Tài liệu Báo cáo khoa học: Vaccines against malaria – an update doc

MINIREVIEW
Vaccines against malaria – an update
Kai Matuschewski
1
and Ann-Kristin Mueller
1,2
1 Department of Parasitology, Heidelberg University School of Medicine, Germany
2 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, UK
Malaria is a preventable and treatable vector-borne
infectious disease that is caused by single-cell eukary-
otic parasites of the genus Plasmodium. According to
recent estimates by the World Health Organization
(WHO), malaria remains one of the major causes of
mortality and morbidity, with 3.2 billion people at
risk, 300–500 million clinical cases and more than one
million deaths annually, particularly in young children
in sub-Saharan Africa [1].
Plasmodium transmission occurs by the injection of
infectious sporozoites during the probing phase for a
blood meal by an infected female Anopheles mosquito
[2]. Sporozoites actively move away from the site of
injection, enter a capillary and within minutes reach
the liver where they transform into liver stages and
commit to continuous replication resulting in the
generation of tens of thousands of pathogenic merozo-
ites [3]. Malaria-associated pathology is exclusively
restricted to the asexual replication of the parasite
within erythrocytes, a rather unique environment for
an intracellular pathogen. This terminally differenti-
ated host cell offers the advantage of complete absence
of MHC I-restricted antigen presentation, and, hence

cellular immunity against the host cell. Protective
mechanisms operate by neutralizing antibodies
against the merozoite surface proteins and surface
Keywords
attenuated live parasite; malaria; MSP1;
Plasmodium; protective immunity; RTS ⁄ S;
severe disease; transmission-blocking
antibodies; vaccine; var2CSA
Correspondence
K. Matuschewski, Department of
Parasitology, Heidelberg University School
of Medicine, Im Neuenheimer Feld 324,
69120 Heidelberg, Germany
Fax: +49 6221 564643
Tel: +49 6221 568284
E-mail: Kai.Matuschewski@med.
uni-heidelberg.de
(Received 27 May 2007, accepted 19 July
2007)
doi:10.1111/j.1742-4658.2007.05998.x
Malaria vaccine discovery and development follow two principal strategies.
Most subunit vaccines are designed to mimic naturally acquired immunity
that develops over years upon continuous exposure to Plasmodium trans-
mission. Experimental model vaccines, such as attenuated live parasites
and transmission-blocking antigens, induce immune responses superior to
naturally acquired immunity. The promises and hurdles of the different
tracks towards an effective and affordable vaccine against malaria are dis-
cussed.
Abbreviations
CSP, circumsporozoite protein; FMP1, falciparum malaria protein-1; GAP, genetically attenuated parasite; MSP1, merozoite surface protein 1;

PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; Pfs25, Plasmodium falciparum surface protein with apparent molecular
mass of 25 kDa; RTS ⁄ S, recombinant P. falciparum CSP vaccine, which includes the central repeat sequence ‘R’ and major T-cell epitopes
‘T’, fused to the entire hepatitis B surface antigen ‘S’ and coexpressed in yeast with the ‘S’ antigen; TRAP, thrombospondin-related
anonymous protein; var2CSA, variant surface antigen 2, chondroitin sulphate A-binding.
4680 FEBS Journal 274 (2007) 4680–4687 ª 2007 The Authors Journal compilation ª 2007 FEBS
proteins of the Plasmodium falciparum-infected eryth-
rocyte (Fig. 1), typically resulting in parasite reduction
rather than clearance [4]. A lower parasite burden may
then account for some of the antidisease effects.
In analogy to virtually any vector-borne disease,
vector control and exposure prophylaxis are two basic
public health tools that, when combined with clinical
management, protect the individual from fatal disease
and limit the spread of malaria [5]. However, examples
of other arthropod-transmitted infectious diseases,
such as the mosquito-transmitted yellow fever virus
and tick-borne encephalitis, teach us that for an effi-
cient eradication of the disease a safe vaccine is
needed. In the absence of a licensed malaria vaccine,
numerous different strategies are currently being tested
to develop one [6–10]. In this review, we highlight
some of the most recent developments towards a
malaria vaccine.
Naturally acquired immunity
)
imitate
nature to advance the immune
responses of malaria-naı
¨
ve individuals

