Nitric oxide controls proliferation of Leishmania major by inhibiting the recruitment of permissive host cells.

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.

Plasmodium sporozoite disintegration during skin passage limits malaria parasite transmission.

During transmission of malaria-causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane-associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localizes at the cytoplasmic periphery and concavin(-) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(-) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(-) sporozoites are transmitted. Strikingly, motile concavin(-) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection.

MAEBL Contributes to Plasmodium Sporozoite Adhesiveness.

The sole currently approved malaria vaccine targets the circumsporozoite protein-the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by Plasmodium sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces Plasmodium berghei hemolymph sporozoite infectivity to mice. Moreover, we found that maebl knockout (maebl-) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The maebl- defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions.

CD11c-expressing Ly6C+CCR2+ monocytes constitute a reservoir for efficient Leishmania proliferation and cell-to-cell transmission.

The virulence of intracellular pathogens such as Leishmania major (L. major) relies largely on their ability to undergo cycles of replication within phagocytes, release, and uptake into new host cells. While all these steps are critical for successful establishment of infection, neither the cellular niche of efficient proliferation, nor the spread to new host cells have been characterized in vivo. Here, using a biosensor for measuring pathogen proliferation in the living tissue, we found that monocyte-derived Ly6C+CCR2+ phagocytes expressing CD11c constituted the main cell type harboring rapidly proliferating L. major in the ongoing infection. Synchronization of host cell recruitment and intravital 2-photon imaging showed that these high proliferating parasites preferentially underwent cell-to-cell spread. However, newly recruited host cells were infected irrespectively of their cell type or maturation state. We propose that among these cells, CD11c-expressing monocytes are most permissive for pathogen proliferation, and thus mainly fuel the cycle of intracellular proliferation and cell-to-cell transfer during the acute infection. Thus, besides the well-described function for priming and activating T cell effector functions against L. major, CD11c-expressing monocyte-derived cells provide a reservoir for rapidly proliferating parasites that disseminate at the site of infection.

HMGB1 Is Involved in IFN-α Production and TRAIL Expression by HIV-1-Exposed Plasmacytoid Dendritic Cells: Impact of the Crosstalk with NK Cells.

Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover, Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection, but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here, we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α, TNF-α, IFN-γ and IL-12, and CCR5-interacting chemokines (MIP-1α and MIP-1ß) in NK-pDCs co-cultures. At high HIV-1BaL concentrations, the addition of NK cells did not promote the release of these mediators, suggesting that once efficiently triggered by the virus, pDCs could not integrate new activating signals delivered by NK cells. However, high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly, we identified the alarmin HMGB1, released at pDC-NK cell synapse, as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover, HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1, HMGB1-specific antibodies, sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether, these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells, and they suggest a novel mechanism of innate control of HIV-1 infection.

Yeast lysates carrying the nucleoprotein from measles virus vaccine as a novel subunit vaccine platform to deliver Plasmodium circumsporozoite antigen.

BACKGROUND: Yeast cells represent an established bioreactor to produce recombinant proteins for subunit vaccine development. In addition, delivery of vaccine antigens directly within heat-inactivated yeast cells is attractive due to the adjuvancy provided by the yeast cell. In this study, Pichia pastoris yeast lysates carrying the nucleoprotein (N) from the measles vaccine virus were evaluated as a novel subunit vaccine platform to deliver the circumsporozoite surface antigen (CS) of Plasmodium. When expressed in Pichia pastoris yeast, the N protein auto-assembles into highly multimeric ribonucleoparticles (RNPs). The CS antigen from Plasmodium berghei (PbCS) was expressed in Pichia pastoris yeast in fusion with N, generating recombinant PbCS-carrying RNPs in the cytoplasm of yeast cells. RESULTS: When evaluated in mice after 3-5 weekly subcutaneous injections, yeast lysates containing N-PbCS RNPs elicited strong anti-PbCS humoral responses, which were PbCS-dose dependent and reached a plateau by the pre-challenge time point. Protective efficacy of yeast lysates was dose-dependent, although anti-PbCS antibody titers were not predictive of protection. Multimerization of PbCS on RNPs was essential for providing benefit against infection, as immunization with monomeric PbCS delivered in yeast lysates was not protective. Three weekly injections with N-PbCS yeast lysates in combination with alum adjuvant produced sterile protection in two out of six mice, and significantly reduced parasitaemia in the other individuals from the same group. This parasitaemia decrease was of the same extent as in mice immunized with non-adjuvanted N-PbCS yeast lysates, providing evidence that the yeast lysate formulation did not require accessory adjuvants for eliciting efficient parasitaemia reduction. CONCLUSIONS: This study demonstrates that yeast lysates are an attractive auto-adjuvant and efficient platform for delivering multimeric PbCS on measles N-based RNPs. By combining yeast lysates that carry RNPs with a large panel of Plasmodium antigens, this technology could be applied to developing a multivalent vaccine against malaria.

Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity.

Introduction: Plasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites. Methods: Considering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development. Results and Discussion: Though IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.

Plasmodium sporozoite search strategy to locate hotspots of blood vessel invasion.

Plasmodium sporozoites actively migrate in the dermis and enter blood vessels to infect the liver. Despite their importance for malaria infection, little is known about these cutaneous processes. We combine intravital imaging in a rodent malaria model and statistical methods to unveil the parasite strategy to reach the bloodstream. We determine that sporozoites display a high-motility mode with a superdiffusive Lévy-like pattern known to optimize the location of scarce targets. When encountering blood vessels, sporozoites frequently switch to a subdiffusive low-motility behavior associated with probing for intravasation hotspots, marked by the presence of pericytes. Hence, sporozoites present anomalous diffusive motility, alternating between superdiffusive tissue exploration and subdiffusive local vessel exploitation, thus optimizing the sequential tasks of seeking blood vessels and pericyte-associated sites of privileged intravasation.

Cytotoxicity of human antibodies targeting the circumsporozoite protein is amplified by 3D substrate and correlates with protection.

Human monoclonal antibodies (hmAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on the sporozoite surface are a promising tool for preventing malaria infection. However, their mechanisms of protection remain unclear. Here, using 13 distinctive PfCSP hmAbs, we provide a comprehensive view of how PfCSP hmAbs neutralize sporozoites in host tissues. Sporozoites are most vulnerable to hmAb-mediated neutralization in the skin. However, rare but potent hmAbs additionally neutralize sporozoites in the blood and liver. Efficient protection in tissues mainly associates with high-affinity and high-cytotoxicity hmAbs inducing rapid parasite loss-of-fitness in the absence of complement and host cells in vitro. A 3D-substrate assay greatly enhances hmAb cytotoxicity and mimics the skin-dependent protection, indicating that the physical stress imposed on motile sporozoites by the skin is crucial for unfolding the protective potential of hmAbs. This functional 3D cytotoxicity assay can thus be useful for downselecting potent anti-PfCSP hmAbs and vaccines.

Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target.

The uptake and digestion of host hemoglobin by malaria parasites during blood-stage growth leads to significant oxidative damage of membrane lipids. Repair of lipid peroxidation damage is crucial for parasite survival. Here, we demonstrate that Plasmodium falciparum imports a host antioxidant enzyme, peroxiredoxin 6 (PRDX6), during hemoglobin uptake from the red blood cell cytosol. PRDX6 is a lipid-peroxidation repair enzyme with phospholipase A2 (PLA2) activity. Inhibition of PRDX6 with a PLA2 inhibitor, Darapladib, increases lipid-peroxidation damage in the parasite and disrupts transport of hemoglobin-containing vesicles to the food vacuole, causing parasite death. Furthermore, inhibition of PRDX6 synergistically reduces the survival of artemisinin-resistant parasites following co-treatment of parasite cultures with artemisinin and Darapladib. Thus, PRDX6 is a host-derived drug target for development of antimalarial drugs that could help overcome artemisinin resistance.

Longitudinal proliferation mapping in vivo reveals NADPH oxidase-mediated dampening of Staphylococcus aureus growth rates within neutrophils.

Upon the onset of inflammatory responses, bacterial pathogens are confronted with altered tissue microenvironments which can critically impact on their metabolic activity and growth. Changes in these parameters have however remained difficult to analyze over time, which would be critical to dissect the interplay between the host immune response and pathogen physiology. Here, we established an in vivo biosensor for measuring the growth rates of Staphylococcus aureus (S. aureus) on a single cell-level over days in an ongoing cutaneous infection. Using intravital 2-photon imaging and quantitative fluorescence microscopy, we show that upon neutrophil recruitment to the infection site and bacterial uptake, non-lethal dampening of S. aureus proliferation occurred. This inhibition was supported by NADPH oxidase activity. Therefore, reactive oxygen production contributes to pathogen containment within neutrophils not only by killing S. aureus, but also by restricting the growth rate of the bacterium.

Recombinant measles vaccine expressing malaria antigens induces long-term memory and protection in mice.

