Title of Invention	"A VACCINE EFFECTIVE AGAINST TYPHOID"
Abstract	The present invention relates to the development of a typhoid vaccine using heat shock protein 70 (Hsp70) of Salmonella typhi. The method of invention involves administering a composition comprising an effective amount of a complex consisting of a Hsp, in part or whole, either alone or covalently or non-covalently bound to an "antigenic molecule", which when administered elicits specific immunological responses in the host. "Antigenic molecule" used herein refers to the exogenous antigens/immunogens viz LPS, peptide, nucleic acid or polysaccharide or antigenic/immunogenic fragments and derivatives thereof.

Title of Invention

"A VACCINE EFFECTIVE AGAINST TYPHOID"

Abstract

The present invention relates to the development of a typhoid vaccine using heat shock protein 70 (Hsp70) of Salmonella typhi. The method of invention involves administering a composition comprising an effective amount of a complex consisting of a Hsp, in part or whole, either alone or covalently or non-covalently bound to an "antigenic molecule", which when administered elicits specific immunological responses in the host. "Antigenic molecule" used herein refers to the exogenous antigens/immunogens viz LPS, peptide, nucleic acid or polysaccharide or antigenic/immunogenic fragments and derivatives thereof.

Full Text	A VACCINE FIELD OF INVENTION The present invention relates to the development of a typhoid vaccine using heat shock protein 70 (Hsp70) of Salmonella typhi. The method of invention involves administering a composition comprising an effective amount of a complex consisting of a Hsp, in part or whole, either alone or covalently or non-covalently bound to an 'antigenic molecule', which when administered elicits specific immunological responses in the host. 'Antigenic molecule' used herein refers to the exogenous antigens/immunogens viz lipopolysaccharides (LPS), peptide, nucleic acid or polysaccharide or antigenic/immunogenic fragments and derivatives thereof. BACKGROUND OF INVENTION Typhoid fever continues to be a major health problem despite the use of antibiotics and the development of newer antibacterial drugs. Typhoid, caused by the infection with Salmonella typhi, is spread by faecal or oral route and is closely associated with poor inadequate sanitation and food hygiene. Salmonella typhi has rapidly gained resistance to antibiotics like ampicillin, chloramphenicol and cotrimoxazole and also to previously efficacious drugs like ciprofloxacin. The incidence of multidrug resistant (MDR) S. typhi is reported to be as high as 60% (Sanghavi et al, 1999) and resurgence of resistant strains has also been reported (Kumar et al, 2002). A US based study (Ackers et al, 2000) has shown an increase in the number of MDR strains and nalidixic acid resistant S. typhi (NARST). The virulence of specific strains in humans and other animals is serovar specific. S. typhimurium is a leading cause of gastroenteritis in humans, an acute localized inflammation of intestine. It is generally self limiting and without complication, provided supportive therapy is available. However, in mice infection with this serovar causes typhoid fever like disease. Intestinal and extra intestinal lesions in this model highly resemble those observed in humans suffering from typhoid fever, therefore, this model has been widely used to mimic infection caused by S. typhi in humans (Santos et al, 2001). Typhoid fever, caused by S. typhi in humans, is marked by protracted fever and a variable clinical presentation depending on the organ system affected (Keusch, 1994). Salmonella paratyphi induces paratyphoid fever, a disease which is similar to, but usually milder than typhoid fever but no disease is associated with experimental infections of mice by serovar typhi or paratyphi A and B. (Carter and Collins, 1974). The type of disease associated with any particular serovar of Salmonella is linked to the ability of that serovar to enter, survive and replicate within and to elicit damage in a specific host's macrophage cells (Alpuche-Aranda et al, 1995; Schwan and Kopecko, 1997; Schwan et al, 2000). Serovar typhimurium and enteritidis do not survive well in human macrophages, generally producing localized infection which results in mild gastroenteritis but they can grown and cause disease in mice (Collins, 1972). In contrast, Serovars. typhi and paratyphi, which can infect mice via the oral route, do not survive well in murine macrophages and can not cause disease in mice. On the other hand, they survive and multiply within human macrophages, causing only slight macrophage cell death, cross the ileal epithelium silently, moving stealthily within macrophage vehicles to deep tissues, where acute multiplication in selected tissues (e.g. live) ultimately results in typhoid fever (Schwan et al, 2000). Humans are the sole reservior of S. typhi infection as well as the only natural host. An important property of S. typhi is its ability to survive the environment for weeks or even months in the absence of a host. This explains how epidemics of typhoid fever occur even where no apparent cause of contamination can be found. Non availability of relevant drugs and rapid development of microbial drug resistance have led to the need of efficacious and affordable vaccines to control typhoid fever. When entering the host from the environment, a microbial pathogen is confronted by several changes, some of which are highly stressful. These include alterations in temperature, pH, and pO2 (Kaufman 1990, 1991). Moreover, the pathogen is exposed to natural host resistance mechanisms such as phagocytosis by professional phagocytes (Kaufman 1998). Once engulfed by phagocytes, the pathogen is confronted with reactive oxygen and nitrogen intermediates and attack by lysosomal enzymes. To protect itself against the host, the pathogen activates various evasion mechanisms including stress protein synthesis. Heat shock proteins (Hsps) or stress proteins (Sps) are synthesized by all living cells in response to various types of environmental stress and physiological insults like elevated temperatures, heavy metals, toxins, oxidants, bacterial and viral infections (Morimoto and Milarski, 1990; Young 1990). They are widely distributed in nature and are amongst the most highly conserved molecules of the biosphere. They ensure survival under stressful conditions that, if left unchecked, can lead to irreversible cell damage and ultimately to cell death. Although they are classified as Sps, Hsps also have essential functions in the cell under normal growth conditions. Hsps perform important functions in the folding and unfolding or translocation of proteins, as well as in the assembly and disassembly of protein complexes. Because of these helper functions, hsps have been termed molecular chaperones. (Ang et al 1991, Gething and Sambrook 1992, Rothman 1989). They are divided into a series of families based on their molecular mass in kilodaltons. Hsps 60, 70 and 90 are generally found in cytosol and mitochondria. Gp96 and calreticulum are located in the endoplasmic reticulum. In non-stressed cells hsps are present in low concentrations (1 to 2% of the total protein content), while in stressed cells their concentration is increased many folds. They are released into the environment during pathological situations such as necrotic cell death. However these proteins under certain circumstances are released extracellular to perform a range of immuno-modulatory activities. In pathogenic bacteria, Hsps are thought to be maximally induced during the infectious process in response to host resistance. Microbial Hsps are the dominant antigens for the host immune response to a variety of pathogens and the immune recognition of Hsps of pathogens serves as first line of defense. They stimulate humoral and cell mediated responses, do not require adjuvants and are effective even in immuno-compromised conditions. These properties permit the use of Hsps for development of a new generation of prophylactic and therapeutic agents against infectious agents. Pathogen-Derived Heat Shock Proteins as Targets for the Immune Response: Infection is a bimodal process determined by the host and pathogen. Both host cells and microbes are confronted with dramatic alterations in their living conditions during infection. Induction of host Hsp synthesis in response to encounter with a pathogen has at least two major causes. First, infected macrophages are confronted with antimicrobial mechanisms which they have activated themselves during infection. Efficient protection against their own effector molecules (e.g., reactive radicals) becomes vital for macrophage survival. Second, once inside a phagocyte, many microbes especially those which persist in the host, interfere with intracellular host cell metabolism. Not surprisingly, many of these pathogens are potent inducers of Hsp synthesis in mammalian cells. Increased pathogen Hsp levels in cells lead to rapid degradation of Hsp by the host processing machinery. Pathogen-derived determinates are efficiently presented by host cells and promote recognition of infected cells by the immune system. Although the exact role of Hsps in immunity to microbial infection is incompletely understood, Hsps apparently serve as important antigens in defense against infectious agents. In fact, immune responses to Hsp have been observed in infectious diseases caused by bacteria, protozoa, fungi, and nematodes, as well as in various experimental infection models (Cohen and Young 1991, Kaufmann and Schoel 1994, Shinnick 1991). Evidently, due to their high conservation among various microbial pathogens, Hsp are major antigens. Hsp60 (GroEl) and Hsp70 (DnaK) of a number of bacteria, including Mycobacterium, Borrelia, Chlamydia, Legionella spp., have been recognized as common antigens in the response to bacterial infection (Anzola et al 1992, Zhong and Bruham 1992, Zhang et al 1998). They are known to induce very strong humoral and cellular immune responses in numerous infections. Studies have revealed that bacterial Hsp60 and 70 modulate immunity by directly inducing cytokine mRNA production in macrophages (Retzlaff et al 1994). Immunological protection against typhoid requires both cell mediated and humoral responses. Salmonella infection induces the generation of specific CD4 and CD8 T cells and both these T lymphocyte populations are important for control of primary infection and protection against secondary infection (Mittrucker and