A key observation in malaria-endemic regions is the
gradual acquisition of protective immune responses as
infants grow older and continue to be exposed to Plas-
modium transmission. By the time children reach
adolescence they typically mount strong antibody
responses against the surface proteins of merozoites
and infected erythrocytes. Indeed, an influential study
demonstrated early on that the passive transfer of
immunoglobulins from semi-immune adults cures the
clinical complications of malaria [11]. This finding pro-
vided the conceptual framework for malaria vaccine
development, i.e. to speed up the generation of protec-
tive immune responses by active immunization with
protective Plasmodium antigens.
Apart from the undesirable slow kinetics of immune
acquisition observed in endemic areas, the fundamental
limitation of this strategy is the identification of pro-
tective as opposed to immunogenic antigens. Assays
that permit the identification of correlates of protec-
tion are largely limited to cytoadhesion of P. falcipa-
rum-infected erythrocytes. Therefore, malaria vaccine
research in the past was largely empirical and driven
by vaccine development rather than by vaccine discov-
ery, which typically comes first. Moreover, as candi-
date formulations progress to larger clinical studies,
another critical obstacle emerges: subunit vaccines typ-
ically have little, if any, impact on overall parasitemia
[12,13], which would constitute an ideal endpoint –
being both highly desirable and easy to measure.
Another endpoint that does matter is disease severity,

which serves as a predictor for morbidity and mortal-
ity. However, ‘severe malaria’ is a very complex multi-
system disorder [14] and remains an ill-defined
endpoint. Moreover, vaccine trials differ fundamentally
from clinical management studies in their study popu-
lation, i.e. healthy individuals versus patients. Because
the incidence of severe malaria is relatively low in any
given study population, most studies remain consider-
ably underpowered for this outcome, except for large
cohort studies such as the recent successful recombi-
nant P. falciparum circumsporozoite protein (CSP)
vaccine (RTS ⁄ S) trial in Mozambique [13].
The first subunit vaccine, which was rapidly acceler-
ated to phase III clinical trials, was SPf66 [12]. This
vaccine, which consists of short peptide sequences of the
two major glycosylphosphatidyl inositol (GPI)-anchored
surface proteins of the invasive stages, merozoite pro-
tein 1 (MSP1) and CSP (Fig. 1), together with two
uncharacterized peptide fragments, initially showed
promising protection in an open human challenge study
with P. falciparum-infected erythrocytes [15] and first
field trials in South America, yet failed to confer substan-
tial protection against natural malaria transmission in
subsequent clinical trials in other endemic countries [12].
The two parasite surface proteins remain the major
candidate antigens that are being developed and tested
in various formulations. Strong support for these anti-
gens comes from two landmark studies that addressed
the relative importance of CSP and MSP1 in protective
immunity. In an engineered PyCSP-tolerant mouse

system Plasmodium yoelii CSP was shown to contri-
bute to protection in the irradiated sporozoite vaccine
model (see below) [16]. These PyCSP-transgenic mice
can now be explored to identify additional protective
pre-erythrocytic antigens that together with CSP con-
fer sterile long-lasting protection in the rodent malaria
model system. Using a population genetics approach
a small N-terminal oligomorphic region of MSP1,
termed block 2, was identified as a likely target of
acquired immunity in endemic populations [17]. Anti-
body responses against this region appeared to be
strongly associated with protection against clinical
malaria.
The RTS
⁄
S vaccine
)
leading present
subunit vaccine research
Currently, the RTS ⁄ S vaccine ) a CSP fragment cov-
ering the central repeat peptide and the C-terminal
T-cell epitope fused to the hepatitis B surface antigen
(Fig. 1) formulated in the proprietary adjuvant
AS02A ) is the most advanced candidate [18,19].
RTS ⁄ S was moved on to proof-of-concept field trials
K. Matuschewski and A K. Mueller Anti-malaria vaccine development
FEBS Journal 274 (2007) 4680–4687 ª 2007 The Authors Journal compilation ª 2007 FEBS 4681
after phase I⁄ IIa trials, where initially 6 of 7 and, more
recently, 40% of malaria-naı
¨

ve individuals remained
protected against a single mosquito challenge [20–22].
Moreover, a consistent delay in patency in those indi-
viduals that became infected indicates that the vaccine
eliminates 90% of the sporozoite inoculum. The antici-
pated outcomes of two large phase IIb trials, one in
adult men in Gambia [23] and one in young children
in Mozambique [13,24], were partial delay of infection
and a trend towards reduction of clinical malaria.
Unexpectedly, the vaccine also showed a 58% efficacy
in reducing the incidence of severe disease, an impor-
tant finding that awaits confirmation in other epi-
demiological settings. Together the outcomes were
interpreted as indicating significant protection against
natural P. falciparum infection and the vaccine was
advanced to a large-scale, multicenter phase III trial
[19]. As expected, RTS ⁄ S did not induce T-cell epitope
selection indicating that cell-mediated immunity may
not be the major protective mechanism [25]. Somewhat
uncommonly, the elementary findings of corresponding
phase I studies on the safety and immunogenicity of
Sporozoite invasion
Anti-parasite:
Anti-parasite:
Anti-parasite:
Inhibitory antibodies reduce inoculation dose
Merozoite invasion
Inhibitory antibodies prevent high parasitemia
Cytoadhesion
Anti-disease:

Inhibitory antibodies block sequestration
TargetsPlasmodium life cycle stage Vaccine
CSP
RI RIII TSRGPI
AMA-1
I II III
TRAP
A-domainTSR CTD
RTS/S
HbS
HbS
MSP1
GPI
var2CSA
5
ε
6
ε
4
ε
3x2x1x
DBL
Research
PfEMP1s
ATSDBL CIDR
α β
γ
δ ε
12
Preclinical

