INFECTION AND IMMUNITY, Nov. 2007, p. 5500–5508
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 11
Protective Properties and Surface Localization of
Plasmodium falciparum Enolase?
Ipsita Pal-Bhowmick,1Monika Mehta,1Isabelle Coppens,2Shobhona Sharma,1* and Gotam K. Jarori1
Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005,
India,1and Johns Hopkins Bloomberg School of Public Health, Malaria Research Institute, Baltimore, Maryland2
Received 17 April 2007/Returned for modification 20 June 2007/Accepted 15 August 2007
The enolase protein of the human malarial parasite Plasmodium falciparum has recently been characterized.
Apart from its glycolytic function, enolase has also been shown to possess antigenic properties and to be
present on the cell wall of certain invasive organisms, such as Candida albicans. In order to assess whether
enolase of P. falciparum is also antigenic, sera from residents of a region of Eastern India where malaria is
endemic were tested against the recombinant P. falciparum enolase (r-Pfen) protein. About 96% of immune
adult sera samples reacted with r-Pfen over and above the seronegative controls. Rabbit anti-r-Pfen antibodies
inhibited the growth of in vitro cultures of P. falciparum. Mice immunized with r-Pfen showed protection
against a challenge with the 17XL lethal strain of the mouse malarial parasite Plasmodium yoelii. The
antibodies raised against r-Pfen were specific for Plasmodium and did not react to the host tissues. Immuno-
fluorescence as well as electron microscopic examinations revealed localization of the enolase protein on the
merozoite cell surface. These observations establish malaria enolase to be a potential protective antigen.
Malaria continues to be a life-threatening infectious disease
in the tropical world. Despite tremendous efforts to control the
malaria epidemic, current prophylaxis and drug treatments are
proving insufficient. The extensive spreading of drug-resistant
Plasmodium strains as well as insecticide-resistant mosquitoes
makes it urgent to develop an effective malaria vaccine. Long
years of antigen identification and characterization have
yielded many potential vaccine candidates, but developing an
effective malaria vaccine has remained an incredibly difficult
challenge (17). It has been observed that immunity to the
disease develops gradually, after many attacks and over many
years, in adults living in areas where malaria is endemic (2).
The successful passive transfer of this immunity by injecting
antibodies from malaria-immune persons to children suscepti-
ble to malaria has demonstrated that antibodies alone can
trigger protection (5, 9, 58). These experiments have worked
across geographical borders, as immunoglobulin G (IgG) from
malaria-immune West Africans have cured East Africans as
well as Thai malaria patients (5). The nature of this immunity
is poorly understood at the molecular level. However, attempts
have been made to identify antigens, the humoral response
against which leads to protection. Seroepidemiological studies
have identified several specific malarial blood-stage antigens
including ring-infected erythrocyte surface antigen (10), apical
membrane antigen (53), and PfP0, a conserved ribosomal pro-
tein (7, 18, 24), as protective antigens.
Enolase has been reported to be present on the cell surface
of several organisms (38). It is also considered to be a major
immunostimulatory protein in the case of visceral leishmania-
sis (19). Enolase has been demonstrated to play a protective
role in Candida albicans infection (31, 41, 55). Recently, it has
also been identified as a potential vaccine candidate in Chla-
mydia pneumoniae infection (13). The single enolase gene of
Plasmodium falciparum has been reported to possess certain
plant-like features (42). We have recently expressed the re-
combinant P. falciparum enolase protein (r-Pfen) and studied
its enzymatic properties (36). There has been one report re-
garding the presence of anti-enolase antibodies among malaria
patients (45). Therefore, we decided to examine the immuno-
genic and protective properties of Pfen. In this paper, we
report the prevalence of anti-enolase antibodies in sera of
immune adults resident in regions of India where malaria is
endemic, the surface localization of enolase protein on the
merozoites, the growth-inhibitory properties of anti-enolase
antibodies, and the protective capacity of Pfen in mice chal-
lenged with the lethal 17XL strain of the mouse malaria par-
asite Plasmodium yoelii.
MATERIALS AND METHODS
Human serum samples. Human serum samples were collected from healthy
adult residents and symptomatic children living in areas of the Phulbani district,
Orissa, Eastern India, where P. falciparum is endemic, as described earlier (24).
