608 • JID 2007:196 (15 August) • Tarun et al.
M A J O R A R T I C L E
Protracted Sterile Protection
with Plasmodium yoelii Pre-erythrocytic
Genetically Attenuated Parasite Malaria Vaccines
Is Independent of Significant Liver-Stage
Persistence and Is Mediated by CD8+T Cells
Alice S. Tarun,1Ronald F. Dumpit,1Nelly Camargo,1Mehdi Labaied,1Pu Liu,1Akihide Takagi,1Ruobing Wang,1
and Stefan H. I. Kappe1,2
1Seattle Biomedical Research Institute and
2Department of Pathobiology, University of Washington, Seattle
(See the article by Jobe et al., on pages 599–607.)
Irradiation-attenuated sporozoite vaccinations confer sterile protection against malaria infection in animal
models and humans. Persistent, nonreplicating parasite forms in the liver are presumably necessary for the
maintenance of sterile immunity. A novel vaccine approach uses genetically attenuated parasites (GAPs) that
undergo arrested development during liver infection. The fate of GAPs after immunization, their persistence
in vaccinated animals, and the immune mechanisms that mediate protection are unknown. To examine the
developmental defects of genetically attenuated liver stages in vivo, we created deletions of the UIS3 and UIS4
loci in the Plasmodium yoelii rodent malaria model (Pyuis3[?] and Pyuis4[?]). The low 50% infectious dose
of P. yoelii in BALB/c mice provides the most sensitive infectivity model. We show that P. yoelii GAPs reach
the liver, invade hepatocytes, and develop a parasitophorous vacuole but do not significantly persist 40 h after
infection. A single dose of Pyuis4(?) sporozoites conferred complete protection, but full protection by
Pyuis3(?) sporozoites required at least 2 immunizations. CD8+T cells were essential for protection, but CD4+
T cells were not. Our results show that genetically distinct GAPs confer different degrees of protective efficacy
and that live vaccine persistence in the liver is not necessary to sustain long-lasting protection. These findings
have important implications for the development of a P. falciparum GAP malaria vaccine.
Plasmodium parasite species that are transmitted by the
bite of infected anopheline mosquitoes cause malaria
in humans and other mammals. The toll malaria in-
fection takes on the human population of developing
Received 10 November 2006; accepted 16 February 2007; electronically pub-
lished 9 July 2007.
Potential conflicts of interest: S.H.I.K. is partially supported by the Bill and
Melinda Gates Foundation through the Foundation at the National Institutes of
Health Grand Challenges in Global Health Initiative. He is an inventor listed on
US Patent 7,22,179 and international patent application PCT/US2004/043023,both
titled “Live Genetically Attenuated Malaria Vaccine.” All other authors report no
Reprints or correspondence: Stefan H. I. Kappe, Seattle Biomedical Research
Institute, 307 Westlake Ave. N., Ste. 500, Seattle, Washington 98109-5219(Stefan
The Journal of Infectious Diseases
? 2007 by the Infectious Diseases Society of America. All rights reserved.
countries is staggering, killing millions each year .
Mosquito-inoculated sporozoite stages of the parasite
invade the liver and undergo anintracellularreplication
phase in hepatocytes to produce merozoite stages,
which initiate the erythrocytic cycle responsible for the
pathology of malaria . The growth and replication
of liver stages is not associated with any clinical symp-
toms and in humans requires ∼7 days, which provides
a good target for anti-infection immune responses. In-
deed, immunization with whole live parasitescompletely
protects against the initial hepatic stage. In mammals,
this was first shown using rodent malaria irradiation-
attenuated sporozoite immunizations, which induced
protection against challenge with infectioussporozoites
. The irradiated sporozoite rodent malaria models
have made critical contributions to our understanding
of the basic immune mechanisms that mediate sterile
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Complete Protection with GAP Malaria Vaccines • JID 2007:196 (15 August) • 609
quences used in the study.
