| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 01.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 11.01.00 |
| PATENT TITLE |
Methods for enhancement of protective immune responses |
| PATENT ABSTRACT |
Methods for eliciting or enhancing immune responses to antigens, including tumor antigens, and/or DNA vaccines are provided. The methods employ polypeptides or nucleic acid compositions that contain at least a biologically active portion of a Leishmania braziliensis or Leishmania major homologue of the eukaryotic initiation factor 4A, or a variant thereof. Such polypeptides and compositions are useful for enhancing or eliciting a patient's cellular and/or humoral immune response, for instance within methods for treating tumors. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 12.12.97 |
| PATENT REFERENCES CITED |
Cornelissen et al, FEMS Immunol & Med. Microbiol. 15:61-72, 1996. Xu et al Vaccine 12(16): 1534-1536, 1994. Skeiky et al. J Exp. Med. 181:1527-1537, 1995. Xu et al, Immunol. 84:173-176, 1995. Kalinna, Immunology & Cell Biology 75:370-75, 1997. Gurunathan et al, J. Exptal. Medicine, 186/7:1137-1147, 1997. Paillard, Human Gene Therapy, 9:1849-1850, 1998. Handman, Parasitology Today, 13/6:236-238, 1997. Dusanic, Southeast Asian J. Trop. Med. Public Health 19/1:11-20, 1988. Gurunathan et al, Nature Medicine, 4/12:1409-1415, 1998. Walker et al, J. Am. Acad. Dermatol. 37/5 pt 1:776-777, 1997. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A method of enhancing or eliciting an immune response to a DNA vaccine in a patient, comprising administering to a patient a DNA vaccine and an LmeIF4A polypeptide comprising an amino acid sequence encoded by a DNA sequence selected from the group consisting of: (a) nucleotides 117 through 1325 of SEQ ID NO:3; and (b) DNA sequences that hybridize to a nucleotide sequence complementary to nucleotides 117 through 1325 of SEQ ID NO:3 under the following conditions, prewashing in a solution of 5.times. SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50.degree. C.-65.degree. C., 5 .times. SSC, overnight; followed washing twice at 65.degree. C. for 20 minutes with each of 2.times., 0.5.times. and 0.2.times. SSC containing 0.1% SDS, wherein the DNA sequence encodes a polypeptide that stimulates a Th1 immune response in peripheral blood mononuclear cells obtained from a Leishmania-infected individual. 2. The method of claim 1 wherein said LmeIF4A polypeptide comprises amino acids 49-403 of SEQ ID NO:4. 3. The method of claim 1 wherein said LmeIF4A polypeptide comprises amino acids 1-403 of SEQ ID NO:4 or a portion thereof. 4. The method of claim 1 wherein said LmeIF4A polypeptide additionally comprises an immunoglobulin Fc region. 5. A method of enhancing or eliciting an immune response to a DNA vaccine in a patient, comprising administering to a patient a DNA vaccine and an LmeIF4A polypeptide comprising amino acids 1-403 of SEQ ID NO:4 or a variant thereof that differs only in conservative substitutions modifications, or combinations thereof. 6. The method of any of claims 1-4 and 5 wherein said DNA vaccine and said LmeIF4A polypeptide are present in the same composition. 7. The method of any of claims 1-4 and 5 wherein said DNA vaccine is encapsulated in biodegradable microspheres. 8. The method of any of claims 1-4 and 5 wherein said LmeIF4A polypeptide is encapsulated in or associated with the surface of biodegradable microspheres. 9. The method of any of claims 1-4 and 5 wherein said DNA vaccine and the LmeIF4A polypeptide are encapsulated in biodegradable microspheres. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
TECHNICAL FIELD The present invention relates generally to compounds and methods for enhancing immune responses in patients, as well as in isolated cells and cell cultures. The invention is more particularly related to compounds comprising all or a portion of a Leishmania antigen that is ai homologue of the eukaryotic initiation factor 4A (eIF4A), and to the use of such compounds in vaccines for stimulating immune responses. BACKGROUND OF THE INVENTION Vaccines commonly induce immunity to an infection or a disease by generating an immune response in a patient to a specific antigen associated with the infection or disease. Modern techniques for the identification and use of appropriate antigens have the potential to lead to the testing and development of a large number of vaccines specific for common infections (including bacterial, viral and protozoan infections), as well as diseases such as cancer. However, in many cases, purified antigens are weak immunogens, i.e., the immune response generated by a specific antigen, while directed against the desired target, is not of a sufficient magnitude to confer immunity. In such cases, an immunomodulating agent, such as an adjuvant or immunostimulant, must be employed to enhance the immune response. Adjuvants are substances that enhance a specific immune response to an antigen when injected with the antigen or at the same site as the antigen. Such substances function by a variety of mechanisms, including (1) trapping the antigen, and releasing it slowly, (2) stimulating migration of cells to the injection site, (3) stimulating or trapping lymphocytes, or stimulating lymphocyte proliferation and (4) improving antigen dispersion within the patient's body. For example, oils, polymers, mineral salts and liposomes have been used as adjuvants in this regard. By comparison, immunostimulants are substances that induce a general, temporary increase in a patient's immune response, whether administered with the antigen or separately. Typical immunostimulants are bacterial, such as BCG (an attenuated strain of Mycobacterium tuberculosis) or a nonviable form of Corynebacterium parvum. By either mechanism, the adjuvant or immunostimulant serves to enhance the desired specific immune response by non-specific means. A serious drawback of many of the adjuvants currently available is their toxicity. In general, the best adjuvants (i.e., those that provide the greatest enhancement of the desired immune response) are also the most toxic. Thus, practitioners must continually balance the level of stimulation against the toxicity of the adjuvant. Accordingly, there is a need in the art for the identification of compounds that provide a desired enhancement of specific immune responses, but with low levels of toxicity. The present invention fulfills these needs and further provides other related advantages. SUMMARY OF THE INVENTION Briefly stated, the present invention provides compounds and methods relating to the Leishmania antigen LbeIF4A or LmeIF4A, which is homologous to the eukaryotic ribosomal protein eIF4A. In one aspect of the invention, methods are provided for enhancing or eliciting an immune response to an antigen and/or an antigen encoded by a DNA vaccine in a patient, comprising administering to a patient an antigen and/or a DNA vaccine, and an LbeIF4A polypeptide comprising an amino acid sequence encoded by a DNA sequence selected from the group consisting of: (a) nucleotides 115-1323 of SEQ ID NO. 1; and (b) DNA sequences that hybridize to a nucleotide sequence complementary to nucleotides 115-1323 of SEQ ID NO. 1 under moderately stringent conditions, wherein the DNA sequence encodes a polypeptide that stimulates a Th1 immune response in a peripheral blood mononuclear cells obtained from a Leishmania-infected individual. In another aspect of the invention, methods are provided for