Kaufmann, 2000), although the mechanisms underlying T cell mediated protection are not yet completely understood. Studies have shown that human genetic deficiencies in IFN-λ or interleukin 12 production or T cell receptor signaling result in increased susceptibility to Salmonella infection (Jouanguy et al 1999). Infection of vaccinated mice with virulent salmonellae has led to similar conclusions, requiring the activation of Thl like population of T cells (Mastroeni et al 1992, 1996). Since S. typhimurium is a facultative intracellular bacterium, the requirement of B cells in the immune response against £ typhimurium is a long standing matter of debate. By infecting the mice deficient in B cells (Igµ-/-) with different strains of S. typhimurium, Mittrucker et al (2000) were the first to clarify the distinct role of B cells in the response against S. typhimurium. In mycobacterial infections, reactivity to Hsp predominates, with Hsp60 as an immunodominant target of the antibody and T-cell response in mice and humans. Hsp60-specific antibodies have been detected in patients with tuberculosis and leprosy, and also in mice after infection with M. tuberculosis (Shinnick 1991, Young et al 1988). In patients with leprosy or in persons vaccinated with M. bovis BCG, CD4 apT cells specific for the cells specific for the mycobacterial hsp60 have been found (Mustafa et al 1993). This finding points to an important role for Hsp60 specific T cells in mycobacterial infection. Similarly, in infants, levels of antibodies against Hsp60 are significantly increased after vaccination with a trivalent vaccine against tetanus, diphtheria, and pertussis (Del Giudice et al 1993). These findings further show that priming of the immune system to Hsp60 is a common phenomenon, occurring at an early stage of life. Similarly increased antibody levels to Hsp70, have been identified in sera of patients suffering from malaria, leishmaniasis, schistosomiasis, filiariasis, and candidiasis (Shinnick 1991). In contrast to Hsp60, responses to pathogen-derived Hsp70 seem to be more restricted, sometimes exclusively species specific. Shinnick (1991) has demonostrated an important role of the humoral response against Hsp90 in systemic candidiasis. The Hsp90-specific antibodies contribute directly to protection against Candida albicans infection (Matthews and Burnie 1992). Vaccination with Pathogen Heat Shock Proteins: Today Hsps are the subject of intense work by scientists all over the world as a potential means of vaccines to treat cancer and other diseases. Hsps as vaccines are a novel approach to disease treatment. Epitope analysis indicates the presence of multiple B and T cell epitopes in many of these Hsps. They can be used as carriers, vectors and in that regard offer a promising future. As Hsps represent dominant antigens in numerous microbial infections, a potential use of pathogen-derived Hsp for vaccination has been suggested. In fact, in various infectious models different vaccination strategies using Hsp have induced significant protection. For example, immunization of mice with recombinant GroES and GroEL from Helicobacter pylori protected the animals against subsequent infection and development of gastroduodenal disease (Ferrero et al 1995). Vaccination of mice with recombinant Hsp60 from Histoplasma capsulatum induces protection against pulmonary histoplasmosis (Gomez et al 1995). Another example of a protective anti-Hsp immune response has been shown in murine infection with Y. enterocolitica. Immunization of mice with yersinia- Hsp60 induces a strong yersinia- Hsp60-reactive T-cell response which conferres protection against a challenge with yersiniae (Noll and Autenrieth 1996). Studies by Lowrie and coworkers (1997) suggest a protective role of mycobacterial Hsp60 in murine infection with M. tuberculosis. Mycobacterial Hsp60 is first transfected into APC, which is then used successfully to vaccinate mice against subsequent infection with M. tuberculosis (Silva and Lowrie 1994). The immunogenic properties of Hsp have been demonstrated in particular for Mycobacterium tuberculosis Hsp70, which has been used successfully as an adjuvant free carrier molecule (Barrios et al, 1992). It has been shown that (Suzue and Yound 1996) a recombinant protein consisting of HIV-1 p24 antigen fused to the amino terminus of M tuberculosis Hsp70 elicits both humoral and cellular immune responses to p24 in mice, in the absence of adjuvants. Splenocytes from mice immunized with the fusion protein proliferate and produce IFN-y, II-2 and IL-5 in response to in vitro stimulation with p24. Most vaccines require adjuvants to provoke effective and protective immune responses, where as Hsp70 fusion proteins induce these immune responses without adjuvants. Most adjuvants used in research cause powerful and unpleasant side effects in humans. Thus only alum, a very week adjuvant is used in human vaccines. Similarly, immunization of mice with a soluble fusion protein, consisting of an ovalbumin fragment (OVA), a well characterized T cell antigen, covalently linked to amino terminus of mycobacterial HspTO, induce a strong MHC class I-restricted CD8 T-cell response against a dominant ovalbumin T-cell epitope and partially protect mice from tumor challenge (Suzue et al 1997). This is unexpected as immunization with soluble proteins, especially in absence of adjuvants, rarely elicits CTL responses. Huang et al (2000) has shown the ability to elicit CTL is independent of CD4 + lymphocytes, and this function resides in a 200 amino acid domain of Hsp70 (aa 161-362), concluding that the ability of the fusion proteins to elicit CD8+ T cell does not depend on the Hsps' chaperone properties. The capacity of Hsp to serve as carrier molecules has been studied intensively in murine tumor models. The observatuion that tumorgenicity of a murine macrophage tumor cell line decreases after transfection with mycobacterial Hsp60 has led to the suggestion that Hsp60 promotes the delivery of immuno-dominant tumor antigens to the cell surface and consequently facilitates the recognition and eradication of tumors by specific T cells (Lukacs et al 1993). Li (1997, 2001) and Srivastava and his colleagues over the last two decades (1986, 1998) have demonstrated that structurally unaltered Hsps, purified from tumor cells, can render the mice immune to that particular tumor, but not to antigenically distinct tumors, whereas corresponding preparations from normal tissues does not grant immunity. There is now substantial evidence that native Hsp (Hsp70, gp96, calreticulum) isolated from tumors can be used as adjuvant free anti tumor vaccines in animal models (Udono and Srivastava 1993, 1994; Udono et al 1994; Suto and Srivastava 1995). The immunogenicity of tumors derived Hsps is shown to be dependent on the peptides associated with Hsp molecules and not against Hsps per se. Not surprisingly, Hsps isolated from the cells infected with viruses or bacteria can be used to immunize against the respective viruses or intracellular organisms because of the ability of Hsps to chaperone corresponding antigens. The ability of Hsps to potentially bind to the whole cellular peptide repertoire makes them attractive candidates for cancer vaccines. Tang et al (1997) have shown the presence of antibodies against 58, 68, 88 Kda proteins of S. typhi in sera of typhoid patients and 58 and 68 kd proteins have been found to be analogous to E coli GroEl and DnaK respectively. Numerous studies have documented antibody responses to S. typhi proteins, and the major antigenie components include the somatic O antigen (endotoxin, lipopolysaccharide), flagellar H antigen, Vi antigen and outer membrane proteins). In contrast very little is known about host immune response to Salmonella Hsps. The pathogenesis of typhoid fever and the role of various components of the human immune response to Salmonella typhi remain poorly understood. There is little information about S. typhi Hsps and genes which are involved in virulence or which are important in eliciting a host immune response. The ability of Hsps to chaperone peptides, including antigenic peptides, interact with APC through a receptor, stimulating Ag presenting cells to secret inflammatory cytokines and mediate motivation of dendritic cells, makes them a unique starting point for generation of immune responses. These properties permit the use of Hsps for development of a new generation of prophylactic and therapeutic vaccines against infectious agents. Although, there have been several vaccination strategies against serovar typhi, however none of them is optimal in all aspects. The old inactivated whole cell typhoid vaccines are highly reactogenic, causing high typhoid fever, pain in abdomen, vomiting and diarrhoea. Also these vaccines are only up to 70% effective and the immunity does not persist for more than 3-5 years (Hornick et al 1967, Ivanoff et al 1994). In many countries these have been replaced by two currently licensed vaccines: purified Vi polysaccharide parenteral vaccine and Ty21a, used as a live oral vaccine. Ty21a, a mutant S. typhi strain, was isolated by Germanier et al (1982) and has been used as an orally administered, live, attenuated vaccine. It is in the form of capsules, 3 capsules given orally on alternate days and the capsules need to be swallowed intact. Though the vaccine is effective after 2 years of age, practically a child below 4-6 years of age cannot swallow capsules. Hence it is recommended after the age of 4-6 years. It is contraindicated in immune compromised host as it is a live vaccine. However, this strain Ty21a has lost an epimerase capable of converting glucose to galactose, a loss resulting in defective synthesis of the polysaccharide component of LPS. As a result Ty21a is not well adapted to survive and multiply in the intestinal tract (Gilman et al, 1977). These days a parenteral vaccine, which is made of purified Vi capsular polysaccharide, is being widely used. Vi polysaccharide is a well standardized antigen that is effective in a single subcutaneous or intramuscular dose and is safer than whole cell vaccine. But this vaccine can be given only after completing 2 years of age and it confers protection seven days after injection. Besides, it can lead to side effects like pain, swelling, redness, tenderness etc and sometimes mild fever lasting for 24 hours. The protective efficacy lasts for 2-3 years in most of the vaccines. Hence one has to revaccinate every three years. Hsps as vaccines are a novel approach