RESEARCH ARTICLE◥

MALARIA

Transferrin receptor 1 is areticulocyte-specific receptor forPlasmodium vivaxJakub Gruszczyk,1 Usheer Kanjee,2 Li-Jin Chan,1,3 Sébastien Menant,1

Benoit Malleret,4,5 Nicholas T. Y. Lim,1 Christoph Q. Schmidt,6 Yee-Foong Mok,7

Kai-Min Lin,8 Richard D. Pearson,9,10 Gabriel Rangel,2 Brian J. Smith,11

Melissa J. Call,1,3 Michael P. Weekes,8 Michael D. W. Griffin,7 James M. Murphy,1,3

Jonathan Abraham,12 Kanlaya Sriprawat,13 Maria J. Menezes,14 Marcelo U. Ferreira,14

Bruce Russell,15 Laurent Renia,5 Manoj T. Duraisingh,2 Wai-Hong Tham1,3*

Plasmodium vivax shows a strict host tropism for reticulocytes.We identified transferrinreceptor 1 (TfR1) as the receptor for P. vivax reticulocyte-binding protein 2b (PvRBP2b).Wedetermined the structure of the N-terminal domain of PvRBP2b involved in red blood cellbinding, elucidating the molecular basis for TfR1 recognition.We validated TfR1 as thebiological target of PvRBP2b engagement by means of TfR1 expression knockdown analysis.TfR1 mutant cells deficient in PvRBP2b binding were refractory to invasion of P. vivax butnot to invasion of P. falciparum. Using Brazilian and Thai clinical isolates, we show thatPvRBP2bmonoclonal antibodies that inhibit reticulocyte binding also block P. vivax entry intoreticulocytes.These data show that TfR1-PvRBP2b invasion pathway is critical for therecognition of reticulocytes during P. vivax invasion.

Of the hundreds of Plasmodium species,onlyP. falciparum,P. vivax,P. ovale curtisi,P. ovale wallikeri, P. malariae, andP. knowlesi are known to infect humans.Within the human host, malaria parasites

invade liver and red blood cells for replicationand transmission. Blood stage infection is themajor cause of all clinical symptoms in malaria,and therefore the therapeutic prevention of par-asite entry into red blood cells could alleviatemalarial disease. Entry into red blood cells de-pends on the interactions between parasite in-vasion ligands and their cognate red blood cellreceptors, of which only a handful have beenidentified (1–7). These ligand-receptor interac-tions initiate a cascade of molecular events thatprogress from initial attachment, recognition,commitment, and last, penetration of the para-site into red blood cells (8, 9).P. vivax is themost widely distributed human

malaria parasite. This parasite has a strict pref-

erence for invasion into reticulocytes, which arevery young red blood cells that are formed inthe bonemarrow after enucleation and releasedinto the circulation. The reticulocyte-specificreceptor involved in P. vivax entry has not beenidentified (10).Most studies have focused on theinteraction between the P. vivax Duffy bindingprotein (PvDBP) and the red blood cell Duffyantigen receptor for chemokines (DARC) be-cause individuals fromwestern and central Africalacking DARC are resistant to P. vivax invasion(11).However, recent reports have highlightedthe presence of P. vivax in apparently DARC-negative individuals, suggesting that P. vivaxmay enter reticulocytes by binding to other re-ceptors (12–14). Furthermore, DARC is presenton both normocytes and reticulocytes, and there-fore this ligand-receptor interaction cannot gov-ern selective entry into reticulocytes (15). Toidentify other parasite proteins involved in retic-ulocyte recognition, we focused on the P. vivax

reticulocyte-binding protein family (PvRBP). Thisprotein family comprises 11 members, of whichseveral have been shown to bind reticulocytes;however, their cognate receptors have not beenidentified (16–19).

PvRBP2b binds transferrin receptor 1 tomediate recognition of reticulocytes

P. vivax preferentially invades reticulocytes thatexpress high levels of transferrin receptor 1 (TfR1or CD71) (20). TfR1 is an essential housekeepingprotein involved in cellular transport of iron intocells through binding of iron-loaded transferrin(Tf) (21). On circulating red blood cells, TfR1 isexpressed only on reticulocytes and is progres-sively lost from theirmembranes as theymatureinto erythrocytes (22, 23). TfR1 is a type II trans-membrane glycoprotein that forms a dimer, andits ectodomain consists of three subdomains: a“protease-like domain” resembling the structureof zinc metalloproteinases, an “apical domain,”and a “helical domain” responsible for dimeriza-tion (24). TfR1 is also a cellular receptor for NewWorld hemorrhagic fever arenaviruses, includ-ing Machupo (MACV), Junin, Guanarito, andSabiá viruses (25, 26). Residues 208 to 212 of theTfR1 apical domain provide a critical recogni-tion site for these viruses (25, 26).PvRBP2b is expressed in late-stage P. vivax

