antibodies to plasmodium circumsporozoite protein (csp) inhibit sporozoite's cell traversal...

Journal of Immunological Methods 377 (2012) 47–52

Contents lists available at SciVerse ScienceDirect

Journal of Immunological Methods

j ourna l homepage: www.e lsev ie r .com/ locate / j im

Research paper

Antibodies to Plasmodium circumsporozoite protein (CSP) inhibitsporozoite's cell traversal activity

Satish Mishra⁎, Ruth S. Nussenzweig, Victor NussenzweigMichael Heidelberger Division of Immunology, Department of Pathology, New York University School of Medicine, NY 10016, USA

a r t i c l e i n f o

⁎ Corresponding author at: Department of MolecuImmunology, Johns Hopkins Bloomberg School ofHopkins University, Baltimore, MD 21205, USA. Telfax: +1 410 955 0105.

E-mail address: samishra@jhsph.edu (S. Mishra).

0022-1759/$ – see front matter © 2012 Elsevier B.V. Adoi:10.1016/j.jim.2012.01.009

a b s t r a c t

Article history:Received 18 January 2012Accepted 19 January 2012Available online 28 January 2012

Plasmodium sporozoites are deposited in the skin of the mammalian host by Anophelesmosqui-toes. To continue the life cycle, the sporozoites have to invade the host's hepatocytes, wherethey transform into exoerythrocytic forms (EEFs) inside a parasitophorous vacuole. Duringtheir route from the skin to the liver, the parasites traverse the capillary epithelium in thedermis to enter the blood circulation, and cross the endothelium of liver sinusoids to enterthe parenchyma. Cell traversal by sporozoites is usually measured by quantifying dyes thatenter or are released from cells during incubation with salivary gland sporozoites. Thesemethods do not distinguish cell traversal from cell wounding. Here we validate an assay thatquantifies cell traversal of sporozoites through monolayers of MDCK cells that form tight junc-tions. We compared cell traversal of wt sporozoites and of parasites lacking the Type I mem-brane protein TLP (TRAP-like protein) previously implicated in cell traversal. We providedirect evidence that TLP ko sporozoites are defective in cell traversal and that they are retainedinside the MDCK cytoplasm. We then used the MDCK assay to study the effect of a monoclonalantibody (3D11) to the circumsporozoite protein (CSP) on the parasite's cell traversal. Weshow that 3D11 inhibits cell traversal at nanomolar concentrations. We conclude that anti-bodies elicited by CSP-based vaccines are likely to inhibit the migration of sporozoites fromthe skin to the liver.

© 2012 Elsevier B.V. All rights reserved.

Keywords:PlasmodiumSporozoitesMDCK cell assayTRAP-like protein (TLP)Cell traversal

1. Introduction

The sporozoites of malaria parasites undergo profoundgenetic changes while they move from the mosquito oocystsinto the salivary glands (Matuschewski, 2006), where theycan remain dormant for days (Zhang et al., 2010). After deliv-ery into the skin of the mammalian host by mosquito bite, theparasites start a long journey to the liver, where they enterhepatocytes and develop into the exoerythrocytic forms.During this migration the parasites need to cross the vascularendothelium to enter the blood or lymphatic circulation, andthe endothelium of liver sinusoids to enter the parenchyma

lar Microbiology andPublic Health, Johns.: +1 443 287 0129;

ll rights reserved.

