INFECTION AND IMMUNITY, Mar. 2008, p. 1193–1199
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 3
Chemical Attenuation of Plasmodium berghei Sporozoites Induces
Sterile Immunity in Mice?
Lisa A. Purcell,1,2Stephanie K. Yanow,3Moses Lee,4Terry W. Spithill,1,5* and Ana Rodriguez2
Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue,
Quebec, Canada H9X 3V91; Department of Medical Parasitology, New York University School of Medicine,
341 E. 25th Street, New York, New York 100102; Provincial Laboratory for Public Health, 8440 112th Street,
Edmonton, Alberta, Canada T6G 2J23; Division of Natural and Applied Sciences and Department of
Chemistry, Hope College, 35 E. 12th Street, Holland, Michigan 494234; and School of Animal and
Veterinary Sciences, Charles Sturt University, Wagga Wagga, Australia 26785
Received 17 October 2007/Returned for modification 16 November 2007/Accepted 19 December 2007
Radiation and genetic attenuation of Plasmodium sporozoites are two approaches for whole-organism
vaccines that protect against malaria. We evaluated chemical attenuation of sporozoites as an alternative
vaccine strategy. Sporozoites were treated with the DNA sequence-specific alkylating agent centanamycin, a
compound that significantly affects blood stage parasitemia and transmission of murine malaria and also
inhibits Plasmodium falciparum growth in vitro. Here we show that treatment of Plasmodium berghei sporozoites
with centanamycin impaired parasite function both in vitro and in vivo. The infection of hepatocytes by
sporozoites in vitro was significantly reduced, and treated parasites showed arrested liver stage development.
Inoculation of mice with sporozoites that were treated in vitro with centanamycin failed to produce blood stage
infections. Furthermore, BALB/c and C57BL/6 mice vaccinated with treated sporozoites were protected against
subsequent challenge with wild-type sporozoites. Our findings demonstrate that chemically attenuated sporo-
zoites could be a viable alternative for the production of an effective liver stage vaccine for malaria.
The development of an effective vaccine is critical to curb
the significant health, social, and economic impacts caused
annually by malaria in countries where the disease is endemic
(39). Malaria infection involves injection of Plasmodium sporo-
zoites from a mosquito into humans. The sporozoites migrate
to the liver, invade hepatocytes, and transform into exoeryth-
rocytic forms (EEFs) that replicate to produce schizonts con-
taining thousands of merozoites (35). These merozoites are
released into the host bloodstream and invade erythrocytes.
The blood stages of malaria are responsible for producing the
symptoms of the disease. Many attempts have been made in
recent years to develop effective subunit vaccines composed of
recombinant Plasmodium antigens. Due to the complexity of
Plasmodium, these vaccines have been only partially effective
(2, 12, 14, 38).
Recently, there has been renewed interest in the attenuated
whole-organism vaccine strategy (16, 22, 38, 53). The whole-
organism approach has historically used radiation-attenuated
sporozoites (RAS) to obtain sterile immunity experimentally in
both mice and humans (16, 30). The RAS invade hepatocytes
in a susceptible host and begin to develop into EEFs, but the
majority of parasites fail to undergo nuclear division and do
not progress to the merozoite form (44, 45). Using mice, RAS
dosing regimens that generate protective immunity have var-
ied, although most regimens require a prime-boost schedule
(4, 11, 32, 50). A meta-analysis of 10 years of immunization of
human volunteers using irradiated Plasmodium falciparum
sporozoites showed a dose response in terms of the immuni-
zation dose required for protection (16, 22). One key issue with
RAS has been the delivery of the correct irradiation dose to
ensure adequate attenuation of the parasite (16, 23, 38, 41, 51).
A strategy to overcome this issue has been to generate genet-
ically attenuated sporozoites (GAS) in which genes essential to
sporozoite function in parasite strains are deleted. Since the
publication of the Plasmodium genome (13), there have been
several studies using this strategy in rodent models of malaria.