parasites, and recombinant PvRBP2b (residues161 to 1454; PvRBP2b161–1454) binds preferentiallyto reticulocytes that express TfR1 (19, 27). Weobserved that binding by recombinant PvRBP2bwas abolished when reticulocytes were treatedwith trypsin and chymotrypsin (fig. S1, A andB). We confirmed that the combination of theseproteases cleaves TfR1 and complement recep-tor 1 (CR1) from the surface of reticulocytes, withother known malaria receptors—including gly-cophorin A, basigin, and DARC—being suscep-tible to different sets of protease treatment (fig.S1, A and B). The profile of PvRBP2b binding isstrikingly similar to the TfR1 surface expressionon reticulocytes (Fig. 1A, bottom), and we showthat the level of PvRBP2b binding is directlycorrelated with the levels of TfR1 on the surfaceof reticulocytes (fig. S1, C and D).To determine whether PvRBP2b161–1454 binds

to the population of reticulocytes that expressTfR1 on their surfaces, we tested a panel of com-mercially available anti-TfR1 monoclonal anti-bodies (mAbs) for their ability toblock recombinantPvRBP2b binding. Indeed, anti-TfR1 mAbs 23D10,L01.1, LT71,M-A712,MEM-189, andOKT9 inhibitedPvRBP2b binding to reticulocytes by 78, 76, 33,75, 92, and 90%, respectively (Fig. 1A). M-A712also prevents MACV pseudovirus entry (25, 28).Anti-TfR1 mAbs 2B6, 13E4, andMEM-75 did notinhibit PvRBP2b binding; although their epitopeshave not been mapped, we propose that thesethree antibodies may bind to a site on TfR1 thatis not involved in the PvRBP2b interaction (Fig. 1A).To determine whether this inhibition was spe-cific to PvRBP2b161–1454 binding, we analyzed thebinding of P. falciparum reticulocyte bindingprotein-like homolog 4 (PfRh4) to its cognate re-ceptor CR1 (4).Whereas addition of the first three

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1The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia. 2Department of Immunology andInfectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA 02115, USA. 3Department of Medical Biology, TheUniversity of Melbourne, Melbourne, Victoria 3010, Australia. 4Department of Microbiology and Immunology, Yong Loo LinSchool of Medicine, National University of Singapore, 117597 Singapore. 5Singapore Immunology Network, A*STAR, 138648Singapore. 6Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Germany. 7Department ofBiochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne,Melbourne, Victoria 3010, Australia. 8Cambridge Institute for Medical Research, Cambridge CB2 OXY, UK. 9Wellcome TrustSanger Institute, Hinxton, Cambridge, UK. 10Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, Oxford,UK. 11La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia. 12Department ofMicrobiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA. 13Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand. 14Department ofParasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil. 15Department of Microbiology andImmunology, University of Otago, Dunedin 9054, New Zealand.*Corresponding author. Email: tham@wehi.edu.au

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complement control protein modules of CR1(CCP 1–3) inhibited PfRh428–766 binding as ex-pected (29), addition of anti-TfR1 mAb OKT9did not significantly reduce PfRh4 binding (Fig.1B). Because anti-TfR1 did not affect PfRh4 bind-ing, these results show that TfR1 is a specific re-ticulocyte receptor for PvRBP2b.To evaluate whether PvRBP2b161–1454 interacts

directly with TfR1, we performed immunoprecip-itation experiments using purified recombinantTfR1, Tf, and PvRBP2b161–1454 proteins (Fig. 1C)

(30). Using an anti-PvRBP2b mAb, we immuno-precipitated PvRBP2b in complex with TfR1 andTf. PvRBP2b and TfR1 also formed a binary com-plex in the absence of Tf, demonstrating thatPvRBP2b binds directly to TfR1 (Fig. 1C). Theinteraction between PvRBP2b and TfR1 is spe-cific; immunoprecipitation of PvRBP1a, PvRBP1b,or PvRBP2a did not show evidence of complexformation with TfR1 (fig. S2A).We developed a fluorescence resonance energy

transfer (FRET)–based assay tomonitor PvRBP2b-

TfR1 complex formation in which TfR1 labeledwith DyLight-594 could be shown to interact withPvRBP2b161–1454 labeled with DyLight-488 (fig.S2B). The addition of 10-fold molar excess ofunlabeled PvRBP2b161–1454 and TfR1 competedout the labeled proteins and reduced the signalof the PvRBP2b-TfR1 FRET pair. By contrast,proteins that were unable to bind TfR1, suchas PfRh4, had no effect on the FRET signal.Using this assay, we observed that anti-TfR1mAbs 23D10, M-A712, MEM-189, and OKT9 that

Gruszczyk et al., Science 359, 48–55 (2018) 5 January 2018 2 of 8

Fig. 1. PvRBP2bbinds TfR1 on thereticulocyte surface.(A) PvRBP2b161–1454binding in the presenceof anti-TfR1 mAbs ana-lyzed by means of flowcytometry. (Left) Dotplots of PvRBP2b161–1454binding (y axis) toreticulocytes stainedwith thiazole orange(TO; x axis). (Right)Normalized bindingresults in whichPvRBP2b161–1454binding in the absenceofmAbswas arbitrarilyassigned to be 100%.(B) PvRBP2b161–1454and PfRh428–766bindingwere evaluatedby means of flowcytometry with theaddition of anti-TfR1mAbOKT9orCCP 1–3.PvRBP2b161–1454 andPfRh4 binding in bufferwere arbitrarily assignedto be 100%. (C) Eluatesof individual or mix-tures of proteinsimmunoprecipitatedwith anti-PvRBP2bmAbanalyzedbymeansof SDS-PAGE. Plus andminus signs indicateprotein present andabsent, respectively. M,molecular weightmarker. (D) Anti-TfR1mAbs inhibit PvRBP2b-TfR1 complex forma-tion in the FRET-basedassay.The FRETsignalwas relative to “nomAb”control. (E) Binding ofPvRBP2b161–1454 and

