FIELD OF INVENTION:
The invention is related to the field of biotechnology, more specifically to bacterial proteins.
BACKGROUND AND PRIOR ARTS:
Salmonella enterica serovar typhi (S. typhi), a gram negative pathogenic bacterium endemic to the Indian subcontinent, remains a major threat to public health in the developing world. It causes an acute febrile illness known as enteric fever (Germanier, 1984), and in a chronic carrier state has been implicated in the development of adenocarcinoma of the gall bladder. Although, effective antimicrobials against S. typhi are available, development of multi-drug resistant strains is a challenge to an effective therapeutic outcome.
Vaccination is found to be effective in preventing infection and contain the disease. Commercially available vaccines against Salmonella typhi include oral (live attenuated Salmonella enterica serovar typhi Ty21a strain) as well as parenteral (purified Vi polysaccharide) preparations (Germory et al, 2002; Guzman et al, 2006; Fraser et al, 2007). Both the live oral and parenteral Vi vaccines have disadvantages that interfere with their success in the mass vaccination trials. Oral (Ty21a) vaccine requires at least 3-4 doses for optimal activity. The major problems inherent with live attenuated vaccines are retained immunogenicity and bacteraemia as has been observed with human volunteers who received a higher dose. Cost effectiveness is another problem with whole cell vaccines.
In recent years, Vi polysaccharide has replaced parenteral killed whole-cell vaccine as the recommended prophylaxis against typhoid fever. Capsular polysaccharides are present on the surface of many gram negative bacteria and may be the major targets of the host immune system including antibodies, complement and phagocytes. As a polysaccharide, Vi is a T cell-independent antigen and does not stimulate helper T cell responses. Hence, no immunologic memory is generated against Vi polysaccharide vaccine. Antibody response depends on the prolonged persistence of the Vi antigen in the reticulo endothelial system of the host. Sometimes, human immune system fails to induce any immune response against bacterial polysaccharides, as they structurally resemble polysaccharides of human neural cell adhesion molecules or oligosaccharides present in foetal tissues. In addition, Meta analysis studies show that the Vi-polysaccharide based vaccine is very effective in the short-term

(85% protective efficacy at the end of 1 year, C.I. 95%), but only modestly efficacious to confer long-term immunity (~50% protective efficacy after 2 years) (Guzman et al, 2006; Fraser et al, 2007). In addition, the safety of the Vi-based vaccine is not confirmed in very young children (< 2 years) (Fraser et al, 2007) where the disease is increasingly reported nowadays.
Thus, there is a need for a vaccine which will elicit T cell -dependent responses conferring long term immunologic memory. Protein subunit vaccines being T cell-dependent antigens, confer specific protection by inducing cell-mediated immune responses and are expected to provide long-term immunologic memory, although duration of protection varies widely (Salerno-Goncalves, et al. 2006; Sable et al, 2007).
Considering the high prevalence of infection in certain geographical locations like India, the serious nature of the disease and its sequelae, increasing drug resistance among the prevalent strains and to overcome the limitations of the available vaccines, the object of the invention is to develop a recombinant protein as a subunit vaccine
OBJECT OF THE INVENTION:
The object of the present invention is to develop a recombinant protein as a subunit vaccine. This novel protein is encoded by the un-annotated sequence of the S. typhi genome and was found to be immunogenic in mice and rabbits.
Yet another object of the present invention is to develop a modified recombinant polypeptide sequence for developing a therapeutically effective vaccines and vaccine compositions.
BRIEF DESCRIPTION OF THE DRAWINGS:
Figure 1 depicts (Left panel) SDS-PAGE analysis of recombinant t2544 protein expressed in E. coli
BL21 (DE3) and purified by metal affinity chromatography. Right panel-Western blot analysis using
anti-His monoclonal antibody (SIGMA).
Figure 2 is a diagrammatic representation of the In-vitro bactericidal activity of t2544 antiserum.
Figure 3 is a graphical representation of the different isotypes of anti-t2544 antibody in the mice
immune sera as measured by ELISA.
Figure 4 A- an illustration of the immunogenicity of t2544 in S. typhi-infected patients.
Figure 4B-Western blot analysis of t2544 probed with typhoid positive (S1, S2, S3, S4) and negative
(S5, S6, S7) human serum

