Functional characterization of Plasmodium berghei PSOP25 during ookinete development and as a malaria transmission-blocking vaccine candidate
Zheng et al. Parasites & Vectors
Functional characterization of Plasmodium berghei PSOP25 during ookinete development and as a malaria transmission-blocking vaccine candidate
Fei Liu 0
Yiwen He 0
Qingyang Liu 0
Gregory B. Humphreys
Yaming Cao 0
0 Department of Immunology, College of Basic Medical Sciences, China Medical University , Shenyang, Liaoning 110001 , China
Background: Plasmodium ookinete surface proteins as post-fertilization target antigens are potential malaria transmission-blocking vaccine (TBV) candidates. Putative secreted ookinete protein 25 (PSOP25) is a highly conserved ookinete surface protein, and has been shown to be a promising novel TBV target. Here, we further investigated the TBV activities of the full-length recombinant PSOP25 (rPSOP25) protein in Plasmodium berghei, and characterized the potential functions of PSOP25 during the P. berghei life-cycle. Methods: We expressed the full-length P. berghei PSOP25 protein in a prokaryotic expression system, and developed polyclonal mouse antisera and a monoclonal antibody (mAb) against the recombinant protein. Indirect immunofluorescence assay (IFA) and Western blot were used to test the specificity of antibodies. The transmission-blocking (TB) activities of antibodies were evaluated by the in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). Finally, the function of PSOP25 during Plasmodium development was studied by deleting the psop25 gene. Results: Both polyclonal mouse antisera and anti-rPSOP25 mAb recognized the PSOP25 proteins in the parasites, and IFA showed the preferential expression of PSOP25 on the surface of zygotes, retorts and mature ookinetes. In vitro, these antibodies significantly inhibited ookinetes formation in an antibody concentration-dependent manner. In DFA, mice immunized with the rPSOP25 and those receiving passive transfer of the anti-rPSOP25 mAb reduced the prevalence of mosquito infection by 31.2 and 26.1%, and oocyst density by 66.3 and 63.3%, respectively. Genetic knockout of the psop25 gene did not have a detectable impact on the asexual growth of P. berghei, but significantly affected the maturation of ookinetes and the formation of midgut oocysts. Conclusions: The full-length rPSOP25 could elicit strong antibody response in mice. Polyclonal and monoclonal antibodies against PSOP25 could effectively block the formation of ookinetes in vitro and transmission of the parasites to mosquitoes. Genetic manipulation study indicated that PSOP25 is required for ookinete maturation in P. berghei. These results support further testing of the PSOP25 orthologs in human malaria parasites as promising TBV candidates.
Plasmodium berghei; PSOP25; Ookinete; Transmission-blocking vaccine
Malaria remains one of the most prevalent tropical
infectious diseases and is endemic in nearly 95 countries
and territories around the world, with estimated > 3.2
billion people being at risk. In 2015, there were
approximately 214 million new malaria cases resulting in
438,000 deaths, ~ 80% of which occurred in Africa .
Currently, due to the spread of insecticide-resistant
mosquitoes and multidrug-resistant parasites, major malaria
control efforts including vector control and
chemotherapy are becoming increasingly ineffective [2–4]. These
trends highlight the need for developing an integrated
malaria control strategy to eliminate malaria
transmission. A transmission-blocking vaccine (TBV) targeting
the sexual stages of the Plasmodium has the potential to
reduce malaria transmission and prevent the spread of
resistant parasites. It is predicted that TBV
administration can reduce child mortality even in areas of high
endemicity . Additionally, TBV can slow down the
spread of mutant parasites, which will prolong the
effective lives of antimalarial drugs and vaccines .
Mathematical models further predict that TBVs will be
an effective tool for malaria elimination .
TBV is designed to target the Plasmodium antigens
expressed during sexual development or Anopheles
midgut proteins that interact with sexual stages and allow
ookinetes to traverse the Anopheles midgut epithelial
cells. Research on TBVs has led to the identification and
experimental validation of several potential TBV
candidates, but only a few including Pfs48/45 [8, 9], Pfs230
[10, 11] and Pfs25  in P. falciparum, and Pvs25 and
Pvs28 in P. vivax , have been found effective in
blocking parasite transmission. Investigations on the two
6-cysteine domain protein family members, Pfs48/45
and Pfs230, have shown that anti-Pfs48/45 monoclonal
and polyclonal antibodies in experimental animals can
effectively inhibit the transmission of P. falciparum to
mosquitoes [9, 14, 15], while Pfs230-raised antibodies are
sufficient to block development of the oocysts and
competent to induce complement-dependent
transmissionblocking (TB) activity . Furthermore, antibodies
against both Pfs48/45 and Pfs230 have been detected in
natural infections, thereby bringing the potential to
boost and/or enhance antibody titers with TBVs against
these antigens . Unlike pre-fertilization proteins,
post-fertilization antigens are expressed solely after the
formation of the zygotes within the mosquito midgut.