These studies have included deletion of the uis3 (28), uis4 (26),
and P36p (50) genes and simultaneous deletion of the uis3 and
uis4 (18) genes in Plasmodium berghei, as well as deletions of
uis3 and uis4 (46) and simultaneous deletion of the P52 and
P36 genes (19) in Plasmodium yoelii. These GAS resemble
RAS in terms of invasion of host hepatocytes and arrested
development, but GAS-infected hepatocytes disappear almost
completely after 24 to 36 h in culture (26, 28, 46, 50), while
RAS persist for longer times in the arrested form (20, 45). Like
RAS, most GAS need to be delivered using a multiple-dose
strategy in order to induce sterile immunity.
We have developed a new strategy to generate attenuated
parasites based on the in vitro chemical treatment of sporozo-
ites. We previously reported the antimalarial activity of AT-
specific DNA binding agents that exploit the AT richness of
the Plasmodium genome (56) and showed that the compound
centanamycin has a significant effect both on blood stages and
on transmission of malaria to mosquitoes (55). Here we used
centanamycin to attenuate P. berghei sporozoites in vitro.
Chemically attenuated sporozoites (CAS) showed a significant
reduction in hepatocyte infection, and in the hepatocytes that
were infected, the sizes of EEFs were greatly reduced. We
showed that CAS do not generate blood stage infections in
* Corresponding author. Present address: School of Animal and
Veterinary Sciences, Charles Sturt University, Locked Bag 588, Wagga
Wagga, NSW, Australia 2678. Phone: 61-2-6933 2439. Fax: 61-2-6933
2991. E-mail: firstname.lastname@example.org.
?Published ahead of print on 3 January 2008.
mice and that immunization of BALB/c and C57BL/6 mice
with CAS produced sterile immunity against challenge with
MATERIALS AND METHODS
Treatment of sporozoites with centanamycin. Anopheles stephensii mosquitoes
were maintained at 70% humidity and 22°C and infected with P. berghei ANKA
wild-type parasites as described previously (48). Infected mosquito salivary
glands were dissected on or about day 18 postfeeding and kept on ice. Sporo-
zoites were quantified by microscopic counting with a hemocytometer. Centana-
mycin (2 M) was prepared in a PET (polyethylene glycol 400, ethanol, Tween
80)-glucose solution (40). Each group of sporozoites was treated with 2 mM
centanamycin diluted in Dulbecco modified Eagle medium (DMEM), while
control groups received the same volume of vehicle. Incubation was performed
at room temperature for 30, 60, or 90 min, and the sporozoites were centrifuged
at 21,000 ? g for 5 min at room temperature and resuspended in the appropriate
medium for each assay.
Analysis of viability and infectivity of treated sporozoites in vitro. Mouse
hepatoma cells (Hepa 1-6) (25) were grown in DMEM with 10% fetal bovine
serum with 1% PSG (penicillin, streptomycin, gentamicin) at 37°C and 5% CO2,
and 2 ? 105cells were seeded on glass coverslips in 24-well plates 24 h prior to
Membrane integrity. Sporozoites were incubated with 10 ?g/ml propidium
iodide for 5 min at room temperature after incubation with the vehicle or
centanamycin. Sporozoites were washed three times, resuspended in 10 ?l
DMEM, and then wet mounted on a microscope slide and covered with a glass
coverslip. The number of fluorescent sporozoites was determined using a Nikon
Eclipse E600 microscope. As a control, freshly dissected sporozoites were la-
beled and quantified soon after dissection to ensure that the dissected sporozo-
ites were viable. Sporozoites that were heat killed at 65°C for 15 min served as
a control. Incubation was performed in triplicate in two independent experi-
ments, and 100 sporozoites were counted per well.
Gliding motility. Glass, eight-chamber Lab-Tek chamber slides (Nalgene)
were coated with 5 ?g/ml 3D11, a monoclonal antibody directed against the
repeat region of P. berghei circumsporozoite protein (57), in phosphate-buffered
saline (PBS) overnight at room temperature. The 3D11 antibody was used to
capture shed circumsporozoite protein. The wells were washed three times with
PBS. For each well 2 ? 104sporozoites were treated as described above. Sporo-
zoites were centrifuged, the medium was replaced with DMEM containing 3%
bovine serum albumin (BSA), and the cultures were incubated at 37°C in 5%
CO2for 1 h, after which the sporozoites were fixed with 4% paraformaldehyde
at 4°C overnight. Each well was washed with PBS and blocked with 1% BSA in PBS.