Following the RTS,S malaria vaccine, which showed only partial protection with short-term memory, there is strong support to develop second-generation malaria vaccines that yield higher efficacy with longer duration. The use of replicating viral vectors to deliver subunit vaccines is of great interest due to their capacity to induce efficient cellular immune responses and long-term memory. The measles vaccine virus offers an efficient and safe live viral vector that could easily be implemented in the field. Here, we produced recombinant measles viruses (rMV) expressing malaria "gold standard" circumsporozoïte antigen (CS) of Plasmodium berghei (Pb) and Plasmodium falciparum (Pf) to test proof of concept of this delivery strategy. Immunization with rMV expressing PbCS or PfCS induced high antibody responses in mice that did not decrease for at least 22 weeks post-prime, as well as rapid development of cellular immune responses. The observed long-term memory response is key for development of second-generation malaria vaccines. Sterile protection was achieved in 33% of immunized mice, as usually observed with the CS antigen, and all other immunized animals were clinically protected from severe and lethal Pb ANKA-induced cerebral malaria. Further rMV-vectored malaria vaccine candidates expressing additional pre-erythrocytic and blood-stage antigens in combination with rMV expressing PfCS may provide a path to development of next generation malaria vaccines with higher efficacy.

Looking for blood.

In vivo imaging has revealed new details about how the malaria parasite enters the bloodstream.

Loss of host cell plasma membrane integrity following cell traversal by Plasmodium sporozoites in the skin.

Plasmodium sporozoites are able to migrate through host cells by breaching their plasma membrane and gliding inside their cytoplasm. This migratory activity, called cell traversal (CT), was studied in vivo mainly using mutant sporozoites lacking the ability to wound host cells, and thus to perform CT. However, direct evidence of CT activity in host tissues by wild-type sporozoites remains scarce. Here, we describe a double-wounding assay to dynamically image CT activity in vivo and monitor cell membrane integrity over time. Based on the incorporation kinetics of a first live cell-impermeant dye, propidium iodide, we could determine whether traversed cells repair their wounded membranes or not. A second impermeant dye, SYTOX Green, was used to confirm the transient or the permanent loss of membrane integrity of traversed cells. This assay allowed, for the first time, the direct observation of sporozoites wounding and traversing host skin cells and showed that, while some traversed cells resealed their membrane, most became irreversibly permeable to these live cell-impermeant dyes. In combination with the study of CT-deficient sporozoites and the use of specific host cell markers, this intravital assay will provide the means to identify the nature of the cells traversed by sporozoites and will thus contribute to elucidating the role of CT by apicomplexan parasites in the vertebrate host.

ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development.

The malaria parasite, Plasmodium, requires iron for growth, but how it imports iron remains unknown. We characterize here a protein that belongs to the ZIP (Zrt-, Irt-like Protein) family of metal ion transport proteins and have named ZIP domain-containing protein (ZIPCO). Inactivation of the ZIPCO-encoding gene in Plasmodium berghei, while not affecting the parasite's ability to multiply in mouse blood and to infect mosquitoes, greatly impairs its capacity to develop inside hepatocytes. Iron/zinc supplementation and depletion experiments suggest that ZIPCO is required for parasite utilization of iron and possibly zinc, consistent with its predicted function as a metal transporter. This is the first report of a ZIP protein having a crucial role in Plasmodium liver-stage development, as well as the first metal ion transporter identified in Plasmodium pre-erythrocytic stages. Because of the drastic dependence on iron of Plasmodium growth, ZIPCO and related proteins might constitute attractive drug targets to fight against malaria.

Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.

Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries.

Imaging sporozoite cell traversal in the liver of mice.

Intravital fluorescence microscopy is an invaluable tool to study a dynamic phenomenon through its direct observation in living organisms. This technique can combine qualitative and quantitative analysis and has been capital to address long-standing questions about Plasmodium biology. Beyond a descriptive view of the parasite life cycle, the possibility to image infection in transgenic animals in which a specific cell type, molecule or process is labeled opens new possibilities to study host cell-parasite interactions in cellular and molecular details. An additional layer of refinement can be achieved with the use of fluorescent knockout mutants (parasite, mice, or both) to dissect the molecular basis of the process of interest. Here, we present a basic protocol for imaging the sporozoite behavior in the liver, emphasizing the detection of the sporozoite's ability to traverse host cells.

Role of host cell traversal by the malaria sporozoite during liver infection.

Malaria infection starts when the sporozoite stage of the Plasmodium parasite is injected into the skin by a mosquito. Sporozoites are known to traverse host cells before finally invading a hepatocyte and multiplying into erythrocyte-infecting forms, but how sporozoites reach hepatocytes in the liver and the role of host cell traversal (CT) remain unclear. We report the first quantitative imaging study of sporozoite liver infection in rodents. We show that sporozoites can cross the liver sinusoidal barrier by multiple mechanisms, targeting Kupffer cells (KC) or endothelial cells and associated or not with the parasite CT activity. We also show that the primary role of CT is to inhibit sporozoite clearance by KC during locomotion inside the sinusoid lumen, before crossing the barrier. By being involved in multiple steps of the sporozoite journey from the skin to the final hepatocyte, the parasite proteins mediating host CT emerge as ideal antibody targets for vaccination against the parasite.