Figure 5 depicts passive immunization assay. Mice (n=10 per group) were immunized with t2544
antiserum (200 µg) i.v. 1 hr prior to i.p. challenge with live Ty2. Cumulative mortality over the next 7
days is presented.
Figure 6 depicts In vitro bactericidal assay. Mean plus 95% confidence interval of bacterial survival at
OD 600 nm is plotted, which shows mean values of 0.312, 0.748, 0.987, 0.490 and 0.378 along the X-
axis. Respective confidence intervals are shown in the figure. Numbers in the graph indicates the
number of patients whose sera were analyzed. NSL, non-Salmonella sera; >128 and <128, antibody
titers; AS > 128 + T2544 represents Ty2At2544-adsorbed AS further adsorbed with recombinant
T2544 before the bactericidal assay.
Figure 7 depicts the rising titres of t2544-specific IgG in the paired sera. A.S- acute phase sera
(collected 3-7 days after the onset of fever); C.S-convalescent sera (collected ~ 2 months from the
onset of fever).
Figure 8A, 8B and 8C depicts the expression of t2544 protein by clinical Salmonella isolates
recovered over the years (2003-2010) from different geographical locations (East, West and south) of
India as detected by ELISA. Log phase cultures of bacteria were fixed on microtitre wells, incubated
with anti-t2544 antiserum followed by HRP-conjugated secondary antibody. OD was measured at 492
nm after addition of OPD substrate. 1-35 = S Typhi; 36-45 = S. paratyphi.
Figure 9 depicts the expression of t2544 protein by clinical isolates of several enteric pathogens.
Figure 10 is an illustration of In vitro bactericidal assay performed with anti-t2544 rabbit antiserum
against different Salmonella sp. and other enteropathogens.
Figure 11A and 11B- Kaplan-Meier plot of the survival assay. Mice were immunized with
recombinant t2544 as stated above and subsequently challenged with Ty2, LT2 or LT2ASTM0306.
DETAILED DESCRIPTION OF THE INVENTION:
Accordingly, the present invention discloses a recombinant polypeptide sequence (SEQ ID 1), and a modified recombinant polypeptide sequence (SEQ ID 2) for developing a therapeutically effective vaccines and vaccine compositions.
Thus the invention is for a vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or 2 , which confers cell mediated immunity against Salmonella typhi.
Further, the vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or 2 is emulsified with adjuvants, wherein the adjuvant be Freund's complete adjuvant or Freund's incomplete adjuvant.

The amount of vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or 2 for eliciting immunogenic response is 25µg (for mice) to 150µg (for rabbits).
The vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or 2, wherein the vaccine is an aqueous solution.
The vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or 2, wherein the vaccine elicits cell mediated immunity against Salmonella typhi.
Further, the invention comprises amplification of the target sequence in the genome of the reference S. typhi strain (Ty2) by Polymerase Chain Reaction using proof-reading polymerase (Hot start Pfu polymerase, Stratagene Inc, USA). The amplicon was cloned into pBlueScript followed by sub-cloning into pET28a expression vector and was transformed into an expression host E coli BL21 (DE3) according to standard procedures. Recombinant t2544 (SEQ ID 1) and (SEQ ID 2 - The signal peptide of the sequence has been deleted in the recombinant protein) generated as insoluble inclusion body was solubilized with 6M guanidium hydrochloride and purified by affinity chromatography using Ni2+ conjugated agarose.
The present invention also relates to a vaccine, or a vaccine composition comprising a polypeptide, including an immunogenic fragment thereof, wherein said polypeptides and fragments are in a pharmaceutically acceptable carrier and wherein said polypeptide and/or fragments are present in an amount effective to elicit antibodies, in an animal against S. typhi.
In yet another embodiment, the present invention discloses a modified recombinant polypeptide sequence for developing a therapeutically effective vaccines and vaccine compositions. The signal peptide of the native sequence has been deleted in the recombinant protein.
The vaccine compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, and the like. A thorough discussion of