The criteria used for the set of healthy adults were the following: (i) permanent
residency of the area, (ii) established record of suffering from symptomatic
malaria earlier in childhood, and (iii) lack of clinical symptoms of malaria for a
minimum of the previous 3 years. These parameters were determined through an
extensive questionnaire and also from records of the primary health care center
(24). From this sample set, 24 samples were selected and tested for reactivity with
r-Pfen protein. The sex ratio of the adult sample set was matched (11 samples
from males and 13 from females), and samples represented adults 16 to 65 years
old. Most of the adult samples (67%) were from people 25 to 50 years old. This
distribution reflected the whole collection and was similar to a profile presented
earlier (24). Six serum samples from symptomatic children were collected from
the same area. The age range of the children was 2 to 7 years, and the samples
were matched for the sex ratio (3 from males and 3 from females). Upon
examination of thick blood smears, 3 out of 24 (12.5%) adult samples and 3 out
of 6 (50%) samples from children showed the presence of P. falciparum rings and
gametocytes. The samples were collected with appropriate ethical clearance after
obtaining subject consent. Approximately 0.1 to 1.0 ml of blood was collected
using heparinized capillaries or tubes, and plasma samples were prepared. As
* Corresponding author. Mailing address: Department of Biological
Sciences, TIFR, Homi Babha Road, Colaba, Mumbai 400 005, India.
Phone: 91 22 2278 2625. Fax: 91 22 2280 4610. E-mail: sharma@tifr
?Published ahead of print on 4 September 2007.
seronegative controls, 34 Caucasian serum samples (kind gifts from Chris King,
United States, and Pierre Druilhe, France) were used.
r-Pfen protein preparation and ELISA. A His-tagged fusion r-Pfen protein
was generated using the vector pQE30 (QIAGEN, Hilden, Germany), induced
and purified from Escherichia coli using protocols described earlier (36). About
200 ng of antigen was used to coat microtiter plates (Nunc, Roskilde, Denmark),
as reported earlier (24), for probing with human serum from Orissa. For com-
paring the reactivity of rabbit anti-r-Pfen antiserum against r-Pfen and rabbit
muscle enolase (RMen), both of these proteins were coated in equimolar quan-
tities (100 ?l of 0.03 ?M). After blocking, the samples were treated with human
serum (1:250 dilution) or rabbit serum samples at dilutions of 1:100 and 1:1,000
in phosphate-buffered saline (PBS). An enzyme-linked immunosorbent assay
(ELISA) was performed using horseradish peroxidase-conjugated anti-human
and anti-mouse IgG at a 1:2,000 dilution.
Maintenance of Plasmodium and parasite protein preparation. Asexual stages
of P. falciparum strain 3D7 parasites were cultured in vitro as described earlier
(18). Briefly, the parasites were maintained at 5% hematocrit in complete RPMI
medium (RPMI medium plus 0.5% albumax) (GIBCO BRL, NY) at 37°C. The
cultures were grown in the presence of a calibrated gas mixture (5% CO2, 2% O2,
with the balance N2) in sealed flasks. P. yoelii strain 17XL was maintained by
passaging asexual stages through Swiss mice by intraperitoneal injections. Para-
sitemia was monitored by making periodic peripheral thin smears stained with
Giemsa (Sigma Chemical Co., St. Louis, MO) or Field stain (Biolab Diagnostics,
Parasite protein extraction. P. falciparum culture was allowed to reach ?5 to
7% parasitemia; cultures were then harvested and washed with incomplete
RPMI solution. For P. yoelii parasite extract, mice were infected with P. yoelii
strain 17XL and were allowed to reach 20 to 40% parasitemia. At this stage, 1 to
2 ml of blood was collected in equal volumes of anticoagulant containing 136 mM
glucose, 42 mM citric acid, and 75 mM sodium citrate. Red blood cells (RBCs)
were pelleted and washed with PBS. Infected erythrocytes from both types of
parasites were then treated with 0.05% saponin (Sigma Chemical Co., St. Louis,
MO) for 10 min at 4°C to release the parasites from the host erythrocyte
membrane (20). The parasite pellets were then sonicated in PBS containing 1%
Triton X-100 and cocktail protease inhibitor (Roche Applied Science, Indianap-
olis, IN). The supernatant was used for sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and immunoblot analysis.
Protein extraction from lymphocytes and liver. Human blood was collected
with the anticoagulant EDTA, and the lymphocytes were separated by centrif-
ugation at 600 ? g for 15 min at 4°C on a Histopaque 1077 density gradient
(Sigma Diagnostics, Inc.). The buffy coat was used as the source of human
leukocytes. Crude protein extract of the leukocytes was obtained by lysing the
cells in a buffer containing 1% Triton X-100 in PBS in the presence of pepstatin
(1 ?g/ml) and leupeptin (1 ?g/ml) (Sigma Chemical Co., St. Louis, MO). For
liver protein extraction, liver was dissected from the mouse, and the tissue was
homogenized in the presence of pepstatin (1 ?g/ml) and leupeptin (1 ?g/ml) and
centrifuged. Supernatants from both the preparations were used for SDS-PAGE
and immunoblot analysis.
