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RESA
Oral Sessions I Parasitology
256
44. Immunology-5 O-0501
International
47 (Suppl.) (1998)
133-281
(Malaria)
HUMAN ANTIBODIES TO A NON-REPEAT REGION OF Plasmodium falciparum ANTIGEN Pfl55/RESA
Siddiaue A. B, Ahlborg N, Warsame M, Perlmann P, Berzins K Department of Immunology, Stockholm University, Stockholm, Sweden Antibodies to the repeat sequences of the Plasmodium asexual blood stage antigen Pfl5YRESA interfere with parasite growth in vitro. Less is known with regard to the effects of antibodies to the regions of the antigen lacking repeats. We investigated the levels of antibodies to a nonrepeat region of Pfl55/RESA in 108 individuals from malaria endemic areas in Liberia, Tanzania and Senegal. Synthetic peptides, in the form of multiple antigen peptides (MAPS) together overlapping residues 199-227 of the antigen were used for ELISA analysis of sera. High levels of antibodies were observed in a majority of the sera to two of the peptides (residue 199-211 and 202-214, respectively) whereas the recognition of the other peptides was poor. The level of antibodies in Liberian sera to these two peptides was significantly higher than in sera from the other areas. Human antibodies were affinity purified from Liberian sera using peptides corresponding to residue 199-211 and 202-216. The speciticity of these antibodies for Pfl55/RESA was confirmed by immunofluoresence and immunoblottine. Furthermore. the antibodies were efficient inhibitors of para&e growth in h-o. The results thus indicate that non-repeated epitopes in Pfl55/RESA are exposed to the immune system and elicit antibodies that are potentially protective against malaria infections.
falciparum
O-0502 CHARACTERIZATIONOF PROTECTIVE EPITOPES IN THE
ACIDIC-BASIC REPEAT ANTIGEN (ABRA) PROTEIN OF THE HUMAN MALARIAPARASITE, PLASMODRIM FALCIPARUM Sharma Pawan, Kumar A, Singh B, Bharadwaj A, Malhotra P, Kushwaha A, Chauhan VS, Adak T’ International Centrc for Genetic engineering and Biotechnology (ICGEB). New Delhi; *Malaria Research Centre, Delhi, India.
By virtue of its role in the formation of the ‘immune clusters of merowites’, its location on the merozoite surface and its highly conserved nature both at the nucleotide sequence level and at the amino acid sequence level, ABRA protein of the human malaria parasite, Plasmodium falciparum, represents a putatively protective antigen. Based upon predicted amino acid sequence of ABRA. we svnthesized eieht Dentides. six of these IAB-1 to AB-61. _ . . ranging from 12 to 18 residues,coveringthe most hydrophihc regionsof the protein,and two more peptidesrepresenting its repetitive sequences. We found that alleightconstructsbound appreciable amountofantibody in serafrom a largeproportion of cases of falciparummalana, indicating that thesewere, indeed, recognized as B epitopesduringnatural exposure to the parasite; two of these peptides(AB-1 and AB-3) also elicited a strong proliferation responsein peripheral bloodmononuclearcellsfrom allelevenhuman subjects recovering from malaria. When usqd as carrier-free immunogens, six pcptidesinduced a strong. boostable. IEG-~?x antibodv rcsnonse in rabbits. indicatine the presence bf lboth ‘B cell detcknin&ts and Th epitopes in these six constructs. These antibodies specifically cross-reacted with the and in a” protein(s) in a” parasite immunoblot immunofluorescence assay. In another immunoblot. rabbit antipeptide sera also recognizedrecombinant fragments of ABRA umressed in bacteria. Most sienilicantlv. ourificd rabbit 1eG r&bodies against two constructs-(Ab-1 and AB-5) Inhibited t?le merozoite rc-invasion of human erythrocytes in vitro upto 90 percent. ‘fhesc results favor furtherstudiesso as to determine possible inclusion of these two constructs in a multi-component sub-unit vaccine againstasexualblood-stagesofP,falcl~arum.