Concealed from the host’s immune system, these antigens
have limited diversity among the parasite populations
[17, 18]. The major ookinete surface protein Pfs25 is a
well-characterized 25-kDa glycosyl-phosphatidylinositol
(GPI)-anchored protein with four epidermal growth
factorlike domains. Pfs25 is involved in adhesion of ookinete and
plays an important role in subsequent penetration of the
mosquito midgut [19, 20]. Mouse antiserum against native
Pfs25 , heterologously expressed Pfs25, or the P. vivax
ortholog Pvs25 proteins can effectively inhibit parasite
development in mosquitoes [22–24]. Though Pfs25 and
Pvs25 provide evidence for the efficacy of post-fertilization
antigens in TBVs, more TBV candidate antigens and
higher levels of TB activities are needed for an effective
With efforts for identifying new TBV candidates, we
have recently identified a post-fertilization antigen
PSOP25 (PBANKA_111920) in the rodent parasite
Plasmodium berghei. Psop25 encodes a 350 amino acid (aa)
protein with a signal peptide, and the native protein is
predicted to be 40 kDa. Psop25 transcript is highly
expressed in ookinetes and occupied in the 99th
percentile in the transcriptome of ookinetes .
Ookinetespecific expression of this protein was confirmed in our
previous study . Antisera from mice immunized with
a partial PSOP25 domain (aa 45–245), which included
ten predicted antibody epitopes, inhibited ookinete
formation by 53.0% in in vitro ookinete cultures.
Mosquitoes fed on this partial PSOP25 domain-immunized
mice also resulted in modestly decreased oocyst
prevalence (25.0%) and significantly reduced oocyst densities
(64.3%) , suggesting that PSOP25 could be a new
promising target for TBVs. Here we set out to further
investigate the TBV activities of the full-length PSOP25
protein in P. berghei, and characterize the functions of
PSOP25 by genetic knockout (KO).
Mice, parasites and mosquitoes
Female BALB/c mice (six- to eight-week-old; Beijing
Animal Institute, Beijing, China) were used for all animal
experiments. P. berghei (ANKA strain 2.34) and Δpsop25
lines (psop25 gene knockout line) were maintained in
mice and used for challenge infection. Adult
Anopheles stephensi mosquitoes of the Hor strain were fed
with 10% (w/v) glucose solution and maintained in an
insectary with a surrounding of 50–80% relative
humidity, at 25 °C.
Expression and purification of rPSOP25
For the expression of full-length PSOP25, a psop25
fragment encoding aa 25–350 (excluding the signal
peptide) was amplified from P. berghei genomic DNA
with psop25-F and psop25-R primers (Additional file 1:
Table S1). The psop25 fragment and the prokaryotic
expression vector pET30a (+) (Novagen, Darmstadt,
Germany) were digested with restriction enzymes NdeI
and HindIII, then ligation was performed using the
Ligation High Kit (Toyobo, Osaka, Japan). The
recombinant plasmid was transformed in Escherichia coli BL-21
(Novagen, Darmstadt, Germany) and the His-tagged
recombinant PSOP25 (rPSOP25) was expressed at 20 °C
for 12 h after induction with 1 mM
isopropyl-β-D-thiogalactopyranoside (Sigma-Aldrich, St. Louis, USA). For
protein purification, cultures were harvested and lysed
using binding buffer containing 10 mM imidazole,
300 mM NaCl and 50 mM sodium phosphate (pH 8.0)
and treated by sonication (15 cycles of 20 s pulses
and 30 s intervals). The soluble rPSOP25 was purified
by Ni-NTA His-Bind Superflow (Novagen, Darmstadt,
Germany), according to the manufacturer’s
instructions. Purified rPSOP25 was extensively desalted in
0.1 M phosphate buffered saline (PBS, pH 7.4)
overnight at 4 °C, and then analyzed by SDS-PAGE.
Animal immunization and monoclonal antibody (mAb)
To obtain polyclonal antisera against rPSOP25, a group
of six female BALB/c mice were subcutaneously
immunized with the purified protein (50 μg/mouse) emulsified
in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis,
USA), which has been used to produce high-titer
antibodies . Subsequently, the mice were given two
booster immunizations of 25 μg of rPSOP25 at 3-week
intervals with the rPSOP25 protein emulsified in
incomplete Freund’s adjuvant (Sigma-Aldrich, St. Louis, USA).
A group of negative control mice (n = 6) were
immunized with PBS and same adjuvant formulations. For the
final bleed, mouse blood was collected at 10 days after
the final immunization by cardiac puncture and the
antisera were obtained after the blood had clotted at room
temperature. Antisera from individual mice were mixed
together and used in the subsequent trials.