pharmaceutically acceptable carriers, diluents, and other excipients are presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).
Vaccine compositions may further incorporate additional substances to stabilize pH, or to function as adjuvants, wetting agents, or emulsifying agents, which can serve to improve the effectiveness of the vaccine.
Vaccines are generally formulated for parenteral administration and are injected either subcutaneously or intramuscularly.
The amount of vaccine sufficient to confer immunity is determined based upon the characteristics of the vaccine recipient and the level of immunity required to pathogenic bacteria, viruses, or other microbes. Where vaccines are administered by subcutaneous or intramuscular injection, the purified proteins used is in the range of 25µg (for mice) to 150µg (for rabbits).
EXAMPLES
The following examples are for the purpose of illustration of the invention and are not intended in any way to limit the scope of the invention.
EXAMPLE 1:
Production of recombinant t2544 protein:
The histidine-tagged recombinant protein (t2544) (SEQ ID 1 or 2) was isolated using bacterial expression system. To this end, target sequence in the genome of the reference S. typhi strain (Ty2) was amplified by Polymerase Chain Reaction using proof-reading polymerase (Hot start Pfu polymerase, Stratagene Inc, USA). Amplicon was cloned into pBlueScript followed by sub-cloning into pET28a expression vector. This newly generated construct was transformed into an expression host E coli BL21 (DE3). Protein (t2544) expression was induced by culturing the bacteria in LB medium at 37 °C for 4hrs in the presence of 1mM Isopropyl p-D-1-thiogalactopyranoside (IPTG). Recombinant t2544 was generated as insoluble inclusion body. Insoluble protein was solubilized with 6M guanidium hydrochloride and purified by affinity chromatography using Ni2+ conjugated agarose. After purification, protein was re-natured by gradual removal of the denaturing agent by dialysis using a 10 kD membrane. Purified protein was quantified by Bradford Reagent and the purity was confirmed by SDS-PAGE stained with Coomassie Brilliant Blue and western blot analysis with anti-HIS monoclonal antibody (Figure 1).

The target protein is shared by multiple serovars of Salmonella enterica and the molecular weight of the recombinant protein as calculated from the sequence information as well as migration in the SDS-PAGE is around 27 kDa (Figure 1).
EXAMPLE 2:
Preparation of antiserum against recombinant t2544 and assessment of its protective efficacy : Antiserum to purified t2544 was raised by injecting male albino rabbit intramuscularly with 100 ug of protein emulsified with Freund's complete adjuvant (Sigma) followed by three booster doses of 150 µg each of the same protein emulsified with Freund's incomplete adjuvant (Sigma). Boosters were administered at intervals of seven days and the animal was bled 7 days after last injection to collect the antiserum. Serum antibody titer was determined by an ELISA-based assay. Briefly, a 96-well assay plate was pre-coated with recombinant t2544. Serial dilutions of the antiserum was added to the wells and incubated for 1 hr at 37 °C. Anti-t2544 antibody was detected using HRP-conjugated anti-rabbit secondary antibody. Ortho-phenylene diamine was used as a substrate for HRP and absorbance was measured at 492 nm.
To determine the protective efficacy, rabbit antiserum along with complement was incubated at different dilutions with early log phase cultures of different S. enterica serovars in a 96 well microtiter plate. After 3hrs of incubation at 37 °C, BHI broth was added to each well and incubated for additional 4 hrs. Bacterial growth was measured by absorbance at 600 nm. Significant death was found up to l/80th dilution of the antiserum as determined by comparing bacterial growth with the control samples where no antiserum/complement was added. (Figure 2)
EXAMPLE 3:
Confirmation of immunogenicity of recombinant t2544 :
Immunogenicity of t2544 was studied by immunizing BALB/c mice subcutaneously with 4 injections of the recombinant protein administered at 7 days intervals. This was followed by measurement of different isotypes of anti-t2544 antibody using an ELISA-based assay as described above. Mice antisera were collected till 120th day after the 1st immunization and incubated with purified t2544 in an ELISA plate. Concentrations of different isotypes were detected using HRP-conjugated secondary antibodies against mouse IgGl, IgG2a, IgA and IgM. An initial increase of IgM titre (up to 1/16000 dilution) was observed followed by rise of IgGl (peak titre 1/32000 dilution) and IgA (peak titre