(Mota et al., 2001; Amino et al., 2008; Ejigiri and Sinnis,2009). Sporozoite migration and infection of hepatocytesare achieved by the concerted activity of an actin–myosinmotor located beneath the parasite's plasma membrane(Kappe et al., 2004; Dowse and Soldati, 2004) and by productsof specialized secretory organelles named rhoptries, densegranules andmicronemes (Carruthers and Sibley, 1997). Spo-rozoites do not have flagella, cilia or lamellipodia, neverthe-less they use an actin/myosin motor to move very rapidlyover solid substrates by gliding motility (Heintzelman, 2006;Tardieux and Ménard, 2008; Daher and Soldati-Favre, 2009;Munter et al., 2009). The first sporozoite membrane proteinshown to link the motor to the extracellular substrate wasthe thrombospondin related anonymous protein (TRAP)(Sultan et al., 1997). In the absence of TRAP sporozoites donot glide. Other members of the TRAP family were identifiedlater (Lacroix and Ménard, 2008), including TLP (that is thesubject of this paper; Moreira et al., 2008; Heiss et al., 2008).

48 S. Mishra et al. / Journal of Immunological Methods 377 (2012) 47–52

All members bear a cytoplasmic tail that is acidic and inter-changeable. This tail contains a sub-terminal tryptophanthat is essential for motility (Kappe et al., 1999). The extracel-lular domains of TRAP family members consist of two adhe-sive modules that bind to substrates: the thrombospondintype I repeat and the A domain.

Two methods are generally used to study cell traversal. Inthe calcein green assay, target cells loaded with a fluorescentdye are incubated with sporozoites and the amounts of fluores-cence released into the supernatant are measured (Coppi et al.,2007). This assay was used in the studies of the TLP knockout(Moreira et al., 2008). However, the calcein assay measurescell wounding rather than cell traversal. A second cell traversalassay uses polarizedmonolayers ofMDCK cells seeded betweentwo chambers (Mota et al., 2001). The sporozoites are placed inthe upper chamber, and after an appropriate time the parasitesin the bottom chamber are counted. As described, this assaydoes not exclude the possibility that sporozoites migratebetween cell junctions as is the case of Toxoplasma tachyzoites(Barragan et al., 2005). Here we ruled this out and used theMDCK assay to confirm the role of TLP in cell traversal. In addi-tion, we studied the effect of antibodies to the sporozoite'scircumsporozoite protein (CSP) on their ability to traverse cells.

2. Material and methods

2.1. Parasites, cells and antibodies

The Plasmodium berghei wt and the mutant lacking TLP[PbTLP ko] were described elsewhere (Moreira et al., 2008).TheMDCK and HepG2 cells weremaintained in DMEM supple-mented with 10% fetal calf serum (FCS) and 1 mM glutamine(DMEM/FCS). The following antibodieswere used:monoclonalantibody (mAb), directed against the repeat region of P. bergheiCSP (Yoshida et al., 1980) and the anti-occludin monoclonalantibody (Invitrogen).

2.2. Mosquito infection with PbTLP ko and wt parasites

All procedures involving animals were performed accordingto US National Institutes of Health guidelines, as approved bytheAnimal Care andUse Committee of theNewYorkUniversitySchool of Medicine laboratory animal protocol #010202-01.Wild type (wt) and PbTLP ko parasite cycles were performedas described (Moreira et al., 2008). Briefly, Anopheles stephensimosquitoes were reared at 27 °C and 80% humidity under a12/12 h light/dark cycle, and adults were allowed to feed on10% sucrose solution. TheA. stephensimosquitoeswere allowedto feed on anaesthetized Swiss Webster mice infected with wtparasites or with the PbTLP ko. The mosquitoes were rinsed in70% ethanol and washed in medium, and the salivary glandsremoved. The glands were gently ground in a tissue homoge-nizer and centrifuged at 800 rpm for 4 min to removemosquitodebris, and sporozoites were counted in a haemocytometerand kept on ice in complete medium (DMEM containing 10%FCS) until use.