Biotinylated 3D11 monoclonal antibody (9) was added, followed by addition of
streptavidin-fluorescein isothiocyanate (Sigma) and incubation for 1 h at 37°C. The
percentage of gliding motility was determined by counting both the number of
sporozoites with trails and the number of circles that each trail contained using a
Nikon Eclipse E600 microscope. Incubation was performed in triplicate in two
independent experiments, and 100 sporozoites were counted per chamber.
Invasion of hepatoma cell line in vitro. A total of 5 ? 104sporozoites were
treated as described above, resuspended in cell medium, and added to wells
containing semiconfluent Hepa 1-6 cells. Plates were incubated for 1 h at 37°C,
fixed in 4% paraformaldehyde at 4°C, and stained using 3D11 and a double
staining technique (36). Intracellular and extracellular sporozoites were differ-
entially stained and counted using a Nikon Eclipse E600 microscope. Incubation
was performed in triplicate in two independent experiments, and 100 sporozoites
were counted per well.
Liver stage development in vitro. A total of 5 ? 104sporozoites were treated
as described above, resuspended in cell medium, and added to wells containing
semiconfluent Hepa 1-6 cells. Plates were incubated for 42 h at 37°C to allow
EEF development and then fixed in 4% paraformaldehyde at 4°C overnight and
washed with PBS. Each coverslip was blocked and permeabilized in a solution
containing 10% goat serum, 1% BSA, 100 mM glycine, 0.05% NaN3(pH 7), and
0.2% saponin for 30 min at room temperature. The coverslips were then incu-
bated with 2E6 (a monoclonal antibody that recognizes Plasmodium HSP70) (47)
for 1 h at room temperature, washed with PBS, and then incubated with anti-
mouse fluorescein isothiocyanate-conjugated antibodies (Sigma) for 1 h. Cover-
slips were washed with PBS and mounted on microscope slides, and the number
of EEFs was counted using a Nikon Eclipse E600 microscope. Images were taken
with a Leica TCS SP2 AOBS confocal microscope using Leica LCS software
(version 5). Incubation was performed in triplicate in two independent experi-
ments, and 50 random fields were counted per coverslip.
Liver stage development in vivo. Procedures for animal experiments were
approved by New York University School of Medicine Institutional Animal Care
and Use Committee. Eight-week-old female C57BL/6 mice were inoculated
intravenously (i.v.) with 2 ? 104sporozoites treated with centanamycin for 30
min and resuspended in DMEM. Livers were harvested from infected mice 40 h
later, as well as from one uninfected mouse. Total RNA was isolated using
TRIzol (Invitrogen), and cDNA was synthesized according to the manufacturer’s
instructions (Applied Biosystems). Malaria infection was quantified using quan-
titative real-time PCR with primers specific for P. berghei 18S rRNA (5, 6).
Tenfold dilutions of a plasmid construct containing the 18S rRNA sequence were
used to create a standard curve. Two independent experiments were performed,
and each sample was analyzed in triplicate with four mice per treatment group.
Blood stage development from treated sporozoites. Eight-week-old female
BALB/c and C57BL/6 mice were inoculated i.v. with 5 ? 104or 2 ? 104CAS
which had been treated with centanamycin for 30 min and resuspended in
DMEM. Parasitemia was evaluated from day 3 postinfection (p.i.) onward by
using Giemsa-stained thin blood smears. The percentage of parasitemia was
calculated by using 1,000 cells per slide. Animals were evaluated for 7, 10, or 21
days after injection of treated sporozoites as indicated below.
Challenge of mice with wild-type sporozoites. The same groups of mice that
were inoculated with CAS to determine blood stage development were chal-
lenged by i.v. inoculation using 5 ? 103untreated, wild-type P. berghei ANKA
sporozoites (for the BALB/c mice) and 1 ? 103or 1 ? 104untreated wild-type
sporozoites (for the C57BL/6 mice) 21, 14, or 10 days after the immunization
regimen. Age-matched, naı ¨ve mice were inoculated with the same number of
sporozoites as infection controls to assess the infectivity of the untreated sporo-
zoites. Parasitemia was evaluated from day 3 p.i. onward by using Giemsa-stained
thin blood smears. The percentage of parasitemia was calculated by using 1,000
cells per slide. Animals were evaluated for at least 30 days postchallenge.