2b TfR

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PfRh428–766 in the presence of anti-TfR1 mAb MEM-189, CCP 1–3, andMACVGP1. (Left) Dot plots showing PvRBP2b161–1454 (top) and PfRh428–766

binding (bottom). (Right) Normalized binding results in which PvRBP2b161–1454and PfRh428–766 binding in the presence of buffer was arbitrarily assignedto be 100%. (F) MACV GP1 inhibits PvRBP2b161–1454–TfR1 complex

formation monitored by means of FRETassay. For (A), (B), (D), (E), and (F),mean ± SEM, n ≥ 3 biological replicates; open circles represent biologicalreplicates. Mann-Whitney test was used for (A) and (D), where MEM-75was considered noninhibitory, and t tests were used for (B), (E), and (F).*P ≤ 0.05, **P ≤ 0.001.

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inhibited PvRBP2b161–1454 reticulocyte bindingalso blocked PvRBP2b-TfR1 complex formation(Fig. 1D).

MACV GP1 and PvRBP2b bind to theapical domain of TfR1

The arenavirus envelope glycoprotein is theonly protein on the virion surface and, duringmaturation, is processed into three subunits:the stable signal peptide, GP1, and GP2. The GP1subunit interacts with cellular receptors, andthe structure of aMACVGP1–TfR1 complex showsthat MACV GP1 binds to the apical domain ofTfR1 (31, 32). TodeterminewhetherPvRBP2b inter-acts with a similar surface on TfR1, we exam-ined whether soluble MACV GP1 competes withPvRBP2b161–1454 for binding to TfR1 on retic-ulocytes (Fig. 1E). Indeed, the addition of MACVGP1 reduced PvRBP2b161–1454 binding to reticu-locytes, albeit at a lower level of inhibition as

compared with the addition of anti-TfR1 mAbMEM-189. This inhibition was specific; PfRh4binding was unaffected by addition of MACVGP1 or MEM-189 but clearly reduced with theaddition of CCP 1–3 (Fig. 1E). The addition ofMACV GP1 inhibited PvRBP2b-TfR1 complexformation and reduced the FRET signal to asimilar extent as unlabeled PvRBP2b161–1454,whereas addition of CCP 1–3 had negligibleeffect (Fig. 1F). These results indicate that MACVGP1 and PvRBP2b161–1454 bind to an overlappingsite on TfR1.

Crystal structure of the N-terminaldomain of PvRBP2b

PvRBP2b is a 326-kDa protein with a putative redblood cell–binding domain and a C-terminal trans-membrane region (Fig. 2). We determined thecrystal structure of the N-terminal domain ofPvRBP2b (residues 169 to 470; PvRBP2b169–470),

refined to 1.71-Å resolution (Fig. 2A; fig. S3, A toD; and table S1). The surface of the domain ismostly positively charged (Fig. 2B). It is predom-inantly ana-helical protein, comprising 10a-helicesand two very short antiparallel b-sheets, eachcomprising two b-strands. The crystal structureof PvRBP2b169–470 has two disulfide bonds: onebetween Cys312 and Cys316 and the other betweenCys240 and Cys284. This structure closely resem-bles the homologous domain of PvRBP2a andPfRh5, with a root mean square deviation of 1.7and 3.7 Å over 268 and 225 aligned Ca atoms,respectively (Fig. 2C and fig. S4) (18, 33, 34). Thetheoretical x-ray solution scattering pattern cal-culated from the PvRBP2b169–470 crystal struc-ture coordinates shows excellent agreement withthe experimental small-angle x-ray scattering(SAXS) data (c = 0.35) (fig. S5, A to F, and tableS2), with concordance between the crystal andsolution conformations apparent from the overlay

Gruszczyk et al., Science 359, 48–55 (2018) 5 January 2018 3 of 8

Fig. 2. Crystal structure of the N-terminal domain of PvRBP2b and itsfunctional requirement. (A) Structure of the N-terminal domain of PvRBP2bfrom amino acid 169 to 470 shown in two orthogonal views. (B) Electrostaticsurface potential on the PvRBP2b structure. (C) Superimposition of thePvRBP2b structure (green) with PvRBP2a (purple) and PfRh5 (orange).TheProtein Data Bank (PDB) ID codes for PfRh5 and PvRBP2a are 4WATand4Z8N, respectively. (D) Crystal structure of the N-terminal domainsuperimposed with SAXS ab initio bead model of PvRBP2b169–470 (left) andPvRBP2b169–652 (right). (E) Sliding window analysis showing nucleotidediversity (p) values and Tajima’s D statistic in PvRBP2b.The gray boxes referto a highly polymorphic region at amino acid positions 169 to 470 that appears