to disease prevention. Srivastava in U.S. Patent. No. 6,410,028 has described the methods of prevention and treatment of cancer and infectious disease by the administration of complexes of human hsp-antigenic molecules to individuals but there is little information about S. typhi proteins and genes which may be involved in virulence or which are important in eliciting a host immune response. The Hsp based vaccines, unlike other recombinant protein based vaccines, stimulate both humoral and cell mediated immune responses and are currently not available against microbial infections. The present invention, therefore, relates to the development of microbial Hsp based Recombinant vaccine for the prevention of typhoid in humans. DnaK (Hsp70) of S. typhi either alone or in combination with antigenic molecules is used in the present invention as a vaccine to augment the immune response against S. typhi in mice. OBJECTS OF THE INVENTION The main object of the present invention is to develop a potent and cost effective vaccine against typhoid and salmonellosis using DnaK (Hsp70) protein of S. typhi which obviates the drawbacks detailed above. Further object of the present invention is to use the immunogenicity of DnaK protein by conjugating it with other antigenic molecules. Still another object of the present invention is to use exogenous antigens/immunogens viz LPS, peptide, nucleic acid or polysaccharide or antigenic/immunogenic fragments and derivatives thereof as antigenic molecules. Yet another object of the present invention is to the evaluate the immune response of the vaccine in animals and humans. Still another object of the present invention is to challenge animals with S. typhimurium to study the efficacy of DnaK after booster immunization with the vaccine. SUMMARY OF THE INVENTION The present invention provides a cost effective vaccine against typhoid and salmonellosis using Hsp70 either alone or bound to an antigenic molecule that elicits immunological responses in animals and humans. In one embodiment of the present invention the Hsp70 protein is of Salmonella typhi and its closely related serovars. In another embodiment of the present invention the Hsp70 is bound to the antigenic molecule covalently or non-covalently. In yet another embodiment of the present invention the antigenic molecule is exogenous antigen or immunogen selected from a group consisting of lipo-polysaccharides, peptides, nucleic acids, polysaccharides, antigenic fragments, immunogenic fragments and derivatives thereof. In still another embodiment of the present invention the vaccine is administered to humans or animals in an amount in the range of 1-100 µg/kg BW. In yet another embodiment the present invention describes a method of vaccinating mammals consisting of administering a pharmaceutical acceptable quantity of vaccine, sufficient to elicit an immune response in animals and humans. The present invention also provides a pharmaceutical composition comprising an effective amount of the vaccine and one or more pharmaceutically acceptable additives. In another embodiment of the invention, the pharmaceutically acceptable additives are selected from the group consisting of carriers diluents, stabilizing agents, solvents, flavoring agents and the like. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Fig. 1: Effect of DnaK of S. typhi vaccination on immune response in mice. Fig. 2: Effect of DnaK of S. typhi vaccination on survival of mice. DETAILED DESCRIPTION OF THE INVENTION The Genomic DNA is isolated from the overnight grown culture of Salmonella typhi using Genomic DNA isolation kit. DnaK gene is amplified by Polymerase Chain Reaction (Taq : Pfu, 1:1) using primers designed in the lab. Conditions of PCR are: 95°C for 5 minutes (Pre heating lid), 95°C for 1 minutes (denaturation), 55°C for 1 minutes (annealing), 72°C for 90 sec. (extension) for total 36 cycles. The PCR product is purified by commercially available kit. Plasmid DNA of pQE-30 expression vector is isolated by kit as per manufacturers instructions. An overnight grown E. coli culture is harvested by centrifugation and subjected to modified alkaline SDS lysis procedure followed by absorption of DNA onto silica in the presence of high salts. Contaminants are removed by washing and the bound DNA is eluted in distilled water (DW) or Tris EDTA (TE) buffer. Restriction digestion: The PCR product and pQE-30 plasmid DNA are restricted with Bam HI and Hind III to generate cohesive ends and purified by spin column. Ligation and transformation: The PCR product of DnaK and pQE-30 vector DNA are then ligated at 16°C overnight, using T4 DNA ligase. E. coli DH 5a cells are transformed with ligated products and the resultant transformants are selected on Ampicillin plates. Briefly, competent cells from DH Set cells are prepared by CaCl2 treatment. The ligated mixture is added to competent cells and incubated on ice for 30 min. After giving heat shock at 42°C for 2 min, the mixture is immediately chilled on ice for 2-3 min. 1ml LB medium is added to cells, incubated at 37°C for 30-45min and plated on Amp+ plates. Screening of Recombinants: Recombinants obtained are screened for the presence of desired insert by restriction digestion and PCR followed by DNA sequencing. The recombinant plasmids are isolated and subsequently introduced into an expression strain of E. coli. BL-21 (DE-3) by electroporation. Transformants are selected on ampicillin plates and recombinants are screened as before. Expression of proteins: Overnight grown cultures of recombinant colonies obtained above are re-inoculated and allowed to grow till O.D. 600 reach 0.5-0.6. Cultures in this logarithmic phase are induced with IPTG (0.5 mM to 4 mM) for 1 to 4 hrs. After induction, cells are lysed in sample buffer and analyzed by SDS-PAGE. Localization and isolation of recombinant protein in induced cells: To determine the solubility of DnaK protein, induced cells (ODeoo ~ 1 -0) are first lysed in native lysis buffer (50 mM Tris HCI, 200mM NaCI, pH 7.5, 1 mM phenyl methyl sulfonyl fluride (PMSF) and incubated on ice for 30 min, followed by sonication on ice to lyse the cells. Lysate is then centrifuged at 15,000g for 20 min and the resulting supernatant (SI) is transferred into a fresh tube. To the pellet, urea lysis buffer (50 mM Tris, HCI, 200mM NaCI, 5M urea, pH 7.5, 1 mM PMSF) is added and again sonicated as before. Supernatant (S2) is collected after centrifugation. Both the fractions, native lysate supernatant SI and urea lysate supernatant S2 are analyzed by SDS PAGE. The protein band is observed in soluble phase SI. This is confirmed by Western blotting using anti-His antibody as primary antibody and Rabbit antimouse IgG/HRP conjugate as secondary antibody. Purification of recombinant DnaK protein: The recombinant DnaK protein is purified by Ni-NTA (Nickel-nitrilotriacetate) chromatography using Chelating Sepharose Fast Flow gel. Briefly, the protein sample is added to the charged gel and allowed to bind to the gel at room temperature with gentle agitation. Gel is sedimented by centrifugation at 500g for 2-5 minutes and the supernatant is carefully decanted. The gel is washed with wash buffers I (20mM Na2HPO4; 0.5 M NaCI; 20mM Imidazole) and II (20mM Na2HPO4; 0.5 M NaCI; 20mM Imidazole). The protein is then finally eluted with elution buffer containing 20mM Na2HPO4; 0.5 M NaCI; 0.5 mM Imidazole. The protein preparation is dialyzed extensively against dialysis buffer containing 50 mM Tris and ImM EDTA for 24 hrs, with buffer changes every 12 hrs. The dialyzed sample is centrifuged and concentrated by AMICON filteration. Immune Response: The immune response of recombinant DnaK is evaluated by injecting 50µg protein/mouse, intraperitonially on 0, 7th and 28th day. Seven days after the last injection, the mice are sacrificed and serum was collected for antibody titre. Antibody titre is determined by ELISA. A significant increase in antibody title (20,00,000) is observed in mice immunized with DnaK after third immunization (fig 1), indicating that DnaK protein is highly immunigenic and stimulates B lymphocytes. Challenge and Protection studies: The study has the approval of Ethics Committee of the Institute. Animal experiments are conducted according to principles set forth in the Declaration of Helsinki and in the Guide for the Care and Use of Laboratory animals. Eight to Twelve week old female BALB/c mice are used in all cases. S. typhi is a host restricted pathogen causing typhoid in humans only. It does not cause disease in mice, whereas S. typhimurium which causes mild gastroenteritis in humans, is lethal in mice. Thus, infection of mice with S. typhimurium provides a murine model for typhoid fever as it bears many similarities to human serovar typhi infection. Mice are vaccinated with DnaK protein (50 µg/mouse) on 0,7th and 28th day. Two weeks after the last booster immunization, both control and vaccinated mice are challenged with S. typhimurium intraperitonially (1xlO5 cells.ml) to study the efficacy of DnaK as vaccine. The mice are monitored daily after infection for at least 2 months. There is 100% death in control mice after challenge with S. typhimurium within 48 hrs. However, all the mice vaccinated with DnaK survive the lethal infection of S. typhimurium even 60 days after challenge (fig 2). Method of development of the vaccine using Hsp70 protein of Salmonella is described hereinafter with an example, which is illustrative and is not intended to be taken restrictively to imply any limitation on the scope of the present invention. WORKING EXAMPLE: Genomic DNA is isolated from the overnight grown culture of Salmonella typhi using Genomic DNA isolation kit. DnaK gene is amplified by PCR using primers designed in the lab. Conditions of PCR are: 95°C for 5 minutes (Pre heating lid), 95°C for 1 minutes (denaturation), 55°C for 1 minutes (annealing), 72°C for 90 sec. (extension) for total 36 cycles. The PCR product is purified by PCR DNA purification kit. Plasmid DNA of pQE-30 expression vector is isolated by miniprep kit. The PCR product and pQE-30 are restricted with Bam HI and Hind III to generate cohesive ends and are purified by Gel extraction. The PCR product of DnaK and pQE 30 vector DNA are then ligated at 16°C overnight, using T4 DNA ligase. Transformation is carried out with ligated products using E coli DH 5a cells and the resultant transformants are selected on Ampicillin plates. Recombinants obtained are screened for the presence of desired insert by restriction digestion