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A STUDY OF ANTIBODY AND T CELL RECOGNITION OF RHOPTRY-ASSOCIATED PROTEIN1 (RAP1) AND RAP2 RECOMBINANT PROTEINS AND PEPTIDES OF PLASMODIUM FALCIPARUMIN MIGRANTS AND RESIDENTS OF THE STATE OF RONDONIA, BRAZIL

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Humoral and cellular responses were examined among natives and migrants in an area of the Amazon region of Brazil. Rhoptry-associated protein-1 (RAP-1) and RAP-2 expressed in Escherichia coli expression systems, a peptide corresponding to the epitope
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  208  Am. J. Trop. Med. Hyg.,  59(2), 1998, pp. 208–216Copyright    1998 by The American Society of Tropical Medicine and Hygiene A STUDY OF ANTIBODY AND T CELL RECOGNITION OF RHOPTRY-ASSOCIATEDPROTEIN-1 (RAP-1) AND RAP-2 RECOMBINANT PROTEINS AND PEPTIDES OF PLASMODIUM FALCIPARUM   IN MIGRANTS AND RESIDENTS OFTHE STATE OF RONDONIA, BRAZIL KYM C. JACOBSON, JOYCE THURMAN, CHERYL M. SCHMIDT, ERIKA RICKEL, JOSELI OLIVIERA DE FERREIRA,MARIA DE FA´TIMA FERREIRA-DA-CRUZ, CLAUDIO T. DANIEL-RIBEIRO,  AND  RANDALL F. HOWARD Seattle Biomedical Research Institute, Seattle, Washington; Department of Immunology, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil; Department of Pathobiology, University of Washington, Seattle, Washington  Abstract.  Humoral and cellular responses were examined among natives and migrants in an area of the Amazonregion of Brazil. Rhoptry-associated protein-1 (RAP-1) and RAP-2 expressed in  Escherichia coli  expression systems,a peptide corresponding to the epitope bound by inhibitory anti-RAP-1 antibodies, and four other RAP-1 and RAP-2 synthetic peptides were used in these studies. Plasma from the native population had greater IgG reactivity to theN-terminal third of RAP-1 than the migrant population; both populations had low levels of IgM to this region of RAP-1. The IgG reactivity to RAP-2 and to the C-terminal third of RAP-1, as well as for all the peptides, includingthe peptide from the inhibitory domain, were low or absent in both populations. In contrast, there were a high numberof subjects with an IgM response to the peptides. Cellular responses were measured by proliferation of peripheralblood mononuclear cells (PBMC) and, in some subjects, by reverse transcription–polymerase chain reaction forinterleukin-2 (IL-2), interferon-   (IFN-  ), IL-4, and IL-10. Proliferation of PBMC was low when stimulated byrecombinant proteins, peptides, or parasite lysate. Both RAP-1 and RAP-2 stimulated cytokine production by donorT cells; IL-2, IL-4, and IFN-   RNA transcripts were observed in response to recombinant proteins and parasite lysate,but with no uniform trends. From the observed antibody responses, RAP-1 appears to be more immunogenic thanRAP-2.Malaria is still endemic in more than 90 countries, withmore than 120 million clinical cases and more than one mil-lion deaths each year (http://www.who.cl/ctd/diseases/mala/ malasit.htm). Immunity to malaria infection is acquiredslowly and is rarely, if ever, complete. Although, our under-standing of the development of immunity to malaria in hu-mans is limited, a number of potential blood-stage vaccinecandidates for  Plasmodium falciparum  have been identifiedbased upon immunogenicity, sequence conservation, and de-gree of protection conferred in animal models. 1 Among thevaccine candidates are proteins localized to the rhoptry or-ganelle. Several of these molecules have a high potential foruse in polyvalent vaccines against the asexual blood-stageof   P. falciparum. Located near the apical end of the merozoite, the rhoptriesappear to be involved in merozoite release, attachment, andinvasion of erythrocytes. 