EP0981624A2 - Leishmania antigene zur verwendung in leishmaniasis therapie und diagnose - Google Patents

Leishmania antigene zur verwendung in leishmaniasis therapie und diagnose

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Publication number
EP0981624A2
EP0981624A2 EP98907489A EP98907489A EP0981624A2 EP 0981624 A2 EP0981624 A2 EP 0981624A2 EP 98907489 A EP98907489 A EP 98907489A EP 98907489 A EP98907489 A EP 98907489A EP 0981624 A2 EP0981624 A2 EP 0981624A2
Authority
EP
European Patent Office
Prior art keywords
glu
ala
leu
lys
asp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98907489A
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English (en)
French (fr)
Inventor
Steven G. Reed
Antonio Campos-Neto
John R. Webb
Davin C. Dillon
Yasir A. Skeiky
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Corixa Corp
Original Assignee
Corixa Corp
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Filing date
Publication date
Priority claimed from US08/798,841 external-priority patent/US6709661B1/en
Application filed by Corixa Corp filed Critical Corixa Corp
Priority to EP04000341A priority Critical patent/EP1422238B1/de
Priority to EP01101398A priority patent/EP1113073A3/de
Priority to EP10177490A priority patent/EP2284186A1/de
Publication of EP0981624A2 publication Critical patent/EP0981624A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to compositions and methods for preventing, treating and detecting leishmaniasis, and for stimulating immune responses in patients.
  • the invention is more particularly related to polypeptides comprising an immunogenic portion of a Leishmania antigen or a variant thereof, and to vaccines and pharmaceutical compositions comprising one or more such polypeptides.
  • the vaccines and pharmaceutical compositions may be used, for example, for the prevention and therapy of leishmaniasis, as well as for the detection of Leishmania infection.
  • Leishmania organisms are intracellular protozoan parasites of macrophages that cause a wide range of clinical diseases in humans and domestic animals, primarily dogs. In some infections, the parasite may lie dormant for many years. In other cases, the host may develop one of a variety of forms of leishmaniasis.
  • the disease may be asymptomatic or may be manifested as subclinical visceral leishmaniasis, which is characterized by mild symptoms of malaise, diarrhea and intermittent hepatomegaly. Patients with subclinical or asymptomatic disease usually have low antibody titers, making the disease difficult to detect with standard techniques.
  • leishmaniasis may be manifested as a cutaneous disease, which is a severe medical problem but is generally self-limiting, or as a highly destructive mucosal disease, which is not self-limiting.
  • the disease may be manifested as an acute visceral infection involving the spleen, liver and lymph nodes, which, untreated, is generally a fatal disease.
  • Symptoms of acute visceral leishmaniasis include hepatosplenomegaly, fever, leukopenia, anemia and hypergammaglobulinemia.
  • Leishmaniasis is a serious problem in much of the world, including Brazil, China, East Africa, India and areas of the Middle East.
  • the disease is also endemic in the Mediterranean region, including southern France, Italy, Greece, Spain, Portugal and North Africa.
  • the number of cases of leishmaniasis has increased dramatically in the last 20 years, and millions of cases of this disease now exist worldwide.
  • About 2 million new cases are diagnosed each year, 25% of which are visceral leishmaniasis.
  • the present invention provides compositions and methods for preventing, treating and detecting leishmaniasis, as well as for stimulating immune responses in patients.
  • polypeptides are provided which comprise at least an immunogenic portion of a Leishmania antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications.
  • the Leishmania antigen comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 2, 4, 20, 22, 24, 26, 36-38, 41, 50-53 and 82.
  • DNA sequences encoding the above polypeptides, recombinant expression vectors comprising these DNA sequences and host cells transformed or transfected with such expression vectors are also provided.
  • the present invention provides pharmaceutical compositions which comprise one or more of the polypeptides described herein, or a DNA molecule encoding such polypeptides, and a physiologically acceptable carrier.
  • Vaccines which comprise one or more such polypeptides or DNA molecules, together with a non-specific immune response enhancer are also provided.
  • the Leishmania antigen has an amino acid sequence selected from the group consisting of SEQ ID Nos: 2, 4, 20, 22, 24, 26, 36-38, 41, 50-53 and 82.
  • the pharmaceutical compositions and vaccines comprise at least two different polypeptides, each polypeptide comprising an immunogenic portion of a Leishmania antigen having an amino acid sequence selected from the group consisting of sequences recited in SEQ ID Nos: 2, 4, 6, 8, 10, 20, 22, 24, 26, 36-38, 41, 50-53, 82, and variants thereof that differ only in conservative substitutions and/or modifications.
  • the inventive pharmaceutical compositions comprise one or more of the inventive polypeptides in combination with a known Leishmania antigen.
  • compositions and vaccines comprise soluble Leishmania antigens.
  • the present invention provides methods for inducing protective immunity against leishmaniasis in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above.
  • methods and diagnostic kits are provided for detecting Leishmania infection in a patient.
  • the methods comprise: (a) contacting dermal cells of a patient with a pharmaceutical composition as described above; and (b) detecting an immune response on the patient's skin, therefrom detecting Leishmania infection in the patient.
  • the diagnostic kits comprise: (a) a pharmaceutical composition as described above; and (b) an apparatus sufficient to contact the pharmaceutical composition with the dermal cells of a patient.
  • the present invention provides methods for stimulating a cellular and/or humoral immune response in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above.
  • methods for treating a patient afflicted with a disease responsive to IL-12 stimulation, comprising administering to a patient a pharmaceutical composition or vaccine as described above.
  • Figure 2 illustrates representative HPLC profiles of peptides isolated from MHC class II molecules of P388D1 macrophages.
  • Panel A shows peptides isolated from uninfected macrophages and panel B shows peptides isolated from L. donovani infected macrophages.
  • the arrows in panel B indicate peptide peaks present only in the infected macrophage preparation.
  • Figure 3 illustrates the expression and purification of the Leishmania antigen Ldp23 as a recombinant fusion protein.
  • Panel A shows a Coomassie blue- stained SDS-PAGE gel of lysed E. coli without (lane 1) and with (lane 2) IPTG induction of Ldp23 expression. Arrow indicates the recombinant fusion protein.
  • B shows the fusion protein following excision from a preparative SDS-PAGE gel, electroelution, dialysis against PBS and analytical SDS-PAGE.
  • Figure 4 presents a Northern blot analysis of total RNA prepared from L. donovani, L. major, L. amazonensis and L. pifanoi with a 32 P labeled Ldp23 gene.
  • 1, 2 and 3 refer to RNA obtained from promastigotes at the logarithmic growth phase, promastigotes at the stationary growth phase and amastigote forms, respectively.
  • Figure 5 shows a Western blot analysis of L. donovani promastigote antigens incubated with pre-immune rabbit serum (lane A) or with anti-Ldp23 rabbit antiserum (lane B).
  • FIG 6 illustrates the surface expression of Ldp23 on live L. donovani promastigotes.
  • the dotted line shows the indirect immunofluorescence performed using pre-immune mouse serum and the solid line shows the result obtained with mouse anti- GST-Ldp23 antiserum. Fluorescence intensity was analyzed by FACScan.
  • Figure 7 shows the stimulation of Leishmania-specific T-cell proliferation by Ldp23. The results are presented as relative cell number as a function of fluorescence intensity.
  • T-cells (10 5 /well) were purified from lymph nodes of BALB/c mice immunized in the foot pad with L.
  • Figure 8 illustrates Ldp23-induced cytokine production by lymph node cells of BALB/c mice. Cultures were incubated with varying amounts of Ldp23 or Leishmania lysate, presented as ⁇ g/mL, and were assayed by ELISA for the production of interferon- ⁇ (panel A) or interleukin-4 (panel B), both of which are shown as ng/mL.
  • Figure 9 shows the PCR amplification of cytokine mRNAs isolated from mucosal leishmaniasis (Panel A) and cutaneous leishmaniasis (panel B) patient PBMC before and after stimulation with representative polypeptides of the present invention.
  • Lanes O and - indicate the level of PCR products at the initiation of culture and after 72 hours of culture, respectively, in the absence of added polypeptide; lanes Lb, 83a and
  • 83b indicate the level of PCR products following culturing of PBMC with L. braziliensis lysate, and the Leishmania antigens Lbhsp83a and Lbhsp83b, respectively.
  • Figure 10 presents a comparison of the levels of interferon- ⁇ (panel A) and TNF- ⁇ (panel B) in the supernatants of 72 hour PBMC cultures from Leishmania- infected and control individuals in response to stimulation with parasite lysate or the indicated polypeptides.
  • Figure 11 illustrates the levels of IL-10 p40 (in pg/mL) in the supernatant of PBMC cultures from L. braziliensis '-infected individuals and uninfected controls 72 hours following stimulation with parasite promastigote lysate (Lb), Lbhsp83a or Lbhsp83b.
  • Figure 12 presents the reactivities of sera from L. braziliensis infected- patients with representative polypeptides of the present invention in a standard ELISA. Values are expressed as absorbance at 405 nm.
  • Figures 13A and 13B illustrate the level of secreted IL-4 and IFN- ⁇ (in pg/mL) stimulated in mouse lymph node cultures by the addition of representative polypeptides of the present invention.
  • Figure 14 shows the level of IFN- ⁇ (in pg/mL) secreted by Leishmania- infected and uninfected human PBMC stimulated by the Leishmania antigen Ml 5, as compared to the levels stimulated by L. major lysate and L-Rack, an antigen that does not appear to be recognized by Leishmania-mf ⁇ ctQd humans.
  • Figure 15 shows the level of IFN- ⁇ (in pg/mL) secreted by infected and uninfected human PBMC stimulated by soluble Leishmania antigens (S antigens), as compared to the levels stimulated by L. major lysate and L-Rack.
  • Figure 16 illustrates the proliferation of murine lymph node cultures stimulated by the addition of representative polypeptides of the present invention. Values are expressed as cpm.
  • Figure 17 shows the proliferation of human PBMC, prepared from Leishmania-im ane and uninfected individuals, stimulated by Ml 5 as compared to the proliferation stimulated by L. major lysate and L-Rack. Values are expressed as cpm.
  • Figure 18 illustrates the proliferation of human PBMC, prepared from Leishmania-inf ected and uninfected individuals, stimulated by soluble Leishmania antigens as compared to the proliferation stimulated by culture medium, L. major lysate and L-Rack. Values are expressed as cpm.
  • Figure 19 presents a comparison of a Lbhsp83 sequence (SEQ ID NO:6) with homologous sequences from L. amazonensis (Lahsp83) (SEQ ID NO: 16), T. cruzi (Tchsp83) (SEQ ID NO: 17) and humans (Huhsp89) (SEQ ID NO: 18).
  • Figure 20 illustrates the reactivity of rabbit sera raised against soluble Leishmania antigens with Leishmania promastigote lysate (lane 1) and soluble Leishmania antigens (lane 2).
  • Figure 21 shows the cDNA and predicted amino acid sequence for the Leishmania antigen Lmspla.
  • Figure 22 shows a Southern blot of genomic DNA from L. major digested with a panel of restriction enzymes (lanes 1 to 7) and six other Leishmania species digested with Pstl (lanes 8 to 13) probed with the full-length cDNA insert of Lmspla.
  • Figure 23 shows a Southern blot of genomic DNA from L. major digested with a panel of restriction enzymes, six other Leishmania species digested with Pstl and the infectious pathogens T. cruzi and T. brucei, probed with the full-length cDNA insert of the Leishmania antigen MAPS- 1 A.
  • Figure 24 illustrates the proliferation of PBMC isolated from uninfected- individuals, patients with active mucosal leishmaniasis and patients post kala-azar infection, stimulated by MAPS- 1 A.
  • Figure 25 illustrates the proliferation of murine lymph node cultures stimulated by MAPS- 1 A.
  • Figure 26 illustrates the reactivity of MAPS- 1 A with sera from human leishmaniasis patients.
  • Figure 27 illustrates the reactivity of MAPS- 1 A with sera from mice immunized against and/or infected with leishmaniasis.
  • Figure 28 illustrates the effectiveness of immunization with either soluble Leishmania antigens or a mixture of Ldp23, LbeiF4A and Ml 5 plus adjuvant in conferring protection against infection (as measured by footpad swelling) in a murine leishmaniasis model system, as compared to the administration of adjuvant alone.
  • Figure 29 illustrates the effectiveness of immunization with MAPS- 1 A plus adjuvant in conferring protection against infection (as measured by footpad swelling) in a murine leishmaniasis model system, as compared to the administration of adjuvant alone.
