WO2016103273A1 - A dna vaccine formulation in cationic liposome vehicle useful for maximizing vaccine potency for leishmaniasis - Google Patents

Abstract

The present invention discloses DNA vaccination with plasmid encoding the cysteine protease C (cpc) gene of Leishmania donovani to induce effective protection against visceral leishmaniasis (VL). The DNA vaccine entrapped in the cationic liposomes comprises of a plasmid vector pVAX1 encoding L. donovani cpc gene operably linked to transcriptional regulatory sequences. The invention relates to efficacy of liposomal formulation containing the bilayer-associated adjuvant MPLA, encapsulating the DNA construct pVAX1-cpc, against lethal L. donovani infection model in BALB/c mice. This formulation of liposomal DNA vaccine is highly effective in inducing robust cell-mediated immune response against leishmanial parasites upon intramuscular administration.

Description

A DNA VACCINE FORMULATION IN CATIONIC LIPOSOME VEHICLE USEFUL FOR MAXIMIZING VACCINE POTENCY FOR LEISHMANIASIS

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a DNA vaccine formulation in cationic liposome vehicle useful for maximizing vaccine potency for Leishmaniasis. More particularly, it relates to cationic liposomes containing intrabilayer monophosphoryl lipid A (MPLA) and cysteine protease C (cpc) gene useful for vaccination for Leishmaniasis. The present invention further relates to method of preparation, characterization arid use of DSPC (1, 2-distearoyl-sn- glycero-3-phosphocholine) bearing cationic liposomes containing intrabilayer MPLA and cpc gene for vaccination against Leishmania sp.

The proposed adjuvant formulation of cationic liposomes containing MPLA is highly promising immunostimulatory agent and efficient delivery vehicle maximizing vaccine potency against various pathogens including Leishmania. This vaccine formulation combines safety, efficacy alongwith cost-effectiveness to provide new possibilities for prophylactic and therapeutic vaccines. BACKGROUND AND PRIOR ART OF THE INVENTION

Protozoan parasites of the genus Leishmania cause a wide spectrum of diseases collectively termed leishrnaniasis that varies in their clinical manifestations in the form of cutaneous, mucocutaneous and visceral Leishmaniasis. Visceral leishmaniasis (VL) or kala- azar, caused by Leishmania donovani, is a fatal parasitic disease causing 25,000-40,000 cases and 200-300 deaths annually in Indian subcontinent alone [Joshi et al., 2008]. VL is endemic in tropical and subtropical areas of Latin America, Europe, Africa and Asia and has emerged as an important opportunistic HIV coinfection. Control of leishmaniasis thus remains a source of grave concern worldwide. There are few drugs available for chemotherapy and treatments are still long-drawn, highly toxic and expensive. Combined therapies advocated to combat the drug resistant parasites remain costly for the majority of patients [Croft and Olliaro, 2011]. Leishmania vaccines continue to hold promise due to the fact that cured patients from VL are subsequently protected against reinfection. Impressive clinical responses to cutaneous leishmaniasis have been observed in the few recent human clinical trials, although the overall track record for Leishmania vaccines, particularly VL is less impressive [Evans and Kedzierski, 2012]. VL is of higher priority than CL since anthroponotic VL foci are the origin of frequent and deadly epidemics. Although a series of vaccine candidates have been tested against Leishmania in the past 10 years, none has successfully emerged for human use. Improvements in vaccine delivery systems are thus an important component of current research efforts against Leishmania to realize their full potential. Previous VL vaccine strategies have included immunization with whole parasite or cell lysate vaccines, peptide vaccines, dendritic cell (DC) vaccines, viral vectored and plasmid DNA vaccines [Modabber 2010; Das and Ali, 2012]. Relative ease of production, stability, safety, low cost and ability to induce cellular immunity has made the plasmid DNA vaccines more attractive over- conventional protein vaccines. Although DNA vaccines are able to elicit both humoral and cellular immune responses, genetic immunization against Leishmania has often been hampered by the failure of the vaccine to elicit a cytotoxic T-cell response strong enough for protection against the disease [Melby et al., 2001; Saldarriaga et al., 2006]. This can probably be overcome by coupling the DNA vaccine with an efficient vaccine delivery system and immunomodulator capable of greater activation of innate immunity during vaccination. A better protection for the plasmid DNA against degradation by cellular nucleases is also achieved by cationic liposomal entrapment due to DNA condensation and covering of DNA strands with lipid bilayers [Radler etal., 1997]. Cationic liposomes have been previously shown to markedly potentiate the ability of plasmid DNA to activate innate immune responses by endosomal targeting, thereby increasing the TLR9 activation and efficient MHC class I-restricted antigen presentation [Zuhorn et al., 2002; Yasuda et al., 2005]. Despite variations in lipids, cationic surface charge enhance antigen uptake when delivered via such liposomes [Korsholm et al., 2007]. However, protection with cationic liposomal protein vaccine was limited mostly to intraperitoneal route of administration [Afrin et al., 2002; Ravindran et al., 2010; Bhowmick et al., 2010] .Therefore, additional adjuvant or immunomodulator was required along with the liposomes for successful protection in human administrable route of vaccination such as subcutaneous, intramuscular and intravenous. Monophosphoryl lipid A (MPLA), which, targets intracellular TLR4, have previously exhibited considerable potency and safety in human trials with a variety of candidate vaccines, including vaccines to malaria, hepatitis B, HIV-1 and several different types of cancer [Agnandji et al., 2012; Cluff 2010; Di Paolo etal., 2010; Fries et al., 1992; Garcon, 2011 ]. Moreover, liposomal antigen presentation and macrophage recruitment have been shown to be augmented by intraperitoneal administration of liposomes containing MPLA compared to those lacking MPLA [Verma et al., 1992]. To optimize the route of immunization in addition to improved immune stimulation, MPL-TDM (monophosphoryl lipid - trehalose dicorynomycolate) has been used for liposomal gp63 antigen which resulted in robust Thl immune responses for protection against VL [Majumder et al., 2011]. The present invention shows further advancement of this strategy by incorporating MPLA within lipid bilayer of cationic liposomes for delivery of a DNA vaccine, thereby making the delivery system more compact and combining all the advantages of DNA vaccination Over protein vaccines.

Cysteine proteases (CPs) are enzymes known to play critical roles in the pathogenesis of Leishmania and other parasitic infections [Vermelho et al., 2010; Silva- Almeida et al., 2012]. Extensive studies have shown that leishmanial CPs are involved in parasitic survival, replication, autophagy, metacyclogenesis and transformation to amastigotes essential for onset of disease. Thus CPs have been identified as putative vaccine candidates and as potential drug targets against leishmaniasis. Although cysteine protease A, B and C have shown to be protective singly or in combination against L. major and L. infantum in different studies, their efficacy has not been studied against L. donovani.

OBJECTIVES OF THE INVENTION

The main object of the present invention is to provide a DNA vaccine formulation in cationic liposome vehicle useful for maximizing vaccine potency for leishmaniasis.

Another object of the present invention is to provide a process for the preparation of an adjuvant cum vaccine delivery system containing liposomal vesicles with intralamellar MPLA thus combining antigen presentation along with TLR 4 signaling.

