Identification of two new protective pre-erythrocytic malaria vaccine antigen candidates.

Abstract

Despite years of effort, a licensed malaria vaccine is not yet available. One of the obstacles facing the development of a malaria vaccine is the extensive heterogeneity of many of the current malaria vaccine antigens. To counteract this antigenic diversity, an effective malaria vaccine may need to elicit an immune response against multiple malaria antigens, thereby limiting the negative impact of variability in any one antigen. Since most of the malaria vaccine antigens that have been evaluated in people have not elicited a protective immune response, there is a need to identify additional protective antigens. In this study, the efficacy of three pre-erythrocytic stage malaria antigens was evaluated in a Plasmodium yoelii/mouse protection model.
Mice were immunized with plasmid DNA and vaccinia virus vectors that expressed one, two or all three P. yoelii vaccine antigens. The immunized mice were challenged with 300 P. yoelii sporozoites and evaluated for subsequent infection.
Vaccines that expressed any one of the three antigens did not protect a high percentage of mice against a P. yoelii challenge. However, vaccines that expressed all three antigens protected a higher percentage of mice than a vaccine that expressed PyCSP, the most efficacious malaria vaccine antigen. Dissection of the multi-antigen vaccine indicated that protection was primarily associated with two of the three P. yoelii antigens. The protection elicited by a vaccine expressing these two antigens exceeded the sum of the protection elicited by the single antigen vaccines, suggesting a potential synergistic interaction.
This work identifies two promising malaria vaccine antigen candidates and suggests that a multi-antigen vaccine may be more efficacious than a single antigen vaccine.

Full text

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RESEARCHOpen Access
Identification of two new protective pre-
erythrocytic malaria vaccine antigen candidates
Keith Limbach1,2*, Joao Aguiar1,2, Kalpana Gowda1, Noelle Patterson1,2, Esteban Abot1,2, Martha Sedegah1,
John Sacci3and Thomas Richie1
Abstract
Background: Despite years of effort, a licensed malaria vaccine is not yet available. One of the obstacles facing the
development of a malaria vaccine is the extensive heterogeneity of many of the current malaria vaccine antigens.
To counteract this antigenic diversity, an effective malaria vaccine may need to elicit an immune response against
multiple malaria antigens, thereby limiting the negative impact of variability in any one antigen. Since most of the
malaria vaccine antigens that have been evaluated in people have not elicited a protective immune response,
there is a need to identify additional protective antigens. In this study, the efficacy of three pre-erythrocytic stage
malaria antigens was evaluated in a Plasmodium yoelii/mouse protection model.
Methods: Mice were immunized with plasmid DNA and vaccinia virus vectors that expressed one, two or all three
P. yoelii vaccine antigens. The immunized mice were challenged with 300 P. yoelii sporozoites and evaluated for
subsequent infection.
Results: Vaccines that expressed any one of the three antigens did not protect a high percentage of mice against
a P. yoelii challenge. However, vaccines that expressed all three antigens protected a higher percentage of mice
than a vaccine that expressed PyCSP, the most efficacious malaria vaccine antigen. Dissection of the multi-antigen
vaccine indicated that protection was primarily associated with two of the three P. yoelii antigens. The protection
elicited by a vaccine expressing these two antigens exceeded the sum of the protection elicited by the single
antigen vaccines, suggesting a potential synergistic interaction.
Conclusions: This work identifies two promising malaria vaccine antigen candidates and suggests that a multi-
antigen vaccine may be more efficacious than a single antigen vaccine.
Background
Malaria kills approximately 863,000 people every year [1].
Although a variety of anti-malarial drugs exist, the cost
of these drugs can be prohibitive in the relatively poor
areas of the world where malaria is endemic. The wide-
spread use of the most commonly employed drugs has
also resulted in the expansion of drug-resistant parasites,
rendering many of these drugs ineffective [2]. In the
absence of inexpensive, highly potent drugs, vaccination
represents the most cost-effective way of supplementing
traditional malaria interventions.
