J Mol Med (2004) 82:600–611DOI 10.1007/s00109-004-0573-9

O R I G I N A L A R T I C L E

Javier E. Garc�a · Hernando Curtidor ·Ramses L�pez · Luis Rodr�guez · Ricardo Vera ·John Valbuena · Jaiver Rosas · Marisol Ocampo ·Alvaro Puentes · Martha Forero ·Manuel A. Patarroyo · Manuel Elkin Patarroyo

Liver stage antigen 3 Plasmodium falciparum peptidesspecifically interacting with HepG2 cellsReceived: 30 September 2003 / Accepted: 26 May 2004 / Published online: 7 August 2004� Springer-Verlag 2004

Abstract Binding assays were carried out with 20 aminoacid long peptides covering the complete 200-kDa Liverstage antigen (LSA) 3 protein sequence to identify its

HepG2 cell binding regions. Seventeen HepG2 cell high-activity binding peptides (HABPs) were identified in theLSA-3 protein. Seven HABPs were found in the nonre-peat (NRA) region A; five of these formed a 100 aminoacid long HepG2 cell binding region located betweenresidues 21Ile and 120Thr. Six HABPs were found in theR2 region and another four in the NRB2 region. LSA-3protein HABPS bound saturably to HepG2 cells havingnanomolar affinity constants and bound specifically to 31,44, and 70 kDa HepG2 cell membrane proteins. Some ofthem were located in antigenic and immunogenic LSA-3protein regions. Immunofluorescence and immunoblot-ting assays using goat sera immunized with LSA-3 pro-tein peptides recognized P. falciparum (FCB-2 strain)erythrocyte stage proteins (58, 68, 72, 81, 86, 160, and175 kDa). This reactivity was due mainly to the VEES-VAEN motif present in some erythrocyte stage proteins.However, our results suggest that antibodies against LSA-3 regions had a crossed reaction with another 86-kDaprotein, and that this crossed reaction was due to a motifpresent in the NRA region.

Keywords P. falciparum · Liver stage antigen-3 · Highactivity binding peptides

Abbreviations HABP: High-activity binding peptide ·LSA: Liver stage antigen · NRA: Nonrepeated region A ·NRB: Nonrepeated region B · NRC: Nonrepeated regionC · PfLSA-3: P. falciparum LSA-3 · RESA: Ring infectederythrocyte surface antigen · SDS-PAGE: Sodium dodecylsulfate polyacrylamide gel electrophoresis ·TBST: Tris-buffered saline solution

Introduction

The preerythrocyte stage of the Plasmodium falciparumlife cycle begins when sporozoites are released from thefemale mosquito’s salivary glands using the host’s cir-culating blood; they enter the hepatocytes within the next

Javier E. Garcia

graduated in chemistry fromthe Universidad Nacional deColombia in 1994. He is cur-rently studying for a Ph.D. inchemistry in the UniversidadNacional de Colombia and isworking as a researcher inFundacion Instituto de In-munologia de Colombia in thereceptor-ligand section. Thehost interaction with patho-genous cells is the main focusin his research directed towardsmalaria caused by P. falcipa-rum.

Manuel E. Patarroyo

received his M.D. degree fromthe Universidad Nacional deColombia in 1971 where he isFull Professor for MolecularPathology. He received furthertraining as an immunologist atthe Rockefeller University,USA, and Karolinska Institute,Sweden. He is also Founderand current Director of theFundaci�n Instituto de In-munolog�a de Colombia. Hismain research interest includessynthetic vaccines mainlyagainst malaria and modulatingthe immune response.

J. E. Garc�a · H. Curtidor · R. L�pez · L. Rodr�guez · R. Vera ·J. Valbuena · J. Rosas · M. Ocampo · A. Puentes · M. Forero ·M. A. Patarroyo · M. E. Patarroyo ())Fundaci�n Instituto de Inmunolog�a de Colombiaand Universidad Nacional de Colombia,Carrera 50 #26-00, Bogot�, Colombiae-mail: mepatarr@mail.comTel.: +57-1-4815219, Fax: +57-1-4815269

30–45 min. However, it is not clear how sporozoitessqueeze through the sinusoid lining into the Disse space(or, as might be the case, pass through Kupffer cells onthe walls of the sinusoids) to reach the hepatocytes [1].Very little is known about the precise nature of the spo-rozoite-hepatocyte ligand-receptor interaction enablingthe parasite to recognize the host cell or about P. falci-parum parasite proteins involved in sporozoite-hepatocyteinvasion processes and parasite intrahepatic developmentup to the liberation of hepatic merozoites which then in-vade erythrocytes. The immunological and protection-inducing relevance of several P. falciparum hepatic stageantigens, such as the circumsporozoite protein, serine-threonine antigen rich protein, sporozoite and liver stageantigen, and liver stage antigen (LSA) 1 and 3 proteinsstudied to date [2, 3, 4, 5], indicates these proteins’ im-portance as vaccine candidates against infection by P.falciparum.

P. falciparum LSA-3 (PfLSA-3) is the first preery-throcyte stage antigen selected by screening differences inthe immunological responses between protected andnonprotected volunteers immunized with irradiated spo-rozoites [2]. LSA-3 has a 200-kDa molecular weight and1,786 amino acids [2, 6]. This protein possesses threenonrepeat regions: NRA (residues 1–222), NRB (residues819–1535), and NRC (residues 1578–1786) and threeregions with repeated sequences named R1 (residues 222–278), R2 (residues 279–818), and R3 (residues 1536–1577; Fig. 2) [2].

The NRA region has a 17 amino acid, noncharged,hydrophobic fragment considered to be a peptide having apotential signal sequence for LSA-3 subcellular local-ization in sporozoite and liver forms. The NRB region ishighly conserved in parasite clone T9/96 and K1 se-quences; HLA-B53 restricted epitope la90 is found in thisregion [6]. The R1 region presents a series of tetrapep-tides which are conserved in clones T9/96 and 3D7. TheR2 region contains a series of repeat sequences formed byoctapeptides containing the VEESVAEN sequence, vary-ing in number (between two and seven depending on thestrain). The R3 region is characterized by the presence ofregularly spaced valine and isoleucine, favoring an a-helix secondary structure; this region also presents a highdegree of conserved sequences in the 3D7 clone andisolates from several geographical areas [2] (http://www.pasteur.Fr/parmed/lsa3).

