Direct venous inoculation of Plasmodium falciparum sporozoites for controlled human malaria infection: a dose-finding trial in two centres

BACKGROUND: Controlled human malaria infection (CHMI) accelerates development of anti-malarial interventions. So far, CHMI is done by exposure of volunteers to bites of five mosquitoes carrying Plasmodium falciparum sporozoites (PfSPZ), a technique available in only a few centres worldwide. Mosquito-mediated CHMI is logistically complex, exact PfSPZ dosage is impossible and live mosquito-based interventions are not suitable for further clinical development. METHODS: An open-labelled, randomized, dose-finding study in 18-45 year old, healthy, malaria-naive volunteers was performed to assess if intravenous (IV) injection of 50 to 3,200 aseptic, purified, cryopreserved PfSPZ is safe and achieves infection kinetics comparable to published data of mosquito-mediated CHMI. An independent study site verified the fully infectious dose using direct venous inoculation of PfSPZ. Parasite kinetics were assessed by thick blood smear microscopy and quantitative real time PCR. RESULTS: IV inoculation with 50, 200, 800, or 3,200 PfSPZ led to parasitaemia in 1/3, 1/3, 7/9, and 9/9 volunteers, respectively. The geometric mean pre-patent period (GMPPP) was 11.2 days (range 10.5-12.5) in the 3,200 PfSPZ IV group. Subsequently, six volunteers received 3,200 PfSPZ by direct venous inoculation at an independent investigational site. All six developed parasitaemia (GMPPP: 11.4 days, range: 10.4-12.3). Inoculation of PfSPZ was safe. Infection rate and pre-patent period depended on dose, and injection of 3,200 PfSPZ led to a GMPPP similar to CHMI with five PfSPZ-infected mosquitoes. The infectious dose of PfSPZ predicted dosage of radiation-attenuated PfSPZ required for successful vaccination. CONCLUSIONS: IV inoculation of PfSPZ is safe, well tolerated and highly reproducible. It shall further accelerate development of anti-malarial interventions through standardization and facilitation of CHMI. Beyond this, rational dose selection for whole PfSPZ-based immunization and complex study designs are now possible. TRIAL REGISTRATION: ClinicalTrials.gov NCT01624961 and NCT01771848

In vitro variant surface antigen expression in Plasmodium falciparum parasites from a semi-immune individual is not correlated with var gene transcription

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus vargene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific vargene primer set showed that the surface recognition signal was not correlated with transcription of individual vargenes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections

Real-time measurement of Plasmodium falciparum-infected erythrocyte cytoadhesion with a quartz crystal microbalance

Additional file 2. Example frequency shift after adding RBCs to a PLL-coated quartz. For this experiment RBCs were added to a PLL-coated quartz. It can clearly be seen that cells attached rapidly (within minutes) to the quartz indicated by a very strong frequency shift (−3389 Hz). This shows that PLL is unspecifically sticky to cells and RBCs attach rapidly to it

Broad-spectrum antimalarial activity of peptido sulfonyl fluorides, a new class of proteasome inhibitors

Despite declining numbers of cases and deaths, malaria remains a major public health problem in many parts of the world. Today, case management relies heavily on a single class of antimalarial compounds: artemisinins. Hence, development of resistance against artemisinins may destroy current malaria control strategies. Beyond malaria control are elimination and eradication programs that will require drugs with good activity against acute infection but also with preventive and transmission-blocking properties. Consequently, new antimalarials are needed not only to ensure malaria control but also for elimination and eradication efforts. In this study, we introduce peptido sulfonyl fluorides (PSF) as a new class of compounds with antiplasmodial activity. We show that PSF target the plasmodial proteasome and act on all asexual stages of the intraerythrocytic cycle and on gametocytes. PSF showed activities at concentrations as low as 20 nM against multidrug-resistant and chloroquine-sensitive Plasmodium falciparum laboratory strains and clinical isolates from Gabon. Structural requirements for activity were identified, and cytotoxicity in human HeLa or HEK 293 cells was low. The lead PSF PW28 suppressed growth of Plasmodium berghei in vivo but showed signs of toxicity in mice. Considering their modular structure and broad spectrum of activity against different stages of the plasmodial life cycle, proteasome inhibitors based on PSF have a great potential for further development as preclinical candidate compounds with improved species-specific activity and less toxicity

No correlation of FACS and <i>var</i> signals in MOA bulk and clones at >65 generations.

<p>(A) The MOA bulk culture exhibits low <i>var</i> gene transcription signals, but a high MFI (201). (B) Clone D2 exhibits the highest MFI (489) in the entire population and high <i>var</i> gene transcription signals for transcripts P_D2 and D2_18. (C) and (D) Clones D5 and C3 display low <i>var</i> gene transcription signals but show MFIs in the medium range (MFI of 63.67 and 139.67 respectively).</p