enhancing or eliciting an immune response to an antigen and/or an antigen encoded by a DNA vaccine, in a patient, comprising administering to a patient an antigen and/or a DNA vaccine, and an LmeIF4A polypeptide comprising an amino acid sequence encoded by a DNA sequence selected from the group consisting of: (a) nucleotides 117 through 1325 of SEQ ID NO:3; and (b) DNA sequences that hybridize to a nucleotide sequence complementary to nucleotides 117 through 1325 of SEQ ID NO:3 under moderately stringent conditions, wherein the DNA sequence encodes a polypeptide that stimulates a Th1 immune response in peripheral blood mononuclear cells obtained from a Leishmania-infected individual. In related aspects, the present invention provides methods for enhancing an immune response to an antigen and/or an antigen encoded by a DNA vaccine in a patient, comprising administering to a patient an antigen and/or a DNA vaccine, and an LbeIF4A polypeptide comprising amino acids 49-403 of SEQ ID NO: 2, or a variant thereof that differs only in conservative substitutions and/or modifications, or an antigen and/or a DNA vaccine and an LmeIF4A polypeptide comprising amino acids 49-403 of SEQ ID NO: 4, or a variant thereof that differs only in conservative substitutions and/or modifications. In another related aspect, methods are provided for enhancing an immune response in a biological sample, comprising contacting a biological sample with an antigen and/or an antigen encoded by a DNA vaccine, and an LbeIF4A polypeptide as described above, wherein the biological sample comprises cells selected from the group consisting of peripheral blood mononuclear cells, monocytes, B cells. dendritic cells, and combinations thereof. In yet another related aspect, methods are provided for enhancing or eliciting an immune response in a biological sample, comprising contacting a biological sample with an LmeIF4A polypeptide as described above, wherein the biological sample comprises cells selected from the group consisting of peripheral blood mononuclear cells, monocytes, B cells, dendritic cells and combinations thereof. In another aspect, methods are provided for enhancing an immune response to a tumor in a patient, comprising administering to a patient a tumor antigen or antigens and/or a DNA vaccine, and an LbeIF4A or an LmeIF4A polypeptide as described above. Within further aspects, methods are provided for treating a tumor in a patient, comprising administering to a patient an LbeIF4A or LmeIF4A polypeptide, as described above. Within each of the aspects noted above, as an alternative to utilizing an LbeIF4A or LmeIF4A polypeptide, one can utilize viral vectors or nucleic acid molecules (collectively, the "nucleic acid compositions") directing the expression of the polypeptide in patient cells infected or transfected with the nucleic acid compositions. The step of administering the nucleic acid composition may be performed in vivo or ex vivo, the latter including the subsequent administration of the infected/transfected cells. In addition, where an antigen or tumor antigen is administered, it will be evident that the nucleic acid composition may also be designed to direct the expression of such antigens (either on the same or different vectors or molecules). Within further aspects, methods are provided for treating a Th2-mediated disease in a patient, comprising administering to a patient (a) an LeIF4A polypeptide; (b) a nucleic acid molecule directing the expression of an LeIF4A polypeptide in patient cells transfected with the nucleic acid molecule or (c) a viral vector directing the expression of an LeIF4A polypeptide in patient cells infected with the viral vector. Th2-mediated diseases include asthma, allergy, Th2 mediated autoimmune disease and Helminth infection. The present invention further provides methods for decreasing production of one or more Th2-associated cytokines in a patient, comprising administering to a patient (a) an LeIF4A polypeptide; (b) a nucleic acid molecule directing the expression of an LeIF4A polypeptide in patient cells transfected with the nucleic acid molecule or (c) a viral vector directing the expression of an LeIF4A polypeptide in patient cells infected with the viral vector. Within certain embodiments, the Th2-associated cytokine is IL-4 or IL-5. Methods for stimulating or enhancing IL-18 production in a patient are also provided, comprising administering to a patient (a) an LeIF4A polypeptide; (b) a nucleic acid molecule directing the expression of an LeIF4A polypeptide in patient cells transfected with the nucleic acid molecule or (c) a viral vector directing the expression of an LeIF4A polypeptide in patient cells infected with the viral vector. Within further aspects, methods are provided for enhancing or eliciting an immune response to an antigen in a biological sample, comprising contacting a biological sample with an LeIF4A polypeptide in combination with one or more Th1-associated cytokines. The present invention also provides method for enhancing or eliciting an immune response to an antigen in a patient, comprising administering to a patient one or more Th1-associated cytokines in combination with (a) an LeIF4A polypeptide; (b) a nucleic acid molecule directing the expression of an LeIF4A polypeptide in patient cells transfected with the nucleic acid molecule or (c) a viral vector directing the expression of an LeIF4A polypeptide in patient cells infected with the viral vector. The Th1-associated cytokines may be IL-2, IL-1 2, IL-15 and/or IL-18. These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 presents the results of Southern blot analysis of Leishmania spp. DNA, indicating that the Leishmania eIF4A homologue is conserved and that L. braziliensis genomic DNA contains at least two copies of LbeIF4A. FIG. 2 shows the results of an immunoblot analysis which demonstrates that LbeIF4A immune rabbit serum reacts with one dominant protein species of size .about.45 kDa in different Leishmania species. FIG. 3 illustrates the ability of purified recombinant LbeIF4A to stimulate proliferation of PBMCs from L. braziliensis-infected individuals. FIGS. 4A and 4B present the results obtained by analysis of cytokine mRNA expression patterns of PBMCs from patients with confirmed cases of L. braziliensis infection. FIG. 5 illustrates the supernatant levels of secreted IFN-.gamma. from PBMCs from L. braziliensis-infected individuals following stimulation with LbeIF4A or parasite lysate. FIG. 6 shows the levels of TNF-U-detected in the supernatants of PBMCs from L. braziliensis-infected individuals following stimulation with LbeIF4A or parasite lysate. FIG. 7, Panels A-D, shows that LbeIF4A also stimulates patient PBMCs to secrete IL-12 in the cultured supernatant with a magnitude significantly higher than the IL-12 level stimulated by parasite lysate and that IL-10 inhibits this IL-12 production. FIG. 8, Panels A and B, demonstrates that in all patient PBMCs tested, IFN-.gamma. production was IL-12 dependent and inhibited by IL-b 10. FIGS. 9A and 9B show that LbeIF4A stimulates IL-12 production in cultured human macrophages and adherent PBMCs. FIG. 10 indicates that LbeIF4A stimulates IL-12 p40 production in the human myeloid leukemia cell-line, THP-1, and synergizes with IFN-.gamma. to stimulate THP-1 cells to secrete IL-12. FIG. 11 presents results that indicate that lymph node cells of mice primed with LbeIF4A proliferate and secrete an almost exclusive Th1 cytokine profile. FIG. 12 demonstrates