LSA-3 is expressed on sporozoites and is present in theparasitophorous vacuole and the periphery of maturinghepatic merozoites [2]. Antibodies induced by lipopep-tides (palmitic acid peptide) from LSA-3 protein inocu-lated in mice and chimpanzees detect LSA-3 expressionin sporozoites and P. falciparum-infected hepatocytes [2,7]. Immune sera from Aotus monkeys inoculated withLSA-3 peptides has recognized native protein expressedon the sporozoite surface, in liver schizonts from twodistinct strains and all the sporozoites from 30 wild iso-lates that developed in mosquitoes fed in vitro on Thaigametocytes [2, 8]. LSA-3 mRNA has not been detectedin blood-stage parasites. Anti-LSA-3 antibodies have not

shown cross-reaction with infected red blood cells.However, antibodies against the LSA-3 glutamine-richrepeat region have a crossed-reaction against the D260homologue antigen glutamine repeat region which hasbeen identified in the parasite’s intraerythrocyte stages;this region is homologous to a ring infected erythrocytesurface antigen (RESA) protein region [2]. It has beenfound that LSA-3 presents immunological cross-reactivitywith Plasmodium yoelli proteins. Immunizing mice withPfLSA-3 DNA induces a potent Th1 response, conferringprotection against experimental challenge with P. yoelli;sera produced in BALB/c mice immunized with PfLSA-3DNA specifically reacts with P. yoelli or P. falciparumsporozoite surface in a similar way [9, 10].

Some LSA-3 recombinant proteins and peptides be-longing to LSA-3 protein have presented antigenic andprotection-inducing immunogenic properties in animalmodels; LSA3-NRI, LSA3-NRII-Lp, LSA3-RE, andLSA3-CT1-Lp induced interferon g production, whilepeptides LSA-3NRI and LSA-3 RE have presented T-cellproliferation in Aotus monkey lymphocytes [8]. Chim-panzees immunized with LSA-1 and LSA-3 recombinantproteins have been protected against three challenges byP. falciparum [2].

LSA-3 is a preerythrocyte antigen which has beenconsidered to be a candidate for a vaccine against P.falciparum due to its antigenic and protection-inducingimmunogenic properties in animal models, making thisprotein of interest to us. Binding assays were carried outwith 20 amino acid long, nonoverlapping synthetic pep-tides covering the whole length of the LSA-3 proteinto identify HepG2 cell high-activity binding peptides(HABPs). Some of the 17 HepG2 cell HABPs thus iden-tified were located in antigenic and protection-inducingimmunogenic regions in experimental animal models.These HepG2 HABPs did not bind to red blood cells,indicating that these sequences bound specifically toHepG2 cells. Goat sera immunized with a mixture of sixpeptides from nonrepeat and repeat regions producedantibodies recognizing 175, 160, 86, 81, 72, 68, and 58-kDa proteins in P. falciparum intraerythrocyte schizontlysate. These sera reacted with schizonts in immunoflu-orescence assays; this was perhaps due to the cross-re-action between LSA-3 protein and D260 protein, at-tributable to the VEESVEENVEESVAENVEESVAENmotif located in LSA-3 protein R2 region.

Materials and methods

Cell culture

The HepG2 human cell line [11, 12] was kept in RPMI 1640supplemented with 10% fetal bovine serum (ICN), penicillin(100 IU/ml, ICN), streptomycin (100 �g/ml, ICN), amphotericin B(0.25 �g/ml, ICN), vitamin (ICN), and nonessential amino acidsolution (Gibco). The cells were grown as monolayers in 75–150 cm2 culture flasks coated with a 2% collagen solution (Nuncand Falcon) at 37�C in a 5% CO2 atmosphere. The monolayerswere removed by 0.25% trypsin, 0.1% EDTA solution after 4 days.The culture was then expanded and kept in the conditions described

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above. The HepG2 cells were harvested by adding PBS-EDTA andcentrifuging. The cells were washed five times with PBS andcounted in a Newbauer chamber before being used in binding as-says.

Peptides

The 88 peptides (sequence reported by [2], Gene data Bank, ac-cession no. CAB65343) were synthesized by solid-phase metho-dology [13], purified by reversed-phase HPLC, and characterizedby matrix-assisted laser desorption/ionization time-of-flight. Thepeptides were 20 amino acids long and covered the whole of theLSA-3 protein sequence. One Tyr residue was added at the C-terminal sequence (see Fig. 2) to those peptides which did notcontain this residue in their sequences to enable 125I-radiolabeling[14]. The polymeric peptides (for goat immunization) were ob-tained by using the following methodology: monomeric peptidesequences containing glycine-cysteine residues at the carboxy andN-terminals were synthesized by solid-phase methodology [13].The synthesized peptide was lyophilized and dissolved in water andoxidized at pH 7.5 by slowly passing oxygen through it until anegative reaction was achieved with Ellman reagent. The polymericpeptides were analyzed by size exclusion chromatography.

Radiolabeling

The peptides were radiolabeled according to Urquiza et al. [15]. Inbrief, 2 nmol purified peptides were radiolabeled with 5 �l Na125I(100 mCi/ml, ICN) and 0.3 �mol chloramine-T at a final 25 �lvolume and incubated for 15 min at room temperature. The reactionwas stopped with 0.3 �mol sodium metabisulfite [14]. The 125I-peptides were purified on a Sephadex G-10 column; these peptideshad 50–400 �Ci/nmol specific activity.

Cell binding assays

Binding assays were performed according to Garc�a et al. [16] witha few modifications. In brief, HepG2 cells (1.2�106 cells) wereincubated with different 125I-peptide concentrations (0, 250, 500,750 and 1,000 nM) in the presence (50–200 excess) or absence ofunlabeled peptide for 1 h at 4�C, with constant shaking. An aliquotof this reaction mixture was passed through a 60:40 dioctylftalate-dibutylftalate cushion (density 1.015 g/ml), spinning at 15,000 g for1.5 min. Cell-associated radioactivity was quantified in an auto-matic gamma counter (4/200 plus, ICN). The hepatitis B, 27-mer,4980 (PLGFFPDHQLDPAFGANSNNPDWDFNP) peptide, report-ed as specifically binding to HepG2 cells [17], was used as positivecontrol for binding assays, according to our established bindingassay protocol [16]. Binding assays were performed in triplicate(Figs. 1, 2A).