Yet another object is to provide a process wherein the cationic liposomes composed of DSPC, stearylamine, cholesterol and MPLA, are designed to encapsulate desired antigen/ vaccine candidate. Yet another object is to provide a process wherein the vaccine formulation will augment T cell response thereby enhancing immunogenicity of a subunit protein/ DNA vaccine. Yet another object is to provide a stable, safe and biodegradable submicron adjuvant system for entrapment of both lipophilic and hydrophilic biomolecules (particularly vaccines) for their delivery and sustained release.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a DNA vaccine formulation in cationic liposome vehicle useful for maximizing vaccine potency for leishmaniasis

In accordance with a first of its aspects, the invention provides a cationic liposomal formulation containing intralamellar MPLA, encapsulating Ldcpc (Leishmania donovani cpc) DNA (GenBank Accession no. JX968801.1, Seq ID 3) for targeting leishmanial parasites prophylactically and which may also be therapeutically active. Cationic liposomes thus provided, incorporating TLR-4 agonist in membrane, possess immunostimulatory activity which is further improved by entrapping DNA vaccine construct into them. Further, the present disclosure provides a pharmaceutical composition comprising MPLA coupled cationic liposomes, as physiologically acceptable carrier as well as adjuvant for DNA and protein vaccine formulations.

In a preferred embodiment, the present invention provides a novel MPLA containing DSPC bearing cationic liposomes as vaccine carriers as well as promising adjuvants. Site- specific delivery of charged molecules like plasmid DNA can be effectively mediated by the cationic liposomes. This invention primarily focuses on a DNA vaccine using plasmid encoding cpc gene of L. donovani whose site specific delivery and efficacy is due to its liposomal encapsulation.

The liposome, containing MPLA that has been used in the present invention can also be effectively used as efficient delivery vehicle for various other biomolecules, particularly drugs and vaccines against other diseases requiring predominantly cell mediated response. Additional immunomodulatory activities of such liposomes can further be exploited to generate strong humoral and cellular immunity required to combat various pathogens and cancer.

In another embodiment of the present invention, it provides a process of cloning a pVAXl-cpc plasmid comprising amplification of cpc gene with primers; ligating cpc gene into pVAXl vector; transforming the said pVA l vector into competent E. coli bacteria; transcription and translation of pVAXl-cpc clones.

Yet another embodiment of invention provides the liposomal DNA vaccine formulation described herein as a cost-effective and health-promoting intervention for control, prevention and/or treatment of Leishmaniasis. The vaccine can substantially ameliorate morbidity, mortality and difficulties in providing medical care globally wherever there is prevalence of VL. The vaccine can also be useful for decreasing the severity of the disease when administered after initial infection with L. donovani.

Yet another embodiment of invention provides a candidate cross-protective gene useful as a DNA vaccine, or for production of a recombinant protein in various expression systems against Leishmania. Yejt another embodiment of invention provides a DNA vaccine formulation in cationic liposome vehicle useful for maximizing vaccine potency for leishmaniasis characterized in containing intralamellar monophosphoryl lipid A (MPLA) and cysteine protease C (cpc) gene.

Yet another embodiment of invention provides a DNA vaccine formulation wherein a plasmid DNA containing Seq ID 3 encoding cysteine protease C (cpc) gene ofL. donovani is encapsulated in the cationic liposome vehicle.

Yet another embodiment of invention provides a DNA vaccine formulation wherein the cationic liposome is a multilamellar vesicle comprising of: a) a neutral lipid preferably distearylphosphatidylcholine (DSPC);

b) a cationic lipid preferably stearylamine (S A); c) a second neutral lipid preferably cholesterol; and

d) a TLR agonist preferably monophosphoryl lipid A (MPLA); wherein the neutral lipid, the cationic lipid, the second neutral lipid and the TLR agonist are formulated in the range of 7:1.5:1.5:0.0016 to 7:2.5:2.5:0.003 molar ratio..

Yet another embodiment of invention provides a DNA vaccine formulation wherein the plasmid DNA is present in the range of 700 to 1000 μg/ml.

- Yet another embodiment of invention provides a DNA vaccine formulation is administered to a subject via intra- venous, intra-muscular or intranasal route wherein said subject is a mammal including human.

Yet another embodiment of invention provides a cationic liposome containing MPLA is useful as efficient delivery vehicle for biomolecules and vaccines

Yet another embodiment of invention provides a process for the preparation of the DNA vaccine formulation wherein said process comprising the steps of a. cloning cysteine protease C (cpc) gene havjing Seq Id. no. 3 into pET28a vector;

b. sub-cloning cpc clone obtained in step a into pVAXl vector to make pVAXl-cpc plasmid construct;

c. dissolving a neutral lipid, a cationic lipid, a second neutral lipid and a TLR agonist in the range of 7:1.5:1.5:0.0016 to 7:2.5:2.5:0.003 molar ratio in an organic solvent preferably chloroform to obtain a solution followed by removing solvent using evaporator to obtain a lipid film;

d. desiccating the lipid film as obtained in step (c) at a temperature in the range of 25 to 28°C for a period in the range of 12 to 16hours to obtain a dried lipid film;

e. dissolving the plasmid DNA in the range of 1 to 2 mg/ml obtained in step b in a physiologically acceptable aqueous medium;

f. hydrating the dried lipid film as obtained in step (d) with the solution obtained in step

(e); g. vortexing the solution obtained in step (f) for a period ranging between 10 to 15 min and sonicating for a period in the range of 30 to 45 sec at 4 to 5 Hz at a temperature in the range of 3 to 4°C to obtain a liposomal formulation; h. removing the excess free plasmid DNA from the liposomal formulation obtained in step (g) by ultracentrifugation to obtain a DNA vaccine formulation.

Yet another embodiment of invention provides a process for the preparation of the DNA vaccine formulation wherein physiologically acceptable aqueous medium is Phosphate Buffer Saline solution in a molar ratio in the range of 0.01 to 0.02.

Yet another embodiment of invention provides use of the DNA vaccine formulation as a vaccine against leishmaniasis for maximizing vaccine potency in mammals against Leishmania parasite Yet another embodiment of invention provides use of_. the cationic liposome to generate strong humoral and cellular immunity required to combat various pathogens and cancer.

Yet another embodiment of invention provides use of the cationic liposome for drug screening against leishmaniasis.

Yet another embodiment of invention provides a pharmaceutical composition comprising a therapeutically effective amount of the DNA vaccine formulation in a pharmaceutically acceptable carrier.

The following ABBREVIATIONS have been used:

DSPC- 1, 2-distearoyl-sn-glycero-3-phosphocholine

SA- stearylamine

PBS- Phosphate buffer saline

VL- Visceral leishmaniasis

MPLA- monophosphoryl lipid A TLR- Toll-like receptor MLV- multilamellar vesicles DNA- deoxyribonucleic acid pDNA- plasmid DNA rCPC- recombinant cysteine protease C protein cpc - cysteine protease C gene MPL-LIP - MPLA containing liposome LIP - Liposome

LDU - Leishmania Donovani Units DTH - Delayed Type Hypersensitivity

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts cloning, overexpression and purification of recombinant cysteine protease C protein (rCPC). a. Schematic representation of pET28a-cpc; b. Clone confirmation of cpc in pET28a vector. Lane 1 & 7, GeneRuler™ 1 kb DNA ladder (Fermentas, Canada) as marker, lane 2, vector pET28a , lane 3, Ndel/BamHI digested pET28a-cpc construct, lane 4, PCR of cpc from genomic DNA (insert), lanes 5 & 6, PCR of cloned construct; c. Coomassie Blue staining of 10% SDS-PAGE, Lane 1, molecular mass marker, lane 2, overexpressed recombinant cpc in E. coli BL21 (DE3), lane 3, purified recombinant cpc.

