Experimental infection and protection against ... - TI Pharma

NF135.C10: a new Plasmodium falciparum clone for controlled human malaria
infections
magnification by assessing 123 high-power fields, equal to approximately 0.5µl
of blood. A smear was considered positive if two unambiguously identifiable
parasites were found. Pre-patent period was defined as the period between
exposure to infected mosquitoes and first positive blood smear. Additionally,
parasitemia was measured by real-time quantitative PCR (qPCR), performed
retrospectively on all samples collected after challenge, as described previously
[24] with minor changes: using the MGB probe AAC AAT TGG AGG GCA AG
instead of the turbo TaqMan probe sequence.
In vitro immunological assays
For antigen preparation, asynchronous asexual-stage cultures of NF135.C10 and
NF54 parasites were harvested at parasitemias of approximately 5-10% and
mature asexual stages were purified by centrifugation on a 27% and 63% Percoll
density gradient [25] resulting in preparations of 80-90% parasitemia with >95%
schizonts/mature trophozoites. Preparations of parasitized red blood cells
(PfRBC) were washed twice in PBS and cryopreserved at 150x10 6 /ml in 15%
glycerol/PBS in aliquots for use in individual stimulation assays. Mock-cultured
uninfected erythrocytes (uRBC) were obtained similarly and served as controls.
For cellular immunology, venous whole blood was collected into citrated
vacutainer CPT cell preparation tubes (Becton and Dickinson) on the day prior to
challenge (C-1), on days 5, 35 and 140 after challenge and on the first day of
treatment (DT). Peripheral blood mononuclear cells (PBMCs) were isolated by
density gradient centrifugation, washed twice in PBS, enumerated, frozen in
foetal-calf serum containing 10% dimethylsulfoxide and stored in liquid nitrogen.
After thawing, 500.000 PBMCs were cultured in the presence of NF135.C10 or
NF54 PfRBC at a 1:2 (PBMC:PfRBC) ratio for 24 hours with addition of Brefeldin A
(Sigma-Aldrich) for the last four hours. Cells were harvested and subsequently
stained for viability (Live/Dead fixable dead cell stain kit Aqua, Invitrogen) and
surface markers; either 1) CD4 PE (SK3, BioLegend), CD45RO ECD (UCHL1,
Beckman-Coulter), CD3 PerCP (UCHT1, BioLegend), CD62L PeCy7 (DREG-56,
eBioscience) and CD8a Alexa Fluor 700 (HIT8A, BioLegend) or 2) anti-TCR Pan
γ/δ-PE (IMMU510, Beckman-Coulter), CD45RO ECD, CD3 PerCP, CD4 PECy7
(RPA-T4, BioLegend), Biotin CD56 (HCD56, BioLegend) and CD8a Alexa Fluor 700.
Cells were washed and the second staining panel was incubated with
Streptavidin eFluor450 (eBioscience). After washing, all cells were incubated
with fixation Medium A (Caltag) and subsequently stained with either 1) IFNγ
FITC (4S.B3, eBioscience), TNF Pacific Blue (MAb11, BioLegend), IL-2 APC (MQ1-
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