Immunogenic antigens of shigella

Abstract

A vaccine for protection against multiple serotypes of Shigella sp., comprising a putative heat shock protein (EL PGI II), and Hypothetical Protein (EL PGIV) is provided.

Claims

1. A method of inducing an immune response in an organism, comprising administering to the organism an immunogenic amount of an immunogenic composition comprising a putative heat shock protein having the sequence consisting of SEQ ID NO: 1 and/or SEQ ID NO: 2, and a hypothetical protein having the sequence consisting of SEQ ID NO: 3 and/or SEQ ID NO: 4.

2. A method of detecting the presence of antibodies against Shigella in a sample obtained from an organism comprising: contacting a sample derived from the blood of an organism suspected of being infected by Shigella with a protein comprising a putative heat shock protein and a hypothetical protein; and detecting binding of the protein to one or more antibodies against Shigella.

3. The method of claim 2, wherein the putative heat shock protein has the sequence of SEQ. ID No: 1 and the hypothetical protein has the sequence of SEQ. ID No: 3.

4. The method of claim 1, wherein the composition is administered at least twice, and wherein a first dose includes an adjuvant including inactivated mycobacteria, and wherein a second dose includes an adjuvant that does not include an inactivated mycobacteria.

5. The method of claim 2, where the detecting step comprises use of an immunosorbent assay.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

(1) FIG. 1: Titres of IgG antibody (1:160) in BALB/c mice against the putative heat shock protein (EL PGI II) antigen inoculated by three different routes: (i/p, s/c and i/n); (P value<0.05 i/p, s/c, i/n)

(2) FIG. 2: Titres of IgA antibody (1:10) in BALB/c mice against the putative heat shock protein (EL PGI II) antigen inoculated by three different routes: (i/p, s/c and i/n); (P value<0.05 i/p, s/c, i/n)

(3) FIG. 3: Titres of IgG antibody (1:160) in BALB/c mice against the putative hypothetical protein (EL PGI V) antigen inoculated by three different routes: (i/p, s/c and i/n); (P value>0.05 i/p, i/n; P value<0.05 s/c)

(4) FIG. 4: Titres of IgA antibody (1:10) in BALB/c mice against the putative hypothetical protein (EL PGI V) antigen inoculated by three different routes: (i/p, s/c and i/n); (P value>0.05 i/p; P value<0.05 s/c, i/n)

(5) FIG. 5: Results with mixture of peptides: mixture produced significant IgG response by all three routes

(6) FIG. 6: Results with mixture of peptides: Mixture produced significant IgA response by all routes

(7) FIG. 7: Results of TNF alpha with mixture of peptides: Mixture produced significant TNF alpha response by all routes

(8) FIG. 8: Results of IFN gamma with mixture of peptides: the mixture was found to produce significant IFN gamma response by all routes

(9) FIG. 9: Sequence IDs of peptides and amino acids EL PGI II (full length peptideSEQ ID NO: 1; synthesized peptideSEQ ID NO: 2), and EL PGI V (full length peptideSEQ ID NO: 3; synthesized peptideSEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

(10) Thus according to this invention is provided novel immunogenic protein antigens for use in preparing vaccines or developing sero-diagnostic test for identification of Shigella.

(11) According to this invention is further provided a vaccine for protection against multiple serotypes of Shigella sp.

(12) In accordance with this invention, the complete proteome databases of most common serotypes of Shigella (S dysenteriae serotype 1, S sonnei, S flexneri 2a) have been searched on NCBI and a local database of approx 17,000 proteins has been made. Then protein BLAST has been performed on selected database to find proteins common in most prevalent serotypes such as S. dysenteriae serotype 1, S. sonnei and S. Flexneri 2a.

(13) After BLAST, 7038 proteins were obtained and for all these proteins, protein localization prediction has been done.

(14) After this localization study, those proteins were targeted which are either secreted out or are present on the surface of the bacterium as immune response is generated against these proteins when infection occurs as these proteins come in contact with host cells. 250 outer-membrane or secreted proteins were obtained and epitope prediction for 250 selected proteins was done. B-cell epitope prediction, T-cell epitope prediction and MHC binding score was done for all proteins so that highly immunogenic proteins could be selected for further use.

(15) Finally 48 peptides which have B-cell and T-cell epitopes and MHC binding properties were arrived at. Out of these, 5 peptides (putative lipoprotein (EL PGI I), putative heat shock protein (EL PGI II), Spa32 (EL PGI III), IcsB (EL PGI IV), and hypothetical protein (EL PGI V)) were artificially synthesized which had maximum MHC binding score. Immunogenicity of these peptides was checked in BALB/c mice and human sera of patients suffering from shigellosis. The antibody response was checked by ELISA and T-cell response by cytokine analysis.