Statistical analyses. All statistical analyses were completed using Prism (ver-
sion 4.0a). When differences in toxicity, gliding motility, invasion, EEF forma-
tion, and quantitative PCR were assessed, normality was tested using the Kol-
mogorov-Smirnov goodness-of-fit test. Data with a P value of ?0.10 were
considered normal. The differences were then tested using an analysis of variance
(ANOVA) with Tukey’s multiple comparison post hoc test. Only the assays
showing significant differences were noted.
Chemical attenuation does not result in sporozoite death.
Whole-organism malaria vaccine strategies depend on the par-
asite being replication deficient, yet still capable of invading
host cells and producing antigens to induce an immune re-
sponse. Incubation of sporozoites for 30, 60, and 90 min with
centanamycin in vitro did not result in decreased viability of
sporozoites compared to controls (Fig. 1). Propidium iodide,
which is membrane impermeable, is frequently used to stain
FIG. 1. Treatment of sporozoites with centanamycin in vitro does not
affect sporozoite membrane integrity. P. berghei sporozoites were incu-
bated with vehicle (gray bars) or 2 mM centanamycin (black bars) for 30,
60, or 90 min before addition of propidium iodide. Control sporozoites
were either tested immediately following dissection (open bar) or heat
killed (striped bar) for 15 min at 65°C before counting. For each sample,
100 sporozoites were counted in two separate experiments, and the aver-
age percentage of staining with propidium iodide is shown.
1194 PURCELL ET AL.INFECT. IMMUN.
viable cells in a population. Similar labeling with propidium
iodide was observed in control and centanamycin-treated
sporozoites, suggesting that centanamycin did not affect mem-
brane integrity. As incubation times increased from 30 to 90
min, the number of nonviable sporozoites increased similarly
in the drug-treated population compared with controls. A de-
crease in the viability of sporozoites is expected after dissection
from mosquito salivary glands (34).
Gliding motility and invasion of hepatocytes by drug-treated
sporozoites. Gliding motility is a feature of Plasmodium sporo-
zoites and is required for invasion of hepatocytes (42). Treat-
ment of sporozoites for 30, 60, or 90 min with centanamycin
reduced the motility of sporozoites in a time-dependent man-
ner (Fig. 2A). While the motility of vehicle-treated sporozoites
also decreased, the motility of centanamycin-treated sporozo-
ites was approximately 24% less than that of vehicle-treated
sporozoites throughout the time course. The sporozoites that
remained motile after treatment with centanamycin produced
trails whose quality was similar to that of vehicle-treated sporo-
zoite trails (Fig. 2B). The gliding motility of RAS and all GAS
reported so far is not different from that of wild-type sporozo-
ites (19, 26, 28, 49, 50).
RAS are able to invade hepatocytes with the same efficiency
as wild-type sporozoites, even if their development is later
impaired (26, 43, 44). We therefore analyzed the ability of
centanamycin-treated sporozoites to invade hepatocytes (Hepa
1-6) in vitro. Treated sporozoites invaded hepatocytes effi-
ciently (Fig. 3), although there was an apparent mean reduc-
tion in invasion. However, the difference was significant only at
the 60-min treatment time when the average for all three
experiments was considered. The cause of the reduced inva-
sion rates is not known, but since sporozoite motility is re-
quired for invasion (42), the reduced invasion could be a con-
sequence of the decreased motility observed with CAS.
