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Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test.

Silva BC, Grassi MF, Coutinho R, Mascarenhas RE, Olavarria VN, Coutinho-Borgo A, Kalil J, Cunha Neto E, Fonseca SG - Mem. Inst. Oswaldo Cruz (2014)

Bottom Line: We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm.Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors.The response to GroEL2 (463-477) was only observed in the TST-positive group.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunologia, Instituto do Coração, São Paulo, SP, Brasil.

ABSTRACT
The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

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: number of Mycobacterium tuberculosis (MTB) peptidesrecognised by tuberculin skin test (TST)-positive (TST+) and TST-negative(TST-) donors. Peripheral blood mononuclear cells (PBMCs) from 16 TST-positiveand 16 TST-negative healthy donors were assayed with seven human leukocyteantigen-DR promiscuous peptides derived from MTB protein sequences. Eachpeptide was tested individually at concentration of 5 µM and the number of spotforming cells (SFC) interferon-γ/106 PBMC were calculated according to thefollowing equation: (average spots in duplicate peptide wells - average spotsin duplicate control wells) x 5. Positive responses were ≥ 30 SFC/106 PBMC. Thecolumns represent the number of TST-positive and TST-negative donors testedthat were negative or able to show positive responses in the ELISPOT assay inresponse to different numbers of peptides (p = 0.01, Mann-WhitneyU test).
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f01: : number of Mycobacterium tuberculosis (MTB) peptidesrecognised by tuberculin skin test (TST)-positive (TST+) and TST-negative(TST-) donors. Peripheral blood mononuclear cells (PBMCs) from 16 TST-positiveand 16 TST-negative healthy donors were assayed with seven human leukocyteantigen-DR promiscuous peptides derived from MTB protein sequences. Eachpeptide was tested individually at concentration of 5 µM and the number of spotforming cells (SFC) interferon-γ/106 PBMC were calculated according to thefollowing equation: (average spots in duplicate peptide wells - average spotsin duplicate control wells) x 5. Positive responses were ≥ 30 SFC/106 PBMC. Thecolumns represent the number of TST-positive and TST-negative donors testedthat were negative or able to show positive responses in the ELISPOT assay inresponse to different numbers of peptides (p = 0.01, Mann-WhitneyU test).

Mentions: Next, we tested if the IFN-γ response to the seven peptides could discriminate betweenTST-positive and TST-negative healthy donors. The mean size (± standard deviation) ofPPD induration detected for TST-negative individuals was 0.71 ± 2.02 mm and forTST-positive individuals was 11.14 ± 4.07 mm. PBMCs from the 16 TST-positive and 16TST-negative individuals were tested in the presence of each peptide for an IFN-γELISPOT assay. The T cell responses are shown in TableII. The frequency of peptide recognition by the TST-positive group wassignificantly higher than the TST-negative group. Eighty-eight percent (14 out of 16donors) of TST-positive donors recognised at least one peptide and 31% (5 out of 16donors) had responses to three or more peptides, while in the TST-negative group, 31% (5out of 16 donors) responded to any peptides and only 12% (2 out of 16 donors) respondedto three or more peptides (p = 0.002) (Fig.1).


Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test.

Silva BC, Grassi MF, Coutinho R, Mascarenhas RE, Olavarria VN, Coutinho-Borgo A, Kalil J, Cunha Neto E, Fonseca SG - Mem. Inst. Oswaldo Cruz (2014)

: number of Mycobacterium tuberculosis (MTB) peptidesrecognised by tuberculin skin test (TST)-positive (TST+) and TST-negative(TST-) donors. Peripheral blood mononuclear cells (PBMCs) from 16 TST-positiveand 16 TST-negative healthy donors were assayed with seven human leukocyteantigen-DR promiscuous peptides derived from MTB protein sequences. Eachpeptide was tested individually at concentration of 5 µM and the number of spotforming cells (SFC) interferon-γ/106 PBMC were calculated according to thefollowing equation: (average spots in duplicate peptide wells - average spotsin duplicate control wells) x 5. Positive responses were ≥ 30 SFC/106 PBMC. Thecolumns represent the number of TST-positive and TST-negative donors testedthat were negative or able to show positive responses in the ELISPOT assay inresponse to different numbers of peptides (p = 0.01, Mann-WhitneyU test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325617&req=5

f01: : number of Mycobacterium tuberculosis (MTB) peptidesrecognised by tuberculin skin test (TST)-positive (TST+) and TST-negative(TST-) donors. Peripheral blood mononuclear cells (PBMCs) from 16 TST-positiveand 16 TST-negative healthy donors were assayed with seven human leukocyteantigen-DR promiscuous peptides derived from MTB protein sequences. Eachpeptide was tested individually at concentration of 5 µM and the number of spotforming cells (SFC) interferon-γ/106 PBMC were calculated according to thefollowing equation: (average spots in duplicate peptide wells - average spotsin duplicate control wells) x 5. Positive responses were ≥ 30 SFC/106 PBMC. Thecolumns represent the number of TST-positive and TST-negative donors testedthat were negative or able to show positive responses in the ELISPOT assay inresponse to different numbers of peptides (p = 0.01, Mann-WhitneyU test).
Mentions: Next, we tested if the IFN-γ response to the seven peptides could discriminate betweenTST-positive and TST-negative healthy donors. The mean size (± standard deviation) ofPPD induration detected for TST-negative individuals was 0.71 ± 2.02 mm and forTST-positive individuals was 11.14 ± 4.07 mm. PBMCs from the 16 TST-positive and 16TST-negative individuals were tested in the presence of each peptide for an IFN-γELISPOT assay. The T cell responses are shown in TableII. The frequency of peptide recognition by the TST-positive group wassignificantly higher than the TST-negative group. Eighty-eight percent (14 out of 16donors) of TST-positive donors recognised at least one peptide and 31% (5 out of 16donors) had responses to three or more peptides, while in the TST-negative group, 31% (5out of 16 donors) responded to any peptides and only 12% (2 out of 16 donors) respondedto three or more peptides (p = 0.002) (Fig.1).

Bottom Line: We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm.Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors.The response to GroEL2 (463-477) was only observed in the TST-positive group.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunologia, Instituto do Coração, São Paulo, SP, Brasil.

ABSTRACT
The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Show MeSH
Related in: MedlinePlus