1/16000 dilution). After 60 days of immunization, IgGl and IgA titres reached plateau at 1/16000 and 1/8000 dilutions, respectively and persisted till 120 days. No significant induction of IgG2 was observed in our experiment (Figure 3).
Immunogenicity of t2544 in humans was studied by measuring anti-t2544 antibody titer in the individuals naturally infected with S. typhi. Sera were collected from 15 culture-positive patients suffering from typhoid fever and antibody against t2544 was detected in an ELISA-based assay as described earlier. Significant antibody titer (1/256 dilution) was found in majority of the patients (Figure 4A). In the subsequent experiment, t2544 was run in SDS-PAGE and probed with human sera in a western blot analysis. The results showed specific band corresponding to the size of t2544 when patients' samples were used while normal sera were un-reactive, suggesting presence of antibody in the sera of infected individuals (Figure 4B).
EXAMPLE 4:
Protective efficacy of anti -t2544 antiserum: Opsonization assay:
For efficient phagocytosis, microorganisms need to be opsonized. To determine whether anti-t2544 antisera can enhance opsono phagocytosis, an in vitro opsonization assay was performed. Mouse peritoneal macrophages were harvested by flushing the peritoneal cavity with ice cold PBS. Collected cells were centrifuged and re-suspended in pre warmed RPMI supplemented with 10% fetal bovine serum. Approximately 5 x 105 cells were added to each well of a 24 well tissue culture plate and incubated for 2h at 37°C. Non-adherent cells were removed, medium was replaced with fresh one and incubated overnight at 37°C. Prior to inoculation, log phase culture of bacteria were mixed with anti-t2544 antisera or pre immune sera and each well were inoculated with 106 number of opsonised bacteria. After 30 min, wells were washed with PBS and medium was replaced by gentamycin containing RPMI. Samples were incubated for 0, 60, 90 and 120 min, following which cells were lysed with 0.1% TritonX-100 and number of viable intracellular bacteria was determined by CFU count on LA plates with appropriate selection. Ty2 pre-incubated with mouse t2544 antiserum as opposed to the pre-immune serum were killed more efficiently by macrophages in vitro (mean CFU of intracellular bacteria: 23.4± 5.07 vs 93.4± 11.5 after 1 hr incubation; p<0.001)
Passive immunization assay
To investigate whether antibody against t2544 may confer protection in vivo, a passive immuniation assay was performed as described by Iankov et.al. Briefly, groups of 10 iron-overload mice were

injected intravenously with 200 µg / per mouse of either heat-inactivated mouse anti-t2544 antisera or normal mouse serum. After 1 hour, animals were challenged intra peritoneally with 104, 105, 5 x 105 and 106 of Salmonella typhi Ty2 bacteria. Protection was evaluated as percent survival during next 7 days. Anti-t2544 serum provided 100% protection with subsequent peritoneal challenge with 10 fold higher dose of Ty2 compared with the dose that killed all immunized mice. However, 50 and 100-fold higher doses resulted in 90% and 70% protection, respectively (Figure 5 1).
In vitro bactericidal assay
Human serum isolated form Typhoid infected individuals were pre incubated with Ty2At2544 and analyzed for in vitro bactericidal assay. Anti-t2544 antisera produced after natural infection in human proved to be bactericidal. It was further confirmed by reduced bactericidal activity when the above mentioned serum was absorbed with purified t2544 protein (Figure 6 2).
Immunogenicity and wide spread distribution of t2544 protein:
Immunogenicity of t2544 in human was further determined by measuring rising titer of anti-t2544 IgG from acute and convalescent sera in an ELISA-based assay. All acute sera were collected within 3-7 days of onset of fever and convalescent sera were collected approximately 2 months from the onset of fever. A sharp rise of IgG titer was observed (froml/16 to 1/512) in paired sera collected form Typhoid patient (Figure 7).
Wide distribution of t2544 protein was determined by ELISA-based detection using multiple clinical isolates of S. typhi and S. paratyphi from different geographical locations of India (Figure 8A, 8B and 8C). Surface expression of t2544 protein was detected only in the S. enteric subspecies, i.e. enterica, arizonae, enteritidis and indica, but not in other enteric pathogens excluding cross-reactivity of the antisera (Figure 9). In vitro bactericidal activity of rabbit anti-t2544 antisera against diverse S. enterica isolates suggested that protective antibody response against broad range Salmonellosis may be generated in humans (Figure 10).
Protective efficacy of immunization with t2544:
Protection of animals immunized with the candidate vaccine was studied in a mouse model. S. typhi may cause systemic disease in mice in an iron-overload state while excess iron and iron-chelator is present. BALB/c mice aged between 6 and 8 weeks were pre-immunized by 4 subcutaneous injections of the recombinant t2544 (25 µg/mice) 7 days apart. Mice were pre-treated for consecutive 6 days with a mixture of antibiotics and anti-fungal agents to sterilize the gut. An iron-overload state was