For monoclonal antibody (mAb) production,
rPSOP25immunized BALB/c mice were obtained as described
above, then the spleen cells of the immunized mice were
extracted and fused with Sp2/0-Ag14 myeloma cells to
produce the anti-PSOP25 mAb . The fused hybridoma
cells were generated using the traditional polyethylene
glycol method, and then selected in the
hypoxanthineaminopterin-thymidine medium. The antibodies were
screened by indirect antibody capture enzyme-linked
immunosorbent assay (ELISA). The IgG fractions were
prepared by ammonium sulfate precipitation, and then
purified on a Protein A column (ThermoFisher Scientific,
Waltham, USA), according to the manufacturer’s
instructions. The mAb isotype was determined by using the SBA
Clonotyping™ System-HRP (Southern Biotechnology
Associates, Birmingham, USA).
with blocking buffer (0.05% Tween 20 in 0.1 M PBS, 1%
bovine serum albumin, pH 7.4) for 2 h at 37 °C. The
plates were then washed twice with PBS-T (0.05%
Tween 20 in 0.1 M PBS, pH 7.4) and incubated with
pooled mouse anti-rPSOP25 sera (1:200 dilution) in
blocking buffer at 37 °C for 2 h. After two washes, the
wells were incubated for 2 h at 37 °C with a 1:5000
dilution of HRP-conjugated goat anti-mouse IgG antibody
(Invitrogen, Waltham, USA). After five final washes,
tetramethyl benzidine (Amresco, Solon, USA) was added
and the reaction was stopped by 2 mM H2SO4. The
absorbance at 490 nm was measured with an ELISA
For estimating the end point titer of immunized mice,
sera from all mice in each immunization and control
group were pooled and diluted from 1:200 to 1:204800
in a blocking buffer and incubated at 37 °C for 2 h. The
end point titers of the total IgG corresponded to the
highest dilution at which the OD490 value was higher
than the cut-off value, which was defined as the mean
of the pooled negative control antisera + 3 × standard
Ookinete enrichment and lysate preparation
The enrichment of ookinetes was referred to a modified
protocol . Briefly, 1.2 mg phenylhydrazine
(SigmaAldrich, St. Louis, USA) in 0.9% NaCl were
intraperitoneally (i.p.) injected into BALB/c mice 3 days before P.
berghei infection. These treated mice were then i.p.
injected with 5 × 106 P. berghei-infected red blood cells
(iRBCs) to initiate the blood-stage infection. Parasitemia
was allowed to reach approximately 1–3% at three days
post-infection (p.i.), when the mice were anesthetized.
After removal of the white blood cells, the infected
blood was diluted 1:10 with the ookinete culture
medium (100 mg/l neomycin, 50 mg/l streptomycin,
50 mg/l penicillin, 20% (v/v) FBS, and 1 mg/l heparin in
RPMI 1640, pH 8.3) in a petri dish and maintained at
19 °C for 24 h. The culture was then diluted in 45 ml of
0.17 M NH4Cl on ice for 10 min to lyse erythrocytes.
After a wash with 0.1 M PBS, ookinetes were separated
on a 62% (v/v) Nycodenz/PBS cushion by centrifugation
(1300× g) for 25 min at 25 °C, treated with 0.15%
saponin and washed once with 0.1 M PBS. The ookinete
lysate was prepared by resuspending the ookinetes in 2%
SDS containing 1% Triton X-100 and 1 × protease
inhibitor cocktail (Roche, Castle Hill, Australia) for 30 min
at room temperature.
Antibody titers to rPSOP25 were determined by ELISA
on day 14, 35 and 52 after the first immunization as
previously described . Briefly, 96-well plates were coated
overnight with purified rPSOP25 at 4 °C, and blocked
The parasite antigens (10 μg) or purified rPSOP25
(500 ng) were subjected to electrophoresis under
reducing or non-reducing conditions using a 10% SDS-PAGE
gel and electro-transferred to PVDF membrane
(BioRad, Hercules, USA). Western blot was performed
essentially as described . Primary antibodies were the
pooled mouse anti-rPSOP25 serum (1:200) or
antirPSOP25 mAb (1:1000), and HRP-conjugated goat
antimouse IgG antibodies (1:10,000) (Invitrogen, Waltham,
USA) were used as the secondary antibodies. Pbs21
mAb (clone 13.1, 1:1000) was included as a positive
control . The sera (1:200) obtained from mice
immunized with the PBS-adjuvant formulations were used as
the negative control. The blot was developed using an
ECL Western Blotting Kit (ThermoFisher Scientific,
Indirect immunofluorescence assay (IFA)
Parasites containing asexual stages, gametocytes, zygotes
and ookinetes of P. berghei were fixed on slides .
Anti-rPSOP25 mAb (1:500) or Pbs21 mAb (clone 13.1,
1:500, positive control) or negative control sera (1:500)
was incubated in 5% skim milk and labeled with
FITCconjugated goat anti-mouse IgG (1:500; Invitrogen,
Waltham, USA) at 37 °C for 1 h. After staining of nuclei
with 4′, 6-diamidino-2-phenylindole (DAPI; Invitrogen,
Waltham, USA), the slides were examined under
Olympus BX53 (Olympus Corporation, Center Valley, USA)
and the images were processed using Adobe Photoshop
(Adobe Systems Inc., San Jose, USA).
