Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice

Abstract

Malaria kills >1 million people each year, in particular in sub-Saharan Africa. Although asexual forms are directly responsible for disease and death, sexual stages account for the transmission of Plasmodium parasites from human to the mosquito vector and therefore the spread of the parasite in the population. Development of a malaria vaccine is urgently needed to reduce morbidity and mortality. Vaccines against sexual stages of Plasmodium falciparum are meant to decrease the force of transmission and consequently reduce malaria burden. Pfs48/45 is specifically expressed in sexual stages and is a well established transmission-blocking (TB) vaccine candidate. However, production of correctly folded recombinant Pfs48/45 protein with display of its TB epitopes has been a major challenge. Here, we show the production of a properly folded Pfs48/45 C-terminal fragment by simultaneous coexpression with four periplasmic folding catalysts in Escherichia coli. This C-terminal fragment fused to maltose binding protein was produced at medium scale with >90% purity and a stability over at least a 9-month period. It induces uniform and high antibody titers in mice and elicits functional TB antibodies in standard membrane feeding assays in 90% of the immunized mice. Our data provide a clear perspective on the clinical development of a Pfs48/45-based TB malaria vaccine.

Full text

Correctly folded Pfs48/45 protein of
Plasmodium
falciparum
elicits malaria transmission-blocking
immunity in mice
Nikolay S. Outchkourov*
†
, Will Roeffen
‡
, Anita Kaan*, Josephine Jansen*, Adrian Luty
‡
, Danielle Schuiffel
‡
,
Geert Jan van Gemert
‡
, Marga van de Vegte-Bolmer
‡
, Robert W. Sauerwein
‡
, and Hendrik G. Stunnenberg*
§
Departments of *Molecular Biology and ‡Medical Microbiology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center,
P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands
Communicated by Louis H. Miller, National Institutes of Health, Rockville, MD, January 16, 2008 (received for review December 21, 2007)
Malaria kills >1 million people each year, in particular in sub-
Saharan Africa. Although asexual forms are directly responsible for
disease and death, sexual stages account for the transmission of
Plasmodium parasites from human to the mosquito vector and
therefore the spread of the parasite in the population. Develop-
ment of a malaria vaccine is urgently needed to reduce morbidity
and mortality. Vaccines against sexual stages of Plasmodium fal-
ciparum are meant to decrease the force of transmission and
consequently reduce malaria burden. Pfs48/45 is specifically ex-
pressed in sexual stages and is a well established transmission-
blocking (TB) vaccine candidate. However, production of correctly
folded recombinant Pfs48/45 protein with display of its TB epitopes
has been a major challenge. Here, we show the production of a
properly folded Pfs48/45 C-terminal fragment by simultaneous
coexpression with four periplasmic folding catalysts in Escherichia
coli. This C-terminal fragment fused to maltose binding protein
was produced at medium scale with >90% purity and a stability
over at least a 9-month period. It induces uniform and high
antibody titers in mice and elicits functional TB antibodies in
standard membrane feeding assays in 90% of the immunized mice.
Our data provide a clear perspective on the clinical development of
a Pfs48/45-based TB malaria vaccine.
Malaria parasites are spread in the population by Plasmo-
dium-infected Anopheles mosquitoes. Successful transmis-
sion of malarial parasites from humans to mosquitoes depends
on the presence and infectiousness of gametocytes in the pe-
ripheral blood and the number of Anopheles mosquitoes in the
area. Transmission of Plasmodium falciparum can be blocked
inside the mosquito by antibodies that have been ingested
together with the gametocytes as part of a blood meal, inter-
rupting the sporogonic cycle inside the mosquito (1).
Pfs48/45 is a transmission-blocking (TB) target protein ex-
pressed by gametocytes (2–4) and present on the surface of the
sporogonic (macrogametes) stages of the malaria parasites.
Pfs48/45 plays a key role in parasite fertilization (5) and anti-
bodies that exclusively target conformational epitopes of
Pfs48/45 protein prevent fertilization (6, 7). Furthermore, anti-
Pfs48/45 antibodies are present in human sera from endemic
areas (8) and correlate with TB activity (8 –10). The induction of
antibodies after natural infection as observed in the field creates
the highly beneficial potential of vaccine boosting in the endemic
setting. TB vaccines might be applied alone or more likely as part
of a combination vaccine or package of control measures
depending on the intensity of malaria transmission (11).