that LbeIF4A provides significant protection against L. major infection in an animal model recognized as having relevance to human disease. FIG. 13 illustrates the elicitation of anti-ovalbumin CTL using a representative LmeIF4A polypeptide. FIG. 14 illustrates the use of a representative LmeIF4A polypeptide as an adjuvant for the induction of antibodies specific for trinitrophenol. FIG. 15 shows the enhancement of anti-MUC-1 antibody production by a representative LmeIF4A polypeptide. FIG. 16 illustrates the enhancement of specific CTL activity by a representative LmeIF4A polypeptide in cultured cells. FIG. 17 shows the in vitro stimulation of CTL activity with IL-2, with and without an LmeIF4A polypeptide. FIG. 18 illustrates the induction of murine alloreactive CTL by an LmeIF4A polypeptide. FIG. 19 shows tumor regression following administration of tumor antigen and LmeIF4A polypeptide contained in microspheres. FIG. 20 shows tumor regression following administration of tumor antigen and LmeIF4A polypeptide contained in microspheres. FIG. 21 shows tumor regression following administration of soluble LmeIF4A polypeptide and tumor antigen contained in microspheres. FIG. 22 presents a comparison of the predicted amino acid sequences of L. major eIF4A (LmeIF), with the homologous proteins from L. brazilienses (LbeIF), mouse (MeIF) and human (HeIF). Positions of identical residues to LmeIF are shaded black. Boxed sequences represent identity between the mouse and human proteins that are distinct from the Leishmania homologue or conservative substitutions. Regions of similarity with conserved elements found in RNA helicases are indicated (I-VI). I and II(DEAD) represent specialized versions of the A and B motifs described in other ATP binding proteins. Cysteine residues are indicated by * and potential N-linked glycosylation sites are underlined. FIGS. 23A-C illustrate the expression and purification of recombinant LmeIF. FIG. 23A is a photograph of coomassie blue-stained 12% SDS-PAGE of E. coli lysates before (lane 1) and after (lane 2) induction with IPTG to express rLeIF with 6 amino-terminal histidine tag residues. rLmeIF following purification from the inclusion body by affinity chromatigraphy on Ni-NTA column is shown in lane 3. FIG. 23B is a photograph of coomassie blue-stained 12% SDS-PAGE of overlapping LeIF deletions. The recombinant clones were designed to encode the N-terminal half (26 kDa, residues 1-226, lane 1), the middle portion (16 kDa, residues 129-261, lane 2) and the C-terminal half (25 kDa, residues 196-403) of LeIF with six His-tag residues and the proteins purified over NiNTA resin. Protein molecular weight markers (lane M) are indicated to the left. FIG. 23C is a schematic representation of the full length cDNA clone of L. major LeIF comprising of a 0.13 kb sequence of 5' untranslated (5'UTR) segment, an open reading frame of 1.209 kb coding for 403 amino acid long protein, and a 1.25 kb of 3' UTR terminating with a stretch of poly A tail. The arrows below show the location and sizes of both the full-length and overlapping fragments of the LeIF constructs. FIGS. 24A-C are graphs illustrating the analysis of the Th1/Th2 cytokine profile of draining lymph node cells from L. major infected BALB/c mice against rLeIF. Draining popliteal lymph node cells (2.times.10.sup.6 /ml) isolated at (A) 10 and (B), 28 days of infection were stimulated in vitro with 10 .mu.g/ml each of rLeIF or SLA and the supernatants analyzed 72 hours later for the amount of IFN-.gamma. and IL-4. In FIG. 24C, L. major infection sera from BALB/c mice (28 day post-infection) were analyzed and titrated for the presence of anti-rLeIF or rLmSTI1 specific antibody and compared with total promastigote lysate (SLA). Bound antibodies were detected with HRP conjugated goat anti-mouse IgG secondary antibody. FIG. 25 is a histogram illustrating the abrogation of the SLA-induced IL-4 secretion by LeIF. Lymph node cells were obtained from BALB/c mice infected with L. major (4 weeks post-infection) and were stimulated with SLA (10 .mu.g/ml) alone or in the presence of various concentration of LeIF. Cells were cultured for 3 days and supernatants were collected and assayed for the production of IL-4 and IFN-.gamma. by ELISA. FIG. 26 is a histogram illustrating the profile of T cell clones isolated from rLeIF or r8E-primed BALB/c mice. Mice were immunized subcutaneously with 70 .mu.g of the respective antigens without adjuvant. Ten days later, their lymph node cells were restimulated in vitro under limiting dilution with the same antigen, irradiated antigen presenting cells and IL-2. The resulting clones were re-stimulated with anti-CD3 mAb and supernatant cytokine patterns of the Th1-(IFN-.gamma.), Th2-(IL-4) or Th0 (IFN-.gamma. and IL-4) were determined by ELISA. The result is presented as the percentage of clones expressing a Th1, Th2, or Th0 cytokine profile. FIGS. 27A-C are histograms illustrating LeIF stimulation of the production of IFN-.gamma. by splenocytes from naive C3H- and Balb/c-SCID mice. In FIG. 27A, splenocytes from SCID mice of both Balb/c and C3H background were cultured at 2.times.10.sup.6 per well and stimulated with 10 .mu.g/ml of the indicated antigen. Supernatants were harvested at 12, 24, and 72 hours and assayed for the production of IFN-.gamma.. In FIG. 27B assays were performed as above using C3H SCID splenocytes in the presence or absence of anti-1L-12 antibody. Supernatants were harvested at 72 hours. In FIG. 27C, stimulation was performed using three overlapping LeIF recombinants comprising amino acid residues 1-226, 129-261 and 196-403 at 2.5, 5.0, and 10 .mu.g/ml in splenocyte cultures from C3H SCID mice. As control, LPS was used at two concentrations, 100 ng and 1 .mu.g/ml. FIG. 28 is a photograph depicting the electrophoresis of RT-PCR experiments to determine the presence of various cytokines in SCID mouse splenocytes cultured for 24 hours in the absence (-) or presence (+) of LeIF(10 .mu.g/ml). Primers were specific for .beta.-actin as a control, or for IFN-.gamma., IL-18 or IL-10, as indicated. FIG. 29 is a graph showing the level of IFN-.gamma. in SCID mouse splenocytes stimulated for 72 hours with varying amounts of IL-18 as indicated, in the presence or absence of LeIF (10 .mu.g/ml). FIG. 30 is a graph showing the level of IFN-.gamma. in SCID mouse splenocytes stimulated for 72 hours with varying amounts of IL-18 as indicated, in the presence or absence of LeIF (10 .mu.g/ml), IL-15 (ng/ml) or both. FIG. 31 is a graph showing the level of IFN-.gamma. in SCID mouse splenocytes stimulated for 72 hours with varying amounts of IL-18 as indicated, in the presence or absence of LeIF (10 .mu.g/ml), IL-15 (100 ng/ml) or both. FIG. 32 is a graph illustrating the cytotoxic activity (expressed as % Specific Lysis) of SCID mouse splenocytes stimulated with IL-15 (100 ng/ml), IL-12 (10 U/ml) or both in the presence or absence of LeIF (10 jig/ml) at varying effector:target ratios, as indicated. The target cells were YAC-1 cells. FIG. 33 is a graph illustrating the cytotoxic activity (expressed as % Specific Lysis) of SCID mouse splenocytes stimulated with 11-18 (100 ng/ml), IL-12 (10 U/ml) or both in the presence or absence of LeIF (10 .mu.g/ml) at varying effector:target ratios, as indicated. The target cells were YAC-1 cells. DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention is generally directed to the enhancement of immune responses, which may be humoral and/or cell-mediated, in a