Experimental infection and protection against ... - TI Pharma

Long-term protection against malaria after experimental sporozoite inoculation 199
21 after re-challenge presumptively received the same curative treatment.
Complete cure was always confirmed by the occurrence of two consecutive
parasite-negative blood-slides.
Outcomes
The primary outcome of the study was microscopic detection of parasites in a
blood-slide, with sampling performed twice daily on days 5 and 6 post-challenge,
thrice daily on days 7-11, twice daily on days 12-15 and once on days 16-21 postchallenge.
Thick blood smears were made from 15µl of EDTA-anti-coagulated
blood, spread over the standardised surface of one well of a 3-well glass slide
(CEL-LINE Diagnostic Microscope Slides, 30-12A-black-CE24). After drying, wells
were stained with Giemsa for 45 minutes. Slides were read at 1000x
magnification by assessing 200 high-power fields, equal to approximately 0.5µl
of blood. The smear was considered positive if two unambiguously identifiable
parasites were found. The pre-patent period was defined as the period between
challenge and first positive blood smear. Additionally, parasitemia was
measured by real-time quantitative PCR (Q-PCR), performed retrospectively on
all samples collected after challenge, as previously described [14].
Secondary outcomes comprised immunological parameters. After preparation of
thick blood smears, remaining plasma was collected and stored at -70°C.
Antibody titres on the day prior to challenge were assessed by ELISA as
previously described [9]. Plasma concentrations of IL-6 and IFNγ were measured
every other day using the Bio-Plex system (Bio-Rad) [15].
For cellular immunology, venous whole blood was collected into cell-preparation
tubes (Becton and Dickinson, Basel) on the day before challenge. Peripheral
blood mononuclear cells (PBMC) were isolated by density gradient
centrifugation, frozen in fetal-calf serum containing 10% dimethylsulfoxide and
stored in liquid nitrogen. Cellular responses against cryopreserved NF54
sporozoite and NF54 asexual-stage parasites were assessed by 24-hour in vitro
stimulation as previously described for the asexual-stage [9] followed by
intracellular cytokine staining.
Preparation of sporozoite- and asexual-stage parasites for immunological assays
NF54 strain P. falciparum sporozoites (PfSpz) were obtained from Anopheles
stephensi mosquitoes that were reared according to standard procedures in our
insectary. Infected mosquitoes were obtained by feeding on gametocytecontaining
cultures of NF54 strain P. falciparum, as described previously [15]. On
day 21–28 after infection, the salivary glands of the mosquitoes were collected