# Patent

## Title
Hemoglobin receptor as novel vaccine for leishmaniasis

## Abstract

The present invention in general relates Hemoglobin receptor or its part as a novel vaccine candidate against Leishmaniasis. Specifically, the present invention envisages HbR DNA for eliciting immune response in a mammal against Leishmaniasis. Additional aspect of the present invention is related to a vaccine composition for inducing immune response against Leishmaniasis in mammals. In a preferred aspect, the present invention relates to use of HbR-polypeptide as marker for diagnosis of Leishmania in kala-azar patients.

## Background
RELATED APPLICATION
This application is a national phase entry under 35 USC 371 of International Patent Application No PCT/IN2014/000231 filed on 11 Apr. 2014, which claims priority from India Patent Application No. 1449/DEL/2013 filed on 15 Mar. 2013, the diclosures of which are incorporated in their entirety by reference herein.
FIELD OF THE INVETION
The present invention in general relates to a Leishmania vaccine using active or inactive hemoglobin receptor either in DNA or protein forms for immunizing mammals.
BACKGROUND OF THE INVENTION
Leishmania, a protozoan pathogen, is the causative agent of various forms of leishmaniasis like cutaneous, mucocutaneous and visceral leishmaniasis; of which visceral Leishmaniasis is fatal (Kedzierski, et al., 2009). Leishmaniasis affects about 12 million people worldwide and 500,000 new cases are reported annually (WHO bulletin 2012). Drugs used for chemotherapy of leishmaniasis, such as antimonials, miltefosine, paromomycin and amphotericin B are very toxic, expensive and frequent resistance occurs against these drugs in endemic areas (Croft & Coombs, 2003). Moreover, no effective vaccine against leishmaniasis is available (WHO bulletin 2012). Now, it is well established that this disease is associated with the lack of Th1 response characterized by down-regulation of IL-12, IFN-γ while disease promoting Th2 cytokines like IL-4, IL-10 are up-regulated (Bacellar, et. al., 2000; Nylen & Sacks, 2007). Therefore, the major focus is to generate protective T-cell responses using appropriate antigen.
Thus, there is an immense requirement to develop an effective vaccine against leishmaniasis. Recently, considerable progress has been made and large numbers of Leishmania antigens have been tried as potential vaccine candidates including surface expressed antigens like gp63, gp46, PSA-2, receptors of activated C kinase (LACK), cysteine proteases (CP), kinetoplastid membrane protein-11 (KMP-11) etc with varied immune response and diverse species specific protection (Kedzierski, 2010; Singh & Sundar, 2012). However, vaccines protective against leishmaniasis are still not available. Therefore, there is need to identify a novel antigen which is physiologically important for the biology of the parasite.
In addition, no cost effective appropriate diagnostic procedure for detection of leishmaniasis is available. Microscopic examination of the Giemsa stained lesion biopsy smears, lymph node, bone marrow or spleen aspirates remain the standard test in areas of endemicity which require hospitalization. Moreover, owing to the low socio-economic status of the people who are largely affected by these diseases, the tests need to be simple and affordable. Molecular approaches like PCR for parasite detection, though specific, remain restricted to hospitals and research centers owing to its high cost (Franceschi et al., 2007). Recently, a specific immunochromatographic test has been developed based on k39 (a repetitive immunodominant epitope in a kinesin-related protein that is highly conserved among viserotropic Leishmania species) and sufficiently validated for field use (Hailu, 2002; Mathur et al., 2005). The test checks for the presence of anti-k39 IgG/M antibodies in serum and has shown excellent sensitivity (93-100%) and specificity (97-98%) in many VL-endemic countries (Alborzi et al., 2006; Pedras et al., 2008). However, there is a need to develop simple and cost effective diagnostic procedure against Leishmania infection.
Leishmania require heme from exogenous sources for growth due to lack of complete heme biosynthetic pathway (Sah, et. al., 2002). As heme, a critical prosthetic group required by the parasites for several metabolic pathways, thus, heme acquisition process in Leishmania could be a potential target (Kelly, et. al., 2003). Leishmania endocytosed hemoglobin (Hb) through a high affinity hemoglobin receptor (HbR) located on the cell surface (Sengupta, et. al., 1999) and internalized Hb is targeted to the lysosomal compartment where it is degraded to generate intracellular heme (Singh, et. al., 2003; Patel, et. al., 2008) which is used by Leishmania for their survival. This receptor is a surface localized hexokinase and N-terminal end of the receptor is extracellular hemoglobin binding region (Krishnamurthy, et. al., 2005). Thus, this molecule has dual roles; firstly, it acts as Hb receptor on cell surface and secondly, being hexokinase, it regulates glycolysis in parasites. Thus, HbR is bi-functional molecule performing two major functions in Leishmania. The present invention relates to active or inactive hemoglobin receptor or its parts either in DNA or protein forms as a novel immunogen for vaccine against leishmaniasis in mammals.
The present innovation demonstrates that HbR-DNA offers following major advantages over other vaccine candidates against VL.