(16) Artificial Peptide Synthesis

(17) Five peptides which were immunodominant by in-silico analysis were artificially synthesized from USV (United States Vitamins Ltd., Mumbai, India). These peptides were of more than 95% purity. We describe the peptides (ELPGI-II and ELPGI-V) that have been filed for the patent and are identified by their sequences below:

(18) TABLE-US-00001 1.Putativeheatshockprotein(ELPGIII)-size28KDa >gi|82777568|ref|YP_403917.1| putativeheatshockprotein[Shigella dysenteriaeSd197] MINQRMIHMKNTKLLLAIATSAALLTGCQNTHGIDTNMAISSGLNAYKAATLSDADAKAIANQGCAEMDS GNQVASKSSKYGKRLAKIAKALGNNINGTPVNYKVYMTSDVNAWAMANGCVRVYSGLMDMMNDNEIEGVL GHELGHVALGHSLAEMKASYAIVAARDAISATSGVASQLSRSQLGDIAEGAINAKYSRDKESEADDFSFD LLKKRGISTQGLVGSFEKLASLDGGRTQSMFDSHPPSTERAQHIRDRIASGK(SEQIDNO:1) Peptidesynthesized-DSGNQVASKSSKYGK(SEQIDNO:2) 2.HypotheticalProtein(ELPGIV)-Size28KDa >gi|30062956|ref|NP_837127.1|hypotheticalproteinSI556[Shigella flexneri2astr.2457T] MTKLKLLALGVLIATSAGVAHAEGKFSLGAGVGVVEHPYKDYDTDVYPVPVINYEGDNFWFRGLGGGYYL WNDATDKLSITAYWSPLYFKAKDSGDHQMRHLDDRKSTMMAGLSYAHFTQYGYLRTTLAGDTLDNSNGIV WDMAWLYRYTNGGLTVTPGIGVQWNSENQNEYYYGVSRKESARSGLRGYNPNDSWSPYLELSASYNFLGD WSVYGTARYTRLSDEVTDSPMVDKSWTGLISTGITYKF(SEQIDNO:3) Peptidesynthesized-YGVSRKESARSGLRGYN(SEQIDNO:4)

(19) FIG. 9: sequence ID.

(20) Immunogenicity Check of Selected Peptides in Balb/c Mice

(21) TABLE-US-00002 Intraperitoneal Subcutaneous (s/c) Mice used (i/p) route route Intranasal (i/n) Dosing Schedule Female Balb/c Control PBS only Control PBS only Control PBS only 3 doses at two weeks interval Female Balb/c Control PBS + Control PBS + Control PBS + 3 doses at two adjuvant adjuvant adjuvant weeks interval 1.sup.st dose with CFA and subsequent with IFA Female Balb/c Test group = Test group = Test group = 3 doses at two Peptide + Peptide + Peptide + weeks interval adjuvant adjuvant adjuvant 1.sup.st dose with CFA and subsequent with IFA

(22) 6 weeks old female Balb/c Mice were taken and divided into three groups

(23) i.e one for intraperitoneal injections (i/p) another for subcutaneous injections(s/c) and yet another for intranasal (i/n) route. Five mice were used in each group for one antigen, two groups of control and one group test (15 mice used per antigen). The Schedule is shown in Table 1 above.

(24) Dosing Schedule:

(25) Three groups of 6 weeks old female balb/c mice were taken. Each group i/p, s/c were or i/n divided into subgroups of control and test. Two types of control were used in the study, PBS only (Phosphate buffer saline) and second control of PBS+ adjuvant. Test group were given peptide with adjuvant. First dose was given with CFA (Freund's complete adjuvant) and two subsequent doses were given with IFA (incomplete Freund's adjuvant). Three doses were given at two weeks interval and after three weeks of last dose blood was collected from mice by retro-orbital plexus. Serum was stored at 20 C. and 80 C. for ELISA and Cytokine analysis respectively.

(26) Collection of Human Patient Sera:

(27) Sera was collected from 15 patients who were suffering from dysentery and who were culture positive for Shigella: Both IgG and IgA ELISA were performed for these sera with the same peptide antigens used for injecting the mouse.

