Identification of New Antigens in Visceral Leishmaniasis by Expression Cloning and Immunoblotting with Sera of Kala-Azar Patients from Bihar
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感染与免疫杂志 2005年第10期
Clinical Research Group, Department of Dermatology, Charite University Medicine Berlin, Humboldt University, D-10089 Berlin, Germany
Indian Institute of Chemical Biology, Jadavpur, Calcutta 700032, India
Kala-Azar Medical Research Center, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India
ABSTRACT
Sera of kala-azar patients from Bihar, India, were used to identify Leishmania donovani antigens encoded by a phage expression library. Ten antigens were identified, five of which have not been described as leishmania antigens before. The antigens specifically react with sera of leishmania-infected patients but not of toxoplasma- or plasmodium-infected patients.
TEXT
Leishmania donovani is the causative agent of visceral leishmaniasis (VL) in India. Like other parasites, Leishmania donovani induces vigorous immune responses which, however, do not control the infection in patients with acute disease. If not treated, VL is lethal. Notwithstanding, there are indications of immunity to VL in populations cured of VL and in individuals in disease-endemic regions who are seropositive without signs of disease. Knowledge of antigens targeted during infection may, therefore, be instrumental in the development of Leishmania donovani vaccines and immunodiagnostics (6, 8). To identify serological antigens in VL, a Leishmania donovani TriplEx2 phage expression library consisting of 1.5 x 106 primary PFU was established from oligo(dT)-primed cDNA of mid-log-phase promastigotes of Leishmania donovani strain AG83 (3). By use of 10 antisera from VL patients (5 obtained from untreated patients and 5 obtained under or shortly after therapy) from highly endemic foci around Muzaffarpur, Bihar, India, 30 positive phage plaques were identified and subcloned to monoclonality as determined by PCR. The inserts were recloned in pTriplEx2 plasmids and sequenced by standard protocols. A comparison of the sequences with the National Center for Biotechnology Information, EMBL, and Leishmania Genome Network databases revealed that they correspond to 10 different genes each represented by 1 to 8 clones of various lengths (Table 1). Sequences for LDHSP70 (5), HASPB1 (4), P0 (10), ARP-1 (11), and -tubulin (9) genes had been published as Leishmania donovani antigen gene sequences before. However, the sequences of our LDHSP70 and P0 inserts deviate from the database entries. One of our LDHSP70 gene sequences covers 1,002 bp, of which the 5' 762 bp code for 254 amino acids of the known LDHSP70. The 3' 240 bp are located 1,630 bp downstream of the hsp70 gene and correspond to no known translation product. Since these 240 bp are in-frame with the LDHSP70 gene sequence, the insert can be translated into a continuous polypeptide. All four LDHSP70 clones differ at the 3' end and deviate from the published cDNA sequence X52314. The database entry for the Leishmania donovani P0 gene (GenBank accession number AY180912) includes a 220-bp insertion missing from our clone as well as from Leishmania chagasi and Leishmania infantum database sequences. L. infantum has two P0 genes, LiP0-A and LiP0-B, of which LiP0-B shows highest similarity to our sequence and may be its homologue. This suggests that Leishmania donovani, like L. infantum, has two P0 genes. In the case of these two antigens, it is not clear whether the published antigenic determinants are the same as ours. Five of the 10 antigens had not been identified before. LePa2 is an subunit of the 20S proteasome, of which another subunit, LePa, had been described as a Leishmania donovani antigen (2). Our sequence was not assigned for Leishmania donovani but is identical to the sequence of the gene for L. infantum Y13059 gene sequence. This antigen, therefore, is a new 20S proteasome subunit of Leishmania donovani. The second new antigen is the putative elongation factor 1 (EF1B). The other three new antigens are products of unique genes without identifiable homologues in other species. We tentatively named them LDAG1, LDAG2, and LDAG3 for Leishmania donovani antigens 1, 2, and 3.