Growth inhibition assay. Asexual stages of P. falciparum 3D7 parasites were
cultured in vitro as described earlier (6). An invasion-blocking assay was per-
formed in triplicate in 96-well sterile tissue culture plates (Nunc, Roskilde,
Denmark) in a total culture volume of 200 ?l. P. falciparum culture, synchronized
by repeated sorbitol treatment at a parasitemia level of 1 to 2% with 5%
hematocrit, was incubated with different preparations and dilutions of rabbit
anti-enolase antibodies. Two rabbits were immunized with r-Pfen as described
earlier (36), and the sera were pooled for this assay. Rabbit antiserum was used
at dilutions of 1:200 and 1:500, and the preimmune antiserum was used at a
dilution of 1:100. IgG fractions were also purified from immunized rabbits and
their preimmune sera using protein A columns and were used at a final concen-
tration of 1 mg/ml each. RBC smears were made at different time points (24, 48,
and 60 h postsynchronization) and stained with Giemsa (Sigma Chemical Co., St.
Louis, MO), and parasitemia was measured by microscopic counting. All the
treatments were done in triplicate, and at least three sets of 2,000 RBCs were
counted for each treatment at every time point. Results of the growth inhibition
assay were statistically analyzed using one-way analysis of variance (GraphPad
InStat, San Diego, CA). The means were considered statistically significantly
different if the P values were ?0.05.
Indirect IFA. An immunofluorescence assay (IFA) was performed at room
temperature with the air-dried blood smears. The slides were fixed with 4%
formaldehyde in PBS for 10 min, washed five times, permeabilized with 0.25%
Triton X-100 in PBS for 10 min, washed, fixed with 3% bovine serum albumin-
PBS for 45 min, and incubated for 1 h with various antibodies. Anti-r-Pfen
antiserum was used at a dilution of 1:200. AlexaFluor 568-conjugated anti-rabbit
IgG and AlexaFluor 488-conjugated anti-mouse IgG (Molecular Probes, NJ)
were used as secondary antibodies at dilutions of 1:500. All antibody dilutions
were made in 1% bovine serum albumin-PBS. Parasite nuclei were stained with
4?,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, NJ) at a final concen-
tration of 1 ?g/ml.
Vaccination studies with r-Pfen in mice. Experiments were performed on
three groups of 8-week-old male Swiss mice (five mice per group). Mice were
injected intraperitoneally with r-Pfen emulsified in Freund’s adjuvant at 21-day
intervals (the first injection was 100 ?g of r-Pfen in complete Freund’s adjuvant,
followed by 50 ?g for the two boosters in incomplete Freund’s adjuvant). In one
control group, mice were injected in parallel with a recombinant Drosophila
odorant binding protein OSF (as an irrelevant His-tagged protein control) emul-
sified in complete Freund’s adjuvant. The other control group received no in-
jections. After three immunizations, the antibody titers against r-Pfen were
monitored. Mice having anti-r-Pfen antibody titers greater than 1:300,000 were
then challenged with the lethal strain of P. yoelii (strain 17XL; 106parasites per
mouse), and parasitemia was monitored daily. Results of the parasitemia profiles
on days 4, 5, and 6 were statistically analyzed using one-way analysis of variance
(GraphPad InStat, San Diego, CA).
Solution IFA. Intact merozoites for the IFA were prepared as described
previously (7). Mice were infected with P. yoelii strain 17XL and were allowed to
develop to ?50 to 60% parasitemia, with the majority of parasites in segmented
schizont stage. Infected blood was incubated at room temperature for 5 to 6 h in
RPMI 1640 medium with 10% albumax. The liberated merozoites were har-
vested at 4°C, washed, and resuspended in complete RPMI 1640 medium (?107
merozoites/ml). All the subsequent steps were also carried out on ice. Merozoites
were incubated with rabbit anti-enolase antiserum (1:50 dilution in complete
RPMI 1640 medium) for 30 min on ice. These samples were then washed three
times with incomplete RPMI medium, resuspended at a 1:500 dilution of Alex-
aFluor 488-conjugated anti-rabbit IgG for 30 min in complete RPMI 1640 me-
dium, and washed again seven to eight times. Parasite nuclei were stained with
DAPI at a final concentration of 1 ?g/ml. Merozoites were washed and mounted
under glass coverslips in 5 ?l of Vectashield mounting medium (Vector Labo-
ratories, CA) on glass slides. The slides were observed under a Nikon microscope
using a 100? phase-contrast objective.
