Title of Invention	PEPTIDE ANTIGENS WHICH ELICIT HIGH HUMORAL IMMUNE RESPONSE AND LOW T-CELL RESPONSE
Abstract	ABSTRACT The present invention relates to peptide antigens of SEQ ID Nos. 1 to 10 and a repeat motif Gly-X-Gly- Asn-X-Gly of SEQ ID No. 11. wherein the peptide antigens and the repeat motif elicits high humoral immune response and low T-eell response. Figures 1-5.

Full Text

FIELD OF THE INVENTION The present invention relates to peptide antigens of SPQ II) Nos. 1 to 10 and repeat motif Gly-X-Gly-Asn-X-Gly of SPQ ID No. 11. I'he peptide antigens and the repeat motif elicits high humoral immune response and low "1 -cell response. BACKGROUND OF THE INVENTION AND PRIOR ART The existence of PF/PPE gene families was evident even before the Mycobacterium tuberculosis genome was sequenced with occasional reports of occurrence of glycine and alanine rich multiple repetitive sequences in the genome [1] or the identification of a few fibronectin binding proteins |2j. Sequencing categorized the PH'PPF gene families as tun large unrelated families of highly acidic glycine rich proteins that constitute about 10% of the coding capacity of the genome [3\. Comparative genome sequencing in various mycobacterial species revealed that by and large PF. and PPH gene families are unique to Mycobacterium tuberculosis with few liomologucs in M leprae. M bovis. M. marinum etc |"4|, Amongst the M leprae homo log ues. a major serine rich antigen is expressed in leprosy patients \5]. It is »enerally believed that the PF. and PPL senes could be a source of antigenic variability. A recombinant PE_PGRS (Kvl759e) protein was shown to possess fibronectin binding properties and was also recognized by patient area [6J. The same group also reported immense intra-strain variability in the PGRS domain with the N-terminal region staving constant. Transposon insertion in the PFPGRS gene (RvlN18e) was shown to reduce macrophage infection ability of Mtb \7\. Surface localization of a PPF protein (Rvl()17c) and many other PH_PGRS proteins has been reported [8.9|. few PFPGRS genes have also been shown lo be expressed during preclinical infection (10|. Dissection of the PF PGRS genes into Ph and the PGRS domains to study their specific immunological response during mice infection revealed that the PF region can elicit an effective cellular immune response and the humoral response is largely directed against the Gly- Ala rich PGRS domain j 111. I lie involvement of PE'PPF genes in the virulence of the pathogen has also been reported |12J. We recently described the biophysical and biochemical properties ot" a PPE gene. Rv2430c and further showed that it is an immunodominant antigen of Mlh [13. 14. 15j. fn this study, we used an in-silico approach to identify probable antigens from the PPEMPTR (Major Polymorphic Tandem Repeat! subfamily and studied the humoral and cellular immune response to the same using well characterized patient samples. Synthetic peptides corresponding to regions of high antigenic index of the protein were used to map the antigenic domains and assess the antigenic potential of the GL\-X-Gly-Asn-X-Gly repeal motif in eliciting a differential immune response. Our results suggest thai the PPE_MPTR ORE R\2608 could be involved in directing the host towards development aid more humoral type of immune response. OBJECTS OF THE INVENTION The main object of the invention is to identify peptide antigens of SEQ ID Nos. 1 to 10. wherein the antigens elicit high humoral immune response and low T-cell response. Another main object of the invention is to identity a repeat motif Gly-X-Gl;-Asn-X-Gl\ of SEQ ID Nlo. 1!. having "X" represented by any of the amino acid residues, wherein the repeat motif elicits high humoral immune response and low T-cell response. STATEMENT OF THE INVENTION Accordingly, the present invention relates to peptide antigens of SEQ ID Nos. 1 to 10. wherein the antigens elicit high humoral immune response and low T-cell response and a repeat molif Gly-X-Gly-As-n-X-Gly of SEQ ID No. 11. having "X" represented by imy of the amino acid residues, wherein the repeat motif elicits high humoral immune response and low T-eell response. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS Figure I: Sau3.\\ Restriction map ot PPE ORE. R\ 2608. Arrowheads point to the SaiuA sites in the 1743bp ORE. Numbers above the line indicate the size of the restriciion fragments (in base pairs) generated afler Suu3.