EEF development is impaired after drug treatment. EEF
formation is a critical phase in the Plasmodium life cycle and is
responsible for the generation of thousands of merozoites that
infect erythrocytes. Hepatocytes infected with wild-type sporo-
zoites develop EEFs whose size increases, reaching approxi-
mately 10 ?m after 48 h of culture (25). There was a decrease
in EEF formation by control sporozoites after 60 and 90 min of
incubation in vitro, which occurs when sporozoites are not
immediately used for infection assays, since sporozoite infec-
tivity is progressively lost after dissection from salivary glands
(7). Treatment with centanamycin for 30, 60, and 90 min sig-
nificantly reduced the number of EEFs formed in vitro after
42 h (Fig. 4A), although a low number of EEFs could still be
observed even after the 90-min treatment. This is in sharp
contrast to GAS, which, in general, do not produce EEFs
persisting beyond 24 to 36 h (19, 26, 28, 50), and it is more
similar to RAS, which produce EEFs that persist at least until
48 h (20, 41, 45). While the sizes of vehicle-treated EEFs were
smaller (Fig. 4B). These results show that centanamycin treat-
ment significantly affects EEF formation in vitro.
FIG. 2. Gliding motility of sporozoites treated with centanamycin
in vitro. P. berghei sporozoites were incubated with vehicle or 2 mM
centanamycin for 30, 60, or 90 min and then incubated for 1 h at 37°C
to allow parasites to move, and then gliding motility was assessed.
(A) A significant reduction in the percentage of centanamycin-treated
sporozoites (black bars) that exhibited gliding motility was observed at
all time points compared to controls (gray bars) (P ? 0.0001, ANOVA;
n ? 100). (B) Quality of the trails, indicated by the number of circles
that each sporozoite generated (open bars, 1 trail; black bars, 2 to 10
trails; gray bars, ?10 trails). Incubation was performed in triplicate in
two independent experiments, and 100 sporozoites were counted per
FIG. 3. Invasion of hepatoma cells in vitro is not significantly re-
duced after treatment with centanamycin. P. berghei sporozoites were
incubated with vehicle (gray bars) or 2 mM centanamycin (black bars)
for 30, 60, or 90 min. Sporozoites were stained with 3D11, followed by
secondary antibodies, both before and after permeabilization to deter-
mine the number of sporozoites that invaded the cells. A significant
reduction in invasion was observed only after the 60-min treatment
(P ? 0.0065, ANOVA; n ? 100). Incubation was performed in tripli-
cate in two independent experiments, and 100 sporozoites were
counted per well.
VOL. 76, 2008 STERILE IMMUNITY GENERATED FROM CAS1195
Centanamycin-treated sporozoites fail to establish infection
in mice. Since the number of EEFs formed in culture was
significantly reduced when sporozoites were treated with cen-
tanamycin, we analyzed the ability of sporozoites treated for 30
min to establish a liver stage infection in C57BL/6 mice. A total
of 2 ? 104treated sporozoites were inoculated into mice i.v.,
and parasites were allowed to develop for 40 h. Using real-time
PCR to quantify the level of infection in the liver, mice inoc-
ulated with vehicle-treated sporozoites produced nearly 60
times more copies of P. berghei 18S rRNA than the mice
inoculated with centanamycin-treated sporozoites (Fig. 5). The
number of copies in mice that received the drug-treated sporo-
zoites was not significantly different than the number of copies
in an uninfected control mouse.
Since the CAS reduced the extent of liver stage infection, we
tested the ability of centanamycin-treated sporozoites to block
a blood stage infection in both BALB/c and highly susceptible
C57BL/6 mice. Mice were infected with 2 ? 104or 5 ? 104
sporozoites that were treated with vehicle or centanamycin for
only 30 min. BALB/c mice that received the vehicle-treated
sporozoites developed hyperparasitemia and were euthanized
by day 15 p.i. In contrast, mice that received the centanamycin-
treated sporozoites never developed a patent parasitemia dur-
ing the 21 days of observation (Fig. 6). C57BL/6 mice that
received the vehicle-treated sporozoites were euthanized after
developing symptoms of cerebral malaria by day 8 p.i., while the
mice that received the centanamycin-treated sporozoites never
developed patent parasitemia during the 14 days of observation
(data not shown). This is in agreement with the in vitro data
suggesting that EEFs derived from centanamycin-treated sporo-
zoites do not differentiate into infectious merozoites.
Immunization with treated sporozoites protects mice
against subsequent infection. Some RAS and GAS immuniza-
tion schemes generate sterile protection in mice against sub-
sequent challenge with wild-type, nonattenuated sporozoites.
