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The current paradigm in the pathogenesis of several hemolytic red blood cell disorders is that reduced cellular deformability is a key determinant of splenic sequestration of affected red cells. Three distinct features regulate cellular deformability: membrane deformability, surface area-to-volume ratio (cell sphericity), and cytoplasmic viscosity. By perfusing normal human spleens ex vivo, we had previously showed that red cells with increased sphericity are rapidly sequestered by the spleen. Here, we assessed the retention kinetics of red cells with decreased membrane deformability but without marked shape changes. A controlled decrease in membrane deformability (increased membrane rigidity) was induced by treating normal red cells with increasing concentrations of diamide. Following perfusion, diamide-treated red blood cells (RBCs) were rapidly retained in the spleen with a mean clearance half-time of 5.9 minutes (range, 4.0-13.0). Splenic clearance correlated positively with increased membrane rigidity (r = 0.93; P < .0001). To determine to what extent this increased retention was related to mechanical blockade in the spleen, diamide-treated red cells were filtered through microsphere layers that mimic the mechanical sensing of red cells by the spleen. Diamide-treated red cells were retained in the microsphilters (median, 7.5%; range, 0%-38.6%), although to a lesser extent compared with the spleen (median, 44.1%; range, 7.3%-64.0%; P < .0001). Taken together, these results have implications for understanding the sensitivity of the human spleen to sequester red cells with altered cellular deformability due to various cellular alterations and for explaining clinical heterogeneity of RBC membrane disorders.

Splenic sequestration of RBCs with reduced surface area and cellular deformability has long been recognized as contributing to pathogenesis of several RBC disorders, including hereditary spherocytosis. However, the quantitative relationship between the extent of surface area loss and splenic entrapment remains to be defined. To address this issue, in the present study, we perfused ex vivo normal human spleens with RBCs displaying various degrees of surface area loss and monitored the kinetics of their splenic retention. Treatment with increasing concentrations of lysophosphatidylcholine resulted in a dose-dependent reduction of RBC surface area at constant volume, increased osmotic fragility, and decreased deformability. The degree of splenic retention of treated RBCs increased with increasing surface area loss. RBCs with a > 18% average surface area loss (> 27% reduced surface area-to-volume ratio) were rapidly and completely entrapped in the spleen. Surface-deficient RBCs appeared to undergo volume loss after repeated passages through the spleen and escape from splenic retention. The results of the present study for the first time define the critical extent of surface area loss leading to splenic entrapment and identify an adaptive volume regulation mechanism that allows spherocytic RBCs to prolong their life span in circulation. These results have significant implications for understanding the clinical heterogeneity of RBC membrane disorders.

Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 ( PArt ), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium -specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins.

THERAVECTYS is a Paris-based, privately-owned, fully integrated discovery & clinical development biotech company. We are developping lentiviral vectors for human and veterinary vaccination and immunotherapy applications.We have have proudly achieved the first-ever human vaccination clinical trial conducted with lentiviral vectors with our anti-HIV vaccine candidate. We are now pursuing our clinical development of further therapeutic innovative vaccine candidates, both in oncology & infection diseases. Particularly, our first oncology clinical trial, which targets Adult T-cell Leukemia/Lymphoma virally-induced by HTLV-1, is programmed before Q3 2015. In addition, we are developing our own T-cell therapy programs (CAR and TCR) based on its proprietary new lentiviral-based system allowing a real switch of the production of the CAR or of the TCR by the modified T-cellsTo control costs, respect timelines and maintain the high quality and diversity of all ongoing and future clinical development programs, THERAVECTYS has decided to internalize the bio-production of its lentiviral vectors and the associated quality controls. To achieve this goal, THERAVECTYS has built a GMP facility in compliance with the requirements of regulatory agencies (EMA and FDA). The 340m2 plant is equipped with state-of-the-art bioreactors (from 20 to 1000L), semi-automated downstream skids and a fill-and-finish system to conform to our fully integrated single use process. This process implies a 16day-USP step corresponding to the expansion and transfection of HEK293SF cells, and a 2day-DSP step comprising 3 purifications. This GMP facility will allow us to produce batches from a minimum of 300 up to 5 000 vials complying with up to phase III clinical trial requirements. Additionally, our GMP facility which is composed of defined distinct independent rooms will allow us to develop our current validated technics, lentiviral vector production and CAR T cells engineering, without any cross-contamination.Our Quality Control unit was architecturally designed to house a vast array of analytical equipment. With more than 30 different analytical methods including environmental monitoring of the GMP facility, contaminants evaluation and RCL testing, THERAVECTYS has gained full independence by keeping all analytical and microbiology testing in house.This cutting-edge GMP facility (production and QC unit) had been built in 9 months, and should be operational in Q1 2015 (the certification process is currently underway). It will allow THERAVECTYS to protect its unique and advanced know-how in the manufacturing of lentiviral vectors and CAR-T cells.