and PCR followed by sequencing. The recombinant plasmids are isolated and subsequently introduced into an expression strain of E. coll BL-21(DE-3) by electroporation. Briefly, electro-competent cells of BL-21 are prepared and electric shock of 1500 V is given to the mixture of cells in a medium containing recombinant plasmid DNA. Transformants are selected on ampicillin plates. The recombinants E. coli BL-21 cells are grown overnight in LB medium at 37°C. The cells are then re-inoculated in fresh medium (1 L culture) and grown till OD6oo reached 0.5. The cells are induced with 0.5 mm IPTG ((isopropyl \|3-D-1-thiogalactopyranoside) for 3 h, lysed in native lysis buffer and incubated on ice for 30 min. The supernatant is collected and analyzed by SDS PAGE and Western blotting for the presence of recombinant protein. The DnaK protein is purified by Ni-NTA chromatography. Briefly, the protein sample is added to the charged gel and allowed to bind to gel for 30 min at room temperature with gentle agitation. Gel is sedimented by centrifugation at 500g for 2 minutes and the supernatant is carefully decanted. The gel is washed with wash buffers and the protein is finally eluted with elution buffer containing 0.5M imidazole. The eluted protein is analyzed by SDS-PAGE followed by western blotting. The protein preparation is dialyzed extensively against dialysis buffer for 24 h, with buffer changes every 12 h. The dialyzed sample is centrifuged and concentrated by AMICON filtration. The immune response of recominant DnaK is evaluated by injecting (50µg protein/mouse) intraperitonially on 0, 7th and 28th day. Seven days after the last injection, the mice are sacrificed and serum is collected for antibody titre Antibody titre is determined by ELISA using 96-well microtitre plates previously coated with 1 µg of DnaK protein/well and blocked with BSA. A significant increase in antibody titre (20,00,000.) is observed in animals immunized with DnaK after third immunization (fig 1). The study revealed that DnaK protein is highly imminogenic and stimulates B lymphocytes. Challenge and Protection studies: Eight to twelve week old female BALB/c mice are used in all cases. Mice(n=10) are vaccinated with DnaK (50µg/mouse) on 0, 7th and 28th day. Two weeks after the last booster immunization, mice are challenged with S. typhimurium intraperitonially (IxlO5 cells/ml) to study the efficacy of DnaK as vaccine. The mice are monitored daily after infection for at least 2 months. All the control mice die within 48 hrs of challenge with S. typhimurium. However, all the vaccinated mice survive the infection even after 60 days of challenge (fig 2). The outline of the methodology adopted is shown in the form of flow chart. FLOW CHART (Figure Removed) It is to be understood that the structure of the present invention is susceptible to modifications, changes and adaptations by those skilled in the art. Such modifications, changes and adaptations are intended to be within the scope of the present invention which is further set forth under the following claims. The main advantages of the present invention are: 1. The present invention discloses a method to develop a potent vaccine against typhoid and salmonellosis in animals and humans using Hsp70 protein of S. typhi and its closely related serovars. 2. The Hsp70 based vaccine disclosed in the present invention stimulates both humoral and cell mediated immune responses and is currently not available against microbial infections. 3. The vaccine disclosed in the present invention is free from unpleasant side effects and toxicity. 4. The vaccine which can be mass produced in short time. 5. Development of the vaccine against typhoid and salmonellosis as disclosed in the present invention has no ethical problems. 6. The method used in the present invention is cost effective and economical compared to the prior art. The running cost is also very low. REFERENCE CITED 1. Ackers, M.L., Puhr, N.D., Tauxe, R.V., Mintz, D. (2000). Laboratory based surveillance of Salmonella serotype typhi infections in the United States:antimicrobial resistance on the rise. JAMA. 283: 2668-73. 2. Alpuche-Aranda, C.M., Berthiaume, E.P., Mock, B., Swanson, J.A., Miller, S.I. (1995). Spacious phagosome formation within mouse macrophages correlates with Salmonella serotype, pathogenicity and host susceptibility. Infect. Immun. 63:4456-4462. 3. Ang, D., Liberek, K., Skowyra, D., Zylicz, M., Georgeopoulou, C. 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Cold Spring Harbour Laboratory, Cold Spring Harbour, NY. 35. Mustafa, A. S., Ludin, K. E.., Oftung, F. (1993). Human T cells recognize mycobacterial heat shock proteins in the context of multiple HLA-DR molecules: studies with healthy subjects vaccinated Mycobacterium bovis BCG and Mycobacterium leprae. Infect. Immun. 61:5294-5301. 36. Noll, A. and Autenreith, I.B. (1996) Immunity against Yersinia enterocolitica by vaccination with Yersinia hsp60 immunostmulating complexes or Yersinia hsp60 plus interleukin-12. Infect. Immun. 64:2955-2961. 37. Retzlaff, C., Yamamoto, Y., Hoffman, P.S., Friedman, H., Klein, T.W. (1994). Bacterial heat shock proteins directly induce cytokine mRNA and interleukin-1 secretion in macrophage cultures. Infect. Immune. 62:5689-5693. 38. Rothman, J. E. (1989) Polypetide chain binding proteins-catalysts of protein folding and related processes in cells. Cell. 59:591-601. 39. Sanghavi SK, Mane MP, Niphadkar KB (1999) Multi drug resistance in Salmonella serotypes. Indian J Med Microbioi. 17: 88-90. 40. Santos, R.L., Zhang, S. Tsolis, R.M., Kingsley, R.A., Adams, L.G., Baumler, A.J. (2001) Animal models of Salmonella infections: enteritis versus typhoid fever. Microbes Infect. 3: 1335-1344. 41. Schwan, W.R, Huang, X.Z., Hu,L., Kopecko, DJ. (2000). Differential bacterial survival, replication and Apoptosis inducting ability of Salmonella serovars within human and murine macrophages. Infect. Immun. 68 (3): 1005-1013. 42. Schwan, W.R. and Kopecko, D.J. (1997). Serovar specific differences in Salmonella survival within macrophage cells. Adv. Exp. Med. Biol. 412:277- 278. 43. Shinnick, T.M. 1991. Heat shock proteins as antigens of bacterial and parasitic pathogens. Curr. Top. Microbioi. Immunol. 167:145-160. 44. Silva, C.L. and Lowrie, D.B. (1994). A single mycobacterial protein (hsp65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis. Immunology. 82: 244-248. 45. Srivastava, P.K. cLeo, A.B., Old, L.J. (1986). Tumor rejection antigens of chemically induced sarcomas of inbred mice. Proc. Natl. Acad. Sci. USA. 83: 3407-3411. 46. Srivastava,P.K., Menoret,A., Basu,S., Binder,R.J., McQuade,K.L., (1998). Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world. Mmunity, 8: 657-665. 47. Suto,R., Srivastava, P.K., (1995) A mecanism for the specific immunogeniciy of heat shock protein-chaperoned peptides. Science. 269:1585-1588. 48. Suzure,K. Young,R.A., (1996) Adjuvant free Hsp 70 fusion protein systemelicits humoral and celluler immune responses to HIV I p24. J. Immunol. 154:873-879. 49. Suzure, K., Zhou, X., Eisen, H.N., Young,R.A. (1997) Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class 1 presentation pathway. Proc. Natl. Acad. Sci. USA. 94: 13146- 13151. 50. Tang,S.W., Abubakar, S., Devi, S., Puthucheary, S., Pang, T. (1997). Induction and characterization of heat shock proteins of Salmonella typhi and their reactivity with sera from patients with typhoid fever. Infect. Immun. 65(7): 2983-2986. 51. Udono, H., Levey, D.L., Srivastava, P.K. (1994). Cellular requirements for tumour specific immunity elicited by heat shock proteins: Tumour rejection antigen gp96 primes CD8+ T cells in vivo. Proc. Natl. Acad. Sci. USA 91: 3077-3081. 52. Udono, H., and Srivastava, P.K., (1993) Heat shock protein 70 associated peptides elicits specific cancer immunity. J. Exp. Med. 178: 1391-1396. 53. Udono, H., and Srivastava, P.K. (1994) Comparison of tumor specific immunogenicities of stress induced proteins gp96, hsp90 and hsp70. J. Immunol. 152: 5398-5403. 54. Young, D.B., Lathigra,R., Hendrix, R., Sweetser, D., Young, R.A. (1988) Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85: 4260-4270. 55. Young, R. A. (1990) Stress proteins and immunology. Ann. Rev. Immunol. 8: 401-420. 56. Zhang, Y., Ohashi,N., Rikihisa,Y. (1998). Clonign of Hsp70 gene of Ehrlichia sennetsu and differential expression of Hsp70 and Hsp70 gene of Ehrlichia sennetsu and differential expression of Hsp70 and Hsp60 mRNA after temperature upshift. Infect. Immune. 66(7): 3106-3112. 57. Zhong,G. and Brunham, R.C. (1992). Antigenic analysis of the chlamydial 75- Kd protein. Infect. Immun. 60: 1221-1224. We claim: 1. A vaccine comprising Hsp70 protein (heat shock protein) of Salmonella typhi or its closely related serovars either alone or in combination with antigenic molecules which elicit immunological responses in the host. 2. The vaccine as claimed in claim 1 wherein said Hsp70 protein is DnaK protein. 3. The vaccine as claimed in claim 1 or 2 wherein said Hsp70 is bound to the antigenic molecule covalently or non-covalently. 4. The vaccine as claimed in claim 1 wherein said antigenic molecule is exogenous antigen or exogenous immunogen selected from a group consisting of lipo- polysaccharides, peptides, nucleic acids, polysaccharides, antigenic fragments, immunogenic fragments and derivatives thereof. 5. The vaccine as claimed in claim 1 or 2 wherein said Hsp70 is administered to humans and animals in an amount in the range of 1 -100 µg/kg B W. 6. A method of vaccinating a mammal against typhoid and salmonellosis, which comprises administering the said mammal a pharmaceutical acceptable quantity of vaccine as claimed in any of preceding claims 1 to 6, that elicit an immune response in the mammal. 7. A vaccine composition which comprises of an effective amount of the vaccine as claimed in any of preceding claims and one or more pharmaceutically acceptable additives. 8. A vaccine composition as claimed in Claim 7 wherein said pharmaceutically acceptable additives are selected from the group consisting of carriers, diluents, stabilizing agents, solvents, flavoring agents, binders, lubricants and the like. 9. A vaccine comprising Hsp70 protein of Salmonella typhi or its closely related serovars Hsp70 protein either alone or bound to an antigenic molecule substantially as hereinbefore described and with reference to the foregoing example. 10. A vaccine composition as claimed in claims 7 and 8 substantially as hereinbefore described and with reference to the foregoing example.