\l digestion. B: Summary of .S' Figure 2: Expression and purification of .1/ tuberculosis protein corresponding to the PPE ORF Rv2608. The left panel shows the unindueed and induced cell ]\ sates and proteinmoleeular size marker (Lanes I. 2. M). I he right panel shows the purified recombinant protein (Pane 3) and the protein molecular size marker (M). Figure 3: in-silico analysis of Rv2608 reveals regions of high antigenic index (potential antigenic determinants). Overall antigenic index of the protein was calculated using the James Wolllnson method of the Protean software of I.asergcne Navigator1 Vl. The boxed areas indicate the regions selected for designing synthetic peptides to map the region that was actually eliciting a variable immune response. As can be seen, one of the peptides (37mer) is largely composed of Cily-Asn repeals which is lesser in number in the other peptide (23mer). B: Stretches of overlapping peptides used for EP1SA and T cell proiileralion assa\. These peptides were used to further map the region that was antigenic. Figure 4: Antibody response of different categories of TB patients to rRv260H is equivalent to the response to rllsplO. a well documented antigen of M. tuberculosis. Serum reactivity was measured by [-LISA and the graph was plotted as patient response (O. P. at 492nm) to rMsplO and rRv2dl)8. The difference between patient response to HsplO and rRv2608 was not significant for all patient categories (p>0.0o using paired t tests). However, the response of health\ controls was lower and differed significant!; from the patients (p^0.0002 using paired t test). (IIC - Health; Controls. Cat= Category) Figure 5: Antibody response of different categories of PB patients to different synthetic peptides (regions of high antigenicitv w ithin Rv2608) as determined by ELiSA. Response to all the peptides was plotted as absorbance at 492nm (mean ± SD). The response of Category II patients was significant!;' higher than Category 1 or 111 (p-' 0.001 for both. using paired t tests) with respect to each peptide antigen. DETAILED DESCRIPTION OF THE INVENTION Flic present invention relates To peptide antigens ol' SFQ II.) Nos. I to 10. wherein the antigens elicit high humoral immune response and low 1-eell response. The present invention also relates to a repeat motif Gly-X-Gly-Asn-X-Gly of SFQ ID No. I I. having '"X" represented by any of the amino acid residues, wherein the repeal motif elicits high humoral immune response and low T-cell response. In this study, vve used an in-silieo approach to identify probable antigens from the PPF_MPTR (Major Polymorphic Tandem Repeal) subfamily and studied the humoral and cellular immune response to the same using well characterized patient samples. Synthetic peptides corresponding to regions of high antigenic index of the protein were used to map the antigenic domains and assess the antigenic potential of the Gly-X-GIy-Asn-X-Gly repeat motif in eliciting a differential immune response. Our results suggest thai the PPF MPTR ORF Rv2608 could be involved in directing the host towards development of a more humoral type of immune response. Ihe ORF Rv260fi selected for the present study is a member of the PPF_MPTR class which is characterized by the presence of a conserved N-terminal region and a C-terminal domain with major polymorphic tandem repeats (MPTR) of Glv -X-Gly-Asn-X-Gk residues. Apart from this, the ORF also possesses regions of high antigenic index, which is a measure of overall hydrophilicily and surface probability. To lest if polymorphism of the C-terminal region of this ORF exists in different clinical isolates of M. tuberculosis, PCR amplified Rv2608 was subjected to PCR-RFFP analysis. The observed variation in the band pattern lends weight to the hypothesis that PE/PPE genes, notably Rv2608 are perhaps a source of antigenic variability in the otherwise conserved genome of ,1/. tuberculosis. The rR\2608 protein was used in Id.IS \s to determine its reaelixily to patient sera. ! he primordial obscrxation thai the recombinant protein reacted with patient sera indicates thai this protein is definitely expressed during infection. Serum response o\' patients as well as healthy controls to rRv2608 was equivalent to or greater than the response to llsplO. a well documented antigen of Mlh [17]. While category