A strategy for vaccine development requires the production of
correctly folded recombinant Pfs48/45 protein. Proper folding of
many cysteine-rich proteins, including Pfs48/45, depends on
correct formation of disulphide bridges. In eukar yotes the
oxidizing environment of the endoplasmic reticulum (ER) pro-
vides a milieu for disulphide bonds formation. Plasmodium
parasites are one of the few eukaryotes that lack the N-linked
glycosylation machinery, and many Plasmodium proteins contain
multiple potential glycosylation sites that are aberrantly glyco-
sylated when expressed in any of the available eukaryotic hosts.
On the other hand, prokaryotic expression systems such as
Escherichia coli, which lacks N-glycosylation also lack the so-
phisticated ER machinery of disulphide bond formation. In E.
coli correct disulphide bonds are formed in the periplasmic space
and catalyzed by a set of periplasmic oxidoreductases, termed
Dsb (12, 13). These proteins function in two separate pathways:
(i) oxidation by DsbA/DsbB, responsible for introducing S-S
bonds, and (ii) reduction and isomerization of aberrant disulfide
bonds by DsbC/DsbD. Previous studies (12) have shown that
overproduction of the enzymes DsbA and DsbC greatly improve
proper disulfide bond formation in cysteine-rich proteins.
Another well known rate-limiting step of the folding of
proteins in vivo is the cis/trans isomerization of prolyl-
iminopeptide bonds that is catalyzed by peptidyl-prolyl cis/
transisomerases (PPIases). The actions of PPIases such as FkpA
and SurA have already been shown to improve the production of
recombinant proteins in the periplasm of E. coli. Previously, the
genes for the oxidoreductase, PPIase, and the general chaperone
activities of DsbA, DsbC, FkpA, and SurA have been combined
on a expression plasmid called pTUM4 (14). Coexpression of the
periplasmic folding catalysts was shown to improve the folding of
two recombinant proteins carrying several disulfide bonds and
showing poor folding efficiency in the periplasm of E. coli.
In this study, we investigated the effect of coexpression of the
four periplasmic folding catalysts on the folding, yield, and
immunogenicity of the recombinant Pfs48/45 protein and frag-
ments thereof. Our results demonstrate that the yield of recom-
binant Pfs48/45 protein is significantly improved as compared
with the recombinant 10C as described (15). A Pfs48/45 fragment
of 10C cysteines retained the highest stability in terms of
conformation and resistance to proteases and elicited high titers
of functional TB antibodies. Our data provide an efficient
production and rapid purification of properly folded Pfs48/45–
10C and a clear perspective on the clinical development of a
Pfs48/45-based TB malaria vaccine.
Results and Discussion
Effect of Folding Catalysts on the Yield of Correctly Folded Pfs48/45.
We investigated the effect of coexpression of the protein folding
catalysts DsbA, DsbC, FkpA, and SurA on the yield and con-
Author contributions: N.S.O. and W.R. contributed equally to this work; N.S.O., W.R.,
R.W.S., and H.G.S. designed research; N.S.O., W.R., A.K., J.J., D.S., G.J.v.G., M.v.d.V.-B., and
R.W.S. performed research; N.S.O., W.R., A.L., R.W.S., and H.G.S. analyzed data; and N.S.O.,
W.R., R.W.S., and H.G.S. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
†Present address: Department of Physiological Chemistry, University Medical Center, 3584
CG, Utrecht, The Netherlands.
§To whom correspondence should be addressed. E-mail: h.stunnenberg@ncmls.ru.nl.
© 2008 by The National Academy of Sciences of the USA
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formation of recombinant Pfs48/45 protein and fragments
thereof in the periplasm of E. coli. Previous unpublished results
had indicated that Pfs48/45 fused to the PelB leader peptide
could not be detected in E. coli periplasm and there was little
effect of the coexpression of chaperones. To attain periplasmic
localization Pfs48/45 full-length (16C) or C-terminal (10C)
(residues 26– 428 and 159 –428, respectively) were fused to a
periplasmic maltose binding protein (MBP) as a carrier mole-
cule. As shown in Fig. 1B, both proteins M-Pfs16C and M-Pfs10C
were detected in the periplasmic extracts of E. coli at low levels.