Erythrocyte binding assays were carried out according to Ur-quiza et al. [15]. In brief, 109 erythrocytes were incubated withvarying concentrations of 125I-peptide in the presence or absenceof unlabeled peptide at a final volume of 150 �l for 1 h at roomtemperature. Cells were then washed five times with PBS. Cell-associated radioactivity was quantified in an automatic gammacounter (4/200 plus, ICN); binding assays were performed in trip-licate. Erythrocyte binding assays were performed with some LSA-3 peptides.

Saturation assays

HepG2 saturation assays were performed with LSA-3 HABPs26238–26242, 26244, 26247, 26283, 26284, and 26293. HepG2cells (1.2�106 cells) were incubated with 125I-peptide at concen-trations ranging from 100 to 2,500 nM, in the presence or absenceof unlabeled peptide (20–500 excess). The curves thus obtainedwere analyzed and affinity constant values were determined by Hill

equation, curves being drawn according to our established protocol[16, 18]. Saturation assays were performed in triplicate.

HepG2 cell membranes

Membranes were obtained by a previously reported methodology[19]. Briefly, 2.5�106 cells were suspended in 6 ml buffer A(0.25 M sucrose, 5 mM Tris-HCl, 1 mM MgCl2, pH 8.0) and passedten times through a 0.27-needle syringe. The lysed cells werecentrifuged at 250 g for 5 min at 4�C; the supernatant was skimmedoff and stored at 4�C. The pellet was suspended in buffer A and theprocess repeated twice. The obtained pool of supernatants wascentrifuged at 1,500 g for 10 min at 4�C, and the pellets weresuspended in buffer A again, then pooled and centrifuged at15,000 g for 30 min at 4�C. The supernatant was discarded and thepellet suspended in buffer B (1.42 M sucrose, 5 mM Tris-HCl,1 mM MgCl2, pH 8.0). This suspension was poured into the bottomof a tube; 2 ml buffer B and 1 ml buffer A were then added onto thetop. The sucrose gradient was centrifuged at 80,000 g for 60 min at4�C. The ring observed in the interface was suspended in PBS andspun at 15,000 g for 60 min. Plasmatic membranes were collectedfrom the pellet, which was then suspended in 1 ml PBS and storedat �20�C [16].

Membrane binding assays and SDS-PAGE

The methodology used for the membrane binding assays was thatreported by us [16], slight modifications being made to it. HepG2cell membranes (50 �l) were incubated with 200 �l 125I-peptide(1000 nM) for 2 h at 4�C in the presence or absence of 50 �l un-labeled peptide (50 �M). The suspension was spun at 15,000g for5 min and the supernatant discarded; the pellet was then washedthree times with PBS. Membrane-bound peptide was cross-linkedwith 20 �M Bis (sulfosuccinimidyl suberate) (BS3, Pierce) for15 min at 4�C and the reaction was stopped with 0.1 M Tris-HCl,pH 6.8. The pelleted membranes were washed three times withPBS, centrifuged, suspended in Lemmli buffer and then boiled at90�C for 2 min. Membrane proteins were separated in 12% sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).The gel was dried, exposed for 7 days at �70�C, and developed onKodak film (X_OMAT). The 125-I-labeled band’s molecular weightwas determined by comparing it with the apparent standard weightmarkers’ low range (Bio-Rad). HABP binding was also tested incross-linking assays to C32 endothelial cell membrane using thesame methodology.

Goat immunization

Polymeric peptides were used for immunization in goats to obtaina large quantity of LSA-3 protein region-immune sera. Two goats(B65 and B98, previously determined to be nonreactive to P. fal-ciparum lysate by western blotting) were injected with a mixturemade up of 500 �g of each one of the following LSA-3 poly-meric peptides corresponding to monomeric peptide amino acidsequences: 28726 (81CGKKLNKLFNRSLGESQVNGELGC100),28730 (993CGSGESLENNEMDKAFFSEIFDGC1012), 28732(1193CGIKDKEKDVSLVVEEVQDNDMGC1213), 28734 (1433CG-LKGSILDMLKGDMELGDMDKGC1452), 28736 (1709CGKNKE-RPFYSFVFDIFKNLKHGC1728), and 28728 (461CGEIVVPTVE-ESVAENVEESVAGC480) (NB: this sequence was obtained fromisolate 3D7, Gene Data Bank, accession no. NP_473111). Thispeptide cocktail was inoculated with Freund’s complete adjuvanton day 0 and incomplete adjuvant on days 20 and 40. Bleeding wasperformed on days 0 (preimmune), 20 (I-20) and 40 (II-20) forantibody production analysis. Final bleeding was carried out onday 60 (III-20) and sera were collected. Immunizations andbleeding were done according to Colombian Ministry of PublicHealth animal-handing procedures.

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Goat sera adsorption with E. coli and M. smegmatis sonicateand Spf66-Sepharose

Escherichia coli (DH5a strain) proteins were obtained from over-night culture in Luria Bertani medium, washed, suspended, andsonicated for 2 min at 4�C and 10 min at 4,500 g. M. smegmatisproteins were obtained from 5-day-old Middlebrook 7H9 brothculture, washed, suspended, sonicated for 10 min (as describedabove) and centrifuged for 10 min at 4,500 g. Both pellets weresuspended in 0.1 M NaHCO3 , 0.5 M NaCl, pH 8.3 (couplingbuffer). The suspended lysates and Spf66 vaccine [20] were col-lected and used individually for coupling to CNBr-activated Se-pharose 4B (Pharmacia Biotech), according to the manufacturer’srecommendations. Each goat serum (preimmune and immune) waspreadsorbed with E. coli–Sepharose, M. smegmatis–Sepharose, andSpf66–Sepharose affinity columns to eliminate cross-reactivity.Briefly, 5 ml of each serum were added to 4 ml lysate (or Spf66)–Sepharose affinity columns and left in a gently rotating/shakingmode for 20 min at room temperature. This procedure was carriedout twice using a new lysate–Sepharose affinity column each timeand the sera were stored at �70�C (the sera which were preadsorbedwith E. coli–Sepharose, M. smegmatis–Sepharose and Spf66–Se-pharose affinity columns were called B65-A and B98-A).

