WO2006058243A2

WO2006058243A2 - Leishmania antigens and related compositions and uses

Info

Publication number: WO2006058243A2
Application number: PCT/US2005/042795
Authority: WO
Inventors: Diane Mcmahon-Pratt
Original assignee: Yale University
Priority date: 2004-11-24
Filing date: 2005-11-23
Publication date: 2006-06-01
Also published as: WO2006058243A3

Abstract

The disclosure provides methods and formulations for eliciting an immune response to a Leishmania protein in a mammal, particularly a human or a canine.

Description

LEISHMANIA ANTIGENS AND RELATED COMPOSITIONS AND USES

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 60/630,986, filed November 24, 2004, entitled "Vaccines, Diagnostics, Treatments and Delivery Systems for Leishmaniasis." The provisional application is incorporated by reference herein.

STATEMENT REGARDING FEDERAL FUNDING Work described herein was funded, in part, by the National Institutes of

Health Grant Nos. AI45044 and AI27811. The United States government has certain rights in the invention.

BACKGROUND The leishmanias are a group of vector-borne parasitic diseases caused by protozoa of the genus Leishmania. The disease is endemic in the tropics, subtropics and southern Europe. The clinical forms range from the self-healing cutaneous leishmaniasis (CL) to visceral leishmaniasis (VL) in which the parasite spreads into the reticuloendothelial system with fatal outcome. The causative parasite is transmitted by sandflies. The sandfly vector becomes infected when feeding on the blood of an infected individual or an animal reservoir host. The leishmania parasites live in the macrophages as round, non- motile amastigotes (3-7 micrometers in diameter). The macrophages are ingested by the fly during the blood-meal and the amastigotes are relesed into the stomach of theinsect. Almost immediately, the amastigotes transform into the motile, elongated (10-20 micrometers), flagellate promastigote form. The promastigotes then migrate to the alimentary tract of the fly, where they multiply in the extracellular space. Four to five days after feeding, the promastigotes move to the esophagus and the salivary glands of the insect. When the sandfly next feeds on a mammalian host, the proboscis pierces the skin and saliva containing anti-coagulant is injected into the wound along with the leishmania promastigotes. Once in the host, the promastigotes are taken up by macrophages where they rapidly revert to the amastigote form. The leishmania are resistant to the microbicidal action of the macrophage acid hydrolases, allowing the parasites to survive and multiply inside the macrophages, eventually lysing the host cells. The released amastigotes are taken up by additional macrophages, thus continuing the infective cycle. Ultimately all organs containing macrophages and phagocytes are infected, especially the spleen, liver and bone marrow. The World Health Organization (WHO) has estimated that some 12 million people worldwide are infected and some 350 million people are at risk of infection. In Mediterranean countries the infection is zoonotic and the domestic dog is the main reservoir. The endemic strain of Leishmania in the Mediterranean area is Leishmania infantum, which infects both humans and dogs, producing cutaneous and visceral leishmaniasis.

Although drug treatment exists for VL, alternative approaches for the control of this disease, including improved vaccine technology, are still needed.

SUMMARY OF THE INVENTION In part, the disclosure provides single antigens and combinations of antigens that may be used to elicit immune responses to leishmania, and to generate vaccines for leishmaniasis, particularly for VL. The disclosure further demonstrates that NKT-active agents may be used as effective adjuvants to enhance the immune response to a leishmania antigen in both CL and VL. In certain aspects, the disclosure provides formulations for eliciting an immune response to a leishmania antigen in a mammal, and, in a preferred embodiment, such formulation is a vaccine. A formulation may comprise a single antigen, or at least two antigens. Antigens may be selected from the group consisting of: GP46, P4, GP63, P36-LACK and P8. A formulation may also comprise at least two nucleic acids encoding at least two antigens selected from the group consisting of: GP46, P4, GP63, P36-LACK. The antigen-encoding nucleic acids may be joined into a single construct, such as a vector, and may even be joined so as to express two or more antigens as a single fusion protein. A formulation may also comprise one or more antigens selected from the group consisting of: GP46, P4, GP63, P36-LACK and P8, and one or more nucleic acids encoding an antigen selected from the group consisting of: GP46, P4, GP63, P36-LACK. GP46, P4, GP63 and P36-LACK are polypeptides and may therefore be used as complete proteins, fusion proteins, or antigenic portions thereof. Variants and fragments of the antigens, and the encoding nucleic acids, are also acceptable for use insofar as such variants and fragments elicit an immune response. Certain P4 polypeptides disclosed herein are novel P4 polypeptides from L. infantum, and accordingly, the disclosure provides isolated or purified polypeptides comprising the amino acid sequence of SEQ ID NO.l or SEQ ID NO:2, as well as formulations comprising such protein or an immunogenic portion thereof. The antigens may be derived from any Leishmania spp. For example, antigens may be derived from a causative agent for visceral leishmaniasis. Preferred antigens may be derived from an organism selected from the group consisting of: Leishmania donovani, Leishmania pifanoi, Leishmania chagasi, Leishmania amazonensis, Leishmania mexicana, Leishmania tropica and Leishmania infantum. A formulation may further comprise an adjuvant, such as an NKT-active adjuvant. An adjuvant may be selected from the group consisting of: a glycosyl ceramide, a P-8 antigen and P. acneslC. parvmn.

In certain aspects, the disclosure provides formulations for eliciting an immune response to a leishmania protein in a mammal, particularly vaccines, wherein such formulations may comprise an NKT-active adjuvant and an antigen from a causative agent for leishmaniasis (or a nucleic acid encoding such antigen). The adjuvant may be, for example, glycosyl ceramide. The glycosyl ceramide may be selected from the group consisting of: a c-GalCer and an alpha-GalCer. Another example of an adjuvant is the P-8 antigen.

In certain aspects, the disclosure provides a formulation for eliciting an immune response to a leishmania protein in an outbred canine, particularly a vaccine, the formulation comprising an antigen selected from the group consisting of: D-13, D-2, D-6/D-15, and D-14. The outbred canine maybe a domestic dog. D- 13, D-2, D-6/D-15, and D-14 maybe used as complete antigens or as antigenic portions thereof. The antigens may be derived from any Leishmania spp. For example, antigens may be derived from a causative agent for visceral leishmaniasis. Preferred antigens may be derived from an organism selected from the group consisting of: Leishmania donovani, Leishmania pifanoi, Leishmania chagasi, Leishmania amazonensis, Leishmania mexicana, Leishmania tropica and Leishmania infantum. A formulation may further comprise an adjuvant, such as an NKT-active adjuvant. An adjuvant may be selected from the group consisting of: a glycosyl ceramide, a P-8 antigen and P.acneslC. parvum. In certain aspects, the disclosure provides methods for eliciting an immune response to a leishmania protein in a mammal by administering, to the mammal, any of the antigens, nucleic acids or formulations disclosed herein. The antigen and/or nucleic acid may be administered as a single formulation or may be administered separately. In certain aspects, the disclosure provides methods for eliciting an immune response to a leishmania protein in a mammal, the method comprising administering to a subject an NKT-active adjuvant and an antigen from a causative agent for leishmaniasis. hi certain aspects, the disclosure provides methods for reducing the likelihood that a mammal will contract leishmaniasis, particularly a visceral leishmaniasis, by administering, to the mammal, any of the vaccines disclosed herein. The vaccine may be administered as a single formulation or may include a plurality of components that are administered together or separately.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Wild type (C57BL/6) and β₂M^"Λ (beta-2 microglobulin knockout) mice were immunized with 9 ^" -%+C.parvumlP. acnes (adjuvant), adjuvant alone, or not immunized, and then infected with 1x10⁵ L. amazonensis parasites and (A) development of the lesion monitored with time as the ration of the size of the infected/non-infected foot as measured with a micrometer. (B) Parasite burdens were determined at 10 weeks post-infection. Each bar in the graph is an averaged value from 6 mice in panel A and 3 mice in panel B. Figure 2. Wild type (C57BL/6) and IFNγ^"7" (interferon-gamma knockout) mice were immunized with ?-8+C.parvum/P. acnes (adjuvant) or adjuvant alone, or not immunized (Control). Mice were infected 5 weeks after the final immunization in the right hind foot with IxIO⁵ L. amazonensis parasites. Parasite burdens were determined at 10 weeks post-infection. Each bar is the mean value from 3 mice.