Phase III
Clinical Phase
AMA-1
IIIIII
EGF
p83 p30 p38 p42
p42
FMPI
bII
MSP3
SPAM
Phase I
Phase I
Phase I
Phase II
Phase IIb
Ookinete penetration
Altruistic vaccine:
Inhibitory antibodies reduce transmission rate
Pfs25
EGF
Pfs28
EGF
Liver stage maturation
IFNγ-secreting T-cells destroy infected hepatocytes
Research
Research
IFNγ
GAP
Anti-malaria vaccine development K. Matuschewski and A K. Mueller

4682 FEBS Journal 274 (2007) 4680–4687 ª 2007 The Authors Journal compilation ª 2007 FEBS
RTS ⁄ S ⁄ AS02A in children were reported only after the
phase IIb trial [26,27]. Importantly, the formulation
was safe and highly immunogenic for antibody
responses against both P. falciparum CSP and the hep-
atitis B surface antigen.
The anticipated continued success of the
RTS ⁄ S ⁄ AS02A formulation in the induction of signifi-
cant protective immune responses will greatly influence
next-generation subunit vaccine developments. Critical
issues are: (a) the selection of additional antigens to
build on the CSP fragment; (b) adjuvant selection,
which made a major contribution to the efficacy of
RTS ⁄ S [19]; and (c) whether a partially protective vac-
cine would be a valuable public health tool. Notably,
there are numerous alternative CSP-based strategies
under preclinical and clinical development [7], such as
linear peptides that contain minimal T- and B-cell epi-
topes [28], and Plasmodium vivax long synthetic pep-
tides [29].
Catching up
)
MSP1-based vaccines
Prior to formulations with recombinant proteins
native, affinity-purified MSP1 was tested in three Aotus
monkeys in a pilot challenge study and was shown to
confer complete protection against inoculation with
the blood stages of a lethal P. falciparum strain [30].
Because of its central function for merozoite invasion
MSP1 is under high natural selection resulting in the

maintenance of allelic variation [17]. However, MSP1
is composed of modules that constitute the four
subunits and most natural variants are derived from
two prototypes only. Therefore, a mixture of recombi-
nant codon-optimized full-length MSP1 constructs is
feasible and is currently in the preclinical phase [31].
Using a formulation that is conceptually similar to
RTS ⁄ S ⁄ AS02, recent progress has been made to
advance a vaccine based on the C-terminal p42 frag-
ment, termed falciparum malaria protein-1 (FMP1)
[32,33]. The encouraging safety and immunogenicity
profiles of FMP1 ⁄ AS02A allowed its entry into proof-
of-concept phase IIb trials. However, choice of the
C-terminal p42 fragment remains problematic in the
absence of a clear association with protection.
Two additional targets stand out among the numer-
ous potential merozoite surface and secretory proteins
and are currently being developed further for vaccine
trials: merozoite surface protein 3 (MSP3) fulfills many
crucial criteria for a potential vaccine candidate:
(a) induction of protective immune responses in the
Aotus monkey challenge model [34], (b) direct proof of
an effector mechanism through a process termed
antibody-dependent cellular inhibition [35], and (c)
association of allele-specific natural responses with
protection from clinical malaria [36]. Apical membrane
antigen 1 constitutes a potential multistage vaccine in
itself because it appears to play important roles both
during merozoite and sporozoite host cell entry
(Fig. 1) [37].