Polymerase chain reaction oligonucleotide primer se-
F , forward; R, reverse.
protection against infection [4, 5]. The success of the experi-
mental sporozoite vaccine model led to trials with irradiated
sporozoites of Plasmodium falciparum. The trials demonstrated
complete and long-lasting sterile protection in humans as well
, indicating that protection by vaccination with live atten-
uated sporozoites is a universal principle. A vaccine based on
a single protein component of sporozoites—the circumspo-
rozoite protein (CSP) of P. falciparum—has undergone ex-
tensive clinical testing  but has not achieved complete pro-
tection. Recently it was proposed that because attenuated
sporozoites remain the only fully protective experimental vac-
cine, their large-scaledevelopmentandlicensureasadeployable
malaria vaccine should be pursued . Gamma irradiation
causes random damage to DNA and, when correctly dosed,
allows sporozoites to maintain their infective properties[9,10].
DNA damage, however, likely results in cell-cycle arrest of the
replicating liver stage and, thus, in aborted development early
during infection . Inactivated sporozoites alone are not
protective , which shows that biological functionality dur-
ing the initial stages of sporozoite infection is critical for the
induction of protection. Furthermore, in P. berghei, the per-
sistence of intrahepatocytic parasites is necessary for the main-
tenance of long-lasting protection .
Using the P. berghei model, we have recently shown that
defects in early parasite liver-stage development in in vitro he-
patocyte infections [13, 14]. These studies and other work 
showed that attenuation of the malaria parasite by precise ge-
netic manipulation is possible (genetically attenuated Plasmo-
dium parasites [GAPs]). Immunization of mice with P. berghei
GAPs leads to complete protection against subsequent infec-
tious sporozoite challenge [13–15].
To further study GAP infections in vivo and to compare the
protective efficacy of distinct GAPs, we constructed UIS3 and
UIS4 deletions in the P. yoelii rodent malaria parasite(Pyuis3[?]
and Pyuis4[?]). P. yoelii is highly infectious to BALB/c mice,
exhibiting a low ID50of !10 sporozoites [16, 17]. This models
the high infectivity of P. falciparum sporozoites for humans 
better than the P. berghei model, which requires at least 10 times
more sporozoites to infect mice . Hence, P. yoelii is arguably
a morerelevant modelformalariavaccinestudies.Here,weshow
that P. yoelii GAP vaccines do not persist in the liver of im-
munized mice but confer sterile protection for at least 6 months.
GAP-mediated protection is strictly dependent on CD8+T cells.
Our studies also reveal that differences in the protective efficacy
of distinct GAP vaccines are made apparent when single-dose
immunization regimens are used.
MATERIALS AND METHODS
Experimental animals and cell lines.
Female Swiss Webster
(SW) and BALB/c mice (6–8 weeks old) for in vivo infection
experiments were purchased from Harlan, and BALB/c mice
(6–8 weeks old) for immunization experiments werepurchased
from Jackson Laboratories. Animal handling was conducted in
accordance with Institutional Animal Care and Use Commit-
tee–approved protocols. P. yoelii 17 XNL (a nonlethal strain)
clone 1.1, Pyuis3(?), and Pyuis4(?) parasites were cycled be-
tween Anopheles stephensi mosquitoes and SW mice. Infected
mosquitoes were maintained on sugar water at 24?C and 70%
humidity. Hepa1-6 (ATCC CRL-1830) and HepG2:CD81 
hepatoma cells were used for in vitro assays.
Generation of Pyuis3(5) and Pyuis4(5) parasites.