The frequencies of seropositivity for the 10 antigens in VL patients were determined by spotting the phages that code for the antigens onto a growing bacteria layer as shown in Fig. 1A, blotting the antigens onto nitrocellulose, and probing the filters with sera from 44 VL patients, 10 control sera from healthy relatives of the patients, and 11 sera from healthy donors from areas in which disease was not endemic (1 from India and 10 from Germany). Eleven of the VL patients had received chemotherapy at the time of serum preparation, and 33 had not. All sera had been tested before against the total amount of Leishmania donovani lysate separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see the supplemental material). Thirty-six of the patient sera were positive at a dilution of 1:640. Except for one serum from an Indian donor who was the healthy relative of a patient, none of the control sera were positive. At 1:60, faint signals were detectable with sera of healthy Indian donors. The statistics of seropositivity for the 10 identified antigens are summarized in Fig. 1B and C. Sixty-four percent of the 44 tested patients and 80% of the 36 seroreactive patients were seropositive for at least one of the defined antigens. LDHSP70 was most prominent, with 64% of the 36 immune sera reacting. The frequencies of seropositivity among the seroreactive patients for the other previously known antigens HASPB1, P0, ARP-1, and -tubulin were 42, 36, 22, and 17%, respectively. The newly identified antigens LePa2, EF1B, LDAG1, LDAG2, and LDAG3 were detected in 39, 28, 6, 3, and 3% of seroreactive patients, respectively. There is no correlation of the extent of seropositivity with the ages or sexes of the patients. The average age of all the 44 VL patients was 24 years (range, 6 to 50 years), that of the 28 patients with responses to the antigens defined herein was 21.8 years (range, 6 to 50 years), and that of the remaining nonreactive 16 patients was 27.8 years (range, 10 to 50 years). Fourteen of the 28 patients who responded to the defined antigens were male, and 12 were female. For two patients, no gender information was available.
As specificity controls, eight sera each were collected from Toxoplasma gondii-, Plasmodium falciparum-, or Plasmodium vivax-infected patients in Germany who had been infected while traveling in disease-endemic areas and, most likely, had not been exposed to Leishmania donovani. Full-length expression clones coding for the identified Leishmania donovani antigens, with the exception of EF1B, were isolated from Leishmania donovani DNA and overexpressed in Escherichia coli. For EF1B, the original insert that codes for a 10-kDa fragment was used. For HSP70, in addition to the full-length clone, the 36-kDa fusion protein (see above) coded for by the corresponding phage clone was tested. The recombinant bacteria were homogenized, and the solutions were cleared of debris, blotted onto nitrocellulose, and probed with the antisera. None of the sera from healthy donors or the sera from Toxoplasma- or Plasmodium-infected patients reacted with the 10 Leishmania donovani antigens described herein (Fig. 2B, C, and D), although the sera from Toxoplasma- and Plasmodium-infected patients cross-reacted with other Leishmania donovani antigens as detected by Western blot analysis of Leishmania donovani lysates after SDS-PAGE (Fig. 2A). The elevated background seen with all patient sera correlates with the low serum dilution of 1:60 in these analyses, and hypergammaglobulinemia typically associated with parasitic infections was confirmed for the sera of this study.
The role of the strong antibody responses in leishmaniasis has been a controversial issue. As recently reported, high immunoglobulin G titers can cause antigen-independent suppression of immunity to leishmaniasis via induction of interleukin-10 (1, 7). In contrast, an earlier report demonstrated a correlation of protective cellular immune responses with antibody responses to the leishmania antigen HASBP-1 (12). Together, these reports suggest that the specificity of the serological responses might determine the outcome of the infection. Ranges and specificities of natural antileishmania responses can provide important information on the suitability of vaccine candidate antigens. As shown with this report, the serological responses in leishmaniasis are very heterogeneous (see the supplementary material). Even LDHSP70, which is rated among the most dominant antigens in leishmaniasis, induces specific antibodies in no more than 60% of the patients. Seropositivity for HASBP1, which, based on studies with mice, is considered a promising vaccine candidate (13), is found in less than 40% of the patients. Future studies will follow up the patients described herein to see whether they are protected against reinfection and whether such protection correlates with serological responses to such previously known antigens and the antigens described in this report.
Nucleotide sequence accession numbers. Sequences for the genes encoding the newly discovered antigens have been deposited in GenBank under the accession numbers given in Table 1.
ACKNOWLEDGMENTS
The technical assistance by Ulrike Fritz and Arthur O'Connor is gratefully acknowledged. We also thank Kaushik Roychoudhury for his help in collecting sera and parasites and Patricia Zambon for her assistance in preparing the manuscript. We thank Thomas Weitzel, Institut für Tropenmedizin, Diagnostisches Labor, Charite, for Plasmodium sera and PD Renate Bollmann, Institut für Mikrobiologie und Hygiene, Abt. Mikrobiologie-Serologie, Charite, for Toxoplasma sera.
The project was supported by the Volkswagen Foundation, Hannover, Germany (grant I/77 908), and the Deutsche Forschungsgemeinschaft, Germany (grant 446 IND 121/3/03).
Supplemental material for this article may be found at http://iai.asm.org/.
REFERENCES
1. Buxbaum, L. U., and P. Scott. 2005. Interleukin 10- and Fc receptor-deficient mice resolve Leishmania mexicana lesions. Infect. Immun. 73:2101-2108.
2. Christensen, C. B., L. Jorgensen, A. T. Jensen, S. Gasim, M. Chen, A. Kharazmi, T. G. Theander, and K. Andresen. 2000. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit. Biochim. Biophys. Acta 1500:77-87.
3. Ghosh, A. K., F. K. Bhattacharyya, and D. K. Ghosh. 1985. Leishmania donovani: amastigote inhibition and mode of action of berberine. Exp. Parasitol. 60:404-413.
4. Jensen, A. T., S. Gasim, T. Moller, A. Ismail, A. Gaafar, M. Kemp, A. M. el Hassan, A. Kharazmi, T. M. Alce, D. F. Smith, and T. G. Theander. 1999. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP. Trans. R. Soc. Trop. Med. Hyg. 93:157-160.
5. MacFarlane, J., M. L. Blaxter, R. P. Bishop, M. A. Miles, and J. M. Kelly. 1990. Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family. Eur. J. Biochem. 190:377-384.
6. Martin-Sanchez, J., J. A. Pineda, F. Morillas-Marquez, J. A. Garcia-Garcia, C. Acedo, and J. Macias. 2004. Detection of Leishmania infantum kinetoplast DNA in peripheral blood from asymptomatic individuals at risk for parenterally transmitted infections: relationship between polymerase chain reaction results and other Leishmania infection markers. Am. J. Trop. Med. Hyg. 70:545-548.
7. Miles, S. A., S. M. Conrad, R. G. Alves, S. M. Jeronimo, and D. M. Mosser. 2005. A role for IgG immune complexes during infection with the intracellular pathogen Leishmania. J. Exp. Med. 201:747-754.
8. Riera, C., R. Fisa, M. Udina, M. Gallego, and M. Portus. 2004. Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods. Trans. R. Soc. Trop. Med. Hyg. 98:102-110.
9. Sheppard, H. W., and D. M. Dwyer. 1986. Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis. Mol. Biochem. Parasitol. 19:35-43.
10. Soto, M., J. M. Requena, and C. Alonso. 1993. Isolation, characterization and analysis of the expression of the Leishmania ribosomal PO protein genes. Mol. Biochem. Parasitol. 61:265-274.
11. Soto, M., J. M. Requena, M. Garcia, L. C. Gomez, I. Navarrete, and C. Alonso. 1993. Genomic organization and expression of two independent gene arrays coding for two antigenic acidic ribosomal proteins of Leishmania. J. Biol. Chem. 268:21835-21843.
12. Stager, S., J. Alexander, A. C. Kirby, M. Botto, N. V. Rooijen, D. F. Smith, F. Brombacher, and P. M. Kaye. 2003. Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses. Nat. Med. 9:1287-1292.
13. Stager, S., D. F. Smith, and P. M. Kaye. 2000. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J. Immunol. 165:7064-7071.(Stephan M. Theinert, Raja)
Indian Institute of Chemical Biology, Jadavpur, Calcutta 700032, India
Kala-Azar Medical Research Center, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India
ABSTRACT
Sera of kala-azar patients from Bihar, India, were used to identify Leishmania donovani antigens encoded by a phage expression library. Ten antigens were identified, five of which have not been described as leishmania antigens before. The antigens specifically react with sera of leishmania-infected patients but not of toxoplasma- or plasmodium-infected patients.
TEXT
Leishmania donovani is the causative agent of visceral leishmaniasis (VL) in India. Like other parasites, Leishmania donovani induces vigorous immune responses which, however, do not control the infection in patients with acute disease. If not treated, VL is lethal. Notwithstanding, there are indications of immunity to VL in populations cured of VL and in individuals in disease-endemic regions who are seropositive without signs of disease. Knowledge of antigens targeted during infection may, therefore, be instrumental in the development of Leishmania donovani vaccines and immunodiagnostics (6, 8). To identify serological antigens in VL, a Leishmania donovani TriplEx2 phage expression library consisting of 1.5 x 106 primary PFU was established from oligo(dT)-primed cDNA of mid-log-phase promastigotes of Leishmania donovani strain AG83 (3). By use of 10 antisera from VL patients (5 obtained from untreated patients and 5 obtained under or shortly after therapy) from highly endemic foci around Muzaffarpur, Bihar, India, 30 positive phage plaques were identified and subcloned to monoclonality as determined by PCR. The inserts were recloned in pTriplEx2 plasmids and sequenced by standard protocols. A comparison of the sequences with the National Center for Biotechnology Information, EMBL, and Leishmania Genome Network databases revealed that they correspond to 10 different genes each represented by 1 to 8 clones of various lengths (Table 1). Sequences for LDHSP70 (5), HASPB1 (4), P0 (10), ARP-1 (11), and -tubulin (9) genes had been published as Leishmania donovani antigen gene sequences before. However, the sequences of our LDHSP70 and P0 inserts deviate from the database entries. One of our LDHSP70 gene sequences covers 1,002 bp, of which the 5' 762 bp code for 254 amino acids of the known LDHSP70. The 3' 240 bp are located 1,630 bp downstream of the hsp70 gene and correspond to no known translation product. Since these 240 bp are in-frame with the LDHSP70 gene sequence, the insert can be translated into a continuous polypeptide. All four LDHSP70 clones differ at the 3' end and deviate from the published cDNA sequence X52314. The database entry for the Leishmania donovani P0 gene (GenBank accession number AY180912) includes a 220-bp insertion missing from our clone as well as from Leishmania chagasi and Leishmania infantum database sequences. L. infantum has two P0 genes, LiP0-A and LiP0-B, of which LiP0-B shows highest similarity to our sequence and may be its homologue. This suggests that Leishmania donovani, like L. infantum, has two P0 genes. In the case of these two antigens, it is not clear whether the published antigenic determinants are the same as ours. Five of the 10 antigens had not been identified before. LePa2 is an subunit of the 20S proteasome, of which another subunit, LePa, had been described as a Leishmania donovani antigen (2). Our sequence was not assigned for Leishmania donovani but is identical to the sequence of the gene for L. infantum Y13059 gene sequence. This antigen, therefore, is a new 20S proteasome subunit of Leishmania donovani. The second new antigen is the putative elongation factor 1 (EF1B). The other three new antigens are products of unique genes without identifiable homologues in other species. We tentatively named them LDAG1, LDAG2, and LDAG3 for Leishmania donovani antigens 1, 2, and 3.
The frequencies of seropositivity for the 10 antigens in VL patients were determined by spotting the phages that code for the antigens onto a growing bacteria layer as shown in Fig. 1A, blotting the antigens onto nitrocellulose, and probing the filters with sera from 44 VL patients, 10 control sera from healthy relatives of the patients, and 11 sera from healthy donors from areas in which disease was not endemic (1 from India and 10 from Germany). Eleven of the VL patients had received chemotherapy at the time of serum preparation, and 33 had not. All sera had been tested before against the total amount of Leishmania donovani lysate separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see the supplemental material). Thirty-six of the patient sera were positive at a dilution of 1:640. Except for one serum from an Indian donor who was the healthy relative of a patient, none of the control sera were positive. At 1:60, faint signals were detectable with sera of healthy Indian donors. The statistics of seropositivity for the 10 identified antigens are summarized in Fig. 1B and C. Sixty-four percent of the 44 tested patients and 80% of the 36 seroreactive patients were seropositive for at least one of the defined antigens. LDHSP70 was most prominent, with 64% of the 36 immune sera reacting. The frequencies of seropositivity among the seroreactive patients for the other previously known antigens HASPB1, P0, ARP-1, and -tubulin were 42, 36, 22, and 17%, respectively. The newly identified antigens LePa2, EF1B, LDAG1, LDAG2, and LDAG3 were detected in 39, 28, 6, 3, and 3% of seroreactive patients, respectively. There is no correlation of the extent of seropositivity with the ages or sexes of the patients. The average age of all the 44 VL patients was 24 years (range, 6 to 50 years), that of the 28 patients with responses to the antigens defined herein was 21.8 years (range, 6 to 50 years), and that of the remaining nonreactive 16 patients was 27.8 years (range, 10 to 50 years). Fourteen of the 28 patients who responded to the defined antigens were male, and 12 were female. For two patients, no gender information was available.
As specificity controls, eight sera each were collected from Toxoplasma gondii-, Plasmodium falciparum-, or Plasmodium vivax-infected patients in Germany who had been infected while traveling in disease-endemic areas and, most likely, had not been exposed to Leishmania donovani. Full-length expression clones coding for the identified Leishmania donovani antigens, with the exception of EF1B, were isolated from Leishmania donovani DNA and overexpressed in Escherichia coli. For EF1B, the original insert that codes for a 10-kDa fragment was used. For HSP70, in addition to the full-length clone, the 36-kDa fusion protein (see above) coded for by the corresponding phage clone was tested. The recombinant bacteria were homogenized, and the solutions were cleared of debris, blotted onto nitrocellulose, and probed with the antisera. None of the sera from healthy donors or the sera from Toxoplasma- or Plasmodium-infected patients reacted with the 10 Leishmania donovani antigens described herein (Fig. 2B, C, and D), although the sera from Toxoplasma- and Plasmodium-infected patients cross-reacted with other Leishmania donovani antigens as detected by Western blot analysis of Leishmania donovani lysates after SDS-PAGE (Fig. 2A). The elevated background seen with all patient sera correlates with the low serum dilution of 1:60 in these analyses, and hypergammaglobulinemia typically associated with parasitic infections was confirmed for the sera of this study.
The role of the strong antibody responses in leishmaniasis has been a controversial issue. As recently reported, high immunoglobulin G titers can cause antigen-independent suppression of immunity to leishmaniasis via induction of interleukin-10 (1, 7). In contrast, an earlier report demonstrated a correlation of protective cellular immune responses with antibody responses to the leishmania antigen HASBP-1 (12). Together, these reports suggest that the specificity of the serological responses might determine the outcome of the infection. Ranges and specificities of natural antileishmania responses can provide important information on the suitability of vaccine candidate antigens. As shown with this report, the serological responses in leishmaniasis are very heterogeneous (see the supplementary material). Even LDHSP70, which is rated among the most dominant antigens in leishmaniasis, induces specific antibodies in no more than 60% of the patients. Seropositivity for HASBP1, which, based on studies with mice, is considered a promising vaccine candidate (13), is found in less than 40% of the patients. Future studies will follow up the patients described herein to see whether they are protected against reinfection and whether such protection correlates with serological responses to such previously known antigens and the antigens described in this report.
Nucleotide sequence accession numbers. Sequences for the genes encoding the newly discovered antigens have been deposited in GenBank under the accession numbers given in Table 1.
ACKNOWLEDGMENTS
The technical assistance by Ulrike Fritz and Arthur O'Connor is gratefully acknowledged. We also thank Kaushik Roychoudhury for his help in collecting sera and parasites and Patricia Zambon for her assistance in preparing the manuscript. We thank Thomas Weitzel, Institut für Tropenmedizin, Diagnostisches Labor, Charite, for Plasmodium sera and PD Renate Bollmann, Institut für Mikrobiologie und Hygiene, Abt. Mikrobiologie-Serologie, Charite, for Toxoplasma sera.
The project was supported by the Volkswagen Foundation, Hannover, Germany (grant I/77 908), and the Deutsche Forschungsgemeinschaft, Germany (grant 446 IND 121/3/03).
Supplemental material for this article may be found at http://iai.asm.org/.
REFERENCES
1. Buxbaum, L. U., and P. Scott. 2005. Interleukin 10- and Fc receptor-deficient mice resolve Leishmania mexicana lesions. Infect. Immun. 73:2101-2108.
2. Christensen, C. B., L. Jorgensen, A. T. Jensen, S. Gasim, M. Chen, A. Kharazmi, T. G. Theander, and K. Andresen. 2000. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit. Biochim. Biophys. Acta 1500:77-87.
3. Ghosh, A. K., F. K. Bhattacharyya, and D. K. Ghosh. 1985. Leishmania donovani: amastigote inhibition and mode of action of berberine. Exp. Parasitol. 60:404-413.
4. Jensen, A. T., S. Gasim, T. Moller, A. Ismail, A. Gaafar, M. Kemp, A. M. el Hassan, A. Kharazmi, T. M. Alce, D. F. Smith, and T. G. Theander. 1999. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP. Trans. R. Soc. Trop. Med. Hyg. 93:157-160.
5. MacFarlane, J., M. L. Blaxter, R. P. Bishop, M. A. Miles, and J. M. Kelly. 1990. Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family. Eur. J. Biochem. 190:377-384.
6. Martin-Sanchez, J., J. A. Pineda, F. Morillas-Marquez, J. A. Garcia-Garcia, C. Acedo, and J. Macias. 2004. Detection of Leishmania infantum kinetoplast DNA in peripheral blood from asymptomatic individuals at risk for parenterally transmitted infections: relationship between polymerase chain reaction results and other Leishmania infection markers. Am. J. Trop. Med. Hyg. 70:545-548.
7. Miles, S. A., S. M. Conrad, R. G. Alves, S. M. Jeronimo, and D. M. Mosser. 2005. A role for IgG immune complexes during infection with the intracellular pathogen Leishmania. J. Exp. Med. 201:747-754.
8. Riera, C., R. Fisa, M. Udina, M. Gallego, and M. Portus. 2004. Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods. Trans. R. Soc. Trop. Med. Hyg. 98:102-110.
9. Sheppard, H. W., and D. M. Dwyer. 1986. Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis. Mol. Biochem. Parasitol. 19:35-43.
10. Soto, M., J. M. Requena, and C. Alonso. 1993. Isolation, characterization and analysis of the expression of the Leishmania ribosomal PO protein genes. Mol. Biochem. Parasitol. 61:265-274.
11. Soto, M., J. M. Requena, M. Garcia, L. C. Gomez, I. Navarrete, and C. Alonso. 1993. Genomic organization and expression of two independent gene arrays coding for two antigenic acidic ribosomal proteins of Leishmania. J. Biol. Chem. 268:21835-21843.
12. Stager, S., J. Alexander, A. C. Kirby, M. Botto, N. V. Rooijen, D. F. Smith, F. Brombacher, and P. M. Kaye. 2003. Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses. Nat. Med. 9:1287-1292.
13. Stager, S., D. F. Smith, and P. M. Kaye. 2000. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J. Immunol. 165:7064-7071.(Stephan M. Theinert, Raja)