Antibodies to Plasmodium falciparum merozoite surface protein-1p19 malaria vaccine candidate induce antibody-dependent respiratory burst in human neutrophils
Study sites, subjects and ethics statement
This study is part of a longitudinal study conducted in Dielmo and Ndiop, two Senegalese
villages with perennial and seasonal transmission, respectively. The sites, population
endemicity and the longitudinal surveys carried out have been described previously
23], 24]. In July 2002, 119 Dielmo and 114 Ndiop villagers were enrolled in a longitudinal
and cross-sectional study. At the time of recruitment, no villagers were symptomatic
for malaria. The mean age of the Ndiop and Dielmo cohorts was 25.3 years (range 3.4–80.5)
and 21.8Â years (range 3.9-76.9), respectively; the distribution in the different age
groups is shown in Table 1. Blood samples were collected by venous puncture, and sera were stored at ?20 °C.
Table 1. Antibody responses against MSP1p19 in Dielmo and Ndiop villagers tested for ADRB
The project protocol and objectives were carefully explained to the assembled villagers,
and informed consent, annually renewed, was obtained individually from all subjects
either by signature or by thumbprint on a voluntary consent form written in both French
and in the local language (Wolof and Serere) 24]. This study was examined and approved by the Senegalese National Health Research
Ethics Committee.
Urban samples were from hospitalized adults with confirmed severe malaria, living
in the unstable hypo-endemic urban area of Dakar. They were treated at Hôpital Principal,
Dakar. Samples were collected day 0 of hospitalization after their use for routine
biological investigations. This study was approved by ad hoc Ethics Committee and informed consent was obtained from all participants.
Antigens and antibodies
The soluble recombinant protein corresponding to PfMSP1p19 was produced in the baculovirus/insect
cell expression system in High Five (Invitrogen) insect cells. The construct has a
C-terminal hexa-histidine tag that replaces the GPI-modification signal sequence of
the parasite protein. Recombinant PfMSP1p19 was purified by metallo-affinity chromatography,
as described previously 25].
A hyper-immune serum pool (HIS) from 30 immune, primarily adult residents of Dielmo
(mean age 36 years, range 9–73 years), and non-immune serum pool (NIS) obtained commercially
(Calbiotech, France), were the positive and negative controls, respectively.
ELISA analysis
The ELISA protocol used to measure PfMSP1p19 antibodies at a 1:200 serum dilution
was essentially as described 9], 26], 27], using baculovirus PfMSP1p19 coated on Immulon-4 plates (Dynatech) at 0.5 µg mL
?1
. For inter-assay comparisons, results were expressed as OD-ratios corresponding to
OD-sample/OD-naïve. Positive responders (PR) were individuals with an OD-ratio over
2, corresponding to the mean OD of naïve controls + 2SD. High responders (HR) were
individuals with an OD-ratio 7, i.e. the threshold level previously shown to be significantly
associated with anti-parasite activity in re-infection study in Ndiop 26].
To monitor specific antibody depletions quantitatively, samples were analysed at 1:200,
1:400 and 1:800 dilutions, and an arbitrary titre was extrapolated using a four-parameter
logistic fit from a standardized positive control regression curve on each plate,
determined using the HIS pool and serial twofold dilutions starting at 1:200 28], 29].
Serum depletion
Sera from high responder individuals to PfMSP1p19 were selected for the depletion
studies. Each serum (100 µL) was diluted 1:3 in PBS and incubated with 50 µg of recombinant
hexa-histidine tagged PfMSP1p19 protein for 30 min at RT to allow antigen–antibody
binding. Packed TALON Metal Affinity Resin (Ozyme) pre-equilibrated with PBS (200 µL),
was added and incubated with gentle mixing for 3Â h at room temperature (RT), to allow
antigen–antibody complex binding via the C-terminal hexa-histidine tag. Depleted sera
were recovered in the supernatant after centrifugation without further dilution, so
that initial and depleted sera were directly comparable. Effective depletion was checked
by ELISA.
Parasite culture and merozoite preparation
Plasmodium falciparum parasites (PAM, an FCR3-like background) and P. falciparum D10 (D10-PfM3?) or transgenic D10 merozoites, in which PfMSP1p19 is replaced by the
non-cross reactive Plasmodium chabaudi orthologue, PcMSP1p19 (also called PcMEGF) 21], 30] were maintained in continuous culture on O
+
erythrocytes in RPMI supplemented with 0.5 % Albumax and 1 ?g mL
?1
gentamycin, in candle jars 31]. Merozoites were collected as described previously 19] from cultures with greater than 5 % parasitaemia after centrifugation 5 min at 400×g, to remove red blood cells (RBCs), followed by a second centrifugation of the supernatant
for 20 min at 1500×g.
PMN preparation
PMNs were prepared as described previously 19]. Briefly, blood samples from six to seven healthy donors were collected into EDTA-K3
tubes, layered onto Ficoll-Histopaque (density 1.077, Sigma) and centrifuged at RT
for 30 min at 400×g. PMNs were harvested at the Ficoll-RBC interface and residual RBCs were lysed by
incubation in 8.32 g L
?1
NH
4
Cl, 0.8 g L
?1
sodium bicarbonate, and 0.043 g L
?1
EDTA for 8 min at 4 °C. PMNs were washed twice with Hank’s balanced salt solution
(HBSS), enumerated using Trypan blue, and resuspended in PBS at 1–5 × 10
7
cells mL
?1
.
Chemiluminescence monitoring and determination of standardized ADRB index
Chemiluminescence was measured as described previously 19] using opaque 96-well plates (Berthold), and a MicroLumat Plus 96 luminometer (Berthold).
Briefly, merozoite pellets (40 µL) were incubated with 10 µL of test or control sera
for at least 30 min at 37 °C. PMN (100 µL at 1–5 × 10
7
cells mL
?1
) and isoluminol (100 µL of 1:100 dilution in PBS of 4 mg mL
?1
stock in DMSO) were loaded rapidly using an Eppendorf multipipette 4780. To facilitate
rapid handling, only 40–50 wells per plate were used using the HIS as systematic internal
control in the first and last wells. Plate reading started immediately, and continued
for 1Â h.
Data are presented as standardized activity index of merozoite ADRB calculated as
where rlu maximum HIS is an average of the first and last wells on the plate. Only
experiments in which the rlu maximum HIS was ?100 (?6 × background), were included
in the analyses. An additional internal control with the same positive serum was included
in each run.
Chemiluminescence assay using antigen coated on plates
Baculovirus PfMSP1p19 was coated on white Nunc opaque Maxisorp plates (Dynatech) at
1 µg/mL overnight at 4 °C. Plates were then washed three times with PBS-Tween-0.05 %
and blocked for 1Â h with PBS-BSA2Â % before a second wash. Native (undecomplemented)
sera diluted 1:5 in PBS were then added and incubated for 1 h at 37 °C. PMN and isoluminol
were then added as for classical ADRB (see above) 19], 22], before reading in the MicroLumat Plus 96 luminometer (Berthold).
Statistical analysis of ADRB assay data
ELISA and/or ADRB data were analysed for statistical significance using the Wilcoxon
signed rank test and the Spearman rank correlation test for non-normally distributed
data, and P values 0.05 were considered significant. Multiple regression analysis including
age of individuals, Ab response to PfMSP1p19 and ADRB was done using R software.