  • Figures 30A and B illustrate the proliferation of murine lymph node cultures stimulated with either LcgSP8, LcgSPIO or LcgSP3.
  • compositions of the subject invention include polypeptides that comprise at least an immunogenic portion of a Leishmania antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications.
  • compositions of the present invention include multiple polypeptides selected so as to provide enhanced protection against a variety of Leishmania species.
  • Polypeptides within the scope of the present invention include, but are not limited to, polypeptides comprising immunogenic portions of Leishmania antigens comprising the sequences recited in SEQ ID NO:2 (referred to herein as Ml 5), SEQ ID NO:4 (referred to herein as Ldp23), SEQ ID NO:6 (referred to herein as Lbhsp83), SEQ ID NO:8 (referred to herein as Lt-210), SEQ ID NO: 10 (referred to herein as LbeIF4A), SEQ ID NO: 20 (referred to herein as Lmspla), SEQ ID NO: 22 (referred to herein as Lmsp9a), SEQ ID NOs: 24 and 26 (referred to herein as MAPS-1A), and SEQ ID NO: 36-42, 49-53 and 55.
  • SEQ ID NO:2 referred to herein as Ml 5
  • SEQ ID NO:4 referred to herein as Ldp23
  • SEQ ID NO:6 referred to herein as Lb
  • polypeptide encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent bonds.
  • a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences.
  • the additional sequences may be derived from the native Leishmania antigen or may be heterologous, and such sequences may (but need not) be immunogenic.
  • An antigen "having" a particular sequence is an antigen that contains, within its full length sequence, the recited sequence.
  • the native antigen may, or may not, contain additional amino acid sequence.
  • An immunogenic portion of a Leishmania antigen is a portion that is capable of eliciting an immune response (i.e., cellular and/or humoral) in a presently or previously Leishmania- ⁇ nfected patient (such as a human or a dog) and/or in cultures of lymph node cells or peripheral blood mononuclear cells (PBMC) isolated from presently or previously Leishmania-infected individuals.
  • the cells in which a response is elicited may comprise a mixture of cell types or may contain isolated component cells (including, but not limited to, T-cells, NK cells, macrophages, monocytes and/or B cells).
  • immunogenic portions are capable of inducing T-cell proliferation and/or a dominantly Thl -type cytokine response (e.g., IL-2, IFN- ⁇ , and/or TNF- ⁇ production by T-cells and/or NK cells; and/or IL-12 production by monocytes, macrophages and/or B cells).
  • a dominantly Thl -type cytokine response e.g., IL-2, IFN- ⁇ , and/or TNF- ⁇ production by T-cells and/or NK cells; and/or IL-12 production by monocytes, macrophages and/or B cells.
  • Immunogenic portions of the antigens described herein may generally be identified using techniques known to those of ordinary skill in the art, including the representative methods provided herein.
  • compositions and methods of the present invention also encompass variants of the above polypeptides.
  • a polypeptide "variant,” as used herein, is a polypeptide that differs from the native antigen only in conservative substitutions and/or modifications, such that the ability of the polypeptide to include an immune response is retained.
  • Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90% and most preferably at least about 95 % identity to the identified polypeptides.
  • such variants may be identified by modifying one of the above polypeptide sequences and evaluating the immunogenic properties of the modified polypeptide using, for example, the representative procedures described herein.
  • a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenic properties, secondary structure and hydropathic nature of the polypeptide.
  • a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein.
  • the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • nucleotide “variant” is a sequence that differs from the recited nucleotide sequence in having one or more nucleotide deletions, substitutions or additions. Such modifications may be readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA, 2:183, 1983). Nucleotide variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant nucleotide sequences preferably exhibit at least about 70%, more preferably at least about 80% and most preferably at least about 90% identity to the recited sequence.
  • stringent conditions refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65 °C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65 °C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65 °C.
  • Polypeptides as described herein also include combination polypeptides.
  • a “combination polypeptide” is a polypeptide comprising at least one of the above immunogenic portions and one or more additional immunogenic Leishmania sequences, which are joined via a peptide linkage into a single amino acid chain.
  • the sequences may be joined directly (i.e., with no intervening amino acids) or may be joined by way of a linker sequence (e.g., Gly-Cys-Gly) that does not significantly diminish the immunogenic properties of the component polypeptides.
  • Leishmania antigens having immunogenic properties may be prepared using any of a variety of procedures from one or more Leishmania species including, but not limited to, L. donovani, L. chagasi, L. infantum, L. major, L. amazonensis, L. braziliensis, L. panamensis, L. mexicana, L. tropica, and L. guyanensis.
  • Leishmania species including, but not limited to, L. donovani, L. chagasi, L. infantum, L. major, L. amazonensis, L. braziliensis, L. panamensis, L. mexicana, L. tropica, and L. guyanensis.
  • ATCC American Type Culture Collection
  • peptides isolated from MHC class II molecules of macrophages infected with a Leishmania species may be used to rescue the corresponding Leishmania donor antigens.
  • MHC class II molecules are expressed mainly by cells of the immune system, including macrophages. These molecules present peptides, which are usually 13-17 amino acids long, derived from foreign antigens that are degraded in cellular vesicles. The bound peptide antigens are then recognized by CD4 T-cells. Accordingly, foreign peptides isolated from MHC class II molecules of, for example, Leishmania-int ected murine macrophages may be used to identify immunogenic Leishmania proteins.
  • peptides derived from Leishmania antigens may be isolated by comparing the reverse phase HPLC profile of peptides extracted from infected macrophages with the profile of peptides extracted from uninfected cells. Peptides giving rise to distinct HPLC peaks unique to infected macrophages may then be sequenced using, for example, Edman chemistry as described in Edman and Berg, Eur J. Biochem, 50:116-132 (1967). A DNA fragment corresponding to a portion of a Leishmania gene encoding the peptide may then be amplified from a Leishmania cDNA library using an oligonucleotide sense primer derived from the peptide sequence and an oligo dT antisense primer.
  • the resulting DNA fragment may then be used as a probe to screen a Leishmania library for a full length cDNA or genomic clone that encodes the Leishmania antigen.
  • Screen may generally be performed using techniques well known to those of ordinary skill in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1989).
  • This approach may be used to identify a 23 kD Leishmania donovani antigen (referred to herein as Ldp23).
  • Ldp23 23 kD Leishmania donovani antigen
  • the sequence of a DNA molecule encoding Ldp23 is provided in SEQ ID NO:3 and the amino acid sequence of Ldp23 is provided in SEQ ID NO:4.
  • Ldp23 has been shown to induce a Thl immune response in T-cells prepared from Leis hmania-inf ected mice.
  • Leishmania cDNA or genomic expression library may be screened with serum from a Leis hmania-inf ected individual, using techniques well known to those of ordinary skill in the art. DNA molecules encoding reactive antigens may then be used to express the recombinant antigen for purification. The immunogenic properties of the purified Leishmania antigens may then be evaluated using, for example the representative methods described herein.
  • sera from Leis hmania-inf ected mice may be used to screen a cDNA library prepared from Leishmania amastigotes. Reactive clones may then be expressed and recombinant proteins assayed for the ability to stimulate T-cells or NK cells derived from Leishmania-immxme individuals (i.e., individuals having evidence of infection, as documented by positive serological reactivity with Leishmania-specific antibodies and/or a Leishmania-specific DTH response, without clinical symptoms of leishmaniasis). This procedure may be used to obtain a recombinant DNA molecule encoding the Leishmania antigen designated Ml 5. The sequence of such a DNA molecule is provided in SEQ ID NO:l, and the amino acid sequence of the encoded protein is provided in SEQ ID NO:2.
  • Lbhsp83 A similar approach may be used to isolate a genomic DNA molecule encoding an immunogenic Leishmania braziliensis antigen, referred to herein as Lbhsp83. More specifically, a genomic clone encoding Lbhsp83 may be isolated by screening a L. braziliensis expression library with sera from a Leis hmania-inf ected individual. The DNA encoding Lbhsp83 is homologous to the gene encoding the eukaryotic 83 kD heat shock protein. The sequence of a DNA molecule encoding nearly all of Lbhsp83 is presented in SEQ ID NO:5, and the encoded amino acid sequence is provided in SEQ ID NO:6.
  • Lbhsp83 has been found to stimulate proliferation, and a mixed Thl and Th2 cytokine profile, in PBMC isolated from L. braziliensis-infected patients. Accordingly, Lbhsp83 is an immunogenic Leishmania antigen. Regions of Lbhsp83 that are not conserved with the mammalian gene have been found to be particularly potent for T-cell stimulation and antibody binding. Such regions may be identified, for example, by visual inspection of the sequence comparison provided in Figure 19.
  • Lt-210 a DNA molecule encoding a 210 kD immunogenic L. tropica antigen, referred to herein as Lt-210.
  • Lt-210 The preparation and characterization of Lt-210, and immunogenic portions thereof (such as Lt-1 and immunogenic repeat and non-repeat sequences), is described in detail in U.S. Patent Application Serial No. 08/511,872, filed August 4, 1995.
  • the sequence of a DNA molecule encoding Lt-1 is provided in SEQ ID NO: 7 and the encoded amino acid sequence is presented in SEQ ID NO:8.
  • LbeIF4A L. braziliensis antigen referred to herein as LbeIF4A.
  • a clone may be isolated by screening a L. braziliensis expression library with sera obtained from a patient afflicted with mucosal leishmaniasis, and analyzing the reactive antigens for the ability to stimulate proliferative responses and preferential Thl cytokine production in PBMC isolated from Leishmania-infected patients, as described below.
  • the preparation and characterization of LbeIF4A is described in detail in U.S. Patent Application Serial Nos. 08/454,036 and 08/488,386, which are continuations-in-part of U.S. Patent Application Serial No.
  • LbeIF4A The sequence of a DNA molecule encoding LbeIF4A is provided in SEQ ID NO: 9 and the encoded amino acid sequence is presented in SEQ ID NO: 10. Homologs of LbeIF4A, such as that found in L. major, may also be isolated using this approach, and are within the scope of the present invention.
  • compositions of the present invention may also, or alternatively, contain soluble Leishmania antigens.
  • soluble Leishmania antigens refers to a mixture of at least 8 different Leishmania antigens that may be isolated from the supernatant of Leishmania promastigotes of any species grown for 8-12 hours in protein-free medium. Briefly, the organisms are grown to late log phase in complex medium with serum until they reach a density of 2-3 x 10 7 viable organisms per mL of medium.
  • the organisms are thoroughly washed to remove medium components and resuspended at 2-3 x 10 7 viable organisms per mL of defined serum- free medium consisting of equal parts RPMI 1640 and medium 199, both from Gibco BRL, Gaithersburg, MD. After 8-12 hours, the supernatant containing soluble Leishmania antigens is removed, concentrated 10 fold and dialyzed against phosphate-buffered saline for 24 hours. The presence of at least eight different antigens within the mixture of Leishmania antigens may be confirmed using SDS-PAGE (i.e., through the observation of at least 8 different bands).
  • the immunogenic properties of the soluble Leishmania antigens may be confirmed by evaluating the ability of the preparation to elicit an immune response in cultures of lymph node cells and/or peripheral blood mononuclear cells (PBMC) isolated from presently or previously Leishmania-infected individuals. Such an evaluation may be performed as described below.
  • PBMC peripheral blood mononuclear cells
  • Lmspla Lmsp9a
  • MAPS- 1 A DNA sequences encoding Lmspla, Lmsp9a and MAPS-IA are provided in SEQ ID NO: 19, 21 and 23, respectively, with the corresponding predicted amino acid sequences being presented in SEQ ID NO: 20, 22 and 24, respectively.
  • mice or rabbits immunized with L. major culture supernatant may be used to screen an L. major genomic DNA library. As detailed below, this procedure may be used to isolate DNA molecules encoding the L. major antigens referred to herein as LmgSPl, LmgSP3, LmgSP5, LmgSP8, LmgSP9, LmgSP13, LmgSPl 9, and DNA molecules encoding the L. chagasi antigens LcgSPl, LcgSP3, LcgSP4, LcgSP8, and LcgSPlO.
  • the DNA sequences encoding these antigens are provided in SEQ ID NO:29-35 and 44-48, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 36-42 and 49-53.
  • the L. major antigens referred to herein as 1G6-34, 1E6-44, 4A5-63, 1B11-39, 2A10-37, 4G2-83, 4H6-41 and 8G3-100 may be isolated by means of CD4+ T cell expression cloning as described below.
  • DNA sequences encoding these antigens are provided in SEQ ID NO: 72-79, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 80-87.