to be under balancing selection. (F) Schematic representation of full-lengthPvRBP2b and recombinant protein fragments (left). Signal peptide (SP),transmembrane domain (TM), and N-terminal domain (yellow) are indicated.(G) PvRBP2b binding results by means of flow cytometry, in whichPvRBP2b161–1454 binding was arbitrarily assigned to be 100%. (H) Unlabeledrecombinant PvRBP2b fragments or PfRh4weremixed at 10-foldmolar excessrelative to the labeled PvRBP2b161–1454–TfR1 FRETpair. The FRET intensitywas relative to buffer control. For (G) and (H), mean ± SEM, n = 4 biologicalreplicates; open circles represent biological replicates.The Mann-Whitney testwas used to calculate the P value by using the binding of 2b474–1454 that wasconsidered no binding. *P ≤ 0.05, **P ≤ 0.001.

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of the crystal structure and the ab initio calculatedmolecular envelope (Fig. 2D, left). We also ob-tained SAXS data for a longer fragment ofPvRBP2b including residues 169 to 652 (fig. S6and table S2). The reconstructed molecular en-

velope has a rodlike shape, with a C-terminal partforming a continuous extension of the N-terminaldomain (Fig. 2D, right). SAXS data for a largerfragment of PvRBP2b encompassing residues 161to 969 indicate that the molecule adopts an

elongated, boomerang-like shape, similar to thatpreviously reported for PvRBP2a (figs. S6, A toF, and S7, A to D, and table S2) (18).We calculated nucleotide diversity (p) and

Tajima’s D within PvRBP2b using data from

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Fig. 3. PvRBP2b binds to TfR1-Tf to form a stable ternary complex.(A) PvRBP2b161–1454 and (B) PvRBP2b161–969 were coupled covalently to abiosensor chip to probe binding of TfR1 (concentration range assayed, 2 mMto 7.5 nM, top) and TfR1-Tf complexes [concentration range of TfR1-Tfcomplexes assayed, 2:4 mM to 1.8:3.9 nM (A) and 2:4 mM to 7.5:15 nM (B),bottom]. (C and D) Complex formation between PvRBP2b,TfR1, and Tfanalyzed by means of analytical SEC. PvRBP2b-TfR1-Tf ternary complex canbe observed for PvRBP2b161–1454 [(C), top] and PvRBP2b161–969 [(D), top].Two corresponding truncations of the N-terminal domain, PvRBP2b474–1454

[(C), bottom] and PvRBP2b474–969 [(D), bottom], do not interact with theTfR1-Tf binary complex.The exclusion volume (V0) of the columns and theelution volumes of selected marker proteins are indicated with blackarrowheads. (Bottom) Coomassie blue–stained SDS-PAGE gels of thefractions obtained from SEC. (E and F) Continuous sedimentation coefficientdistributions derived from fitting sedimentation velocity data to a c(s)sedimentation model. (E) c(s) distributions for TfR1 (black line),Tf (magentaline), and PvRBP2b161–969 (blue line). (F) c(s) distributions for the TfR1-Tfcomplex (red line) and PvRBP2b161-969–TfR1-Tf complex (green line).

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theMalariaGEN P. vivaxGenome Variation proj-ect (35). Therewas a peak in bothmetrics betweenamino acid positions 169 and 470, suggestingbalancing selectionwithin theN-terminal domain(Fig. 2E). Such signatures of balancing selectionare often associated with genes or proteins ex-pressed on the surface ofmerozoites and are likelydue to interaction with the immune system.To determine the importance of the N-terminal

domain for PvRBP2b function, we generated aseries of purified recombinant PvRBP2b proteinfragments (Fig. 2F and tables S3 and S4). Allproteins were soluble and properly folded asindicated by high a-helical content in CD spectra,which is in agreement with the secondary struc-ture predictions (fig. S8, A to D). We observed

that all fragments with the N-terminal domainbound reticulocytes (PvRBP2b161–1454, PvRBP2b161–969,PvRBP2b169–813, and PvRBP2b169–652), whereas theircorresponding fragments without the domaindid not (PvRBP2b474–1454 and PvRBP2b474–969)(Fig. 2G).However, the isolatedN-terminal domainPvRBP2b169–470 was unable to bind reticulocyteson its own (Fig. 2G), indicating that this frag-ment of PvRBP2b is necessary but not sufficientfor reticulocyte binding. The shortest PvRBP2bfragment that showed binding to reticulocytesencompasses residues 169 to 652 (Fig. 2G). OurFRET-based assay showed that unlabeled recom-binant fragments that bind reticulocytes inhibitedPvRBP2b-TfR1 complex formation,whereas recom-binant fragments that did not bind reticulocytes

had a negligible effect (Fig. 2H). Collectively, ourstructural and functional analyses indicate thatthe N-terminal domain is necessary for bindingbut requires the presence of the elongated C-terminal fragment to form a fully functionalbinding site.