wise differentiation of serum reactivity towards the full length recombinant protein was not very apparent, it was significant to note that the extrapulmonary TB patients showed less reactivity with rR\2608 protein as compared TO Category 1 or II (p=0.048). It will be worthwhile to explore whether R\ 26(18 represents a protein(s) required by the bacterium to establish a pulmonary infection. Since the serum response to the recombinant PPK protein was equal to or greater than HsplO. it was decided to possibly map the antigenic domains of the probable PPli antigen using a synthetic peptide approach 118-20]. Peptides corresponding to regions of high antigenic index were according designed. Our analyses of the comparative humoral immune responses indicate thai the serum response of patients to all the ten peptides is similar. This could he explained b\ the fact that all the peptides have a common repeat motif thereby eliciting similar response. While this negated our efforts to map the immunodominant epitope required for eliciting a strong humoral immune, a difference in the response of patients categorized according to different states of infection was surprisingly evident. Category II patients (relapsed infection cases) demonstrate the highest B cell response to the peptides followed by extrapulmonary TB cases. The synthetic peptides were also used for T cell proliferation assays with the peripheral blood mononuclear cells of all category patients, it has been earlier shown that in about 90% of patients with active TB. there is a significant antibody response and.or T cell proliferate response to peptide specific single antigens of Mlh [21]. The 38 kDa antigenic protein of M tuberculosis is a potent stimulus for both T cell and B cell responses in humans |"22. 23 |. The T cell proliferate response to the synthetic peptides was of the order of fresh infection cases - relapsed TB -• extrapulmonary TB cases at least for peptide 2 and its derivatives. However, the observed Stimulation Index (SI) with ail the peptides was very low in all categories of TB patients (S.l. response and a low cellular immune response to the peptides in category II patients points to an important possible function of the PF.'/PPL- gene families. It is likely that these antigens play a role in evading the host immune response and prevent the establishment of an effective cellular response, which is required to contain the disease. I he positive T cell response in some eases could be explained by the fact that IgCi antibody responses again require the involvement of helper "I cells. Antibody levels usually decrease in cured i'B cases hut dramatical!} increase in patients .showing poor compliance [24]. High anlibods response to the peptides and a low T cell response hence explain the relapse of infection in category I! patients. In vivo, it is possible that the responsive T cells are not able to expand as the glycine, asparagine repeat motifs somehow prevent antigen processing. The situation can be equated with the Lpstein Barr Virus Nuclear antigen, where again the Glv-Ala repeat regions play an important role in preventing antigen processing [25]. Peptides 2 and 2c, which have lesser number of Gly-Asn repeats show a comparatively higher T cell response. in conclusion, we have been able to establish a relationship between immune responses to the PPK antigen and the status of the disease (fresh or relapsed TB). The present stud} is the first report wherein we demonstrate, in a clinical setting, that the repeat sequences present within R\2608 elicit a high humoral immune response and a low T cell response. Since PPK MP fR is a gene family of Mlh of which R\2608 is a member sharing the MPTR motif, it is likely that other members of the same family may also serve the same function in the bacterium. Our data contribute towards a better understanding of humoral as well as cellular immune responses elicited by PPE antigens. The practical utility of using these peptides for differentiating fresh infection from relapsed or reactivation cases is another interesting proposition. The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of the invention. Example 1: PCR-RKLP analysis of the PPK OttF, Rv2608: PC'R-RIT P was carried out to examine if Rv2608 exhibited polymorphism in different clinical isolates of M. tuberculosis. Brief]}. R\2608 was PCR amplified from about 30 