Protective mechanisms operate by neutralizing antibodies against the merozoite surface proteins and surface Keywords attenuated live parasite; malaria; MSP1; Plasmodium; protective immun
Trang 1Vaccines against malaria – an update
Kai Matuschewski1and Ann-Kristin Mueller1,2
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.
Trang 2proteins 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 acprotec-tive 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
Trang 3after 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
Targets
CSP
RI RIII TSR GPI
AMA-1 I II III
TRAP
A-domainTSR CTD
RTS/S HbS
HbS
MSP1
GPI
var2CSA 1x 2x 3x 4ε 5 ε 6ε
DBL
Research
PfEMP1s
ATS DBL CIDR
α 1 β γ δ 2 ε Preclinical
Phase III Clinical Phase
AMA-1 I II III
EGF p83 p30 p38 p42 FMPI p42 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
Trang 4RTS⁄ 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 Aotusmonkey 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 sursur-face 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 eryery-throcytes 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 epider-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.
Trang 5clinical 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
Trang 6[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 Anophelesvector 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
Trang 713 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 falciparummalaria 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 falciparumdisease 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 falciparumcircumsporozoite 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
Trang 8phase 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 falciparummerozoite 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
falciparumsporozoites 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
falciparuminvolved 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 falciparumsporozoites 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 bergheiliver-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 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