Quantification of TB activities
For the in vitro assay, phenylhydrazine pre-treated mice
were infected as described above. On day 3 p.i.,
parasitemia was determined, and 10 μl of blood were taken
from appropriate hosts and added to 90 μl ookinete
culture medium containing anti-rPSOP25 serum or
negative control mouse serum at final dilutions of 1:5, 1:10,
and 1:50. Additionally, anti-rPSOP25 mAb was added to
the ookinete culture at 10, 5 and 1 μg/100 μl
(concentration of mAb was 0.5 μg/μl) of ookinete culture,
respectively. Ookinete cultures were incubated at 19 °C for 24 h
and the ookinete conversion rates were determined as
described previously [26, 32].
For direct mosquito feeding assays (DFA),
experimental mice (n = 3) were immunized with rPSOP25 and
negative control mice (n = 3) were immunized with the
PBS-adjuvant formulations as described above. Ten days
after the final immunization, mice were infected i.p. with
5 × 106 P. berghei ANKA iRBC. For the antibody transfer
experiment, three normal mice were injected
intravenously with 150 μg of anti-rPSOP25 mAb/mouse 1 h
before mosquito feeding. Four-day-old female A. stephensi
mosquitoes (starved for 12 h) were allowed to feed on
rPSOP25 immunized mice or antibody-transferred mice
for 30 min. Engorged mosquitoes were maintained in an
insectary at 21 °C and 70% relative humidity. Ten days
after feeding, ~ 30 mosquitoes were dissected, and
midguts were stained with 0.5% mercurochrome
(SigmaAldrich, St. Louis, USA) to count the number of oocysts
Generation of psop25 KO parasites
To knock out all the protein-coding sequence of the
psop25 gene, the target vector containing an hdhfr
selection cassette was used (kindly provided by plasmoGEM,
vector design ID, PbGEM-042760; http://plasmogem.
sanger.ac.uk/). Before P. berghei transfection, vector
DNA was digested by NotI followed by ethanol
precipitation. The linearized plasmid (10 μg) was electroporated
into purified schizonts using the Nucleofector device as
described previously . After transfection, the
complete parasite suspension was injected intravenously
via the tail vein into mice. Following a 24 h incubation
period, infected mice were treated for 3–4 days with
pyrimethamine (Sigma-Aldrich, St. Louis, USA) via
drinking water (70 μg/ml). Infected blood was collected
and confirmed by integration-specific PCR (Additional
file 1: Table S1). Monoclonal parasite lines were then
obtained by limiting dilution.
Phenotypic analysis of the Δpsop25 line
To study whether deletion of psop25 affected parasite
growth, five phenylhydrazine-treated mice were
inoculated i.p. with either 5 × 106 wildtype (WT) or Δpsop25
iRBCs. For each genotype, blood smears were used to
monitor daily parasitemia, gametocytemia (mature
gametocytes per 100 RBCs), and the gametocyte sex ratio
(female: male ratio) . To quantify male gamete
exflagellation, 10 μl of P. berghei-infected blood collected
from mouse tail vein on day 3 p.i. were added into 90 μl
of ookinete culture medium and incubated for 15 min at
25 °C, and the exflagellation centers were counted as
previously described . At the same time, ookinetes
were cultured in vitro as described above , and the
ookinete conversion rates were determined by IFA using
a Pbs21 mAb . For oocyst quantifications, mice at
3 days p.i. with each parasite line were fed to starved A.
stephensi mosquitoes for 30 min [36, 37]. Ten days after
feeding, ~ 30 fed mosquitoes from each genotype were
dissected for counting of the number of oocysts per
infected mosquito and to determine the prevalence and
intensity of infection.
Parasitemia, gametocytemia, gametocyte sex ratio and
ookinete conversion rates between groups were
analyzed by the Student’s t-test, using GraphPad Prism
software. The prevalence of infection (proportion of
infected mosquito) was analyzed by the Fisher’s exact
test, while the intensity (number of oocysts per
midgut) was analyzed by the Mann-Whitney U-test ,
using SPSS version 17.0. P-values less than 0.05 were
considered statistically significant.
The full-length rPSOP25 is immunogenic
A PSOP25 fragment which corresponded to aa 25–350
excluding the signal peptide was expressed in E. coli.