different clinical isolates and the amplified product was digested with SaitiAI enzyme. The digested product was separated on a 10% polyacrylamide gel and visualized under UV after ethidium bromide staining. Example 2: Cloning, over expression and purification of Rv2608, a PIT. MPTR subfamily member of M. tuberculosis: The PPE ORF. Rv2608 was amplified from .1-/. tuberculosis lB7Rv genomic DNA using primers earning specific restriction enzyme sites to enable directional cloning. The amplified gene was first cloned in pCiHMT eas} \eetor followed b\ subcloning in pRSHTu expression \eclor. Lixpression of the 5().6 kDa recombinant Rv2608 protein in K. co/i BI.2I cells was achieved as described earlier [14|. The recombinant protein was purified to homogeneity on a Nickel NTA affinity column (Qiagen |nc). Example 3: Synthetic peptides: The PPK ORf. Rv2608 was scanned to identify regions of high antigenic index using the Protean software of Lasergene Navigator™ (DNA STAR). Ten synthetic peptides of varying lengths corresponding to in-silico predicted regions of high antigenic index were commercially obtained as lyophilized powders. Peptide stocks of concentration 0.1 mgrnil were prepared in carbonate bicarbonate buffer and stored in aliquotsat-70"C. Example 4: Subjects: lift; one TB patients confirmed by tuberculin skin test, radiographic examination and obsenation of Acid fast Bacilli (AFB) in sputum lor pulmonary TB and at the site of presumed TB in case of extrapulmonary infection were selected for this study. These patients were reporting to the Out Patient Department o\' the Mahavir Hospital and Research Centre Hyderabad. India. All the patients with confirmed diagnosis of IB were culture positive as well. We categorized the patients as follows: Categon. 1: Individuals (n=22) diagnosed for 'IB for the first time: Category II: Individuals (n=21) with a relapsed TB and Category III: Extrapulmonary IB patients (n=8). Sera were collected from all the subjects during earlv stage of infection when chemotherapy had just started. Health) control (n-TO) sera were taken from the laboratory staff of CDFI). These were individuals who had not had a prolonged direct contact with a I B patient. As this slud\ was carried on a PPI: gene family member of Mih. members of which are unique to mveobacteria [3|. cross reactivity to this protein would not be expected and therefore control subjects with other bacterial infection were not considered necessary for inclusion in our study. Example 5: ELISA and Lymphocyte proliferation assay: All the 51 patients were tested against each of the 10 peptide antigens to evaluate for a B cell as well as a T cell response. ELlSAs with 2ug/ml of rHsplO/rRv2608 protein /svnthetie peptides were carried oul as described earlier [14], The l.ymphoeyte proliferation assays were carried out essentially as per method described earlier with a few modifications 11 6|. 1 leparinised blood was drawn and diluted with equal volume of RPM1 164(1 medium without serum. Diluted blood was layered on Rcoll gradient in 1:3 proportion. Alter a low speed (SOOg) eenlrifugalion for 3D minutes, the peripheral blood mononuclear cells (PBMCs) were isolated and washed twice for 10 minutes at SOOg to remove the cell debris and platelets. Cell concentration was adjusted to lO'Vml. Viability of the cells was checked using Trypan Blue. To each well of the microliter plates. 0.1ml of cell suspension and 0.1 ml of antigen (2(.ig ml) was added. ConA (Concanavalin A) was used as a positi\e control antigen. Control and experimental cultures were run in triplicate. The plate was incubated at 37"C with 5% CO? for a period of 72 hours. At the end of the 3"" day. 15ul of the tetrazolium salt MTT (2mg/ml) was added and incubated for another 4 hours. I he culture was terminated and the MTT crystals were dissolved in 100 ul of acidified isopropanoi. After one hour, the optical density was recorded using IT.ISA plate reader at a dual wavelength of 570 nm and 620 nm reference filter. Data were expressed as Stimulation Index (S.I.) i.e. ratio of the mean O. D. of experimental cultures (with lest antigen) to the mean 0.1). of control cultures (without antigen). S.I. ureatcr than or equal to 2 was considered as positive stimulation index. Example 6: Statistical methods: Analvsis of variance (ANOVA) as a test of statistical significance was performed using an online