var2CSA
)
a case for a tailor-made
subunit vaccine
A hallmark of P. falciparum blood-stage infections is
the presence of parasite-encoded antigens on the sur-
face of infected erythrocytes. These variant surface
antigens (VSAs) mediate the adhesion of infected ery-
throcytes to endothelial cells and cause many of the
Fig. 1. Vaccine strategies against malaria. Natural transmission to the human host (upper) may be reduced by high titers of sporozoite-neu-
tralizing antibodies that act prior to hepatocyte entry. In addition, vaccination with sporozoite antigens may induce cell-mediated responses
to the infected hepatocyte. The progression of pathogenic blood stages can be reduced during the brief phase of merozoite entry into ery-
throcytes (second row). P. falciparum-infected erythrocytes adhere to endothelial cells (center) in capillaries and the placenta through para-
site-encoded surface proteins that eventually lead to antibody recognition. Maturation of liver-stage schizonts (lower center) and ookinete
pentration of the mosquito midgut (lower) represent two immunology silent stages of the Plasmodium life cycle. GAPs elicit long-lasting
complete protection in experimental models. These genetically defined parasites are inoculated as sporozoites and invade and transform nor-
mally, but arrest during subsequent liver-stage development. At this time they likely display protective antigens (yellow) in the context of
MHC class I presentation (green) that, in turn, activate interferon-c-secreting effector T cells (blue). Two partially redundant ookinete surface
proteins, Pfs25 and Pfs28, constitute attractive targets for the development of transmission blocking vaccines. Targets for vaccine develop-
ment include surface proteins (red) or adhesion proteins (green) of invasive stages. Shown are the primary structures and known protein
domains (colored boxes) for selected vaccine candidates and lead vaccines. Cleavable signal peptides and transmembrane spans are boxed
in red and black, respectively. The current developmental status is shown to the right. I-III, AMA-1 domains; A-domain, von Willebrand factor
A-domain; AMA-1, apical membrane antigen 1; ATS, acidic terminal segment; bII, block 2 oligomorphic region of MSP1; CIDR, cysteine-rich
interdomain region; CSP, circumsporozoite protein; CTD, TRAP-family cytoplasmic tail domain; DBL, Duffy-binding like domain; EGF, epider-
mal growth factor domain; FMP1, falciparum malaria protein-1; GPI, glycosylphospatidyl inositol anchor; HbS, Hepatitis B surface antigen;
MSP, merozoite surface protein; PfEMP1, P. falciparum erythrocyte membrane protein 1; RI, region I; RIII, region III; SPAM, secreted poly-
morphic antigen associated with merozoites; TSR, thrombospondin type I repeat; var2CSA, variant surface antigen 2, chondroitin sulphate
A-binding.
K. Matuschewski and A K. Mueller Anti-malaria vaccine development
FEBS Journal 274 (2007) 4680–4687 ª 2007 The Authors Journal compilation ª 2007 FEBS 4683

clinical complications of malaria infection. Nonethe-
less, they also elicit strong protective immune
responses [38]. The most straightforward explanation
is that iterative recognition of individual VSAs upon
continuous Plasmodium exposure eventually results in
naturally acquired immunity to severe disease. The
best-characterized family of VSAs is the var gene
family, which encodes for  60 different P. falciparum
erythrocyte membrane proteins (PfEMP1s; Fig. 1)
and undergoes clonal antigenic variation [38]. This
remarkable antigenic repertoire partially explains the
slow kinetics of naturally acquired immunity and
poses tremendous problems for direct vaccine
research. Unless a subfraction of the most deleterious
PfEMP1s can be identified, mimicking natural immu-
nity with PfEMP1-based subunit vaccines remains a
distant vision ) except for one unique, structurally
distinct PfEMP1 variant, termed variant surface anti-
gen 2, chondroitin sulphate A-binding (var2CSA)
(Fig. 1) [39]. High antibody titers correlate specifically
with protection against pregnancy-associated malaria
[40], a serious complication with poor outcomes such
as low birthweight and preterm delivery due to
sequestration in the placenta. Although additional
VSAs are likely contribute to the pathology, a
var2CSA-based vaccine may induce substantial pro-
tective maternal immune responses similar to those
detected in women after multiple pregnancies [41].
Composed strategies
)

better than
nature?
One central obstacle in malaria vaccine discovery is
the absence of sterilizing immunity during natural
infection. Our current portfolio of successful vaccines
acts against acute viral or bacterial infections. The cor-
responding whole-organism vaccines mimic an acute
pathogen infection, which were known to function as a
natural vaccination after the host immune system
resolved the first infection. There is no such model of
acquired immunity against the Plasmodium parasite.
Yet, a malaria vaccine will only become an efficient
public health tool if it provides protection for several
years with no more than three immunizations.
One potential, yet challenging, solution to this prob-
lem may be the composition of vaccine strategies that
aim at inducing protective immune responses against
immunological silent Plasmodium life cycle stages, i.e.
those that are not the typical targets of naturally
acquired immunity (Fig. 1). Recent insights into the par-
asite biology and technological advancements open the
possibility to explore such alternative vaccine strategies.
Whole-parasite vaccines
The first, and as yet unsurpassed, success in inducing
protective immune responses against malaria was
achieved with irradiated sporozoites in a rodent
malaria model system [42]. Immunization of mice with
three doses of c-irradiated sporozoites results in atten-
uated liver-stage development and elicits complete
sustained protection against sporozoite challenge.

Analogous to other live-attenuated vaccines, arrested
Plasmodium liver stages likely induce protective cell-
mediated immune responses against the entire anti-
genic repertoire of the liver stage and may be the most
potent malaria vaccine. But is it worth investing in
a complex live, attenuated liver-stage vaccine, as
opposed to an economically more viable subunit strat-
egy that is only limited by the number of potential tar-
get proteins?
Large-scale production of an attenuated parasite
vaccine may indeed become feasible, because some
challenges, such as sterility, cryopreservation, and
route of immunization, have either already been met
or are under active investigation [43]. Other roadblocks
related to the safety and batch-to-batch variation of
genetically undefined irradiated sporozoites have
recently been removed in the rodent malaria model
system by the generation of genetically attenuated par-
asites (GAPs) [44]. Although translation to the P. falci-
parum system may take several years, early human
challenge studies with irradiated sporozoites indicate
that complete attenuation of liver-stage development
elicits protection [45] –to date the gold-standard in
P. falciparum vaccine development. GAPs differ from
c-irradiated sporozoites in their consistent production,
genetic stability, and higher potency [46]. A fundamen-
tal issue is whether natural exposure to Plasmodium
transmission would boost GAP-induced immune
responses. If this was the case a GAP vaccine would
be feasible for individuals from malaria-endemic coun-