PvRBP2b, TfR1, and Tf form a stablecomplex at nanomolar concentrations

Using surface plasmon resonance, we found thatPvRBP2b161–1454 interacts with TfR1 alone orwith the binary complex of TfR1-Tf (Fig. 3A, topand bottom, respectively). We also observed sim-ilar results for the PvRBP2b161–969 fragment withTfR1 and TfR1-Tf (Fig. 3B, top and bottom, re-spectively). These results indicate that Tf was

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Fig. 4. Deletions in TFRC reduce TfR1 surface expression, abolishPvRBP2b binding, and inhibit P. vivax invasion. (A) Expression of TfR1,BSG, and GypA on the surface of jkRBCs, TfR1 mutants, DBSG null,and cultured erythrocytes (cRBCs) as measured with flow cytometry.(Right) Cytospin analysis of cells stained with May-Grünwald Giemsastaining technique. (B) TfR1DG217 mutation in TfR1 abrogates PvRBP2bbinding as observed by using analytical SEC. (C) Quantitative surfaceproteomics demonstrate specific reduction in TfR1 protein levels in TfR1mutants compared with wild-type jkRBCs. Levels of Tf, the bindingpartner for TfR1, are similarly reduced. Significance A was used to estimateP values, and a minimum of two peptides were required for protein

quantitation. (D) Binding of recombinant PvRBP2b fragments tojkRBCs, TfR1 mutants, DBSG, and cRBCs are shown in blue. Negativecontrols of unstained cells and isotype control stained cells are shownin the gray and orange lines, respectively. (Right) Compilation ofresults from PvRBP2b fragment binding to jkRBCs, TfR1 mutants, DBSG,and cRBCs. Mean ± SEM, n = 3 biological replicates. (E) Comparison ofinvasion efficiency between jkRBCs and TfR1 mutant cell lines with eitherP. vivax or P. falciparum. The data shown are averages and SEM frombetween four to five biological replicates shown as open circles. P valuewas calculated by using a paired, two-tailed t test. ****P ≤ 0.0001; ns,nonsignificant.

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not required for the PvRBP2b-TfR1 complex for-mation because the addition of Tf resulted insimilar binding responses than for TfR1 alone.We analyzed a PvRBP2b, TfR1, and Tf ternarycomplex using analytical size exclusion chroma-tography (SEC) and used SDS–polyacrylamide gelelectrophoresis (SDS-PAGE) analyses to confirmcomigration of complex components. The ter-nary complex was detected for PvRBP2b161–1454and PvRBP2b161–969 (Fig. 3, C and D, top, re-spectively, and table S5). By contrast, their cor-responding fragments without the N-terminaldomain (PvRBP2b474–1454 and PvRBP2b474–969)did not form any observable ternary complexes(Fig. 3, C and D, bottom). The interaction be-tween PvRBP2b and TfR1-Tf binary complexis similar in the presence of either the iron-depleted or iron-loaded form of human transferrin(fig. S2C). Furthermore, the homologous memberof the same protein family, PvRBP2a, did notform a ternary complex with TfR1-Tf (fig. S2D).Sedimentation velocity analyses of TfR1, Tf,

and PvRBP2b161–969 indicated that the isolatedproteins are homogenous, with weight-averagesedimentation coefficients of 7.3, 4.9, and 3.6 S,respectively (Fig. 3E). These values are consistentwith a stable dimer of TfR1 andmonomeric formsof both Tf and PvRBP2b161–969. The empiricallyfitted shape parameter value (frictional ratio)calculated for PvRBP2b161–969 was ~1.8, which isconsistent with a highly elongated structure insolution. Mixtures of TfR1-Tf and PvRBP2b161–969–TfR1-Tf yielded single symmetrical peaks withweight-average sedimentation coefficients of 11.5and 11.2 S, respectively, with no peaks observedfor the individual components in these samples(Fig. 3F and fig. S9). These results indicate that Tfand TfR1 form a stable binary complex in solu-tion and that PvRBP2b161–969 binds to this binarycomplex. The frictional ratio (f/f0) for the ternaryPvRBP2b161–969–TfR1-Tf was higher than for thebinary TfR1-Tf complex, resulting in a reductionin the sedimentation coefficient on formation ofthe ternary complex and indicating that it has anelongated structure in solution.

Deletions in TfR1 generated viaCRISPR/Cas9 abolishes PvRBP2bbinding and P. vivax invasion

To investigate whether loss of TfR1 surface ex-pression on red blood cells would affect PvRBP2bprotein binding, we attempted to generate aknockout of the TFRC gene using CRISPR/Cas9genome editing of the JK-1 erythroleukemia cellline. We obtained single-cell clones that displayedreduced expression of TfR1 and validated themutation in two independent clones (TfR1 mut1and TfR1 mut2) (fig. S10, A to C). Both clones con-tained an identical –3-bp deletion that resultedin the loss of amino acid Gly217 in the TfR1 apicaldomain but left the rest of the protein in-frame.TfR1 mut1 was homozygous for this deletion,whereas TfR1 mut2 has a –3-bp deletion, as de-scribed above, on one allele and a –11-bp deletionon the other allele, the latter leading to a pre-mature stop codon. Deletion of TFRC in a mousemodel is embryonic lethal and leads to severe