Adsorption of preabsorbed sera with peptide-Sepharose columns

B65-A and B98-A sera were adsorbed in Sepharose columns cou-pled to peptides belonging to LSA-3 protein to determine whetherthe antibodies present in these sera (presenting a crossed reactionwith schizont lysate) are retained by these peptides. The coupledpeptides were: peptide 26271 (this peptide is known to possessthe PSVEESVEENVEESVAENVE repeat motif present in D260protein [21]), 6685 (from RESA protein, 421QKYMDMLDTSEE-ESVEENE440 [22]), 28826 (CGFLTYYNYDTFSKTKFNNNIK-GC, from the P. falciparum hypothetical NP-703322.1 protein,unrelated peptide) and the mixture of peptides used to inoculate thegoats (28726, 28728, 28730, 28732, 28734 and 28736, 1 �mol ofeach one). The LSA-3 peptides were coupled to Sepharose columnsusing 1 �mol of peptide for coupling to 1 ml CNBr-activated Se-pharose 4B (Pharmacia Biotech), according to the manufacturer’srecommendations. Then 500 �l of each serum (B65-A, B98-A) wasadded to 500 �l peptide–Sepharose affinity columns and left in agentle rotating/shaking mode for 20 min at room temperature. Thisprocedure was carried out twice using a new peptide-Sepharoseaffinity column each time and the sera were stored at �70�C.

Fig. 1 Binding assays. Thethree types of behavior obtainedin HepG2 cell binding assaysusing peptides from LSA-3protein can be observed. LeftGraphs of peptide 26244,26248, and 26323 binding,where the total binding curvecan be seen (filled squares),representing binding to HepG2cells incubated with only 125I-peptide and inhibited binding(empty squares), representingbinding to HepG2 cells incu-bated with 125I-peptide in thepresence of 50–200 excess ofthe same unlabeled peptide.Right Specific binding curvesfor these peptides (total bindingminus inhibited binding, filledtriangles). It can be seen thatpeptide 26244 presented highspecific binding activity (2.0slope) and was considered to bean HABP, while peptide 26248presented high nonspecificbinding (<0.1 slope) and pep-tide 26323 presented low spe-cific binding to HepG2 cells(<0.1 slope)

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Fig. 2 Screening assays. A Re-sults of binding assays usingpeptides from LSA-3 proteinbinding to HepG2 cells. Thepeptide number (used in ourInstitute’s numbering system)can be seen, as well as its po-sition and the amino acid se-quence of peptides from syn-thesized LSA-3 protein. Blackbars Slope values obtained fromthe specific HepG2 cell bindingcurves for each one of the pep-tides assayed. Right Diagramsof the different LSA-3 proteinnonrepeat and repeat regions.When this sequence was divid-ed into 20 amino acid frag-ments, the amino acid se-quences for peptides 26251 and26267 completely coincided (asdid those for peptides 26254and 26258); peptides 26267(residues 601–620) and 26258(residues 421–440) were thusdeleted from A. The tyrosineresidues added to the peptidesnot containing this residue inthe native sequence and allow-ing radiolabeling are shownunderlined in the last column.B Results of the erythrocytebinding assays for some HABPpeptides from LSA-3 protein.Low specific HepG2 cell bind-ing peptides belonging to theNRB region were: 26286,26287, 26296, 26297, 26308,and 26309. Peptide 6685 fromRESA protein possessed theSEEESVEENE motif

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SDS-PAGE and immunoblotting

Proteins from P. falciparum intraerythrocyte schizont lysate wereseparated in a discontinuous SDS-PAGE system using a 7.5–15%(wt/vol) acrylamide gradient. A total of 500 �g/ml lysate wasloaded per gel and then transferred to nitrocellulose membrane(Hybond 203c, Pharmacia) using the semidry blotting technique[23]. Commercial molecular mass markers (NEB) were used forcalibration.

Nitrocellulose membrane were incubated with a 1:100 dilutionof pretreated sera (B65-A and B-98-A), adsorbed on Sepharosecolumns coupled to each one of peptides 26271, 6685, 28826 andthe inoculated peptide mixture) in Tris-buffered saline solution(TBST: 0.02 M Tris-HCl pH 7.5, 0.05 M NaCl, 1% Tween 20) and5% skimmed milk. One hour’s incubation with 1:5,000 alkalinephosphatase conjugated anti-goat IgG antibody (ICN) was carriedout after five TBST washes. The reaction was developed with NBT/BCIP (KPL).

Each of the peptides used above was incubated with B65-A andB-98-A to determine whether these peptides could block P. falci-parum schizont lysate recognition of sera in immunoblotting as-says. Nitrocellulose membranes were incubated with a 1:100 di-lution of B65-A sera with each one of the following peptides:26241, 26271, 6685, 28726, and 28728 (in 100, 200, and 800 �Mconcentrations) in TBST and 5% skimmed milk. One hour’s in-cubation with 1:5,000 alkaline phosphatase conjugated anti-goatIgG antibody (ICN) was carried out after five TBST washes. Thereaction was then developed with NBT/BCIP (KPL). The wholeprocedure was then repeated with B-98-A sera.

Immunofluorescence assay

Immunofluorescence was performed with Sera B65-A and B98-A(III-20). Late-stage continuous P. falciparum culture (FCB-2)schizonts, presenting 10% parasitemia, were used. They weresynchronized according to Lambros and Vanderberg’s [24] method,collected and then washed in sterile PBS (0.15 M phosphate buffer,containing 0.15 M NaCl, pH 7.2). The infected cell pellet wassuspended in fetal bovine serum/PBS (1:1 v/v) and left to dry on theslide. Air-dried, fixed sporozoites (a kind gift from Patricia de laVega, NIMR, Washington) were also used for immunofluores-cence. The slides were then blocked for 10 min with 1% nonfatmilk and incubated for 30 min with twofold sera dilutions, startingat 1:40 dilution. They were then washed five times for 5 min, in-cubated with rabbit anti-goat IgG (H+L; Kirkegaard and Perrylaboratories, 02-13-06) in the dark for 30 min at 1/100 dilution.Reactivity was observed by fluorescence microscopy. Preimmunegoat sera were used as negative controls.