Full Text

A VACCINE FIELD OF INVENTION
The present invention relates to the development of a typhoid vaccine using heat shock protein 70 (Hsp70) of Salmonella typhi. The method of invention involves administering a composition comprising an effective amount of a complex consisting of a Hsp, in part or whole, either alone or covalently or non-covalently bound to an 'antigenic molecule', which when administered elicits specific immunological responses in the host. 'Antigenic molecule' used herein refers to the exogenous antigens/immunogens viz lipopolysaccharides (LPS), peptide, nucleic acid or polysaccharide or antigenic/immunogenic fragments and derivatives thereof. BACKGROUND OF INVENTION
Typhoid fever continues to be a major health problem despite the use of antibiotics and the development of newer antibacterial drugs. Typhoid, caused by the infection with Salmonella typhi, is spread by faecal or oral route and is closely associated with poor inadequate sanitation and food hygiene. Salmonella typhi has rapidly gained resistance to antibiotics like ampicillin, chloramphenicol and cotrimoxazole and also to previously efficacious drugs like ciprofloxacin. The incidence of multidrug resistant (MDR) S. typhi is reported to be as high as 60% (Sanghavi et al, 1999) and resurgence of resistant strains has also been reported (Kumar et al, 2002). A US based study (Ackers et al, 2000) has shown an increase in the number of MDR strains and nalidixic acid resistant S. typhi (NARST).
The virulence of specific strains in humans and other animals is serovar specific. S. typhimurium is a leading cause of gastroenteritis in humans, an acute localized inflammation of intestine. It is generally self limiting and without complication, provided supportive therapy is available. However, in mice infection with this serovar causes typhoid fever like disease. Intestinal and extra intestinal lesions in this model highly resemble those observed in humans suffering from typhoid fever, therefore, this model has been widely used to mimic infection caused by S. typhi in humans (Santos et al, 2001).
Typhoid fever, caused by S. typhi in humans, is marked by protracted fever and a variable clinical presentation depending on the organ system affected (Keusch, 1994). Salmonella paratyphi induces paratyphoid fever, a disease which is similar to, but usually milder than typhoid fever but no disease is associated with experimental
infections of mice by serovar typhi or paratyphi A and B. (Carter and Collins, 1974). The type of disease associated with any particular serovar of Salmonella is linked to the ability of that serovar to enter, survive and replicate within and to elicit damage in a specific host's macrophage cells (Alpuche-Aranda et al, 1995; Schwan and Kopecko, 1997; Schwan et al, 2000). Serovar typhimurium and enteritidis do not survive well in human macrophages, generally producing localized infection which results in mild gastroenteritis but they can grown and cause disease in mice (Collins, 1972). In contrast, Serovars. typhi and paratyphi, which can infect mice via the oral route, do not survive well in murine macrophages and can not cause disease in mice. On the other hand, they survive and multiply within human macrophages, causing only slight macrophage cell death, cross the ileal epithelium silently, moving stealthily within macrophage vehicles to deep tissues, where acute multiplication in selected tissues (e.g. live) ultimately results in typhoid fever (Schwan et al, 2000).
Humans are the sole reservior of S. typhi infection as well as the only natural host. An important property of S. typhi is its ability to survive the environment for weeks or even months in the absence of a host. This explains how epidemics of typhoid fever occur even where no apparent cause of contamination can be found. Non availability of relevant drugs and rapid development of microbial drug resistance have led to the need of efficacious and affordable vaccines to control typhoid fever.
When entering the host from the environment, a microbial pathogen is confronted by several changes, some of which are highly stressful. These include alterations in temperature, pH, and pO2 (Kaufman 1990, 1991). Moreover, the pathogen is exposed to natural host resistance mechanisms such as phagocytosis by professional phagocytes (Kaufman 1998). Once engulfed by phagocytes, the pathogen is confronted with reactive oxygen and nitrogen intermediates and attack by lysosomal enzymes. To protect itself against the host, the pathogen activates various evasion mechanisms including stress protein synthesis.
Heat shock proteins (Hsps) or stress proteins (Sps) are synthesized by all living cells in response to various types of environmental stress and physiological insults like elevated temperatures, heavy metals, toxins, oxidants, bacterial and viral infections (Morimoto and Milarski, 1990; Young 1990). They are widely distributed in nature and are amongst the most highly conserved molecules of the biosphere. They ensure survival under stressful conditions that, if left unchecked, can lead to irreversible cell damage and ultimately to cell death.
Although they are classified as Sps, Hsps also have essential functions in the cell under normal growth conditions. Hsps perform important functions in the folding and unfolding or translocation of proteins, as well as in the assembly and disassembly of protein complexes. Because of these helper functions, hsps have been termed molecular chaperones. (Ang et al 1991, Gething and Sambrook 1992, Rothman 1989). They are divided into a series of families based on their molecular mass in kilodaltons. Hsps 60, 70 and 90 are generally found in cytosol and mitochondria. Gp96 and calreticulum are located in the endoplasmic reticulum. In non-stressed cells hsps are present in low concentrations (1 to 2% of the total protein content), while in stressed cells their concentration is increased many folds. They are released into the environment during pathological situations such as necrotic cell death. However these proteins under certain circumstances are released extracellular to perform a range of immuno-modulatory activities.
In pathogenic bacteria, Hsps are thought to be maximally induced during the infectious process in response to host resistance. Microbial Hsps are the dominant antigens for the host immune response to a variety of pathogens and the immune recognition of Hsps of pathogens serves as first line of defense. They stimulate humoral and cell mediated responses, do not require adjuvants and are effective even in immuno-compromised conditions. These properties permit the use of Hsps for development of a new generation of prophylactic and therapeutic agents against infectious agents. Pathogen-Derived Heat Shock Proteins as Targets for the Immune Response:
Infection is a bimodal process determined by the host and pathogen. Both host cells and microbes are confronted with dramatic alterations in their living conditions during infection. Induction of host Hsp synthesis in response to encounter with a pathogen has at least two major causes. First, infected macrophages are confronted with antimicrobial mechanisms which they have activated themselves during infection. Efficient protection against their own effector molecules (e.g., reactive radicals) becomes vital for macrophage survival. Second, once inside a phagocyte, many microbes especially those which persist in the host, interfere with intracellular host cell metabolism. Not surprisingly, many of these pathogens are potent inducers of Hsp synthesis in mammalian cells. Increased pathogen Hsp levels in cells lead to rapid degradation of Hsp by the host processing machinery. Pathogen-derived determinates are efficiently presented by host cells and promote recognition of infected cells by the immune system.
Although the exact role of Hsps in immunity to microbial infection is incompletely understood, Hsps apparently serve as important antigens in defense against infectious agents. In fact, immune responses to Hsp have been observed in infectious diseases caused by bacteria, protozoa, fungi, and nematodes, as well as in various experimental infection models (Cohen and Young 1991, Kaufmann and Schoel 1994, Shinnick 1991). Evidently, due to their high conservation among various microbial pathogens, Hsp are major antigens. Hsp60 (GroEl) and Hsp70 (DnaK) of a number of bacteria, including Mycobacterium, Borrelia, Chlamydia, Legionella spp., have been recognized as common antigens in the response to bacterial infection (Anzola et al 1992, Zhong and Bruham 1992, Zhang et al 1998). They are known to induce very strong humoral and cellular immune responses in numerous infections. Studies have revealed that bacterial Hsp60 and 70 modulate immunity by directly inducing cytokine mRNA production in macrophages (Retzlaff et al 1994).
Immunological protection against typhoid requires both cell mediated and humoral responses. Salmonella infection induces the generation of specific CD4 and CD8 T cells and both these T lymphocyte populations are important for control of primary infection and protection against secondary infection (Mittrucker and Kaufmann, 2000), although the mechanisms underlying T cell mediated protection are not yet completely understood. Studies have shown that human genetic deficiencies in IFN-λ or interleukin 12 production or T cell receptor signaling result in increased susceptibility to Salmonella infection (Jouanguy et al 1999). Infection of vaccinated mice with virulent salmonellae has led to similar conclusions, requiring the activation of Thl like population of T cells (Mastroeni et al 1992, 1996). Since S. typhimurium is a facultative intracellular bacterium, the requirement of B cells in the immune response against £ typhimurium is a long standing matter of debate. By infecting the mice deficient in B cells (Igµ-/-) with different strains of S. typhimurium, Mittrucker et al (2000) were the first to clarify the distinct role of B cells in the response against S. typhimurium.
In mycobacterial infections, reactivity to Hsp predominates, with Hsp60 as an immunodominant target of the antibody and T-cell response in mice and humans. Hsp60-specific antibodies have been detected in patients with tuberculosis and leprosy, and also in mice after infection with M. tuberculosis (Shinnick 1991, Young et al 1988). In patients with leprosy or in persons vaccinated with M. bovis BCG, CD4 apT cells specific for the cells specific for the mycobacterial hsp60 have been found
(Mustafa et al 1993). This finding points to an important role for Hsp60 specific T cells in mycobacterial infection. Similarly, in infants, levels of antibodies against Hsp60 are significantly increased after vaccination with a trivalent vaccine against tetanus, diphtheria, and pertussis (Del Giudice et al 1993). These findings further show that priming of the immune system to Hsp60 is a common phenomenon, occurring at an early stage of life.