Gruszczyk et al., Science 359, 48–55 (2018) 5 January 2018 6 of 8

Fig. 5. Anti-PvRBP2b mAbs inhibit reticulocyte binding and P. vivax invasion in Brazilian andThai clinical isolates. (A) ELISA plates were coated with equimolar concentrations of eachrecombinant fragment, and detection with anti-PvRBP2b mAbs 3E9, 6H1, 8G7, and 10B12 are shown.(B) Competition ELISA by using immobilized PvRBP2b incubated with unconjugated anti-2b mAbs(x axis) and detected with 3E9-HRP, 6H1-HRP, 8G7-HRP, and 10B12-HRP as indicated. For (A) and (B), errorbars represent range showing the variability of duplicate measures. (C) PvRBP2b161–1454 binding in thepresence of anti-PvRBP2b mAbs 3E9, 6H1, 8G7, and 10B12 was analyzed by means of flow cytometry.Normalized binding results inwhichPvRBP2b161–1454 binding in the absence ofmAbswas arbitrarily assignedto be 100%.The anti-PvRBP2a mAb 3A11 was used as a negative antibody control. Mean ± SEM, n = 5biological replicates, open circles represent biological replicates.The Kruskal-Wallis test was used tocalculate the P value by using 8G7 binding as no inhibition. *P ≤ 0.05, ***P ≤ 0.0001. (D) Invasion of P. vivaxin Brazilian (blue open circles) and Thai (black open circles) clinical isolates in the presence of anti-PvRBP2b3E9, 6H1, 8G7, and 10B12, pooled mAbs (each mAb at one-third of final concentration), mouse isotypecontrol, purified rabbit prebleed IgG, purified total IgG of anti-PvRBP2b polyclonal antibody R1527, andcamelid anti-Fy6 mAb. Antibodies were added in concentrations from 25 to 125 mg/ml, except for thecamelid anti-Fy6 mAb, which was used at 25 mg/ml. Mean ± SD, n = 2 to 6 biological replicates; opencircles represent biological replicates. For experiments with n > 2 biological replicates, we used theKolmogorov-Smirnov test to compare 8G7 with 3E9, 6H1, and 10B12. *P ≤ 0.05, **P ≤ 0.001.

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disruption of erythropoiesis (36), suggesting thatcomplete deletion of TFRC in erythroid-lineagecells, such as JK-1, may not be possible.Differentiated polychromatic JK-1 cells (termed

jkRBCs) express surface proteins (including TfR1)at levels comparable with those of differentiatedCD34+ bone marrow–derived cultured red bloodcells (cRBCs) (37). The jkRBCs, cRBCs, and differ-entiated jkRBCs with a knockout within thebasigin receptor (DBSG) show normal levels ofTfR1, whereas TfR1 mutant clones displayed anintermediate level of TfR1 surface staining, witha panel of anti-TfR1 mAbs (Fig. 4A and fig. S11).Levels of glycophorin A (GypA) and basigin (BSG)on these TfR1 mutant clones were similar to allcontrol cells, showing that only TfR1 surface ex-pression is affected on these cells (Fig. 4A). Todetermine whether deletion of Gly217 affectsPvRBP2bbinding,wegenerateda recombinantTfR1protein that lacks this amino acid (TfR1DG217).Using SEC,we show that althoughTfR1DG217wasstill able to bind Tf, its binding to PvRBP2b wascompletely abolished (Fig. 4B). Gly217, which re-sides on the lateral surface of the TfR1 apicaldomain, is close to the MACV GP1 interactionsurface (fig. S10D) (31).To confirm that themutation in TFRC did not

result in changes in expression of other redblood cell proteins, we compared the abundanceof cell surface proteins betweenwild-type jkRBCsand the two TfR1 mutants using tandem masstag–based quantitative surface proteomics (Fig.4C). Out of 237 quantified surface proteins, onlyTfR1 and Tf were significantly modified, con-firming the specificity of the TFRCmutations.Wenextwanted todeterminewhetherPvRBP2b

binding was affected in the TfR1 mutant clones.PvRBP2b161–1454 and PvRBP2b161–969 bound jkRBCsand cRBCs, whereas recombinant fragmentsPvRBP2b474–1454 and PvRBP2b474–969 that lackedthe N-terminal domain did not (Fig. 4D). Bycontrast, we did not detect any PvRBP2b161–1454and PvRBP2b161–969 binding to TfR1mutant cells.This abolition of binding was specific to deletionsin TFRC because PvRBP2b bindingwas unaffectedon DBSG null cells or on cRBCs (Fig. 4D, right).We also compared the invasion efficiency betweenjkRBCs and TfR1 mutant cell lines with eitherBrazilian P. vivax isolates or P. falciparum 3D7(fig. S10E). A significant (>10-fold) reduction ininvasion efficiency was observed in the TfR1mutant line compared with the jkRBCs line withP. vivax, whereas no significant difference wasobserved with P. falciparum (Fig. 4E). Theseresults validate TfR1 as the cognate receptor forPvRBP2b and that TfR1 is an essential host factorfor P. vivax invasion.