A successful malaria vaccine will need to protect people
against a large population of antigenically diverse malaria
parasites. A vaccine based on a single isolate of a single
antigen may not be able to elicit an immune response that
is broad enough to protect individuals against this hetero-
geneous population. Therefore, an efficacious malaria
vaccine may need to induce an immune response against
multiple malaria antigens, a belief that has propelled the
development of whole-organism malaria vaccines, such as
the irradiated sporozoite vaccine and the genetically atte-
nuated sporozoite vaccine [3,4].
A variety of malaria vaccine candidates are being eval-
uated in clinical trials throughout the world. The most
advanced vaccine candidate, RTS,S, is currently being
evaluated in a phase 3 trial at 11 sites in seven African
countries. RTS,S is a recombinant protein vaccine based
on the Plasmodium falciparum circumsporozoite pro-
tein (CSP). It has protected malaria-naïve adults against
an experimental P. falciparum challenge and reduced
* Correspondence: keith.limbach@med.navy.mil
1U.S. Military Malaria Vaccine Program, Naval Medical Research Center, 503
Robert Grant Avenue, Silver Spring, MD, USA
Full list of author information is available at the end of the article
Limbach et al. Malaria Journal 2011, 10:65
http://www.malariajournal.com/content/10/1/65
© 2011 Limbach et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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malaria-associated episodes in children living in malaria
endemic areas [5,6]. The level and duration of immunity
induced by RTS,S, however, is relatively modest.
One way to potentially enhance the efficacy of RTS,S,
or any other subunit malaria vaccine, would be to incor-
porate additional malaria antigens into the vaccine,
thereby broadening the immune response elicited by the
vaccine. At least one other malaria antigen has protected
volunteers against a malaria challenge. A prime-boost
regimen with adenovirus and poxvirus vectors expres-
sing P. falciparum thrombospondin-related adhesive
protein (TRAP) has protected volunteers against an
experimental P. falciparum challenge [7]. A prime-boost
regimen with DNA and adenovirus vectors expressing
CSP and apical membrane antigen 1 (AMA1) has also
protected volunteers against an experimental P. falci-
parum challenge [8]. Although the data from both of
these clinical trials are not yet published, these studies
indicate that CSP, TRAP and possibly AMA1 can induce
protective immune responses in people. Unfortunately,
most of the other malaria vaccine antigens evaluated in
people have not induced significant levels of protection.
For example, recombinant protein vaccines containing
the C-terminal end of the merozoite surface protein 1
(MSP142), the AMA1 ectodomain or a combination of
three P. falciparum antigens (MSP1, MSP2 and ring-
infected erythrocyte surface antigen (RESA)) have not
induced significant levels of protection against natural
infection in children living in malaria endemic regions
[9-11]. Each of these vaccines, however, may have
induced some level of strain-specific protection against
the P. falciparum strain from which the vaccine antigen
was derived [11,12]. Since an immune response against
multiple malaria antigens may be necessary to protect a
high percentage of people against the large number of
antigenically diverse P. falciparum strains throughout
the world, there is a great need to identify new malaria
vaccine antigens.
In this report, the efficacy of three malaria vaccine
antigens was evaluated in a P. yoelii/mouse model.
Although these three pre-erythrocytic stage antigens,
PY03011, PY03424 and PY03661, were independently
identified by our bioinformatic and genomic analyses,
two of the antigens (or their orthologs) were previously
described (PY03011 = PyUIS3, PY03424 = falstatin)
[13,14]. Protection studies with DNA and vaccinia virus
vaccine vectors expressing these antigens suggest that
two of the antigens, PY03011 and PY03424, can protect
mice against a P. yoelii sporozoite challenge.