Figure 3. Mice (5-6/group) were immunized 3 times at biweekly intervals with P- 8+C.parvum/P. acnes (adjuvant), adjuvant alone, or not immunized, and 7 weeks after the final immunization mice were infected with 10⁵ L. amazonensis. Lesion development was monitored, and 10 weeks post-infection parasite burdens (3 mice/group) were determined.

Figure 4. Protection against visceral leishmaniasis in LACK DNA-LACK vaccinia virus-vaccinated mice. Shown are the results of parasite burden analyses of BALB/c mice vaccinated with a prime-boost regimen (DNA genes and recombinant vaccinia virus [VV]) using the LACK antigen and then infected intradermally with L. infantum promastigotes. Parasite burdens were determined using limiting dilution analyses and represent the averaged values for at least four mice/group. (A) Spleen; (B) liver; (C) lymph node. Statistical analyses were performed using Student's t test comparing vaccine groups to a vector control group (pCI-neo-WRLuc). ***, P < 0.001; **, P < 0.01; *, P < 0.05. PBS, phosphate-buffered saline. Figure 5. Antigen-specific IFN-γ, TNF-α, and IL-10 responses in prime-boost vaccinated mice prior to infection. Shown are the IFN-γ, TNF- α, and IL-IO responses found for the vaccinated and control groups of mice (as indicated) prior to infection. The results of (A) ELISAs of IFN-γ from culture supernatants of splenic cells in response to recombinant LACK antigen, (B) corresponding enzyme-linked immunospot analyses of IFN-γ, and ELISAs of (C) TNF-α /LT and (D) IL- 10 are shown. PBS, phosphate-buffered saline.

Figure 6. Shown are (A) the parasite burdens in the draining lymph nodes in LACK (DNA-VV) vaccinated mice infected with L. major, and (B) lesion development over 13 weeks of infection. Figure 7. EFN-γ production by LACK-stimulated splenocytes prior to infection with Leishmania infantum. Figure 8. Parasite burden five weeks post-infection in (A) the spleen and liver, and (B) the lymph node.

Figure 9. BALB/c mice were vaccinated with either D- 13 or P-8 and infected 5 weeks after the last immunization with 10⁴ L. infantum intravenously. Parasite burdens in the liver (panel A, left) or spleen (panel A, right) were evaluated one month after infection. Alternatively, BALB/c mice were infected with 10⁵ L. infantum intravenously. Parasite burdens in the liver were evaluated 1 month after infection.

Figure 10. BALB/c mice were vaccinated with either D- 13 or P-8 and infected 5 weeks after the last immunization with 10⁵ L. infantum intravenously. Parasite burdens in the liver (panel A, left) or spleen (panel A, right) were evaluated 4.5 months after infection. Alternatively, BALB/c mice were infected with 10⁴ L. infantum intravenously. Parasite burdens in the spleen were evaluated 4.5 months after infection. Figure 11. Comparison of P4 amino acid sequences from six different Leishmania species. (SEQ ID Nos. 1-6). SEQ ID No. 1, labeled "453Al" is from Leishmania infantum. SEQ ID No. 2, labeled "Linf29" is from Leishmania infantum. SEQ ID No. 3, labeled "L.pif" is from Leishmania pifanoi. SEQ ID No. 4, labeled "Lmajcl" is from Leishmania major. SEQ ID No. 5, labeled "Lama" is from Leishmania amazonensis. SEQ ID No. 6, labeled "Ldcl" is from Leishmania chagasi.

Figure 12. Comparison of LACK amino acid sequences from six different Leishmania species. The sequences, obtained from public databases, are identical. (SEQ ID No. 7).

Figure 13. Comparison of GP46 amino acid sequences from six different Leishmania species. (SEQ ID Nos. 8-13). SEQ ID No. 8, labeled "MAQQP2" is from Leishmania donovani. SEQ ID No. 9, labeled "GP46/M2" is from Leishmania amazonensis. SEQ ID No. 10, labeled "PSA-2" is from Leishmania major. SEQ ID No. 11, labeled "PSA.1" is from Leishmania tropica. SEQ ID No. 12, labeled "Li PSA" is from Leishmania infantum. SEQ ID No. 13, labeled "Lch" is from Leishmania chagasi. Figure 14. Comparison of GP63 amino acid sequences from six different Leishmania species. (SEQ ID Nos. 14-19). SEQ ID No. 14, labeled "C fasc" is from Crithidia fascicidata. SEQ ID No. 15, labeled "L amaz" is from Leishmania amazonensis. SEQ ID No. 16, labeled "L chag" is from Leishmania chagasi. SEQ ID No. 17, labeled "L don" is from Leishmania donovani. SEQ ID No. 18, labeled "L maj" is from Leishmania major. SEQ ID No. 19, labeled "L mex" is from Leishmania mexicana.

DETAILED DESCRIPTION OF THE INVENTION 1. Leishmania AntiRens

In certain aspects, the disclosure provides leishmania antigens that may be used singly or in combination to provide effective vaccines or other formulations that elicit an immune response in a mammal, particularly a canine or a human. A vaccine will generally provide protection to the vaccinated mammal against future challenges with a leishmania parasite. A combination may include two or more of the following antigens: P4, P8, LACK, GP46 and GP63. Of these, P4, LACK, GP46 and GP63 are polypeptides. P8 is a glycolipid, the preparation of which is described below. Polypeptide antigens 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 Nos. 1-6 (P4), SEQ ID No. 7 (LACK), SEQ ID Nos. 8-13 (GP46) and SEQ ID Nos. 15-19 (GP63). Preferred antigens are those from a causative agent for visceral leishmaniasis, including, for example, SEQ ID Nos. 2, 7, 12 and 17. Polypeptide antigens maybe used as full- length proteins or as truncated portions, so long as immunogenic activity is retained. For example, polypeptide antigens corresponding to 25%, 50%, 75%, 85%, 95% or more of the subject protein may be used.

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 mammal (such as a human or a dog). 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). The 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 a native protein in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is not substantially diminished. The ability of a variant to react with antigen-specific antisera may be enhanced or unchanged, relative to the native protein, or may be diminished by, for example, less than 50%, and preferably less than 20%, relative to the native protein. Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein. Certain variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other preferred variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein. With respect to GP46, one or more repeat portions (as indicated in Figure 13) may be removed or used in duplicate or higher multiples. Polypeptide variants encompassed by the present invention include those exhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity (determined as described below) to the polypeptides disclosed herein. A variant may contain conservative substitutions. 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. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (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, tip, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the immunogenicity of the encoded polypeptide is not diminished. Immunogenic polypeptides may be produced as fusion proteins. A fusion protein comprises at least one of the immunogenic polypeptides disclosed herein and one or more additional sequences, particularly an immunogenic sequence from Leishmania, 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.

Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non- fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in frame. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

When comparing polynucleotide or polypeptide sequences, two sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted using

BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. MoI. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

The polypeptides to be used herein may be administered as polypeptides, or by introduction of the encoding nucleic acid into the individual. Mixtures of nucleic acids and proteins may also be prepared and administered, or nucleic acid and protein components may be administered separately. Administration of DNA is advantageous because DNA itself acts as an adjuvant, stimulating certain immune responses. As demonstrated herein, DNA- vaccinia virus delivery systems are effective for producing the desired immune response. See Example 3. The polynucleotides encoding the polypeptide antigens disclosed herein, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length maybe employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative DNA segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention. Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides may bear limited homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that differ from one another as a result of one or more deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison). In general, Leishmania antigens having immunogenic properties, and DNA sequences encoding such antigens, 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. braziliemis, L. panamensis, L. mexicana, L. tropica, and L. guyanensis. Such species are available, for example, from the American Type Culture Collection (ATCC), Rockville, Md.

The immunogenic properties of the 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. The response to be measured may be the secretion of one or more cytokines (such as interferon-gamma; DFN-γ), and/or tumor necrosis factor-alpha (TNF-α) or the change in the level of mRNA encoding one or more specific cytokines.

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. For example, an immunogenic portion of an antigen may generate at least about 25%, and preferably at least 25 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. Polypeptides may also be isolated directly from cultures of the parasitic organism. 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. For example, 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. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/ Applied BioSystemsDivision, Foster City, Calif., 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. For example, 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.

In general, 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 polynucleotide that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, 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. For example, 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. P4 has been purified as a membrane-associated Leishmania pifanoi amastigote protein and has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. Homologues of the P-4 gene are found in all other species of the genus Leishmania that have been examined. The P-4 protein is related to the Pl zinc-dependent nuclease of

Penicillium citrinum and the C-terminal domain of the 3' nucleotidase of Leishmania donovani. Purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity. P-4 protein is localized in the endoplasmic reticulum of the amastigote. The gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. See, e.g., Kar et al., J Biol Chem. 2000 Dec l;275(48):37789-97.

P36-LACK Antigen (LACK), which is expressed by both promastigotes and amastigotes, has been shown to protect mice from infection. LACK is also the major target for Th2 responses in susceptible BALB/c mice, and BALB/c mice made tolerant to LACK are resistant to infection.

GP46 Antigen is also known as gp46/M2 or parasite surface antigen 2. As with gp63, PSA-2 belongs to a multigene family expressed in all Leishmania species except L. braziliensis. Similar but distinct gene products are found in amastigotes and promastigotes of L. major and L. donovani, but in L. mexicana expression seems to be restricted to promastigotes. This antigen provides cross-protection between species. Immunization with the L. donovani GP46 protects mice against infection with L. major and that, conversely, immunization with the L. major proteins afforded partial protection against infection with L. donovani. Recombinant DNA- derived GP46 protein is variable in its ability to confer protection, while the protein derived from the yeast Pichia pastoris provides good protection. GP63 is also known as leshmaniolysin. It is a glycoprotein membrane protease with an Mr 65,000 that is present in promastigotes of all species. GP63 is one of the parasite receptors for host macrophages, and parasite mutants lacking the protein are avirulent. In certain embodiments, the disclosure provides P-8 antigen for use as a component of a formulation for eliciting an immune response. As demonstrated herein, mice inoculated with P-8 are protected against infection with the VL casuative agent L. infantum. Accordingly, P-8 may be used as the sole antigen, or in combination with other antigens, in a formulation for eliciting an immune response. Additionally, P-8 has a surprisingly selective tendency to stimulate Natural Killer T cells (NKT). Thus, P-8 may be used in the role of an adjuvant in a formulation so as to assist in eliciting a protective immune response by eliciting an NKT response. While P-8 from a member of the L. donovani complex may be used as a preferred antigen in a formulation designed to elicit an immune response against VL causative agents, P-8 from other forms of parasite, such as the L. mexicana complex, may also be used in such a formulation as a general NKT-active adjuvant.

P-8 is a glycolipid complex from amastigote forms of Leishmania spp. P-8 maybe prepared as described in Soong et al., Infect Immun. 1995 Seρ;63(9):3559- 66, and in Colmenares et al., Infect Immun. 2001 Nov;69(l l):6776-84. In brief, P-8 antigen may be purified by obtaining surface membranes of Z,. pifanoi (or other Leishmania spp.) amastigotes. Membrane proteins are then solubilized, incubated with reducing agent (e.g., 2-mercaptoethanol) and subsequently alkylated by addition of iodoacetamide. The reduced and alkylated solubilized membranes may be fractionated by Sephadex G-25 (Pharmacia, Piscataway, NJ.) gel exclusion chromatography to remove the excess of reagents. The sample may then be subjected to P-8 immunoaffmity chromatography and eluted. Any available P-8 antibody may be employed in affinity purification, including the CXVI-3G11-ClO. See Pan et al. J Immunol. 1988 Apr l;140(7):2406-14 and Traub-Cseko et al. MoI Biochem Parasitol. 1993 Jan;57(l):101-15. In certain embodiments, the disclosure provides formulations for eliciting an immune response to a leishmania antigen in outbred canines, and particularly in outbred domestic dogs. Antibodies that react with antigens termed D- 13, D-2, D-6 and D- 14 were previously identified in inbred dogs challenged with L. donovani. Jaffe et al, J Immunol. 1984 JuI; 133(1 ):440-7. The antibodies disclosed therein can be used to prepare purified antigen by affinity purification from the membranes of L. donovani promastigotes. See, e.g., Ahmed et al., Infect Immun. 2003 Jan;71(l):401- 10; Rachamim et al., J. Immunol. 150:2322-2331; White et al., J. Infect. Dis. 161:1313-1314. Applicants have determined that these antigens are also immunogenic in outbred dog populations, which is a more reliable predictor of the likelihood that such an antigen will produce a desired immune response in the general domestic dog population.

2. Leishmania Adjuvants

As shown in the Examples, adjuvants that stimulate a NKT cell reaction ("NKT-active adjuvants") may be used as effective adjuvants in compositions for eliciting an immune response to a Leishmania antigen in a mammal. Examples of NKT-active adjuvants include P-8 and glycosyl ceramides. Given that alpha-GalCer is an agonist of the CDId receptor found on NKT cells, it is expected that other agonists of this protein will be effective as adjuvants for eliciting an immune response to a Leishmania antigen. It has been demonstrated that NKT cells can be activated both in vitro and in vivo by alpha-galactosyl-ceramide (alpha-GalCer), a glycolipid originally extracted from Okinawan marine sponges (Natori et al., Tetrahedron, 50:2771-2784, 1994) or its synthetic analog KRN 7000 [(2S,3 S,4R)-l-0-(.alρha.-D-galactoρyranosyl)-2-(N- hexacosanoylamino)-l,3- ,4-octadecanetriol] which can be obtained from Pharmaceutical Research Laboratories, Kirin Brewery (Gurnna, Japan) or synthesized as described previously (see, e.g., Kobayashi et al. 1995, One. Res. 7:529-534).

In one aspect, the disclosure provides compositions for eliciting an immune response to a Leishmania antigen in a mammal, comprising administering said antigen conjointly with an adjuvant composition comprising a glycosyl ceramide. For examples of glycosyl ceramides, see U.S. Pat. Pub. Nos. 20050222048 and 20050192248 and U.S. Pat. Nos. 6,747,010; 6,555,372; 6,531,453; 6,417,167; 6,071,884; 5,936,076; 5,849,716; 5,780,441; and 5,767,092. Preferred glycosyl ceramides are alpha-GalCer and the more hydrophobic form c-GalCer.