tries. Otherwise only short-term visitors would benefit
and GAPs would fall into the category of ‘boutique
vaccines’.
Irrespective of large-scale application, GAPs may
also become an excellent model to study sterilizing cel-
lular immunity and may thus lead to the identification
of potential protective liver-stage antigens. These anti-
gens could then be delivered intracellularly as DNA or
viral vectors. Such a strategy was advanced for throm-
bospondin-related anonymous protein (TRAP) and
tested in proof-of-concept phase IIb trials [47,48]. The
observed lack of protection correlates with the rapid
decrease of TRAP expression after sporozoite invasion
Anti-malaria vaccine development K. Matuschewski and A K. Mueller
4684 FEBS Journal 274 (2007) 4680–4687 ª 2007 The Authors Journal compilation ª 2007 FEBS
[49] and highlights the need to prioritize vaccine
targets based on immunological as well as biological
criteria.
Transmission-blocking antibodies
Induction of neutralizing antibody responses against
gametocyte and ookinete surface proteins that can
block the obligatory parasite fertilization, zygote
transformation and subsequent traversal of the mos-
quito midgut is an attractive strategy that would
result in interruption of the Plasmodium life cycle.
Two major ookinete surface proteins, termed Pfs25
and Pfs28 (Fig. 1), together perform essential func-
tions prior to oocyst development [50]. Because these
proteins are expressed only during transmission to
the mosquito vector, malaria-exposed individuals do

not mount Pfs25 ⁄ 28-specific immune responses [51].
The absence of immune pressure correlates with
remarkable sequence conservation. However, Pfs25 is
an intrinsically poor immunogenic antigen. This hur-
dle was recently overcome by the generation of pro-
tein–protein conjugates that proved to be highly
immunogenic in mice [52]. High antibody levels per-
sisted over months and these antibodies, when fed to
mosquitoes, blocked oocyst formation. The small size
and conservation of the Pfs25 ⁄ 28 proteins will expe-
dite vaccine development. Such a transmission-block-
ing vaccine is highly likely to be efficient against
malaria transmission and may prove to be an effi-
cient tool in combination with vector control and
exposure prophylaxis.
Projections
Recent promising developments have spurred new
hopes that development of a malaria vaccine may be
realistic. In an attempt to mimic naturally acquired
immunity, an impressive portfolio of subunit vaccines
against the major sporozoite and merozoite surface
proteins has been developed over the past two decades
[7]. One of them, the pre-erythrocytic CSP-based sub-
unit vaccine RTS ⁄ S, has recently entered phase III
clinical trials throughout Africa [19]. Genetically atten-
uated parasites [44] and transmission-blocking anti-
bodies [52] offer the advantage that they induce
complete inhibition of the Plasmodium life cycle, a
scenario not seen in the field. If these composed strate-
gies can be translated to disease-endemic countries,

and are safe and affordable, they may ultimately
become important public health tools against one of
the deadliest and most elusive infectious diseases. In
the meantime, global coverage of the conventional
triad, i.e. vector-control programs, exposure prophy-
laxis and clinical management, as suggested by Ronald
Ross nearly a century ago, must be supported.
Acknowledgements
We thank two anonymous reviewers for critical and
valuable suggestions. The work in the authors’ labora-
tory is supported by the research focus ‘Tropical Medi-
cine Heidelberg’ of the Medical Faculty of Heidelberg
University, and in part by grants from the Deutsche
Forschungsgemeinschaft (Ma 2161 ⁄ 3-2), the European
Commission (BioMalPar, #23), the Grand Challenges
in Global Health initiative, the Joachim Siebeneicher
Foundation and the Chica and Heinz Schaller Founda-
tion. AKM is a recipient of an EMBO long-term
fellowship.
References
1 World Health Organization (2005) World Malaria
Report 2005. WHO, Geneva.
2 Matuschewski K (2006) Getting infectious: formation
and maturation of Plasmodium sporozoites in the
Anopheles vector. Cell Microbiol 8, 1547–1556.
3 Prudencio M, Rodriguez A & Mota MM (2006) The
silent path to thousands of merozoites: the Plasmodium
liver stage. Nat Rev Microbiol 4, 849–856.
4 Marsh K & Kinyanjui S (2006) Immune effector mecha-
nisms in malaria. Parasite Immunol 28, 51–60.