FIG. 2 shows the cloning and expression of L. donovani cpc in mammalian expression vector: (a). Schematic representation of pVAXl-cpc; (b) Clone confirmation of cpc in pVAXl vector. Lane 1, λ DNA digested with Hind III marker, lane 2 & 3, Hindlll/BamHI digested pVAXl-cpc construct, lane 4, PCR of cloned construct, lanes 5, GeneRuler™ 1 kb DNA ladder (Fermentas, Canada) as marker; (c) RT-PCR analysis for cpc in the pVAXl-cpc transfected HEK293T cells. Lane 1, GeneRuler™ 1 kb DNA Ladder, lane 2, HEK293T cells without transfection, Tane 3, HEK293T cells transfected with vector pVAXl, lane 4 & 5, HEK293T cells transfected with pVAXl-cpc; (d) Western blot analysis for expression of cpc in transfected HEK293T cell line, using rabbit anti-CPC antibody. Lane 1, western blot of pVAXl-cpc construct transfected in HEK293T cell line, lane 2, western blot of pVAXl vector transfected in HEK293T cell line.

FIG. 3 is the representative of Acoustic Alternative current (AAC) mode atomic forced microscopy (AFM) images of empty DSPC liposomes with intralamellar MPLA and same liposomes entrapping plasmid pV AX 1-cpc.

FIG. 4 shows the resistance of cationic liposomes to DNAse. Lane 1 : naked plasmid DNA (p VAX 1-cpc). Lanes 2 &3: naked plasmid DNA (pVAX 1-cpc) incubated with DNAse I for 5min and 1 hour respectively. Lane 4: liposomal pV AX 1-cpc incubated with DNAse I for 1 hour and subsequently extracted with phenol: chloroform (1 :1). Lane 5: Liposomal pVAXl- cpc incubated with DNAse I for 1 hour and run in gel without phenol: chloroform extraction.

FIG. 5 represents in vitro cell viability assay. Empty cationic liposomes with intrabilayer MPLA (3.2-200 ^g/ml with respect to DSPC) were incubated with murine peritoneal macrophages and lh, 4 h; 12h later MTT assay was performed. Values presented are the mean ± S.E. of four replicates.

FIG. 6 is the toxicity analysis after intravenous administration of liposomal DNA. Serum samples were collected from mice (n=5), fifteen days after intravenous administration of liposomal DNA vaccine formulation and assayed for the enzymes aspartate transferase (AST), alanine transferase (ALT) and alkaline phosphatase (ALP) as indicators of hepatotoxicity. FIG. 7 shows the DTH responses in differently vaccinated mice. Mice were immunized twice at 2-week interval. Ten days after immunization with recombinant CPC protein -specific DTH responses were measured. The response is expressed as the difference (in mm) between the thickness of the test (recombinant CPC protein -injected) and control (PBS-injected) footpads at 24 h. Each bar represents the mean ± S.E. for five individual mice per group at designated time point. The results are those from one experiment representative of two performed. Asterisks over each bar indicate significant differences in comparison to control groups. Asterisks over line indicate significant differences between groups. * - P < 0.05; *** - P < ^' 0.001. FIG. 8 shows the antibody production by liposomal DNA vaccine. Mice were immunized with PBS, empty liposomes containing intrabilayer MPL, vector pVAXl, naked or liposomal pVAXl-cpc DNA. Individual mouse sera from each group (n=5) were collected before infectious challenge. Recombinant CPC protein -specific production of IgG, IgGl and IgG2a was assayed by ELISA and an average of duplicate absorbance values of each serum sample recorded. Values are given as mean of four measurements. *, P < 0.05; significant differences from each other (Tukey post test).

FIG. 9 shows the cytokine responses in vaccinated mice. Mice were immunized twice at 2- week intervals. Ten days after last immunization spleens were collected from mice and restimulated in vitro with recombinant CPC protein (5 μ^πύ). After 72 h supernatants were collected and concentrations of released IFN-γ, IL-4, IL-2, lL-10 and IL-12 levels were determined by ELISA. Each sample was examined in duplicate. Each bar represents the mean ± S.E. for five individual mice per group. The results are those from one experiment representative of two performed. Asterisks over each bar indicate significant differences in comparison to PBS control group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 10 is evaluation of protection against L. donovani in vaccinated mice. Kinetics of liver and spleen parasite burden of mice immunized intramuscularly twice at 2-week interval with naked pDNA (pVAXl-cpc), only vector (pVAXl) and pDNA entrapped in cationic liposomes (pVAXl-cpc/MPL-LIP). Control animals received PBS or adjuvant (MPL-LIP) only. At 10 days after the last immunization, mice were challenged intravenously with 2.5 x 10⁷ promastigotes of L. donovani. At the designated time mice were sacrificed and LDU were- calculated from the weight and microscopic examination of impression smears of liver and spleen tissues. Each bar represents the mean ± S.E. for five individual mice per group. The results are those from one experiment representative of two performed. Asterisks over each bar indicate significant differences in comparison to control groups. ***, P < 0.001.

DETAILED DESCRIPTION OF THE INVENTION

Despite the past 15 years of research, human DNA vaccines have yet to fulfill their clinical success in the areas of infectious disease and cancer. Use of traditional adjuvants like alum, lipopolysaccharides (LPS), CpG motifs etc. to increase the immunogenicity of DNA vaccines have been modestly effective showing mixed results. Vaccine formulation comprising of plasmid DNA entrapped in cationic liposomal vesicles with intralamellar immunomodulator MPLA, as disclosed in the present invention, gets closer to the level of immunogenicity required for human use. In general, any vaccine or drug molecule must reach its intended site of action, mostly inside the cell for its action avoiding non-specific interactions. Therefore, the problem of intracellular gene delivery involves not only the cellular uptake of large charged molecules like DNA but also their intracellular availability at the target sites, traversing the biological barrier of plasma membrane, lysosomal degradation and nuclear envelop. Multilamellar positively charged liposomes have emerged as ideal delivery system carrying DNA in its core, interacting with the negatively charged cell surface for successful gene delivery after endocytosis-mediated uptake [Almofti et al., 2003)]. In addition, cationic liposomes provide a protective role against extra- and intracellular nucleases due to the compaction and covering of DNA by the lipid bilayers [Ibanez et al., 1996, Eastman et al., 1997]. In brief, manipulating DNA into liposomal delivery system makes it more efficient to stimulate both humoral and cell-mediated responses. Since liposomal efficacy largely depends on suitable immunostimulatory adjuvants to generate the appropriate adaptive immune response, the adjuvant monophosphoryl lipid A (MPLA) has been incorporated in the lipid-bilayer of delivery vesicles. MPLA is a detoxified form of the endotoxin lipopolysaccharide from Salmonella minnesota, and is among the first of a new generation of Toll-like receptor (TLR) agonists to be used in several human clinical trials [Garcon and Van Mechelen, 2011].