Various glycosyl ceramides are defined as follows (U.S. Pat. No. 6,747,010):

wherein:

R¹ represents H or OH,

X represents an integer between 7 and 27,

R² represents a substituent selected from the group consisting of the following (a) to (e) (wherein Y represents an integer between 5 and 17):

(a) -CH₂ (CH₂)γCH₃

(b) -CH(OH)(CH₂)γCH₃

(c) -CH(OH)(CH₂)_YCH(CH₃)₂

(d) -CH=CH(CH₂)γCH₃

(e) -CH(OH)(CH₂)YCH(CH₃)CH₂CH₃, and

R to R represent substituents as defined in any one of the following i) to v): i) when R³, R⁶ and R⁸ represent H,

R⁴ represents H, OH, NH₂, NHCOCH₃, or a sugar (preferably a pentose or hexose);

R⁵ represents OH or a sugar (preferably a pentose or hexose derivative); R⁷ represents OH or a sugar (preferably a pentose or hexose); R⁹ represents H, CH₃, CH₂OH or a sugar (preferably a pentose or hexose); ii) when R³, R⁶ and R⁷ represent H,

R⁵ represents OH or a sugar (preferably a pentose or hexose);

R⁸ represents OH or a sugar (preferably a pentose or hexose);

R⁹ represents H, CH₃, CH₂OH or a sugar (preferably a pentose or hexose); and pharmaceutically acceptable salts or esters thereof.

In a preferred embodiment, a glycosyl ceramide is (2S,3S,4R)-l-(αD- galactopyranosoyloxy)-2-hexacosanoylamino-3,4-octadecanediol.

Modified glycosyl ceramides that have increased hydrophobicity (generally, c-GalCer) are described in U.S. Pat. Pub. No. 20050222048 as compounds of formulae I, II and III:

wherein X is O or NH;

R³ is OH or a monosaccharide and R⁴ is hydrogen, or R³ is hydrogen and R⁴ r a monosaccharide;

R⁵ is hydrogen or a monosaccharide; and pharmaceutically acceptable salts or esters thereof.

A compound of formula I is, for example, formula I-a:

A compound of formula II is, for example, formula II-a:

(II-a) Compounds of formula III is, for example, are shown as IΙI-a(cis) and III- a(trans).

(III-a)(trans) A preferred c-GalCer is α-C-GalCer [(2S,3S,4R)-l-CH2-(-D- galactopyranosyl)-2-(N-hexacosanoylamino)- 1 ,3,4-octadecanetriol] (see, e.g., Schmieg et al. J Exp Med. 2003 Dec 1;198(11):1631-41). According to the present invention, the use of an NKT-active adjuvant, such as P-8 or glycosyl ceramide, results in an enhancement and/or extension of the duration of the protective immunity induced by the antigen, whether administered as a protein or as a nucleic acid encoding the protein. For example, as disclosed herein, conjoint administration of such adjuvants with polypeptide antigens or nucleic acid encoding polypeptide antigen increases the protection against leishmania challenge in many different tissues. An adjuvant and antigen can be administered either as two separate formulations or as part of the same composition. If administered separately, the adjuvant and antigen can be administered either sequentially or simultaneously. Simultaneous administration of adjuvant with the antigen is preferred and generally permits the most efficient immunostimulation.

The adjuvant of the invention can be administered as part of a pharmaceutical or vaccine composition comprising an antigen or as a separate formulation, which is administered conjointly with a second composition containing an antigen. In any of these compositions the compounds of the invention can be combined with other adjuvants and/or excipients/carriers, as described below.

3. Formulations Pharmaceutical compositions for eliciting an immune response to a leishmania polypeptide may comprise one or more antigens, such as P-8, antigenic leishmania polypeptides and/or encoding nucleic acids, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. Immunogenic compositions may comprise one or more of the above antigens and an immunostimulant, such as an adjuvant, particularly an NKT- active adjuvant, such as a glycosyl ceramide or a P-8 antigen. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A, and cytokines, such as GM-CSF, interleukm-2,-7,-12, and other like growth factors, may also be used as adjuvants.

Immunogenic compositions may additionally contain a delivery vehicle, such as a biodegradable microsphere (disclosed, for example, in U.S. Pat. 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 or immunogenic composition may contain an immunostimulant, such as an adjuvant, and DNA encoding one or more of the polypeptides or fusion proteins described above, such that the polypeptide is generated in situ. In such compositions, 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 individual (such as a suitable promoter and terminating signal). In a preferred embodiment, 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. 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 uptake of naked DNA maybe increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. 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. 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.

Routes and frequency of administration, as well as dosage, for the above aspects of the present invention will vary from individual to individual. In general, the pharmaceutical 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. 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. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 100 ng to about 1 mg per kg of host, typically from about lOμg to about lOOμg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

The disclosure also provides a pharmaceutical pack or kit comprising one or more of the ingredients of the immunogenic formulations of the invention, in a related embodiment, the present invention provides a kit for the preparation of a pharmaceutical or vaccine composition comprising at least one antigen and an adjuvant compound, said kit comprising the antigen in a first container, and the adjuvant in a second container, and optionally instructions for admixing the antigen and the adjuvant and/or for administration of the composition. Each container of the kit may also optionally include one or more physiologically acceptable carriers and/or excipients and/or auxiliary substances. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human or canine administration.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient (i.e., an antigen and/or an adjuvant compound). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

The application will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present application, and are not intended to limit the application. EXAMPLES

Example 1. P-8 antigen protects against L. amazonensis in mice in an NK cell- dependent manner.

This example confirms that the P-8 antigen protects against L. amazonensis infection in mice. The mechanism of P-8 action was investigated in various knockout mouse strains. The data indicate that P-8 stimulates an NKT cell response which is necessary for protection against subsequent challenge with L. amazonensis.

NK cells are generally characterized by their ability to kill certain tumor cells without prior sensitization and to produce pro-inflammatory cytokines, especially interferon gamma (IFNγ), following activation. A unique subset of T cells, designated NKT cells, express the NKl.1 marker, as well as other typical NK receptors and upon stimulation through their TCR, rapidly produce substantial amounts of cytokines especially IL4. Unlike NK cells, NKT cells develop in the thymus and express a rearranged TCR. In contrast to typical T cells, NKT cells respond to antigen presented by the atypical MHC Class I molecule, CDlD, and express intermediate levels of TCR. In addition, NKT cells are either CD4+ or CD4- CD8-, in contrast to typical CD8+ Class I restricted T cells.

Transgenic mice which exhibit decreases in the level or function of NK or NK T cells can be used to probe the contribution of these cell types to certain immune responses. Cell surface expression of MHC class I (including CDlD) is dependent on association with Beta 2 microglobulin (β2M). Since Class I proteins are essential for the maturation of conventional Class I restricted CD8+ cytolytic T cells and CdId restricted NKT cells, mice homozygous for the null mutation of B2M have severe depletion of both conventional CD8+ T cells, as well as NKT cells. In this case there is an almost complete deletion of NKT cells, but the deletion is not specific. Complete and specific deletion of the NKT population is obtained in mice that are homozygous for the CdId targeted mutation. The CDId mutation leaves NK cells are unaffected. Incomplete loss of function is also found in perforin- deficient mice in which natural killing capability is absent, but other functions are likely to be intact. By using mice deficient for β₂M, IFN-γ, TAP-I, CDId and perforin, it was possible to demonstrate that NKT cells play a critical role in the protective effect of P-8 immunization.

Wild type (C57BL/6) and β₂M^"Λ (beta-2 microglobulin knockout) mice were immunized with P- 8+C.parvum/P. acnes (adjuvant), adjuvant alone, or not immunized, and then infected with IxIO⁵ L. amazonensis parasites. As shown in Fig. 1, P-8 provided substantial protection against parasite in wild type mice. In β₂M^"7" mice, P-8-mediated protection was attenuated. Cell surface expression of MHC class I receptors is dependent on β₂-microglobulin. Thus, the effectiveness of P-8 immunization depends upon MHC class I receptor activity. Wild type (C57BL/6) and IFNγ^"7" (interferon-gamma knockout) mice were immunized withP ^' -8+C.parvum/P. acnes (adjuvant) or adjuvant alone, or not immunized, and then infected with IxIO⁵ L. amazonensis parasites. As shown in Fig. 2, the protective effect of P-8 immunization was abolished in the IFNγ^{" "} mice.