2.3. Cell traversal assay in vitro

The ability of mutant and wt sporozoites to cross cell layerswas determined by a modification of the method described by

Mota et al. (2001). MDCK cells (105) were cultured in 3-μmpore diameter Transwell filters (Costar) for 3 days to form acontinuous monolayer. The wells underneath the filters eitherwere left empty or 2×105 HepG2 cells were placed in thelower chamber. Sporozoites (105)were then added to the filterinset. After 1 h the filter was removed and the sporozoitesthat crossed the MDCK monolayer were collected from thebottom well and counted. The wells that contained HepG2cells were incubated for additional 48 h, and the HepG2 cellsremoved from wells. The parasite's RNA was then quantifiedby real-time PCR (Bruña-Romero et al., 2001). Other MDCK-containing filterswere fixedwith paraformaldehyde, embeddedin paraffin, sectioned and stained with 3D11, and intra-cellularparasites counted. In addition we counted directly sporozoitesinside and outside of the monolayer by another approach, i.e.,paraformaldehyde-fixing followed by permeabilization of theMDCK cells (Renia et al., 1988). The monolayers were stainedfor the tight junction marker occludin following the manu-facturer's instructions. Trans-monolayer Electrical Resistance(TER) was measured across the monolayer during the transmi-gration of sporozoites using an ohmmeter (World PrecisionInstruments, Bradenton, FL).

2.4. Gliding motility assay

Glidingmotility assays were performed as described (Coppiet al., 2006). Briefly, sporozoites were added to Lab-Tek wellspre-coated with 3D11 for 1 h at 37 °C. After removal of theme-dium, fixative was added, followed by the biotinylated 3D11and streptavidin-FITC (Zymed) to visualize the CSP-containingtrails. Gliding motility was quantified by counting the numberof sporozoites associated with trails and the number of circlesin each trail.

2.5. Preparation of 3D11 Fab

Monovalent Fabwas prepared using the Fabmicro prepara-tion kit (Pierce), following the manufacturer's instructions.Briefly, immobilized papainwas equilibratedwith Fabdigestionbuffer. The IgG sample was desalted using Zeba desalt spin col-umn. Desalted IgGwas incubatedwith immobilized papain anddigestion reaction was performed for 5–6 h with an end-over-end mixer at 37 °C. Fc fragments and remaining intact anti-bodies were removed by passing the sample through Agarose-protein-G column.

2.6. Treatment of P. berghei sporozoites with 3D11

P. berghei sporozoites, 5×104 for gliding motility experi-ments or 105 for the cell traversal assay were pre-incubatedfor 30 min at room temperature in medium containing 10%FCS and variable concentrations of 3D11. As controls, sporo-zoiteswere incubated inmedium in the absence of antibodies,or added to wells in the presence of 1 μM cytochalasin D toinhibit gliding motility.

2.7. Statistical analysis

Results are shown as means±SD or percentage±SD.Unpaired two-tailed Student's t-test was used to determinethe statistical significance.

49S. Mishra et al. / Journal of Immunological Methods 377 (2012) 47–52

3. Results

3.1. PbTLP ko sporozoites are defective in cell traversal

As shown in Fig. 1A and B about 70% fewer PbTLP ko spo-rozoites than wt crossed the MDCK monolayer. As controls,we verified that MDCK cells formed tight junction visually(Fig. 1C) and were polarized by measuring the Transmono-layer Electrical Resistance (TER). The TER did not changeduring the migration of the sporozoites (Fig. 1D), as wouldbe expected if the parasites disrupted the junctions. To deter-mine whether the mutant parasites were retained inside thecytoplasm of MDCK cells, we sectioned the monolayer,stained the sections with 3D11, and counted the parasitesinside the cytoplasm of the MDCK cells. We found that 70%more PbTLP ko than wt sporozoites were inside the cyto-plasm of the MDCK cells (Fig. 2A). Representative microscopysections are shown in Fig. 2C and D. This result was con-firmed by counting sporozoites inside and outside of theMDCK cells as described by Renia et al. (1988). Again, 69%more mutants than wt were inside the MDCK cells (Fig. 2B).As an additional control for the integrity of the monolayer,we added large numbers of motile Trypanosoma brucei tothe upper chamber, and found none in the bottom chamber(data not shown). These results provide direct evidencethat TLP plays an important role in Plasmodium sporozoite

Fig. 1. PbTLP ko sporozoites are impaired in cell-passage activity. Wild type and PbT1 h at 37 °C. (A) The percentage of sporozoites that cross the MDCK monolayer andthe MDCK monolayer and develop into EEFs inside HepG2 cells seeded in the bottomMDCK cells formed tight junctions. (D) TER was relatively constant during sporozoittwo-tailed Student's t-test.

passage through cell barriers. It also validates the MDCKassay as a reliable method to measure cell traversal.