For trypan blue staining of the merozoites, 0.1 ml of merozoites (?105cells
per ml) was suspended in incomplete RPMI medium and stained with 20 ?l of
0.4% trypan blue (Sigma Chemical Co., St. Louis, MO). The suspension was
mixed thoroughly and allowed to stand for 5 min at room temperature. These
cells were then observed under a microscope.
Immunoelectron microscopy. Preparations of P. falciparum-containing RBCs
and human leukocytes were fixed in 4% paraformaldehyde (Electron Microscopy
Sciences, PA) in 0.25 M HEPES (pH 7.4) for 1 h at room temperature and then
in 8% paraformaldehyde in the same buffer overnight at 4°C. Cells were infil-
trated, frozen, and sectioned as described previously (14). The sections were
immunolabeled with mouse anti-r-Pfen antibodies (1:100 in PBS–1% fish skin
gelatin) and then with anti-mouse IgG antibodies, followed directly by 15-nm
protein A gold particles (Department of Cell Biology, Medical School, Utrecht
University, The Netherlands) before examination with a Philips CM120 electron
microscope (Eindhoven, The Netherlands) under 80 kV.
A His-tagged r-Pfen fusion protein was generated using pro-
tocols described earlier (36). An ELISA was carried out with
r-Pfen as the substrate, using serum from adults and children
from Orissa, Eastern India, along with control serum samples
from Caucasians who had not been exposed to malaria. The
histogram presented in Fig. 1 shows the individual reactivity of
24 serum samples from malaria-immune adults. The average
values of the reactivity of the 34 serum samples from Cauca-
sians and the 6 serum samples from children were 0.078 ?
0.007 and 0.088 ? 0.019, respectively. Using the Caucasian
serum average reactivity plus 3 standard deviations (SDs) as
the cutoff value, it was observed that 96% of immune adults
showed reactivity to r-Pfen protein. Of the six samples from
children from Orissa, none showed reactivity above the cutoff
value of the average of the Caucasian sera plus 3 SDs (data not
shown). These results demonstrated that anti-enolase antibod-
VOL. 75, 2007ENOLASE AS A PROTECTIVE ANTIGEN AGAINST MALARIA5501
ies were prevalent among malaria-immune adults resident in
Immunoblotting, growth inhibition assays, and indirect
IFAs. In order to test whether antibodies against parasite eno-
lase can interfere with parasite growth, it was decided to test
the specificity of the antibodies raised in rabbits against r-Pfen.
Whole-cell extracts prepared from P. falciparum and from hu-
man erythrocytes and purified r-Pfen were subjected to SDS-
PAGE and blotted on a membrane, and the blot was treated
with rabbit anti-r-Pfen antibodies (Fig. 2A). The rabbit anti-
serum did not react with human erythrocyte proteins although
it recognized the recombinant 50-kDa r-Pfen protein. Among
the proteins in the whole-cell extract from P. falciparum, the
antiserum recognized a single protein with a molecular mass of
?49 kDa (Fig. 2A). The size matches well with the expected
size of Pfen (48.6 kDa). To test the specificity of the serum to
native protein, an ELISA was carried out using r-Pfen and
commercially available rabbit enolase protein (Fig. 2B). The
Coomassie-stained SDS gel shows that the Plasmodium and
mammalian enolase proteins used for the ELISA were pure
proteins (Fig. 2B). The ELISA and immunoblotting data es-
tablished the specificity of the rabbit anti-r-Pfen antibodies
A growth inhibition assay was then performed using the
asexual stages of P. falciparum parasites (Fig. 2C). The syn-
chronized cultures were examined through development of the
trophozoite (around 24 h), schizont (around 48 h), and subse-
quent ring (?48 h) stages in the presence of anti-r-Pfen anti-
bodies. No significant difference in parasitemia levels was
observed during the trophozoite and schizont stages. For in-
stance, the culture treated with purified anti-r-Pfen IgG
showed parasitemia levels of 1.25% ? 0.07% and 1.39% ?
0.09% versus the control parasitemia levels of 1.62% ? 0.30%
and 1.61% ? 0.24% at 24 and 48 h, respectively. However,
significant differences were observed at 60 h in the numbers of
freshly infected ring stages in the presence of anti-r-Pfen an-
tibodies (Fig. 2C). While the percent parasitemia with anti-
r-Pfen IgG remained 1.24% ? 0.11%, the control reached
FIG. 1. Reactivity of r-Pfen (200 ng) with 24 serum samples col-
lected from immune adults resident in the Phulbani district of Orissa
where malaria is endemic. The horizontal line represents the cutoff
determined by the mean reactivity plus 3 SDs determined from 34
normal Caucasian serum samples. OD, optical density.