This fragment included 14 predicted B cell epitopes 
(Additional file 2: Figure S1). The purified rPSOP25
protein had a molecular size of ~ 36.5 kDa from
SDSPAGE analysis, which was consistent with the
predicted size of PSOP25 protein (Fig. 1a). To determine
the immunogenicity of purified rPSOP25, we
performed ELISA using pooled serum generated from
mice immunized with the recombinant protein. The
results showed that immunization with rPSOP25
induced strong antibody responses as compared to the
negative control; the rPSOP25-specific IgG titers
increased continuously during the course of vaccination
(Student’s t-test: t(10) = 35.13, P < 0.0001; Fig. 1b). The
antisera collected 10 days after the final immunization
with rPSOP25 reached a titer of 1:1024000 (Fig. 1c).
Meanwhile, an anti-rPSOP25 mAb was produced from
a selected hybridoma line, which was determined to
be the IgG1 isotype (Additional file 2: Figure S2).
Anti-rPSOP25 antisera and mAb recognize the ookinete
The specificity of the pooled anti-rPSOP25 antisera and
mAb was determined by Western blot against the
rPSOP25 or protein lysate from ookinetes. On Western
blots, both the antisera and mAb detected the 36.5 kDa
rPSOP25 (Fig. 2a) and a band of approximately 40 kDa
in the lysate of purified ookinetes under reducing
conditions (Fig. 2a) and non-reducing conditions (Additional
file 2: Figure S3), which is close to the predicted size of
PSOP25. In a previous study, IFA using antisera raised
against a partial domain of the PSOP25 protein indicated
that PSOP25 is expressed on the surface of ookinetes
. Consistently, IFA with the anti-rPSOP25 mAb
using zygotes, retorts and ookinetes without membrane
permeabilization revealed strong fluorescence of the
parasite body, which agrees with the surface localization
of PSOP25 (Fig. 2b).
Antibodies against PSOP25 show obvious TB activities
Anti-rPSOP25 antisera and mAb were used in ookinete
conversion assay to study the TB activity of the
antibodies against PSOP25. When incubated with pooled
mouse anti-rPSOP25 antisera or mAb, ookinete
conversion was inhibited in a dose-dependent manner. In
Fig. 1 a Mouse immunization and analysis scheme. b rPSOP25 was purified from E. coli and analyzed on a 10% SDS-PAGE gel. c Antibody titers
in immunized mice during immunization, experimental mice were immunized with rPSOP25 formulated in Freund’s adjuvant; control mice were
immunized with only Freund’s adjuvant and 0.1 M PBS. The data represent two separate experiments. Error bar shows mean ± standard deviation
(SD). SD indicates the assay variance. **P < 0.01 (Student’s t-test). d Anti-rPSOP25 total IgG titer at 10 days after the final immunization analyzed by
ELISA. Mean of control antisera + 3 × SD is shown by the broken lines. IgG titers correspond to the last dilution of the anti-rPSOP25 sera where in
OD490 values were above the cut-off values. Cut-off value was defined as that of the pooled sera from control mice. The experiment was performed
three times. Error bar shows mean ± SD. *P < 0.05, **P < 0.01 (Student’s t-test). Abbreviations: M, molecular weight marker; rPSOP25, purified rPSOP25
under reducing conditions
Fig. 2 a Western blot analysis of purified rPSOP25 protein and P. berghei ookinete (Ook) lysates with anti-rPSOP25 sera (PolyAb) and anti-rPSOP25
mAb. Lysates were subjected to electrophoresis under reducing conditions by SDS-PAGE. Pbs21 mAb was used as positive control; a control
mouse serum (PBS) was used as negative control. b IFA was performed on zygote, retort and ookinete at different time points of ookinete culture
using anti-rPSOP25 mAb (green - FITC). Positive control - Pbs21 mAb, negative control - a control mouse serum (PBS). Nuclei were labelled with
DAPI (blue). BF, bright field. Scale-bars: 5 μm
ookinete cultures supplemented with the pooled immune
sera at 1:5, 1:10 and 1:50 dilutions, ookinete conversion
rates were reduced by 62.5% (Student’s t-test: t(4) = 22.52,
P < 0.0001), 47.9% (Student’s t-test: t(4) = 21.44, P <
0.0001), and 22.5% (Student’s t-test: t(4) = 9.11, P = 0.0008),
respectively (Fig. 3a). Compared with the control sera,
ookinete conversion rates in cultures with mAb added at
10, 5 and 1 μg/100 μl were reduced by 71.6% (Student’s
ttest: t(4) = 32.04, P < 0.0001), 60.8% (Student’s t-test: t(4) =
27.84, P < 0.0001) and 32.0% (Student’s t-test: t(4) = 18.60,
P < 0.0001), respectively (Fig. 3a).
To further examine the TB effect of anti-rPSOP25
antibodies in vivo, mice were immunized with rPSOP25
or passively transferred with the mAb and used in DFA.