3.2. Comparison of the capacity of 3D11 IgG to inhibit celltraversal and gliding

P. berghei sporozoites were incubated with different con-centrations of 3D11 IgG for 30 min at room temperature andused for cell traversal or gliding motility assays. 3D11 signifi-cantly inhibited the cell traversal (p=0.003). Glidingmotilitywas significantly inhibited (p=0.02) only when comparingthe numbers of >10 circles generated by antibody-treatedand non-treated sporozoites. Both gliding and cell traversalwere abolished at 50 μg/ml (Fig. 3A and B). However, evenat 50 μg/ml the monovalent Fab fragments of 3D11 did notinhibit significantly cell crossing (data not shown).

4. Discussion

Here we validate the MDCK assay to measure cell tra-versal by sporozoites and use it to support the findings ofMoreira et al. (2008) suggesting that TLP plays a role in celltraversal. To this end, we compared the ability of wt andTLP ko sporozoites to cross the monolayer of MDCK cells thatseparates two chambers. We found that significantly fewerTLP ko traverse the monolayer. To ensure that the MDCK cells

LP ko salivary gland sporozoites were added to filter inset and incubated forare present in the bottom of the well was counted. (B) Sporozoites that crosschamber were quantified by real time PCR. (C) Occludin staining shows that

e migration. Data (mean±SD) are from quadruplicates analyzed by unpaired

A B

C D

0

2

4

6

8

10

wt PbTLP ko

Num

ber

of s

poro

zoit

es in

side

MD

CK

cel

ls (

x102 )

Num

ber

of s

poro

zoit

es in

side

MD

CK

cel

ls (

x102 )

0

0.5

1

1.5

2

2.5

3

3.5

4

wt PbTLP ko

(P< 0.0001)

(P< 0.0001)

wt PbTLP ko

Fig. 2. A greater number of PbTLP ko sporozoites are retained in the cytoplasm of MDCK cells compared to wt. (A) The number of wt and PbTLP ko sporozoitesfound in the cytoplasm of MDCK cells. (B) The number of wt and PbTLP ko sporozoites present in the MDCK cells estimated by inside/outside staining. (C) Pho-tograph of wt MDCK filter section. (D) Photograph of PbTLP ko MDCK filter section. Data (mean±SD) are from quadruplicates analyzed by unpaired two-tailedStudent's t-test.

A B

0

10

20

30

40

50

60

70

80

1 circle

2-10 circles

>10 circles

3D11 µg/ml

% w

ith

indi

cate

d nu

mbe

r of

cir

cles

0102030405060708090

100

3D11 µg/ml

% s

poro

zoit

es t

rave

rse

MD

CK

mon

olay

er c

ompa

red

to c

ontr

ol

111 103 65.5 57.5 45

Fig. 3. Monoclonal antibody 3D11 inhibits cell traversal and gliding motility of sporozoite. (A) 3D11 significantly inhibits cell traversal (p=0.003). (B) Glidingmotility was also decreased significantly when comparing the number of >10 circles of treated and nontreated sporozoites (p=0.02). The total number of circlesis presented on top of the bar. Data (mean±SD) are from duplicates.