We used mice that received one dose of the noninfectious CAS
described above and challenged them with 5 ? 103or 1 ? 103
FIG. 6. Treatment of sporozoites with centanamycin for 30 min in
vitro before injection of mice prevents blood stage infection. P. berghei
sporozoites were incubated with vehicle (Œ) or 2 mM centanamycin
(f) for 30 min before injection of washed sporozoites into BALB/c
mice. Development of detectable blood stage parasites was followed
for 21 days p.i. Mice that received vehicle-treated sporozoites devel-
oped parasites on day 4 p.i. and were euthanized by day 15 p.i. Exper-
iments were performed twice with four mice per group. The results of
one representative experiment are shown.
FIG. 4. Treatment of sporozoites with centanamycin decreases the
formation of EEFs in hepatoma cells. P. berghei sporozoites were
incubated with vehicle (gray bars) or 2 mM centanamycin (black bars)
for 30, 60, or 90 min. Sporozoites were added to Hepa 1-6 cells for 42 h
at 37°C. (A) The number of EEFs was significantly reduced in all
treatment groups (asterisks, P ? 0.0001, ANOVA). (B) Phase-contrast
images (upper images) and fluorescent images (lower images) of rep-
resentative EEFs from vehicle-treated (left images) and centanamycin-
treated (right images) sporozoites. The outline of the EEF is shown in
the upper images. Scale bars ? 10 ?m. Scale bar in inset ? 1 ?m.
FIG. 5. Sporozoites treated with centanamycin for 30 min do not
establish a significant liver stage infection. P. berghei sporozoites were
incubated with vehicle or 2 mM centanamycin for 30 min, washed, and
then inoculated i.v. into C57BL/6 mice. Forty hours later, mice were
sacrificed and total liver RNA was extracted. An uninfected mouse
served as a negative control. Malaria infection was determined by
quantitative reverse transcription-PCR. Infection is expressed as the
number of copies of P. berghei 18S rRNA (Pb18S rRNA). Treatment
of sporozoites with centanamycin resulted in significant reduction (as-
terisks, P ? 0.0001, ANOVA) in 18S rRNA levels compared with the
vehicle-treated controls. Shown are the results of one of two indepen-
dent experiments with four mice per treatment group.
1196PURCELL ET AL.INFECT. IMMUN.
untreated sporozoites to evaluate the efficacy of CAS as a
vaccine. BALB/c mice that received the CAS did not develop
any detectable parasitemia over 30 days of observation, while
naı ¨ve mice inoculated with the same challenge doses devel-
oped high levels of parasitemia and were euthanized by day
16 p.i. (Table 1). In C57BL/6 mice given a single immunization
there was a 2-day delay in the development of detectable par-
asitemia with the challenge dose compared to naı ¨ve mice. A
multiple immunization schedule consisting of 5 ? 104CAS and
two doses of 2 ? 104CAS 7 days apart was used to generate
sterile immunity in C57BL/6 mice with a challenge dose of 104
untreated sporozoites. This dosing schedule also produced
sterile immunity using RAS from the same mosquito batches
(Table 1). Most GAS and RAS vaccine schedules require some
combination of multiple-dose periods in order to consistently
obtain complete protection. CAS fully protected BALB/c mice
in two independent experiments using a single-dose schedule.
Interestingly, mice that were challenged again with 5 ? 103
sporozoites 30 days after the initial challenge did not develop
detectable blood stage parasitemia (data not shown).
Our results show that P. berghei CAS are completely arrested
at the liver stage, fail to produce blood stage parasites in mice,
and induce sterile protection in BALB/c and C57BL/6 mice
following vaccination. A previous study attempted a chemical
attenuation strategy by treating P. berghei NK65 sporozoites
with high doses (0.8 mg/ml) of chloroquine (33). It was found
that using five immunizing doses of 2.5 ? 104sporozoites
treated with 0.8 mg/ml of chloroquine for 60 min produced
78.6% protection in mice, although the viability of the sporo-
zoites was not reported. This malaria vaccine strategy, as well
as the RAS vaccine approach, was then abandoned in favor of
the subunit vaccine approach (22).