Antibodies to PvRBP2b blockreticulocyte binding and P. vivax invasion

To examine whether PvRBP2b antibodies couldinhibit P. vivax invasion, we raisedmousemono-clonal antibodies to PvRBP2b161–1454 and ob-tained four mAbs. 3E9, 6H1, and 10B12 boundepitopes within the N-terminal domain presentin PvRBP2b169–470 with high affinities (Fig. 5A;fig. S12, A to C; and table S6), whereas mAb 8G7

recognized an epitope outside the N-terminaldomain within amino acids 813 to 969 (Fig. 5A).Competitionenzyme-linked immunosorbent assay(ELISA) experiments using mAbs directly conju-gated to horseradish peroxidase (HRP) show thateach mAb only competed with itself for bindingto PvRBP2b, showing that 3E9, 6H1, and 10B12bind to distinct epitopes in theN-terminal domain(Fig. 5B). Neither polyclonal nor monoclonal anti-bodies to PvRBP2b recognize recombinant PfRh4and five other recombinant PvRBPs, indicatingthat these antibodies are specific to PvRBP2b(fig. S12B) (19). Using flow cytometry, we showthat addition of anti-PvRBP2b mAbs 3E9, 6H1,and 10B12 abolished the PvRBP2b161–1454 bindingto reticulocytes, whereas anti-PvRBP2b mAb8G7 and anti-PvRBP2amAb 3A11 had no effect(Fig. 5C).WetestedtheabilityoftheantibodiestoPvRBP2b

to inhibit P. vivax invasion into human retic-ulocytes,usingashort-termP.vivaxexvivoassaywith Brazilian and Thai clinical isolates (Fig. 5D,blue and black open circles, respectively). As acontrol,weusedacamelid antibody toFy6,whichis a single monovalent VHH domain (15 kDa)(38,39) that targets a surface-exposedepitopeonDARC and blocks its interaction with PvDBP.Theadditionof the25mg/mlcamelid antibody toFy6 in ex vivo assays by using Thai isolatesresulted in 85% inhibition of P. vivax invasion(Fig. 5D).Using fourThai isolates, theadditionofinhibitoryanti-PvRBPmAbs3E9,6H1,and10B12at25mg/mlresulted in49, 45,and42%inhibitionof P. vivax invasion, respectively. To determinewhether inhibition could be improved by in-creasingtheconcentrationofanti-PvRBP2bmAbsto match the molarity and valency of the singleVHH domain, we used 125 mg/ml of inhibitoryanti-PvRBP2bmAbs. Under these conditions, wetested the invasion efficiency of two Brazilianisolates. We observed that addition of inhibitoryanti-PvRBPmAbs3E9,6H1,and10B12at125mg/mlresulted in 68, 45, and 57% inhibition of P. vivaxinvasioninBrazilianisolates,respectively(Fig.5D).To enable quantitative analyses of the ex vivo as-says, we combined our initial results of Thai andBrazilianisolatesattheirrespectivemAbconcen-trations. Increased concentration of the inhibitoryanti-PvRBP2b mAbs resulted in an equivalent orsmall increase in inhibition of P. vivax invasion(Thai at 25 mg/ml versus Brazilian at 125 mg/ml).Thus,ourcombinedsamplesetunderestimatesthelevel of inhibition for antibody concentrations of125 mg/ml. As additional controls, we includedthe noninhibitory anti-PvRBP2b mAb 8G7 andan immunoglobulinG1 (IgG1)mouse isotype con-trol, which displayed only 8 and 9% inhibition ofP. vivax invasion, respectively (Fig. 5D). Theseresults show that addition of anti-PvRBP2b in-hibitory mAbs 3E9, 6H1, and 10B12 resulted insignificant reduction of P. vivax invasion com-paredwith the noninhibitory anti-PvRBP2bmAb8G7 (Fig. 5D).We show that the inhibitory anti-PvRBP2b

mAbs target a domain that appears to be underbalancing selection (Figs. 2E and 5A), which mayresult in differences in inhibition between clinical

isolates owing to the presence of polymorphicepitopes. To circumvent inter-isolate differences,we further tested the combination of all threeinhibitory mAbs, 3E9, 6H1, and 10B12 pooledtogether (mAb pool) and polyclonal antibodies toPvRBP2b. The mAb pool resulted in significant48% reduction in P. vivax invasion in both Thaiand Brazilian isolates comparedwith that of anti-PvRBP2bmAb 8G7 (Fig. 5D). Addition of purifiedtotal IgG of polyclonal antibodies to PvRBP2bR1527 resulted in 53% reduction inP. vivax invasion,whereas the rabbit prebleed IgG showed only5% inhibition (Fig. 5D). A previous study usingrabbit antibodies to PvDBP shows that P. vivaxinvasion was reduced up to 64% (40), a level ofinhibition comparable with what has been ob-served with our antibodies to PvRBP2b (Fig. 5D).These results show that anti-PvRBP2bmAbs thatblock binding to reticulocytes also inhibit P. vivaxinvasion and highlight the important role of thePvRBP2b-TfR1 invasion pathway in P. vivax fieldisolates.Our results reveal a stable interaction be-