FIG. 2. (A) SDS-gel electrophoresis of ?100 ?g of cell lysates from
P. falciparum and noninfected RBCs (lanes 1 and 3) and ?5 ?g of
purified r-Pfen protein (lane 2) was followed by Coomassie blue stain-
ing (a) and immunoblotting using rabbit anti-enolase antisera (1:800
dilution) (b). (B) ELISA. Reactivity of equimolar amounts (100 ?l of
30 nM solutions) of r-Pfen and RMen, checked with rabbit anti-r-Pfen
antiserum at 1:100 and 1:1,000 dilutions. Inset shows the SDS-PAGE
analysis and Coomassie blue staining of 10 ?l of 30 ?M stock solutions
of r-Pfen (P) and RMen (R), which were used for the ELISAs. OD,
optical density. (C) Effect of polyclonal rabbit antibodies on in vitro
growth of synchronized cultures of P. falciparum. Synchronized cul-
tures were treated with rabbit anti-r-Pfen antiserum at dilutions of
1:200 and 1:500 and with rabbit preimmune (Pre Imm) antiserum at a
1:100 dilution. IgG purified from rabbit anti-r-Pfen and preimmune
serum was used at a final concentration of 1 mg/ml each. Parasitemia
is shown as a percentage of the untreated control.**, P ? 0.01;***,
P ? 0.001. (D) IFA of the schizont stage of P. falciparum using DAPI
(a), rabbit anti-r-Pfen antiserum (1:200) with secondary anti-rabbit
IgG conjugated with AlexaFluor 568 (b), and mouse anti-MSP-1 an-
tibodies (1:100) with secondary anti-mouse IgG conjugated with Alex-
aFluor 488 (c). An overlay of the images from frames a, b, and c is
shown in frame d.
5502 PAL-BHOWMICK ET AL.INFECT. IMMUN.
2.63% ? 0.30%, showing about 50% inhibition with purified
IgG. The amount of purified IgGs required for 50% inhibition
was much greater than that of serum at a 1:200 dilution. This
suggests that other classes of Igs may also contribute to growth
inhibition. In our attempts to raise monoclonal antibodies
against Pfen, we observed that several of the parent clones
secreted the IgM class of antibodies (37). These results dem-
onstrate that the antibodies against Pfen inhibit the growth of
the parasite. Since the inhibition was observed specifically at
the ring stage, it can be surmised that the antibodies interfered
with the invasion of merozoites into fresh red cells.
These results also indicate that enolase is likely to be ex-
pressed on the surface of P. falciparum merozoites. Therefore,
we studied the localization of enolase in mature segmented
schizonts of P. falciparum along with a known merozoite sur-
face marker, merozoite surface protein 1 (MSP-1) (Fig. 2D).
An indirect IFA showed that in addition to the cytoplasmic
presence of enolase, the typical beehive pattern of a surface
protein was also observed when P. falciparum schizonts were
stained with anti-enolase antibodies, and there was significant
colocalization of enolase with MSP-1 protein (Fig. 2D). These
results support the view that enolase is localized on the surface
of P. falciparum merozoites.
Vaccination studies with r-Pfen in mice. Next, we wanted to
evaluate the ability of the Pfen to protect mice against a chal-
lenge with mouse malaria parasites. Since P. falciparum and P.
yoelii enolases share 90% sequence homology and since anti-
bodies to r-Pfen cross-react with P. yoelii enolase in all the
erythrocytic stages of the parasite (37), we examined the effect
of immunization of mice with r-Pfen, followed by a challenge
with lethal P. yoelii strain 17XL. Figure 3A to C show the
parasitemia profile of each mouse from the three groups over
a period of 15 days after the P. yoelii challenge. All the control
mice and the mice immunized with the irrelevant His-tagged
protein developed a high degree of parasitemia (?17% on
average) by day 4 postchallenge (Fig. 3A and B), whereas
r-Pfen-immunized mice showed ?1% parasitemia at that time
point (Fig. 3C and D). Figure 3D shows the average para-
FIG. 3. Vaccination study using r-Pfen as an immunogen. Groups of five mice each were either nonimmunized (Œ) (A), or immunized with an
irrelevant His-tagged protein (Drosophila odorant binding protein OSF) (F) (B) or r-Pfen (f) (C). The parasitemia profile of each mouse is shown
in panels A to C. The average parasitemia and survival pattern for each group of mice are shown in panels D and E.
VOL. 75, 2007 ENOLASE AS A PROTECTIVE ANTIGEN AGAINST MALARIA5503
sitemia values for each treatment. The highest average para-
sitemia values were 70% and 40% for nonimmunized mice and
mice injected with irrelevant His-tagged protein, respectively.