With the renewed interest in whole-organism vaccines (22,
38, 53), we evaluated the chemical attenuation of parasites
using the DNA sequence-specific alkylating agent centanamy-
cin and characterized its effects on sporozoites both in vitro
and in vivo. Centanamycin has been shown to block P. falcip-
arum blood stage growth in vitro, to inhibit blood stage infec-
tions with Plasmodium chabaudi adami and P. berghei in mice,
and to significantly reduce the transmission potential of P.
berghei, with a 99% reduction in sporozoite production (55).
Our studies show that treatment of P. berghei ANKA sporozo-
ites with centanamycin for 30, 60, or 90 min in vitro does not
affect membrane integrity. We found that there was a moder-
ate decrease in gliding motility of treated sporozoites that
probably caused the small decrease observed in hepatocyte
invasion in vitro. However, this small decrease in hepatocyte
invasion did not seem to affect the capacity of treated sporo-
zoites to induce protective responses in mice.
Hepatocyte invasion by sporozoites is an important step in
eliciting an immune response to the parasite. Inactivated
sporozoites that are not able to infect hepatocytes have con-
sistently failed to induce protective immune responses (1, 23,
31), although they can efficiently prime the immune system
(15). Conversely, malaria-infected hepatocytes and their ex-
tracts induce significant protection when they are injected into
rats or mice (37, 41). Previous experiments suggested that
when animals are immunized with RAS, the protection against
a challenge dose of sporozoites is dependent upon the persis-
tence of irradiated sporozoites in the liver (23, 41). However,
more recent data indicate that GAS do not require persistence
in the liver to induce protective immunity (26, 28, 46, 50). Our
study shows that CAS do produce liver stages in vitro, albeit at
significantly lower levels (?85% reduction) than control sporo-
zoites, and these liver stages were much smaller than those of
the controls. Whereas RAS and GAS both invade liver cells
and transform into the rounded trophozoite stage, they gener-
ally do not enter schizogony (51). GAS-infected hepatocytes
normally do not persist longer than 24 to 36 h (26, 28, 46, 50),
compared to RAS-infected hepatocytes, which persist much
longer (20, 41, 45). Our study showed that CAS persist in
cultured hepatocytes for at least 42 h. Taken together, these
results suggest that the CAS strategy is an effective attenuation
strategy that can produce the infective liver stages needed to
elicit an immune response.
A dose of 2 ? 104CAS in BALB/c and C57BL/6 mice failed
to establish a blood stage infection, and the CAS-vaccinated
BALB/c mice exhibited protective immunity when they were
challenged with 5 ? 103untreated, wild-type sporozoites. A
multiple-dose regimen was employed to produce sterile immu-
nity in C57BL/6 mice. The genetic restriction observed, where
sterile immunization requires more doses of attenuated sporo-
zoites in C57BL/6 mice than in BALB/c mice, has been re-
ported previously (10, 11, 26, 50). This is probably a conse-
TABLE 1. Protection of mice immunized with CAS against challenge with wild-type sporozoites
Group Mouse strain
Immunization with RAS or CAS
No. of mice protected
aGroups of mice were immunized with P. berghei ANKA control (vehicle-treated) sporozoites, CAS, or RAS as indicated, using sporozoites isolated from the same
mosquito batches; in group 4, mice were immunized three times with CAS or RAS with 7-day intervals between doses.
bGroups of mice were challenged with P. berghei ANKA wild-type sporozoites isolated from the same mosquito batches.
cNaı ¨ve, age-matched mice were used at the time of all immunizations and challenges.
dNA, not applicable.
eNI, the treatment group was not included.
fNaı ¨ve, age-matched mice developed patent parasitemia 3 days p.i.