Results

Binding assays

Twenty amino acid long synthetic peptides spanning theLSA-3 protein were used in HepG2 cell binding assays,incubating a number of cells with increasing concentra-tions of radiolabeled peptide in the absence (total binding)or presence (inhibited binding) of the same unlabeledpeptide. Specific binding was calculated by subtractingtotal binding from inhibited binding (Fig. 1) [16]. Threetypes of behavior were found in the binding assays: (a)specific high binding peptides (e.g., peptide 26244), (b)nonspecific high-binding peptides (e.g., peptide 26248),and (c) nonbinding peptides (e.g., peptide 26323). Thespecific binding curves represent the ratio of added ra-diolabeled peptide to bound radiolabeled peptide (slope).

Those peptides presenting a slope value greater than orequal to control peptide were considered to be HABPs, asshown in Fig. 2A.

Seventeen HepG2 cell HABPs were identified in LSA-3 protein; these were located in the NRA (7/17), R2 (6/17), and NRB (4/17) regions. No HABPs were found inthe R1 or R3 regions. A HepG2 cell binding region wasfound in the NRA (residues 21–120) spanning 100 aminoacids. This consisted of the following peptides: 26238-26242 (21ITTIFNRYNMNPIKKCHMREYKINKYFFLI-KILTCTILIWAVQYDNNSDINKS WKKNTYVDKKL-NKLFNRSLGESQVNGELYASEEVKEKILDLLEEGNT-LTY120). The following HABPs were found in the NRAregion’s C-terminal: 26244 (141LSNIEEPKENIIDNLL-NNIGY160) and 26247 (201SQVNDDIFNSLVKSVQQE-QQY220). HABPs 26240, 26241, and 26247 presentedthe greatest slope values: 5.6, 3.8, and 3.4, respectively(Fig. 2A).

Six HepG2 cell HABPs were identified in the R2region: 26251 (281PTVEEIVAPSVVESVAPSVEY300),26263 (521PTVEEIVAPTVEEIVAPSVVY540), 26270(661EIVAPTVEEIVAPSVVESVAY680), 26274 (741EIVA-PSSVVESVAPSVEESVEY760) 26276 (781ESVAENVE-ESVAPTVEEIVAY800), and 26277 (801PSVEESVA-PSVEESVAENVAY820). Peptide 26270 presented thehighest slope value (>6.0; Fig. 2A).

The following HABPs were found in the NRB region:26283 (921DVIEEVKEEVATTLIETVEQY940), 26284(941AEEKSANTTITEIFENLEENAY960), 26290 (1061GL-LNKLENISSTEGVQETVTY1080), and 26293 (1121FK-SESDVITVEEIKDEPVQKY1140). HABP 26284 present-ed the greatest slope value (5.0; Fig. 2A).

Our results show that homologous sequences present-ed differences in HepG2 cell binding. HABPs 26251(281PTVEEIVAPSVVESVAPSVEY300) and 26263 (521PT-VEEIVAPTVEEIVAPSVVY540), belonging to the R2region, possessed the same amino acid sequence, exceptfor 290Ser, 292Val, 294Ser, and 300Glu amino acid beingreplaced by 530Thr, 532Glu, 534Ile, and 540Val (under-lined), presenting 2.0 and 3.6 slope values, respectively.Peptides 26266 (581ESVAENVEEIVAPTVEEIVAY600)and 26276 (781ESVAENVEESVAPTVEEIVAY800) weredifferentiated in terms of amino acid sequence in 590Ileand 790Ser residues (underlined), presenting 3.0 and 1.8slope values, respectively. Low specific binding pep-tide 26262 (501ESVAENVEESVAENVEEIVAY520) (0.8slope) was differentiated from HABP 26266 in 510Ser,513Glu and 514Asn residues and from HABP 26276 in513Glu and 514Asn residues.

Binding assays were performed to determine whetherHepG2 cell HABPs or LSA-3 peptides (being homolo-gous to some of the intraerythrocyte stage proteins)present erythrocyte binding activity. Our results show thatHepG2 cell HABPs do not present high erythrocytebinding activity; however, peptides 26242, 26283 and26308 presented intermediate binding activity, havingslopes of 1.4, 1.6, and 1.4, respectively. The other pep-tides presented slope values less than 1.0. Peptides 26271and 6685, possessing D260 and RESA protein homolo-

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gous sequences, had low erythrocyte binding activity(Fig. 2B). No HABP binding to C32 endothelial cellmembrane was presented (data not shown).

Saturation and cross-linking assays

Saturation assays carried out with LSA-3 HABPs be-longing to nonrepeat regions showed that only peptides26238, 26239, 26240, 26241, and 26244 (which weresaturable in assay conditions) presented nanomolar af-finity constants (480, 680, 650, 880, and 720 nM, re-spectively) and had 0.9�106, 1.6�106, 0.7�106, 0.9�106,and 0.5�106 binding sites, respectively. Their Hill coef-ficients (greater than 1) indicated that the receptor-ligandinteraction presented positive cooperativity (Fig. 3).Cross-linking assays with HABP 26239 to HepG2 cell

membranes showed that these bound specifically to pro-teins having 31, 44, and 70 kDa molecular weight (Fig. 4).HABP binding to HepG2 cell membranes was not in-hibited by unrelated peptides 1585 [15] and 4980 [17];these HABPs also did not present C32 endothelial cellmembrane binding, indicating LSA-3 protein HABPspecificity for HepG2 cell membranes (data not shown).