patient or cell culture. Within the context of this invention, an immune response to an antigen, including an immunostimulating antigen (i.e., an antigen against which a patient raises an immune response), may be initiated or enhanced by administering to the patient the antigen and one or more LbeIF4A-derived or LmeIF4A-derived polypeptides as described herein. Antigens and immunostimulating antigens are in general protein molecules and include molecules derived from viruses, such as HIV, HBV, influenza virus, respiratory syncytial virus, bacteria, such as Hemophilus influenza, Pneumoccocus pneumoniae, and parasites, such as Leishmania, and Trypanosoma. In addition, an immune response to a tumor may be enhanced or elicited by administering to the patient a tumor antigen (i.e., an antigen that stimulates an immune response (e.g., CTL) to a tumor). Within the context of this invention, tumor antigens include virally encoded molecules, MAGE-1, Her-2. PSA, and other molecules. Accordingly, the methods of this invention involve the co-administration of a specific antigen or immunostimulating antigen and an LeIF4A-derived polypeptide as disclosed herein. A tumor may also be treated by administering to the patient an LbeIF4A or LmeIF4A polypeptide in the absence of such an exogenously administered tumor antigen. The LbeIF4A and LmeIF4A polypeptides of the present invention may also be used to elicit or enhance an immune response to an antigen encoded by a DNA vaccine. DNA vaccines encode one or more immunostimulating antigens, such that the antigen is generated in situ. For instance, the DNA vaccine may encode a tumor antigen and, optionally, an LeIF4A-derived polypeptide as described herein. In such vaccines, 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). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an epitope of a prostate cell antigen on its cell surface. The DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., PNAS 91:215-219, 1994; Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. 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 published PCT application WO 90/11092, and Ulmer et al., Science 259:1745-1749, 1993, reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. The compounds of this invention generally comprise a polypeptide that stimulates a Th1 or CTL (cytotoxic T lymphocyte) immune response in peripheral blood mononuclear cells (PBMCs). In particular, polypeptides comprising all or a stimulatory portion of a Leishmania braziliensis or Leishmania major homologue of the eukaryotic ribosomal protein eIF4A are disclosed. Such proteins may be referred to herein as LbeIF4A and LmeIF4A, or as LbeIF and LmeIF, respectively. As used herein, the term "PBMCs" refers to preparations of nuclear cells that are present in peripheral blood. The term "polypeptide," in the context of this invention, encompasses amino acid chains of any length, including full length proteins and portions thereof, wherein amino acid residues are linked by covalent peptide bonds. Therefore, an "LbeIF4A polypeptide" comprises LbeIF4A, or a portion or other variant thereof that retains stimulatory activity. Similarly, an "LmeIF4A polypeptide" comprises LmeIF4A, or a portion or other variant thereof that retains stimulatory activity. As used herein, "LeIF4A" or "LeIF" refers to either LbeIF4A or LmeIF4A. Although LbeIF4A is described herein for exemplary purposes, within the context of this invention, LmeIF4A, portions thereof, and variants of the polypeptide (or portions thereof) may also be used. An LeIF4A polypeptide may consist entirely of one or more stimulatory portions of LeIF4A, or the stimulatory portion(s) may be supplied in the context of a larger protein that contains additional LeIF4A sequences and/or amino acid sequences heterologous to LeIF4A. Preferably, the polypeptides are substantially free of contaminating endogenous materials. The polypeptides of the present invention include variants of LbeIF4A or LmeIF4A that retain the ability to stimulate a Th1 or CTL immune response in PBMCs. Such variants include various structural forms of the primary protein. Due to the presence of ionizable amino and carboxyl groups, for example, a LbeIF4A polypeptide may be in the form of an acidic or basic salt, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction. Variants within the scope of this invention also include polypeptides in which the primary amino acid structure of LeIF4A or a fragment thereof is modified by forming covalent or aggregative conjugates with other polypeptides or chemical moieties such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared, for example, by linking particular functional groups to amino acid side chains or at the N- or C-termini. Alternatively, for derivatives in which a polypeptide is joined to a LeIF4A polypeptide, a fusion protein may be prepared using recombinant DNA techniques, as described below. In one such embodiment, the LeIF4A polypeptide may be conjugated to a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast .alpha.-factor leader). Protein fusions within the present invention may also comprise peptides added to facilitate purification or identification of LeIF4A polypeptides (e.g., poly-His). For example, the peptide described by Hopp et al., Bio/Technology 6:1204 (1988) is a highly antigenic peptide that can be used to facilitate identification. Such a peptide provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. The sequence of Hopp et al. is also specifically cleaved by bovine mucosal enterokinase, allowing removal of the peptide from the purified protein. Fusion proteins capped with such peptides may also be resistant to intracellular degradation in E. coli. Protein fusions encompassed by this invention further include, for example, LeIF4A polypeptides linked to an immunoglobulin Fe region. If LbeIF4A fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a protein oligomer with as many as four LbeIF4A protein regions. Also within the scope of the present invention are LbeIF4A polypeptides linked to a leucine zipper domain. Leucine zipper domains are described, for example, in published PCT Application WO 94/10308. LbeIF4A polypeptides comprising leucine zippers may, for example, be oligomeric, dimeric or trimeric. All of the above protein fusions may be prepared by chemical linkage or as fusion proteins, as described below. Preferred protein fusions include polypeptides that comprise sequences useful for stimulating immunity to infectious pathogens (e.g., antigens). Such sequences may be derived, for example, from viruses, tumor cells, parasites or bacteria. The present invention also includes LeIF4A polypeptides with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems may be similar to or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. For instance, expression of DNA encoding LbeIF4A polypeptides in bacteria such as E. coli provides non-glycosylated molecules. N-glycosylation sites of eukaryotic proteins are characterized by the amino acid triplet Asn-A.sub.1 -Z, where Al is any amino acid except Pro, and Z is Ser or Thr. Variants of LbeIF4A polypeptides having inactivated N-glycosylation sites can be produced by techniques known to those of ordinary skill in the art, such as oligonucleotide synthesis and ligation or site-specific mutagenesis techniques, and are within the scope of this invention. Alternatively, N-linked glycosylation sites can be added to a LbeIF4A polypeptide. The polypeptides of this invention also