Experimental infection and protection against ... - TI Pharma

178 Chapter 9
followed by five identical doses at 8, 24, 36, 48 and 60 hours) and followed
closely for three days. Complete cure was confirmed by thick blood smears. All
volunteers still negative by day 21 post-challenge were presumptively treated
with artemether/lumefantrine.
Haematological and biochemical parameters were determined in routine fashion
at the hospital’s central clinical laboratory. Nucleic Acid Sequence Base
Amplification (NASBA) and real-time QT-PCR to determine the densities of P.
falciparum parasites have been described in detail [15,16]. Chloroquine levels
were measured by liquid chromatography as previously described [17,18].
Minimum therapeutic concentrations for plasma chloroquine levels maintained
by the laboratory were 30 µg/l, in accordance with Rombo et al. [14].
Immunological analysis
Venous whole blood was collected into CPT vacutainers (Becton and Dickinson,
Basel) prior to first immunization and again prior to challenge. Plasma was
collected and stored at -70°C. Peripheral Blood Mononuclear Cells [PBMCs)
were isolated by density gradient centrifugation, frozen down in 10% DMSO/FCS
and stored in liquid nitrogen. Antibody titres were assessed by ELISA and
immunofluorescence assay according standard protocols as previously described
[19-21]. Cellular responses to cryopreserved asexual parasites were assessed by
24-hour in vitro PBMC cell stimulation assays as previously described [22],
followed by intracellular cytokine staining (Fix & Perm kit, Caltag Laboratories)
and flow-cytometry. A more detailed description of these immunological assays
is provided in the web-only supplementary methods.
Statistical analysis
Flowcytometric analysis was performed using CellQuest and data analyzed in
SPSS; differences in responses within volunteers between time points and
between vera and control volunteers were analyzed by non-parametric tests
(Wilcoxon and Mann-Whitney-U, respectively), two-sided p-values