However, among the mice immunized with r-Pfen, there was
significant delay in the increase in parasitemia, and the highest
average parasitemia was about 20% on day 8 postchallenge.
The averages of these groups were compared using one-way
analysis of variance, which showed that the mice immunized
with enolase were significantly protected (P ? 0.01) for days 4,
5, and 6, while there were no significant differences seen be-
tween the control and the group immunized with irrelevant
His-tagged protein (P ? 0.05). Figure 3E shows the survival
profile of the mice. The immunized mice also had a signifi-
cantly longer survival period. The reduction in parasitemia was
found to correlate with the antibody titer present in each
mouse. The sera from mice immunized with the His-tagged
OSF protein showed no cross-reactivity to the His-tagged r-
Pfen (data not shown), indicating that the protection was spe-
cific for r-Pfen. These results establish partial protection of the
mice upon vaccination with the Plasmodium enolase protein. It
is documented that combinations of different parasite strains
with different mouse strains give different susceptibility pat-
terns of malaria (47) and that the protection depends on not
only the immunity to a specific antigen but also various other
factors such as diet (1). Our results with enolase are similar to
those obtained with other malaria candidate antigens and P.
yoelii lethal strain combinations (7, 32, 59).
Specificity of enolase as an antigen and surface localization.
In order to use Plasmodium enolase as a vaccine candidate, the
immune response has to be specific for the antigen, and the
enolase protein must be present on the surface of P. yoelii
merozoites. The specificity of the antibodies raised in r-Pfen-
immunized mice was examined against P. yoelii and P. falcip-
arum as well as murine and human cells (Fig. 4). An immuno-
blot assay showed specific recognition of the serum with both
P. falciparum and P. yoelii proteins at the expected molecular
sizes of 48.6 and 50.3 kDa (Fig. 4, top panel, b). Neither the
murine liver and leukocyte preparations nor the human leu-
kocyte extract exhibited any reactivity with serum from immu-
nized mice, demonstrating the specificity of the antibodies
generated in mice toward the parasite enolase protein.
P. falciparum- and P. yoelii-infected blood cells, permeabil-
ized with Triton X-100, were also examined by IFA. This assay
not only determined the specificity of anti-r-Pfen antibodies
but also assesses the localization of parasite enolase at differ-
ent stages of infection (Fig. 4, bottom panel). No staining of
Plasmodium enolase was seen in the red cell compartment of
the infected erythrocytes, nor was any staining observed with
uninfected mammalian erythrocytes (Fig. 4, bottom panel, A
and B) or with mouse monocytes (Fig. 4, bottom panel, B).
Dominant cytoplasmic localization of enolase was observed in
all the erythrocytic substages: the rings, trophozoites, and schiz-
onts (Fig. 4, bottom panel, A and B). There was also some
evidence of localization within the nucleus in some of these
stages (Fig. 4, bottom panel, B). Since permeabilized cell prep-
arations cannot resolve the issue of surface localization un-
equivocally, a solution IFA was performed on freshly prepared
nonpermeabilized merozoites from P. yoelii to assess the pres-
ence of enolase on the surface of the merozoites (Fig. 4, bot-
tom panel, C). Distinct anti-enolase signal was observed on the
free merozoites (Fig. 4, bottom panel, C), whereas no signal
was observed with preimmune serum (data not shown). The
accepted criterion for viability of merozoites is their ability to
invade red cells in culture. Since such an assay is not possible
for rodent species, we decided to try trypan blue staining to
assess the permeability of the dye in fixed merozoites. It was
found that trypan blue did not stain the merozoites either with
or without fixation with 1% formaldehyde. Thus, trypan blue
does not appear to be an exclusion criterion for P. yoelii mero-
FIG. 4. (Top panel) Specificity of polyclonal antibodies raised
against r-Pfen in mouse: ?100 ?g of proteins from the crude extract of
P. yoelii (lane 1), P. falciparum (lane 2), mouse liver (lane 3), human
(lane 4), and mouse leukocytes (lane 5) was analyzed on a 12% SDS
gel followed by Coomassie blue staining (a) or Western blotting using
mouse anti-r-Pfen antisera at 1:800 dilution (b). (Bottom panel) IFA
of permeabilized P. falciparum-infected and noninfected human red
cells (A), P. yoelii-infected mouse blood smear (B), and nonpermeabi-
lized free P. yoelii merozoites with anti-r-Pfen antiserum (C). Panel A
shows the same field of P. falciparum-infected erythrocytes stained
with DAPI (blue) (a) and with mouse anti-r-Pfen antiserum (green)
(b) and a bright-field image of the area showing infected (arrow) and
uninfected erythrocytes (c). (B) Merged confocal image of P. yoelii-
infected mouse blood smear stained with DAPI (red) and mouse
anti-r-Pfen antiserum (green). The arrow indicates a monocyte.