Methods
Down-selection of vaccine candidate genes
P. falciparum and P. yoelii express approximately 5,800
genes. It is not feasible to evaluate the vaccine potential
of that many genes. Therefore, various methods were
used to down-select the most promising vaccine candi-
dates. Assuming that a vaccine based on a pre-erythro-
cytic antigen is more likely to be successful than a
vaccine based on an erythrocytic antigen, the down-
selection process focused on sporozoite and liver stage
antigens. To identify promising sporozoite antigens,
genomic and proteomic information contained in pre-
existing malaria databases was evaluated [15,16]. To
identify promising liver stage antigens, an expression
library created with material isolated from P. yoelii-
infected liver cells was evaluated [17]. The P. falciparum
genes encoding the down-selected sporozoite and liver
stage antigens were cloned using a high-throughput
cloning strategy [18]. Evaluation of the proteins encoded
by these genes with antisera from volunteers who had
received a P. falciparum irradiated sporozoite vaccine
identified 20 promising vaccine candidates [19].
Generation of DNA and vaccinia virus vaccine vectors
The re-annotated single exon PY03011 gene was isolated
from P. yoelii (17XNL) genomic DNA by PCR with the
primers, 5’-TGGATCCATGAAAGTGTATAAAATGAA-
CACTCTC-3’ and 5’-TGGATCCTCATTTTGGTTGA-
TATTGTTCTTTAAG-3’. The DNA-PY03011 vaccine
vector was generated by cloning the PY03011 gene from
this PCR reaction into the BamHI site of the DNA vaccine
vector, VR1020 (Vical Inc., San Diego, CA). This cloning
reaction positions the full length PY03011 gene down-
stream from a cytomegalovirus (CMV) immediate-early
(IE) promoter and in-frame with a human tissue plasmino-
gen activator (TPA) signal sequence. Since the PY03011
protein contains a signal sequence, cloning the PY03011
gene into VR1020 downstream from an in-frame TPA sig-
nal sequence results in a PY03011 construct that contains
two signal sequences. The vaccinia-PY03011 vaccine vec-
tor was generated using a host range selection system [20].
The full length PY03011 gene in this vector is inserted
into the vaccinia virus A-type inclusion body (ATI) locus
and is under the transcriptional control of a synthetic
early/late (E/L) promoter [21].
Exon 2 of the re-annotated PY03424 gene was isolated
from P. yoelii (17XNL) genomic DNA by PCR with the
primers, 5’-TGGATCCTACTCTTTTGACATTGTAAA
CGAG-3’ and 5’-TGGATCCTTATTGGACAGTTACG-
TATAAAATTTTAG-3’. The DNA-PY03424 vaccine
vector and the vaccinia-PY03424 vaccine vector were
generated with the same reagents and techniques used
to generate the DNA-PY03011 and vaccinia-PY03011
vectors. The DNA and vaccinia vaccine vectors expres-
sing PY03424 (exon 2) do not contain the first 26
codons of the PY03424 gene. Since the first 21 codons
of the PY03424 gene encode a signal sequence, the
PY03424 proteins expressed by the DNA-PY03424 and
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vaccinia-PY03424 vectors do not contain the native
PY03424 signal sequence. To enhance expression, the
DNA-PY03424 vector was engineered to express a
PY03424 protein with a TPA signal sequence and the
vaccinia-PY03424 vector was engineered to express a
PY03424 protein with a human decay accelerating factor
(DAF) signal sequence.
The PY03661 gene was isolated from P. yoelii (17XNL)
genomic DNA by PCR with the primers, 5’-TGGATC-
CATGTTTCGATCTGATTCCCATTTCC-3’ and 5’-
TGGATCCTTATGTTTGATGATAATTTTCTTTCG-3’.
The DNA-PY03661 vaccine vector and the vaccinia-
PY03661 vaccine vector were generated with the same
reagents and techniques used to generate the other DNA-
P. yoelii and vaccinia-P. yoelii vectors. Since the native
PY03661 gene does not contain a signal sequence, the vac-
cinia-PY03661 expression cassette was constructed with a
DAF signal sequence. Therefore, the DNA-PY03661 vec-
tor expresses a PY03661 protein with a TPA signal
sequence and the vaccinia-PY03661 vector expresses a
PY03661 protein with a DAF signal sequence.
