Benjamin Mordmüller

Hemozoin (Malarial Pigment) Inhibits Differentiation and Maturation of Human Monocyte-Derived Dendritic Cells: A Peroxisome Proliferator-Activated Receptor-γ-Mediated Effect

Acute and chronic Plasmodium falciparum malaria are accompanied by severe immunodepression possibly related to subversion of dendritic cells (DC) functionality. Phagocytosed hemozoin (malarial pigment) was shown to inhibit monocyte functions related to immunity. Hemozoin-loaded monocytes, frequently found in circulation and adherent to endothelia in malaria, may interfere with DC development and play a role in immunodepression. Hemozoin-loaded and unloaded human monocytes were differentiated in vitro to immature DC (iDC) by treatment with GM-CSF and IL-4, and to mature DC (mDC) by LPS challenge. In a second setting, hemozoin was fed to iDC further cultured to give mDC. In both settings, cells ingested large amounts of hemozoin undegraded during DC maturation. Hemozoin-fed monocytes did not apoptose but their differentiation and maturation to DC was severely impaired as shown by blunted expression of MHC class II and costimulatory molecules CD83, CD80, CD54, CD40, CD1a, and lower levels of CD83-specific mRNA in hemozoin-loaded iDC and mDC compared with unfed or latex-loaded DC. Further studies indicated activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) in hemozoin-loaded iDC and mDC, associated with increased expression of PPAR-γ mRNA, without apparent involvement of NF-κB. Moreover, expression of PPAR-γ was induced and up-regulation of CD83 was inhibited by supplementing iDC and mDC with plausible concentrations of 15(S)-hydroxyeicosatetraenoic acid, a PPAR-γ ligand abundantly produced by hemozoin via heme-catalyzed lipoperoxidation.

Nitric oxide synthase 2Lambaréné (G-954C), increased nitric oxide production, and protection against Malaria

A point mutation in the promoter of the nitric oxide synthase 2 gene (NOS2), termed NOS2(Lambaréné) (NOS2-G954C), protects heterozygous carriers against severe malaria as effectively as the sickle cell trait. In a prospective longitudinal study, 841 individual infections of initially 200 children (151 wild-type vs. 49 NOS2(Lambaréné) carriers) were monitored for 4 years, to assess the rates of malarial attacks in the 2 groups; carriers of the NOS2(Lambaréné) polymorphism were significantly less likely to experience malarial attacks than were others (P=.002). The distribution of the NOS2(Lambaréné) polymorphism was investigated in malaria-endemic areas. It was found to be present with the highest frequency in Africa and at a lower frequency in Asia. Ex vivo studies showed that cells isolated from people with this polymorphism have a 7-fold higher baseline NOS activity, compared with the levels detected in cells from subjects with the wild-type gene (P=.003).

The IκB Kinase Complex and NF-κB Act as Master Regulators of Lipopolysaccharide-Induced Gene Expression and Control Subordinate Activation of AP-1

ABSTRACT Toll-like receptors (TLRs) recognize conserved products of microbial pathogens to initiate the innate immune response. TLR4 signaling is triggered upon binding of lipopolysaccharides (LPS) from gram-negative bacteria. Using comparative gene expression profiling, we demonstrate a master regulatory role of IκB kinase (IKK)/NF-κB signaling for immediate-early gene induction after LPS engagement in precursor B cells. IKK/NF-κB signaling controls a large panel of gene products associated with signaling and transcriptional activation and repression. Intriguingly, the induction of AP-1 activity by LPS in precursor B cells and primary dendritic cells fully depends on the IKK/NF-κB pathway, which promotes expression of several AP-1 family members, including JunB, JunD, and B-ATF. In pre-B cells, AP-1 augments induction of a subset of primary NF-κB targets, as shown for chemokine receptor 7 (CCR7) and immunoglobulin κ light chain. Thus, our data illustrate that NF-κB orchestrates immediate-early effects of LPS signaling and controls secondary AP-1 activation to mount an appropriate biological response.

Lymphotoxin and lipopolysaccharide induce NF‐κB‐p52 generation by a co‐translational mechanism

The ‘classical’ NF‐κB activation pathway proceeds via IκB kinase (IKK)‐β/γ‐mediated phosphorylation, induced ubiquitination and the degradation of small IκBs. An alternative, NF‐κB‐inducing kinase and IKK‐α‐dependent pathway, which stimulates the processing of NF‐κB2/p100, has recently been suggested. However, no physiological stimulus has been shown to trigger the activation of this pathway. Here we demonstrate that persistent stimulation with lymphotoxin β (LT‐β) receptor agonists or lipopolysaccharide (LPS), but not with interleukin‐1β, tumour necrosis factor‐α or 12‐O‐tetradecanoylphorbol‐13‐acetate, induces the generation of p52 DNA‐binding complexes by activating the processing of the p100 precursor. Induction of p52 DNA‐binding activity is delayed in comparison with p50/p65 complexes and depends on de novo protein synthesis. p100 is constitutively and inducibly polyubiquitinated, and both ubiquitination and p52 generation are coupled to continuing p100 translation. Thus, both LT‐β receptor agonists and LPS induce NF‐κB/p100 processing to p52 at the level of the ribosome.

Selection of a trioxaquine as an antimalarial drug candidate.