(C) Solution IFA of free P. yoelii merozoites treated with DAPI (a) or
rabbit anti-r-Pfen antiserum followed by goat anti-rabbit IgG conju-
gated with fluorescein isothiocyanate (b). Each antiserum was used at
a 1:50 dilution.
5504PAL-BHOWMICK ET AL.INFECT. IMMUN.
zoites. Therefore, in order to assess the presence of enolase on
the merozoite surface, we decided to carry out immunoelec-
tron microscopy (IEM).
IEM was carried out on human leukocytes as well as on
various stages of P. falciparum-infected erythrocytes (Fig. 5).
Figures 5A and B show IEM images of P. falciparum seg-
mented schizont and young trophozoite stages, respectively,
while Fig. 5C shows an IEM image of a human leukocyte.
There was an absence of anti-r-Pfen activity in the human
leukocyte or the red cell compartment of the infected eryth-
rocyte, confirming the specificity of anti-enolase antibodies.
While the surface localization of enolase at the trophozoite
stage is not unequivocal, the presence of enolase on the par-
asite cell surface (marked with arrows) in the segmented schiz-
onts is evident. The localization of enolase on the parasite
plasma membrane is seen on several of the segmented mero-
zoites, whereas neither the RBC cytoplasm nor the RBC
plasma membrane of the infected cells shows any reactivity
(Fig. 5A and B). The presence of enolase in the parasite
cytoplasm and the nucleus is also observed.
In this paper we have demonstrated that the Plasmodium
enolase protein is immunogenic and is present on the cell
surface of merozoites and that anti-enolase antibodies can
protect against the malarial parasite. The in vitro parasite
growth inhibition observed in the P. falciparum culture is un-
likely to be due to the inhibition of the glycolytic activity of
enolase, since the rabbit anti-enolase antibodies did not inhibit
the enzymatic activity of enolase (data not shown). Thus, the
observed growth inhibition may be due either to the direct
disruption of some nonglycolytic, but surface-related, func-
tion(s) of Plasmodium enolase or to indirect effects of antibody
binding to the merozoite surface. Protective anti-r-Pfen anti-
bodies raised in animals were specific for Plasmodium. These
reacted with the enolase from both human and murine Plas-
modium species but did not cross-react with the host proteins.
Thus, Pfen possesses the properties suitable for a potential
candidate malaria vaccine antigen.
Our results show that anti-enolase antibodies are wide-
spread among adult residents of the area of Eastern India
where malaria is endemic. We observed that a high frequency
of adults (96%) tested positive for enolase while none of the
samples from children tested positive, indicating that there is
an age correlation with the buildup of anti-enolase antibodies.
Of the adult population examined, only 12.5% of the immune
adults showed the presence of parasites through examination
of blood smears, but 96% were positive for anti-enolase anti-
bodies. This difference could be due to either the persistence
of anti-enolase antibodies after the clearance of parasites or
the lack of sensitivity of the blood smear to detect low levels of
parasites present in immune adults. The sensitive methods of
PCRs detect low levels of the parasite in about a threefold
higher frequency in the population (28). It would be useful to
employ such methods in order to establish if anti-enolase an-
tibodies may be used for detection of parasites, as has been
previously suggested (45). It is also possible that the high
frequency of anti-enolase antibodies may be due to cross-re-
activities with anti-enolases induced by exposure to other
pathogens such as Candida (41), Chlamydia (13), and Leish-
Antigenic variation and allelic polymorphisms render the
malaria vaccine candidate antigens ineffective for use as a
long-lived and broadly available vaccine. In rodent malaria
challenge studies, two leading vaccine candidates, MSP-119
(44) and AMA-1 (apical membrane antigen 1) (11), were
FIG. 5. IEM image using mouse anti-r-Pfen antiserum at a 1:100
dilution with a P. falciparum-infected red cell at segmented schizont
stage (A), a P. falciparum-infected cell at young trophozoite stage (B),
and a human leukocyte (C). In panel A, the presence of enolase on the
parasite plasma membrane (ppm) is marked with arrows. Hpm, host
cell plasma membrane; n, nucleus.