software (http://www.phvsics.csbsiu.edu/stats/ANOVA.html) to calculate the p values and determine if there was any difference between different patient categories with respect to each antigen tested. Ihc 95% confidence intervals for means were also determined for each set of data. Differences between groups were considered statistical^ significant if the 95% confidence interval limits did not overlap. To ascertain the results obtained h\ ANOVA. we also carried out Kruskal Wallis non-parametric test 1 http://department.obQ.cuhk.edu.hk/ResearchSupport/KruskallWallis.ASP). Additionally. v\e also carried out t tests for paired comparison of means. p-'0.05 was considered statistically significant. Example 7: Genetic variation in the PPE ORE, Rv26G8 PH/PPE genes are predicted to be a source of antigenic variability of M. tuberculosis and polymorphism in a few of them based upon variation in the number of repeat sequences has already been reported [6]. We analyzed the PPE gene. Rv2608 of the major polymorphic tandem repeat (MPTR) subclass by PCR-R1T.P to score for the presence of genetic variation in different clinical isolates. The 1,7 kb amplicon was digested with Sau?\\l and the digest was electrophoresed on a 10% polyacrylamide gel. 16% of the clinical isolates showed a deviation trotn the normal band pattern. Figure I gives the complete summary of the polymorphism obtained in 30 different clinical isolates, fhe disappearance of restriction fragments \\a is the predicted variable region ol'the 1'1'F ORFs. It was therefore important to further e\aluate R\260K in terms of its ahtlitj to elicit B and T cell response in order to studv its role as a possible antigen for immune suneiDance. Example S: Expression and purification of the rPI'E protein: To evaluate the antigenic ahiliu of Rv2608. the corresponding gene was expressed in A" coli BF21 cells and purified as a 6X llis-tag fusion protein. Purified recombinant Rv2d()8 was fractionated by electrophoresis on a 12% polyaerylamide gel. A single band corresponding to 59.6 kDa protein was observed upon staining the gel with Coomassie Brilliant Blue dye (Figure 2). 'I he expression of the gene was confirmed by probing the membrane containing the total cellular protein of /-.'. coli BI.21 cells harboring the Rv2608 construct with anti-llistidine antibody. There v\as no leaky expression of the protein in uninduced cells. The recombinant protein was largely present in the insoluble fraction and was therefore purified in the presence of 8M urea (Figure 2. LaneH). The \ield of the protein was 6mg.'litrc of culture. The recombinant protein was dialy/ed o\ernight and used for immunoreaetix it\ analysis. Example 9: Design of synthetic peptides based on antigenicity prediction of Rv26U8: hi-siiico analysis of Rv2608 revealed the presence of two regions ol'high antigenicity: I wo amino acid stretches (37 amino acids and 25 amino acids) corresponding to important antigenic epitopes within R\2(S08 were selected for peptide synthesis (Figure 3a). Additional eight overlapping regions (Figure 3b) which were essentially the subsets of the two main peptides were also selected for peptide synthesis. These peptides were u>ed to map the antigenic domains of the protein. Tabic 1 shows the amino acid sequences of all the 10 synthetic peptides used in the present study. 1 he peptides were part of the C terminal region of Rv2608 and apan from the high antigenic index also possessed the repeat motif Gly-X-Cily-Asn-X-GIy. characteristic of the PPKMPTR gene lamih. Example 10: rl'PK protein slums positive reactivity to sera from different categories of TB patients: The humoral response to the recombinant PPF protein was characterized by measuring serum IgG antibodies to the protein using F.LISA. Antibody response was analyzed as a function of mean absorbance at 492nni. Recombinant HspIO. a major antigen of M. tuberculosis v\as used tor comparison u\" the response to the rl'PF protein. It was observed that for all the patient categories, .serum reactivity to rRv2608 was equal to or higher than the response to HspIO (p>0.05. indicating no difference between the response to HSPIO and Rv2608) (Figure 4). Healthy controls also showed some reactivity to the recombinant protein, however the response however was significant!