2, 3 Monoclonal antibodies haveidentified a number of rhoptry proteins, including complexeswith proteins of 86–37 kD and 140–105 kD. 4, 5 Rhoptry-associated protein 1 (RAP-1) is processed into 86-, 82-, 70-,and 67-kD polypeptides. 4, 6, 7 The RAP-1 products associatenoncovalently with either p39 (RAP-2) or p37 (RAP-3). 6, 8, 9 This study focuses on RAP-1 and RAP-2. Promise fortheir potential role in vaccination comes not only from evi-dence of conservation, but also from indications of their im-munogenicity and protective capacity in both animal trialsand  in vitro  studies. Sequence conservation of the RAP-1gene is high among both culture-adapted isolates and fieldsamples. 10–12 Also, monoclonal antibodies (MAbs) againstcomponents of these rhoptry protein complexes have suc-cessfully inhibited erythrocyte invasion  in vitro. 13–16 Further-more,  Saimiri  monkeys immunized with parasite-derivedRAP-1/RAP-2/RAP-3 have been protected from a lethalchallenge of   P. falciparum. 17, 18 Involvement of both antibody-mediated and cell-mediatedimmune mechanisms have been demonstrated in malaria in-fection. Elaborate studies in rodent malaria models have de-lineated crucial roles for T cells and B cells during infectionand recovery. 19–21 In humans, however, the roles of antibod-ies and other immune effectors responsible for the devel-opment of immunity, including the dichotomy of T helpercell subtypes and related cytokines, are not as easily defined.Previously, we demonstrated the production of antibodies toregions of RAP-1 in immune and semi-immune patients inSudan and Nigeria. In the nearly 30 sera tested, the highestreactivity was observed to a region of RAP-1 containingamino acids 1-294 (RAP-1 1-294 ). 11 A T cell epitope, predictedin the octameric repeat region of RAP-1, was confirmed byJakobsen and others, who also detected serum antibodies toRAP-1 1-294 . 22 Recent reports have also demonstrated naturallyoccurring antibody responses to full length RAP-1 and RAP-2 proteins 23 and the C-terminal one-third of RhopH3, a com-ponent of the 140–105-kD complex of   P. falciparum  rhoptryproteins. 24 In the present study, we characterize human im-mune responses to RAP-1 and RAP-2, using recombinantproteins and peptides to examine the antibody reactivity andthe T cell response (measured by cell proliferation and cy-tokine induction) in peripheral blood samples obtained fromtwo populations of donors located in the state of Rondonia,Brazil. One donor population consists primarily of migrantswho moved to the Amazon Basin from areas not endemicfor  P. falciparum.  The other consists of a population nativeto the Amazon Basin that reside in a fishing area along thebanks of the Rio Madeira, a tributary of the Amazon River. SUBJECTS ,  MATERIALS ,  AND METHODS Study subjects.  Individuals from the Brazilian state of Rondonia were recruited for participation in this study. The  209 HUMAN IMMUNE RESPONSES TO RAP-1 AND RAP-2 PRODUCTS T ABLE  1 Plasmodium falciparum  rhoptry-associated protein-1 (RAP-1) and RAP-2 synthetic peptides and recombinant proteins* Peptide Residues Sequence p39Ap39Bp82Ap82BiB-1RAP-2:a.a.59-75RAP-2:a.a.377-394RAP-1:a.a.355-372RAP-1:a.a.746-761RAP-1:a.a.198-211IYNHFSSADELIKYLEKYSRQYSNRAAE-NFKAIRENNSNLFDTIESLQGRIIDKKFKAEIRDFFKEMRIQYAKCPKNTLTPLEELYPT Protein Residues A-2-21-1-4MBP-RAP-1 1-294 MBP-RAP-1 293-707 rRAP-1rRAP-2rp39RAP-1:a.a.1-294RAP-1:a.a.514-769RAP-1:a.a.1-294RAP-1:a.a.293-707RAP-1:a.a.23-608RAP-2:a.a.22-398RAP-2:a.a.22-398 * a.a.    amino acids; iB-1    inhibitory B cell epitope 1; MBP    maltose binding protein. study groups included the farming area of Colina and theriverside fishing community of Ribeirinha, both of which arein close proximity to the city of Porto Velho and the MadeiraRiver, a tributary of the Amazon River. This region of theAmazon Basin has seen a steady increase in malaria inci-dence over the past three decades, presumably due to defor-estation and an influx of people from areas not endemic forthe parasite. 