Finally, mice with deficiencies in various proteins of the immune system were immunized with V-S+C.parvum/P. acnes (adjuvant) or adjuvant alone, or not immunized, and infected with 1x10⁵ L. amazonensis parasites. P-8 immunization was not dependent on TAP-I. TAP-I deficient mice have severely reduced levels of surface class I molecules, a reduced ability to present cytosolic antigens to class I - restricted cytotoxic T cells and a reduced number of CD4-8+ T cells. Accordingly, the P-8 protection appears to be independent of these components. By contrast,

CDId and perforin deficiencies caused substantial reductions in the effectiveness of the P-8 immunization. CDId is an essential component of the NKT cell response. Perforin is a component of the cell lysis machinery of NKT cells and other cytotoxic cells. See Fig. 3. Taken together, these data indicate that the P-8 antigen stimulates an NKT response against leishmania parasites. Therefore, it is expected that other agents that stimulate an NKT response will provide protective effects and may be useful as adjuvants in leishmania vaccines. This conclusion is further supported by experiments with the NKT-stimulating agents, glycosyl ceramides, presented in Example 3, below. Example 2. Heterologous Prime-Boost Vaccination with the LACK Antigen Protects against Murine Visceral Leishmaniasis

This example demonstrates the efficacy of a heterologous prime-boost vaccination using DNA and vaccinia viruses (Western Reserve [WR] virus and modified [attenuated] vaccinia virus Ankara [MVA]) expressing the LACK antigen (Leishmania homologue of receptors for activated C kinase) and an intradermal murine infection model employing Leishmania infantum.

Visceral leishmaniasis (VL) is a protozoan parasitic disease, fatal in the absence of treatment. Although drug treatment exists for VL, alternative approaches for the control of this disease (vector control, immunotherapeutic, chemotherapeutic, and vaccine) are still needed. Vaccine studies of VL have been less extensive, and the level of protection found is generally poorer than those found for murine cutaneous leishmaniasis. However, studies utilizing a murine intradermal infection model of VL indicate that this is, in part, due to the animal model employed. Immunological studies of the mechanisms of pathogenesis as well as immunotherapeutic studies of VL indicate tissue site-specific mechanisms (for the spleen, liver, and lymph node). Consequently, one of the challenges in the development of a vaccine against VL is the induction of protection at multiple and distinct tissue sites. The LACK antigen (Leishmania analogue of the receptors of activated C kinase) (36 kDa) is highly conserved among Leishmania species and expressed by both the promastigote and amastigote forms of the parasite. Studies indicate that DNA coding for the LACK antigen provides protection against Leishmania major. However, a LACK DNA vaccine failed to protect against L. mexicana. Further, a LACK DNA vaccine, although highly immunogenic, failed to protect against murine VL in either intradermal or intravenous infection, suggesting that LACK may not be a useful antigen for a general DNA-based vaccine against leishmaniasis.

A heterologous prime-boost regimen using DNA and vaccinia viruses expressing the LACK antigen has been shown to be highly immunogenic and protective against murine L. major infection. A heterologous prime-boost regimen using DNA and the replication-competent Western Reserve (WR) strain of vaccinia virus expressing the LACK antigen was recently explored in canine VL. However, the immune response in the canine model is known to differ significantly from those in the murine and human hosts of leishmaniae in terms of their regulation by interleukin-13 (IL-13), IL-12, and IL-IO. Previous leishmaniasis vaccine studies have demonstrated that the murine model can be predictive for vaccine outcomes in nonhuman-primate models. Therefore, in the current study, the potential of a prime- boost regimen using DNA-vaccinia virus was explored using the murine intradermal model for VL. In order to assess the potential use of this vaccination regimen against murine VL, the efficacies of priming were examined using an L. infantum DNA- LACK construct (previously employed for vaccine studies against cutaneous leishmaniasis caused by L. major) and the highly attenuated modified vaccinia virus Ankara (MVA) strain as well as the replication-competent WR strain, given the abilities of these viruses to induce both strong ThI and CD8+ T-cell responses.

BALB/c mice (4 to 6 weeks of age) were vaccinated intradermally with 100 μg of DNA encoding the LACK antigen (DNAp36) and then boosted 2 weeks later intraperitoneal^ with 1 x 10⁷ or 5 x 10⁷ PFU of either recombinant Western Reserve-wild-type (WR-LACK or WRρ36) or Ankara-MVA (MVA-LACK or MVAp36) vaccinia viruses expressing the LACK antigen. Three and one-half weeks after boosting, mice were infected intradermally in the ear pinnae using 10⁷ metacyclic promastigotes of L. infantum. One month after infection, the parasite burdens were evaluated by limiting dilution analysis in vaccinated and control groups of mice. This evaluation of protection in the spleen, the liver, and the draining lymph node demonstrated that the mice receiving a prime-boost vaccination using the LACK (p36) antigen were significantly protected against infection (Fig. 4). The levels of protection at each tissue site were comparable among the various vaccinated groups of mice and did not statistically differ between mice receiving the WRp36 or the MVAp36 virus. However, the level of protection did vary with the target organ site, with the highest levels of protection achieved in the draining lymph node (Fig. 4C). The level of protection in the draining lymph node was evidenced by a 144- to 244-fold reduction in the parasite burdens in comparison to those of control mice. Lower levels of protection were achieved when the parasite burdens were evaluated in the spleen and the liver. These results ranged from 6- to 9-fold reductions in parasite burdens in the liver and 9- to 30-fold reductions in the spleen, hi the spleen, a slight protective effect was also observed for the mice receiving control DNA and vaccinia virus (WR-Luc), which may be due to the low gamma interferon (IFN-γ) response observed for these mice (Fig. 5). However, in other vaccine experiments employing WR-Luc (107 PFU) an IFN-γ response and a reduction of the splenic parasite burden were not consistently observed, nor were reductions in parasite burdens observed in the livers and lymph nodes of the WR- Luc-vaccinated mice (Fig. 4). However, the differences between the parasite burdens observed for the WR-Luc-vaccinated mice and those for mice receiving WR-LACK or MVA-LACK were significant (P < 0.02 to 0.05) (Fig. 4A), demonstrating a LACK antigen-specific effect.

IFN-γ and tumor necrosis factor alpha/lymphotoxin (TNF-α/LT) have been found to be involved in resistance to infection in murine VL, while IL-10 correlates with susceptibility. The levels of IFN-γ, IL-10, and TNF-α/LT produced by spleen cells of vaccinated and nonvaccinated mice in response to LACK antigen were evaluated before infection and at 1 month after infection (Fig. 5). Before infection, mice receiving 10⁷ WRp36 or 5 x 10⁷ MVAp36 PFU appeared to produce somewhat higher levels of IFN-γ (100 to 113 ng/ml) than mice boosted with either 5 x 10⁷ WRp36 or 10⁷ MVAp36 PFU (55 to 67 ng/ml) (Fig. 5A). As shown in Fig. 5B, enzyme-linked immunospot analyses indicated that the number of IFN-γ-secretmg cells correlated with the levels of IFN-γ found by enzyme-linked immunosorbent assay (ELISA), with the frequency of IFN-γ -producing cells ranging from 380 to 640/10⁶ spleen cells. In addition, significant levels of TNF-α/LT (58 and 134 pg/ml) were observed for mice boosted with recombinant wild-type WRp36, while lower levels of TNF-α/LT were produced in response to LACK antigen by mice receiving MVAp36 (27 pg/ml and 8 pg/ml, respectively). These differences in the levels of induction of TNF-α may reflect, in part, the different abilities of WR and attenuated MVA vaccinia viruses to induce an inflammatory response and NF-κB activation, which result in distinct cytokine profiles. MVA has been shown to enhance NF-κB activation, while WR appears to inhibit it. The amounts of LACK-specific IL-IO produced by splenocytes before challenge varied from 0.1 ng/ml in mice boosted with 5 x 10⁷ WRρ36 PFU to 0.7 ng/ml in those receiving 10⁷ WRp36 or MVAp36 PFU (Fig. 5D).