Similarly increased antibody levels to Hsp70, have been identified in sera of patients suffering from malaria, leishmaniasis, schistosomiasis, filiariasis, and candidiasis (Shinnick 1991). In contrast to Hsp60, responses to pathogen-derived Hsp70 seem to be more restricted, sometimes exclusively species specific. Shinnick (1991) has demonostrated an important role of the humoral response against Hsp90 in systemic candidiasis. The Hsp90-specific antibodies contribute directly to protection against Candida albicans infection (Matthews and Burnie 1992). Vaccination with Pathogen Heat Shock Proteins:
Today Hsps are the subject of intense work by scientists all over the world as a potential means of vaccines to treat cancer and other diseases. Hsps as vaccines are a novel approach to disease treatment. Epitope analysis indicates the presence of multiple B and T cell epitopes in many of these Hsps. They can be used as carriers, vectors and in that regard offer a promising future. As Hsps represent dominant antigens in numerous microbial infections, a potential use of pathogen-derived Hsp for vaccination has been suggested. In fact, in various infectious models different vaccination strategies using Hsp have induced significant protection. For example, immunization of mice with recombinant GroES and GroEL from Helicobacter pylori protected the animals against subsequent infection and development of gastroduodenal disease (Ferrero et al 1995). Vaccination of mice with recombinant Hsp60 from Histoplasma capsulatum induces protection against pulmonary histoplasmosis (Gomez et al 1995). Another example of a protective anti-Hsp immune response has been shown in murine infection with Y. enterocolitica. Immunization of mice with yersinia- Hsp60 induces a strong yersinia- Hsp60-reactive T-cell response which conferres protection against a challenge with yersiniae (Noll and Autenrieth 1996). Studies by Lowrie and coworkers (1997) suggest a protective role of mycobacterial Hsp60 in murine infection with M. tuberculosis. Mycobacterial Hsp60 is first transfected into APC, which is then used successfully to vaccinate mice against subsequent infection with M. tuberculosis (Silva and Lowrie 1994).
The immunogenic properties of Hsp have been demonstrated in particular for Mycobacterium tuberculosis Hsp70, which has been used successfully as an adjuvant free carrier molecule (Barrios et al, 1992). It has been shown that (Suzue and Yound 1996) a recombinant protein consisting of HIV-1 p24 antigen fused to the amino terminus of M tuberculosis Hsp70 elicits both humoral and cellular immune responses to p24 in mice, in the absence of adjuvants. Splenocytes from mice immunized with the fusion protein proliferate and produce IFN-y, II-2 and IL-5 in response to in vitro stimulation with p24. Most vaccines require adjuvants to provoke effective and protective immune responses, where as Hsp70 fusion proteins induce these immune responses without adjuvants. Most adjuvants used in research cause powerful and unpleasant side effects in humans. Thus only alum, a very week adjuvant is used in human vaccines.
Similarly, immunization of mice with a soluble fusion protein, consisting of an ovalbumin fragment (OVA), a well characterized T cell antigen, covalently linked to amino terminus of mycobacterial HspTO, induce a strong MHC class I-restricted CD8 T-cell response against a dominant ovalbumin T-cell epitope and partially protect mice from tumor challenge (Suzue et al 1997). This is unexpected as immunization with soluble proteins, especially in absence of adjuvants, rarely elicits CTL responses. Huang et al (2000) has shown the ability to elicit CTL is independent of CD4 + lymphocytes, and this function resides in a 200 amino acid domain of Hsp70 (aa 161-362), concluding that the ability of the fusion proteins to elicit CD8+ T cell does not depend on the Hsps' chaperone properties.
The capacity of Hsp to serve as carrier molecules has been studied intensively in murine tumor models. The observatuion that tumorgenicity of a murine macrophage tumor cell line decreases after transfection with mycobacterial Hsp60 has led to the suggestion that Hsp60 promotes the delivery of immuno-dominant tumor antigens to the cell surface and consequently facilitates the recognition and eradication of tumors by specific T cells (Lukacs et al 1993). Li (1997, 2001) and Srivastava and his colleagues over the last two decades (1986, 1998) have demonstrated that structurally unaltered Hsps, purified from tumor cells, can render the mice immune to that particular tumor, but not to antigenically distinct tumors, whereas corresponding preparations from normal tissues does not grant immunity. There is now substantial evidence that native Hsp (Hsp70, gp96, calreticulum) isolated from tumors can be used as adjuvant free anti tumor vaccines in animal models (Udono and Srivastava 1993,
1994; Udono et al 1994; Suto and Srivastava 1995). The immunogenicity of tumors derived Hsps is shown to be dependent on the peptides associated with Hsp molecules and not against Hsps per se. Not surprisingly, Hsps isolated from the cells infected with viruses or bacteria can be used to immunize against the respective viruses or intracellular organisms because of the ability of Hsps to chaperone corresponding antigens. The ability of Hsps to potentially bind to the whole cellular peptide repertoire makes them attractive candidates for cancer vaccines.
Tang et al (1997) have shown the presence of antibodies against 58, 68, 88 Kda proteins of S. typhi in sera of typhoid patients and 58 and 68 kd proteins have been found to be analogous to E coli GroEl and DnaK respectively. Numerous studies have documented antibody responses to S. typhi proteins, and the major antigenie components include the somatic O antigen (endotoxin, lipopolysaccharide), flagellar H antigen, Vi antigen and outer membrane proteins). In contrast very little is known about host immune response to Salmonella Hsps. The pathogenesis of typhoid fever and the role of various components of the human immune response to Salmonella typhi remain poorly understood. There is little information about S. typhi Hsps and genes which are involved in virulence or which are important in eliciting a host immune response. The ability of Hsps to chaperone peptides, including antigenic peptides, interact with APC through a receptor, stimulating Ag presenting cells to secret inflammatory cytokines and mediate motivation of dendritic cells, makes them a unique starting point for generation of immune responses. These properties permit the use of Hsps for development of a new generation of prophylactic and therapeutic vaccines against infectious agents.
Although, there have been several vaccination strategies against serovar typhi, however none of them is optimal in all aspects. The old inactivated whole cell typhoid vaccines are highly reactogenic, causing high typhoid fever, pain in abdomen, vomiting and diarrhoea. Also these vaccines are only up to 70% effective and the immunity does not persist for more than 3-5 years (Hornick et al 1967, Ivanoff et al 1994). In many countries these have been replaced by two currently licensed vaccines: purified Vi polysaccharide parenteral vaccine and Ty21a, used as a live oral vaccine. Ty21a, a mutant S. typhi strain, was isolated by Germanier et al (1982) and has been used as an orally administered, live, attenuated vaccine. It is in the form of capsules, 3 capsules given orally on alternate days and the capsules need to be swallowed intact. Though the vaccine is effective after 2 years of age, practically a child below 4-6 years of age
cannot swallow capsules. Hence it is recommended after the age of 4-6 years. It is contraindicated in immune compromised host as it is a live vaccine. However, this strain Ty21a has lost an epimerase capable of converting glucose to galactose, a loss resulting in defective synthesis of the polysaccharide component of LPS. As a result Ty21a is not well adapted to survive and multiply in the intestinal tract (Gilman et al, 1977).
These days a parenteral vaccine, which is made of purified Vi capsular polysaccharide, is being widely used. Vi polysaccharide is a well standardized antigen that is effective in a single subcutaneous or intramuscular dose and is safer than whole cell vaccine. But this vaccine can be given only after completing 2 years of age and it confers protection seven days after injection. Besides, it can lead to side effects like pain, swelling, redness, tenderness etc and sometimes mild fever lasting for 24 hours. The protective efficacy lasts for 2-3 years in most of the vaccines. Hence one has to revaccinate every three years.
Hsps as vaccines are a novel approach to disease prevention. Srivastava in U.S. Patent. No. 6,410,028 has described the methods of prevention and treatment of cancer and infectious disease by the administration of complexes of human hsp-antigenic molecules to individuals but there is little information about S. typhi proteins and genes which may be involved in virulence or which are important in eliciting a host immune response.
The Hsp based vaccines, unlike other recombinant protein based vaccines, stimulate both humoral and cell mediated immune responses and are currently not available against microbial infections. The present invention, therefore, relates to the development of microbial Hsp based Recombinant vaccine for the prevention of typhoid in humans. DnaK (Hsp70) of S. typhi either alone or in combination with antigenic molecules is used in the present invention as a vaccine to augment the immune response against S. typhi in mice. OBJECTS OF THE INVENTION
The main object of the present invention is to develop a potent and cost effective vaccine against typhoid and salmonellosis using DnaK (Hsp70) protein of S. typhi which obviates the drawbacks detailed above.
Further object of the present invention is to use the immunogenicity of DnaK protein by conjugating it with other antigenic molecules.
Still another object of the present invention is to use exogenous antigens/immunogens viz LPS, peptide, nucleic acid or polysaccharide or antigenic/immunogenic fragments and derivatives thereof as antigenic molecules.
Yet another object of the present invention is to the evaluate the immune response of the vaccine in animals and humans.
Still another object of the present invention is to challenge animals with S. typhimurium to study the efficacy of DnaK after booster immunization with the vaccine. SUMMARY OF THE INVENTION
The present invention provides a cost effective vaccine against typhoid and salmonellosis using Hsp70 either alone or bound to an antigenic molecule that elicits immunological responses in animals and humans.
In one embodiment of the present invention the Hsp70 protein is of Salmonella typhi and its closely related serovars.
In another embodiment of the present invention the Hsp70 is bound to the antigenic molecule covalently or non-covalently.