Vaccine formulation comprising of plasmid DNA entrapped in cationic liposomal vesicles with intralamellar immunomodulator MPLA, as disclosed in the present invention, gets closer to the level of immunogenicity required for human use. Liposomes and MPLA were chosen as adjuvants i this study as they are human-compatible inducers of cell- mediated immunity. Although liposomes containing MPL have shown considerable safety and potency in human trials in several diseases including malaria, HIV-1 and cancer [Alving et al., 2012], their adjuvant potency has never been tried against VL. The present invention relates to the development of vaccination strategies with cationic liposomal vesicles containing intrabilayer MPLA through intramuscular route of administration for DNA vaccination against experimental VL. Cysteine proteinase has been known to be protective against L. infantum infection in BALB/c mice [Khoshgoo et al., 2008} and thus, was chosen as the vaccine candidate. The potentiating effects of distearoylphosphatidyl choline (DSPC)-bearing cationic liposomes containing MPLA have been analyzed for formulating a DNA vaccine. It has been observed that current adjuvant formulation of cationic liposomes with intrabilayer MPLA enhanced the Thl based cellular immunity mediated by DNA vaccine alone without compromising the safety. This is consistent with some early studies [Gustafson et al., 1992; Rickman et al., 1991; Majumder et al., 2011] which relates the adjuvant activity of MPL to activation of macrophages and its ability to induce IFN-γ, IL-2, and IL-12 known to be essential for the induction of Thl - derived cell mediated immunity [Nijkamp and Parnham, 2011]. Predominant activation of the Thl -derived immune system elicits a strong DTH response and IgG2a production in liposomal pVAXl-cpc vaccinated mice as shown in the results. Findings from the current invention suggest the advantages of using liposomal carriers with immunomodulatory activity over naked pDNA for inducing strong systemic -mimunity against expressed antigen. Liposomal encapsulation protects DNA against nuclease present in serum thereby increasing their retention time, transfection efficacy and enhanced antigen presentation. Intramuscular immunization with naked pDNA (pVAXl-cpc) alone could not elicit optimum protection. The liposome-mediated pDNA delivery on the other hand resulted in robust protection, 34.18 and 54.25 times higher than naked pVAXl-cpc in liver and spleen respectively. Hence, genetic vaccination using pDNA entrapped in immunomodulatory liposomes containing MPLA represents an important advantage in inducing protection against leishmaniasis.

Statistical analysis

One-way ANOVA analysis (Multiple comparisons Tukey's post hoc test) was performed using the GraphPad Prism 5.0 software for Windows (GraphPad Software, San Diego CA). Results were considered significant when probability values were < 0.05.

EXAMPLES

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1

Cloning and purification of recombinant CPC protein from L. donovani Genomic DNA isolated from L. donovani promastigotes (MHOM/IN/1983/AG83 originally isolated from an Indian kala-azar patient and maintained in hamsters and BALB/c mice at the Indian Institute of chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, West Bengal, India ,Pin:-700 032) was subjected to polymerase chain reaction (PCR) with sets of gene specific primers corresponding to cpc (~1 kb) gene. The primers used were Seq ID 1 (forward): 5' GGA ATT CCA TAT GCCA GCG ACG TCA AGC GCC GCT 3' and Seq ID 2 (reverse): 5'CGG GAT CC CTA CTC CTG CGC GGG TAT GCC AGC3' PCR conditions were one cycle of 5 min at 94°C, 40 cycles of 1 min at 94°C, 1 min 20 s at 58°C, and 1 min 10 s at 72°C, followed by a final cycle of 7 min at 72°C. The PCR amplified fragment was cloned in Hindlll/BamHI site of bacterial expression vector pET28a (Novagen). Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37°C in 5.0ml of LB broth (with 50μg/μl kanamycin). Cells were pelleted and plasmid DNA extracted using the QIAprep Spin Miniprep Kit (Qiageri, Valencia, CA), following the manufacturer's instructions. For clone confirmation, approximately ^g plasmid DNA from individual miniprep was double digested with the appropriate restriction enzymes and the digest loaded onto a 1% agarose gel, in parallel with the molecular weight marker: GeneRuler™ 1 kb DNA ladder (Fermentas, Canada). Positive clones were selected on the basis of the size of insert and confirmed by DNA sequencing (ABI Prism, Model 377; Applied Biosystems). Escherichia coli BL21 (DE3) (parental strain for BL21 deficient in the Ion and ompT proteases, from Novagen, Cat No 69450 ) transformed with pET28a-cpc construct was grown in 500ml culture medium at 37 °C until OD at 600nm reached 0.6. The host includes BL21 (DE3) and/or any of its precursors. Protein production was induced by adding isopropyl β-d-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and incubating for an additional 4 h at 30 °C. The culture was then harvested by centrifugation at 6,000g, for 6 min, at 4 °C, and the cell pellet was resuspended in 6 ml of resuspension buffer (25 mM Tris-HCl, 500 mM NaCl, and 1 mg/ ml of Lysozyme, pH 8.0). The cell lysate was sonicated on ice for 5 min with 1 min pulse and 1 min interval between pulses using an ultrasonicator (Misonix). The pellet containing inclusion bodies was solubilised with solubilization buffer [50 mM CAPS {3-(Cyclohexylamino)-l-propanesulfonic acid} buffer (pH 11.0), 300 mM NaCl, and 0.5 % sarkosyl], kept at room temperature for 30 min and finally centrifuged at 12,000 g for 30 min at 4°C. The supernatant containing solubilised proteins were loaded separately onto Ni²⁺-nitrilotriacetic acid-agarose (Ni-NTA) column (Qiagen, Valencia, CA) and purified under denaturing condition. The Ni-NTA column was pre equilibriated with equilibriation buffer [50 mM CAPS buffer (pH 11.0), 150 mM NaCl, 0.5 % sarkosyl and 10 mM imidazole]. The column was washed with wash buffer [50 mM CAPS buffer (pH 11.0), 150 mM NaCl, 0.5 % sarkosyl and 20 mM imidazole] and eluted with elution buffer [50 mM CAPS buffer (pH 11.0), 150 mM NaCl, 0.5 % sarkosyl and 300 mM imidazole]. To refold, the purified materials were diluted 2 fold in dilution buffer containing 50 mM CAPS buffer (pH 11.0), 150 mM NaCl and 300 mM imidazole, and then dialyzed against 25 mM Tris-HCl, 250 mM NaCl, pH 8.0 and finally in 0.02M PBS for 6 hrs at 4 °C [Tao et al., 2010]. Protein concentrations were determined using Lowry's method [Lowry et al., 1951]. Purity and homogeneity of purified proteins were checked by using SDS-PAGE, and the gel was subsequently stained with Coomassie Brilliant Blue R-250 (Bio- Rad Laboratories, Hercules, CA).

The over expressed protein from E. coli BL21 (DE3) cells containing plasmid pET28a-cpc was purified through Ni -NTA agarose column under denaturing conditions finally yielding refolded protein in native state. The recombinant protein was refolded, dialyzed and finally concentrated by freeze-drying. The yield of purified protein was approximately 2.5 mg per liter of culture. Analysis of the purified recombinant CPC protein showed that the protein was essentially homogeneous (Figure lc).