} less when compared to that of patients (p=0.00()2 using student's t test as a test of statistical significance for paired comparison of means between the patients and healthy controls). Example 11: Synthetic peptides corresponding to regions of high antigenicity elicit strong humoral immune response in patients with relapsed TB infection: Having shown that the recombinant protein coded by R\2608 elicited an antibody response which was equal to or higher than that elicited by HspIO antigen in all the categories of I B patients selected for the study, we tried to dissect differential responses if any as a function of patient category For this, synthetic peptides spanning the two major antigenic regions within Rv2608 (PI and P2) were used in }■',[.ISA (fable 1). '['he results suggest that these peptides strongly react with patient sera (Figure 5) and hence the protein must be generating a strong humoral response in the host. Since a positive response was obtained with the peptides ] and 2. patient sera were also tested for reactivity against the short overlapping peptide sequences la. lb. le. Id. 1c which were all components of peptide 1 and 2a. 2b and 2c which were a part of peptide 2. j he results obtained indicate that e\en these overlapping peptide stretches react equally well with patient sera. Fxaet mapping of the antigenic region was not possible as most of the peptides showed a similar response, fhis was a reflection of the fact that the (ily-X-tih- '\sn-X-Gl\ repeat motifs were present in all the peptides. Very interestingly, there was a significantly varied response to the peptides in different categor\ of TB patients which was not so when the complete recombinant Rv260X protein was used. The peptides could clearly distinguish between the patient categories (pO.001 using ANOVA for each peptide antigen) (Table 2). While humoral response observed in ease of fresh infection cases (Category I) was similar to that of extrapulmonary TB patients (Category 111), category II or the relapsed eases showed an unusually high antibody response to all the peptides. The response of Category II patients was significantly higher than Categon I or III ip Kvamplc 12: The T cell response of TB patients to Rv26()8 peptide antigens was low and the differences between various categories of patients were not evident: 1 cell proliferation assays were carried out to evaluate the response to different s\nlhetie peptides. The overall T cell response of patients to these peptides was very low (S.l.0.05. using ANOVA and Kruskal Wall is test) at least for peptide 1 and its derivatives. Peptide 2 and its derivatives exhibited a higher response in fresh infection cases as against relapsed and extrapulmonary cases (p INFERENCES 1. Poulet S and Cole ST. Characterization of the highly abundant polymorphic GC-rieh-repetitive sequence (PGRS) present in Mycobacterium tuberculosis. Arch Microbiol 1995: 163:87-95. 2. Abou-/eid C. Garbc T. Lathigra R. W'iker IIG. Harboe M. Rook G.V Young DB. Genetic and immunological analysis of Mycobacterium luberculosis tibronectin- binding proteins. Infect Iminun 1991: 59:27I2-K. 3. Cole ST. Broseh R. Parkhill J. Gamier T ei ul. Deciphering the biology of Mycobacterium luberculosis from the complete genome sequence. Nature 1998: .393:537- 44. 4. Cole ST. Comparative and functional genomics of the Mycobtn^er'mm tuhennlosis complex. Microbiology 2002: 148:2919-28. 5. Vega-Lopez [•'. Brooks LA. Dockrell HM. Dc Smet K.A. Thompson JR. Ilussain R. Stoker \'G. Sequence and immunological characterization of a serine-rich antigen from Mycobacterium leprae. Infect Immun 1993: 61:2145-53. 6. Fspitia C. I.aclctte ,IP. Mondragon-Palomino M. Amador A. Campuzano J. Martens A. Singh M. Cicero R. /hang Y. Moreno C. The PF-PGRS glycinc-rich proteins of Mycobacterium tuberculosis: a new family of llbronectin-binding proteins'.' Microbiology 1999: 145:34X7-95. 7. Brennan M.I. Delogu G. Chen Y. Bardarov S. Kriakov ,1. Alavi M. Jacobs WR Jr. L\idence that mycobacterial PL_PGRS proteins are cell surface constituents that influence interactions with other cells, infect Immun 2001: 69:7326-33. 8. Sampson SL. Lukey P. Warren RM. \an Llelden PP. Richardson M. Lvcrett MJ. 2001. Lxpression. characterization and subcellular localization ol the Mycobacterium tuberculosis PPL L>ene Rvl9l 7e. Tuberculosis (Fdinb) 2001: XI :305-l 7. 9. Banu S. Ilonore N: Smnl-Jounis B. 1'hilpoll I.). Pre\ost MC. Cole ST. 2002. Are the PE-PGRS proteins of Mycobacterium luherculosis \ariable surface antigens.' Mol Microbiol 2002:44:4-19. 