In yet another embodiment of the present invention the antigenic molecule is exogenous antigen or immunogen selected from a group consisting of lipo-polysaccharides, peptides, nucleic acids, polysaccharides, antigenic fragments, immunogenic fragments and derivatives thereof.
In still another embodiment of the present invention the vaccine is administered to humans or animals in an amount in the range of 1-100 µg/kg BW.
In yet another embodiment the present invention describes a method of vaccinating mammals consisting of administering a pharmaceutical acceptable quantity of vaccine, sufficient to elicit an immune response in animals and humans.
The present invention also provides a pharmaceutical composition comprising an effective amount of the vaccine and one or more pharmaceutically acceptable additives.
In another embodiment of the invention, the pharmaceutically acceptable additives are selected from the group consisting of carriers diluents, stabilizing agents, solvents, flavoring agents and the like.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Fig. 1: Effect of DnaK of S. typhi vaccination on immune response in mice. Fig. 2: Effect of DnaK of S. typhi vaccination on survival of mice.
DETAILED DESCRIPTION OF THE INVENTION
The Genomic DNA is isolated from the overnight grown culture of Salmonella typhi using Genomic DNA isolation kit. DnaK gene is amplified by Polymerase Chain Reaction (Taq : Pfu, 1:1) using primers designed in the lab. Conditions of PCR are: 95°C for 5 minutes (Pre heating lid), 95°C for 1 minutes (denaturation), 55°C for 1 minutes (annealing), 72°C for 90 sec. (extension) for total 36 cycles. The PCR product is purified by commercially available kit.
Plasmid DNA of pQE-30 expression vector is isolated by kit as per manufacturers instructions. An overnight grown E. coli culture is harvested by centrifugation and subjected to modified alkaline SDS lysis procedure followed by absorption of DNA onto silica in the presence of high salts. Contaminants are removed by washing and the bound DNA is eluted in distilled water (DW) or Tris EDTA (TE) buffer. Restriction digestion:
The PCR product and pQE-30 plasmid DNA are restricted with Bam HI and Hind III to generate cohesive ends and purified by spin column. Ligation and transformation:
The PCR product of DnaK and pQE-30 vector DNA are then ligated at 16°C overnight, using T4 DNA ligase. E. coli DH 5a cells are transformed with ligated products and the resultant transformants are selected on Ampicillin plates. Briefly, competent cells from DH Set cells are prepared by CaCl2 treatment. The ligated mixture is added to competent cells and incubated on ice for 30 min. After giving heat shock at 42°C for 2 min, the mixture is immediately chilled on ice for 2-3 min. 1ml LB medium is added to cells, incubated at 37°C for 30-45min and plated on Amp+ plates. Screening of Recombinants:
Recombinants obtained are screened for the presence of desired insert by restriction digestion and PCR followed by DNA sequencing. The recombinant plasmids are isolated and subsequently introduced into an expression strain of E. coli. BL-21 (DE-3) by electroporation. Transformants are selected on ampicillin plates and recombinants are screened as before. Expression of proteins:
Overnight grown cultures of recombinant colonies obtained above are re-inoculated and allowed to grow till O.D. 600 reach 0.5-0.6. Cultures in this logarithmic
phase are induced with IPTG (0.5 mM to 4 mM) for 1 to 4 hrs. After induction, cells are lysed in sample buffer and analyzed by SDS-PAGE. Localization and isolation of recombinant protein in induced cells:
To determine the solubility of DnaK protein, induced cells (ODeoo ~ 1 -0) are first lysed in native lysis buffer (50 mM Tris HCI, 200mM NaCI, pH 7.5, 1 mM phenyl methyl sulfonyl fluride (PMSF) and incubated on ice for 30 min, followed by sonication on ice to lyse the cells. Lysate is then centrifuged at 15,000g for 20 min and the resulting supernatant (SI) is transferred into a fresh tube. To the pellet, urea lysis buffer (50 mM Tris, HCI, 200mM NaCI, 5M urea, pH 7.5, 1 mM PMSF) is added and again sonicated as before. Supernatant (S2) is collected after centrifugation. Both the fractions, native lysate supernatant SI and urea lysate supernatant S2 are analyzed by SDS PAGE. The protein band is observed in soluble phase SI. This is confirmed by Western blotting using anti-His antibody as primary antibody and Rabbit antimouse IgG/HRP conjugate as secondary antibody. Purification of recombinant DnaK protein:
The recombinant DnaK protein is purified by Ni-NTA (Nickel-nitrilotriacetate) chromatography using Chelating Sepharose Fast Flow gel. Briefly, the protein sample is added to the charged gel and allowed to bind to the gel at room temperature with gentle agitation. Gel is sedimented by centrifugation at 500g for 2-5 minutes and the supernatant is carefully decanted. The gel is washed with wash buffers I (20mM Na2HPO4; 0.5 M NaCI; 20mM Imidazole) and II (20mM Na2HPO4; 0.5 M NaCI; 20mM Imidazole). The protein is then finally eluted with elution buffer containing 20mM Na2HPO4; 0.5 M NaCI; 0.5 mM Imidazole. The protein preparation is dialyzed extensively against dialysis buffer containing 50 mM Tris and ImM EDTA for 24 hrs, with buffer changes every 12 hrs. The dialyzed sample is centrifuged and concentrated by AMICON filteration. Immune Response:
The immune response of recombinant DnaK is evaluated by injecting 50µg protein/mouse, intraperitonially on 0, 7th and 28th day. Seven days after the last injection, the mice are sacrificed and serum was collected for antibody titre. Antibody titre is determined by ELISA. A significant increase in antibody title (20,00,000) is observed in mice immunized with DnaK after third immunization (fig 1), indicating that DnaK protein is highly immunigenic and stimulates B lymphocytes.
Challenge and Protection studies:
The study has the approval of Ethics Committee of the Institute. Animal experiments are conducted according to principles set forth in the Declaration of Helsinki and in the Guide for the Care and Use of Laboratory animals. Eight to Twelve week old female BALB/c mice are used in all cases. S. typhi is a host restricted pathogen causing typhoid in humans only. It does not cause disease in mice, whereas S. typhimurium which causes mild gastroenteritis in humans, is lethal in mice. Thus, infection of mice with S. typhimurium provides a murine model for typhoid fever as it bears many similarities to human serovar typhi infection.
Mice are vaccinated with DnaK protein (50 µg/mouse) on 0,7th and 28th day. Two weeks after the last booster immunization, both control and vaccinated mice are challenged with S. typhimurium intraperitonially (1xlO5 cells.ml) to study the efficacy of DnaK as vaccine. The mice are monitored daily after infection for at least 2 months. There is 100% death in control mice after challenge with S. typhimurium within 48 hrs. However, all the mice vaccinated with DnaK survive the lethal infection of S. typhimurium even 60 days after challenge (fig 2).
Method of development of the vaccine using Hsp70 protein of Salmonella is described hereinafter with an example, which is illustrative and is not intended to be taken restrictively to imply any limitation on the scope of the present invention. WORKING EXAMPLE:
Genomic DNA is isolated from the overnight grown culture of Salmonella typhi using Genomic DNA isolation kit. DnaK gene is amplified by PCR using primers designed in the lab. Conditions of PCR are: 95°C for 5 minutes (Pre heating lid), 95°C for 1 minutes (denaturation), 55°C for 1 minutes (annealing), 72°C for 90 sec. (extension) for total 36 cycles. The PCR product is purified by PCR DNA purification kit.
Plasmid DNA of pQE-30 expression vector is isolated by miniprep kit. The PCR product and pQE-30 are restricted with Bam HI and Hind III to generate cohesive ends and are purified by Gel extraction. The PCR product of DnaK and pQE 30 vector DNA are then ligated at 16°C overnight, using T4 DNA ligase. Transformation is carried out with ligated products using E coli DH 5a cells and the resultant transformants are selected on Ampicillin plates.
Recombinants obtained are screened for the presence of desired insert by restriction digestion and PCR followed by sequencing. The recombinant plasmids are
isolated and subsequently introduced into an expression strain of E. coll BL-21(DE-3) by electroporation. Briefly, electro-competent cells of BL-21 are prepared and electric shock of 1500 V is given to the mixture of cells in a medium containing recombinant plasmid DNA. Transformants are selected on ampicillin plates.
The recombinants E. coli BL-21 cells are grown overnight in LB medium at 37°C. The cells are then re-inoculated in fresh medium (1 L culture) and grown till OD6oo reached 0.5. The cells are induced with 0.5 mm IPTG ((isopropyl |3-D-1-thiogalactopyranoside) for 3 h, lysed in native lysis buffer and incubated on ice for 30 min. The supernatant is collected and analyzed by SDS PAGE and Western blotting for the presence of recombinant protein. The DnaK protein is purified by Ni-NTA chromatography. Briefly, the protein sample is added to the charged gel and allowed to bind to gel for 30 min at room temperature with gentle agitation. Gel is sedimented by centrifugation at 500g for 2 minutes and the supernatant is carefully decanted. The gel is washed with wash buffers and the protein is finally eluted with elution buffer containing 0.5M imidazole. The eluted protein is analyzed by SDS-PAGE followed by western blotting. The protein preparation is dialyzed extensively against dialysis buffer for 24 h, with buffer changes every 12 h. The dialyzed sample is centrifuged and concentrated by AMICON filtration.
The immune response of recominant DnaK is evaluated by injecting (50µg protein/mouse) intraperitonially on 0, 7th and 28th day. Seven days after the last injection, the mice are sacrificed and serum is collected for antibody titre Antibody titre is determined by ELISA using 96-well microtitre plates previously coated with 1 µg of DnaK protein/well and blocked with BSA. A significant increase in antibody titre (20,00,000.) is observed in animals immunized with DnaK after third immunization (fig
1).
The study revealed that DnaK protein is highly imminogenic and stimulates B lymphocytes. Challenge and Protection studies:
Eight to twelve week old female BALB/c mice are used in all cases. Mice(n=10) are vaccinated with DnaK (50µg/mouse) on 0, 7th and 28th day. Two weeks after the last booster immunization, mice are challenged with S. typhimurium intraperitonially (IxlO5 cells/ml) to study the efficacy of DnaK as vaccine. The mice are monitored daily after infection for at least 2 months. All the control mice die within
48 hrs of challenge with S. typhimurium. However, all the vaccinated mice survive the
infection even after 60 days of challenge (fig 2).
The outline of the methodology adopted is shown in the form of flow chart.