Example 2

Cloning and expression of L. donovani cpc for DNA vaccine The gene encoding full-length of L. donovani cpc (Seq ID 3; GenBank accession number JX968801.1) was subcloned from pET28a in frame into pVAXl (Invitrogen, San Diego, CA) at the Hindlll/BamHI restriction sites. The full length cpc was amplified with cpc-specific primers. The primers used were Seq ID 4 (forward): 5' CCC AAG CTT GGA ATG GGA GCC CTC CGC GCC AAG TCT 3', and Seq ID 2 (reverse) 5' CGG GAT CC CTA CTC CTG CGC GGG TAT GCC AGC 3' in a Thermocycler (Gene Amp PCR System 9700; Applied Biosystems) using pfx Taq DNA polymerase (Invitrogen, San Diego, CA). PCR conditions were one cycle of 5 min at 94°C, 40 cycles of 1 min at 94°C, 1 min 20 s at 58°C, and 1 min 10 s at 72°C, followed by a final cycle of 7 min at 72°C. Amplified PCR product was electrophoresed in agarose gel and eluted from the gel (QIA quick gel extraction kit, Qiagen, Valencia, CA). The eluted product was subsequently cloned into mammalian expression vector pVAXl and transformed into competent E. coli TOP 10 cells. The transformants were screened for the presence of recombinant plasmids. Growth was initiated by adding 20 ml of an overnight culture from a single colony to 1 L LB broth and incubating overnight in presence of 50 μg/mL kanamycin (Himedia, Mumbai, India) at 37^°C with constant shaking at 225 rpm. Isolated positive clones were sequenced by DNA sequencer (ABI Prism, Model 377; Applied Biosystems). Recombinant plasmids were then maintained and propagated in TOP 10 E. coli. Endotoxin- free plasmid DNA was isolated using Endo-free plasmid isolation kit (Qiagen, Valencia, CA) according to manufacturer's protocol and used

HCL4517) were maintained in DMEM medium (Invitrogen, San Diego, CA) supplemented with 10% FBS. The expression of cpc was detected in mammalian cell by transfecting pVAXl-cpc construct in HEK293T cells using lipofectamine 2000 (Invitrogen, San Diego, CA) according to the manufacturer's instructions with slight modifications. Briefly, HEK293T cells were cultured at 2 x 10⁵ per well in 12-well plates to produce 85-90% confluence on the day of transfection. Lipofectamine 2000 and both pVAXl vector and pVAXl-cpc construct were diluted in serum-free Opti-MEM media (Invitrogen, San Diego, CA) at 1μ^200 μΐ and 2μ /200 μΐ, respectively. The diluted lipofectamine 2000 and plasmid DNA were mixed together and incubated for 30 min at room temperature. The mixture was then added drop wise onto the cell under gentle rocking condition, and incubated for 45 min at room temperature. The transfected cells were incubated 4-6 h at 37 °C with 5% C0₂ and medium was replaced by 1ml of DMEM (Gibco) supplemented with 10% FCS (Gibco). The cells were incubated at 37°C in a C0₂ incubator for 18-24 hours post-transfection before assaying for transgene expression. The media was replaced 24 h later with fresh media and transfected cells were maintained in presence of 250 μg/ml of G418. The transcription level of the cpc gene was detected by reverse transcription-PCR

(RT-PCR) using the cpc -specific primers mentioned above (Seq ID 4 and Seq ID 2) and the transient expression was analyzed by Western blotting. Briefly, total cellular RNA from pVAXl-cpc-transformed HEK293T cells was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The extracted RNA was treated with DNase I (Roche, Mannheim, Germany) to completely remove any residual , DNA. cDNA was synthesized from lOOng of total RNA using Superscript^® III First-Strand Synthesis kit (Invitrogen, San Diego; CA). The cDNA was amplified by PCR with L. donovani cpc specific primers mentioned above (Seq ID no 4 and 2). After amplification, 10 μΐ of each PCR product was applied to 1% agarose gel and visualized by ethidium bromide staining. For Western blotting, the protein extracts from the transfected 293 T cells were separated by SDS- PAGE and transferred onto a polyvinylidenefiuoride (PVDF) membrane. To detect the expressed protein, a primary polyclonal antibody against recombinant cpc was used at 1:1000 dilution followed by 1 : 1000 dilution of HRP-conjugated goat anti-rabbit IgG secondary antibody (Bangalore Genei, Bangalore, India).

Example 4

Preparation of cationic liposomes with MPLA and entrapment of plasmid DNA within it:

Lipids used herein were obtained from Sigma and Fluka. MPLA was bought from Invivogen, San Diego, California. All other chemicals were of analytical reagent grade. Initially the solution of lipid was prepared by dissolving 20 mg DSPC, 3mg cholesterol, 2mg SA and l(^g MPLA in approximately 1ml chloroform. The molar ratio of DSPC: Cholesterol: SA is 7:2:2 with lC^g/ml MPLA dissolved in chloroform followed by evaporating the organic solvents to form a thin film. The uniform lipid film is made in round bottomed flask with rotary evaporator. The lipid film is then desiccated in vacuum desiccator for almost 16 hours at a temperature of 25°C. Empty and plasmid DNA entrapped liposomes were prepared by dispersion of lipid film in either 1 ml of 0.02 M PBS (pH 7.4) alone or containing lmg/ml of pVAXl-cpc. The mixture was then vortexed for 15 mins and sonicated in an ultrasonicator (Misonix, New York, USA) for 30 s at 5 Hz, followed by incubation at 4°C for 2 h. The excess free plasmid DNA was removed by ultracentrifugation at 105,000xg for 1 h at 4°C twice. Estimation of percent liposomal DNA entrapment was based on its quantification in the unentrapped fraction by absorbance at 260 nm. The level of DNA incorporation ranged between 75-80%. The liposomes thus prepared, with or without DNA, were stored at 4°C until characterized and/or used. Example 5

Atomic forced microscopy for structural analysis of lipososmes

For AFM imaging of liposomal samples, ΙΟμΙ of the sample was deposited onto freshly cleaved muscovite Ruby mica sheet (ASTM VI Grade Ruby Mica from MICAFAB, Chennai) for 15-30 minutes. Mica sheets are basically negatively charged so samples binds strongly on the mica surface. After 15 min, the sample was dried by using vacuum dryer. The sample was gently washed with 0.5ml Milli-Q water to remove the molecules that were not firmly attached to the mica and the sample was dried as mentioned above.

' . _.

AAC mode AFM was performed using a Pico plus 5500 ILM AFM (Agilent Technologies, USA) with a piezoscanner maximum range of 9μπι. Micro fabricated silicon cantilevers of 225 μτη in length with a nominal spring force constant of 21-98 N/m were used from Nano sensors, USA. Cantilever oscillation frequency was tuned into resonance frequency. The cantilever resonance frequency was 150-300 kHz. The images (512 by 512 pixels) were captured with a scan size of between 0.5 and 2 μπι at the scan speed rate of 0.51ines/S. Images were processed by flatten using Pico view 1.4 version software (Agilent Technologies, USA). Image processing and analyzation has been done through Pico Image Advanced version software (Agilent Technologies, USA).

The AFM images of liposomes on mica are shown in Figure 3 AFM in the acoustic alternative current (AAC) mode approaches allows the observation of the liposomal morphology avoiding any sample manipulation such as staining, labeling, or fixation. Flattening of vesicles on the mica support just few minutes after deposition indicates a moderate stability of the liposomes on a mica substrate. AFM images clearly depict the spherical, well-defined shape of liposomes with visible multilamellar structures. The liposomal DNA complex was seen to be compacted with very few aggregations.

Example 6

Zeta potential Measurement

The zeta potential, which is an indirect measurement of the vesicle surface charge, was measured at room temperature by half-diluting the liposomes in 20 mM PBS using Nano ZS Zetasizer (Malvern Instruments, Worcestershire, UK). A polydispersity index value of 0.0 represents a homogeneous particle population, while a value of 1.0 indicates the heterogeneity of the liposome preparations.