include variants of LeIF4A polypeptides that have an amino acid sequence different from the native LeIF4A protein because of one or more deletions, insertions, substitutions or other modifications. Such variants should be substantially homologous to the native LeIF4A and should retain the ability to stimulate a Th1 or CTL immune response in PBMCs. "Substantial homology," as used herein, refers to amino acid sequences that may be encoded by DNA sequences that are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding, for instance, LbeIF4A. Suitable moderately stringent conditions include prewashing in a solution of 5.times. SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times. SSC, overnight; followed by washing twice at 65.degree. C. for 20 minutes with each of 2.times., 0.5.times. and 0.2.times. SSC containing 0.1% SDS). Such hybridizing DNA sequences are also within the scope of this invention. The effect of any such modifications on the activity of a LbeIF4A polypeptide may be readily determined by analyzing the ability of the mutated LbeIF4A peptide to induce a Th1 or CTL response using, for example, any of the methods described herein. A preferred variant of LbeIF4A is the Leishmania major homologue of LbeIF4A (LmeIF4A). Generally, amino acid substitutions should be made conservatively; i.e., a substitute amino acid should replace an 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. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, 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 within the scope of this invention may also, or alternatively, contain other modifications, including the deletion or addition of amino acids, that have minimal influence on the stimulatory properties, secondary structure and hydropathic nature of the polypeptide. In general, fragments of LeIF4A may be constructed by deleting terminal or internal residues or sequences. Additional guidance as to suitable modifications may be obtained by a comparison of the sequence of LeIF4A to the sequences and structures of other eIF4A family members. For example, terminal or internal residues or sequences of LeIF4A not needed for biological activity may be deleted. Cysteine residues may be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation. Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. An LeIF4A full length protein may generally be obtained using a genomic or cDNA clone encoding the protein. A genomic sequence that encodes full length LbeIF4A is shown in SEQ ID NO:1, and the deduced amino acid sequence is presented in SEQ ID NO:2. A genomic sequence that encodes full length LmeIF4A is shown in SEQ ID NO:3 and the deduced amino acid sequence in SEQ ID NO:4. Such clones may be isolated by screening an appropriate Leishmania braziliensis or Leishmania major expression library for clones that express antigens that react with sera from a patient afflicted with mucosal leishrnaniasis, and then analyzing the reactive antigens for the ability to stimulate proliferative responses and preferential Th1 cytokine production in patient T cell assays or for the ability to stimulate a CTL response in patient T cells. The library preparation and screen may generally be performed using methods known to those of ordinary skill in the art, such as methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, which is incorporated herein by reference. Briefly, a bacteriophage expression library may be plated and transferred to filters. The filters may then be incubated with serum and a detection reagent. In the context of this invention, a "detection reagent" is any compound capable of binding to the antibody-antigen complex, which may then be detected by any of a variety of means known to those of ordinary skill in the art. Typical detection reagents contain a "binding agent," such as Protein A, Protein G, IgG or a lectin, coupled to a reporter group. Preferred reporter groups include enzymes, substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. More preferably, the reporter group is horseradish peroxidase, which may be detected by incubation with a substrate such as tetramethylbenzidine or 2,2'-azino-di-3-ethylbenzthiazoline sulfonic acid. Plaques containing genomic or cDNA sequences that express a protein which binds to an antibody in the serum are isolated and purified by techniques known to those of ordinary skill in the art. Appropriate methods may be found, for example, in Sambrook et al., Molecular Cloning.: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989. Patient T cell assays may generally be performed by treating patient PBMCs with the reactive antigens and analyzing the cells for a suitable response. For example, the PBMC supernatant may be assayed for the level of secreted cytokines. Preferably, the cytokine assayed is interferon-.gamma., interleukin-2, interleukin-12 (either the p40 subunit or biologically active p70), interleukin-1 or tumor necrosis factor-.alpha.. The cytokines interleukin-4 and interleukin-10 may also be assayed, since the levels of these representative Th2-type cytokines generally decrease in response to treatment with a polypeptide as described herein. Cytokines may be assayed, for example, using commercially available antibodies specific for the cytokine of interest in an ELISA format, with positive results determined according to the manufacturer's instructions. Suitable antibodies may be obtained, for example, from Chemicon, Temucula, Calif. and PharMingen, San Diego, Calif. Alternatively, the treated PBMCs may be assayed for mRNA encoding one or more of the cytokines interferon-.gamma., interleukin-2, interleukin-12 p40 subunit, interleukin-1 or tumor necrosis factor-.alpha., or the PBMCs may be assayed for a proliferative response as described herein. Alternatively, cytokines may be measured by testing PBMC supernatants for cytokine-specific biological activities. Variants of LeIF4A that retain the ability to stimulate a Th1 immune response in PBMCs may generally be identified by modifying the sequence in one or more of the aspects described above and assaying the resulting polypeptide for the ability to stimulate a Th1 response. Such assays may generally be performed by treating patient PBMCs with the modified polypeptide and assaying the response, as described above. Naturally occurring variants of LeIF4A may also be isolated from other Leishmania species by, for example, screening an appropriate cDNA or genomic library with a DNA sequence encoding LeIF4A or a variant thereof. The above-described sequence modifications may be introduced using standard recombinant techniques or by automated synthesis of the modified polypeptide. For example, mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analogue having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide a gene in which particular codons are altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al., Gene 42:133, 1986; Bauer et al., Gene 37:73, 1985; Craik, BioTechniques, January 1985, 12-19; Smith et al., Genetic Engineering: Principles and Methods, Plenum Press, 1981; and U.S. Pat. Nos. 4,518,584 and 4,737,462. Mutations in nucleotide sequences constructed for expression of such LeIF4A polypeptides must, of course, preserve the reading frame of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the receptor mRNA. Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed LeIF4A protein mutants screened for the desired activity. Not all mutations in a nucleotide sequence which encodes a LeIF4A protein will be expressed in the final product. For example, nucleotide substitutions may be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA (see, e.g., European Patent Application 75,444A), or to provide codons that are more readily translated by the selected host, such as the well-known E. coli preference codons for E. coli expression. The polypeptides of the present invention, both naturally occurring and modified, are preferably produced by recombinant DNA methods. Such methods include inserting a DNA sequence encoding a LeIF4A polypeptide into a recombinant expression vector and expressing the DNA sequence in a recombinant microbial, mammalian or insect cell expression system under conditions promoting expression. DNA sequences encoding the polypeptides provided by this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being inserted in a recombinant expression vector and expressed in a recombinant transcriptional unit. Recombinant expression vectors contain a DNA sequence encoding a LeIF4A polypeptide operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation, as described in detail below. An origin of replication and a selectable marker to facilitate recognition of transformants may additionally be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, in reading frame. DNA sequences encoding LeIF4A polypeptides which are to be expressed in a microorganism will preferably contain no introns that could prematurely terminate transcription of DNA into mRNA. Expression vectors for bacterial use may comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. Promoters commonly used in recombinant microbial expression vectors include the .beta.-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and European Patent Application 36,776) and the tac promoter (Maniatis, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, p.412, 1982). A particularly useful bacterial expression system employs the phage .lambda. P.sub.L promoter and cI857ts thermolabile repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the .lambda. P.sub.L promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082). Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J Biol. Chem. 255.2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldchyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., European Patent Application 73,657. Preferred yeast vectors can be assembled using DNA sequences from pBR322 for selection and replication in E. coli (Amp.sup.r gene and origin of replication) and yeast DNA sequences including a glucose-repressible ADH2 promoter and .alpha.-factor secretion leader. The ADH2 promoter has been described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724. 1982). The yeast .alpha.-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed (see, e.g., Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984). The leader sequence may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes. The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from polyoma, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl II site located in the viral origin of replication is included. Further, viral genomic promoter, control and/or signal sequences may be utilized, provided such control sequences are compatible with the host cell chosen. Exemplary vectors can be constructed as disclosed by Okayama and Berg, Mol. Cell. Biol. 3:280, 1983. A useful system for stable high level expression of mammalian receptor cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A preferred eukaryotic vector for expression of LbeIF4A protein DNA is pDC406 (McMahan et al., EMBO J. 10:2821, 1991), and includes regulatory sequences derived from SV40, human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV). Other preferred vectors include pDC409 and pDC410, which are derived from pDC406. pDC410 was derived from pDC406 by substituting the EBV origin of replication with sequences encoding the SV40 large T antigen. pDC409 differs from pDC406 in that a Bgl II restriction site outside of the multiple cloning site has been deleted, making the Bgl II site within the multiple cloning site unique. A useful cell line that allows for episomal replication of expression vectors, such as pDC406 and pDC409, which contain the EBV origin of replication, is CV-1/EBNA (ATCC CRL 10478). The CV-L/EBNA cell line was derived by transfection of the CV-1 cell line with a gene encoding Epstein-Barr virus nuclear antigen-I (EBNA-1) and constitutively express EBNA-1 driven from human CMV immediate-early enhancer/promoter. Transformed host cells are cells which have been transformed or transfected with expression vectors constructed using recombinant DNA techniques and which contain sequences encoding a LeIF4A polypeptide of the present invention. Transformed host cells may express the desired LeIF4A polypeptide, but host cells transformed for purposes of cloning or amplifying LeIF4A DNA do not need to express the LeIF4A protein. Expressed LeIF4A proteins will preferably be secreted into the culture supernatant, depending on the DNA selected, but may also be deposited in the cell membrane. Suitable host cells for expression of recombinant proteins include prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or Bacilli. Higher eukaryotic cells include established cell lines of insect or mammalian origin as described below. Cell-free translation systems could also be employed to produce LbeIF4A proteins using RNAs derived from the DNA constructs disclosed herein. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, by Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985. Prokaryotic expression hosts may be used for expression of LeIF4A polypeptides that do not require extensive proteolytic and disulfide processing. Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although other hosts may also be employed. Recombinant LeIF4A polypeptides may also be expressed in yeast hosts, preferably from the Saccharomyces species, such as S. cerevisiae. Yeast of other genera, such as Pichia or Kluyveromyces may also be employed. Yeast vectors will generally contain an origin of replication from the 2.mu. yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the LeIF4A polypeptide, sequences for polyadenylation and transcription termination and a selection gene. Preferably, yeast vectors will include an origin of replication and selectable marker permitting transformation of both yeast and E. coli , e.g., the ampicillin resistance gene of E. coli and the S. cerevisiae trp1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, and a promoter derived from a highly expressed yeast gene to induce transcription of a structural sequence downstream. The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Suitable yeast transformation protocols are known to those of skill in the art. An exemplary technique described by Hind et al. (Proc. Natl. Acad. Sci. USA 75:1929, 1978), involves selecting for Trp.sup.+ transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 mg/ml adenine and 20 mg/ml uracil. Host strains transformed by vectors comprising the ADH2 promoter may be grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4.degree. C. prior to further purification. Various mammalian or insect (e.g., Spodoptera or Trichoplusia) cell culture systems can also be employed to express recombinant protein. Baculovirus systems for production of heterologous proteins in insect cells are reviewed, for example, by Luckow and Summers, Bio/Technology 6:47, 1988. Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, CV-1/EBNA (ATCC CRL 10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), COS, NS-1, HeLa and BHK cell lines. Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Purified LeIF4A polypeptides may be prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts. For example, supernatants from systems which secrete recombinant protein into culture media may be first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate may be applied to a suitable purification matrix. For example, a suitable affinity matrix may comprise a counter structure protein (i.e., a protein to which LeIF4A binds in a specific interaction based on structure) or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Gel filtration chromatography also provides a means of purifying LeIF4A. Affinity chromatography is a particularly preferred method of purifying LeIF4A polypeptides. For example, a LeIF4A polypeptide expressed as a fusion protein comprising an immunoglobulin Fe region can be purified using Protein A or Protein G affinity chromatography. Moreover, a LeIF4A protein comprising a leucine zipper domain may be purified on a resin comprising an antibody specific to the leucine zipper domain. Monoclonal antibodies against the LeIF4A protein may also be useful in affinity chromatography purification, by utilizing methods that are well-known in the art. Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media (e.g., silica gel having pendant methyl or other aliphatic groups) can be employed to further purify a LbeIF4A protein composition. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein. Recombinant LeIF4A polypeptide produced in bacterial culture is preferably isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) may be employed for final purification steps. Microbial cells employed in expression of recombinant LeIF4A protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Fermentation of yeast which express LeIF4A polypeptide as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). This reference describes two sequential, reverse-phase HPLC steps for purification of recombinant human GM-CSF on a preparative HPLC column. Preparations of LeIF4A polypeptides synthesized in recombinant culture may contain non-LeIF4A cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover the LeIF4A protein from the culture. These components ordinarily will be of yeast, prokaryotic or non-human eukaryotic origin. Such preparations are typically free of other proteins which may be normally associated with the LeIF4A protein as it is found in nature in its species of origin. Automated synthesis provides an alternate method for preparing polypeptides of this invention having fewer than about 100 amino acids, and typically fewer than about 50 amino acids. For example, any of the commercially available solid-phase techniques may be employed, such as the Merrifield solid phase synthesis method, in which amino acids are sequentially added to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963.) Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied Biosystems, Inc. of Foster City, Calif., and may generally be operated according to the manufacturer's instructions. As an alternative to the presentation of LeIF4A polypeptides, the subject invention includes compositions capable of delivering nucleic acid molecules encoding an LeIF4A polypeptide or portion thereof. Such compositions include recombinant viral vectors (e.g., retroviruses (see WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (see Berkner, Biotechniques 6:616-627, 1988; Li et al., Hum. Gene Ther. 4:403-409, 1993; Vincent et al., Nat. Genet. 5:130-134, 1993; and Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994), pox virus (see U.S. Pat. No. 4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973)), naked DNA (see WO 90/11092), nucleic acid molecule complexed to a polycationic molecule (see WO 93/03709), and nucleic acid associated with liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA 84:7851. 1987). In certain embodiments, the DNA may be linked to killed or inactivated adenovirus (see Curiel et al., Hum. Gene Ther. 3:147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89:6094, 1992). Other suitable compositions include DNA-ligand (see Wu et al., J. Biol. Chem. 264:16985-16987, 1989) and lipid-DNA combinations (see Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989). In addition, the efficiency of naked DNA uptake into cells may be increased by coating the DNA onto biodegradable latex beads. In addition to direct in vivo procedures, ex vivo procedures may be used in which cells are removed from an animal, modified, and placed into the same or another animal. It will be evident that one can utilize any of the compositions noted above for introduction of LeIF4A nucleic acid molecules into tissue cells in an ex vivo context. Protocols for viral, physical and chemical methods of uptake are well known in the art. As noted above, the subject invention provides methods of using the polypeptides or related nucleic acid compositions disclosed herein for enhancing or eliciting immune responses. It has been found within the present invention that LeIF4A contains epitope(s) that stimulate proliferation of PBMCs from Leishmania-infected individuals. LbeIF4A also stimulates PBMCs from infected individuals to generate an exclusive Th1 cytokine profile. A Th1 response is characterized by the production of the cytokines interleukin-1 (IL-I), interleukin-2 (IL-2), interleukin-12 (IL-12) or interferon-.gamma. (IFN-.gamma.), as well as tumor necrosis factor-.alpha. (TNF-.alpha.). IL-12 is a heterodimeric molecule comprising p40 and p35 subunits, which must be coexpressed for the production of biologically active IL-12 p70. The p40 subunit is produced only by IL-12-producing cells and is induced in vitro and in vivo after bacterial and parasite stimulation, whereas the p35 subunit is both ubiquitous and constitutively expressed. Therefore, cells producing IL-12 also have a large excess (10-100 fold) of biologically inactive free p40 chains. The stimulation of IL-12 production is particularly significant as this cytokine has the ability to influence T cells towards a Th1 response (IFN-.gamma. and IL-2 production). The ability of a protein to stimulate IL-12 production is therefore an important adjuvant property. LeIF4A also stimulates a Th1 profile of mRNAs encoding IFN-.gamma., IL-2, IL-12 p40 subunit, and TNF-.alpha., in PBMCs from Leishmania infected patients. No detectable IL-4 or IL-10 mRNA, indicative of a Th2 response, is present in such stimulated PBMCs. In fact, LeIF4A generally down-regulates the expression of such Th2-associated cytokines. In addition, LeIF4A stimulates expression of IL-18 mRNA. These properties of LeIF4A suggest a role for LeIF4A in generating a protective or therapeutic immune response in leishmaniasis patients. In addition, LeIF4A stimulates the production of IL-12 and IL-2 in PBMCs obtained from uninfected control individuals, as well as in cultured human macrophages, in the human myeloid leukemia cell line THP-1 and in mice. LeIF4A also synergizes with IFN-.gamma. to stimulate THP-1 cells to secrete IL-12, and the induction of IFN-.gamma. production by patient PBMCs is abrogated by the presence of anti-IL-12 antibody. The ability to stimulate IL-12 and IL-2 production indicates that LeIF4A has the ability to induce an immune response, and that the polypeptides described herein have a wide applicability in the