  • the immunogenic properties of the isolated Leishmania antigens may be evaluated using, for example, the representative methods described herein.
  • the antigens described herein are immunogenic.
  • the antigens (and immunogenic portions thereof) are capable of eliciting an immune response in cultures of lymph node cells and/or peripheral blood mononuclear cells (PBMC) isolated from presently or previously Leishmania-infected individuals.
  • PBMC peripheral blood mononuclear cells
  • the antigens, and immunogenic portions thereof have the ability to induce T-cell proliferation and/or to elicit a dominantly Thl -type cytokine response (e.g., IL-2, IFN- ⁇ , and/or TNF- ⁇ production by T-cells and/or NK cells; and/or IL-12 production by monocytes, macrophages and/or B cells) in cells isolated from presently or previously Leishmania-infected individuals.
  • a Leishmania-infected individual may be afflicted with a form of leishmaniasis (such as subclinical, cutaneous, mucosal or active visceral) or may be asymptomatic. Such individuals may be identified using methods known to those of ordinary skill in the art.
  • PBMC PBMC
  • PBMC may be isolated by density centrifugation through, for example, FicollTM (Winthrop Laboratories, New York).
  • Lymph node cultures may generally be prepared by immunizing BALB/c mice (e.g., in the rear foot pad) with Leishmania promastigotes emulsified in complete Freund's adjuvant.
  • the draining lymph nodes may be excised following immunization and T-cells may be purified in an anti-mouse Ig column to remove the B cells, followed by a passage through a Sephadex G10 column to remove the macrophages.
  • lymph node cells may be isolated from a human following biopsy or surgical removal of a lymph node.
  • a polypeptide e.g., a Leishmania antigen or a portion or other variant thereof
  • the amount of polypeptide that is sufficient for the evaluation of about 2 x 10 5 cells ranges from about 10 ng to about 100 ⁇ g, and preferably is about 1-10 ⁇ g.
  • the incubation of polypeptide with cells is typically performed at 37°C for about 1-3 days. Following incubation with polypeptide, the cells are assayed for an appropriate response. If the response is a proliferative response, any of a variety of techniques well known to those of ordinary skill in the art may be employed.
  • the cells may be exposed to a pulse of radioactive thymidine and the incorporation of label into cellular DNA measured.
  • a polypeptide that results in at least a three fold increase in proliferation above background i.e., the proliferation observed for cells cultured without polypeptide is considered to be able to induce proliferation.
  • the response to be measured may be the secretion of one or more cytokines (such as interferon- ⁇ (IFN- ⁇ ), interleukin-4 (IL-4), interieukin- 12 (p70 and/or p40), interleukin-2 (IL-2) and/or tumor necrosis factor- ⁇ (TNF- ⁇ )) or the change in the level of mRNA encoding one or more specific cytokines.
  • cytokines such as interferon- ⁇ (IFN- ⁇ ), interleukin-4 (IL-4), interieukin- 12 (p70 and/or p40), interleukin-2 (IL-2) and/or tumor necrosis factor- ⁇ (TNF- ⁇ )
  • the secretion of interferon- ⁇ , interleukin-2, tumor necrosis factor- ⁇ and/or interleukin- 12 is indicative of a Thl response, which is responsible for the protective effect against Leishmania.
  • Assays for any of the above cytokines may generally be performed using methods known to those of ordinary skill in the art, such as an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Suitable antibodies for use in such assays may be obtained from a variety of sources such as Chemicon, Temucula, CA and PharMingen, San Diego, CA, and may generally be used according to the manufacturer's instructions.
  • the level of mRNA encoding one or more specific cytokines may be evaluated by, for example, amplification by polymerase chain reaction (PCR).
  • a polypeptide that is able to induce, in a preparation of about 1-3 x 10 5 cells, the production of 30 pg/mL of IL-12, IL-4, IFN- ⁇ , TNF- ⁇ or IL-12 p40, or 10 pg/mL of IL- 12 p70, is considered able to stimulate production of a cytokine.
  • Immunogenic portions of the antigens described herein may be prepared and identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native antigen for immunogenic properties using, for example, the representative techniques described herein.
  • An immunogenic portion of a polypeptide is a portion that, within such representative assays, generates an immune response (e.g., proliferation and/or cytokine production) that is substantially similar to that generated by the full length antigen.
  • an immunogenic portion of an antigen may generate at least about 25%), and preferably at least about 50%, of the response generated by the full length antigen in the model assays described herein.
  • Portions and other variants of immunogenic Leishmania antigens may be generated by synthetic or recombinant means.
  • Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids may be generated using techniques well known to those of ordinary skill in the art.
  • such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 55:2149-2146, 1963.
  • Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/ Applied BioSystemsDivision, Foster City, CA, and may be operated according to the manufacturer's instructions.
  • Recombinant polypeptides containing portions and/or variants of a native antigen may be readily prepared from a DNA sequence encoding the antigen.
  • supernatants from suitable host/vector systems which secrete recombinant protein into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant protein.
  • any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention.
  • Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide.
  • Suitable host cells include prokaryotes, yeast and higher eukaryotic cells.
  • the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.
  • the DNA sequences expressed in this manner may encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.
  • variants of a native antigen may generally be prepared using standard mutagenesis techniques, such as oligonucleotide- directed site-specific mutagenesis, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides.
  • the present invention provides epitope repeat sequences, or antigenic epitopes, of a Leishmania antigen, together with polypeptides comprising at least two such contiguous antigenic epitopes.
  • an "epitope" is a portion of an antigen that reacts with sera from Leishmania-infected individuals (i.e. an epitope is specifically bound by one or more antibodies present in such sera).
  • epitopes of the antigens described in the present application may be generally identified using techniques well known to those of skill in the art.
  • antigenic epitopes of the present invention comprise an amino acid sequence provided in SEQ ID NO:43, 56, 57 or 58.
  • antigenic epitopes provided herein may be employed in the diagnosis and treatment of Leishmania infection, either alone or in combination with other Leishmania antigens or antigenic epitopes.
  • Antigenic epitopes and polypeptides comprising such epitopes may be prepared by synthetic means, as described generally above and in detail in Example 15.
  • polypeptides, antigenic epitopes and/or soluble Leishmania antigens may be incorporated into pharmaceutical compositions or vaccines.
  • polypeptide will be used when describing specific embodiments of the inventive therapeutic compositions and diagnostic methods.
  • antigenic epitopes of the present invention may also be employed in such compositions and methods.
  • compositions comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier.
  • Vaccines comprise one or more of the above polypeptides and a non-specific immune response enhancer, such as an adjuvant (e.g., LbeIF4A, interieukin- 12 or other cytokines) or a liposome (into which the polypeptide is incorporated).
  • Vaccines may additionally contain a delivery vehicle, such as a biodegradable microsphere (disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109).
  • Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other Leishmania antigens, either incorporated into a combination polypeptide or present within one or more separate polypeptides.
  • a pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerri ⁇ ) that expresses an immunogenic portion of the polypeptide on its cell surface.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retro virus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus.
  • a viral expression system e.g., vaccinia or other pox virus, retro virus, or adenovirus
  • a non-pathogenic (defective), replication competent virus e.g., vaccinia or other pox virus, retro virus, or adenovirus
  • a non-pathogenic (defective), replication competent virus e.g., vaccinia or other pox virus, retro virus, or adenovirus
  • Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art.
  • the DNA may also be "naked,” as described, for example, in Ulmer et al., Science 259:1745-1749 (1993) and reviewed by Cohen, Science 259:1691-1692 (1993).
  • the type of carrier will vary depending on the mode of administration.
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactic galactide
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
  • adjuvants may be employed in the vaccines of this invention to nonspecifically enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a nonspecific stimulator of immune responses, such as lipid A, Bordella pertussis or Mycobacterium tuberculosis.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), alum, biodegradable microspheres, monophosphoryl lipid A and quil A.
  • compositions of the present invention include multiple polypeptides selected so as to provide enhanced protection against a variety of Leishmania species. Such polypeptides may be selected based on the species of origin of the native antigen or based on a high degree of conservation of amino acid sequence among different species of Leishmania.
  • a combination of individual polypeptides may be particularly effective as a prophylactic and/or therapeutic vaccine because (1) stimulation of proliferation and/or cytokine production by individual polypeptides may be additive, (2) stimulation of proliferation and/or cytokine production by individual polypeptides may be synergistic, (3) individual polypeptides may stimulate cytokine profiles in such a way as to be complementary to each other and/or (4) individual polypeptides may be complementary to one another when certain of them are expressed more abundantly on the individual species or strain of Leishmania responsible for infection.
  • a preferred combination contains polypeptides that comprise immunogenic portions of M15, Ldp23, Lbhsp83, Lt-1 and LbeIF4A. Alternatively, or in addition, the combination may include one or more polypeptides comprising immunogenic portions of other Leishmania antigens disclosed herein, and/or soluble Leishmania antigens.
  • compositions and vaccines may be used, for example, to induce protective immunity against Leishmania in a patient, such as a human or a dog, to prevent leishmaniasis.
  • Appropriate doses and methods of administration for this purposes are described in detail below.
  • the pharmaceutical compositions and vaccines described herein may also be used to stimulate an immune response, which may be cellular and/or humoral, in a patient.
  • the immune responses that may be generated include a preferential Thl immune response (i.e., a response characterized by the production of the cytokines interieukin- 1, interleukin-2, interieukin- 12 and/or interferon- ⁇ , as well as tumor necrosis factor- ⁇ ).
  • the immune response may be the production of interieukin- 12 and/or interleukin-2, or the stimulation of gamma delta T-cells. In either category of patient, the response stimulated may include IL-12 production.
  • Such responses may also be elicited in biological samples of PBMC or components thereof derived from Leishmania-infected or uninfected individuals.
  • assays for any of the above cytokines may generally be performed using methods known to those of ordinary skill in the art, such as an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Suitable pharmaceutical compositions and vaccines for use in this aspect of the present invention are those that contain at least one polypeptide comprising an immunogenic portion of a Leishmania antigen disclosed herein (or a variant thereof).
  • the polypeptides employed in the pharmaceutical compositions and vaccines are complementary, as described above. Soluble Leishmania antigens, with or without additional polypeptides, may also be employed.
  • the pharmaceutical compositions and vaccines described herein may also be used to treat a patient afflicted with a disease responsive to IL-12 stimulation.
  • the patient may be any warm-blooded animal, such as a human or a dog.
  • diseases include infections (which may be, for example, bacterial, viral or protozoan) or diseases such as cancer.
  • the disease is leishmaniasis, and the patient may display clinical symptoms or may be asymptomatic.
  • the responsiveness of a particular disease to IL-12 stimulation may be determined by evaluating the effect of treatment with a pharmaceutical composition or vaccine of the present invention on clinical correlates of immunity.
  • polypeptide administration may be as described below, or may extend for a longer period of time, depending on the indication.
  • polypeptides employed in the pharmaceutical compositions and vaccines are complementary, as described above.
  • a particularly preferred combination contains polypeptides that comprise immunogenic portions of M15, Ldp23, Lbhsp83, Lt-1 and LbeIF4A, Lmspla, Lmsp9a, and MAPS- IA. Soluble Leishmania antigens, with or without additional polypeptides, may also be employed.
  • compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 12 doses may be administered over a 1 year period.
  • 12 doses are preferably administered, at one month intervals.
  • 3 doses are preferably administered, at 3 month intervals. In either case, booster vaccinations may be given periodically thereafter.
  • a suitable dose is an amount of polypeptide or DNA that, when administered as described above, is capable of raising an immune response in an immunized patient sufficient to protect the patient from leishmaniasis for at least 1 -2 years.
  • the amount of polypeptide present in a dose ranges from about 100 ng to about lmg per kg of host, typically from about 10 ⁇ g to about 100 ⁇ g.
  • Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • this invention provides methods for using one or more of the polypeptides described above to diagnose Leishmania infection in a patient using a skin test.
  • a skin test is any assay performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as induration and accompanying redness) is measured following intradermal injection of one or more polypeptides as described above.
  • DTH delayed-type hypersensitivity
  • Such injection may be achieved using any suitable device sufficient to contact the polypeptide or polypeptides with dermal cells of the patient, such as a tuberculin syringe or 1 mL syringe.
  • the reaction is measured at least 48 hours after injection, more preferably 72 hours after injection.
  • the DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to a test antigen (i.e., an immunogenic portion of a polypeptide employed, or a variant thereof).
  • the response may measured visually, using a ruler.
  • induration that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of Leishmania infection, which may or may not be manifested as an active disease.
  • polypeptides of this invention are preferably formulated, for use in a skin test, as pharmaceutical compositions containing at least one polypeptide and a physiologically acceptable carrier, as described above.
  • Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 ⁇ g to
  • inventive polypeptides may also be employed in combination with one or more known Leishmania antigens in the diagnosis of leishmaniasis, using, for example, the skin test described above.
  • individual polypeptides are chosen in such a way as to be complementary to each other. Examples of known Leishmania antigens which may be usefully employed in conjunction with the inventive polypeptides include K39 (Burns et al., Proc. Natl. Acad. Sci. USA. 1993 90:775-779).
  • This Example illustrates the preparation of a Leishmania antigen Ml 5, having the sequence provided in SEQ ID NO:2.
  • An L. major (Friedlan strain) amastigote cDNA expression library prepared in the ⁇ ZAP II vector (Stratagene, La Jolla, CA) was screened according to manufacturer's instructions using sera obtained from L. major infected BALB/c mice (8 weeks post inoculation). Approximately 40,000 plaques were screened and four clones expressing reactive antigens were purified to homogeneity by two subsequent rounds of low density screening. Bluescript phagemid inserts were excised from positive clones for further analysis.
  • DNA sequence analyses were performed on an Applied Biosystems automated sequencer using Taq polymerase and dye coupled ddNTP terminators or dye- labeled sequencing primers.
  • the complete sequence of the 2685 bp insert was determined using a combination of primer-directed sequencing and by sequencing a series of overlapping ⁇ xonuclease III deletion subclones generated using the ⁇ rase-a- base system (Promega, Madison, WI).
  • the sequence of this insert is provided in S ⁇ Q ID NO:l
  • the deduced amino acid sequence is provided in S ⁇ Q ID NO:2.
  • the complete insert of clone pfl-1 was excised by digestion with
  • BamRl/Kpnl was subcloned in frame into BamHI/Kpnl digested pQ ⁇ 31 (QUIAGEN) to generate the construct pM151A.
  • E. coli containing this construct inducibly expressed high levels of the L. major antigen encoded by pfll-1 (designated as Ml 5) with the addition of a 6-histidine tag at the amino terminus.
  • Large volume cultures (500 ml) of E. coli host cells containing the pM151A construct were induced to express recombinant protein by the addition of 2mM IPTG at mid-log phase of growth. Growth was continued for 4 to 5 hours and bacteria were then pelleted and washed once with cold PBS.
  • lysis buffer 50 mM Na j HPO ⁇ pH 8.0, 300 mM NaCl, 10 mM -mercaptoethanol
  • lysis buffer 50 mM Na j HPO ⁇ pH 8.0, 300 mM NaCl, 10 mM -mercaptoethanol
  • Insoluble material was removed by centrifugation at 10,000xg for 10 minutes and although the recombinant protein was found to be evenly distributed between the soluble and insoluble fractions the insoluble material was discarded at this point.
  • Recombinant protein containing the amino terminal histidine tag was affinity purified using Ni-NTA resin (QIAGEN) according to the manufacturer's recommendations.
  • Ni-NTA resin resuspended in lysis buffer was added to the soluble lysate fraction and binding was conducted with constant mixing for 1 hour at 4°C. The mixture was then loaded into a gravity flow column and the non-binding material was allowed to flow through.
  • the Ni-NTA matrix was washed 3 times with 25 ml of wash buffer (50 mM pH 6.0, 300 mM NaCl, 10 mM ?-mercaptoethanol) and bound material was eluted in 25 ml of elution buffer (50 mM Na ⁇ PO ⁇ pH 5.0, 300 mM NaCl, lOmM ⁇ - mercaptoethanol).
  • the eluted material was then dialyzed against 3 changes of PBS, sterile filtered and stored at -20°C.
  • the purified recombinant protein was shown by SDS-PAGE analysis to be free of any significant amount of E. coli protein.
  • a small number of bands of lower molecular weight were assumed to be proteolytic products of the L. major antigen based on their reactivity by western blot analysis.
  • a high titre polyclonal antisera against Ml 5 was generated in rabbits by repeated subcutaneous injection of recombinant protein.
  • Western blot analysis of lysates from L. major promastigotes and amastigotes using this antisera indicated that the protein is constitutively expressed throughout the parasite lifecycle.
  • This Example illustrates the preparation of a Leishmania antigen Ldp23, having the sequence provided in SEQ ID NO:4.
  • lymph node T-cells were infected as indicated above and incubated at 26°C for 6 hours, and then as 37°C for either 24, 48 or 72 hours. At each of these time points the non- adherent cells and free parasites were washed out and the adherent cells were mechanically dislodged, washed and fixed with paraformaldehyde. These cells were then used as antigen presenting cells (APCs) for purified lymph node T-cells from BALB/c mice immunized with L. donovani promastigotes. To generate these anti- .
  • APCs antigen presenting cells
  • mice H-2 d mice of both sexes (The Jackson Laboratory, Bar Harbor, ME) were immunized at 8 to 14 weeks of age in the rear foot pad with 5-10 x 10 6 L. donovani promastigotes emulsified in complete Freund's adjuvant (CFA) (Difco Laboratories, Madison, MI) as described in Rodrigues et al., Parasite Immunol. 14:49 (1992).
  • CFA complete Freund's adjuvant
  • the draining lymph nodes were excised 8 days after the immunization and T-cells were purified in an anti-mouse Ig column to remove the B cells, as described in Bunn-Moreno and Campos-Neto, J. Immunol. 727:427 (1981), followed by a passage through a Sephadex G10 column to remove the macrophages.
  • Stimulation index was calculated by dividing the cpm obtained for the cells cultured in the presence of infected P388D1 macrophages by the cpm obtained for the cells cultured in the presence of non-infected macrophages, but subjected to the same conditions as the infected macrophages.
  • the results shown Figure 1 indicate that L. donovani-infected P388D1 macrophage process parasite antigens and that optimal presentation occurs after 48 hours of infection. No stimulation of the T-cells by the non-infected macrophages was observed.
  • P388D1 macrophages were infected with L. donovani promastigotes for an initial incubation of 6 hours at room temperature. The cultures were then transferred to 37°C for the remainder of the 48 hour incubation period. At a ratio of 3-5 parasites per macrophage nearly 90% of the macrophages were infected after 24 hours of incubation at 37°C.
  • the MHC class II molecules were then affinity-purified. Approximately 1.5 x 10 10 L. donovani-infected or an equal number of non-infected P388D1 macrophages were used for each purification. The cells were harvested, washed with PBS and incubated for 30 minutes in cold lysis buffer (PBS, 1% Nonidet P40, 25mM iodoacetamide, 0.04% sodium azide, ImM aprotinin and lmM PMSF).
  • PBS cold lysis buffer
  • H-2 d 5ml anti-MHC class II molecules
  • H-2 d Sepharose column
  • Culture supernatants of MK-D6 hybridoma cells (American Type Culture Collection, Rockville, MD) were employed as the source for anti-MHC class II (H-2 d ) monoclonal antibody.
  • the column was washed with 50ml of lysis buffer and then with 50ml of PBS containing 0.5% octyl glucopyranoside detergent.
  • Bound molecules were eluted from the column with 1M acetic acid in 0.2%> NaCl.
  • the MHC/peptide molecules were separated from the IgG (MK-D6 monoclonal antibody) using a Centricon 100 filter unit (Amicon Division, W.R. Grace & Co., Beverly, MA).
  • the peptides were then dissociated from the class II molecules by the addition of acetic acid to 2.5M, followed by separation using a Centricon 10 filter unit.
  • the resulting peptide preparation present in the low molecular weight sample, was then dried using a speed vac concentrator (Savant Instrument Inc., Farmingdale, NY).
  • peptides were redissolved in 200 ⁇ l of 0.05% TFA and separated by reverse-phase high performance liquid chromatography (RP-HPLC) using a 2.1mm x 25cm Vydac C-18 column at a flow rate of 0.15ml/min employing a 1 to 30% acetonitrile gradient (60 min) followed by a 30 to 60% gradient (30 min) and then a 60 to 80% gradient (90-110 min).
  • Non-infected P388D1 cells were similarly processed to serve as background control for endogenous MHC class II associated peptides.
  • Figure 2 shows a representative experiment; four distinct peaks which are present only in the material isolated from infected macrophages (panel B), and not in the material isolated from uninfected macrophages (panel A) are indicated.
  • sequences could not be obtained for some peaks because the peptides were blocked. Notwithstanding, three peptides sequences were determined. Amino-acid sequences were searched for identity with proteins in the GenBank database using the GENPETP, PIR and SWISSPROT programs. The sequence data base analysis revealed that one of the peptides was highly homologous to glyceraldehyde-3 -phosphate dehydrogenase of various species. Another peptide had homology with elongation factor of several species, including Leishmania. The third sequence was not clearly related to any known proteins, and is shown below: XQXPQ(L/K)VFDEXX (SEQ ID NO:l 1).
  • the peptide sequence of uncertain origin was chosen to guide the strategy for cloning the corresponding parasite gene.
  • a DNA fragment was initially amplified from L. donovani promastigote cDNA by PCR.
  • the sense primer was a peptide derived oligonucleotide (5' > GGAATTCCCCInCAGCTInGTInTTCGAC ⁇ 3') (SEQ ID NO: 12) containing an EcoRI restriction endonuclease site (underlined).
  • the bases were selected following the preferential codon usage of L. donovani, as described in Langford et al., Exp.
  • the antisense primer was a poly-thymidine oligonucleotide (oligo dT, downstream primer) containing a Xhol restriction endonuclease site.
  • the gene fragment was amplified from a L. donovani promastigote cDNA preparation using the following reaction conditions: one cycle of 3 min at 94°C immediately followed by 35 cycles of 1 min at 94°C, 1 min at 45°C and 1 min at 72°C.
  • the L. donovani cDNA was prepared from 5 x 10 7 washed promastigote forms harvested at the log growth phase (3 days culture).
  • the cDNA was obtained using an Invitrogen cDNA cycleTM kit (Invitrogen Co., San Diego, CA). Oligonucleotide primers were synthesized by the DNA Synthesis Laboratory, Department of Pathology, Yale University School of Medicine. The PCR products were analyzed by gel electrophoresis. Only one band of approximately 300 bp was obtained. This fragment was cloned and its sequence confirmed the sequence of the peptide-based primer including the glutamic acid codon, deliberately not included in the primer sequence.
  • the PCR amplified gene fragment was ligated into the pCRTM vector using the TA cloning system (Invitrogen Co., San Diego, CA). Transformants were selected in LB medium containing lOO ⁇ g/ml ampicillin and the plasmid DNA was isolated using the WizardTM Minipreps DNA purification kit (Promega Co., Madison, WI). Insert DNA was released with the restriction enzymes EcoRI and Xhol (New England Biolabs, Beverly, MA), purified from an agarose gel electrophoresis and labeled with 32 P using a random priming method (Megaprime Labeling Kit, Amersham Life Science, Buckinghamshire, England).
  • This DNA fragment was used as probe to screen a L. donovani promastigote cDNA library as described in Skeiky et al., Infect. Immun. 62:1643 (1994).
  • An approximately 650 bp cDNA (Ldp23) was excised from the phagemid by in vivo excision using the Stratagene protocol.
  • DNA sequencing was performed using the Sequenase version 2 system (DNA sequencing kit) in the presence or absence of 7- deaza-GTP (United States Biochemical, Cleveland, OH). The sequence is provided as SEQ ID NO:3, and shows complete homology with the original 300 bp PCR fragment.
  • a 525 bp open reading frame containing an ATG codon that follows the last 4 bases of the spliced leader sequence and 3 stop codons adjacent to the poly A tail was identified.
  • This frame also codes the carboxyl terminal sequence (KVFDE) (SEQ ID NO: 13) of the purified MHC class II associated peptide.
  • KVFDE carboxyl terminal sequence
  • the sequence analysis of the deduced protein sequence revealed one potential glycosylation site (Asn-Cys-Ser) at positions 68-70.
  • PCR was used to subclone the cloned gene in frame into the expression vector pGEX 2T.
  • Primers containing the appropriate restriction site enzymes, initiation and termination codons were: 5' > GQATC£ATGGTCAAGTCCCACTACATCTGC ⁇ 3' (SEQ ID NO: 14) for the upstream primer and 5' > G ATT£AGACCGGATAGAAATAAGCCAATGAAA ⁇ 3' (SEQ ID NO: 15) for the downstream primer (restriction sites of BamHl and EcoRI are underlined respectively).