generated by injecting intra-peritoneally Fe3+ as FeCl3 in 10-4N HC1 (0.32 mg per gm of body weight) along with Desferoxamine (25 mg/Kg body weight) 4 hrs prior to the bacterial challenge with Ty2, LT2 or LT2ASTM0306 [STM0306 is the homologue of t2544]. Protection was observed against Ty2 and LT2 but not against LT2ASTM0306, which indicates the specific nature of protection. As expected, no protection was observed in the unimmunized mice. (Figure 11A and B)
EXAMPLE 5:
Preparation of the vaccine:
The vaccine formulation contained the recombinant protein emulsified with the Freund's complete and incomplete adjuvants for the first immunization and the booster doses, respectively. Each dose contains 25 ug of protein in 50 µl of protein-adjuvant mixture. Freund's adjuvants are water-in-oil emulsions. The so-called complete form (CFA) is composed of inactivated and dried Mycobacteria, usually Mycobacterium tuberculosis while the incomplete form (FIA) is the same adjuvant without the Mycobacterial components.
ADVANTAGES
1. The protein subunit vaccine of the invention elicits T cell-dependent responses conferring long term immunological memory.
2. The protein of invention can also be used to develop a Vi polysaccharide conjugate vaccine.
3. The newly developed vaccine may be produced in a cost effective manner and hence will be significantly cheaper than the existing ones.
Reference:
Fraser, A. et al (2007). Cochrane Database Syst. Rev. 3:CD001261
Garmory H.S., et al (2002) FEMS Microbiology Reviews 26 : 339-353
Germanier R., (1984) Bacterial vaccines (London : Academic Press)
Guzman CA et al (2006). Vaccine 24: 3804-11
Salerno-Gonc.alves R, et al (2006) TRENDS in Microbiology 14:536-542
Sable SB et al (2007). Clinical Immunology 122:239-251.

We Claim:
1. A vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or SEQ ID 2, which confers cell mediated immunity against Salmonella typhi.
2. An expression vector for the expression of recombinant protein t2544 of Salmonella typhi as claimed in claim 1.
3. A host cell transformed with the vector as claimed in claim 2 for the expression of recombinant protein t2544 of Salmonella typhi, wherein the host is a bacterium.
4. The vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or SEQ ID 2 as claimed in claim 1, optionally emulsified with adjuvants.
5. The vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or SEQ ID 2 as claimed in claim 4, wherein the adjuvant is Freund's complete adjuvant.
6. The vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or SEQ ID 2 as claimed in claim 4, wherein the adjuvant is Freund's incomplete adjuvant.
7. The vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or SEQ ID 2 as claimed in claim 4, wherein the amount of vaccine for eliciting the immunogenic response is 0.25µg- 150µg.
8. The vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or SEQ ID 2 as claimed in claim 4, wherein the vaccine is an aqueous solution.
9. The vaccine comprising of recombinant protein t2544 of Salmonella typhi of SEQ ID 1 or SEQ ID 2 as claimed in claim 4, wherein the vaccine elicits cell mediated immunity against Salmonella typhi.