    
    
        a. HbR-DNA vaccination induces sterile protection against Leishmania infection in both mice and hamster.
        b. It not only evokes protective Th1-response without any adjuvant but also suppresses disease promoting cytokines to confer sterile protection.
        c. HbR is a bifunctional antigen possibly interferes with two major pathways in Leishmania. 
        d. Anti-HbR antibody inhibits both promastigote and amastigote growth, therefore, anti-HbR antibody present in vaccinated animals might kills extracellular parasites during infection process.
        e. Successful protection of VL with vaccination of truncated HbR (HbR-N) indicates the feasibility of developing subunit vaccine of HbR against leishmaniasis.
        f. HbR is conserved across Leishmania species and hence could also be potential candidate against different forms of leishmaniasis.
        g. HbR protein or fragments thereof as marker for diagnosis of Leishmania in kala-azar patients.
    
    


SUMMARY OF THE INVENTION
One aspect of the present invention is related to a HbR DNA or its part for inducing sterile protection against virulent Leishmania, wherein said DNA comprises nucleotide sequence selected from the group consisting of: a) nucleotide sequence as set forth in any one of SEQ ID NO: 1 (HbR-FL), SEQ ID NO: 2 (HbR-N) or fragments thereof; b) a nucleotide sequence that encodes polypeptide having amino acid sequence as set forth in SEQ ID NO: 3.
In a preferred embodiment, the said DNA consists of nucleotide sequence as set forth in. SEQ ID NO: 2 (HbR-N).
A further aspect of the present invention is related to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 3; b) a polypeptide that is encoded by the nucleotide sequence as set forth in any of SEQ ID NO: 1 or SEQ ID NO: 2; and c) a polypeptide that is a fragment of SEQ ID NO: 3.
In a preferred embodiment, the said polypeptide is having the amino acid sequence as set forth in SEQ ID NO: 3.
Another aspect of the present invention is related to a recombinant DNA vector comprising the DNA as defined as hereinabove operably linked to a promoter.
Additional aspect of the present invention is related to a vaccine composition comprising any one of the polypeptide as defined hereinabove along with a pharmaceutically acceptable diluents or carrier wherein the said vaccine composition induces immune response against leishmaniasis in mammals.
In a preferred embodiment, the said vaccine composition comprises an adjuvant.
Another aspect of the present invention is related to a method for eliciting an immune response in mammals, comprising administering a vaccine composition as defined hereinabove to a mammal to be treated.
A further aspect of the present invention is related to use of the vaccine composition so obtained to induce immune response against leishmaniasis in mammals.
Another aspect of the present invention is related to a HbR-polypeptide as marker for diagnosis of Leishmania in kala-azar patients, wherein said polypeptide is selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 3 or fragments thereof ; b) a polypeptide that is encoded by the nucleotide sequence as set forth in any of SEQ ID NO: 1 or fragments thereof.
A further aspect of the present invention is related to use of HbR-polypeptide as marker for diagnosis of Leishmania in kala-azar patients.