Inmunofluorescence assays

Inmunofluorescence assays with B65-A and B98-A seraobtained from goats immunized with the mixture of LSA-3 protein polymeric peptides revealed that only B65-A(III-20) serum reacted with schizont-infected erythrocytesand sporozoites (Fig. 5; B-98-A III-20 serum did notpresent any reaction) while B65-A preimmune serum did

Fig. 3 Saturation assays. Satu-ration assays were carried outwith HABPs obtained fromHepG2 cell binding assays us-ing peptides from LSA-3 pro-tein. Affinity constants werecalculated from the saturationcurves defined as being theconcentration of free 125I-pep-tide necessary for obtaining halfthe 125I-peptide maximumbinding. The maximum numberof ligand binding sites per cellwas calculated from the maxi-mum 125I-peptide binding. Thebinding peptides were saturable,and the affinity constants werein the nanomolar range. TheHill plot is placed within themain graph; abscissa log F; or-dinate log(B/Bmax�B); B pmolbound peptide; F free peptide

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not present any reaction. When B65-A (III-20) serum wasthen adsorbed on Sepharose–peptide columns (peptide26271 or a mixture of inoculated peptides) it presentedsimilar behavior to that of preimmune serum, while B65-A (III-20) serum adsorbed on a Sepharose–peptide 28826column (unrelated peptide) continued reacting withschizont-infected erythrocytes (data not shown).

Immunoblotting assays

Only goat B65-A (III-20) sera recognized several bands inP. falciparum lysate in immunoblot assays. This serumrecognized 175, 160, 86, 81, 72, 68, and 58-kDa proteinsand some proteins with molecular weights greater than175 kDa that could not be determined. Proteins havinglower molecular weight greater than 175 kDa were ofinterest in this work as such weight represented the pos-sible weight of RESA protein, while values greater than175 kDa could possibly have represented D260 or proteinfragments (Fig. 6A, lane 2). Recognition of all proteinswas lost when B65-A (III-20) serum was adsorbed on aSepharose column coupled to a mixture of peptides in-oculated into both goats (Fig. 6A, lane 3). B65-A (III-20)serum adsorbed on a Sepharose–peptide 26271 column(possessing the PSVEESVEENVEESVAENVE repeatmotive present in D260 protein) only recognized 86 and81-kDa proteins (Fig. 6A, lane 4). B65-A (III-20) serumadsorbed on columns coupled to peptides 6685 (belongingto RESA protein containing the SEEESVEENE motif,lane 5) and 28826 (belonging to P. falciparum hypo-

thetical protein NP-703322.1, unrelated peptide, lane 6)similarly recognized the same two proteins migrating as86 and 81-kDa proteins, just as the goat sera absorbed onpeptide 26271 (lane 4).

When B65-A (III-20) serum was incubated with pep-tide 26271 (at 100, 200, and 800 �M, but only 100 �M isshown in Fig. 6B, as the rest presented the same result), itonly recognized an 86-kDa protein in P. falciparumschizont lysate (Fig. 6B, lane 3). The same behavior waspresented when B65-A serum was incubated with poly-meric peptide 28728 which was part of the inoculatedmixture (Fig. 6B, lane 4). B65-A (III-20) serum incubatedwith peptide 26241 (a HABP from the NRA region)recognized most proteins (Fig. 6B, lane 5); however, B65-A (III-20) serum incubated with polymeric peptide 28726(26241 is the monomeric form) did not recognize the 86-kDa protein.

Discussion

PfLSA-3 protein is considered to be a vaccine candidateas it possesses B- and T-epitopes which have inducedprotection in challenges against P. falciparum in experi-mental animal models. These epitopes are located indifferent protein regions. Our laboratory has identified P.falciparum CSP, STARP, and LSA-1 protein binding re-gions in previous studies using HepG2 cells [18, 25, 26]as part of our strategy in designing and developing mul-

Fig. 4 Cross-linking assays. HABP 26239 binding to HepG2 cellmembranes can be seen. Lane 1 Total binding (membranes incu-bated only with 125I-peptide). Lane 2 Inhibited binding (membranesincubated with 125I-peptide in the presence of the same unlabeledpeptide). The different HABPs bound specifically to proteins fromHepG2 cell membrane having 31, 44, and 70 kDa molecular weighthaving less intensity; the differences in band intensity were due toradiolabeled peptide specific activity (50–400 �Ci/nmol). C32 en-dothelial cell membranes were used as negative control (data notshown)

Fig. 5 Immunofluorescence assays. Immunofluorescence assayswere performed with sera obtained from goat B65-A immunizedwith polymerized peptides belonging to LSA-3 protein nonrepeatand repeat regions. A Reaction of B65-A serum (III-20) withsporozoites in immunofluorescence assays. B Reaction of B65-Aserum (III-20) with P. falciparum intraerythrocyte schizonts inimmunofluorescence assays. The preimmune B65-A serum (B65-API) did not show reactivity in either P. falciparum sporozoites orintraerythrocyte schizont immunofluorescence assays (data notshown)

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tistage, multiantigen vaccines against P. falciparum in-fection. Seventeen HepG2 cell HABPs were found inHepG2 cell binding assays using LSA-3 protein syntheticpeptides located in the protein’s repeat and nonrepeatregions. No HABPs were found in the protein’s C-ter-minal (R3 and NRC regions) or in the R1 region locatedin LSA-3 protein’s N-terminal, suggesting that the LSA-3C-terminal is not involved in HepG2 cell binding(Fig. 2A). Only those peptides located in nonrepeat re-gions were of interest and were used in saturation andcross-linking assays (Figs. 3, 4). HepG2 HABPs did notpresent high erythrocyte binding activity, suggesting thatthese peptides bind specifically to HepG2 cells. Peptides26271 (D260 protein) and 6685 (RESA protein), pos-sessing repeat sequence homologous motifs did not bindto erythrocytes, suggesting that repeat regions responsiblefor LSA-3 crossed reaction with intraerythrocyte stageproteins no erythrocyte binding activity.