(28) Cytokines Assay

(29) Cytokine analysis was done by Flow cytometry using BD Biosciences Ltd. India, mouse Th1 and Th2 cytokine kit. Serum samples stored at 80 C. were processed according to manufacture's instructions. Cytokine assays were performed for both Th1 and Th2 cytokines (TNF, IFN, IL-4, IL-1, and IL-10).

(30) Enzyme-Linked Immunosorbent Assay (ELISA)

(31) Protocol for In-House Indirect ELISA for Detection of Antibody IgG & IgA against Selected Synthetic Peptides:

(32) The antibody detection (IgG and IgA) was done by micro ELISA technique. Optimum antigen dilution (10 g/well), used for coating the wells of the micro-titer plates was determined by checker board titration method. The optimum serum dilutions of the test samples (1:20, 1:40, 1:80 for IgG. and 1:5, 1:10, 1:20 for IgA) which were used were determined in the same way using various dilution of known positive and negative sera with plates coated with optimum dilution of antigen. ELISA was carried out according to the standard technique with certain modifications wherever required. The steps followed were as follows:

(33) 1) One hundred micro liter of antigen diluted in coating buffer (10 g/well) was used for coating the well of micro-titer plate and the plate was kept at 4 C. overnight covered with tin foil and ELISA plate cover, so as to minimize evaporation.

(34) 2) Next morning the antigen coated plate was washed thrice with washing buffer PBST (Phosphate buffer saline with 0.02% tween 20)

(35) 3) 2% bovine serum albumin (BSA, 100 l) was added to each well to block the remaining unbound sites on the plate. Plate was incubated at 37 C. for 1 hour and was washed thrice with washing buffer.

(36) 4) Test and control sera was diluted in PBST to optimum dilution and 1000 of dilution was added to appropriate labelled wells of micro titer plate and was incubated at 37 C. for 1 hour.

(37) 5) After washing with PBST 100 l of enzyme labelled anti-mouse IgG horse radish peroxidise (HRP) conjugate (Sigma-Aldrich), diluted as 1:10,000 (optimum dilution will be added to each well. The plate was incubated at 37 C. for 1 hour.

(38) 6) This was followed by washing thrice with PBST and 100 l of ortho phenylene diamine (OPD) and H.sub.2O.sub.2 was added in each well as substrate in dark. The plate was kept at room temperature for 15 minutes in dark.

(39) 7) The reaction was stopped with 1M H.sub.2SO.sub.4 and absorbance was read with ELISA reader at 490 nm.

(40) Similarly IgA antibody was detected by ELISA after checker board titration.

(41) TABLE-US-00003 TABLE 2 Results of ELISA and Cytokine analysis: Human antibody Peptide Name Mouse antibody and cytokine response putative lipoprotein (EL PGI I) IgA (i/p), TNF, IFN Negative putative heat shock protein IgG, IgA and TNF by all three IgG and IgA (Very good (EL PGI II) routes response) Spa32 TNF, IFN Negative (EL PGI III) IcsB IgA (only by S/c route) and TNF, IgG and IgA in some (EL PGI IV) IFN patients hypothetical protein (EL PGI IgG, IgA and TNF by all three IgG and IgA (Very good V) routes response)

(42) Out of these five tested antigens, either humoral or cytokine response was seen in all the antigens, whereas except spa32 (EL PGI III), four antigens showed both antibody and cytokine response in Balb/c mice. Two antigens putative heat shock protein (EL PGI II), IcsB (EL PGI IV) and hypothetical protein (EL PGI V)) came out to be very promising. The results are depicted in the FIGS. 1-8

(43) The results are summed up as follows (Table 3 shows for the ELPGI-II and ELPGI-V)

(44) TABLE-US-00004 TABLE 3 Peptide Route IgG IgA TNF INF (ELPGI-2) Ip Y Y Y Sc Y Y In Y Y (ELPGI-5) ip Y sc Y Y Y in Y Y Y Mix ip Y Y Y Y sc Y Y Y Y in Y Y Y Y Y: Yes

(45) The antigens showed antibody response with human sera of patients suffering from shigellosis. This shows these antigens are immunogenic for humans also. Discovery of these immunodominant antigens common to major serotypes of Shigella (S dysenteriae serotype 1, S sonnei, S flexner Ua) is of further immunodiagnostic importance for diagnosis of Shigella and serves as a vaccine candidate.

