A panel of recombinant proteins from human-infective Plasmodium species for serological surveillance.

BACKGROUND: Malaria remains a global health problem and accurate surveillance of Plasmodium parasites that are responsible for this disease is required to guide the most effective distribution of control measures. Serological surveillance will be particularly important in areas of low or periodic transmission because patient antibody responses can provide a measure of historical exposure. While methods for detecting host antibody responses to Plasmodium falciparum and Plasmodium vivax are well established, development of serological assays for Plasmodium knowlesi, Plasmodium ovale and Plasmodium malariae have been inhibited by a lack of immunodiagnostic candidates due to the limited availability of genomic information. METHODS: Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species. RESULTS: Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38. CONCLUSIONS: Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites

SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells

Background: Compounds mimicking the inhibitory effect of SMAC / DIABLO on X-linked inhibitor of apoptosis (XIAP) have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFkB system and TNF signaling. In view of the overwhelming importance of the NFkB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells. Principal Findings: BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFkB pathway, but it also diminished the stronger NFkB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12. Significance: The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies

Basigin is a druggable target for host-oriented antimalarial interventions

Plasmodium falciparum is the parasite responsible for the most lethal form of malaria, an infectious disease that causes a large proportion of childhood deaths and poses a significant barrier to socioeconomic development in many countries. Although antimalarial drugs exist, the repeated emergence and spread of drug-resistant parasites limit their useful lifespan. An alternative strategy that could limit the evolution of drug-resistant parasites is to target host factors that are essential and universally required for parasite growth. Host-targeted therapeutics have been successfully applied in other infectious diseases but have never been attempted for malaria. Here, we report the development of a recombinant chimeric antibody (Ab-1) against basigin, an erythrocyte receptor necessary for parasite invasion as a putative antimalarial therapeutic. Ab-1 inhibited the PfRH5-basigin interaction and potently blocked erythrocyte invasion by all parasite strains tested. Importantly, Ab-1 rapidly cleared an established P. falciparum blood-stage infection with no overt toxicity in an in vivo infection model. Collectively, our data demonstrate that antibodies or other therapeutics targeting host basigin could be an effective treatment for patients infected with multi-drug resistant P. falciparum.Wellcome Trust (London, England) (Grant 098051)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology

Systematic identification of genes encoding cell surface and secreted proteins that are essential for in vitro growth and infection in Leishmania donovani.

Leishmaniasis is an infectious disease caused by protozoan parasites belonging to the genus Leishmania for which there are no approved human vaccines. Infections localise to different tissues in a species-specific manner with the visceral form of the disease caused by Leishmania donovani and L. infantum being the most deadly in humans. Although Leishmania spp. parasites are predominantly intracellular, the visceral disease can be prevented in dogs by vaccinating with a complex mixture of secreted products from cultures of L. infantum promastigotes. With the logic that extracellular parasite proteins make good subunit vaccine candidates because they are directly accessible to vaccine-elicited host antibodies, here we attempt to discover proteins that are essential for in vitro growth and host infection with the goal of identifying subunit vaccine candidates. Using an in silico analysis of the Leishmania donovani genome, we identified 92 genes encoding proteins that are predicted to be secreted or externally anchored to the parasite membrane by a single transmembrane region or a GPI anchor. By selecting a transgenic L. donovani parasite that expresses both luciferase and the Cas9 nuclease, we systematically attempted to target all 92 genes by CRISPR genome editing and identified four that were required for in vitro growth. For fifty-five genes, we infected cohorts of mice with each mutant parasite and by longitudinally quantifying parasitaemia with bioluminescent imaging, showed that nine genes had evidence of an attenuated infection although all ultimately established an infection. Finally, we expressed two genes as full-length soluble recombinant proteins and tested them as subunit vaccine candidates in a murine preclinical infection model. Both proteins elicited significant levels of protection against the uncontrolled development of a splenic infection warranting further investigation as subunit vaccine candidates against this deadly infectious tropical disease

A physical wiring diagram for the human immune system.

The human immune system is composed of a distributed network of cells circulating throughout the body, which must dynamically form physical associations and communicate using interactions between their cell-surface proteomes1. Despite their therapeutic potential2, our map of these surface interactions remains incomplete3,4. Here, using a high-throughput surface receptor screening method, we systematically mapped the direct protein interactions across a recombinant library that encompasses most of the surface proteins that are detectable on human leukocytes. We independently validated and determined the biophysical parameters of each novel interaction, resulting in a high-confidence and quantitative view of the receptor wiring that connects human immune cells. By integrating our interactome with expression data, we identified trends in the dynamics of immune interactions and constructed a reductionist mathematical model that predicts cellular connectivity from basic principles. We also developed an interactive multi-tissue single-cell atlas that infers immune interactions throughout the body, revealing potential functional contexts for new interactions and hubs in multicellular networks. Finally, we combined targeted protein stimulation of human leukocytes with multiplex high-content microscopy to link our receptor interactions to functional roles, in terms of both modulating immune responses and maintaining normal patterns of intercellular associations. Together, our work provides a systematic perspective on the intercellular wiring of the human immune system that extends from systems-level principles of immune cell connectivity down to mechanistic characterization of individual receptors, which could offer opportunities for therapeutic intervention

BV6 induces apoptotic and necrotic cell death in monocytes.