FLOW CHART
(Figure Removed)
It is to be understood that the structure of the present invention is susceptible to modifications, changes and adaptations by those skilled in the art. Such modifications, changes and adaptations are intended to be within the scope of the present invention which is further set forth under the following claims. The main advantages of the present invention are:
1. The present invention discloses a method to develop a potent vaccine against
typhoid and salmonellosis in animals and humans using Hsp70 protein of
S. typhi and its closely related serovars.
2. The Hsp70 based vaccine disclosed in the present invention stimulates both
humoral and cell mediated immune responses and is currently not available
against microbial infections.
3. The vaccine disclosed in the present invention is free from unpleasant side
effects and toxicity.
4. The vaccine which can be mass produced in short time.
5. Development of the vaccine against typhoid and salmonellosis as disclosed in
the present invention has no ethical problems.
6. The method used in the present invention is cost effective and economical
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We claim:
1. A vaccine comprising Hsp70 protein (heat shock protein) of Salmonella typhi or its
closely related serovars either alone or in combination with antigenic molecules which
elicit immunological responses in the host.
2. The vaccine as claimed in claim 1 wherein said Hsp70 protein is DnaK protein.
3. The vaccine as claimed in claim 1 or 2 wherein said Hsp70 is bound to the antigenic
molecule covalently or non-covalently.
4. The vaccine as claimed in claim 1 wherein said antigenic molecule is exogenous
antigen or exogenous immunogen selected from a group consisting of lipo-
polysaccharides, peptides, nucleic acids, polysaccharides, antigenic fragments,
immunogenic fragments and derivatives thereof.
5. The vaccine as claimed in claim 1 or 2 wherein said Hsp70 is administered to humans
and animals in an amount in the range of 1 -100 µg/kg B W.
6. A method of vaccinating a mammal against typhoid and salmonellosis, which
comprises administering the said mammal a pharmaceutical acceptable quantity of
vaccine as claimed in any of preceding claims 1 to 6, that elicit an immune response in
the mammal.
7. A vaccine composition which comprises of an effective amount of the vaccine as
claimed in any of preceding claims and one or more pharmaceutically acceptable
additives.
8. A vaccine composition as claimed in Claim 7 wherein said pharmaceutically acceptable
additives are selected from the group consisting of carriers, diluents, stabilizing agents,
solvents, flavoring agents, binders, lubricants and the like.
9. A vaccine comprising Hsp70 protein of Salmonella typhi or its closely related serovars
Hsp70 protein either alone or bound to an antigenic molecule substantially as
hereinbefore described and with reference to the foregoing example.
10. A vaccine composition as claimed in claims 7 and 8 substantially as hereinbefore
described and with reference to the foregoing example.

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Patent Number

259393

Indian Patent Application Number

2648/DEL/2005

PG Journal Number

11/2014

Publication Date

14-Mar-2014

Grant Date

11-Mar-2014

Date of Filing

04-Oct-2005

Name of Patentee

DIRECTOR GENERAL, DEFENCE RESEARCH & DEVELOPMENT ORGANISATION

Applicant Address

MINISTRY OF DEFENCE, GOVERNMENT OF INDIA, WEST BLOCK-VIII, WING-I, SECTOR-1, R. K. PURAM, NEW DELHI-110066, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	MUSTOORI SAI RAM	DIPAS, TIMARPUR, LUCKNOW ROAD, DELHI-110054, INDIA.
2	ANJU BANSAL	DIPAS, TIMARPUR, LUCKNOW ROAD, DELHI-110054, INDIA.
3	S. SARADA SURYA KUMARI	DIPAS, TIMARPUR, LUCKNOW ROAD, DELHI-110054, INDIA.
4	PIYUSH PALIWAL	DIPAS, TIMARPUR, LUCKNOW ROAD, DELHI-110054, INDIA.

PCT International Classification Number

C12N 15/31

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA