Glycoconjugate vaccine for <i>leishmaniasis </i>

Abstract

Certain embodiments are directed to an immunogenic composition comprising an immunogenic glycoconjugate comprising a glycan having the chemical formula of galactopyranose (Galp)-α(1,2)-R, Galp-α(1,3)-R, Galp-α(1,4)-R, or Galp-α(1,6)-R, wherein in R is any monosaccharide, oligosaccharide, or polysaccharide, coupled to a carrier peptide or protein. Certain aspects described herein are directed to compounds and therapies for treating Leishmania infections. In certain aspects, the glycoconjugates as described herein, are incorporated into pharmaceutical compositions or vaccines

Claims

1. An immunogenic glycoconjugate, wherein the immunogenic glycoconjugate is a glycoinositolphospholipid (GIPL)-2-derived Galpα(1,3)Galfβ-tetanus toxoid peptide (TTP) (G2-TTP) glycoconjugate or a GIPL-3-derived Galpα(1,6)Galpα(1,3)Galfβ-TTP glycoconjugate (G3-TTP).

2. The immunogenic glycoconjugate of claim 1, wherein the tetanus toxoid peptide consists of the amino acid sequence of EQYIKANSKFIGITE (SEQ ID NO:1).

3. The immunogenic glycoconjugate of claim 1, wherein the immunogenic glycoconjugate is formulated as a vaccine composition.

4. A method of inducing an immune response in a mammal to comprising administering the immunogenic glycoconjugate of claim 1.

5. The method of claim 4, wherein the immune response is induced in a human or canine.

6. The method of claim 4, wherein the mammal has cutaneous leishmaniasis (CL).

7. The method of claim 4, wherein the immunogenic composition is administered two or more times.

8. A method for treating Leishmaniasis by inducing in a mammal an immune response against Leishmaniasis comprising the step of administering to the mammal a composition comprising the glycoconjugate of claim 1.

9. The method of claim 8, wherein the tetanus toxoid peptide consists of the amino acid sequence of EQYIKANSKFIGITE (SEQ ID NO: 1).

10. The method of claim 8, wherein the mammal is a human or canine.

11. The method of claim 8, wherein the mammal is infected with Leishmania major or Leishmania mexicana.

12. A glycoconjugate having the chemical formula of (a) (Galp)α(1,6)(Galp)α(1,3) Galfβ coupled to a carrier protein or a carrier peptide comprising a helper T cell epitope; or (b) Galpα(1,3)Galfβ disaccharide coupled to a carrier protein or a carrier peptide comprising a helper T cell epitope, wherein carbohydrate portion of the glycoconjugate consists of the disaccharide.

13. The glycoconjugate of claim 12, wherein the carrier peptide is a tetanus toxoid peptide or the carrier protein is bovine serum albumin (BSA).

14. The glycoconjugate of claim 13, wherein the carrier peptide is a tetanus toxoid peptide.

15. The immunogenic glycoconjugate of claim 14, wherein the tetanus toxoid peptide consists of the amino acid sequence of EQYIKANSKFIGITE (SEQ ID NO:1).

Description

DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

(2) FIG. 1. Schematic representation of two synthetic neoglycopeptides. G2-TTP, GIPL-2-derived Galpα(1,3)Galfβ coupled to TTP; and G3-TTP, GIPL-3-derived Galpα(1,6)Galpα(1,3)Galfβ glycan coupled to TTP. TTP=EQYIKANSKFIGITE (SEQ ID NO:1)

(3) FIG. 2. Immunization of a1,3GalT-KO mice with Galα1,3Galfβ1,4GlcNAc-BSA (Galα3LN-BSA) induces partial protection against L. major. Lesion in the foot pad was measured with a caliper at days 30, 35, and 37 post-infection.

(4) FIG. 3. Anti-α-Gal Ab titers in α1,3GalT-KO mice immunized with Galα3LN-BSA. RLU, relative luminescence units.

(5) FIG. 4. Synthesis of two glycosyl amino acid building blocks for solid phase peptide synthesis.

(6) FIG. 5. Solid-phase peptide synthesis of two neoglycopeptides. TTP=EQYIKANSKFIGITE (SEQ NO:1)

(7) FIG. 6. Vaccination strategy.

