expressed alpha1-antichymotrypsin in contrast to macrophages in other zones of the onchocercoma. Furthermore, the multifunctional cytokine TGF-beta was only weakly expressed by macrophages and lymphocytes in the secondary follicles. Only few tryptase-positive mast cells, calprotectin-positive young macrophages, eosinophils and neutrophils occurred in the secondary follicles, although these cells were abundant in the onchocercomas. In conclusion, the ectopic secondary lymph follicles in onchocercomas and lymph nodes from hyperreactive onchocerciasis patients are equally composed.

Transforming growth factor-beta (TGF-β) is a highly conserved cytokine that has a well-known regulatory role in immunity, but also in organ development of most animal species including helminths. Homologous tgf-b genes and mRNA have been detected in the filaria Brugia malayi. The in situ protein expression is unknown for filariae. Therefore, we examined several filariae for the expression and localization of latent (stable) TGF-β in adult and larval stages. A specific goat anti-human latency associated protein (LAP, TGF-β 1) antibody, purified by affinity chromatography, was used for light and electron microscopic immunohistochemistry. Adult Onchocerca volvulus, Onchocerca gibsoni, Onchocerca ochengi, Onchocerca armillata, Onchocerca fasciata, Onchocerca flexuosa, Wuchereria bancrofti, Dirofilaria sp., B. malayi, and infective larvae of W. bancrofti reacted with the antibody. Labeling of worm tissues varied between negative and all degrees of positive reactions. Latent TGF-β was strongly expressed adjacent to the cell membranes of the hypodermis, epithelia, and muscles and adjacent to many nuclei in all organs. TGF-β was well expressed in worms without Wolbachia endobacteria eliminated by doxycycline treatment. Pleomorphic neoplasms in O. volvulus were also labeled. We conclude that latent TGF-β protein is expressed by filariae independently of Wolbachia, possibly regulating worm tissue homeostasis.

Immunosuppression in human filarial disease involves regulatory T cells. We hypothesized that natural or worm antigen-induced FOXP3 regulatory T cells could be involved locally, suppressing effector cells via granzymes. Natural and treatment-induced death of worms implies enhanced exposure to worm antigens. Therefore, we examined FOXP3+T cells and granzyme expression in onchocercomas harbouring adult Onchocerca volvulus worms, with respect to worm viability, productivity, the patient's immune status and filaricidal treatment. The immunohistological analysis revealed that dead adult worms were strongly associated with FOXP3+T cells in generalized hyporeactive onchocerciasis. FOXP3+ cells hardly expressed granzymes, but cell contacts with granzyme A+ or B+ cells were frequent. While suramin directly kills most adult worms within 6 months, the Wolbachia depleting antibiotic doxycycline indirectly causes adult worm degeneration within 18 months. Contrary to suramin, depletion of Th1-driving endobacteria most strongly promoted FOXP3+T cells and granzyme-expressing cells. In hyperreactive patients, FOXP3+ cells were less frequent. This is the first demonstration of local FOXP3+Treg cells in human filariasis and their induction by natural worm death and anti-parasitic treatment. We newly report granzyme responses to helminths and their association with immunosuppression. FOXP3+Treg and granzyme+ cells might locally suppress defence against newly acquired worms.

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ABSTRACTUnderstanding the protective mechanism in the liver induced by recombinant vaccines against the pre-erythrocytic stages of malaria is important for vaccine development. Most studies in mice have focused on splenic and peripheral blood T cells and identified gamma interferon (IFN-γ)-producing CD8+T cells as correlates of protection, which can be induced by prime-boost vaccination with recombinant poxviruses. Invariant natural killer T (Vα14iNKT) cells can also protect against liver stage malaria, when activated, and are abundant in the liver. Since poxviruses have nonspecific immunomodulating effects, which are incompletely understood, we investigated whether recombinant poxviruses affect the protective properties of hepatic Vα14iNKT cells and thus vaccine efficacy. We show that intradermal vaccination with recombinant poxviruses activated Vα14iNKT cells and NK cells in the livers of BALB/c mice while inducing IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing pre-erythrocytic stage antigen-specific CD8+T cells. Greater numbers of hepatic Vα14iNKT cells secreted interleukin-4 than IFN-γ. Vaccinated Vα14iNKT-cell-deficient mice had lower, but still protective levels of hepatic and splenic IFN-γ+and TNF-α+CD8+T cells and better protection rates later after challenge withPlasmodium bergheisporozoites. Therefore, vaccine-activated hepatic Vα14iNKT cells help in generating specific T cells but are not required for protection induced by recombinant poxviruses. Furthermore, double-positive INF-γ+/TNF-α+CD8+T cells were enriched in protected livers, suggesting that cells expressing both of these cytokines may be most relevant for protection.