‘-OH3
ROLE OF EXTRATHYMIC T CELLS IN A RODENT MALARIAL INFECTION
j&g&&l& Watanabe H, Sekikawa H, Mannoor K, Abo T Department of Immunology, Niigata University School of Medicine, Japan Intermediate TCR cells (Int TCR), which express IL-2R B and intermediate intensity of CD3iTCR, are shown to be extrathymic in origin. We studied their role in immunocompetent C3H/He and C57BL/6 and nude mice infected with Plasmodium yoelii (17X, lethal and 17XNL, non-lethal) by intraperitoneal passage of lo4 infected RBC. Phenotypic characterization of mononuclear cells from liver, spleen and thymus showed expansion of Int TCR cells with both strains of malaria. Furthermore, they were found to possess self reactive forbidden clones and cytotoxicity against parasitized RBC. Depletion of IL-ZR fi +cells from infected mice by treatment with mAb resulted in exacerbation of infection during the early phase of the nonlethal malaria. These findings indicate that extrathymic T cells play a role during the erythocytic stage of the rodent malarial infection. O-0504
RECONBINATION-BASEDGENETIC DIVERSITY OF PLASMODIUNFALCIPARUMHEROZOITE SURFACE PROTEIN 1 IN FIELD ISOLATES FROH THAILAND
Naoko Sekihama’, Masatsugu Kimura**, Kenji Nirayarsa***, Resara Na-Bangchang****, Kazuyuki Tanabe’ 'Lab. of E&l., Osaka Inst. of Technol., Osaka, Japan, "Lab. of Biopbys., Osaka City Univ. Med. School, Osaka, Japan, “‘Dep. of Med. Zool., Saitama Med. School, Saitama, Japan and ****Clinical Pharmacology Unit, Fat. of Trop. Med., Hahidol Univ., Bangkok, Thailand The genetic diversity of Plasmodium falciparum meropoite surface protein 1 (MSPl) is generated by recombination of dimorphic allelic types in variable blocks, except block 2, a polymorphic region. using a PCR method that distinguishes 24 different HSPl gene types, we investigated frequency distribution of the gene types in field isolates from northwestern Thailand for comparison with our previous results from southern Vietnam (Kaneko et al., 1997) and Amazon (Ferreira et al., 1998). The overall frequency distribution pattern differed significantly, suggesting differentiation of the genetic structure of P.falciparum natural population among different geographic areas. Chi-square analysis of pairwise associations between particular two blocks (i.e. “linkage” of different loci) dersonstrated random recombination in blocks 4a-4b and nonrandom association in blocks 2-4 and 2-6. Complete association was found for concordant allelic types in blocks 6-16. These results suggest that (a) recombination within the HSPl gene specifically occurs at the 5' portion of the gene or that (b) there is some mechanism of selec,tion that favors particular MSPl gene type after recombination.
256
44. Immunology-5 O-0501
International
47 (Suppl.) (1998)
133-281
(Malaria)
HUMAN ANTIBODIES TO A NON-REPEAT REGION OF Plasmodium falciparum ANTIGEN Pfl55/RESA
Siddiaue A. B, Ahlborg N, Warsame M, Perlmann P, Berzins K Department of Immunology, Stockholm University, Stockholm, Sweden Antibodies to the repeat sequences of the Plasmodium asexual blood stage antigen Pfl5YRESA interfere with parasite growth in vitro. Less is known with regard to the effects of antibodies to the regions of the antigen lacking repeats. We investigated the levels of antibodies to a nonrepeat region of Pfl55/RESA in 108 individuals from malaria endemic areas in Liberia, Tanzania and Senegal. Synthetic peptides, in the form of multiple antigen peptides (MAPS) together overlapping residues 199-227 of the antigen were used for ELISA analysis of sera. High levels of antibodies were observed in a majority of the sera to two of the peptides (residue 199-211 and 202-214, respectively) whereas the recognition of the other peptides was poor. The level of antibodies in Liberian sera to these two peptides was significantly higher than in sera from the other areas. Human antibodies were affinity purified from Liberian sera using peptides corresponding to residue 199-211 and 202-216. The speciticity of these antibodies for Pfl55/RESA was confirmed by immunofluoresence and immunoblottine. Furthermore. the antibodies were efficient inhibitors of para&e growth in h-o. The results thus indicate that non-repeated epitopes in Pfl55/RESA are exposed to the immune system and elicit antibodies that are potentially protective against malaria infections.
falciparum
O-0502 CHARACTERIZATIONOF PROTECTIVE EPITOPES IN THE
ACIDIC-BASIC REPEAT ANTIGEN (ABRA) PROTEIN OF THE HUMAN MALARIAPARASITE, PLASMODRIM FALCIPARUM Sharma Pawan, Kumar A, Singh B, Bharadwaj A, Malhotra P, Kushwaha A, Chauhan VS, Adak T’ International Centrc for Genetic engineering and Biotechnology (ICGEB). New Delhi; *Malaria Research Centre, Delhi, India.