Ten days after feeding, mosquitoes were dissected and
midgut oocysts were counted. Mosquitoes fed on the
rPSOP25-immunized mice showed a 31.2% reduction in
the prevalence of oocysts, as compared to the control
groups. The mean prevalence was 68.7% in mosquitoes
fed on the rPSOP25-immunized mice, whereas it was
99.9% in mosquitoes fed on the control mice (Fisher’s
exact test: OR = 40.72, 95% CI = 5.38–307.91, P < 0.001;
Fig. 3b, Table 1). Moreover, mosquitoes fed on
rPSOP25-immunized mice revealed a 66.3% reduction in
oocyst density compared to the control group
(MannWhitney U-test: Z = -8.32, P < 0.0001; Fig. 3b, Table 1).
Similarly, mosquitoes fed on the mice passively
transferred with the mAb against PSOP25 had a 26.1%
Fig. 3 a TB activities of anti-rPSOP25 serum and anti-rPSOP25 mAb on P. berghei ookinete formation in vitro. Anti-rPSOP25 serum, anti-rPSOP25 mAb, or
normal mouse serum (control) were diluted at 1:5, 1:10 and 1:50, respectively. Means were representative of three independent experiments. Error bar
shows mean ± SD. ** indicate significant difference compared with the control sera (P < 0.01). # indicate significant difference between anti-rPSOP25
serum and mAb group (P < 0.05), ## P < 0.01 (Student’s t-test). b Direct mosquito feeding assay to assess the TB activity of polyclonal antisera
in rPSOP25-immunized mice (3 mice per group). c Passive antibody transfer experiment to assess the TB activity of the anti-PSOP25 mAb.
For b and c, mosquito midguts were dissected at ten days post-infection, the number of oocysts was counted under a microscopy. The
data are collated from three experiments. The mean number of oocysts and the SEM in each group are shown. **P < 0.01 (Mann-Whitney U-test)
Table 1 Evaluation of TB effect of anti-rPSOP25 serum and mAb in mosquito feeding assays
No. of mosquitoes infected/dissected
Prevalence of infection (%)c
Reduction in prevalence (%)d
Reduction in oocyst intensity (%)g
aTB activity assay was carried out using rPSOP25-immunized mice
bTB activity assay was carried out using BALB/C mice transferred with the anti-PSOP25 mAb
cThe prevalence of infection was calculated by the number of mosquitoes with oocysts/total mosquitoes dissected in each group × 100%
dThe percent reduction of prevalence was calculated as % mean prevalence control – % mean prevalence PSOP25
eMean number of oocysts per mosquito midgut
fStandard error of the mean
gThe percent reduction in oocyst intensity was calculated as (mean oocyst intensity control – mean oocyst intensity PSOP25)/mean oocyst intensity control × 100%
*P < 0.001 for comparisons between the experimental group and the control group
reduction in the prevalence of oocysts (Fisher’s exact
test: OR = 16.46, 95% CI = 3.76–72.13, P < 0.001; Fig. 3b,
Table 1) and a 63.3% reduction in density of oocysts
(Mann-Whitney U-test: Z = -6.97, P < 0.0001; Fig. 3b,
PSOP25 is required for the maturation of ookinetes
To determine the function of PSOP25 during
Plasmodium development, a psop25 gene KO line (Δpsop25)
was generated in P. berghei (Fig. 4a) . Genotypes of
the cloned pyrimethamine-resistant parasites were
confirmed by integration-specific PCR (Fig. 4b). To
determine if psop25 gene knockout led to any deficiencies
in parasite development, we compared parasitemia,
gametocytemia and gametocyte sex ratio between groups
of BALB/c mice infected with 5 × 106 Δpsop25 or WT P.