50 S. Mishra et al. / Journal of Immunological Methods 377 (2012) 47–52

51S. Mishra et al. / Journal of Immunological Methods 377 (2012) 47–52

formed tight junctions, we measured the TER of the MDCKsduring sporozoite migration. The TER did not change. Wethen used two methods to demonstrate that TLP ko parasiteswere retained inside the cytoplasm of the MDCK cells: first,we sectioned the monolayer, stained the parasites with anti-bodies to CSP, and counted those inside the MDCK cells;second, we directly counted the inside/outside parasites asdescribed in Renia et al. (1988). By either method we docu-mented the greater retention of the TLP ko in the cytoplasmof the MDCK cells as compared to wt. We conclude that TLPindeed plays an important role in cell traversal. Some TLP kosporozoites, however, crossed the monolayer. We speculatethat an additional sporozoite surface molecule, perhaps TRAPitself, complements the TLP function. One key question, how-ever, remains to be elucidated. Since gliding motility is strictlysubstrate-dependent, what is the nature of the substrate(s)used by sporozoites for moving rapidly through the cell cyto-plasm? Cytoskeletal elements such as F-actin and tubulin areobvious candidates to interact with adhesive domains of TLP(and TRAP?), but this remains to be investigated.

The monoclonal antibody 3D11 abolished cell traversalat a concentration of 0.3 μM and significant inhibition wasdetected at 100 times lower concentrations. As previouslyreported (Stewart et al., 1986) gliding motility was alsoinhibited by 3D11 although its effect was less striking(Fig. 3). One possible reason is that gliding motility is verydifficult to measure with precision. The trajectories of sporo-zoites on glass or plastic slides vary widely. While mostexhibit a stop–go circular gliding, some have straight unpre-dictable directions, others move in a pendulum fashion, andsome remain attached and make waving movements. OnMatrigel, the movement is circular but three-dimensionallyit is helical (Amino et al., 2008). To bypass these complicatedproblems, gliding motility is usually measured indirectly byenumerating CSP trails left on the glass slides during move-ment. Therefore what is reported as “gliding motility” is inreality an imperfect measurement of the parasite's secretionof CSP during gliding. Most importantly, gliding on glass oron artificial matrices is unlikely to reflect the sporozoite'smovement in vivo between cells or over cell surfaces.

Our findings do not explain why antibodies to CSP inter-fere with sporozoite cell traversal. The antibodies do cross-link CSP and lead to “circumsporozoite protein reaction” athread-like precipitate that increases in length toward theposterior end of the parasite (Vanderberg et al., 1969). Themonovalent Fab fragments do not trigger the CSP reaction(Potocnjak et al., 1980) and they do not prevent cell crossingat high concentrations. Perhaps CSP cross-linking inhibits theactin–myosin motor leading to the paralysis of the parasite.This is speculative because CSP has no direct connectionwith the parasite's cytoplasm; it is attached to the sporo-zoite's plasma membrane by a glycosylphosphatidylinositol(GPI) anchor that only crosses the outer membrane of thebilayer. Nevertheless, it has been argued that the associationof GPI anchors with lipid rafts in prions and in the comple-ment receptor CD59 is associated with signaling properties(Taylor and Hooper, 2006; Kimberley et al., 2007).

Finally, we point out that CSP is a component of a humanvaccine named RTS,S that protected about 50% of individualsin a recent phase III trial in Africa (Agnandji et al., 2011). Pro-tection is at least in part mediated by antibodies against CSP

(Cohen et al., 2010) known to inhibit the attachment phaseof sporozoite infection of hepatocytes (Yoshida et al., 1980;Potocnjak et al., 1980). The present findings highlight anadditional effector mechanism of anti-CSP antibodies: theyinhibit cell traversal and should interfere with the migrationof the parasites from the host's skin to the liver.

Acknowledgments

We thank Dr. Photini Sinnis for providing the knockout ofPlasmodium berghei TLP, and for reading the manuscript criti-cally. V.N. and R.S.N. were supported by the Bill and MelindaGates Foundation, NIH R56 AI073658, GlaxoSmithKline (GSK)and the Dana Foundation. We would like to thank SandraGonzalez and Jean Nonon for their help with mosquito rearingand infection. Also, we thank the NYUHistology Core for expertassistance with MDCK monolayer sectioning. The authorsacknowledge no competing financial interests.