25 Most of the samples can be separated into twodistinct groups: the non-native farmers of Colina (henceforthtermed the migrant or Colina population) and the natives of the region who live in Ribeirinha (called native or Ribeirin-ha). Blood samples were collected by venipuncture. An oralhistory and signed consent form was obtained from donorsat the time of blood donation; blood smears were made fordetection of parasitemia. Peripheral blood mononuclear cells(PBMC) were also collected from a few patients in Wash-ington state that had contracted  P. falciparum  abroad. Cellsfrom nonexposed individuals were isolated from buffy coatsobtained from the American Red Cross (Portland NorthwestRegional Blood Services, Portland, OR). Ethical review of the project was obtained from the Ethical Committee of theFundacao Oswaldo Cruz, the Institutional Review Board of the Seattle Biomedical Research Institute, and the Washing-ton State Department of Social and Human Services/De-partment of Health Human Research Review Board. Isolation and freezing of PBMC and plasma.  ThePBMC were purified from 10–30 ml of whole blood by Fi-coll-Paque density gradient centrifugation. Cells not imme-diately used in assays were frozen in RPMI 1640 mediumsupplemented with 50% AB heat-inactivated normal humanserum and 7% dimethylsulfoxide, and transported and main-tained in liquid nitrogen, as was plasma. Some assays wereperformed on site in Porto Velho.  Plasmodium falciparum  culture and antigen prepara-tion.  The Honduras I/CDC strain (K  ) of   P. falciparum  (ob-tained from Dr. Susan Langreth, Uniformed Services Uni-versity of the Health Sciences, Bethesda, MD) was culturedat 37  C in mixed gas using TES-buffered RPMI 1640 me-dium supplemented with sodium bicarbonate, L-glutamine,gentamicin, and 10% A  human serum and A  or O  humanerythrocytes, as previously described. 26 Synchronous cul-tures were obtained with regular treatments of sorbitol and/ or sedimentation in modified gelatin. 6, 27 Parasite antigen wasprepared from cultures with parasitemias (of late stage par-asites)    60% by lysing parasitized and normal red bloodcells (RBC) in 10 mM phosphate buffer, washing in phos-phate-buffered saline (PBS) with or without protease inhib-itors, sonicating, and pelleting membranes and parasites bycentrifugation at 30,000    g  for 20 min at 4  C. Sampleswere frozen in liquid nitrogen until needed. Synthetic peptides and peptide ELISAs.  The T-Sitescomputer program (Medimmune, Gaithersburg, MD) wasused to predict potential T cell epitopes within the RAP-1and RAP-2. Four peptides, two from RAP-1 (p82A and B)and two from RAP-2 (p39A and B), were chosen for syn-thesis based upon the results of the T-sites computer programand the feasibility of synthesis. Amino acid numbers andsequences for the peptides are given in Table 1. Althoughthe T-Sites program identified several possible amino acidsequences within the proteins that were predicted to repre-sent T cell epitopes, many were not feasible for synthesisdue to their protein chemistry. None of the peptides werelocated in regions of repeats. These peptides were synthe-sized commercially and used without further purification(Genosys, Woodlands, TX).An epitope bound by several anti-RAP-1 MAbs that in-hibit  P. falciparum  invasion of RBC  in vitro  was identifiedby deletion and peptide scanning methods. 