Trioxaquines are antimalarial agents based on hybrid structures with a dual mode of action. One of these molecules, PA1103/SAR116242, is highly active in vitro on several sensitive and resistant strains of Plasmodium falciparum at nanomolar concentrations (e.g., IC50 value = 10 nM with FcM29, a chloroquine-resistant strain) and also on multidrug-resistant strains obtained from fresh patient isolates in Gabon. This molecule is very efficient by oral route with a complete cure of mice infected with chloroquine-sensitive or chloroquine-resistant strains of Plasmodia at 26–32 mg/kg. This compound is also highly effective in humanized mice infected with P. falciparum. Combined with a good drug profile (preliminary absorption, metabolism, and safety parameters), these data were favorable for the selection of this particular trioxaquine for development as drug candidate among 120 other active hybrid molecules.

Atypical and classical memory B cells produce Plasmodium falciparum neutralizing antibodies

Plasmodium falciparum infection leads to the development of protective classical and atypical memory B cell antibody responses.

Notch ligands Delta-like1, Delta-like4 and Jagged1 differentially regulate activation of peripheral T helper cells.

The Notch pathway is involved in cell differentiation processes in various organs and at several developmental stages. The importance of Notch for early T lymphocyte development is well established. Recently, Notch has been implicated in directing naive T helper cell differentiation towards the Th1, Th2 or regulatory T cell lineages. However, the molecular events underlying these processes are poorly understood.We show that the Notch ligands Delta‐like1, Delta‐like4 and Jagged1 differentially affect early T cell activation and proliferation following T cell receptor cross‐linking. Delta‐like1 and Jagged1 induce a dose‐dependent inhibition of early activation markers CD69 and CD25, as well as inhibition of proliferation after anti‐CD3 stimulation of purified CD4+ T cells. Similarly, the rapid activation of transcription factors NF‐AT, AP‐1 and NF‐κB is suppressed. In contrast, triggering of Notch by Delta‐like4 enhances T cell activation and proliferation. The observed effects are dependent on simultaneous cross‐linking of TCR and Notch but independent of γ‐secretase‐mediated cleavage of Notch. These data suggest direct interference between Notch and early TCR signal transduction events, independent of the classical Notch pathway via release of the Notch intracellular domain. A Notch‐mediated alteration of TCR signaling strength may contribute to the recently described modulation of naïve T cell differentiation by Notch ligands.

Safety and immunogenicity of GMZ2 — a MSP3–GLURP fusion protein malaria vaccine candidate

Malaria is a major public health problem in Sub-Saharan Africa. In highly endemic regions infants, children and pregnant women are mostly affected. An effective malaria vaccine would complement existing malaria control strategies because it can be integrated in existing immunization programs easily. Here we present the results of the first phase Ia clinical trial of GMZ2 adjuvanted in aluminium hydroxide. GMZ2 is a malaria vaccine candidate, designed upon the rationale to induce immune responses against asexual blood stages of Plasmodium falciparum similar to those encountered in semi-immune individuals. Ten, 30 and 100 microg of GMZ2 were well tolerated in 30 healthy malaria-naïve German volunteers when given three times in monthly intervals. Antigen-specific antibodies as well as memory B-cells were induced and detectable throughout the one year follow-up of the study. We conclude that GMZ2 is a safe and immunogenic malaria vaccine candidate suitable for further clinical development.

Fixed-Dose Pyronaridine-Artesunate Combination for Treatment of Uncomplicated Falciparum Malaria in Pediatric Patients in Gabon

BACKGROUND The development of novel artemisinin-combination therapies suitable for the treatment of pediatric patients suffering from malaria is a research priority. The aim of this study was to investigate a novel fixed-dose pyronaridine-artesunate combination for the treatment of uncomplicated falciparum malaria in Gabonese patients 2-14 years old. METHODS The study was designed as an open-label dose-escalation study recruiting 60 pediatric patients sequentially in 4 treatment cohorts: study drugs were administered once daily for 3 days, as tablet coformulations (pyronaridine:artesunate ratios of 6:2, 9:3, and 12:4 mg/kg) and as a granule coformulation (pyronaridine:artesunate ratio of 9:3 mg/kg). The primary end points were tolerability, safety, and pharmacokinetics of pyronaridine-artesunate treatment. Efficacy was treated as a secondary outcome measure. RESULTS The drugs had a good tolerability and safety profile, at all dose levels. Pharmacokinetic analysis revealed a dose-dependent increase in the maximum plasma/blood concentration and the area under the curve, as well as comparable relative bioavailability for the granule coformulation. Polymerase chain reaction-corrected cure rates at day 28 were 100% in per-protocol analysis, at all dose levels. CONCLUSIONS Pyronaridine-artesunate is a promising novel artemisinin-combination therapy for pediatric patients with uncomplicated Plasmodium falciparum malaria, and the development of both the tablet and the granule coformulations is warranted.

Sterile protection against human malaria by chemoattenuated PfSPZ vaccine

A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ (‘PfSPZ Vaccine’); or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine or mefloquine (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ (‘PfSPZ Challenge’) to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac). Three doses of 5.12 × 104 PfSPZ of PfSPZ Challenge at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 103 (group I) or 1.28 × 104 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 104 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.