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention and its various embodiments will now be described with the help of the accompanying drawings.
FIG. 1. PCR amplification of hemoglobin receptor from various. Leishmania species. Lane M, 1 kb DNA ladder (Promega) Lane 1, L. amazonensis; Lane 2, L. amazonensis var.7; Lane 3, L. amazonensis var.5; Lane 4, L. infantum; Lane 5, L. infantum var.2; Lane 6, L. infantum var.6; Lane 7, L. donovani; Lane 8, L. major; Lane 9, L. major var.1; Lane 10, L. major var. 4; Lane 11, L. major var. 4-2; Lane 12, L. turanica; Lane 13, L. turanica var.16; Lane14, L.gerbilli; Lane 15, L. tropica; Lane 16, L. tropica ver. 1; Lane 17, L. tropica ver. 7; Lane 18, L. enrietti. 
FIG. 2A. Promastigotes were incubated in presence equimolar amount of indicated proteins in M199 medium supplemented with [3H]-thymidine for 12 h at 23° C. Cells were washed and the cell growth was measured by determining the amount of radioactivity incorporated by cells. Results are expressed as mean ±S.D. of three independent experiments.
FIG. 2B. Fluorescent and phase images of HbR-GFP expressing L. donovani promastigotes (green) incubated in the presence of indicated antibodies. Pre-immune serum was used as control. Results are representative of three independent observations.
FIGS. 3A, 3B, 3C, 3D, and 3E. HbR-DNA vaccination completely protects animals against challenge of virulent L. donovani. BALB/C mice (n=8) or hamster (n=6) were challenged with virulent Leishmania donovani after immunization with indicated DNA construct. PBS injected infected animals were used as control. Animals were sacrificed 60 days after infection and parasite load in spleen and liver from respective animals were determined. The results are expressed as LDU±S.E.M. of respective organ in mice and as median values in hamster. Data were analyzed by t test, and levels of significance are indicated by p values.

    
    
        FIG. 3A. Splenic parasite burden in mice
        FIG. 3B. Hepatic parasite burden in mice.
        FIG. 3C. Splenic parasite burden in hamster.
        FIG. 3D. Hepatic parasite burden in hamster.
        FIG. 3E. Survival kinetics of vaccinated and parasite challenged hamsters during experimental period of 260 days post-infection.
    
    