10.1128/IAI.73.2.849-858.2005. 2005, 73(2):849. DOI: Infect. Immun. Taniguchi and Adrian V. S. Hill Eric G. Sheu, Robert Sinden, Stephan Gadola, Masaru Simone Korten, Richard J. Anderson, Carolyn M. Hannan, Vaccination of Mice T Cells in the Liver after Poxvirus + CD8 Tumor Necrosis Factor Alpha-Producing Protein-Specific Gamma Interferonand Circumsporozoite Plasmodium berghei 14 Chain NKT Cells Promote α Invariant V

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-gamma-secreting CD8(+) T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8(+) T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster's FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8(+) T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8(+) T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.

Ly49 antigens, interacting with MHC classI molecules, enable NK cells to distinguish “self” from “non-self”. Here, we investigated the activating receptor Ly49D after allogeneic bone marrow transplantation (BMT). After transplantation of B6bone marrow (BM) into BALB/c recipients we observed a significant reduction of Ly49D+ NK cells and a decreased density of expression. We found a nonstochastic distribution of Ly49D with Ly49G2. In contrast to reduced coexpression with Ly49A, a constant rate of Ly49G2 on Ly49D+ NK cells was observed in allogeneic chimeras. Cytotoxicity was reduced during the first two months after BMT. After this time allogeneic chimeras showed tolerance against host-specific targets. We conclude that NK cells are able to shape their Ly49 repertoire fitting to a new environment after allogeneic BMT. This alteration seems to depend on the presence of new corresponding MHC classI molecules resulting in downregulation of respective receptors on donor cells. Analysing coexpression of Ly49D and Ly49G2, we found a relationship between these two receptors, showing a distinct effect after allogeneic BMT. Functional data indicate that a time of reduced NK cell cytotoxicity after BMT is followed by in vitro tolerance of allogeneic chimeras.

Natural killer cell-associated direct cytotoxicity and cytokine production are crucial mechanisms for early innate host resistance against viruses, bacteria, or protozoa. The engagement of inhibitory NK cell receptors can influence host responses to viruses. However, these receptors have not been investigated to date in parasitic infections, and little is known about the role of NK cells in the defense against helminths. Therefore, we have correlated the frequencies of cells expressing the pan-NK marker DX5 and subsets bearing inhibitory Ly-49 receptors with worm survival and cytokine production during infection with Litomosoides sigmodontis in BALB/c mice (H2(d)), the only fully permissive model of filariasis. A marked influx of DX5(+)/CD3(-) NK cells and DX5(+)/CD3(+) T cells into the pleural cavity, where the parasites were located, was observed. The frequency of pleural NK cells expressing the H2(d)-reactive inhibitory receptors Ly-49A, Ly-49C, or Ly-49G2 declined most strongly compared with spleen and blood. In the peripheral blood, longitudinal analysis revealed an early and stable reduction of Ly-49C(+) and Ly-49G2(+) NK cells, a subsequent significant increase of the entire NK cell and DX5(+)/CD3(+) T cell populations, and a reduction in the Ly-49A(+) subset. The in vivo depletion of NK cells strongly enhanced the worm load and influenced IL-4 and IL-5 plasma levels. These data demonstrate a new role for NK cells in the host defense against filariae and, for the first time, alterations of Ly-49 receptor-expressing NK cell subsets in a parasitic infection.

We investigated the role of IFN-gamma in host defense during murine filariasis. Using the fully permissive infection of BALB/c mice with the rodent filaria Litomosoides sigmodontis, we show that interferon (IFN)-gamma is essential for encapsulation of adult filarial worms in inflammatory nodules and for normal worm clearance. IFN-gamma knockout (KO) mice had only one third of the nodules of wild-type mice but displayed a more than twofold increase in worm burden and increased microfilaremia. Neutrophil granulocytes, but not macrophages or eosinophils, appear to directly control worm load and nodule formation. Neutrophils, which we showed earlier to be essential for the encapsulation process in the thoracic cavity, where the worms reside, were diminished at this location in IFN-gamma KO compared to wild-type mice; they also displayed strongly reduced chemotactic and phagocytic activity compared to neutrophils of controls. This argues for a distinct defect in neutrophil activation accounting for the low formation of inflammatory nodules. Tumor necrosis factor-alpha, a major neutrophil-activating cytokine expressed by macrophages in the thoracic cavity around the worms, was highly induced in wild-type but absent in KO mice. Diminished activation of neutrophils seems to be a general hallmark of IFN-gamma KO mice, since neutrophils from uninfected KO mice also showed a reduction in chemotactic and phagocytic activity when induced by casein. In conclusion, these data are the first to define an IFN-gamma-dependent immune effector mechanism in murine filarial infection, i.e. neutrophil-mediated control of the adult worm load.