VOL. 75, 2007 ENOLASE AS A PROTECTIVE ANTIGEN AGAINST MALARIA5505
found to protect only from homologous, but not heterologous,
challenge. In several other studies, protection has been docu-
mented to apply across a limited number of strains (11, 27). A
subunit vaccine with chimeric antigens composed of multiple
epitopes from different antigens of P. falciparum is thought to
be an ideal way to address these difficulties. However, a recent
study showed that variants of MSP-1 can mutually turn off
CD4?T cells, implying that the inclusion of multiple allelic
variants in a vaccine may be detrimental to both the initial
priming as well as the in vivo restimulation of preexisting
effector T cells (23). For malaria vaccine candidates, it is often
the conserved subdomains and regions that have shown prom-
ising protective effects, for example, region II of the circum-
sporozoite protein (8) and the thrombospondin-related anon-
ymous protein (49). Thus, candidate antigens conserved across
different strains of Plasmodium may serve as better candidates,
as long as these are immunogenic and do not cross-react with
human proteins. r-Pfen is perhaps one such candidate. Al-
though the Plasmodium enolase protein shows considerable
homology (?68% identity and ?78% homology) with the hu-
man orthologue, it possesses certain novel plant-like features
(42), and there are distinct structural differences (35). The lack
of cross-reactivity of anti-r-Pfen with host enolases has been
demonstrated in this paper through immunoblotting, IFAs,
and electron microscopic examinations.
Antibodies against human enolase protein have been de-
tected in certain autoimmune disorders such as Hashimoto’s
encephalopathy (16, 33), retinopathy (26, 60), rheumatoid ar-
thritis (21, 46), systemic sclerosis (29), autoimmune premature
ovarian failure (51), relapsing polychondritis (52), and lung
adenocarcinoma (54). Detection of anti-enolase antibodies has
been suggested as a means of specific diagnosis for some of
these disorders (16, 51, 54, 60). Although the pathological
significance of these anti-enolase antibodies is not very clear,
one has to exercise caution in the advocacy of Pfen as a malaria
vaccine candidate and use only those regions of the protein
that are protective and also very specific for the parasite.
Epitope mapping of protective antibodies, as well as compar-
ative structural analysis of host and parasite enolase proteins,
will help toward defining such domain(s) on the parasite eno-
lase protein. We are working toward identification of the pro-
tective parasite-specific epitopes of enolase. Monoclonal anti-
bodies have been generated by immunizing mice with r-Pfen.
Two of the Plasmodium-specific clones obtained mapped at the
N-terminal 165 aminoacyl region of r-Pfen, which contains the
plant-specific peptide insert (37). These studies indicate that
such parasite-specific epitopes can be identified.
Presence of enolase on the surface of a cell has been re-
ported in some mammalian cell lines, such as the U937 mono-
cytoid line (30), neutrophils, T cells, B cells, peripheral blood
monocytes (43), and human brain tumor cells (56, 57). Neither
the function(s) nor the regulation of the surface-localized eno-
lase is understood at present. Cell surface association of eno-
lase is also reported in the case of pathogens such as Strepto-
cocci (3, 39, 40), C. albicans (31, 41, 55), and other bacteria (4,
15, 48). Recently, different experimental approaches such as
genetic, cellular biology, and proteomic have been used to
show that yeast enolase can reach the cell surface (25). Secre-
tion of enolase and its involvement in the invasion process have
been reported for an apicomplexan avian parasite, Eimeria
tenella (22). In Plasmodium, enolase is present on the surface
as well as in the nucleus, as observed through IFA and IEM
data presented in this paper. Our biochemical analyses of sub-
cellular fractions of P. yoelii have also provided similar results
(34). The nuclear presence of enolase has also been reported
in the closely related apicomplexan Toxoplasma gondii. How-
ever, the significance of such translocation is not yet under-
stood (12). Increasingly, metabolic enzymes are being reported
to have diverse functions (50). Clearly, the housekeeping pro-
teins are regulated and utilized for other functions, perhaps
when glycolysis is not the main objective of the cell, such as in
the invasive merozoite stage. What determines the transloca-
tion of enolase to regions other than the cytoplasm, as well as
the function(s) of enolase protein at these locations, remains to
In this paper we have shown that a housekeeping enolase
protein of Plasmodium can protect mice against malaria. An
effective malaria vaccine has been difficult to achieve for sev-
eral reasons, predominant among which are antigenic variation
and diversity. An additional problem is the emerging resistance
of the parasite to most antimalarial drugs. Thus, it may be
worthwhile to evaluate enolase, a protein critical to the survival
of the parasite, for the immunoprophylactic and immunother-
apeutic control of malaria.
We are indebted to Nirbhay Kumar of Johns Hopkins University for
help with parasite cultures for IEM and to the Malaria Research and
Reference Reagent Resource for MSP-1 antibodies. We are also grate-
ful to B. Ravindran, Chris King, and Pierre Druilhe for the human
serum samples. We thank Marc Pypaert and the technical personnel in
the Yale Center for Cell and Molecular Imaging for excellent assis-
tance in electron microscopy.
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Editor: W. A. Petri, Jr.
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