FIGS. 4A, 4B, 4C, and 4D. HbR-DNA vaccination reverses T-cell anergy. Proliferation of splenocytes isolated from indicated group of animals (a,b) were determined in response to HbR. B. Functional significance of splenocyte proliferation in response to indicated antigens was determined by measuring the secretion of IL-2 in the culture supernatant by splenocytes isolated from different groups of mice (c) and hamsters (d) after 48 h of incubation at 37° C. by cytometric bead array.
FIGS. 5A, 5B, 5C, 5D, and 5E. HbR vaccination induced Th-1 cytokine in mice. Levels of different cytokine secreted by splenocytes isolated from indicated group of mice in the culture supernatant were measured after 48 h of incubation. a. IL-12; b. IFN-γ; c. TNF-α; d. IL-10; e. IL-4. Results are expressed as mean ±S.D. of pg/ml of cytokines secreted by spenocytes isolated from 12 mice in each group. Results are expressed as mean unit of absorption ±S.D. All data were analyzed by t test, and levels of significance are indicated by p values.
FIGS. 6A, 6B, 6C, 6D, and 6E. HbR vaccination in hamster induced the expression of Th1 cytokines and simultaneously suppressed the expression of disease promoting Th2 cytokines. Levels of different cytokine in splenocytes isolated from indicated group of hamster were measured by Real Time PCR using cytokine specific primers after 48 h of incubation. a. IL-12; b. IFN-γ; c. TNF-α; d. IL-10; e. IL-4. Results are expressed as mean ±S.D. of pg/ml of cytokines secreted by spenocytes isolated from 12 mice in each group. Results are expressed as mean unit of absorption ±S.D. All data were analyzed by t test, and levels of significance are indicated by p values.
FIGS. 7A and 7B. Determination of HbR specific antibody in vaccinated and control groups of mice (a) and hamster (b). Bloods was collected from vaccinated and control groups of indicated animals and serum was analyzed for the presence of HbR-specific antibody by ELISA. Results are expressed as mean unit of absorption ±S.D. All data were analyzed by t test, and levels of significance are indicated by p values.
FIG. 8. Detection of anti-HbR antibody in kala-azar patient serum.
FIG. 9. Sequence ID Number 1: Full length Hemoglobin receptor DNA sequence (HbR-FL): Sequence ID Number 2: N-terminal Hemoglobin receptor DNA sequence (HbR-N): Sequence ID Number 3: Full length Hemoglobin receptor protein sequence (HbR-FL):.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is related to a HbR DNA for inducing sterile protection against virulent Leishmania, wherein said DNA comprises nucleotide sequence selected from the group consisting of: a) nucleotide sequence as set forth in any one of SEQ ID NO: 1 (HbR-FL), SEQ ID NO: 2 (HbR-N) or fragments thereof; b) a nucleotide sequence that encodes polypeptide having amino acid sequence as set forth in SEQ ID NO: 3.
In a preferred embodiment, the said DNA consists of nucleotide sequence as set forth in SEQ ID NO: 2 (HbR-N).
The present invention also relates to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 3; b) a polypeptide that is encoded by the nucleotide sequence as set forth in any of SEQ ID NO: 1 or SEQ ID NO: 2; and c) a polypeptide that is a fragment of SEQ ID NO: 3.
Preferably, the said polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 3.
The present invention also envisages a recombinant DNA vector comprising the DNA as defined hereinabove operably linked to a promoter.
The said promoter is selected from a group consisting of mammalian expression vectors but not limiting to CMV promoter.
Furthermore, the present invention is related to a vaccine composition comprising any one of the polypeptide as defined hereinabove along with a pharmaceutically acceptable diluents or carrier wherein the said vaccine composition induces immune response against Leishmaniasis in mammals.
The said diluents or carriers are selected from but not limited sterile saline.
In another preferred embodiment, the said mammals include but are not limited to mice, hamster, dogs, humans.
In a further preferred embodiment, the said vaccine optionally composition also comprises an adjuvant.
Preferably, the said adjuvant is selected from IL-12, GLA-SE (stable emulsion of monophosphoryl lipid A) and other related preparations.
The present invention is additionally related to a method for eliciting an immune response in mammals, comprising administering a vaccine composition as defined hereinabove to a mammal to be treated.
A further embodiment of the present invention is related to use of the vaccine composition so obtained to induce immune response against leishmaniasis in mammals.
In another embodiment, a HbR-polypeptide as marker for diagnosis of Leishmania in kala-azar patients, wherein said polypeptide is selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 3 or fragments thereof ; b) a polypeptide that is encoded by the nucleotide sequence as set forth in any of SEQ ID NO: 1 or fragments thereof is envisaged.
In a further embodiment, use of HbR-polypeptide as marker for diagnosis of Leishmania in kala-azar patients is enunciated.
In the present innovation, immunization of mammalian host with HbR-DNA or its part provides sterile protection against virulent Leishmania donovani infection in both BALB/c mice and hamster by inducing Th1 response.