References

Agnandji, et al., 2011. First results of phase 3 trial of RTS,S/AS01 malariavaccine in African children. N. Engl. J. Med. 365, 1863.

Amino, R., Giovannini, D., Thiberge, S., Gueirard, P., Boisson, B., Dubremetz,J.F., Prevost, M.C., Ishino, T., Yuda, M., Menard, R., 2008. Host cell traversalis important for progression of the malaria parasite through the dermisto the liver. Cell Host Microbe 3, 88.

Barragan, A., Brossier, F., Sibley, L.D., 2005. Transepithelial migration ofToxoplasma gondii involves an interaction of intercellular adhesion mol-ecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell. Microbiol. 7, 561.

Bruña-Romero, O., Hafalla, J.C., González-Aseguinolaza, G., Sano, G., Tsuji, M.,Zavala, F., 2001. Detection of malaria liver-stages in mice infectedthrough the bite of a single Anopheles mosquito using a highly sensitivereal-time PCR. Int. J. Parasitol. 31, 1499.

Carruthers, V.B., Sibley, L.D., 1997. Sequential protein secretion from threedistinct organelles of Toxoplasma gondii accompanies invasion of humanfibroblasts. Eur. J. Cell Biol. 73, 114.

Cohen, J., Nussenzweig, V., Nussenzweig, R., Vekemans, J., Leach, A., 2010.From the circumsporozoite protein to the RTS,S/AS candidate vaccine.Hum. Vaccines 6, 90.

Coppi, A., Cabinian, M., Mirelman, D., Sinnis, P., 2006. Antimalarial activity ofallicin, a biologically active compound from garlic cloves. Antimicrob.Agents Chemother. 50, 1731.

Coppi, A., Tewari, R., Bishop, J.R., Bennett, B.L., Lawrence, R., Esko, J.D., Billker,O., Sinnis, P., 2007. Heparan sulfate proteoglycans provide a signal toPlasmodium sporozoites to stop migrating and productively invadehost cells. Cell Host Microbe 2, 316.

Daher, W., Soldati-Favre, D., 2009. Mechanisms controlling glideosome func-tion in apicomplexans. Curr. Opin. Microbiol. 12, 408.

Dowse, T., Soldati, D., 2004. Host cell invasion by the apicomplexans: the sig-nificance of microneme protein proteolysis. Curr. Opin. Microbiol. 7, 388.

Ejigiri, I., Sinnis, P., 2009. Plasmodium sporozoite–host interactions from thedermis to the hepatocyte. Curr. Opin. Microbiol. 12, 401.

Heintzelman, M.B., 2006. Cellular and molecular mechanics of gliding loco-motion in eukaryotes. Int. Rev. Cytol. 251, 79.

Heiss, K., Nie, H., Kumar, S., Daly, T.M., Bergman, L.W., Matuschewski, K.,2008. Functional characterization of a redundant Plasmodium TRAPfamily invasin, TRAP-like protein, by aldolase binding and a geneticcomplementation test. Eukaryot. Cell 7, 1062.

Kappe, S., Bruderer, T., Gantt, S., Fujioka, H., Nussenzweig, V., Menard, R.,1999. Conservation of a gliding motility and cell invasion machinery inApicomplexan parasites. J. Cell Biol. 147, 937.

Kappe, S.H., Buscaglia, C.A., Nussenzweig, V., 2004. Plasmodium sporozoitemolecular cell biology. Annu. Rev. Cell Dev. Biol. 20, 29.

Kimberley, F.C., Sivasankar, B., Paul Morgan, B., 2007. Alternative roles forCD59. Mol. Immunol. 44, 73.