non-specific enhancement of immune responses. Accordingly, the present invention discloses methods for enhancing or eliciting, in a patient or cell culture, a cellular immune response (e.g. the generation of antigen-specific cytolytic T cells). The present invention also discloses methods for enhancing or eliciting a humoral immune response to an antigen (e.g., antigen-reactive antibody production) using a LeIF4A polypeptide (i.e., LbeIF4A, LmeIF4A or a variant thereof) as described above. As used herein, the term "patient" refers to any warm-blooded animal, preferably a human. A patient may be afflicted with a disease, such as leishmaniasis (or other infectious diseases) or cancer, such as melanoma, breast cancer, prostate cancer, lymphoma, colon cancer or other tumor, or may be normal (i.e., free of detectable disease and infection). A patient may also, or alternatively, be afflicted with any Th2-mediated disease including, but not limited to, asthma, allergy, Th2-mediated autoimmune disease or Helminth infection. A "cell culture" is any preparation of PBMCs or isolated component cells (including, but not limited to, macrophages, monocytes, B cells and dendritic cells). Such cells may be isolated by any of a variety of techniques well known to those of ordinary skill in the art (such as Ficoll-hypaque density centrifugation). The cells may (but need not) have been isolated from a patient afflicted with leishmaniasis, or another disorder, and may be reintroduced into a patient after treatment. Within these methods, the LeIF4A polypeptide (or nucleic acid composition) is administered to a patient or cell culture along with an antigen, such that it functions as an immunomodulating agent to enhance or elicit the immune response to the antigen. Within certain embodiments, one or more Th1-associated cytokines (e.g., IL-2, IL-12, IL-15 and/or IL-18) may also be administered in combination with the LeIF4A polypeptide. The LeIF4A polypeptide may be administered within the same preparation (e.g., vaccine) as the antigen, or may be administered separately. In one embodiment, the antigen and the LeIF4A polypeptide are administered to a patient at the same time and site. In this manner, LeIF4A polypeptides may be used, for example, as adjuvants in vaccine preparations for heterologous agents. In another embodiment, the antigen and LeIF4A polypeptide are administered at different sites on the patient. For example, the LeIF4A polypeptide could be administered (e.g., injected) in one arm, and the antigen administered in the other arm. Such administrations may, but need not, take place at the same time. Alternatively, the LeIF4A polypeptide may be administered before or after the antigen. For example, the LeIF4A polypeptide could be administered 24 hours prior to antigen administration. Suitable doses and methods of administration are presented in detail below. The immune response generated by a patient to whom a LeIF4A polypeptide is administered may vary, depending on the condition of the patient. For Leishmania-infected patients, the immune responses that may be generated include a preferential Th 1 immune response (which includes stimulation of IL-12 production) and the down-regulation of expression of Th2-associated cytokines, such as IL-4, IL-5 and/or IL-10. For uninfected individuals, the immune response may be the production of IL-12, the production of IL-2, the stimulation of gamma T cells, the production of interferon, the generation of antigen-reactive CTL, the production of antigen-specific antibodies or any combination thereof. Either type of response provides enhancement of the patient's immune response to the antigen administered with the LeIF4A polypeptide. In addition, for patients with diagnosed cancer, such as melanoma, breast cancer, lymphoma, colon cancer, prostate cancer and the like, the immune response may include a preferential CTL response. For treatment of a tumor, the immune response should result in a reduction in tumor mass. The LeIF4A polypeptide (or nucleic acid composition) is preferably formulated for use in the above methods as a pharmaceutical composition or a vaccine. Pharmaceutical compositions generally comprise one or more LbeIF4A polypeptides in combination with a physiologically acceptable carrier, excipient or diluent. Such carriers will be nontoxic to recipients at the dosages and concentrations employed. The vaccines comprise one or more LbeIF4A polypeptides and one or more additional antigens appropriate for the indication. The use of LbeIF4A proteins in conjunction with soluble cytokine receptors, cytokines, and chemotherapeutic agents is also contemplated. Routes and frequency of administration and polypeptide (or nucleic acid composition) doses will vary from individual to individual and may parallel those currently being used in immunization or treatment of other infections. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. The amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth. Typically, between 1 and 4 doses may be administered for a 2-6 week period. Preferably, two doses are administered, with the second dose 2-4 weeks later than the first. A suitable dose is an amount of LeIF4A polypeptide that stimulates the production of IL-12 in the patient, such that the amount of IL-12 in supernatants of PBMCs isolated from the patient is between about 10 ng and pg per mL. In general, the amount of IL-12 may be determined using any appropriate assay known to those of ordinary skill in the art, including the assays described herein. The amount of LbeIF4A polypeptide present in a dose typically ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 .mu.g. Suitable dose sizes will vary with the size of the animal, but will typically range from about 0.01 mL to about 5 mL for 10-60 kg animal. Specific appropriate dosages for a particular indication can be readily determined. Alternatively, cells, preferably peripheral blood mononuclear cells, are removed from a patient and stimulated in vitro with one of the LeIF4A polypeptides and an antigen (including a tumor antigen). Upon generation of an antigen-specific immune response, such as a CTL response, the cells may be expanded and reinfused into the patient. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration and whether a sustained release administration is desired. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, 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) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109 and in U.S. patent application Ser. Nos. 08/116,484 and 08/116,802 (incorporated by reference herein). The polypeptide or polypeptide/antigen combination may be encapsulated within the biodegradable microsphere or associated with the surface of the microsphere. In this regard, it is preferable that the microsphere be larger than approximately 25 microns. Pharmaceutical compositions and vaccines may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. Preferably, product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Optionally, any of a variety of additional agents may be employed in the vaccines or pharmaceutical compositions of this invention, in addition to the LeIF4A polypeptide, to further nonspecifically enhance the immune response. Such agents usually 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, Bortadella pertussis or Mycobacterium tuberculosis. Such agents are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). |
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