(46) These two antigens, putative heat shock protein (EL PGI II), and hypothetical protein (EL PGI V) which are immunogenic in Balb/c mice and with human sera of patients suffering from shigellosis are novel immunogenic antigens as they are common to multiple serotypes of Shigella. The immune response of these antigens against Shigella spp. Has been tested. Serotype specific immunity is a major drawback for vaccine development against Shigella till now so these three antigens have overcome this aspect also. So this finding can be of great importance in future for developing serodiagnostic test for identification of Shigella or for developing an effective vaccine against multiple serotypes of Shigella. Amino acid sequences of whole proteins as well as sequences of synthetic peptides of these immunogenic antigens used in this invention are shown in FIG. 9. Overall these documents does not exist any patent relating to these novel common immunogenic antigens against Shigella spp.

(47) The results are being described for the EL PGI II & EL PGI V antigens only. They were found to be immunogenic by the intranasal route also (FIG. 1-8 and Table 3, 4)

(48) Human Experiment Data

(49) Collection of Human Patient Sera

(50) 15 patients sera who were suffering from dysentery and who were culture positive for Shigella. Both IgG and IgA ELISA were performed for these sera with the same peptide antigens used for injecting the mouse. Same number of age and sex matched healthy controls were also selected who did not suffer from diarrhea in past six months

(51) The results are highlighted in Table 4

(52) TABLE-US-00005 TABLE 4 EL EL EL EL Age in Days of PGI PGI PGI PGI years diarrhoea/ II II V V Patient no. & sex dysentery IgG IgA IgG IgA 1. 5/F 5-6 +ve +ve +ve +ve 2. 45/M 6-7 +ve +ve +ve +ve 3. 65/M 10 +ve +ve ve ve 4. 41/M Chronic ve +ve ve +ve diarrhoea 5. (BMT) 54/M 6 ve +ve +ve +ve 6. 28/M 10 +ve +ve ve ve 7. (HIV +ve) 50/M Chronic ve ve +ve +ve diarrhoea 8. 63/F Chronic +ve +ve ve +ve diarrhoea 9. 57/M 7-8 +ve +ve +ve +ve 10. 1/M 8 +ve ve ve +ve 11. 14/M 7 +ve +ve +ve 12. 14/M 7-8 ve +ve +ve +ve 13. (Renal transplant) 40/M 6 ve +ve ve +ve 14. 2/F 5-6 +ve ve ve ve 15. 45/M 5-6 ve +ve +ve +ve

(53) ELPGI II IgA antibody response in 12/15 patients

(54) ELPGI V IgA antibody response in 11/15 patients

(55) Therefore protective antibodies are present in human sera against these antigens

(56) Table 5 shows the cytokine responses in these patient sera: As can be seen IL-1beta, IL-10, TNFalpha and IFN gamma responses were significant

(57) TABLE-US-00006 TABLE 5 Control samples sample no. IL-1 IL-2 IL-4 IL-10 TNF- IFN- 1 17.23 19.05 16.99 12.89 20.76 23.09 2 12.68 5.09 20.99 68.09 11.98 23.34 3 12.23 37.9 17.21 9.89 12.67 22.02 4 9.76 8.09 19.89 45.9 11.9 21.89 5 5.64 12.9 23.98 58.23 18.09 27.83 6 14.73 11.56 1.58 22.9 12.23 24.45 7 21.89 1.34 45.02 27.74 12.67 10.98 8 27.83 17.34 32.23 11.12 11.99 22.87 9 24.45 2.9 23.87 11.91 12.9 23.83 10 10.98 9.02 11.87 33.9 23.81 35.68 11 21.76 17.09 15.23 15.44 18.82 21.19 12 19.01 26.01 1.89 20.04 21.09 27.23 13 8.04 5.08 12.01 28.78 18.89 24.45 14 14.71 17.98 19.23 12.09 18.02 22.9 15 9.86 28.02 11.89 34.9 15.05 15.84 P value 0.03 0.15 0.82 0.017 0.0004 0.0001 Signif- NS NS Signif- Signif- Signif- icant icant icant icant S. no. 1 2.98 8.19 11.9 12 12.09 2.89 2 8.34 2.98 8.01 8.72 3.78 15.91 3 12.08 11.56 40 2.04 1.98 3.98 4 10.78 1.34 9.87 7.78 2.98 12.12 5 15.93 17.39 23.9 11.09 8.34 13.72 6 12.09 2.9 18.33 4.9 12.08 11.28 7 4.01 9.02 11 19.02 10.78 10.34 8 7.9 17.09 23.01 20.9 15.93 8.45 9 2.87 6.01 12 22.9 12.09 18.72 10 18.9 5.08 45.9 20.4 4.01 15.64 11 12.87 17.98 19.809 10.74 7.9 10.38 12 10.8105 8.02 10.60106 9.62 8.9 3.98