The DNA-P. yoelii vaccines were manufactured to
pre-clinical grade specifications by Puresyn, Inc. (Mal-
vern, PA). The vaccinia-P. yoelii vaccines were propa-
gated in RK-13 cells (rabbit kidney cells; ATCC CCL37)
using standard laboratory procedures [22].
Mice and parasites
Female CD1 outbred mice (5-6 weeks old) were pur-
chased from Charles River Laboratories (Wilmington,
MA). P. yoelii (17XNL non-lethal strain) parasites were
maintained by alternating passage in Anopheles stephensi
mosquitoes and female CD1 outbred mice.
Production of recombinant P. yoelii proteins
PY03011, PY03424 (exon 2) and PY03661 recombinant
proteins were generated with a wheat germ cell-free
expression system [23]. In brief, P. yoelii gene-specific
RNA was generated from a plasmid containing an SP6-
promoted P. yoelii-glutathione S-transferase (GST) tagged
construct with SP6 RNA polymerase. The P. yoelii RNA
transcripts were translated into recombinant P. yoelii pro-
tein in a wheat germ cell-free extract (CellFree Sciences
Co., Yokohama, Japan). The GST-tagged P. yoelii proteins
were affinity purified with a glutathione sepharose resin
and cleaved from the GST tag with tobacco etch virus pro-
tease (Invitrogen Corp., Carlsbad, CA).
Generation of P. yoelii protein-specific antisera with
recombinant P. yoelii proteins
Female CD1 mice were injected subcutaneously in the
tail and scruff of the neck on days 0 and 29 with 10 μg
of PY03661, PY03424 (exon 2) or PY03661 recombinant
protein adjuvanted in Montanide™ ISA720. On day 38,
the mice were bled and P. yoelii protein-specific antisera
prepared.
Indirect fluorescent antibody analyses
Indirect fluorescent antibody (IFA) assays with sporo-
zoite and blood stage parasites were performed as pre-
viously described [24]. In brief, serial dilutions of
P. yoelii protein-specific antisera were incubated with
P. yoelii (17XNL) sporozoites or blood from P. yoelii-
infected mice. Parasites were visualized with a fluores-
cein isothiocyanate (FITC) conjugated goat anti-mouse
IgG (KPL Inc., Gaithersburg, MD). IFA analyses with
liver stage parasites were performed as previously
described [25]. In brief, mice were infected with P. yoelii
sporozoites and livers were harvested 48 hours post-
infection. P. yoelii-infected liver sections were prepared
and incubated with P. yoelii protein-specific antisera.
Parasites were visualized with a FITC conjugated goat
anti-mouse IgG. Evans blue (0.02%) counterstain was
added to the secondary antibody, providing a red back-
ground to contrast the green FITC fluorescence when
excited at the same wavelength.
In vitro expression analyses
Protein expression from the DNA and vaccinia vectors
was evaluated by Western blot analyses. DNA-P. yoelii
plasmids were transfected into RK-13 cells with lipofec-
tamine 2000CD (Invitrogen Corp., Carlsbad, CA). Vacci-
nia-P. yoelii infections were performed in RK-13 cells.
Cell lysates were run on 4-20% Tris-Glycine acrylamide
gels (Invitrogen Corp., Carlsbad, CA), transferred to
Immobilon-P polyvinylidene difluoride (PVDF) mem-
branes (Millipore Corp., Bedford, MA) and probed with
antisera from mice immunized with PY03011, PY03424
or PY03661 vaccines. Proteins were detected with an
alkaline phosphatase Western-Light Chemiluminescent
Detection System (Tropix Inc., Bedford, MA) and an
alkaline phosphatase colorimetric substrate (KPL Inc.,
Gaithersburg, MD).