Leishmaniasis is a complex diseases varying from cutaneous to visceral leishmaniasis, caused by diverse species of Leishmania. Ideally, an antigen use for developing effective vaccine against different forms of leishmaniasis should be present in different species of Leishmania. In the present innovation, HbR is found to be conserved among different species of Leishmania. Thus, the present invention is related to use HbR or its part as vaccine composition against different forms of leishmaniasis.
For the purpose of illustration, the invention will now be described non-limitatively in the following examples which are provided in order to demonstrate and further illustrate the preferred embodiments and aspects of the present invention.
EXAMPLES
The following Reagents and Chemicals were Used to Carry Out the Study as Set Forth in the Forthcoming Examples
Medium RPMI-1640, M199, FCS (Sigma), Penicillin-Streptomycin solution (Invitrogen), Endo free Plasmid Mega/Giga Kit (Qiagen), anti-mouse CD4, CD8, CD3, TNF-α, IL-2 (BD Biosciences), anti-mouse IFN-γ (Biolegend), BDTM mouse Th1/Th2 cytokine kit II (BD Pharmingen) and BDTM CBA mouse Inflammatory kit (BD Pharmingen), Aqua Live/dead staining dye (Invitrogen) were used in the present study.
Animals and parasite
Four- to six-week-old BALB/c mice (Jackson Laboratory, Bar Harbour, Me., USA) and golden hamsters (Mesocricetus auratus), reared in pathogen free institute animal facilities, were used for experimental purposes with prior approval of the animal ethics committee of the Indian Institute of Chemical Biology (Kolkata, India). AG83 (MHOM/IN/83/AG83) strain of L. donovani was used for experimental purposes. Parasites were maintained in golden hamsters. Promastigotes obtained after transforming amastigotes from infected spleen were maintained in M199 (Sigma) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS. at 22° C.
Example 1
Presence of HbR in Different Species of Leishmania 
Genomic DNA isolated from 18 different species and sub-strains of Leishmania were used. Full-length HbR were amplified from genomic DNA by RT-PCR from all 18 species and sub-strains of Leishmania using forward (5′-GTGGATCCATGGC CACCCGCGTGAAC-3′) and reverse (5′-GTGAAT TCCTACTTCTTGTTGACGGCCA-3′) primers designed against the start and stop codons of HbR by RT-PCR.
The results in the present innovation showed that full-length HbR from genomic DNAs of various strains of different Leishmania species namely, L. amazonensi, L. infuntum, L. donovani, L. major, L. turanica, L. gerbilli, L. tropica, L. enrietti are amplified (FIG. 1) and sequence comparison showed 95-100% identity.
Example 2
Evaluation of HbR as a Novel Vaccine Candidate against Leishmaniasis
HbR-FL, HbR1-126 (HbR-N) and HbR270-471 (HbR-C) were previously cloned, expressed as GST fusion proteins and specific antibodies were raised against HbR-N and HbR-C. Further characterization revealed that N-terminal of HbR, the HbR1-126 (HbR-N) is extracellular hemoglobin binding domain and anti-HbR-N antibody or HbR1-126 peptide specifically blocks hemoglobin uptake by Leishmania like HbR full-length (HbR-FL) protein (Krishnamurthy, et. al., 2005). Therefore, in the present investigation, effect of different fragments of HbR on the growth of Leishmania promastigotes were determine. Briefly, log phase promastigotes were washed, resuspended in M199 medium (106 cells/200 μl) containing equimolar, amount of GST-HbR-N or GST-HbR-C peptides (942 pmol/ml) along with 3H-thymidine (0.6 μCi/50 μl/well) and incubated for 12 h at 23° C. Cells were harvested using cell harvester and radioactivity incorporated by cells were determined. Our results show that HbR1-126 (HbR-N) fragment specifically inhibits the complete growth of the parasites like HbR-FL protein (FIG. 2a).
To determine effect of HbR-fragment-specific antibodies, log phase Leishmania promastigotes (1×107 cells) were washed, resuspended in M199 medium containing anti-HbR-N, anti-HbR-C or pre-immune sera (1:100) and incubated for 10 min at 23° C. in 6-well tissue culture plates. Subsequently, the promastigotes were visualized using confocal microscopy. These results show that anti-HbR-N specifically induce the aggregation of the cells and thereby blocks the parasite growth (FIG. 2b). Therefore, in the present innovation, DNA constructs containing HbR-FL or HbR-N are used to evaluate the efficacy of HbR as potential vaccine candidate in experimental models visceral leishmaniasis.
Example 3
HbR Elicited Complete Protection against Visceral Leishmaniasis Both in Mice and Hamster
BALB/c mice and hamsters models of visceral leishmaniasis are widely used to determine the efficacy of vaccine. Mice model is useful in understanding the immune dysfunction whereas golden hamster mimics the clinical features of visceral leishmaniasis and ultimately death. Therefore, in the present innovation, HbR-DNA as vaccine candidate was determined in both mice and hamster models of visceral leishmaniasis.
HbR-FL or HbR-N coding sequence was sub-cloned into Barn HI and Eco RI sites of mammalian expression vector (pcDNA3.1) downstream to the CMV promoter using respective primers.