15 We call thisdomain iB-1, short for inhibitory B-cell epitope 1. A peptidecorresponding to the iB-1 domain (amino acids 198-211)was synthesized by standard Fmoc chemistry at Molecu-metics (Bellevue, WA) and included an additional N-termi-nal Cys to aid coupling.Peptide ELISAs were performed in triplicate using 50   l(5   g/ml) of p82A and B and p39A and B peptides or 50  l (1   g/ml) of iB-1 peptide bound to Nunc (Roskilde, Den-mark) Maxisorp 96 well plates overnight. The human plasmawere used after dilution (1:50) in PBS/0.1% Tween 20/0.1%bovine serum albumin. The secondary antibody was horse-radish peroxidase (HRP)–conjugated goat anti-human IgG orgoat anti-human IgM (Boehringer Mannheim Biochemicals,Indianapolis, IN). 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Zymed, South San Francisco, CA)was used as substrate in the HRP reaction. Reactions wereread at 405 nm in an ELISA plate reader. A sample wasconsidered positive when the optical density (OD) value was  210  JACOBSON AND OTHERS higher than the average OD of the normal plasma plus threestandard deviations. Expression and purification of recombinant proteins. The RAP-1 1-294  region of RAP-1 was expressed in two forms,one with a carboxyl (C-) terminal 6 His-tag (hexameric his-tidines) sequence and a second as a dual fusion to both themaltose binding protein (MBP) and 6 His-tag. The produc-tion and purification of these 6 His-tagged recombinant pro-teins are described in more detail elsewhere. 16 Briefly, thisregion of RAP-1, and all other constructs devised in ourlaboratory to express RAP-1 and RAP-2, were amplifiedfrom genomic DNA of the  P. falciparum  isolate HondurasI/CDC in a polymerase chain reaction (PCR) and insertedinto the  Escherichia coli  expression vector pQE70 (Qiagen,Valencia, CA). The recombinant protein, A-2-2, was ex-pressed after induction with isopropyl   -D-thiogalactoside(IPTG) as a 31-kD recombinant protein. The 6 His sequencepermitted purification by Ni  -chelation chromatography ac-cording to the manufacturer’s recommendations (Qiagen).Purified A-2-2 was used in immunoblot analyses.To increase the yield of recombinant RAP-1 1-294 , the RAP-1 insert in pA-2-2 was fused to MBP in the bacterial ex-pression vector pMALc (New England Biolabs, Beverly,MA) and a 6 His sequence was inserted at the C-terminusof the RAP-1 coding region. This fusion, termed MBP-RAP-1 1-294 , was purified from IPTG-induced bacterial cultures bysequential use of the amylose binding ability of MBP, fol-lowing the manufacturer’s protocols, and the Ni  bindingproperties of the 6 His-tag domain. The purified MBP-RAP-1 1-294 was essentially homogeneous for a 80-kD band by so-dium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE). The purified protein was dialyzed againstPBS, concentrated in SpectroPor   dialysis tubing (SpectrumMedical Industries, Los Angeles, CA) using Aquacide (Cal-biochem, La Jolla, CA), and redialyzed against PBS. Proteinconcentration was determined by a Coomassie protein assay(Pierce, Rockford, IL). The cleavage site for Factor Xa wasnot accessible in any of our MBP fusion proteins. The MBPfusion proteins were used in T cell stimulation experiments.The MBP-RAP-1 293-707  protein was another MBP fusionprotein containing amino acids 293 to 707 of the RAP-1coding region. This protein was also expressed from pMALcafter a 6 His sequence was added to the C-terminus of theRAP-1 coding region by ligation into the expression vector.The MBP-RAP-1 293-707  protein was poorly soluble in the ab-sence of guanidine or urea, which prevented a primary pu-rification step on amylose. The protein was purified on che-lated Ni  after solubilization in buffer containing 6 M gua-nidine-HCl using the batch purification procedure suggestedby the manufacturer (Qiagen). Protein was eluted with 250mM imidazole in buffer containing 8 M urea. Urea was re-moved by stepwise dialysis against PBS/urea to maintainsoluble protein although typically some protein was lost dueto precipitation during dialysis.The recombinant protein 1-1-4, containing RAP-1 514-769 ,was the C-terminal most region of RAP-1 expressed. Thisregion was amplified with the sense primer 5  -CCCGGGCATGCACGAAGCATCTCAGGATATTGCTGCTG-3  and the antisense primer, 5  -GCTCTAGATTCTTAATCTA-ATCTCTTGAAGGCA-3  . The PCR product was clonedinto pQE70 and expressed after induction with IPTG. Ex-pression and purification of protein 1-1-4 followed the meth-odology used to isolate A-2-2. Protein 1-1-4 was used forimmunoblot analyses.The mature RAP-2 protein, containing amino acids 22-398, was expressed from pQE60 (Qiagen) after inductionwith IPTG. The RAP-2 gene was amplified with the primerssense 5   CCATGGATAAGTGTGAAACTGAATTTTCC 3  and anti-sense 5   GGATCCAAGAACATTTAATTCT CTA-ATAGC 3  . Purification of recombinant RAP-2 (rp39) fol-lowed the procedures described above for MBP-RAP-1 293-707 except for the following differences. After induction, the cellpellet was resuspended in sonication buffer (50 mM sodiumphosphate, pH 7.8, 300 mM NaCl). After freeze/thawing, themixture was sonicated and centrifuged at 300–350    g  for10 min. The clarified extract was batch purified on Ni  aga-rose using 250 mM imidazole for elution. Much of the rp39precipitated during stepwise dialysis into PBS. Protein in theprecipitate was quantitated and used in experiments.Recombinant RAP-1 and RAP-2 were also kindly provid-ed by Dr. Allen Saul (Queensland Institute of Medical Re-search, Brisbane, Australia). This recombinant RAP-1 mol-ecule (here called rRAP-1) contains amino acids 23-608 andwas expressed as a 6 His C-terminal protein. The recombi-nant RAP-2 (rRAP-2) consists of the entire mature protein(amino acids 22-398) 9 and was expressed as a 6 His N-ter-minal protein. Both proteins were purified over a Ni  che-lating resin as an initial purification, followed by further pu-rification by isotachophoresis. 28 Western blot analysis.  Purified recombinant proteins(50–150 ng per linear centimeter of gel) were resolved bySDS-PAGE (8% polyacrylamide gel) and transferred to ni-trocellulose membrane (Schleicher and Schuell, Keene, NH).Human plasma was diluted 1:10 in 50 mM Tris/0.01% so-dium azide/150 mM NaCl with 1% casein and used as pri-mary antibody. Goat anti-human IgG-HRP and goat anti-human IgM-HRP conjugated antibodies were used as sec-ondary antibodies. The reactions were developed with ami-noethyl carbazole (Zymed). Scoring of Western blots wasvisual and relative to other samples. Proliferation of PBMC.  Frozen PBMC were thawed at37  C and washed three times in incomplete RPMI 1640 me-dium. Fresh or thawed PBMC were plated in triplicate in96-well, U-bottom plates (Corning, Corning, New York) incomplete RPMI 1640 (10% human AB serum, 50   M 2-mercaptoethanol, 50   g/ml of gentamicin, and 2 mM L-glu-tamine) in the presence of parasite lysate, recombinant pro-tein, or synthetic peptide. Recombinant proteins with MBP(MBP-RAP-1 1-294  and MBP-RAP-1 293-707 ) were used at a con-centration of 23   g/ml to provide 10   g/ml of the parasiteprotein with 1.5    10 5 cells/well. Purified MBP and recom-binant proteins expressed with only the 6 His tag were usedat a concentration of 10   g/ml. Lysates of infected or un-infected RBC were used to give 10 6 or 10 7 cell equivalents,as indicated. Peptides were used at a concentration of 20   g/ ml, with 2    10 4 cells/well. Parasite lysate was used at aconcentration of 20   g/ml in the peptide experiments insteadof using cell equivalents. Cells stimulated with 1% phyto-hemaglutinin (PHA) served