Protection studies
Female CD1 mice were injected intramuscularly in the
tibialis anterior muscle with 100 μl of vaccine (50 μl in
each leg) using a 0.3 ml syringe and a 29G1/2 needle
(Becton Dickinson Co., Franklin Lakes, NJ) fitted with a
plastic collar cut from a micropipette tip [26]. The DNA
vaccine vectors were prepared in 1X Phosphate Buffered
Saline (PBS) and diluted to the appropriate concentra-
tion for vaccination in 1X PBS. The vaccinia vaccine
vectors were prepared in 1 mM Tris (9.0) and diluted to
the appropriate concentration for vaccination in 1X
PBS. Mice were challenged intravenously in the tail vein
with 300 P. yoelii (17XNL) sporozoites using a 1 ml syr-
inge and 26G1/2 needle (Becton Dickinson Co., Franklin
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Lakes, NJ). Sporozoites were hand dissected from
infected mosquito salivary glands and diluted for chal-
lenge in M199 medium containing 5% normal mouse
serum (Gemini Bio-Products, West Sacramento, CA).
In protection study 1, 14 mice per group were primed
on day 0 with 100 μg of the appropriate DNA-P. yoelii
vaccine vector and boosted on day 40 with 5 × 107
plaque forming units (pfu) of the corresponding vacci-
nia-P. yoelii vaccine vector. Mice immunized with a
combination of vectors expressing PY03011, PY03424
and PY03661 were primed with a total of 300 μg of the
DNA-P. yoelii vectors and boosted with a total of 1.5 ×
108pfu of the vaccinia-P. yoelii vectors. Vaccine vectors
expressing PyCSP were included in each study as a posi-
tive control. On day 50, the mice were bled and sera
prepared. On day 54, the mice were challenged with 300
P. yoelii sporozoites. On days 61-68, parasitaemia was
evaluated by examining Giemsa-stained blood smears.
Mice were considered positive if parasites were observed
in any sample. To gauge the severity of the challenge,
four groups of naïve CD1 mice were challenged with
four suboptimal doses of P. yoelii sporozoites (calculated
through serial dilution to be approximately 100, 33.3,
11.1 or 3.7 sporozoites per mouse). From these infectiv-
ity control mice, an ID50was calculated. (An ID50, or
infectious dose 50, equals the dose of sporozoites
required to infect 50% of the mice.) Extrapolation from
these results indicated that the mice injected with 300
P. yoelii sporozoites were challenged with a dose equiva-
lent to seven times the ID50dose.
In protection study 2, 14 mice per group were primed
on day 0 with 100 μg of the appropriate DNA-P. yoelii
vaccine vector and 30 μg of a DNA vector expressing
murine granulocyte-macrophage colony-stimulating fac-
tor (mGM-CSF) and boosted on day 42 with 3.3 × 107
pfu of the corresponding vaccinia-P. yoelii vaccine vec-
tor. Mice immunized with two or three DNA-P. yoelii
vectors were primed with a total of 200 μg or 300 μg of
the DNA-P. yoelii vectors and 30 μg of the DNA-mGM-
CSF vector and boosted with a total of 6.6 × 107pfu or
1 × 108pfu of the vaccinia-P. yoelii vectors. Three sepa-
rate groups of negative control mice were immunized
with three different doses of DNA and vaccinia vectors
that do not express a P. yoelii antigen. One group was
primed with 100 μg of an “empty” DNA vector and 30
μg of a DNA-mGM-CSF vector and boosted with 3.3 ×
107pfu of an “empty” vaccinia vector. A second group
was primed with 200 μg of an “empty” DNA vector and
30 μg of a DNA-mGM-CSF vector and boosted with 6.6
× 107pfu of an “empty” vaccinia vector. A third group
was primed with 300 μg of an “empty” DNA vector and
30 μg of a DNA-mGM-CSF vector and boosted with 1
× 108pfu of an “empty” vaccinia vector. On day 52, the
mice were bled and sera prepared. On day 57, the mice
were challenged with 300 P. yoelii sporozoites. On days
64-71, parasitaemia was evaluated by examining
Giemsa-stained blood smears. Mice were considered
positive if parasites were observed in any sample. To
gauge the severity of the challenge, four groups of naive
mice were challenged with four suboptimal doses of
P. yoelii sporozoites (100, 33.3, 11.1 or 3.7 sporozoites).
From these infectivity control mice, it was calculated
that the mice injected with 300 P. yoelii sporozoites in
this study were challenged with a dose equivalent to
13.6 times the ID50dose.