The altered deformability of erythrocytes infected with Plasmodium falciparum is central in malaria -pathogenesis, as it influences the hemodynamic properties of the infected cell and its retention in the spleen. Exported parasite proteins, as well as the shape and volume of the parasite itself, influence the deformability of the infected erythrocyte. To explore changes in erythrocyte deformability, we have developed a new method, called microsphiltration, based on filtration of erythrocytes through a mixture of metal microspheres that mimic the geometry of inter-endothelial splenic slits. As P. falciparum develops in its host cell, the retention rates observed in microspheres correlate with the progressive decrease of erythrocyte deformability and with the retention rates in the spleen. The yields of microsphiltration separation allow for molecular analyses of subpopulations with distinct mechanical phenotypes.

The altered deformability of erythrocytes infected with Plasmodium falciparum is central in malaria -pathogenesis, as it influences the hemodynamic properties of the infected cell and its retention in the spleen. Exported parasite proteins, as well as the shape and volume of the parasite itself, influence the deformability of the infected erythrocyte. To explore changes in erythrocyte deformability, we have developed a new method, called microsphiltration, based on filtration of erythrocytes through a mixture of metal microspheres that mimic the geometry of inter-endothelial splenic slits. As P. falciparum develops in its host cell, the retention rates observed in microspheres correlate with the progressive decrease of erythrocyte deformability and with the retention rates in the spleen. The yields of microsphiltration separation allow for molecular analyses of subpopulations with distinct mechanical phenotypes.

Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.

Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host., Singapore-MIT Alliance for Research and Technology Center, National Institutes of Health (U.S.). (Grant R01HL094270), National Center for Research Resources (U.S.) (9P41-EB015871-26A1)