as positive controls, and mediumalone served as a negative control. Cells were incubated at37  C in CO 2  for six days and pulsed during the last 16 hrof culture with 1   Ci/well of   3 H-thymidine (ICN, Costa  211 HUMAN IMMUNE RESPONSES TO RAP-1 AND RAP-2 PRODUCTS T ABLE  2Characteristics of the 1995 study populations in the state of Ron-donia, Brazil PopulationAge distribution,yearsMean (range)Residence time,yearsMean (range)Males/ femalesInfection at timeof sampling RibeirinhaColina38.430.0(13–74)(8–59)38.411.7(13–74)(6–20)14/612/51 subject*1 subject† *  Plasmodium vivax. †  P. falciparum. T ABLE  3Reactivities of anti–rhoptry-associated protein-1 (RAP-1) and RAP-2 antibodies in plasma from the Colin and Ribeirinha populations Antigen* 2   Ab†Colina patients3 4 14 15 22 23 26 28 31 32 A-2-2rp39  IgG  IgM  IgG  ‡  ND§  Ribeirinha patients1 7 9 10 13 20 24 28 36 A-2-2rp39  IgG  IgM  IgG  ND  ND  * A-2-2 contains amino acids 1-294 of RAP-1; rp39 contains amino acids 22-398 of RAP-2.† The secondary antibody (2   Ab) used in the immunoblots was anti-IgG (  IgG) or anti-IgM (  IgM).‡ Reactivities were scored visually:     weak reaction;     medium reaction;     strong reaction.§ ND    not determined. Mesa, CA), harvested, and incorporated radioactivity wasdetermined. Results are expressed as the stimulation index(SI): the mean cpm in experimental cultures/mean cpm inthe medium controls. An SI    4 was considered positive. Isolation of RNA and reverse transcription PCR (RT-PCR) for cytokine analysis.  The PBMC were cultured for72 hr with antigen as described above for our proliferationstudies, except that two wells were used per antigen treat-ment, instead of three, resulting in a total of 3    10 5 cellsper antigen treatment. Isolation of total RNA from cell pel-lets followed a modified method of Chomczynski and Sac-chi. 29 Synthesis of cDNA followed the manufacturer’s rec-ommendations for Superscript Reverse Transcriptase usingoligo(dT) as a primer (Life Technologies, Gaithersburg,MD).The cDNA was normalized with respect to the constitu-tively expressed   -actin gene prior to the cytokine PCR. Se-rial dilutions of cDNA were used to verify that the   -actinPCR conditions were within the linear range. NormalizedcDNA were amplified by PCR using  Taq  polymerase with0.2   M of the respective 5’ and 3’ external primers in a 50-  l reaction. Interleukin-2 (IL-2), IL-4, and IL-10 amplifica-tion reactions were as follows: denaturation at 94  C for 30sec, annealing at 55  C for 30 sec, and elongation at 72  C for90 sec; 30 cycles for   -actin, IL-2, and IL-4 and 25 cyclesfor IL-10. Interferon-   (IFN-  ) was amplified for 30 cycles:30 sec at 94  C, 45 sec at 60  C, and 1.5 min at 72  C. Thenucleotide sequences for the sense and anti-sense oligonu-cleotide PCR primers for IL-2, IL-4, IL-10, and IFN-   havebeen published elsewhere. 30, 31 For comparative analysis of the cytokine transcripts, PCRswere probed by Southern blot hybridization. Blots were hy-bridized with DNA sequences for human   -actin, IL-2, IL-4, IFN-  , and IL-10 labeled with  32  -P-ATP in a primer ex-tension reaction with a random nonamer (Stratagene, La Jol-la, CA). 32 Where indicated, Southern blots were scanned forquantitation on a Storm Phosphorimager (Molecular Dynam-ics, Sunnyvale, CA). Statistical analysis.  Analysis of variance was performedto determine the significance of differences in time of resi-dence between populations. The significance of differencesbetween populations in the prevalence of positive responsesto peptides was determined by   2 analysis. RESULTS Study populations.  