berghei parasites. Consistent with no expression of the
PSOP25 protein in asexual erythrocytic stages, Δpsop25
had no evident effect on asexual parasitemia (Fig. 5a). In
addition, on day 3 p.i., gametocytemia and gametocyte
sex ratio did not differ significantly between the WT
parasite and the Δpsop25 line (Fig. 5b, c). However,
mean male gamete exflagellation events were slightly but
significantly reduced in the Δpsop25 line, as compared
to the WT parasites (Student’s t-test: t(10) = 4.01, P =
0.0024; Fig. 5d). Furthermore, in vitro ookinete cultures
established from parasites at day 3 p.i. showed that
ookinete conversion rate was significantly reduced in the
Δpsop25 line, although the mature ookinetes in the
Δpsop25 line appeared morphologically normal (data not
shown). Using reverse-transcriptase-PCR, we determined
that there was no psop25 expression in the ookinetes of
Fig. 4 a Schematic representation of the WT locus, the construct used for transfection and the recombined locus with psop25 replaced with the
hdhfr cassette. Primers QCR1, QCR2, GW2, hdhfr1, hdhfr2, GW1 and GT used for diagnostic PCR of the WT locus or knockout are marked. b Lane 1:
primers QCR1 + QCR2 (696 bp) are used for diagnostic PCR of the WT locus. Lanes 2, 3 and 4 are PCR products from primers GW2 + QCR2 (1,002 bp),
hdhfr1 + hdhfr2 (561 bp), GW1 + GT (1,831 bp) for PCR verification of psop25 KO, respectively
Fig. 5 a Average parasitemia was calculated in mice after infection with the wild-type (WT) or Δpsop25 parasites (n = 5). b Gametocytemia in mice
infected with WT or Δpsop25 parasites (n = 3). c Female: male gametocyte ratios of WT or Δpsop25 parasites (n = 3). d Exflagellation of WT and Δpsop25
microgametes (n = 3), *P < 0.05 (Student’s t-test). e Ookinete conversion rates in vitro of WT and Δpsop25 parasites. For c, d and e, characteristic
morphologies of parasites are shown on the right (n = 3), **P < 0.01 (Student’s t-test). f Oocyst number per midgut in mosquitoes 10 days after
feeding on mice infected with the WT and the Δpsop25 parasites. The horizontal bar shows the mean number of oocysts per midgut in mosquito
(± SEM). **P < 0.01 (Mann-Whitney U-test). For a-f, all the data are representative of three separate experiments. Scale-bars: 5 μm
the Δpsop25 line, further confirming that psop25 was
deleted (data not shown). In the WT line, the zygote,
retort, and ookinete conversion rates was 2.0, 8.8 and
87.74%, respectively. Whereas in the Δpsop25 line,
13.3 and 41.0% parasites progressed to the zygote and
retort stages, respectively, further maturation to
ookinetes was reduced by 60.9% (Student’s t-test: t(4) =
31.69, P < 0.0001; Fig. 5e), indicating that PSOP25
might play a crucial role in ookinete maturation. The
oocyst density was reduced to 29.7 per mosquito
midgut in those fed on the Δpsop25 parasites as
compared to 96.9 in WT parasites, reflecting a 69.4%
reduction (Mann-Whitney U-test: Z = -4.25, P < 0.0001;
Fig. 5f, Table 2).
Disrupting the parasite life-cycle to prevent the disease
from being transmitted to other individuals represents a
key component of an integrated malaria control strategy
. Despite investigations on several TBV antigens over
the last 40 years, there is still a need to discover new TBV
vaccine candidates for malaria elimination purpose .
In our previous study, we evaluated the
transmissionblocking activities of a partial 200 aa PSOP25 domain
. Here we expressed the full-length rPSOP25 protein
and raised polyclonal antisera as well as mAb for this
protein, which were found to possess effective TB activities in
an in vitro ookinete formation assay and in vivo DFA.
Antibody concentrations against TBV candidates, as
measured by conventional ELISA, have been shown to
be associated with the effectiveness of TB activities in
mosquito membrane feeding assays [19, 43]. In our
previous study, the 200 aa PSOP25 fragment including ten
predicted antibody epitopes had elicited obvious
antibody responses, and immunized mouse antisera
produced TB activities with 25% reduction in prevalence
Table 2 Oocyst number per midgut in mosquitoes 10 days after feeding on mice infected with the WT and the Δpsop25 parasites
Mean oocyst (IQR)b SEMc P
aThe prevalence of infection was calculated by the number of mosquitoes with oocysts/total mosquitoes dissected in each group × 100%
bIQR, inter-quartile range
cStandard error of the mean
dThe percent reduction in oocyst intensity was calculated as (mean oocyst intensity WT – mean oocyst intensity Δpsop25)/mean oocyst intensity WT × 100%
and 64.3% reduction in oocyst density . The
fulllength PSOP25 is predicted to contain four additional
antibody epitopes, and the full-length rPSOP25 indeed
induced high antibody titers in mice. In parallel
comparison experiments, mosquitoes fed on mice
immunized with the full-length and partial rPSOP25 showed
similar levels (~60%) of reduction in oocyst density as
compared to those fed on control mice. However, there
was a greater degree of reduction in oocyst prevalence in
mosquitoes fed on mice immunized with the full-length
protein (31.2%) as compared to that in mosquitoes fed
on mice immunized with the partial rPSOP25 (25%)
. The TB activities of PSOP25 were comparable to
those of PSOP12 , PSOP7 and PSOP26  in the
reduction of oocyst density and infection prevalence in
DFA. Furthermore, monoclonal antibodies have been a
valuable tool for the characterization of TBVs [45, 46].
Previous studies have explored passive transfer of
transmission blocking mAbs (e.g. Pbs21 mAb clone 13.1) for
TB activities [31, 47]. In this study, an IgGl-type mAb
against PSOP25 significantly inhibited the development
of ookinetes and oocyst when administered through
passive transfer prior to mosquito feeding, and the TB
activities were comparable to those from the full-length
rPSOP25 immunization group. Passive immunization
with TB mAbs may be of additional values as an
intervention in specific circumstances, including malaria
epidemic settings [48, 49].
Transmission-blocking strategies require improved
knowledge of the basic biology of the parasite .