Thus, MBP was an efficient vehicle in targeting Pfs48/45 to the
periplasm. Furthermore, pTUM4 encoded periplasmic chaper-
ones accumulated at high levels in the periplasmic fraction and
had a profound effect of at least 10-fold enhancement on protein
accumulation and epitope recognition of both M-Pfs16C and
M-Pfs10C. Note that in addition to the full-length M-Pfs16C and
M-Pfs10C we observed a degradation product with apparent
mobility of ⬇43–45 kDa (⬇43 kDa in the case of M-Pfs16C and
⬇45 kDa for the M-Pfs10C) that reacted to the MBP antibody
(data not shown) in the Coomassie-stained SDS/PAGE. Thus
the Pfs48/45 part of the MBP fusion degraded rapidly in the E.
coli periplasm, and the protease-resistant MBP part accumulated
as a prominent product. Coexpression of the chaperones in-
creased significantly the amount of full-length M-Pfs16C and
M-Pfs10C with an concomitant increase of reactivity with the
conformation-dependent mAb (Fig. 1B). Apparently, proper
folding of the Pfs48/45 protein is essential for its stability and
accumulation.
Purification and Primary Characterization of M-Pfs16C and M-Pfs10C.
Both M-Pfs16C and M-Pfs10C were extracted from the E. coli
periplasm and purified on a DEAE FF column. Although the
M-Pfs10C remained soluble after purification, the M-Pfs16C
showed a strong tendency to aggregate upon storage for 1 week
at 4°C especially at protein concentrations of ⬇0.25 mg/ml or
more (Fig. 2A). Further purification of the M-Pfs16C and
M-Pfs10C on Superdex 75 yielded ⬎95% pure protein prepa-
rations as judged from a nonreduced SDS/PAGE (Figs. 2 Aand
3A). In a two-site capture ELISA, purified M-Pfs10C showed
similar epitope recognition as native gametocyte-derived
Pfs48/45 protein (Fig. 3 Band C), whereas the recognition of the
M-Pfs16C was much weaker (Fig. 2 Band C). Different com-
binations of Pfs48/45-specific mAbs for capture and detection
gave similar results (data not shown). Thus, we obtained a ⬎90%
properly folded homogeneous M-Pfs10C preparation and a less
well folded M-Pfs16C preparation. Because of poor solubility
and much weaker epitope recognition of the M-Pfs16C prepa-
rations, we focused for further analysis mainly at the M-Pfs10C
protein preparation. The final yield of this correctly folded
M-Pfs10C protein was ⬇1 mg/liter. Purified M-Pfs10C protein
was stable at 4°C for at least 9 months, i.e., repetitive confor-
mation-dependent two-site capture ELISA and mobility on a
nonreduced SDS/PAGE yielded nearly identical results (data
not shown). A second batch of M-Pfs10C was produced with
similar yields and ELISA values.
Immunogenicity of Recombinant M-Pfs10C. Immunogenicity of two
independently produced batches of the purified M-Pfs10C pro-
tein with ⬎90% proper folding was assessed in BALB/c mice.
A
Domain III
(ep. I)
Domain II
(ep. IIb and III)
Domain I
(ep. V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
10C
16C
Domain III
(ep. I)
Domain II
(ep. IIb and III)
Domain I
(ep. V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
10C
16C
Domain III
(ep. I)
Domain II
(ep. IIb and III)
Domain I
(ep. V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
10C
16C
Domain III
(ep. I)
Domain II
(ep. IIb and III)
Domain I
(ep. V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
10C
16C
Domain III
(ep. I)
Domain II
(ep. IIb and III)
Domain I
(ep. V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
10C
16C
Domain III
(ep. I)
Domain II
(ep. IIb and III)
Domain I
(ep. V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
10C
16C
mAb IIb
mAb III
mAb V
mAb I
Coomassie
M-Pfs16C, pTUM4
M-Pfs16C
M-Pfs10C,pTUM4
M-Pfs10C
16C
10C
B
82
64
49
37
26
19
**
KDa
Fig. 1. Effect of chaperones expression from the pTUM4 plasmid on the
MBP-fused Pfs48/45 full-length (M-Pfs16C) and 10C fragment (M-Pfs10C). (A)
A schematic representation of the Pfs48/45 protein with three putative do-
mains recognized by different TB mAb as described (15). The coding sequence
contains a total of 448 aa. Bars indicate the relative position of cysteine
residues. (B) Periplasmic E. coli extracts were screened with mAbs against
epitope I, IIb, III, and V or Coomassie blue. Arrows indicate the position of
M-Pfs16C (16C) and M-Pfs10C (10C) proteins. Samples were mixed with SDS
sample buffer without reducing agent and separated on SDS/PAGE. Note that
the blot with the mAb against epitope I was overexposed to show the
expression in the lanes without chaperones.