5 Rieckmann KH (2006) The chequered history of
malaria control: are new and better tools the ultimate
answer? Ann Trop Med Parasitol 100, 647–662.
6 Richie TL & Saul A (2002) Progress and challenges for
malaria vaccines. Nature 415, 694–701.
7 Ballou WR, Arevalo-Herrera M, Carucci D, Richie TL,
Corradin G, Diggs C, Druilhe P, Giersing BK, Saul A,
Heppner DG et al. (2004) Update on the clinical devel-
opment of candidate malaria vaccines. Am J Trop Med
Hyg 71 (Suppl. 2), 239–247.
8 Good MF (2005) Vaccine-induced immunity to malaria
parasites and the need for novel strategies. Trends
Parasitol 21, 29–34.
9 Matuschewski K (2006) Vaccine development against
malaria. Curr Opin Immunol 18, 449–457.
10 Malkin E, Dubovsky F & Moree M (2006) Progress
towards the development of malaria vaccines. Trends
Parasitol 22, 292–295.
11 Cohen S, McGregor GI & Carrington S (1961)
Gamma-globulin and acquired immunity to human
malaria. Nature 192, 733–737.
12 Graves P & Gelband H (2006) Vaccines for preventing
malaria (SPf66). Cochrane Database System Rev
CD005966.
K. Matuschewski and A K. Mueller Anti-malaria vaccine development
FEBS Journal 274 (2007) 4680–4687 ª 2007 The Authors Journal compilation ª 2007 FEBS 4685
13 Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E,
Milman J, Mandomando I, Spiessens B, Guinovart C,
Espasa M et al. (2004) Efficacy of the RTS,S ⁄ AS02A
vaccine against Plasmodium falciparum infection and

disease in young African children: randomized con-
trolled trial. Lancet 364, 1411–1420.
14 Mackintosh CL, Beeson JG & Marsh K (2004) Clinical
features and pathogenesis of severe malaria. Trends
Parasitol 20, 597–603.
15 Patarroyo ME, Amador R, Clavijo P, Moreno A, Guz-
man F, Romero P, Tascon R, Franco A, Murillo LA,
Ponton G et al. (1988) A synthetic vaccine protects
humans against challenge with asexual blood stages of
Plasmodium falciparum malaria. Nature 332, 158–161.
16 Kumar KA, Sano G-I, Boscardin S, Nussenzweig RS,
Nussenzweig MC, Zavala F & Nussenzweig V (2006)
The circumsporozoite protein is an immunodominant
protective antigen in irradiated sporozoites. Nature 444,
937–940.
17 Conway DJ, Cavanagh DR, Tanabe K, Roper C, Mikes
ZS, Sakihama N, Bojang KA, Oduola AMJ, Kremsner
PG, Arnot DE et al. (2000) A principal target of human
immunity to malaria identified by molecular population
genetic and immunological analyses. Nat Med 6,
689–692.
18 Heppner DG, Kester KE, Ockenhouse CF, Tornieporth
N, Ofori O, Lyon JA, Stewart VA, Dubois P, Lanar
DE, Krzych U et al. (2005) Towards an RTS,S-based
multi-stage, multi-antigen vaccine against falciparum
malaria: progress at the Walter Reed Army Institute of
Research. Vaccine 23, 2243–2250.
19 Bojang KA (2006) RTS,S ⁄ AS02A for malaria. Expert
Rev Vaccines 5, 611–615.
20 Stoute JA, Slaoui M, Heppner DG, Momin P, Kester

KE, Desmons P, Wellde BT, Garcon N, Krzych U,
Marchand M et al. (1997) A preliminary evaluation of
a recombinant cicrumsporozoite vaccine against Plas-
modium falciparum malaria. N Engl J Med 336,
86–91.
21 Kester KE, McKinney DA, Torniepoth N, Ockenhouse
CF, Heppner DG, Hall T, Krzych U, Delchambre M,
Voss G, Dowler MG et al. (2001) Efficacy of recombi-
nant circumsporozoite protein vaccine regimens against
experimental Plasmodium falciparum malaria. J Infect
Dis 183, 640–647.
22 Kester KE, McKinney DA, Tornieporth N, Ockenhouse
CF, Heppner DG, Hall T, Wellde BT, White K, Sun P,
Schwenk R et al. (2007) A phase I ⁄ IIa safety, immuno-
genicity, and efficacy bridging randomized study of a
two-dose regimen of liquid and lyophilized formulations
of the candidate malaria vaccine RTS,S ⁄ AS02A in
malaria-naı
¨
ve adults. Vaccine 25, 5359–5366.
23 Bojang KA, Milligan PJM, Pinder M, Vigneron L,
Alloueche A, Kester K, Ballou WR, Conway DJ, Reece
WHH, Gothard P et al. (2001) Efficacy of RTS,S ⁄ AS02
malaria vaccine against Plasmodium falciparum infection
in semi-immune adult men in the Gambia: a random-
ized trial. Lancet 358, 1927–1934.
24 Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E,
Aide P, Sigauque B, Milman J, Mandomando I, Bassat
Q et al. (2005) Duration of protection with RTS,S ⁄
AS02A malaria vaccine in prevention of Plasmodium