Recent efforts in genomics, transcriptomics and proteomics
have revealed a large number of molecules that may play
key roles in ookinete development [51–53]. Further,
screening for novel vaccine candidates based on gene
KO and phenotypic analysis will undoubtedly yield
valuable information regarding the cell biology of the
ookinetes . Several ookinete proteins which play various
roles in midgut colonization have been characterized,
including the GPI-anchored P25 and P28 proteins [20, 54],
circumsporozoite TRAP-related protein (CTRP) [55, 56],
Plasmodium von Willebrand factor A domain-related
protein (WARP) , secreted ookinete apical protein
(SOAP) [55, 58], and the recently described putative
secreted ookinete proteins (PSOPs) . Here, we
generated a Δpsop25 line, and detected slightly reduced
exflagellation activity of male gametocytes, but significantly
reduced ookinete conversion rate in vitro. Whereas most
Δpsop25 parasites progressed normally to zygote and
retort stages, further maturation to ookinetes was
retarded, which resulted in a 60.9% reduction in the
number of ookinetes as compared to the WT parasite.
This phenotype shows some similarity with that of psop2
knockout parasites, which appeared morphologically
normal but showed reduced in vivo ookinete numbers
. The blockade in ookinete maturation in the
Δpsop25 parasite was further reflected in the reduction
of oocyst density in DFA. The oocysts per mosquito
midgut in those fed on the Δpsop25 parasites was
reduced by 69.4%, like that with the Δpsop9 line .
Given that other PSOP proteins such as PSOP26 showed
ookinete surface localization  and there is a
possibility that these surface proteins interact, it would be
interesting to determine whether psop25 disruption could
affect the expression of other PSOP proteins.
This study confirmed that the full-length recombinant
protein of a newly identified TBV candidate PSOP25
expressed in ookinetes of the rodent parasite P. berghei
could also elicit a strong antibody response in mice.
Both polyclonal mouse antisera and mAb against this
protein recognized the surface of zygotes, retorts and
ookinetes and possessed similar TB activities as the
polyclonal antisera generated against the truncated version
of this protein. Genetic KO study indicated that PSOP25
in P. berghei is required for ookinete formation and
maturation. Collectively, PSOP25 is an excellent TBV
candidate targeting the post-fertilization stages, and further
assessment of TB activities in P. falciparum and P. vivax
Additional file 1: Table S1. Primer information and sequences.
(DOCX 15 kb)
Additional file 2: Figure S1. Predicted B cell epitopes of the PSOP25
protein (http://tools.iedb.org/bcell). Below is the protein domain architecture
of PSOP25 with signal peptide highlighted in red, low complexity in pink, and
transmembrane region in blue. Figure S2. The isotype of anti-rPSOP25 mAb
was identified by ELISA using by the SBA Clonotyping™ System-HRP.
The data represent two separate experiments. Error bar shows mean
+ standard deviation. Figure S3. Western blot analysis of P. berghei
ookinete lysates with anti-rPSOP25 sera (PolyAb) and anti-rPSOP25
mAb. Lysates were subjected to electrophoresis under non-reducing
conditions by SDS-PAGE. Pbs21 mAb was used as positive control; a
control mouse serum (PBS) was used as negative control. (ZIP 2413 kb)
CTRP: Circumsporozoite TRAP-related protein; ELISA: Enzyme-linked
immunosorbent assay; GPI: Glycosyl-phosphatidylinositol; i.p.: Intraperitoneally;
IFA: Indirect immunofluorescence assay; iRBCs: Infected red blood cells;
KO: Knockout; mAb: Monoclonal antibody; PBS: Phosphate-buffered saline;
PBS-T: 0.05% Tween 20 in phosphate-buffered saline; PSOPs: Putative secreted
ookinete proteins; RBCs: Red blood cells; SOAP: Secreted ookinete apical
protein; TBS-T: 0.1% Tween 20 in Tris-buffered saline; TBV: Transmission-blocking
vaccine; WARP: von Willebrand factor A domain-related protein; WT: Wildtype
We are grateful to Ms. Jun Liu for technical support and to Dr. Hiroyuki Matsuoka
for providing the Pbs21 mAb clone 13.1. We thank plasmoGEM for kindly
providing the target vector PbGEM-042760 (http://plasmogem.sanger.ac.uk/).
Availability of data and materials
The data supporting the conclusions of this article are included within the article.
YC and LC conceived the study and helped draft the manuscript. EL, GBH and TT
helped with the bioinformatics analysis and drafted the manuscript. WZ carried
out the rPSOP25 protein expression, mAb development, TB activity studies of
PSOP25 and drafted the manuscript. FL and YH carried out function studies by
genetic knockout, statistical analysis. QL and QF participated in the antibodies
specificity detection and statistical analysis. All authors contributed to the writing
of the manuscript, read and approved the final manuscript.
Ethics approval and consent to participate
Animal use was carried out according to the guidelines of the animal ethics
committee of China Medical University.
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