Human T-cell Lymphotrophic Virus 1 (HTLV-1) is the etiologic agent of Adult-T cell Leukemia/Lymphoma (ATL). Therapeutic options for ATL patients are very limited and in aggressive forms of the disease survival rate is only 10% to 30% with conventional chemotherapies and bone marrow transplantation. Although some clinical trials gave encouraging results regarding the efficacy of new treatments, most of them are lifelong, aggressive and failed to achieve a significant impact on long-term survival. Consequently, new treatments for ATL patients are needed to limit relapses and side effects. Specific HTLV-1 cellular immune response is dramatically impaired in ATL patients, which could favor the initiation and the progression of the disease. Hence, stimulating immune responses against HTLV-1 can be an appropriate therapeutic option to treat ATL. THERAVECTYS has developed an anti-HTLV-1 vaccine, based on its lentiviral vector technology inducing a broad, intense and long-lasting cellular immune response after intra-muscular injection. THERAVECTYS was the first company to have launched a clinical trial based on lentiviral vectors technology with the THV01 vaccine for the treatment of HIV (NCT02054286). Results obtained demonstrated both safety and immunogenicity of THV01 in human, with polyfunctional and multi-specific CD4 and CD8 T-cells responses. The anti-HTLV-1 lentiviral vector, THV02 vaccine, encodes for a unique polypeptide derived from Tax, HBZ, p12I and p30II proteins, involved in HTLV-1 pathogenicity and known to be recognized by the immune system of HTLV-1 infected patients. Our preclinical results have demonstrated that THV02 can induce a cellular immune response in C57Bl/6j and BalbC mice and in Sprague Dawley rats, as demonstrated by IFN-γ Elispot. Safety of the THV02 vaccine has been demonstrated during carcinogenicity and regulatory GLP preclinical toxicity studies. Biodistribution and shedding studies demonstrated the very limited diffusion of THV02 after injection, its fast clearance and a non-dissemination in body fluids. As no relevant ATL immunocompetent animal model is available to assess the anti-tumor effect of THV02, THERAVECYTS is developing an ex-vivo efficacy model using blood samples of ATL patients. Briefly, monocyte-derived dendritic cells (MDDC) from blood of ATL patients are purified by isolation of CD14 positive cells from PBMC and differentiation in the presence of IL4 and GM-CSF. MDDC are then transduced with lentiviral vectors encoding for the anti-HTLV-1 antigen and maturation is induced upon TNFa and PGE2 exposure before the co-culture with autologous CD8+ T-cells for stimulation of the cellular immune response. Then, stimulated CD8+ are co-cultured with autologous CD4+ CD25+ ATL cells and the cytotoxic activity is monitored by flow cytometry. Preliminary results demonstrated that MDDC from a chronic ATL patient can be efficiently transduced and matured as attested by the CD40, CD86, HLA-DR, -A, -B and C markers on their surface. In addition, we have observed a specific stimulation of the CD8+, ie an increase of IFNg, TNFa, IL2 and perforin in the media of the co-culture of CD8+ with MDDC expressing anti-HTLV-1 antigen. These data are very encouraging and demonstrate for the first time the feasibility to develop an ex vivo model to assess vaccine efficacy using ATL blood sample. The development of this model is ongoing using several ATL donors representing the different subtypes of the disease and will be presented at the meeting. Regarding the indication and the safety profile of THV02, THERAVECTYS plans to begin a clinical trial in Q4 2015. This assay will be an open-label, dose escalation phase I/II study to assess the safety and the immunogenicity (cellular immune response) of the THV02 vaccination as a treatment of ATL patients. All ATL subtypes will be considered since THV02 vaccine can be combined with conventional ATL treatments. In addition, as the THV02 antigen contains peptides derived from Tax but also HBZ, p12I and p30II viral proteins, all ATL patients can be treated whatever the status of Tax expression. As secondary objectives, both humoral immune response and clinical effect will be assessed. HTLV-1 RNA expression and clonality of HTLV-1 infected cells will be studied as exploratory objectives. Finally, up to 16 patients will be enrolled in France, UK, French Guiana, Martinique and Guadeloupe before doing a phase of extension cohort in US. Disclosures No relevant conflicts of interest to declare.

Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum–infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-μm-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deformable RBC subpopulations could be separated and characterized at the molecular level, and the device was used to deplete a stored RBC population from its subpopulation of rigid RBCs. This experimental approach may contribute to a better understanding of the role of the spleen in the pathogenesis of inherited and acquired RBC disorders.

Clinical manifestations of Plasmodium falciparum infection are induced by the asexual stages of the parasite that develop inside red blood cells (RBCs). Because splenic microcirculatory beds filter out altered RBCs, the spleen can innately clear subpopulations of infected or uninfected RBC modified during falciparum malaria. The spleen appears more protective against severe manifestations of malaria in naïve than in immune subjects. The spleen-specific pitting function accounts for a large fraction of parasite clearance in artemisinin-treated patients. RBC loss contributes to malarial anemia, a clinical form associated with subacute progression, frequent splenomegaly, and relatively low parasitemia. Stringent splenic clearance of ring-infected RBCs and uninfected, but parasite-altered, RBCs, may altogether exacerbate anemia and reduce the risks of severe complications associated with high parasite loads, such as cerebral malaria. The age of the patient directly influences the risk of severe manifestations. We hypothesize that coevolution resulting in increased splenic clearance of P. falciparum–altered RBCs in children favors the survival of the host and, ultimately, sustained parasite transmission. This analysis of the RBC–spleen dynamic interactions during P falciparum infection reflects both data and hypotheses, and provides a framework on which a more complete immunologic understanding of malaria pathogenesis may be elaborated.