10. Singh KK. Zhang X. Patihandla AS. Cliien P Jr. I.aal S. Antigens of Mycobacterium luherculosis expressed during preclinical tuberculosis: serological immunodominance of proteins wilh repetitive amino acid sequences. Infect Immun 2001; 69:4185-91. 11. Delogu O. Brennan MJ. Comparative immune response to PE and PE_PGRS antigens of Mycobacterium tuherculo\is. Infect Immun 2001: 69:5606-1 1. 12. Ramakrishnan I.. I-'ederspiel NA. I'alkou S. Granuloma-specific expression of M\cobaetcrium virulence proteins from the glyeine-rich PE-P(iRS family. Science 2000: 288:1436-9. 13. Choudhan RK. Pullakhandam R. Hhtesham NZ- and Hasnain SI:. Expression and characleri/alion of R\2430c. a novel immunodominant antigen of Mycobacterium tuberculosis. Protein Kxprcsion and Purification 2004: 00:00-000 (In Press). 14. Choudhan RKC. Mukhopadhyay S. Chakhaivar P. Sharma N. Murlhy KJR. Katoch VVI and Hasnain SH. PRE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B cell response. Infect Immun 2003: 71: 6338-43. 15. Chakhaiyar P and Hasnain SK. Defining the mandate of tuberculosis research in a post genomic era. Medical Principles and Practice 2004; 00:00-000 (In Press). 16. Van de l.oosdrecht A A. Beelen RH. Ossenkoppele G.I. Broekhoven MG. Langenhuijsen MM. A tetrazolium-based colorimetric MTT assay to quantitatc human monoevte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods 1994: 174:311-20. 17. Young DB. Garbe I R. Ileal shock proteins and antigens of Mycobacterium luherculosis. Infect Immun 1991: 59:30X6-93. 18. Migue/ J. Faferte J. Tejero Y. Gonzalez G. Otero AJ. Rivera J. Duarte C. F\aluation of the serologic response against two consensus V3 loop peptides from human immunodeficiency virus-1 in Cuban patients, hit .1 Infect Dis 1998: 2:221-5. 19. Filjeipist JA. frybala F. Svennerholm B. Jeansson S. Sjogren-Jansson F. Bergstrom f. Localization of type-specific epitopes of herpes simplex virus Type 2 gKcoprolein G recognized by human and mouse antibodies, ,1 Gen Virol 1998: 79:1215-24, 2(1. Benite/ J. Palenzuela D, Ri\'cro J. Gavilondo JV. A recombinant protein based immunoassay for the combined detection of antibodies to HIV-I. II1V-2 and IITFV-I. J Virol Methods 1998: 70:85-9. 21. Wilkinson RJ. Vordermeier HM. Wilkinson KA. Sjolund A. Moreno C. Pasvol G. ivanvi .1. Peplide-specific T cell response to Mycobacterium tuberculosis: clinical spectrum, compartmenlalization. and effect of chemotherapy, .f Infect Dis 1998: 178:760-8, 22. Young D. Kent L. Rees A. I.amb J. Ivanvi J. Immunological activity of a 38-kilodalton protein purified, from Mycobacterium tuberculosis. Infect Immun 1986: 54:177-83. 23. Andersen AH. Hansen FB. Structure and mapping of antigenic domains of protein antigen b. a 38.000-molecular-weighi protein of Mycobacterium tuberculosis. Infect Immun 1989:57:2481-8. 24. Bothamley GIF Sehreuder GM, de Vries RR. Ivanvi .1. Association of antibody responses to the 19-k.Da antigen of Mycobacterium tuberculosis and the F1LA-DQ locus. .Unfed Dis 1993: 167:992-3, 25. Fcvitskaya ,1. Coram M. Fevitsky V. imreh S. Steigeruald-Mullcn PM. Klein G. Kurilla MG. Masueci MG. Inhibition of antigen processing by the internal repeat region of the Fpstein-Elarv virus nuclear antigen. Nature 1995: 375:685-8. We Claim: 1. Peptide antigens of SEQ II) Nus. I to i 0. wherein the antigens elicit high humoral immune response and low T-cell response. 2. A repeal motif Gly-X-Gly-Asn-X-Gly of'SHQ ID No. 11. having "X" represented by am of the amino acid residues, wherein the repeat motif elicits high humoral immune response and low J -cell response. 3. The peptide antigens of SFQ II) Nos. I to 10 and the repeat motif Gly-X-Gly- Asn-X-GIy of SI/.Q ID No. 11 substantially as herein described with reference to the accompanying drawings and examples.

Documents:

709-che-2004 abstract duplicate.pdf

709-che-2004 abstract.pdf

709-che-2004 claims duplicate.pdf

709-che-2004 claims.pdf

709-che-2004 correspondence others.pdf

709-che-2004 correspondence po.pdf

709-che-2004 description (complete) duplicate.pdf

709-che-2004 description (complete).pdf

709-che-2004 drawings duplicate.pdf

709-che-2004 drawings.pdf

709-che-2004 form-1.pdf

709-che-2004 form-13.pdf

709-che-2004 form-18.pdf

709-che-2004 form-26.pdf

709-che-2004 form-3.pdf

709-che-2004 form-5.pdf

709-che-2004 petition.pdf