HbR-FL:



Forward:



5′-GTGGATCCATGGCCACCCGCGTGAAC-3′



 



Reverse:



5′-GTGAATTCCTACTTCTTGTTGACGGCCA-3′



 



HbR-N:



Forward:



5′-GTGGATCCATGGCCACCCGCGTGAAC-3′



 



Reverse:



5′-GTGAATTCATCGCCAACCAGGGCACT-3′






Finally, respective animals (BALB/c mice and hamsters) were divided into five groups and immunized with endotoxin free indicated plasmid DNA construct. (i.) Unfected control; (ii). Infected control; (iii). Immunized with blank vector (pCDNA3.1) and infected; (iv). immunized with vector containing coding sequence of full length HbR and infected; (v.) immunized with vector containing coding sequence of N-terminal HbR and infected.
Respective animals were immunized by intramuscular injection in the hind thigh using 28-gauge needle with 100 μg of endotoxin-free respective plasmid DNA construct dissolved in sterile saline on day-1 and day-15. On day 27, immunized mice and hamster were challenged with intracardial injection of 1×107 virulent strain of Leishmania donovani (AG83) using 28-gauge needle. Finally, parasite load in spleen and liver from respective animals were determined 60 days after parasite challenge.
Determination of Hepatic and Splenic Parasite Burden in Mice and Hamster
To determine the prophylactic efficacy of HbR vaccination in mice and hamster, animals were sacrificed 60 days after parasite challenge. Liver and spleen from different groups were isolated to determine parasite load. The number of parasite present in the respective organ was determined by microscopic evaluation of Giemsa stained tissue imprints following previously described method (Haldar, et. al., 2009; Mukhopadhyay, et. al., 1989). The parasite burden in respective organs was expressed as Leishman Donovan Unit (LDU), representing the number of amastigotes/1000 host cells X organ weight (Stager, et. al., 2000).
Results obtain in the present innovation showed that immunization of mice with both HbR-N-DNA and HbR-FL-DNA reduce more than 99% of parasite loads in spleen (FIG. 3a) and liver (FIG. 3b) of mice in comparison to unimmunized control mice. No live parasite was detected in the spleen or liver homogenates of most of the mice immunized with HbR-FL or HbR-N indicating complete protection by HbR-DNA vaccination. Similar protection in hamster model of visceral leishmaniasis was also observed. More than 99% of splenic (FIG. 3c) and hepatic (FIG. 3d) parasite load were eliminated by immunizing the hamster with either HbR-FL-DNA or HbR-N-DNA in comparison to control groups. The HbR-FL-DNA or HbR-N-DNA induced sterile protection was supported by the fact that almost all HbR-N-DNA and HbR-FL-DNA vaccinated hamsters were healthy and survived against lethal challenge of virulent Leishmania during experimental period of 260 days (FIG. 3e). In contrast, all hamsters from control groups died within 200 days.
Therefore, present innovation demonstrated that immunization of infected animals with HbR-DNA or its part completely protects VL and therefore, HbR or its part is novel vaccine candidate against leishmaniasis. Similar results can be obtained with immunization of HbR or its truncated proteins. As this antigen is conserved among different species of Leishmania, therefore, it might be used to protect other forms of leishmaniasis.
Example 4
HbR Vaccination Reversed T-cell Anergy
Cellular immune response is found to be suppressed both in visceral leishmaniasis patient as well as in experimental models. Splenocytes isolated from AG83-challenged HbR-DNA vaccinated animals after 21 days of infection were assayed for T cell proliferation. Briefly, spleens were isolated from different experimental groups of mice and hamster after 21 days of infection and single-cell suspensions of splenocytes were prepared after Ficoll density gradient centrifugation. Cells were resuspended in complete RPMI 1640 and plated in triplicate at a concentration of 1×105 cells/well in 96-well plate. Subsequently, cells were allowed to proliferate for 3 days at 37° C. in a 5% CO2 incubator either in the presence or absence of HbR protein (5 μg/ml). Complete soluble antigen from Leishmania was used as control. Cells were pulsed with BrDu 2 h before harvesting on day-3 to measure cell proliferation.
Results obtain in the present innovation showed that HbR-N and HbR-FL DNA immunization significantly enhance the T-cell proliferation in both mice (FIG. 4a) and hamsters (FIG. 4b) in comparison to infected and blank vector-immunized animals in the presence of HbR.