<p>(A) Freshly isolated monocytes were incubated in GM-CSF/IL4 supplemented medium for 1 h with the indicated mixtures of z-VAD-fmk (20 µM), necrostatin-1 (70 µM) and the TNF inhibitor TNFR2-Fc/Enbrel (50 µg/ml). Cells were then challenged overnight with 10 µM BV6 and cell viability was finally evaluated by help of the MTT assay. (B) Freshly isolated monocytes and monocytes cultivated overnight in the presence of the indicated mixtures of GM-CSF/IL4 and 10 µM BV6 were analyzed by FACS for cell surface expression of membrane TNF and the death receptors TNFR1, CD95 and TRAILR2. (C) Monocytes were challenged with BV6 in the presence of soluble Fc fusion proteins of TRAILR2 (5 µg/ml), CD95 (50 µg/ml) and TNFR2 (Enbrel, 20 µg/ml) or a mixture of them. After 24 h viability was determined using the MTT assay (Left panel). The functionality of the three Fc fusion proteins was controlled in cell death assays with HT1080 and recombinant 50 ng/ml TNF, 4 ng/ml Fc-CD95L and 50 ng/mlTRAIL (right panel). Data shown are representative for three independent experiments.</p

Based on peptide 1 which was synthesized using standard Fmoc solid phase methods on SASRIN™ resin, BV6 (peptide 3) and monovalent (peptide 2) and trivalent (peptide 4) variants derived thereof were synthesized.

<p>Based on peptide 1 which was synthesized using standard Fmoc solid phase methods on SASRIN™ resin, BV6 (peptide 3) and monovalent (peptide 2) and trivalent (peptide 4) variants derived thereof were synthesized.</p

Monocyte-derived dendritic cells and macrophages are barely sensitive for BV6-induced cell death.

<p>(A and B) Monocyte-derived macrophages, immature and Fc-CD40L maturated monocyte-derived dendritic cells were challenged for one day with and without 10 µM BV6. Cells were then analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent DC experiments and 6 independent experiments with monocytes and macrophages). p100 processing were determined by western blotting and is shown for one representative experiment (B). (C) Monocytes cultivated overnight in GM-CSF/IL4, iDCs obtained after 7 days of cultivation with GM-CSF/IL4 and mDCs maturated with TNF or Fc-CD40L were analyzed by western blotting with respect to the expression of the indicated proteins (data shown are representative for four independent experiments).</p

GM-CSF and IL4 rapidly induce resistance against BV6-induced cell death in monocytes.

<p>(A and B) Monocytes were cultivated in medium supplemented with GM-CSF/IL4 and were treated immediately or after 1 and 2 days with 10 µM BV6. One day after stimulation BV6 treated cells and a corresponding control sample cultivated without BV6 were analyzed by annexin-V staining (A) and western blotting (B).</p

SMAC mimetic BV6 attenuates TNF- and CD40L-induced proinflammatory signaling and triggers DC maturation.

<p>(A and B) Immature monocyte-derived dendritic cells (iDCs) were generated by cultivation for 7 days with GM-CSF/IL4. To obtain mature dendritic cells (mDCs), iDCs were further treated with Fc-CD40L (200 ng/ml) for 2 days. mDCs were then cultivated in Fc-CD40L–free medium for additional 24 hours with and without 10 µM BV6 and were stimulated for the indicated times with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (A). iDCs were primed overnight with 10 µM BV6 and were then challenged for 5 and 20 min with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (B). iDCs and mDCs were finally analyzed by western blotting to determine the presence of the indicated proteins. The results shown with mDCs are representative of three independent experiments, the results with iDCs for two experiments. (C) iDCs were treated with 10 µM BV6, 300 ng/ml TNF and 1 µg/ml of Flag-CD40L oligomerized with 1 µg of the Flag-specific mAb M2 or as a control remained untreated. After three days cells were analyzed by FACS for the cell surface expression of CD83 and CD86. (upper panel: representative analysis of one individual sample; lower panel: summary of the data of iDCs of 4 independent donors. (D) Cell culture supernatants from “C” were analyzed for the presence of IL12. IL12 production was normalized to the corresponding values of untreated cells. The average of experiments with five independent donors is shown.</p