Samples were taken from studygroups in 1993 and 1995. Characteristics of the 1995 Ron-donian study populations are shown in Table 2 because thesesamples were used in most assays unless noted. There wasno significant difference in the mean ages among the pop-ulations, but differences in mean time of residence were sig-nificantly different ( P    0.01). All residents of Ribeirinhawere natives of the area; thus, age distribution was identicalto the residence time. Binding of antibodies to recombinant RAP-1 and RAP-2.  Anti-RAP-1- and anti-RAP-2-specific IgG and IgM in 10Colina samples and nine Ribeirinha samples were assayedon Western blots (Table 3). His-tagged recombinant proteinsA-2-2 (containing RAP-1 1-294 ), 1-1-4 (containing RAP-1 514-769 ), and rp39 (RAP-2) were used for these analyses. All of the Ribeirinha samples and nine of the 10 Colina sampleshad IgG recognizing RAP-1 1-294 . Three samples from Colinaand two from Ribeirinha were tested for reactivity to RAP-1 514-769 . None of these were positive. Because of previousobservations that African sera have low anti-RAP-1 606-769 an-tibody titers, 11 the screening was not extended to the re-maining Brazilian samples. The IgG antibody reactivity toRAP-2 was tested in nine samples from Colina and sevenfrom Ribeirinha. Only two Colina samples and one Ribei-rinha sample were positive (Table 3), indicating that anti-bodies to denatured recombinant RAP-2 are less common inthese Brazilian subjects than antibodies to denatured recom-binant RAP-1. Because we observed low signal intensity of IgM to the RAP-1 1-294  region (Table 3) and only small vol-umes of plasma were available, the binding of patient IgM  212  JACOBSON AND OTHERS T ABLE  4Percentage of individuals positive for antibodies to rhoptry-associated protein-1 (RAP-1) and RAP-2 peptides* Populationp39A:a.a.59-75% (NP)†p39B:a.a.377-394% (NP)p82A:a.a.355-372% (NP)p82B:a.a.742-761% (NP)† IgG responsesRibeirinha (n    20)Colina (n    16)200(4) 106(2)(1)2012(4)(2)156(3)(1)Total (n    36) 11 (4) 8 (3) 17 (6) 11 (4)IgM responsesRibeirinha (n    20)Colina (n    16)7562(15)(10)6037(12)(6)6544(13)(7)5012(10)(2)Total (n    36) 69 (25) 50 (18) 55 (20) 33 (12) * Positive responses were based on a ratio greater than 1 obtained by dividing the mean patient optical density by the mean of negative controls plus 3 SD. a.a.    amino acids; NP   number of positive individuals.† The number of individuals responding with IgM to peptide 82B were significantly different among the two populations ( P    0.02), and the difference between the number of individualsresponding with IgG to peptide 39A was marginally significant ( P    0.06). to recombinant RAP-2 (using rp39) and RAP-1 514-769  (using1-1-4) was not investigated. Antibody responses of Brazilians and Africans to theinhibitory epitope iB-1.  Several anti - RAP-1 mAbs that in-hibit invasion  in vitro  bind an epitope (iB-1) at residues 200-211 in RAP-1. 15 To determine whether there were significantdifferences in anti-iB-1 antibody levels in the Colina andRibeirnha populations, plasma were tested on peptide iB-1(Table 1) by ELISA. No significant differences were ob-served between these two populations: 10 Colina plasmasamples had a mean OD of 0.1169 (SEM    0.0050) andnine Ribeirinha samples had a mean OD of 0.1143 (SEM   0.0073). In addition, neither population differed significantlyfrom three North American negative control sera (mean OD   0.1050, SEM    0.0098). Two of the ten Colina samples(a 50-year-old and a 17-year-old, both residents in Rondoniafor nine years) and two Ribeirinha samples (a 25-year-oldand a 44-year-old) had OD levels that were greater than 2SD above the mean of the negative controls, but less thanthe 3 SD cut-off level used here to establish significance. Aswith the Brazilian samples, there were no significant anti-iB-1 antibody titers in five Nigerian immune sera (mean OD   0.1112, SEM    0.0050) or one Sudanese serum sample(single value of 0.087) compared with the negative controls.These results do not show a correlation between anti-iB-1antibody titers and exposure to  P. falciparum  infection oranti-malarial immunity. Reactivity of Rondonian antibodies to RAP-1 andRAP-2 synthetic peptides.  