The regimens for the two protection studies were
slightly different. For example, the dose of the individual
vaccinia-P. yoelii vectors was slightly higher in protec-
tion study 1 (5 × 107pfu) than protection study 2 (3.3 ×
107pfu). Consequently, the total dose of the trivalent
vaccine was 1.5 × 108pfu in protection study 1 and 1 ×
108pfu in protection study 2. Additionally, in protection
study 2, the DNA vectors were mixed with a DNA-
mGM-CSF plasmid. Although previous studies had indi-
cated that co-administration of a DNA-PyCSP vector
with a DNA-mGM-CSF plasmid could enhance the
immunogenicity and efficacy of a DNA-vaccinia prime-
boost regimen [27], this enhancement is greater in
inbred mouse strains (BALB/c and C57BL/6) than
outbred strains [28]. Therefore, it is not surprising that
the DNA-mGM-CSF plasmid did not appear to enhance
the efficacy of the PyCSP or trivalent P. yoelii vaccines
in protection study 2, relative to protection study 1.
Statistical analyses
Protection results were analyzed by a Fisher’s Exact Test
with GraphPad Prism 5.03 software (GraphPad Software
Inc., LaJolla, CA).
Results
Genomic characterization of three P. yoelii vaccine
antigens
Analysis of pre-erythrocytic P. falciparum proteins with
sera from human volunteers immunized with a P. falci-
parum irradiated sporozoite vaccine identified 20 pro-
mising vaccine antigens [19]. To evaluate the vaccine
potential of these proteins in a murine protection
model, vaccine vectors that express the P. yoelii ortholog
of three of these antigens, PY03011, PY03424 and
PY03661, were generated.
PY03011
PY03011 is predicted by PlasmoDB [29], a Plasmodium
database, to be the ortholog of the P. falciparum gene,
PF13_0012. PF13_0012 is predicted by PlasmoDB to be a
single exon gene that encodes a protein that is 229 amino
acids long. PY03011 is predicted by PlasmoDB to contain
two exons and encode a protein that is 241 amino acids
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long. The first exon is predicted to encode the first 16
amino acids and the second exon is predicted to encode
the remaining 225 amino acids. A previous study, how-
ever, annotated the PY03011 gene to be a single exon
gene that encodes a protein that is 220 amino acids long
[30]. The re-annotated PY03011 protein is more homolo-
gous to PF13_0012 than the PlasmoDB-annotated
PY03011 protein (30% vs. 28%). The single exon annota-
tion is also consistent with the annotations of the P. falci-
parum, Plasmodium berghei, Plasmodium chabaudi,
Plasmodium knowlesi and Plasmodium vivax orthologs
of this gene, which are predicted by PlasmoDB to be sin-
gle exon genes. Based upon these data, the studies in this
report were performed with a single exon PY03011 gene
that encodes a protein that is 220 amino acids long [30].
The re-annotated PY03011 protein is predicted to
contain a signal sequence with a cleavage site between
amino acids 30-31 and a transmembrane domain
between amino acids 59-81. IFA analyses with PY03011-
specific antisera indicate that PY03011 is expressed in
the sporozoite, but not in the liver or blood stages of
the P. yoelii life-cycle (Figure 1). A previous study indi-
cated that this protein was expressed in the sporozoite
and liver stages [13]. Therefore, it is likely that PY03011
is expressed in the liver, but at levels that are below the
level of detection with the serological reagents used in
the present study. The genetic characteristics of the re-
annotated PY03011 gene are summarized in Table 1.