(8) FIG. 7. Immunization of female α1,3GalT-KO mice (C57BL6) (n=6 per group) with the neoglycoprotein (NGP) Galα1,3-Galβ-BSA (KM17), Galα1,4-Galα-BSA (KM12) or Galα1,6-Galβ-BSA (KM5). The immunizations were performed with 10 μg NGP/animal/dose (subcutaneously) at 7-day intervals, in a total of 4 immunizations. Then, the animals were challenged with 1e5 (100,000) L. major (Friedlin strain) metacyclic promastigotes 41 days after the last immunization. Lesion in the foot pad was measured with a caliper at 30, 40, 48, 55 and 62 days post-infection. Statistical analysis: Student's t-test; ns, non-significant.

DESCRIPTION

(9) Trypanosomatids are a group of kinetoplastid protozoa distinguished by having only a single flagellum. All members are exclusively parasitic, found primarily in insects. A few genera have life cycles involving a secondary host, which may be a vertebrate, invertebrate, or plant. These include several species that cause major diseases in humans. The three major human diseases caused by trypanosomatids are—African trypanosomiasis (Sleeping Sickness, caused by Trypanosoma brucei and transmitted by Tsetse flies), South American trypanosomiasis (Chagas disease, caused by Trypanosoma cruzi and transmitted by triatomine bugs), and leishmaniasis (a set of trypanosomal diseases caused by various species of Leishmania transmitted by sandflies). Certain aspects described herein are directed to compounds and therapies for treating Leishmania infections.

(10) Leishmaniasis has a wide range of clinical symptoms and over 20 species and subspecies of Leishmania can infect humans, causing three different diseases, visceral (VL), cutaneous (CL), or mucocutaneous leishmaniasis (MCL). Approximately 350 million people are at risk in 88 countries around the world (Ameen, Clinical and experimental dermatology 35:699-705, 2010). Current anti-Leishmania drugs show high toxicity and, frequently, marginal efficacy (Launois et al., Expert review of vaccines 7:1277-87, 2008). Moreover, strains resistant to current treatments have been reported and no vaccine is available (Croft et al., Trends in parasitology 21:508-12, 2005; Mutiso et al., Journal of biomedical research 27:85-102, 2013).

(11) Glycoinositolphospholipids (GIPLs) are major molecules on the plasma membrane of all Leishmania spp. (McConville and Ferguson, Biochem J 294(2):305-24, 1993; de Assis et al., Biochimica et biophysica acta 1820:1354-65, 2012). Specifically in L. major (Old World) and L. mexicana (New World), GIPL-2 and GIPL-3 contain terminal α-Gal residues, which are highly immunogenic to humans (Avila et al., J Immunol 142:2828-34, 1989; Avila et al., Journal of clinical microbiology 26:1842-47, 1988; Avila et al., Journal of clinical microbiology 26:126-32, 1988; McConville et al., The Journal of biological chemistry 265:7385-94, 1990). The current invention uses synthetic glycans, derived from these GIPLs and covalently attached to CD4 T cell epitope, as vaccine candidates for CL.

(12) Synthesis of Leishmania-specific α-Gal-containing neoglycopeptides. Neoglycopeptides (NGPs) can be synthesized by solid phase peptide synthesis (Chan and White, Fmoc solid phase peptide synthesis: a practical approach, (Oxford University Press: Oxford, 2000)). One NGP consists of the disaccharide Galα(1,3)Galfβ derived from glycoinositolphospholipid 2 (GIPL-2), and another NGP contains the trisaccharide Galpα(1,6)Galpα(1,3)Galfβ derived from GIPL-3, both found in L. major and L. mexicana (McConville et al., The Journal of biological chemistry, 265:7385-94, 1990; McConville and Ferguson, Biochem J, 294 (Pt 2):305-24, 1993; McConville et al., The Journal of biological chemistry 268:15595-604, 1993). These glycans are covalently attached to a CD4+ T cell epitope, e.g., tetanus toxoid peptide (TTP) (FIG. 1). An unglycosylated TTP can also be synthesized as a reference antigen.

(13) Development of a Leishmania glycopeptide-based vaccine platform. α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice are immunized with synthetic NGPs and evaluated for B cell- and T cell-mediated immune responses. αGal1,3T-KO mouse model (Tearle et al., Transplantation 61:13-19, 1996) is employed because it closely mimics the human humoral response against the highly immunogenic α-Gal epitopes.