A C
B
0
0.5
1
1.5
2
2.5
3
0.1 1 10 100 1000
ng Pfs 48/45 prote in or M-Pfs 16C / we ll
OD 450 nm
M-Pf s 1 6C
gct
0
0.5
1
1.5
2
2.5
3
0.1 1 10 100 1000
ng Pfs48/45 protein or M -Pfs16c / well
OD 450 nm
M-Pf s 1 6C
gct
1 2 3 4
KDa
82
64
49
37
26
19
Fig. 2. Purification and characterization of M-Pfs16C. (A) Coomassie-stained SDS/PAGE analysis after DEAE column purification of the M-Pfs16C protein. Lane
1, aggregated insoluble M-Pfs16C after 1 week at 4°C; lane 2, broad-range protein marker; lane 3, remaining soluble M-Pfs16C; lane 4, soluble M-Pfs16C purified
over Superdex 75 column. (Band C) Two-site ELISA experiment using rat mAbs against epitopes III for capture and peroxidase-conjugated mAb against epitope
I(B) or IIb (C) for detection. Gametocyte extracts (gct) with known concentration of the Pfs48/45 protein were used as a positive control.
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Fig. 4 shows that serum of M-Pfs10C (batch 1)-immunized mice
showed antibody reactivity in an ELISA using whole gametocyte
extract with increasing titers after each subsequent boost. Re-
activity against the M-Pfs10C was already apparent after the first
immunization. In the final bleed (S5), the M-Pfs10C fragment
induced titers of 1/165,000 (SD ⫽1/101,143; range 1/30,000 to
1/300,000) when tested in the Gametocyte-ELISA using en-
riched gametocyte extracts and up to 1/1,000,000 in the
M-Pfs10C ELISA. Control sera from the two protein batches
were negative in the Gametocyte-ELISA. Sera tested in an
ELISA using MBP gave similar results as compared with the
M-Pfs10C ELISA. Sera from the immunization experiment with
batch 2 of M-Pfs10C yielded similar results (data not shown).
The ability of the antiserum to recognize native sexual stage
protein was further assessed by immunofluorescence assays
(IFAs) of air-dried sexual stage parasites and live intact mac-
rogametes/zygotes in suspension IFA (SIFA). All sera reacted
specifically with the antigen present in air-dried gametocytes
(Fig. 5A) and on the surface of live intact gametes and zygotes
of P. falciparum (Fig. 5C) but not against Pfs48/45 knockout
parasites (Fig. 5B) or asexual stage parasites.
To test for recognition of TB epitopes, M-Pfs10C immune sera
(batches 1 and 2) were used in a two-site competition ELISA
with a fixed amount of peroxide-conjugated rat anti-epitope I
and III mAbs for binding to native Pfs48/45 (Fig. 6 and data not
shown).Sera from batch 1 competed effectively for epitope I and
III of Pfs48/45 at serum dilutions from 1/20 to 1/160. Ten of 12
sera of M-Pfs10C-immunized mice competed ⬎50% with con-
jugated anti-epitope I and III mAbs, whereas no competition was
found with adjuvant-immunized control sera or preimmune sera.
Competition with nonconjugated anti-epitope I or III Pfs48/45
mAb was used as positive control and for quantification (data not
A
C
B
0
0.5
1
1.5
2
2.5
3
0.1 1 10 100 1000
ng Pfs48/45 protein or M-Pfs10C/well
OD 450 nm
M-Pf s 1 0 C
gct
0
0.5
1
1.5
2
2.5
3
0.1 1 10 100 1000
ng Pfs48/45 protein or M -Pfs10C/well
OD 450 nm
M-Pf s 1 0 C
gct
KDa
82
64
49
37
26
19
Fig. 3. Immunocharacterization of M-Pfs10C. (A) Coomassie-stained SDS/PAGE analysis of the purified M-Pfs10C protein. (Band C) Two-site ELISA experiment
using rat mAbs against epitopes III for capture and peroxidase-conjugated mAb against epitope I (B) or IIb (C) for detection. Gametocyte extracts with known
concentration of the Pfs48/45 protein were used as a positive control.