Values of the zeta-potential of liposomes indirectly reflect vesicle surface net charge and therefore are used to evaluate the extent of interaction between the surface charges of cationic liposomes and the anionic charges of DNA. On this basis, the question of DNA entrapped versus empty DSPC liposomes with MP LA was investigated before and after encapsulating the desired plasmid DNA. The positively charged liposomes make a strong complex with negatively charged phosphate moiety on the sugar backbone of DNA. Hence, entrapment of plasmid DNA into cationic liposomes probably led to neutralization of cationic charges and compaction leading to clear reduction of the zeta-potential values and size for the formulation studied (Table 01). The liposomal formulations were heterogeneous, size of empty LIP (MPL) = 134 ± 23.86 run; size of pVAXl-cpc/LIP (MPL) = 96.33 ± 14.31 nm and the zeta potential (z-p) of about 24.3 ± 3.1 mV and 22.6 ± 1.2 respectively. Table 1. Incorporation of Plasmid DNA into Liposomes: Vesicle Size, zeta Potential and entrapment. pV AX 1-cpc was entrapped in cationic liposomes as shown. Results represent mean ± S.E., n = 3.

xamp e

DNase Protection Assay The plasmid DNA gets easily degraded by endonucleases such as DNase I, which is one of the obstacles for the delivery of plasmid DNA in vitro or in vivo. Therefore, the stability in the presence of DNase I is one of the essential parameters of systemic gene delivery. To confirm that liposomes described herein protects plasmid from nuclease, DNase I protection assay was carried out using a method modified slightly from that reported previously (Wheeler et al., 1999). The following samples were tested: naked plasmid DNA and plasmid DNA entrapped in liposomal vesicles. A Τμ DNA aliquot of each sample was treated with Ι μί DNase I (lU/μΙ) (Invitrogen, San Diego, CA), Ι μΐ of 1 Ox DNase I reaction buffer, and water to ΙΟμΙ total volume and incubated at 37°C for 15-60 min. The complexes - were subsequently extracted with phenol-chloroform and analyzed on 1% agarose gel (Tris- acetate buffer system, pH 8.2) and stained with ethidium bromide post electrophoresis.

Fig 4. reveals that most of the plasmid DNA incorporated in the cationic liposomes [pVAXl-cpc/LIP (MPL)] could not be degraded by DNAse I. In contrast, naked plasmid DNA (pVAXl-cpc) was degraded with few minutes upon exposure to the enzyme. This may be attributed to condensed. DNA state of the plasmid within the cationic liposomes which is resistant to DNAse action ((Legendre ^"and Szoka, 1995). Results of liposomal DNA vulnerability to degradation by DNase were largely confirmed by agarose gel electrophoresis of samples of naked or liposomal DNA exposed to DNase I (Fig 4.). Based on intensity of staining and the appearance of smearing, it can be seen that, whereas naked plasmid DNA was completely digested (Fig 4. lanes 2 and 3), DNA entrapped in cationic liposomes under study was fully protected (Fig 4. lanes 4 and 5). Most of the DNA was entrapped in the liposomes and retarded in the gel as no free DNA is seen in lane 5.

Example 8

In vitro cytotoxicity assay . To test the effect of liposomes on cell viability, MTT [3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide; Sigma-Aldrich) assay was performed in normal murine peritoneal macrophages. Freshly harvested intraperitoneal macrophages from healthy BALB/c mice were plated at a density of l xlO⁶ cells/well in a 96 well microtiter plates at 37°C in 5% C0₂ in RPMI as growth medium and incubated at 37°C overnight in 5% C0₂ atmosphere. After overnight incubation, empty cationic liposomes (3.2-200 μg/ml with respect to DSPC) were added to the medium and cells were incubated for another 1 , 4 or 24 h. Cells incubated with only medium served as controls (100% viability). 100 μΐ MTT solution (2mg/ml) per 1 ml medium was added to each well after removing the media for 4 h at 37°C. The reduced, insoluble formazan crystals were solubilized in DMSO and absorption was measured at 550 nm on Thermo MULTISCAN EX plate reader. Viability was expressed in percent in comparison to untreated cells (100% survival). Experiments were performed in triplicates and repeated at least twice. Relative cell viability was calculated by dividing [Abs₅₅₀] (mean absorbance) of treated cells to [Abs₅₅₀] of the control cells. Results of the cell viability assays after liposome incubation are expressed in Fig. 5. Very high liposome concentrations between 100-200 μg/ml reduced the viability by 10-15% after 4 h of incubation, but did not affect the cell viability further even after 12h indicating that the invented formulation is totally safe for human use (Fig. 5).

Example 9

,

Toxicity studies in vivo

The toxicity profiling of the developed formulation (liposomal p VAX! -cpc) was carried out using different toxicity markers. 5-week-old male BALB/c mice (n=5) were treated with liposomes containing 50 μg or 100 g plasmid DNA (with 1-2 mg of lipid w.r.t DSPC/animal/dose) in a ΙΟΟμΙ. injection volume in PBS, administered by lateral tail vein injection, in a single, double or triple dose. Animals were sacrificed 15 days after final treatment. Plasma alanine aminotransferase (ALT), aspartate amiotransferase (AST) and alkaline phosphatase (ALP) levels were determined for evaluating hepatotoxicity in plasma samples collected from different animal groups (using diagnostic kits from Randox, Ardmore, UK) [Heyes et al, 2006]. ,

The relative toxicity of the new cationic liposomal formulation is of utmost interest for intended systemic administration. The toxicity, if any, after intravenous delivery of liposomal pVAXl-cpc vaccine was examined by detecting the levels of serum AST, ALT and ALP in mice. AST is an enzyme involved in the transfer of an amino group from aspartate to alpha ketoglutarate to produce oxaloacetic acid and glutamate, present in highest concentration in liver followed by heart, skeletal muscle, kidney etc. Elevation in AST level in serum generally indicates systemic tissue damage. ALP is an enzyme that comes mainly from the cells lining bile ducts and is normally eliminated in bile. Increase in serum ALP level than normal is suggestive of liver or bile-duct disease due to the body's inability to excrete it through bile. ALT is primarily found in the liver, making it a more specific test for detecting hepatocellular damage. The enzyme levels were elevated compared to normal controls only after triple dose of the vaccine which was not statistically significant as shown in Fig 6. AST/ ALT and ALP levels actually remained within the normal limits with 0μg liposomal pVAXl-cpc injected twice within 48 hrs (administered dose) indicating that the invented formulation is totally safe for human use. Example 10

Immunization of mice and challenge infection

For immunization, ten 4-6 weeks-old, healthy BALB/c mice were injected intramuscularly in the hind leg thigh muscle with 50 μg (in 50 μΐ of PBS) of pVAXl-cpc, empty liposome or PBS. For all immunization study, all groups were boosted once at 2-week interval. Ten days after the booster the mice were challenged intravenous with 2.5 x 10 freshly transformed stationary phase promastigotes in 200 μΐ PBS injected intravenously as described earlier [Mazumdar et al., 2004]. Example 11

Delayed type Hypersensitivity

One week after the last vaccination, delayed-type hypersensitivity (DTH) was determined as an index of cell-mediated immunity. The response was evaluated by measuring the difference in the footpad swelling at 24 h following intradermal inoculation of the test footpad with 50 μΐ of recombinant CPC protein (5 μg/animal) from that of control (PBS- injected) footpad with a constant pressure caliper (Starret, Anthol, USA) [Afrin et al., 1997].