Analyzing the sequences of those HABPs belonging tothe R2 region indicated that replacing 290Ser, 292Val,294Ser and 300Glu by 530Thr, 532Glu, 534Ile, and 540Val(respectively) increased peptide 26263 binding by 80%;similarly, replacing 790Ser (peptide 26276) by 590Ile(peptide 26266) increased peptide 26266 binding by 66%,suggesting that detailed amino acid changes in a sequencecould positively or negatively affect binding activity. Thiswas the case with low-binding peptide 26262, presentingfewer differences in its amino acid sequence than HABPs26266 (containing 510Ser, 513Glu, and 514Asn) and 26276(containing 513Glu and 514Asn), suggesting that these re-sidues were critical in HepG2 cell binding. The foregoingsuggested that replacing amino acids as well as the orderof the amino acid sequences are important in peptideHepG2 cell binding activity. We have reported that pep-

tide binding activity to target cells depends on each par-ticular amino acid sequence in previous work [18, 25, 26].Assays using HABP glycine scan analogue peptides withother hepatic stage proteins have shown that peptidesdiffering in only a single amino acid (glycine) presentsubstantially different binding activity [18, 25, 26]. Thisexplains why some peptides present high binding capacityin the LSA-3 protein R2 region while their homologuesdo not. This is also why it is possible to find other HABPsbelonging to other protein regions which do not presenthomology to R2 region peptides or among themselves.HABPs 26283, 26284, 26290, and 26293 were found inthe NRB region while HABPs 26238, 26239, 26240,26241, 26242, 26244, 26247, and 26251 were found at theN-terminal of LSA-3 protein. Five of these HABPsformed a HepG2 cell binding region formed by peptides26238–26242, spanning 100 amino acids (residues 21–120). Our results thus indicate that the LSA-3 N-terminalregion could be important in HepG2 cell binding.

HABP binding was saturable and possessed nanomolaraffinity constants, indicating that these peptides bound toHepG2 cells with high specificity and affinity (Fig. 3).The number of calculated binding sites indicated thatthese peptides presented high HepG2 cell binding ca-pacity. Peptide 26238 presented the highest affinity andpeptide 26239 presented the greatest binding capacity(Fig. 3).

Cross-linking assays showed that the HABPs boundspecifically to HepG2 cell membrane proteins and not toother cell membranes (erythrocytes and C32 cells). This isimportant when it is borne in mind that hepatic cells aretargeted for infection by sporozoites, and that LSA-3protein belongs to the human hepatocyte stage. Bindingwas observed to proteins having 31, 44, and 70 kDa

Fig. 6 Immunoblotting assays. A Sera obtained from goat B65 (III-20) inoculated with LSA-3 protein polymeric peptides were ad-sorbed in Sepharose columns coupled to M. smegmatis, E. coli, andSpf-66 vaccine (B65-A III-20). This sera recognized 175, 160, 86,81, 72, 68, 58, and >175 kDa bands (lane 2) in erythrocyte stageschizont lysate, while preimmune serum (B65-A, PI) did notpresent recognition (lane 1). B65-A serum adsorbed in Sepharosecolumns coupled to different peptides: Sepharose–initial inoculum(lane 3); Sepharose–peptide 26271 (lane 4); Sepharose–peptide

6685 (lane 5); Sepharose–peptide 28826 (lane 6). B Immunoblotassays, using erythrocyte stage schizont lysate, were done withB65-A serum (III-20) incubated with peptides 26271 (lane 3),28728 (lane 4), 26141 (lane 5), and 28726 (lane 6). Peptide con-centrations were 100, 200, 400, and 800 �M; only the result cor-responding to 100 �M is shown as all the rest showed the samebehavior. B-65 A (PI) and B65 A (III-20) sera (lanes 1 and 2,respectively)

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molecular weight. 125I-peptide binding to these proteinsdisappeared when this peptide was incubated with HepG2cell membranes in the presence of the same unlabeledpeptide, indicating that these peptides bound specificallyto 31, 44, and 70-kDa proteins. In turn, this indicates thatunlabeled peptide present in greater concentration inhibits125I-peptide binding (Fig. 4). All of the other HABPsbound to the same bands but with less intensity (data notshown). Our results show that these peptides boundspecifically to HepG2 cell membrane proteins with highaffinity and high binding capacity, suggesting that pep-tides having sequences located in different LSA-3 proteinregions bound specifically to three HepG2 cell membraneproteins. Similar behavior has been reported for STARPand PfSSP2 protein HABPs which bind to several proteinson HepG2 cell membrane [26, 27]. However, the possi-bility that HABPs may be binding to cleavage subprod-ucts from a single protein cannot be discarded.

Amino acid sequences from these HepG2 cell HABPswere found to be part of T- and B-epitopes, as reportedby others [6, 8, 28]. The HABP 26244 sequence (under-lined) formed part of peptide LSA-3CT1 sequence(LLSNIEEPKENIIDNLLNNIG); HABP 26247 (under-lined) can be found in the LSA-3NRII peptide sequen-ce (LEESQVNDDIFNSLVKSVQQEQQHNV). Similar-ly, HABPS 26251, 26263, 26270, 26274, 26276, and26277 possessed part of the LSA-3RE peptide sequen-ce (VESVAPSVEESVAPSVEES), while peptide 26246(NEL-LNSVDVNGEVKENILEEY) (1.6 slope) was seento be part of the LSA3-NRI peptide sequence.

The immunological importance of peptides LSA-3CT1, LSA-3 NRI, LSA-3NRII, and LSA-3RE (wherethe HABPs mentioned in the previous paragraph werefound) has been found to induce T-cell proliferation inchimpanzees, and LSA-3NRI, LSA-3NRII, and LSA-3REhave stimulated interferon g production [28, 29]. Anti-bodies from chimpanzees immunized with peptides LSA-3CT1, LSA-3 NRI, LSA-3NRII, and LSA-3RE recog-nized all peptides except LSA-3CT1, suggesting thatthese are B-epitopes. Sera from chimpanzees immunizedwith hepatic stage antigens and LSA-3 peptides reactedstrongly with proteins expressed in P. falciparum sporo-zoite and hepatic stages [28]. Sera from Aotus monkeysimmunized with LSA-3CT1, LSA-3NRI, LSA-3NRII,and LSA-3RE peptides recognized native (LSA-3) proteinexpressed on sporozoite surface and hepatic stage sch-izonts. These four LSA-3 peptides induced interferon gproduction, but only LSA-3 NRI and LSA-3RE producedT-cell proliferation in Aotus monkeys [8]. CTL epi-topes (underlined) identified in LSA-3 protein, present-ing HLA2 restriction, were contained in some HABPsas follows: HABP 26242 contained epitope la20(101ASEEVKEKILDLLEEGNTLTY120), HABP 26290contained epitope la30 (1061GLLNKLENISSTEG-VQETVTY1080), and peptide 26286 (1.6 slope) containedepitope la26 (981TVLDKVEETVEISGESLENNY1000)[6]. The reported data, together with results obtained frombinding assays, indicate that some HABPs are located inantigenic, immunogenic, and protection-inducing regions,

suggesting that HABPs are related to P. falciparumpreerythrocyte intrahepatic binding, invasion and devel-opment processes and could be considered as possiblecandidates for a multistage, multiantigenic vaccine.