Parasite M-Pfs10C
Fig. 4. Immunogenicity of sera from mice immunized with M-Pfs10C. Anti-
body reactivity of sera from six mice immunized with recombinant M-Pfs10C
from the first immunization experiment in the gametocyte-ELISA (Left) and
M-Pfs10C ELISA (Right). S0, S1, S3, and S5 indicate preimmune sera, day 14, day
56, and day 98 of the time course of the immunization, respectively; see
Materials and Methods. Boxes extend from the 25th and 75th percentiles with
the median value of the six mice sera.
B
B
A
C
Fig. 5. IFA and SIFA analysis of sera from mice immunized with M-Pfs10C. (A
and B) Immunofluorescence microscopy with anti-M-Pfs10C mouse sera on P.
falciparum gametocytes air-dried on a multispot slide (IFA) with wild-type
parasites (A) and Pfs48/45 knockout parasites (B). (C) SIFA using live intact
macrogametes/zygotes. (Magnification: ⫻400.)
Outchkourov et al. PNAS
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March 18, 2008
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MEDICAL SCIENCES

shown). Estimates based on unlabeled mAb added to the assay
revealed concentrations of anti-epitope I and anti-epitope III
antibodies in the sera of the M-Pfs10C-immunized mice between
20 and 100
␮
g/ml, which is in the range in which TB activity can
be expected (15). Briefly, a fixed amount of labeled mAb
competed for Pfs48/45 binding with either test serum or a known
concentration range of the same but unlabeled mAb. An esti-
mate of anti-epitope-specific antibodies in the test serum was
made based on comparison of OD values between test serum and
labeled mAb.
TB Activity Induced by M-Pfs10C. Individual sera of mice immu-
nized with M-Pfs10C (batches 1 and 2) after bleed 5 (S5) were
analyzed for TB activity in the standard membrane-feeding assay
(SMFA). Table 1 shows a near-complete blockade of transmis-
sion in 11/12 sera at a standard dilution of 1:10 (Mann–Whitney
test P⬍0.0001). Sera of control mice immunized with PBS and
MBP alone showed no reducing activity in the SMFA. Finally, a
clear correlation was observed between TB activity of the sera
from M-Pfs10C-immunized mice including results obtained with
a recombinant 10C fragment without the MBP fusion (15) and
the percentage of competition in the epitope I competition
ELISA (r
2
⫽0.873) (Fig. 7). The curve shows that ⬎90%
inhibition in the SMFA correlated with ⬎40% competition for
epitope I.
Concluding Remarks. Our data show that an N-terminally trun-
cated Pfs48/45 protein fused to MBP, coined M-Pfs10C, and
coexpressed with four periplasmic folding catalysts in the
periplasm of E. coli yielded a properly folded homogeneous
Pfs48/45 protein that is stable over for at least 9 months. The two
independent batches of M-Pfs10C protein elicited functional TB
antibodies (transmission reducing activity ⬎90%) in ⬎90% of
the mice.
Clinical development of malaria vaccines has accelerated over
recent years with a clear increase in the portfolio of candidates.
Although most efforts are concentrated on preeythrocytic and
blood-stage vaccines, progress in TB vaccine development has
lagged behind and was basically limited to two postfertilization
proteins, P25 and P28 (16).
From a biological perspective and supported by experimental
data, Pfs48/45 has been for a long time considered as an
attractive prefertilization target for inhibition of sporogonic
development. However, technological constraints related to
protein folding have hampered its development for many years.
The high TB activity of the C-terminal fragment of Pfs48/45
described here provides a solid basis for the clinical development
of a prefertilization TB malaria vaccine.