Moreover, depressed splenic T cell response could be due to impaired IL-2 production. To determine the functional significance of induced T-cell proliferation in HbR-DNA vaccinated animals, the levels of IL-2 secretion by spenocytes isolated from different groups of infected mice was measured. Isolated spenocytes from 21 days post-infected mice were stimulated in the presence of 5 μg/ml of HbR protein and culture supernatants were used to measure the levels of IL-2 by ELISA. Results showed that culture supernatants from splenocytes from HbR-FL and HbR-N DNA immunized mice contained about 5 and 6 folds more IL-2, respectively, compared to infected control and vector immunized mice (FIG. 4c). Higher levels of IL-2 transcripts in HbR-N and HbR-FL DNA vaccinated infected hamsters was observed in comparison to control animals by real time RT PCR (FIG. 4d). These results indicated that full length and N-terminal HbR-DNA vaccination overcome impaired. T-cell proliferation and induce the production of IL-2 to generate appropriate T cell response for parasite clearance.
Example 5
HbR Induced Multifunctional Th-1 Protective Immune Response in Animals
Any vaccine to be successful against VL should skew the immune response towards Th1 type. Thus, the levels of these Th1 cytokines secreted by splenocytes isolated from vaccinated and control groups of animals 21 days after infection was compared by cytometric bead array (CBA). Single-cell suspension of splenocytes from different groups of mice 21 days post-infection was prepared as described previously. Subsequently, 2.5×105- cells/well in complete RPMI 1640 medium were plated in 96 well plates and incubated for 48 h at 37° C. in 5% CO2 incubator in the presence or absence of 5 ug/ml of indicated proteins. After 48 h culture supernatant were collected by centrifugation. Levels of different cytokine in the culture supernatant were measured by cytometric bead array (multiplexing). Mouse Th1/Th2 and Mouse Inflammatory kit (BD Biosciences) were used for cytometric Bead Array (CBA). All the data were analyzed using Fcap Array in a FACS ARIAII Flow Cytometer (BD).
The results presented in the current innovation show that spenocytes isolated from HbR-FL-DNA immunized mice in response to HbR protein induce about 2.27 folds increase in IFN-γ production, 2 folds increase in IL-12 and 2.37 folds increase in TNF-α production whereas cells from HbR-N-DNA vaccinated mice produced about 1.88, 2 and 2.69 folds more IFN-γ, IL-12 and TNF-α, respectively, compared to that of infected and vector control groups (FIG. 5a-c). In contrast, vaccination with HbR-FL-DNA and HbR-N-DNA suppressed about 2.15 and 5 folds less production of Th1 suppresive cytokine IL-10, respectively, compared to that of infected controls (FIG. 5d). In addition, HbR-FL-DNA and HbR-N-DNA vaccination resulted in 2.42 and 2.77 folds decrease in production of Th2 signature cytokine IL-4, respectively, in comparison to control groups (FIG. 5e). These results indicated that full length and N-terminal HbR-DNA vaccination induce TH1 protective response. Interestingly, significantly more amount of IL-12 and higher IFN-γ/IL-10 ratio were obtained in HbR-N-DNA than HbR-FL-DNA vaccination, indicating greater Th1 bias protective immune response generated by HbR-N-DNA.
Example 6
Measurement of mRNA Levels of Different Cytokines in Splenocytes Isolated from Hamster by Real-time PCR
Splenocytes were isolated from 8 randomly chosen animals from different group of hamsters. Total RNA was isolated using Trizol reagent (Invitrogen) and used for cDNA synthesis of using maxima first-strand cDNA synthesis kit (Fermentas). Real Time PCR was performed following the method described earlier using cytokine specific primers. Briefly, it was carried out with 12.5 μl of SYBR green PCR master mix (ABI), 2 μl of cDNA from RT reaction mix, and gene specific primers in a final volume of 25 μl. PCR was conducted under the following conditions: initial denaturation at 95° C. for 2 min followed by 40 cycles, each consisting of denaturation at 95° C. for 30 s, annealing at 55° C. for 40 s, and extension at 72° C. for 40 s per cycle using the ABI 7500 Real time PCR system. All quantifications were normalized to the housekeeping gene HGPRT. A no-template control cDNA was included to eliminate contaminations. Differences in gene expression were calculated by the comparative CT method. Results are expressed as the degrees of difference between ΔCT values of test and comparator samples.
Transcript profiles of different cytokines in splenocytes isolated from vaccinated and control hamsters after 21 days of infection was compared by RT-PCR. Results also showed that levels of IFN-γ, IL-12 and TNF-α transcript are up-regulated whereas IL-10 expression is lower in the vaccinated hamsters in comparison to control groups. No significant change was observed in IL-4 expression between different groups of hamster (FIG. 6a-e). Thus, HbR-DNA vaccination not only induced protective Th1 response but also suppressed disease promoting cytokines like IL-10 and IL-4, to achieve complete protection both in mice and hamster models of VL.