tween PvRBP2b and TfR1 and that antibodies toPvRBP2b that block binding to reticulocytes alsoinhibit P. vivax invasion into human reticulocytes.P. vivax invasion is significantly inhibited in thepresence of TfR1 mutant cells, showing that TfR1is a critical host factor for entry into reticulo-cytes. We propose that the PvRBP2b-TfR1 inter-action is important for the initial recognition ofthe target reticulocyte cells, which results in thecommitment of P. vivax parasites for reticulo-cyte invasion and the subsequent engagement ofPvDBP-DARC in tight junction formation, lead-ing to the successful completion of the invasionprocess. Identification of the molecular entitiesrequired for P. vivax invasion offer the possi-bility to target multiple invasion pathways forsynergistic inhibition of P. vivax blood stageinfection.

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ACKNOWLEDGMENTS

We thank J. Newman from the Commonwealth Scientific andIndustrial Research Organization Collaborative Crystallization Centrefor assistance with setting up the crystallization screens, the Walterand Eliza Hall Institute’s Monoclonal Antibody Facility for productionof antibodies, J. Williamson for assistance with mass spectrometry,and MX and SAXS beamline staff at the Australian Synchrotron fortheir assistance during data collection. We thank F. Nosten, the staffand patients attending the Mae Sot Malaria Clinic in Thailand, andclinics associated with the Shoklo Malaria Research Unit (SMRU), TakProvince, Thailand. We also thank Y. Colin and O. S. Bertrand(INSERM/University Paris 7) for the generous gift of the antibodies toDARC. W.-H.T. is a Howard Hughes Medical Institute–Wellcome TrustInternational Research Scholar (208693/Z/17/Z). This work wassupported in part by the Australian Research Council FutureFellowships to W.-H.T. and M.D.W.G., a Speedy Innovation Grant toW.-H.T., and a National Health and Medical Research Councilfellowship (1105754) to J.M.M. U.K. was supported by a CanadianInstitutes of Health Research Postdoctoral Fellowship. R.D.P. isfunded by Wellcome Trust 090770. M.P.W. was supported by aWellcome Trust Senior Clinical Research Fellowship (108070/Z/15/Z). This study received funding from Singapore National MedicalResearch Council (NMRC) (NMRC/CBRG/0047/2013) and theAgency for Science, Technology and Research (A*STAR, Singapore).SMRU is sponsored by The Wellcome Trust of Great Britain as part ofthe Oxford Tropical Medicine Research Programme of Wellcome

Trust–Mahidol University. Work in the M.T.D. laboratory wassupported by National Institutes of Health grant 1R01HL139337. Wealso acknowledge the support of the B.R. laboratory from the MarsdenFund 17-UOO-241.The authors acknowledge the Victorian StateGovernment Operational Infrastructure Support and AustralianGovernment National Health and Medical Research CouncilIndependent Research Institute Infrastructure Support Scheme. Alldata and code to understand and assess the conclusions of thisresearch are available in the main text, supplementary materials, andvia the following repositories: The atomic coordinates and structurefactors for PvRBP2b have been deposited in PDB with accessionnumber 5W53. Genotypes were derived from sequence datagenerated at the Wellcome Trust Sanger Institute (Wellcome Trust206194 and 098051).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/359/6371/48/suppl/DC1Materials and MethodsFigs. S1 to S12Tables S1 to S6References (41–83)Data Set S1

6 March 2017; resubmitted 29 September 2017Accepted 16 November 201710.1126/science.aan1078

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Plasmodium vivaxTransferrin receptor 1 is a reticulocyte-specific receptor for

Russell, Laurent Renia, Manoj T. Duraisingh and Wai-Hong ThamD. W. Griffin, James M. Murphy, Jonathan Abraham, Kanlaya Sriprawat, Maria J. Menezes, Marcelo U. Ferreira, Bruce

MichaelYee-Foong Mok, Kai-Min Lin, Richard D. Pearson, Gabriel Rangel, Brian J. Smith, Melissa J. Call, Michael P. Weekes, Jakub Gruszczyk, Usheer Kanjee, Li-Jin Chan, Sébastien Menant, Benoit Malleret, Nicholas T. Y. Lim, Christoph Q. Schmidt,

DOI: 10.1126/science.aan1078 (6371), 48-55.359Science 

, this issue p. 48Science infection of red blood cells.P. vivax protein successfully hindered P. vivaxMonoclonal antibodies to the

could not.P. vivax could still infect cells in which TfR1 expression was knocked down, but P. falciparumas a receptor: , does not share TfR1P. falciparum surface protein. However, the parasite that causes cerebral malaria, P. vivaxspecific

. TfR1 binds to aP. vivax identified TfR1 (host transferrin receptor 1) as an alternative receptor for et al.Gruszczyk , which causes relapsing malaria, specifically parasitizes immature red blood cells called reticulocytes.P. vivaxbiology.

protozoan parasites, each with distinctivePlasmodiumHuman malaria is caused by half a dozen species of Vivax malaria host receptor

ARTICLE TOOLS http://science.sciencemag.org/content/359/6371/48

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2018/01/03/359.6371.48.DC1

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