Deletions of PyUIS3 and PyUIS4 were performed by construct-
ing replacement plasmids in vector b3D.DT.H Db (provided
by Dr. A. Waters, Leiden University, Leiden, The Netherlands)
containing the pyrimethamine-resistant Toxoplasma gondii
dhfr/ts gene. Using oligonucleotide primers PY3REP1 forward
(F) and PY3REP2 reverse (R), a 500-bp fragment was amplified
encoding the 5?untranslated region (UTR) of PyUIS3, and a
similar-sized fragment encodingthe3?UTRwasamplifiedusing
PY3REP3F and PY3REP4R primers with P. yoelii genomicDNA
as the template (table1showsoligonucleotideprimersequences
used in the study). Two fragments were amplifiedusingprimers
PY4REP1F and PY4REP2R for the 1-kb fragment containing
the 5?UTR sequence and PY4REP3F and PY4REP4R for the
600-bp fragment containing the 3?UTR sequence of PyUIS4,
with P. yoelii genomic DNA as template. The 5?UTR fragments
were cloned into the KpnI and HindIII sites of the vector. The
3?UTR fragments were cloned into the EcoRI and BamHI sites.
The resulting plasmids were digested with KpnI and XbaI to
release the replacement fragment used for transfection. Re-
placement knockout parasites, as indicated above, are referred
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616 • JID 2007:196 (15 August) • Tarun et al.
faces many formidable obstacles , it may ultimatelyprovide
the only opportunity to completely protect humans against
malaria infection by vaccination.
We thank J. Whisler, M. Roberts, and Chelsea Kungkagam for expert
technical assistance withmosquitorearingandparasiteinfections,theNaval
Medical Research Center for providing the HEP17 monoclonal antibody,
and Lawrence Bergman for providing the merozoite surface protein 1
1. Guinovart C, Navia MM, Tanner M, Alonso PL. Malaria: burden of
disease. Curr Mol Med 2006;6:137–40.
2. Miller LH, Baruch DI, Marsh K, Doumbo OK. The pathogenic basis
of malaria. Nature 2002;415:673–9.
3. Nussenzweig RS, Vanderberg J, Most H, Orton C. Protectiveimmunity
produced by the injection of X-irradiated sporozoites of Plasmodium
berghei. Nature 1967;216:160–2.
4. Doolan DL, Martinez-Alier N. Immune response to pre-erythrocytic
stages of malaria parasites. Curr Mol Med 2006;6:169–85.
5. Hafalla JC, Cockburn IA, Zavala F. Protective and pathogenic roles of
CD8+ T cells during malaria infection. Parasite Immunol 2006;28:
6. Hoffman SL, Goh LM, Luke TC, et al. Protection of humans against
malaria by immunization with radiation-attenuated Plasmodium fal-
ciparum sporozoites. J Infect Dis 2002;185:1155–64.
7. Alonso PL, Sacarlal J, Aponte JJ, et al. 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
randomised controlled trial. Lancet 2005;366:2012–8.
8. Luke TC, Hoffman SL. Rationale and plans for developing a non-
replicating, metabolically active, radiation-attenuated Plasmodium fal-
ciparum sporozoite vaccine. J Exp Biol 2003;206:3803–8.
9. Mellouk S, Lunel F, Sedegah M, Beaudoin RL, Druilhe P. Protection
against malaria induced by irradiated sporozoites. Lancet 1990;335:
10. Silvie O, Semblat JP, Franetich JF, Hannoun L, Eling W, Mazier D.
Effects of irradiation on Plasmodium falciparum sporozoite hepatic
development: implications for the design of pre-erythrocytic malaria
vaccines. Parasite Immunol 2002;24:221–3.
11. Alger NE, Harant J. Plasmodium berghei: heat-treated sporozoite vac-
cination of mice. Exp Parasitol 1976;40:261–8.
12. Scheller LF, Azad AF. Maintenance of protective immunity against
malaria by persistent hepatic parasites derived from irradiated spo-
rozoites. Proc Natl Acad Sci USA 1995;92:4066–8.
13. Mueller AK, Camargo N, Kaiser K, et al. Plasmodium liver stage de-
velopmental arrest by depletion of a protein at the parasite-host in-
terface. Proc Natl Acad Sci USA 2005;102:3022–7.