The cytokine responses at 1 month postinfection paralleled but were somewhat higher than those found prior to infection. IFN-γ levels ranged from 20 ng/ml in mice receiving 5 x 10⁷ WRp36 PFU to 204 ng/ml in those boosted with 5 x 10⁷ MVAp36 PFU. The levels of TNF-α/LT in response to LACK antigen stimulation in vaccinated mice ranged from 64 pg/ml in mice boosted with 10⁷ MVAp36 PFU to 120 pg/ml in the group boosted with 5 x 10⁷ WRp36 PFU. Significant levels of IL-10 (0.04 ng/ml to 0.54 ng/ml) were also produced in response to LACK antigen at 1 month postinfection. Both the level of IFN-γ and the IFN-γ /IL-IO ratio found at 1 month postinfection appeared to correlate with the protection levels found (Table 1).

TABLE 1. Cytokine levels and corresponding reductions (n-fold) in parasite burden at 1 month postinfection

Both IFN-γ and TNF-α have been implicated in the macrophage killing of intracellular L. donovani, through the up-regulation of inducible NO synthase (iNOS) and production of nitrite oxide. Nitric oxide has been demonstrated to be critical for the leishmanicidal activity of murine macrophages (4, 16, 19) and also has been shown to enhance, at low levels, a ThI response. However, IL-IO is known to down-regulate macrophage NO production. To further determine the ongoing immune response dynamics in the vaccinated versus control mice, the induction of NO/nitrite at 1 month postchallenge was examined. Significant amounts of this antimicrobial agent were observed and ranged from 6 to 7 μM in the vaccinated mice, with control group NO/nitrite levels ranging from 0.6 μM to 1 μM. Therefore, the vaccinated mice, consistent with the findings for levels of IFN-γ (Fig. 5; Table 1), had higher levels of nitric oxide induction and potential leishmanicidal activity. These results were consistent with the protection found in the LACK-DNA-LACK- WR- or MVA-vaccinated mice. hi conclusion, this study demonstrated that a heterologous prime-boost regimen using DNA and vaccinia virus, both expressing the same antigen, LACK, is highly immunogenic and confers protection against murine L. infantum infection. Notably, the highly attenuated MVA and the replication-competent WR strain vaccinia viruses achieved comparable levels of protection. This heterologous prime- boost approach resulted in higher levels of IFN-γ (up to 200 ng/ml) than those that have been reported for DNA-DNA vaccination (6 to 12 ng/ml) (23), where protection was not achieved. This observation prompts a question as to the biologically effective amount of IFN-γ required to induce protection against VL. Although additional effector mechanisms may be involved, these results suggest that higher levels of IFN-γ may be required for protection against visceral disease than are needed against cutaneous leishmaniasis.

Materials and Methods for Example 2: Parasite strains and animals:

L. infantum was provided by Dr. Jorge Alvar (Centro Nacional de Microbiologia Instituto de Salud Carlos III, Majadahonda, Spain). Promastigotes were grown at 24°C in Schneider's Drosophila medium (Sigma, St. Louis, MO) supplemented with 20% heat inactivated fetal bovine serum and antibiotics. The virulence of the strain was preserved by periodic passage through BALB/c mice. All mice used in this study were female BALB/c mice of 4-6 weeks of age purchased from the NIH.

Plasmids, recombinant p36 protein and recombinant vaccinia viruses:

The gene encoding Z. infantum p36 protein was obtained and inserted downstream of the cytomegalovirus (CMV) promoter into the Smal site of the pCI- neo vector (Promega, Madison, WT). The empty plasmid pCI-neo (Promega) was used as control (DNA control). Plasmid DNA was purified using the Qiagen Endofree Plasmid Maxi Kit (Qiagen Inc., Valencia, CA) using pyrogen- free material and eluted in pyrogen-free deionized water. The endotoxin level of the purified plasmid was tested using LAL Kt (Bio-Wittaker, Walkersville, MD) before immunization. Preparations had a maximum of 0.1 ng LPS/μg DNA. rVVs were derived from the wild-type WR and MVA strains; MVA-LACK (MVAp36) and WRJLACK (WRρ36) were prepared using standard methods.

Prime-boost vaccination and infection of mice with L. infantum:

Six experimental groups of BALB/c mice (n=7) were constituted. Mice were primed intradermally with p36 (LACK) plasmid DNA or control vector DNA (100 μg/mouse in 100:1 of phosphate buffered saline). Mice were boosted intraperitoneally two weeks later (14 d.p.i.) with two different doses of WRp36 or MVAp36 (IxIO⁷ or 5xlO⁷ PFU). Mice immunized earlier with the control DNA were boosted with 5x10⁷ PFU of the control virus encoding for the luciferase gene (WR-Luc). Infection control mice received PBS alone. Three weeks after boosting (35 days after the initial immunization), three mice per group were sacrificed and serum, spleen ad draining lymph node were collected for immunological analysis. Three and a half weeks after viral boosting, the mice were infected intradermally on the ear pinnae with 1x10⁷ metacyclic L. infantum promastigotese. This method of infection, witch mimics the situations in vivo where ingestion is transmitted through the bite of a phlemobotomine sand fly, has been previously described and the results in successful visceralization of the parasite. Mice were sacrificed at one month post- challenge and draining lymph node, spleen, liver and serum were harvested for parasitological and immunological assays.

Antibody Response:

Antibody levels to LACK antigen were assessed by ELISA. Total IgG as well as IgGl and IgG2a isotype responses were evaluated for sera collected from each group of animals before challenge and at one month post-challenge. The ELISA was performed as previously described with some modifications. Briefly, 96-well Maxisorp plates (Nunc, USA) were coated overnight at 4⁰C with 100:1 of purified recombinant p36 proteins (5 μg/ml) or unstimulated. Supernatants were collected after 72 h of incubation and stored at -20 ⁰C until used. Cytokine levels were measured from culture supernatant by sandwich ELISA, according to manufacturers' specifications. Background cytokine levels were determined using supernatant from unstimulated cell populations.

ELISPOT Assay:

The ELISPOT assay to quantify LACK-specific IFN-γ producing cells from the spleen was conducted. The 96-well cellulose plats (Millipore, Bedford, MA) were coated with 6 μg/ml of anti-mouse IFN (BD-Pharmingen, San Diego, CA) monoclonal antibody and incubated overnight at room temperature. Subsequently, two-fold serial dilution of splenic cells (beginning with 10⁶) were added to each well in RPMI medium supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin. Recombinant p36 antigen was added (5 μg/ml) and the plates were incubated at 37°C with 5% CO2. After 72 h, the plates were washed and then incubated for 2 h at room temperature with 2 μg/ml of biotinylated anti-mouse IFN-γ (BD_Pharmingen, San Diego, CA) followed by incubation with 1/800 dilution streptavidin-horseradish peroxidase conjugate (Pharmingen, Palo Alto, CA). After 1 h at room temperature, plates were washed and the presence of IFN-γ producing cells was revealed by adding 1 mg/ml of fluorescent substrate. Measurement of nitrite/nitric oxide Nitric oxide (NO) production was measured as nitrite using the Greiss reaction. Briefly to each 96-well flat bottom microtiter plate (Nunc, USA) containing 50:1 of culture supernatant equal volumes of the Greiss reagents (1% sulfanilamide and 0.1% napthylethylenediamine di chloride in 2.5% phosphoric acid) were added and incubated at room temperature for 10 minutes. Standards of NaNO2 using concentrations from 1.56 to 100 μM NaNO2 were employed with each assay. Absorbance was measured at 540 nm with the Bio-tek plate reader (Bio-tek Instruments, Winooski, VT). Background concentrations were assessed using supernatant from unstimulated splenocytes. Evaluation of parasite burden:

The total number of parasites in the various infected tissues (spleen, liver and draining lymph node) was evaluated by limiting dilution analysis using Schneider's Drosophila medium. The individual parasite burdens from four mice per group were determined; results are expressed at the average ± standard errors of the parasite burdens obtained from each group.