HABP 26247 (underlined) was contained in peptideLSA-3NRII (198LEESQVNDDIFNSLVKSVQQEQQH-NV223), considered to be a potent T- and B-epitope whichprotects chimpanzees against P. falciparum infection [2,7, 28]. This in turn suggests that HABP 26247 could berelevant in the parasite-cell interaction and possiblyconsidered as being a prospect for inducing an immuneresponse which could be protection-inducing against P.falciparum hepatic stage infection.

The results of the immunofluorescence assays withgoat sera inoculated with LSA-3 protein polymeric pep-tides showed that only B65-A (III-20) serum presented apositive reaction to either P. falciparum schizont-infectederythrocyte and sporozoites (Fig. 5), while preimmuneB65-A sera (PI) did not react. Immunofluorescence assayswith B65-A (III-20) sera adsorbed on Sepharose–peptidecoupled columns (26271 or a mixture containing pep-tide 28728) possessing the VEESVAEN motif presentednegative reactions in immunofluorescence assays with P.falciparum schizont-infected erythrocyte, indicating thatthe antibodies responsible for the positive reaction re-mained bound to Sepharose-coupled peptides, in turnsuggesting that the immune response generated by theinitial inoculum was mainly directed against the VEES-VAEN motif present in peptide 28728, and that the pos-itive reaction of sera B65-A (III-20) in these assays(Fig. 6A, lane 2) was due to the presence of proteinspossessing this motif (VEESVAEN) showing cross-reac-tivity with inoculated sequences derived from LSA-3protein. This agrees with the findings of Daubersiers et al.[2] insofar as LSA-3 had a cross-reaction with the D260protein during the erythrocyte stage.

Immunoblotting assays were carried out with P. fal-ciparum schizont lysate to identify which proteins wererecognized by B65-A serum (III-20). The results indicatethat B65-A serum recognizes several intraerythrocytestage 175, 160, 86, 81, 72, 68, 58 kDa and greater than175 kDa P. falciparum lysate proteins (schizont lysate;Fig. 6A, lane 2). The molecular weights of some proteinsrecognized in immunoblot assays were similar to themolecular weight of RESA (155 kDa), LSA-3 (175 kDa),and possibly D260 (260 kDa) as well as fragments de-rived from the D260 protein.

When B65-A (III-20) serum was adsorbed on Sepha-rose–inoculum columns (inoculated peptide cocktail), itdid not recognize any of the proteins in the P. falciparumschizont lysate, presenting similar behavior to preimmuneB65-A sera; this indicates that antibodies retained by theSepharose–inoculum column were responsible for recog-nizing the different proteins (Fig. 6A).

Peptide 28728 (forming part of the peptide mixtureinoculated into the two goats) has a sequence containingthe VEESVAENVEESVA motif found in P. falciparumerythrocyte stage D260 protein [2, 21]. When B65-A (III-20) serum was incubated with peptide 28728, it recog-

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nized only the 86-kDa protein, indicating that recognizingother proteins is mainly due to antibodies binding to thispeptide. B65-A (III-20) serum was adsorbed on a Se-pharose–peptide 26271 column (peptide 26271 possessesthis motif in its sequence, but was not part of the inocu-lated mixture) to determine whether these antibodiesspecifically recognize the VEESVAEN octapeptide. Theadsorbed Sepharose–peptide 26271 sera presented similarbehavior, recognizing only 86- and 81-kDa proteins. Thisresult indicates that the antibodies were mainly directedagainst the repeat VEESVAEN motif which is found in P.falciparum D260 intraerythrocyte protein. It is thus un-likely that this motif will be found in these proteins (86-and 81-kDa proteins). It should be borne in mind that suchproteins recognized by antibodies directed against thismotif could possibly be D260 protein fragments con-taining this motif or motifs from other erythrocyte stageproteins containing this octapeptide.

Immunoblot assays were performed with B65-A(III-20) serum incubated with peptide 26271 or 28728(monomer and polymer, respectively, possessing theVEESVAEN motif) showing that only the 86-kDa proteinwas recognized (Fig. 6B, lanes 3 and 4, respectively).This indicates that these peptides blocked recognition ofmost proteins, except for recognizing the 86-kDa protein,agreeing with previously obtained results. These resultsconfirm that those antibodies recognizing the majority ofproteins in P. falciparum schizont lysate were directedagainst the VEESVAEN sequence (Fig. 6B, lane 2) andcould be involved in a crossed-reaction due to having thismotif. However, when this serum was incubated withpolymer peptide 28726 belonging to the NRA region, thispeptide blocked recognition by the 86-kDa protein, sug-gesting that these antibodies recognize peptide 28726sequence, and that this sequence could possibly be foundin the 86-kDa protein fragment, presenting a crossed-re-action with LSA-3. However, when B65-A serum wasincubated with HABP 26241 (monomeric form), it did notblock recognition of 86-kDa protein, suggesting that onlythe polymeric form can block recognition of 86-kDaprotein (Fig. 6B).

Our results indicate that recognition of goat sera in-oculated with peptides from LSA-3 protein in schizont(erythrocyte stage) lysate may be due to the VEESVAENmotif being present in D260 and LSA-3 proteins. How-ever, the same results show that recognizing the 86-kDaband is not due to the VEESVAEN motif but could be dueto a cross-reaction with a motif belonging to the LSA-3protein NRA region. Our study shows that peptides lo-cated in the LSA-3 protein’s N-terminal and central re-gions specifically and saturably interact with HepG2cells, suggesting the possible role of this protein in in-teractions between sporozoites from the P. falciparumparasite and hepatic cells during the infection’s preery-throcyte stage.

Acknowledgements This study was supported by the Office of thePresident of Colombia and by the Colombian Ministry of PublicHealth. We thank Jason Garry for reading the manuscript.

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