Experimental tools to identify human red blood cells (RBC) prone to mechanical retention upstream from the spleen venous sinus inter-endothelial slits are currently suboptimal. We designed a micro-bead device mimicking the geometry of the human narrow and short inter-endothelial slits. Upon filtration through a mixture of 5-25 μm diameter micro-beads, Plasmodium falciparum-hosting RBC (Pf-RBC) were retained in a parasite developmental stage-dependent way, the retention rates of a subset of ring-RBC being similar in micro-beads and in isolated-perfused human spleens. We found that this retention might be linked principally to the reduced surface-area-to-volume ratio of Pf-RBC. Interestingly, other rigid RBC, such as heat-treated RBC, and RBC from hereditary spherocytosis patients were also retained in micro-beads without any hemolysis. Micro-beads allow (i) depletion of heterogeneous RBC population from its rigid-RBC subpopulation ii) characteriziation of distinct molecular signatures of rigid versus deformable RBC subpopulations. This simple method portends wide medical applications, such as improving the quality of stored RBC concentrates prior to transfusion.

Background Translation of the genome sequence of Plasmodium sp. into biologically relevant information relies on high through-put genomics technology which includes transcriptome analysis. However, few studies to date have used this powerful approach to explore transcriptome alterations of P. falciparum parasites exposed to antimalarial drugs. Results The rapid action of artesunate allowed us to study dynamic changes of the parasite transcriptome in synchronous parasite cultures exposed to the drug for 90 minutes and 3 hours. Developmentally regulated genes were filtered out, leaving 398 genes which presented altered transcript levels reflecting drug-exposure. Few genes related to metabolic pathways, most encoded chaperones, transporters, kinases, Zn-finger proteins, transcription activating proteins, proteins involved in proteasome degradation, in oxidative stress and in cell cycle regulation. A positive bias was observed for over-expressed genes presenting a subtelomeric location, allelic polymorphism and encoding proteins with potential export sequences, which often belonged to subtelomeric multi-gene families. This pointed to the mobilization of processes shaping the interface between the parasite and its environment. In parallel, pathways were engaged which could lead to parasite death, such as interference with purine/pyrimidine metabolism, the mitochondrial electron transport chain, proteasome-dependent protein degradation or the integrity of the food vacuole. Conclusion The high proportion of over-expressed genes encoding proteins exported from the parasite highlight the importance of extra-parasitic compartments as fields for exploration in drug research which, to date, has mostly focused on the parasite itself rather than on its intra and extra erythrocytic environment. Further work is needed to clarify which transcriptome alterations observed reflect a specific response to overcome artesunate toxicity or more general perturbations on the path to cellular death.

The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum–infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.

Since lentiviral vectors are incrinsingly used in various applications (vaccination, gene therapy, T-cell therapies …) and given that so far the best way to keep these vectors is a -80°C storage, more adapted ways of formulation and storage must be developped.Previous attempts have been made to freeze-dry viral vectors: adenovectors and Moloney Monkey Leukemia Virus-based vectors (Cruz et al., 2006) and lentiviral vectors (Shin et al, 2010). None of these attempts have proved satisfactory as the final lyophilized products were not suitable for pharmaceutical applications (mainly due to unacceptable levels of contaminants observed in the final products).We developped an new method to freeze-dry our clinical grade batches of lentiviral vectors. This method allows us to store the batches at -20°C and +4°C while maintaining the characteristics of the particules after reconstitution and the ability to use them as intended (direct injection, T-cell modification …). Our method allows the lyophilization of lentiviral vectors either produced in adherent cells cultures (with FBS) or in suspension cells cultures (without FBS and with synthetic culture medium). The vectors are protected during the freeze-drying process by the addition of a lyoprotectant (sucrose or threalose) and stability is evaluated over time. View Large Image | Download PowerPoint SlideQuality controls have been performed to evaluate the impurity profiles of the lentivector batches before the freeze-drying process and after the reconstitution of the particules (titer, total proteins, total DNA, HEK 293T proteins), and we demonstrated that the profiles were not impacted by the process and that the batches were still fitting the international pharmacopea recomandations..We also made some immunogenicity evaluation of our vaccine candidates before and after the lyophilization process, and we were able to show that the process had no impact on the breadth and intensity of the cellular immune response we are able to elicit after a direct injection of the reconstituted particles in mice.THERAVECTYS is a Paris-based, privately-owned, fully integrated discovery & clinical development biotech company. THERAVECTYS develops lentiviral vectors used for human vaccination and immunotherapy applications (CAR-T cells and TCR). An in house cGMP facility is now up and running, allowing us to produce and charaterize our own lentiviral vector clinical batches. A qualified freeze-dryer has been installed in the plant, to systematically encompass this formulation and repartition step in our bioproduction processes.