  • PCR conditions were as indicated above for the amplification of the original peptide related DNA fragment.
  • the template used was pBluescript plasmid containing the cloned gene from the cDNA library.
  • Overexpression of the recombinant fusion protein was accomplished by growing the transformed E. coli (DH5 ⁇ ) and inducing the tac promoter with lmM isopropyl- ⁇ -thiogalactopyranoside (IPTG) (Stratagene, La Jolla, CA). Cells were collected, centrifuged, and analyzed for the presence of the fusion protein by SDS- PAGE. A glutathione-S-transferase fusion protein of 43-44 kD was produced, indicating a leishmanial protein of approximately 18 kD, as glutathione-S-transferase (GST) has a MW of 26 kD.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the fusion protein was very insoluble and therefore could not be purified by affinity chromatography using a glutathione column.
  • the use of low concentrations of detergents like SDS, sarcosyl, deoxycolate, and octyl- glucopyranoside during the extraction steps was efficient to solubilize the protein but unfortunately prevented its binding to the glutathione column.
  • Other maneuvers such as the growth of the E. coli and incubation and induction of the tac promoter with IPTG at 33°C, did not improve the protein solubility.
  • the purification was achieved by preparative SDS-PAG ⁇ . The band was visualized with 0.1M KC1, cut and electroeluted from the gel followed by extensive dialysis against PBS and concentration on Centricon 10 filters.
  • the band corresponding to the overexpressed recombinant fusion protein was identified by KC1, cut out, electroeluted from the gel strip, dialyzed against PBS and submitted to analytical SDS-PAGE (12%). Numbers on the left side indicate the molecular weights of the markers. Attempts to further purify the leishmanial protein by cleaving it out from the fusion protein GST with thrombin were unsuccessful.
  • RNA prepared from different promastigote growth phases (logarithmic and stationary) and from the amastigote form of these parasites.
  • the RNA was prepared from 2 x 10 7 parasite cells using the Micro RNA isolation kit (Stratagene, La Jolla, CA) according to the company's recommended instructions.
  • RNA was prepared from L. donovani promastigotes (logarithmic growth phase); from L. major promastigotes (logarithmic and stationary growth phases); from L.
  • L. donovani IS strain
  • L. amazonensis MHOM/BR/77/LTB0016
  • L. major MHOM/IR/79/LRC-L251
  • L. pifanoi MHOM/VE/60/Ltrod promastigotes were grown and maintained at 26°C in Schneider's medium containing 20%) FCS and 50 ⁇ g/ml gentamicin.
  • L. pifanoi amastigotes were obtained from axenic culture as previously reported by Pan et al, J. Euk. Microbiol. 40:213 (1993).
  • the hybridization was carried out at 45°C in the presence of 50%> formamide, 5x Denhardt's solution, 0.1 %> SDS, lOO ⁇ g/ml single stranded salmon sperm DNA and 5x SSPE using 0.45 ⁇ m Nytran membrane filters (Schleicher & Schuell, Keene, NH).
  • the probe was the 32 P labeled Ldp23 gene.
  • Figure 4 shows that one single RNA band of 680 bp was observed for all growth phases and forms of all tested Leishmania.
  • the numbers 1, 2 and 3 refer to RNA obtained from promastigotes at the logarithmic growth phase, promastigotes at the stationary growth phase and amastigote forms, respectively, and the numbers on the left side indicate the molecular weights of the markers in base pairs. This result is consistent with the corresponding gene size (525 bp) and with the molecular weight of the expressed protein and points to the ubiquitous distribution and expression of this gene within the genus Leishmania.
  • mice and rabbits were immunized with the GST-fusion protein in CFA.
  • BALB/c mice were immunized in the rear foot pad with 5-10 ⁇ g of protein emulsified in CFA. Protein concentration was determined using the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Richmond, CA). The mice were boosted 7 days later with 5-10 ⁇ g of protein emulsified in incomplete Freund's adjuvant (IF A) inoculated into the peritoneal cavity.
  • IF A incomplete Freund's adjuvant
  • mice were bled 7 days after the second immunization.
  • New Zealand white rabbits (Millbrook Farm, Amherst, MA) were immunized according to the following protocol: one intra- muscular (IM) injection of 25-30 ⁇ g of purified recombinant protein emulsified in CFA into each thigh on day one; one IM injection of 25-30 ⁇ g of purified protein emulsified in IFA into each shoulder on day 7; on day 15, 25-30 ⁇ g of the purified protein in PBS was injected into the subcutaneous tissue.
  • the rabbit was bled 7 days after the last immunization.
  • Sera were prepared and the anti-Leishmania antibody response was measured by Western blot analysis and by FACScan. In both cases L.
  • donovani promastigotes were used as antigen. Approximately 2 x 10 6 L. donovani promastigotes were grown in Schneider's medium for 3 days (log phase), were washed with PBS, lysed with SDS-PAGE loading buffer and submitted to electrophoresis under reducing conditions using a 15%> polyacrylamide gel. The proteins were transferred onto 0.45 ⁇ Immobilon-P transfer membrane (Millipore Co., Bedford, MA) using a wet-type electroblotter (Mini Trans-Blot Electrophoretic Transfer Cell, Bio Rad Life Science Division, Richmond, CA) for 2 hours at 50 V.
  • the membranes were blocked overnight at room temperature with PBS containing 3%> normal goat serum (NGS), 0.2% Tween- 20 and 0.05%> sodium azide, followed by 3 washes with PBS. The blots were then incubated for 3-4 hours at 4°C with a 1/200 dilution of pre-immune rabbit serum (lane A, Figure 5) or with the same dilution of anti-fusion protein rabbit antiserum (lane B, Figure 5).
  • NBS normal goat serum
  • Tween- 20 0.2% Tween- 20 and 0.05%> sodium azide
  • the sera was previously absorbed 2x with non-viable desiccated Mycobacterium tuberculosis H-37 RA (Difco Laboratories, Detroit, MI) and were diluted in PBS containing 1%> NGS and 5% powdered non-fat bovine milk (Carnation, Nestle Food Company, Glendale, CA).
  • PBS containing 1%> NGS and 5% powdered non-fat bovine milk
  • the membranes were then washed with PBS, incubated for 1 hour at room temperature with goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (Promega, Madison, WI), washed once with PBS and 2x with veronal buffer pH 9.4.
  • reaction was visualized using the substrate mixture 5- bromo-4-chloro-3-indoyl-phosphate and nitroblue tetrazolium (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) according to the manufacturer's instructions.
  • Figure 5 shows that the rabbit anti-recombinant protein antiserum detects a single protein of 23 kDa (Ldp23) in the Leishmania crude extract antigen preparation. No bands were observed when an anti-GST antiserum was used (not shown).
  • the FACScan analysis ( Figure 6) shows that the antibody induced by the recombinant Ldp23 reacts with intact live L. donovani promastigotes, thus pointing to a cell surface expression of this molecule on these organisms.
  • the dotted line in Figure 6 shows the indirect immunofluorescence performed using pre-immune mouse serum and the solid line in Figure 6 shows the result obtained with mouse anti-GST-Ldp23 antiserum. Both sera were diluted at 1/100. Parasites were washed with staining buffer and incubated with FITC conjugated goat anti-mouse immunoglobulin antibody. Fluorescence intensity was analyzed by FACScan.
  • lymph node T-cells (lOVwell) from BALB/c mice immunized with L. donovani promastigotes (as described above) were stimulated to proliferate with 2 x 10 5 Mitomycin C-treated normal mononuclear spleen cells (APC) and pulsed with the purified recombinant fusion protein. Proliferation of T-cells was measured at 72 hours of culture. Values are expressed in Figure 7 as cpm and represent the mean of [ 3 H]TdR incorporation of triplicate cultures. Background cpm of cells (T cells + APC) cultured in the presence of medium alone was 1291.
  • Figure 7 shows that Leishmania specific T-cells proliferate well and in a dose response manner to recombinant Ldp23. No response was observed when purified GST was added instead of the recombinant fusion protein nor when lymph node T-cells from mice immunized with CFA alone were stimulated to proliferate in the presence of the Leishmanial fusion protein (not shown).
  • Ldp23 The recognition of the recombinant Ldp23 protein by Leishmania- specific T-cells was also tested using two murine models of leishmaniasis, the L. major highly susceptible BALB/c mice and the L. amazonensis susceptible CBA/J mice as described in Champsi and McMahon-Pratt, Infect. Immun. 56:3212 (1988). These models were selected to investigate the cytokine pattern induced by Ldp23. In the mouse model of leishmaniasis, resistance is associated with Th 1 cytokines while susceptibility is linked to Th 2 responses.
  • Lymph node cells were obtained 3 weeks after the initiation of infection of BALB/c mice with L. major and the ability of these cells to recognize the recombinant Ldp23 was measured by proliferation and by the production of the cytokines IFN- ⁇ and IL-4.
  • 2 x 10 6 cells obtained from the draining popliteal lymph node of infected mice were cultured for 72 hours in the presence of recombinant Ldp23 or Leishmania lysate.
  • the levels of IFN- ⁇ and IL-4 in culture supernatants were measured by ELISA as previously described (Chatelain et al, J. Immunol. 148:1172 (1992), Curry et al, J. Immunol. Meth. 104:131 (1987), and Mossman and Fong, J. Immunol. Meth. 116: 151 (1989)) using specific anti IFN- ⁇ and IL-4 monoclonal antibodies (PharMingen, San Diego, CA).
  • Ldp23 did stimulate these cells to proliferate (not shown) and induced a typical Th 1 type of cytokine response as indicated by the production of high levels of IFN- ⁇ (panel A of Figure 8) and no IL-4 (panel B of Figure 8). Stimulation of these cells with a Leishmania crude lysate yielded a mixed Th cytokine profile. Exactly the same pattern of cytokine production was obtained from the CBA/J mice infected with L. amazonensis (not shown). These results clearly indicate that Ldp23 is a powerful and selective activator of the Th 1 cytokines by mouse cells. EXAMPLE 3 PREPARATION OF HSP83
  • This Example illustrates the preparation of a Leishmania antigen Hsp83, having the sequence provided in SEQ ID NO:6.
  • a genomic expression library was constructed with sheared DNA from L. braziliensis (MHOM/BR/75/M2903) in bacteriophage ⁇ ZAP II (Stratagene, La Jolla, CA). The expression library was screened with Escherichia coli preadsorbed serum from an L. braziliensis-infected individual with ML. Immunoreactive plaques were purified, and the pBSK(-) phagemid was excised by protocols suggested by the manufacturer. Nested deletions were performed with exonuclease III to generate overlapping deletions for single-stranded template preparations and sequencing.
  • Single- stranded templates were isolated following infection with VCSM13 helper phage as recommended by the manufacturer (Stratagene, La Jolla, CA) and sequenced by the dideoxy chain terminator method or by the Taq dye terminator system using the Applied Biosystems automated sequencer model 373 A.
  • Recombinant antigens produced by these clones were purified from 500 ml of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG)-induced cultures as described in Skeiky et al., J. Exp. Med. 176:201-211 (1992). These antigens were then assayed for the ability to stimulate PBMC from Leishmania-infected individuals to proliferate and secrete cytokine. Peripheral blood was obtained from individuals living in an area (Corte de Pedra, Bahia, Brazil) where L.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • braziliensis is endemic and where epidemiological, clinical, and immunological studies have been performed for over a decade, and PBMC were isolated from whole blood by density centrifugation through Ficoll (Winthrop Laboratories, New York, N.Y.).
  • PBMC peripheral blood mononuclear cells
  • 2 X 10 5 to 4 X 10 5 cells per well were cultured in complete medium (RPMI 1640 supplemented with gentamicin, 2-mercaptoethanol, L-glutamine, and 10% screened pooled A+ human serum; Trimar, Hollywood, Calif.) in 96-well flat-bottom plates with or without 10 ⁇ g of the indicated antigens per ml or 5 ⁇ g of phytohemagglutinin per ml (Sigma Immunochemicals, St.
  • cytokine mRNA PCR analysis total RNA was isolated from PBMC and cDNA was synthesized by using poly(dT) (Pharmacia, Piscataway, NJ) and avian mycloblastosis virus reverse transcriptase.
  • diluted cDNA was amplified by PCR using Taq polymerase (Perkin-Elmer Cetus, Foster City, CA) with 0.2 ⁇ M concentrations of the respective 5' and 3' external primers in a reaction volume of 50 ⁇ l.
  • Taq polymerase Perkin-Elmer Cetus, Foster City, CA
  • the nucleotide sequences of the primary pairs and the PCR conditions used were as described in Skeiky et al., J. Exp. Med. 181:1521-1531 (1995). We verified that our PCR conditions were within the semiquantitative range by initially performing serial dilutions of the cDNAs and varying the number of cycles used for PCR.
  • Plasmids containing the human sequences for IL-2, IFN- ⁇ , IL-4, IL-10, and ⁇ -actin were digested, and the DNA inserts were purified after separation on 1% agarose gels. Radiolabeled 32 P probes were prepared by the random priming method. PCR products were analyzed by electrophoresis on 1.5% agarose gels, transferred to nylon membranes, and probed with the appropriate 32 P-labeled DNA insert.
  • Lbhsp83 a Leishmania braziliensis homolog of the eukaryotic 83 kD heat shock protein.
  • the sequence of the clone is provided in SEQ ID NO:5 and the deduced protein sequence is provided in SEQ ID NO:6.
  • this clone designated Lbhsp83a, appears to lack the first 47 residues of the full length 703 amino acid residues.
  • Lbhsp83 has an overall homology of 94% (91% identity and 3%> conservative substitution), 91%o (84% identity and 7% conservative substitution) and 77% (61%> identity and 16% conservative substitution) with L. amazonensis hsp83, T. cruzi hsp83 and human hsp89, respectively.
  • a second clone (designated Lbhsp83b), which contained the 43 kD C-terminal portion of hsp83 (residues 331 to 703) was also isolated.
  • Figure 19 presents a comparison of the Lbhsp83 sequence with L. amazonensis hsp83(Lahsp83), T. cruzi hsp83 (Tchsp83) and human hsp89 (Huhsp89).
  • cytokine patterns of PBMC from ML patients was performed by reverse transcriptase PCR. Cytokine mRNAs were evaluated in cells prior to culturing ( Figure 9, lanes O) or following culturing in the absence (lanes -) or presence of the indicated antigen for 48 and 72 h.
  • Figure 4A shows the results for five of the six ML patients whose PBMC were analyzed. In about half of the ML patients, noncultured (resting) PBMC had detectable levels of mRNA for IFN- ⁇ , IL-2, and IL-4 but not IL-10.
  • CL patient PBMC had IL-10 mRNA in the resting state in addition to mRNAs for the other cytokines tested (Figure 4B).
  • Figure 4B Following in vitro culture without antigen, the levels of mRNA for IFN- ⁇ , IL-2, and IL- 4 in resting cells from ML patients decreased to background levels while IL-10 mRNA levels increased.
  • PBMC of most CL patients had stable or increased IL-10 mRNA, while the mRNAs for IL-2, IFN- ⁇ , and IL-4 were reduced to barely detectable levels in the absence of antigen stimulation.
  • Lbhsp83 polypeptides elicited the production of mRNA for IFN- ⁇ and IL-2 from all ML patient PBMC tested.
  • profiles of mRNA for IL-10 and IL-4 differed for the two hsp83 polypeptides.
  • Lbhsp83a stimulated the production of IL-10 but not IL-4 mRNA (patients I, II, III, and IV), while Lbhsp83b stimulated the production of IL-4 but not IL-10 mRNA in all six patients.
  • PBMC supernatants were also assayed for the presence of secreted IFN- ⁇ , TNF- ⁇ , IL-4, and IL-10.
  • Cells from all ML and self-healing CL patients (seven and six patients, respectively) and from four of seven CL patients were analyzed for secreted IFN- ⁇ following stimulation with both rLbhsp83 polypeptides, parasite lysate and Lbhsp70, an L. braziliensis protein homologous to the eukaryotic 70 kD heat shock protein (Figure 10A).
  • rLbhsp83a stimulated patient PBMC to secrete higher levels of IFN- ⁇ than did rLbhsp83b (0.2 to 36 and 0.13 to 28 ng/ml, respectively).
  • the presence of secreted IFN- ⁇ correlated well with the corresponding mRNA detected by PCR
  • PBMC from four of five ML patients had supernatant TNF- ⁇ levels (0.8 to 2.2 ng/ml) higher than those detected in cultures of PBMC from uninfected controls following stimulation with parasite lysate ( Figure 10B).
  • the same PBMC were stimulated by rLbhsp83 to produce levels of TNF- ⁇ in supernatant ranging from 0.61 to 2.9 ng/ml.
  • PBMC from three (I, V, and VI), five (I, II, IV, V, and VI), and two (II and V) of six individuals analyzed produced higher levels of TNF- ⁇ in response to parasite lysate, rLbhsp83a, and rLbhsp83b, respectively.
  • the levels of TNF- ⁇ produced by PBMC from CL patients in response to parasite lysate were comparable to those produced by uninfected controls.
  • rLbhsp83 stimulated TNF- ⁇ production in the PBMC of two of these patients.
  • rLbhsp83a stimulated higher levels of TNF- ⁇ production than did rLbhsp83b.
  • PBMC from ML patients only PBMC from ML patients (five of six) produced detectable levels of supernatant TNF- ⁇ (60 to 190 pg/ml).
  • IL-10 was detected by ELISA in the antigen-stimulated PMBC culture supernatants from ML and CL patients.
  • the levels (49 to 190 pg) were significantly higher (up to 10-fold) following stimulation with rLbhsp83a compared with those after parallel stimulation of the same cells with rLbhsp83b ( Figure 11).
  • Parasite lysate also stimulated PMBC from some of the patients to produce IL-10.
  • rLbhsp83 stimulated PMBC from uninfected individuals to produce IL-10, with one exception, the levels were lower than those observed with patient PMBC.
  • IL-4 was not detected in any of the supernatants analyzed.
  • the level of any secreted IL-4 is below the detection limit of the ELISA employed (50 pg/ml).
  • This Example illustrates the preparation of clones encoding portions of the Leishmania antigen Lt-210, and which has the sequence provided in SEQ ID NO:8.
  • An expression library was constructed from L. tropica
  • DNA was sheared by passage through a 30-gauge needle to a size range of 2-6 kilobase, and was repaired by incubation with DNA poll in the presence of 100 ⁇ M each dATP, dCTP, dGTP, and dTTP.
  • EcoRI adapters were ligated to the DNA fragments. After removal of unligated adapters by passage over a G-25 SephadexTM column, the fragments were inserted in EcoRI cut Lambda ZapII (Stratagene, La Jolla, CA).
  • VTL viscerotropic leishmaniasis
  • Lambda phage expressing reactive proteins were detected after antibody binding by protein A-horseradish peroxidase and ABTS substrate.
  • Three clones, Lt-1, Lt-2, and Lt-3, containing a portion of the Lt-210 gene were identified and purified. The clones ranged in size from 1.4 to 3.3 kb and encoded polypeptides of 75 kD, 70 kD, and 120 kD, respectively. These three clones contain partial sequences of the Lt-210 gene.
  • Lt-1 and Lt-2 are overlapping clones and were chosen for further study. The DNA sequences of Lt-1 and Lt-2 were determined.
  • ⁇ xonuclease III digestion was used to create overlapping deletions of the clones (Heinikoff, Gene 25:351-359, 1984).
  • Single strand template was prepared and the sequence determined with Applied Biosystems Automated Sequencer model 373A or by Sanger dideoxy sequencing. The sequence on both strands of the coding portion of Lt-1 clone was determined. The partial sequence of one strand of Lt-2 clone was determined.
  • S ⁇ Q ID NO:7 presents the DNA sequence of Lt-1
  • S ⁇ Q ID NO:8 provides the predicted amino acid sequence of the open reading frame.
  • the DNA sequence of the coding portion of the Lt-1 clone includes a repeated nucleotide sequence at the 5' portion of the clone containing eight copies of a 99 bp repeat, three copies of a 60 bp repeat unit, which is part of the larger 99 bp repeat, and 800 bp of non-repeat sequence.
  • the deduced amino acid sequence of the 99 bp repeat contains limited degeneracies.
  • the mass of the predicted recombinant protein is 67,060 Daltons.
  • a database search of PIR with the predicted amino acid sequence of the open reading frame yielded no significant homology to previously submitted sequences.
  • Predicted secondary structure of the repeat portion of the clone is entirely ⁇ -helical.
  • Lt-2 Sequence analysis of Lt-2 revealed that the 3' portion of the clone consisted of a mixture of 60 and 99 bp repeats that were identical, excepting occasional degeneracies, to the 60 and 99 bp repeats observed in Lt-1. Collectively, the sequencing data suggest that Lt-1 and Lt-2 are different portions of the same gene, Lt-2 being upstream of Lt-1, with possibly a small overlap.
  • Hybridization analysis confirmed that rLt-2 and rLt-1 contain overlapping sequences. Genomic DNAs of various Leishmania species were restricted with a variety of enzymes, separated by agarose gel electrophoresis, and blotted on Nytran membrane filter (Schleicher & Schuell, Keene, NH). Inserts from rLt-1 and rLt- 2 were labeled with 32 P-CTP by reverse transcriptase from random oligonucleotide primers and used as probes after separation from unincorporated nucleotides on a Sephadex G-50 column.
  • Hybridizations using the rLt-1 or the rLt-2 probe are performed in 0.2M Na ⁇ PO ⁇ . ⁇ M NaCl at 65°C, whereas hybridization using the rLt- lr probe is performed in 0.2 M NaH 2 PO 4 /3.6 M NaCl 0.2 M EDTA at 60°C overnight. Filters are washed in 0.075 M NaCl/0.0075 M sodium citrate pH 7.0 (0.15 M NaCl/0.0150 M sodium citrate for the Lt-lr probe), plus 0.5% SDS at the same temperature as hybridization.
  • Genomic DNA from a number of Leishmania species including L. tropica were analyzed by Southern blots as described above using the Lt-1, Lt-2, and Lt-lr inserts separately as probes. Collectively, various digests of L. tropica DNA indicate that this gene has a low copy number. A similar, overlapping pattern was observed using either the Lt-1 or Lt-2 insert as a probe, consistent with the premise that these two clones contain sequences near or overlapping one another. In addition, sequences hybridizing with these clones are present in other Leishmania species. L. tropica isolates have limited heterogeneity. Southern analyses of digested genomic DNA from four L. tropica parasite strains isolated from VTL patients and three L.
  • Lt-1 and Lt-2 The recombinant proteins of Lt-1 and Lt-2 were expressed and purified.
  • the nested deletion set of Lt-1 formed for sequencing included a clone referred to as Lt-lr, which contains one and one-third repeats. This polypeptide was also expressed and purified.
  • In vivo excision of the pBluescript SK " phagemid from Lambda Zap II was performed according to the manufacturer's protocol. Phagemid virus particles were used to infect E. coli XL-1 Blue. Production of protein was induced by the addition of IPTG.
  • Protein was recovered by first lysing pellets of induced bacteria in buffer (LB, 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, lO mM ⁇ DTA) using a combination of lysozyme (750 ⁇ g/mL) and sonication.
  • LB buffer
  • rLt-1, rLt-2, and rLt-lr were recovered from the inclusion bodies after solubilization in 8M urea (rLt-1 and rLt-2) or 4M urea (rLt-lr).
  • Proteins rLt-1 and rLt-2 were enriched and separated by precipitation with 25%-40%> ammonium sulfate and rLt-lr was enriched by precipitation with 10%>-25%> ammonium sulfate.
  • the proteins were further purified by preparative gel electrophoresis in 10%> SDS-PAG ⁇ . Recombinant proteins were eluted from the gels and dialyzed in phosphate-buffered saline (PBS). Concentration was measured by the Pierce (Rockford, IL) BCA assay, and purity assessed by Coomassie blue staining after SDS- PAGE.
  • PBS phosphate-buffered saline
  • This example illustrates the molecular cloning of a DNA sequence encoding the L. braziliensis ribosomal antigen LbeIF4A.
  • a genomic expression library was constructed with sheared DNA from L. braziliensis (MHOM/BR/75/M2903) in bacteriophage ⁇ ZAPII (Stratagene, La Jolla, CA).
  • the expression library was screened with E. co/t-preadsorbed patient sera from an L. braziliensis-infected individual with mucosal leishmaniasis. Plaques containing immunoreactive recombinant antigens were purified, and the pBSK(-) phagemid excised using the manufacturer's protocols. Nested deletions were performed with Exonuclease III to generate overlapping deletions for single stranded template preparations and sequencing.
  • Single stranded templates were isolated following infection with VCSM13 helper phage as recommended by the manufacturer (Stratagene, La Jolla, CA) and sequenced by the dideoxy chain terminator method or by the Taq dye terminator system using the Applied Biosystems Automated Sequencer Model 373A.
  • the immunoreactive recombinant antigens were then analyzed in patient T-cell assays for their ability to stimulate a proliferative and cytokine production, as described in Examples 7 and 8 below.
  • a recombinant clone was identified in the above assays which, following sequence comparison of its predicted amino acid sequence with sequences of other proteins, was identified as a Leishmania braziliensis homolog of the eukaryotic initiation factor 4A (eIF4A).
  • the isolated clone (pLeIF.1) lacked the first 48 amino acid residues (144 nucleotides) of the full length protein sequence.
  • the pLeIF.1 insert was subsequently used to isolate the full length genomic sequence.
  • SEQ ID NO: 9 shows the entire nucleotide sequence of the full-length LbeIF4A polypeptide.
  • the open reading frame (nucleotides 115 to 1323) encodes a 403 amino acid protein with a predicted molecular weight of 45.3 kD.
  • a comparison of the predicted protein sequence of LbeIF4A with the homologous proteins from tobacco (TeIF4A), mouse (MeIF4A), and yeast (YeIF4A) shows extensive sequence homology, with the first 20-30 amino acids being the most variable.
  • LbeIF4A shows an overall homology of 75.5%> (57%> identity, 18.5% conservative substitution) with TeIF4A, 68.6% (50% identity, 18.6% conservative substitution) with MeIF4A and 67.2% (47.6% identity, 19.6% conservative substitution) with YeIF4A.
  • This Example illustrates the preparation of soluble Leishmania antigens from an L. major culture supernatant.
  • L. major promastigotes were grown to late log phase in complex medium with serum until they reached a density of 2-3 x 10 7 viable organisms per mL of medium. The organisms were thoroughly washed to remove medium components and resuspended at 2-3 x 10 7 viable organisms per mL of defined serum-free medium consisting of equal parts RPMI 1640 and medium 199, both from Gibco BRL, Gaithersburg, MD. After 8-12 hours, the supernatant was removed, concentrated 10 fold and dialyzed against phosphate-buffered saline for 24 hours. Protein concentration was then determined and the presence of at least eight different antigens confirmed by SDS-PAGE. This mixture is referred to herein as "soluble Leishmania antigens.”
  • Example 2 illustrates the immunogenic properties of the antigens prepared according to Examples 1, 2, 5 and 6, as determined by their ability to stimulate IL-4 and IFN- ⁇ in lymph node cultures from infected mice and in human PBMC preparations.
  • Lymph node cultures for use in these studies were prepared from L. major-infected BALB/c mice 10 days after infection, as described in Example 2.
  • PBMC peripheral blood obtained from individuals with cured L. donovani infections who were immunologically responsive to Leishmania. Diagnosis of the patients was made by clinical findings associated with at least one of the following: isolation of parasite from lesions, a positive skin test with Leishmania lysate or a positive serological test.
  • IFN- ⁇ was quantitated by a double sandwich ELISA using mouse anti-human IFN- ⁇ mAb (Chemicon, Temucula, CA) and polyclonal rabbit anti-human IFN- ⁇ serum. Human rIFN- ⁇ (Genentech Inc., San Francisco, CA) was used to generate a standard curve.
  • IL-4 was quantitated in supernatants by a double sandwich ELISA using a mouse anti-human IL-4 mAb (Ml) and a polyclonal rabbit anti-human IL-4 sera (P3).
  • Human IL-4 (Immunex Corp., Seattle, WA) was used to generate a standard curve ranging from 50 pg/ml to 1 ng/ml.
  • Figures 13A and 13B illustrate the mean level of secreted IL-4 and IFN- ⁇ , respectively, 72 hours after addition of 10 ⁇ g/mL of each of the following antigens to a lymph node culture prepared as described above: soluble Leishmania antigen (i.e., an extract prepared from ruptured promastigotes which contains membrane and internal antigens (SLA)), Ldp23, LbeIF4A (LeIF), Lbhsp83, Ml 5 and LmelF (the L. major homolog of LbeIF4A).
  • SLA membrane and internal antigens
  • Ldp23 LbeIF4A
  • LeIF LbeIF4A
  • Ml 5 and LmelF the L. major homolog of LbeIF4A
  • Figure 14 shows the level of secreted IFN- ⁇ in culture filtrate from infected and uninfected human PBMC preparations 72 hours after addition of 10 ⁇ g/mL L. major lysate, Ml 5 or L-Rack, an immunodominant leishmanial antigen in murine leishmaniasis.
  • Figure 15 illustrates the level of secreted IFN- ⁇ in culture filtrate from infected and uninfected human PBMC preparations 72 hours after addition of lO ⁇ g/mL L. major lysate, soluble Leishmania antigens (prepared as described in Example 6) or L-Rack.
  • Ml 5 and soluble Leishmania antigens are potent stimulators of IFN- ⁇ production in patient PBMC, but not in PBMC obtained from uninfected individuals.
  • Ml 5 and soluble Leishmania antigens elicit a dominant Thl cytokine profile in both mice and humans infected with Leishmania.
  • This Example illustrates the immunogenic properties of the antigens prepared according to Examples 1, 2, 5 and 6, as determined by their ability to stimulate proliferation in lymph node cultures from infected mice and in human PBMC preparations.
  • 2 - 4 x 10 ⁇ cells/well were cultured in complete medium (RPMI 1640 supplemented with gentamycin, 2-ME, L-glutamine, and 10%) screened pooled A+ human serum; Trimar, Hollywood, CA) in 96-well flat bottom plates with or without 10 ⁇ g/ml of the indicated antigens or 5 ⁇ g/ml PHA (Sigma Immunochemicals, St. Louis, MO) for five days. The cells were then pulsed with 1 ⁇ Ci of [ ⁇ H] thymidine for the final 18 hours of culture.
  • Figure 16 illustrates the proliferation observed after addition of 10 ⁇ g/mL or 20 ⁇ g/mL of each of the following antigens to a lymph node culture prepared as described in Example 7: SLA, Ldp23, LbeIF4A, Lbhsp83, and Ml 5. The level of proliferation without the addition of antigen is also shown. Data are represented as mean cpm. These results demonstrate that a variety of leishmanial antigens are capable of stimulatory lymph node cell proliferation from Leishmania-infected mice.
  • Figures 17 and 18 illustrate the proliferation observed in human PBMC preparations from Leishmania-immune and uninfected individuals following the addition of 10 ⁇ g/mL Ml 5 and soluble Leishmania antigens, respectively. These values are compared to the proliferation observed following the addition of culture medium, L. major lysate or L-Rack.
  • Ml 5 and soluble Leishmania antigens stimulate proliferation in Leishmania-immune PBMC, but not in PBMC obtained from uninfected individuals, demonstrating that Ml 5 and soluble antigens (but not L-Rack) are recognized by PBMC from individuals immune to Leishmania due to a previous infection.
  • This Example illustrates the preparation of two soluble Leishmania antigens, Lmspla and Lmsp9a.
  • a high titer rabbit sera was raised against L. major soluble antigens, prepared as described above in Example 6. Specifically, a New Zealand white rabbit was immunized subcutaneously at multiple sites with 180 ⁇ g of L. major soluble antigens in a suspension containing 100 ⁇ g muramyl dipeptide and 50 % incomplete Freund's adjuvant. Six weeks later the rabbit was given a subcutaneous boost of 100 ⁇ g of the same soluble antigen preparation in incomplete Freund's adjuvant. This was followed by two intravenous boosts spaced two weeks apart, each with 100 ⁇ g of the soluble antigen preparation. Sera was collected from the rabbit 11 days after the final boost.
  • Anti E. coli antibody reactivities were removed from the rabbit sera by pre-adsorbing on nitrocellulose filters containing lysed E. coli.
  • Adsorbed sera were evaluated by Western blot analysis using 10 ⁇ g Leishmania promastigote lysate (lane 1) and 1 ⁇ g soluble L. major antigen mixture (lane 2).
  • the rabbit sera was found to be reactive with seven dominant antigens of the soluble L. major antigen mixture with molecular weights ranging from 18 to >200 kDa.
  • a four times longer exposure of the same blot revealed three additional immunoreactive species with molecular weights less than 18 kDa.
  • the rabbit sera described above was subsequently used to screen an L. major cDNA expression library prepared from L. major promastigote RNA using the unidirectional Lambda ZAP (uni-ZAP) kit (Stratagene) according to the manufacturer's protocol. A total of 70,000 pfu of the amplified cDNA library was screened with the rabbit sera at a 1 :250 dilution. Nineteen positive clones were confirmed in the tertiary screening. The phagemid were excised and DNA from each of the 19 clones was sequenced using a Perkin Elmer/Applied Biosystems Division automated sequencer Model 373 A. All 19 clones were found to represent two distinct sequences, referred to as Lmspla and Lmsp9a.
  • Lmspla and Lmsp9a are provided in S ⁇ Q ID NO: 19 and 21, respectively, with the corresponding amino acid sequences being provided in S ⁇ Q ID NO: 20 and 22, respectively.
  • Fig. 21 shows the full-length cDNA (SEQ ID NO: 19) and predicted amino acid sequence (SEQ ID NO: 20) for the antigen Lmspla.
  • the EcoRI/XhoI insert is 1019 bp long and contains the following features: a) the last 17 nt of the spliced leader sequence characteristic of all trypanosoma nuclearly encoded mRNA; b) 39 nt of 5' untranslated sequence; c) an open reading frame of 453 nt long coding for a 151 deduced amino acid sequence with a predicted molecular mass of 16.641 kDa; and d) 471 nt of 3' untranslated sequence terminating with a poly A tail.
  • Lmspla contains three potential phosphorylation sites at amino acid residues 3, 85 and 102.
  • Lmspla contains an RGD sequence at residue 104, a sequence that may play a role in parasite invasion of the macrophage. RGD sequences have been shown to mediate the binding of various adhesion proteins to their cell surface receptors. There is no obvious leader sequence (secretory signal) at the amino terminal portion suggesting that the protein might be shed or excreted. Lmspla appears to be one of the most abundant antigens found in the culture supernatant of live promastigote, since 17 of the 19 clones contain sequences of variable lengths identical to Lmspla.
  • Lmsp9a The remaining two cDNA clones isolated from the soluble L. major antigen mixture represent identical sequences (referred to as Lmsp9a; SEQ ID NO: 21), suggesting that the two copies resulted from amplification of the primary library. Sequencing of the Lmsp9a cDNA revealed that the clone does not contain the full length 5' sequence since it is lacking both the spliced leader and 5' untranslated sequences. The 3' end of the cDNA contains a poly A stretch, as would be expected for a Leishmania mRNA. Of the predicted translated sequence (SEQ ID NO: 22), 34 of the 201 amino acids (17%>) represent cysteine residues. Comparison of the predicted protein sequence with those of known proteins as described above, revealed some homology with other cysteine rich proteins such as the major surface trophozoite antigen of Giardia lamblia and furin proteases.
  • This Example illustrates the preparation and characterization of the Leishmania antigen MAPS-IA (SEQ ID NO: 24).
  • mice A pool of sera was obtained from 5 BALB/c mice that had been given a primary immunization and two boosts with crude L. major promastigote culture supernatant as described below in Example 12. These mice were subsequently shown to be protected when challenged with a dose of live L. major promastigotes generally found to be lethal.
  • the mouse sera thus obtained were used to screen an L. major amastigote cDNA expression library prepared as described in Example 1.
  • Several seroreactive clones were isolated and sequenced using a Perkin Elmer/Applied Biosystems Division automated sequencer Model 373A (Foster City, CA).
  • MAPS-IA One of these clones, referred to herein as MAPS-IA, was found to be full-length. Comparison of the cDNA and deduced amino acid sequences for MAPS- IA (SEQ ID Nos: 23 and 24, respectively) with known sequences in the gene bank using the DNA STAR system revealed no significant homologies to known Leishmania sequences, although some sequence similarity was found to a group of proteins, known as thiol-specific antioxidants, found in other organisms. Recombinant MAPS-IA protein having an amino-terminal HIS-Tag was prepared using a high level E. coli expression system and recombinant protein was purified by affinity chromatography as described in Example 1. Southern blot analysis of genomic DNA from L.
  • MAPS-IA cDNA sequence provided in SEQ ID NO: 23, the corresponding gene was isolated from L. tropica by means of PCR (using 30 cycles of the following temperature step sequence: 94 °C, 1 minute; 50 °C, 1 minute; 72 °C, 1 minute)
  • the determined cDNA sequence for the L. tropica MAPS-IA protein is provided in SEQ ID NO: 25, with the corresponding amino acid sequence being provided in SEQ ID NO: 26.