Materials and Methods
Construct Preparation. A synthetic Pfs48/45 gene optimized for expression in
E. coli (GenBank accession no. EU366251) encoding wild-type Pfs48/45 protein
(17) was designed and used throughout this study. Fragments corresponding
to processed Pfs48/45 protein residues 26–428 (16C) and N-terminally trun-
0.1
20.1
40.1
60.1
80.1
5 2.5 1.25 0.61
% v/v dilution of the mice sera
Percentage Competition
Fig. 6. Two-site competition ELISAs. mAb against epitope IIb (85RF45.2b)
was used to capture Pfs48/45 molecule from gametocyte extract. Percentage
competition of sera from mice immunized with the M-Pfs10C protein (batch
1) was calculated by using a fixed amount of peroxidase-conjugated mAb
anti-epitope I (Ep I) or anti-epitope III (Ep III) at serum dilutions [ranging from
1/20 to 1/160 (5–0.62% vol/vol)]. Data are presented as mean percentage
competition ⫾1 SD of the six mice.
Table 1. TB activity of sera from mice immunized with M-Pfs10C
Sera AM, mean no. TN, nTBA, %
Immunization batch 1
1A1 0 0 100
1A2 0.05 1 99.6
1A3 0.30 6 97.4
1A4 0 0 100
1A5 0.75 15 94.5
1A6 0 0 100
Control 13.6 263
Immunization batch 2
2A1 30.7 614 27.1
2A2 0 0 100
2A3 5.1 91 88.0
2A4 0.05 1 99.9
2A5 0.15 3 99.6
2A6 0 0 100
Control 42 843
Sera (1:10 diluted) were tested in the SMFA. Seven days after membrane
feed, the number of infected mosquitoes and oocyst density were deter-
mined. Sera, mouse serum from M-Pfs10C-immunized group; Control, mean
of five feeders with serum from control immunization; AM, arrhythmic mean
oocyst number in 20 dissected mosquitoes; TN, total number of oocysts in 20
dissected mosquitoes; TBA, percentage of TB activity (19).
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
0.0 20.0 40.0 60.0 80.0 100.0
Percentage competition against epitope I
Percentage inhibition of oocysts
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
0.0 20.0 40.0 60.0 80.0 100.0
Fig. 7. TB activity correlates with the concentration of the anti-epitope I
antibodies. Percentage inhibition of oocysts are related to the percentage
competition of the anti-epitope I antibodies in the sera from mice after five
immunizations with the rec10C (
䊐
) (15) and M-Pfs10C (}) protein as calculated
by using a fixed amount of peroxidase-conjugated mAb anti-epitope I
(85RF45.1) at a dilution of 1/20 of the serum. The line represents the regression
of results by use of a polygon equation (y⫽⫺1E ⫺06x5⫹0.0003x4⫺0.0322x3
⫹1.2884x2⫺17.369x⫹43.997; Rvalue of 0.93).
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cated (10C) residues 159–428 were cloned into pMAL-p2x (New England
Biolabs), resulting in N-terminal MBP fusions. The pTUM4 plasmid has been
described (14).
Expression and Purification of M-Pfs10C. Expression and preparations of
periplasmic fractions were performed essentially as described (14). The re-
combinant proteins were purified on a DEAE FF (Amersham Biosciences)
column equilibrated in 20 mM Tris䡠HCl, pH 8.6. The column was washed with
50 mM NaCl, and bound proteins were eluted with a linear gradient of 50 –400
mM NaCl in the same buffer. Fractions containing the M-Pfs10C protein as
analyzed by Coomassie staining and ELISA were concentrated on a Vivaspin
20, 30-kDa molecular mass cut-off utrafiltration unit (Vivascience). M-Pfs10C
protein was further purified over a Superdex 75 HR 10/30 column (Amersham
Biosciences) in PBS that contained 0.01% N-dodecyl-N-N-dimethyl-3-
ammonio-1-propane sulfonate.
Parasites. Mature P. falciparum gametocytes (NF54 strain) were produced in
an automated static culture system as described (7, 18), isolated (19), and
stored at ⫺70°C until used. NF54 gametocytes were extracted in 25 mM
Tris䡠HCl (pH 8.0) supplemented with 150 mM NaCl, 1.0% sodium desoxy-
cholate, and 1 mM phenylmethylsulphonyl fluoride. Insoluble debris was
pelleted by centrifugation (13,000 ⫻gfor 5 min at room temperature); the
supernatant provided antigen for Western blot analysis and ELISAs.
Antibodies. To detect epitopes of Pfs48/45, various mAbs of mouse origin, 32F5
(epitope I) and 32F1 (epitope IIb) (6, 20), or rat origin, 85RF45.1 (epitope I),
85RF45.2b (epitope IIb), 85RF45.3 (epitope III), and 85RF45.5 (epitope V) (7,
19), were used. HRP-conjugated anti-mouse IgG was purchased from Dako
(P0161). Alexa Fluor488 anti-mouse IgG (A21200) was purchased from Molec-
ular Probes.