falciparum disease in Mozambican children: single-blind
extended follow-up of a randomized control trial. Lan-
cet 366, 2012–2018.
25 Enosse S, Doban
˜
o C, Quelhas D, Aponte JJ, Lievens
M, Leach A, Sacarlal J, Greenwood B, Milman J, Dub-
ovsky F et al. (2006) RTS,S ⁄ AS02A malaria vaccine
does not induce parasite CSP T cell epitope selection
and reduces multiplicity of infection. PLoS Clinical
Trials 1, e5.
26 Bojang KA, Olodude F, Pinder M, Ofori-Anyinam O,
Vigneron L, Fitzpatrick S, Nije F, Kassanga A, Leach
A, Milman J et al. (2005) Safety and immunogenicity of
RTS,S ⁄ AS02A candidate malaria vaccine in Gambian
children. Vaccine 23, 4148–4157.
27 Macete E, Aponte JJ, Guinovart C, Sacarlal J, Ofori-
Anyinam O, Mandomando I, Espasa M, Bevilacqua C,
Leach A, Dubois MC et al. (2007) Safety and immuno-
genicity of the RTS,S ⁄ AS02A candidate malaria vaccine
in children aged 1–4 in Mozambique. Trop Med Int
Health 12, 37–46.
28 Calvo-Calle JM, Oliveira GA, Watta CO, Soverow J,
Parra-Lopez C & Nardin EH (2006) A linear peptide
containing mininmal T- and B-cell epitopes of Plasmo-
dium falciparum circumsporozoite protein elicits protec-
tion against transgenic sporozoite challenge. Infect
Immun 74 , 6929–6939.
29 Herrera S, Bonelo A, Perlaza BL, Fernandez OL, Victo-
ria L, Lenis AM, Soto L, Hurtado H, Acuna LM, Velez

JD et al. (2005) Safety and elicitation of humoral and
cellular responses in Colombian malaria-naı
¨
ve volun-
teers by a Plasmodium vivax circumsporozoite protein-
derived synthetic vaccine. Am J Trop Med Hyg 73
(Suppl. 5), 3–9.
30 Siddiqui WA, Tam LQ, Kramer KJ, Hui GSN, Case SE,
Yamaga KM, Chang SP, Chan EBT & Kan S-C (1987)
Merozoite surface coat precursor protein completely
protects Aotus monkeys against Plasmodium falciparum
malaria. Proc Natl Acad Sci USA 84, 3014–3018.
31 Pan W, Ravot E, Tolle R, Frank R, Mosbach R, Tur-
bachova I & Bujard H (1999) Vaccine candidate MSP-1
from Plasmodium falciparum
: a redesigned 4917 bp poly-
nucleotide enables synthesis and isolation of full-length
protein from Escherichia coli and mammalian cells.
Nucleic Acids Res 27, 1094–1103.
32 Withers MR, McKinney D, Ogutu BR, Waitumbi JN,
Milman JB, Apollo OJ, Allen OG, Tucker K, Soisson
LA, Diggs C et al. (2006) Safety and reactogenicity of
an MSP-1 malaria vaccine candidate: a randomized
Anti-malaria vaccine development K. Matuschewski and A K. Mueller
4686 FEBS Journal 274 (2007) 4680–4687 ª 2007 The Authors Journal compilation ª 2007 FEBS
phase Ib dose-escalation trial in Kenyan children.
PLoS Clinical Trials 1, e32.
33 Thera MA, Doumbo OK, Coulibaly D, Diallo DA,
Sagara I, Dicko A, Diemert DJ, Heppner DG, Stewart
VA, Angov E et al. (2006) Safety and allele-specific

immunogenicity of a malaria vaccine in mammalian
adults: results of a phase I randomized trial. PLoS
Clinical Trials 1, e34.
34 Hisaeda H, Saul A, Reece JJ, Kennedy MC, Long CA,
Miller LH & Stowers AW (2002) Merozoite surface pro-
tein 3 and protection against malaria in Aotus nancymai
monkeys. J Infect Dis 185, 657–664.
35 Druilhe P, Spertini F, Soesoe D, Corradin G, Mejia P,
Singh S, Audran R, Bouzidi A, Oeuvray C & Roussil-
hon C (2005) A malaria vaccine that elicits in human
antibodies able to kill Plasmodium falciparum. PLoS
Medicine 2, e344.
36 Polley SD, Tetteh KKA, Lloyd JM, Akpogheneta OJ,
Greenwood BM, Bojang KA & Conway DJ (2007)
Plasmodium falciparum merozoite surface protein 3 is a
target of allele-specific immunity and alleles are main-
tained by natural selection. J Infect Dis 195, 279–287.
37 Silvie O, Franetich JF, Charrin S, Mueller MS, Siau A,
Bodescot M, Rubinstein E, Hannoun L, Charoenvit Y,
Kocken CH et al. (2004) A role for apical membrane
antigen 1 during invasion of hepatocytes by Plasmodium
falciparum sporozoites. J Biol Chem 279, 9490–9496.
38 Kyes S, Horrocks P & Newbold C (2001) Antigenic var-
iation at the infected red cell surface in malaria. Annu
Rev Microbiol 55, 673–707.
39 Salanti A, Staalsoe T, Lavstsen T, Jensen AT, Sowa
MP, Arnot DE, Hviid L & Theander TG (2003) Selec-
tive upregulation of a single distinctly structured var
gene in chondroitin sulphate A-adhering Plasmodium
falciparum involved in pregnancy-associated malaria.