To examine the antibody re-sponses to other epitopes in RAP-1 and RAP-2, ELISAswere performed with 20 Ribeirinha plasma samples and 16Colina samples using four synthetic peptides. Two of thesepeptides contained sequence from RAP-1 and two fromRAP-2 sequence (Table 1). On average, fewer than 20% of the individuals had IgG antibodies that bound any of the fourpeptides (Table 4). However, for each of the four peptidesused, the percentage of the native (Ribeirinha) populationwith antibodies to these peptides was higher than that of themigrant population. The percentage of samples with IgMthat bound the peptides was greater than that seen for IgG.As with IgG, the percentage of individuals with an IgM re-sponse was higher in the native population (Table 4). Forboth populations, peptide p39A gave the greatest number of IgM-positive responders. No cross-reaction was seen withplasma from individuals with toxoplasmosis or leishmania-sis. These results show that 10–20% of Ribeirinha samplesand a lower percentage of Colina samples contain IgG thatbind any of four different RAP-1 and RAP-2 peptides. Theseresults contrast with those above describing reactivity to theiB-1 peptide in which no titers greater than 3 SD of meanof negative controls were observed. T cell proliferation in response to recombinant RAP-1and RAP-2 proteins and synthetic peptides.  The prolifer-ation of T cells from seven samples collected in 1993 wasexamined  in vitro  in the presence of recombinant RAP-1fusion proteins MBP-RAP-1 1-294 , MBP-RAP-1 293-707 , and par-asite lysate. The PBMC responses were low when stimulatedwith MBP-RAP-1 1-294  and MBP-RAP-1 293-707 , both fusionproteins failing to stimulate proliferation above that ob-served with MBP alone. Purified, unfused MBP was used asa control for the MBP-fusion proteins, and incubation of PBMC with MBP alone did not produce an SI    4 in anyof the subjects examined. Parasite lysate stimulated PBMC(SI    4) in three of the samples tested in this experiment.Of these, one from Ribeirinha had an SI of 11.3 followingincubation with 10 6 infected RBC equivalents, and two fromColina had an SI    4 (SI of 4 and 6) following incubationwith 10 7 infected RBC equivalents. The PBMC from twoUS residents who had contracted falciparum malaria whiletraveling abroad were also tested; their cells failed to prolif-erate to parasite lysate.Proliferative responses of Colina and Ribeirinha PBMCwere also examined using the peptides p39A, p39B, andp82B, which were predicted to contain T cell epitopes.Twenty samples from the native population (Ribeirinha) and17 from the migrant population (Colina) were tested withindividual peptides, PHA, or  P. falciparum  blood-stage an-tigens. All samples proliferated with PHA. Only two sampleshad an SI    four (considered positive) when exposed to anyof the peptides. Both positive samples were PBMC collectedfrom subjects in the migrant population. Colina sample 30(CL30) proliferated with peptide p39A (SI    4.3) and sam-ple CL15 proliferated with p82B (SI    9.2). Parasite lysateproduced an SI    4 in a total of 13 patients (5 migrant[29%], and 8 native [40%]). Sample CL30 had an SI of 3.5,while sample CL15 had an SI of 85. Cytokine responses of patient PBMC to recombinantRAP-1 and RAP-2.  In addition to examining proliferation
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