Immunization of BALB/c Mice. Groups (n⫽6) of female BALB/c mice were
immunized s.c. with 50
␮
g of recombinant M-Pfs10C fragment per mouse or
with adjuvant alone (control, n⫽5) as described (15). Briefly, mice were
immunized with a total volume of 0.1 ml per mouse emulsified in complete
Freund’s adjuvant on day 1 and boosted with the same amount of antigen on
21, 42, 63, and 84 days with incomplete Freund’s adjuvant. Blood was taken on
days 0 (preimmune serum), 14, 35, 56, 77, and 98 and tested for specific
antibody reactivity and TB activity in the SMFA.
IFA. An indirect IFA was done with cultured sexual-stage parasites (NF54
isolate of P. falciparum) air-dried on multispot slides as described (7, 8). Briefly,
parasites were incubated with a 1:100 dilution of the test sera in PBS, rinsed
with PBS, and incubated with Alexa-conjugated goat-anti-mouse Ig (IgG). The
cells were examined for clear sexual-stage parasite-specific green fluores-
cence. For SIFA analysis, gametocytes were allowed to undergo gametogen-
esis as described (7, 8, 19), and the cells were examined as above under IFA.
ELISAs. All incubation steps, except antigen coating, were done at room
temperature.
Detection of Pfs48/45. Pfs48/45-specific ELISA was performed by a two-site
ELISA as described (7, 8, 15). Briefly, 96-well microtiter plates were coated with
rat mAbs recognizing particular epitopes of Pfs48/45 (19). After blocking with
5% milk in PBS, the antigen (starting with 250,000 parasites per well or 20
␮
g/ml M-Pfs10C) was captured by incubation in a serial dilution with PBS, and
the bound antigen was detected by HRP-conjugated rat mAbs.
Gametocyte and M-Pfs10C ELISA. Serum antibody analysis was conducted by
ELISA with whole gametocyte extracts or M-Pfs10C ELISA as described (15).
Briefly, ELISA plates were coated with enriched gametocyte antigen extract
(250,000 parasites per well) or 2
␮
g/ml M-Pfs10C diluted in PBS, stored at 4°C
overnight, and then blocked with 5% skimmed milk/PBS. Diluted sera were
added and incubated for 2 h, and bound antibodies were detected by HRP-
conjugated goat anti-Mouse IgG.
Competition ELISA. The competition ELISA was performed by a two-site ELISA
as described (7, 8, 15, 19). Briefly, gametocyte extract was captured essentially
as for the Pfs48/45-specific ELISA by using mAb 85RF45.2b. Competition of
HRP-labeled mAb 85RF45.1 or 85RF.45.3 with sera antibodies from M-Pfs10C-
immunized mice was performed by incubation of 30
␮
l of HRP-labeled mAb
(2.5
␮
g/ml, diluted with PBS containing 1% milk) and 30
␮
l of mice serum
(dilutions ranging from 1/20 to 1/160) or 30
␮
l of unlabeled mAb (5-fold serially
diluted started at 5
␮
g/ml) for 2 h.
TB Assay. Antisera obtained from mice immunized with the M-Pfs10C frag-
ment were tested for their TB activities in a SMFA as described (15, 21, 22).
Briefly, 27
␮
l of the mice sera was mixed with 63
␮
lofnaı¨ve human serum and
180
␮
lofin vitro gametocyte culture of P. falciparum (NF54 line). This mixture
was fed to Anopheles stephensi (Nijmegen strain) mosquitoes through a
membrane feeding apparatus, and 7 days later at least 20 mosquitoes (⬎90%
survival) were dissected and oocysts were counted from extracted midguts of
test and control mosquitoes.
ACKNOWLEDGMENTS. We thank Dr. Arne Skerra (Technische Universitat,
Munich) for pTUM4 plasmid, Karina Teelen for technical assistance, and
Rumyana Karlova for stimulating discussions. This work was supported by
European Malaria Vaccine Initiative Grant 041222 and BioMalPar.
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Outchkourov et al. PNAS
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vol. 105
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no. 11
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MEDICAL SCIENCES