By virtue of its role in the formation of the ‘immune clusters of merowites’, its location on the merozoite surface and its highly conserved nature both at the nucleotide sequence level and at the amino acid sequence level, ABRA protein of the human malaria parasite, Plasmodium falciparum, represents a putatively protective antigen. Based upon predicted amino acid sequence of ABRA. we svnthesized eieht Dentides. six of these IAB-1 to AB-61. _ . . ranging from 12 to 18 residues,coveringthe most hydrophihc regionsof the protein,and two more peptidesrepresenting its repetitive sequences. We found that alleightconstructsbound appreciable amountofantibody in serafrom a largeproportion of cases of falciparummalana, indicating that thesewere, indeed, recognized as B epitopesduringnatural exposure to the parasite; two of these peptides(AB-1 and AB-3) also elicited a strong proliferation responsein peripheral bloodmononuclearcellsfrom allelevenhuman subjects recovering from malaria. When usqd as carrier-free immunogens, six pcptidesinduced a strong. boostable. IEG-~?x antibodv rcsnonse in rabbits. indicatine the presence bf lboth ‘B cell detcknin&ts and Th epitopes in these six constructs. These antibodies specifically cross-reacted with the and in a” protein(s) in a” parasite immunoblot immunofluorescence assay. In another immunoblot. rabbit antipeptide sera also recognizedrecombinant fragments of ABRA umressed in bacteria. Most sienilicantlv. ourificd rabbit 1eG r&bodies against two constructs-(Ab-1 and AB-5) Inhibited t?le merozoite rc-invasion of human erythrocytes in vitro upto 90 percent. ‘fhesc results favor furtherstudiesso as to determine possible inclusion of these two constructs in a multi-component sub-unit vaccine againstasexualblood-stagesofP,falcl~arum.
‘-OH3
ROLE OF EXTRATHYMIC T CELLS IN A RODENT MALARIAL INFECTION
j&g&&l& Watanabe H, Sekikawa H, Mannoor K, Abo T Department of Immunology, Niigata University School of Medicine, Japan Intermediate TCR cells (Int TCR), which express IL-2R B and intermediate intensity of CD3iTCR, are shown to be extrathymic in origin. We studied their role in immunocompetent C3H/He and C57BL/6 and nude mice infected with Plasmodium yoelii (17X, lethal and 17XNL, non-lethal) by intraperitoneal passage of lo4 infected RBC. Phenotypic characterization of mononuclear cells from liver, spleen and thymus showed expansion of Int TCR cells with both strains of malaria. Furthermore, they were found to possess self reactive forbidden clones and cytotoxicity against parasitized RBC. Depletion of IL-ZR fi +cells from infected mice by treatment with mAb resulted in exacerbation of infection during the early phase of the nonlethal malaria. These findings indicate that extrathymic T cells play a role during the erythocytic stage of the rodent malarial infection. O-0504
RECONBINATION-BASEDGENETIC DIVERSITY OF PLASMODIUNFALCIPARUMHEROZOITE SURFACE PROTEIN 1 IN FIELD ISOLATES FROH THAILAND
Naoko Sekihama’, Masatsugu Kimura**, Kenji Nirayarsa***, Resara Na-Bangchang****, Kazuyuki Tanabe’ 'Lab. of E&l., Osaka Inst. of Technol., Osaka, Japan, "Lab. of Biopbys., Osaka City Univ. Med. School, Osaka, Japan, “‘Dep. of Med. Zool., Saitama Med. School, Saitama, Japan and ****Clinical Pharmacology Unit, Fat. of Trop. Med., Hahidol Univ., Bangkok, Thailand The genetic diversity of Plasmodium falciparum meropoite surface protein 1 (MSPl) is generated by recombination of dimorphic allelic types in variable blocks, except block 2, a polymorphic region. using a PCR method that distinguishes 24 different HSPl gene types, we investigated frequency distribution of the gene types in field isolates from northwestern Thailand for comparison with our previous results from southern Vietnam (Kaneko et al., 1997) and Amazon (Ferreira et al., 1998). The overall frequency distribution pattern differed significantly, suggesting differentiation of the genetic structure of P.falciparum natural population among different geographic areas. Chi-square analysis of pairwise associations between particular two blocks (i.e. “linkage” of different loci) dersonstrated random recombination in blocks 4a-4b and nonrandom association in blocks 2-4 and 2-6. Complete association was found for concordant allelic types in blocks 6-16. These results suggest that (a) recombination within the HSPl gene specifically occurs at the 5' portion of the gene or that (b) there is some mechanism of selec,tion that favors particular MSPl gene type after recombination.