14. Mueller AK, Labaied M, Kappe SH, Matuschewski K. Geneticallymod-
ified Plasmodium parasites as aprotectiveexperimentalmalariavaccine.
15. van Dijk MR, Douradinha B, Franke-Fayard B, et al. Genetically at-
tenuated, P36p-deficient malarial sporozoites induce protective im-
munity and apoptosis of infected liver cells. Proc Natl Acad Sci USA
16. Belmonte M, Jones TR, Lu M, et al. The infectivity of Plasmodium
yoelii in different strains of mice. J Parasitol 2003;89:602–3.
17. Weiss WR. Host-parasite interactions and immunity to irradiated spo-
rozoites. Immunol Lett 1990;25:39–42.
18. Verhage DF, Telgt DS, Bousema JT, et al. Clinical outcome of exper-
imental human malaria induced by Plasmodium falciparum-infected
mosquitoes. Neth J Med 2005;63:52–8.
19. Tarun AS, Baer K, Dumpit RF, et al. Quantitative isolation and in vivo
20. Renia L, Miltgen F, Charoenvit Y, et al. Malaria sporozoitepenetration:
a new approach by double staining. J Immunol Methods 1988;112:
21. Tsuji M, Mattei D, Nussenzweig RS, Eichinger D, Zavala F. Demon-
stration of heat-shock protein 70 in the sporozoite stage of malaria
parasites. Parasitol Res 1994;80:16–21.
22. Charoenvit Y, Mellouk S, Sedegah M, et al. Plasmodium yoelii: 17-kDa
hepatic and erythrocytic stage protein is the target of an inhibitory
monoclonal antibody. Exp Parasitol 1995;80:419–29.
23. Bergman LW, Kaiser K, Fujioka H, et al. Myosin A tail domain inter-
acting protein (MTIP) localizes to the inner membrane complex of
Plasmodium sporozoites. J Cell Sci 2003;116:39–49.
24. Wang R, Charoenvit Y, Corradin G, De La Vega P, Franke ED,Hoffman
SL. Protection against malaria by Plasmodium yoelii sporozoite surface
protein 2 linear peptide induction of CD4+ T cell- and IFN-gamma-
dependent elimination of infected hepatocytes. J Immunol 1996;157:
25. Gwadz RW, Cochrane AH, Nussenzweig V, Nussenzweig RS. Prelim-
inary studies on vaccination of rhesus monkeys with irradiated spo-
rozoites of Plasmodium knowlesi and characterization of surface anti-
gens of these parasites. Bull World Health Organ 1979;57(Suppl 1):
26. Collins WE, Skinner JC, Millet P, et al. Reinforcement of immunity
in Saimiri monkeysfollowingimmunizationwithirradiatedsporozoites
of Plasmodium vivax. Am J Trop Med Hyg 1992;46:327–34.
27. Silvie O, Greco C, Franetich JF, et al. Expression of human CD81
differently affects host cell susceptibility to malaria sporozoites de-
pending on the Plasmodium species. Cell Microbiol 2006;8:1134–46.
28. Baer K, Roosevelt M, Clarkson AB Jr, van Rooijen N, Schnieder T,
Frevert U. Kupffer cells are obligatory for Plasmodium yoelii sporozoite
infection of the liver. Cell Microbiol 2007;9:397–412.
29. Amino R, Thiberge S, Martin B, et al. Quantitative imaging of Plas-
modium transmission from mosquito to mammal. Nat Med 2006;12:
30. Doolan DL, Hoffman SL. The complexity of protective immunity
against liver-stage malaria. J Immunol 2000;165:1453–62.
31. Belnoue E, Costa FT, Frankenberg T, et al. Protective T cell immunity
against malaria liver stage after vaccination with live sporozoitesunder
chloroquine treatment. J Immunol 2004;172:2487–95.
32. Renia L, Gruner AC, Mauduit M, Snounou G. Vaccination against
malaria with live parasites. Expert Rev Vaccines 2006;5:473–81.
by guest on December 28, 2015