Mol Microbiol 49, 179–191.
40 Salanti A, Dahlba
¨
ck M, Turner L, Nielsen MA, Barford
L, Magistrado P, Jensen ATR, Lavstsen T, Ofori MF,
Marsh K et al. (2004) Evidence for the involvement of
VAR2CSA in pregnancy-associated malaria. J Exp Med
200, 1197–1203.
41 Fried M, Nosten F, Brockman A, Brabin BJ & Duffy
PE (1998) Maternal antibodies block malaria. Nature
395, 851–852.
42 Nussenzweig RS, Vanderberg J, Most H & Orton C
(1967) Protective immunity produced by the injection of
X-irradiated sporozoites of Plasmodium berghei . Nature
216, 160–162.
43 Luke TC & Hoffman SL (2003) Rationale and plans
for developing a non-replicating, metabolically active,
radiation-attenuated Plasmodium falciparum sporozoite
vaccine. J Exp Biol 206, 3803–3808.
44 Mueller A-K, Labaied M, Kappe SHI & Matuschewski
K (2005) Genetically modified Plasmodium parasites as
a protective experimental malaria vaccine. Nature 433
,
164–167.
45 Hoffman SL, Goh LM, Luke TC, Schneider I, Le TP,
Doolan DL, Sacci J, de la Vega P, Dowler M, Paul C
et al. (2002) Protection of humans against malaria by
immunization with radiation-attenuated Plasmodium
falciparum sporozoites. J Infect Dis 185, 1155–1164.
46 Jobe O, Lumsden J, Mueller A-K, Williams J, Silva-

Rivera H, Kappe SHI, Schwenk RJ, Matuschewski K &
Krzych U (2007) Genetically-attenuated Plasmodium
berghei liver-stages induce sterile protracted protection
that is mediated by MHC class I-dependent IFN-c pro-
ducing CD8+ T cells. J Infect Dis 196, 599–607.
47 Moorthy VS, Imoukuede EB, Milligan P, Bojang K,
Keating S, Kaye P, Pinder M, Gilbert SC, Walraven G,
Greenwood BM & Hill AS (2004) A randomized,
double-blind, controlled vaccine efficacy trial of
DNA ⁄ MVA ME-TRAP against malaria infection in
Gambian adults. PLoS Medicine 1, e33.
48 Bejon P, Mwacharo J, Kai O, Mwangi T, Milligan P,
Todryk S, Keating S, Lang T, Lowe B, Gikonyo C
et al. (2006) A phase 2b randomized trial of the candi-
date malaria vaccines FP9 ME-TRAP and MVA ME-
TRAP among children in Kenya. PLoS Clinical Trials
1, e29.
49 Wang Q, Brown S, Roos DS, Nussenzweig V & Bhanot
P (2004) Transcriptome of axenic liver stages of Plasmo-
dium yoelii. Mol Biochem Parasitol 137, 161–168.
50 Tomas AM, Margos G, Dimopoulos G, van Lin LH,
de Koning-Ward TF, Sinha R, Lupetti P, Beemtsma
AL, Rodriguez MC, Karras M et al. (2001) P25 and
P28 proteins of the malaria ookinete surface have multi-
ple and partially redundant functions. EMBO J 20,
3975–3983.
51 Carter R, Graves PM, Quakyi IA & Good MF (1989)
Restricted or absent immune responses in human popu-
lations to Plasmodium falciparum gamete antigens that
are targets of malaria transmission-blocking antibodies.

J Exp Med 169, 135–147.
52 Kubler-Kielb J, Majadly F, Wu Y, Narum DL, Guo C,
Miller LH, Shiloach J, Robbins JB & Schneerson R
(2007) Long-lasting and transmission-blocking activity
of antibodies to Plasmodium falciparum elicited in mice
by protein conjugates of Pfs25. Proc Natl Acad Sci
USA 104, 293–298.
K. Matuschewski and A K. Mueller Anti-malaria vaccine development
FEBS Journal 274 (2007) 4680–4687 ª 2007 The Authors Journal compilation ª 2007 FEBS 4687