Example 7
Determination of HbR Specific IgG1 and IgG2 Antibody Response in HbR-DNA Vaccinated Mice and Hamster
To determine the levels of anti-HbR specific IgG1 and IgG2a antibody in vaccinated and control groups of animals, blood was collected on 21 days post-infection and sera samples were analyzed for the presence of HbR-specific antibody. The 96-well ELISA plates (BD) were coated with full-length HbR protein (2 μg/ml) in PBS overnight at 4° C. Plates were washed and blocked with 5% FCS in PBS at room temperature for 1 h to prevent nonspecific binding. Sera (1:10 dilution) from different groups of mice was added into HbR-coated wells and incubated for 2 h at room temperature. Well were washed and incubated with biotin-conjugated Rat anti-mouse IgG for 1 h at room temperature followed by another 1 h incubation with avidin-conjugated HRP. ABTS as peroxidase substrate in citrate buffer (0.1 M, pH 4.3) with 0.1% H2O2 was added and absorbance was read on ELISA plate reader at 405 nm. Similar assay using biotin-conjugated mouse anti-hamster IgG1 and mouse anti-Armenian and anti-Syrian hamster IgG2 was carried out to detect the levels of HbR specific IgG1 and IgG2 levels in hamster sera.
These results showed that immunization of HbR-FL and HbR-N DNA generated about 3-4 folds higher IgG2a levels vaccinated mice (FIG. 7a) and hamster (FIG. 7b) compared to controls Higher level of IgG2a should trigger pathogen protection in vaccinated animals. Thus, HbR-DNA vaccination induced both humoral and cell-mediated immune response.
HbR Vaccination Induces both CD4+ and Cytotoxic CD8+ T-cells Response
Generation of multifunctional CD4+ T-cells response by vaccination was found to induce better protection (Darrah, et. al., 2007). Multiparameter flow cytometry analysis revealed that HbR-DNAs vaccination generated significantly higher population of both CD4+ and cytotoxic CD8+ T-cells with very high levels of single positive cytokines like IFN-γ+, TNF-α+ and IL-2+ compared to infected controls (data not shown). IFN-γ was shown to mediate pathogen killing (Cooper, et. al., 1993; Flynn, et. al., 1993) and the effect was additive by TNF-α (Liew, et. al., 1990), thus, enhanced production IFN-γ+TNF-α+ CD4+ and CD8+ T-cells by HbR-DNA vaccination rendered better protection against VL. Similarly, higher frequency of antigen specific IFN-γ+IL-2+ CD4+ T-cells by HbR-DNA immunization might provide durable protection by developing memory cells (Wu, et. al., 2002) in comparison to IFN-γ or IL-2 alone. IL-12 was reported as strong-Th1-inducing adjuvant (Heinzel, et. al., 1993; Sypek, et. al., 1993) and also enhanced targeting intracellular pathogens to lysosome (Bhattacharya, et. al., 2006). Thus, increased levels of IL-12 in HbR-DNA vaccinated animals contributed to parasite clearance. Taken together, the sterile protection observed in the present innovation by HbR-DNA vaccination in animals is due to simultaneous activation of both CD4+-Th1 and cytotoxic CD8+ T-cell response of immune system.
Example 8
HbR as Diagnostic Marker
To evaluate, HbR as potential marker for diagnosis of Leishmania, HbR was purified from Leishmania as described previously. Confirmed kala-azar patient sera were used with approval of the human ethics committee from Rajendra Memorial Research Institute of Medical Sciences, Patna, India. Purified HbR protein (1 μg) was subjected to SDS-PAGE, transferred onto nitrocellulose membrane and incubated with appropriate dilution of patient sera. Normal sera prepared from healthy individuals were used as control. Membranes were washed and probed with anti-human IgG+IgM antibody conjugated with HRP.
Interestingly, we found that 27 out of 30 VL patient sera possess antibody-against HbR demonstrating that HbR is a potential candidate for developing a diagnostic procedure for Leishmania. 
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## Claims

1. A GST HbR-DNA for eliciting an immune response against virulent Leishmania, wherein said GST HbR-DNA comprises GST and a nucleotide sequence selected from the group consisting of: a) nucleotide sequence as set forth in any one of SEQ ID NO: 1 (HbR-FL), SEQ ID NO: 2 (HbR-N) or fragments thereof; and b) a nucleotide sequence that encodes polypeptide having amino acid sequence as set forth in SEQ ID NO: 3.

2. The GST-HbR DNA as claimed in claim 1, wherein the said HbR-DNA consists of nucleotide sequence as set forth in SEQ ID NO: 2 (HbR-N).

3. A recombinant DNA vector comprising the DNA as claimed in claim 1 operably linked to a promoter.