PY03424
PY03424 is predicted by PlasmoDB to be the ortholog of
the P. falciparum gene, PFI0580c. PFI0580c is predicted
by PlasmoDB to contain two exons and encode a protein
that is 413 amino acids long. The first exon is predicted to
encode the first 22 amino acids and the second exon is
predicted to encode the remaining 391 amino acids.
PY03424 is predicted by PlasmoDB to contain two exons
and encode a protein that is 1,856 amino acids long. The
first exon is predicted to encode the first 1,521 amino
acids and the second exon is predicted to encode the
remaining 335 amino acids. We believe that PY03424 is
not annotated correctly and have re-annotated this gene.
The re-annotated PY03424 gene is predicted to contain
two exons and encode a protein that is 357 amino acids
long. The first exon of the re-annotated gene encodes 22
amino acids and the second exon encodes the remaining
335 amino acids. The re-annotated PY03424 protein is sig-
nificantly more homologous to PFI0580c than the Plas-
moDB-annotated PY03424 protein (33% vs. 6%). A
comparison of PY03424 with other Plasmodium orthologs
also suggests that the PlasmoDB annotation is not correct.
The re-annotated PY03424 protein is predicted to
contain a signal sequence with a cleavage site between
amino acids 21-22. IFA analyses with PY03424-specific
antisera indicate that PY03424 is expressed in sporo-
zoites, on the parasitophorous vacuole of the liver stage
and in the blood stage (Figure 1). This profile is similar
to the expression profile of the P. falciparum PFI0580c
ortholog, which is expressed in the sporozoite, liver and
blood stages of the P. falciparum life-cycle [14,15,19].
The genetic characteristics of the re-annotated PY03424
gene are summarized in Table 1.
PY03661
PY03661 is predicted by PlasmoDB to be the ortholog of
the P. falciparum gene, PFC0555c. PFC0555c is predicted
by PlasmoDB to be a single exon gene and encode a pro-
tein that is 233 amino acids long. PY03661 is predicted to
be a single exon gene and encode a protein that is 225
amino acids long. The homology between the PFC0555c
and PY03661 proteins is 60%. PY03661 does not appear to
contain a signal sequence or a transmembrane domain.
IFA analyses with PY03661-specific antisera indicate that
PY03661 is expressed in sporozoites, but not in the liver
or blood stages (Figure 1). The P. falciparum PFC0555c
ortholog is expressed in the sporozoite and liver stages of
the P. falciparum life-cycle [19]. Since PFC0555c is
expressed in the liver stage, it is likely that PY03661 is also
expressed in the liver, but at levels that are below the level
of detection with the serological reagents used in this
study. The genetic characteristics of PY03661 are summar-
ized in Table 1.
Construction and analyses of DNA and vaccinia virus
vaccine vectors expressing PY03011, PY03424 or PY03661
DNA and vaccinia virus vaccine vectors expressing the
re-annotated PY03011 gene, the second exon of the re-
annotated PY03424 gene or the PY03661 gene were
generated. Western blot analyses with antigen-specific
antisera indicate that the DNA (Figure 2) and vaccinia
virus (Figure 3) vectors express the appropriate P. yoelii
protein.
Protection studies in P. yoelii/mouse model
To evaluate the vaccine potential of the three P. yoelii
antigens, CD1 outbred mice were immunized in a hetero-
logous prime-boost regimen with DNA and vaccinia
virus vectors that express PY03011, PY03424 or
PY03661, or a combination of these vectors (Table 2). As
a positive control, mice were immunized with DNA and
vaccinia vectors that express P. yoelii CSP (PyCSP). As a
negative control, mice were immunized with “empty”
DNA and vaccinia vectors that do not express a P. yoelii
protein. Two weeks after the vaccinia vector boost, the
mice were challenged with 300 P. yoelii sporozoites.
Seven through fourteen days after the challenge, protec-
tion against blood stage parasitaemia was evaluated by
examining Giemsa-stained blood smears. None of the 14
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