  • MAPS-IA The ability of recombinant MAPS-IA to stimulate cell proliferation was investigated as follows. PBMC from 3 L. braziliensis-infected patients having active mucosal leishmaniasis, from 4 patients post kala-azar infection (previously infected with L. chagasi and/or L. donovani) and from 3 uninfected-individuals were prepared as described above in Example 7. The ability of MAPS-IA to stimulate proliferation of these PBMC was determined as described in Example 8 above. As shown in Figure 24, significant levels of MAPS-IA specific PBMC proliferation were seen in 2 of the 7 Leishmania patients. The ability of MAPS-IA to stimulate proliferation in mice lymph node cultures was determined as described in Example 8.
  • Figure 25 shows the amount of proliferation stimulated by MAPS-IA (at 25 ⁇ g/ml, 5 ⁇ g/ml and 1 ⁇ g/ml) as compared to that stimulated by the positive control ConA and by crude L. major promastigote supernatant proteins, 20 days post-infection with L. major.
  • Cells isolated 20 days post- infection were highly responsive to MAPS-IA, whereas cells isolated 10 days post- infection were unresponsive.
  • MAPS-IA The reactivity of MAPS-IA with sera from uninfected individuals, from human leishmaniasis patients with cutaneous infection, from human patients with acute visceral leishmaniasis, and from L. major-infected BALB/c mice was determined as follows.
  • Assays were performed in 96-well plates coated with 200 ng antigen diluted to 50 ⁇ L in carbonate coating buffer, pH 9.6. The wells were coated overnight at 4 °C (or 2 hours at 37 °C). The plate contents were then removed and the wells were blocked for 2 hours with 200 ⁇ L of PBS/1% BSA. After the blocking step, the wells were washed five times with PBS/0.1% Tween 20TM. 50 ⁇ L sera, diluted 1:100 in PBS/0.1% Tween 20TM/0.1% BSA, was then added to each well and incubated for 30 minutes at room temperature. The plates were then washed again five times with PBS/0.1% Tween 20TM. The enyzme conjugate (horseradish peroxidase - Protein A, Zymed, San
  • TMB tetramethylbenzidine peroxidase
  • Figure 26 shows the reactivity of MAPS-IA with increasing dilutions of sera from BALB/c mice previously administered either (i) saline solution; (ii) the adjuvant B. pertussis; (iii) soluble Leishmania antigens plus B. pertussis; (iv) live L. major promastigotes; or (v) soluble Leishmania antigens plus B. pertussis followed by live L. major promastigotes (as described below in Example 12).
  • saline solution saline solution
  • the adjuvant B. pertussis soluble Leishmania antigens plus B. pertussis
  • live L. major promastigotes live L. major promastigotes
  • soluble Leishmania antigens plus B. pertussis followed by live L. major promastigotes as described below in Example 12
  • mice Two weeks after the last immunization, the mice were challenged with 2 x 10 5 late-log phase promastigotes of L. major. Infection was monitored weekly by measurement of footpad swelling. The amount of footpad swelling seen in mice immunized with either crude soluble Leishmania antigens, a mixture of Ldp23, LbeiF4A and Ml 5 ( Figure 28), or MAPS-IA ( Figure 29) was significantly less than that seen in mice immunized with C. parvum alone. These results demonstrate that the Leishmania antigens of the present invention are effective in conferring protection against Leishmania infection.
  • This example illustrates the isolation of seven soluble Leishmania antigen genes from an L. major genomic DNA library.
  • An L. major genomic DNA expression library was prepared from L. major promastigotes using the unidirectional Lambda ZAP (uni-ZAP) kit (Stratagene) according to the manufacturer's protocol. This library was screened with a high titer rabbit sera raised against L. major soluble antigens, as described above in Example 9. Seven positive clones were identified. The phagemid were excised and DNA from each of the seven clones was sequenced using a Perkin Elmer/ Applied Biosystems Division automated sequencer Model 373A.
  • LmgSPl The DNA sequences for these antigens, referred to as LmgSPl, LmgSP3, LmgSP5, LmgSP8, LmgSP9, LmgSP13, LmgSP19, are provided in SEQ ID NO:29-35, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 36-42, respectively.
  • LmgSPl 3 was found to contain a 39 amino acid repeat sequence shown in SEQ ID NO:43.
  • the full-length DNA sequence is provided in SEQ ID NO: 54, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 55.
  • the amino acid sequence was found to contain six 14 amino acid repeat units (SEQ ID NO: 56), with each unit being further divided into two 7 amino acid units, provided in SEQ ID NO: 57 and 58.
  • LmgSP5 was found to be related to the known PSA2 family.
  • LmgSP8 was found to bear some homology to a sequence previously identified in E. coli (2- succinyl-6-hydroxy-2,4-cyclohexadiene-l -carboxylic acid synthase).
  • LmgSP9 and LmgSPl 9 were found to be homologous to a L. major hydrophilic surface protein referred to as Gene B (Flinn, H.M. et al. Mol. Biochem. Parasit. 65:259-210, 1994), and to ubiquitin, respectively.
  • Gene B L. major hydrophilic surface protein
  • LmgSP9 In order to obtain a higher specificity for the detection of antibodies in sera from visceral leishmaniasis patients, a homologue of LmgSP9 was isolated from L. chagasi, one of the causative agents of visceral leishmaniasis. A total of 80,000 pfu of an amplified L. chagasi genomic library were screened with the entire coding region of LmgSP9 (amplified from L. major genomic DNA). Seven hybridizing clones were purified to homogeneity.
  • Lc Gene A and LcGene B The determined DNA sequences for two of these clones, referred to as Lc Gene A and LcGene B, are provided in SEQ ID NO: 59 and 60, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 61 and 62, respectively.
  • the open reading frame for Lc Gene A was found to show some homology to Gene A/C, previously isolated from L. major (McKlean et al., Mol. Bio. Parasitol., 55:221-231, 1997).
  • the open reading frame for Lc Gene B showed some homology to Gene B of L. major, discussed above, and was found to contain eleven repeats of a 14 amino acid repeat unit (SEQ ID NO: 63), with each repeat being further divided into two 7 amino acid units, provided in SEQ ID NO: 64 and 65.
  • Lc Gene A and Lc Gene B were evaluated by ELISA as described above using sera from visceral leishmaniasis patients from Sudan and Brazil, and from uninfected controls. Absorbance values were compared to those obtained using LmgSP9. Much higher absorbance values were obtained with Lc Gene A and Lc Gene B than with LmgSP9, with Lc Gene B appearing to be more effective that Lc Gene A in detecting antibodies in certain cases. These results indicate that Lc Gene B is highly effective in the diagnosis of visceral leishmaniasis.
  • This example illustrates the preparation of five soluble Leishmania antigen genes from an L. chagasi genomic DNA library.
  • L. chagasi genomic DNA expression library was prepared from L. chagasi promastigotes using the unidirectional Lambda ZAP (uni-ZAP) kit (Stratagene) according to the manufacturer's protocol. This library was screened with a high titer rabbit sera raised against L. major soluble antigens, as described above in Example 9. Five positive clones were identified. The phagemid were excised and DNA from each of the Five clones was sequenced using a Perkin Elmer/ Applied Biosystems Division automated sequencer Model 373A.
  • LcgSPl The DNA sequences for these antigens, referred to as LcgSPl, LcgSP3, LcgSP4, LcgSP8, and LcgSPIO are provided in SEQ ID NO:44- 48, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO:49-53, respectively.
  • FIGS 30A and B illustrate the proliferative response of murine lymph nodes to recombinant LcgSP8, LcgSPIO and LcgSP3. Lymph nodes were taken BALB/c mice 17 days after infection with L. major. Infection occurred by footpad injection of 2 x 10 6 parasites/footpad. The cells were stimulated with recombinant antigen and proliferation was measured at 72 hours using 3 H-thymidine.
  • Figure 30A shows the CPM, a direct measurement of mitotic activity in response to the antigens
  • Figure 3 OB shows the stimulation index, which measures the proliferative response relative to the negative control.
  • This example illustrates the isolation of T cell antigens of J. major using a direct T cell screening approach.
  • Leishmania-specific CD4+ T cell lines were derived from the PBMC of an individual who tested positive in a leishmania skin test but had no clinical history of disease. These T cell lines were used to screen a L. major amastigote cDNA expression library prepared as described in Example 1. Immunoreactive clones were isolated and sequenced as described above. The determined cDNA sequences for the 8 isolated clones referred to as 1G6-34, 1E6-44, 4A5-63, 1B11-39, 2A10-37, 4G2-83, 4H6-41, 8G3-100 are provided in SEQ ID NO: 72-79, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 80-87, respectively. The cDNA sequences provided for 1E6-44, 2A10-37, 4G2-83, 4H6-41 and 8G3-100 are believe to represent partial clones. All of these clones were shown to stimulate T cell proliferation.
  • Polypeptides may be synthesized on a Perkin Elmer/ Applied Biosystems Division 430 A peptide synthesizer using FMOC chemistry with HPTU (O-
  • Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide.
  • Cleavage of the peptides from the solid support may be carried out using the following cleavage mixture: trifiuoroacetic acid:ethanedithiol:thioanisole:water:phenol
  • the peptides may be precipitated in cold methyl-t-butyl-ether. The peptide pellets may then be dissolved in water containing
  • TFA trifiuoroacetic acid
  • lyophilized prior to purification by C18 reverse phase HPLC.
  • a gradient of 0%-60%> acetonitrile (containing 0.1 %> TFA) in water (containing 0.1 %> TFA) may be used to elute the peptides.
  • the peptides may be characterized using electrospray or other types of mass spectrometry and by amino acid analysis.
  • ADDRESSEE SEED and BERRY LLP
  • ATC CGC GAC CCT TCG CAG GGC CGG CTG TAC ATG GAA GAC CAG CGC TTT 948 lie Arg Asp Pro Ser Gin Gly Arg Leu Tyr Met Glu Asp Gin Arg Phe 165 170 175
  • ATC GAT CCT GAG ATC GCG AAG CAG AAG AAA GAC GAA GGT AAC CAG TAC 1524 lie Asp Pro Glu lie Ala Lys Gin Lys Lys Asp Glu Gly Asn Gin Tyr 355 360 365
  • ATC AAG CGC AAC CCT GCC GAG CAC ACC TCC TAC AGC AAT CGC GCG GCC 1620 lie Lys Arg Asn Pro Ala Glu His Thr Ser Tyr Ser Asn Arg Ala Ala 385 390 395 400
  • GCTATCTTCT CAACGGAGAC ATGAAAGGCG TTTGTTAGGA AAAGAGGGGG GGGGGAAGAG 2648
  • CAG GCC CGC GTC CAG GCC CTC GAG GAG GCA GCG CGT CTC CGC GCG GAG 48

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US6013268A (en) * 1994-04-22 2000-01-11 Corixa Corporation Methods for enhancement of protective immune responses
US6500437B1 (en) 1995-09-22 2002-12-31 Corixa Corporation Leishmania antigens for use in the therapy and diagnosis of leishmaniasis
US6638517B2 (en) 1995-09-22 2003-10-28 Corixa Corporation Leishmania antigens for use in the therapy and diagnosis of leishmaniasis
US6613337B1 (en) * 1997-02-12 2003-09-02 Corixa Corporation Leishmania antigens for use in the therapy and diagnosis of leishmaniasis
US6607731B1 (en) 1995-09-22 2003-08-19 Corixa Corporation Leishmania antigens for use in the therapy and diagnosis of leishmaniasis
US6365165B1 (en) * 1995-09-22 2002-04-02 Corixa Corporation Leishmania antigens for use in the therapy and diagnosis of Leishmaniasis
US6673351B1 (en) * 2001-03-16 2004-01-06 Astralis, Llc Compositions and methods for the treatment and clinical remission of psoriasis
EP1436325A1 (de) * 2001-10-12 2004-07-14 U.S. Army Medical Research and Material Command Mikrofluidisiertes leishmania lysat und verfahren zu deren herstellung und verwendung
BRPI0913972B1 (pt) 2008-07-03 2018-06-26 Infectious Disease Research Institute Polipeptídeo de fusão compreendendo antígenos de leishmania, polinucleotídeo, composição farmacêutica, bem como métodos para detecção de infecção e para identificação de leishmaniose e kit diagnóstico para detecção e identificação de leishmaniose

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US5075109A (en) 1986-10-24 1991-12-24 Southern Research Institute Method of potentiating an immune response
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US5912166A (en) * 1995-04-21 1999-06-15 Corixa Corporation Compounds and methods for diagnosis of leishmaniasis
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