VOL. 76, 2008STERILE IMMUNITY GENERATED FROM CAS 1197
quence of the fact that vaccination with attenuated sporozoites
induces different mechanisms of protection in these two mouse
strains (10). The highly susceptible C57BL/6 mice (24) require
booster doses of vaccine to induce fully effective immune re-
sponses. In general, both RAS and GAS require higher initial
doses of P. berghei ANKA sporozoites in BALB/c mice (at least
2 ? 104sporozoites) to provide complete protection against
lower or similar challenge doses of wild-type parasites (1 ? 103
sporozoites) (8, 11, 50). This suggests that the CAS approach
could be an efficient approach for producing a whole-organism
The immune responses against both RAS and GAS are
complex and involve both cell-mediated and humoral immu-
nity (10, 18, 27, 29, 37, 46). In addition, some RAS and GAS
seem to induce long-lasting, cross-species protection (11, 31,
32). Attenuation of irradiated sporozoites presumably occurs
due to double-strand breaks in the DNA that lead to a block in
liver stage development. Each sporozoite would contain a
number of strand breaks randomly distributed in its DNA. In
the case of CAS treated with centanamycin, the attenuated
sporozoites would contain a set of adducts covalently bound to
adenine nucleotides (40). This compound, like other AT-spe-
cific binding compounds, recognizes selective DNA sequences,
and the potential number of adducts can be defined bioinformati-
cally (52, 54). Given that both the CAS and RAS approaches
disrupt the integrity of the parasite DNA, it is possible that the
immune responses generated by RAS and CAS would be similar,
but further studies are necessary to confirm this.
Many issues have been raised concerning the feasibility of
both GAS and RAS as whole-organism sporozoite vaccines,
including mass production of sterile parasites, proper storage
to maintain viability, and the safety of a mosquito-derived
vaccine (3, 16, 22, 38). Both types of attenuation have individ-
ual inherent weaknesses. In the case of RAS, the overattenu-
ation of sporozoites has been shown to block liver stage devel-
opment at the trophozoite stage (23, 44) and generate poor
protection (23), suggesting that the dose of irradiation is piv-
otal to the success of each lot of RAS. Uniform exposure of
parasites to the radiation source is essential to prevent the
escape of sporozoites that could generate a malaria infection
following vaccination (16, 38, 51). In contrast, the risk of
“breakthrough infections” with GAS is low due to the gene
knockout strategy employed (17, 26, 50). Yet the widespread
distrust of genetically modified products, especially for a vac-
cine that would be inoculated into humans and invade host
cells, may complicate efforts to utilize GAS in the field. Our
proposed CAS vaccine has the advantage that the chemical
attenuation process can be strictly controlled, leading to a
vaccine that is reproducibly attenuated. Given that centanamy-
cin shows covalent DNA sequence specificity similar to that of
adozelesin and that the frequency of binding sites for adozele-
sin has been estimated to be 440 sites per kb of genomic
Plasmodium DNA (52), treatment of sporozoites with centana-
mycin could potentially saturate these binding sites to obtain a
maximal effect on the parasite. Since the generation of viable,
cryopreserved sporozoites is currently being optimized (22),
chemical attenuation using centanamycin could be considered
an additional strategy for the production of whole-organism
vaccines against malaria. Although the potential toxicity of
residual centanamycin in humans is a concern, the risk may be
minimal since free drug can be washed from the parasites
before vaccine delivery and the drug that is present in treated
sporozoites is covalently bound to parasite DNA and thus not
available to modify host DNA. Nevertheless, the risk of toxicity
needs to be addressed by in-depth pharmacokinetic and mutage-
nicity studies. More generally, our results suggest that chemical
attenuation with drugs such as centanamycin may be a feasible
approach for generating live attenuated vaccines for other major
parasites with AT-rich DNA, such as Theileria (21, 54).
We thank J. Noonon, S. Gonzalez, and A. Coppi for assistance with
mosquito experiments and E. Bettiol for assistance with confocal mi-
croscopy. We thank A. Waters, who kindly provided the P. berghei
ANKA strain. We thank Spirogen Ltd. and Centana Pharmaceuticals
for use of centanamycin.
This work was supported by a Canada Graduate Scholarship from
the Natural Sciences and Engineering Research Council of Canada (to
L.P.), by Canadian Foundation for Innovation grant 201221 (to T.S.),
by Le fonds que ´be ´cois de la recherche sur la nature et les technologies
(FQRNT) Centre for Host-Parasite Interactions grant 87902 (to T.S.),
by Canada Research Chair in Immunoparasitology grant 201221 (to
T.S.), and by NIH grant RO1 AI 053698 (to A.R.).
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Editor: W. A. Petri, Jr.
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