Example 3. Glycosyl ceramides are effective adjuvants for immunization with leishmania antigens.

It has been demonstrated that NKT cells can be activated both in vitro and in vivo by alpha-galactosyl-ceramide (alpha-GalCer), a glycolipid originally extracted from Okinawan marine sponges (Natori et al., Tetrahedron, 50:2771-2784, 1994) or its synthetic analog KRN 7000 [(2S.3 S,4R)-l-0-(alpha-D-galactoρyranosyl)-2-(N- hexacosanoylamino)-l,3- ,4-octadecanetriol] which can be obtained from Pharmaceutical Research Laboratories, Kirin Brewery (Gumna, Japan) or synthesized as described previously (see, e.g., Kobayashi et al. 1995, One. Res.

7:529-534). Other glycosyl ceramides, particularly c-GalCer, have similar effects on NKT cells.

The data presented in this Example demonstrate that glycosyl ceramides act as effective adjuvants in the immunization of mice against antigen from the causative agents for cutaneous and visceral leishmaniasis. In particular, the data demonstrate that the protection elicited using a DNA-vaccinia virus prime-boost system with the LACK antigen (see Example 2) can be substantially increased using alpha-GalCer or c-GalCer during the DNA priming.

BALB/c mice were primed either with DNA-LACK (100 μg/mouse) alone or DNA-LACK (100 μg/mouse) and alpha-GalCer (2μg/mouse). They were boosted two weeks later with vaccinia virus expressing LACK (1x10⁷ pfu/mouse). Control mice received either PBS twice or control DNA and alpha-GalCer, and boosted with the control virus (VVluc). Mice were infected three weeks later in the right hind footpad with 5x10⁴ stationary phase promastigotes of L. major. Prior to challenge, immunological data were collected from immunized and control mice (3 mice/group). Parasite burden in the draining lymph nodes evaluated by limiting dilution assay 13 weeks post-infection showed significantly higher protection in the vaccinated mice primed with DNA-LACK and alpha-GalCer (2000-fold reduction in parasite burden versus controls) than those primed with DNA-LACK alone (258- fold reduction. See Fig. 6 A. The footpad lesion size was monitored weekly.

Although smaller compared to control groups, lesions of mice primed with DNA- LACK alone appeared progressive while those of mice primed with DNA-LACK and alpha-GalCer were healing at 13 weeks post-infection.

Cytokines such as IFN-γ, IL-10 and TNF-α were measured from in vitro LACK- and SLA- (soluble leishmanial antigen) stimulated spleen and lymph node cells. The pre-challenge and post-challenge levels of IFN-γ were significantly higher in the protectively vaccinated groups. An enhanced production of these cytokines was observed in the vaccinated mice receiving alpha-GalCer as adjuvant. Consequently, the enhanced protection correlates with the increased levels of IFN-γ and TNF-α produced in response to the antigen. Notably, the enhanced cytokine levels persist for as long as five months post-boost. Therefore these experiments suggest that activation of NKT cells results in enhanced effector (protection) and memory T cells and should be useful as an adjuvant for vaccine against cutaneous leishmaniasis. The effectiveness of alpha-GalCer and c-GalCer as adjuvants for a vaccine against visceral leishmaniasis was also examined. This disease is particularly challenging because the parasite disseminates to multiple tissue sites. In this experiment, BALB/c mice were primed with DNA-LACK (100 μg/mouse) alone, DNA-LACK (100 μg/mouse) and alpha-GalCer (2μg/mouse), DNA-LACK (100 μg/mouse) and c-GalCer diluted with either polysorbate (Tween20) or DMSO (2μg/mouse). Mice were boosted two weeks later with vaccinia virus expressing LACK (IxIO⁷ pfu/mouse). Control mice received either PBS twice or control DNA and alpha-GalCer, and boosted with the control virus (VVluc). Mice were infected three weeks later intradermally with 1x10⁷ metacyclic promastigotes of L. infantum. This method of infection allows visceralization of the parasite and mimics the natural course of infection, caused by the bite of a phlebotomine sandfly.

Pre- and post-challenge data showed higher levels of IFN-γ produced by spleen cells stimulated with LACK antigen. See Fig. 7. However, these levels of EFN-γ did not consistently correlate to the level of protection observed at 5 weeks post-infection. See Fig. 8. Note that the enhanced protection caused by c-GalCer is tissue-site specific (evident in the liver and spleen but not in the draining lymph node). In a similar experiment using only alpha-GalCer and a different intradermal site for vaccination, enhanced protection resulted in the draining lymph node and spleen but not the liver. It should be noted that the LACK-DNA vaccine alone fails to provide protection in this model, and therefore the effects of glycosyl ceramides are substantial.

Data from a subsequent experiment in the L. infantum system are shown in Table 2. These data confirm that c-GalCer improves the protective effect of immunization in multiple tissues, including the spleen and the liver.

Table 2: Intradermal Infection Leishmania infantum - 1 Month Post-Infection

* Values are parasite burdens xlO^"3 (fold reduction).

Example 4. P-8 and D- 13 Antigens Protect Against L. infantum infection. BALB/c mice were vaccinated with either D-13 or P-8 and infected 5 weeks after the last immunization with 10⁴ or 10⁵ L. infantum intravenously. See figures 9 and 10. Parasite burdens in the liver and spleen were evaluated one month and 4.5 months after infection. The immunization diminished the degree of infection in both the liver and the spleen.

INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, hi case of conflict, the present application, including any definitions herein, will control.

Fuertes-Lopez et al. U.S. Pat. Pub. No. 2004-0235772-A1 Larraga Rodriguez De Vera et al. U.S. Pat. Pub. No. 2004-0156866-A1 Bedate et al. U.S. Pat. No. 6,458,359 Gonzalez-Aseguinolaza et al. 2003-0157135-A1 Taniguchi et al. U.S. Pat. No. 6,747,010

Taniguchi et al. U.S. Pat. No. 6,531,453

Bachmann et al. U.S. Pat. Pub. No. 2003-0099668-A1

Jaffe et al. J Immunol. 1984 Jul;133(l):440-7. Belli, et al., 1999, Exp. Parasitol. 91:151-160.

Campos-Neto et al., 2001, Infect. Immun. 69:4103-4108.

Dondji et al. Infection and Immunity, August 2005, p. 5286-5289, Vol. 73, No. 8.

Dumonteil et al., 2003, Vaccine 21:2161-2168.

Masina et al., 2003, J. Infect. Dis. 188:1250-1257. Gonzalo et al., 2002, Vaccine 20:1226-1231.

Lopez-Fuertes et al., 2002, Vaccine 21:247-257.

Tapia et al., 2003, Microbes Infect. 5:73-84.

Pinelli, et al., 1999, Vet. Immunol. Immunopathol. 69:121-126.

Quinnell, et al., 2001, J. Infect. Dis. 183:1421-1424. Ramiro et al., 2003, Vaccine 21 :2474-2484.

Santos et al., 2002, Vaccine 21:30-43.

Santos-Gomes, et al., 2002, Vet. Immunol. Immunopathol. 88:21-30.