DTH response was measured in vaccinated mice 10 days after the last immunization. Vaccinated mice with naked pVAXl-cpc pDNA and its encapsulation in cationic liposomes displayed significant DTH responses in comparison to control groups (Figure 7; P < 0.05 and P < 0.01 respectively). However, the response by pVAXl-cpc was significantly lower (P < 0.05) than the response induced by liposomal pVAXl -cpc immunization.

Example 12 rCFC specific humoral response

Serum samples of individual mice were obtained before infection, . ten days post vaccination. Sera of individual mice were assayed for the presence of cpc-specific total IgG, IgGland IgG2a antibodies using enzyme-linked immunosorbent assay (ELISA) as described earlier [Afrin et al., 1997]. In brief, 96-well mi crotiter plates (Nunc, Naperville, IL) were coated with recombinant CPC protein (50 μg/ml) and blocked to prevent nonspecific binding. The plates were then incubated with sera at a 1 :200 dilution, followed by horseradish peroxidase (HRP)-conjugated goat IgGl, and IgG2a (1:1000) (BD Pharmingen, San Diego, CA). The color reaction was developed, and the absorbance was read in an ELISA plate reader (Thermo, Waltham, MA) at 450 nm [Afrin et al, 1997]

In VL, increased IgGl would characterize the Th2 response and disease progression in L. donovani -infected animals, while increase in IgG2 is indicative of Thl response and resistance to infection [Deplazes et al., 1995]. Moreover, antibody isotype profile correlates with type of cytokines produced from antigen-specific T cells. Switching of the IgG isotype to IgG2a is brought about by IFN-γ while IL-4 is associated with IgGl production [Coffman et al., 1993]. IgG levels did not differ significantly between controls and liposomal DNA vaccinated animals. The combined IgGl and IgG2a production with significantly high ratio of IgG2a:IgGl as a measure of Thl :Th2 balance, discloses a Thl -biased immune protection with liposomal pVAXl-cpc DNA vaccine. In contrast, mice immunized with empty liposomes and naked pVAXl-cpc had similar IgGl and IgG2a ratio lower than pVAXl-cpc entrapped in liposomes revealing a lack of Thl dominance (Figure 8). . Example 13

Cytokine assay

For determination of recombinant CPC protein specific total cytokine production, spleens were removed aseptically from experimental mice of each group at 10 days after the last immunization. Each spleen was teased between 20 um pore size sieve to prepare single cell suspension in complete medium prepared with RPMI 1640 (Sigma) supplemented with 10 mM NaHC0₃, 10 mM HEPES, lOoJu/ml penicillin, 100 μg/ml streptomycin sulphate, 50 μΜ 2-ME (Sigma) and 10% heat inactivated fetal bovine serum. Erythrocytes were removed by lysis with 0.14 M Tris buffered NHUC1. The splenocytes were washed twice, resuspended in the culture medium and viable mononuclear cell number was determined by Trypan blue exclusion. Splenocytes were then incubated in a 96- well ELISA plate (Nunc) at a density of 2 x 10⁵ cells/well in a final volume of 200 μΐ with or without recombinant CPC protein (2^g/well). The cells were incubated for 96 h at 37 °C in a humified chamber containing 5% C0₂. After 72 h incubation supernatants were collected and cytokine concentrations of IFN-γ, interleukin (IL)-4, IL-2, IL-10, and IL-12 were quantitated using Opt EIA kits (BD Pharmingen) in accordance with manufacturer's instructions.

The quality and magnitude of the immune response is probably regulated by the synergistic effect of the cytokines. We evaluated the Thl and Th2 cytokine responses in groups of vaccinated mice. Splenocytes from immunized mice were isolated 10 days after immunization and IFN-γ, IL-2, IL-10, IL-12 and IL-4 levels were measured in vitro following restimulation with recombinant CPC protein. While IL-12, IFN-γ and IL-2, are regarded as crucial for parasite control and resolution of disease, growing evidences suggests importance of IL-10 in down modulating type 1 responses, resulting in parasite persistence in the host and progressive VL. CD4⁺ and CD8⁺ Thl cells, via IFN-γ and IL-2, direct the response towards cell -mediated immunity involving cytotoxicity and macrophage activation, whereas Th2 cells, via IL-4 and IL-10, direct the response towards antibody production. The two poles are counter-regulatory such that antibody formation is inhibited by IFN-γ and macrophage activation is largely inhibited by IL-4 and IL-10. Strong Thl based immune protection was achieved with liposomal DNA formulation [pVAXl-cpc/LIP(MPL)] compared to PBS, adjuvant control and naked plasmid DNA (pVAXl-cpc) as reflected by increased IFN-γ, IL- 2 and IL-12 along with low levels of Th2 promoting cytokines like IL-4 and IL-10 (Figure 9 ), after immunization. Even the mice immunized with empty liposomes containing MPLA shows increased IL-12 and IFN-γ as markers for Thl response which enhanced with entrapping the plasmid DNA construct.

Example 14

. -

Evaluation of parasite burden in liver and spleen

I

The efficacy of naked and liposome-encapsulatqd pVAXl-cpc plasmids to protect animals against lethal L. donovani challenge, by intramuscular immunization, was evaluated in murine model of progressive VL (Fig 10. After 3 months of challenge infection clearance of organ parasite burden was quantitated as LDU in liver and spleen biopsies. The course of infection was monitored by the microscopic examination of Giemsa-stained impression smears of liver and spleen. The parasite load was expressed as Leishman-Donovan units (LDU) and was calculated by the following formula: number of amastigotes per 1,000 cell nuclei x organ weight (in milligrams) (Stauber et al., 1958).

As shown in Figure 10, control mice receiving PBS or empty liposomes developed highest parasite load in the liver and spleen due to progressive disease. Both naked and liposomal pDNA (pVAXl-cpc) conferred protection against parasite challenge upon intramuscular immunization in spleen. However, protection was maximum and significantly increased in mice vaccinated with liposomal vaccine formulation over naked pDNA in liver (P < 0.05) and spleen (P < 0.05). In contrast, only vector and adjuvant control provided no protection in spleen. Interestingly, there was significant decrease in parasite load in mice receiving only adjuvant (LIP-MPL) in liver (P < 0.05) after 3 months showing some protective capacity of cationic liposomes with MPLA alone. Liposomal p-DNA conferred 82.10 and 79.9 percent higher protection than empty liposomes, in liver and spleen respectively.

ADVANTAGES OF THE INVENTION

1. The adjuvant formulation comprising of MPLA incorporated in the lipid bilayer of cationic liposomes is a novel and safe strategy of combining TLR signaling with efficient delivery vehicle for antigen presentation and to promote CD4⁺ and CD8⁺ T cell-mediated protective immunity against Leishmania, through a route compatible with human administration.

2. In a further aspect, there is provided a composition comprising plasmid DNA with Thl promoting adjuvant and/or carrier to prevent and/or treat any parasitic infection. The results obtained with encapsulating DNA can be extrapolated to other molecules like proteins/ peptides and may find application against intracellular pathogens like Leishmania and also against cancer. j ^'

3. The present invention is a breakthrough in the field of medical immunology and further to methods of preparation of vaccines and immunomodulators to combat parasitic diseases and cancer.