(14) Immunization of αGalT-KO mice with Galα1,3Galβ1,4GlcNAcβ-bovine serum albumin (Galα3LN-BSA) resulted in high levels of anti-α-Gal Abs, accompanied by partial protection from disease (FIG. 2 and FIG. 3) (Tearle et al., Transplantation 61:13-19, 1996). Briefly, α1,3GalT-KO mice (8 weeks old, 5 mice per group) were immunized intraperitoneally (i.p.) four times, at 10-day intervals, with 20 μg Galα3LN-BSA or with BSA (control-placebo). Twelve days after the last immunization, animals were challenged with a lethal dose of 10.sup.6 L. major metacyclic promastigotes. FIG. 2 shows that 37 days post-infection (dpi) immunized mice showed a foot pad lesion 2.3-fold smaller than the control group. Both groups maintained similar weight, suggesting that no toxic effect was caused by the vaccine or placebo. Taken together, these results provide strong evidence of the potential of an α-Gal-based vaccine for CL.

(15) Synthesis of two Leishmania-specific α-Gal-containing neoglycopeptides. The two glycosyl amino acid building blocks 6 and 7 will be synthesized as shown in FIG. 4 (scheme 1). Galactofuranosyl donor 1 was synthesized following known procedures (Completo and Lowary, J Org Chem. 73(12):4513-25, 2008). Glycosylation of linker 2 (Wu et al., Org. Lett. 6:4407-10, 2004) followed by removal of the protecting groups and regioselective benzoylation furnishes acceptor 3, which upon α-galactosylation with the galactosyl donor 4 (Kimura et al., Synlett, 2379-82, 2006; Imamura et al., Chem. Eur. J. 12:8862-70, 2006) produces the fully protected disaccharide 5. Reduction of the azido group by Staudinger reaction, coupling to the amino acid Fmoc-Glu-OAll, and Pd(0)-catalyzed deallylation gives glycosyl amino acid building block 6. Selective ring opening of the DTBS group of compound 5 with HF-pyr complex, followed by glycosylation with donor 4, reduction of the azido group, coupling to the amino acid Fmoc-Glu-OAll and Pd(0)-catalyzed deallylation gives glycosyl amino acid building block 7.

(16) TTP and the two proposed NGPs, G2 (Galpα(1,3)Galfβ) and G3 (Galpα(1,6)Galpα(1,3)Galfβ) glycan coupled to TTP (G3-TTP) will be synthesized by solid phase peptide synthesis using the Fmoc strategy, Sieber amide resin, and standard coupling conditions (FIG. 5, scheme 2).

(17) Cleavage from the resin is achieved with 1% TFA. The NGPs are purified by silica column and/or size exclusion chromatography. After deprotection of the amino acid side chains with TFA, and removal of the ester groups with sodium methoxide, the neoglycopeptides are chromatographed by reversed phase HPLC, characterized by ESI-TOF-MS, and are ready to be used as immunogens.

(18) Leishmania glycopeptide-based vaccine platform. FIG. 6 illustrates a vaccination strategy. Four experimental groups are used to test the NGPs described herein. Briefly, animals (n=10, each group) receive four immunizations of 20 μg G2-TTP (group 3) or G3-TTP (group 4) per animal per dose, with 10-day interval between immunizations. The control groups (n=10, each) receives phosphate-buffered saline (PBS; immunization vehicle) (group 1) or 20 μg TTP (group 2) (FIG. 6). All immunizations are done by intraperitoneal (i.p.) injection (max. vol. 200 μl).

(19) Ten days after the 4.sup.th immunization (day 30), individual sera from all animal groups is obtained and analyzed for anti-α-Gal Ab titers by CL-ELISA, using G2-TTP and G3-TTP as antigens. Immunoglobulin isotyping is performed, using commercially available kit (BD Pharmingen). All animals are challenged by i.d. injection in the left footpad with 10.sup.7 infective L. major metacyclic promastigotes. The animals are monitored 3× per week. Lesions start to appear 20 dpi. Mice are then monitored 3× per week, the size of the lesion and the animal weight is assessed during 50 days. To assess parasite load in the footpad quantitative real-time PCR (qRT-PCR) is performed at day 120 (experimental endpoint) (Wortmann et al., The American journal of tropical medicine and hygiene 73:999-1004, 2005). Upon observation of any abnormal physical appearance (e.g., weight loss, febrile state, distress, etc.), control animals (groups 1 and 2) are humanely euthanized and the footpad harvested for qRT-PCR. Groups 3 and 4 are euthanized at day 120 and the left footpad collected for qRT-PCR analysis. The immunogenicity of both NGPs is evaluated by chemiluminescent-ELISA (Almeida et al., Transfusion 37:850-57, 1997) and lytic anti-α-Gal antibody assay (Almeida et al., Biochem. J. 304:793-802, 1994), using sera of the immunized mice. Sera from patients with L. major infection, which contain high levels of anti-α-Gal Abs, are used as positive controls. The cell-mediated immune response to the two NGPs and controls is evaluated first by measuring the profile of proinflammatory (IFN-γ, TNFα, IL-2, IL-12, and IL-17) and anti-inflammatory/regulatory (IL-4 and IL-10) cytokines in the serum (Sema et al., Vaccine 32:3525-32, 2014), and by intracellular cytokine staining (ICS) of spleen cells (ProImmune). These assays are performed before and after challenge with lethal dose of parasites of the immunized mice.