Lacroix, C., Ménard, R., 2008. TRAP-like protein of Plasmodium sporozoites:linking gliding motility to host-cell traversal. Trends Parasitol. 24, 431.

Matuschewski, K., 2006. Getting infectious: formation and maturation ofPlasmodium sporozoites in the Anopheles vector. Cell. Microbiol. 8, 1547.

Moreira, C.K., Templeton, T.J., Lavazec, C., Hayward, R.E., Hobbs, C.V., Kroeze,H., Janse, C.J., Waters, A.P., Sinnis, P., Coppi, A., 2008. The Plasmodium

52 S. Mishra et al. / Journal of Immunological Methods 377 (2012) 47–52

TRAP/MIC2 family member, TRAP-Like Protein (TLP), is involved intissue traversal by sporozoites. Cell. Microbiol. 10, 1505.

Mota, M.M., Pradel, G., Vanderberh, G., Hafalla, J.C., Frevert, U., Nussenzweig,R.S., Nussenzweig, V., Rodríguez, A., 2001. Migration of Plasmodiumsporozoites through cells before infection. Science 291, 141.

Munter, S., Sabass, B., Selhuber-Unkel, C., Kudryashev, M., Hegge, S., Engel,U., Spatz, J.P., Matuschewski, K., Schwarz, U.S., Frischknecht, F., 2009.Plasmodium sporozoite motility is modulated by the turnover of discreteadhesion sites. Cell Host Microbe 6, 551.

Potocnjak, P., Yoshida, N., Nussenzweig, R.S., Nussenzweig, V., 1980. Monova-lent fragments (Fab) ofmonoclonal antibodies to a sporozoite surface anti-gen (Pb44) protect mice against malarial infection. J. Exp. Med. 151, 1504.

Renia, L., Miltgen, F., Charoenvit, Y., Ponnudurai, T., Verhave, J.P., Collins,W.D., Mazier, D., 1988. Malaria sporozoite penetration: a new approachby double staining. J. Immunol. Methods 112, 201.

Stewart, M.J., Nawrot, R.J., Schulman, S., Vanderberg, J.P., 1986. Plasmodiumberghei sporozoite invasion is blocked in vitro by sporozoite-immobilizing antibodies. Infect. Immun. 51, 859.

Sultan, A.A., Thathy, V., Frevert, U., Robson, K.J., Crisanti, A., Nussenzweig, V.,Nussenzweig, R.S., Ménard, R., 1997. TRAP is necessary for gliding motil-ity and infectivity of Plasmodium sporozoites. Cell 90, 511.

Tardieux, I., Ménard, R., 2008. Migration of Apicomplexa across biologicalbarriers: the Toxoplasma and Plasmodium rides. Traffic 9, 627.

Taylor, D.R., Hooper, N.M., 2006. The prion protein and lipid rafts. Mol.Membr. Biol. 23, 89.

Vanderberg, J., Nussenzweig, R., Most, H., 1969. Protective immunity pro-duced by the injection of x-irradiated sporozoites of Plasmodium berghei.V. In vitro effects of immune serum on sporozoites. Mil. Med. 134, 1183.

Yoshida, N., Nussenzweig, R.S., Potocnjak, P., Nussenzweig, V., Aikawa, M.,1980. Hybridoma produces protective antibodies directed against thesporozoite stage of malaria parasite. Science 207, 71.

Zhang,M., Fennell, C., Ranford-Cartwright, L., Sakthivel, R., Gueirard, P., Meister,S., Caspi, A., Doerig, C., Nussenzweig, R.S., Tuteja, R., Sullivan Jr., W.J., Roos,D.S., Fontoura, B.M., Ménard, R., Winzeler, E.A., Nussenzweig, V., 2010. ThePlasmodium eukaryotic initiation factor-2α kinase IK2 controls the latencyof sporozoites in the mosquito salivary glands. J. Exp. Med. 207, 1465.