4. In the present invention incorporation of MPLA in the liposomal formulation provided better protection that too in very low dose of MPLA (~^g/animal/dose) in comparison to the dose of MPL-TDM whose dose requirement is

animal [Majumder et al., 2011].

5. Compared to previous DNA vaccination with type I (cpa) and II (cpb) cysteine protease genes from Leishmania major within solid-lipid nanoparticles (J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(3) 320 - 335, 2010), the present invention shows better immunopotentiation by incorporating MPLA in liposomes. The liposome-mediated pDNA (pVAXl-cpc construct) delivery resulted in robust protection, 34.18 and 54.25 times higher than naked pVAXl-cpc in liver and spleen respectively.

References

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Seq ID 1 (forward): 5'GGA ATT CCA TAT G GCC CTC CGC GCC AAG TCT

GCG 3'

Seq ID 2 (reverse): 5'CGG GAT CC CTA CTC CTG CGC GGG TAT GCC AGC3'

Seq ID 3:

LOCUS JX968801 1038 bp DNA linear INV 28-OCT-2012

DEFINITION Leishmania donovani strain MHOM/IN/1983/AG83 cysteine protease C gene, complete cds.

ACCESSION JX968801

VERSION JX968801.1 GI: 409905639

SOURCE Leishmania donovani

ORGANISM Leishmania donovani

Eukaryota; Euglenozoa; Kinetoplastida; Trypanosomatidae;

Leishmaniinae; Leishmania.

ORIGIN

1 atgggtcgcg gatccatggc cctccgcgcc aagtctgcgc tgtgcctggt ggccgtgttt

61 gccgtgttgc tggccaccac ggtgagcggc ctctacgcca agccgagtga ctttccgctt

121 ctcggcaaga gttttgtggc ggagatcaac tcaaaggcga ggggtcagtg gaccgcctcg

181 gccgataatg^ gctacctggt cagcggcaag agcctcgagg aggtgcgcaa gctgatgggt

241 gtgaccgaca tgagcaccga ggctgttcct ccccgcaact tctctgtggt ggaaatgcag

301 caagacctgc cagagttctt cgacgccgcc gagcactggc ccatgtgcgt gacaatcagc

361 gagatccgtg accaatcgaa ctgcggctcg tgctgggcca tcgccgcggt ggaggctatt

421 tcggaccgct actgcaccct cggtggcgtt ccggatcgcc gcatatcgac cagcaacctt

481 ctctcctgct gcttcatatg cggctttggc tgctacggcg gcattccgac gatggcgtgg

541 ctgtggtggg tgtgggtggg cataacgacg gaggtctgcc agccctaccc ctttggccca

601 tgcagccatc acgggaacag cgacaagtac ccgccctgcc cgaacaccat ctacgatacc

661 cctaaatgca ataccacctg cgagaaaagc gagatggatc tggtcaagta caagggcggc

721 acatcttact ccgtcaaagg cgagaaggag ctcatgatcg agctcatgac caacggcccc

781 ttggaggtga ccatgcaggt gtactccgac ttcgtcggct acaagagtgg agtgtacaag

841 cacgtctctg gtgaccttct cggtggacac gccgtaaaac tggtcggctg gggaacccaa

901 ggcggtgtcc cgtactggaa gatcgccaac agctggaaca ccgactgggg tgacaaaggc

961 tacttcctga tccagcgcgg cagcaatgag tgcggtattg agagcggcgg cgttgctggc

1021 acacccgcgc aggagtag

Seq ID 4 (forward): 5' CCC AAG CTT GGA ATG GGA GCC CTC CGC GCC AAG TCT 3'

Claims

A DNA vaccine formulation in cationic liposome vehicle useful for maximizing vaccine potency for leishmaniasis characterized in containing intralamellar monophosphoryl lipid A (MPLA) and cysteine protease C (cpc) gene.

A DNA vaccine formulation as claimed in claim 1 wherein a plasmid DNA containing Seq ID 3 encoding cysteine protease C (cpc) gene of L. donovani is encapsulated in the cationic liposome vehicle.

A DNA vaccine formulation as claimed in claim 1 wherein the cationic liposome is a multilamellar vesicle comprising of: a) a neutral lipid preferably distearylphosphatidylcholine (DSPC);

b) a cationic lipid preferably. stearylamine (SA);

c) a second neutral lipid preferably cholesterol; and

d) a TLR agonist preferably Monophosphoryl lipid A (MPLA).

A DNA vaccine formulation as claimed in claim 3 wherein the neutral lipid, the cationic lipid, the second neutral lipid and the TLR agonist are formulated in a range of 7:1.5:1.5:0.0016 to 7:2.5:2.5:0.003 molar ratio.

A DNA vaccine formulation as claimed in claim 2 wherein the plasmid DNA is present in the range of 700 to 1000 μ^πιΐ.

A DNA vaccine formulation as claimed in claim 1 is administered to a subject via intra- venous, intra-muscular or intranasal route wherein said subject is a mammal including human.

The cationic liposome containing MPLA as claimed in claim 3 is useful as efficient delivery vehicle for biomolecules and vaccines.

A process for the preparation of the DNA vaccine formulation as claimed in claim 1 wherein said process comprising the steps of a) cloning cysteine protease C (cpc) gene having Seq ID 3 into pET28a vector; b) sub-cloning cpc clone obtained in step (a) into pVAXl vector to make pVAXl-cpc plasmid DNA construct; c) dissolving a neutral lipid, a cationic lipid, a second neutral lipid and a TLR agonist as claimed in claim 3 in a range of 7:1.5:1.5:0.0016 to 7:2.5:2.5:0.003 molar ratio in an organic solvent preferably chloroform to obtain a solution followed by removing solvent using evaporator to obtain a lipid film; d) desiccating the lipid film as obtained in step (c) at a temperature in the range of 25 to 28°C for a period in the range of 12 to 16 hours to obtain a dried lipid film; e) dissolving the plasmid DNA as obtained in step (b) in the range of 1 to 2 mg/ml in a physiologically acceptable aqueous medium to obtain a solution; f) hydrating the dried lipid film as obtained in step (d) with the solution as obtained in step (e) to obtain a solution; g) vortexing the solution obtained in step (f) for a period ranging between 10 to 15 min and sonicating for a period in th range of 30 to 45 sec at 4 to5 Hz at a temperature in the range of 3 to 4°C to Obtain a liposomal formulation; h) removing the excess free plasmid DNA from the liposomal formulation as obtained in step (g) by ultracentrifugation to obtain a DNA vaccine formulation.

A process for the preparation of the DNA vaccine formulation as claimed in step (e) of claim 8 wherein physiologically acceptable aqueous medium is Phosphate Buffer Saline solution in a molar ratio in the range of 0.01 to 0.02.

Use of the DNA vaccine formulation as claimed in claim 1 as a vaccine against leishmaniasis for maximizing vaccine potency in mammals against Leishmania parasite.

Use of the cationic liposome as claimed in claim 7 to generate strong humoral and cellular immunity required to combat various pathogens and cancer.

12. Use of the cationic liposome as claimed in claim 7 for drug screening against leishmaniasis.

13. A pharmaceutical composition comprising a therapeutically effective amount of the DNA vaccine formulation of claim 1 in a pharmaceutically acceptable carrier.