(20) Immunization of female αGal1,3T-KO mice (C57BL6) (n=6 per group) with the neoglycoprotein (NGP) Galα1,3-Galfβ-BSA (KM17), Galα1,4-Galα-BSA (KM12) or Galα1,6-Galβ-BSA (KM5) (FIG. 7). The immunizations were performed with 10 μg NGP/animal/dose (subcutaneously) at 7-day intervals, in a total of 4 immunizations. Then, the animals were challenged with 1000,000 L. major (Friedlin strain) metacyclic promastigotes 41 days after the last immunization. Lesion in the foot pad was measured with a caliper at 30, 40, 48, 55 and 62 days post-infection. Statistical analysis: Student's t-test; ns, non-significant.

(21) Pharmaceutical and Vaccine Compositions

(22) In certain aspects, the glycoconjugates as described herein, are incorporated into pharmaceutical compositions or vaccines. Pharmaceutical compositions generally comprise one or more glycoconjugates as described herein in combination with a physiologically acceptable carrier. Vaccines, also referred to as immunogenic compositions, generally comprise one or more of the glycoconjugates as described herein, in combination with an immunostimulant, such as an adjuvant. In particular embodiments, the pharmaceutical compositions comprise glycoconjugates as described herein that are capable of providing protection against, for example in an in vivo assay as described herein, Leishmania species such as L. donovani, L. major, or L. infantum.

(23) An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. In certain aspects an immunostimulant can be covalently attached and/or co-formulated with an antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccine preparation is generally described in, for example, Powell & Newman, eds., Vaccine Design (the subunit and adjuvant approach) (1995).

(24) Any of a variety of immunostimulants may be employed in the vaccines of this invention. For example, an adjuvant may be included. 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 (natural or synthetic), Bordatella pertussis or Mycobacterium species or Mycobacterium-derived proteins. Suitable 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, N.J.); AS-2 and derivatives thereof (GlaxoSmithKline Beecham, Philadelphia, Pa.); CWS, TDM, LeIF, 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 quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

(25) The vaccine and pharmaceutical compositions of the invention may be formulated using any of a variety of well-known procedures. In certain embodiments, the vaccine or pharmaceutical compositions are prepared as stable emulsions (e.g., oil-in-water emulsions) or as aqueous solutions.

(26) In the compositions of the invention, formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intradermal, subcutaneous and intramuscular administration and formulation.

(27) In certain applications, the compositions disclosed herein may be delivered via oral administration to a subject. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

(28) In certain circumstances it will be desirable to deliver the compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described, for example, in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

(29) The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In most cases the form must be sterile and must be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

(30) For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards.

(31) Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

(32) As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known to one of ordinary skill in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

(33) The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood to one of ordinary skill in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

(34) In certain embodiments, the compositions of the present invention may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, polynucleotides, and peptide compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

(35) The pharmaceutical compositions and vaccines of the invention may be used, for example, to induce protective immunity against Leishmania species such as L. donovani, L. major and/or L. infantum in a patient, such as a human or a dog, to prevent leishmaniasis or diminish its severity. The compositions and vaccines may also be used to stimulate an immune response, which may be cellular and/or humoral, in a patient, for treating an individual already infected.

(36) Appropriate doses and methods of administration for these purposes can be readily determined by a skilled artisan using available knowledge in the art and/or routine techniques. Routes and frequency of administration, as well as dosage, for the above aspects of the present invention may vary from individual to individual and may parallel those currently being used in immunization against other infections, including protozoan, viral and bacterial infections. For example, in one embodiment, between 1 and 12 doses of composition having glycoconjugate are administered over a 1 year period. Booster vaccinations may be given periodically thereafter as needed or desired. Of course, alternate protocols may be appropriate for individual patients. In a particular embodiment, a suitable dose is an amount of glycoconjugate that, when administered as described above, is capable of eliciting an immune response in an immunized patient sufficient to protect the patient from leishmaniasis caused by Leishmania species such as L. donovani, L. major and/or L. infantum for at least 1-2 years. In general, the amount of glycoconjugate present in a dose ranges from about 100 ng to about 1 mg per kg of host, typically from about 10 μg to about 100 μg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

(37) The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.