EQUIVALENTS While specific embodiments of the subject inventions are explicitly disclosed herein, the above specification is illustrative and not restrictive. Many variations of the inventions will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the inventions should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

WE CLAM:

1. A formulation for eliciting an immune response to a Leishmania antigen in a mammal, the formulation comprising at least two antigens selected from the group consisting of:

(a) GP46 or an antigenic portion thereof;

(b) P4 or an antigenic portion thereof;

(c) GP63 or an antigenic portion thereof; (d) P36-LACK or an antigenic portion thereof; and

(e) P8.

2. A formulation for eliciting an immune response to a Leishmania antigen in a mammal, the formulation comprising two or more nucleic acids encoding at least two antigens selected from the group consisting of: (a) GP46 or an antigenic portion thereof;

(b) P4 or an antigenic portion thereof;

(c) GP63 or an antigenic portion thereof;

(d) P36-LACK or an antigenic portion thereof.

3. A formulation for eliciting an immune response to a Leishmania antigen in a mammal, the formulation comprising

(a) an antigen selected from the group consisting of: i. GP46 or an antigenic portion thereof; ii. P4 or an antigenic portion thereof; iii. GP63 or an antigenic portion thereof; iv. P36-LACK or an antigenic portion thereof; and v. P8; and (b) A nucleic acid encoding an antigen selected from the group consisting of: i. GP46 or an antigenic portion thereof; ii. P4 or an antigenic portion thereof; iii. GP63 or an antigenic portion thereof; and iv. P36-LACK or an antigenic portion thereof.

4. The formulation of any of claims 1-3, wherein each antigen is derived from a causative agent of visceral leishmaniasis.

5. The formulation of any of claims 1-3, wherein each antigen is derived from an organism selected from the group consisting of: Leishmania donovani, Leishmania pifanoi, Leishmania chagasi and Leishmania infantum.

6. The formulation of any of claims 1-3, further comprising an adjuvant.

7. The formulation of claim 6, wherein the adjuvant is an NKT-active adjuvant.

8. The formulation of claim 6, wherein the adjuvant is selected from the group consisting of: a glycosyl ceramide, a P-8 antigen and P.acnes/C. parvum.

9. A formulation for eliciting an immune response to a Leishmania antigen in a mammal, the formulation comprising an NKT-active adjuvant and an antigen from a causative agent of leishmaniasis.

10. The formulation of claim 9, wherein the adjuvant is a glycosyl ceramide.

11. The formulation of claim 10, wherein the glycosyl ceramide is selected from the group consisting of: a c-GalCer and an alpha-GalCer.

12. The formulation of claim 10, wherein the adjuvant is a P-8 antigen.

13. A formulation for eliciting an immune response to a Leishmania antigen in an outbred canine, the formulation comprising an antigen selected from the group consisting of: D-13, D-2, D-6/D-15, and D-14.

14. A method for eliciting an immune response to a Leishmania antigen in a mammal, the method comprising administering to a mammal at least two antigens selected from the group consisting of:

(a) GP46 or an antigenic portion thereof; (b) P4 or an antigenic portion thereof;

(c) GP63 or an antigenic portion thereof;

(d) P36-LACK or an antigenic portion thereof; and

(e) P8.

15. A method for eliciting an immune response to a Leishmania antigen in a mammal, the method comprising administering to the mammal one or more nucleic acids encoding at least two antigens selected from the group consisting of:

(a) GP46 or an antigenic portion thereof;

(b) P4 or an antigenic portion thereof; (c) GP63 or an antigenic portion thereof;

(d) P36-LACK or an antigenic portion thereof; and

16. A method for eliciting an immune response to a Leishmania antigen in a mammal, the method comprising administering to the mammal:

17. The method of any of claims 14-16, wherein antigen and/or nucleic acid are administered as a single formulation.

18. The method of any of claims 14-16, wherein antigen and/or nucleic acid are administered separately.

19. A method for eliciting an immune response to a Leishmania antigen in a mammal, the method comprising administering to the mammal an NKT- active adjuvant and an antigen from a causative agent for leishmaniasis.

20. A purified P4 polypeptide, comprising an amino acid sequence selected from the group consisting of: SEQ ED No. 1, an antigenic portion of SEQ ID No. 1, and SEQ ID No. 2, an antigenic portion of SEQ ID No. 2.

21. A formulation for eliciting an immune response to a Leishmania antigen in a mammal comprising a purified polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID No. 1 and SEQ ED No. 2.

22. A formulation for eliciting an immune response to a Leishmania antigen in a mammal, the formulation comprising P-8 antigen.

23. The formulation of claim 22, wherein the P-8 antigen is derived from a causative agent for visceral leishmaniasis.

24. The formulation of claim 22, wherein the P-8 antigen is derived from an organism selected from the group consisting of: Leishmania donovani, Leishmania pifanoi, Leishmania chagasi, Leishmania amazonensis, Leishmania mexicana, and Leishmania infantum.

25. The formulation of any of claims 22-24, further comprising an adjuvant.

26. A vaccine for protecting a mammal from visceral leishmaniasis, the vaccine comprising at least two antigens selected from the group consisting of: (a) GP46 or an antigenic portion thereof;

(b) P4 or an antigenic portion thereof;

(c) GP63 or an antigenic portion thereof;

(d) P36-LACK or an antigenic portion thereof; and (e) P8.

27. A vaccine for protecting a mammal from visceral leishmaniasis, the vaccine comprising two or more nucleic acids encoding at least two antigens selected from the group consisting of:

(c) GP63 or an antigenic portion thereof;

(d) P36-LACK or an antigenic portion thereof.

28. A vaccine for protecting a mammal from visceral leishmaniasis, the vaccine comprising (a) an antigen selected from the group consisting of: i. GP46 or an antigenic portion thereof; ii. P4 or an antigenic portion thereof; iii. GP63 or an antigenic portion thereof; iv. P36-LACK or an antigenic portion thereof; and v. P8; and

(b) A nucleic acid encoding an antigen selected from the group consisting of: i. GP46 or an antigenic portion thereof; ii. P4 or an antigenic portion thereof; iii. GP63 or an antigenic portion thereof; and iv. P36-LACK or an antigenic portion thereof.

29. The vaccine of any of claims 26-28, wherein each antigen is derived from a causative agent of visceral leishmaniasis.

30. The formulation of any of claims 26-28, further comprising an adjuvant.

31. The formulation of claim 30, wherein the adjuvant is an NKT-active adjuvant.

32. The formulation of claim 30, wherein the adjuvant is selected from the group consisting of: a glycosyl ceramide, aP-8 antigen and P. acnes/ C. parvum.

33. A vaccine for protecting a mammal from visceral leishmaniasis, the vaccine comprising an NKT-active adjuvant and an antigen from a causative agent of visceral leishmaniasis.

34. The formulation of claim 9, wherein the adjuvant is a glycosyl ceramide.

35. The formulation of claim 10, wherein the glycosyl ceramide is selected from the group consisting of: a c-GalCer and an alpha-GalCer.

36. The formulation of claim 10, wherein the adjuvant is a P-8 antigen.

37. A formulation for eliciting an immune response to a Leishmania antigen in a mammal comprising a purified polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID No. 1 and SEQ

ID No. 2.

38. A formulation for eliciting an immune response to a Leishmania antigen in a mammal, the formulation comprising P-8 antigen.

39. The formulation of claim 22, wherein the P-8 antigen is derived from a causative agent for visceral leishmaniasis.

40. The formulation of claim 22, wherein the P-8 antigen is derived from an organism selected from the group consisting of: Leishmania donovani,

Leishmania pifanoi, Leishmania chagasi, Leishmania amazonensis, Leishmania mexicana, and Leishmania infantum.